Orchids Phytochemistry, Biology and Horticulture: Jean-Michel Mérillon Hippolyte Kodja

Reference Series in Phytochemistry
Series Editors:
J.-M. Mérillon · K. G. Ramawat
Jean-Michel Mérillon
Hippolyte Kodja
Editors
Orchids
Phytochemistry,
Biology and
Horticulture
Fundamentals and Applications
Reference Series in Phytochemistry
Series Editors
Jean-Michel Mérillon, Faculty of Pharmaceutical Sciences, Institute of Vine and
Wine Sciences, University of Bordeaux, Villenave d’Ornon, France
Kishan Gopal Ramawat, Department of Botany, University College of Science,
M. L. Sukhadia University, Udaipur, Rajasthan, India
This series provides a platform for essential information on plant metabolites and
phytochemicals, their chemistry, properties, applications, and methods.
By the strictest definition, phytochemicals are chemicals derived from plants.
However, the term is often also used to describe the large number of secondary
metabolic compounds found in and derived from plants. These metabolites exhibit a
number of nutritional and protective functions for human wellbeing and are used
e.g. as colorants, fragrances and flavorings, amino acids, pharmaceuticals, hor-
mones, vitamins and agrochemicals.
The series offers extensive information on various topics and aspects of phyto-
chemicals, including their potential use in natural medicine, their ecological role,
role as chemo-preventers and, in the context of plant defense, their importance for
pathogen adaptation and disease resistance. The respective volumes also provide
information on methods, e.g. for metabolomics, genetic engineering of pathways,
molecular farming, and obtaining metabolites from lower organisms and marine
organisms besides higher plants. Accordingly, they will be of great interest to readers
in various fields, from chemistry, biology and biotechnology, to pharmacognosy,
pharmacology, botany and medicine.
The Reference Series in Phytochemistry is indexed in Scopus.
More information about this series at http://link.springer.com/series/13872

Jean-Michel Mérillon • Hippolyte Kodja
Editors
Orchids Phytochemistry,
Biology and Horticulture
Fundamentals and Applications
With 122 Figures and 50 Tables

Editors
Jean-Michel Mérillon Hippolyte Kodja
Faculty of Pharmaceutical Sciences Qualisud, Université de La Réunion
Institute of Vine and Wine Sciences Université Montpellier
University of Bordeaux CIRAD, Institut Agro, Avignon Université
Villenave d’Ornon, France Montpellier, France
ISSN 2511-834X ISSN 2511-8358 (electronic)

ISBN 978-3-030-38391-6 ISBN 978-3-030-38392-3 (eBook)
ISBN 978-3-030-38393-0 (print and electronic bundle)
https://doi.org/10.1007/978-3-030-38392-3
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Preface
Orchidaceae is one of the largest flowering plant families along with Asteraceae,
comprising of 28000 species in about 1000 genera. Orchids have attained significant
economic importance as potted plants and cut flowers due to very attractive flowers
in many species, and are finding their way in cosmetic, food (as fragrance, e.g.,
vanilla), and medicine industry. This book encompasses all aspect of biodiversity,
biology, biotechnology, and varied applications of orchids from horticulture to
medicine.
The book is a timely compilation of recent developments on orchids and is
divided into six parts, covering the entire gamut of orchid research and applications:
Part I: Biogeography, Biodiversity, and Environmental Factors

Part II: Biology
Part III: Horticulture
Part IV: Agri-food Applications
Part V: Phytochemistry and Medicinal Properties
Part VI: Cosmetic Applications
The 26 well-illustrated chapters are prepared by experts working in this field, who
have been selected from all over the world. This book is envisioned as a reference
work providing comprehensive information on orchids. It is intended to serve the
needs of graduate students, scholars, and researchers in the field of botany, horticul-
ture, pharmacy, cosmetology, biotechnology, and phytochemistry; industrial scien-
tists; and those involved in marketing flowers and phytochemicals, plants, and plant
extracts.
We would like to acknowledge the cooperation, patience, and support of our
contributors who have put their serious efforts to ensure the high scientific quality of
this book with up-to-date information. We are thankful to the staff at Springer,
namely Dr. S. Blago and J. Klute, for their professional support in this project.
Villenave d’Ornon, France Jean-Michel Mérillon

Saint Denis, La Réunion, France Hippolyte Kodja
January 2022 Editors
v
Contents
Part I Biogeography, Biodiversity, and Environmental Factors ... 1
1 The Role of Ecological Factors in Distribution and Abundance of

Terrestrial Orchids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
Vladan Djordjević and Spyros Tsiftsis
2 Which Environmental Factors Drive Distribution of Orchids?

A Case Study from South Bohemia, Czech Republic . . . . . . . . . . . 73
Zuzana Štípková, Dušan Romportl, and Pavel Kindlmann
3 Diversity, Ecology, and Conservation of Mauritius Orchids ..... 107

Cláudia Baider and F. B. Vincent Florens
4 Diversity of Orchids from Continental Sub-Saharan Africa . . . . . 135

Adama Bakayoko, Noufou Doudjo Ouattara, Akoua Clémentine Yao,
Djah François Malan, Danho Fursy-Rodelec Neuba,
Bi Fézan Honora Tra, and Tanoh Hilaire Kouakou
5 Orchid Biodiversity and Genetics . . . . . . . . . . . . . . . . . . . . . . . . . . 153

Seeja G and Sreekumar S
Part II Biology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 173
6 Orchid-Associated Bacteria and Their Plant Growth

Promotion Capabilities . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 175
Héctor Herrera, Alejandra Fuentes, Javiera Soto, Rafael Valadares,
and Cesar Arriagada
7 Mycorrhiza in Orchids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 201

Saranjeet Kaur
8 Phytoalexins in Orchids ................................. 215

Saranjeet Kaur
vii
viii Contents
Part III Horticulture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 223
9 Micropropagation of Some Orchids and the Use of

Cryopreservation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 225
Kanchit Thammasiri, Nipawan Jitsopakul, and Sasikarn Prasongsom
10 Cymbidium: Botany, Production, and Uses . . . . . . . . . . . . . . . . . . . 261

Ram Pal, N. K. Meena, R. P. Pant, and M. Dayamma
11 Biotechnology Approaches on Characterization, Mass Propagation,

and Breeding of Indonesian Orchids Dendrobium lineale (Rolfe.)
and Vanda tricolor (Lindl.) with Its Phytochemistry . . . . . . . . . . . . 299
Endang Semiarti, Aziz Purwantoro, and Ika Puspita Sari
12 Preferences of Orchid Consumers and the Substitute

Products’ Influences . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 313
Adilson Anacleto and Luciane Scheuer
Part IV Agri-food Applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 327
13 Vanilla: Culture, Reproduction, Phytochemistry, Curing,

Pest, and Diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 329
Keshika Mahadeo, Tony L. Palama, Bertrand Côme, and
Hippolyte Kodja
14 Vanillin: Biosynthesis, Biotechnology, and Bioproduction . . . . . . . 341

Shahnoo Khoyratty, Rob Verpoorte, and Hippolyte Kodja
Part V Phytochemistry and Medicinal Properties ............. 359
15 Ethnobotany and Recent Advances in Indian Medicinal

Orchids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 361
Ram Pal, N. K. Meena, M. Dayamma, and D. R. Singh
16 Traditionally Used Medicinal Dendrobium: A Promising Source

of Active Anticancer Constituents . . . . . . . . . . . . . . . . . . . . . . . . . . 389
Mukti Ram Paudel, Hari Datta Bhattarai, and Bijaya Pant
17 A Review on Phytochemistry, Nutritional Potential, Pharmacology,

and Conservation of Malaxis acuminata: An Orchid with
Rejuvenating and Vitality Strengthening Properties . . . . . . . . . . . 415
Renu Suyal, Sandeep Rawat, R. S. Rawal, and Indra D. Bhatt
18 Phytochemistry, Pharmacology, and Conservation of

Ansellia africana: A Vulnerable Medicinal Orchid of Africa . . . . . 435
Paromik Bhattacharyya, Shubhpriya Gupta, and Johannes Van Staden
Contents ix
19 Dendrobium sp.: In vitro Propagation of Genetically Stable

Plants and Ethnomedicinal Uses . . . . . . . . . . . . . . . . . . . . . . . . . . . 453
Leimapokpam Tikendra, Nandeibam Apana,
Angamba Meetei Potshangbam, Thoungamba Amom,
Ravish Choudhary, Rajkumari Sanayaima, Abhijit Dey, and
Potshangbam Nongdam
20 Eulophia spp.: In Vitro Generation, Chemical Constituents, and
Pharmacological Activities . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 495
Varsha Shriram and Vinay Kumar
21 Cyrtopodium glutiniferum, an Example of Orchid Used in Folk
Medicine: Phytochemical and Biological Aspects . . . . . . . . . . . . . . 517
Carlos Fernando Araujo-Lima, Israel Felzenszwalb, and
Andrea Furtado Macedo
22 Phenanthrenes from Orchidaceae and Their Biological
Activities . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 533
Andrea Vasas
23 Orchids of Genus Bletilla: Traditional Uses, Phytochemistry,
Bioactivities, and Commercial Importance . . . . . . . . . . . . . . . . . . . 573
Hari Prasad Devkota, Rajan Logesh, Anjana Adhikari-Devkota, and
Mukti Ram Paudel
24 Orchids of Genus Vanda: Traditional Uses, Phytochemistry,
Bioactivities, and Commercial Importance . . . . . . . . . . . . . . . . . . . 591
Hari Prasad Devkota, Anjana Adhikari-Devkota, Rajan Logesh,
Tarun Belwal, and Bijaya Pant
Part VI Cosmetic Applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 607
25 Orchid Extracts and Cosmetic Benefits . . . . . . . . . . . . . . . . . . . . . 609

Mayuree Kanlayavattanakul and Nattaya Lourith
26 Orchids from Basilicata: The Scent . . . . . . . . . . . . . . . . . . . . . . . . 627
Maurizio D’Auria, Simonetta Fascetti, Rocco Racioppi,
Vito Antonio Romano, and Leonardo Rosati
Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 649
About the Editors
Professor Dr. Jean-Michel Mérillon received his MPharma (1979) and PhD
(1984) from the University of Tours, France. He joined the University of Tours as
assistant professor in 1981, became associate professor in 1987. In 1993, he moved
to the Faculty of Pharmacy, University of Bordeaux, France, accepting a position as
full professor. He has been the director of the research group on biologically active
plant substances for over 15 years, at the Institute of Vine and Wine Sciences
(University of Bordeaux, France), which comprises 25 scientists and research
students. The group has been working on phenolic compounds from vine and
wine for many years, mainly complex stilbenes and their involvement in health.
He is involved in developing courses on plant biology, natural bioactive compounds,
and biotechnology. Prof. Mérillon has published more than 180 research papers in
internationally recognized journals, and has co-edited books and reference works on
secondary metabolites and biotechnology. In 2004, he founded the technology
transfer unit “Polyphenols Biotech,” providing support for R&D programs for
SMEs and major groups in the cosmetic, pharmaceutical, agricultural, and health-
nutrition sectors. He is currently the manager of this unit.
Professor Dr. Hippolyte Kodja received his PhD in plant cell biotechnology from
the Faculty of Pharmacy, University of Tours, France, in 1988, and later, he joined
the University of La Reunion as Professor of Plant Physiology. With a vast teaching
and research experience, Prof. Kodja has been exploring the contribution of fungal
and bacterial endophytes of vanilla on biosynthesis of vanilla flavor and aroma
metabolites, and he is also interested in the characterization of diversity in vanilla
flavor production from Madagascar, Maurice, and La Réunion by phytochemical,
microbiology, and sensory analyses, and in the comparative inventories of mycor-
rhizal fungi of vanilla. Prof. Kodja has published several research papers in interna-
tionally recognized journals on plant biology, physiology, and biochemistry, and has
co-authored a chapter in the volume Fungal Metabolites from the Reference Series
in Phytochemistry.
xi
Contributors
Anjana Adhikari-Devkota Graduate School of Pharmaceutical Sciences, Kuma-

moto University, Kumamoto, Japan
Thoungamba Amom Department of Biotechnology, Manipur University,

Canchipur, Manipur, India
Adilson Anacleto State University of Paraná, Paranaguá, Brazil
Nandeibam Apana Department of Biotechnology, Manipur University, Canchipur,

Manipur, India
Carlos Fernando Araujo-Lima Laboratory of Environmental Mutagenesis,
Department of Biophysics and Biometry, Rio de Janeiro State University, Rio de
Janeiro, Brazil
Roberto Alcantara Gomes Institute of Biology, Universidade do Estado do Rio de
Janeiro, UERJ, Rio de Janeiro, Brazil
Cesar Arriagada Laboratorio de Biorremediación, Facultad de Ciencias

Agropecuarias y Forestales, Departamento de Ciencias Forestales, Universidad de
La Frontera, Temuco, Chile
Cláudia Baider The Mauritius Herbarium, Agricultural Services, Ministry of

Agro-Industry and Food Security, Réduit, Mauritius
Adama Bakayoko UFR des Sciences de la Nature (SN), Université NANGUI
ABROGOUA, Abidjan, Ivory Coast
Centre Suisse de Recherches Scientifiques en Côte d’Ivoire, Abidjan, Ivory Coast
Tarun Belwal College of Biosystems Engineering and Food Science, Key Labo-
ratory for Agro-Products Postharvest Handling of Ministry of Agriculture and Rural
Affairs, Zhejiang Key Laboratory for Agro-Food Processing, Zhejiang University,
Hangzhou, China
Indra D. Bhatt G.B. Pant National Institute of Himalayan Environment, Almora,
Uttarakhand, India
xiv Contributors
Paromik Bhattacharyya Research Centre for Plant Growth and Development,

School of Life Sciences, University of KwaZulu-Natal Pietermaritzburg, Scottsville,
South Africa
Hari Datta Bhattarai Central Department of Botany, Tribhuvan University, Kath-
mandu, Nepal
Ravish Choudhary DSST, Indian Agricultural Research Institute, New Delhi,
India
Bertrand Côme La Vanilleraie, Sainte-Suzanne, La Réunion, France
Maurizio D’Auria Dipartimento di Scienze, Università della Basilicata, Potenza,
Italy
M. Dayamma ICAR-National Research Centre for Orchids, Darjeeling Campus,
Darjeeling, West Bengal, India
Hari Prasad Devkota Graduate School of Pharmaceutical Sciences, Kumamoto
University, Kumamoto, Japan
Abhijit Dey Department of Life Sciences, Presidency University, Kolkata, India
Vladan Djordjević Faculty of Biology, Institute of Botany and Botanical Garden,
University of Belgrade, Belgrade, Serbia
Simonetta Fascetti School of Agricultural Forestry, Food, and Environmental
Science, Università della Basilicata, Potenza, Italy
Israel Felzenszwalb Laboratory of Environmental Mutagenesis, Department of
Biophysics and Biometry, Rio de Janeiro State University, Rio de Janeiro, Brazil
Roberto Alcantara Gomes Institute of Biology, Universidade do Estado do Rio de
Janeiro, UERJ, Rio de Janeiro, Brazil
F. B. Vincent Florens Tropical Island Biodiversity, Ecology and Conservation Pole
of Research, Department of Biosciences and Ocean Studies, University of Mauritius,
Réduit, Mauritius
Alejandra Fuentes Laboratorio de Biorremediación, Facultad de Ciencias
Seeja G Department of Plant Breeding and Genetics, College of Agriculture,
Ambalavayal, India
Shubhpriya Gupta Research Centre for Plant Growth and Development, School of
Life Sciences, University of KwaZulu-Natal Pietermaritzburg, Scottsville, South
Africa
Héctor Herrera Laboratorio de Biorremediación, Facultad de Ciencias
Contributors xv
Nipawan Jitsopakul Department of Plant Science, Textile and Design, Faculty of

Agriculture and Technology, Rajamangala University of Technology Isan, Surin,
Thailand
Mayuree Kanlayavattanakul School of Cosmetic Science, Mae Fah Luang Uni-
versity, Chiang Rai, Thailand
Phytocosmetics and Cosmeceuticals Research Group, Mae Fah Luang University,
Chiang Rai, Thailand
Saranjeet Kaur Department of Chemistry, University Institute of Sciences, Chan-
digarh University, Mohali, Punjab, India
Shahnoo Khoyratty Natural Products Laboratory, Institute of Biology, Leiden
University, Leiden, The Netherlands
Qualisud, Université de La Réunion, Université Montpellier, CIRAD, Institut Agro,
Avignon Université, Montpellier, France
Pavel Kindlmann Global Change Research Institute, Academy of Sciences of the
Czech Republic, České Budějovice, Czech Republic
Institute for Environmental Studies, Faculty of Science, Charles University,
Benátská 2/Prague 2, Czech Republic
Hippolyte Kodja Qualisud, Université de La Réunion, Université Montpellier,
CIRAD, Institut Agro, Avignon Université, Montpellier, France
Tanoh Hilaire Kouakou UFR des Sciences de la Nature (SN), Université
NANGUI ABROGOUA, Abidjan, Ivory Coast
Vinay Kumar Department of Biotechnology, Modern College of Arts, Science and
Commerce, Savitribai Phule Pune University, Pune, India
Rajan Logesh TIFAC-CORE in Herbal Drugs, Department of Pharmacognosy and
Phytopharmacy, JSS College of Pharmacy (JSS Academy of Higher Education and
Research), Udhagamandalam, Tamil Nadu, India
Nattaya Lourith School of Cosmetic Science, Mae Fah Luang University, Chiang
Rai, Thailand
Andrea Furtado Macedo Integrated Laboratory of Plant Biology, Department of
Botany, Institute of Biosciences, Federal University of Rio de Janeiro State,
UNIRIO, Rio de Janeiro, Brazil
Keshika Mahadeo Qualisud, Université de La Réunion, Université Montpellier,
CIRAD, Institut Agro, Avignon Université, Montpellier, France
Laboratoire de Chimie des Produits Naturels, Université de la Réunion, Faculté des
Sciences et Technologies, 15 Avenue René Cassin, CS 92 003, 97 744 St Denis
Cedex 9, La Réunion, France
xvi Contributors
Djah François Malan UFR des Sciences de la Nature (SN), Université NANGUI
N. K. Meena ICAR-National Research Center on Seed and Spices, Tabiji, Ajmer,
India
Danho Fursy-Rodelec Neuba UFR des Sciences de la Nature (SN), Université
Potshangbam Nongdam Department of Biotechnology, Manipur University,
Noufou Doudjo Ouattara UFR des Sciences de la Nature (SN), Université
Ram Pal ICAR-National Research Centre for Orchids, Darjeeling Campus, Dar-
jeeling, West Bengal, India
Tony L. Palama Université Sorbonne Paris Nord, Laboratoire de Chimie, Struc-
tures, Propriétés de Biomatériaux et d’Agents Thérapeutiques, CSPBAT, CNRS,
UMR 7244, Villetaneuse, France
Bijaya Pant Central Department of Botany, Tribhuvan University, Kathmandu,
Nepal
R. P. Pant ICAR- Indian Agricultural Research Institute, Pusa Campus, New Delhi,
India
Mukti Ram Paudel Central Department of Botany, Tribhuvan University, Kath-
mandu, Nepal
Angamba Meetei Potshangbam Department of Biotechnology, Manipur Univer-
sity, Canchipur, Manipur, India
Sasikarn Prasongsom Pathum Wan District, Bangkok, Thailand
Aziz Purwantoro Faculty of Agriculture, Universitas Gadjah Mada, Yogyakarta,
Indonesia
Ika Puspita Sari Faculty of Pharmacy, Universitas Gadjah Mada, Yogyakarta,
Indonesia
Rocco Racioppi Dipartimento di Scienze, Università della Basilicata, Potenza,
Italy
R. S. Rawal G.B. Pant National Institute of Himalayan Environment, Almora,
Uttarakhand, India
Sandeep Rawat G.B. Pant National Institute of Himalayan Environment, Sikkim
Regional Centre, Gangtok, Sikkim, India
Contributors xvii
Vito Antonio Romano Dipartimento di Scienze, Università della Basilicata,

Potenza, Italy
Dušan Romportl Department of Physical Geography and Geoecology, Faculty of
Science, Charles University, Albertov 6, Czech Republic
Leonardo Rosati School of Agricultural Forestry, Food, and Environmental Sci-
ence, Università della Basilicata, Potenza, Italy
Sreekumar S Biotechnology and Bioinformatics Division, KSCSTE- Jawaharlal
Nehru Tropical Botanic Garden and Research Institute, Thiruvananthapuram, India
Rajkumari Sanayaima DDU College, University of Delhi, New Delhi, India
Luciane Scheuer State University of Paraná, Paranaguá, Brazil
Endang Semiarti Faculty of Biology, Universitas Gadjah Mada, Yogyakarta,
Indonesia
Varsha Shriram Department of Botany, Prof. Ramkrishna More Arts, Commerce
and Science College, Savitribai Phule Pune University, Pune, India
D. R. Singh ICAR-National Research Centre for Orchids, Pakyong, East Sikkim,
Sikkim, India
Javiera Soto Laboratorio de Biorremediación, Facultad de Ciencias Agropecuarias
y Forestales, Departamento de Ciencias Forestales, Universidad de La Frontera,
Temuco, Chile
Zuzana Štípková Global Change Research Institute, Academy of Sciences of the
Czech Republic, České Budějovice, Czech Republic
Renu Suyal G.B. Pant National Institute of Himalayan Environment, Almora,
Uttarakhand, India
Kanchit Thammasiri Department of Plant Science, Faculty of Science, Mahidol
University, Bangkok, Thailand
Leimapokpam Tikendra Department of Biotechnology, Manipur University,
Bi Fézan Honora Tra UFR des Sciences de la Nature (SN), Université NANGUI
Spyros Tsiftsis Department of Forestry and Natural Environment, International
Hellenic University, Drama, Greece
Global Change Research Institute, Academy of Science of the Czech Republic,
Brno, Czech Republic
Rafael Valadares Instituto Tecnologico Vale, Belém, PA, Brazil
xviii Contributors
Johannes Van Staden Research Centre for Plant Growth and Development,
School of Life Sciences, University of KwaZulu-Natal Pietermaritzburg, Scottsville,
South Africa
Andrea Vasas Department of Pharmacognosy, University of Szeged, Szeged,
Hungary
Rob Verpoorte Natural Products Laboratory, Institute of Biology, Leiden Univer-
sity, Leiden, The Netherlands
Akoua Clémentine Yao UFR des Sciences de la Nature (SN), Université NANGUI
Part I
Biogeography, Biodiversity, and
Environmental Factors
The Role of Ecological Factors in
Distribution and Abundance of Terrestrial 1
Orchids
Vladan Djordjević and Spyros Tsiftsis
Contents
1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
2 Elevation, Latitude, Longitude, and the Orchid Species-Area Relationship . . . . . . . . . . . . . . . 7
2.1 Elevation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
2.2 Latitude and Longitude . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
2.3 Orchid Species-Area Relationship . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
3 Climate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
3.1 Temperature . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
3.2 Precipitation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14
3.3 Atmospheric Humidity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
3.4 Light . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16
3.5 Orchid Species Distribution and Its Patterns in a World Subjected to Climate
Change . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19
4 Geological Substrates and Soil Properties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
4.1 Geological Substrates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21
4.2 Soil Properties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25
5 Vegetation Types . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39
5.1 Forest and Scrub Vegetation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40
5.2 Herbaceous Vegetation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45
5.3 Anthropogenic Vegetation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49
6 Effects of Disturbance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 52
7 Orchid Specialists and Generalists . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54
V. Djordjević (*)
Faculty of Biology, Institute of Botany and Botanical Garden, University of Belgrade,
Belgrade, Serbia
e-mail: vdjordjevic@bio.bg.ac.rs
S. Tsiftsis
Department of Forestry and Natural Environment, International Hellenic University, Drama, Greece
Global Change Research Institute, Academy of Science of the Czech Republic, Brno,
Czech Republic
e-mail: stsiftsis@for.ihu.gr
© Springer Nature Switzerland AG 2022 3

J.-M. Mérillon, H. Kodja (eds.), Orchids Phytochemistry, Biology and Horticulture,
Reference Series in Phytochemistry, https://doi.org/10.1007/978-3-030-38392-3_4
4 V. Djordjević and S. Tsiftsis
8 Orchid Mycorrhizal Fungi . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 56

9 Pollination Systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58
10 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 62
Abstract
Distributed throughout the continents, terrestrial orchids are known for their great
species richness and specificity in relation to pollinators and mycorrhizal symbi-
onts. Moreover, a large number of them are rare and sensitive to environmental
changes. This chapter is mainly focused on the terrestrial orchids of Europe and
reviews the major environmental factors affecting the patterns of their distribu-
tion, abundance, and richness (elevation, latitude, longitude, area size, climatic
factors, geological substrates, soil characteristics, vegetation types, effects of
disturbance), as well as the significance of mycorrhizal fungi and pollination
systems. Some new data, especially regarding the responses of orchids to climate
change and their occurrence on specific geological and soil substrates and
vegetation types, are presented. Although the distribution and abundance of
terrestrial orchids are associated with the joint effects of most of the examined
factors, some factors have emerged as crucial, especially on the northern and
southern borders of their distribution. Furthermore, the role of environmental
factors depends largely on the belowground strategies of orchids. The chapter
highlights the importance of exploring the level of specialization of orchids with
respect to habitat conditions as an important basis for their conservation.
Keywords
Orchidaceae · Ecology · Distribution · Elevation · Climate · Geological
substrates · Soil characteristics · Vegetation · Effects of disturbance · Specialists
and generalists
1 Introduction
The family Orchidaceae is one of the most species-rich families in the plant kingdom
and includes approximately 26,000 species within 749 genera [1]. Epiphytic orchids of
tropical and subtropical areas have the largest number of representatives, whereas
terrestrial orchids comprise either one-fourth or one-third of the total number of
described orchid taxa, depending on the resolution of taxonomic issues [2, 3]. Terres-
trial orchids are classified into three out of five subfamilies of the family Orchidaceae
(Cypripedioideae, Orchidoideae, and Epidendroideae) [4]. Representatives of the
family occur on all continents, and the most important centers of their diversity are
Indochina, Southwest Australia, Europe, Northern Asia, and North America [2, 3, 5].
A wide variety of life histories have been identified among terrestrial orchids,
both in terms of their development, morpho-anatomical, and physiological
adaptations, life expectancy and survival of adverse periods during the year and
with respect to their pollination systems and mycorrhizal associations [6, 7].
1 The Role of Ecological Factors in Distribution and Abundance of Terrestrial. . . 5
All representatives of the family Orchidaceae in Europe are terrestrial, belonging to

the life form of cryptophytes, i.e., geophytes. This life form category encompasses
an ecological group of plants whose aboveground parts are completely extinct
during the climatically unfavorable season, whereas the buds from which new
sprouts emerge the next year are found in underground organs (tubers or rhizomes)
in the soil. The evolutionary development of underground organs in terrestrial
orchids has produced variants ranging from rhizomes to tubers (Fig. 1), the most
primitive representatives of European orchids being species of the genera
Cephalanthera, Epipactis, and Cypripedium with short rhizomes [8]. In addition to
the ones mentioned above, other known European genera that include rhizomatous
species are Corallorhiza, Epipogium, Goodyera, Limodorum, and Neottia (Fig. 1a)
[9]. An important moment in the evolution of terrestrial orchids was the development
of tubers, the organ for storing reserves of matter for survival during unfavorable
periods of the year [2]. Among tuberoid orchids, Pseudorchis albida is the most
primitive species, whereas representatives of the genera Coeloglossum, Dactylorhiza,
Nigritella, and Gymnadenia have palmate tubers (Fig. 1b), and species of the genus
Platanthera are characterized by fusiform tubers (Fig. 1c) [2, 8, 10, 11]. Representa-
tives of the genera Anacamptis, Herminium, Himantoglossum, Neotinea, Ophrys,
Orchis, Spiranthes, and Traunsteinera are particularly prominent among the group
of orchids with ovoid tuberoids (Fig. 1d) [2, 8, 11].
Over 300 species of orchids have been reported in Europe, North Africa, and
the Middle East [5]. The estimated orchid species richness in Europe varies
depending on the applied taxonomic concept. According to one author, about
250 species and subspecies from 35 genera of orchids occur in Europe [12].
However, orchids occurring in Europe are not uniformly distributed throughout
the area. The most important center of orchid diversity in Europe is the eastern
Mediterranean area, i.e., the area around the Aegean Sea [5, 13, 14]. Significant
centers of orchid diversity in Europe also include other parts of the Balkan
Peninsula, the Alps, Sicily, and the Caucasus [5]. Among countries with a high
diversity of orchids in Europe, it is especially important to single out Greece, with
193 species and subspecies [13, 14]. Considerable richness of orchid species has
also been reported in Italy (c. 175 species and subspecies) [16]; in Turkey (c. 170
species and subspecies) [17]; on the Iberian Peninsula (122 species and subspe-
cies) [19]; and in Croatia (113 species and subspecies) [15]. On the island of
Corsica, 77 orchid species and a density of 88 orchid species per 1000 km2 have
been recorded, making it one of the areas with the highest known value of the
orchid diversity index [18].
Ophrys, Dactylorhiza, Orchis, Anacamptis, and Epipactis are the genera with the
largest number of species and subspecies in Europe [9, 12, 20]. The genus Ophrys
includes c. 90 species and subspecies [21], which are distributed in Europe, North
Africa, and Southwest Asia [9]. The genus Dactylorhiza is represented by 86 species
and subspecies, which are widespread in Europe, Asia, North Africa, and Alaska,
whereas the genus Orchis is represented by 32 species and subspecies, mostly
distributed in Europe, temperate Asia, and North Africa [12, 13, 20, 21]. The
genus Anacamptis includes 11 species and 7 subspecies, which are distributed in
Europe, North Africa, and Southwest and West Asia [13, 21]. Finally, the genus
Fig. 1 Root systems of terrestrial orchids. (a) rhizome Neottia ovata, (b) palmate tubers
Gymnadenia conopsea, (c) fusiform tubers Platanthera bifolia, (d) ovoid tubers Anacamptis
coriophora (photos V. Djordjević)
Epipactis includes a total of 79 species and subspecies, which are widespread in

Europe, Asia, North Africa, and North America [21, 22].
Terrestrial orchids are known for their complex ecology, rarity, and ability to
inhabit almost all terrestrial ecosystems [3, 6]. Many representatives have limited
distribution due to their mycorrhizal specificity, pollinator specialization, and special
needs when it comes to habitat conditions [7]. Numerous orchids are sensitive to
changes in the ecosystem’s balance, especially changes in the light regime, moisture
content, availability of nutrients, and the level of competition. All of these changes
have a significant impact on the survival and ability of seedlings to germinate and
successfully reach adulthood [3]. Due to changes in ecological factors, land-use
practices, and habitat loss, the ranges and abundance of many terrestrial orchids have
declined [18, 23]. Consequently, many terrestrial orchids are protected by laws and
included in Red Data Books [6]. In general, rarer terrestrial species and those at high
risk of extinction are geographically restricted to a small area and are characterized
by a small population size, have a higher level of specialization in terms of habitat
conditions, and exhibit greater dependence on mycorrhizal symbionts and pollina-
tors [3, 24]. Understanding the role of environmental conditions in determining the
distribution and abundance of orchid species is therefore necessary in order to
properly and adequately organize the protection of these plants [11, 25, 26].
In this chapter, we summarize knowledge about the ecological preferences of
terrestrial orchids, with major emphasis on European orchids. Our main objective
was to provide an overview of environmental factors that significantly affect the
distribution, richness, and abundance of terrestrial orchids. The chapter highlights
the role of elevation, latitude, longitude, the species-area relationship, climatic
factors, geological substrates, soil factors, vegetation types, and effects of distur-
bance. The importance of mycorrhizal associations and pollination systems in orchid
distribution and abundance is also discussed.
2 Elevation, Latitude, Longitude, and the Orchid Species-

Area Relationship
2.1 Elevation
Elevation is one of the most important factors affecting the richness and composition
of orchid species [11, 27]. Investigations of orchid diversity patterns along elevation
gradients have been performed mainly outside Europe, more specifically in the
countries of Asia [27–29], Africa [30], and America [31, 32]. However, several
studies on the relationship between elevation and orchids in Europe indicated that
elevation influences orchid abundance and distribution patterns and affects the
separation of ecological niches of orchid species [11, 24, 25, 33, 34]. Other studies
showed that orchid breeding systems and floral traits [30] and the relative proportion
of food-deceptive orchids [35] also changed with elevation. It can be asserted that the
most important factors responsible for variation of orchid species richness along the
elevation gradient are climatic conditions [27, 28], bearing in mind that as elevation
increases, the air temperature, total atmospheric pressure, and partial pressure of all
atmospheric gasses decrease, whereas precipitation, relative humidity, and UV
radiation usually increase [36]. Researchers found that mean annual temperature
and mean annual rainfall are the most important factors influencing the abundance of
orchid species along the elevational gradient in the Yunnan province of China [28]
and in the central and eastern Himalayas [27]. Other important factors affecting the
variation of orchid species abundance and composition along the elevational gradi-
ent are reduction of land area per bioclimatic unit, differences of nutrient availability,
changes in vegetation types, evolutionary history, ecotone effects, different kinds of
disturbance, and biotic processes [30, 36].
A mid-elevational bulge in orchid diversity was noted in several orchid studies
[27, 37], whereas a decrease in the number of orchid species with increasing
elevation was observed less frequently [30]. In the central Balkans (western Serbia),
it was found that most orchid species and subspecies occur at middle elevations
(1000–1100 m) [26, 34]. In Greece, the total orchid species richness increased with
elevation up to 2000 m and then slightly decreased [11]. Moreover, recent research
has revealed differences in the distribution of specific life forms of orchids and their
root types along the elevational gradient [11, 27, 28, 34]. In the Himalayas and China
(Yunnan), researchers found that species richness peaked at a higher elevation in the
case of terrestrial orchids than in that of epiphytic species [28]. In Greece, tuberous
orchids showed a significantly unimodal response to the elevation gradient with a
peak at c. 1000 m, whereas the richness of rhizomatous orchids and orchids with
palmate and fusiform tubers monotonously increased with elevation [11].
It is assumed that orchid species richness is highest in the mid-elevation zones of
most European countries. This can be explained by several hypotheses. The concept
of the mid-domain effect (MDE) predicts that orchid richness will peak in the mid-
elevation zone because geometric constraints result in an increased overlap of
species ranges near the midpoint of the center of the domain [38]. The climate-
gradient hypothesis predicts that maximum orchid species richness occurs at a
particular elevation where the combination of growing conditions proves optimal
for the species [28]. Finally, the species-area relationship (SAR) hypothesis posits
that the greatest number of species grow in elevation zones that cover the largest area
[28]. A smaller number of orchid species in high-elevation areas can also be
attributed to lower pollinator diversity and lower pollinator visitation rates [30].
Among European orchids that prefer lower elevations, several Mediterranean
species with ovoid tubers from the genera Serapias and Ophrys stand out, along
with certain species from the genera Himantoglossum, Anacamptis, Neotinea, and
Orchis [13, 20]. On the other hand, orchid species known to grow in the highest-
elevation zones are mainly orchids with palmate and fusiform tubers – Chamorchis
alpina, Coeloglossum viride, Pseudorchis albida, Traunsteinera globosa, species
from the genus Nigritella, and many Dactylorhiza species (Fig. 2) [9, 20].
Recent studies have emphasized that the altitudinal ranges of terrestrial orchids in
Europe can vary greatly depending on geographical regions. Thus, many species
primarily characteristic of Central and Northern Europe have found similar ecological
conditions at higher elevations in the southern parts of their ranges [25, 26]. For
instance, the altitudinal ranges in Greece for the species Coeloglossum viride,
Corallorhiza trifida, Epipactis purpurata, Epipogium aphyllum, Neottia cordata,
Orchis militaris, and Pseudorchis albida are 700–2200 m, 800–1900 m,
1200–1500 m, 1150–1800 m, 1300–1800 m, 1200–1800 m, and 1800–1900 m,
Fig. 2 Terrestrial orchids occurring mostly in high-altitude areas. (a) Coeloglossum viride, (b)
Nigritella rhellicani, (c) Pseudorchis albida, (d) Traunsteinera globosa, (e) Chamorchis alpina
(a–d photos V. Djordjević; e photo M. Bobocea)
respectively [13]. By way of contrast, the altitudinal ranges of these species in Europe
are 0–2970 m, 0–2350 m, 50–1400 m, 80–1900 m, 10–2300 m, 0–2000 m, and
0–2700 m, respectively [20].
Interestingly, in some studies, the influence of habitat heterogeneity on orchid
diversity was expressed by the difference between elevation of the highest peak and
that of the lowest point in the country [32]. Researchers found that increase in the
total number of orchid species with increasing altitudinal amplitude plays an impor-
tant role in tropical but not in temperate areas of Latin America [32]. The authors
hypothesized that this is because in temperate countries, only a few orchid species
occur in high-elevation zones, so that there is no significant increase in the number of
orchid species with increasing elevation [32].
2.2 Latitude and Longitude
It should be emphasized that latitude is one of the most important determinants of

orchid richness, distribution, and abundance [11, 32, 39, 40]. Several hypotheses
about the significance of latitude (postulation of a latitudinal diversity gradient, the
latitude niche-breadth hypothesis, and Rapoport’s latitude rule) were recently tested
to determine the patterns of diversity of orchid taxa [11]. Latitude and elevation have
a similar effect on the climatic conditions of an area: areas at high latitudes and in
high-elevation areas are both characterized by lower temperatures, greater season-
ality, and higher environmental stochasticity than areas at low latitudes and in low-
elevation zones [11, 36]. In general, the number of terrestrial orchid species increases
with decreasing latitude from the poles to tropical areas, and this pattern is among the
most consistent ones in biogeography [32]. In Europe, it is expressed from the North
Pole to the Mediterranean. Thus, Northern Europe has the smallest number of orchid
species, which is a consequence of its climate, geology, vegetation, and history,
whereas the number of orchid species gradually increases toward the southern areas
of Europe, being highest in the Mediterranean area [5, 9]. However, many specific
orchid species belonging to the boreal and Central European chorological groups
occur mainly in Northern and Central Europe. Among boreal orchids, Corallorhiza
trifida, Epipogium aphyllum, Goodyera repens, and Neottia cordata should be
emphasized. Central Europe is one of the important centers of diversity and abun-
dance of the genera Epipactis and Dactylorhiza [9, 20, 22]. On the other hand, the
Mediterranean area of Southern Europe represents the center of diversity of
the genera Ophrys, Serapias, Anacamptis, Orchis, Neotinea, and Himantoglossum
[9, 11, 16].
In general, latitude when viewed on a large scale has a more important role than
energy availability in determining patterns of orchid diversity [40]. It is important to
note that a significant influence of latitude on orchid species richness was recorded
for Europe and the Americas, whereas no significant influence of latitude was
identified in Africa and Asia [40]. The importance of latitude and longitude in the
distribution and abundance of orchids has also been demonstrated on regional scales.
Thus, the spatial distribution of Greek orchids is associated with a combination of
latitude, elevation, and climate [11]. With increasing latitude, orchids in Greece were
more widely distributed and had greater mean niche breadths. The same study
indicated that the numbers of orchid species with palmate and fusiform tubers
increased with increasing latitude and slightly decreased with increasing longitude.
At the same time, the numbers of tuberous orchid species and the total number of
orchid species decreased with increasing latitude and showed an inverse unimodal
trend with respect to longitude [11]. In northeastern Greece and western Serbia, both
latitude and longitude (but especially latitude) were found to have a significant
impact on the distribution and abundance of orchids, as well as on the separation
of ecological niches of orchids [24–26]. Studies of orchid species richness on islands
around the world showed that effects of latitude exist, but are weak compared to
those of other factors, such as island size and elevation [39]. Moreover, for relatively
small archipelagos, the effects of latitude can be ignored [39].
Attention should be called to a study of morphological variation in populations of

some orchid species (Cypripedium calceolus, Neottia ovata, Platanthera bifolia, and
Gymnadenia conopsea) along a latitudinal gradient in three European countries, viz.,
Russia [Murmansk (66–70 N) and Tver (55–59 N)], the Netherlands (50–54 N),
and Italy (35–47 N) [41]. This study showed that the mean values of shoot structural
characters in the majority of orchid species increase from the peripheral parts to the
centers of their ranges and are higher on their southern than on their northern
boundaries [41]. The results of this study also indicated that the number of flowers
in inflorescences and the number of bracts were the most labile traits in most
investigated species, whereas Cypripedium calceolus had the most conservative
shoot structure, with little variation between different parts of its range [41].
2.3 Orchid Species-Area Relationship
The species-area relationship hypothesis postulates that the number of species increases
with size of the area. Specifically, greater habitat heterogeneity and greater availability
of resources in vast areas contribute to greater species diversity. The relative importance
of area size has been tested in several orchid studies [32, 39, 40]. One study treated both
total areas and protected areas of 67 countries from 5 continents and demonstrated that
area viewed on a large scale is always very important and that size of a protected area
gives a better fit than the total area of a country in most cases [40]. However, the results
of this study indicated that in Europe, where the orchid flora consists solely of terrestrial
species, total area size predicted the number of orchid species better than protected area
size [40]. A possible explanation of this lies in the fact that many orchids in Europe
grow in semi-natural habitats such as grasslands and meadows, which are maintained
by regular mowing or grazing [42, 43]. In addition, numerous orchids in Europe grow
in forest ecosystems, which are often not protected [40].
3 Climate
Climatic factors strongly affect the distribution, richness, abundance, and population
dynamics of terrestrial orchids [11, 28, 29, 44, 45]. The patterns of diversity of
orchid species on large geographic scales are influenced mainly by macroclimate,
whereas on regional and local scales, meso- and macroclimate play an important role
affecting the richness and abundance of orchids [44]. Among the most important
climatic factors are temperature and precipitation, as well as atmospheric humidity
and light.
3.1 Temperature
The influence of temperature on orchid populations varies depending on altitude and

latitude, which is especially evident when comparing the centers and boundaries of
species ranges [44, 46]. From a phytogeographical point of view, it is known that in
temperate regions, orchid species richness is lower in areas of cold compared to
warm climatic conditions, which is consistent with the general pattern observed
among vascular plant species. The greatest number of terrestrial orchid species in
Europe grows in the Mediterranean area, which is characterized by warm and dry
summers and mild, humid winters, whereas the smallest number of species occurs in
the northernmost, coldest areas [5, 8]. However, it is unclear whether temperature,
precipitation, or the combination of these two factors most strongly affects orchid
distributions.
Most terrestrial orchid species occurring in Europe that can tolerate colder
conditions with great success belong to the group of orchids with palmate and
fusiform tubers, e.g., some species from the genera Dactylorhiza, Coeloglossum,
Gymnadenia, Chamorchis, Nigritella, and Pseudorchis [2, 8, 13, 20, 47]. This is
understandable considering the evolutionary development of tuberoid orchids. Spe-
cifically, the formation and development of the first orchids with palmate and
fusiform tubers are associated with the Alpine orogenesis and the formation of
mountain habitats with low temperatures [8]. The same author claims that these
orchids significantly expanded their distribution areas at the end of the Neogene and
in the Pleistocene, when temperatures were low. The fact that these orchids inhabit
colder areas with high precipitation is confirmed by their phytogeographical affili-
ation, since they are mostly representatives of the Boreal, Central European Moun-
tain, Arctic-Alpine, and Central European chorological groups [34]. In addition,
among cold-adapted orchids, there are some rhizomatous species (Neottia cordata
and Corallorhiza trifida) [48, 49] and Malaxis monophyllos, a species that forms one
basal pseudobulb [50].
On the other hand, most orchids that best tolerate high temperatures and dry
conditions belong to a group of orchids with ovoid tubers, especially species of the
genera Ophrys, Orchis, Serapias, Neotinea, and Himantoglossum, as well as some
species of the genus Anacamptis (e.g., A. papilionacea and A. pyramidalis) (Fig. 3).
Orchids with this root system represent a terminal phase in the evolution of under-
ground organs of terrestrial orchids that enable orchids to survive in warm and dry
conditions [8, 10].
It is known that most orchid species require relatively stable temperature condi-
tions [29], whereas it has been established that extreme temperatures within the
season (high temperatures during summer and low temperatures during winter)
equally adversely affect orchid populations in Europe [44, 51]. Studies about the
effect of temperature on the abundance of orchids at the northern boundaries of orchid
distribution in Russia showed that air temperature during the previous and current
growing season strongly affects orchid populations [44, 52]. At the same time, it has
been proved that temperatures both at the beginning and end of the growing season
strongly influence the performance of orchid populations. Among climatic factors,
seasonal temperature changes and low winter temperatures especially are factors that
exert significant influence on orchid species richness in China [29]. Furthermore,
scientists have determined that most terrestrial orchids in China prefer a lower
temperature (10.1–13.3 C) compared to epiphytic orchids (15.0–17.4 C) [28].
1
The Role of Ecological Factors in Distribution and Abundance of Terrestrial. . .
Fig. 3 Terrestrial orchids that tolerate high temperatures and dry conditions. (a) Anacamptis pyramidalis, (b) Anacamptis papilionacea, (c) Orchis italica, (d)
Himantoglossum robertianum, (e) Serapias orientalis, (f) Ophrys apifera, (g) Ophrys argolica, (h) Ophrys homeri, (i) Ophrys regis-ferdinandii, (j) Ophrys
13
umbilicata (a, f photos V. Djordjević; b–e, g–j photos S. Tsiftsis)

Air temperature affects germination and seedling establishment, photosynthetic

activity, the flowering period, fruiting, abundance, and the density of orchid
populations [33, 53–57]. Climatic variables, including air temperature, have a
significant influence on the fruiting of orchids since they are related to weather
conditions favorable for pollinating insects [53]. Studies have shown that very warm
weather in spring and early summer can lead to insufficient water supply, which
causes abortion of inflorescences in Dactylorhiza sambucina [55], Himantoglossum
hircinum [58], and Ophrys apifera [59]. During our field investigations, we found
that this is also the case with the Epipactis taxa occurring in Southern Europe.
Specifically, under warm climatic conditions during late spring and early summer,
flowers of these species do not open and in several cases dry up and fall on the
ground without being pollinated. Contrary to the situation in spring and summer, low
temperatures in winter may negatively influence orchid populations, since seed
germination and flowering of orchids are sensitive to temperature [27]. Moreover,
most pollinators of orchids are specific insects that are very sensitive to winter
coldness [29].
A study in the north of Russia indicates that the abundance of Dactylorhiza
maculata, Gymnadenia conopsea, Neottia ovata, and Epipogium aphyllum is neg-
atively correlated with the rise in temperature in the previous few seasons, indicating
that lower temperatures are favorable for these orchids [44]. Furthermore, a study
from the Czech Republic shows that Dactylorhiza fuchsii is very likely to be present
in areas with more frost days per year and that the species is almost absent in areas
where there are only a few frost days per year [60]. Cool seasons may have positive
effects on the performance of orchid species by exerting negative influence on their
competitors [54, 61]. Furthermore, species of the genus Dactylorhiza and many
other European orchid species require a chilling period to develop their leaves,
whereas most species of the genera Serapias and Ophrys from Southern Europe
do not require vernalization [62]. In Central Norway, a positive and statistically
significant correlation was found between the population density of Dactylorhiza
lapponica and temperature of the previous growing season [54].
3.2 Precipitation
The results of numerous studies indicate that a second, very important climatic factor
that affects the distribution patterns of orchids, their population dynamics, flowering
period, and the height of individuals is the amount of precipitation [63]. Scientists
have found that reduced precipitation combined with high temperatures, especially
in May and June, are the most important factors that lead to decrease in the size of
individuals and decrease in the number of individuals of Himantoglossum hircinum
at the southern boundaries of its distribution [46]. The same authors found that
drought during May and June may lead to a reduction in the flowering of this species,
whereas an increased amount of precipitation in May most likely leads to an increase
of photosynthetic efficiency, resulting in higher heights of individuals, higher prob-
abilities of flowering, and increased flower production. The importance of
precipitation and its effects on orchid populations were also demonstrated by an

investigation in England, where it was established that height of the inflorescence
and the number of flowers in Ophrys apifera were positively correlated with the
amount of precipitation in the current and previous growing season [63]. A positive
correlation between annual precipitation of the previous growing season and the
number of flowering specimens and flowers was also found for Himantoglossum
adriaticum, whereas these traits are negatively correlated with the number of frost
days in the previous growing season [64]. It has been shown that the probability of
flowering of Orchis simia strongly depends mainly on weather conditions in the
current year [65], whereas according to the results of a study conducted in Sweden,
the flowering of Dactylorhiza sambucina and Neottia ovata is negatively correlated
with the summer drought period of the previous growing season [55]. Similarly, a
decline in the proportion of flowering individuals of Neottia ovata in a particular
year as a result of drought in the previous year was reported by Brzosko [66]. A
negative effect of drought in combination with high temperature on future growth
and flowering was also found in Herminium monorchis [67] and Gymnadenia
conopsea [54], whereas the typical wetland species Epipactis palustris does not
tolerate drought [68]. In general, drought is linked not only with soil moisture but
also with the loss of the orchid mycorrhizal fungi [61]. Changes in precipitation and
temperature directly affect orchid seedlings and indirectly influence the carbon
source and availability of fungi [56].
Several scientists have investigated the impact of precipitation and temperature
on the morphological characteristics of individual orchids. Thus, it was determined
that June precipitation is positively correlated and May precipitation negatively
associated with the total leaf area of Dactylorhiza majalis, whereas a highly signif-
icant positive correlation was determined between May temperatures and total leaf
area of this orchid species [42]. Furthermore, lip size and spur length in
Himantoglossum jankae were found to be influenced by precipitation during the
warmest quarter, larger lips being recorded in Greek areas with more precipitation
during the flowering period of this species [69].
A study in the north of Russia showed that the correlation between the number of
individuals and snow depth in most populations of the species Cypripedium
calceolus, Dactylorhiza maculata, Platanthera bifolia, and Gymnadenia conopsea
was negative but the abundance of populations of the two species Dactylorhiza
maculata and Coeloglossum viride was positively correlated with snow depth [44].
3.3 Atmospheric Humidity
The relationship between atmospheric humidity and abundance of orchid

populations has been insufficiently explored. However, the results indicate that
different species react differently to changes in atmospheric humidity. Specifically,
according to the results of an investigation conducted at the northern border of orchid
populations in Russia, the number of individuals of Dactylorhiza maculata and
D. incarnata was negatively correlated with atmospheric humidity recorded in the
previous growing season, whereas populations of Gymnadenia conopsea and

Cypripedium calceolus showed a positive correlation [44]. The same author found
that high atmospheric humidity in the current growing season negatively influences
the abundance of many orchid populations and that an especially negative correla-
tion between these parameters was recorded in Neottia ovata. Some authors have
determined favorable values of relative humidity for the cultivation of European
terrestrial orchids in greenhouses. Thus, the recommended humidity for orchids is
50–60% in winter and 60–70% in summer [70].
3.4 Light
The light regime is also an important factor in defining patterns of orchid distribution
and abundance [60, 71–73]. This explains the abundance of orchids mainly at
the microhabitat level, i.e., in an area smaller than 4 m2 [72]. Studies have shown
that morphological and physiological characteristics of terrestrial orchids, as well
as the density of orchid populations, depend on the light regime of their habitats
[57, 71, 74]. It is known that sunlight is one of the major environmental factors
influencing photosynthesis, growth, and reproduction of terrestrial orchids [75].
However, specific light requirements of orchid species may depend on their life
form, developmental phase, and nutritive regime [57]. Furthermore, light plays a
significant role in determining the population dynamics and flowering patterns of
orchid species [76]. Moreover, specific morphological features were found to be
related to light conditions of the studied sites. It has been discovered that differences
of light conditions from site to site can cause differences in length of the inflores-
cence and the number of flowers in specific orchids, but this was not always the case.
The hypothesis of light influence is supported by findings that individuals of Orchis
punctulata occurring in shade and semi-shade in forest habitats had larger inflores-
cences with more flowers compared to those recorded under conditions of full light
in grassland habitats [77]. In contrast, no significant differences were recorded in the
cases of Orchis purpurea and O. mascula [77–80].
It is assumed that most terrestrial orchids grow at sites with illumination of
50–100% [49], whereas a significantly smaller number of orchid species grow
in habitats with conditions of deep shade (e.g., Cephalanthera damasonium,
Corallorhiza trifida, Epipogium aphyllum, Epipactis helleborine, E. purpurata,
Neottia cordata, and Goodyera repens) (Fig. 4). Among the orchids that can tolerate
shade conditions, Neottia nidus-avis and Epipogium aphyllum are characterized by
special ecological and biological adaptations. They do not photosynthesize and can
be said to be extremely adapted to heterotrophy in view of the fact that throughout
their life cycle they depend on mycorrhizal symbionts and are associated with
basidiomycete fungi which form ectomycorrhizae on tree roots [81, 82]. In general,
mycoheterotrophic orchids are usually independent of the light regime [57].
However, in the case of partially mycoheterotrophic orchids, low illumination
leads to strong mycoheterotrophy, whereas greater illumination stimulates orchids
to autotrophy [83]. It should be noted that the photosynthetic apparatus of orchid
Fig. 4 Terrestrial orchids growing in habitats with deep shade conditions. (a) Neottia nidus-avis,
(b) Epipogium aphyllum, (c) Goodyera repens, (d) Corallorhiza trifida (a–c photos V. Djordjević;
d photo S. Tsiftsis)
species inhabiting a forest floor can acclimate to reduced illumination by changing

the Chl a/b ratio to manage coordination between photosystem II (PSII) and photo-
system I (PSI) [57, 75].
Some species that require full light conditions are Anacamptis morio [84],
Epipactis palustris [68], and Pseudorchis albida [85]. On the other hand, a
significant number of orchids have a wide ecological valence in relation to light
(e.g., Epipactis helleborine, Cephalanthera longifolia, Dactylorhiza fuchsii, Neo-
ttia ovata, and Platanthera bifolia) [9, 73]. A good example is Cypripedium
guttatum, which grows in both shady and open habitats where illumination can
vary between 22 and 76% of full sunlight [75]. The successful acclimatization of
this species to a wide range of light levels probably allows for its wide geograph-
ical distribution [75]. These are general trends, but in some cases, especially at the
boundaries of species ranges, orchids can have specific ecological requirements.
Although it is known that Platanthera bifolia occurs both in open habitats with
full light conditions and at shady sites within forest ecosystems, a study from the
Jeseníky Mountains in the Czech Republic showed that solar radiation is the most
important factor associated with distribution of the given species, which occurs
mainly in places with low values of this factor [60]. Furthermore, on the south-
ernmost border of the range of Pseudorchis albida in Europe (northeastern
Greece), this species is found only in semi-shadow and shadow conditions of
dense Pinus heldreichii forest patches on north-facing slopes, whereas it is absent
from well-lit neighboring sites [86]. In addition, the well-known light-demanding
species Epipactis palustris, which rarely occurs at places where the illumination
in summer falls to below 40% of full sunlight, in some cases was found to grow in
damp forests and scrub ecosystems where light penetration is significantly lower
than in open habitats [68]. Recent studies from Serbia showed that certain typical
grassland species (e.g., Anacamptis morio, A. pyramidalis, Dactylorhiza maculata
subsp. transsilvanica, Gymnadenia conopsea, Neotinea ustulata, and N. tri-
dentata) also occur in semi-shadow and even shadow conditions in forests of
Pinus sylvestris, P. nigra, Quercus, and Ostrya carpinifolia [34]. Such examples
are not rare in the existing literature, and within the range of distribution of each
species, differences between sites with respect to ecological requirements are
often recorded.
The way that light conditions affect orchid species can also be seen in cases where
habitats are altered due to specific natural or artificial changes (e.g., natural succes-
sions of vegetation and management treatments). For example, natural deforestation
and a certain degree of disturbance of forest ecosystems, leading to opening of the
forest structure, positively influence the development of populations of some forest
orchids [73, 87, 88]. Thus, the probability of flowering and fruiting of Cypripedium
calceolus increased at intensively harvested sites of spruce forests, whereas its
survival and population density increased at moderately harvested broadleaf forest
sites [89]. On the other hand, reduction of light intensity caused by closing of the
forest structure leads to an extension of the dormant period and postponement of the
flowering period in some orchids (Cephalanthera rubra and Cypripedium calceolus)
[74]. Furthermore, it has been found that weakened illumination leads to a reduction
of flower production in the species Cephalanthera longifolia and to a decrease in the

height of individuals of Cypripedium calceolus [90].
3.5 Orchid Species Distribution and Its Patterns in a World

Subjected to Climate Change
In the last few decades, we have been experiencing the phenomenon of climate
change, which affects different regions of the world differently. The most commonly
observed consequences are increasing temperature, less pronounced seasonality, and
lower environmental stochasticity. Influencing different areas of the world, all these
changes, regardless of their degree, are expected to strongly affect the distribution of
orchids and their population dynamics [91, 92]. Recent studies explore the possibil-
ity of predicting distributional, ecological, and evolutionary consequences of climate
change [91–95]. A study in southwestern China showed that climate warming in the
region, along with a reduced level of soil moisture, has a negative influence on most
of the investigated orchid species [93]. The same authors found that orchid
populations that occur on limestone are especially highly subject to the danger of
extinction due to the lack of high places to which they can migrate, whereas heavy
rainfall can initiate stronger erosion, which also adversely affects orchid populations
[93]. However, a study treating the pattern of distribution of Sardinian orchids under
conditions of climate change has shown that a consequence of the trend of increasing
temperature and decreasing precipitation is a widening of areas suitable for orchids
[95]. Moreover, although prognoses regarding climate change and the abundances of
orchid populations on the northern boundaries of orchid distribution in Russia
(Murmansk region) show that global warming is generally favorable for many orchid
species, the predicted global warming and aridization could have negative impacts
on the species Dactylorhiza incarnata, Hammarbya paludosa, and Epipogium
aphyllum, since they are sensitive to changes in moisture conditions [44].
Some authors used herbarium records to explore climatic patterns of orchid
diversification [96] and clarify phenological cues that reveal the consequences of
climate change on pollination and reproductive success [97, 98]. Scientists have
found that orchids generally flower earlier than in the past in Hungary and that
deceptive, autogamous, early flowering and long-lived terrestrial orchids with
mainly Mediterranean distributions (e.g., Orchis simia and Anacamptis pyramidalis)
follow global warming more closely, whereas nectar-rewarding, later-flowering, and
short-lived orchids with non-Mediterranean distributions (e.g., Coeloglossum viride)
do not respond or respond less markedly to changing climate [97]. The first indica-
tion that climate change upsets the relationships between orchid species and their
pollinators was obtained, thanks to a study led by Prof. Michael Hutchings at the
University of Sussex, who studied how rising temperatures since the mid-seven-
teenth century have disturbed the relationship between the deceptive orchid Ophrys
sphegodes and its pollinator – male bees of the species Andrena nigroaenea [98].
The results of this study showed that global warming has changed the timing of
phenological events that are critical to the reproductive success of the given orchid
species. For optimal pollination of this orchid species, male bees have to emerge
before female bees and before orchid flowering, whereas orchid flowering has to
occur before female bees emerge. The increase in temperature led to earlier orchid
flowering and earlier flying of male and female bees. However, the timing of these
events did not change to the same extent. To be specific, female bees now fly before
orchid flowering occurs and male bees consequently will mate with female bees
rather than pseudocopulate with the orchid flower. The pollination of this orchid
species therefore is less likely today than during the spring season in the past when
temperatures were lower, suggesting that global warming will increase the period in
which this orchid species experiences reproductive failure [98].
Recent studies explore the importance of climatic variables in determining the
distribution of orchid species [11, 29, 45, 60, 99]. Climatic data freely available
through specific databases (e.g., WorldClim database) [100, 101] constitute a very
useful tool in exploring trends of orchid distribution and the effects of climatic
conditions. Thus, most recent research carried out in Greece showed unimodal
associations between the total number of orchid species and the maximum temper-
ature in the warmest month and indicated that the number of rhizomatous and
intermediate orchid species decreases with increasing maximum temperature in the
warmest month [11]. Moreover, this study showed that the number of rhizomatous
orchids and ones with palmate and fusiform tubers is greatest in the areas of Greece
with the coldest winters, the highest orchid richness occurring in areas with harsh
and mild winters, whereas the lowest orchid richness is in areas with moderately cold
winters. Furthermore, scientists explored the probability of orchid occurrence in
relation to climatic variables such as annual precipitation and annual mean temper-
ature on Crete [99]. The results of this study showed Anacamptis coriophora subsp.
fragrans, A. papilionacea subsp. heroica, Ophrys bombyliflora, O. mammosa,
Serapias bergonii, and S. orientalis to be highly positively correlated with the
mean annual temperature, whereas Epipactis cretica, Cephalanthera cucullata,
C. damasonium, Neotinea tridentata, and Orchis prisca were highly negatively
correlated [99]. Moreover, these latter orchids were also found to prefer habitats
with high annual precipitation. Important studies have also explored the key climatic
factors that affect survival of orchid species on the southernmost border of their
distribution in Europe. For example, the most significant factors for the distribution
of Neottia cordata on the southernmost border of its distribution were found to be
precipitation during the warmest quarter and the seasonality of precipitation, thereby
highlighting the importance of the amount of summer rainfall [45].
4 Geological Substrates and Soil Properties
The geological substrate and soil characteristics are important factors affecting
the distribution and abundance of orchids primarily at the regional and local levels
[11, 24–26, 99, 102–104]. In general, variation in the availability of soil resources
(water and nutrients) across geological substrates significantly influences the rich-
ness and composition of orchid species [24, 26, 34].
4.1 Geological Substrates
Calcareous geological substrates and soils can be considered to be the most important
substrates on which terrestrial orchids grow in Europe, which means that the
greatest number of species occur on limestone, chalk, dolomite, and carbonate
clastites (carbonate shales, sands, sandstones, clays, gravels, conglomerates, and
marls) [11, 13, 25, 105–107]. This pattern represents one of the most consistent
features of species richness, and it also applies to other vascular plants in Europe
[108]. While the richness of orchids on carbonate substrates is very prominent in
Central Europe, the patterns of distribution and abundance of orchids in Southern and
Southeast Europe have somewhat different shapes. To be specific, recent research from
the central Balkans (western Serbia) and Greece indicated that a large number of
orchid species grow on non-calcareous bedrock types, i.e., on various silicate sub-
strates, including felsic, intermediate, mafic, and even ultramafic igneous rocks, as
well as on metamorphic and silicate sedimentary rocks [24–26, 107]. These studies
highlighted the important role of the gradient of geological substrates in separating
niches of orchid species. Differences in the chemical and physical composition of
geological substrates and soils not only lead to differences in the richness and
composition of species; they also affect the size of orchid populations [24].
Terrestrial orchids that grow exclusively or mainly on carbonate geological sub-
strates include the following species: Anacamptis papilionacea, Orchis militaris,
O. pauciflora, O. anthropophora, Gymnadenia conopsea, G. odoratissima, Nigritella
rhellicani, Neotinea ustulata, Dactylorhiza fuchsii, Epipogium aphyllum, Epipactis
palustris, E. purpurata, E. muelleri, and many Ophrys and Himantoglossum species
(Fig. 5) [9, 12, 20, 61, 68, 109, 110]. However, most of these species were
also recorded on different silicate substrates in Greece [13, 25], in the central Balkans
[24, 26, 34], and in Italy [16]. Some orchid species are known to occur on a large
number of bedrock types, indicating their great ecological plasticity (Anacamptis
coriophora, Cephalanthera longifolia, Dactylorhiza sambucina, Gymnadenia
conopsea, Epipactis helleborine, Neottia nidus-avis, N. ovata, Platanthera bifolia,
etc.) [9, 12, 26, 34, 107, 111, 112].
Recent studies have highlighted the importance of ophiolitic mélanges as an impor-
tant bedrock type for the survival and development of many terrestrial orchids in the
Balkans [26, 34, 107]. Great orchid richness on this bedrock type can be attributed to its
heterogeneous composition, bearing in mind that these volcanogenic-sedimentary
formations usually contain diabase (basic igneous rock) and cherts (silicate sedimentary
rocks, primarily composed of quartz, the mineral form of silicon dioxide). Although it is
known that Si plays an important role in the alleviation of abiotic and biotic stress, its
significance for orchid growth has been insufficiently investigated. Some orchids that
have significant abundance on ophiolitic mélanges are the Balkan endemic
Himantoglossum calcaratum subsp. calcaratum, two subendemics (Dactylorhiza
maculata subsp. transsilvanica and D. cordigera), certain species of Central or North
European origin (Anacamptis morio, Traunsteinera globosa, Neotinea ustulata,
Dactylorhiza incarnata, Coeloglossum viride, Epipactis leptochila subsp. neglecta,
and E. purpurata), as well as Dactylorhiza saccifera [26, 34, 107].
Fig. 5 Terrestrial orchids that grow mainly or exclusively on calcareous geological substrates. (a)
Ophrys oestrifera (syn. Ophrys scolopax subsp. cornuta), (b) Orchis militaris, (c) Orchis
anthropophora, (d) Orchis pauciflora (a, b photos V. Djordjević; c, d photos S. Tsiftsis)
A significant number of terrestrial orchid species in the Balkans occur on

metamorphic rocks, including schists, gneisses, and phyllites [25, 26, 34, 107].
Among orchid species found on these bedrock types, due to the significant abun-
dance and frequency of findings on them, two species (Nigritella rhellicani and
Gymnadenia frivaldii) are defined as indicators of these geological substrates [26]. In
addition, a study from the Czech Republic shows that Dactylorhiza fuchsii has a high
probability of occurring on phyllites [60]. Although the species Epipactis pontica is
known to occur mainly on calcareous substrates, in recent studies populations of this
species were recorded on gneisses in Bulgaria and Greece [25, 113]. Furthermore,
many terrestrial orchids also occur on intermediate igneous rocks (andesite, dacite,
and porphyrite) and flysch, whereas some species were found to grow on Quaternary
sediments, including proluvial and alluvial deposits, eluvial-deluvial sediments, and
river terraces [26, 107]. The majority of species found on Quaternary sediments
prefer habitats with high soil moisture (e.g., Dactylorhiza incarnata, D. saccifera,
and Anacamptis palustris) [26].
Recent studies show that many terrestrial orchids grow on ultramafic rocks in the
Balkans [24, 26, 34, 107], Malaysia [114, 115], and Russia [116], which at first glance
is unexpected considering the stressful conditions of these substrates. To be specific,
soils derived from serpentine (ultramafic) rocks are known for high concentrations of
Ni, Cr, and Co; low concentrations of macronutrients (P, K, and N); low Ca/Mg ratios;
and properties unfavorable for plant life, such as a wide range of diurnal temperature
fluctuations and poor water-retaining capacity [24]. In the central Urals (Russia),
scientists found Epipactis atrorubens on this bedrock type [116]. Investigations in
the central Balkans (western Serbia) showed that 29 orchid species and subspecies
occur on ultramafic rocks, the following orchids having notably large populations
on this type of substrate: Anacamptis morio, Gymnadenia conopsea, Dactylorhiza
sambucina, D. maculata subsp. transsilvanica, and Platanthera bifolia (Fig. 6)
[34, 107]. Interestingly, two species (Gymnadenia conopsea and Anacamptis morio)
have statistically significantly larger populations in serpentine areas than in non-
serpentine areas of the Valjevo Mountains in Serbia [24].
All orchid species recorded on serpentine substrates in the central Balkans are
serpentine-facultative plant species, since they are also present on other bedrock types
[24]. However, due to notably large populations and high frequency of occurrences,
two orchids (Dactylorhiza maculata subsp. transsilvanica and Platanthera bifolia) are
defined as indicator species of serpentine substrates in western Serbia [26]. The
explanation for the existence of a significant number of orchid species and large
orchid populations on this bedrock type lies in the physical and chemical properties
of serpentine soils, primarily their low content of nutrients [116], considering that most
orchid species are sensitive to high levels of phosphorus and nitrogen in the soil [103].
Furthermore, it is also assumed that mycorrhizal fungi play a key role in increasing
tolerance to high content of heavy metals in serpentine soils.
It should be noted that serpentine substrates allow for the development of open
habitats with a generally low degree of competition, which makes possible the
survival of poorly competitive orchid species that have a high demand for light
[24]. Furthermore, scientists found that conditions of the open structure of forest
24
Fig. 6 Orchid species that grow on ultramafic substrates in the central Balkans (western Serbia). (a) an ultramafic landscape on Mt. Zlatibor (Serbia), (b)
Dactylorhiza sambucina, (c) Anacamptis morio, (d) Gymnadenia conopsea, (e) Platanthera bifolia, (f) Dactylorhiza maculata subsp. transsilvanica (photos V.
Djordjević)
V. Djordjević and S. Tsiftsis
ecosystems on serpentine substrates are an important factor influencing the richness

of both terrestrial and epiphytic orchids in Kinabalu Park in Malaysia [114]. These
authors assume that adaptations to the stressful chemical conditions of ultramafic
(serpentine) substrates may be an important factor for the high level of endemism of
terrestrial orchids in this part of the world [114]. Two terrestrial orchids found on
serpentine substrates in Malaysia are Crepidium metallicum and Paphiopedilum
dayanum [114]. It is interesting that Rothschild’s slipper orchid (Paphiopedilum
rothschildianum), one of the most famous orchid species in the world, now widely
available thanks to its propagation in culture, in fact originated and grows on
serpentine substrates in Kinabalu Park in Malaysia [115]. Detailed ecological
study showed that extreme substrate conditions are reflected in the leaf chemistry
of this species, while experiments involving translocation onto non-ultramafic sub-
strates were successful, indicating that substrate chemistry is not a factor limiting the
growth of this species [115].
A smaller number of orchid species are recorded to grow on acidic igneous
rock [107]. Species found on quartz latite include Anacamptis coriophora,
Cephalanthera longifolia, Dactylorhiza incarnata, D. maculata subsp. maculata,
D. sambucina, Nigritella rhellicani, Gymnadenia conopsea, Coeloglossum viride,
Neottia cordata, and Traunsteinera globosa [26, 34, 111]. Ones recorded on grano-
diorites are mainly forest orchids such as Cephalanthera damasonium, C. longifolia,
C. rubra, Epipactis helleborine, E. microphylla, Limodorum abortivum, Neottia
nidus-avis, Platanthera bifolia, and P. chlorantha [34, 107]. Among species grow-
ing on acidic igneous rocks in Greece, many species were found to occur on rhyolite,
quartz-monzonite, granodiorite, and granite (e.g., Epipactis pontica, Goodyera
repens, Neottia cordata, etc.) [13, 25, 45].
Overall, recent studies suggest that non-calcareous bedrock types, i.e., different
siliceous substrates, are favorable for many Central European and Boreal orchid
species that occur in the central Balkans and Greece [25, 26, 34, 45, 107]. This can
be attributed to the higher water-retaining capacity of these substrates in comparison
with calcareous ones and to the fact that they are present mainly in high-altitude
areas where the humidity is greater [26]. Therefore, future research should examine
whether areas with silicate substrates can represent significant orchid reserves.
Further research should also explore the impact of physical and chemical character-
istics of the soils of different bedrock types on certain characteristics of orchids, such
as their potential for accumulation of trace elements and the phytochemical proper-
ties of these plants.
4.2 Soil Properties
Physical and chemical properties of the soil are known to have a significant impact
both on the growth of terrestrial orchids and on their abundance and distribution
[25, 103, 104, 117]. Among numerous physical and chemical properties of the soil,
scientists have primarily studied its moisture, pH, nutrients (nitrogen, phosphorus,
and potassium), content of calcium and magnesium, and organic content in soils that
support terrestrial orchid species (Tables 1 and 2). These properties vary in response
to differences in bedrock types, climatic conditions, and vegetation, and they affect
the performances of orchid populations [102, 117]. Because many orchid species are
ecologically specialized to limited ranges of soil factors, adequate ecophysiological
adaptations can be expected [103].
4.2.1 Soil Moisture

Soil moisture represents an important factor influencing orchid distribution patterns
mainly on a microsite scale [72, 118]. Studies have shown that soil moisture up to
10 cm depths significantly affects orchid population density and distribution,
whereas the absence or presence of orchids depends on soil moisture to a lesser
extent below a depth 10 cm [119, 120]. Of course, this is because of the depth where
orchids have their root system, which rarely exceeds 10–15 cm below the surface of
the ground [62]. Detailed data on soil moisture have been obtained for some orchid
species, for example, in nine forest populations of Neottia ovata in Belgium, where
measured soil moisture ranged from 8.8% to 28.6% [121].
An important property of the soil influencing moisture content – through its
retaining capacity – is soil texture, but corresponding analyses focusing exclusively
on orchids are lacking in the existing literature. Apart from other advantages of its
availability, dissolvable cations and nitrogen compounds can be transported through
the underground flow of water. This transport can potentially affect pH and nitrogen
content in the soil, and as a result the dynamics of moisture in space and time can
indirectly influence the composition of vegetation, its diversity, and species distri-
bution [118]. Moreover, the effects of soil moisture can be seen in the growth of
orchids, their germination, establishment of their seedlings, diversity of mycorrhizal
fungi, and soil temperature characteristics [118, 122]. As regards orchids specifi-
cally, a recent study from the central Balkans indicated that soil moisture is one of the
key factors affecting the distribution and abundance of orchid species within grass-
lands and herbaceous wetlands [26]. Moreover, the results of this research showed
that the number of orchid species and their occurrences decrease at the extreme ends
of the moisture gradient and that the majority of the orchid species occur in habitats
with moderately moist soils (the data follow a unimodal distribution).
However, such a unimodal trend is not the case for every region or country, as
orchid trends are strongly dependent on the orchid flora of the studied region or
country. In Europe, orchids can be found from totally dry sites of the Mediterranean
region to moist and wet places of the North European countries [5]. Specifically, many
orchid species are totally dependent on high levels of water availability, and such
orchids can become extinct when their habitats dry up or when the amount of soil
moisture is below a specific threshold for some months. Some of the best-known
terrestrial orchids that grow exclusively on high-moisture soils in Europe are
Anacamptis palustris, Epipactis palustris, Hammarbya paludosa, Liparis loeselii,
Gymnadenia frivaldii, and most species of the genus Dactylorhiza [9, 26, 47]. On
the other hand, orchids that tolerate dry soil conditions are most representatives of the
genera Serapias, Ophrys, Orchis, Himantoglossum, and Anacamptis (e.g., Anacamptis
papilionacea and A. pyramidalis) (Fig. 3) [9, 13, 26].
Table 1 Soil pH at sites with some terrestrial orchid species

The soil
Orchid taxon pH range Soil pH values/literature sources
Anacamptis boryi 7.1–8.1 7.1–7.2 [131], 7.2–8.1 [132]
Anacamptis collina 6.7–8.6 7.5–8.4 [131], 6.7–8.6 [132]
Anacamptis coriophora 4.74–8.3 4.74–5.72 [25], 6.0–8.3 [126], 6.3 [131], 5.5–8.0
subsp. coriophora [133]
Anacamptis coriophora 4.58–8.1 4.58–7.74 [25], 7.7–8 [131], 8.1 [132], 7.33–7.53
subsp. fragrans [134]
Anacamptis laxiflora 3.5–8.0 4.89–7.13 [25], 7.9–8.0 [132], 3.5–5.5 [133],
7.32–7.7 [134], 5.0–8.0 [135]
Anacamptis morio subsp. 4.13–8.0 4.13–7.70 [25], 5.5–8.0 [133], 4.7–7.21 [134]
caucasica
Anacamptis morio subsp. 4.5–8.1 4.5–7.5 [49], 5.5–8.1 [126], 4.5–8.0 [133], 4.9–6.8
morio [136]
Anacamptis morio subsp. 4.5–7.5 4.5–7.5 [135]
champagneuxii
Anacamptis palustris subsp. 5.5–8.0 5.91 [25], 7.05–7.75 [126], 5.5–8.0 [133]
elegans
Anacamptis palustris subsp. 6.4–8.01 6.4–8.01 [126]
palustris
Anacamptis papilionacea 4.67–7.43 4.67–7.43 [25], 5.04–6.4 [134]
subsp. papilionacea
Anacamptis pyramidalis 4.94–8.7 4.94–7.99 [25], 7.17–8.25 [126], 7.5–8.5 [131],
8.0–8.7 [132], 5.5–8.0 [133], 5.75–7.58 [134],
5.5–7.5 [135]
Calypso bulbosa 4.5–7.5 6.9 [131], 4.5–7.5 [133]
Cephalanthera damasonium 4.3–8.0 4.45–7.65 [25], 5.76–7.98 [126], 6.0 [131],
5.5–8.0 [133], 6.0–7.5 [135], 4.3–7.4 [137]
Cephalanthera longifolia 4.0–8.6 7.85 [25], 4.59–8.0 [126], 6.5–8.6 [131], 5.5–8.0
[133], 4.34–5.07–7.03 [134], 4.0–7.0 [135],
4.3–7.4 [137]
Cephalanthera rubra 4.3–8.2 4.38–7.74 [25], 5.16–7.83 [126], 5.9–8.2 [131],
5.5–8.0 [133], 4.3–7.4 [137]
Chamorchis alpina 5.5–8.0 5.5–7.5 [131], 5.5–8.0 [133]
Coeloglossum viride 3.5–8.0 4.58–7.54 [25], 4.4–8.0 [49], 5.0–7.9 [126],
5.7–7.9 [131], 3.5–7.5 [133]
Corallorhiza trifida 3.5–7.5 4.14–7.48 [25], 5.4 [117], 6.0–7.5 [126], 6.5–7.5
[131], 3.5–7.5 [133], 4.1–4.4 [137]
Cypripedium calceolus 4.5–8.26 5.7–8.26 [126], 7.3–7.9 [131], 4.5–8.0 [133],
7.1–7.4 [136], 5.4–7.4 [138]
Dactylorhiza aristata 4.5–7.5 4.5–7.5 [133]
Dactylorhiza baltica 4.5–8.0 4.5–8.0 [133]
Dactylorhiza cordigera 4.03–7.5 4.03–6.64 [25], 4.5–7.5 [133]
Dactylorhiza cruenta 4.5–7.5 6.7 [131], 4.5–7.5 [133]
Dactylorhiza elata 5.0–7.5 5.0–7.5 [135]
Dactylorhiza euxina 4.5–8.0 4.5–8.0 [133]
(continued)
Table 1 (continued)
The soil
Dactylorhiza flavescens 3.5–8.0 3.5–8.0 [133]
Dactylorhiza foliosa 4.9–5.9 4.9–5.9 [131]
Dactylorhiza fuchsii 4.5–7.5 5.8–7.5 [126], 4.5–7.5 [133]
Dactylorhiza iberica 7.3–8.2 7.3–8.2 [131]
Dactylorhiza incarnata 4.5–8.3 5.71–5.97 [25], 5.5–8.3 [126], 4.5–7.5 [133], 5.0
subsp. incarnata [135]
Dactylorhiza incarnata 7.59–7.7 7.59–7.7 [126]
subsp. ochroleuca
Dactylorhiza insularis 4.5–7.0 4.5–7.0 [135]
Dactylorhiza lapponica 7.5 7.5 [126]
Dactylorhiza macedonica 5.58–5.71 5.58–5.71 [25]
Dactylorhiza maculata 3.5–7.6 3.5–7.5 [133], 5–7.6 [136]
Dactylorhiza majalis 4.5–7.8 5.1–7.8 [126], 4.5–7.5 [133]
Dactylorhiza markusii 5.0–5.5 5.0–5.5 [135]
Dactylorhiza romana 4.13–8.0 4.13–4.6 [25], 6.7 [132], 5.5–8.0 [133]
Dactylorhiza russowii 4.5–8.0 4.5–8.0 [133]
Dactylorhiza saccifera 5.26–7.6 5.26–7.6 [25]
Dactylorhiza sambucina 3.5–8.0 4.29–7.68 [25], 4.2–8.0 [111], 4.3–6.4 [126],
3.5–8.0 [133], 5.0 [135], 4.9–5.0 [136], 4.0–5.6
[139]
Dactylorhiza traunsteineri 4.5–7.5 4.5–7.5 [133]
Dactylorhiza urvilleana 4.5–8.0 4.5–8.0 [133]
Epipactis albensis 4.5–7.65 4.5–7.65 [126], 6.98 [140]
Epipactis atrorubens 5.5–9.0 6.52–7.7 [25], 6.0–8.1 [126], 7.5–9.0 [131],
5.5–8.0 [133], 5.6–7.0 [141]
Epipactis bugacensis 7.9–8.1 7.9–8.1 [126]
Epipactis exilis 4.27–7.48 4.27–7.48 [25], 4.5 [126], 7.17 [134], 4.3–7.4
[137]
Epipactis futakii 5.9–7.1 5.9–7.1 [126]
Epipactis helleborine subsp. 4.27–8.0 4.27–7.74 [25], 5.2–7.7 [126], 4.5–8.0 [133],
helleborine 7.1–7.86 [134], 4.3–7.4 [137]
Epipactis leptochila subsp. 5.9–7.6 5.9–7.6 [126]
leptochila
nauosaensis
neglecta
Epipactis mecsekensis 5.2–7.7 5.2–7.7 [126]
Epipactis microphylla 5.12–8.0 5.12–7.39 [25], 5.2–7.9 [126], 6.3–7.9 [131],
5.5–8.0 [133], 5.6 [137]
Epipactis moravica 6.6–7.7 6.6–7.7 [126]
Epipactis muelleri 6.3–7.9 6.3–7.9 [126]
Epipactis nordeniorum 4.5–7.9 4.5–7.9 [126]
(continued)
Table 1 (continued)
The soil
Epipactis palustris 4.97–8.5 4.97–7.94 [25], 6.6–8.3 [126], 6.5–8.5 [131],
5.5–8.0 [133]
Epipactis placentina 5.7–6.7 5.7–6.7 [126]
Epipactis pontica 4.48–7.5 4.54–5.47 [25], 5.1–7.5 [126], 4.48–5.65 [127],
5.7–6.3 [128], 6.2–7.1 [131]
Epipactis purpurata 5.2–8.0 5.2–6.8 [126], 5.5–8.0 [133]
Epipactis tallosii 5.5–7.9 5.5–7.9 [126]
Epipactis tremolsii 7.0–7.5 7.0–7.5 [135]
Epipactis voethii 7.2–7.8 7.2–7.8 [126]
Epipogium aphyllum 4.5–8.0 4.72–6.75 [25], 4.5 [82], 4.6–7.6 [126], 6.0–7.6
[131], 4.5–8.0 [133]
Goodyera repens 4.27–7.5 4.27–7.03 [25], 4.27–7.03 [126], 4.6–7.5 [131],
4.5–7.5 [133]
Gymnadenia conopsea 4.5–8.5 4.88–7.73 [25], 5.0–8.0 [126], 4.5–8.5 [131],
5.5–8.0 [133]
Gymnadenia densiflora 7.6–7.8 7.6–7.8 [126]
Gymnadenia frivaldii 4.97 4.97 [25]
Gymnadenia odoratissima 5.4–8.0 7.5–8.0 [126], 5.4–7.7 [131], >6.5 [133]
Hammarbya paludosa 3.5–7.5 4.0–5.9 [126], 4.5–6.0 [131], 3.5–7.5 [133],
4.73–5.35 [142]
Herminium monorchis 5.5–8.1 7.4–8.1 [126], 5.7–8.1 [131], 5.5–8.0 [133]
Himantoglossum adriaticum 6.3–7.85 6.3–7.5 [64], 7.35–7.85 [126]
Himantoglossum 5.5–8.0 5.5–8.0 [133]
comperianum
Himantoglossum hircinum 5.0–8.0 5.0–8.0 [135], 6.9–7 [136]
Himantoglossum jankae 4.43–8.0 4.43–7.72 [25], 7.34–8.0 [126], 5.5–8.0 [133],
7.19–7.51 [134]
Himantoglossum 5.0–8.5 7.6–8.5 [131], 8.2 [132], 5.0–8.0 [135]
robertianum
Limodorum abortivum 4.19–8.5 4.19–7.99 [25], 5.38–8.01 [126], 7.1–8.5 [131],
6.9 [132], 5.5–8.0 [133], 5.9 [134], 5.0–7.5 [135]
Limodorum trabutianum 5.5 5.5 [135]
Liparis loeselii 6.77–8.5 6.77–7.72 [126], 7.3–8.5 [131]
Malaxis monophyllos 3.5–8.1 6.7–8.1 [126], 6.9–7.7 [131], 3.5–8.0 [133]
Neotinea lactea 8.4 8.4 [132]
Neotinea maculata 4.0–8.3 6.2–8.3 [131], 7.4 [132], 4.0–7.5 [135]
Neotinea tridentata 4.59–8.0 4.59–7.99 [25], 5.7–8.0 [126], 5.5–8.0 [133],
5.04–7.3 [134], 6.5 [135], 7.0–7.4 [136]
Neotinea ustulata var. 5.0–7.1 5.0–7.1 [126]
aestivalis
Neotinea ustulata var. 4.32–8.5 4.32–7.6 [25], 4.9–7.6 [110], 5.46–8.0 [126],
ustulata 5.3–8.5 [131], 5.5–8.0 [133], 5–7.5 [135], 5.2–7.3
[136], 6.0–8.5 [143]
Neottia cordata 2.8–6.0 2.8–5.5 [48], 4.0–6.0 [131], 3.0–4.5 [133]
(continued)
Table 1 (continued)
The soil
Neottia nidus-avis 4.14–8.5 4.14–7.74 [25], 5.2–8.0 [126], 6.4–8.5 [131],
5.5–8.0 [133]
Neottia ovata 4.29–8.5 4.29–7.63 [25], 5.5–7.5 [112], 5.74–7.54 [121],
4.6–7.98 [126], 6.4–8.5 [131], 4.5–7.5 [133]
Neottianthe cucullata 3.5–8.0 3.5–8.0 [133]
Nigritella rhellicani 4.5–7.5 5.23–6.18 [25], 4.5–7.5 [144]
Ophrys apifera 4.89–8.5 4.89–7.6 [25], 7.4–7.9 [126], 7.4–8.5 [131],
5.5–8.0 [133], 7.37–7.48 [134], 5.0–8.0 [135]
Ophrys apulica 7.5 7.5 [135]
Ophrys atlantica 7.5 7.5 [135]
Ophrys attica 6.5 6.5 [135]
Ophrys argolica 7.1–8.2 7.1–8.2 [131]
Ophrys bertolonii 7.3–8.4 7.3–8.4 [131], 7.5 [135], 7.83 [145]
Ophrys bombyliflora 5.0–9.4 7.1–9.4 [131], 5.0–7.5 [135]
Ophrys cretica subsp. cretica 8.1–8.2 8.1–8.2 [132]
Ophrys ferrum-equinum 7.0–8.2 7.5–8.2 [131], 7.0 [135]
Ophrys fuciflora s.str. 7.0–7.7 7.2–7.7 [126], 7.0–7.3 [136]
Ophrys grammica 6.12–7.85 6.12–7.85 [25]
Ophrys heldreichii 8.0–8.6 8.0–8.6 [132]
Ophrys helenae 6.5 6.5 [135]
Ophrys insectifera 5.5–8.5 7.8–8.0 [126], 7.1–8.5 [131], 5.5–8.0 [133],
7.0–7.5 [135], 7.1 [136]
Ophrys mammosa 4.87–8.1 4.87–7.85 [25], 8.1 [132], 6.5–8.0 [135]
Ophrys oestrifera (syn. 5.5–8.0 5.64–7.77 [25], 7.6–8.0 [126], 5.5–8.0 [133],
Ophrys scolopax subsp. 7.19–7.51 [134]
cornuta)
Ophrys reinholdii 5.15–7.8 5.15–6.29 [25], 7.5–7.8 [131], 7.0 [135]
Ophrys speculum 7.1–8.1 7.1–8.1 [131]
Ophrys sphegodes 5.0–8.3 7.2–8.3 [126], 5.0–7.5 [135]
Ophrys spruneri 6.5–8.0 6.5–8.0 [135]
Ophrys tenthredinifera 5.5–8.4 6.4–8.4 [131], 7.3 [132], 5.5–8.0 [135]
Ophrys zeusii 5.64–5.81 5.64–5.81 [25]
Orchis anatolica 6.8–8.2 6.9–8.2 [131], 6.8–7.2 [132]
Orchis anthropophora 5.5–8.6 7.0–8.6 [131], 5.5–8.0 [133], 6.5–8.0 [135]
Orchis italica 5.1–9.1 5.1–7.6 [25], 6.8–9.1 [131], 5.26–7.6 [134],
6.5–8.0 [135]
Orchis mascula subsp. 4.18–8.5 4.18–7.99 [25], 4.8–8.5 [131], 5.5–8.0 [133],
mascula 6.9–7.27 [134], 5.0–7.0 [135]
Orchis mascula subsp. 5.0–8.0 5.0–8.0 [135]
olbiensis
Orchis mascula subsp. 5.6–7.7 5.6–7.7 [126]
speciosa
Orchis militaris subsp. 5.5–9.0 7.25–7.54 [25], >7.5 [109], 6.0–8.3 [126], 7.4–9.0
militaris [131], 5.5–8.0 [133], 7.5 [135]
(continued)
Table 1 (continued)
The soil
Orchis pallens 4.83–8.0 4.83–7.54 [25], 6.4–7.5 [126], 6.7–7.0 [131],
5.5–8.0 [133], 6.3–7.3 [136]
Orchis pauciflora 7.04–8.3 7.04–7.85 [25], 7.5–8.3 [131], 8.2 [132], 8.0 [135]
Orchis provincialis 4.59–8.0 4.59–5.87 [25], 5.5–7.8 [131], 5.5–8.0 [133], 7.0
[135]
Orchis punctulata 5.5–8.0 5.5–8.0 [133]
Orchis purpurea subsp. 4.67–8.7 4.67–7.84 [25], 6.2–8.0 [126], 7.5–8.7 [131],
purpurea 5.5–8.0 [133], 5.5–7.0 [135], 6.9–7.3 [136]
Orchis quadripunctata 5.09–8.0 5.09–7.85 [25], 7.5–8.0 [131], 7.8 [132], 7.1–7.3
[134], 6.5–8.0 [135]
Orchis simia subsp. simia 4.74–8.5 4.74–7.85 [25], 7.4–7.9 [126], 7.5–8.5 [131], 7.9
[132], 5.5–8.0 [133], 7.3–7.33 [134], 5.5–7.5
[135]
Orchis spitzelii subsp. 5.0–8.1 7.6–8.1 [131], 5.0–8.0 [135]
spitzelii
Platanthera bifolia 4.3–8.0 4.45–6.73 [25], 4.6–8.0 [126], 4.3–7.5 [131],
4.5–8.0 [133]
Platanthera chlorantha 3.98–8.4 3.98–7.85 [25], 5.3–8.0 [126], 5.4–8.4 [131],
5.5–8.0 [133], 6.9–7.1 [134], 7.4 [137]
Pseudorchis albida 3.5–8.0 4.7–6.2 [131], 3.5–8.0 [133], 4.5–7.2 [146],
4.1–4.6 [147]
Serapias bergonii 4.67–7.2 4.67 [25], 5.27–7.2 [134]
Serapias cordigera subsp. 4.0–5.0 4.67–4.84 [25], 4.0–5.0 [135]
cordigera
Serapias lingua 4.0–9.0 7.1–9.0 [131], 4.0–7.5 [135]
Serapias orientalis subsp. 7.0–8.0 7.0–8.0 [135]
orientalis
Serapias parviflora 4.0–8.0 5.7–7.8 [131], 4.0–8.0 [135]
Serapias perez-chiscanoi 5.0 5.0 [135]
Serapias strictiflora 7.5 7.5 [135]
Serapias vomeracea 4.5–7.5 4.99–6.44 [25], 4.5–7.5 [133], 5.27–7.37 [134],
6.5–7.0 [135]
Spiranthes aestivalis 5.5–7.9 5.5–7.9 [126], 6.8 [131]
Spiranthes spiralis 4.47–8.1 4.47–7.52 [25], 5.7–8.1 [126], 5.0–7.6 [131],
4.5–7.5 [133], 7.4 [136]
Steveniella satyrioides 5.5–8.0 6.4–7.9 [131], 5.5–8.0 [133]
Traunsteinera globosa 5.0–8.0 5.0–6.27 [126], 5.5–8.0 [133]
Traunsteinera sphaerica 5.5–8.0 5.5–8.0 [133]
The proportion of orchids belonging to each one of these categories strongly

depends on both the latitudinal gradient and the altitudinal gradient [11]. Around the
Mediterranean Sea, species that are totally dependent on wet meadows and other
habitat types characterized by high levels of soil moisture are few and restricted to
microsites of suitable land patches, whereas in the countries of Northern Europe,
32
Table 2 Chemical elements and organic matter in soils at sites with some terrestrial orchid species
Orchid taxa C N K P Ca Mg Organic matter
Cypripedium – 1–2 mg 100 g 1 9–29 mg 100 g 1 8–62 mg 100 g 1 300–980 mg 100 g 1 6–12 mg 100 g 1 –
calceolus [136], [136], [136], [136], [136],
5.9–55.3 mg kg 1 30–100 mg kg 1 4.6–10 mg kg 1 [138] 3500–10,000 mg kg 1 60–990 mg kg 1
[138] [138] [138] [138]
Dactylorhiza – 1 mg 100 g 1 [136] 1.5–14 mg 100 g 1 7–20 mg 100 g 1 129–181 mg 100 g 1 5–34 mg 100 g 1 –
maculata [136] [136] [136] [136]
Dactylorhiza – 1 mg 100 g 1 [136], 0.6–8.9 g kg 1 [111], 1.17–6.68 mg 100 g 1 0.3–40.4 g kg 1 [111], 11–16 mg 100 g 1 0.97–35.93%
sambucina 160.3–418.5 mg 14–23 mg 100 g 1 [25], 79–83 mg 100 g 1 [136] [25]
100 g 1 [139] [136], 2.1–14.6 mg kg 1 [136],
0.05–0.2 g kg 1 [111], 0.5–1.8 g kg 1 [139]
[139] 5–8 mg 100 g 1 [136],
1.8–14.9 mg kg 1
[139]
1
Epipactis – 28.3 2.3 mg kg – 22.0 2.1 mg kg 1 – – 7.93–30.18%
atrorubens [116], [116], [25],
1
59.0 4.0 mg kg 71.3 5.2 mg kg 1 0.3–1.0% [141]
[116] [116],
1.69–4.39 mg 100 g 1
[25]
Epipactis pontica 4.10–5.30% 0.38–0.49% 13.07–18.34 mg 2.61–8.78 mg 100 g 1 – – 2.97–15.32%
[128] [128] 100 g 1 [25], [25],
[128] 1.08–1.40 mg 100 g 1 Humus:
[128] 7.01–9.09%
[128]
1
Hammarbya – – 0.2–0.37 g kg 1 0.32–0.76 g kg 1 0.6–2.89 g kg [142] 0.37–0.89 g kg 1 96.5–98.3%
paludosa [142] [142] [142] [142]
Himantoglossum – 1–1.5 mg 100 g 1 28 mg 100 g 1 [136] 41–46 mg 100 g 1 162–203 mg 100 g 1 10–42 mg 100 g 1 –
hircinum [136] [136] [136] [136]
Neotinea – 1–2 mg 100 g 1 24–30 mg 100 g 1 21–29 mg 100 g 1 448–484 mg 100 g 1 8–32 mg 100 g 1 –
tridentata [136] [136] [136] [136] [136]
1
Neotinea ustulata – 0.261–0.516% [110], 25.1–263.2 mg kg 1 14.81–23.54 mg kg 1 1636–6001 mg kg 1 69.7–368.2 km kg 5.4–10.76%
1–2 mg 100 g 1 [110], [110], [110], [110], [110],
[136] 12–44 mg 100 g 1 1.69–4.66 mg 100 g 1 357–780 mg 100 g 1 7–65 mg 100 g 1 1.68–27.99%
[136] [25], [136] [136] [25]
1
8–27 mg 100 g 1
[136]
1
Neottia ovata – 41.9–194.2 mg kg – 3.1–31.6 mg kg 1 – – 8.8–28.6% [121],
[121] [121], 0.78–28.41%
1.17–8.78 mg 100 g 1 [25]
[25]
Ophrys fuciflora – 1–2 mg 100 g 1 26–44 mg 100 g 1 5–46 mg 100 g 1 162–620 mg 100 g 1 7–42 mg 100 g 1 –
s.str. [136] [136] [136] [136] [136]
Ophrys – 2 mg 100 g 1 [136] 44 mg 100 g 1 [136] 27 mg 100 g 1 [136] 620 mg 100 g 1 [136] 7 mg 100 g 1 [136] –
insectifera
1 1 1
Orchis militaris – 120–360 μg g 68–160 mg kg 0.037–0.10% [109], 54–94% [109] 50–91 mg kg [109] 20.23–21.03%
1
[109] [109] 3.92–4.71 mg 100 g [25]
[25]
1 1 1 1
Orchis pallens – 2–3 mg 100 g 31–38 mg 100 g 12–28 mg 100 g 1 420–665 mg 100 g 14–16 mg 100 g 1.68–24.03%
[136] [136] [136], [136] [136] [25]
1
1.76–4.25 mg 100 g
[25]
1 1 1
Orchis purpurea – 1–3 mg 100 g 16–30 mg 100 g 1 8–41 mg 100 g 1 203–510 mg 100 g 6–12 mg 100 g –
[136] [136] [136] [136] [136]
1
Pseudorchis 7.2–16.3% 0.4–1.1% 4.0–112.0 mg 0.4–20.6 0 mg 100 g – – 4.3–89.4%
albida [147] [147] 100 g 1 [146] [146]
[146],
1 1 1
Spiranthes – 1 mg 100 g [136] 24 mg 100 g 1 [136] 21 mg 100 g 1 [136], 440 mg 100 g [136] 8 mg 100 g [136] 1.28–7.45% [25]
spiralis 1.5–5.33 mg 100 g 1
[25]
The Role of Ecological Factors in Distribution and Abundance of Terrestrial. . .
33
these orchids can prevail or be present in a proportion notably higher than in the
countries of Southern Europe. For example, in the British Isles, 32.7% of the
orchid species and subspecies (19 out of 58 taxa) belong to the group of orchids
which require wet conditions, while the corresponding figure is 25.7% (18 out of 70
taxa) for the Czech Republic and 0.07% (14 out of 193 orchid taxa) for Greece
[13, 14, 123, 124].
4.2.2 Soil pH
Soil pH is one of the most important soil factors affecting the distribution and
abundance of terrestrial orchids [25, 26, 105, 117]. Since the soil reaction controls
the uptake of minerals, either directly or through the mycorrhizal association, the pH
gradient can be considered to be a resource gradient [108]. In general, it can be
considered that most terrestrial orchids in Europe occur on soils that range from
slightly acidic to slightly alkaline, whereas just a small portion of orchids can tolerate
and survive in conditions of low (<4–4.5) or high (>8.5) pH values. The smaller
number of orchids on strongly acidic soils can be attributed to the fact that acidic
soils with pH < 4.5 contain high concentrations of harmful H+ and Al3+ ions
[103, 108]. Increasing richness of orchid species with increase of soil pH can be
attributed to the fact that increased pH can positively affect the availability of
nutrients. However, in highly alkaline soils, and also in very acidic ones, mycorrhiza
cannot survive, which in turn can cause a reduction in the number of orchids
occurring at a site [103].
Soil pH is often considered to be one of the most important factors separating
habitats of similar and related species. Thus, Dactylorhiza fuchsii is generally linked to
soils with higher pH values than Dactylorhiza maculata, which is often associated
with acidic soils [125]. In addition to D. maculata, other well-known terrestrial orchids
that occur on acidic soils in Europe are Neottia cordata, Anacamptis coriophora,
Dactylorhiza romana, D. cordigera, Hammarbya paludosa, Corallorhiza trifida,
Malaxis monophyllos, Epipactis pontica, E. purpurata, and Serapias cordigera
(Fig. 7, Table 1), whereas the group of species that prefer alkaline soils includes
Orchis militaris, O. pauciflora, Epipactis atrorubens, Anacamptis pyramidalis, Cyp-
ripedium, and most Ophrys species (Table 1) [9, 25, 48, 49, 109, 126–128].
A significant number of species of orchids that prefer acidic soils grow in high-altitude
areas, which is understandable in view of the fact that soil pH is negatively correlated
with altitude, since at higher altitudes the decomposition of organic matter is slower
and the acidification process more intense due to higher precipitation [26, 129].
Although this is a general rule and is frequently observed in Central Europe, it is not
the case in the southernmost areas of Europe, where values of soil pH in many high
mountains are greater than 7.0 [130].
It should be noted that several orchids have a wide range of soil pH at which they
occur, while others have a narrower pH range (Table 1). Specifically, Anacamptis
laxiflora, Coeloglossum viride, Dactylorhiza flavescens, D. incarnata, D. sambucina,
Epipactis helleborine subsp. helleborine, Malaxis monophyllos, Neotinea maculata,
Neottia nidus-avis, N. ovata, Neottianthe cucullata, Orchis mascula subsp. mascula,
O. purpurea subsp. purpurea, Platanthera chlorantha, Pseudorchis albida, and
Fig. 7 Terrestrial orchids occurring mostly on acidic soils. (a) Anacamptis coriophora, (b)
Dactylorhiza romana, (c) Serapias cordigera, (d) Epipactis pontica, (e) Neottia cordata (a photo
V. Djordjević; b–e photos S. Tsiftsis)
Serapias lingua are orchids that occur over a wide range of soil pH values, thereby
indicating their considerable ecological plasticity. Furthermore, the recorded pH
values can differ from one geographical area to another. Thus, the pH ranges indicated
by Sundermann [131] for the species Orchis simia, Corallorhiza trifida, and
Cephalanthera longifolia were 7.5–8.5, 6.5–7.5, and 6.5–8.6, respectively, whereas
pH values measured in northeastern Greece were 4.74–7.85, 4.14–7.48, and
4.34–7.85, respectively [25].
4.2.3 Nitrogen, Phosphorus, and Potassium

According to the results of numerous studies, the content of nutrients in the soil
plays an important role in the growth, development, and distribution of orchids
[25, 57, 62, 103, 117, 148]. There are several ways in which the availability of
nutrients can affect the growth of orchids [103]. Nutrient availability can directly
affect the growth of orchids, a situation determined by ecophysiological charac-
teristics of the orchids. In addition, the content of nutrients in the soil influences
the growth of associated mycorrhizal fungi and affects the relationship between
mycorrhizal fungi and orchids [103]. Furthermore, increased content of nutrients in
the soil can also increase the growth of surrounding strongly competitive plant
species that endanger the survival of orchids [42].
Although
it is known that terrestrial orchids can absorb both nitrate nitrogen
NO3 and ammonium nitrogen (NH4+), the absorption rate is higher in the case of
NO3 [57]. Some studies explored the transfer of nutrients from mycorrhizal fungi to
orchids [149]. While all orchids in the early growth stages are entirely dependent on
mycorrhizal fungi, many autotrophic adult orchids still obtain nutrients through
mycorrhizal fungi [57]. Furthermore, the transfer of nitrogen and carbon from sub-
strates or trees to orchids through mycorrhizal fungi has been demonstrated using
radiocarbon and stable isotopes [150].
The widespread distribution of some terrestrial orchids (e.g., Platanthera bifolia
and Neottia ovata) can be explained by the fact that they tolerate high nitrogen
content in the soil, i.e., they are euryvalent with respect to the amount of nitrogen in
the soil [151]. Interesting results of research were obtained in Greece, where it was
established that the content of phosphorus is the most important factor affecting the
distribution of Goodyera repens on the southern border of its distribution [104].
Although most terrestrial orchids are sensitive to increased content of nutrients in
the soil [103], species respond differently to changes of nutrient content. Thus, soil
enrichment with phosphorus, nitrogen, and potassium has a negative effect on
populations of Dactylorhiza majalis [152]. It has been concluded that Dactylorhiza
maculata, Platanthera bifolia, and Neottia ovata can survive if the content of
calcium or calcium together with nitrogen in the soil increases but that they cannot
survive when the levels of calcium, nitrogen, and phosphorus increase together
[151]. The negative effect of high content of nutrients is frequently reflected in
disturbance of mycorrhizal relationships [103]. To be specific, studies have shown
that a high concentration of nitrogen in the soil negatively influences the develop-
ment of protocorms in Dactylorhiza incarnata [148]. Moreover, in soil with high
carbon and nitrogen content, protocorms of some orchid species rejected mycorrhi-
zal fungi [148]. A negative impact of soil fertilization was shown by research in
Flanders and the Netherlands, where investigators found a significant decrease in the
abundance of Anacamptis morio due to soil enrichment with phosphorus [153].
Several studies provided detailed insight into nitrogen, phosphorus, and potassium
content in the soil on which terrestrial orchids grow (Table 2). Overall, it is considered
that the majority of orchid species occur on soils that are relatively poor in nutrients
[103]. Well-known terrestrial orchid species that occur on oligotrophic soils are
Goodyera repens, Epipactis atrorubens, and Spiranthes spiralis [49]. Moreover,
Dactylorhiza maculata and D. praetermissa were found to grow primarily on low-
phosphorus soils [154]. Among orchid species that grow on soils with very low
phosphorus content in northeastern Greece were Anacamptis pyramidalis, Neotinea
tridentata, Orchis quadripunctata, Goodyera repens, Epipogium aphyllum, and

Serapias species, whereas species that grow on soils with high phosphorus content
were Cephalanthera damasonium, C. longifolia, C. rubra, Corallorhiza trifida,
Epipactis helleborine, E. microphylla, and Neottia nidus-avis [25].
4.2.4 Calcium and Magnesium

Most terrestrial orchids in Europe are known to grow on calcium-rich soils [9, 11, 34].
Calcium in the soil is found primarily in the form of calcium carbonate, but also as
phosphoric and silicic acid salts, as well as salts of various organic acids [155].
Terrestrial orchids mostly occur on typical calcium-rich soils that are developed on
the sedimentary rock limestone, and little is known about orchids that grow on soils
containing high concentrations of calcium derived from other geological substrates
[34]. In addition, a significant number of species grow on soils developed on
dolomite, a sedimentary rock composed mostly of the mineral dolomite, i.e., calcium
magnesium carbonate – CaMg(CO3)2 [34, 107]. Recent studies suggested that
numerous terrestrial orchids can also be found on serpentine soils that are rich in
magnesium, with lower calcium content [24, 26, 107, 116]. The calcium and mag-
nesium content in soils with some terrestrial orchid species has been presented in
several studies (Table 2).
Calcium affects both physical and chemical properties of the soil. Most often,
calcareous soils are permeable to water and therefore are warm, dry, and well aerated.
Consequently, these soils are suitable for most orchids that prefer warm and dry habitat
types, especially ones from the genera Orchis, Ophrys, Anacamptis, Himantoglossum,
Serapias, and Neotinea [9]. Since these soils contain large amounts of Ca2+ and HCO3
ions, the negative effects of H+ and Al3+ ions are suppressed. At the same time,
calcareous soils are characterized by high activity of nitrogen-fixing bacteria. On the
other hand, due to their high calcium concentration and high pH, some elements
become less available for plants (phosphorus is converted to insoluble calcium
phosphate, whereas iron and manganese are poorly soluble), which is a situation
favorable to a large number of orchids that are sensitive to high levels of phosphorus
and heavy metals [103, 104]. Free calcium at high concentrations may be toxic to
orchid cells. Therefore, orchids often produce calcium oxalate crystals to remove
excess calcium or oxalic acid [156]. The presence of calcium oxalate crystals has
been reported in almost all orchid parts, especially in tubers, roots, stems, anthers, and
lips. Moreover, calcium oxalate crystals have a prominent role in plant defense,
osmotic regulation, and maintenance of ionic balance [156].
4.2.5 Organic Matter

Orchids at their initial growing stages are fully mycoheterotrophic, obtaining carbo-
hydrates and mineral nutrients through the mycorrhizal fungi with which they are
associated [62]. Contrary to this fully mycoheterotrophic relationship, orchids at
maturity obtain carbon both through photosynthesis and from ectomycorrhizal fungi,
thereby becoming only partially mycoheterotrophic. Soil organic matter contains the
main source of carbon and nutrients that are transferred to orchids through their
mycorrhizal fungi [62]. For example, albino specimens of Cephalanthera longifolia

depend on their fungi for carbon (mycoheterotrophy), whereas photosynthetic green
specimens recover just 33% of their carbon from fungal partners (myxotrophy) [157].
So far it has been found that the germination rate of orchids is positively correlated
with the content of organic matter in the soil [158]. Moreover, the content of organic
matter in soils significantly affects the abundance of orchids at the population level,
i.e., in areas larger than 400 m2 [72]. Species that grow on soil with a high percentage
content of organic matter include Orchis simia (1.46–47.99%), Dactylorhiza
cordigera (2.96–45.10%), D. incarnata (2.03–41.72%), Cephalanthera damasonium
(0.78–38.96%), C. rubra (0.97–38.96%), and Corallorhiza trifida (1.86–38.96%),
whereas other species grow on soil with low percentage content of organic matter –
Ophrys reinholdii (1.85–2.47%), O. zeusii (2.07–3.57%), O. apifera (1.87–2.47%),
and Dactylorhiza romana (3.35–4.09%) [25].
The humus content of the soil, i.e., specific mixtures of organic and mineral
materials, mainly in the colloidal state, formed by the decomposition of organic
residues in soil under the influence of microorganisms, was highlighted in a recent
study treating the orchid species Epipactis pontica [128].
4.2.6 Orchids Growing on Anthroposols

In other studies, some terrestrial orchid species were found to grow on anthroposols,
i.e., azonal soils highly modified or constructed by human activities (e.g., fly ash, mine
waste deposits and soils in the vicinity of roads) [159–162]. Orchids encounter a range
of stressful and growth-limiting factors on anthroposols, factors such as drought, high
temperature, and light intensity; low/high pH; unfavorable mechanical composition;
high concentrations of soluble salts; lack of essential nutrients (N, P, K); toxic
concentrations of As, B, Cd, Cr, Cu, Hg, Mn, Mo, Ni, Zn, Pb, and Se; reduced
presence of mycorrhizal fungi; and reduced abundance of nitrogen-fixing microorgan-
isms [163]. It is assumed that some orchids grow on anthroposols primarily due to
favorable light regimes and reduced competition between plants [164].
Orchids that are found to occur on anthroposols primarily belong to the genera
Epipactis and Dactylorhiza [160–162, 164, 165]. Among orchids which have the
ability to colonize and grow on soils in the vicinity of roads, it is especially important
to emphasize species from the genus Himantoglossum [166, 167], as well as
Epipactis helleborine, Neottia ovata, Dactylorhiza saccifera, Ophrys apifera, and
Cephalanthera longifolia [26, 165, 167]. In southern Poland, Dactylorhiza majalis,
Epipactis atrorubens, and E. helleborine were found to grow on zinc mine tailings
consisting of post-flotation material and characterized by high concentrations of Zn,
Pb, and Cd [159]. In that study, mycorrhizal fungi were found to play an important
role in the detoxification of heavy metals in the case of E. atrorubens, in which Pb
and Zn are accumulated in the roots [159]. The same authors showed that heavy
metals are mostly stored in the fungal cell walls, a circumstance which is probably
associated with the presence of fungal pigments such as melanin, known for the fact
that it is involved in detoxification mechanisms [159]. In this way, it was demon-
strated that the mycorrhizal mycelium takes heavy metals from the soil and stores
them, thereby reducing their toxicity to the orchid plant host. However, heavy metals
can also be transported directly through plant root cells, which may explain the high
concentration of heavy metals in the shoots of E. atrorubens [159].
Several orchid species were found growing on mine tailings in northeastern
Estonia, viz., Epipactis atrorubens, Orchis militaris, and Dactylorhiza baltica
[160]. It has been established that these orchids and their mycorrhizal fungi are not
limited to environmentally polluted sites compared to natural sites. Moreover, the
same fungi associated with E. atrorubens and O. militaris were found on mine
tailings and in natural habitats [160]. Furthermore, Epipactis palustris was found to
grow in former lignite mining areas in eastern Germany [161]. The results of
molecular investigation indicated that genetic diversity values for populations of
this species from mining areas do not differ from those of populations from natural
habitats [161]. Epipactis palustris was found on Pb-Zn polymetallic deposits of the
Rudnik orefield in Serbia (unpublished data). To be specific, specimens of this
species were discovered on flotation tailings that are in the initial phase of coloni-
zation and sparsely covered with vegetation.
Interesting findings come from Australia, where orchids were found to grow on
bauxite-mined land, suggesting that mycorrhiza fungi also play an important role in
orchid colonization [168, 169]. The latter authors determined that orchids occur for
the first time in 2-year-old rehabilitated areas, but increase in abundance over time
[169]. Especially at rehabilitated sites more than 10 years old, the density of orchids
was found to have increased significantly [168]. Among orchids growing in the
rehabilitated areas, the most abundant species were Caladenia flava, Disa bracteata,
Diuris longifolia, Microtis media, Pterostylis mint, and Pterostylis vittata [168].
In general, there have been few studies on the ecophysiological responses of
orchid species to the stressful conditions of anthroposols, and it is unclear whether
habitats developed on these substrates can act as refuges for endangered and rare
orchid species.
5 Vegetation Types
Terrestrial orchids occur in almost all known ecosystems with various types of
vegetation from coasts to highlands, usually in forest and grassland communities,
as well as in wetlands, on steppes, and in desert oases [5, 9]. However, orchids are
absent or less abundant in salt marshes, in extremely dry deserts, and on cultivated
agricultural lands [5]. Although certain species in Europe occur exclusively in
forests and scrub vegetation, and some inhabit only herbaceous vegetation, a signif-
icant number of species grow in both forests and herbaceous communities [9, 26].
However, a large number of orchids grow in forest-grassland and scrub-grassland
ecotones and on the very edges of forests and herbaceous communities, which makes
it harder to determine vegetation units more precisely. In general, vegetation types
significantly affect the distribution and abundance of orchids, representing one of the
key gradients influencing separation of the ecological niche of orchids [24–26].
The degree of representation of orchids in plant communities is rarely greater than
30% [49]. Therefore, they rarely represent diagnostic species of plant communities.
However, some orchid species appear as dominant species in several plant commu-
nities on the Balkan Peninsula. For example, Ophrys sphegodes, Anacamptis
coriophora, and A. papilionacea have significant abundance in the Orchido-
Chrysopogonetum community of the thermo-Mediterranean calcicolous phrygana
of northern Greece (Dorycnio-Coridothymion capitati) [34]. Furthermore, the
Trolio-Orchidetum bosniacae (Calthion palustris) community was described in
Bosnia and Herzegovina, whereas the Orchido-Schoenetum nigricantis community
of the alliance Caricion davallianae is represented in Slovenia and Croatia [34]. It
should be noted that the association of orchid species and surrounding species within
plant communities is determined not only by abiotic factors, such as moisture, the
light regime, and soil properties, but also by many complex biotic factors, as well as
by age and evolutionary development of the ecosystem. For example, the association
between orchid species and the surrounding forest trees is further strengthened by
the fact that many orchids form associations with fungi that are ectomycorrhizal on
the roots of neighboring trees [7, 81, 170], indicating that the phylogeographic
dynamics of many orchid species can be linked to the phylogeographic patterns of
trees [22].
In this section, we present an overview of the main vegetation types at sites with
terrestrial orchids in Europe, with special reference to the Balkan Peninsula. The
names of syntaxonomic units follow the phytocenological nomenclature proposed
by Mucina et al. [171].
5.1 Forest and Scrub Vegetation
5.1.1 Deciduous Forests

Among the types of forest vegetation in Europe, plant communities of the class
Carpino-Fagetea sylvaticae are particularly important owing to the great diversity of
terrestrial orchids, in terms of both the number of species and their abundance [34].
These are mesic deciduous and mixed forests of temperate Europe, Anatolia, the
Caucasus, and Southern Siberia, consisting primarily of different beech and mixed
fir-beech forests, as well as oak-hornbeam and mesic oak forests [171]. Orchid
studies indicate that the largest number of Epipactis, Cephalanthera, and Neottia
species occurs in beech-dominated forests in Europe [9, 12, 20, 106, 126, 127]. The
great richness of orchid species in these forest types can be attributed to (i) the strong
presence of these forests, both horizontally and vertically; (ii) the fact that they
appear on different geological substrates (calcareous and non-calcareous) and in
different climatic zones; and (iii) the age of these forests. In general, the beech-
dominated forests of Southern Europe, including the Iberian, Apennine, and Balkan
Peninsulas, are important refugia of many orchid species [22]. To be specific, during
the last glaciations, many orchid species were distributed in beech forests and
restricted to these peninsulas as well as the Caucasus. However, during the climate
amelioration that began c 10,000 BC, beech forests moved slowly to the north and to
the west, reaching Scandinavia around AD 500 [9]. Scientists believe that this
second arrival of beech forests in Central Europe made possible the process of
evolutionary radiation of many orchid species, especially ones of the genus
Epipactis. Some recent research provides an insight into the exact number of orchid
taxa growing in these forest types. For example, 26 orchid species and subspecies
were found to grow in beech-dominated forests in western Serbia, representing the
forest communities richest in orchid taxa in this part of the central Balkans [34].
Furthermore, 22 orchid species were found to inhabit these forests in northeastern
Greece [13]. It should be noted that many boreal and Central European orchids that
have the southern boundaries of their distribution on the Balkan Peninsula (e.g.,
Epipogium aphyllum, Epipactis leptochila subsp. neglecta, E. pontica, and
E. purpurata) were found in such beech and beech-fir forests [13, 25, 128, 172].
The most common orchid species that grow in oak-hornbeam forests
(Carpinetalia betuli) are Epipactis helleborine, E. purpurata, E. pontica, Neottia
nidus-avis, N. ovata, Orchis mascula, O. pallens, O. purpurea, and Platanthera
bifolia [9, 34, 112, 127, 173]. Overall, these forests are inhabited by a large number
of Central European orchid species that require mesophilic conditions. In the central
Balkans (western Serbia), ten orchid species were found in oak-hornbeam forests,
especially in the community Querco-Carpinetum betuli [34].
Terrestrial orchids have significant representation in open oak, mixed deciduous,
and conifer forests of warm regions in the temperate zone, i.e., in communities of the
class Quercetea pubescentis. These forests are important vegetation types for many
orchid species in Central and Southern Europe and in Mediterranean regions, Asia
Minor, and the Middle East [171]. Within this vegetation class, orchids are especially
well represented in the order Quercetalia pubescenti-petraeae and the alliances
Fraxino orni-Ostryion, Quercion petraeo-cerridis, Quercion confertae, Quercion
petraeae, Aceri tatarici-Quercion, and Carpinion orientalis [34]. Studies from the
Balkan Peninsula provide precise data on how many orchid species grow in oak
forests. For example, 21 orchid species were found to occur in forest communities
dominated by the oaks Quercus frainetto and Q. petraea subsp. medwediewii in
northeastern Greece [174]. Furthermore, 21 orchid species and subspecies were
registered in thermophilous montane oak forests of the alliance Quercion petraeo-
cerridis, whereas 16 orchid species were found to inhabit thermophilous deciduous
oak forests on slightly acidic deep soils (Quercion confertae) in western Serbia [34].
Other studies highlighted the importance of amphi-Adriatic calcareous sub-Med-
iterranean and inland oak and hop-hornbeam forests of the alliance Fraxino orni-
Ostryion [24, 25, 34]. The large number of orchids in these forests is explained by
the fact that they are mainly widespread on limestones, especially in gorges and
canyons, where they are sheltered from extreme climatic influences. The importance
of Tertiary relict Ostrya carpinifolia forests is reflected in the great abundance of
orchid species from the sub-Mediterranean and Mediterranean chorological groups
(e.g., many Ophrys, Orchis, and Himantoglossum species) and in the presence of
certain orchids that are primarily cenobionts of grassland ecosystems [34]. Not
strongly influenced by anthropogenic factors, these forests include some of the
most highly specialized orchid species [24]. In western Serbia, 24 orchid species
and subspecies were found to grow in these forest types [34]. Similarly, 22 orchid
species were registered in communities consisting of Ostrya carpinifolia, Carpinus
orientalis, and Fraxinus ornus in northeastern Greece [174].
Terrestrial orchids in Europe also occur in acidophilous oak and oak-birch forests
on nutrient-poor soils of Europe (Quercetea robori-petraeae) [171]. For example, in
Ukraine, Dactylorhiza sambucina was found to grow in Quercion robori-petraeae
forests [111]. Within this vegetation class, orchids inhabit acidophilous chestnut-oak
forests (Castaneo-Quercion petraeae) of Southeast Europe. In forests dominated by
chestnut (Castanea sativa) in northeastern Greece, the following species were
recorded: Cephalanthera longifolia, C. rubra, Dactylorhiza saccifera, Epipactis
helleborine, E. microphylla, Limodorum abortivum, Neottia nidus-avis, and
Platanthera chlorantha [174]. Orchids inhabiting similar forest communities in
western Serbia include Epipactis microphylla, Neottia nidus-avis, Ophrys scolopax
subsp. cornuta, and Orchis purpurea (authors’ unpublished data).
Several terrestrial orchids were found to grow in forest communities of the class
Brachypodio pinnati-Betuletea pendulae. These forests represent hemiboreal pine and
birch-pine forests on fertile soils of the southern Urals and Southern Siberia and relict
birch-poplar forests of Europe [171]. For example, Epipactis helleborine and
Platanthera bifolia were found to have significant populations in birch forests of
Russia and the former Soviet Union [133]. Furthermore, some orchid species inhabit
relict extrazonal temperate deciduous birch-poplar forests on mineral soils (Fragario
vescae-Populion tremulae). In western Serbia, orchid representatives include mainly
species that are characteristic cenobionts of grassland communities (e.g., Anacamptis
morio, Dactylorhiza sambucina, Gymnadenia conopsea, and Platanthera bifolia)
[34], whereas Dactylorhiza sambucina, Epipactis helleborine, Neottia ovata, and
Platanthera chlorantha were recorded in birch forests in northeastern Greece [174].
The smaller number of orchids in birch forests on the Balkan Peninsula is explained by
the limited distribution of these forests and the fact that they generally represent an
unstable stage in the succession of forest vegetation [34]. To be specific, birch forests
in western Serbia and Greece are widespread within the belt of oak, pine, and beech
forests and arise mainly after forest fires or logging.
5.1.2 Coniferous and Mixed Broadleaved-Coniferous Forests

A significant number of orchids grow in communities of the vegetation class
Vaccinio-Piceetea, i.e., in Holarctic coniferous and boreo-subarctic birch forests of
the boreal zone and in high-elevation areas of mountains in the nemoral zone of
Eurasia [9, 12, 13, 19, 34, 73, 133, 175]. Within this vegetation class, many orchid
species occur in European boreo-montane and subalpine spruce and pine forests on
nutrient-poor soils (Piceetalia excelsae); in boreo-temperate pine forests on nutrient-
poor and hydromorphic soils (Pinetalia sylvestris); in boreo-montane spruce, fir, and
pine forests on nutrient-rich soils (Athyrio filicis-feminae-Piceetalia); and in boreal
mesophilous coniferous forests developed on podzolic soils in easternmost European
Russia, the Urals, and Siberia (Piceo obovatae-Pinetalia sibiricae) [34, 171]. These
forests are inhabited by many orchids from the boreal chorological group, which
tolerate shade and cooler temperatures, species such as Epipogium aphyllum,
Goodyera repens, Corallorhiza trifida, and Neottia cordata [9]. In Southern Europe,
most of these orchid species grow in forests of spruce (Picea abies), pine (Pinus
sylvestris), and mixed forests of spruce, fir, and beech at higher altitudes on the
southernmost boundaries of their distribution in Europe [34, 45, 104, 175]. In the
central Balkans (western Serbia), 26 orchid species and subspecies were found to
grow in communities of the class Vaccinio-Piceetea [34]. In this region, many
orchids were found to grow in stands of these communities at lower altitudes,
primarily due to temperature inversion and higher rainfall [34]. Recent research
from Greece treated the importance of Picea abies and Pinus sylvestris forests for the
growth and survival of Neottia cordata, and the results indicated that Picea abies
forests are more suitable for its conservation than those of Pinus sylvestris [45].
Members of the family Orchidaceae are also represented in different pine forests
and related scrub communities [9, 13, 24, 34, 133]. Numerous orchid species were
found in such communities, especially in pine forests on calcareous and ultramafic
geological substrates in the Balkans, the Alps, the Carpathians, and the Crimea within
the vegetation class Erico-Pinetea. Furthermore, orchids are also registered in com-
munities of the vegetation class Roso pendulinae-Pinetea mugo, which include pine
krummholz in subalpine belts of the nemoral mountain ranges of Europe [48, 133].
Orchids are significantly abundant in relict Pinus sylvestris forests on calcareous
substrates of the Alps, the Hercynicum, and the Massif Central (Erico carneae-
Pinion). Moreover, the high abundance of some orchids (e.g., Cephalanthera rubra
and Epipactis muelleri) resulted in corresponding names of syntaxa, such as
Cephalanthero rubrae-Pinion sylvestris and Epipactido muelleri-Pinion sylvestris
[171]. On the Balkan Peninsula, orchids inhabit Pinus sylvestris forests on calcare-
ous substrates of the central and eastern Dinarides (Seslerio rigidae-Pinion); Pinus
nigra forests on calcareous substrates of the central and southern Balkans (Fraxino
orni-Pinion nigrae); Pinus nigra forests on dolomite and ultramafic geological
substrates of the Dinarides (Erico-Fraxinion orni); and Pinus heldreichii forests on
calcareous and ultramafic substrates of the southern Balkans (Pinion heldreichii)
[24, 25, 34]. Particularly high diversity of orchids in pine forests has been reported in
Greece [13]. Specifically, in northeastern Greece, 19 orchid species were found in
Pinus sylvestris forests, whereas 18 orchid species were recorded in Pinus nigra
forests [174]. In communities of the vegetation class Erico-Pinetea in western
Serbia, a total number of 22 orchid species was registered [34]. In this area, orchids
were often found in pine forest communities developed on ultramafic substrates [34].
Due to significant illumination, many orchids characteristic of grassland ecosystems
have been recorded in these pine forests, species such as Anacamptis morio,
Dactylorhiza sambucina, D. maculata subsp. transsilvanica, Gymnadenia
conopsea, Platanthera bifolia, and Traunsteinera globosa [24, 34]. In addition,
G. conopsea has been registered in Central Europe in communities of the alliance
Erico-Pinion and the Molinio-Pinetum community in eastern Switzerland [61].
In the central Balkans, some orchid species were registered in Picea omorika
forests (Erico carneae-Piceion omorikae) within the vegetation class Erico-Pinetea.
Among the 11 orchid species recorded in these forests in western Serbia, especially
the boreal species Goodyera repens and Neottia cordata are significantly abundant
[34, 176]. Interestingly, the community Goodyero-Piceetum omorikae has been

described in Bosnia and Herzegovina, which confirms the association between
Goodyera repens and paleoendemic coniferous species (Picea omorika) [34, 176].
5.1.3 Mire and Swamp Forests

Among damp forest types, orchids are especially common in communities within the
classes Alnetea glutinosae (alder swamp forests and willow scrub occurring in
habitats with a permanently high water table) and Franguletea (willow carr of
Western Europe and the sub-Atlantic regions of Central Europe) [19, 34]. Thus,
Dactylorhiza majalis was found to inhabit Alnetea glutinosae communities in
Central Europe [42], whereas Neottia ovata was registered in the Stellario
nemorum-Alnetum glutinosae community [112]. In western Serbia, Dactylorhiza
saccifera and Neottia ovata were found to occur in similar communities with Alnus
glutinosa as a diagnostic species [34]. In Slovakia and Romania, Epipactis albensis
was found to grow in alluvial forests and on banks with willows and poplars [140].
5.1.4 Broadleaved Evergreen Forests, Coniferous Forests of the

Mediterranean, and Scrub Vegetation
Communities of the class Quercetea ilicis (thermo-Mediterranean pine and oak
forests and associated macchia) are inhabited by a significant number of orchids in
the Mediterranean region of Europe [16, 19, 174]. Furthermore, Junipero-Pinetea
sylvestris communities (relict oro-Mediterranean and sub-Mediterranean pine for-
ests, juniper woods, and related scrub of the Mediterranean) also support many
terrestrial orchids [19]. A great number of orchids from the Mediterranean and sub-
Mediterranean chorological groups are distributed in scrub vegetation of types such
as Ononido-Rosmarinetea (Mediterranean scrub on calcareous substrates under the
names garrigue, phrygana, tomillar, espleguer, romeral, and batha), Cisto-
Lavanduletea stoechadis (Mediterranean scrub on siliceous and ultramafic substrates
known as matorral, garrigue, phrygana, and jaral), and Cytisetea scopario-striati
(Mediterranean and sub-Atlantic scrub on acidic substrates) [19].
Scrub and mantle vegetation seral or marginal to broadleaved forests in the sub-
Mediterranean regions and nemoral zone of Europe (class Crataego-Prunetea) [171]
also represent an important vegetation type for growth of many terrestrial orchid
species. The communities richest in orchid species on the Balkan Peninsula belong
to the vegetation order Paliuretalia (thermophilous mantle, pseudomaquis, and
shrubbery fringing oak forests of the sub-Mediterranean regions of Southeast
Europe) [171]. These vegetation types are distributed mainly in low- and middle-
altitude areas, and they contain a large number of sub-Mediterranean and Mediter-
ranean orchids of the genera Ophrys, Orchis, Anacamptis, Serapias, and
Himantoglossum [25].
Representatives of the orchid family also inhabit communities of the vegetation
class Robinietea [34]. This vegetation type represents seral forest clearings and
anthropogenic successional scrub and thickets on nutrient-rich soils in temperate
Europe [171]. Specifically, Dactylorhiza saccifera and Neottia ovata were found in
communities of elder, willow, and hazel scrub on nutrient-rich soils in forest

clearings (Sambuco-Salicion capreae) [34].
5.2 Herbaceous Vegetation
Terrestrial orchids are widely represented in various types of herbaceous vegeta-

tion, including grasslands, meadows, heaths, montane-subalpine tall-herb vegeta-
tion, and tall-grass perennial swards on mobile coastal dunes, as well as fens, bogs,
and marshes [19, 26, 133, 177]. Recent studies of herbaceous communities in
Europe indicated that herbaceous vegetation types significantly influence the
patterns of orchid diversity, in regard to both composition and abundance of
species [26, 105, 178, 179].
5.2.1 Grasslands, Meadows, and Heaths

Many terrestrial orchids grow in communities of the class Festuco-Brometea
[24, 26, 34, 105, 133, 178, 180–182]. This is dry and semi-dry grassland and steppe
vegetation in the sub-Mediterranean, nemoral, and hemiboreal zones of Europe
[171]. The large number of orchids within communities of the Festuco-Brometea
class indicates that these represent a major herbaceous vegetation type for orchids,
which can be explained by the significant distribution of this vegetation from
lowland to highland areas, as well as by the diversity of geological substrates and
soils on which the communities are developed. Although these communities are
mainly distributed on calcareous substrates [171], many orchids are also widely
represented in communities on various siliceous and ultramafic substrates [24, 26].
In communities of this vegetation class, a total of 31 orchid species and subspecies
were found in western Serbia, suggesting that the given vegetation class is the
richest with orchid species in this region of the central Balkans [34]. Moreover,
according to the European Union’s Habitats Directive (92/43/EEC), it is also
emphasized that “semi-natural dry grasslands and scrubland facies on calcareous
substrates (Festuco-Brometalia) ( important orchid sites)” are one of the priority
vegetation types [179].
Within the class Festuco-Brometea, orchids are especially well represented in the
order Brachypodietalia pinnati, which encompasses meso-xerophytic basiphilous
grasslands of Western Europe and sub-Atlantic Central Europe (Bromion erecti);
meso-xerophytic basiphilous grasslands of the subcontinental regions of Central and
Southeast Europe (Cirsio-Brachypodion pinnati); and dry and semi-dry grasslands on
deep soils over siliceous bedrocks in the colline to submontane belts of the southern
and central Balkans (Chrysopogono-Danthonion calycinae) [2, 34, 171, 182]. Well-
known orchids registered on carbonate substrates in communities of the alliance
Bromion erecti (often called Mesobromion) in Central Europe include Anacamptis
pyramidalis, A. morio, Gymnadenia conopsea, Herminium monorchis, Neotinea
tridentata, N. ustulata, Neottia ovata, Ophrys apifera, O. sphegodes, O. holoserica,
O. insectifera, Orchis mascula, O. militaris, O. purpurea, O. simia, Platanthera
bifolia, and Spiranthes spiralis [61, 112, 180–182]. However, orchids from
Chrysopogono-Danthonion calycinae communities have only recently been studied,

in investigations where researchers found that orchids growing in these communities
in the central Balkans are common both on ultramafic substrates and on calcareous as
well as siliceous soils [24, 26]. Orchids that form large colonies in these grassland
communities are Anacamptis morio, A. coriophora, A. papilionacea, A. pyramidalis,
Gymnadenia conopsea, Neotinea tridentata, N. ustulata, Platanthera bifolia, and
Dactylorhiza sambucina [24, 26, 34]. A significant number of orchids were found to
inhibit the meso-xerophytic basiphilous open grassland communities of sub-Atlantic
and sub-Mediterranean Europe (order Artemisio albae-Brometalia erecti), also known
as Xerobrometalia [171, 178]. In Italy, common species that inhabit these communities
are Anacamptis morio, A. pyramidalis, Himantoglossum adriaticum, Neotinea
tridentata, N. ustulata, Ophrys sphegodes, O. benacensis, and Serapias vomeracea
[178, 179].
Another important vegetation order is Festucetalia valesiacae, representing
steppes and rocky steppe grasslands in the steppe and forest-steppe zones of Europe
and northwestern Central Asia [26, 171]. Orchids were recorded especially on
calcareous substrates in communities of the alliance Festucion valesiacae
[26, 182, 183]. Some of the most common orchids found in these communities in
the central Balkans (western Serbia) are Anacamptis morio, A. pyramidalis,
Gymnadenia conopsea, Himantoglossum calcaratum subsp. calcaratum, Neotinea
tridentata, N. ustulata, and Ophrys scolopax subsp. cornuta [26, 34].
Within the class Festuco-Brometea, a small number of orchid species have been
registered in communities of the Halacsyetalia sendtneri order (ultramafic and sili-
ceous xeric rocky grasslands in continental regions of the Balkan Peninsula), which
can be attributed to extreme habitat conditions [34]. Specifically, Anacamptis morio
was the only species registered in the community Poo molinieri-Plantaginetum
holostei, of which it represents an indicator species [26]. Furthermore, a few species
such as Dactylorhiza saccifera and Gymnadenia conopsea were recorded in com-
munities of the order Scorzoneretalia villosae (amphi-Adriatic dry steppic sub-Med-
iterranean pastures of the Prealpine, Illyrian, and Dinaric regions) [34].
Terrestrial orchids in Europe that require mesophilous and hygro-mesophilous
habitat conditions have especially significant abundance in communities of the vege-
tation class Molinio-Arrhenatheretea, which includes meadows, pastures, and tall-herb
meadow fringes [19, 24, 26, 34, 42, 111, 112, 133, 154]. Within this vegetation class,
many orchid species were found to occur in communities of the order
Arrhenatheretalia elatioris (meadows and pastures on well-drained mineral soils)
and the alliance Arrhenatherion elatioris [34]. Some of the orchids most frequently
observed in these communities are Anacamptis morio, A. coriophora, A. pyramidalis,
Dactylorhiza sambucina, D. maculata, D. majalis, D. saccifera, Epipactis palustris,
Gymnadenia conopsea, Neotinea ustulata, Neottia ovata, Orchis mascula,
Platanthera bifolia, and Traunsteinera globosa [26, 34, 110–112, 180, 183, 184].
The vegetation order Molinietalia caeruleae (mown meadows on mineral and peaty
soils) also supports many terrestrial orchid species, including a significant number of
Dactylorhiza species, which have large populations in this vegetation type [26, 34].
The most common orchids that were found to grow in communities of the alliance
Molinion caeruleae are Dactylorhiza cordigera, D. incarnata, D. majalis, D. maculata

subsp. maculata, Epipactis palustris, Gymnadenia conopsea, G. densiflora, Neotinea
ustulata, and Neottia ovata [9, 61, 68, 112, 184]. Recent studies indicated that stands
of Molinion caeruleae communities on ultramafic substrates are especially suitable for
Dactylorhiza maculata subsp. transsilvanica and Platanthera bifolia, whereas
Molinion caeruleae communities on Quaternary sediments support significant
populations of Dactylorhiza incarnata and Anacamptis palustris [26]. In addition,
orchid species that frequently occur in communities of the alliance Calthion palustris
include Dactylorhiza incarnata, D. maculata, D. majalis, D. praetermissa,
D. saccifera, D. cordigera, Epipactis palustris, Gymnadenia conopsea, and Neottia
ovata, whereas Dactylorhiza incarnata, D. saccifera, Epipactis palustris, Gymnadenia
conopsea, and Platanthera bifolia are orchids that have significant populations in
Deschampsion cespitosae communities [26, 34, 42, 61, 68, 112, 154, 185].
Based on recent studies, it can be stated that some orchids are significantly
represented in communities of the vegetation orders Trifolio-Hordeetalia (wet
meadows distributed on the Apennine and Balkan Peninsulas) and Filipendulo
ulmariae-Lotetalia uliginosi (tall-herb wet meadow fringe vegetation on mineral
soils) [34]. For example, Dactylorhiza incarnata and Epipactis palustris have partic-
ularly high abundance in Mentho longifoliae-Juncion inflexi communities within the
second mentioned vegetation order [26, 34]. Furthermore, certain species have been
reported to grow in high-altitude mesofilous meadows and pastures in the mountain
ranges of Europe (Poo alpinae-Trisetetalia) [34, 171]. Within this vegetation order,
several high-altitude orchid species (Traunsteinera globosa, Dactylorhiza cordigera,
D. sambucina, Gymnadenia conopsea, and Orchis mascula subsp. speciosa) were
found to grow in communities of the endemic alliance Pancicion serbicae in the
central Balkans [26, 34].
Among herbaceous vegetation, tall-herb vegetation at high altitudes of
Europe, Siberia, and Greenland (class Mulgedio-Aconitetea) supports the growth
and development of some terrestrial orchids [26, 34]. Thus, recent studies
showed that Gymnadenia conopsea inhabits communities of the alliance
Calamagrostion villosae [34], i.e., tall-grass and herb-rich communities on dry
acidic soils in the upper montane and subalpine belts of the investigated moun-
tain ranges [171].
Secondary mat-grass swards on nutrient-poor soils of the temperate, boreal,
and subarctic regions of Europe (class Nardetea strictae) also represent an
important vegetation type for the existence of populations of numerous orchid
species [34]. Some of the most frequently recorded species in these communities
in Central and Western Europe are Gymnadenia conopsea, Dactylorhiza
maculata, D. majalis, D. sambucina, Neottia ovata, Orchis militaris, and Pseud-
orchis albida [42, 61, 85, 109, 111, 112, 125]. In the central Balkans, 23 orchid
species were found to grow in similar communities, especially within the alliance
Nardo-Agrostion [34]. The most common species in these communities are
Dactylorhiza sambucina, Traunsteinera globosa, Anacamptis coriophora,
A. morio, Gymnadenia conopsea, Orchis mascula subsp. speciosa, and Neotinea
ustulata [26, 34].
Terrestrial orchids requiring acidic soils have significant representation within the
vegetation class Juncetea trifidi, which includes acidophilous grasslands in the
alpine belt of Europe, in the Caucasus, and in the boreo-arctic zones of Northern
Europe and Greenland [171]. The importance of this type of vegetation is reflected in
the presence of significant populations of high-altitude orchid species such as
Coeloglossum viride, Nigritella gabasiana, N. rhellicani, Pseudorchis albida,
Traunsteinera globosa, and many taxa of the genus Dactylorhiza (D. incarnata,
D. maculata subsp. maculata, D. maculata subsp. transsilvanica, and D. sambucina)
[34, 85, 111, 125, 171]. Within this vegetation class, certain orchid species are
cenobionts in communities of the order Nardetalia strictae (secondary mat-grass
swards on nutrient-poor soils at low and middle altitudes of temperate, boreal, and
subarctic regions of Europe), the order Festucetalia spadiceae, and especially the
alliance Nardion strictae (mat-grass swards in the subalpine and alpine belts of the
Alps, Carpathians, and northern Apennines) and the alliance Potentillo ternatae-
Nardion (oligotrophic mat-grass swards of mountain ranges of the southern and
central regions of the Balkan Peninsula) [34, 171]. Furthermore, a few species were
found to grow in communities of the order Seslerietalia comosae and the alliance
Poion violaceae, which represent alpine and subalpine silicicolous grasslands of the
Balkan Peninsula [34]. According to the most recent studies, Dactylorhiza
sambucina represents an indicator species of this vegetation type in the central
Balkans (western Serbia) [26, 34].
Orchids are less prevalent in communities of the vegetation class Calluno-
Ulicetea [34]. This vegetation represents a heath on acidic and nutrient-poor soils
in the temperate and boreal zones of Europe [171]. In the central Balkans, commu-
nities of the alliance Bruckenthalion spiculifoliae (dwarf heath on siliceous sub-
strates of the southern Carpathians and the Dinarides) are inhabited by Dactylorhiza
maculata subsp. maculata and Neotinea tridentata [34].
5.2.2 Vegetation of Bogs and Fens

Fens, transitional mires, and bogs in the temperate, boreal, and Arctic zones of
the Northern Hemisphere (class Scheuchzerio palustris-Caricetea fuscae) represent
important vegetation types for many moisture-demanding orchid species
[19, 25, 26, 133, 186]. Orchids are primarily registered in communities of the
orders Caricetalia fuscae (sedge-moss vegetation of acidic fens in the boreal and
temperate zones and in the supra-Mediterranean belt of mountains in Southern
Europe) and Caricetalia davallianae (sedge-moss vegetation of calcareous and
extremely mineral-rich fens in Eurasia) [34, 171]. The following orchid taxa were
recognized as diagnostic taxa of this vegetation class: Anacamptis palustris subsp.
palustris, Dactylorhiza cordigera, D. fuchsii, D. incarnata subsp. cruenta,
D. incarnata subsp. incarnata, D. incarnata subsp. ochroleuca, D. incarnata
subsp. pulchella, D. lapponica subsp. angustata, D. lapponica subsp. lapponica,
D. maculata subsp. maculata, D. maculata subsp. savogiensis, D. majalis subsp.
majalis, D. majalis subsp. sphagnicola, D. russowii, D. traunsteineri subsp.
curvifolia, D. traunsteineri subsp. traunsteineri, Epipactis palustris, Gymnadenia
densiflora, G. frivaldii, Hammarbya paludosa, Herminium monorchis, Liparis
loeselii, Spiranthes aestivalis, and S. sinensis (Fig. 8) [13, 42, 68, 122, 126, 142,
171, 186, 187]. Other orchid taxa that are cited as cenobionts of these vegetation
communities are Dactylorhiza maculata subsp. transsilvanica, Gymnadenia
conopsea, Neottia ovata, Platanthera bifolia, Pseudorchis albida, and Malaxis
monophyllos (Fig. 8h) [26, 50, 61, 85, 112, 186]. Recent research from the central
Balkans indicated that this vegetation type is inhabited by 12 orchid species and
subspecies [34], including 4 indicator species (Dactylorhiza cordigera,
D. maculata subsp. maculata, D. saccifera, and Gymnadenia frivaldii) (Fig. 8)
[26]. Moreover, it has been found that endemic orchids (Dactylorhiza cordigera
subsp. bosniaca and Gymnadenia frivaldii) occur mainly in moderately mineral-
rich relict oro-Mediterranean fens of the Balkans (Narthecion scardici), calcareous
mineral-rich fens (Caricion davallianae), and moderately calcium-rich to low-
calcium slightly acidic fens (Caricion fuscae) [34].
Members of the family Orchidaceae are less frequently found in communities of
the class Oxycocco-Sphagnetea, which represent dwarf-shrub, sedge, and peat-moss
vegetation of Holarctic bogs and wet heaths on extremely acidic soils [171]. This
vegetation type is inhabited by a few orchid species that require strongly acidic soils.
In Poland, Hammarbya paludosa was recorded in the community Sphagnetum
magellanici [142], whereas in Central Europe Neottia cordata was found in the
community Andromedo polifoliae-Sphagnetum magellanici [48] from the alliance
Sphagnion medii. It should be noted that Dactylorhiza majalis subsp. sphagnicola is
recognized as a diagnostic taxon of this vegetation class [171].
5.2.3 Marshland Vegetation

Some water-demanding orchid species were found to grow in communities of the
vegetation class Phragmito-Magnocaricetea [34]. This is reedy swamp, sedge bed,
and herb-land vegetation of freshwater or brackish water bodies and streams of Eurasia
[171]. For example, it was found that Epipactis palustris inhabits communities of
Magnocaricion elatae (marsh vegetation on oligotrophic to mesotrophic organic
sediments of temperate Europe) [68]. In Germany, the Czech Republic, and Hungary,
Dactylorhiza incarnata, D. majalis, Epipactis palustris, Hammarbya paludosa, and
Liparis loeselii have been reported to grow in communities where Phragmites
australis is highly represented [122, 185]. In the central Balkans, this vegetation
type is inhabited especially by Dactylorhiza incarnata and Epipactis palustris within
the vegetation orders Magnocaricetalia and Phragmitetalia [26, 34].
5.3 Anthropogenic Vegetation
Terrestrial orchid species have significant representation in various anthropogenic

habitat types such as roadsides; abandoned quarries; railway embankments; forests
influenced by industrial emissions, industrial terrains, and wasteland; city parks and
hedges; plantations of ecologically alien and non-native trees; sandpits; and clay pits
[162, 164, 166]. Of particular interest are studies showing that a large number of
orchid species grow in cemeteries: 86 species in Turkey [188] and 29 species in
50
Fig. 8 Orchids inhabiting the vegetation of bogs and fens. (a) Epipactis palustris, (b) Gymnadenia frivaldii, (c) Dactylorhiza cordigera, (d) Dactylorhiza
incarnata, (e) Hammarbya paludosa, (f) Herminium monorchis, (g) Liparis loeselii, (h) Malaxis monophyllos (a–d photos V. Djordjević; e–h photos
M. Bobocea)
Albania [189]. Moreover, in research on 3 Mediterranean islands (Cyprus, Crete, and

Lesbos), investigators found 77 orchid species and almost 7,000 individuals of these
plants in 2 anthropogenic habitat types (cemeteries and roadside verges) [167]. The
same authors found that Serapias bergonii, Anacamptis sancta, A. coriophora subsp.
fragrans, and Himantoglossum robertianum were the most abundant species. These
studies suggest that graveyards and roadside verges have a great conservation potential
and can be considered as important refuges for orchids [167, 188]. Linear habitats
along roadsides act as dispersion corridors that can contribute to linking of landscapes
and create better connection between orchid populations [167]. Generally, the expla-
nation for why roadside habitats are important for the survival and growth of many
orchid species lies in the fact that competitively weak orchid species often inhabit
newly created sites that are poorly covered by dominant plant species and that
roadsides can act as ecotones, which are favorable for many orchid species [26, 167].
Several studies provide detailed insights into the performances of orchids
growing in anthropogenic vegetation types, including reproductive success, the
height of individuals, and inflorescences and pollinator diversity [165, 190]. For
example, it has been found that Epipactis helleborine in ruderal vegetation in
Poland had higher reproductive success than in natural vegetation types, which can
be attributed to greater diversity of pollinator species and higher frequency of
visits by pollinators, as well as to the larger size of plants growing in such habitats
[165]. Investigations of morphology and size of the genome of this species in
anthropogenic and natural habitats showed that ten biometric features of flowers
differed significantly between habitats [190]. These plants were taller in anthro-
pogenic habitats than those from natural populations, and genome size in some
populations was significantly different between plants growing in natural and
anthropogenic habitats [190]. In addition, it was found that reduced seed weight
is characteristic of E. helleborine in anthropogenic habitats, which is in part
associated with its inability to adapt to the lower nutrient availability in soils of
these habitats [191]. Moreover, scientists found that orchids inhabiting anthropo-
genic vegetation most often have rapid growth and flower production [164]. In the
case of Himantoglossum species, it has been established that their reproductive
success is weaker near roads [166]. It is nevertheless considered that traditionally
managed roadside verges can ensure long-term persistence of the species in
question and be important refuges for them [166, 167].
Representatives of the family Orchidaceae often inhabit non-native poplar plan-
tations. For example, in Poland, Cephalanthera longifolia, Epipactis helleborine,
Epipactis schmalhausenii, Platanthera bifolia, Neottia ovata, and Dactylorhiza
majalis were registered in fast-growing hybrid poplar (Populus canadensis)
plantations [164]. The authors emphasized that orchid populations occupying these
anthropogenous habitats often have a high population density and abundance, which
is explained primarily by good light conditions, limited competition from other
plants, and disturbance of the upper soil levels [164]. For example, 677 individuals
of Platanthera bifolia per 100 m2 and 275 individuals of Epipactis
schmalhausenii per 100 m2 were recorded in poplar plantations in Poland [164].
Moreover, orchids growing under a canopy in poplar plantations reached a
significant height (up to 130 cm in the case of E. schmalhausenii and up to 92 cm

in the case of Platanthera bifolia) [164].
Some studies provide detailed knowledge about the anthropogenic vegetation
units in which orchids grow. Thus, in the central Balkans (western Serbia), orchids
were reported in some ruderal communities of the class Artemisietea vulgaris [34].
Specifically, Anacamptis morio and Neotinea ustulata were found in communities of
the order Agropyretalia intermedio-repentis (semiruderal grasslands and herblands
and weed segetal vegetation of perennial crops in the nemoral, forest-steppe, and
subboreal zones of Europe), whereas A. morio and Orchis purpurea were registered
in communities of the order Onopordetalia acanthi (subxeric ruderal vegetation
dominated by short-lived perennials) [34, 171]. Moreover, O. purpurea was the only
species recognized as an indicator species of the ruderal vegetation type in western
Serbia [26].
Disa bracteata (the South African orchid) and Microtis media (the common
mignonette orchid) are two relatively ruderal (weedy) orchid species in Australia
[192]. In the case of M. media, it can be asserted that mycorrhizal association with
species of the fungal genus Tulasnella enables this orchid to inhabit anthropogenic
vegetation types, bearing in mind that fungal pathways contribute significantly to its
ability to exploit the minimal phosphorus reserves in the soil [193].
6 Effects of Disturbance
Based on Grime’s theory of population strategies [194], orchids are classified as

stress tolerators [2], whereas other authors classify them between ruderal plants and
stress tolerators [5]. This position in Grime’s C-S-R (competitors – stress tolerators –
ruderals) triangle indicates that orchids can grow under conditions of certain stress
and disturbance and are not competitively strong. Moreover, specific studies have
shown that some degree of disturbance may reduce the competition between orchids
and other plants and thus have a beneficial effect on the development of orchid
populations [42], whereas the growth and survival of some terrestrial orchids require
natural disturbances such as fires that maintain the forest openings necessary for
long-term orchid survival [195].
Since many terrestrial orchids grow in grasslands and meadows, i.e., secondary
forest-free areas created by humans, appropriate management of these habitats is
needed for the existence of these orchid populations [42, 196]. Activities and
management practices such as mowing and grazing actually help to maintain the
relatively low-growing herbaceous vegetation and are favorable for most terrestrial
orchids of these habitats. Thus, regular annual mowing, especially late in the season,
is essential for the optimal development of many species of the genus Dactylorhiza
[42, 43]. Scientists indicate that mowing improves the performance of Dactylorhiza
majalis because the presence of constantly larger individuals is maintained and the
rate of disappearance of individuals is reduced [42, 43]. The negative effects of
unmown grasslands on orchids have to do with reduction of light reaching the lower
parts of grassland vegetation, which negatively affects orchid populations and their
viability [42]. Community stands with a high degree of coverage of dominant

Filipendula ulmaria were identified as especially bad sites for growth and develop-
ment of D. majalis [42]. This can be explained by the fact that orchid species with
shorter stature are often unable to overcome the increased competition from associ-
ated tall-growing species and consequent decrease of light availability within these
stands [23]. It has been shown that Dactylorhiza incarnata is capable of withstand-
ing reduced light availability to a certain extent by increasing its vertical growth
[185]. Since populations of this species increased exponentially during the first
10 years after the re-introduction of mowing, the authors recommended an initially
higher mowing frequency at previously abandoned sites [185]. Mowing of roadside
verges is suggested as a highly favorable practice from the point of view of orchid
conservation because the regularly mowed 0–2 m margin of roads is the part of the
roadside verge most important for the growth of orchid individuals [167]. Certain
research has provided insights into when and to what extent mowing best influences
the performance of orchid populations, highlighting the importance of modified
mowing regimes. Thus, in Merseyside (England), it was found that delaying
spring/summer mowing until 15 July each year led to an increase in the population
of Anacamptis morio, whereas populations of adjacent habitats subjected to frequent
regular mowing or early and heavy grazing showed no such increase [197]. In the
Netherlands, site management with mowing in July and sheep grazing in winter is
beneficial to populations of Coeloglossum viride [196]. Scandinavian authors
suggested that Dactylorhiza lapponica in central Norway would benefit the most
from traditional mowing performed after seed dispersal, but would also have a high
probability of future survival in the absence of mowing [198].
It is therefore important to note that abandoning traditional practices such as
mowing or grazing can influence the survival of many orchid species, bearing in
mind that species which require open habitats are threatened by vegetation succes-
sion (development of forest and scrub vegetation). In the absence of traditional land
management practices, orchids growing in fen communities are particularly endan-
gered in view of the fact that vegetation overgrowth with woody trees occurs under
such conditions. Moreover, research in the UK showed that orchids inhabiting
calcareous grasslands are the most endangered ones, a circumstance associated
with the loss of a very high percentage of these traditionally sheep-grazed grassland
ecosystems [23]. In agreement with the results obtained in Europe, controlled
grazing was found to have a positive effect on certain orchids in North America.
Specifically, it was found that reduction in the level of competition from shrubs and
trees has a direct beneficial effect on the development of populations of Spiranthes
species and Cypripedium reginae in North America [5].
There are studies showing that some degree of natural and anthropogenic distur-
bance of forest communities has a positive impact on the survival and development
of orchid populations [88]. For example, the authors highlighted the importance of
regular coppicing of forests in maintaining viable populations of Orchis mascula
over the long term, as it increases the probability of flowering, fruit and seed set, and
seedling recruitment [87]. Furthermore, it was found that fencing in Pinus sylvestris
forests of Scotland favors the establishment of Goodyera repens colonies [199]. It
seems that the maintenance of a complete canopy cover is necessary to restrict

competition from other plants and provides the shade conditions to which this
species is adapted [104]. In Greece, however, pine forests, which constitute the
main habitat of G. repens, are in rapid decline, primarily due to their replacement
with beech and spruce forests, which is a consequence of natural succession and
reduced levels of disturbance caused by grazing and fires [104]. Scientists investi-
gating the response of the lady’s slipper orchid (Cypripedium calceolus) to tree
removal [89] demonstrated that tree removal sites had greater orchid density, higher
odds of orchid survival, and better flowering and fruiting of orchids than the control
sites. Although most of these effects disappeared after 3 years, when the canopy gaps
closed, this study suggests that selective tree harvesting represents a suitable man-
agement practice that increases the size of populations of the given orchid species
[89]. Other investigators explored the influence of clear-cutting, green-tree retention
and artificial drainage on the abundance of 11 terrestrial orchid species in Estonia
[73]. Although the highest number of species was found on artificially drained plots
and in mature stands, most sites were inhabited by shade-tolerant orchid species that
disappeared after timber harvesting. Moreover, this study showed that cut-over areas
(3–7 years after harvest) did not host any species that were not present from uncut
forest stands and that keeping solitary trees had no impact on orchid abundance in
comparison with clear-cut areas ones [73].
Fire is an important disturbance factor, especially in areas characterized by the
Mediterranean climate [2]. In Europe, the effects of fire have not been well studied,
and there is little information about its effects on orchids. However, it is known that
forest fires favor orchid populations, as they maintain open space in phrygana,
maquis, and other scrublands. In contrast to the European continent, the effects of
fire on orchid performance have been investigated in detail on some terrestrial
orchids in Australia. Thus, it was established that management of the habitats of
Prasophyllum correctum should involve frequent burning, the benefits of which for
this species include reduction of competition from grasses, shortening of dormancy
periods, reduction of mortality, and enhancement of flowering [195]. Moreover,
there are some orchids that need fire for their growth, so that accidental fires provide
conditions favorable for their flowering. Burnettia cuneata, Pyrorchis nigricans,
Leptoceras menziesii, and Prasophyllum australe are such fire-dependent orchid
species [200]. The same author states that there are also fire-stimulated species
(many species of the genera Caladenia, Diuris, Prasophyllum, and Thelymitra),
fire-neutral species (some representatives of the genus Pterostylis), fire-sensitive
species (Pterostylis alveata and Corunastylis despectans), and fire-killed species
(ones that die due to intense fire, e.g., Thynninorchis huntianus) [200].
7 Orchid Specialists and Generalists
Terrestrial orchids, depending on the degree of habitat specialization, can be divided

into two major groups, generalists and specialists [25, 26]. However, these two
categories can only be considered as the extreme ends of the continuum. It should be
noted that the extent of tolerance of orchid species to different environmental factors
depends on the level of competition, geographical and historical factors, the level of
pollinator specialization, and the level of specialization with associated mycorrhizal
fungi [3].
Generalist orchid species are those occurring across a wide range of vegetation
types with widely varying conditions, such as different geological substrates, differ-
ent light conditions, varying degrees of soil moisture and atmospheric humidity, and
a wide range of altitudes. Specifically, the altitudinal range where a particular species
occurs is directly related to breadth of the ecological niche of a given species, which
means that species occurring from lowland to high-altitude areas have a greater
physiological tolerance and ability to inhabit different vegetation types and thus to
grow sympatrically with different species. Moreover, generalists are known to be
usually widespread species, whereas specialists tend to occur in more restricted areas
[201, 202]. The most significant orchids in Europe considered to be generalists are
Epipactis helleborine, Neottia ovata, Platanthera bifolia, Cephalanthera longifolia,
Anacamptis morio, Dactylorhiza sambucina, D. fuchsii, D. saccifera, Gymnadenia
conopsea, and Neotinea ustulata [9, 12, 24–26, 73, 112, 133]. Recent studies in the
central Balkans have particularly highlighted species that inhabit a wide range of
vegetation units (Gymnadenia conopsea, Anacamptis morio, Dactylorhiza saccifera,
D. sambucina, Neottia ovata, and Platanthera bifolia) and species growing on a
large number of geological substrates (Cephalanthera longifolia, Dactylorhiza
sambucina, Epipactis helleborine, Gymnadenia conopsea, Neottia nidus-avis, and
Platanthera bifolia) [34, 107]. The obtained results emphasize the high ecological
plasticity and adaptability of these orchids.
On the other hand, specialist terrestrial orchids are ones having a narrow range of
ecological requirements, inhabiting a small number of vegetation communities, occur-
ring in a narrow range of altitudes and represented on a small number of geological
substrates. Among the most common specialist orchids in Europe are the following:
Dactylorhiza traunsteineri, D. lapponica, D. macedonica, D. cordigera, Gymnadenia
frivaldii, Nigritella rhellicani, Hammarbya paludosa, Herminium monorchis, Liparis
loeselii, Spiranthes aestivalis, Cephalanthera cucullata, and Epipactis cretica [9, 25,
26, 99]. It is noticeable that a large number of orchid specialists are those which inhabit
wet habitats, such as wet meadows or fen communities, as well as species that inhabit
warm and dry habitats. This is consistent with the general statement that the majority
of highly specialized species occur at the extreme ends of environmental gradients and
in extreme and rare vegetation types [201, 202].
Certain authors found that orchid taxa are relatively common and have large
populations close to the center of their geographic distribution, whereas they are
rarer and characterized by smaller populations toward the boundaries of their ranges
[46]. At the same time, the levels of specialization of particular orchid species vary
depending on the geographical area, i.e., on the position of populations of different
species in relation to the center/edges of their ranges. Although this is a situation
perceived rather intuitively by many orchidologists, recent studies from the Balkan
Peninsula provide rigorous and thoughtful arguments to substantiate it on the basis
of numerical analyses [24–26, 99]. Thus, it was found that orchids belonging to the
Central European and boreal chorological groups generally have higher levels of
specialization in northeastern Greece and on Crete than in the central Balkans, which
is attributable primarily to differences in the climates of these areas [24, 26]. In the
central Balkans (western Serbia), there is a humid and continental climate that allows
the widespread distribution of wet and mesophilous habitat types, whereas in
northeastern Greece, and especially on Crete, the Mediterranean climate has strong
influence [26]. At the same time, most orchids of Central or North European origin
that have the southern boundaries of their distribution on the Balkan Peninsula occur
at high altitudes in Greece, whereas in Central and Northern Europe, they grow in
habitats ranging from lowlands to highlands [25]. On the other hand, orchids
belonging to the Mediterranean and sub-Mediterranean chorological groups (e.g.,
Anacamptis papilionacea, A. pyramidalis, A. laxiflora, Orchis simia, and Ophrys
scolopax subsp. cornuta) have a higher level of specialization in the central Balkans
compared to the degree of their specialization in northeastern Greece and on Crete
[24–26, 99].
Particularly noticeable differences of orchid specialization are evident when
orchids growing in Central Europe are compared with those occurring on the Balkan
Peninsula. Thus, some moisture-demanding orchids (e.g., Epipactis palustris,
Dactylorhiza incarnata) have a higher level of specialization in Southern Europe
(e.g., in Greece) [25] than in Central Europe [9, 68]. Epipactis pontica grows
exclusively in communities of Fagion sylvaticae and is considered a specialist
species in the central Balkans [128], but it is less specialized in Slovakia, where it
grows in forest communities of the vegetation alliances Luzulo-Fagion sylvaticae,
Fagion sylvaticae, Quercion confertae-cerris, and Carpinion betuli [127]. In addi-
tion, Dactylorhiza fuchsii is considered to be one of the least specialized species in
Central Europe [9], but it is very rare and specialized in the central Balkans
[26, 177]. This general trend is obvious in the case of Crete, where some of the
most widespread species of European shrimp (Cephalanthera damasonium,
C. longifolia, and Neottia ovata) are categorized as specialists [99]. Overall, the
results of these analyses suggest that levels of specialization of orchid species
increase from the center to the edges of species ranges [24, 26].
8 Orchid Mycorrhizal Fungi
Mycorrhizal fungi play a crucial role in the growth of orchids, especially in the early
stage of development that is during germination and phases of seedling establishment
[62]. Mycorrhizal dependency varies depending on the stage of orchid development.
While the need for specialized fungi during seed germination is well documented, the
degree of dependence in adult orchid specimens is less known and varies throughout
autotrophic and heterotrophic phases of the orchids [3]. As orchid plants mature, their
dependency changes to partial mycoheterotrophy, in which plants utilize carbon both
from their fungal partners and from photosynthesis [203]. However, recent studies
showed that partial mycoheterotrophy plays a great role in rhizoctonia-associated
orchids, even under full light conditions in open meadow habitats [203]. Some
terrestrial orchids do not photosynthesize at all throughout their lives, so they are
completely mycoheterotrophic. It should be noted that mycoheterotrophy exists in

several plant families but is most common in the family Orchidaceae, in which over
100 fully mycoheterotrophic species have been recorded [7]. Since orchids have stages
that depend fully or partially on fungi for carbon and other elements, the distribution
and abundance of orchid populations depend on the distribution and abundance of
orchid mycorrhizal fungi [204, 205]. Scientists found that seed germination and
protocorm development reflect the distribution and abundance of mycorrhizal fungi
and that fungal “hot spots” represent a key factor in the dynamics of orchid
populations [206]. The distribution of mycorrhizal fungi is probably independent of
the distribution of orchids and the ability of orchids to engage in symbiosis with fungi
that otherwise live freely is their unique feature [7]. Most fungi found to be associated
with terrestrial orchids are basidiomycetes (Ceratobasidiaceae, Sebacinales, and
Tulasnellaceae) [7, 204, 207]. Among the best-known genera, Rhizoctonia,
Ceratobasidium, Sebacina, and Tulasnella [208] should be particularly emphasized.
Some orchids are associated with ascomycetes, primarily members of the genera
Tuber, Peziz, Wilcoxina, Tricharina, and Phialophora [204, 207, 209]. The majority
of fully mycoheterotrophic orchids and certain partially mycoheterotrophic ones
mainly associate with ectomycorrhizal agaricomycetes, including many species of
Russulales, Thelephorales, and Sebacinales [204], whereas some orchids associate
with ectomycorrhizal ascomycetes [209].
Many studies showed that orchids use carbohydrates, minerals, and water of
mycorrhizal fungi without adequate reciprocal benefit for the fungi [3]. However,
other studies suggested that there are mutually beneficial interactions between
orchids and mycorrhizal fungi: carbon produced by plant photosynthesis is obtained
in exchange for access to nutrients from the soil such as nitrogen, phosphorus,
vitamins, and amino acids [7]. Fungi supply orchids with carbon, vitamins, hor-
mones, minerals, and other physiologically active substances necessary for growth
by decomposing organic compounds from the substrate, especially cellulose and
lignin. However, the relationship between orchids and fungi ranges from parasitism
on fungi to commensalism and may approach neutralism [210]. On the other hand,
mutualism was recorded in Goodyera repens [149, 210]. The dependence of orchids
on their symbiotic fungi is obvious in the case of albino orchids and when orchids
spend long periods of dormancy. Both cases demonstrate that the fungi do not rely on
carbon produced by orchids through their photosynthesis and can grow well even
without these orchid plants [204].
It is worth mentioning that the interactions between orchid plants and fungi show
a higher degree of specialization than those recorded in other plants [3]. Thus, the
rarity and long-term survival of some orchid species can be explained by the degree
of mycorrhizal specificity and the fact that the mycorrhizal association is restricted to
fungal species limited in distribution [7, 211]. The fact is that a high level of
dependency between orchid species and mycorrhizal fungi is seen as an increased
risk of extinction [3]. Moreover, this risk is linked not only to the level of special-
ization but also to the ability of orchid species to switch from one fungal partner to
another under changed environmental conditions [3].
It has been hypothesized that orchids that can swap and share mycorrhizal fungi
have a broader distribution than those species that are highly specialized for fungi with
a limited distribution [3]. However, recent research indicated that the rarity and
persistence of orchids are not necessarily related to fungal diversity, and it is suggested
that other factors may play a more important role in determining the persistence of
orchids [211]. This has been proved in the case of endemic Australian terrestrial
orchids with limited ranges, whose rarity and distribution patterns are affected to a
greater extent by soil characteristics and pollination systems than by the narrow
distributions of fungal species [212]. In Europe, study of Cypripedium calceolus
showed that this orchid has a fungal partner with one of the narrowest of ranges, but
is nevertheless a widespread species [213]. In general, orchids with a high degree of
exploitation of mycorrhizal fungi exhibit a high degree of specialization toward
mycorrhizal symbionts and above all toward fungi that form ectomycorrhizal relation-
ships with trees [3, 7]. These orchids therefore can be considered plant epiparasites
because they exploit ectomycorrhizal networks between fungi and other neighboring
plant species [7]. Examples include Corallorhiza maculata and C. mertensiana, which
specialize in ectomycorrhizal species from the family Russulaceae [7], and Neottia
nidus-avis, which specializes in fungi from the family Sebacinaceae, orchids that form
ectomycorrhizae with trees [81]. Overall, high mycorrhizal specialization occurs
primarily in species that are completely mycoheterotrophic, whereas the specificity
of photosynthetic orchids can vary [7]. Thus, it has been found that 16 photosynthetic
species of Mediterranean orchids from the genera Anacamptis, Orchis, Ophrys, and
Serapias have a low degree of specialization in mycorrhizal fungi [214]. Mycorrhizal
specialization is most likely associated with the one-sided nature of the relationship
between orchids and fungi [7]. To be specific, if the fungi have little or no benefit from
the symbiosis, then orchids can be considered parasites, and they often exhibit high
specialization due to selection driven by evolutionary “arms-races” [7]. On the other
hand, some authors assumed that the degree of specialization is correlated with the
degree of heterotrophy [170].
It is important to emphasize that narrow orchid specificity toward fungi has a
major impact on the ecology and distribution of orchids [7, 204, 215]. At the same
time, some authors believe that there may be an impact on orchid diversity [7, 213].
Specifically, mycorrhizal specialization can encourage orchid diversification by
affecting orchid distribution patterns. Fragmentary fungal distribution, together
with high mycorrhizal specialization, may be responsible for extremely dispersed
orchid populations [7]. The consequences of long-range orchid seed transmission
can be small effective population sizes and reduced gene flow, results that create
conditions favorable for the drift-selection model of orchid speciation [7, 216]. The
mycorrhizal relationship between orchids and fungi thus may increase the potential
for faster emergence of new species in the family Orchidaceae [7].
9 Pollination Systems
Orchid pollination has intrigued scientists from the time of Darwin, primarily
because of its complexity and great diversity [3]. Orchid pollination systems are
often mistakenly considered to be the outcome of co-evolutionary processes [217].
However, co-evolution between orchid species and their pollinators is most likely
rare, and evolutionary changes in orchids are largely unilateral, with no evolutionary
changes in pollinators [218]. Insects of the order Hymenoptera represent the most
numerous pollinators of European orchids, followed by butterflies (Lepidoptera),
whereas members of the order Diptera represent a smaller number of pollinators
[217, 219]. Furthermore, the fertilization of some terrestrial orchids can result from
autogamy or even cleistogamy.
The most common pollination system among orchids is the system of rewarding,
where insects receive mostly nectar as a reward for pollination. However, a specific
characteristic of the family Orchidaceae is very high representation of non-rewarding
pollination systems. Specifically, as many as one-third of the total number of orchid
species (between 6500 and 9000 species) deceive their pollinators [218]. There are
several mechanisms by which orchids deceive their pollinators, mechanisms such as
generalized food deception for example, where orchids advertise floral signals that
are characteristic of rewarding plant species [7, 218]. In sexual deception, orchid
flowers imitate the mating signals of female insects, especially using odors that
mimic female insect pheromones and tactile or visual cues, and are then pollinated
by male insects trying to copulate with the flowers [7, 218]. Other deception
mechanisms include Batesian floral mimicry, brood-site imitation, shelter imitation,
pseudo-antagonism, and rendezvous attraction [218]. The pollination system that an
orchid species uses has a direct effect on pollination success. Specifically, it has been
found that non-rewarding orchids, on average, have lower frequencies of visitation
by pollinators and lower reproductive success than rewarding orchids [220]. Darwin
therefore suggested that low levels of fruit set, which are typical of non-rewarding
species, might be a key factor in determining orchid rarity [221]. However, results
obtained in a recent study conducted in Belgium and the Netherlands showed that
orchid distribution patterns are not related to nectar reward and that the relationship
between nectar rewards and extinction of orchids is not significant [222]. Another
recent study indicated that the relative occurrence of food-deceptive orchids
decreases with increasing altitude on the territory of Switzerland and in the Vaud
Mountains [35]. The authors of that study found that this may be linked to altitude-
dependent climatic factors such as temperature and precipitation and factors that
cause decrease in the pollinator visitation rate at high altitudes. In high-altitude areas,
the growing season becomes shorter, and consequently plant species tend to flower
simultaneously, which results in increasing the levels of competition among plants
for access to pollinators and reduced access to pollinators that have not learned to
recognize rewarding and non-rewarding orchid flowers [35].
Many authors have stressed that pollinator limitation and specialization may be
an important factor affecting the distribution of orchids, especially near the margins
of their distribution [3, 6, 219]. It has been found that orchids that are pollinated by a
large number of diverse pollinators most often have a widespread distribution. An
example of such a species, Epipactis helleborine, has great pollinator diversity, and
among other things, this fact allows it to grow in both natural and anthropogenic
habitats and helps to explain why it is one of the few orchids to have successfully
colonized North America [165]. On the other hand, some species of the genus
Ophrys are highly specialized and this causes their rarity. Ophrys pollinators can
be divided into three groups: food generalists, food specialists, and parasitic
specialists [219]. The sexual deception present in this genus imposes a high degree
of specialization, as insect pheromones are species-specific in most cases. Moreover,
a large number of Ophrys pollinators include solitary bees that have pollen special-
ization (oligolecty) [219]. In general, researchers found that sexually deceptive
orchids are usually pollinator specialists, whereas the majority of food-deceptive
orchids are pollinator generalists [7]. Some authors suggested that specialized
pollination strategies influence the diversification of orchids and increase the risk
of extinction, especially if environmental changes affect their long-term survival and
evolutionary potential [3]. It should be noted that a great number of orchids are
pollinated by specialized insects, which often require specific conditions (e.g.,
specific nesting sites, the presence of surrounding nectar plants and host plants for
egg-laying, existence of brood cell parasites, and pollen specialization), suggesting
that the relationship between orchids and pollinators is fragile and that many orchids
are thereby rendered prone to extinction [3, 219].
Self-pollination occurs in a small number of representatives of the family
Orchidaceae, more precisely in about 3% [217] or between 5 and 20% of the total
number of orchid species [223]. The number of completely self-pollinating orchid
species is small, and self-pollinating species more often are ones that have cross-
pollination in addition to self-pollination [2]. Facultative autogamy occurs in many
orchid species and is an appropriate strategy when the frequency of cross-pollination
is low [223]. In Europe, self-pollination is present primarily in species from the
genera Epipactis, Cephalanthera, and Neottia, followed by members of the genera
Corallorhiza (C. trifida), Limodorum (L. trabutianum), Neotinea (N. maculata),
Ophrys (O. apifera), and Serapias (S. parviflora) [13, 219]. It has been found that
the number of self-pollinated orchids increases with increasing latitude, as well as in
isolated geographical areas [216]. Moreover, most self-pollinated orchids occur in
high-altitude areas and by self-pollination overcome the lack of pollinator availabil-
ity there [30, 223]. The highest percentage of self-pollinating orchids (about 50%)
was found in boreal regions [223] and on Réunion Island [30]. In eastern Canada,
self-pollination was reported in 17% of the total number of orchid species, whereas
in Europe it occurs in 27–50% of orchid species [216, 223].
Self-pollination in orchids may be advantageous in habitats where high levels of
disturbance cause uncertain activity of pollinators [223]. It is important to emphasize
that self-pollination reduces the rate of pollen export and the number of seed
embryos. At the same time, autogamy reduces the level of genetic variation and
can lead to inbreeding depression, resulting in fewer offspring and a lower survival
rate [216].
10 Conclusions
Terrestrial orchids include a great variety of species that are widespread on all
continents and characterized by specific life histories and varying sensitivity to
changing habitat conditions. The importance of environmental factors affecting the
distribution, abundance, and richness of orchids varies depending on the geographical
scale. It can be concluded that latitude, area size, macroclimate, and evolutionary
history have an important impact on the distribution and abundance of orchids on a
large scale. On the other hand, soil moisture, the light regime, geological substrates,
soil characteristics, the disturbance regime, and the specificity of mycorrhizal fungi
and pollinators play an important role on a fine scale. The importance of the elevation
range is recognized both on the macroscale level and at regional levels.
In general, the number of terrestrial orchid species increases with decreasing
latitude, which is among the most consistent of discernible patterns. Thus, the
northern countries of Europe have the smallest number of orchid species, while
the number of orchid species gradually increases toward southern areas of the
continent, reaching a peak in the Mediterranean area. It is assumed that orchid
species richness in most European countries has a hump-shaped pattern with the
highest species number at middle elevations. This unimodal pattern can be caused by
climatic factors along the elevational gradient, by some spatial aspects like geomet-
ric constraints, by size of the region, and by biotic processes.
Temperature, precipitation, and the light regime play an important role in deter-
mining patterns of growth, development, flowering, population dynamics, abun-
dance, and distribution of terrestrial orchids. The role of temperature and
precipitation is particularly pronounced on the southern and northern borders of
species distribution. Studies have indicated that climatic parameters, most often in
the previous and current seasons, influence the number of flowering individuals. As
a result of global warming and climate change, many terrestrial orchids have altered
their performances. However, the degree of such changes in the performances of
orchids depends on the type of their life history and especially on pollination
systems. Furthermore, the importance of climate variables in predicting the distri-
bution of terrestrial orchids should be emphasized.
Variation in availability of soil resources (water and nutrients) across geological
substrates significantly affects the richness and composition of orchid species. Most
orchids in Europe grow on calcareous geological substrates and soils, moderately
damp soils, slightly acidic to slightly alkaline soils, and soils that are relatively poor
in nutrients. However, a surprising number of orchids have recently been discovered
on non-calcareous substrates, primarily felsic, intermediate, mafic, and even ultra-
mafic igneous rocks, as well as on metamorphic and silicate sedimentary rocks in the
central Balkans. Future research should therefore be focused on the study of
ecophysiological characteristics, the potential for trace element accumulation, and
the phytochemistry of orchids growing on these substrates.
Vegetation types significantly affect patterns of distribution and abundance of
orchids, as well as separation of their ecological niches. Although terrestrial orchids
inhabit almost all known vegetation types in Europe, the greatest species diversity is
recorded in various deciduous forests (beech, oak, and hornbeam forests); coniferous
and mixed coniferous-deciduous forests (spruce, fir, and pine forests); Mediterra-
nean vegetation types, especially scrubs; different grassland and meadow types
including heaths; montane-subalpine tall-herb vegetation; and fens, bogs, and
marshes. Moreover, anthropogenic vegetation types are also inhabited by terrestrial
orchids, and it is often asked whether they can play an important role as refuges for
endangered species. The importance of disturbance effects should be emphasized,

since traditional management practices such as mowing and grazing may reduce the
competition between orchids and other plants and provide favorable light conditions,
thus having a beneficial effect on the development of orchid populations.
The patterns of distribution and abundance of terrestrial orchids are largely
determined by characteristics of their life histories, especially by the existing three
belowground strategies determined by their root systems. Most terrestrial orchids
that can tolerate colder conditions and high moisture soils in Europe are species with
palmate and fusiform tubers. At the same time, these species are the ones most
prevalent at higher altitudes. On the other hand, most orchids that best tolerate high
temperatures and dry conditions are species with ovoid tubers. Consequently, these
species are dominant in low- to mid-elevation areas.
Although the influence of environmental factors on the distribution and abun-
dance of terrestrial orchids can be viewed individually, these factors in reality act
cumulatively in nature. According to the degree of specialization in relation to
habitat conditions, terrestrial orchids can roughly be divided into two large groups
– specialists and generalists. It has been pointed out that the levels of specialization
of orchid species increase from the center to the edges of their ranges. Moreover, the
abundance and distribution of orchids largely depend on the abundance and distri-
bution of adequate mycorrhizal fungi, pollinator limitation, and specialization. To
sum up, it can be stated that awareness of the relationships between terrestrial
orchids and ecological factors is a necessary prerequisite for the success of future
research and conservation of this intriguing group of plants.
Acknowledgments This study was supported by the Ministry of Education, Science and Techno-
logical Development of the Republic of Serbia under Grant [number 173030]. ST was partially
supported by the Ministry of Education, Youth and Sports of the Czech Republic within the
National Sustainability Program I (NPU I) [LO1415]. The authors would like to thank Mihai
Bobocea for providing photos of specific orchids. We are grateful to Prof. Dr. Vladimir Stevanović,
Prof. Dr. Slobodan Jovanović, and Prof. Dr. Dmitar Lakušić for useful suggestions and information.
We are very grateful to Mr. Raymond Dooley, native English editor for the proofreading of the
manuscript.
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Which Environmental Factors Drive
Distribution of Orchids? A Case Study from 2
South Bohemia, Czech Republic
Zuzana Štípková, Dušan Romportl, and Pavel Kindlmann
Contents
1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 74
2 Materials and Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 75
3 Results and Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 79
3.1 Anacamptis morio (L.) R.M. Bateman, A.M. Pridgeon & M.W. Chase 1997 . . . . . . 79
3.2 Cephalanthera rubra (Linne) L.C.M. Richard 1818 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 80
3.3 Dactylorhiza fuchsii (Druce) Soó 1962 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 83
3.4 Epipactis palustris (Linne) Crantz 1769 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 86
3.5 Neottia nidus-avis (Linne) L.C.M. Richard 1817 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 90
3.6 Neottia ovata (L.) Bluff & Fingerh. 1838 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 94
3.7 Platanthera chlorantha (Custer) Rchb. 1828 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 96
4 Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 100
5 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 100
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 103
Abstract
Species distribution models are a useful tool applied in many branches of biology,
especially when dealing with threatened organisms. In combination with GIS
techniques, these models are especially important and valuable for predicting
Z. Štípková (*) · P. Kindlmann

Global Change Research Institute, Academy of Sciences of the Czech Republic,
České Budějovice, Czech Republic
e-mail: zaza.zuza@seznam.cz; pavel.kindlmann@centrum.cz
D. Romportl
Department of Physical Geography and Geoecology, Faculty of Science, Charles University,
Albertov 6, Czech Republic
e-mail: dusan.romportl@natur.cuni.cz

74 Z. Štípková et al.
occurrence of rare species, for example, orchids. Orchids are an endangered plant
group, protected in the whole world. Questions of their conservation are therefore
highly discussed, but not all factors affecting their survival and distribution are
known so far. Here we show an example of using SDMs for analysis of orchid
species occurrence data from the region of South Bohemia in the Czech Republic.
Our data were analyzed using the MaxEnt program, which produces species
distribution maps and thus allows predicting potential occurrence of orchids in
yet unknown localities. This program also determines the environmental factors
affecting species distribution. This is important for better protection of orchids,
because we can improve management plans that are crucial for maintaining
orchid localities to stay alive. We determined the most important factors affecting
studied species occurrence and areas, where new sites are most likely to be
discovered. This approach can help us to find new localities of orchids and
to understand which environmental factors influence the occurrence of these
endangered plants.
Keywords
Orchids · Distribution · Environmental variables · Species distribution models ·
MaxEnt
1 Introduction
Questions concerning species diversity have attracted ecologists for over a century.
Recently, this issue became even more important, because the diversity of life on
Earth is in rapid decline [1]. Therefore, one of the most pressing tasks facing the
global conservation community is trying to understand the main factors determining
diversity of species [2] and identifying important areas for their conservation [3].
This effort is often followed by creation of a network of protected areas, wherein
negative human influence is considerably limited [4–7]. This especially holds for
threatened groups of organisms, such as orchids [8, 9].
The orchid family, with estimates of about 20,000–35,000 species [10–12], is an
important group with respect to conservation biology [13], being at the front line of
extinction [14]. Many characteristics, such as great species richness, its specific role
in ecosystem, or endangered situation, make it crucial to explore the distribution and
conservation status of Orchidaceae [15]. Orchids are also known for their sensitivity
to environmental changes [16], as well as to their high extinction risk, compared
to other plant families, as a result of natural and/or anthropogenic causes [17, 18].
However, decrease of many orchid species occurred in whole Europe, mainly as
a result of the loss or even alteration of their natural habitats [8, 19, 20]. The most
effective methods for conserving orchids undoubtedly involve protection of their
habitats [12, 21].
Species distribution models (SDMs) are a useful tool, which is often applied
in many branches of biogeography, conservation biology, and ecology in the
2 Which Environmental Factors Drive Distribution of Orchids? A Case Study. . . 75
last decades [22], especially when threatened species are concerned [23]. These
numerical tools combine species occurrence records with environmental data [22].
In combination with GIS techniques, these models are especially important and
useful for predicting occurrence of rare species [24]. Despite the fact that the results
of species distribution models often suffer from high levels of uncertainty to several
factors, concerning biased species distribution data, errors in environmental vari-
ables used as predictors, spatial resolution, and the modeling process [25, 26], SDMs
have become widely accepted tools to predict species distributions [27].
The maximum entropy algorithm in the MaxEnt application [28–31] is often used
for modeling species distributions from presence-only species records [31]. This
approach was used by conservation practitioners for predicting the distribution of
a species from a set of occurrence records and environmental variables [31, 32].
MaxEnt is one of the most robust approaches of species distribution in terms of
successfully estimating the area from only a few records of occurrence [33, 34].
Despite long history of studies on orchids, only a minute part of previous
papers concerning distribution, phytogeography, or conservation strategies of this
taxonomic group included application of species distribution models (e.g., see
[35–38]). Presence-only modeling methods require exclusively a set of known
species occurrences together with predictor variables such as topographic, climatic,
edaphic, biogeographic, and/or remotely sensed data [29, 30].
Here, we show an example of using the species distribution models and MaxEnt
for analyses of orchid species distribution in the region of South Bohemia, Czech
Republic. Using MaxEnt analysis, we estimated which environmental factors affect
the distribution of selected orchid species and tried to find new suitable localities for
orchid occurrence in the area selected.
2 Materials and Methods
This study was conducted in the region of South Bohemia, in the south of the Czech
Republic. This area with about 10,057 km2 stretches from 400 m as the lowest parts
to more than 1300 m above sea level as the highest parts of the Šumava National
Park. This region is quite rich in orchid flora; it includes also critically endangered
species of the Czech Republic such as Liparis loeselii, Neottia cordata, or Malaxis
monophyllos.
As a source of information, we used data about orchid occurrence from five
databases: (1) the database of the Nature Conservation Agency of the Czech
Republic [39]; (2) the Czech National Phytosociological Database and (3) the
Floristic Documentation, both deposited at the Department of Botany and Zoology,
Faculty of Science, Masaryk University in Brno [40]; (4) the database of the South
Bohemian Branch of the Czech Botanical Society [41]; and (5) the database of the
inheritance of the late František Procházka (10,000 items, digitized from original
cards). The whole database that consists of all data from these five databases is
deposited at the Global Change Research Institute, Department of Biodiversity
Research in České Budějovice. However, there is no public access to either of these

databases in order to protect the existing localities of orchid species.
Our field studies took place within this region during 2014–2016 (Fig. 1) and
were based on information taken from databases described above. During the field
studies, we checked localities listed in databases in order to determine whether
a selected orchid species is still present there or not. To do this, we visited as
many localities as we were able to and recorded any important notes that we were
able to. If an orchid species was found, the number of flowering individuals was
counted, and other important information was registered, such as accurate GPS
position of the locality, notes about how the locality looked like, or if the locality
is somehow maintained or not. At the end of our field studies, the total of 428 local-
ities were visited and checked.
As was mentioned above, there are many orchid species occurring in the region of
South Bohemia. To describe which environmental factors affect their distribution,
we used only species with an adequate number of records [42, 43], which is
required to obtain reliable predictions. Two most numerous species in the region,
Dactylorhiza majalis and Platanthera bifolia, have been tested before and described
in Štípková et al. [44], and other two abundant species, Epipactis atrorubens and
Cephalanthera damasonium, were analyzed in Kosánová [45]. In this study, other
seven orchid species were analyzed:
• Anacamptis morio, a representative of thermophilous orchid species

• Cephalanthera rubra, preferring forest habitats
• Dactylorhiza fuchsii, preferring forest habitats
• Neottia nidus-avis, preferring forest habitats
• Epipactis palustris, thriving in wet habitats with stable water regime
• Neottia ovata, species that can be found in various types of habitats
• Platanthera chlorantha, which prefers place in higher altitude
A set of possible important environmental variables was chosen according to our

knowledge and available information published in the literature about ecological
demands and factors affecting distribution of studied species [46–49]. These factors
might be important for individual orchid species distribution, and therefore they
were included into the analysis. Environmental factors we used in analyses of all
species are summarized in Table 1. One of the factors we used needs more descrip-
tion: the “consolidated layer of ecosystems,” KVES [50]. It is a list of 40 habitat
types (KVES 1, KVES 2, . . . KVES 40), such that each KVES number refers to
a specific type of habitat. For example, KVES 6 means mesophilic meadows, KVES
10 means oak and oak-hornbeam forests, KVES 30 means mixed forests, and so
on. The list and meaning of KVES we used are shown in Table 1. A KVES without
a number means that the species occurrence depends on a certain habitat type. KVES
is a categorical variable.
It is clear that the intensity of agriculture in the vicinity of an orchid locality
affects its distribution in a broad scale. Previous studies about factors affecting
orchid distribution [44, 45] indicate that the lower the amount of arable land is in
2
12° 13° 14° 15° 16° 17° 18° 19°

36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80
48 47
49 48
51°
50 49
51°
51 50
52 51
53 52
54 53
55 54
56 55
57 56
58 57
59 58
50°
60 59
50°
61 60
62 61
63 62
64 63
65 64
66 65
67 66
68 67
69 68
49°
70 69
49°
71 70
72 71
73 72
74 73
Which Environmental Factors Drive Distribution of Orchids? A Case Study. . .
74
75
76 75
38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79
13° 14° 15° 16° 17° 18° 19°
77
Fig. 1 Map of the study site in region of South Bohemia in the Czech Republic where field study has taken place
Table 1 The list of all environmental variables that were used in our analysis
Code Description
dem Altitude
frost_days Number of freezing days per year
KVES Consolidated layer of ecosystems
4 Alluvial and wet meadows
5 Dry grasslands
6 Mesophilic meadows
10 Oak and oak-hornbeam forests
12 Beech forests
13 Dry pine groves
17 Natural scrublands
19 Wetlands and coastal vegetation
23 Swamps and marshes
24 Ponds
29 Deciduous forests
30 Mixed forests
31 Coniferous forests
33 Urban green areas, gardens, parks, cemeteries
36 Discontinuous urban development
39 Agricultural meadows
40 Arable land
precipitation Total precipitation per year (mm)
slope Slope of terrain (degrees)
solar_rad Solar radiation – total amount of incoming solar insolation (WH/m2)
summer_days Number of summer days (with temperature exceeding 25 C) per year
temp_1 Mean annual temperature ( C)
temp_2 Temperature variability during year ( C)
trop_days Number of tropical days (with temperature exceeding 30 C) per year
veg_season Duration of vegetation season
the vicinity of an orchid locality, the higher is the probability of presence of an

orchid. For this reason, the amount of arable land was not tested separately again in
our study, but the amount of arable land is included in the list of KVES under
the number 40 (see Table 1).
The climatic data were obtained from the Global Change Research Institute of
the CAS, and a climate character from a timeline of 1981–2011 was created. The
resolution of these data was 500 500 m. Slope of the terrain [51] was also added
into the analysis as additional factor that could influence the distribution of studied
species.
The ecological niche modeling was conducted using maximum entropy
method implemented in MaxEnt program version 3.3.2 [29–31] based on the
species presence-only observations. Environmental data (described above) with
spatial resolution of 500 m was used. The maximum number of iterations was set
to 10,000 and convergence threshold to 0.00001. The “random seed” option,
which provided random test partition and background subset for each run, was
applied.
As an output from MaxEnt program, jackknife pictures, importance of

each environmental factor, and potential distribution maps were obtained. In the
resulting figure of jackknife procedure, two different blue bars are always displayed.
The length of the dark-blue bar is telling us how large is the impact of selected
variable. The length of light-blue bar is showing how much information would be
lost if the corresponding factor were excluded from the analysis. The most important
thing is to look on the combination of lengths of both blue bars, because these factors
play an important role in the distribution of studied species. The most relevant
factors are often displayed as at least a bit of dark-blue bar length in combination
with shorter light-blue bar in comparison with other factors in the jackknife figure.
Thus, deletion of such factor would cause a large loss of explanatory power of the
model. Resolution of potential distribution maps was set to 500 500 m to make the
map precise and detailed for determining possible new localities of studied species
with suitable conditions.
In the description of important factors for studied species, percentage contribu-
tions of all factors were displayed in a table, but only the first three most important
factors were described in more details in the text belonging to each studied species.
3 Results and Discussion
3.1 Anacamptis morio (L.) R.M. Bateman, A.M. Pridgeon &

M.W. Chase 1997
The results of jackknife procedure in Fig. 2 revealed that the consolidated layer of
ecosystems (KVES) is the most important factor that influences the distribution of
Anacamptis morio in the South Bohemian region. Other important factors for this
species are precipitation and slope of terrain (slope).
A closer look at the pictures of the most important environmental variables
(Fig. 3) that play an important role in the distribution of A. morio reveals some
interesting patterns. Consolidated layer of ecosystems (KVES) was determined as
the most important factor with the contribution of 61.2%. Figure 3a of the analysis
of KVES indicates that the highest probability of presence of this species is in oak
and oak-hornbeam forests (KVES 10), mixed forests (KVES 30), discontinuous
urban development (KVES 36), and agricultural meadows (KVES 39). According to
our personal observation in the field, discontinuous urban development and agricul-
tural meadows may be suitable habitats for A. morio when suitable management is
applied. However, oak and oak-hornbeam forests and mixed forests are not suitable
habitats for this species. These inconsistencies may be caused by border zone of two
or more different habitat types. Plants may be present close to a forest border, and
this place could have been identified as a forest during a monitoring of habitats and
not as mesophilic meadow, agricultural meadow, or dry grassland, which A. morio
can favor. On one hand, this is not a precise result, but, on the other hand, we can also
identify negative aspects of MaxEnt analysis in prediction of a particular species
distribution and possible gaps in procedure of habitat or biotope monitoring.
Jackknife test of variable importance for Anacamptis morio

dem
frost_days
KVES
precipitation
slope
Variable
solar_rad
summer_days
temp_1
temp_2
trop_days
veg_season
Total gain
0 0,1 0,2 0,3 0,4 0,5 0,6
Regularized training gain
With only variable Without variable With all variables
Fig. 2 Graph of the jackknife procedure for Anacamptis morio
Another factor that was determined as important is the amount of precipitation

per year (mm). It is clearly visible in Fig. 3b that A. morio prefers places with
low amount of precipitation. It is in agreement with ecological demands of this
species mentioned in the literature – we can find it on dry or slightly humid
substrates [46–48].
The last factor with an important effect on distribution of A. morio is the slope of
terrain. Figure 3c shows that this species prefers rather flat or slightly inclined places,
which was confirmed by our personal observation on its localities. We would
probably not find this species in steep or exposed habitats.
The potential distribution map of Anacamptis morio is depicted in Fig. 4. It shows
that there are many ecologically suitable places for distribution of this species but not
all of them are suitable indeed because of inappropriate management. Other vege-
tation overgrows many of these suitable places, and it is impossible for A. morio to
set a new population on such places [52, 53]. In the past, there were many thriving
localities of this species in the region of South Bohemia, but the majority of them is
now extinct (about 95%) [54] because of inappropriate management (overgrowing or
even no management) or conversion of suitable places into agricultural fields that
happened in the past [49, 55].
3.2 Cephalanthera rubra (Linne) L.C.M. Richard 1818
The jackknife procedure in Fig. 5 revealed that more factors have a similar impact on
the distribution of this species. The first one is mean annual temperature (temp_1),
then precipitation, and consolidated layer of ecosystems (KVES). Other important
factors affecting its distribution are altitude (dem) and slope of the terrain (slope).
Using Fig. 5, it may be hypothesized that many factors have a certain impact on
a Response of A. morio to KVES

0.85
Logistic output (probability of presence)

0.80
0.75
0.70
0.65
0.60
0.55
0.50
0.45
0.40
0.35
1 3 5 7 9 11 13 17 19 22 24 29 32 34 37 39
kves
b Response of A. morio to precipitation

0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0.0
400 500 600 700 800 900 1000 1100 1200 1300
precipitation
c Response of A. morio to slope

0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
−2 0 2 4 6 8 10 12 14 16
slope
Fig. 3 Responses of Anacamptis morio to (a) consolidated layer of ecosystems (KVES),

(b) precipitation, (c) slope of a terrain
Fig. 4 Potential distribution map of Anacamptis morio in the region of South Bohemia
Jackknife test of variable importance for Cephalanthera rubra

dem
frost_days
KVES
precipitation
slope
Variable
solar_rad
summer_days
temp_1
temp_2
trop_days
veg_season
Total gain
0 0,2 0,4 0,6 0,8 1 1,2 1,4 1,6
Fig. 5 Graph of the jackknife procedure for Cephalanthera rubra

distribution of C. rubra; they are co-affecting its presence together to some extent,
and no single one factor has such an outstanding impact that KVES had in the case
of Anacamptis morio.
The most important factor affecting the distribution of C. rubra is the mean
annual temperature (temp_1). Figure 6a shows that C. rubra thrives in places where
mean annual temperature is high. Intuitively, this is connected with altitude because
high mean annual temperatures are in lowlands. Therefore, this means that this
species will flourish in lower altitudes and we should not find it in high mountains.
It is confirmed by information in the literature [46, 48] that says that we can find this
species from lowlands to middle altitudes in the Czech Republic.
Second factor that was found to be important for distribution of C. rubra is
the amount of precipitation per year, and its effect is depicted in Fig. 6b. It is clearly
visible that this species prefers low amount of precipitation. This is again connected
to altitude, as in the case of the previous factor – C. rubra should occur in the
lower altitudes where there is only a little rain, which is congruent with the literature
[46, 48].
Consolidated layer of ecosystems (KVES) was found to be the third most
important factor affecting the presence of C. rubra in South Bohemian region.
Figure 6c indicates that the most suitable habitats for this species are oak and
oak-hornbeam forests (KVES 10), mixed forests (KVES 30), and agricultural
meadows (KVES 39). It is said in the literature [46–49] that this species grows in
bright forests, so both oak/oak-hornbeam forests and open mixed forests could be
suitable for this species. To compare with one European country, Hungary, this
species also prefers deciduous or mixed forests with Pinus nigra (Pacsai, pers.
com.). Agricultural meadows may be considered as suitable, if a proper management
is applied or if an agricultural meadow neighbors a suitable open forest.
The potential distribution map of Cephalanthera rubra (Fig. 7) shows potential
suitable places for distribution of this species in the region of South Bohemia. Such
places may be found around the city of Český Krumlov and toward the southern
borders from this town and in the southeastern part bordering Austria. It may be also
present in smaller limestone areas, because C. rubra prefers places where limestone
is present [46, 48] such as abandoned limestone quarries. Such places were found,
for example, around Horažďovice and Sušice, Tábor, Milevsko, and Písek city.
3.3 Dactylorhiza fuchsii (Druce) Soó 1962
Figure 8 shows the result of jackknife procedure generated by MaxEnt. It revealed

that the most important factor affecting the distribution of D. fuchsii is consolidated
layer of ecosystems (KVES). The second most important factor was slope followed
by annual year temperature (temp_1).
Responses of the most important factors affecting the distribution of D. fuchsii are
shown in Fig. 9. It represents the most important factor that affects the distribution of
this species – consolidated layer of ecosystems (KVES). It is clearly visible that in
this region, D. fuchsii prefers coniferous forests (KVES 31). It is known from the
a Response of C. rubra to temp_1

0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
45 46 47 48 49 50 51 52 53 54 55 56
temp_1
b Response of C. rubra to precipitation

0.65
0.60
0.55
0.50
0.45
0.40
0.35
0.30
0.25
0.20
0.15
0.10
400 500 600 700 800 900 1000 1100 1200 1300
precipitation
c Response of C. rubra to KVES

0.85
0.80
0.75
0.70
0.65
0.60
0.55
0.50
0.45
0.40
0.35
0.30
1 3 5 7 9 11 13 17 19 22 24 29 32 34 37 39
kves
Fig. 6 Responses of Cephalanthera rubra to (a) mean annual temperature (temp_1),

(b) precipitation, (c) consolidated layer of ecosystems (KVES)
Fig. 7 Potential distribution map of Cephalanthera rubra in the region of South Bohemia
literature [46–48] that this species can be found in meadows and pastures as well as
in forests. Coniferous forests are suitable [49] especially if they are not dense and
the species has enough light to grow. From our personal observations in the field,
suitable habitats are mainly on the borders of coniferous forests with other habitats
(meadows or pastures) and in sparse coniferous forests, too.
Slope of terrain was determined as the second most important factor for distri-
bution of D. fuchsii. From Fig. 9b, it is clearly visible that this species prefers a bit
hilly landscape, and we will probably not find it in flat areas. It is in accordance with
the literature, because D. fuchsii can be found from middle altitudes [46, 48], where
the landscape is a bit wavy, not completely flat, and it disappeared from the low
altitudes in South Bohemia [49].
The important factor that has an effect on the distribution of this species
is mean annual temperature (temp_1). From Fig. 9c, it may be assumed that
D. fuchsii prefers from middle to higher values of annual mean temperature.
It means it will not be probably found in the highest places that are most exposed
and cold, but it may be found in middle altitudes, where the temperatures are still
high enough for its presence.
Jackknife test of variable importance for Dactylorhiza fuchsii

dem
frost_days
KVES
precipitation
slope
Variable
solar_rad
summer_days
temp_1
temp_2
trop_days
veg_season
Total gain
0 0,2 0,4 0,6 0,8 1 1,2
Fig. 8 Graph of the jackknife procedure for Dactylorhiza fuchsii
In Fig. 10, the map of potential distribution of Dactylorhiza fuchsii is displayed.

The most suitable places, where new localities of this species may be found, are
distributed mainly in the southernmost part of the South Bohemian region around
Vyšší Brod city near the borders of Austria. Other suitable places can be found
around Prachatice city toward Boletice Military Training Area and in middle alti-
tudes in Šumava National Park. However, smaller suitable habitats are scattered
across the whole region of South Bohemia.
3.4 Epipactis palustris (Linne) Crantz 1769
In Fig. 11, the results of jackknife procedure of Epipactis palustris are displayed.
From this picture, it is clearly visible that consolidated layer of ecosystems (KVES)
was revealed as the most important factor affecting its distribution in the region of
South Bohemia. Other two factors that have an impact on its distribution were slope
of the terrain (slope) and solar radiation (solar_rad).
According to the results of jackknife procedure, the most important factor was the
consolidated layer of ecosystem (KVES). In a closer look at picture of this factor
(Fig. 12a), we can clearly distinguish that most probably we will find this species in
habitats of dry pine forests (KVES 13), mesophilic meadows (KVES 6), and partly
in agricultural meadows (KVES 39). The information in the literature says that this
species prefers habitats, mainly meadows, with stable water level and regime [49],
and it may also be present in secondary habitats that were somehow altered by
people in the past [46–48]. According to our personal observations in the field, it was
found that this species is often present in the near vicinity of a forest (e.g., at the
border zone between meadow and pine forest) – often in a kind of terrain depression
a Response of D. fuchsii to KVES

0.60
0.55
0.50
0.45
0.40
0.35
0.30
0.25
0.20
0.15
0.10
1 3 5 7 9 11 13 17 19 22 24 29 32 34 37 39
kves
b Response of D. fuchsii to slope

0.9
0.8
0.7
0.6
0.5
0.4
0.3
−2 0 2 4 6 8 10 12 14 16
slope
c Response of D. fuchsii to temp_1

0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
45 46 47 48 49 50 51 52 53 54 55 56
temp_1
Fig. 9 Responses of Dactylorhiza fuchsii to (a) consolidated layer of ecosystems (KVES),

(b) slope of a terrain, (c) mean annual temperature (temp_1)
Fig. 10 Potential distribution map of Dactylorhiza fuchsii in the region of South Bohemia
Jackknife test of variable importance for Epipactis palustris

dem
frost_days
KVES
precipitation
slope
Variable
solar_rad
summer_days
temp_1
temp_2
trop_days
veg_season
Total gain
0 0,2 0,4 0,6 0,8 1
Fig. 11 Graph of the jackknife procedure for Epipactis palustris

a Response of E. palustris to KVES

1.0

0.9
0.8
0.7
0.6
0.5
0.4
0.3
1 3 5 7 9 11 13 15 18 21 24 29 32 35 38
kves
b Response of E. palustris to slope

0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0.0
−2 0 2 4 6 8 10 12 14 16
slope
c Response of E. palustris to solar_rad

0.70
0.65
0.60
0.55
0.50
0.45
0.40
0.35
0.30
0.25
3550 3600 3650 3700 3750 3800 3850 3900 3950
solar_rad
Fig. 12 Responses of Epipactis palustris to (a) consolidated layer of ecosystems (KVES),

(b) slope of a terrain (slope), (c) solar radiation (solar_rad)
where water regime is stable and higher. Both mesophilic and agricultural meadows
are also suitable for distribution of E. palustris if a suitable water regime is
maintained.
Slope of a terrain (slope) was determined as the second most important factor
affecting the distribution of this orchid. Figure 12b shows the probability of presence
of E. palustris on a different slope. According to the results in Fig. 12b, this species
will be most probably found in flat terrain that is in accordance with the previous
statements about water regimes and possible terrain depressions. We will not find
this species in steep slopes, because water regime varies here a lot during the year
and is not stable.
The last factor – the amount of incoming solar radiation (solar_rad) – goes in
hand with the first factor. From the graph generated by MaxEnt (Fig. 12c), it is
clearly visible that E. palustris prefers shady places which is in accordance with its
suitable habitats in this region which are pine groves. It was also monitored during
our field studies that E. palustris was found in humid meadows, where surrounding
vegetation was quite high, so orchid plants were not exposed to direct sunbeams
in such places. According to Fig. 12c, E. palustris can be also found in semi-shaded
places.
The potential distribution map of Epipactis palustris is depicted in Fig. 13. It
shows suitable places, where it is possible to find new localities of this species in the
future if their management and climate will not change. Such suitable localities are
quite scattered in this region, and the majority of this area is not much suitable for this
species. However, few suitable places can be still found mainly in the vicinity
of Veselí nad Lužnicí city, where some Special Areas of Conservation (SAC) are
present, and toward the southeastern borders with Austria near Suchdol nad Lužnicí
and Chlum u Třeboně villages in Třeboňsko Nature Conservation Area. Some smaller
scattered suitable places were also found between Dolní Dvořiště and Vyšší Brod city
in the southernmost part of this region and in the Šumava National Park.
3.5 Neottia nidus-avis (Linne) L.C.M. Richard 1817
Figure 14 shows the effect of various factors tested that influence the distribution of
Neottia nidus-avis in the South Bohemian region, according to the jackknife proce-
dure. Clearly, consolidated layer of ecosystem (KVES) has the main impact on the
distribution of this species. The following two main important environmental vari-
ables were slope of a terrain (slope) and mean annual precipitation.
The pictures from the results of the three most important variables affecting the
distribution on N. nidus-avis in the region of South Bohemia (Fig. 15) revealed some
interesting patterns. According to MaxEnt analysis, the most important factor was
set to KVES (consolidated layer of ecosystem). From Fig. 15a, it is clearly visible
that the most suitable habitats for this species are oak and oak-hornbeam forests
(KVES 10); however, it is possible to find it partly also in coniferous forests
(KVES 31) and in agricultural meadows (KVES 39). Suitable habitat of oak
and oak-hornbeam forests is in accordance with the information from literature
[47, 48, 56]; partly also coniferous forests may be suitable but only in case that
Fig. 13 Potential distribution map of Epipactis palustris in the South Bohemian region
Jackknife test of variable importance for Neottia nidus-avis

dem
frost_days
KVES
precipitation
slope
Variable
solar_rad
summer_days
temp_1
temp_2
trop_days
veg_season
Total gain
0 0,2 0,4 0,6 0,8 1
Fig. 14 Graph of the jackknife procedure for Neottia nidus-avis

a Response of N. nidus-avis to KVES

0.9
0.8
0.7
0.6
0.5
0.4
0.3
1 3 5 7 9 11 13 15 18 21 24 29 32 35 38
kves
b Response of N. nidus-avis to slope

0.9
0.8
0.7
0.6
0.5
0.4
0.3
−2 0 2 4 6 8 10 12 14 16
slope
c Response of N. nidus-avis to precipitation

0.70
0.65
0.60
0.55
0.50
0.45
0.40
0.35
0.30
0.25
0.20
0.15
0.10
0.05
400 500 600 700 800 900 1000 1100 1200 1300
precipitation
Fig. 15 Responses of Neottia nidus-avis to (a) consolidated layer of ecosystems (KVES), (b) slope
of a terrain (slope), (c) mean annual precipitation
particular forest is not too dense. But agricultural meadows are not suitable for
N. nidus-avis. This inconsistency may be caused by a borderline between forest
habitat and agricultural meadow. Dykyjová stated that this species is able to reach
also atypical habitats from forests edges, such as grass meadows or scrublands [46].
Also Lepší et al. confirmed forest edges as a partly suitable habitat [49].
Slope of a terrain was revealed as the second most important factor affecting the
presence of N. nidus-avis. According to Fig. 15b, it may be assumed that this species
will be found in at least a bit hilly countryside and not in completely flat places.
Based on the literature, it prefers the altitudinal range from lower places or foothills
to mountains [46, 48].
Another important factor for distribution of N. nidus-avis is annual mean precip-
itation. Figure 15c indicates that this species prefers places with lower amount of
precipitation during the whole year. Intuitively, these places can be found in lower
altitudes but also in higher altitudes in rain shadow, and these may be suitable for
presence of N. nidus-avis.
In Fig. 16, the map of potential distribution of Neottia nidus-avis is shown. It
indicates that there are many places with suitable conditions for this species in the
Fig. 16 Potential distribution map of Neottia nidus-avis in the region of South Bohemia
South Bohemian region. They can be found in the area between Český Krumlov and
Vyšší Brod cities in the southern part of this region, in foothills of Šumava National
Park, and in the central part of South Bohemia around Písek city.
3.6 Neottia ovata (L.) Bluff & Fingerh. 1838
In Fig. 17, the results of jackknife procedure for Neottia ovata are displayed. From
this picture, it is clearly visible that the most important factor affecting the distribu-
tion of this species is again the consolidated layer of ecosystems (KVES). Other two
factors with the second and third highest percent contribution were slope of a terrain
(slope) and solar radiation (solar_rad).
A closer look at pictures of environmental variables that had the most important
effect on the distribution of N. ovata (Fig. 18) reveals certain patterns. Figure 18a
shows that most suitable habitats for this species are present in urban green areas,
gardens and parks (KVES 33); in oak and oak-hornbeam forests (KVES 10), beech
forests (KVES 12); and in agricultural meadows (KVES 39). All of the habitats that
were revealed as suitable by the analysis of MaxEnt are places proved by the
information from literature. It says that N. ovata is one of the species that has
broad ecological niche, so it may be found in various types of habitats [46–48, 56].
Slope of a terrain was also determined as important factor that may affect the
distribution of N. ovata. Figure 18b indicates that we will probably not find it in
completely flat places, but it prefers at least a bit hilly countryside. The center of its
occurrence in the region of South Bohemia is in a foothill level [49]. However, as it
was stated above, this species has no specific ecological demands, so we can find it in
various places.
Jackknife test of variable importance for Neottia ovata

dem
frost_days
KVES
precipitation
slope
Variable
solar_rad
summer_days
temp_1
temp_2
trop_days
veg_season
Total gain
0 0,1 0,2 0,3 0,4 0,5
Fig. 17 Graph of the jackknife procedure for Neottia ovata

a Response of N. ovata to KVES

1.0

0.9
0.8
0.7
0.6
0.5
0.4
1 3 5 7 9 11 13 15 18 21 24 29 32 35 38
kves
b Response of N. ovata to slope

0.90
0.85
0.80
0.75
0.70
0.65
0.60
0.55
0.50
0.45
0.40
0.35
−2 0 2 4 6 8 10 12 14 16
slope
c Response of N. ovata to solar_rad

0.60
0.55
0.50
0.45
0.40
0.35
0.30
0.25
3550 3600 3650 3700 3750 3800 3850 3900 3950

solar_rad
Fig. 18 Responses of Neottia ovata to (a) consolidated layer of ecosystems (KVES), (b) slope of
a terrain (slope), (c) solar radiation
Fig. 19 Potential distribution map of Neottia ovata in the region of South Bohemia
Figure 18c shows the impact of solar radiation on the distribution of N. ovata. It is
clearly visible from the picture that the more solar radiation is present, the higher is
the probability of presence of this species on such places. It does not prefer entirely
shady places.
The potential distribution map of Neottia ovata is displayed in Fig. 19. From this
picture, it can be assumed that there are many suitable places in the region of South
Bohemia for presence of N. ovata and there are almost no places that would be
completely unsuitable (dark-blue color). This also proved the statement above that
this species may be found in many different types of habitats and climatic conditions;
it is not specialized in this term. The most suitable places (red and yellow colors) are
present in southwestern part of the South Bohemian region, in the foothills of
Šumava National Park in the area from Český Krumlov toward Prachatice city.
3.7 Platanthera chlorantha (Custer) Rchb. 1828
The results of jackknife procedure are displayed in Fig. 20. It implies that there are
more environmental variables that have the main impact on the distribution of
Jackknife test of variable importance for Platanthera chlorantha

dem
frost_days
KVES
precipitation
slope
Variable
solar_rad
summer_days
temp_1
temp_2
trop_days
veg_season
Total gain
0 0,5 1 1,5 2 2,5
Fig. 20 Graph of the jackknife procedure for Platanthera chlorantha
P. chlorantha. There are many factors that cooperate with each other and influence
where this species occurs. According to Fig. 20, the most important factors affecting
its distribution in the region of South Bohemia are number of tropical days per year
(trop_days) followed by total amount of solar radiation that enters a locality
(solar_rad) and amount of precipitation per year (precipitation). It is also worth to
say that consolidated layer of ecosystems (KVES) was not evaluated as one of the
most important factors affecting species distribution, as was the case with species
described above, so it is the only species that do not rely strongly on a type of habitat
that is present on its localities. Literature says that this species has no clear relation
with a particular habitat type [47].
A closer look at pictures of the most important factors that had a significant
impact on the distribution of P. chlorantha (Fig. 21) shows interesting results. In
Fig. 21a, the impact of the number of tropical days per year is displayed. It is
visible that there is a high probability of occurrence of this species in places with
zero or only a few tropical days per year and almost zero probability in places
where many tropical days are present. These findings imply that P. chlorantha
prefers higher altitudes and we will probably not find this species in lowlands in
the studied region. Also according to the literature, it prefers higher and colder
places [49].
Figure 21b shows a response of the studied species to solar radiation
(solar_rad), a typical mesoclimatic factor. In general, the extent of solar radiation
is not different throughout the whole Czech Republic, so this factor tells us
whether P. chlorantha prefers shady or sunny places. From the graph, it is clearly
visible that it is more likely to find this species in shady places and it tries to
avoid places in full sunlight. This is in accordance with the literature; it may
occur also in mountain meadows that may represent the middle part of the curve
presented.
a Response of P. chlorantha to trop_days

0.8

0.7
0.6
0.5
0.4
0.3
0.2
0.1
0.0
−2 0 2 4 6 8 10 12 14 16
trop_days
b Response of P. chlorantha to solar_rad

0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0.0
3550 3600 3650 3700 3750 3800 3850 3900 3950
solar_rad
c Response of P. chlorantha to precipitation

0.64
0.62
0.60
0.58
0.56
0.54
0.52
0.50
0.48
0.46
400 500 600 700 800 900 1000 1100 1200 1300
precipitation
Fig. 21 Responses of Platanthera chlorantha to (a) amount of tropical days per year (trop_days),
(b) total amount of solar radiation (solar_rad), (c) mean annual precipitation
The impact of mean annual precipitation on the distribution of P. chlorantha is

displayed in Fig. 21c. This picture shows that the more precipitation per year, the
higher is the probability of presence of this species, and it does not prefer places with
low amount of rainfalls throughout a year. Generally speaking, high amount of
precipitations is a characteristic feature for higher altitudes and mountain areas, so
this result indicates that such places are suitable for the presence of P. chlorantha.
It is similar to the case of the impact of tropical days on this species, and it is also
stated in the literature [46, 47, 49].
In Fig. 22, the potential distribution map of Platanthera chlorantha is
depicted. This map shows that there are a few suitable places for potential
occurrence of the studied species, but climatic and environmental conditions
are not suitable for P. chlorantha in most of the region. Potential suitable places
for this species may be found mainly in the southeastern edge at the borders with
Austria around Nová Bystřice city, in smaller scattered areas in the south around
Vyšší Brod city, in a middle-to-higher-altitude belt stretching from Český
Fig. 22 Potential distribution map of Platanthera chlorantha in the South Bohemian region
Krumlov toward Prachatice, and partly also in the northeastern part of the region
in the vicinity of Chýnov.
4 Summary
As stated in other papers, the amount of arable land is an important factor that affects
the orchid distribution. In our study, no orchid species studied prefer any kind of
arable land (not presented here). It proved the previous statements that the high
amount of arable land in the vicinity of an orchid locality has a negative effect on its
distribution [44, 45].
The most important and most common environmental factor affecting the
distribution of numerous orchid species in the region of South Bohemia was
the consolidated layer of ecosystems (KVES) as it played the most important role
for 10 out of 11 species studied (see Table 2). Only for Platanthera chlorantha,
KVES was not found as important factor affecting its occurrence. The other two
most important variables were mean annual precipitation and slope of a terrain
that was important for 7 out of 11 species studied (Table 2). According to the fact
that KVES was set as the most important factor that can influence the distribu-
tion and presence of many orchids species, evaluation of a particular habitat type
(KVES) was also done (see Table 3). Based on our analysis, the most important
KVES types (habitat type) are oak and oak-hornbeam forests (KVES 10)
followed by agricultural meadows (KVES 39). However, forests as well as
meadows in general should be protected as they host many endangered species
of orchids. The duration of vegetation season (veg_season) was also added into
our analysis, but it has no important effect on the distribution of studied species
because the length of the vegetation season does not differ a lot across the whole
country. The small differences in the length of vegetation season are more
connected with altitude.
5 Conclusions
The MaxEnt program is a useful tool for predicting potential distribution of species
in general but is especially effective when working with threatened and endangered
species. According to the results of our study, the most important factors for many
orchid species in the South Bohemian region are habitat type (represented by
consolidated layer of ecosystems, KVES), precipitation, and slope of a terrain.
Our results are important and helpful in determination of possible new localities
in the region of South Bohemia but may be also used in larger scale. Without
potential distribution maps, searching of new localities would be only a random
choice of researchers. Our findings may help in orchid conservation by preserving
suitable habitats for chosen orchid species.
2
Table 2 The list of the most important environmental factors (the first three) affecting distribution of studied species in the region of South Bohemia in the
Czech Republic ( impact of a factor is less than 50%, • impact of a factor is 50% and more). Results for species in red are described in Štípková et al. [44] and
Kosánová [45]
dem KVES precipitation slope solar_rad temp_1 temp_2 trop_days veg_season
Anacamptis morio
Cephalanthera damasonium
Cephalanthera rubra
Dactylorhiza fuchsii
Dactylorhiza majalis
Epipactis atrorubens
Epipactis palustris
Neottia nidusavis
Neottia ovata
Platanthera chlorantha
Platanthera bifolia
Which Environmental Factors Drive Distribution of Orchids? A Case Study. . .
101
102
Table 3 The most important habitat types (KVES) for studied species (Platanthera chlorantha was not included in the table because it does not strongly rely on
a specific habitat type)
KVES KVES KVES KVES KVES KVES KVES KVES KVES KVES KVES KVES KVES KVES KVES
4 5 6 10 12 13 17 19 23 24 30 31 33 36 39
Anacamptis morio • • • •
Cephalanthera • • • •
damasonium
Cephalanthera • • •
rubra
Dactylorhiza •
fuchsii
Dactylorhiza • • • • • • • •
majalis
Epipactis • • • •
atrorubens
Epipactis palustris • • •
Neottia nidus-avis • • •
Neottia ovata • • • •
Platanthera • • • • •
bifolia
Z. Štípková et al.
Acknowledgments This work was supported by the Ministry of Education, Youth and Sports of
CR within the National Sustainability Program I (NPU I), grant number LO1415. We also thank the
South Bohemian Branch of the Land Office in Ceske Budejovice for their kind cooperation and
Kristina Kosánová for her help in the field.
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Diversity, Ecology, and Conservation
of Mauritius Orchids 3
Cláudia Baider and F. B. Vincent Florens
Contents
1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 108
1.1 Mass-Extinction and Island Biodiversity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 108
1.2 Mauritius . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 109
2 Diversity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111
2.1 Background . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111
2.2 Diversity and Endemism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 112
2.3 Types and Distribution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 113
3 Ecology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 116
4 Threats and Conservation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 117
4.1 Deforestation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 117
4.2 Harvesting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 118
4.3 Alien Plants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 119
4.4 Alien Animals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 121
4.5 Indirect Effects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 123
4.6 Other Threats . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 123
5 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 127
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 128
Abstract
Mauritius was one of the last places on Earth to be colonized by humans offering
one of the most complete history of what native species occurred originally and
what was lost, when, and why. This situation can therefore serve as a laboratory to
C. Baider (*)
The Mauritius Herbarium, Agricultural Services, Ministry of Agro-Industry and Food Security,
Réduit, Mauritius
e-mail: cbaider@govmu.org
F. B. V. Florens
Tropical Island Biodiversity, Ecology and Conservation Pole of Research, Department of
Biosciences and Ocean Studies, University of Mauritius, Réduit, Mauritius
e-mail: Vin.Florens@uom.ac.mu

108 C. Baider and F. B. V. Florens
study human impacts in the current age of human-driven species extinction.

Mauritius is also one of the most human-impacted places, thereby reflecting
what awaits much of the tropics as human impacts intensify. We used available
literature, herbarium samples, and personal observations and studies on the
Orchidaceae to characterize its diversity, distribution and ecology, and the
human-induced threats they face, to better inform their conservation in Mauritius.
There are 91 native orchid species from 30 genera recorded on the island. Twenty
species (22%) appear extinct, although some may survive undetected. New
species and records continue to be added. Only 10% of the species are endemic
to Mauritius, and 80% are unique to the south-west Indian Ocean islands. Most
species are epiphytic, and the highest diversity occurs in native forests of the wet
uplands. Mauritian orchids, particularly the larger ones, face many threats, some
inexorably worsening. There exists much room to improve knowledge about
Mauritian orchids that would better inform their conservation which is today
still very neglected. This includes taxonomic research, detection of ecological
patterns and trends, ecology of the species, as well as quantification and hierar-
chization of threats to prioritize conservation management. Studying Mauritius
native orchids helps understand how devastating, sustained, and accelerating the
many threats that human activities pose to orchid biodiversity can be and which
await other countries currently less human-impacted than Mauritius.
Keywords
Mascarenes · Biodiversity · Invasive alien species · Island · Extinction
1 Introduction
1.1 Mass-Extinction and Island Biodiversity
Biodiversity worldwide is rapidly being lost under a wide array of threats from human
activities driven principally by human overpopulation and unsustainable consump-
tion patterns of natural resources [1]. The situation is such that it has been described as
the sixth mass extinction [2]. Such an extent and worsening biodiversity loss is
particularly damaging to humans and their societies given the dependence on biodi-
versity in many ways including for the provision of food, water, or a swath of
ecosystem services [3, 4]. Biodiversity loss also shares its root causes with and
positively feeds back into global climate change, which is the greatest existential
threat facing humanity today [5]. These global problems must be tackled to stop the
degradation and reverse the trend, and the implementation of solutions often has a
local or regional theatre [6, 7]. In turn, the success of locally applied solutions
depends on a good understanding of the threats driving degradation at that level
because conservation management is as sound as the science on which it is based [8].
In this context, oceanic islands are particularly informative places to study
biodiversity, ecology, and conservation [9, 10]. They have played and continue to
3 Diversity, Ecology, and Conservation of Mauritius Orchids 109
play an important role in the development and refining of the theory of evolution by
natural selection [11] and in understanding species distribution and how communi-
ties assemble through time [12], as well as, the biogeographical factors that strongly
shape biotas [13–15]. Furthermore, oceanic islands contribute a disproportionately
large share of global biodiversity relative to their area by virtue of the often-high
endemism that characterizes their biota [16], particularly when they are relatively
isolated, high and old, and occur in tropical or subtropical areas. With the advent of
the widespread and profound impacts of human activities, humans have now com-
bined the high degree of endemism of oceanic islands to a high degree of threat to
biodiversity, resulting in a disproportionately large representation of oceanic island
among the world’s biodiversity hotspots [17, 18].
Interestingly, oceanic islands are often among the last places on Earth to have
been reached by humans and be subjected to their impacts and modifications
[19, 20]. This means that oceanic islands can provide us with some of the most
accurate accounts and best understanding of how human activities influence nature
and drive biodiversity loss, because they enable a better understanding of what
biodiversity was initially present when the place was pristine, and what was lost,
when and why following human colonization, a situation absent from places which
have been inhabited by humans for eons like the continents. A more accurate
understanding of human impacts on biodiversity is itself crucial if we are to devise
sound responses to the global mass extinction. Oceanic island can therefore serve as
useful laboratories to study the impacts and consequences of human activities on
biodiversity, and by extension the solutions that are required to reverse
biodiversity loss.
Orchids, in particular, make for an interesting model to study biodiversity patterns
and threats besetting species and the corresponding conservation solutions, because
they form one of the most diverse family of flowering plants present globally
(>29,500 species, summing about 8% of all known flowering plants [21]) and
on many tropical archipelagos [22], including the Mascarenes (around 166 species
[23–28]). The occurrence of many species gives the best chances for any pattern to
be more reliably established than would be the case if one were to be dealing with
smaller groups of species, as the latter are more highly subjected to possible spurious
conclusions caused, for example, by sampling error. Furthermore, orchids have been
a group of plants that attracted much interest [29, 30] and since long [31], including
being part of culture and iconography of older cultures (e.g., Greek, Roman,
Chinese; see [32]). Importantly, today the whole family is listed under Appendix II
of CITES, even though some orchids are invasive [33, 34].
1.2 Mauritius
Mauritius (1865 km2, 828 m maximum elevation) is one of the three main volcanic
oceanic islands comprising the Mascarenes along with La Réunion (2512 km2) some
175 km to the west-south-west and Rodrigues (108 km2) located about 595 km to the
east. Mauritius is centered around 20 200 S and 57 350 E some 900 km east of
Madagascar. It emerged some 7.8 million years ago and experienced its last volcanic
activity in its north-east region about 20,000 years ago [35]. The bedrock is mainly
basaltic with limited calcarenitic areas confined to small patches on the coastline or
on lagoonal islets in the south and southeast. Annual rainfall varies from about
800 mm on parts of the western coast to about 4000 in the wettest highlands, with an
average of about 2100 for the island [36]. The temperature varies from an average of
16.4 C at night in July–August to an average of 29.2 C during the day in January–
February. The original native vegetation varied from a palm-rich woodland in the
drier coastal areas behind the coastal vegetation fringe, to small mossy forest patches
on the highest grounds, and comprised in majority, of a wet forest which covered
about half of the island [37]. The vegetation ecosystems did not appear to have
shifted or changed majorly from the end of the Pleistocene (38,000 years ago) to the
time of human colonization in 1638 but underwent a series of relatively limited
reassortment of species dominance instead [38].
Although Mauritius was among the last places on Earth to be settled by humans
(in 1638), it underwent one of the most rapid and advanced levels of native habitat
destructions that spared only about 4.4% of its original terrestrial habitats [39] within
the following 375 years or so of human presence under the Dutch (1638–1710),
French (1715–1810), British (1810–1968), and Mauritian (1968–present) sover-
eignty. Some types of habitat, like the palm-rich drier forests, have been completely
destroyed from the mainland and only survive as highly degraded small patches on
tiny offshore islets [37] which are fortunately undergoing ecological restoration. The
remaining habitats on Mauritius are also highly fragmented [40], and despite their
small extent, habitat destruction continues and has been recorded even within Nature
Reserves protected by law [41]. Furthermore, the overwhelming majority of the
80 or so km2 of native habitats that have so far escaped deforestation are currently
highly invaded by encroaching alien plants [42]. In effect, Mauritius may arguably
be regarded as representing a “window” into the future of many other tropical places
as the latter catch up, in line with current trends, with the already advanced levels of
habitat destruction and fragmentation, alien species invasion, native species extinc-
tion and endangerment rates, human overpopulation, and urban sprawl, among
others [39, 41]. Mauritius, therefore, approximates what other places are increas-
ingly approaching and can thus serve as an informative laboratory for them of how
biodiversity will be lost, but also of possible solutions to stem this biodiversity loss.
Here we discuss the diversity, ecology, and conservation of biodiversity using the
native orchid flora of Mauritius as a model. We used the available literature,
herbarium samples, and notes thereon as well as personal observations and studies
on the field to characterize the diversity of native orchids in terms of the number of
genera and species, the patterns of species discoveries through time, species distri-
bution (whether island endemic, archipelago endemic, or of wider distribution),
ecology (pollination, habit, elevational distribution, etc.), the various human-
induced threats that they face (e.g., habitat destruction, overcollection, the impact
of invasive alien plants and animals, etc.), and the rate of extinction and species
rarity that ensued, in order to propose conservation measures to try to reverse the
current ongoing loss of the orchid flora of Mauritius.
2 Diversity
2.1 Background
The oldest traceable samples of orchids collected in Mauritius were two native species
of Nervilia, a genus of ground orchid, placed on the same herbarium sheet, and
collected in 1769 by the French botanist Philibert Commerson (1727–1773; https://
en.wikipedia.org/wiki/Philibert_Commerson). Although this first collection was made
over 130 years after the island was first colonized by humans, it is unlikely that orchid
species could have already been lost as a result of human activities by then (in contrast
with vertebrates [37]) because 82.5% of the island’s native cover still subsisted and
none of the broad types of vegetation communities had yet been destroyed
[37, 43]. After Commerson, who botanized on Mauritius until 1773, came Louis-
Marie Aubert du Petit Thouars (1758–1831; https://en.wikipedia.org/wiki/Louis-
Marie_Aubert_du_Petit-Thouars), another French botanist who is most well-known for
his work on collecting orchids of Mauritius and also of nearby Réunion island and
Madagascar from 1793 to 1802. Du Petit Thouars produced a small book with six
paintings (believed to have been published between1804 and 1819 [44]), followed by
an article that contained tables with the description of dozens of species present on
Mauritius in 1809 [45] and, later, a more comprehensive first book on Mauritius orchids
(along with species from Réunion and Madagascar) in 1822 [46] and described
11 orchid genera, four of which still stand today, including Bulbophyllum, the largest
genus of orchids with over 2000 species [47], which makes it among the largest genera
of flowering plants. Du Petit Thouars recognized 51 orchid species for Mauritius.
Another notable botanist of the nineteenth century, who contributed significantly to
expanding knowledge on Mauritius native orchids, was the Czech Wenceslas Bojer
(1795–1856; https://en.wikipedia.org/wiki/Wenceslas_Bojer) who lived on Mauritius
for over 30 years [48] and published the first Flora of the island [49].
Additions to the native flora of Mauritius orchids continue to be made despite the
very limited extent of native habitats that survive the rapid deforestation experienced
[41]. Thus, during the last 16 years alone (which represents 7% of the total period of
orchid study on the island), seven species have been added, representing about 8% of
the total known Mauritian native orchid flora. This suggests that several species
might have been driven extinct before they had a chance of being discovered, while
the 95% or so of the island’s native habitats were being destroyed. New additions of
native species which were already known to exist elsewhere but recorded or con-
firmed for the first time on Mauritius include the diminutive aphyllous
Taeniophyllum coxii [50] and the fairly large – by Mauritius standard – epiphytic
Jumellea exilis and J. rossi [51]. In addition, four new species were described
including two that are also shared with La Réunion, namely, the epiphytic Poly-
stachya jubaultii [52] and Bulbophyllum mascarenense [27], and two that are
endemic to Mauritius, namely, Angraecum jeannineanum [53] and A. baiderae
[25]. Various taxa (genera or sections) of the orchid flora of Mauritius are also
currently undergoing reviews in the context of the updating of the Flora of the
Mascarenes [25, 54].
2.2 Diversity and Endemism
To date, 91 native species of orchids belonging to 30 genera have been recorded in

Mauritius. The most diverse genera are the predominantly epiphytic Angraecum
(18 species) and Bulbophyllum (17 species) and the terrestrial Cynorkis (9 species)
(Fig. 1). The flora is dominated by species that are endemic to the biodiversity
hotspot region (80%), followed by those endemic to the Mascarene archipelago
(41%) (Fig. 2), with a rate of endemism to the island of only 10%, which is relatively
low compared to other families of native angiosperms (up to 100% in families like
the Ebenaceae, or nearly so for Pandanaceae). About 67% of the island’s orchid flora
had been discovered within the first 40 years of the 250 or so years of botanical
history of Mauritius orchids, with some more (ca. 12% of today’s known species)
collected later by Bojer and Louis Hyacinthe Boivin (https://plants.jstor.org/stable/
10.5555/al.ap.person.bm000000840) over a period of 30 years (1821–1851) (Fig. 3).
After a hiatus of more than 70 years, interest in orchids restarted with the arrival in
1924 of the British botanist Reginald E. Vaughan (1895–1987). Vaughan started
botanizing as soon as he established himself on the island (sadly part of his early
collections were lost in a fire in 1937), and a decade later he re-organized existing
herbarium collections, which led to the creation of a centralized national herbarium
(The Mauritius Herbarium). He also initiated the ongoing project of updating the
account of the Mascarene flora [56]. Vaughan, together with his assistant (and
later the first curator of The Mauritius Herbarium, Joseph Guého [57], and the
scientific director of the Flore des Mascareignes, Jean Bosser (1922–2013;
Fig. 1 Number of species and their respective percentage within each native genus of native
orchids recorded for Mauritius. Only genera with three or more species are listed by name, others
being lumped
Fig. 2 Number of species and respective percentages of species of orchids present on Mauritius
that are endemic to the island, to the Mascarene archipelago and the Western Indian Islands hotspot.
Figure made with GeoMapApp (www.geomapapp.org [55])
https://en.wikipedia.org/wiki/Jean_Marie_Bosser), collected an additional 15% of

the known species. David Roberts [58], while doing his PhD on the orchids of the
Mascarenes, added some new records (Fig. 3).
Although the collection and description of the orchid flora of the Mascarenes
started relatively early, several problems have been delaying a greater understanding
of its diversity. For example, some closely related species had previously been
misidentified (e.g., Jumellea fragrans and J. rossii, see [51, 59] (but see also [60]),
or not recognized as different entities (e.g., Angraecum cadetii and A. jeannineanum
[53]; Bulbophyllum nutans and B. mascarenense [27]). Also, orchid specimens can
easily deteriorate, and many type specimens and other collections were lost, espe-
cially those of Thouars, Commerson, Boivin, and Bojer. Finally, a number of species
were described from different islands of the region, but only more recently, with
better available tools (digitization and imaging of collections, and larger sampling of
specimens on phylogenetic studies), many of them are being synonymized and being
recognized to have larger distribution than previously thought (e.g., [25, 51, 54],
decreasing the so-called “taxonomic inflation” as the group gets better studied and
understood [61, 62].
2.3 Types and Distribution
Like is often the case in the wet tropics [63, 64], most orchids of Mauritius are
chiefly epiphytic (66%) (e.g., most species of the genera Angraecum and
Fig. 3 Cumulative number of first collection of orchid specimens of species native to Mauritius by
date (in periods of 20 years). Data is based on available herbarium records, or date of publication of
the basionym
Fig. 4 Habit of Mauritius native orchids by number of species, and their respective percentages
Bulbophyllum), the rest being ground orchids (Fig. 4), which comprise either
perennial species (12 species, e.g., Calanthe and Phaius) or seasonally emerging
species (19 species, 61% of the ground orchids, e.g., Nervilia and Disperis). A
number of these typically epiphytic species are, however, often also found growing
on rock as lithophytes, good examples of which may be found on the cliff face by the
south access path towards the Le Pouce Mountain Nature Reserve (Angraecum
pectinatum), along Montagne Longue crestline (Polystachya concreta) or on the
southern steep cliff of Trois Mamelles mountain’s highest peak (Bulbophyllum sp.).
Similarly, some typically ground orchids are often encountered growing in moss
patches on trees in very wet regions, like some Cynorkis and Benthamia. No
saprophytic orchid species, those depending on root mycorrhizal fungi for uptake
of carbon [65], are known from Mauritius like occurs on nearby island of Réunion
(Gastrodia similis [66]) or Madagascar. There are two aphyllous species recorded
from the island, namely, the predominantly moist to dry forest species Microcoelia
aphylla, and the more recently discovered Taeniophyllum coxii, which has a more
restricted distribution in parts of the Black River Gorges National Park [50].
A very marked pattern in Mauritius is the fact that native orchids are almost
exclusively confined to native vegetation or their remnants, where, in the case of
epiphytic species, they grow predominantly on a variety of native trees. Although no
quantitative studies of preferred phorophytes have been done, observations suggest
that only one species of native epiphyte is found nearly all the time on a single tree
species (the African orchid Angraecopsis parviflora on the Mascarene endemic tree
Nuxia verticillata). In very rare cases, native orchid species may be found growing
outside native habitats. An example is the native Angraecum calceolus observed
growing on planted roadside Jacaranda (Jacaranda mimosifolia) or Aeranthes
arachnites growing on planted camphor (Cinnamomum camphora) or litchi (Litchi
chinensis) trees. These, and other species, may also be found growing naturally in
certain private gardens, for example, in Curepipe, but then they would most often be
found on native trees or native tree clumps remnants of the original native forest, or
on trees planted in their close vicinity.
Of the orchid species with known intra-island distribution, few on Mauritius can
grow from sea level (or close to it, at around 100 m), to the island’s highest elevation
(N ¼ 5, 6%). Similarly, few species are found to be restricted to the upper limits of
the island’s elevation (> 700 m; N ¼ 2, 2.4%). A majority of species (N ¼ 50, 61%)
occur at least at elevations of 300–600 m, and an even greater majority grow
between 600 m elevation to the island’s culminating point of 828 m (N ¼ 70,
85%) (Fig. 5). Although one could suspect that such patterns could be artifactual
because most lowland native vegetation has been destroyed early after human
colonization [37], and that specimens with more precisely recorded locality are
those that were more recently collected, it has also been shown that orchid diversity
(including many of the same species as in Mauritius) drops with elevation on the
sister island of Réunion [67], where much more intact elevational gradient of native
vegetation subsists and that both elevation and climate do influence distribution of
orchids and the epiphytic species [68–70], among other factors (e.g., area in case of
islands). In general, on Mauritius, elevation above 300 m would be mostly within
transitional and wet forests.
Orchid species richness on Mauritius is strongly related with elevation, with sites
at 600 m elevation or more harboring higher species richness despite their smaller
area and greater isolation. Because of risks associated with such information, we
opted not to disclose the name of species and locations (for more on this see
“Threats” below). The most species-rich areas include a variety of habitat types
with low to high vegetation canopy. Also, they tend to be areas that are least invaded
Fig. 5 Number of species of native orchids per bracket of elevation range, in terms of both
minimum and maximum known elevation per species
by alien plant species and/or receiving conservation management, sometimes since

50 years. Of the extant species, the majority have at least one locality within legally
protected areas – either a Nature Reserve or National Park, which also represent a
large part of the surviving habitat remnants. However, the distribution of several
species does not coincide with protected areas. Worryingly, a few species or
populations appear to have gone extinct in the last 50–60 years, even within
protected areas on the island.
3 Ecology
Although orchid seeds tend to be among the smallest and lightest of flowering plants
[71], long-distance dispersal might be limited on islands [72], with climatic and
environment factors being more important diversification factors [64, 68–70, 72]. On
Mauritius, the endemism of orchids is relatively low at island scale (~10%), but
higher at the level of the Western Indian Ocean Islands hotspot (~80%, Fig. 2). In
fact, at this scale, most species of this region are endemic [26], a pattern resulting
from factors operating through time and space, including island hopping facilitated
by sea level drops during the Pleistocene [15], as well as regional forcing [13].
Based mostly on literature (e.g., [67], the majority of orchid of Mauritius are
pollinator-dependent (N ¼ 52, 57%), with just over a third (N ¼ 32, 35%) being self-
pollinated, although a few have unknown pollination systems (N ¼ 7, 8%) as these
species are known only from drawings or few old specimens. Nevertheless, the
percentage of auto-pollinating species on Mauritius is higher than on Réunion at

similar elevation (around 23%) [67]. Among the pollinator-dependent species, one
section (Hadrangis) of the genus Angraecum, which is endemic to the Mascarenes,
evolved unique pollination patterns. Indeed, the two Réunion endemic species are
bird pollinated [73, 74], while a species shared with Mauritius is pollinated on
Réunion by an endemic nocturnal orthopteran [75]. On Mauritius, observations on
this same orchid species (and on the sister Mauritius endemic species) were done but
no pollinator was seen during the period of the study [53].
4 Threats and Conservation
4.1 Deforestation
The most immediate and damaging impact of human activities on biodiversity is

habitat destruction, something that happened in Mauritius at one of the fastest
rates recorded worldwide [37, 41]. Indeed Mauritius was one of the last places
on Earth to be colonized by humans, when Dutch settlers established on the
island in 1638 and, by the 1990s, its native habitat extent had been reduced
20 folds [40, 76]. The island of Mauritius now has one of the lowest extents of
surviving original native cover of any country in the tropics. Native habitat
destruction has continued beyond the 1990s, although on a much smaller scale
given the small fragments of native habitats that survive as a confetti of remnants
over parts of the island [77]. Early twenty-first-century deforestation occurred,
for example, in what was the best surviving remnant of coastal native forest at
Roches Noires on the east coast or in a Nature Reserve harboring a hardwood
forest that was supposed to be strictly protected [41], and which contain a fair
diversity of orchids. Other small-scale destruction of native habitat also con-
tinues on mountain reserves, mostly for ranching of alien deer (for hunting and
meat) and for ecotourism facilities, along river reserves (mainly for cultivation),
and for road enlargement, as happened at Le Pétrin, one of the hotspots of
orchids inside the Black River Gorges National Park and Chamarel in 2021.
An often-overlooked threat for epiphytic and terrestrial orchids comes from
thinning of trees within native forests for illicit cannabis plantations. The last
estimate puts native habitats as covering 4.4% of Mauritius [39].
The rapid loss of habitat cover on Mauritius corroborates relatively well with the
last records or disappearance of the 20 species (22%) of Mauritian native orchids that
are currently considered to have been driven extinct by human activities, as almost
all of them (N ¼ 17) were last collected between 1769 and 1888. However, it must
also be kept in mind that a swath of other threats, although relatively less severe, as
described below, has also been operating alongside deforestation, even within
habitats that have not been destroyed. Some of these threats, like the negative
impacts of alien long-tailed macaques (Macaca fascicularis), likely introduced in
1602 [37], would have started even before humans finally colonized the island (see
below).
4.2 Harvesting
Mauritian native orchids are typically unremarkable relative to showy commer-

cial orchids like those of the Neotropical genus Cattleya or, the mostly southeast
Asian Dendrobium. Nevertheless, harvesting of wild plants still appears to pose
problems, although it has not been quantified. Some species like Angraecum
eburneum or Cryptopus elatus have some degree of horticultural interest, and
indeed the latter is often collected from the wild. Collection is likely to have
contributed to decline of species that have been used as beverage enhancer (e.g.,
J. rossii and J. fragrans used to give fragrance to rum [54]), or as a medicinal
plant (Angraecum mauritianum as stated on specimen MAU 0016681 at The
Mauritius Herbarium). However, these along with some other species of no real
horticultural interest for their few and inconspicuous greenish flowers are still
being collected and sold, for example, in the market of the capital city of Port
Louis (pers. obs., Z Jhumka, pers. comm. 2019). Some plants seen on sale were
clearly recently uprooted from their host and sold as such without potting,
possibly benefiting from the buyers’ ignorance that the plants would in most
likelihood not survive, and that if they do, any flower would be most
unremarkably inconspicuous. Rarity increases the willingness for keeping sam-
ples of species in personal collections [78, 79], and this might have been the
reason why the largest individual of Taeniophyllum coxii, which was growing by
a forest track inside the Black River Gorges National Park and illustrated in fruit
in 2017 [80], ended up being peeled off its host tree as witnessed by the sharp
blade marks left behind. This species, recently recorded in the country, is
extremely rare on Mauritius, and it would be considered locally as Critically
Endangered [50], according to the Red List criteria of the International Union for
the Conservation of Nature [81].
Harvesting of other native species also poses a more indirect threat to indig-
enous orchids. There is abundant evidence in wet forests of Mauritius like at
Macchabé and Brise Fer in the Black River Gorges National Park that illegal
harvesting of the larger specimens of the native tree fern Alsophila excelsa was
common in the past [82]. The wide base of the stem of these tree ferns, apart
from having some medicinal uses, was commonly harvested to make flower pots,
and their fibrous roots often used as a growth medium for orchids since the time
Mauritius was a French colony in the eighteenth century [83]. Many cut stumps
can be observed especially in the vicinity of access roads, largely because the
fern bases are often massive and doubtless hard to carry over longer distances. In
the wild, A. excelsa is an important host to many orchid species to which its
harvest is therefore harmful. Furthermore, A. excelsa provides the best microsites
of germination to a few native tree species including Nuxia verticillata, a large,
long-lived tree often host to many epiphytic orchid species including
Angraecopsis parviflora. The illegal harvesting of tree ferns thus triggers both
a direct and an indirect loss of highly favorable hosts to many native epiphytic
orchids.
4.3 Alien Plants
The small areas of native vegetation that escaped deforestation so far [39], and which
constitute the stronghold or sole habitat of all native orchids, are today for the most
part invaded by many alien plant species either in the understory (e.g., the strawberry
guava, Psidium cattleyanum in tall forests) or in the canopy (e.g., the cinnamon
Cinnamomum verum, or the emergent travellers palm Ravenala madagascariensis),
or both [42]. Alien plants exert intense competition for light on native orchids [84],
which greatly reduces orchid diversity (expressed as both species richness and
abundance) by virtue of their extreme density [42] (but see more below). Forest
areas where alien plants have been controlled for about a decade as part of conser-
vation management [85] harbor a much higher diversity of orchids than adjacent
alien invaded forests [84]. Monitoring of permanent plots indicated that the epi-
phytic orchid community progressively recovers within areas cleared of alien plants
while simultaneously declining in adjacent areas invaded by alien plants
(unpublished data). Control of invasive alien plants on Mauritius is therefore
extremely important for the conservation of native epiphytic orchids. However, for
each hectare of forest undergoing such ecological restoration, there are about
20 hectares where invasion continues to progress. Consequently, there exists no
reasonable doubt today that native epiphytic orchids are declining as a whole over
the island particularly that it has been shown that the invasion by alien plants has not
stabilized but is in fact worsening with time [42].
Alien woody weeds also harm native orchids in indirect ways. Alien-invaded
native forests are losing native host trees at an alarming rate. For instance, native
trees with stem of at least 10 cm diameter at breast height (DBH) have diminished by
half within 68 years from what were some of the best preserved native forests of
Mauritius in the 1930s [86] despite their presence in protected areas [87]. A decline
among smaller diameter woody native plants (>2.5 to <10 cm DBH) is also
apparent when comparing with studies done in the 1980s [87, 88]. A closer tree by
tree monitoring through time between adjacent weeded and nonweeded native
forests showed native tree mortality to be increased by the presence of invasive
alien plants at both community [89] and population levels [90]. Even native trees that
largely overtop the alien plants are dying faster when growing within stands of alien
plants [90], indicating that root interactions alone suffice in increasing mortality of
native trees. This elevated native tree mortality is generating an inexorable loss of
suitable host plants for epiphytic orchids.
Competition for light is not the only direct harm delivered by invasive alien plant
on native orchids. As alien plant invasion progressively worsens through time [42],
native tree densities progressively drop [87], such that a long-term gradual replace-
ment of native by alien trees is ongoing in invaded native forests. One might think
that this would be of little consequence for epiphytic orchids as they need a woody
host (phorophyte) to support themselves and that it would not matter much if that
host is native or alien. But epiphytes are not randomly distributed because conditions
offered by a particular host species matter and, in turn, influence, among other
factors, the microclimatic conditions for the orchid development and availability of
resources (light and water) which vary through time, for example, when host trees
seasonally shed leaves, or if the bark of the host has the right rugosity for the orchid
roots to grip, or if the orchid is able to develop as the stem or branch where they rest
grows [91, 92]. Psidium cattleyanum is by far the main alien plant invading the most
orchid-rich forests of Mauritius (79% of sampled alien stems [42]). It is an under-
story tree of small diameter that frequently sheds its bark, making it an exceptionally
poor host for virtually all native epiphytic orchids (Fig. 6) although a few species
may still establish and mature on strawberry guava [50]. Several other invasive alien
tree species, for example, C. verum and Ligustrum robustum, also present particu-
larly poor substratum to epiphytic orchids, although not as extreme as
P. cattleyanum. The latter species also creates a very thick root mass at the soil
surface which appears to substantially change soil texture and porosity away from
what is conducive for ground orchids. Indeed, only after this weed had been removed
from a patch of forest in the Black River Gorges National Park, was a Nervilia
species that was hitherto thought extinct on Mauritius, relocated as a strongly
regenerating small population [93].
The invasion by alien plants has also been shown to be reducing the extent of
flowering of native woody plants [90, 94] as well as the production of fruits [90, 94,
95] hence of seeds too, resulting in a much lowered native plant regeneration in
forests invaded by alien plants [93]. Invasive plants also reduce growth rate of native
trees [90], which in turn is expected to result in smaller trees with fewer opportunities
for epiphytic orchids to grow on, as well as feeding back into reduced seed
Fig. 6 (a) Stems of native tree species often form suitable substratum for a variety of plants,
including lichens, mosses, ferns, and orchids compared to (b) the smooth stems of the invasive alien
strawberry guava, Psidium cattleyanum whose outer surface is frequently shed. (Photos: F. B.
Vincent Florens)
production and regeneration because smaller trees typically carry fewer fruits.
Invasion also decreases the density of tree ferns [82] and litter basket ferns [96],
suggesting that establishment of new orchids could be reduced if mycorrhizal fungi
availability is harmed by lower phorophyte diversity or ideal microclimate produced
by mosses and ferns that enable this symbiotic relation (fungi-orchid) to occur.
Finally, there is evidence also that invasive alien plants greatly harm some insect
groups [97] including the native butterfly community [98]. Butterflies are often used
as an indicator group of insect diversity, itself important for native plant regeneration
as pollinators. In conclusion, through several direct and indirect mechanisms, inva-
sive alien plants are harmful to the establishment, growth, and reproduction of native
trees, which themselves are important as hosts for a large number of epiphytic
orchids of Mauritius.
4.4 Alien Animals
Invasive alien animals like the long-tailed macaque (Macaca fascicularis) whose
introduction predates human colonization also threaten orchids. They chew on the
meristem of predominantly the larger terrestrial orchids of the genera Calanthe and
Phaius, killing the individuals affected (Fig. 7). Monkeys also chew on meristems of
Fig. 7 (a) The invasive alien long-tailed macaque (Macaca fascicularis) in the canopy of native
vegetation in the highlands of Mauritius, (b) disarticulated leaves (about 30 cm long) of a native
terrestrial orchid (Calanthe sp.) that has been attacked by a long-tailed macaque, (c) details of leaves
in b showing chewed bases of the leaves. The plant’s meristem was completely destroyed. (Photos:
F. B. Vincent Florens, February 2021)
epiphytic species of Jumellea [51], which might have significant parts of the plant
removed. Indeed, four on six (67%) Jumellea species shared between Mauritius and
the nearby Réunion, which is free of alien macaques, are more threatened with
extinction on Mauritius than on the latter island [54]. But more interestingly, the
larger colonies of Mauritian Jumellea are clearly today primarily confined to places
that are inaccessible or most difficult of access to macaques, like cliffs or high up on
isolated large trees with straight boles that are difficult to climb [51]. Another
example of the contrast between macaque-infested Mauritius and macaque-free
Réunion is illustrated by Bulbophyllum longiflorum, a species known to be chewed
by macaques, and down to about seven known individuals at two sites on Mauritius,
but very common in drier forests on Reunion [99]. Also noteworthy is that the largest
Mascarene orchid, Angraecum palmiforme (up to 1 or 1.2 m tall), was last recorded
on Mauritius in the early 1800s [49], and although rare [100], it has been seen on
Réunion less than three decades ago [101]. Similarly, A. eburneum now occurs in the
wild in Mauritius in very small numbers and almost exclusively in inaccessible steep
cliffs, whereas on Réunion it often grows in highly accessible spots [99, 101].
Macaques also pose substantial indirect threats by hindering the growth and
reproduction of host trees of epiphytic orchids. They do so by commonly breaking
and chewing on many young shoots of saplings to adults of many native trees and
reducing the vitality of the plants [89, 102], thereby incidentally destroying flower
buds, flowers, or unripe fruits [90, 95]. Macaques also directly attack large quantities
of fruits before they have time to ripen their seeds. For example, 95% of the fruits of
the canopy endemic Sideroxylon grandiflorum are destroyed by macaques before the
seeds within have time to ripen contributing to the tree to virtually stop regenerating
in the wild [90]. Similar types of damages are known to happen to various other large
native trees and large-fruited species of the genus Mimusops (Sapotaceae) and
Diospyros (Ebenaceae) (pers. obs.; [95, 102], which collectively form a sizeable
proportion of trees in Mauritian native moist to wet forests [103]. Some smaller
understorey trees are also impacted in a similar fashion, one example being Eugenia
alletiana (Myrtaceae) [104].
Native orchids also face several threats from other invasive alien species in
addition to the ones described above. For example, the invasive feral pigs (Sus
scrofa) destroy terrestrial orchids by uprooting and smothering them, especially
when rooting, which apart from reducing plant cover is known to also change soil
physical structure and chemical composition [105]. Damage by feral pigs is also
influenced by their density. In Australia, higher density of feral pigs is known to
increase damage to ground orchids [106]. On Mauritius, pigs seasonally converge to
the upland wet forests during the fruiting of the alien strawberry guava [89] to eat the
abundant fallen guavas, indicating that this alien plant species is an important
resource for the pig. Some ground orchid species might be more at risk of negative
effects from feral pigs, such as species of Habenaria (a genus counting an endemic
extinct species) which tend to grow in swampy and very wet soils, which are
particularly impacted by feral pigs, especially for wallowing.
Several other alien animals also negatively affect orchids. Terrestrial orchids,
especially those with soft leaves and succulent stems (e.g., Platylepis), have their
stem grazed and chopped by the alien invasive Giant African snails (Lissachatina
fulica and Achatina immaculata). The very rare native orchid, Nervilia, which
seasonally forms a single leaf appearing above ground for 4–5 months during a
year, is also attacked by these same mollusks, but also by alien slugs (pers. obs.). The
invasive java deer (Rusa timorensis), introduced in 1608 [37], impacts orchids by
grazing, soil compacting, direct removal of epiphytes during antler rubbing, which
itself can kill even large trees by triggering wood rot that causes the tree to eventually
snap, reducing the number of available phorophytes hosts for epiphytic orchids.
Invasive alien rats (Rattus rattus and R. norvegicus) can chew on orchids [27], and
the alien hare (Lepus nigricollis) may graze on ground orchids in more open
vegetation.
4.5 Indirect Effects
All large frugivores of Mauritius (>1 kg) were quickly driven extinct following
human colonization [37]. Such losses are known to impact the dynamics and
composition of forests [107, 108]. Today, the role of maintaining seed dispersal,
and consequently, fostering regeneration of adequate phorophyte for orchids,
depends almost exclusively, especially for large seeded-species, on the Mauritius
flying fox (Pteropus niger), a Mascarenes endemic and Endangered species
[109]. This single species has an ecological keystone role of seeds dissemination
on Mauritius as it is known to feed on fruits of, on average, about 53% of individual
woody plants in various Mauritian forests, and particularly those of the larger trees
[110], which are known to have important physical ecosystem engineer roles that
enable the survival of many other forest species [111], including orchids. Yet,
Mauritian authorities have long been planning to cull flying foxes [112, 113] and
since 2015, implemented multiple mass-culling campaigns on spurious justifications
[114–116]. These recurrent mass-culling campaigns are unnecessarily heightening
the risk of extinction of the flying fox [109] and must already be weakening its
ecological keystone role given the way such roles are played out by this group of
fruit bats [117]. Indeed, the detrimental effects on regeneration of native forest trees
that the weakening and losing of the seed dissemination role of P. niger bring are
emerging and being confirmed [118]. Loss of frugivores, including P. niger, is
known to have strong detrimental effects on forest resilience [119].
4.6 Other Threats
A rather wide variety of unusual threats to native orchids have also come to light in
Mauritius often unexpectedly originating from certain conservation managers them-
selves. Perhaps the most direct one was the targeted removal of a ground orchid
inside conservation management areas as part of the program of maintenance
weeding meant to control invasive alien plants. A wide diversity of native plants
recover by regenerating much better [93] following the initial weeding of the
principally woody invasive alien plants [42] that invade native forests, when con-
servation management areas are created [85]. However, alien plants tend to reinvade
the weeded areas, necessitating their regular maintenance weeding. One of the rare
ground orchids of Mauritius, a Platylepis, was observed to benefit much from alien
plants weeding only to then be mistakenly taken for an alien weed itself by managers
during maintenance weeding and weeded (Fig. 8). Many years later, the population
of the species appears to have still not yet recovered from this unfortunate event.
This situation underscores the importance of conservation managers to develop
appropriate levels of identification skills and of implementing effective supervision
on the field, so that similar events do not recur on this or other species, the more so
that it is not an isolated event because several other native species are often weeded
during maintenance weeding, including woody plants like Cnestis glabra,
Mussaenda arcuata, or Scutia myrtina themselves serving as phorophytes for
orchids.
Another activity that is damaging to native orchids within protected areas, and
which is more recurrent this time, concerns indiscriminate maintenance weeding.
Indeed, the use of hoes to remove alien grasses also destroys many species of ground
orchids growing along the alien plants being removed (Fig. 8). A much better
solution would be to remove the alien plants by hand alongside putting a definitive
stop to the ongoing practice of cutting native pioneer trees [85] or lopping their
Fig. 8 (a) The native ground orchid Platylepis occulta was mistakenly weeded by the hundreds
during maintenance weeding operations within a protected area receiving conservation manage-
ment. (b) All individuals from small juveniles (on the right) to large adults (on the left) that were
spotted ended up being uprooted. (c) Maintenance weeding of alien plants being carried out using
hoes, an indiscriminate technique that destroys many native plants including ground orchids. (d) A
pile of freshly weeded alien plants removed with hoes and containing native seedlings, ferns, and
ground orchids. (Photos: F. B. Vincent Florens)
branches, because these activities not only, at a cost, set back ecological restoration,
but also they themselves increase light at ground level which boosts the growth of
the same alien grasses which the managers are trying to control with hoes inside
conservation management areas.
Brush cutting is also a common practice along forest tracks within protected
areas. Many orchid species grow better at track edges because the high invasion
levels of alien plants further inside the vegetation [42] make it too dark for many
orchids to grow. While brush cutting has been observed to damage some perennial
orchids growing on edges of tracks, such as Angraecum mauritianum or
A. ramosum, along with many other native plants, the activity appears to be much
more damaging to seasonal ground orchids like Disperis or Cynorkis because the
brush cutting is often carried out precisely when these orchids are growing leaves,
flowers, and fruits above ground. Brush cutting edges would itself become largely
redundant if managers allowed overtopping vegetation to link up above the forest
tracks thereby shading them, but the opposite management of widening tracks and
lopping branches is done instead, which create more influx of light which favors
thicker alien plants growth, which is then dealt with by brush cutting. If ever brush
cutting is truly necessary, it should be done in seasons where the ground orchids are
dormant underground, or else avoiding areas where colonies are growing, flowering,
and fruiting.
A new threat has been noted recently within the country’s main National Park
which is damaging ground orchids particularly the smaller seasonal species that
grow on rock on the forest floor, such as Disperis spp. and Cynorkis spp. During
forest track maintenance works, tons of rocks were gathered from the adjacent
protected forest floor and used for leveling the tracks. Several species of seasonal
orchid grow on these rocks, which are also colonized by various bryophytes and
ferns. They are all destroyed when the extracted rocks are used on the tracks. Many
orchids, because they are seasonal, are not visible on these rocks for the most part of
the year, as they occur as dormant tubers in the rocks’ cracks and depressions or
within the moss cover. They only do sprout into view in the growing season. These
forest floor rocks form an integral part of the forest and are important for native
biodiversity both for plants growing on top of them and for animals hiding from
predators below them and should therefore not be harvested for building purposes,
particularly within protected areas.
Another unusual threat to orchids, which is this time more persistent and long-
lasting, concerns the practice of conservation managers cutting native pioneer trees
(Harungana madagascariensis) that grow predominantly in areas undergoing eco-
system restoration after alien plant weeding [85]. Although this practice endured for
many years and was more widespread before subsiding in around 2013, the native
pioneer trees are still occasionally cut by conservation managers in areas undergoing
restoration as happened in the Conservation Management Area of Pétrin in 2019.
Lopping of the branches of this pioneer tree is more commonly practiced in recent
years, but still represents inappropriate management because it involves investing
resources in an activity that reduces native biomass and increases re-infestation rates
of alien plants due to increased influx of light [85]. As far as orchids are concerned,
the native pioneer tree that is cut or pruned makes for a good phorophyte for
epiphytic species [84], particularly also because it typically grows in forest gaps,
where other phorophytes are rare [120].
Many of the unusual threats highlighted above, could be reduced or avoided if
decision-makers, managers and technicians working in conservation were better
trained in ecology and conservation, in particular of tropical native forest biodiver-
sity, as this would have led to more informed evidence-based management, com-
pared to the current situation, where many staff, for example of the authorities, are
primarily trained in agriculture-related fields (https://civilservice.govmu.org/Pages/
SOS/SOS/agro.pdf). With the help of foreign institutions and scientists, Mauritius
has nonetheless achieved several conservation successes [41], some of which of
worldwide acclaim [121]. Sustaining such successes rests on sufficient appropriately
trained conservation professionals, an increasing number of whom are now
Mauritians compared to three to four decades ago. Worryingly, however, there has
recently been a recrudescence of obstacles to capacity building in ecology and
conservation in Mauritius. For instance, the country’s main university, and only
one locally producing graduates in biology, many of whom with a particular interest
in conservation [122], has decided in 2015 to replace its biology undergraduate
program with an equivalent that effectively reduced the ecology-related component
three-folds, removing all of it from the final year of studies and removing conser-
vation biology altogether from the program for at least 4 years (V.F. pers. obs),
despite this field counting among the research strengths of the university. The new
program contains one of the lowest ecology/conservation components of comparable
programs worldwide and incidentally, it coincided with a severe drop in student
intake that culminated with just four students graduating in 2019, a ten-fold drop
over the long-term average.
To complicate matters, several other barriers to local ecology and conservation
training and research have also emerged, including a policy by the authorities to
restrict such research in the National Parks to office hours on week days, and
selectively applied to the country’s main university [122]. Another restriction of
the policy, similarly selectively applied, included payments being associated with
permission to carry out ecology/conservation research in the National Parks
[122]. The policy effectively selectively blocked university students from accessing
field stations of government for their research, a situation that has not yet returned to
normal, although the policy was shelved after several years. The policy was also
followed by an attempt of academic gagging [123], suggesting that conservation
authorities sometimes have pursuits other than promoting research into conservation.
Another barrier concerns the delays for obtaining permits from the authorities for
carrying out research in ecology and conservation, which changed from the officially
stated 3 weeks to at least 3 months, often more, effectively crippling many research
projects and capacity building. Examples include a foreign student in Mauritius for
about 5 months to study native orchids conservation and whose research permission
was granted a few days before the end of her stay, and a PhD candidate also seeking
to study orchid ecology and conservation, but who could not because his request for
research permission was ignored.
5 Conclusion
Even in a small island with a rich and long-lasting heritage of natural history studies
and very little native habitat surviving, recent dedicated surveys and research are still
revealing new species of orchids and improving the understanding of orchid taxon-
omy, distribution, ecology, and conservation. It could be expected that further
research would reveal more species and useful information for conservation man-
agers including relocation of species currently believed extinct on the island, such as
the ground orchid Nervilia that was relocated after a lapse of 239 years after it was
last collected [93]. The orchids comprise the largest family of native flowering plants
in Mauritius, and one with the highest rate of extinction so far and on those bases
should be considered the current top priority for such surveys.
Furthermore, given the numerous threats besetting the biodiversity of Mauritius
and in particular its native orchids, there is an urgent need to greatly strengthen
ecological and conservation research and capacity building to rise to the challenges
of improving evidence-based management to appropriate standards, to give Mauri-
tius a chance to not just slow but rather stem and reverse the current trend of loss of
native biodiversity, particularly in the context of the worsening and longer-lasting
threats imposed by anthropogenic climate change. Local institutions involved in
conservation should seek to strengthen their collaboration and work in a more
complementary manner than at present as well as build stronger links with interna-
tional institutions and scientists to encourage and attract more research being done
locally. While increasing the training and capacity of many existing conservation
professional in fields like taxonomy, ecology, or conservation is urgent, there is also
a need for creating a much larger number of stable and sufficiently valorized posts of
conservation professionals with clear career paths so as to attract, retain and motivate
highly trained and able professionals in the field.
Training and capacity building alone would not suffice to adequately deal with
the daunting challenges of the current biodiversity loss in Mauritius. There is also an
urgent need to address the numerous and widespread problems posed by invasive
alien species at a scale that is ecologically meaningful to ensure long-term conser-
vation. Valorizing biodiversity to the appropriate extent for the diversity of services
that it provides, including for the water cycle, carbon sequestration etc. would help
mainstreaming conservation action and stimulate ecosystem restoration and refores-
tation programs to the scale needed to rise to the challenge. More collaboration,
dialogue and trust among local stakeholders and actors is essential in the broader
interest of the country, as well as, identifying and removing barriers that are currently
impeding or distracting from true positive outcomes. The best way by far to conserve
native orchids would be to do so in their restored natural habitats where sufficiently
large enough populations can subsist that will make extinction risks negligible, and
where the orchids ecological function will continue, thereby sustaining the biodi-
versity associated with the various species like pollinators, herbivores etc.
Apart from serving the interests of Mauritius in better conserving its threatened
native orchid biodiversity and the habitats and ecological function that are crucial to
it, with the appropriate actions and levels of involvement, Mauritius is poised to play
an important role for the world beyond as a laboratory of sorts to better understand
threats to biodiversity and experiment corresponding solutions, because the island
combines many attributes, such as high human population densities, high degree of
habitat destruction and fragmentation or high rates of alien species invasion, that
await many other countries with rich biodiversity. If Mauritius is able to take the
right actions to turn things around and become an example of successful orchid
conservation, like it is known for globally with conservation of endemic birds, the
island could well open a path useful for conserving threatened orchid floras else-
where thereby generate a multiplier effect of its conservation efforts.
Acknowledgment We thank the late Jean Bosser and Thierry Pailler for their insights about
Mauritius orchids over the years and the reviewers for their constructive comments on the
manuscript. The National Parks and Conservation Service and the Forestry Service of the Ministry
of Agro-Industry and Food Security are acknowledged for permission of access and research over
the years. Mary-Ann Stanley and Owen L. Griffiths of Bioculture (Mauritius) Ltd. and Ebony Forest
Reserve Chamarel Ltd. gave permission to access and carry out research at Mt. Camizard and
Chamarel and provided substantial logistical support in terms of transport and on-site camping
facilities for some of our surveys.
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Diversity of Orchids from Continental
Sub-Saharan Africa 4
Adama Bakayoko, Noufou Doudjo Ouattara,
Akoua Clémentine Yao, Djah François Malan,
Danho Fursy-Rodelec Neuba, Bi Fézan Honora Tra, and
Tanoh Hilaire Kouakou
Contents
1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 136
2 Method of Data Collection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 137
3 Brief Presentation of Orchidaceae . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 137
3.1 Distribution of Orchids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 137
3.2 Botanical Description and Systematic . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 137
4 The Orchids of Sub-Saharan Africa . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 138
4.1 Richness According to Geographic Areas . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 138
4.2 Endemism of Orchidaceae Species in the Sub-Saharan Countries . . . . . . . . . . . . . . . . . . . 139
5 Uses of Orchidaceae Species in African Countries . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 145
6 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 150
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 151
Abstract
Africa is a vast continent containing many types of ecological environments. It
harbors the second largest forest reserve in the world but its flora is not well
known for lack of financial means to carry out botanical prospecting studies.
While many families have been well studied, others like the Orchidaceae are little
known. This study is intended to contribute to the knowledge of this family
through its use and distribution in continental sub-Saharan Africa.
The overall analysis of the orchid flora was done based on the four major
regions except North Africa and South Africa (West Africa, Central Africa,
Southern Africa, and East Africa). This study is based on the investigation of
the literature. We have consulted previous published studies on orchids, floras,
A. Bakayoko (*) · N. D. Ouattara · A. C. Yao

UFR des Sciences de la Nature (SN), Université NANGUI ABROGOUA, Abidjan, Ivory Coast
D. F. Malan · D. F.-R. Neuba · B. F. H. Tra · T. H. Kouakou
UFR des Sciences de la Nature (SN), Université NANGUI ABROGOUA, Abidjan, Ivory Coast

136 A. Bakayoko et al.
and distribution maps of the targeted areas. We were able to draw up a list of 1373
species belonging to 88 genera. The results show that of the four phytogeographic
zones, Central Africa is the richest with 708 species, followed by Southern Africa
and East Africa with 637 and 583 species, respectively. West Africa, with
413 species, is the least rich area. Several uses have been listed. Mostly, orchids
are using in pharmacopoeia, in feeding and as ornamental plants. We were also
able to establish endemism in several countries (e.g., Tanzania, Zimbabwe, the
Democratic Republic of Congo, Ethiopia, Cameroon, Mozambique, Zambia,
Malawi, Rwanda, Angola, Kenya, Nigeria, Gabon, Central African Republic,
Uganda, and Burundi).
Keywords
Diversity · Distribution · Orchids · Sub-Saharan Africa · Endemic
1 Introduction
Third largest continent in the world in terms of area after Asia and America, African
continent has a great diversity of ecological environments ranging from wet ever-
green forests to vast desert areas [1]. With this multitude of vegetation, the African
continent is rich in more than 62,000 species of flowering plants but still remains
largely unknown. Compared to other continents, the diversity is relatively low. Some
studies estimate this diversity probably at 25% of the world’s species. Several
reasons could explain this ignorance. The fragmentation of Africa due to the history
of colonization is believed to have encouraged sectarianism. The large surface area
of the continent and the lack of financial support to carry out botanical inventories is
also a cause of the ignorance of the flora of the African continent. Most of the
country or regional floras of Africa are old.
African continents contain the second largest forest reserve in the world. While
many botanical families have been well studied, others are very little known. This is
the case of Orchidaceae family which has an estimated global richness of 25,000
species [2]. This family has good representation in tropical and equatorial regions of
the world but in Africa it is little studied. The richness of the genera of the
Orchidaceae is estimated at 700 accord [3]. This family presents the greatest
diversity and number of species in flowering plants [4]. In Africa, the richness of
Orchidaceae is estimated at 1500 species belonging to l 87 genera [5, 6].
The Orchidaceae family is important both in botanical terms for its richness, and
economically through the use of several species as ornamental plants [7]. Many of
these species are sold internationally as an ornamental. This international trade of the
orchids species has an impact on the abundance of the species, in their habitats.
Some species became rare, and others are on the way of extinction, while others have
already disappeared. The main reason for their disappearance is the degradation of
their habitat by human activities. Therefore, orchids represent an excellent indicator
of the quality of biotopes in which they are found [8]. This work, which aims to
4 Diversity of Orchids from Continental Sub-Saharan Africa 137
contribute to the knowledge of the floristic diversity of the Orchidaceae family, will
focus on species from sub-Saharan Africa, except South Africa.
2 Method of Data Collection
This work on the orchids of sub-Saharan Africa was based on the use of literature
and on all the flora that we had at our disposal. We have particularly used the books
presenting the distribution of Orchidaceae in the countries of sub-Saharan Africa
[9, 10]. It should be noted that the work of these authors is based on specimens
deposited in different herbaria around the world. We analyzed the flora of orchids
according to the four major regions except North Africa and South Africa. Thus the
areas considered in this work are: West Africa, Central Africa, Southern Africa (not
the country), and East Africa.
3 Brief Presentation of Orchidaceae
3.1 Distribution of Orchids
The Orchidaceae are a cosmopolitan family with a very large ecological environment.
Orchids are much more present in warm regions [11]. Herbaceous and perennial, the
species of this family have colonized practically all habitats. They can be found in the
coastal areas as well as at higher altitudes. However, they are absent from extreme
environments such as Antarctica, the sea, the most arid deserts, and the coldest
mountain peaks. Orchidaceae are more common in tropical areas where its flora
represent nearly 10% of tropical flora. According to Ref. [11], they can be terrestrial
plants (tropical, temperate, and boreal environments for geophytes with tubers or
rhizomes), epiphytes (generally in tropical environments), and even lianas as species
of Vanilla genera. In Africa, the Orchidaceae constitute an important part of the
epiphytes of tropical forests. Among the 153 species of epiphytes recorded in the
evergreen forests of West Africa, 101 belong to the Orchidaceae family [12].
3.2 Botanical Description and Systematic
The plants belonging to the Orchidaceae family are terrestrial or epiphytic herbs,
rarely helophytes, never halophytes, aquatic, or parasitic. The terrestrial Orchidaceae
are provided with important underground apparatus, in the form of tubers of stem or
root origin or of rhizomes, on which are born the adventitious roots and the leafy and
flowering aerial stems. The stems of Orchidaceae are simple or branched and erect.
The creeping form is rarely observed.
The strongly adherent roots of orchids allow a good attachment to trees and better
withstand the stresses of aerial life. In some species, the leafless vegetative apparatus
is reduced to a system of green roots, from which the inflorescences arise directly.
In Orchidaceae family, the leaves are simple, alternate, more or less fleshy, with a
sheathing base, parallel to the veins. They are often arranged in two rows, rarely
opposite or in whorls. They are sessile or petiolate and sometimes reduced to scales.
The greatest diversity at the leaf level is observed in terrestrial Orchidaceae, hence
their extensive use as decorative plants.
Epiphytic Orchidaceae have both leathery and succulent, green, or spotted leaves
with usually long, hanging aerial roots, gray or white, covered with a velamen, with a
meristematic apex. This system absorbs the moisture and nutrients necessary for the
life of the plant. These thick ligulate leaves reduce water loss and promote photo-
synthesis. The survival of many epiphytes in environments subject to extreme
conditions is due to the development of particularly active photosynthetic tissues
in all organs (stems, roots, and even flowers).
Many tropical and subtropical orchids, both terrestrial and epiphytic, have
pseudo-bulbs as reserve organs for water and nutrients. Those pseudo-bulbs vary,
depending on the species. We can observe from barely swollen stems to very hard, to
apple-shaped bright green organs, from which the leaves emerge. A plant may have
only one pseudo-bulb, but these may also be gathered in a tight group, or, in some
tropical species, be spaced along a creeping or climbing rhizome. Their size varies
from that of a pinhead to a thick cylinder up to 3 m high. Pseudo-bulbs are always on
the ground or above ground level, but in several groups of terrestrial orchids in
temperate regions, there are similar organs growing on roots in the ground.
The Orchidaceae family is constituted by five subfamilies which are the
Epidendroideae, the Apostasioideae, the Vanilloideae, the Cypripedioideae, and
the Orchidoideae [13]. The classification of African orchids has been a real challenge
for a very long time [14]. Taxonomic confusions are not limited only to the specific
level but also to the generic level. For example, the generic delineation within the
African Angraecoid group resulted in the discovery of 6 new species and 2 new
genera [14]. In addition, 30 species were not correctly assigned, from a phylogenetic
point of view, to genera.
The taxonomic insufficiencies of this family, in continental Africa, are justified by
several reasons including the political instability of the countries (making field missions
difficult), logistical problems, the lack of effective protocols for inventories, etc. At the
phylogenetic level, the studies showed that the genera Angraecopsis, Diaphananthe,
and Margelliantha are polyphyletic while the genera Aerangis, Ancistrorhynchus,
Bolusiella, Campylocentrum, Cyrtorchis, Dendrophylax, Eurychone, Microcoelia,
Nephrangis, Podangis, and Solenangis are monophyletic [14].
4 The Orchids of Sub-Saharan Africa
4.1 Richness According to Geographic Areas
The Orchidaceae species inventoried in sub-Saharan Africa are 1373 species

grouped into 88 genera. The genera Habenaria (391 spp.), Polystachya (279 spp.),
Eulophia (232 spp.), and Bulbophyllum (115 spp.) are the important genera. These
genera are followed by Disa, Satyrium, Tridactyle, Angraecum, Disperis,

Rhipidoglossum, and Brachycorythis with more than 50 species. We recorded
108 species common to the 4 major regions (West Africa, Central Africa, East
Africa, and Southern Africa). According to the work of Refs. [9, 10], the richness
of Central Africa phytogeographic zones is 708 species. This zone is followed by
southern Africa and eastern Africa with 637 and 583 species, respectively. These
numbers are higher than those given by the work of Ref. [15]. For this author, East
Africa is the richest region with 679 species followed by Central Africa with
517 species, South Africa with 425 species, and West Africa with 413 species.
The region with the lowest orchid abundance is West Africa. Central Africa appears
to be the most studied area. Indeed, several works have been carried out in this area
by numerous researchers. After Central Africa, the orchids of East and South Africa
which have been the best studied. In West Africa very few studies have been focused
on Orchidaceae. However, in this area, the important work on the orchids of Côte
d’Ivoire should be highlighted [4].
The most abundant species distributed in most countries are: Ansellia africana
Lindl., Bolusiella alinae Szlach., Brachycorythis ovata Lindl., Brachycorythis
pubescens Harv., Brachycorythis tenuior Rchb.f., Bulbophyllum fuscum Lindl.,
Bulbophyllum maximum (Lindl.) Rchb.f., Bulbophyllum scaberulum (Rolfe) Bolus,
Cyrtorchis arcuata (Lindl.) Schltr. (Fig. 10), Eulophia angolensis (Rchb.f.)
Summerh., Eulophia cucullata (Afzel. ex Sw.) Steud., Eulophia guineensis Lindl.,
Eulophia horsfallii (Bateman) Summerh., Habenaria ichneumonea (Sw.) Lindl.,
Habenaria schimperiana Hochst. ex A. Rich., Habenaria zambesina Rchb.f., Liparis
nervosa (Thunb.) Lindl., Nervilia bicarinata (Blume) Schltr., Nervilia kotschyi
(Rchb.f.) Schltr., Nervilia petraea (Afzel. ex Sw.) Summerh., Platylepis glandulosa
(Lindl.) Rchb.f., Polystachya golungensis Rchb.f., Polystachya modesta Rchb.f.,
Polystachya concreta (Jacq.) Garay & H. R. Sweet., Rangaeris muscicola (Rchb.f.)
Summerh., Rhipidoglossum rutilum (Rchb.f.) Schltr., Tridactyle anthomaniaca
(Rchb.f.) Summerh., Tridactyle bicaudata (Lindl.) Schltr., Tridactyle filifolia (Schltr.)
Schltr. et Tridactyle tridactylites (Rolfe) Schltr.
4.2 Endemism of Orchidaceae Species in the Sub-Saharan

Countries
Apart from species widely distributed in all regions, other species seem rather
confined to a given country or region. Recent studies showed that 284 species are
found in East Africa, 259 species in Central Africa, 201 in South Africa, and only
58 species in South Africa [9, 10]. There is thus an endemism in certain countries or
in two border countries.
The richness countries in endemic species based on the distribution map of
herbarium samples are Tanzania (78 species), Zimbabwe (41 species), the Demo-
cratic Republic of Congo (38 species), Ethiopia (32 species), Cameroon (29 species),
Mozambique (27 species), Zambia (24 species), Malawi (23 species), Rwanda
(21 species), Angola (20 species), Kenya (18 species), Nigeria (17 species), Gabon
(16 species), Central African Republic (8 species), and Uganda and Burundi with
5 species each [9, 10]. This number of endemic species could vary with taxonomic
revisions and sampling effort conducted in several countries. The endemic species
found in Tanzania include: Aerangis confusa J. Stewart, Ancistrorhynchus
parviflorus Summerh., Angraecopsis tenerrima Kraenzl.; I. & E. la Croix,
Angraecum brevicornu Summerh., Bonatea volkensiana (Kraenzl.) Rolfe,
Brachycorythis tanganyikensis Summerh, Cynorkis pleistadenia (Rchb.f.) Schltr.,
Disperis elaphoceras Verdc., Epipactis ulugurica Mansf., Eulophia amblyosepala
(Schltr.) Butzin, Habenaria apiculata Summerh., Holothrix hydra P. J. Cribb,
Margelliantha clavata P. J. Cribb, Mystacidium nguruense P. J. Cribb, Nervilia
similis Schltr, Oeceoclades zanzibarica (Summerh.) Garay & P. Taylor, Platycoryne
ambigua (Kraenzl.) Summerh., Polystachya acuminata Summerh., Pterygodium
ukingense Schltr, Rangaeris schliebenii (Mansf.) P. J. Cribb, Rhipidoglossum
melianthum (P. J. Cribb) Senghas, Satyrium comptum Summerh., Tridactyle
brevifolia Mansf., Vanilla zanzibarica Rolfe, Zeuxine lunulata P. J. Cribb &
J. Bowden.
Kenya, which borders Tanzania, has 243 Orchidaceae species divided into
47 genera according to Ref. [16]. Among the species we note as endemic to Kenyan
flora are Ancistrorhynchus paysanii Senghas, Angraecum decipiens Summerh.,
A. spectabile Summerh., Bilabrella kraenzliniana, Polystachya bella Summerh.,
Eulophia endlichiana (Kraenzl)., Habenaria altior Rendle, H. bonateoides Ponsie,
H. haareri Summerh., H. keniensis Summerh., H. thomsonii Rchb. f., Holothrix
pentadactyla (Summerh.) Summerh., Polystachya holstii Kraenzl., P. teitensis P. J.
Cribb, Rhipidoglossum montanum (Piers) Senghas, Ypsilopus longifolius (Kraenzl.)
Summerh, Y. viridiflorus P. J. Cribb & J. Stewart.
According to the distribution map, these two East African countries have in
common 17 endemic species found only on their territory. These are: Aerangis
coriacea Summerh., Angraecopsis breviloba Summerh, Angraecum viride Kraenzl.,
Bonatea rabaiensis (Rendle) Rolfe, Cynorkis uncata (Rolfe) Kraenzl.,
C. usambarae Rolfe, Disperis egregia Summerh., Habenaria helicoplectrum
Summerh., H. plectromaniaca Rchb.f. & S. Moore, Polystachya confusa Rolfe,
P. disiformis J. Cribb, P. leucosepala P. J. Cribb, P. shega Kraenzl., Tridactyle
cruciformis Summerh., T. furcistipes Summerh., T. tanneri P. J. Cribb,
Rhipidoglossum tanneri (P. J. Cribb) Senghas.
Ethiopia has 160 Orchidaceae species, divided into 37 genera [15]. These authors
have recorded 26 endemic species while 35 species have been reported by Refs.
[9, 10]. Among the species indicated, we can cite Angraecopsis trifurca (Rchb.f.)
Schltr, Habenaria lefebureana (A. Rich.) T. Durand & Schinz, Disa pulchella Hochst.
ex A. Rich., Disperis crassicaulis Rchb.f., Eulophia abyssinica Rchb. f., Habenaria
aethiopica S. Thomas & P. J. Cribb, Holothrix praecox Rchb.f., Liparis abyssinica
A. Rich., Polystachya aethiopica P. J. Cribb, Rhipidoglossum candidum (P. J. Cribb)
Senghas, Roeperocharis alcicornis Kraenzl., Satyrium aethiopicum Summerh.
In Central Africa, Cameroon harbors the highest number of endemic species with
29 endemic species recorded [9, 10]. In a previous work in this country, 44 endemic
species were identified [17]. These endemic species are, among others,
Ancistrorhynchus constrictus Szlach. & Olsz., Angraecopsis cryptantha Cribb,

Angraecum curvipes Schltr., Bolusiella zenkeri (Kraenzl.) Schltr., Bulbophyllum
saltatorium Lindl., Chamaeangis letouzeyi Szlach. & Olsz., Cheirostylis divina
(Guinea) Summerh. var. ochyrae Szlach. & Olsz., Diaphananthe garayana Szlach.
& Olsz., Disperis kamerunensis Schltr., Disperis nitida Summerh., Gastrodia afri-
cana Kraenzl., Genyorchis macrantha Summerh., Habenaria alinae Szlach., Liparis
kamerunensis Schltr., Microcoelia sanfordii L. Jónss., Ossiculum aurantiacum P. J.
Cribb & Laan, Platycoryne alinae Szlach., Polystachya albescens Ridl. subsp.
angustifolia (Summerh.) Summerh., Rangaeris trachypus (Kraenzl.) Guillaumin,
Rhipidoglossum ochyrae Szlach. & Olsz., Rhipidoglossum polydactylum (Kraenzl.)
Garay, Vanilla ochyrae Szlach. & Olsz.
Orchids species of Gabon can be checked at http://orchideesgabon.e-monsite.
com/pages/orchidees-liste.html. This site gives a non-exhaustive list of 284 species
divided into 53 genera among which 16 endemic species [9, 10]. These endemics
species are: Angraecopsis hallei Szlach. & Olszewski, Angraecum cribbianum
Szlach. & Olszewski, Bulbophyllum apodum Hook.f., B. subligaculiferum J. J.
Vern., Cyrtorchis henriquesiana (Ridl.) Schltr., Dinklageella villiersii Szlach. &
Olszewski, Rhipidoglossum magnicalcar Szlach. & Olszewski, Taeniorrhiza
gabonensis Summerh., Tridactyle latifolia Summerh., T. minutifolia Stévart &
D’Haijère, T. pentalobata P. J. Cribb & Stévart, Vanilla acuminata Rolfe,
V. chalotii Finet, V. hallei Szlach. & Olszewski, Veyretella hetaerioides (Summerh.)
Szlach. & Olszewski, V. flabellata Szlach.
Gabon and Cameroon have 11 species in common [17]. These species are:
Aerangis gracillima (Kraenzl.) J. C. Arends & J. Stewart, Ancistrorhynchus obovata
Stévart inédit., Angraecum eichlerianum Kraenzl. var. curvicalcaratum Szlach. &
Olsz., Bulbophyllum fayi J. J. Verm., Bulbophyllum minutifolium Stévart in Stévart
& al., Diaphananthe ceriflora J. B. Petersen, Eggelingia gabonensis P. J. Cribb &
Laan, Genyorchis platybulbon Schltr., Halleorchis aspidogynoides Szlach. & Olsz.,
Polystachya kubalae Szlach. & Olsz., Polystachya letouzeyana Szlach. & Olsz.
However, 19 species have been indicated as common endemic species to these
two countries [9, 10].
Cameroon shares with the Republic of Congo, the Democratic Republic of
Congo, Equatorial Guinea, and Ethiopia five endemic species which are Habenaria
phantasma la Croix, Habenaria stenoceras Summerh., Rhipidoglossum monte-
alenense Descourvières, Stévart & P. J. Cribb and Stolzia grandiflora P. J. Cribb.
Habenaria egregia Summerh. is only observed in Cameroon, Ethiopia, and Kenya.
The species Orestias micrantha Summerh., Polystachya batkoi Szlach. &
Olszewski, Polystachya letouzeyana Szlach. & Olszewski, Polystachya
moniquetiana Stévart & Geerinck, Polystachya riomuniensis Stévart & Nguema
and Polystachya stodolnyi Szlach. & Olszewski are only found in Cameroon,
Gabon, and Equatorial Guinea. Cameroon also shares with Gabon and the Republic
of Congo the species Habenaria lisowskii Szlach. and with Gabon and the Demo-
cratic Republic of Congo the species Tridactyle laurentii (De Wild.) Schltr. Two
species Polystachya bifida Lindl. and Polystachya testuana Summerh are endemic to
Gabon and Equatorial Guinea.
The Democratic Republic of Congo has 38 endemic species according to the

distribution maps of Refs. [9, 10]. Some of these species are: Angraecum mofakoko
De Wild., Bulbophyllum horridulum J. J. Vern., Cynorkis summerhayesiana
Geerinck, Diaphananthe divitiflora (Kraenzl.) Schltr., Disa alinae Szlach., Eulophia
fernandeziana Geerinck, Habenaria garayana Szlach. & Olszewski, Polystachya
alicjae Mytnik, Roeperocharis maleveziana Geerinck, Tridactyle fimbriatipetala
(De Wild.) Schltr., Tridactyle stipulata (De Wild.) Schltr., Tridactyle vanderlaaniana
Geerinck.
In République Centrafricaine, we found only eight endemics species which are:
Ancistrorhynchus brevifolius Finet, Eulophia falcatiloba Szlach. & Olszewski,
Habenaria kornasiorum Szlach. & Olszewski, Habenaria letestuana Szlach. &
Olszewski, Microcoelia jonssonii Szlach. & Olszewski, Platycoryne lisowskiana
Szlach. & Kras, Platycoryne ochyrana Szlach., Disperis raiilabris Summerh.
A total of 21 endemic species from Rwanda flora has been noticed [9, 10]. They
are: Bulbophyllum kivuense J. J. Vern., Diaphananthe eggelingii P. J. Cribb,
Diaphananthe liae Ed. Fischer, Killmann, J.-P. Lebel & Delep., Disperis natascha-
oppeltae Eb. Fischer, Killmann, Disperis reklieberae Eb. Fischer, Eulophia pocsii
Eb. Fisch., Gastrodia rwandensis Eb. Fisch. & Killmann, Habenaria bequaertii
Summerh., Margelliantha lebelii Eb. Fischer & Killmann, Polystachya
anastacialynae Eb. Fischer, Polystachya bruechertiae Eb. Fischer, Killmann &
J.-P. Lebel, Polystachya erica-lanzae Eb. Fischer, Polystachya isabelae Mytnik,
Polystachya lawalreeana Geerinck, Polystachya samilae Eb Fisch., Rhipidoglossum
cuneatum (Summerh.) Garay, Rhipidoglossum mildbraedii (Kraenzl.) Garay, Stolzia
heiligenthalii Eb. Fisch., Killmann, Stolzia kalkhof-roseae Eb. Fischer, Killmann,
Tridactyle nanne-ritzkae Eb. Fischer, Killmann, Lebel & Delepierre, Tridactyle
stevartiana Geerinck.
According to the two authors [9, 10], there are five endemic species in Burundi
which are: Habenaria lewallei Geerinck, Polystachya editae Eb. Fisch., Polystachya
lacroixiana Geerinck, Polystachya maculata P. J. Cribb, Polystachya walravensiana
Geerinck & Arbonn.
Burundi and Rwanda share 16 endemic species which are: Habenaria
coeloglossoides Summerh., Habenaria lehae Eb. Fischer, Polystachya tridentata
Summerh, Polystachya troupiniana Geerinck, Polystachya undulata P. J. Cribb &
Podz., Polystachya woosnamii Rendle, Rhaesteria eggelingii Summerh., Polystachya
proterantha P. J. Cribb, Habenaria brachylobos (Summerh.) Summerh., Rhipidog-
lossum ovale (Summerh.) Garay, Tridactyle eggelingii Summerh., Margelliantha burttii
(Summerh.) P. J. Cribb, Polystachya fabriana Geerinck, Polystachya macropoda
Summerh., Rhipidoglossum arbonnieri (Geerinck) Eb. Fischer, Killmann, Rhipidog-
lossum delepierreanum (Lebel & Geerinck) Eb. Fisch., Killmann.
In West Africa, the orchids flora of Nigeria and Côte d’Ivoire is well known
compared to the other countries of the area. Nigeria is rich with 239 species divided
between 34 genera. It is the country with the most endemic species in West Africa
[9, 10]. According to these authors, there are 15 endemic species. But other studies
based on inventories reveal 7 endemic species which are: Diaphananthe dorotheae
(Rendle) Summerh., Genyorchis apertiflora Summerh., Habenaria linguiformis
Summerh., Habenaria nigerica Summerh., Habenaria phylacocheira Summerh.,

Habenaria prionocraspedon Summerh., Alectra virgata Hemsl., which are indicated
to be endemic species of Nigeria [18].
By observing the distribution maps of species, Nigeria (West Africa), neighbor of
Cameroon (Central Africa), shares with this country 26 endemic species
[9, 10]. These are for example: Aerangis gravenreuthii (Kraenzl.) Schltr.,
Ancistrorhynchus serratus Summerh., Angraecopsis cryptantha P. J. Cribb,
Angraecum angustum (Rolfe) Summerh., Diaphananthe lanceolata (Summerh.)
P. J. Cribb & Carlsward, Disperis mildbraedii Schltr. ex Summerh., Genyorchis
micropetala (Lindl.) Schltr, Habenaria alinae Szlach., Liparis letouzeyana Szlach.
& Olszewski, Platycoryne megalorrhyncha Summerh., Polystachya alpina Lindl.,
Tridactyle lagosensis (Rolfe) Schltr., Rhipidoglossum obanense (Rendle) Summerh.
Platylepis xerostele Ormerod is observed on the border of Nigeria and Cameroon.
The book entitled Les Orchidées de Côte d’Ivoire gave for this country a total of
264 species divided into 48 genera [4]. This study increased the number of species
recorder in the frame of previous studies [19]. Indeed three new species
(Ancistrorbyncbus akeassiae Perez-Vera, Bulbupbyllum danii Perez-Vera, and
Cbamaeangis pauciflora Perez-Vera) and two new varieties (Bulbopbyllum
scaberulum (Rolfe) Bolus var. album Perez-Vera and Cyrtorcbis broumii (Rolfe)
Schltr. var. guillaumetii Perez-Vera) have been described [4].
Côte d’Ivoire shares with Liberia and Sierra Leone three species: Polystachya
pseudodisa Kraenzl., Tridactyle fusifera Mansf., and Bulbophyllum denticulatum
Rolfe. Polystachya leonensis Rchb.f. is found in Guinea and Sierra Leone. This
species is not counted among ivorian orchids, although it is collected near the Ivorian
border. This species has been also observed in Cameroon flora. The species Poly-
stachya bancoensis Burg has only been observed in Côte d’Ivoire and Ghana. The
species Polystachya rivae Schweinf. encountered in Ethiopia has been reported in
Côte d’Ivoire [9, 10]. This species is not included in the list compiled by Ref. [4].
Côte d’Ivoire, Liberia, Sierra Leone, and Ghana share Bulbophyllum danii Perez-
Vera, Angraecum modicum Summerh., Diaphananthe suborbicularis Summerh. and
Bulbophyllum parvum Summerh. The species Malaxis melanotoessa Summerh. and
Polystachya bequaertii Summerh. et Polystachya elastica Lindl. are only found in
Liberia and Sierra Leone. Species like Habenaria jacobi Summerh. and Habenaria
jaegeri Summerh. are found in Liberia and Sierra Leone and Guinea. The species
Habenaria angustissima Summerh. is endemic in Senegal and Guinea. The species
Bulbophyllum parvum Summerh. and Diaphananthe suborbicularis Summerh. are
found in Ghana and Sierra Leone. The species Rhipidoglossum laticalcar (J. B. Hall)
Senghas is only found in Ghana and Nigeria. Polystachya monolenis Summerh. is an
endemic species of Sierra Leone and Ghana. The species Rhipidoglossum laxiflorum
Summerh. has been reported in Ghana, Togo, and Côte d’Ivoire in the work of Refs.
[9, 10]. However, this species has not been reported in Côte d’Ivoire [4]. Habenaria
dinklagei Kraenzl. has only been observed in Liberia and Nigeria. It has not been
found in other regions between the two countries.
The well-known Southern African countries for orchids are Angola, Malawi,
Mozambique, Zambia, and Zimbabwe. The flora of Angola is rich of 228 orchids
belonging to 40 genera [20]. This Angolan flora has 20 endemic species according to
Refs. [9, 10]. For example we can mention Angraecopsis gassneri G. Will.,
Brachycorythis mixta Summerh., Diaphananthe welwitschii (Rchb.f.) Schltr.,
Eulophia aloifolia Welw. ex Rchb.f, Eulophia protearum Rchb.f., Habenaria
decaptera Rchb.f., Habenaria robusta Welw. ex Rchb.f., Holothrix klimkoana
Szlach. & Marg., Polystachya angularis Rchb.f., Satyrium aciculare van der Niet
& P. J. Cribb et Satyrium welwitschii Rchb.f.
The site https://www.malawiflora.com/speciesdata/family.php?family_id¼161
gave for Malawi 420 Orchidaceae species grouped into 57 genera. Among these
420 species, 23 are endemic according to the work of Refs. [9, 10]. Three hundred
six species distributed among 48 genera have been counted by Ref. [21]. It is
understandable that this number may be increasing with the work prior to this
study [22]. In this flora of Malawi, 23 species are endemic according to the work
of Refs. [9, 10]. Those species are: Aerangis carnea J. Stewart, Angraecopsis lovettii
P. J. Cribb, Angraecum umbrosum P. J. Cribb, Bonatea stereophylla (Kraenzl.)
Summerh., Disa walleri Rchb.f., Disperis decipiens Verdc.; Habenaria diselloides
Schltr., Holothrix buchananii Schltr., Polystachya mzuzuensis P. J. Cribb & la Croix,
Polystachya suaveolens P. J. Cribb, Rhipidoglossum oxycentron (P. J. Cribb)
Senghas, Satyrium afromontanum la Croix & P. J. Cribb.
Mozambique harbors 232 species and 49 genera of orchids with 7 endemic
species [23]. These species are: Cyrtorchis glaucifolia Summerh., Disperis
mozambicensis Schltr., Eulophia biloba Schltr., Eulophia bisaccata Kraenzl.,
Habenaria hirsutissima Summerh., Habenaria mosambicensis Schltr. et Liparis
hemipilioides Schltr. In addition, 14 other species are shared with Zimbabwe,
Tanzania, Malawi, and South Africa. The country shares 10 endemic species with
Zimbabwe: Bulbophyllum ballii P. J. Cribb, Cynorkis anisoloba Summerh., Disa
chimanimaniensis (H. P. Linder) H. P. Linder, Disa zimbabweensis H. P. Linder,
Neobolusia ciliata Summerh., Oligophyton drummondii H. P. Linder & G. Will.,
Polystachya subumbellata P. J. Cribb & Podz., Polystachya valentina la Croix &
P. J. Cribb, Satyrium flavum la Croix, and Schizochilus lepidus Summerh. Mozam-
bique shares, respectively, with Malawi and Tanzania the species Polystachya
songaniensis G. Will. et Habenaria stylites Rchb.f. & S. Moore subsp. johnsonii
(Rolfe) Summerh.
In Zambia 412 orchids species divided into 46 genera are recorded on the site https://
www.zambiaflora.com/speciesdata/family.php?family_id¼161. Among these species,
22 are endemic. These species are: Bonatea antennifera Rolfe, Bonatea cassidea
Sond., Cynorkis clarae Geerinck, Disa helenae Buscal. & Schltr., Disa praecox
(H. P. Linder) H. P. Linder in Linder & Kurzweil, Disperis bifida P. J. Cribb, Eulophia
brenanii P. J. Cribb & la Croix, Eulophia richardsiae P. J. Cribb & la Croix; Habenaria
argentea P. J. Cribb, Habenaria bertauxiana Szlach. & Olszewski, Habenaria
binghamii G. Will., Habenaria macrotidion Summerh., Habenaria orthocentron P. J.
Cribb, Habenaria pasmithii G. Will., Habenaria petraea Renz & Grosvenor, Habenaria
tubifolia la Croix & P. J. Cribb, Habenaria velutina Summerh., Holothrix villosa Lindl.
var. villosa, Polystachya asper P. J. Cribb & Podz., Polystachya erythrocephala
Summerh., Polystachya mafingensis P. J. Cribb, Pteroglossaspis corymbosa G. Will.
Zimbabwe has 358 species of orchids for 54 genera that can be viewed at the site
https://www.zimbabweflora.co.zw/speciesdata/family.php?family_id¼161.
A list of the plant taxa endemic or nearly endemic to Zimbabwe has not previ-
ously been compiled [24]. The list of endemics identified in this flora will be based
on Refs. [9, 10]. Zimbabwe comes after Tanzania with 41 endemic species. We have
retained Aeranthes parkesii G. Will., Angraecum chimanimaniense G. Will.,
Bulbophyllum chikukwa Fibeck & Mavi, Brownleea galpinii Bolus subsp. galpinii,
Cynorkis anisoloba Summerh., Disa chimanimaniensis (H. P. Linder) H. P. Linder,
Disperis concinna Schltr., Eceoclades quadriloba (Schltr.) Garay & P. Taylor,
Eulophia foliosa (Lindl.) Bolus, Goodyera afzelii Schltr, Habenaria bicolor Conrath
& Kraenzl., Holothrix macowaniana Rchb. f., Mystacidium gracile Harv., Neo-
bolusia ciliata Summerh., Oeceoclades pandurata (Rolfe) Garay & P. Taylor,
Oligophyton drummondii H. P. Linder & G. Will., Platycoryne affinis Summerh.,
Polystachya pubescens (Lindl.) Rchb.f., Satyrium flavum la Croix, Schizochilus
calcaratus P. J. Cribb & la Croix, Stenoglottis woodii Schltr., Tridactyle
hurungweensis W. Fibeck.
5 Uses of Orchidaceae Species in African Countries
Orchids are best known for their beautiful flowers which make them a resource of
great economic importance in the horticultural industry.
Very little information related to the indigenous African ornamental orchids are
available. However, several orchids from other continents or countries have been
introduced and domesticated and constitute important sources of income for African
horticulturalists. Among these species Arachnis hookeriana (Rchb.f.) Rchb.,
Arachnis flos-aeris (L.) Rchb.f. native to Asia, Papilionanthe hookeriana (Rchb.f.)
Schltr. native to Malaysia and Bletilla striata (Thunb.) Rchb.f. from Japan, China,
and Tibet. These species are cultivated and sold in Cameroon [25]. Ansellia africana
Lindl. (Fig. 1), nicknamed the panther orchid because of its remarkable size of
flowers and its yellow color streaked with brown, is an ornamental species in Gabon.
This species is also called the queen of African orchids. Its range stretches from
West Africa to South Africa. This large epiphytic orchid is arguably the most popular
African orchid in the West. In Gabon, we can also see, Eulophia bouliawongo
(Rchb.f.) J.Raynal (Eulophia oedoplectron Summerh.), another ornamental orchid,
named the queen of marshes and coastal savannas prone to flooding. It is a large pink
orchid over 2 m tall. Its flower stalk can produce up to 35 quite spectacular, deep
pink flowers (https://www.lepratiquedugabon.com/les-orchidees-du-gabon/).
Some orchids are also consumed by several communities in Africa. The species
of orchids consumed by people are different from one country to another. However,
Disa robusta N.E.Br. (Fig. 2) and Satyrium buchananii Schltr. are eaten in several
countries. In Cameroon (Central Africa), the underground organs of Habenaria
keayi Summerh. and Habenaria zambesina Rchb.f. (Fig. 3) are eaten [26]. Orchids
are also eaten in East Africa. In Malawi the tubers of several orchids are used
for food. These are, among others, Disa engleriana Kraenzl. (Fig. 4), Disa zombica
Fig. 1 Image of Ansellia africana Lindl. (From Aké-Assi)
Fig. 2 Image of Disa robusta

N.E.Br.
N.E.Br., Habenaria walleri Rchb.f., Satyrium amblyosaccos Schltr. [27]. In Tanza-

nia these are Brachycorythis pleistophylla Rchb.f., Disa erubescens Rendle, Disa
ochrostachya Rchb.f., Eulophia schweinfurthii Kraenzl., Habenaria xanthochlora
Schltr., Satyrium atherstonei Rchb.f., Disa robusta N.E.Br., Satyrium robustum
Schltr., Satyrium sceptrum Schltr., Satyrium acutirostrum Summerh., Roeperocharis
wentzeliana Kraenzl. which are used for the preparation of several meals [28]. In
Benin (West Africa), Eulophia horsfallii (Bateman) Summerh. (Fig. 5), Habenaria
cirrhata (Lindl.) Rchb.f. (Fig. 6), and Nervilia bicarinata (Blume) Schltr. are used in
the preparation of sauces [29].
An often-overlooked value of plants in the Orchidaceae family is their role in
medicine. In many countries of Europe, America, Asia, and Africa, orchids have
been used as traditional medicine for a very long time. Particularly in Africa, they are
Fig. 3 Image of Habenaria

zambesina Rchb.f.
Fig. 4 Image of Disa

engleriana Kraenzl.
used in human and animal medicine. In Benin, in human health, orchids are used to
cure several diseases [29]. Calyptrochilum christyanum (Rchb.f.) Summerh. is used
in the treatment of dysmenorrhea (painful periods), edema of the lower limbs,
malaria, and liver disease (disease of the faith). This species is also used to speed
up the walking of a baby. Eulophia guineensis Lindl. is known to cure edema of the
lower extremities, cough, epigastralgia (stomach pain), and fever. Habenaria
schimperiana Hochst. ex A. Rich. is used to cure visual disturbances. To treat
fever, myalgia (muscle pain), urinary disorders, epigastralgia (stomach pain), people
of Benin use Nervilia bicarinata (Blume) Schltr. Nervilia kotschyi (Rchb.f.) Schltr. is
used to treat dysmenorrhea (painful periods), epigastralgia (stomach pain), and
Fig. 5 Image of Eulophia

horsfallii (Bateman)
Summerh.
Fig. 6 Image of Habenaria

cirrhata (Lindl.) Rchb.f.
cough [29]. In Cameroon, orchids are also used to treat several diseases. Angraecum
angustipetalum Rendle is used for bone fortification of children. It is also used to
perform abortions. This species is also used against snakes. Ancistrorhynchus
serratus Summerh. is used to treat diabetes. Bulbophyllum fuscum var.
melinostachyum (Schltr.) J. J. Verm., Bulbophyllum barbigerum Lindl.,
Bulbophyllum intertextum Lindl., and Bulbophyllum calyptratum Kraenzl. are used
to cure diseases such as side pain, otodynia (ear pain), burns, wounds, and derma-
toses (skin diseases). Cyrtorchis arcuata (Lindl.) Schltr. (Fig. 7) is recommended in
the treatment of diabetes and skin diseases.
Diaphananthe bidens (Afzel. ex Sw.) Schltr. (Fig. 8) is used for fertility.
Graphorkis lurida (Sw.) Kuntze is used to treat coughs and tuberculosis. Habenaria
procera (Afzel. ex Sw.) Lindl. is recommended for the purification of blood,
gastritis, and arthritis. Liparis nervosa (Thunb.) Lindl. is used to treat ulcers and
Fig. 7 Image of Cyrtorchis

arcuata (Lindl.) Schltr.
Fig. 8 Image of
Diaphananthe bidens
(Afzel. ex Sw.) Schltr.
(From Swiss Center for
Scientific Research)
burns. Listrostachys pertusa (Lindl.) Rchb.f. (Fig. 9) is recommended for

epigastralgia (stomach pain), constipation, and measles. Polystachya concreta
(Jacq.) Garay & H. R. Sweet, Polystachya cultriformis (Thouars) Lindl. ex Spreng.,
and Polystachya caloglossa Rchb.f. are used to treat rheumatism, arthritis, measles,
and burns. Tridactyle tridactylites (Rolfe) Schltr. is used for the treatment of demen-
tia or mental disorders [25].
In Benin, orchids are used spiritually. Calyptrochilum christyanum (Rchb.f.)
Summerh. is used to attract good luck, the power of prophecy. It also has a vanishing
power. Eulophia guineensis Lindl. fights the spirits of twins. Nervilia bicarinata
(Blume) Schltr. (Fig. 10) is used for the power of prophecy. This species is also used
to fight against the spirit of the deceased, witchcraft, and the spirits of twins [29]. In
Cameroon, Ansellia africana Lindl. is used to ward off evil spirits and promote
romantic relationships. Angraecum birrimense Rolfe, Bulbophyllum lupulinum
Lindl., and Bulbophyllum falcatum (Lindl.) Rchb.f. are used to fight witchcraft.
These species are also used to make predictions. Calyptrochilum emarginatum
(Afzel. ex Sw.) Schltr. is used for seduction and luck [25].
Fig. 9 Image of
Listrostachys pertusa. (From
the Parc National des Île
Ehotilés)
Fig. 10 Image of Nervilia

bicarinata (Blume) Schltr.
(From Sinfra)
In animal health, Calyptrochilum christyanum (Rchb.f.) Summerh. and

Habenaria cirrhata (Lindl.) Rchb.f. are used to treat diseases in chickens [29].
6 Conclusion
Based on this review of the literature, the contribution of the sub-Saharan African
orchid flora is estimated to be more than 5% of the world flora of orchids. It is clear
that the number of species is underestimated taking into account the fact that several
regions of the continent were not highly inventoried. Also, many species are
epiphytes, thus difficult to be collected. Orchidaceae is a diverse family and impor-
tant for communities for the diversity of its uses (ornamental, food, medicine, etc.).
The fact that some regions are more studied than others should be highlighted. For
instance, Central and East Africa are relatively well known compared to West Africa.
With the high rate of deforestation in Africa, several species could be threatened in
the continent. Intensive prospection of understudied areas should be carried out to
avoid the loss of many species. Also, it is important to assess the conservation status
of the African orchids.
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Orchid Biodiversity and Genetics
5
Seeja G and Sreekumar S
Contents
1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 154
2 Orchid Biodiversity Versus Adaptation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 155
3 Floral Development and Genetics in Orchids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 156
4 Orchid Flower Development and MADS Box Genes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 158
5 Floral Whorl Development in Orchids – Class A MADS Box Genes . . . . . . . . . . . . . . . . . . . 160
6 The Orchids Class B MADS-Box Genes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 160
7 The Orchid Class C and D MADS-Box Genes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 161
8 Ovule Development . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 162
9 Perianth Senescence/Development . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 163
10 Genetic Variation Versus Secondary Metabolite and Adaptations . . . . . . . . . . . . . . . . . . . . . . . . 163
11 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 166
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 167
Abstract
The angiosperm family Orchidaceae comprises ~900 genera and 30,000–35,000
species, which are distributed all over the world. Besides, more than two lakhs
man made hybrids are available. The unparalleled unique characteristic features
and adaptations to thrive on almost all habitats on the earth made this group of
plants as a distinct one from the rest of the plants in the Plant Kingdom. The
diversity in morphology, physiology, and genetic peculiarities induces large level
of speciation with remarkable evolutionary significance. The constructions of
orchid flowers are always a curiosity to both layman and scientists. The unique
flower characters (phenotype) such as wide variations in floral morphology,
S. G
Department of Plant Breeding and Genetics, College of Agriculture, Ambalavayal, India
e-mail: seeja.g@kau.in
S. S (*)
Biotechnology and Bioinformatics Division, KSCSTE- Jawaharlal Nehru Tropical Botanic Garden
and Research Institute, Thiruvananthapuram, India
e-mail: drsreekumar@rediffmail.com

154 S. G and S. S
colors, shapes, enchanting smells to attract pollinators are the expression of genes
(genotype). The construction of orchid flowers as well as the efficiency in
successfully carrying out the function for which they are intended and underlying
genetics is briefly discussed.
Keywords
Orchid · Biodiversity · Genetics · MADS box · Phytomolecules · Pigments ·
Pollinium · Homeotic · Secondary metabolite
1 Introduction
Orchids are one of the natural wonders with exceptional characteristic features, not
only amazing flowers with large diversity but also with remarkable evolutionary
significance. It belongs to the angiosperm (flowering plants) family orchidaceae and
consists of about 900 genera and 30000–35000 species. Many species are commer-
cially exploited as ornamentals, medicinal, and food additives. For examples, many
species under the genus Dendrobium, Vanda, Cymbidium, Phalaenopsis, Oncidium,
Cattleya, Paphiopedilum, Aranda, Renanthera are highly remunerative floriculture/
horticulture crops; Vanda tessellate, Rhyncostylis retusa, and Dendrobium monticola
are used in Indian traditional medicine; Gastrodia sesamoides and Gastridia falconeri
are used as food and vanillin derived from Vanilla planifolia as a food additive. In
floriculture industry, orchid flowers rank top most position as cut flowers and potted
plants. Considering the ornamental value, plant breeders have been explored its
significant traits and produced over two lakhs hybrids, which are multiplied in large
numbers by the application of tissue/seed culture method for flourishing the floricul-
ture industry in world wide. However, illegal harvesting of the wild species for local,
regional, and international trade is ever increasing that leads to the extinction of many
orchid species which grow only in specific micro-agro-climatic zones or specific
ecological niche and limited population. Besides, the rapid deforestation and urbani-
zation/change in land use pattern results devastation of habitat with interacting factors
like pollinating agents and mycorrhizal symbionts which became a great threat to
orchid population and extinction of many species. Therefore, conservation of orchid
biodiversity and sustainable utilization of wild species attain prime importance.
The incomparable amazing floral diversity of over 30,000 orchid species and its
underlying genetic mechanism still remain as a mystery. Even though Orchids’
unveiling species-specific variation in the floral whorl initiation and floral whorl
development is a multifaceted one, its evolutionary schoolwork is worthy and
underlying molecular data is relatively limited. The recently proposed “orchid
code” theory explained the genetic mechanism of floral whorl and its diversity
development in orchids [1]. The uniqueness and diversity is generated by the
combinatorial collaboration of four DEF-like MADS-box genes groups with other
floral homeotic genes. The changes in the expression of DEF-like or CYC-like genes
create floral diversity and promote floral whorl development as the perception of
5 Orchid Biodiversity and Genetics 155
evolutionary developmental biologist (“evo-devo”) that the developmental genetic

system can influence the direction and pace of evolutionary changes [1]. True
changes in organ identity are rare events in the evolution of orchid flowers. Hence,
the unprecedented developmental genetic predisposition that originated early in
orchid evolution may be the cause of floral diversity in orchids [1].
2 Orchid Biodiversity Versus Adaptation
Orchids belonging to the family Orchidaceae consist of ~900 genera and

30,000–35,000 species with exceptional phenotypic and genotypic diversity. The
database, “The Plantlist.org” currently provides accepted names of 27,801 species
under 899 genera. Besides, hundreds of synonyms and unassessed species names are
in the database for further clarification. Orchids are cosmopolitan in distribution and
growing throughout the world from tropical to subtropical and temperate regions.
Due to the specific ecological adaptations evolved through evolution, the members
of orchidaceae have diverse habitat on earth, such as soil (terrestrial), for example,
Spathoglottis, rock surfaces (lithophytic), for example, Paphiopedilum, and on other
plants (epiphytic), for example, Dendrobium. Among the terrestrial forms, some
species are associated with fungus as endotrophic or as ectotrophic. Majority of the
species (70%) are epiphytes and some are saprophytes, for example, Didymoplexis.
In order to thrive on diverse habitat, orchids have variety of designs and adaptations.
Presence of velamen roots, thick, waxy, or leathery leaves, pseudobulbs,
Crassulacean acid metabolism (CAM) photosynthesis mechanism, etc., are some
of the adaptations found among the orchids to grow under water stress condition.
Orchids are popular mainly due to the presence of unique and fascinating flowers
with great complexity, incredible diversity, and long floral lifespan as cut flowers,
which are unrivalled in the plant world. The unique floral characters such as wide
variations in floral morphology, colors, shapes, enchanting smells to attract pollina-
tors are expression of genes (genotype), and these all indicate high degree of
speciation among orchids. It is interesting to note that in many orchids, the flowers
are constructed in such a way to attract specific insect or bird to pollinate the flowers
and in some others produce chemicals to attract specific pollinators. These all
features establish floral and reproductive isolation that facilitate genetic diversity
and consequent speciation.
The special characteristic features of the orchid flowers include except few
species all others have zygomorphic flowers (bilateral symmetry) with trimerous
arrangement, outer whorl of three sepals and inner whorl of three petals, both are
alike in appearance and known as tepals. The posterior petal is modified to form
labellum or lip and twisting of flower on its axis through 180 makes it to the anterior
position such that it forms a convenient landing platform for pollinator insects. The
labellum is distinctly modified and attractive in each species. It is variously cut,
highly colored, may bear warts or out-growths and in various shapes. A spur devel-
oped either from the base of the lip or partly from it and axis which produces nectar
for attracting the pollinators. The staminal filaments and style fused to form a
156 S. G and S. S
cylindrical structure, gynostemium, or column. At the top of the column, pollinia are
attached. A pollinium is a coherent mass of pollen grains put together by glue-like
alkaloid viscidium, a combination of cellulosic strands and mucopolysaccharides.
The pollinium has a caudicle with sticky disc, which may attach on the insects’ legs
when they visit the flower. The column serves the same functions of style and stigma.
It has an opening toward the labellum side and stigma is inside the column. These
features direct the pollinating insects to the flowers. A innumerable number of
minute seeds facilitate the expression of genetic variability and dispersal of seeds
in large proportion across geographical/ecological barriers. All these unique charac-
teristic features of orchids facilitate evolution and speciation [2] leading to high
diversity among orchids. The genetic mechanism involved in the evolution of
different orchid genera and species, and diversity of flowers engenders keenness
exclusively to orchid breeders. It led to the production of over 200,000 man-made
orchid commercial hybrids through hybridization and that made orchids as a
top-ranked cut flower in the global floriculture industry.
3 Floral Development and Genetics in Orchids
In reproductive biology, orchids exhibit distinctive evolutionary mechanism. One is

the fused structure of androecium and gynoecium to form single column known as
gynostemium which facilitates pollination by pollinators evolved simultaneously
naturally through coevolution. Presence of protandry, pollinia, synchronization in
microsporogenesis, pollination-triggered megagametogenesis, effective fertilization
on entire length of placenta, and production of incalculable small immature embryos
known as seeds especially without endosperm in dehiscent capsule are the special
features evolved in orchids and its underlying mechanism; particularly the gene
regulation processes are investigated by different molecular workers.
Generally at a particular stage of plant development due to the activities of
flowering time genes, the apical meristem switches program from a leaf-producing
to a flower-producing tissue and becomes an inflorescence meristem and forms
numerous primitive primordia that later develops into sepal, petal, stamen, and
carpel. This phenomenon is due to homeotic mutations in normal vegetative cells
that lead to develop normal organs in inappropriate position. As in other flowering
plants, orchids also exhibit two stages, floral transition and flower development.
Floral transition is influenced by various factors such as juvenility, ambient temper-
ature, and photoperiod. These factors play pivotal role in determining flower initi-
ation time with respect to ontogeny and season [3]. In Phalaenopsis [4] and
Dendrobium [5], temperature below 26 C promoted flower induction, but in
Dendrobium hybrids, namely Dendrobium Chao Praya Smile and Dendrobium
Madame Thong-In, flower induction occurs in high temperature (30 C) and in
some others like Cypripedium species vernalization below 5 C or subzero temper-
ature is a prerequisite for flowering [6]. Photoperiod also regulates flower induction
in certain orchids, for example, in Phalaenopsis pulcherrima, 30 C day and 20 C
night condition, flowering is more efficient under 9 h light than 12 h light treatment
[3, 7]. Tropical regions are the treasure house of orchids where the day and night
length are almost equal throughout the year; hence, the influence of photoperiod on
orchid flowering may be insignificant. The significant effect of phytohormones on
flower transition in orchids has been demonstrated, for example, in Phalaenopsis
and Doritaenopsis (hybrid Doritis Phalaenopsis) plants sprayed with
6-benzylaminopurine (BAP) produced inflorescence 3–9 days earlier [8]. Similarly,
influence of synthetic cytokinins (BAP, gibberellic acid, abscisic acid) on orchid
flower induction in in vitro has been demonstrated [3].
Flower transition and inflorescence architecture are modulated by two homolo-
gous proteins, FLOWERING LOCUS T (FT) and TERMINAL FLOWER 1 (TFL1).
The FT promotes the transition to reproductive development and flowering, while
TFL1 represses this transition [9]. FT and TFL1 showed ~60% amino acid sequence
similarity but function in opposite manner [9–11]. Photoperiod is a primary trigger
of FT expression. FT facilitates the transition to flowering in response to its induction
by the transcription factor CONSTANS (CO) [9, 12, 13]. Temperature-responsive
FT regulators, which target the FT promoter or noncoding regions, include SHORT
VEGETATIVE PHASE (SVP) [13], PHYTOCHROME INTERACTING FACTOR
4 (PIF4) [14], LIKE HETEROCHROMATIN PROTEIN 1 (LHP1) [15], and
FLOWERING LOCUS C (FLC) [9, 16]. Once the FT is inducted by CO, it will
move from the leaves to shoot apical meristem (SAM) and binds to the bZIP
transcription factor FD (basic region/leucine zipper motif) to form a complex that
regulates meristem identity genes, resulting in flowering induction [9, 17, 18]. The
meristem identity genes induced by the FT-FD complex are APETALA 1(AP1) and
FRUITFULL(FUL) that reprogram the primordia to produce reproductive organs
instead of vegetative ones [9, 19]. In addition to inducing FT, CO is suggested to
induce, directly or indirectly, the expression of TERMINAL FLOWER 1(TFL1),
which controls inflorescence meristem identity and delays the transition to the
reproductive phase at the SAM [9, 20]. TFL1 represses the expression of several
genes downstream of FT such as LEAFY(LFY) and AP1, perhaps by partnering with
FD, with which it weakly interacts [9, 21]. The high sequence homology of TFL1 to
FT suggests conserved biochemical action, but the action and regulation of these
proteins at the molecular level remain unclear [9–11].
In orchids, flower transition study is limited. In Phalaenopsis aphrodite,
FLOWERING LOCUS T1 (PaFT1) was isolated and functionally characterized
[22]. Its expression depends on the ambient temperature and photoperiod has
insignificant role [22]. In Dendrobium nobile, two genes homologous to
FLOWERING LOCUS (FT) and MOTHER OF FT (MFT) were isolated and
designated as DnFT and DnMFT, respectively. Expression and function of these
genes in regulating the transition from vegetative to reproductive stage were studied
[23]. In accordance with maturation, expression of DnFT was increased in leaves and
decreased in buds, while DnMFT level was decreased in leaves and increased in
buds. Low-temperature treatment enhanced DnFT expression in leaves and declined
in buds when compared to normal. But low temperature treatment has insignificant
effect on the expression of DnMFT in both leaves and buds [23]. In Arabidopsis, the
overexpression of DnFT induced flowering, which indicated that DnFT may act as a
158 S. G and S. S
promoter of flowering in D. nobile [23]. Ding et al. [24] isolated similar type of gene,
that is, DOSC1 from Dendrobium Chao Praya Smile; DOSC1 has been particularly
expressed in emerging floral meristems and created seven 35S:DOSOC1 transgenic
Dendrobium orchid lines, which consistently exhibited earlier flowering than wild-
type orchids, and it can be utilized for genetic manipulation of flowering time in
orchids. DOAP1, an ortholog of AP1, was also identified and characterized from
Dendrobium Chao Praya Smile [25], which also promote early flowering. Study on
differential gene expression in shoot apical meristem (SAM) during floral transition
in in vitro cultures of Dendrobium Madame Thong-In revealed that several tran-
scription factors, including a MADS-box gene of the AP1/AGL2 family, a class I
KNOX gene, and a homolog of the Drosophila SEVEN-UP gene, were differentially
expressed in vegetative and transitional SAM. The KNOX gene plays an important
role in the function of SAM and encodes a KNOTTED1-like homeobox (Knox)
protein later designated as Dendrobium Orchid Homeobox 1 (DOH1) [3, 26]. Over-
expression of antisense DOH1 resulted in early flowering in Dendrobium [3]. In
Oncidium, high temperature (30 C) promotes flowering. Both flowering promoter
FT and repressor TERMINAL FLOWER 1 (TFL1) have been identified in Oncidium
Gower Ramsey [3, 27]. The expression of OnFT is regulated by photoperiod but
expression of OnTFL1 is not influenced by photoperiod. The OMADS1, a homolog
of Arabidopsis AGL6, has also been identified in Oncidium Gower Ramsey [3] and
its overexpression in Oncidium resulted in precocious flowering [3, 28]. According
to Chin et al. [29], ascorbate/dehydroascorbate (AsA/DHA) redox ratio may act as
one of the endogenous signals that induce the flowering of Oncidium in response to
high ambient temperature [3]. Reduced GSH redox ratio caused by down-regulation
of GSH metabolism-related genes such as glutathione reductase (GR1),
γ-glutamylcysteine synthase (GSH1), and glutathione synthase (GSH2) was linked
to the decrease in the AsA redo redox ratio for flowering of Oncidium orchid
[3, 29]. A FVE homologue gene was isolated from Doritaenopsis “Tinny Tender”
(Doritaenopsis Happy smile Happy valentine) and designated as DhFVE [3, 30],
which has pivotal role in flowering time and cold response regulation. Besides,
EARLY FLOWERING 4 (EFL4) family genes, DhEFL2, DhEFL3, and DhEFL4,
have been identified in Dortiaenopsis [3, 31]. In Cymbidium and Erycina, many
putative flowering genes have also been identified through transcriptome analyses
[3, 32, 33].
4 Orchid Flower Development and MADS Box Genes
In plants, homeotic genes are called as MADS box, which is a conserved sequence
motif found in genes comprises of MADS-box gene family. The acronym MADS box
is derived from the initials of four loci, MCMI of Saccharomyces cerevisiae, AG of
Arabidopsis thaliana, DEF of Antirrhinum majus, and SRF of Homo sapiens, all of
which contain the MADS-box domain, a conserved 56 amino acid DNA binding
domain [34, 35]. To exemplify the functional activity of floral homeotic, various
flower-development models such as ABC, ABCE, ABCED, and floral quartet have
been suggested. Coen and Meyerowitz [36] described the genetic interactions con-
trolling flower development in Arabidopsis and Antirrhinum and proposed ABC
model. According to them, the genetic control of meristem behavior has two classes
of genes: those control identity of meristems and those determine the identity of
organs. The genes of both classes can be considered homeotic. A series of homeotic
mutations affect the identity of floral organs, sepals, petals, stamens, and carpels.
They define three regions of the floral meristem, each coincides with the domain of
action of one of the three classes of floral homeotic genes. Region A comprises whorl
1 (sepal) and 2 (petal), region B comprises whorl 2 (petal) and 3 (stamen), and region
C comprises whorl 3 (stamen) and 4 (carpel) [36]. The interactions of these genes
express a variety of unique phenotypic expression. For example, genes acting in
regions A, B, and C are required for three regulatory functions a, b, and c, respec-
tively, then the combination of functions in the four whorls of wild type would be a,
ab, bc, c. In principle, this might provide sufficient information to specify the identity
of organs in each whorl [36]. That is, sepals form if a alone is expressed, a and b
together direct petal development, b and c together specify stamens, and c expressed
alone determines carpel formation [36]. Further genetic analysis in Arabidopsis
thaliana that revealed the A function is mediated by APETALA1 (AP1) and
APETALA2 (AP2), the B function by APETALA3 (AP3) and PISTILLATA (PI),
and the C function by AGAMOUS (AG) [37]. All of these genes encode putative
transcription factors [38, 39], suggesting that ABC genes may control the transcrip-
tion of other genes (“target genes”) whose products are directly or indirectly involved
in the formation or function of floral organs. According to Irish [40] except for AP2,
all ABC genes encode MIKC-type MADS-domain proteins [37].
The ABC model has some limitations since it is not sufficient for the speciation of
floral organ identity. Subsequently, based on the studies in Petunia hybrid, ABC
model was extended to an “ABCD” model by addition of a D function specifying
ovule identity [37, 41]. In Arabidopsis thaliana, three genes closely related to AG,
namely, SEEDSTICK (STK; formerly known as AGL11), SHATTERPROOF1
(SHP1; formerly known as AGL1), and SHATTERPROOF2 (SHP2; formerly
known as AGL5) [42, 43], were identified as D-function genes; stk shp1 shp2 triple
mutants are characterized by conversion of ovules into carpel-like or leaf-like
structures [37, 43]. The C-function gene AGAMOUS was also considered as an
additional class D gene [37].
The ABCD model was further extended to ABCDE model based on the study in
Arabidopsis thaliana and Antirrhinum [44]. According to this model, the A genes
APETALA1 (AP1) and APETALA2 (AP2) control sepal development [28] and
together with B genes regulate the formation of petals (LIPLESS 1 & 2). The
B genes PISTILLATA (PI) and APETALA3 (AP3), together with C genes, mediate
stamen development (e.g., DEFICIENS (DEF) and GLOBOSA (GLO)). The C
genes AGAMOUS determine the formation of carpel and the D genes SEEDSTICK
and SHATTERPROOF specify the identity of the ovule. The E genes SEPALLATA
express in the entire floral meristem and are necessary (SEP1, SEP2, SEP3, and
SEP4) [34]. The controlling genes are A sepal and petal, B petal and stamen,
C stamen and carpel, D ovule specification, and E all floral organs.
160 S. G and S. S
Both ABC and ABCDE models relied on genetic data which cannot properly
explain the molecular mechanism of different floral homeotic genes’ interaction. To
overcome from this shortcoming instead of genes level, encoded proteins level is
considered that led to a new model called as floral quartet model (FQM). According
to the floral quartet model (FQM), the identity of the different floral organs is
specified during development by quaternary (tetrameric) protein complexes com-
posed of MIKC type MADS-domain proteins [37, 44]. These quartets are assumed
to function as transcription factors by binding to the DNA of their target genes,
which they either activate or repress to control the development of the respective
floral organs [37, 44].
Floral development in orchids has been investigated based on the forgoing
models by various authors. The ABC model [45] enumerated profiling of gene
expression in the transition of flower development in orchids by in vitro flowering
of Dendrobium species and later examined by the mRNA differential display method
[46]. It revealed that genes involved in the regulation of transcription, cell division,
and other metabolic processes are exhibited close association with transition process
of flower development in orchids [46]. MADS-box genes have been identified in
Aranda cv Deborah and Dendrobium cv Madame Thong-in [46, 47]. Gene expres-
sion investigation revealed that these genes have significant role in flowering.
5 Floral Whorl Development in Orchids – Class A MADS

Box Genes
It includes the phylogenetically related MADS-box genes of class A and class E

[34]. It involved in the floral meristem initiation and development and also deter-
mines floral organ development. Based on the ABCDE model, the class A and class
E MADS-box genes are necessary for the correct development of sepals, petals, and
floral meristem. Class A includes subfamily AP1/SQUA-like (from the APETALA1
and SQUAMOSA locus of Arabidopsis thaliana and Antirrhinum majus, respec-
tively), and class E includes subfamily SEP-like (from the SEPALLATA locus of
A. thaliana), subfamily SEP-like again includes SEP3, SEP1/2/4 (previously known
as AGL 9 and AGL 2/3/4 clades, respectively), and AGL6 clades [34]. Class A genes
have been identified and functionally characterized in many orchids such as
Dendrobium thyrsiflorum, Dendrobium Madame Thong-In, Oncidium Gower Ram-
sey, and Phalaenopsis amabilis, and class E includes Oncidium Gower Ramsey and
Dendrobium Madame Thong-In [34].
6 The Orchids Class B MADS-Box Genes
Based on the ABCDE model, the class B MADS-box genes are necessary for
the correct development of petals and stamens and include two major lineages, the
AP3/DEF-like genes (from the APETALA3 and DEFICIENS loci of A. thaliana and
A. majus, respectively) and the PI/GLO-like genes (from the PISTILLATA and
GLOBOSA loci of A. thaliana and A. majus, respectively) [34]. Class B MADS-box

orchid genes involve in the first whorl of floral organs that may be responsible for the
development of petaloid sepals in orchids. In several orchids viz. Cymbidium hybrid
cultivar, Dendrobium crumenatum, Dendrobium moniliforme, Gongora galeata,
Habenaria radiata, Oncidium Gower Ramsey, Phalaenopsis equestris, Phragmi-
pedium longiflorum, Spiranthes odorata, Vanilla planifolia, Epipactis palustris, and
Orchis italic, class B genes have been functionally characterized [34].
7 The Orchid Class C and D MADS-Box Genes
Within the ABCDE model of flower development, the class C genes regulate the
development of carpels and, together with the class B genes, of stamens. The class D
genes are primarily involved in the development of ovules [34]. Class C includes
AG-like and class D includes STK-like (SEEDSTICK) genes. Class C genes have
been characterized from the orchid species viz. Cymbidium ensifolium, Dendrobium
crumenatum, Dendrobium thyrsiflorum, and Phalaenopsis sp. Class D includes
Dendrobium crumenatum, Dendrobium thyrsiflorum, and Phalaenopsis sp. [34].
In orchids according to the ABCDE model, class B gene involved in the devel-
opment of outer whorl of sepals (tepals), inner whorl of petals (tepals), and third
whorl known as stamens. But in the development of modified petal (labellum), gene
involved has not been satisfactorily explained [34].
The recent theory known as “the orchid code” proposes an elegant model describ-
ing the development and evolution of the orchid perianth [1, 34, 48, 49]. This theory
explained about the genetic code attributes to the class B AP3/DEF-like genes a
pivotal role in tepal and lip identity and leaves unchanged the function of the class
B PI/GLO-like genes and the functions of the A, C, D, and E class genes with respect
to the modified ABCDE model [34].
In Arabidopsis, the identity of petals is realized through the interaction of one
AP3/DEF-like and one PI/GLO-like gene product, and the orchid code theory
suggests that the identity of orchid tepals and lips is determined by the interactions
of the products of four paralogous AP3/DEF-like genes belonging to four different
clades with the product of one PI/GLO-like gene. The orchid AP3/DEF-like genes
are grouped into four well-defined clades: clade 1 (PeMADS2-like) is sister to clade
2 (OMADS3-like), while clade 3 (PeMADS3-like) is sister to clade 4 (PeMADS4-
like). Each clade is characterized by a specific expression pattern [1, 34, 48, 49].
In orchid code theory, the interactions of the clade 1 and clade 2 gene products
mediate the development of the outer tepals (whorl 1). The formation of the two
lateral inner tepals (whorl 2) is specified by the interaction of high levels of the clade
1 and 2 and low levels of the clade 3 and 4 gene products, whereas the development
of the lip, which is a highly modified inner tepal, is determined by the expression of
high levels of the clade 3 and 4 gene products, in addition to low levels of clades
1 and 3 gene products. Thus, the expression of clade 3 genes differentiates between
the inner and outer tepals, whereas the expression of clade 4 genes distinguishes
between the two lateral inner tepals and the lip [1, 34, 48]. This proposed scheme can
162 S. G and S. S
also explain the evolution of the zygomorphic orchid flower, starting from an
actinomorphic flower composed of six nearly identical tepals in which the ancestor
of the current AP3/DEF-like genes was equally transcribed. The duplication and
evolution of different cis regulatory elements played a fundamental role in the
functional diversification of the four AP3/DEF-like orchid clades. An initial dupli-
cation event produced the ancestor of the clade 1 and clade 2 genes and the ancestor
of the clade 3 and clade 4 genes. At this stage, the evolution of a more specialized
expression of the ancestor of the clade 3 and 4 genes, which was excluded from the
outer tepals, might have established an intermediate flower structure, with distinctive
outer and inner tepals. After a second duplication round, clade 3 and clade 4 genes
differentiated, and the modularization of their expression led to the evolution of the
lip [34, 48, 49]. These are well explained through the EVO/DEVO molecular
approach [34].
Similar to MADS-box genes, class 1 knox genes are transcription factors
involved in floral development. During flower development, there is interaction
between these two genes [26]. In cv Madame Thong-In, down-regulation of the
expression of DOH1 gene, a class 1 knox gene, causes multiple shoot apical
meristem formation and early flowering, in association with the expression of
DOMADS 1 which is a MADS-box gene involved in the floral transition of
orchids [45, 46]. But in a wild-type orchid plant, DOMADS 1 gene expression
in shoot apical meristem during floral transition is associated with a marked
reduction in DOH1 gene and later both type genes are located at the same region
in the inflorescence meristem and the developing floral primordia [45, 46]. Com-
pared to other flowering plants, unique and different developmental programs may
be present in orchids due to the highly evolved floral structures, which are being
investigated [46]. In orchids MADS-box genes, DOMADS2 and DOMADS3
have shown novel expression patterns in the shoot apical meristem during floral
transition [45, 46, 50].
8 Ovule Development
Unlike other flowering plants, in orchids ovule maturation is elicited by pollination.

Genes associated with ovule development have been investigated in Phalaenopsis
spp. Isolation and characterization of several genes involved in ovule differentiation
provided that similar function of homologues in Arabidopsis also. Phalaenopsis O39
gene is a member of a new class of plant home box transcription factors designated
HD-GL2 and expressed in ovule from primordium formation at early stages to
various late stages of ovule differentiation. So it is an important regulator gene
involved in the ovule initiation and development [46, 51]. Role of ethylene in ovary
maturation and ovule differentiation in orchids has been extensively investigated by
both physiological and molecular methods [46, 51, 52]. Even though there is
coordinated regulation of ACC synthase and ACC oxidase gene expression in the
ovary [52], the molecular events triggered by pollen-pistil interactions leading to
these gene expressions are not well understood [46].
9 Perianth Senescence/Development
Wilting and drying up of calyx and corolla after pollination is a key indication of
successful pollination in orchids. The exact reason for this senescence is explained as
the resources are directed for ovule development and embryogenesis after fertiliza-
tion. The physiological and molecular mechanisms of pollination-induced senes-
cence have been studied in orchid species Phalaenopsis and Dendrobium, regarding
ethylene sensitivity and production. Initial event triggering is an increase in ethylene
production and the consequent physiological changes of flower [46, 53]. The iden-
tification of sensitivity factors such as GTP binding protein [54], short-chain satu-
rated fatty acids [55], and auxin [56] gives way to the elucidation of the mechanisms
under the regulation of ethylene sensitivity [46]. In orchid flowers, after pollination,
there is an increase in ethylene production. In Phalaenopsis spp., the production of
abundant ethylene in the perianth up to 72 h after pollination was observed but not
the accumulation of ACC synthase. But ACC oxidase expression is up-regulated in
the petals and sepals about 48 h after pollination in parallel with the onset of perianth
senescence [46]. Both ACC synthase and ACC oxidase are positively regulated by
increased ethylene production [46, 52] in orchids.
In some Phalaenopsis species (subdivision stauroglottis), the sepals and petals
turn green and photosynthetic following successful pollination. These organs
become leaf-like and provide photosynthates for the developing ovules/ovary and
the embryos subsequent to fertilization over an extended period of many months
until the capsules are mature. The molecular genetics for this transformation of the
perianth from an energy sink to an energy source during postpollination develop-
ment is interaction of ABC genes [46, 57].
10 Genetic Variation Versus Secondary Metabolite

and Adaptations
Some sudden or gradual changes in traits occurred in individuals, like mutation, as

well as the influence of various types of stresses may induce alteration in plant
metabolism which leads to the production of various types of plant secondary
compounds and those phytomolecules help to overcome the adverse effects or
ensure lively functions. Secondary compounds impart flower color, volatile fra-
grance, food flavor, attract pollinators, interaction with symbiotic microorganisms,
and tolerance against pest and diseases (isofalvonoids and phenylpropanoid deriv-
atives). These are also take part in the mechanism of frost tolerance, nutrient storage,
structural reinforcement, photo protective, UV–Vis absorption, signaling to mutual-
ist, and in the adaptation of the plant under various environmental conditions and
stress [58, 59, 60]. Besides, these phytomolecules have medicinal properties and
majority of the currently used lifesaving drugs are derived from the natural source
like plants. Production of the metabolites by the plants is regarded an adaptive
capacity to cope up with stressful constraints during challenging and changing
environment of growth that may involve production of complex chemical types
164 S. G and S. S
and interactions in the structural and functional stabilization through signaling

processes and pathways [61, 62].
The unique adaptations evolved in orchids are a curiosity among the biologist but
the underlying mechanism is still unclear. For over a century, it has been debated
whether adaptations are likely caused by a large number of mutations of small
phenotypic effect or by very few genetic changes of large effect [63]. The vegetative
modifications among the orchids to thrive as lithophytes and epiphytes or under
drought condition might cause many changes in physiology and biochemistry of
plants, including arrest of cell growth and alteration of photosynthesis mechanism
(CAM plant) with an enhanced respiration [62, 64], which thus may affect biosyn-
thetic pathways for the production of PSMs through provision of precursors or
intermediates from the primary metabolism [62]. Many authors have reported that
plants under drought condition produced higher rate of different categories of plant
secondary metabolites such as terpenes, complex phenols, and alkaloids during
in vitro and in vivo growth through the induction of ionic or osmotic stress
[62, 65–69]. Increasing antioxidant enzyme activities and osmolytes play an impor-
tant role in protecting plants under drought stress [62]. In many orchids, the
foregoing phytochemicals are reported, for example, alkaloids, triterpenoids, tan-
nins, phenols, steroids, flavonoids have been reported from the leaves of
Dendrobium panduratum subsp. villosum [70]. In Dendrobium ochreatum, presence
of glycosides, alkaloids, flavonoids, tannins, and phytosterol were reported by
Banerjee et al. [71]. Leaves and roots of six Phalaenopsis hybrids, namely Green
Field Sweet Valentine “Montclair,” Sakura Hime, Sogo Yukidian “V3,” Chian Xen
Queen, Fusheng‘s Bridal Dress “Meidarland,” and Younghome Golden Leopard
“Peachy,” contain high amounts of phenolic compounds with strong antioxidant
activity. Ferulic acid, p-coumaric acid, and sinapic acid were accumulated largely in
the roots in comparison with the leaves of these hybrids [72]. Hence, the root extracts
of these hybrids may be used as a potential source of natural antioxidants [72]. In
Phalaenopsis Sogo Yukidian “V3” roots, stems and leaves are the rich source of
phenolics, flavonoids, and antioxidants, and also isolated five phenolic compounds
including caffeic acid, syringic acid, vanillin, ellagic acid, and cinnamic acid and
found that the plant parts have potent antioxidant activity and may be utilized
as an efficient and safe antioxidant source [73]. In five different species of
Dendrobium, namely, D. nobile, D. fimbriatum, D. moschatum, D. chrysanthum,
and D. chrysotoxum, content of terpenoids (ursolic acid, β-sitosterol, and lupeol) and
a phenolic compound (gallic acid) were analyzed in different plant parts such as
stem, leaves, and roots, and it was found that all samples were good source of
β-sitosterol with maximum content in D. fimbriatum stem and minimum content in
D. chrysanthum roots. D. nobile (roots and stem) and D. moschatum (stem) were
also found to be a source of ursolic acid and lupeol, respectively [74]. Perusal of the
literature revealed that about 100 compounds from 42 Dendrobium species including
32 alkaloids, 6 coumarins, 15 bibenzyls, 4 fluorenones, 22 phenanthrenes, and
7 sesquiterpenoids constituents have been reported [75]. Williams [76] conducted
a major survey on 142 orchid species belonging to 75 genera and found that the most
common constituents were flavone C-glycoside and flavonols [75].
The diversity of floral color, shape, and fragrance is the key factor that makes the
orchid flowers a distinctive one. In most species, the sepals are just uniformly green
and do not contribute to interesting color patterns. Orchids are exceptional in that the
sepals and the lip are usually as colorful as the petals, which results in an unbalanced
pigment distribution among different segments of the perianths and lip to show
various flower color patterns [77]. The flower color is mainly attributed by pigmen-
tation such as flavonoids, carotenoids, and betalains. Chlorophyll also plays a
significant role in floral whorl pigmentation in orchids. Betalains are red colored
pigments substituted by anthocyanins exclusively in the Angiosperm order
Caryophyllales and certain fungus belonging to the group Basidiomycetes. These
pigments are structurally and biosynthetically distinct from flavonoids and anthocy-
anins [78]. In orchids, presence of betalains is not so far reported. Flavonoids are low
molecular weight natural compounds with varying phenolic structures. They are the
major floral pigments which provide a wide spectrum of coloration. There are about
6000 flavonoids that contribute to the colorful pigments of fruits, herbs, vegetables,
and medicinal plants [79]. Among flavonoids, anthocyanin belongs to the red series
and controls pink to blue-violet flower colors. Other flavonoids belong to the pure
yellow series, among which chalcone and aurone are deep yellow, and flavones,
flavonols, and flavanones are light yellow or nearly colorless [80].
The initial step of flavonoid synthesis is the condensation of three acetate units
and a hydroxycinnamic acid unit and formed chalcone, the key intermediate in the
synthesis of flavonoids [81]. Chalcone itself acts as a pigment having yellow or
orange in color and it may be converted into bright yellow aurone. Usually, however,
chalcone is modified to a colorless flavanone, and flavanone may be directly
converted into flavones, which vary in color from very pale to bright yellow,
depending on their degree of hydroxylation. Alternatively, flavanone can be
converted into dihydro flavonol, which can then be modified by flavonol synthase
to various flavonols. The flavonols are usually colorless, but act as co-pigments,
stabilizing and modifying the color of other pigment molecules. Alternatively,
dihydroflavonols may be modified through a number of steps to make anthocyanins
[82]. In orchids, anthocyanins are the widely distributed pigments and produce red,
pink, purple, black, and blue coloration. Carotenoids are lipophilic isoprenoid
compounds responsible for yellow, orange, and red coloration; they also contribute
to brown and bronze hues in combination with anthocyanin. Although many study
reports are available for describing the role of pigments and floral coloration in many
crops, the study in this line in orchids is limited.
The main pigments (anthocyanin, beta-carotene, and chlorophyll) in the petals of
six different orchid species such as Mokara Pink, Mokara Aranda, Mokara Gold
Nugget, Ascocenda Dong Tarn, Dendrobium Sonia 17, and Dendrobium Shavin
White and the phenylalanine ammonia-lyase (PAL) activity were investigated
[83]. Petals that have intense color have high amount of anthocyanin content,
whereas those pale in color have high amount of chlorophyll content. PAL activity
was shown to be positively correlated with the total anthocyanin content in
the orchid flower petals [83] since anthocyanin is synthesized throughout
phenylpropanoid pathway which is mainly catalyzed by PAL [84]. Matsui and
166 S. G and S. S
Nakamura [85] studied the distribution pattern of flower pigments and shape of
epidermal cells in perianth tissues in 68 orchid species and reported that the yellow,
orange, and purple color of the flowers depend on the differential distribution pattern
of anthocyanins and carotenoids in the epidermal and parenchymatous cells. Red
flowers were ascribed to the coexistence of carotenoids and anthocyanins. Under-
standing of pigmentation and its inheritance patterns may contribute to the breeding
programs [85].
Besides flower color, nectar and volatile compounds produced from the floral
secretary glands/cells and pollen play crucial role in the reproductive biology by
attracting pollinators. Nectar and pollen are the food resources of various insects,
which consist of sugar, amino acids, sterols, lipids, and vitamins. Other minor
secondary metabolites such as organic acids, terpenes, alkaloids, flavonoids, and
glycosides are also present at various level [86]. The specificity of floral scent
emitted by different species of flowers epitomises key floral signals to the particular
insect to direct towards rewarding flower species.
Genes that encode enzymes of the flavonoid pathway affect flower pigmentation
in orchids [87, 88]. These genes include chalcone synthase (CHS), chalcone isom-
erase (CHI), flavanone-3-hydroxylase (F3H), flavonoid 30 -hydroxylase (F30 H) and
flavonoid 30 ,50 -hydroxylase (F30 50 H), dihydroflavonol 4-reductase (DFR),
anthocyanidin synthase (ANS), UDP-glucose: flavonoid 3-o-glucosyltransferase
(UFGT) and methyl transferase (MT) [80]. For examples, based on homology
analysis of the Phalaenopsis genome, a total of 49 genes which include all above
category were identified [89]. DFR genes were cloned from Freesia hybrid [90],
Bromheadia finlaysoniana [87], Cymbidium “Rosannagirl Mild” [88], Oncidium
Gower Ramsey [91], Dendrobium [92], Dendrobium Sonia “Earsakul” and
Dendrobium Red Bull [93], Dendrobium helix x cv. Pomeo Brown hybrid
[94]. DFR, CHS, and F3’5’H genes were isolated from Dendrobium moniliforme
[95], and F3H genes were isolated from Ascocenda orchid, which showed homology
to F3H from Bromheadia finlaysoniana [96]. Identification of additional regulatory
genes in the flavonoid pathway is essential. Further investigations of their regulatory
mechanisms and pathways will provide strategies toward genetic engineering of
color in orchids [57].
Orchid flowers are unique for their specific patterns of colors in sepals, petals, and
the modified dorsal petals (lips). These are discrete spots, streaks, or blotches. The
patterns on the lips of some species are strikingly contrasting to serve as the landing
platform for insect pollinators. The regulation of pigmentation is refined to specific
cells of the different floral organs, and the expression of genes involved in flavonoid
synthesis is the initial steps in the complex regulation of pigmentation [57].
11 Conclusion
The unparalleled adaptive features and evolutionary developments in orchids par-

ticularly to thrive under adverse environmental conditions and its instinctive genetic
mechanism evolved through evolutionary process to survive in different habitats are
always generate curiosity and give significant insights to the biologist. Knowledge
about underlying genetic mechanisms behind the vegetative and reproductive biol-
ogy particularly in floral development, pollination mechanism, seed production,
dissemination, and germination is meager and in-depth studies in this line certainly
ensure novel insights in evolutionary biology of this unique ornamental crop.
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Part II
Biology
Orchid-Associated Bacteria and Their Plant
Growth Promotion Capabilities 6
Héctor Herrera, Alejandra Fuentes, Javiera Soto, Rafael Valadares,
and Cesar Arriagada
Contents
1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 176
2 Plant Growth–Promoting Bacteria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 177
3 Mycorrhiza Helper Bacteria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 179
4 Methods for the Study of Orchid-Associated Bacteria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 181
5 Diversity of Orchid-Associated Bacteria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 182
5.1 Seed-Associated Bacteria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 183
5.2 Phyllosphere-Associated Bacteria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 188
5.3 Rhizosphere-Associated Bacteria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 189
5.4 Root Endosphere–Associated Bacteria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 190
5.5 Fungi-Associated Bacteria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 191
6 Management of Root-Associated Bacteria in Cultural Practices . . . . . . . . . . . . . . . . . . . . . . . . . . . 192
7 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 193
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 193
Abstract
Orchids are one of the most widespread plants inhabiting lands in almost all
terrestrial ecosystems. Most plants from the Orchidaceae family depend on
specific mycorrhizal fungi to germinate their tiny and dust-like seeds. There,
mycorrhizal fungi provide the resources that the embryo needs to advance to
further developmental stages. However, at seed germination and the plantlet stage
orchids need other nutrients to sustain the plantlet development and completion of
its life cycle. There, other soil-borne beneficial microorganisms, such as free-
living and symbiotic bacteria, can associate with orchid roots establishing a
special interaction that has scarcely been explored. Most of the beneficial
H. Herrera · A. Fuentes · J. Soto · C. Arriagada (*)

Laboratorio de Biorremediación, Facultad de Ciencias Agropecuarias y Forestales, Departamento
de Ciencias Forestales, Universidad de La Frontera, Temuco, Chile
e-mail: hector.herrera@ufrontera.cl; alejandra.fuentes@ufrontera.cl; cesar.arriagada@ufrontera.cl
R. Valadares
Instituto Tecnologico Vale, Belém, PA, Brazil

176 H. Herrera et al.
root-associated bacteria can help plants solubilize essential nutrients such as

phosphorous and nitrogen, as well as to produce diverse beneficial metabolites
that can contribute to improving biomass production, stress tolerance, and bio-
control of potential phytopathogenic fungal species. This chapter will summarize
the principal studies performed in orchid-associated bacteria and analyze their
potential plant growth–promoting capabilities.
Keywords
Bacteria · Endosphere · Rhizosphere · Plant growth promotion · Symbiosis
1 Introduction
Orchids belong to the widespread Orchidaceae family, which includes about 899
genera and 27,801 species distributed in almost all terrestrial ecosystems [1–3].
Orchids can grow in soil (terrestrial), rocks (lithophytes), or tree trunks (epiphytes)
[4]. According to their physiological mode of carbon nutrition, orchids can be
classified as: (i) photoautotrophic, which can obtain their own carbon throughout
photosynthesis; (ii) mycoheterotrophic, which lack a photosynthetic apparatus and
fully depend on the carbon obtained via a compatible mycorrhizal fungus; and (iii)
partially mycoheterotrophic, which can obtain carbon from both photosynthesis and
mycorrhizal fungi [5–7]. A common characteristic of orchids is the production of a
capsule with thousands of dust-like seeds, with a nutrient-poor endosperm which
lacks the essential nutrients to sustain the initial plant growth [8, 9]. These seeds are
dispersed into the environment and once they reach the growth substrate they must
find a compatible mycorrhizal fungus able to provide the carbon necessary to start
the seed germination process [10, 11]. After that, a mycoheterotrophic organ known
as protocorm is formed. At the protocorm stage, the mycoheterotrophic processes are
completely active and the fungi provide almost all the nutrition for the embryo [12,
13]. Once the plantlet stage is reached, the mycoheterotrophic processes can be
progressively reduced, especially in autotrophic or partially mycoheterotrophic
species. At seed germination, plantlets develop and in the adult mature plant, orchids
must associate with other fungal and bacterial taxa that can synergistically contribute
to plant growth and development, providing essential nutrients such as nitrogen (N),
protection against disease and phytopathogens, stress tolerance, and others [14–16].
The orchid mycorrhizal fungi live inside root tissues, inhabiting specific symbi-
otic structures known as pelotons [17, 18]. Such structures are essential for orchid
nutrition, playing key roles in resource mobilization and orchid development [19].
Commonly, the mycorrhizal fungi associated with orchids are included in the
Rhizoctonia-like fungi complex, which can involve Basidiomycetes from the genera
Ceratobasidium, Tulasnella, Thanatephorus, Sebacina, and Serendipita [20, 21].
However, nowadays our understanding of mycorrhizal fungi associated with orchids
has grown considerably and they can include different ectomycorrhiza-forming
fungi, wood- or litter-decomposing fungi, and a broad spectrum of other different
6 Orchid-Associated Bacteria and Their Plant Growth Promotion Capabilities 177
endophytic fungi without a specific role for seed germination or development [4, 22,
23]. Despite our understanding about fungi-associated microorganisms and their
role in the orchid metabolism having been largely addressed [24–26], the study
of orchid-associated bacteria and their benefits for orchids are mainly unknown
[14]. Bacteria are commonly isolated from soils and can also live in association
with other organisms, playing essential roles in the ecosystems [27, 28]. Specif-
ically, several studies have demonstrated that plants are commonly associated
with free-living or symbiotic bacteria and these microorganisms can have posi-
tive effects on the metabolisms of the associated plant [28–30]. In this chapter,
we will summarize the principal studies performed on orchid-associated bacteria
and discuss their putative function on orchids, with emphasis on plant growth–
promoting mechanisms.
2 Plant Growth–Promoting Bacteria
Bacteria that positively influence plant growth are categorized as plant growth–
promoting bacteria (PGPB) and include those that are free-living and those that form
specific symbiotic relationships with plants (e.g., Rhizobia spp. and Frankia spp.)
[31]. They can have beneficial effects on plant growth via direct and indirect
mechanisms [32, 33]. Despite the differences between symbiotic and free-living
bacteria, they all utilize the same mechanisms to promote plant growth: (i) directly
by facilitating the supply of nutrients to the plant (N fixation or phosphate solubi-
lization), increasing iron (Fe) bioavailability (through siderophore production),
producing phytohormones like auxins, or modulating hormone levels such as ethyl-
ene through enzyme 1-aminocyclopropane-1-carboxylate (ACC) deaminase; or (ii)
indirectly acting as biocontrol agents by controlling phytopathogenic soil-borne
microbes, for instance, by producing antibiotics, hydrogen cyanide, or enzymes
with the ability to hydrolyze the fungal cell wall, and stimulating mycorrhizal
processes [34, 35].
PGPB should be capable of supplying host plants with additional nutrients or
facilitating the acquisition of existing ones, such as potassium (K), N, phosphorus
(P), or Fe from the growth substrate. Some bacteria are able to fix N2 (conversion of
atmospheric N to ammonium) and supply N to plants. Diverse genera such as
Azotobacter, Azospirillum, and Rhizobium have known roles in plant growth pro-
motion by N fixation [36–38]. Among N-fixing bacteria, some are free-living and do
not require a host to perform the process, whereas others are symbiotic and only fix
N in association with certain plants in specific root structures, such as leguminous
[39]. P is usually found strongly adhered to the soil colloids in insoluble forms, thus
not available for plants. Some bacteria have the ability of dissolving inorganic
phosphates helping plants to cope their P needs. Such bacteria comprise genera
such as Burkholderia and Serratia, among others [40–42]. Several genera of
rhizobacteria (Bacillus, Pseudomonas, Burkholderia, Agrobacterium, and Rhizo-
bium) are involved in the solubilization of K in the soil by converting it to soluble
forms through release of organic acid [43–45]. These microbes play a vital role in the
release of nutrients from soil adhered to mineral surfaces, living in association with
plants or beneficial soil-borne fungi and contributing to growth and development
[46, 47].
Siderophores are low-weight molecules (between 500 and 1500 Da) that possess
great affinity and selectivity to binding and complex Fe [48]. Bacterial siderophores
make Fe available to plants via two methods: one is by a ligand exchange reaction
and the other is by direct uptake of siderophore-Fe complexes [49]. The primary
function of these compounds is to chelate the ferric Fe [Fe(III)] from the environ-
ment and thereby make it available for microbial and plant cells [50]. Bacterial
siderophores excreted by various genera such as Pseudomonas, Rhizobium, Strep-
tomyces, Azotobacter, Bacillus, Burkholderia, and Serratia are known to sequester
Fe and benefit plant growth [51–53]. Studies in orchid nutrition reporting on the
interaction among nutrients and their ideal balance within this plant are scarce [54].
However, it is known that Fe is the third most limiting nutrient for plant growth and
metabolism, primarily due to the low solubility of the oxidized ferric form in aerobic
environments [55]. As a critical component of proteins and enzymes, Fe plays a
significant role in basic biological processes such as photosynthesis, chlorophyll
synthesis, respiration, N fixation, uptake mechanisms, and DNA synthesis [56, 57].
It is a cofactor of many enzymes that are necessary for plant hormone synthesis, such
as ethylene, lipoxygenase, ACC oxidase, or abscisic acid [58].
The phytohormones auxins control and model several aspects on plant, such as
cell expansion and division, cell elongation and differentiation, and a variety of
physiological responses that result in improved plant growth [59]. Among auxins,
the most common is indole-3-acetic acid (IAA), which is known to be produced not
only by plants but also by bacteria [60]. Other phytohormones are salicylic acid and
abscisic acid, which are particularly known for regulating the defense response in
plants against pathogens [61, 62]. Another important plant growth regulator is
ethylene, which controls the growth of roots, leaves, flowers, and fruits [63].
Ethylene is derived from ACC and is produced in plants as a response to multiple
stresses [63]. Thus, different environmental stresses such as metals, chemicals,
water, and extreme temperatures induce ethylene biosynthesis through the induction
of ACC synthase [63]. When in excess, ethylene may be toxic to the plant, causing
defoliation and other harmful effects [64]. ACC deaminase (ACCD) is an enzyme
produced by some bacteria such as Bacillus, Pseudomonas, and Azotobacter, among
others [65–67] and is considered as a key characteristic for PGPB strains [68].
ACCD is beneficial for plants as it decreases ACC levels by cleaving it into ammonia
and α-ketobutyrate. This helps to reduce the adverse effects of ethylene by reducing
its levels. Therefore, PGPB capable of producing ACCD are beneficial for plants
grown under stress conditions such as drought, salt, and heavy metals by regulating
the plant’s ACC levels [65, 69, 70] and thus reducing the ethylene to nontoxic levels.
PGPB can grow in most plant structures (flowers, fruits, stems, leaves, and roots)
[71], and can be localized inside or outside the plant cells (vascular tissues or seeds)
[72, 73]. In the soil, microorganisms play important roles in the ecosystem,
interacting closely with plant roots and soil nutrients [74]. The rhizosphere is an
area of intensive molecular interaction between plant roots and soil colloids. It is
defined as a narrow zone of soil that surrounds the plant roots and which is rich in
nutrients compared to bulk soils, showing intense biological and chemical activities
[75]. Over the years, the rhizosphere definition has been refined to include three
zones which are defined based on their relative proximity to the root [76]: (i) the
endorhizosphere, which includes the endodermis and cortical layers; (ii) the rhizo-
plane, known as the root surface where soil particles and microbes are adhered,
including the epidermis, cortex, and a mucilaginous polysaccharide layer; and (iii)
the ectorhizosphere, the outermost zone which extends from the rhizoplane out into
the bulk soil. The volume of the soil which is not a part of the rhizosphere and is not
influenced by any plant root is known as bulk soil [77]. During the course of growth
and development of plants, a variety of organic compounds are released from the
roots by exudation, secretion, and deposition making the rhizosphere rich in nutri-
ents as compared to the bulk soil [75]. This acts as a driving force for the setup of
active and enhanced microbial populations in the root zone, much higher than the
bulk soil [78, 79]. Plants secrete low-molecular-weight compounds, such as amino
acids, sugars, phenolics, terpenoids, and lipids, and high-molecular-weight com-
pounds, including proteins, polysaccharides, and nucleic acids, depending on the
growth stage and environmental conditions [80]. Upon secretion into the rhizo-
sphere, most metabolites are rapidly degraded by soil microbes, but some, particu-
larly specialized metabolites, remain in the soil and mediate biological
communication [81, 82]. Microbial diversity is reduced near the roots, with further
reduction in the endosphere [83, 84]. In this sense, the communities of microorgan-
isms which reside in plants for at least a specific part of their life cycle are referred to
as endophytes [85, 86]. Endophytes are often classified as obligate or facultative.
Obligate endophytes are inherently dependent on their host, and their transmission to
other plants occurs vertically or via vectors [87], whereas facultative endophytes
have a biphasic lifestyle and can also survive in other habitats, such as the plant
surface or soil [88]. Inside the host plant, endophytes can colonize both intercellular
or intracellular spaces [89]. Endophytic communities are affected by several biotic
and abiotic factors, such as plant species, developmental stage, or interactions with
other plant-associated microorganisms, temperature, radiation, and physical or
chemical attributes of the soil [90, 91]. The roles of endophytes inside the plant
are varied and include nutrient acquisition, such as N fixation and phosphate
solubilization, phytohormone and siderophore production, and protection against
abiotic stresses (i.e., salinity, drought, or pollution) or phytopathogen control [87].
3 Mycorrhiza Helper Bacteria
Other indirect beneficial bacteria associated with plants are mycorrhiza helper
bacteria (MHB). Soil microorganisms have significant effects on the physiological
state of plants and productivity. Root-associated fungi and bacteria can establish
mutualistic associations with plants, the most studied being mycorrhizal symbiosis,
an association between filamentous fungi and most plant roots [92]. More than 90%
of plants can establish these root-fungus associations, being established as the most
widespread and ancient in the biosphere [93, 94]. As a general rule, the host plant
provides the fungal associate carbohydrates from photosynthesis, while the fungus
improves the acquisition of essential nutrients and water for the plants [95].
According to the root colonization strategy, two main types of mycorrhizae are
commonly recognized: (i) ectomycorrhizae, in which the fungus colonizes the
intercellular spaces of the root tissues, basically growing around it [96]; and (ii)
endomycorrhizae, in which the fungus develops within the cortical cells [97].
Endomycorrhizae are further subdivided into specific types, the most recognized
being arbuscular, ericaceous, and orchid mycorrhizae [98].
A group of specific soil bacteria colonizes the mycorrhizosphere, defined as the soil
zone influenced by the roots and hyphae of external mycelium of the symbiotic fungus.
These bacterial communities are known as MHB, establishing a complex physical and
metabolic interaction producing beneficial effects for the mycorrhizal symbiosis [99].
The main effects through which MHB can favor the establishment of mycorrhizal
symbiosis can be summarized as: (i) increasing survival and germination of fungal
spores or other reproductive structures; (ii) stimulating presymbiotic growth mycelium;
(iii) increasing root receptivity since bacteria proliferate in the rhizosphere prior to the
development of the symbiotic fungus; (iv) stimulating the root-mycelium recognition;
(v) changing physicochemical properties in the mycorrhizosphere; and (vi) reducing
soil-mediated stress by biotic and abiotic factors through detoxification and phytopath-
ogen control [100–102]. Although the role of MHB associated with orchid remains
largely unexplored, the high diversity of root-associated bacteria growing in the orchid-
growing substrates may represent an opportunity to study and design strategies to
explore the beneficial potential of such microorganisms for mycorrhizal orchid plants.
MHB can produce diverse bioactive compounds including phytohormones and
enzymes. The enzyme ACCD prevents the ACC produced by the plant from being
transformed into ethylene, a key regulator in the stress response in plants [103]. In
addition to their role in tolerance to environmental stress, ACC deaminase-produc-
ing bacteria improve nodulation and mycorrhizal colonization in the plant [104].
Other enzymes produced by MHB seem to have a positive role in the colonization of
the root cortex due to their ability to secrete enzymes such as endoglucanases,
cellobiose hydrolases, pectatoliases, and xylanases, which softens the cell wall,
improving the receptivity of the plant tissues [105].The phytohormone IAA is the
most important signal molecule and is involved in the processes of cell division,
elongation, and differentiation, promoting root elongation and proliferation of sec-
ondary roots [106]. Some MHB have the ability to produce large amounts of IAA,
which promotes the development and growth of the hypha [107].
MHB can be found in many genera of gram-negative and gram-positive bacteria
[100]. Many of them belong to Proteobacteria (Azotobacter, Klebsiella, and Pseu-
domonas), α-Proteobacteria (mainly Azospirillum, Bradyrhizobium, and Rhizo-
bium), β-Proteobacteria (Burkholderia), Firmicutes (mainly Bacillus, Brevibacillus,
and Paenibacillus), and Actinobacteria (mainly Streptomyces, Rhodococcus, and
Arthrobacter) [105]. These genera are frequently recognized as having a direct effect
on plant growth-promoting solubilization and mineralization of phosphate, N fixa-
tion, secondary metabolite production, phytohormone synthesis, and secretion of
siderophores, among others [31].
The tripartite interaction between plant-mycorrhizal fungi-bacteria is still scarcely

explored. The study of the functions of MHB, their cultivation, and determination of
their potential can be of great utility to improving the growth and development of the
mycorrhizal fungus and the plant. This issue can be relevant for sustainable produc-
tive practices as well as in ecological restoration programs based on an efficient
management of the microbial components of the soil, biodiversity, and the symbiotic
interactions that occur there.
4 Methods for the Study of Orchid-Associated Bacteria
For the isolation and identification of culturable bacteria, it is necessary to have pure
cultures of the strains, for which the technique of serial dilution and spreading onto
agar nutrient media is the most standard [108]. For the isolation of rhizospheric
microorganisms only the soil firmly attached to the roots is considered [109]. The
procedures for isolating endophytic bacteria involve a crucial step of surface disin-
fection to eliminate saprophytes or other microorganisms associated with the surface
of the tissue, including leaves, stems, bark, roots, fruits, and seeds [110]. Common
culture media used for the isolation of plant-associated bacteria are Luria-Bertani
agar, tryptic soy agar, yeast extract sucrose agar, nutrient agar, and others. To limit
fungal growth, the media should be amended with antifungal agents such as cyclo-
heximide, benomyl, benzalkonium chloride, captan, and others [111, 112]. The study
of the diversity of root-associated bacteria has been conducted using mainly these
culture-dependent techniques. The identification of the isolated strains can be carried
out based on the morphological and biochemical characterization. However, the
culture-dependent methods present discrepancies between the observable morpho-
logical and/or phenotypic characteristics of the isolates, which means that the same
strain can generate different results in repeated tests [113]. Currently for the identi-
fication of bacteria, complementary genotypic methods are used and can be divided
mainly into two categories: (i) pattern or fingerprint-based techniques (such as
phospholipid fatty acid, denaturing gradient gel electrophoresis, single-strand con-
formation polymorphism, terminal restriction fragment length polymorphism, and
amplified fragment length polymorphism) and (ii) sequence-based techniques (such
as metagenomics, pyrosequencing, metatranscriptomics, and transcriptomics). Both
techniques generate a database of fingerprints or specific DNA sequences against
which the test organisms or sequences can be compared. The degree of similarity or
coincidence is a measure of how related the two organisms are to each other [114].
For the molecular identification of bacterial species based on the nucleotide
sequence, a wide variety of genes have been used, such as 16S–23S rRNA intergenic
space (ITS), rRNA 23S, RpoB (β subunit of RNA polymerase), and GyrB (ß subunit
of DNA gyrase) [115–117]. However, the 16S rRNA analysis has proven to be the
most efficient gene for identification; due to its highly conserved analysis, it is
possible to obtain information on phylogenetic relationships to classify and identify
different prokaryotic species [118].
The new proteomic tools, based mainly on mass spectrometry, have made it
possible to complement the classical techniques of microbiology and genomics for
the classification, identification, and phenotypic characterization of bacteria [119].

Bacterial identification using ribosomal protein profiling using matrix-assisted laser
desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has been
commonly applied to accurately identify microorganisms given that the mass spec-
trum is specific for each species [120]. The results are expressed as a homology
percentage for sequencing and in an absolute value for the MALDI TOF and allows
the proximity of the proposed profile to the data banks to be determined. Among the
main advantages of this technique is speed and accuracy, in addition to the low
operating cost [121].
In recent decades, the greatest advances in molecular techniques have shown
that only a small fraction of microorganisms can grow in culture media [122] and
95–99% of microorganisms in any environment are uncultured. Regarding soil
bacteria, only 1% can be cultured by traditional techniques, which is not represen-
tative of the total phylogenetic diversity [123]. Culture-independent techniques are
used to investigate the microbial diversity of a variety of environmental niches
including the root-associated bacteria (rhizosphere and endosphere). Denaturing
gradient gel electrophoresis (DGGE) allows as much as 95–99% of the bacterial
community to be detected. This technique separates genes of the same size that differ
in their profile of denaturation due to differences in the nucleotide sequence [124].
However, it presents disadvantages at the time of analysis such as the presence of
artifacts which have been a serious problem for genetic diversity analyses [125].
Currently, next-generation sequencing techniques (NGS) allow massive parallel
sequencing with a high throughput of amplicons at low cost compared to those
based on the Sanger method. Metagenomic approaches analyze the composition and
diversity of a microbial community with the aim of detecting genetic diversity,
metabolic routes, and access to complete genes or sequences of the sample [109,
126]. For a bacterial community study, the 16 rRNA ribosomal subunit gene is used
as a target. It consists of nine hypervariable regions flanked by conserved regions, of
which the hypervariable regions are specific to each genus and species [127]. In
addition, other advantage of 16S rRNA gene is the availability of several databases
of reference sequences and taxonomy [128]. On the other hand, shotgun meta-
genomics and pyrosequencing technologies are alternatives that provide more com-
plete data about diversity and metabolic functional features present in microbial
communities [129]. Additionally, they describe the corresponding genes in an
ecosystem, facilitating the characterization of the potential functionality of the
microbiome in specific environments [130]. Thus, these methodologies can be
important for the study of orchid-associated bacteria and their metabolic effects on
the plant.
5 Diversity of Orchid-Associated Bacteria
Like most land plants, orchids also have associated bacteria inside their structures.
Although non-culture-dependent methods have increased our understanding of root-
associated bacteria in several plants, these NGS methodologies have been scarcely
explored in plants from the Orchidaceae family. Recently, Alibrandi, Schnell [131]
analyzed the endophytic bacterial diversity of the Mediterranean orchids Neottia
ovata (L.) Bluff and Fingerh., Serapias vomeracea (Burm.f.) Briq., and Spiranthes
spiralis (L.) Chevall. using metabarcoding of the 16S rRNA gene. That study
identified Proteobacteria and Actinobacteria as principal phyla associated with the
analyzed orchids, with less richness and diversity in the aerial biomass than in the
roots. Similarly, Li, Xiao [132] used a metagenome pyrosequencing-based approach
to identify bacterial communities associated with Dendrobium catenatum Lindley,
identifying the genera Pseudomonas, Escherichia/Shigella, Delftia, and
Burkholderia as the dominant taxa in roots, leaves, and stems. Additionally, Yu,
Zhou [133] used a nested PCR-DGGE approach to analyze bacterial endophytes in
Dendrobium officinale Kimura et Migo plants, identifying the genus Burkholderia as
the principal orchid-associated bacteria that can contribute mainly to N fixation. Pei,
Mi [134] also studied the diversity of endophytic bacteria associated with D.
officinale through metagenomics, showing that the phylum Proteobacteria,
Actinobacteria, Firmicutes, and Bacteroidetes were commonly identified in the
analyzed structures (roots, stems, and leaves). Lin, Xiong [135] showed that the
terrestrial orchid Gymnadenia conopsea (L.) R.Br. has a particular bacterial root-
associated microbiome including mainly the taxa Proteobacteria, Bacteroidetes,
Acidobacteria, Actinobacteria, Verrucomicrobia, Chloroflexi, and Planctomycete,
showing that geographical location was the main factor affecting the diversity of
bacterial taxa associated with the roots. Overall, such studies involving culture-
independent methods have provided essential information to understand the huge
diversity of orchid-associated bacteria with the ability to grow inside plant structures
and for a better understanding of the ecology of orchids in relation to their bacterial
associates. However, many such genera cannot be cultured using standard laboratory
procedures, limiting the exploration of plant growth–promoting mechanisms, their
use in productive systems. and conservation strategies involving commercial and
threatened species. Therefore, it is essential to study the diversity of culturable
microorganisms in order to assign a putative beneficial role to orchid-associated
bacteria in seed germination and/or plant growth promotion at advanced develop-
mental stages. Consequently, we will summarize some of the principal studies
performed on orchid-associated bacteria involving culture-dependent methods and
their possible plant growth–promoting traits detected (Table 1).
5.1 Seed-Associated Bacteria
An orchid interacts with bacteria throughout its life cycle. Several plants harbor a
selective and specific group of bacteria that can colonize pollen and seeds [2]. Such
bacteria are associated with the initial plant developmental stages, contributing to
processes such as disease prevention, phytohormone production, stress tolerance,
and biological control of phytopathogenic microorganisms. In this sense, the study
of pollen-/seed-associated bacteria with orchids may provide valuable data for the
sustainable management of orchid cultivars.
184
Table 1 Diversity of culturable bacteria isolated from orchid plants and with potential plant growth–promoting capabilities
Plant growth–promoting
Isolated bacteria Host Isolation source capabilities Location Reference
Pseudomonas sp., Pantoea sp., Spiranthes spirali, Serapias Root, stem, leaf, Phosphate solubilization ACC Italy Alibrandi, Lo
Rahnella sp., Staphylococcus vomeracea, and Neottia ovata and capsule deaminase activity IAA Monaco [140]
sp., Microbacterium sp., endosphere production Siderophore
Fictibacillus sp., Streptomyces production potassium
sp., Sphinomonas sp., and solubilization antimicrobial
Bacillus sp. activity
Bacillus sp., Flavobacterium Dendrobium moschatum and Aerial and Phosphate solubilizationa ACC Southeast Tsavkelova,
sp., Nocardia sp., Pseudomonas Acampe papillosa. substrate root deaminase activitya IAA Asia Cherdyntseva
sp., Curtobacterium sp., endosphere productiona Siderophore [152]
Rhodococcus sp., Xanthomonas productiona potassium
sp., Acinetobacter sp., solubilizationa
Aquaspirillum sp., Micrococcus
sp., Streptomyces sp.,
Cellulomonas sp.,
Gluconobacter sp., and
Mycobacterium sp.
Caulobacter vibrioides, Dendrobium moschatum Root endosphere, IAA production Southeast Tsavkelova,
Roseomonas cervicalis, rhizoplane Asia Egorova [138]
Streptomyces sp., Azospirillum
irakense, Enterobacter cloacae,
Agrococcus iejuensis,
Sphingomonas sp., and Bacillus
pumilus
Pseudomonas fluorescens- Thelymitra crinita, Lyperanthus Root endosphere Seed germination Western Wilkinson,
putida nigricans, and Pterostylis Australia Dixon [162]
recurva
H. Herrera et al.
6
Bacillus toyonensis, Bacillus Anacamptis pyramidalis, Ectorhizosphere, IAA production Turkey Altinkaynak
mobilis, Pseudomonas Himantoglossum caprinum, endorhizosphere, Phosphate solubilization ACC and Ozkoc
fluorescens, Acinetobacter Limodorum abortivum, and rhizoplane deaminase activity [143]
calcoaceticus, Bacillus simplex, Platanthera bifolia, Serapias
Pseudomonas baetica, vomeracea, Spiranthes spiralis,
agrobacterium tumefaciens, Ophrys apifera, Ophrys
Bacillus cereus, sphegodes, Orchis coriophora,
Stenotrophomonas maltophilia, Orchis laxiflora, Orchis
Bacillus mobilis, Pseudomonas provincialis, and Orchis
frederiksbergensis, and Bacillus tridentata
thuringiensis
Streptomyces sp., Bacillus sp., Paphiopedilum appletonianum, Endosphere and IAA production Vietnam Tsavkelova,
Pseudomonas sp., Burkholderia and Pholidota articulata rhizoplane Cherdyntseva
sp., Erwinia sp., Nocardia sp., [145]
Flavobacterius sp.,
Stenotrophomonas sp., Pantoea
sp., Chryseobacterium sp.,
Agrobacterium sp., and
Paracoccus sp.
Sphingomonas paucimobilis Dendrobium officinale Endosphere Salicylic acid, IAA, zeatin, China Yang, Zhang
abscisic acid production. [153]
Nitrogen fixation
Bacillus sp., Sporosarcina sp., Dendrobium officinale Root, stem, and Phosphate solubilizationa ACC China Pei, Mi [134]
Paenibacillus sp., Burkholderia leaf endosphere deaminase activitya IAA
sp., Methylobacterium sp., productiona Siderophore
Brevibacterius sp., and productiona potassium
Orchid-Associated Bacteria and Their Plant Growth Promotion Capabilities
Curtobacterium sp. solubilizationa

(continued)
185
186
Table 1 (continued)
Plant growth–promoting
Isolated bacteria Host Isolation source capabilities Location Reference
Bacillus amyloliquefaciens Vanilla phaeantha, V. planifolia Shoot tip Fungal inhibitor production United White, Torres
x V. pompona Seedling growth promotion States of [141]
America
Bacillus sp., Gemella sp., Stanhopea connata Root fungal IAA productiona Ecuador Novotna and
Staphylococcus sp., hyphae Siderophore productiona Suárez [154]
Streptococcus sp., Paracoccus antifungal activity a nitrogen
sp., Achromobacter sp., fixationa
Acidobacter sp.
Collimonas pratensis, Chloraea barbata, Chloraea Root endosphere Phosphate solubilization Chile Herrera,
Pseudomonas sp., Pandoraea collicensis, Chloraea gavilu, Siderophore production IAA Sanhueza [14]
oxalativorans, Pseudomonas Chloraea magellanica, Gavilea production antifungal activity
koreensis, Exiguobacterium araucana, and Gavilea lutea
aurantiacum, Dyella marensis,
Luteibacter rhizovicinus,
Bacillus sp., Pseudomonas
azotoformans,
Chryseobacterium sp.,
Pseudomonas costantinii
Paenibacillus lentimorbus, Cymbidium eburneum Meristem Indole compound production Not Faria, Dias
Paenibacillus macerans Phosphate solubilization specified [142]
H. Herrera et al.
6
Sphingomonas sp., Guarianthe skinneri Root IAA production phosphate Mexico Aguilar Díaz,
Sinorhizobium sp., Bacillus sp., solubilization nitrogen fixation Bertolini
Nocardia cerradoensis. [146]
Bacillus megaterium, and
Burkholderia phytofirmans
Bacillus thuringiensis, Cymbidium sp. Root and leaf Nitrogen fixation, phosphate Brazil Gontijo,
Burkholderia cepacia, endosphere solubilization, and zinc oxide. Andrade [15]
Burkholderia gladioli, Indolic compound production
Herbaspirillum frisingense,
Pseudomonas stutzeri,
rhizobium cellulosilyticum,
rhizobium radiobacter, and
Stenotrophomonas maltophilia
a
Potential plant growth promotion assigned based on the plant growth–promoting capabilities detected in other plant species
Orchid-Associated Bacteria and Their Plant Growth Promotion Capabilities
187
In orchids, the diversity of endophytic bacteria associated with pollen/seeds have

been scarcely explored. Pavlova, Leontieva [136] reported that the rhizobacterial
strains Pseudomonas fluorescens and Klebsiella oxytoca can colonize root tissues
and the surface of protocorms and seeds of the orchid Dendrobium nobile Lindl.
Additionally, the same study demonstrated plant growth–promoting capabilities of
the tested rhizobacterial strains mainly through auxin production. Similarly,
Tsavkelova, Cherdyntseva [137] demonstrated that bacteria isolated from the rhizo-
plane of the epiphytic tropical orchid Dendrobium moschatum Buch.-Ham., such as
the genera Sphingomonas and Mycobacterium, can enhance seed germination
despite the absence of compatible mycorrhizal fungi, demonstrating the crucial
role of certain bacterial taxa at the same level as orchid mycorrhizal fungi. Addi-
tionally, Tsavkelova, Egorova [138] showed that endophytic or rhizoplane bacterial
isolates belonging to the genera Mycobacterium, Bacillus, Agrococcus, and
Sphingomonas can have positive roles in orchid growth, colonizing the seed surface
and inner tissues of protocorms and rhizoids, influencing the seed germination
process and embryo growth. Colonization of endophytic bacteria was also detected
in mature capsules of the terrestrial orchid S. spiralis [88]. In this study, the genera
Bacillus, Sphingomonas, and Staphylococcus were effectively isolated and charac-
terized showing intense plant growth–promoting attributes such as phosphate solu-
bilization, K solubilization, and IAA production. More recently, Alibrandi, Schnell
[131] described the bacterial microbiome associated with orchids seeds after super-
ficial disinfection of the capsule, describing seed microbiota associated with the
S. spiralis as sharing a core of taxa with roots, stems, and leaves, suggesting a
vertical transfer of the core microbiota, which can be essential to stimulating the
early colonization of endophytic-associated bacteria. Strictly speaking, the diversity
of culturable orchid seed–endophytic bacteria still remain unexplored, but our
understanding of seed-associated bacteria has provided essential information to
understand the beneficial role of these microorganisms at the first developmental
stages of the orchid.
5.2 Phyllosphere-Associated Bacteria
Microorganisms inhabiting the phyllosphere can be beneficial for the associated

plants, contributing to resistance to biotic/abiotic stresses and production of antimi-
crobial compounds, among others [139]. Alibrandi, Lo Monaco [140] analyzed the
diversity of endophytic bacteria associated with leaves and stems of S. spiralis, N.
ovata, and S. vomeracea orchids, exploring their plant growth–promoting traits. That
study mainly described to the genera Pseudomonas, Staphylococcus, Pantoea, and
Rahnella as principal bacterial associates to orchid aboveground organs.
The analysis of plant growth–promoting capabilities revealed that most of the
isolated strains have potential beneficial effects on the associated orchid, including
nutrient solubilization, ACC deaminase activities, and IAA biosynthesis. In the same
vein, Pei, Mi [134] identified Burkholderia sp., Bacillus sp., Methylobacterium sp.,
and Curtobacterium sp. as principal bacterial associates of the leaf and stem of D.
officinale. Most of these bacterial genera involve species with clear roles in plant
growth promotion including phosphate solubilization, ACC deaminase activity, IAA
production, siderophore production, and K solubilization. White, Torres [141] ana-
lyzed the colonization pattern of the endophytic bacterial strain Bacillus
amyloliquefaciens in shoot meristems and stomatal areas of stems and leaves of
Vanilla phaeantha Rchb. f., in which a protective and defensive role was expected
for vanilla orchids. Gontijo, Andrade [15] analyzed leaf-associated bacteria of
Cymbidium sp. plants, identifying several genera with beneficial effects on plant
growth including the species Burkholderia cepacia, Burkholderia gladioli, and
Rhizobium radiobacter. Similarly, Faria, Dias [142] identified Paenibacillus spp.
associated with meristems of Cymbidium eburneum Lindl., showing that most of
these isolates can promote plant growth and can be effectively used as a strategy for
improving the acclimatization of orchid plantlets.
The studies conducted on phyllosphere-associated bacteria have increased our
understanding about the potential benefits of these bacteria for orchids plants.
Several of the isolated strains have been identified as PGPB (i.e., Bacillus spp.,
Burkholderia spp., Pseudmonas spp., and Rhizobium spp.), which can have a
potential beneficial role at the first developmental stage, where improved growth
rates, acclimatization, and biocontrol against phytopathogens are the main benefits
expected.
5.3 Rhizosphere-Associated Bacteria
Currently, few studies have characterized the diversity of culturable bacteria associated
with the rhizosphere of orchid roots. In the rhizosphere, several bacteria live under the
influence of the plant root metabolism and can have essential roles in nutrient solubi-
lization, influencing the growth of the associated plants, some of which can also
colonize the inner root tissues. Altinkaynak and Ozkoc [143] characterized bacteria
inhabiting the rhizosphere of native orchids from Turkey, identifying the genera
Bacillus, Paenibacillus, Pseudomonas, Acinetobacter, Agrobacterium, and Stenotro-
phomonas. That study isolated almost 16 PGPB with the ability of phosphate solubi-
lization, ACC deaminase activity, and IAA production. Similarly, Tsavkelova,
Cherdyntseva [144] isolated and characterized auxin-producing bacteria from the
rhizoplane of D. moschatum (Rhizobium, Microbacterium, Sphingomonas, and Myco-
bacterium), which were further analyzed for their potential role in seed germination,
showing that some of these isolates were able to improve the seed germination rates of
the inoculated seeds [137]. Similarly, Tsavkelova, Cherdyntseva [145] analyzed the
diversity of bacteria colonizing the rhizoplane of the terrestrial orchid Paphiopedilum
appletonianum (Gower) Rolfe and the epiphytic orchid Pholidota articulate Lindl. The
main results showed that the culturable bacterial diversity was different in the two
analyzed orchids, identifying Streptomyces, Bacillus, Pseudomonas, Burkholderia,
Erwinia, and Nocardia as principal associates of P. appletonianum, whereas Pseudo-
monas, Flavobacterium, Stenotrophomonas, Pantoea, Chryseobacterium, Bacillus,
Agrobacterium, Erwinia, Burkholderia, and Paracoccus were associated with P.
articulate roots. Similarly, Aguilar Díaz, Bertolini [146] identified rhizospheric bacteria
are associated with Guarianthe skinneri (Bateman) Dressler and W.E. Higgins in
Mexico, identifying Sphingomonas sp., Sinorhizobium sp., Bacillus spp., Nocardia
cerradoensis, and Burkholderia phytofirmans as the main bacterial isolates.
Among the isolated bacteria, several genera with demonstrated roles in plant
growth promotion and nutrient solubilization have been identified. Therefore, rhi-
zosphere-associated bacteria can contribute to vital processes such as auxin produc-
tion (i.e., Sphingomonas spp. and Rhizobium spp.), nutrient solubilization (i.e.,
Sphingomonas spp., Sinorhizobium spp., and Nocardia spp.), siderophore produc-
tion (i.e., Pseudomonas spp.), volatile organic compounds (i.e., Bacillus spp.),
protection against disease (i.e., Bacillus spp., and Pseudomonas spp.), and control
of phytopathogens (i.e., Rhizobium spp., Pseudomonas spp., and Bacillus spp.).
5.4 Root Endosphere–Associated Bacteria
Mycoheterotrophic species like orchids commonly associate with a broad range of

soil-borne fungi, including those accepted as orchid mycorrhizae as well as a broad
range of free-living or endophytic species [112]. Some of these microorganisms can
have a saprophytic or phytopathogenic lifestyle, including species from the genera
Fusarium, Thanatheporus, and dark septate endophytes [147–149]. Therefore,
endophytic bacteria associated with orchids may provide essential information
about the mechanisms of control of intracellular fungal hyphae to avoid overspread
in vital plant tissues [150, 151].
Endophytic bacteria associated with orchids have a strong influence on orchid
metabolism, interacting directly with the cells of the host plant. In this sense,
endophytic bacteria may represent an opportunity to improve the yield of orchid
plants. Tsavkelova, Cherdyntseva [145] identified endophytic bacteria associated
with roots of P. appletonianum, showing Streptomyces, Bacillus, Erwinia, and
Pseudomonas genera as the principal endophytic taxa, whereas Pseudomonas,
Bacillus, and Flavobacterium were the endophytic bacteria isolated from P.
articulata roots. In the same way, Tsavkelova, Egorova [138] explored the role of
the PGPB bacterial genera isolated from D. moschatum on the growth of D. nobile,
showing that some endophytic isolates, such as Agrococcus, Sphingomonas, Myco-
bacterium, and Bacillus pumilus, can have a beneficial effect on seed germination.
Alibrandi, Lo Monaco [140] also isolated endophytic bacteria colonizing the roots of
S. spiralis, N. ovata, and S. vomeracea. The main endophytic taxa associated with
the roots of the analyzed species were Pseudomonas sp., Staphylococcus sp.,
Microbacterium sp., Streptomyces sp., Fictibacillus sp., and Bacillus sp., some of
which showed strong plant growth–promoting traits including ACC deaminase
activity, phosphate solubilization, IAA production, siderophore production, and K
solubilization. Tsavkelova, Cherdyntseva [152] studied the diversity of endophytic
bacteria isolated from the root of the epiphytic orchids Acampe papillosa (Lindl.)
Lindl. and D. moschatum, identifying the bacterial taxa Bacillus sp., Flavobacterium
sp., Pseudomonas sp., Rhodococcus sp., Xanthomonas sp., Alcaligenes sp., and
Gluconobacter sp. as the principal associated taxa. Similarly, Pei, Mi [134] also
analyzed the diversity of endophytic bacteria colonizing roots of D. officinale,
showing to Bacillus sp., Sporosarcina sp., Paenibacillus sp., and Brevibacterium
sp. as principal associates. In the same vein, Yang, Zhang [153] isolated and
characterized a PGPB isolates from the root endosphere of D. officinale. The authors
identified Sphingomonas paucimobilis as the main bacterial associate, which showed
strong plant growth–promoting traits such as N fixation and secretion of plant
growth regulators such as salicylic acid, IAA, and abscisic acid, which were
involved in an improved plant growth of the inoculated seedlings. A recent study
isolated and characterized endophytic bacterial strains by directly colonizing the
mycorrhizal structures (pelotons) of some terrestrial Andean orchids [14]. That study
identified the main endophytic bacterial taxa associated with fungal hyphae of
mycorrhizal fungi-colonizing roots of native orchids from the genera Chloraea
spp. and Gavilea spp. in southern Chile. The bacterial genera Collimonas sp.,
Pseudomonas spp., Pandoraea sp., Exiguobacterium sp., Dyella sp., Luteibacter
sp., Bacillus sp., and Chryseobacterium sp. were commonly detected. Plant growth–
promoting capabilities were detected in almost all of the isolates as well as a
restriction of fungal growth, especially the isolates Collimonas pratensis, Dyella
marensis, Exiguobacterium aurantiacum, and Pseudomonas azotoformans, an attri-
bute that can contribute to the intraradical control of fungal hyphae of mycorrhizal
fungi as well as potential phytopathogenic species.
Until now, several genera have been described as common associates of the
endosphere of plants from the Orchidaceae family. These endophytic isolates include
several taxa also identified as rhizospheric-associated microorganisms, such as
Pseudomonas spp., Bacillus spp., Sphingomonas spp., and Burkholderia spp.,
showing also plant growth-promoting capabilities such as IAA production and
phosphate/K solubilization. Additionally, the potential capabilities of some bacterial
isolates to inhibit the growth of potential phytopathogenic species (i.e., Collimonas
sp., Pseudomonas spp., Exiguobacterium sp., Dyella sp., and Chryseobacterium sp.)
points to essential roles in the control of fungal spread inside the root tissues of
symbiotic orchids.
5.5 Fungi-Associated Bacteria
Bacteria associated with fungal hyphae have been commonly detected in several
terrestrial plants. Similarly, they can be indirectly involved in orchid mycorrhizal
processes, especially helping the extraradical fungal hyphae inhabiting the growth
substrate. In this sense, only one study has reported the direct association of orchid
mycorrhizal fungi mycelia with bacteria. Specifically, Novotna and Suárez [154]
characterized bacteria associated with living hyphae of the mycorrhizal fungi
Serendipita sp., isolated from roots of the epiphytic orchid Stanhopea connata
Klotzsch in southern Ecuador. Several genera such as Bacillus sp., Gemella sp.,
Staphylococcus sp., Streptococcus sp., Paracoccus sp., Achromobacter sp., and
Acidobacter sp. were detected. Some of the detected species match with other
bacterial isolates with the ability to live inside fungal hyphae, such as Bacillus spp.,
Paracoccus spp. and Achromobacter spp. [155–157]. Therefore, this study has
provided essential information on the diversity of bacteria living in association
with fungal hyphae of orchid mycorrhizal fungi.
6 Management of Root-Associated Bacteria in Cultural

Practices
As reviewed, several studies have reported that orchid species establish specific
symbiotic interactions with soil bacteria. In this sense, identifying microhabitats in
which threatened orchid populations grow naturally may be essential to knowing the
reproduction mechanisms in nature and applying the information obtained to seed
germination strategies and plant growth promotion, with a focus on orchid-associ-
ated bacteria. Although most soil root–associated bacterial communities (endophytic
and rhizospheric) cannot be cultured, identifying potential PGPB with a positive
influence on orchid metabolism, especially at the first developmental stages, can be
useful to improving the germination and survival rates of wild orchids, improving
growth rates, plant establishment, and development. Therefore, developing a poten-
tial inoculant involving a single bacterium or consortium may be essential for
cultural management in orchids, especially for plantlets obtained by asymbiotic
seed germination procedures. In this sense, key developmental stages for orchid
bacterization can be seed germination, initial plantlet development, and acclimati-
zation. A compatibility test is necessary because recent studies have shown that
bacterization can have different effects depending on the host [138]. The methods for
inoculation of bacteria can include direct inoculation of the bacterial cells or through
different microencapsulation methodologies [158, 159]. Most studies on orchid-
associated bacteria have tested the plant growth–promoting capabilities under con-
trolled conditions, but information about the in vivo effects of inoculation on plants
are limited. Therefore, studies regarding in vivo effects are needed for a better
understanding of the role of orchid-associated bacteria.
Soil microorganisms can establish synergistic interactions with soil-borne
microbes that usually result in improved plant growth. Therefore, it is necessary to
explore the potential of the noncultured microbiota for a better understanding of the
symbiotic interactions of orchids. The soil microbiome, involving all microbiolog-
ical populations inhabiting a soil, certainly influences the orchid seed germination
processes, because in the microbiome the compatible mycorrhizal fungi and effec-
tive orchid-associated bacteria can colonize the growth substrate. Identifying such
hotspots of orchid reproduction and developing efficient management methodolo-
gies involving the respective associated microbiome can contribute to improving the
seed germination rates in both, nature and controlled conditions. In this sense, recent
studies have shown that the use of soil microbiomes can be an effective strategy to
improve plant growth [160, 161]. Therefore, the use of the original soil microbiome
able to sustain the germination and growth of orchid plants may be essential to
improving reproduction and yield of orchids plants.
7 Conclusion
The presence of diverse bacterial genera in orchid structures denotes that bacteria are
common associates at different life cycle stages (seed, protocorm, plantlet, and
mature plant). Additionally, the isolation and characterization of orchid-associated
bacteria have provided evidence of the benefits for the associated plants. Commonly,
orchid mycorrhizal fungi have been described as essential microorganism associated
with orchids, but nowadays knowledge of orchid-associated bacteria has provided
clues about the beneficial role of bacteria for stress tolerance, plant growth promo-
tion, and protection against disease or phytopathogens. Therefore, orchid-associated
bacteria must be considered essential to orchid development, and some can effec-
tively colonize inner plant structures, having direct contact with the plant cells.
However, further evidence is required to define the optimal developmental stage
and specific strains for an adequate inoculation of plant growth–promoting bacteria,
as well as to test their synergistic or antagonistic effect with orchid mycorrhizal
fungi.
Acknowledgments The authors would like to thank to Fondo Nacional de Desarrollo Científico y
Tecnológico (FONDECYT), grant numbers 1211857 to Cesar Arriagada and 3200134 to Hector
Herrera.
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Mycorrhiza in Orchids
7
Saranjeet Kaur
Contents
1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 202
2 Geological Location and Environment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 203
3 Mycobiont Invasion in Orchid Tissues at Different Stages of Development . . . . . . . . . . . . . 204
4 Root Cortex and Fungal Pelotons . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 204
5 Fungal Members as Orchid Mycorrhiza . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 206
5.1 Rhizoctonia Fungi . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 206
5.2 Basidiomycetous and Ascomycetous Ectomycorrhiza . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 206
6 Orchid Specificity for a Symbiont . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 206
7 Peloton Formation and Mycophagy or Necrotrophy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 207
8 Orchid Mycorrhiza and Nutrient Transport . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 207
9 Nutrient Transfer Mechanism in Orchids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 208
10 Transport of Phosphorus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 209
11 Transfer of Other Micronutrients . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 209
12 Transport of Nitrogen . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 210
13 Transport of Carbon . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 210
14 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 211
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 211
Abstract
The orchids are a highly medicinal and floriferous assemblage of flowering plant
species. Each orchid fruit encloses thousands of dust like minute and highly
reduced seeds. Due to lack of endosperm and presence of complex carbohydrates,
these seeds require specific fungal partner to accomplish germination in nature.
The fungus plays a crucial role in the germination of the orchid seeds and
their growth and development in mature orchid plants. In this manuscript, some
intricacies pertaining to mycorrhizal interactions are being discussed.
S. Kaur (*)
Department of Chemistry, University Institute of Sciences, Chandigarh University, Mohali, Punjab,
India
e-mail: sarana_123@rediffmail.com

202 S. Kaur
Keywords
Endomycorrhiza · Monocot · Orchid seeds · Root cortex · Symbiosis
1 Introduction
The orchids are highly evolved group of angiosperm plants belonging to monocot
family Orchidaceae. In the world, a total of 28,000 species are confined to 763 orchid
genera [1]. Despite of being cosmopolitan in distribution, they do not appear as
dominant vegetation in any part of the world.
The orchids produce a large number of structurally and functionally highly
reduced, microscopic seeds (Fig. 1). Although, the seeds are produced in enormous
quantities in a single capsule, merely 0.2–0.3% germinate in nature, whereas count-
less perish away. Moreover, the seeds lack endosperm tissue and possess little
amount of complex carbohydrates as reserve food material which the seeds are
unable to utilize. The undifferentiated embryos of orchid seeds lack distinct root
and shoot meristem. Therefore, orchid seeds necessarily require specific mycobionts
which could convert complex carbohydrates into simple molecules and provide them
to the germinating entities. Various orchid mycobionts establish a symbiotic rela-
tionship with the roots of orchid plants. All orchid species are myco-heterotrophic at
certain stage in their life cycle. In symbiotic association, the specific fungus invades into
the seed as well as roots. The fungus forms loosely coiled structures called pelotons in the
parenchymatous cells of the cortical region of the roots. The orchids maintain symbiotic
association with fungal endophytes throughout their lifecycle to obtain nutrients, sugars,
and minerals [2]. Conversely, there are some orchid species that establish symbiotic
association with their specific fungal endophytes only in severe conditions [3]. The orchid
seeds germinate into a pyriform structure called protocorm; these get associated with
specific mycorrhizal partners. The achlorophyllous orchid species such as Corallorhiza
maculata and Rhizanthella species known as achlorophyllous myco-heterotrophs main-
tain their fungal symbionts throughout their life cycle to get nutrition.
Fig. 1 Minute orchid seeds

7 Mycorrhiza in Orchids 203
Table 1 Shows endophytic fungi promote enzyme activity in orchid species

Name of the endophytic
Host Plant fungus Activity Reference
Anoectochilus Epulorhiza sp. Enhances activities of following the [7]
formosanus enzymes: chitinase, β-1,3-glucase,
phenylalanine ammonia-lyase,
polyphenol oxidase
Anoectochilus Epulorhiza sp., Mycena Enhance enzyme activities [8, 9]
roxburghii anoectochila
Cymbidium Mycena orchdicola Secretes phytohormones [10]
sinense
Dendrobium Mycena dendrobii Secretes phytohormones [10]
candidum
Dendrobium Epulorhiza sp., Mycena Enhance the absorption of nutrients in [11]
nobile, sp., Tulasnellales, plants, promotes seed germination of
D. chrysanthum Sebacinales, the host plant
Cantharellales
Pecteilis Epulorhiza sp., Fusarium Enhance the absorption of N, P, and K [12]
susannae sp. elements in plants
promoting the seed germination of host
The importance of a variety of phytohormones in the regulation of entire life

process including plant development and their defense response has long been
established in orchids. Besides providing nutrients such as nitrogen and phosphorus
for various life processes in orchid plants, the fungal partners are also reported to be
secreting certain growth hormones, for instance, cytokinins and auxins mainly
(indole-3-acetic acid, indole-3-acetonitrile) [4, 5]. A study in Gastrodia elata dem-
onstrated that the fungal Mycena dendrobii secreted IAA which promoted seed
germination and growth of its host plant [6]. Literature studies also reported that
endophytic fungi promote the growth of the host plants by enhancing the enzyme
activity (Table 1).
2 Geological Location and Environment
Some orchids are extremely specific in selecting their symbionts, as they prefer a
single genus of fungi. Corallorhiza maculata, a myco-heterotroph, associate only
with Russulaceae irrespective of their geological location and presence of other
orchids in its vicinity [13].
Certain orchid species change their symbiotic fungal partner in response to
environmental stress with regard to variations in altitude from tropical to temperate
regions [3]. Goodyera pubescens associates only with one fungal mycobiont, if not
subjected to changes in the environment, like drought, etc. The orchids with high
degree of specialization have fewer fungal associations [14]. Some other orchid
species, for instance, Chloraea collicensis and Chloraea gavilu, make symbiotic
association with only Rhizoctonia [15].
204 S. Kaur
3 Mycobiont Invasion in Orchid Tissues at Different Stages

of Development
The fungus invades into the tissues of orchid at its different stages of development.
Fungal hyphae penetrate inside the testa of seed through the opening and into the
parenchymatous cells of germinating orchid seeds, protocorms, and cortex of the
roots. Certain physiological and cytological changes also take place at the time of the
invasion of fungus into parenchyma cells. Increased number of mitochondria and few
vacuoles enhances metabolic activity of the parenchyma cells of embryo. Fungal
mycobiont enter into the protocorms through the chalazal end of the embryo [16, 17].
4 Root Cortex and Fungal Pelotons
In the present study, it was observed that the fungus penetrated into roots mainly
through root hair tips. Once the fungus enters into the parenchymatous cells of cortex
of the orchid root, its hyphae start coiling loosely inside the cells; these coiled
structures are known as pelotons [18, 19]. Pelotons inhabit the parenchymatous
region of the cortex of roots (Fig. 2), which is an important anatomical feature of
orchid mycorrhiza that clearly distinguishes it from the other forms of fungi [20].The
pelotons vary in their size, packaging, and arrangement of their hyphal mass [21].
The pelotons remain functionally active for certain period. Later, these disintegrate
inside the cortical root cell forming a sort of round clumped or disc-like structure. In
present study, it was also observed that the pelotons of adjacent cells make seen
interconnection with each other (Fig. 3).
As soon as the fungus invades into the root cell, it undergoes biochemical
changes. The cells with disintegrated pelotons lack starch grains, whereas the
newly invaded cortical root cells possess large starch grains, which indicate the
hydrolysis of starch grains after the fungus colonization [14]. When pelotons
Fig. 2 Transverse hand-

section of orchid root showing
fungal invasion in the
parenchyma cells of cortex
Fig. 3 Pelotons
interconnected with the
pelotons of neighboring cells
Fig. 4 Completely lysed

pelotons
disintegrate or are lysed, they appear as brown or yellow clumps in the orchid cells
[22] as also observed in the present study (Fig. 4). At the time of peloton disinte-
gration, certain structural and physiological changes also occur [23]. Certain cyto-
logical changes also occur in the invaded cell. The nucleus becomes conspicuous, it
enlarges in size considerably, and shows increased amount of DNA content [21]. The
increased DNA content is correlated with the differentiation of parenchyma cells
suggesting its role in orchid growth [24].
In orchids, two types of host cells such as digestive cells and host cells are
involved in nutrient transfer [25]. The digestive cells are engaged in dense peloton
development followed by digestion and subsequent reinvasion, whereas the host
206 S. Kaur
cells contain live hyphae in pelotons, which are not digested, or at least not as readily
[25]. There is an additional mode of nutrient transfer known as phytophagy. It
involves lysis of fungal cells. The complete fungal hyphae are not digested, but
only the growing end (tip region) is degraded, and the cell contents are released into
interfacial space between the plant and hyphal membrane and are provided to the
plant [20].
5 Fungal Members as Orchid Mycorrhiza
5.1 Rhizoctonia Fungi
The fungi that act as orchid mycorrhizae belong to class basidiomycetes. These
basidiomycetous fungi include certain genera of fungus, namely, Rhizoctonia,
Sebacina, Tulasnella, and Russula species. Most of the orchid species build up
their association with saprotrophic or pathogenic fungi, whereas a few show prefer-
ence for ectomycorrhizal fungal species. The orchid species show association with
different fungal partners at different developmental stages in their life cycle. These
associations could be at the time of either seed germination or protocorm develop-
ment or could be throughout the life cycle of an orchid plant species [26]. Moreover,
different orchid species show preference for their symbiotic fungi depending on the
type of environmental niches in which they thrive, whether terrestrial or growing on
other plants as an epiphyte [27].
5.2 Basidiomycetous and Ascomycetous Ectomycorrhiza
Few species of basidiomycetous fungi are also reported to be making symbiotic

association with orchid species, but they do not belong to Rhizoctonia. Specific
myco-heterotrophic orchids are also reported to be associated with ectomycorrhizal
basidiomycetes that belong to genera such as Thelephora, Tomentella, and Russula.
Ascomycetous fungi are rarely seen establishing symbiotic connections with orchid
species. A terrestrial orchid species Epipactis helleborine has a specific association
with ectomycorrhizal ascomycetes in the Tuberaceae.
6 Orchid Specificity for a Symbiont
At successive developmental stages, orchid species show preference for their fungal
mycobionts. Terrestrial orchid species build symbiotic association with members of
family Tulasnellaceae, yet a few autotrophic and saprophytic orchids make associ-
ation with several ectomycorrhizal fungi also [28, 29]. Few clades of fungus
Rhizoctonia also show association with some epiphytic species [30]. Rhizoctonia
fungi can form symbiotic relationship with either an epiphytic or terrestrial orchid,
but very rarely, they associate with both [30]. It has been shown through seed baiting
techniques that the seeds of Dendrobium aphyllum germinate when they make
symbiotic association with Tulasnella, but do not germinate when treated with
Trichoderma isolated from orchid plant, which strongly advocates about the speci-
ficity of symbionts to different developmental stages. The preference for symbiosis
with a fungal partner differs at various developmental stages of an orchid species
[31, 32]. With the advancing age of an orchid plant, the fungal associations also
become more complex. Cephalanthera longibracteata, a mixotrophic orchid spe-
cies, symbiotically associates with numerous fungal species belonging to family
Russulaceae, Tricholomataceae, Sebacinales, and Thelephoraceae [33].
7 Peloton Formation and Mycophagy or Necrotrophy
Metabolically active and live pelotons facilitate the transfer of nitrogen and carbon;
however, when fungal pelotons get digested, most of the nitrogen and carbon is
absorbed by the plant itself by the process of mycophagy [25, 34]. Shortly, after the
invasion of fungus into the cortical tissues and peloton formation, their lysis starts
[35]. Pelotons are formed in a unique way. At the time of their development, a thin
membrane surrounds them, which eventually acts as endoplasmic reticulum
surrounded by Golgi apparatus. Afterward, inside this cover, digestive enzymes
are secreted into the space between the plant membrane and peloton to digest them
[36]. Further, the digestion of pelotons starts; at the same time, a secondary mem-
brane is also formed around the fungal peloton which is a large vacuole and allows
the degradation of the peloton in isolated manner [36]. Additionally peroxisomes
accumulate within the digestive plant cells and undergo exocytosis into the newly
formed vacuole; a number of enzymes concentrate such as chitinases, uricases,
peptidases, oxidases, and catalases are secreted which ultimately, breaks down the
peloton [35, 36].The fungal remnants are consumed by the plant itself, thus, trans-
ferring the nutrients to the host plant [25].
Few experimental studies using stable isotope imaging technique reveal that C13
and N15 when applied to mycorrhizal hyphae got readily transported to the host
plant through fungal pelotons, leading to an inconsistent quantity of these isotopes
inside the peloton containing plant cell and the peloton itself. It had also been
observed that the senescing pelotons contain higher concentrations of C13 and
N15 isotopes than in live pelotons [34].The fungal hyphae also undergo morpho-
logical change; they swell before collapsing, most probably due to the increased load
of nutritive compounds [34]. Once the pelotons are completely digested, reinvasion
into the digestive cell occurs shortly after, and a new peloton starts forming again
[25]. Reinvasion and digestion occur cyclically throughout the entire life span of the
symbiotic association [35].
8 Orchid Mycorrhiza and Nutrient Transport
Orchid mycorrhizal associations involve a variety of nutrient transport systems,

structures, and phenomenon which are specifically found in the family
Orchidaceae. These interactions are observed between basidiomycetes and
208 S. Kaur
almost all species of Orchidaceae [37]. In basidiomycete, the fungi Rhizoctonia

is generally found associated with orchid species. Rhizoctonia is known for its
saprophytic abilities also and establishes anomalous associations [37]. The
orchid plant species which inhabit dense and highly shaded forest areas depend
completely on their mycorrhiza for nutrition such as carbon [35, 38]. Orchid
seeds being extremely reduced and non-endospermic show an obligatory para-
sitic stage during germination and draw nutrients with the help of mycorrhizal
fungus [39]. After the orchid seed germination, the orchid fungal interactions
become specific to utilize the carbon and available nutrients. These associations
are often governed by the orchid plant itself [40]. Orchid mycorrhizal interac-
tions could either be completely parasitic on the fungal associate or show
mutualistic interaction, thus, establishing bidirectional nutrient flow between
the plant and mycorrhizal fungus [40]. In the natural habitats, orchid mycorrhiza
shows a specific mycorrhizal nutrient transfer interaction upon which the diver-
sity of the orchid genera depends [35].
9 Nutrient Transfer Mechanism in Orchids
Orchid mycorrhizal interactions show unique flow of nutrients. In the arbuscular

mycorrhizal associations, it is observed that plant species channelize unidirectional
supply to fungus with carbon swapping with either or both, phosphorus or
nitrogen, depending on the environment [41, 42]. There occurs bidirectional flow
of carbon between the fungus and plant, besides flow of nitrogen and phosphorus
from the fungus to plant. Nearly 400 plant species are not able to provide carbon
to their system. In fact, all of the nutrients of the plant are supplied by the fungus
[40]. However, in these interactions, carbon gain by the plant is positive in the
majority of the observed interactions [25]. Peloton starts forming shortly 20–
36 hours, after the invasion of fungus [35]. The plasma membrane of invaded
parenchyma cells also assists with the fungal infection and its further growth [43].
For exchange of nutrient materials between pelotons and surrounding plasma
membrane, extensive surface area is created. The invaginated plasma membrane
surrounds the growing pelotons and creates vast surface area from which nutrients
can be exchanged. The pelotons are highly coiled fungal hyphal mass as compared to
endomycorrhiza of arbuscular mycorrhiza [35, 44, 45]. During fungal invasion,
increase in ribosomes also takes place. Plasma membrane participates in the exchange
of nutrients between plant and fungus besides the enzyme excretion inside the space
termed interfacial apoplast [40, 46].
The pelotons are not permanent structures; these are swiftly digested within a
few hours of their formation in orchid parenchyma cells. The digestion of
pelotons is a universal feature observed in almost all endomycorrhizal associa-
tions; in case of orchid species, these coiled structures get digested sooner after
their formation [34].
10 Transport of Phosphorus
Phosphorus is a macroelement which is required by all plants. Phosphorus is taken

up by the mycorrhizal fungus from the soil particles in three distinct forms: inorganic
phosphorus, organic phosphorus, and phosphate. Deficiency of phosphate in soil
allows the formation of a symbiotic relationship between plant and fungus. Mycor-
rhizal fungi are capable of increasing soil surface area besides initiating the secretion
of a variety of enzymes [41, 42, 47]. Inorganic phosphate is transferred either
through active transport as phosphate through Pi transporters (inorganic phosphorus)
and is moved out of the fungal hyphae into the interfacial apoplast, where it forms
dihydrogen phosphate and then subsequently gets transferred by active Pi trans-
porters into the plant cell or it depends upon passive efflux of Pi out of the fungus
and active absorption by the plant [41, 47]. For efficient transport of phosphorus,
these pathways depend on comparative high concentrations of Pi inside fungal cell
and low concentration of Pi inside the plant cell. The second method is more
dependent on this condition. Certain genes which are Pi transporter genes such as
MtPT4 and StPT3 are known to regulate the exchange of phosphorus in orchid
plants along with H+ ATPase transporters [42]. In orchid species, once mycorrhizal
symbiotic connections are established afterward phosphorus is obtained by the plant
only through the metabolically active pelotons of fungal tissue; once the digestion of
pelotons starts degrading simultaneously, the flow of phosphorus also ceases [47].
11 Transfer of Other Micronutrients
Mostly, passive transport helps in the transfer of micronutrients across the cell
membranes, both during absorption from soil by fungi and further from fungi to
the host plant [41]. But under specific conditions, active transport of micro-
nutrients takes place as well [48]. The upregulation of cation transporters is seen
in orchid D. officinale symbioses, suggesting that fungi make possible the transfer
of nutrients from fungi to plant [49]. Cation, such as iron, mostly found adhered
tightly to the organic substrates and remains out of reach of plants and fungi.
There are certain compounds, for instance, siderophores (small molecule which
have high affinity for Fe3+ utilized by fungal species), which are secreted into the
soil by fungi to acquire these cations [50]. These cations are liberated into the soil
around the hypha and absorb iron from the soil. These siderophore molecules are
reabsorbed into the fungal mycelium where the iron has to be dissociated from the
siderohore and quickly utilized [50]. The orchid species possess siderophores in
association with mycorrhizal fungi within the genus Rhizoctonia which can utilize
the siderophore “basidiochrome” as the major iron-chelating compound [48].
Apart from these known chelating compounds, other vital nutrients may also be
transferred between mycorrhizal fungi and orchid plants through specialized
methods also.
210 S. Kaur
12 Transport of Nitrogen
Nitrogen transport is an equally important and essential process that often occurs
through mycorrhizal associations [51]. Nitrogen is abundant and much easy to obtain
as compared to phosphorus. Mycorrhizal interactions give a significant benefit in the
allocation of nitrogen. Bioavailable nitrogen (nitrate and ammonium) is absorbed
from the soil media by the mycorrhizal fungi and further assimilated into the amino
acids [42]. There are a few proposed mechanisms by which nitrogen is transferred to
the host plant. These pathways are biotrophic; a significant amount of nitrogen may
also be transferred necrotropically but through a distinct process [51].
In pathway-1, the amino acids get transferred into the extra-radical mycelium
where these amino acids are broken down. The amino acid such as arginine is
synthesized and transferred intra-fungally and later on is catabolized into ammonium
ions and is moved into the interfacial space between peloton and surrounding plant
membrane and later on transported into the plant cell through ammonium trans-
porters and incorporated into the plant [46].
In the transportation of nitrogen primarily as ammonium, certain ammonium
transporter genes get regulated in the plant and mycorrhizal fungal associate which
further regulate a class of genes called “protease genes” along with other three
transporters such as an external amino acid permeases, nitrate transporters, and
ammonium transporters [41, 46]. Nitrogen may also be transferred in the form of
other amino acids, namely, arginine, glycine, and glutamine into the cell through
specialized amino acid transporters. The mycorrhizal fungus T. Calospora in sym-
biotic association with orchid plant is able to regulate the expression of SvAAP1 and
SvAAP2. These genes encode amino acid permeases that strongly support amino
acids to be important molecules involved in nitrogen transport [34, 46, 47]. The
transport of inorganic nitrogen in the form of ammonium and the transport of organic
nitrogen as amino acids occur simultaneously [35]. In fact, it has been proved
through isotope (C13 and N15) studies that amino acids may be the primary nitrogen
compound transferred in the orchid [46].
13 Transport of Carbon
As soon as the fungus gets carbon, it converts it into sugar mainly trehalose and
assimilates into the fungal mycelium. The transport of carbon from fungal partner to
plant cells occurs in one of two forms primarily trehalose, but carbon can also get
converted into glucose and sucrose or as an amino acid arginine but can also be
converted into glycine and glutamine [34, 40, 52].The transport of these molecules
occurs through specialized amino acid permeases and carbohydrate transporter
protein molecules. These molecules are fixed into the fungal peloton membrane,
into the interfacial space where they get absorbed into the plant cell by similar
transporter protein molecules in the orchid cell endoplasmic reticulum membrane
surrounding the hyphal coils [34, 52].
The active transport of carbon from symbiotic fungal partner to plant cell is a
biotrophic phenomenon. A significant amount of carbon gets transferred inside plant
cell when the fungal pelotons are degraded and digested. Genes encoding the
transporter proteins get regulated, simultaneously, both in plant and fungi, in the
similar fashion as nitrogen and phosphorus compound transporter genes during
symbiosis [46].
Orchid mycorrhizal interactions consist of various symbiotic fungi, ranging from
myco-heterotrophic plants (Monotropa uniflora) to chlorophyllous orchid species
such as Goodyera repens [40, 52, 53]. As mentioned earlier in the text that during
symbiotic interactions in orchid mycorrhiza carbon is translocated readily from fungi
to the plant tissues. Conversely, this may or may not occur with the transfer of carbon
from plant to fungi [25, 52, 54]. Orchid mycorrhiza is more or less considered to be
showing partial myco-heterotrophic interactions [35]. In myco-heterotrophic
orchids, carbon is taken up, by the fungi (basidiomycetes), in the form of molecules
of peptide and carbohydrate [35, 55, 56]. Specific genes that codes for proteases and
cellulose, lignin digestive enzymes, as well as oligopeptide and carbohydrate trans-
porters get regulated mycelium present in soil to support enhanced carbon uptake [46].
Interestingly, it is also observed that apart from orchids, myco-heterotrophic fungi
interact with roots of beech trees as well [57]. Few studies report that myco-
heterotrophic fungi are also involved in the translocation of photosynthates from
tree to the fungus and then to orchid, but a thorough investigation is required to
unravel such intricacies in orchid fungus interactions [58].
14 Conclusion
The orchids have meticulously evolved to survive in nature in spite of having highly
reduced seed structure. It becomes imperative to understand orchid fungus intrica-
cies for conservation purpose. A few studies have been reported about orchid fungus
interactions. The knowledge of species-specific as well as developmental stage-
specific fungal symbionts would be a step toward saving them from getting extinct
in nature. More morphological and molecular studies are required for the identifica-
tion of fungal symbionts that inhabit at every stage of their development. It would
prove useful in replenishing their natural resources, thus saving them for future use.
Acknowledgments English language assistance from Dr. H.S. Sekhon is acknowledged with deep
gratitude.
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Phytoalexins in Orchids
8
Saranjeet Kaur
Contents
1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 216
2 Biosynthetic Pathways . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 217
3 Role of Phytoalexins in Plants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 218
4 Role of Plant Hormones . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 219
4.1 Phytoalexins in Gymnosperms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 220
5 Phytoalexins in Orchids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 220
6 Phytoalexins and Human Health . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 220
7 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 221
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 221
Abstract
The plant kingdom harbors a number of chemicals called phytoalexins, as natural
products, which are secreted temporarily whenever plants are attacked by any
kind of microbe or pathogen. The family Orchidaceae synthesizes phytoalexins in
their system as defensive compounds. These bioactive compounds act as antimi-
crobials upon attack by any kind of microbe or fungi in orchids. There are few
reports in literature regarding the phytoalexins. This chapter reviews phytoalexins
in the monocot family Orchidaceae.
Keywords
Defense mechanism · Monocot · Bioactive · Orchid root · Symbiosis
S. Kaur (*)
Department of Chemistry, University Institute of Sciences, Chandigarh University, Mohali, Punjab,
India
e-mail: sarana_123@rediffmail.com

216 S. Kaur
1 Introduction
Since the dawn of civilization, the plants have been resonating with human life on
earth. Various diseases of human are treated by the plants. The plants harbor a variety
of bioactive chemical compounds including alkaloids, flavonoids, sesquiterpenes,
etc. At one hand where the plants have the ability to cure human system, they are
efficient enough to combat any type of pathogen attack or disease if that has invaded
into their system.
The plant kingdom harbors a number of chemicals called phytoalexins, as natural
products, which are secreted temporarily whenever plants are attacked by any kind
of microbe or pathogen. These inhibitory compounds are biologically active against
a variety of pathogens such as insects, fungi, bacteria, or nematodes and sometimes
toxicity produced by plants themselves and animals as well [1]. A perusal of
literature reveals that phytoalexins are secondary metabolites having low molecular
weight [2, 3]. Phytoalexins are lipophilic in nature and are capable of traversing
through the plasma membrane. They exhibit their toxicity because of their acidic
character. As soon as the fungus attacks the plant tissues, these phytoalexins starts
disorganizing the cellular contents, ruptures plasma membrane, granulation of cyto-
plasm occurs, and later these phytoalexins inhibit the fungal enzymes thus reducing
or inhibiting the growth of the mycelium [4]. In plant system, their synthesis occurs
de novo and gets accumulated rapidly at the site when and wherever there is any kind
of pathogenic attack or some kind of abiotic cause. Accumulation of phytoalexins at
the site of pathogenic attack itself triggers resistance response to that particular
pathogen. Although these “defense compounds” are synthesized in minute quanti-
ties; however, they are quite efficient as toxins to the infecting agent/s. Phytoalexins
are often found in dicots but hardly ever in monocots and in few gymnosperms.
The Orchids: The orchids belong to one of the highly evolved monocot family
Orchidaceae which is very well known for its floriferous blooms and therapeutically
important herbs. The orchid species for instance Dendrobium chrysanthum is known
to harbor chemical compounds that are highly cytotoxic to cancerous cells. The
orchids are highly priced ornamental plants as species like Cymbidium, Dendrobium
Oncidium, Phalaenopsis, etc. find special place in cut-flower trade world over and
are able to generate handsome revenue.
The orchid species of diverse habits and habitats, similar to other plant species,
are also known to synthesize bioactive chemical compounds identified as “phyto-
alexins.” These are self-defense allelo-chemicals synthesized in plants. Phytoalexins
are low molecular weight compounds that provide resistance to plant system against
microbes. The phytoalexin Pisatin was first discovered in Pisum sativum [1]. The
main phytoalexin discovered in orchid is “orcinol” which is a dihydrophenanthrene
from Orchis militaris when infected with an endophytic fungus Rhizoctonia repens
[5, 6]. The tubers of the orchid species Loroglossum hirsinum, upon infection with
another species of genus Rhizoctonia, that is, Rhizoctonia versicolor synthesized two
potential phytoalexins namely loroglossal which is an isomer of orcinol and the other
phytoalexin from Loroglossum is hircinol [6]. These two compounds were observed
to be structurally related to orcinol. The compound loroglossal was found to be
8 Phytoalexins in Orchids 217
active against Phytophthora infestans and Monilinia fruticola whereas hircinol was
reported for its antifungal activity at ultra-low concentration of 6 106 M [7].
Phytoalexins are the secondary metabolites that are lipophilic in nature. Many
angiosperm families such as Malvaceae, Solanaceae, and Orchidaceae report the
secretion of secondary metabolites. The class of phytoalexins secreted by members
of Malvaceae and Solanaceae are sesquiterpenes, and in family Leguminosae
the phytoalexins found are isoflavonoids or polyacetylenes, and that of family
Orchidaceae are dihydrophenanthrenes (http://www1.biologie.uni-hamburg.de/b-
online/e33/33d.htm).
Some plant species like the potato produce several similar substances at the same
time. Little is known about their mode of action. Some results point an effect that
changes the membrane properties of the fungus cell. Other substances seem to block
the oxidative phosphorylation, and still others can link up DNA molecules. Phyto-
alexins provide no absolute protection against fungus infections. They are mainly
directed at “non-pathogenic” species. Many parasites are able to protect themselves
against these substances or to develop defense mechanisms of their own.
2 Biosynthetic Pathways
A survey of literature indicates that biosynthesis of phytoalexins in plants is regu-

lated by certain compounds synthesized endogenously, for instance, phyto-
hormones (cytokinins, auxins, abscisic acid, ethylene, salicylic acid, jasmonic
acid, and gibberellins), and by other transcriptional regulators, defense-related
genes, phosphorylation relays and cascades [8, 9].
In general, there are pathways that help in synthesizing different phytoalexins.
These pathways are: (a) the phenylpropanoic-polymalonic acid pathway, (b) the
methylerythritol phosphate and geranyl-geranyl diphosphate pathway, and (c) the
indole phytoalexin pathway. Phytoalexins deriving from phenylpropanoic-
polymalonic acid pathway: All kinds of flavonoid phytoalexins such as iso-
flavonoids, isoflavones, pterocarpans, isoflavans, coumestans and arylbenzofurans,
stilbenes and its derivatives (dihydrophenanthrenes) are synthesized by the common
phenylpropanoic-polymalonic acid pathway.
The pathway starts with phenylalanine/phenylalanine ammonia lyase (PAL)/
tyrosine ammonia lyase (TAL)/tyrosine precursors. Subsequently, para-coumaric
acid gets activated in para-coumaroyl-CoA through its ligation to coenzyme A by
4-coumaroyl: CoA ligase (C4L). Later, chalcone synthase (CHS), stilbene synthase
(STS) using same substrate condensed it with three successive units of malonyl-
CoA, further producing naringenin chalcone, the first intermediate which is C15 in
flavonoid pathway and other compound resveratrol, the precursor molecule of
stilbenes [10, 11, 12]. Another pathway is mevalonoid-derived phytoalexin pathway:
In this pathway, sesquiterpene, monoterpene, carboxylic sesquiterpene, and
diterpene phytoalexins are synthesized. In this pathway the gene transcripts
(OsDXS3, OsDXR, OsCMS, OsCMK, OsMCS, OsHDS, and OsHDR) are involved
which were observed in Oryza sativa cells [13].
218 S. Kaur
Indole phytoalexins pathway: The third type of pathway of phytoalexin synthesis

is well studied in Arabidopsis. The pathway starts from chlorismate. This pathway
involves production of camalexin from tryptophan which shows the involvement of
two cytochrome P450 homologs CYP79B2 and CYP79B3, which further form
indole-3-acetaldoxime; it is converted into indole-3-acetonitrile (IAN) through cyto-
chrome P450, CYP71A13. The last step involved in the synthesis of camalexin is
under the control of a gene named Phytoalexin Deficient 3, Pad 3 (CYP71B15),
which codes for camalexin through dihydrocamalexic acid [13].
3 Role of Phytoalexins in Plants
The term phytoalexin was coined by K.O. Müller in 1940. Phytoalexins are consid-
ered to be plant antibiotics that are synthesized de novo when the plant part gets
some microbial infection [14]. Phytoalexins, an antimicrobial compound, could not
be preformed in the tissue or released from preexisting plant constituents [14]. Thus,
in contemporary terms, these antibiotics are produced in response to a microbial
elicitor, and their production requires the expenditure of plant energy, generally in
the form of new transcriptional and or translational activity. The species of family
Brassicaceae show piling up of metabolites such as sulfur-containing tryptophan-
derived alkaloids whenever there is a pathogen attack [15, 16].
Phytoalexin compounds are synthesized in minute quantities over the surface of
plants; if consumed by human system in excess, they can prove harmful to human
beings and animals. For instance, in other plant species, the phytoalexin named
“pisatin” which are produced by garden pea and phaseolin by green bean are capable
of lysing bovine red blood cells at 200 ppm and 17.5 ppm concentration, respec-
tively. Likewise, carrots contain phytoalexin “myristicin,” an insecticidal, which can
induce cerebral excitation in human beings. Damaged Ipomaea batata show the
elevated levels of phytoalexin called Ipomeamarone which can cause damage to
lungs and liver in cattle and human beings.
Blighted white potato causes poisoning and ultimately death due to the forma-
tion of two glyco-alkaloids: alpha-chaconine and alpha-solanine. From consumer
point of view, it becomes essential to trim down the production of phytoalexins
through improved agricultural practices. In family Leguminosae, the phytoalexins
produced by the members belong to six iso-flavonoid classes such as iso-flavones,
iso-flavanones, pterocarpans, pterocarpenes, iso-flavans, and coumestans.
Different plant species harbor a variety of phytoalexins. Some of the types of
phytochemicals synthesized by different genus of angiosperm families are summa-
rized in Table 1.
The compound phytoalexins are low molecular weight antimicrobial compounds,
which gather at site of infection, releases enzymes such as proteases, chitinases, beta-
1,3-glucanases which destroy the pathogens, and also release certain defensive bio-
polymers (peroxidases and phenol oxidases) which restrict the spread of pathogens
for instance lignin, callose, hydroxyproline-rich glycoproteins, and some other
compounds which regulate the induction and/or activity of the protective compounds.
A kind of phytoalexin named trans-resveratrol has been reported, when a fungus
Table. 1 Shows various types of phytoalexins found in angiosperm families [3]

Angiosperm
S. No. Family Phytoalexin Type
1 Amaryllidaceae Flavans
2 Brassicaceae Indole phytoalexins/camalexin Sulfur-containing phytoalexins/
brassinin
3 Chenopodiaceae Flavanones/betagarin Isoflavones/betavulgarin
4 Convolvulaceae Furanosesquiterpenes/Ipomeamarone
5 Compositae Polyacetylenes/safynol
6 Poaceae Diterpenoids: Kauralexins; Momilactones; Phytocassanes
Oryzalexins; Zealexins
Deoxyanthocyanidins/luteolinidin and apigeninidin Flavanones/
sakuranetin Phenylamides
7 Leguminosae Isoflavones, Isoflavanones, Isoflavans, Coumestans Pterocarpans/
pisatin, phaseollin, glyceollin and maiackiain Furanoacetylenes/
wyerone Stilbenes/resveratrol Pterocarpens
8 Malvaceae Terpenoids naphtaldehydes/gossypol
9 Linaceae Phenylpropanoids/coniferyl alcohol
10 Moraceae Gossypol/Terpenoids naphtaldehydes
11 Orchidaceae Loroglossol/Dihydrophenanthrenes
12 Rutaceae Methylated phenolic compounds/xanthoxylin
13 Umbelliferae Falcarinol Phenolics: xanthotoxin 6-methoxymellein/
Polyacetylenes
14 Solanaceae Coumarins/Sesquiterpenoids
15 Rosaceae Auarperin Dibenzofurans/Cotonefurans
16 Vitaceae Resveratrol/Stilbenes
Botrytis cinerea infects Vitis vinifera [17], and another phytoalexin called delta-
viniferin is synthesized when Plasmopara viticola infects the grapevine [18]. A
phytoalexin 6-methoxymellein is known to be induced in carrot slices by UV-C,
[19], and it induces resistance to Botrytis cinerea [20] and other microorganisms [21].
A survey of available reports reveals that the plant Sorghum produces two
discrete phytoalexin molecules of 3-deoxyanthocyanidin chemical group,
apigeninidin and luteolinidin. The precursor molecule for biosynthesis is flavanone
naringenin which differ from marginally from that of the anthocyanin pathway. In
Zea mays, the phytoalexins such as zealexins and kauralexins belong to terpenoid
class [22]. Whenever any pathogenic fungi attacks the plant, the phytoalexins are
produced. These phytoalexins interact with invaded pathogen and start to neutralize
its noxious effects. Alternaria brassicicola which is a necrotrophic fungus can
detoxify brassinin, which is a phytoalexin synthesized in Brassicaceae family [22].
4 Role of Plant Hormones
Plant hormones are also known to activate defense-response related genes to syn-
thesize defensive metabolites such as sesquiterpenoids. Plant hormones play an
important role in controlling plant stress and various diseases. Literature study
220 S. Kaur
reveals that external application of abscicic acid (ABA) in certain way elevates the
resistivity of plant to pathogen attack [23]. In dicot family Solanaceae, the synthesis
of phytoalexin known as capsidiol, a sesquiterpenoid, is regulated by ABA [24].
4.1 Phytoalexins in Gymnosperms
Apart from Angiosperms, Gymnosperms are also known to produce certain phyto-
alexins. A toxin named Pinosylvin which is a stilbenoid toxin is synthesized before
fungal invasion. The heartwood tissues of members of Pinaceae show phytoalexin
synthesis, and it is a fungitoxin protecting the wood from fungal infection [25].
5 Phytoalexins in Orchids
In family Orchidaceae, there exists an inverse relationship between mycorrhiza and

the amount of phytoalexin synthesized. It is noticed that when mycorrhiza invades
into parenchyma cells of roots, the level of endogenous amount of phytoalexin
declines. Phytoalexins are synthesized in almost every part of orchid plant such as
roots, rhizomes, and bulbs as soon as they are invaded by any fungus as observed in
Bletila striata, which showed 100% in enzyme bibenzyl synthase [26]. Literature
survey indicates the isolation of more than 40 dihydrophenanthrene phytoalexins in
orchid species of diverse habits and habitats [27]. Radioactive experimental
study indicated that L-phenylalanine is a precursor molecule for the biosynthesis
of 9,10-dihydrophenanthrenes with intermediate compounds m-coumaric acid,
dihydro-m-coumaric acid, and 3,30 ,5-trihydroxybibenzyl [28]. Since these are deriv-
atives of dihydrostilbenes or bibenzyls so these dihydrophenanthrenes are catego-
rized as stilbenoids [29]. When mycorrhiza is digested completely in orchid root cell,
its production increases again inside the cell.
Molecular investigations reveal that whenever an orchid is attacked or invaded by
any kind of pathogen (fungi or bacteria) intense activation of specific genes that
encodes for the synthesis of phytoalexin enzymes takes place [27]. The key enzyme,
bibenzyl synthase, that is primarily involved in the synthesis of phytoalexins gets
activated as soon as fungus infects the orchid roots [27]. The phytoalexin molecule
precursors were synthesized in Epipactis palustris rhizomes, when these got infected
by Rhizoctonia strain [28]. Literature studies reveal an interesting observation about
stem tissues showing more fungicidal activity than tubers of Dactylorhiza incarnata
and less in roots [29].
6 Phytoalexins and Human Health
The phytoalexins are synthesized in minute quantities over the surface of plants; if
consumed by human system in excess, can prove harmful. For instance, in other
plant species, the phytoalexins named “pisatin,” which is produced by garden pea,
and phaseolin, by green bean, are capable of lysing bovine red blood cells at
200 ppm and 17.5 ppm concentration, respectively. Likewise, carrots contain phy-
toalexin “myristicin,” an insecticidal, which can induce cerebral excitation in human
beings. Damaged Ipomaea batata show the elevated levels of phytoalexin called
Ipomeamarone which can cause damage to lungs and liver in cattle and human
beings. Likewise, blighted white potato causes poisoning and ultimately death due to
the formation of two glyco-alkaloids: alpha-chaconine and alpha-solanine. From
consumer point of view, it becomes essential to trim down the production of
phytoalexins through improved agricultural practices. On the contrary, there are
reports that indicate the positive influence of phytoalexins on human health. A few
of phytoalexins have shown health-promoting effects in human beings [22]. They
are reported to be acting as antioxidants, showing anticarcinogenic and acting as
cardiovascular protective. The topical applications of phytoalexin called resveratrol
shows antitumor activities against skin cancer in vivo. Another phytoalexin named
as maslinic acid, synthesized in olives, exerts a broad spectrum of biological
activities such as antitumoral, antidiabetic, neuroprotective, cardioprotective, anti-
parasitic, and growth stimulating. Synthesis of maslinic acid in olive makes it a food
having nutraceutical value [22].
7 Conclusion
The orchids have smartly evolved themselves to survive in nature in spite of having
highly reduced seed structure. It is crucial to understand orchid fungus intricacies for
conserving them. Reports have been cited in orchid literature about orchid fungus
interactions in relation to phytoalexins. Studies are required to unravel the mode of
interaction in which these biomolecules show their activity against microorganisms.
Furthermore, research in molecular studies would be a step toward understanding the
relationship of symbiont and phytoalexin that is synthesized by various orchid
species upon getting infected by any pathogen attack.
Acknowledgments English language assistance from Dr. H.S. Sekhon is acknowledged with deep
gratitude.
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Part III
Horticulture
Micropropagation of Some Orchids
and the Use of Cryopreservation 9
Kanchit Thammasiri, Nipawan Jitsopakul, and Sasikarn Prasongsom
Contents
1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 226
2 Orchid Micropropagation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 226
2.1 Protocorm and Protocorm-Like Body Formation in Orchids . . . . . . . . . . . . . . . . . . . . . . . . 227
2.2 The Components of Orchid Tissue Culture Media . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 227
3 Orchid Cryopreservation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 239
3.1 Dormant Bud Method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 241
3.2 Slow Freezing Method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 242
3.3 Vitrification Method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 242
3.4 Encapsulation-Dehydration Method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 242
3.5 Encapsulation-Vitrification Method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 245
3.6 Droplet-Vitrification Method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 246
3.7 V Cryo-Plate Method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 247
3.8 D Cryo-Plate Method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 250
4 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 250
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 251
Abstract
Orchid micropropagation is an indispensable tool for seed and clonal propagation,
especially for commercial purposes. It has been interestingly researched and used for
enormous species and hybrids. In contrary, many wild orchid species are in endan-
gered and extinction because of deforestation and natural disaster. Only outstanding
horticultural characteristic orchids are cultivated. Therefore, orchid conservation is
K. Thammasiri (*)
Department of Plant Science, Faculty of Science, Mahidol University, Bangkok, Thailand
e-mail: kanchitthammasiri@gmail.com
N. Jitsopakul
Department of Plant Science, Textile and Design, Faculty of Agriculture and Technology,
Rajamangala University of Technology Isan, Surin, Thailand
S. Prasongsom
Pathum Wan District, Bangkok, Thailand

226 K. Thammasiri et al.
urgently needed by various means, such as living collection, seed and pollen storage,
in vitro conservation, and cryopreservation for small aseptic materials, such as
protocorms, protocorm-like bodies (PLBs), buds, root tips, meristem, callus, and
cell suspension. In this review, orchid cryopreservation which is a long-term storage
with genetic stability is focused for the development and successful use.
Keywords
Orchid · Micropropagation · Protocorms · Cryopreservation · Vitrification ·
Cryo-plate
1 Introduction
Orchids are in the family Orchidaceae which is one of the most diverse, advance, and
largest with about 25,000 species. They are unique and fascinating in characteristics
that make them popular worldwide for plant studies, hobbies, and commercial uses.
They are used as ornamentals, medicine, and cosmetics. Orchid micropropagation is
an indispensable tool for seed and clonal propagation, especially for commercial
purposes; otherwise, orchid industry will not be rapidly progressed. Orchid micro-
propagation has been interestingly researched and used for enormous species and
hybrids. In contrary, many wild orchid species are in endangered and extinction
because of deforestation and natural disaster. Only outstanding horticultural charac-
teristic orchids are cultivated. Therefore, orchid conservation is urgently needed by
various means, such as living collection, seed and pollen storage, in vitro conserva-
tion, and cryopreservation for small aseptic materials, such as protocorms,
protocorm-like bodies (PLBs), buds, root tips, meristem, callus, and cell suspension.
Cryopreservation is a long-term storage with genetic stability. It was developed about
60 years ago from using sophisticated equipment with time-consuming protocols to
simple equipment with fast, efficient, and high survival. For orchids, cryopreserva-
tion was developed for many orchid species for over 20 years.
2 Orchid Micropropagation
Asexual propagation by division or cutting, which is a conventional method for

general plant propagation, as well as for orchids, is also practiced but mainly as
a hobby and not for large-scale production because multiplication is slower in such
cases. This method is; however, unavoidably used when tissue culture (micro-
propagation) fails to work. The beginning of orchid tissue culture experiment is in
the nineteenth century, scientists reported the successful use for both seeds [1] and
explants [2]. Many studies have focused on asymbiotic propagation of orchids from
seeds [3, 4]. Success for propagating orchids through tissue culture was first
achieved in 1949 in Phalaenopsis species. Later in 1967, the successful development
of techniques for tissue culture of Dendrobium provided a breakthrough, especially
for the Thai orchid cut-flower business, since this genus has contributed the most to
9 Micropropagation of Some Orchids and the Use of Cryopreservation 227
cut-flower production. Many types of culture media were used for orchid tissue
culture. Medium composition is an important factor for growth and morphogenesis.
It consists of water, a solid or semisolid support, macronutrients, micronutrients,
vitamins, carbon sources, plant growth regulators or hormones, amino acids, sor-
bents, and other undefined organic supplements [5]. Plant tissue culture is one of the
most important techniques that used for growing plant cells under sterile conditions.
The suitable medium compositions and environment are required for producing
clones of a plant. At present, successful propagation through tissue culture or
micropropagation has been achieved in over 80 genera of orchids [6].
2.1 Protocorm and Protocorm-Like Body Formation in Orchids
The orchid seeds are very small and dust-like because they contain only the small
embryo and without any associated endosperm storage tissue. Most range in length
from 0.05 mm to 6.0 mm. Seed weight extends from 0.31 μg to 24 μg [7]. Single
fruit or pod contains millions of seeds that are suitable for dispersing by wind [8].
Enormous numbers of seeds produced but only a few seeds germinate in nature [9].
Other reasons for seed germination, seeds lack enzyme to metabolize polysaccharides
but utilize liquid as a major nutrient source and the embryos also lack enzyme to
convert liquid to soluble sugar [10]. Orchid seeds require a symbiotic with a suitable
fungus [11]. Under natural conditions, germination of the mature orchid seeds,
partially those from the terrestrial orchids, is dependent on the association of
a mycorrhizal fungus. Since Knudson’s discovery in 1946 [12], orchid seeds can
also germinate in culture medium without mycorrhiza fungus [13]. For germination,
the embryo enlarges to form a small, corm-like structure, called a protocorm, which
possesses a quiescent shoot and root meristem at opposite poles. In nature,
a protocorm becomes green and accumulates carbohydrate reserves through photo-
synthesis. Normal seedling growth then continues utilizing the stored protocorm food
reserves. These somatic protocorms can appear to be similar to seedling protocorms,
and many workers on orchid propagation, have used terms such as “protocorm-like
bodies” (PLBs) to describe them. When a shoot tip of an orchid is transferred to
culture on a suitable medium, PLBs can grow and develop as a mature shoot apex.
PLBs also arise directly on some other orchid explants and proliferate from other
PLBs in a fashion which is exactly comparable to the direct formation of somatic
embryos. Champagnat and Morel [14] and Norstog [15] considered the appearance of
protocorms to be a manifestation of embryogenesis is because they represent a
specialized stage in embryo development and are normally derived directly from
zygotic embryos.
2.2 The Components of Orchid Tissue Culture Media
Artificial media used in plant tissue culture are copied from natural nutrients in soil for
supporting plant growth. Essential inorganic elements can be divided into two types,
first is macro-elements, such as nitrogen (N), potassium (K), calcium (Ca),
phosphorus (P), magnesium (Mg), and sulfur (S). The second is micro-elements, such
as iron (Fe), nickel (Ni), chlorine (Cl), manganese (Mn), zinc (Zn), boron (B), copper
(Cu), and molybdenum (Mo) [16]. Other elements, such as cobalt (Co), aluminum
(Al), sodium (Na), and iodine (I) are essential for some plant species. Other compo-
nents are carbon sources, plant growth regulators, nondefined organic additives
(banana homogenate, potato extract, coconut water, etc.), chitosan, and activated
charcoal.
2.2.1 Culture Media

The most commonly used media are Murashige and Skoog (MS) [17], Vacin and
Went (VW) [18], and Knudson (KC) [12]. For orchid growth media, MS is often
used in many researches because MS was the best liquid medium than VW and KC
for inducing PLBs of Dendrobium [19]. Half-strength MS (½MS) was popularly
used in many orchid species, such as somatic embryogenesis of Coelogyne cristata
formed embryo on ½MS (100%) medium better than cultured on MS (0–30%)
[20]. PLBs of Grammatophyllum speciosum were cultured on ½MS supplemented
with 2 mg/l naphthalene-1-acetic acid (NAA) and 1 mg/l BAP induced shoot and
root formation [21]. Kishor and Sharma [22] reported that ½MS was suitable for
germinating the hybrid of Renanthera imschootiana Vanda coelurea seeds when
compared with VW medium supplemented with 15% coconut water. The results had
significant difference on growth responses of leaf and root between two media.
Kishor and coworkers [23] reported that the best basal medium for culturing
immature embryo of Aerides vandarum and Vanda stangeana was ½MS medium
supplemented with 20% (v/v) coconut water. Seedlings of Paphiopedilum callosum
were cultured on ½MS supplemented with 5 μM thidiazuron (TDZ) gave the highest
number of shoots per explant [24]. VW basal medium was used for inducing the
PLBs in hybrid Cymbidium Twilight Moon “Day Light” [25]. Even though MS
contains more elements and concentration than VW but also some orchids grow the
best on VW, such as seeds of Paphiopedilum villosum var. densissimum germinated
better on VW medium than ½MS and ¼MS media [26]. In different media, SH [27]
medium was developed for culturing calli of monocots and dicots. This medium was
studied in some orchids. Luo et al. [28] reported that SH supplemented with 30 g/l
sucrose was the best medium for proliferating PLBs of Dendrobium huoshanense
when compared with MS, B5 [29], and N6 [30] media. PLBs of Cymbidium that
cultured on SH medium can induce the small of both calli and PLBs [31].
2.2.2 Sugar
Carbohydrates are very important component in in vitro cultures because they
have energy and carbon source, as well as an osmotic agent. They were added in
any nutrient media that is essential for in vitro growth and development because
photosynthesis is insufficient in in vitro culture [32]. Sucrose is mostly used but
glucose, fructose, sorbital, maltose, and other sugars are also used for comparing the
efficacy with sucrose. Germination of Paphiopedilum ciliolare seeds was the best on
a medium containing 5 g/l fructose plus 5 g/l glucose; a mixture of 7.5 g/l of each
sugar was optimal for seedling growth [33]. Glucose showed the best frequency of
germination in Paphiopedilum villosum var. dentissimum seeds on ¼MS; while,

sucrose, maltose, and mannitol provided lower on seed germination [26]. Hong et al.
[34] reported that 30 g/l sucrose for cultivar Grower Ramsey and 20 g/l glucose for
cultivar Sweet Sugar in free PGRs ½MS media for 60 days was suitable for inducing
direct embryo for Oncidium. Maltose (2%) in ½liquid MS medium was suitable
carbon source for enhancing growth rate of Grammatophyllum speciosum PLBs
when compared with the same concentration of sucrose, glucose, trehalose, sorbitol,
and mannitol [21]. Including the results of Luo et al. [35], PLBs of Dendrobium
huoshanense cultured on ½MS medium supplemented with 10 g/l maltose was the
best for inducing number of new shoots and had significant difference from the
results of sucrose and glucose. Different concentrations of sugar gave different
results on orchid growth, such as Dendrobium Second Love explants were increased
in the number of shoot and root length when cultured on modified VW medium
supplemented with 2–4% sucrose under light and dark condition [36].
2.2.3 Plant Growth Regulators

Auxins and cytokinins are two main groups of plant growth regulators that often
used in several plants, including orchids [37, 38]. Auxins are used for inducing plant
root or enhance the cytokinin effects. Cytokinins are popularly used for shoot
proliferation and induce shoot formation. Plant growth regulators (PGRs) with
different types and concentrations play an important role during in vitro propagation
including multiple of shoots, root and shoot formation, elongation of root tips and
callus formation of many orchid species. PGRs have been considered to accelerate
the growth and development of PLBs and plantlets from root-derived calli of
Oncidium [38]. The presence of auxins in the medium is generally essential for
embryo initiation [39]. Auxins play two main roles in plant tissue culture. Firstly,
they can be used to induce root formation, such as indole acetic acid (IAA) and
indole butyric acid (IBA). Secondly, they can initiate callus formation, such as
2,4-dichlorophenoxy acetic acid (2,4-D) and NAA [40]. Jheng et al. [41] showed
that a lower level of 2,4-D seemingly stimulated the increase of cell masses, and a
higher level of TDZ induced granular structure under dark condition. But the
combination of exogenous 2,4-D with TDZ was found very crucial for long-term
maintenance of callus cultures without loss of vigorous growth and regenerative
capability. Cytokinins are widely used for their ability to induce either shoot
proliferation by breaking dormancy in lateral buds or adventitious meristem (shoot
formation), such as kinetin, 6-benzylaminopurine (BAP) [42–44] and thidiazuron
(TDZ) [37, 45, 46]. TDZ is a substituted phenyl urea with cytokinin-like activity. It is
generally believed that TDZ is more active in stimulating shoot formation than BAP
or kinetin, even at extremely low concentration [47]. For some years, TDZ has been
generally used to culture orchid tissue, which could induce organogenesis and
somatic embryo formation [40, 48]. Disadvantage of TDZ in regeneration is the
difficulty in elongation and rooting of the regenerated shoots. This problem was
overcome by transferring regenerated shoots to MS medium supplemented with
BAP and NAA. Wasiksiri [49] studied the effects of six different liquid media
including, modified Vacin and Went [18], Murashige and Skoog [17], Schenk and
Hildebrandt [27], half strength MS (½MS), half strength SH (½SH), and half
strength KNO3 in SH (½KNO3SH) which contained 100 ml/l coconut water and
without sucrose on multiple shoot formation from lateral buds of Vanda coerulea.
After 8 weeks of culture, the survival of lateral buds (100%) was observed on ½SH
and ½KNO3SH but the protocorm-like structure was found only in ½KNO3SH. For
increasing multiple shoots or PLBs, lateral buds were cultured on ½SH and
½KNO3SH media supplemented with different concentrations of plant growth
regulators (TDZ, BAP, and NAA). The highest fresh weight of PLBs (total fresh
weight of 1.4 g) were observed on ½KNO3SH medium supplemented with 1 mg/l
BAP and 0.1 mg/l NAA (Fig. 1). Development of PLBs was established by organic
additives (activated charcoal, potatoes, bananas, and sucrose). The results revealed
that PLBs which cultured on ½KNO3SH supplemented with 1 g/l activated charcoal,
50 g/l potatoes, 50 g/l bananas, and 20 g/l sucrose produced the highest shoots
(434 shoots/lateral bud). These new shoots were able to develop into plantlets. New
shoots were cultured on ½KNO3SH supplemented with 1 g/l activated charcoal,
50 g/l bananas, and 20 g/l sucrose. After 16 weeks of culture, the best growth of
plantlets was found (Fig. 2).
2.2.4 Vitamins
All media used for orchid micropropagation have vitamins. Some are in organic
materials, such as banana, coconut water, potato, etc. Most added vitamins in orchid
culture media are niacin (nicotinic acid), pyridoxin (vitamin B6), and thiamine
(vitamin B1). Biotin, folic acid, and pantothenic acid are also used in some media.
2.2.5 Amino Acids

Glycine which is a component of the MS medium is the most used in orchid culture
media. Other amino acids are also used in some media. All organic materials already
have various amino acids.
2.2.6 Banana Homogenate

Homogenized banana fruit is sometimes added to media of orchid culture and is
often reported to promote plant growth. The reason for its stimulatory effect has not
been explained. One suggestion mentioned earlier is that it might help to stabilize the
pH of the medium. Banana pulp is a rich source of natural cytokinins which inhibit
culture initiation but promote differentiation and growth of shoots at later stages
[33, 50]. Vyas et al. [51] reported that KC medium supplemented with 10% (v/v)
banana homogenate provided high percentage germination of Dendrobium
lituiflorum seeds and gave maximum on regeneration of leaves and roots within
60 days of culture. Lo et al. [52] studied the effect of various additives in MS
medium on seedling growth of Dendrobium tosaense. The results showed that 8%
banana and 8% potato juice gave the same on fresh weight, plant height, stem
diameter, and root length after 20 weeks of culture. When compared to the different
concentrations of banana homogenate (5–30%), 10% gave the best proliferation
of PLBs on Dendrobium hybrid on ½MS medium for 4 weeks and all 4 banana
cultivars and banana powder that were added into the agar media gave significant
difference on fresh weight of PLBs [53]. The addition of 2.5 g/l banana powder
Fig. 1 Micropropagation of a lateral bud of Vanda coerulea in ½KNO3SH liquid medium after
8 weeks of culture in medium; (a) without PGR, (b) 0.1 mg/l TDZ, (c) 0.5 mg/l TDZ, (d) 1 mg/l
TDZ, (e) 1.5 mg/l TDZ, (f) 2 mg/l TDZ, (g) 1 mg/l BAP, (h) 2 mg/l BAP, (i) 0.1 mg/l NAA and
1 mg/l BAP, (j) 0.5 mg/l NAA and 1 mg/l BAP, (k) 1 mg/l NAA and 1 mg/l BAP, (l) 0.1 mg/l NAA
and 2 mg/l BAP, and (m) 0.5 mg/l NAA and 2 mg/l BAP. Bar ¼ 0.5 cm
Fig. 2 Effects of organic additives and combinations with concentrations of sucrose and organic
additives on embryogenesis on PLBs of Vanda coerulea under culture in ½KNO3SH contained
100 ml coconut water and 1 g/l activated charcoal after 8 weeks of culture. (a) ½KNO3SH agar
medium without organic additives and sucrose. (b) ½KNO3SH agar medium supplemented with
10 g/l sucrose. (c) ½KNO3SH agar medium supplemented with 20 g/l sucrose. (d) ½KNO3SH agar
medium in combination with 50 g/l banana without sucrose. (e) ½KNO3SH agar medium in
combination with 50 g/l banana and supplemented with 10 g/l sucrose. (f) ½KNO3SH agar medium
in combination with 50 g/l banana and supplemented with 20 g/l sucrose. (g) ½KNO3SH agar
medium in combination with 50 g/l potato without sucrose. (h) ½KNO3SH agar medium in
combination with 50 g/l potato and supplemented with 10 g/l sucrose. (i) ½KNO3SH agar medium
in combination with 50 g/l potato and supplemented with 20 g/l sucrose. (j) ½KNO3SH agar
medium in combination with 50 g/l banana and 50 g/l potato without sucrose. (k) ½KNO3SH agar
medium in combination with 50 g/l banana and 50 g/l potato and supplemented with 10 g/l sucrose,
and (l) ½KNO3SH agar medium in combination with 50 g/l banana and 50 g/l potato and
supplemented with 20 g/l sucrose. Bar ¼ 1 cm
was the best to enhance the number and length of roots of Phalaenopsis amabilis
PLBs after 4, 6, and 8 weeks on ½MS supplemented with 10% coconut water,
2 g/l peptone, and 1 g/l activated charcoal [54]. The best proliferated PLBs of
Phalaenopsis violacea (80%) was induced when cultured on ½MS medium
supplemented with 10% banana pulp extract; while, other organic additives (tomato,
coconut water, and papaya) provided lower effect for inducing PLBs (35%) in the
same condition [55]. Half strength MS medium containing100 g/1 banana homog-
enate facilitated root development and shoot growth of Rhynchostylis gigantea PLBs
[56]. The in vitro plantlets of Renanthera Tom Thumb “Qilin” provided the highest
number of roots and root length when cultured on VW medium supplemented with
1 g/l peptone, 1 g/l NAA, 1 g/l activated charcoal, and 100 g/l banana homogenate.
While other concentrations of banana homogenate (50 and 200 g/l), coconut water,
and potato homogenate had low effect to regenerate plantlet roots [50]. The seed-
lings of Dendrobium lituiflorum were cultured on KC medium with 20% (v/v)
banana homogenate gave elongated leaves and well-developed roots compared to
the same medium without banana homogenate after 30 days of the fourth subculture
[57]. Hyponex N016 medium supplemented with 1.0 mg/1 NAA, 1.0 g/1 peptone,
100 g/1 banana homogenate, and 1.0 g/1 AC was suitable for 2 cm Paphiopedilum
wardii plantlet growth when compared with 50, 150, and 200 g/l banana homogenate
in the same medium [58]. The PLBs of Vanda coerulea which cultured on
½KNO3SH supplemented with 1 g/l activated charcoal, 50 g/l potatoes, 50 g/l
bananas, and 20 g/l sucrose produced the highest shoots (434 shoots/lateral bud).
These new shoots were able to develop into plantlets. New shoots were cultured on
½KNO3SH supplemented with 1 g/l activated charcoal, 50 g/l bananas, and 20 g/l
sucrose [49].
2.2.7 Potato Extract

Potato is a carbohydrate-rich food which contains about 80% water and 20% dry
matter, with 60–80% of the latter composed of starch [59]. In orchid seed germina-
tion, potato extract showed 100% seed germination in Dendrobium hamaticalcar
after 50 days of culture on ½MS supplemented with 1 g/l potato extract that is the
same result as on ½MS supplemented with 1 g/l yeast extract [60]. VW medium
supplemented with coconut water and 5–20% potato extract gave high proliferation
percentage of Vanda Kasem’s Delight PLBs that was the same effect as supple-
mented with 5–20% tomato extract [59]. Regenerated shoots of Cypripedium
formosanum produced roots when cultured on the basal medium with 1 g/1 activated
charcoal and 20 g/1 potato extract for 60 days [61]. The PLBs of Vanda coerulea
which cultured on ½KNO3SH supplemented with 1 g/l activated charcoal, 50 g/l
potatoes, 50 g/l bananas, and 20 g/l sucrose gave the highest shoots (434 shoots/
lateral bud) [49].
2.2.8 Coconut Water

Coconut water (CW) is a natural growth promoter which contains higher levels of
zeatin, zeatin riboside, 1,3-diphenylurea (contains cytokinin-like activity), auxins,
nitrogenous compounds, inorganic elements, organic acids, sugars and their alco-
hols, peptides, vitamins, amino acids, and many other unknown components in its
composition. CW was demonstrated that physiologically active substances presented

in CW promote the cell division which further enhance shoot multiplication. Amino
acids increase the number of shoots by inducing cell division [62, 63]. In orchid seed
germination, CW showed 100% seed germination in Dendrobium tetrachromum
after 50 days of culture on ½MS supplemented with 15% CW that is the same result
as on ½MS supplemented with 2 g/l peptone [60]. The plantlets of Calanthe hybrids
(Bukduseong Hyesung) that cultured on modified Hyponex medium
supplemented with 50 ml/l CW showed significant difference and gave good results
in fresh weight and dry weight of plantlets after 8 weeks of culture [64]. CW was
reported to enhance the regeneration of Paphiopedilum rothschildianum PLBs to
the new plantlets on ½MS medium supplemented with 20% CW. Ng and Saleh [65]
compared the effect of CW with banana homogenate, potato homogenate, and
tomato homogenate. The results showed that CW was the best organic additive
on the regeneration of plantlet from secondary PLBs of Paphiopedilum
rothschildianum. Asghar et al. [66] studied the effects of CW on Dendrobium nobile
var. Emma White. Plantlet regeneration on phytotechnology medium (O753)
supplemented with 100 ml/l CW gave the highest number of shoots per explant,
shoot length, fresh weight shoot, and dry weight shoot when compared with other
concentrations of CW (50–300 ml/l). The immature seeds of Rhynchostylis retusa
and Aerides maculosa on VW, MS, and Mitra media that added 15% of CW
increased the percentage of callus induction when compared with control
(VW) [67]. CW was the best organic additive to increase PLBs of Dendrobium
Alya Pink on ½MS after 4 weeks of culture when compared with the same percent-
age of banana homogenate, tomato extract, and control group [53].
2.2.9 Chitosan
Chitin, consisting of a copolymer of N-acetyl-D-glucosamine and D-glucosamine
residues linked by β-1,4 glycosidic bonds, is a natural polysaccharide. It is presented
in broad range of species: in shells of crustaceans, in cuticles of insects, and in the
cell wall of fungi and some algae. The deacetylated form of chitin is chitosan.
Chitosan has been extensively used in agriculture, such as in seed, leaf, fruit, and
vegetable coating, as fertilizer and in controlled agrochemical release, to increase
plant product, to stimulate the immunity of plants, to protect plants against micro-
organisms, and to stimulate plant growth [68]. Very diluted chitosan solution was
sprayed on orchid roots showing stimulation of growth, renewed flower production,
and enhanced resistance against fungi and virus [69]. Nge et al. [68] suggested
that fungal chitosan is an attractive candidate to be used as growth stimulator in
VW liquid medium for orchid tissue culture. Sopalun et al. [21] studied effects
of chitosan on growth rate, number of new PLBs per explant, number of shoots
per explant, number of roots per explant, and number of leaves per explant of
Grammatophyllum speciosum PLBs in in vitro culture. The 0, 5, 10, 15, 20, 25,
50, or 100 mg/l of chitosan were supplemented in ½MS liquid or on solid medium
containing 2% (w/v) sucrose. The results showed that liquid medium supplemented
with 15 mg/l chitosan showed the highest growth at 756% after 1 month of culture.
The supplementation of chitosan more than 25 mg/l reduced the growth of
Grammatophyllum speciosum PLBs. Relative growth of Grammatophyllum

speciosum PLBs were significantly reduced when chitosan concentrations were
more than 25 mg/l. Moreover, the supplement of 100 mg/l chitosan caused the
necrosis of PLBs and released some browning compounds into the medium. Various
concentrations of chitosan supplemented in 1/2 MS solid medium showed the
differences in the development of Grammatophyllum speciosum PLBs. The 5, 10,
and 15 mg/l of chitosan supplemented in medium promoted leaf development. The
highest number of leaves per explant was found in the medium supplemented with
15 mg/l chitosan (1.8 leaves per explant). Moreover, supplementation with 5, 10, and
15 mg/l chitosan promoted a higher number of new PLBs and shoots. However,
chitosan supplementation did not promote rooting.
2.2.10 Activated Charcoal

Activated charcoal (AC) is composed of carbon that has numerous pores on the
surface. AC was used as a culture component for adsorption of toxic plant metab-
olites, such as phenolic compound and plant growth regulators [70]. AC provided a
dark environment during in vitro culture that affected shoot and root production.
Dark condition when added the AC in the media can enhance to prolong the activity
of some PLBs that can be degraded by light, such as IAA and IBA in culture media
[71]. For seed germination, three different agar media (Fast, Knudson C, and 0.1
MS) that added 2 g/l AC increased the percentage of seed germination of Encyclia
aff. oncidioides. Znaniecka et al. [72] concluded that activity of AC to accumulate
phenolic compounds and oxidative products promoted the growth of Encyclia
seedling. Calanthe hybrids are popular orchids in Japan and Korea but wild orchids
of this genus had low percentage of seed germination (2%). Shin et al. [73] reported
that Hyponex medium supplemented with 0.1 ppm NAA or 0.5 ppm BAP combined
with 0.1 g/l AC enhanced germination percentage of Calanthe hybrid seeds when
compared with other treatments. For plantlet growth, in vitro cultures of endangered
orchid, Anoectochilus formosanus on H3 medium with different concentrations of
AC showed that 0.5 mg/dm3 gave the highest plant height, root length, fresh mass,
and dry mass. Addition of AC (0.5, 1, 3, and 5 mg/dm3) in culture media gave
stronger plantlets that produced more roots and leaves than control [74]. Thomus and
Michael [75] reported that MS medium supplemented with BAP, NAA, and 1 g/l
of AC showed more multiple shoots per explant of Rhynchostylis retusa seedlings
than in medium without AC. The regeneration of Rhynchostylis rubrum plantlets
from callus after 8 weeks of culture on New Dogashima (ND) and VW medium
supplemented with 15% CW and 0.2% AC showed greener somatic embryo than the
same non-AC additive media [76]. Mass propagation and seedling of Vanda
coerulea on both Phytamax medium and MS medium supplemented with 3 g/l AC
showed healthier plantlets from PLBs than the same medium that added lower
concentration of AC [77]. In vitro plantlets of endangered herb orchid, Malaxis
acuminata on MS medium supplemented with 30 g/l sucrose and 3 g/l AC showed
the highest number of shoots per subculture and number of roots per plant when
compared with different concentrations of AC (0, 1, 2, 4, and 5 g/l) [78]. PLBs of
Phalaenopsis cornu-cervi that cultured on ND medium supplemented with 0.2% AC
and 4% sucrose gave the best result in 100% survival and promoted the plantlet
growth after 5 months of culture [79]. Khatun et al. [80] reported that MS medium
supplemented with different concentrations of PGRs had significant difference to
promote the root production (root numbers and root length) of Dendrobium hybrid
explants when compared with the same medium without AC after 120 days of
culture. In callus culture of Coelogyne cristata, AC could effectively stimulate
formation of somatic embryos on ½MS medium [20]. Graphite and AC have carbon
structures, odorless, and non-toxic substances; therefore, they were used to darken
the culture medium of plant tissue culture. In Cattleya bicolor seedlings, free PGRs
KC agar medium supplemented with 6 or 7.5 g/l of graphite gave the highest number
of buds; while, 6 g/l of AC gave the largest roots; however, in double hybrid “BLC
Pastoral Innocence” seedlings, 4.5 g/l of AC gave the highest number of buds and
roots [81].
2.2.11 Solid or Semisolid Supports

Micropropagation protocols have been established for many orchid species and
hybrids by different media, carbon sources, plant growth regulators, and have been
employed for several orchid species [82]. Knudson [1, 12] developed the culture
media for asymbiotic seed germination by using Knudson B and C media. After that,
other tissue culture media with other supplementations (carbon source, organic
supplementation, etc.) were suggested for orchid germination, such as Vacin
and Went medium [18], Murashige and Skoog medium [17], Malmgren Modified
Terrestrial Orchid Medium [83], tomato culture medium [84], Mitra medium [85],
banana culture medium [84], and PDA medium (potato dextrose agar). Various
species of orchids have different requirements for nutrient and other conditions for
growth. Rasmussen et al. [86] suggested that specific orchid species may have
different limiting factors for growth and development.
However, the relationship between plant growth regulators and plant are investi-
gated by using tissue culture technique. The supplementation of different ratios of
plant growth regulators was observed by measuring the growth rate [16]. Normally,
five major classes are accepted as plant hormones, including auxins, cytokinins,
gibberellins, ethylene, and abscisic acid. The chemicals of hormone are different
based on structure of each group that showed their effects on plant physiology
[87]. Tissue culture technique was used as a model to study the effects of
plant growth regulators, usually cytokinins, auxins, and the combination of both.
However, it has been considered that plant growth regulators may cause somaclonal
variation in plant. Tissue culture in orchids remains an indispensable tool for the
commercial production of elite selections in many orchid producing countries
(Table 1) because of low cost, uniformity, fast propagation, and high yield in a
short period of time [88]. Most cut-flower orchids, Dendrobium, Oncidium, Mokara,
Aranda, Ascocenda, and Cattleya alliances are propagated successfully through
tissue culture using mainly modified VW, MS, and ½MS media. Within 1–2 years,
lateral buds and apical bud from one young pseudobulb multiply to over 10,000
plantlets from the laboratory and are ready to grow in the greenhouse. Figures 3 and 4
Table 1 Micropropagation of some orchid species and hybrids

Orchid species and
hybrids Explant Regenerants Medium composition References
Phalaenopsis Seeds Protocorms VW agar medium [89]
amboinensis
Dimorphorchis Seeds Protocorms Knudson C agar [90]
lowii medium + 150 ml/l
coconut water
Dendrobium Seeds Protocorms VW agar medium + [91]
lasianthera 2 g/l peptone
Cymbidium Seeds Protocorms MS agar medium + [92]
iridioides D.Don 1.0 mg/l NAA
Aerides Seeds Protocorms MS agar medium [93]
odorata Lour.
Coelogyne cristata Seeds Protocorms MS agar medium + [94]
Lindl. 0.5 mg/l
NAA + 1.0 mg/l BAP
Dendrobium Seeds Protocorms MS agar medium + [95]
densiflorum Lindl. 1.0 mg/l BAP
Cymbidium Seeds Protocorms MS agar medium + [95, 96]
elegans Lindl. 1.0 mg/l BAP
Phaius Seeds Protocorms MS agar medium + [97]
tancarvilleae 0.5 mg/l BS
(L’Her) Blume.
dloifolium Lindl. 0.1% casein hydrolysate
+ 10% CW
Coelogyne Seeds Protocorms MS agar medium + [99]
fuscescens Lindl. 1 mg/l BAP + 0.5 mg/l
NAA
aloifolium Lindl. 0.5 mg/l BAP +
SW. 0.5 mg/l NAA
devonianum 2 mg/l BAP + 0.5 mg/l
Paxton NAA
Phaius Shoot tips Shoots MS medium + 1.0 mg/l [102]
tancarvilleae BAP
(L’Her.)
Grammatophyllum Shoot tips PLBs ½Murashige and Skoog [21]
speciosum (MS) liquid medium +
2% (w/v) sucrose
Rhynchostylis Shoot tips Adventitious VW liquid medium + [103]
gigantea shoots 10 g/l sucrose
(Lindl.) Ridl.
Dendrobium Shoot tips PLBs ½Murashige and Skoog [104, 105]
cruentum Rchb. f. (MS) liquid + 1% (w/v)
sucrose + 1.0 mg/l NAA
(continued)
Table 1 (continued)
Orchid species and
Vanda coerulea Shoot tips Adventitious Vacin and Went [106]
shoots (VW) medium + 10 g/l
sucrose
Spathoglottis Shoot tips Shoots ½MS medium + 1 mg/l [107]
eburnea Gagnep NAA + 2 mg/l BAP
Cattleya sp. Shoot tips Rooting Half strength MS [108]
medium + 2.4 mg/l
IBA + 3% sucrose +
60 PPFD
Anacamptis Shoot tips PLBs MS medium + NAA/ [109]
pyramidalis IBA/IAA; 0.5–1 mg/
Lindl. Rich. l + CW
Anoectochilus Shoot tips Shoot buds Hyponex [74]
formosanus Hay. medium + 1 mg/l BAP
Arundina Shoot tips Shoots Raghavan and Torrey’s [110]
bambusifolia Lindl. medium, N and N
medium
Cymbidium Shoot tips PLBs VW medium + 5.0 mg/l [111]
aloifolium Lindl. NAA
Sw.
Cymbidium Shoot tips PLBs MS medium + 2.5 mg/l [112]
atropurpureum BAP
(Lindley) Rolfe.
Dendrobium Shoot tips PLBs VW medium + 1 mg/l [113]
wardianum BAP + 1.5 mg/l NAA
R. Warner
Dendrobium Shoot tips Shoot buds ½MS medium + 1 mg/l [114]
cv. Sonia BAP + 7.5%CW
Dendrobium Shoot tips PLBs VW medium + 15% [115]
Joannie Ostenhault CW
Phaius Shoot tips PLBs Raghavan and Torrey’s [110]
tankervilleae (1964) basal medium
(Banks ex Aiton)
Blume.
Vanilla Shoot tips Shoots MS medium + 1 mg/l [116]
planifolia Andr. BAP + 150 ml/l CW
Aerides crispum Leaf explant PLBs MS medium + 2.0 mM [117]
Lindl. BAP
Aerides Leaf explant PLBs MPR medium + 2 mg/l [118, 119]
multiflora Roxb. BAP + 0.5 mg/l NAA
Dendrobium Leaf explant Somatic ½MS medium + [120]
Cheingmai Pink embryos 18.16 mM TDZ
Dendrobium Leaf explant PLBs MS medium + 44.4 mM [121]
hybrids (Sonia BAP
17 and 28)
(continued)
Table 1 (continued)
Orchid species and
Micropera pallida Leaf explant PLBs ½MS medium + 2 mg/l [122]
Lindl. NAA+ 2 mg/l BAP
Phalaenopsis Little Leaf explant Somatic ½MS medium + [123]
Steve embryos 4.54 mM TDZ
Vanilla Leaf explant Callus MS medium + 4.52 mM [124]
planifolia Andr. Shoots from 2,4-D + 2.22 mM BAP
the callus
Phalaenopsis Leaf explant PLBs MS medium + 15 mg/l [125]
amabilis var. BAP + 3 mg/l NAA
‘Manila’
Aranda Deborah Inflorescence PLBs KC medium + 1 mg/l [126]
(Arachis explants BAP + CW
hookeriana Rchb.
f. Vanda
lamellate Lindl.)
Epidendrum Inflorescence PLBs/shoot ½MS medium + [127]
radicans Pav. explants buds 0.1 mg/l TDZ
Lindl.
Oncidium Sweet Inflorescence PLBs ½MS medium + 5 mg/l [128]
Sugar explants BAP + 5 mg/l NAA
Ponerorchis Inflorescence Shoot buds ½MS medium + [129]
graminifolia explants 4.44 mM
Rchb. f. BAP + 0.54 mM NAA
Vanda hybrid “Dr. Inflorescence Direct ½MS medium + 10 mg/l [130]
Anek” explants shooting BAP+ 2 mg/l TDZ+
30 g/l sucrose +7.5 g/l
agar + 250 mg/l
cefotaxime
showed the establishment of Grammatophyllum speciosum micropropagation and

transfer plantlets to the saranhouse, respectively [21].
3 Orchid Cryopreservation
Cryopreservation is the process of freezing living materials using liquid nitrogen

(LN) at 196 °C or 320 °F that physical and metabolic cellular processes are
effectively stopped, and then they can be recovered and grown to regenerate a whole
plant after cryopreservation. Cryopreservation is an ideal method for a long-term
conservation of plant genetic resources because it demands the least space, labor, and
maintenance; reduces susceptibility to disease and mutation. Orchid materials can
be used for cryopreservation including pollen, seeds, protocorms, PLBs, apical bud,
lateral bud, and meristem.
Fig. 3 Establishment of Grammatophyllum speciosum micropropagation; (a) A shoot tip was

excised under a stereo microscope, (b) PLBs were induced in ½MS liquid medium (2 months after
culture), (c) PLBs were subcultured for multiplication (1 month after culture), (d) PLBs were
cultured on ½MS solid medium supplemented with 0.05% (w/v) of activated charcoal, 2.0 mg/l
NAA and 1.0 mg/l BA, (e, f) Shoots and roots were induced, respectively (3 months after culture).
Bar ¼ 1 mm
Cryopreservation methods are based on the phenomenon of slow freezing

(cell dehydration due to cold temperature) and vitrification (glass formation). Slow
freezing was applied for cryopreservation of dormant buds and slow freezing
methods are concerned. In vitrification-based methods, cells are dehydrated by
exposure to concentrated chemicals or air drying before freezing, followed by
rapid warming to avoid intracellular ice formation. Six different vitrification-based
methods are: (1) vitrification, (2) encapsulation-dehydration, (3) encapsulation-
Fig. 4 Transfer of Grammatophyllum speciosum plantlets to the saranhouse. (a) Plantlets

(6.0–8.0 cm long) were obtained after 3 months of culture, (b) Plantlets were removed from the
bottle and washed with tap water, (c) Plantlets were transplanted to pots filled with small pieces
of coconut husk, (d) Plants grown 6 months in the saranhouse, (e) Plants grown 1 year in the
saranhouse, and (f) Plants grown 2 years in the saranhouse. Bar ¼ 1 cm (a–d)
vitrification, (4) droplet-vitrification, (5) vitrification cryo-plate method (V cryo-

plate), and (6) dehydration cryo-plate (D cryo-plate).
3.1 Dormant Bud Method
This is the first cryopreservation method that dormant buds were collected during
winter when buds were dehydrated at 10 °C to 30 °C before storage in liquid
nitrogen (196 °C). This method was successful in willows (Salix koriyanagi)
and poplar (Populus sieboldii) [131]. Later, dormant bud method was studied in
apple [132–134], cherry [135], blueberry [136], etc. There is no research on orchid
dormant bud method.
3.2 Slow Freezing Method
Slow freezing was the standard method in the initial stages [137], but need program-
mable freezing controller to reduce the temperature at the rate of 0.5–2 °C/min
depending on the plant type and the growth stage until about 40 °C, then store in
liquid nitrogen. The protocol takes many hours to complete and require expensive
tools. This method was popular during 1980–1990 to store undeveloped plant
tissues, such as suspended cells and calli of various plants including ornamental
plants [138] except orchids.
3.3 Vitrification Method
Vitrification is a method developed to reduce the temperature quickly and to warm

quickly, all parts of cells and tissues are in a glass state. Vitrification method involves
treatment of explants with plant vitrification solution (PVS), such as PVS1
(consisted of 22% glycerol + 15% EG + 15% PEG + 7% DMSO + 0.5 M sorbitol)
[139], PVS2 (consisted of 30% glycerol, 15% ethylene glycol, and 15% dimethyl
sulfoxide) [140], PVS3 (consisted of 50% glycerol + 50% sucrose in medium) [141],
and PVS4 (consisted of 35% glycerol + 20% EG + 20.5% sucrose) [142] to induce
dehydration of explants during cooling and warming to avoid intracellular ice-crystal
formation [139, 140]. The key to successful cryopreservation by vitrification method
is to prevent injury by optimizing exposure time to PVS for dehydration [143]
because over exposure time to PVS may result in cell injury and intracellular ice
formation during cooling. The optimum exposure time to PVS depends on explant
size and species specific. The suitable dehydration duration was related to the sample
size, the composition, and loading solution [144]. Sakai et al. [140] succeeded to
cryopreserve nucellar cells of naval orange using PVS2 solution, after that many
plants were experimented with success including orchids (Table 2). Thammasiri
[145] succeeded to cryopreserve seeds, using orchid seeds of Doritis pulcherrima.
The results showed that 50 min exposure time to PVS2 solution gave 62% survival.
3.4 Encapsulation-Dehydration Method
Encapsulation-dehydration method is developed from artificial seed production that

explants are encapsulated in alginate beads, precultured with high sucrose, desic-
cated by air-drying in a laminar air-flow cabinet or with silica gel, and then plunged
into liquid nitrogen [142, 146]. The advantages of this method are easy for manip-
ulation of encapsulated explants [147], preventing direct contact of toxic chemicals
Table 2 Successful cryopreservation of some orchid species and hybrids

Survival/
Material Species Method Dehydration regrowth References
Zygotic Bletilla Vitrification 3 h PVS2 at 60% [181]
embryo striata 0 °C germination
Seeds Dendrobium Drying Drying to 95% survival [182]
candidum <12%
Seeds Doritis Vitrification 50 min 92% [145]
pulcherrima PVS2 at germination
25 2 °C
Immature Bletilla Vitrification 60 min 81% survival [183]
seeds striata PVS2 on ice
Seeds Ponerochis Vitrification 60 min 86% survival [184]
graminifolia PVS2
var.
suzukiana
Seeds Vanda Vitrification 70 min 67% [185]
coerulea PVS2 at germination
25 2 °C
Seeds Vanda Vitrification 180 min 13.6% [186]
tricolor PVS2 on ice germination
Seeds Oncidium Encapsulation- 5 h silica gel 4.8% of the [187]
bifolium dehydration cryopreserved
seeds
produced
complete
plants
Mature Bletilla Droplet- 60 min 93% [167]
seeds striata vitrification PVS2 at germination
25 °C
Seeds Dendrobium D cryo-plate 60 min in 57.8% [105]
cruentum laminar regrowth
air-flow
cabinet
Protocorms Dendrobium Vitrification 15 min 88% survival [182]
candidum PVS2
Cell Doritaenopsis Vitrification PVS2 64% survival [188]
suspension
Protocorms Dactylorhiza Vitrification PVS2 9% survival [189]
fuchsii
Protocorms Vanda Encapsulation- 8 h in 40% [150]
coerulea dehydration laminar
air-flow
cabinet
Protocorms Grammato- Encapsulation- 8 h in 24% regrowth [168]
phyllum dehydration laminar
speciosum air-flow
cabinet
(continued)
Table 2 (continued)
Survival/
Material Species Method Dehydration regrowth References
Protocorms Dendrobium Encapsulation- 60 min 15% survival [190]
cariniferum vitrification PVS2 at
25 2 °C
Protocorm Dendrobium Encapsulation- 50 min 85% survival [191]
like-bodies candidum vitrification PVS2 at
0 °C
Protocorms Grammato- Encapsulation- 60 min 14% regrowth [168]
phyllum vitrification PVS2
speciosum
Protocorms Bletilla Droplet- 40 min 66% survival [167]
striata vitrification PVS2 at
25 °C
Protocorms Grammato- Droplet- 30 min 38% regrowth [168]
phyllum vitrification PVS2
speciosum
Protocorms Arundina D cryo-plate 30 g drying 77% regrowth [180]
graminifolia beads in
laminar
air-flow
cabinet
Protocorms Vanda V cryo-plate 20 min 33.33% [177]
lilacina PVS2 survival
Teijsm. &
Binn
Protocorms Vanda D cryo-plate Silica gel 83.78% [177]
lilacina for 1 h survival
Teijsm. &
Binn
Shoot Vanda pumila Desiccation Precultured 65% survival [192]
primordia in 1 mg/l
ABA for
3 days
Shoot tips Dendrobium Encapsulation- 6–8 h in 13.33% [193]
Walter dehydration laminar survival
Oumae air-flow
cabinet
Pollinia Dendrobium Vitrification 3 h PVS2 on 60% [194]
hybrids ice
Pollinia Luisia Vitrification 10 min 56% [195]
macrantha PVS2 germination
with plant materials, and nontoxic cryoprotectants are applied to protect

during dehydration [148]. However, this method is the longer dehydration procedure
than vitrification method [145]. Fabre and Dereuddre [149] succeeded to cryopre-
serve tomato apical meristems by using this method. There are many plant
species including orchids to be cryopreserved by encapsulation-dehydration.
Fig. 5 Regrowth of Vanda coerulea protocorms after cryopreservation by encapsulation-

dehydration in combination with loading solution. (a) Precultured beads after sterile air-flow
dehydration for 10 h. (b) Cryopreserved protocorms after 20 days of regrowth. (c) 3 months of
culture on modified VW agar medium showing shoot growth. (d) Plantlets after 8 months of culture
on modified VW agar medium (left: noncryopreserved, right: cryopreserved plantlet). (e) Plantlets
derived from cryopreserved protocorms after 5 months, and (f) 15 months of culture in the
greenhouse. Bar for a–c ¼ 1 mm, for d ¼ 0.5 cm, and for e, f ¼ 1 cm
Jitsopakul et al. [150] successfully cryopreserved protocorms of Vanda coerulea by

encapsulation-dehydration in combination with a loading solution (Fig. 5). Pro-
tocorms were selected 70 days after sowing seeds, harvested from 7-month-old
fruits. After encapsulation in an alginate matrix composed of 2% Na-alginate, 2 M
glycerol plus 0.4 M sucrose (loading solution), the protocorms were precultured in
modified VW [18] liquid medium supplemented with 0.7 M sucrose on a shaker
(100 rpm) at 25 3 °C for 20 h. Encapsulated protocorms were then dehydrated in a
sterile air-flow in a laminar air-flow cabinet at 25 3 °C for 1–10 h, and then directly
plunged into liquid nitrogen for 1 day. After thawing at 49 °C for 2 min,
cryopreserved beads were cultured on modified VW agar medium for regrowth.
The highest regrowth of 40% was observed with cryopreserved bead with 35% water
content for 8 h dehydration. No morphological variation was detected between
noncryopreserved and cryopreserved plantlets, and ploidy level was unchanged as
a result of cryopreservation.
3.5 Encapsulation-Vitrification Method
Encapsulation-vitrification method is a combination of encapsulation-dehydration

method and vitrification method. The explants are encapsulated in alginate bead, and
then subjected to dehydration by concentrated plant vitrification solutions, such
as PVS1, PVS2, PVS3, and PVS4. This method gives higher survival than
encapsulation-dehydration [151–153]. This method is used for many orchid species
and hybrids [154].
3.6 Droplet-Vitrification Method
This recent method is the fast freezing from small drops of plant vitrification solution
(PVS) on aluminum foil. Droplet-vitrification method is a combination of droplet-
freezing and solution-based vitrification, then placed on aluminum foil strip in
droplet of vitrification solution and then frozen by rapidly immersion in liquid
nitrogen [155]. Rapid warming was done by dipping the aluminum foil strips
in unloading solution without using a water bath [156]. During the cooling
and warming procedures, rapid heat transfer is needed to avoid freezing injury
[156–158]. Aluminum foil has an efficient thermal conductivity, resulting in quick
and uniform heat distribution among tissue [156, 158, 159]. A high warming rate
was employed to avoid recrystallization of intracellular ice or addition cell dehydra-
tion by extracellular ice [156]. The first success of droplet-vitrification method
was studied in potatoes [160]. Later, successes in papaya [161], prunes [162], jam
[163], chrysanthemum [164], banana [165], rose [166], Bletilla striata [167],
Grammatophyllum speciosum [168], Vanda coerulea [150], Sugar cane [169],
Vanilla orchid [170, 171], Oil Palm [172], Grapes [173], Orange [174], and etc.
Jitsopakul et al. [150] studied droplet-vitrification method for cryopreservation
of Bletilla striata mature seeds (0 day after sowing), zygotic embryos (3 days after
sowing), and protocorms (6, 9, and 12 days after sowing). Mature seeds were
surface-sterilized and sown on solidified ND medium supplemented with 3%
sucrose and cultured under illumination provided at an intensity of 62.0 μmol m2 s1
for 16 h/day at 25 °C for preparation of zygotic embryos and protocorms. Mature
seeds, zygotic embryos, and 6-day-old protocorms were precultured in liquid ND
medium supplemented with 0.3 M sucrose for 3 h on a shaker (110 rpm) and then
dehydrated with 2 M glycerol and 0.4 M sucrose in ND liquid medium (loading
solution) for 15 min, followed by exposure to PVS2 solution for 60 min at 25 °C.
Then, plant materials were soaked in liquid nitrogen by droplet-vitrification method,
and then were cultured on solidified ND medium supplemented with 3% sucrose,
germination rates of cryopreserved mature seeds, cryopreserved zygotic embryo,
and survival rate of cryopreserved 6-day-old protocorms were 93%, 91%, and 84%,
respectively. Cryopreserved 9-day-old protocorms gave the highest survival rate of
66% when precultured with 0.5 M sucrose for 3 h on a shaker, dehydrated with
loading solution for 15 min, followed by exposure to PVS2 solution for 40 min at
25 °C and cultured on solidified ND medium supplemented with 480 mg/l ammo-
nium nitrate and 3% sucrose. No survival was observed in cryopreserved 12-old-day
protocorms (Fig. 5). Figure 6a–e showed development of mature seeds. Mature
seeds developed into zygotic embryos at 3 days after sowing (Fig. 6b). Protocorms
formed green spots on zygotic embryos at 6 days of sowing (Fig. 6c). Protocorms
Fig. 6 Mature seeds of Bletilla striata. (a) Development of mature seeds sown on solidified
ND medium supplemented with 3% sucrose under illumination provided at an intensity of
62 μM m2 s1 for 16 h/day at 25 °C for 3 days, (b) 6 days, (c) 9 days, (d) 12 days, (e) and
Twenty droplets of PVS2 solution (2 μl) with protocorms, placed on sterilized aluminum foil strip
(7 20 mm2) (f). Bar: a–e ¼ 0.5 mm, f ¼ 1 cm
formed apical meristems at 9 days of sowing (Fig. 6d) and formed primary leaves
at 12 days of sowing (Fig. 6e).
3.7 V Cryo-Plate Method
A new method for preserving plant varieties in cold conditions was developed
9 years ago by Yamamoto et al. [175] is the V cryo-plate method. It is the
combination of encapsulation-vitrification and droplet-vitrification methods to
make artificial seeds to attach well (high quality) on aluminum sheets (cryo-
plate) size 7 mm 37 mm 0.5 mm, which has 10–12 holes (hole size
1.5 mm 0.75 mm) in which the aluminum sheet is thicker than the aluminum
foil (aluminum foil) used with the droplet-vitrification method. The thermal
conductivity of the aluminum sheet is better than the aluminum foil which has
a cooling rate of approximately 4,000 °C/min and an increase of temperature of
around 3,000 °C/min; while, aluminum sheets have a temperature reduction of
around 5,000 °C/min and an increase of temperature around 4,500 °C/min.
Therefore, V cryo-plate method gives higher survival than droplet-vitrification
method because the cooling and heating rates are faster. The advantages of the
V cryo-plate method are: (1) The cooling and heating rates are very fast.
Therefore, providing high survival. (2) The protocol is easy and convenient
because the plant materials are attached to the aluminum sheet throughout the
operation, not in a suspended state in a super cool solution, such as PVS2 and
PVS3. Using this method does not need special skills. Jitsopakul et al. [176]
Fig. 7 Dendrobium signatum Rchb. f. (a) blooming flowers, (b) pollinia embedded with 3%
sodium alginate gel on aluminum cryo-plates, (c) survival of pollinia, and (d) no survival of pollinia
after cryopreservation using V cryo-plate method
studied the effect of V cryo-plate and D cryo-plate methods for cryopreservation

of pollinia of Dendrobium signatum Rchb. f., a Thai orchid for pollination.
Pollinia were collected from flowers and then placed on the aluminum cryo-
plate containing 12 wells embedded with 3% sodium alginate gel (Fig. 7). In V
cryo-plate method, cryo-plates with pollinia were immersed in 0.6 M sucrose +
2 M glycerol loading solution (LS) for 15 min, then dehydrated with PVS2
solution for 40 min at room temperature (29 2 °C). In D cryo-plate method,
cryo-plates with pollinia were treated with LS for 15 min, then dehydrated in a
laminar air-flow cabinet for 3 h at room temperature (29 2 °C). In both
methods, cryo-plates were directly plunged into liquid nitrogen for 40 min, and
rapidly warmed in 1.2 M sucrose for 15 min. The cryopreserved and non-
cryopreserved pollinia were used to pollinate flowers of the same species. The
results showed that cryopreserved pollinia retained fertilizing ability, and had
similar fruit formation as those pollinated with noncryopreserved pollinia. The
fruit formation from cryopreserved pollinia in V cryo-plate and D cryo-plate
methods were 55.6% and 50%, respectively. Seeds from noncryopreserved and
cryopreserved pollinia were successfully produced and germinated into plantlets
with well-formed leaves and roots. Multiple stems (4.8 stems/plant) produced
from one plantlet when cultured on modified VW agar medium [18]
supplemented with 100 g/l banana, 150 ml coconut water, 50 g/l potato, and
20 g/l sucrose for 120 days at 25 2 °C.
Imsomboon et al. [177] studied two new cryopreservation methods, the V cryo-
plate and D cryo-plate methods of PLBs of Vanda lilacina Teijsm. and Binn. PLBs
were dehydrated with PVS2 solution (0, 20, 40, 60, and 120 min) and silica gel
(0, 0.5, 1, 1.5, 2, 2.5, and 3 h) for the V cryo-plate and D cryo-plate methods,
respectively. The results showed that the V cryo-plate method, dehydration with
PVS2 solution for 20 min, was a suitable method. It gave a survival score of 0.31,
higher than control (+LN; 0 min) and gave a percentage of survival of 33.33%. For
the D cryo-plate method, the suitable method for cryopreserved PLBs of Vanda
lilacina was dehydration with silica gel for 1 h. It gave the highest survival score of
0.78 and percentage of survival was up to 83.78%. Then, an unpaired t-test was used
to compare data of survival scores of cryopreserved Vanda lilacina PLBs between
the V cryo-plate and the D cryo-plate method using silica gel for 1 h in dehydration
step (Figs. 8 and 9).
Fig. 8 PLBs of Vanda lilacina recovered from V cryo-plate method on ½MS agar medium at the
4th week. (a, c) Noncryopreserved (LN) and (b, d) cryopreserved (+LN), (a, b) without PVS2
solution (0 min), and (c, d) with PVS2 solution (20 min) (bar ¼ 0.2 mm)
Fig. 9 PLBs of Vanda lilacina recovered from D cryo-plate method on ½MS agar medium at the
4th week. (a, c) Noncryopreserved (LN) and (b, d) cryopreserved (+LN), (a, b) without silica gel
(0 min), and (c, d) with silica gel (1 h) (bar ¼ 0.5 mm)
3.8 D Cryo-Plate Method
Niino et al. [178] developed the D cryo-plate method, which is a combination of

encapsulation-dehydration with the V cryo-plate method [179]. As some plants may
be affected by the use of PVS2 solutions, there is low survival. Therefore, in order
to avoid this damage, cells were dried by blowing air from a laminar air-flow cabinet.
In addition, the cryopreserved plant materials can be larger than those using the
V cryo-plate method because there is no problem of dehydration and the toxicity of
the PVS2 solution after prolonged immersion. Both V cryo-plate and D cryo-plate
methods have the same protocol except for the cell dehydration by air-drying, silica
gel, or drying beads, instead of using concentrated chemical solutions, such as PVS2
solution. Later, Cordova II and Thammasiri [180] developed D cryo-plate method
using silica gel and drying beads for dehydrating cells.
In this experiment, protocorms were placed in the preculture solution consisting
of 0.7 M sucrose on a shaker (110 rpm) at 25 3 °C for 1 day. After that, protocorms
were placed one by one in the wells which filled before with the alginate solution
containing 2% (w/v) sodium alginate in calcium-free ½MS basal medium with 0.4 M
sucrose. The cryo-plates were hardened for 20 min by slowly dispensing the calcium
chloride solution containing 0.1 M calcium chloride in ½MS basal medium with
0.4 M sucrose. Then the cryo-plates were surface dried using sterile filter paper,
placed in Petri dishes containing silica gel or drying beads in a laminar air-flow
cabinet (Fig. 10). Cryo-plates were dehydrated for 5 h until 25% moisture content
was achieved. Dehydrated cryo-plates were placed in 2 ml cryotubes and plunged
directly into liquid nitrogen for 1 day. Cryo-plates were removed from cryotubes and
warmed in unloading solution (1.2 M sucrose solution) for 20 min. Protocorms were
then removed from the cryo-plate and placed on ½MS agar medium for regrowth.
Growth conditions were conducted using 16 h light at 25 3 °C. For the cryo-plate
method, regrowth of control treatments dehydrated using silica gel was observed to
be 90%. Regrowth of control treatments dehydrated using drying beads was
observed to be 92.1%. In all other treatments, regrowth was observed to be 73.8%
using silica gel for dehydration. Regrowth for all other treatments dehydrated using
drying beads was observed to be 76.5%. Regrowth was observed at the 2nd week of
transfer to ½MS media. Dehydration using silica gel or drying beads did not
significantly affect regrowth rate. Protocorms dehydrated using silica gel or drying
beads developed into normal plantlets (Table 2).
4 Conclusions
In conclusion, plant tissue culture is a useful tool to increase and conserve orchid
under sterile conditions. But their problems are time-consuming, quite expensive,
and labor intensity and costly on growth of orchids in sterile condition. Furthermore,
the higher somaclonal variation, phenotypically change and losing materials by
microbial contamination were observed by using plant tissue culture technique.
Fig. 10 Cryo-plate method dehydrated with silica gel or drying beads. (a) Protocorm development,
(b) Preculture of protocorms in ½MS liquid medium with 0.7 M sucrose for 1 day, (c) Pour the
alginate solution containing 2% (w/v) sodium alginate in calcium-free ½MS basal medium with
0.4 M sucrose in the wells, (d) Place the precultured protocorms in the wells one by one, (e) Pour the
calcium chloride solution containing 0.1 M calcium chloride in ½MS basal medium with 0.4 M
sucrose, (f) Dehydration with 50 g silica gel, (g) Dehydration with 30 g drying beads, (h) Put
each cryo-plate in a 2 ml cryotube, (i) Plunge 2 ml cryotubes into liquid nitrogen for 1 day,
(j) Warming in 1.2 M sucrose solution for 20 min, (k) Plate on ½MS agar medium, (l) Regrowth,
and (m) Regrowth after 60 days
Plant cryopreservation methods have been developed from the beginning in 1960.
The methods changed from time-consuming protocols to more simple and more
efficient. For orchids, the methods are developed for various plant materials, such
as seeds, embryo, protocorm, protocorm-like bodies, apical bud, lateral bud, root tip,
and meristem. The developed methods are vitrification, encapsulation-dehydration,
encapsulation-vitrification, droplet-vitrification, V cryo-plate, and D cryo-plate,
respectively.
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Cymbidium: Botany, Production, and Uses
10
Ram Pal, N. K. Meena, R. P. Pant, and M. Dayamma
Contents
1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 262
2 Genetic Diversity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 263
3 Cytology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 264
3.1 Chromosome Number . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 264
3.2 Pre- and Postzygotic Barriers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 264
4 Natural Hybridization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 265
5 Cymbidium Breeding . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 266
5.1 Breeding Strategies in Cymbidium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 267
6 Propagation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 269
6.1 Conventional Propagation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 269
6.2 Nonconventional Propagation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 270
6.3 Thin Section Culture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 272
6.4 Shoot and Root Development . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 273
6.5 Acclimatization and Hardening . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 274
7 Cultivating Cymbidiums . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 275
7.1 Agroclimatic Requirements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 275
7.2 Growing Media . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 277
7.3 Nutrient Management . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 277
7.4 Watering . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 279
8 Plant Health Management . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 279
8.1 Insects and Pests . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 279
8.2 Diseases of Cymbidium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 284
9 Other Uses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 290
9.1 Medicinal Use . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 290
9.2 Cosmetics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 291
R. Pal (*) · M. Dayamma

ICAR-National Research Centre for Orchids, Darjeeling Campus, Darjeeling, West Bengal, India
e-mail: rampal_nrco@yahoo.com
N. K. Meena
ICAR-National Research Center on Seed and Spices, Tabiji, Ajmer, India
R. P. Pant
ICAR- Indian Agricultural Research Institute, Pusa Campus, New Delhi, India

262 R. Pal et al.
9.3 Food . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 291

9.4 Other Uses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 291
10 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 291
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 292
Abstract
Cymbidiums enjoy a prime position in the floriculture industry for heavy sub-
stance of the flowers and high flower longevity. They are cultivated for cut
flowers as well as pot plants for house decorations. Besides, they are used in
medicine, cosmetics, and food. The successful cymbidium cultivation requires
effective management of several factors controlling production and productivity.
Various aspects of cymbidium orchid cultivation including genetics and breeding
and plant health management have been discussed in the chapter.
Keywords
Genetic diversity · Breeding · Cultivation · Disease and pest management ·
Propagation · Agroclimatic requirements
1 Introduction
Cymbidiums are in cultivation since the last several centuries in China. The early
Chinese scholars, Wang Kuei Hseh in 1247, Zhang Shi Geng in 1233, Lu Ting Weng
in 1368, and others, wrote about orchids and their cultivation practices [1].
This indicates the popularity of cymbidium orchids during the period. Orchids in
China were not hybridized but collected from the forests, alba forms, inter- and
intraspecific variations, and plants with variegated leaves and fragrance which had
the highest regard. The first artificial cymbidium hybrid, Cymbidium eburno-
lowianum, was evolved by crossing Cymbidium eburneum with Cymbidium
lowianum in 1889 in England [2]. Over the past decades, many efforts have been
made to improve its flower size, color, shape, length and nature of inflorescence, and
flower longevity and to extend the period of cultivation, free-flowering habit, etc. by
transferring genes from different species as well as genera. Nowadays, cymbidium
cultivars are available in a range of colors except blue and black for cut flowers as
well as a house plant. The technological advancement made in the field of biotech-
nology, production technologies, disease and pest management, and postharvest
management has greatly benefitted orchids, including cymbidiums.
Orchid growers in the Netherlands, Australia, New Zealand, and America culti-
vate cymbidium varieties that are cool growing and bear large attractive flowers.
However, growers in China, Japan, Taiwan, and Korea cultivate cymbidiums only
for pot plants and appreciate it for flower, foliage, and fragrance. Cymbidiums for
flowers are preferred for clear colors, suberect or arching foliage, and round petals
with no markings or suffusion. The plants for foliage with various types of variega-
tion are also preferred. The combined character, variegation and flower, have an
10 Cymbidium: Botany, Production, and Uses 263
added advantage. The two species of this region, C. floribundum and C. ensifolium,
are very useful in the breeding of cymbidiums. C. floribundum transfers its trait for
dwarfing, free-flowering, clear coloration, and earliness and C. ensifolium for com-
pact growing, fragrance, and warm tolerance to their progenies [3]. The genus,
Cymbidium, has vast potential in the field of horticulture and medicine. However,
the natural population of some species like C. whiteae in India and C. tortisepalum in
China is dwindling very fast. The species need to be protected and conserved using
modern conservation techniques under in situ and ex situ.
2 Genetic Diversity
The genus Cymbidium shows a large variation in habitat ecology, plant architecture,
and floral morphology. The species of this genus are widely distributed from Sri
Lanka, Nepal, and India to China and Japan south through Malay Archipelago to the
Philippines, New Guinea, and north and east Australia [2]. The number of flower
in this genus has been reported to vary from 1–3 in C. eburneum to 20–60 in C.
madidum or 22–60 in C. canaliculatum and flower size from 2.5 cm in C. suave to
15 cm in C. tracyanum. Likewise, the leaf length ranges from 22 cm in C. tigrinum to
125 cm in C. atropurpureum and pseudobulb length from 50 cm in C. suave to
barely 1 cm in C. tortisepalum. These few morphological characters indicate
that a significant variation exists within the genus Cymbidium. Traditionally, the
diversity in Cymbidium is evaluated for morphological and physiological characters.
The members of the genus were grouped into 3 subgenera, 15 sections, and 53
species [4]. The classification of species is based on morphological observations
primarily floral morphology that cause taxonomic confusion. These are further
supplemented by anatomical, micromorphological, biochemical, and molecular
markers. Obara-Okeyo et al. [5] demonstrated that isozyme markers are helpful in
resolving the ambiguities arising through the morphological characterization of
Cymbidium species. In their study, they found the placement of different species
under different sections agreed with the morphological and karyological classifica-
tion of species proposed by [3] except C. floribundum which is currently under
section Cymbidium. Choi et al. [6] differentiated subtropical species from temperate
species, single terminal flower bud species with many-flowered species, broad-
leaved species with narrow-leaved species, and cultivars from 21 species and
cultivars of Cymbidium native to Korea, China, Taiwan, and Japan. The classifica-
tion using RAPD supported ecological-, physiological-, and morphological-based
classifications. Wang et al. [7] reported that ISSR markers provided valuable data on
the origin and biology of C. ensifolium cultivars. The markers successfully identified
the cultivars and helped in explaining their origin. It was also revealed that about
69% of total genetic variation in this species is due to genetic divergence inside
geographical groups suggesting both germplasm collection and in situ conservation
are essential for conserving the species. Sharma et al. [8] reported low intraspecific
variation in C. aloifolium and C. tigrinum compared to C. mastersii, C. elegans, and
C. eburneum, and these were closely related. Working on C. goeringii population
264 R. Pal et al.
structure, [9] recorded genetic similarity among the individuals within population up
to 14 m distance is partly due to limited pollen and seed dispersal in C. goeringii.
He suggested sampling should be conducted at 14–16-m interval to optimize the
genetic diversity.
3 Cytology
3.1 Chromosome Number
Majority of species under genus Cymbidium have 40 diploid chromosomes except

for Cymbidium macrorhizon Lindl., C. lancifolium Hook., and C. javanicum Blume.
These species have 38 diploid chromosomes. Thus, there are two groups in the genus
Cymbidium [10]. The occurrence of aneuploid has also been noticed in different
Cymbidium species like C. kanran Makino (2n = 40 and 41), C. faberi Rolfe
(2n = 40, 42, 43, and 44), C. javanicum Blume (2n = 38, 43), C. lancifolium
Hook. (2n = 38 and 39), and C. cyperifolium Wall. ex. Lindl. (2n = 36, 40).
Leoonhardt [11] reported chromosomal counts of 119 clones of 88 species and
hybrids of Cymbidium in which 89 were diploid, 15 triploid, 12 tetraploid, 1
hexaploid, and 2 aneuploid. C. insigne “Bierii” and C. pumilum “Yashima” were
found to be triploid with 60 chromosomes each. The other famous triploid grex
were Cymbidium Alexette, Cymbidium Alexfrida, Cymbidium Arabian Nights,
Cymbidium Bengal Bay, and Cymbidium Cassandra. The triploid arose from the
breeding of tetraploids with diploid. The tetraploid clones produce large flower and
have heavier flower substance bred out of crossing tetraploid with a tetraploid. Some
of important tetraploid grexes or clones are Cymbidium Early Bird “Pacific,”
Cymbidium Alexanderi “Westonbirt,” Cymbidium Babylon “Carpentier,”
Cymbidium Babylon “Castle Hill,” Cymbidium Balkis, Cymbidium Cleo’s Sherman,
and Cymbidium Dorchester “Alpha.” Nowadays, Cymbidium breeders use tetraploid
lines in their breeding program.
3.2 Pre- and Postzygotic Barriers
In addition to the delivery of the male nucleus to the ovule for the formation of
zygote, the pollen tube in orchids also serves as a stimulus for megasporogenesis.
Megasporogenesis occurs when the pollen tube reaches the ovarian cavity, after
assurance of fertilization. After delivery of pollens on the stigma, if it fails to
germinate or the pollen tube fails to reach the ovarian cavity, the flowers fall off
within a week [12]. The pre- and postzygotic barriers in flowering plant affect
successful seed formation. The prezygotic barriers include asynchronous flowering,
floral rewards for pollinators, and nonadherence of pollen to the stigmatic surface
and abnormal pollen tube growth. The postzygotic barriers are effective after
successful fertilization, and these include seed abortion and debility or sterility of
F1 hybrids and future generations. The prezygotic barriers like asynchronous
flowering could be overcome by growing the crops in a controlled environment or
storing the pollens for future use. Stort [13] studied microsporogenesis, pollen
germination, and fertility of male gametes in 24 artificial intergeneric and interspe-
cific F1 hybrids of orchids and found that irregular microsporogenesis in parental
species has similar chromosomes due to lack of homology of chromosomes in
parental species. The lack of homology in chromosome resulted in the formation
of tetrads without micronuclei, triads with 1–8 micronuclei, triads, dyads with or
without micronuclei, and monads. He encountered three situations: first pollens do
not germinate on the stigma, second the pollen tube grows down to the style yet do
not penetrate the ovary, and third the pollen tube grows normally though the style
and reaching the ovule. No fruit set occurred when the pollen tube failed to penetrate
the ovary. Thus, in vitro pollen germination and pollen tube growth test may be
insufficient to predict the fertilization. Wimber [14] reported normal cycle of meiotic
and mitotic division and pollen formation in the clones of Cymbidium schroederi, C.
erythraeum, C. grandiflorum, C. tracyanum, and C. lowianum except the clones of
C. insigne and C. lowianum var. concolor that showed varying levels of abnormal-
ities during reduction division. These included formation of univalents and frag-
ments leading to formation of polyads rather than tetrads. Du Puy [15] observed
occasional disruption in the meiotic cycle of C. lowianum and C. tracyanum by
forming univalents or fragments during telophase I of the reduction division. Stort
and Morales [12] reported break down of megasporogenesis at various stages of
megasporogenesis. He observed degeneration of megaspore mother cell at the
beginning of the first meiotic division and attributed it to lack of chromosome
homology that prevented full cell division and, consequently, degeneration of
megasporocyte during megasporogenesis. However, integument developed nor-
mally and ovule without embryo sack formed. Leoonhardt [11] reported production
of apparently viable yet not germinating seeds from 11 intergeneric crosses of
cymbidium. He suggested that this could be due to accumulation of inhibitors;
however, an attempt to leach out the inhibitors in liquid germination media did not
encourage germination. Another kind of endogenous incompatibility was suggested
by [11] where the progenies of complex hybrid grow normally but are incapable of
further hybridization due to chromosomal nonhomology during meiosis. The fertil-
ity of such clones can be restored by doubling of chromosomes. The diploid Cym
Peter Pan (C. ensifolium Cym Miretta) is a good example of such incompatibility.
For restoring the fertility, Cym Peter Pan was converted to tetraploid using colchi-
cines. The Cym Peter Pan has now produced 914 descendants.
4 Natural Hybridization
Plants maintain reproductive isolation through pre- and postzygotic barriers.

The prezygotic barriers include asynchronous flowering, rewards for different pol-
linators, the inability of pollen to adhere on the surface of stigma, and failure of the
pollen tube to fertilize the ovules. The postzygotic barriers cause seed abortion or
sterile seed production. Despite the prevalence of these barriers, several natural
hybrids are known to occur in the Orchidaceae family. Sympatric species in the
family of Orchidaceae maintain reproductive isolation through pollinator specificity.
266 R. Pal et al.
Table. 1 List of natural hybrids of Cymbidium

Year
Sl Area of of
no. Natural hybrid Female parent Male parent occurrence report
1. C. ballianum Rolfe C. eburneum C. mastersii Myanmar 1904
2. C. baoshanense F.Y. C. lowianum C. tigrinum China to N. 2001
Liu & Perner Thailand
3. C. florinda Rolfe C. erythrostylum C. iridioides Vietnam 1913
4. C. gammieanum C. elegans C. Northeast 1895
King & Pantl. erythraeum Himalayas and
Nepal
5. C. glebelandense C. insigne C. Vietnam 1911
Rolfe schroederi
6. C. hillii F.Muell. ex C. canaliculatum C. suave N. Queensland 1879
Regel
7. C. monanthum J.M. C. qiubeiense C. serratum China 2012
H.Shaw
8. C. nishiuchianum C. goeringii C. kanran S. Japan 2002
Makino ex J.M.H.
Shaw
9. C. nomachianum T. C. kanran C. Japan 2008
Yukawa & Nob.Tanaka lancifolium (Shikoku)
10. C. nujiangense X.P. C. cyperifolium C. China 2007
Zhou, S.P.Lei & Z.J. var. tortisepalum
Liu szechuanicum
11. C. oblancifolium Z.J. C. ensifolium C. China 2000
Liu & S.C.Chen lancifolium
12. C. purpuratum L.J. C. kanran C. China 2007
Chen, Li.Q.Li & Z.J. qiubeiense
Liu
13. C. rosefieldense C. insigne C. Vietnam 1912
Rolfe tracyanum
14. C. woodlandense C. mastersii C. Myanmar 1913
Rolfe tracyanum
According to [16], there are 14 natural hybrids of Cymbidium all over the world. The
first natural hybrid in the genus Cymbidium, C. gammieanum, was described
by [17]. It is a cross of C. erythraeum and C. elegans flowering at the same time in
Himalayan region. The natural hybrids reported in the genus Cymbidium are men-
tioned in Table 1.
5 Cymbidium Breeding
Artificial hybridization of cymbidium orchids began in the late nineteenth

century in England. The first artificial Cymbidium hybrid, C. eburno-lowianum (C.
eburneum C. lowianum), flowered in Messrs James Veitch & Sons Nursery,
England, in 1889. In the early years, the gene pool consisted of a few species like C.
tracyanum, C. eburneum, C. lowianum C. hookerianum, C. mastersii, and C.
giganteum (=C. iridioides). These species limited the improvement over color and
size, but the introduction of C. insigne brought a great change in the breeding
of cymbidiums. This species passed on characters like long inflorescence well
above the leaves, floriferousness, and large and long-lasting flowers to its proge-
nies [2]. C. Alexanderi (C. insigne C. eburno-lowianum) is one of the most
significant contributions of this species. Further, various color forms of C. insigne
and C. erythrostylum contributed significantly in evolving new hybrids of cymbid-
ium. Occurrence of natural tetraploids, C. Alexanderi “Westonbirt” and C. Rosanna
“Pinkie,” in two diploid grexes C. Alexanderi and C. Rosanna, respectively, made
the beginning of polyploidy breeding in cymbidiums. Soon after, the importance of
polyploidy in the breeding of cymbidium was realized, and a number of superior
hybrids were converted into tetraploids for using in the breeding program. The gene
pool was expanded further by introducing miniature species, viz., C. pumilum (=C.
floribundum), C. devonianum, C. madidum, and C. ensifolium, in the breeding
program, and many desirable traits such as perfume, cascading inflorescence,
color, and size were improved. The breeders are now utilizing less known species
and related genera for introducing variability in the cultivated hybrids. Over the
years, about 16,000 hybrids of cymbidium have been registered with International
Orchid Registration Authority, Royal Horticultural Society, London.
The important traits for cymbidium breeding include compact in growth habit, free
flowering, fast growing, multiple flower spikes, upright and self-supporting flower
spikes, round flower shape, orientation of flowers on the spike, flower longevity, and
attractive colors including pure color without sustaining. The flowers should have
attractive color, and the traits like fragrance, off-season flowering, extension of the
flowering season, and earliness should also be taken into consideration.
5.1 Breeding Strategies in Cymbidium
5.1.1 Hybridization and Selection

The cultivars of cymbidium are highly heterozygous, and crossing between them
usually leads to the production of fertile seeds that provides a range of genotypes.
The segregating F1 population in these crosses gives a source of useful gene
recombinants for selection and cloning of new, desired individuals. In hybridization
of cymbidiums, selection of parents, direction of cross, time of crossing, and F1
population size are the points taken into consideration. Seed germination and raising
of seedlings in tissue culture, acclimation of seedling and growing them to flowering
size in greenhouses, evaluation of seedlings for desired characters, selection, multi-
plication of selected clones, testing the elite clones in genotype x environment under
trials for testing their stability, and uniformity are the major steps required for
the development of the superior variety through hybridization and selection. It
takes about 12–14 years from crossing to release a commercial variety.
The primary and secondary crosses are developed through crossing
between the species and cultivars or variety. For large for flowers, C. eburneum,
268 R. Pal et al.
C. lowianum, C. mastersii, C. tracyanum, C. iridioides, C. hookerianum, C. insigne,

and C. erythrostylum and, for breeding of miniatures, C. pumilum (= C.
floribundum), C. ensifolium, C. devonianum, C. madidum, C. tigrinum, and C.
canaliculatum were used for developing primary and secondary hybrids. Since the
last few years, Australian species C. canaliculatum, C. suave, and C. madidum, and
Oriental cymbidium species C. faberi and C. goeringii have also been included in
breeding program. The superior diploid and tetraploid clones are used further
to improve desirable traits of the cultivar. Intergeneric hybrids involving two,
three, and four genera in crossing with cymbidium are mentioned below.
Bigeneric
Ansellia Cymbidium = Ansidium (Asdm.)
Bifrenaria Cymbidium = Bifrenidium (Bifdm.)
Bolbidium Cymbidium = Bolbicymbidium (Bby.)
Catasetum Cymbidium = Cymasetum (Cymst.)
Eulophia Cymbidiella = Cymbidilophia (Cdl.)
Cymbidiella Grammangis = Cymbidimangis (Cdn.)
Cymbidium Promenaea = Cymbidinaea (Cbn.)
Cymbidium x Zygopetalum = Cymbipetalum (Cbp.)
Ansellia Cymbidiella= Cymbisellia (Cml.)
Cymbidium Eulophiella = Cymphiella (Cymph.)
Cymbidium Maxillaria = Maxidium (Mxd.)
Cymbidium Phaius = Phaiocymbidium (Phcym.)
Trigeneric
Catasetum Clowesia x Cymbidium = Cymaclosetum (Cma.)
Cyrtopodium Cymbidium x Grammatophyllum = Cyrtogramcymbidium (Cgc.)
Cymbidium Grammangis x Grammatophyllum = Gramcymbimangis (Gbi.)
Cymbidium Eulophia x Grammatophyllum = Gramcymbiphia (Gyp.)
Cymbidium Grammatophyllum = Grammatocymbidium (Grcym.)
Catasetum Cymbidium x Grammatophyllum = Thompsonara (Thmpa.)
Quadrageneric
Catasetum Clowesia x Cymbidium x Grammatophyllum = Kalakauara (Kal.)
5.1.2 Polyploidy Breeding

The importance of polyploidy in cymbidium orchids was recognized soon after the
flowering of Cymbidium Alexanderi “Westonbirt,” a clone of Cymbidium
insigne Cymbidium eburno-lowianum registered in 1911. It was a chance tetra-
ploid with a large flower, heavy substance, superior color, and full shape. This clone
was repeatedly used to develop superior varieties of cymbidium. Cymbidium
Rosanna, a cross of Cymbidium Alexander Cymbidium Kittiwake, was registered
in 1927. Cymbidium Alexander was a tetraploid, while Cymbidium Kittiwake was
a diploid, but a clone “Pinkie” was a tetraploid. It is likely that Cymbidium
Alexanderi “Westonbirt” and Cymbidium Rosanna “Pinkie” have had developed
through the fertilization of unreduced gametes of their parents. Menninger [18]
produced tetraploid cymbidiums by applying colchicine on shoots produced through

the pseudobulbs. Wimber and Cott [19] cultured cymbidium protocorms in liquid
nutrient culture added with colchicine and converted in tetraploid seedlings (40%).
Kim et al. [20] produced triploid and tetraploid of Cymbidium Silky by treating PLBs
with 0.05% concentration colchicines for a week. The popular cultivars are often
tetraploid, while the wild species are diploid. Therefore, it is challenging to transfer
favorable genes from wild species to advanced varieties or hybrids. This problem
can be solved through the conversion of diploid species or primary hybrids
to tetraploid for using with superior cultivars in cymbidium breeding program.
Conversion of diploid to different ploidy levels in cymbidiums is generally realized
through using colchicine. The fertility in the Cymbidium Peter Pan “Greensleeves”
(Cymbidium ensifolium Cymbidium Miretta) was restored by doubling the chro-
mosome number, and this clone produced many beautiful hybrids [2]. The change in
ploidy level is also associated with change in morphological and physiological
characteristics of plant like increase in number and size of stomata, increase in cell
size and flower size, etc. According to [21], tetraploid flowers have a stronger scent
than diploid flowers in Cymbidium Golden Elf “Sundust.” The micropropagation
characteristics such as rate of proliferation, shoot bud and root differentiation
changes after chromosome conversion. The diploid has a higher rate of proliferation
and shoot and root differentiation than tetraploids.
6 Propagation
6.1 Conventional Propagation
Division of mother plant and sprouting of back bulbs are the two conventional
methods practiced for the propagation of cymbidiums. The conventional propaga-
tion methods are slow and take a reasonably long time to produce planting material
in a sufficient quantity. Viral diseases in cymbidium can be transmitted through
the use of the unsterilized cutting tool for division of plant and removal of pseudo-
bulb hence, sterilise the cutting tools before the division of a cymbidium plant.
6.1.1 Division of Plant

The division of the mother plant is the most familiar method for propagation of
sympodial orchids like Cattleya, Dendrobium, Cymbidium, and Epidendrums.
It involves the division of large clumps into smaller segments. The divided segment
should retain at least 1 back bulb, 2–3 leafy pseudobulbs, and 2–4 vegetative shoots.
The plants should be divided when new leads sprout and new roots have begun to
emerge from the base of the lead. The best time to divide orchids is from spring into
early fall because during this time, the plants are in active growth and are likely
to establish quickly with a little shock [22]. For dividing the plant, observe for
natural divisions of the plants and divide from the natural division by pulling them
apart; remove leafless pseudobulbs, dead roots, and leaf from the plant; and repot the
division in growing media. The plant establishes when the new roots anchor
270 R. Pal et al.
the growing medium. Always use sterilized cutting tools as CymMV and ORSV
spread through mechanical means [23].
6.1.2 Propagation Through Back Bulbs

Pseudobulbs are the enlarged and swollen part of the stem from which all leaves and
inflorescences arise. After flowering, leaves on the pseudobulb gradually dry up and
fall from the pseudobulbs. These leafless pseudobulbs are back bulbs and employed
for the regeneration of new plants. Soon [24] described a method for producing
plantlets from the back bulbs of cymbidium where back bulbs hung under the shade
and watered periodically until new shoots sprout and develop into plantlet. If the
bottommost bud is healthy, it will develop into new plantlet; otherwise, any bud
along the length of pseudobulb may develop into a new plantlet. A slightly different
method was proposed by [25]. In this method, washed back bulbs were placed in
plastic bags, and then the bags were hooked under the growing benches in a cool and
shady place. The bags were checked periodically for the emergence of shoots, upon
the emergence of shoot bud planted in small pots. All the above-described methods
do not reuse the pseudobulb in the process of propagation. Pal and Medhi [26]
suggested that back bulbs should be cleaned, washed, and soaked in fungicide
solution (Bavistin 2 g/l) for 30 min. Thereafter, back bulbs are immersed in a
solution containing 100 mg BAP (6-benzylamino purine) for 12 h, removed, air-
dried, and placed layer by layer in polyethylene bag filling empty spaces with
sawdust of Cryptomeria japonica (Dhuppi) and stored for 45 days in a cool and
dark place and monitored periodically for sprouting. After sprouting of pseudobulbs,
they are planted in the sawdust covering 3/4 the portion of the pseudobulb in beds or
planting trays. The sprouted buds develop shoots within 2–3 months. When the
plantlets attain 2–3 leaves and 2–3 roots, these are severed from the back bulb using
a sterilized knife and planted separately. The back bulbs with no sprouts are again
soaked in BAP solution and stored in a cool and shady place as described above.
In this process, back bulbs can be used 4–6 times to produce 6–8 plantlets in their life
span.
6.2 Nonconventional Propagation
Clonal multiplication through traditional methods of propagation is not rapid enough

to meet the demand for disease-free quality planting materials. By micropropagation,
it has become possible to propagate quickly the cultivars that are slow to increase
through conventional means. Because of its importance, micropropagation of cym-
bidiums has been widely studied [27–31]. The technique is based on in vitro culture
of explants capable of plant regeneration through culturing of various organs and
tissues on the media which are suitable for plant regeneration.
6.2.1 Seeds
Orchid seeds are very tiny and produced in thousands per capsule. The seeds lack
metabolic machinery and functional endosperm, with the result of only 0.2–0.3% of
the seeds germinate in nature [32]. Bernard [33] discovered that in nature, orchid
seeds would germinate if infected by a fungus. He described the role of mycorrhiza
in the germination of orchid seeds [34]. Knudson [35], for the first time, demon-
strated that seeds of orchid could germinate bypassing the requirement of fungus.
He germinated orchid seeds on sugar-enriched mineral medium in aseptic condi-
tions. Later, success in asymbiotic germination of orchid seeds and seedling forma-
tion on Knudson’s medium was reported by [36–39]. Arditti et al. [40] opined that
nutritional requirement, pH, source of nitrogen, and light requirement vary with
genus, with species, and even with ecotypes. Besides, the age of the capsule also
affects the seed germination in orchids [41].
A number of Cymbidium species often have been successfully propagated in vitro
through the seeds. Oriental cymbidiums are difficult to propagate than epiphytic
cymbidiums. The seeds of C. sinense (And.) Willd. germinate well on MS medium,
and the one supplemented with 0 mg BA, 1.0 mg l1 NAA, 8.0 g agar, and 30 g
sucrose is best for shoot development. However, a half-strength medium added
with 3.0 mg l1 NAA is best for root development [42]. Cymbidium seeds take
about 8–10 months to mature, and thus culturing at an early stage can save time. [43]
cultured 50-day-old C. faberi Rolfe on hormone-free medium, and the rhizome
developed within 4 months that can be multiplied about five times by subculturing
on the same medium supplemented with 1.0 mg l1 NAA at 40-day interval.
Rhizomes induced shoot buds on medium containing 1.0 mg l1 NAA + 2.0 or
5.0 mg l1 BA and complete plantlet on medium added with 1.0 g l1 activated
charcoal within 50 days. Seeds of C. giganteum Wall ex. Lindl. germinate well
(100%) on [44] medium (M) or Phytamax medium (PM) supplemented with 2.0 g l1
peptone or 1.0 mg l1 BAP. The highest protocorm multiplication occurs in liquid
medium supplemented with 2.0 mg l1 BAP and 1.0 mg l1 NAA. BAP and NAA
2.0 mg l1 each induces multiple shoots, and roots develop in half-strength PM or M
medium [45]. The seeds obtained through the crossing of two cultivars or species are
highly heterozygous and unsuitable for utilization in commercial production of cut
flowers or house plants. The commercial orchid production industry requires iden-
tical and uniform plants for the production of cut flowers or house plant. The seed
culture is only resorted to multiply a particular species or for raising the plants of
crosses by the breeders.
6.2.2 Induction of Protocorm-Like Bodies (PLBs)

Arditti and Ernst [46] reported that like many other higher plants, regeneration
of orchids by tissue culture method takes two main pathways, namely, (i) direct
differentiation of protocorm-like bodies (PLBs) from cultured tissues and organs and
their subsequent development into plants and (ii) indirect differentiation of PLBs
from explant tissues through an intermediary callus phase.
Shoot Tip Culture

Morel [27] produced virus-free cymbidium by culturing shoot tips on modified
Knudson C medium that gave rise to 1–2 mm size colorless PLBs within 2 months
of culture that turned green and produced rhizoids and leaf primordium. These
272 R. Pal et al.
bodies were sectioned to produce more PLBs. Wimber [28] published a detailed
protocol for in vitro production of cymbidium. He could produce the PLBs within
a month on liquid Tsuchiya medium. Sagawa et al. [47] cultured the explants first in
liquid-modified KC medium for 4 weeks and then transferred them on solid medium
to induce PLBs from the explants. Gu et al. [29] produced PLBs from lateral buds on
VW medium added with 1.0 mg BA l1. Likewise, [48] also induced PLBs from
the shoot apices of Cymbidium cv. Horunoumi on MS medium supplemented with
0.1 mg l1 BAP and 0.5 mg l1 NAA. Subramaniam and Taha [49] found that VW
medium and 5.0 mg l1 NAA are the best for PLB induction from the shoot tips of C.
atropurpureum (Lindl.) Rolfe.
Leaf Segment Culture

The foliar explants are readily available and do not require the sacrifice of the mother
plant, and they are available throughout the year. Wimber [50] reported the produc-
tion of PLBs from the leaf explants of cymbidium. It is the only report on the
propagation of cymbidium using a leaf as explants. Probably, leaf explants do not
respond well to callus or PLB induction.
Root Segment Culture

Beechy [51] suggested the possibility of using aerial roots for micropropagation
of orchids. The root meristem consists of highly determinate cells which have
limited morphogenic competence for bud formation [52]. Yasugi et al. [53] induced
PLBs from basal root segments of in vitro-derived root segments of Kenny “Wine
Color” on MS medium supplemented with NAA and BA at 0 or 1 mg l1 under light
conditions. Nayak et al. [54] induced multiple shoots from root segments of C.
aloifolium (L.) Sw., on MS medium, supplemented with 2.2–4.5 μM thidiazuron. In
all the published reports, the explant has been derived from the in vitro-grown plants,
not from the ex vitro-grown plants.
6.3 Thin Section Culture
Thin cell layer (TCL) system refers to excision of small explants excised from plant
organs like stem, leaves, floral inflorescence, or flower primordial either longitudi-
nally (lTCL) or transversally (tTCL). The lTCL consists of only one type of tissue
such as monolayer of epidermal cells, whereas tTCL comprises a small number of
cells from different tissue types, epidermal, cortical, cambium, perivascular, and
medullary tissue, and parenchyma cells [55]. Thin cell layer culture is highly
efficient as compared to the standard tissue culture technique [56]. Recently, thin
cross section of actively growing plant parts such as shoots, leaves, inflorescence
stalk, and developing PLBs has effectively been used by some workers for plantlet
generation in some orchids [57].
Begum et al. [30] reported that outer tissues (OT) from the PLBs of
Cymbidium Thanksgiving cv. Nativity when cultured on medium supplemented
with 0.1 mg NAA and 0.5 mg l1 BA showed highest PLB formation. Culturing
TCS of C. aloifolium PLBs on MS medium supplemented with ZR at 14.0 μM

produced 28.2 PLBs per explant 80% shoot formation frequency [31]. Huan et al.
[58] reported protocorm segments of Cymbidium Great Flower “Rainbow” when
cultured on a medium containing 0.5 mg l1 NAA and 0.1 mg l1 TZD produced
callus that converted to PLBs by culturing on hormone-free medium. Chang and
Chang [59] induced calli in the sections of pseudobulb, rhizome, and the roots of
seed-derived plantlets of C. ensifolium var. misericors on medium supplemented
with 10 mg l1 2,4-D and 0.1 mg l1 TDZ. Le et al. [60] also induced callus on VW
medium containing 0.1 mg l1 NAA and 0.01 mg l1 TDZ within 1 month of culture
that can proliferate at a 4-week interval and can convert into PLBs on culturing at
hormone-free medium. The callus-derived PLBs form complete plant within 4
months.
6.4 Shoot and Root Development
Skoog and Miller [61] reported that a balance between cytokinin and auxin controls
organogenesis. They found a relatively high auxin/cytokinin ratio induced root
formation in tobacco callus, whereas a low ratio of the same hormone favored
shoot production. Paek and Kozoi [62] reported that the growth pattern and regen-
eration ability of rhizomes derived from asymbiotic seeds or shoot tip culture varied
according to media composition, kind and concentration of growth regulators,
culture conditions, and species and varieties. The BA is the best for inducing
shoot formation, switching rhizome in PLBs, and directly forming shoot from
branched rhizomes.
The rhizome tips of C. faberi Rolfe. produced shoots in modified Kyoto solution
incorporated with 0.1 mg l1 BA; however, the higher concentration of BA
(10.0 mg l1) prevented the shoot formation [63]. On the contrary, [64] reported
that shoot formation in C. kanran occurred best when the basal medium (MS) was
supplemented with 10 mg l1 BA. Both the species might have varying requirements
for cytokinins for shoot bud development. Kinetin concentration 10 mg l1 in culture
medium inhibited adventitious root formation in C. goeringii and dependent on the
intensity of light [65]. Ogura and Okubo [66] achieved proper shoot development
in rhizome apices of C. ensifolium (L.) Sw. by supplementing the MS medium with 1
or 10 mg l1 NAA and by reducing ammonium and potassium nitrate to 25% and
50%, respectively, from their original medium. However, in C. kanran, shoot
development occurred by adding 1.0 mg 11 BA and 0.1 mg l1 NAA to the basal
medium. TDZ 0.1–1.0 mg l1 in culture medium initiated shoot bud development in
the rhizomes of C. sinense Willd. but retarded rhizome proliferation. Culturing
regenerated shoot buds on basal medium with 0.5 mg l1 NAA and 1 g l1 activated
charcoal developed complete plant within 4 months. Kusumoto [67] achieved
reliable shoot bud formation and their consistent development to shoots in cymbid-
ium protocorms with kinetin 0.1–1.0 mg l1 and 0.01–0.1 mg l1 NAA or with
0.1–1.0 mg l1 GA3 and 0.01–0.1 mg l1 NAA, but the combination of GA3
(1.0 mg l1) and NAA (0.1 mg l1) was most effective for root formation.
274 R. Pal et al.
Compared to terrestrial cymbidiums, the epiphytic cymbidiums are easy to

propagate. Siddique and Paswan [68] observed the best growth and differentiation
in PLBs of C. longifolium (=Cymbidium erythraeum) on NC medium supplemented
with 0.5 mg l1 BA. Nayak et al. [69] reported the effect of growth regulators on
proliferation and organogenesis of rhizomes derived from seeds of C. aloifolium Sw.
They found that auxins increase the proliferation of rhizomes, but NAA 5 mg l1 is
most effective. The highest shoot formation frequency (91.5%) and maximum
number shoot bud/rhizome (3.5) were obtained when the medium was supplemented
with 1 mg l1 BA.
Apart from growth regulators, altering the compositions of defined culture media
or adding organic supplements is useful in inducing shoot buds from the PLBs.
Kukulczanka and Jastrezebska [70] reported that addition of Heller’s minor elements
to Tsuchiya medium stimulates shoot and root formation in the PLBs. However, Mg
at 250–400 mg l1 retarded the protocorm differentiation but increased protocorm
number. Paek and Chun [71] obtained shoot formation in cymbidium protocorms by
supplementing KC medium with 4 mM NH4 N or 8 mM Ca2+ or 1 mM Mg2+ and the
root formation on medium supplemented with of 4 mM NHþ 4 or 4 mM Ca .
2+
Shimasaki et al. [72] studied the effect of sucrose and trehalose on organogenesis
of PLBs of C. finlaysonianum Wall. ex Lindl. and C. kanran and found that trehalose
at 10 g l1 enhanced shoot bud differentiation, while 5 g l1 increased root formation
in both the species of Cymbidium. Ueda and Torikata [73] studied the effects of
growth substance on organogenesis of C. insigne Rolfe., C. pumilum Rolfe, and C.
goeringii Rchb. f. and found that C. insigne and C. pumilum protocorms develop into
plants in basal medium (KC with NC microelements) within 2 months of culture.
The organic supplements NAA, bacto-tryptone, L-arginine, and L-aspartic acid
promoted growth and development, whereas yeast extract inhibited the growth. In
C. goeringii protocorm developed in rhizome rather than shoots. The basal medium
supplemented with 0.1 mg l1 NAA produced shoots, while yeast extract and 1 g l1
and bacto-tryptone (1–5 g l1) promoted the growth of shoots remarkably.
6.5 Acclimatization and Hardening
The acclimatization of plantlets from in vitro to ex vitro environment is concerned

with the transfer of plantlets in compots and then placing them in high humidity
structures and high shade levels and then slowly reducing the humidity and increas-
ing the intensity of light so that the plantlets could withstand the outside environ-
ment. Successful ex vitro acclimatization of micropropagated plants determines
the quality of end products and its commercial introduction and the economic
viability of enterprise [74]. When the shoots or plantlets are transplanted from
culture to greenhouse, they may desiccate or wilt rapidly and can die as a result
of the change in the environment unless substantial precautions are taken to accom-
modate them [75]. The in vitro environment is characterized by a saturating atmo-
sphere with water vapor, with very low vapor pressure deficit [76, 77], low CO2
concentration during photoperiod [78], high CO2 concentration during dark period
[79], and high ethylene concentration [80]. These characteristics except for light
intensities are primarily due to the low number of air exchanges of the vessel and
relatively small air volume of the vessel headspace. High relative humidity and low
CO2 concentration in the vessel limit the gas exchange [81]. Mani and Nagaraju [82]
suggested that cocopeat alone or in combination with sand is suitable for hardening
micropropagated plantlets of cymbidium.
7 Cultivating Cymbidiums
The commercial cultivation of cymbidium begins with the acquisition of mericlones

or seedlings from the plant propagators. The mericlones are used for the production
of cut flowers or pot plants as they are genetically identical and uniform in size.
The seedling is the product of seeds obtained through artificial hybridization of two
dissimilar plants for evolving a new variety. These plants are grown under optimum
conditions for a certain period to attain maturity. Thus, plants defer sexual repro-
duction until they acquire the capacity to produce flower and seed. In cymbidium
orchids, this period varies from 3 to 4 years from the deflasking and differs
with species or variety. Miniature cymbidium cultivars require a shorter period
than cultivars of standard cymbidiums.
7.1 Agroclimatic Requirements
7.1.1 Temperature
Based on climatic requirements, cymbidiums are divided into two groups: standard
and miniature. The standard cymbidium cultivars have been evolved from the
species occurring in high hills of Himalayas. They bear large flower and require
a long cool period to induce flowering. The two views are often proposed for flower
induction in large-flowered cymbidiums: (i) cooler nights in the Himalayan region
(10–14 C) during summer months and (ii) a temperature difference of 10–12 C
between day and night helps in induction of flowering in large-flowered cymbid-
iums [83]. Went [84] reported that no flowering occurred in C. giganteum (=Cym-
bidium iridioides) when grown for 1–3 months at 26/14 C and 23/14 C day/night
temperatures, and little flowering was induced in plants held at 20/10 C and 20/
14 C. The highest number of plants induced flowering when the plants were grown
under 20/10 C and 20/14 C temperature regime. He concluded that both day and
night temperatures have an effect on the induction of flowering in large size or cool
growing cymbidium. Powell et al. [83] reported that Cymbidium cultivar Astronaut
“Rahah” when exposed to day/night temperatures of 26/12 C and 14-h photoperiod
produced an average of 5.9 inflorescences per plant. The numbers of inflorescence
declined to 0.8 and 1.7 at day/night temperature of 20/12 C or 16/18 C, respec-
tively. The large-flowered cymbidiums grown today have many species in their
ancestry and are likely to influence the flowering behavior of hybrid cultivars. It is
276 R. Pal et al.
likely that contribution of the species to the genome of the hybrid influences the
temperature requirement of the hybrids.
The temperature influences flowering and quality of flowers in cymbidium. Li
et al. [85] transferred the plants of Cymbidium Sazanami in a cold room
(8–28 C), warm room (15–30 C), and hot room (20–30 C) after flower bud
initiation. The flowering in cymbidium was delayed by 1 month in a cold room
and advanced by 1 month in the hot room as compared to the warm room. The
best quality of flowers was in warm rooms. The high temperature (>25 C) at an
early stage of flower development causes bud drop in miniature and intermediate
cymbidium cultivars. To get rid of this disorder, cymbidium growers in Japan
shift their plants from original sites to high hills where the temperature varies
between 20 C and 30 C [86]. Higher temperature (18 C) during winters brings
to the rapid growth of shoots in Cymbidium and results in early flowering
(March–April), but plants suffer from bud blast due to higher summer tempera-
ture. Whereas the low temperature during winters (6 C) suppresses the growth
during winters that get recovered during spring, shoots mature in June and
initiate flower buds in August [86].
The vegetative buds grow normally at 30/25 C day/night temperature. During
winters low night temperature (2 C) encourages the production of leads, and higher
light intensity helps in accumulation of assimilates. The higher temperature during
winter reduces the production of leads [87]. According to [88], warm day/night
temperature (30/25 and 25/20 C) accelerates pseudobulb development and flower
formation in Cymbidium ensifolium var. misericors. Plants with 1-year-old pseudo-
bulb produced 2.3 and 1.6 inflorescences, respectively.
7.1.2 Light
In cymbidium orchids, both vegetative and reproductive stages are affected
by temperature and light. Komori [87] reported that both light and temperature
influence the lead production in Cymbidium cv. Lovely Angel “The Two Virgins,”
a cultivar with difficulty to induce leads. Growing the plants at 2 C in without shade
increased production of leads (1.7 per plant), but these were only 0.9 per plant under
50% shade. The plants cultivated at 15 C without shade had more fresh weight of
shoots and roots compared to plants cultivated under the 50% shade at the same
temperature. Low light intensity in flowering years results in a marked reduction in
flower stalk emergence because of reduced accumulation of assimilatory prod-
ucts [88, 89]. NI has been effective for accelerating the growth and development
of LD herbaceous plants. Kim et al. [90] reported that though night interruption with
low light intensity at 3–7 mol m2 s1 for 4 h (22:00–02:00) promotes flower
induction with increased growth rate during the juvenile stage in Cymbidium Red
Fire and Cymbidium Yokini, but night interruption with high light intensity (120 mol
m2 s1) produced high-quality plants. The increase of starch in leaves during
vegetative growth and soluble sugars in pseudobulbs and roots during reproductive
growth of Cymbidium “Red Fire” under night interruption is crucial for increasing
plant size and thereby promoting flowering [90].
7.1.3 Humidity
Humidity ranging from 50% to 80% in the growing environment is necessary for
proper growth and flowering of Cymbidium. The excess humidity would likely to
increase the incidence of diseases and, therefore, dehumidify the environment by
providing proper air circulation by controlling the vents of the greenhouse. Humidity
below 40% can be managed by installing overhead sprayers or foggers.
7.2 Growing Media
The growing media for cymbidium should have good water holding capacity;
a porous, pH value between 5.5 and 6.5; and electrical conductivity of about
1.05 mhos/cm which is relatively stable. Cymbidium producers use various kinds
of growing media like composted bark, vermicompost, carbonized rice husk, pum-
ice, groundnut shells, leafmold, coconut husk, coir dust, leafmold, and alone or in
combination with inert growing media constituents like perlite, rock wool granules,
expanded clay, and vermiculite. Incorporating 30–40% vermicompost in a growing
medium containing 50% pumice, 30% charcoal, 10% vermiculite, and 10% peat
moss improves growth and flowering in cymbidium orchid [91]. Yamane and
Sakuamoto [92] reported that dusting of the roots of young cymbidium plants with
hydrophilic polymer and then planting in composted bark enhances the growth of
plantlets, whereas plants older than 1 year grow and flower well in a medium
containing composted bark and granular soil containing hydrophilic polymer in
1:1 ratio. The growing substrate that dries quickly would reduce water and nutrient
availability for plants, and high water retentivity in substrate reduced oxygen
availability and encouraged rotting of the roots.
7.3 Nutrient Management
Usage of fertilizers depends on the plant growth stage, cultivar, growing media, and
growing environment. Plants require high nitrogen during the vegetative stage and
less during the reproductive stage. Well-balanced fertilizer is needed for optimum
growth and flowering and can be supplied through organic and inorganic sources.
Naik et al. [93] analyzed growing media (leafmold/coco chips/vermiculite/bricks,
4:2:1:1) at different intervals of growing period and found that NPK content
decreases with the growing period. NP was present in the range of sufficiency. K
was present, but its deficiency symptoms were not observed may be due to its
mobilization to leaves from the storage organs. The required amount of calcium,
magnesium, and sulfur was available from the media content. The microelements
manganese, copper, iron, and zinc also decreased with the advancing of crop growth.
The content of iron was found 30–40% below in young plants. The young plants
show the deficiency by manifesting the symptom chlorosis between the veins in
extreme case necrosis of the leaf margins and tip of the leaves. The deficiency was
corrected by applying 50 ppm iron sulfate at 15-day interval in young plants and
278 R. Pal et al.
100 ppm iron sulfate in 2-year-old plants. Naik et al. [93] reported the NPK
requirement for young plants (1 year old) of Cymbidium Pine Clash “Moon
Venus” and found that NPK in the ratio of 30:10:10 @ 0.1% is best in terms of
growth attributes like leaf length, leaf girth, pseudobulb length, pseudobulb girth,
and number of pseudobulb per clump, and for intermediate size of plants (2 years
old), the NPK ratio of 20:20:20 @ 0.1% is suitable for enhancing the growth
attributes.
Poole and Seeley [94] found that Cymbidium hybrids in nutrient culture
required 100 ppm N, 50–100 ppm K, and 25 ppm Mg for optimal growth. The
nitrogen below 50 ppm shows nitrogen deficiency symptoms. Magnesium at
100 ppm decreased growth in comparison to 50 ppm, and potassium has little
response on the growth of cymbidium plants. Increasing nitrogen concentration
(4, 6, and 8 mM/l) increased shoot production but reduced spike/shoot ratio in
miniature Cymbidium cv. “Pendragon Sikkim.” Further, withholding of nutrient
application during May to June resulted in a reduced shoot formation, but a
higher spike/shoot ratio along with earlier flowering, as compared with a con-
tinuous fertilizer supply in the nutrient solution [95]. Pascale et al. [96] studied
nitrogen in three cultivars of cymbidium, Floripink, Pendragon Irene, and
Traceredway, in soilless culture system and reported that N uptake was 8.0,
7.2, and 3.1 g per plant per year. The leachate volume was 40% of the given
nutrient solution in Floripink and Traceredway, while it was inconsistent and less
in Pendragon Irene (18%), with a mean concentration of 1.68 mmol of NO 3 ,
1
0.055 of PO3 4 , and 0.24 of K+ per liter of leachate. High EC (1.4 dS m )
1
produced more flower spikes per m , as compared with low EC (0.6 dS m ), but
2
fewer per shoot, and the spikes were shorter in Cymbidium Mary Pinchess “Del
Rey.” There was a positive correlation between shoot formation and spike
production per shoot, whereas it was negative with the nitrogen content of
young leaves. The shoots produced during autumn and winter were more pro-
ductive than shoots produced during spring and summer [97]. Song et al. [98]
investigated nitrogen and potash absorption by 2-year-old plants of Cymbidium
Jungfrau. Nitrogen absorption was higher in full sunlight than 60% light,
whereas the phosphorus absorption was higher under 60% of sunlight. The
67% of N absorbed in plants was redistributed to the bulbs (39%) and leaves
(28%), while 46% of P was absorbed and distributed in bulbs (36.2%) and leaves
(10.2%). Accumulation of P in leaves is threefold lower than that of N. N and P
absorption in 1.5- or 1-year-old daughter plants were greater than in immature
daughter plants of the mother plant. The solution culture had more NPK and Mg
content in leaves than pot culture.
Naik et al. [99] studied the different concentrations of Panchgavya on the
growth and flowering of Cym “Sleeping Nymph.” Panchgavya is an organic
product made out mixing five components, namely, cow dung, cow urine, cow
milk, curd, and ghee, in a definite proportion and believed to promote growth and
immunity in plant system. Drenching of growing media content or spraying with
1:30 (Panchgavya/water) improved vegetative and reproductive characters of
Cymbidium.
7.4 Watering
The growing media of cymbidiums should always be moist, but not too wet. Soggy
growing medium would reduce oxygen in the container and encourage the patho-
gens to decay the roots. Cymbidium requires sufficient water during the season of
active growth, particularly during spring and summer. Water thoroughly, once or
twice a week, more often when the weather is warm. A light misting each day will
keep cymbidiums moist enough. Pascale et al. [96] studied water consumption of 6-
year-old plants of three cultivars of three cymbidium, Floripink, Pendragon Irene,
and Traceredway, in a growing medium containing (v/v) urea-formaldehyde (48%),
polyurethane (48%), polyurethane (4%) and pH and EC of the nutrient solution
adjusted at 6.0 and below 0.6 dS m1. The mean water consumption was least in cv.
Pendragon Irene (0.6 L per plant per day) compared to Traceredway (1.6 L per plant
per day) and Floripink (1.4 L). The water consumption was related to leaf area of the
cultivars.
8 Plant Health Management
Cymbidium orchids grow in subtropical to temperate regions of Indian subcontinent.

It produces a unique and high-quality flower in diverse color and pattern. The quality
and quantum of Cymbidium flowers get affected by numerous insect-pests and
diseases at different growth stages, both in artificial natural habitation and inside
protected structures under controlled conditions. Insect-pests associated with
Cymbidium are described below.
8.1 Insects and Pests
8.1.1 Aphids
Aphids are the major problem in Cymbidium orchid. There are two species of aphid,
i.e., black aphid, Toxoptera aurantii (Boyer de Fonscolombe), and yellow aphid,
Macrosiphum luteus (Buckton), which mainly cause damage to orchids.
Black aphid, Toxoptera aurantii (Hemiptera: Aphididae): Black aphid (T. aurantii)
recorded to infests on 120 host plants that include orchids, Hibiscus, coffee, cocoa,
ficus, citrus, mango, pomelo, etc. [100]. It is also known as black citrus aphid found
in South America, Africa, eastern Asia, India, Australia, and the Mediterranean
region. Adults are oval, shiny black or brownish-black in color, winged as well as
wingless, small in size, and measuring about 2–3 mm in length. Nymphs are
wingless and smaller than adults but similar in shape. A pair of black and white
banded antennae is reflected well toward the abdomen. This species formed colonies
on the young shoots, flowers buds, and flowers. T. aurantii completes a life cycle in
6–20 days depending on existing environmental conditions. Lower temperature
increases the developmental period of aphid.
280 R. Pal et al.
Yellow aphid, Macrosiphum luteum (Hemiptera: Aphididae): Yellow aphid, M.

luteum, is reported to feed on Dendrobium, Epidendrum, Vanda, Oncidium, Phal-
aenopsis, Cattleya, and Arundina other than Cymbidium under protected conditions.
Adult of this species is yellow to greenish-yellow in color, while nymphs are pale
green. Pest is small-sized, about 2–3 mm length span; oval-shaped with two blackish
cornicles are present on the tip of the abdomen. Adults are winged or wingless and
the wingless form has a brownish patch on the top of the abdomen.
Damage
Aphids feed on host plants by sucking the cell sap. In Cymbidium orchids, nymphs
as well as adults colonize on tender shoots or flower buds or new flower spike and
even opened flower and suck the cell sap. This kind of damage often causes the
plants to become deformed, and tender shoots curled and shrivelled, and sometimes,
gall is formed on an affected portion. They also secrete honeydew like other soft-
bodied insects, resulting in the development of sooty mold that affects the photo-
synthesis process. High humidity and cloudy weather fasten the buildup of aphid
population. The affected plants retard growth and ultimately deteriorate the quality
of flowers. T. aurantii is a vector of some viral diseases like citrus tristeza virus on
citrus and little leaf and lemon-ribbing virus of lemon and also believed to transmit
some viral diseases in orchids, i.e., Cymbidium mosaic virus from infested plant to
healthy plants.
Management
For producing quality flowers, aphid infestation should be kept under control.
Several natural enemies of aphids are present in the orchid ecosystem, which play
a vital role in maintaining aphid population below a harmful level. These are
Coccinella septempunctata, Menochillus sexmaculata, Cryptolaemus montrouzieri,
Adonia variegata, and Chrysoperla carnea that feed on aphid’s nymph and adult
stages. On account of the artificial need of control, apply Vertilec (Verticillium
lecanii, an entomopathogenic fungus formulation) @ 4 g/l of water or azadirachtin
0.03% EC @ 3 ml/l of water alternatively as a foliar spray to reduce aphid
population.
If chemical control becomes necessary, plants should be treated with any one of
the following insecticides like fipronil 5% SC @ 0.03% or malathion 50 EC @
0.05% or acephate 75 SP @ 0.05% or imidacloprid 17.8 SL @ 0.003% on the
appearance of aphids on the new spikes or flower buds before opening the flowers
and repeat the spray at 10–15-day interval to control the pest and quality production
of flowers.
8.1.2 Scale Insects

Scales are the major pest problem in orchids in the forest and artificial natural
habitation. There are five species of scale insect recorded on various orchid species
and hybrids in Indian subcontinent, i.e., boisduval scale, Diaspis boisduvalii;
ti-scale, Pinnaspis buxi; soft brown scale, Coccus hesperidum; Florida red scale,
Chrysomphalus aonidum; and lecanium scale, Lecanium sp. These scales cause
damage to orchids particularly Cymbidium aloifolium in the tropical region to

Cymbidium gammieanum, Cymbidium tracyanum, and Cymbidium tigrinum and
many hybrids in the temperate region around the year. Scales feed on roots inside
potting media, pseudobulbs, leaves, flower spikes, and flowers, causing economic
loss.
Boisduval Scale, Diaspis boisduvalii (Signoret)

Boisduval scale, Diaspis boisduvalii (Signoret), is an economically important pest of
orchids including various species and the hybrid of Cymbidium in India. It is also
reported as an important pest of orchids in Florida [101]. This scale is circular to oval
in shape, thin and flat, whitish to light yellow in color, and semitransparent on the
removal of waxy material. When insect developed in complete growth, the body is
entirely covered with white waxy growth which protects the insect from its natural
enemies. It caused a 44% extent of damage to the plants and flowers of Dendrobium
nobile [102].
Ti Scale, Pinnaspis buxi (Bouche)

Ti scale, Pinnaspis buxi (Bouche), is a serious pest of Cymbidium orchid. It is sticky,
pear-shaped, small in size which is about 1–2 mm long, flat-bodied, and elongated
without any permanent body organs like wings, legs, or eyes. Ti-scale completes its
life cycle 20–30 days depending on existing temperature. Pests attack on all aerial
parts and prefer old leaves that look like dead ones, brown to dark brown, and dried
rather than plump. Infested plants produce small-sized, deformed flower spikes with
inferior quality flowers like miniatures. Other than Cymbidium, it also feeds on
Dendrobium, anthurium, banana, Hibiscus, coconut, and palm [101].
Soft Brown Scale, Coccus hesperidum Linn.

Coccus hesperidum is a soft scale insect that feeds on the midrib of leaves. It is oval
to round in shape and more flattened than either the black or hemispherical scales.
Adults are pale brown to dirty white or sometimes grayish mottled with dark brown
in color on the back. This species reported on many orchids, citrus, papaya, rubber,
etc. [103].
Florida Red Scale, Chrysomphalus aonidum Linn.

Chrysomphalus aonidum commonly feeds on nursery plants of Cymbidium and few
other orchids and ornamentals in greenhouses. It is round or moderately convex in
shape, is dark reddish brown to almost black or somewhat ash gray in color, and has
a size of almost 2–2.5 mm in diameter. The exuviae are approximately central,
reddish brown or brick red sometimes covered with grayish secretion, surrounded by
a reddish-brown ring.
Damage
Scale insects are generally found on leaves particularly in the midrib, stems, and
pseudobulb and suck the plant sap, resulting in deterioration of plant growth and
causing loss of vigor and deformation of infected plants. Substantial scale
282 R. Pal et al.
infestations, however, can reduce overall plant health and cause yellow leaves and
leaf drop. If infestation reached the flower buds, flowers do not open properly. Scale
insects also excrete sticky honeydew which attracts sooty mold, affecting the
average rate of photosynthesis.
Management
Scales are the major challenge in the harvesting of quality flowers of orchids. It is
challenging to control the pest or keep the plants free from scales by application of
only insecticides; hence, it is indispensable to apply various management approaches
such as cultural, biological, and chemical control for effective management of scales.
Cultural Practices
Exclusion is the first measure to be taken to evade scale infestation; thus, select
scale-free planting material by carefully examining all plant parts while establishing
new orchid farm to prevent early buildup of pest. Keep cleanliness inside the pot and
potting media. Need base pruning and burning of infested plant parts should be done
to reduce the further spread of scales. Keep a proper distance between plants and
isolate infested plants from healthy ones to prevent the scales from moving from
one plant to another. If scale infestation is found on the roots, repotting should be
done to eradicate harboring eggs and crawlers.
Biological Control
Promote natural enemies of scales. Coccinellid beetles feed on larvae of coccids that
help in the suppression of its infestation. Coccidencyrtus sp. (Hymenoptera:
Encyrtidae) has been reported as a parasite of boisduval scale [104].
Chemical Control
Scales can be removed by rubbing the scurf encrustation with toothbrush or cotton
swab and killing them by dipping in 70% isopropyl alcohol or methylated spirit,
or after gentle cleaning, roots should be sprayed with any of these insecticides, i.e.,
malathion 50 EC @ 0.05%, acephate 75 SP @ 0.05%, or carbaryl 0.2%, which
would help to reduce scale infestation. A spray of these insecticides should also
be done at the time of crawler emergence for effective control of scales on orchids.
8.1.3 Thrips, Dichromothrips nakahari (Mound)

Thrips, Dichromothrips nakahari Mound (Thysanoptera: Thripidae), is an important
pest of Cymbidium orchid in all orchid-growing region. It is also reported on
Dendrobium sp., Oncidium, Arundina, Aerides, Calanthe, Coelogyne, and Thunia.
It feeds on leaves, flower buds, and full-bloomed flowers of orchids. Adults are
slender, are dark brown to black in color, have apically pointed wings, and measure
about 1–2 mm in length. Nymphs are resembling adults but are small in size and
have pale yellow color. Wings are absent in the nymphal stage.
Damage
Thrip infestation on Cymbidium is reported in hardened plants in the nursery. In the
later stage, D. nakahari feeds on flower buds and flowers by sucking the cell sap.
Both stages by adult and by the nymphs cause damage. They also suck the cell
sap from the tender portion of plants and on leaves, resulting in plants becoming
discolored and shrivelled. In case of severe infestation, there are malformation in
leaves, flower buds, and flowers. If thrip infestation occurs on young plants,
they may finally dry up.
Management
For effective management of thrips, regular monitoring is recommended. Keep
orchid plants free from weeds; hence, remove weeds and old plant debris and
growing medium. If thrips are observed, immediately remove the infected plant
parts and discard away to reduce the incidence. Spray orchid plants with neem oil
5 ml/l. of water. In case of severe infestation, apply any of these insecticides, i.e.,
malathion 50 EC and fipronil 5% SC @ 0.05% or imidacloprid 17.8 SL 0.003%.
8.1.4 Red Spider Mite, Tetranychus urticae Koch (Acari: Tetranychidae)

Mites are not insects; they are members of the arachnid family, but in India, spider
mite is a most severe problem in Cymbidium orchids under controlled conditions.
It is also reported on many other orchids, viz., Dendrobium, Epidendrum, Arundina,
Luisia, Pholidota, and Thunia. Meena et al. and Nagrare [105, 106] reported that
T. urticae is a serious pest of Cymbidium. It is noticed on the ventral surface of leaves
and flowers. Mite incidence on Cymbidium is active throughout the year under
greenhouses and open polyhouses. Adult mites are oval in shape, about
0.4–0.6 mm in length with well-marked four pairs of legs, and pale greenish to
yellow with pairs of distinct dark lateral patches from orange to brick red; nymphs
are pale green with darker margins [105].
Damage
The damage is caused by both nymphs and adult stages. Just after hatching, nymphs
feed on the undersurface of leaves near the midrib by sucking the cell sap. The loss
of cell sap causes yellowing of leaves. The injuries due to feeding can be seen as
silvery marks left on the surface of leaves which usually turn brown to black. In case
of substantial damage, plant growth is stunted, vigor is lost, and whole plants are
covered with webs. Infested plants produce inferior quality flower buds which
are not open properly, and flowers become abortive, turn brown, and fall before
maturation.
Management
The mite is a serious problem on orchids under controlled conditions. It is more
severe during warm and dry weather; hence, increasing humidity and leaf wetness
and lowering the temperature help in the suppression of its infestation. Remove the
infested plant parts (leaves/flowers) and destroy them to reduce further multiplica-
tion of mite. Clean cultivation and proper ventilation are also helpful in mite control.
284 R. Pal et al.
On heavy incidence, immediately spraying the crop with plain water reduces mite
population. If required, spray the plants with miticides, i.e., dicofol or propargite or
ethion in recommended dose or concentration, to control mite infestation in orchids.
8.2 Diseases of Cymbidium
There are about 130 plant diseases affecting one or more orchid genera caused by
fungi, bacteria, nematodes and viruses. Cymbidium orchids succumb to a number of
diseases and pests under protected conditions. Several diseases have attained signif-
icant status and can lead to severe economic losses. Plants infected by diseases lose
their vigor and production capacity and affect their market value considerably.
Among fungal diseases, black rot (Phytophthora palmivora, P. parasitica, Pythium
ultimum, and P. splendens), anthracnose (Colletotrichum cymbidicola and C. clivae),
orchid wilt (Sclerotium rolfsii), petal blight (Botrytis cinerea), rust (Uredo sp.) and
leaf blight (Fusarium oxysporum), Sclerotinia white rot (Sclerotinia sclerotium),
and leaf spots caused by species of Fusarium, Cercospora, and Haplosporella are
the most common. The bacterial soft rot, caused by Erwinia sp., has been reported
on many Cymbidium hybrids and orchid species. An ectoparasite nematode,
Helicotylenchus microcephalus, causing root necrosis has also been reported on
many Cymbidium hybrids. The most serious problem of orchids is viral diseases.
More than 50 viruses are known to infect orchids all over the world, but in India, at
least 9 viruses have been reported. CymMV and ORSV are the most important and
prevalent viruses. These viruses are widely distributed on all commercial hybrids
and species.
8.2.1 Anthracnose
Causal Pathogens: Colletotrichum cymbidicola and C. clivae
Symptoms
The pathogen attacks all the aboveground parts of the plants, but leaves are more
frequently attacked. Initially, small oblong to circular, oval, sunken, and reddish-
brown to dark brown and gray-colored spots appear at the tip or middle
of the leaf lamina which gradually enlarges and covers a large area of the leaf
surface. It produces dieback symptoms which start from the tip and proceed down-
ward. It produces conidia within black acervuli. It also affects leaf sheaths and floral
spikes. It is found in nature mostly in conidial stage and can overwinter as mycelium
or conidia.
Epidemiology
The pathogen perpetuates when phytosanitary measures are not adequate. It spread
through infested compost, media, or leaf mold and through old contaminated pots.
The disease usually occurs throughout the year. However, during June–September,
when the temperature reaches over 30 C and relative humidity is above 80%, the
incidence is very high.
Management
Immediate removal of the diseased plants from the polyhouse and manually cutting
of infected leaf portions with sterilized scissors are necessary. Repotting of the plant
with properly sterilized potting mixture and fungicidal treatment is recommended.
Contaminated pots, potting mix, and the wooden benches should be sterilized with
2% formalin. Keep the surrounding free from other host plants. Plant should not
be exposed to direct sunlight as direct exposure to sunlight acts as a precursor of the
disease. Spraying of Blitox @ 2.5–3.0 g/l at 10-day interval or spraying
of carbendazim + mancozeb @ 1g each in one liter of water at 7-day interval has
shown good control of the disease.
8.2.2 Black Rot

Causal Pathogens: Phytophthora palmivora and Phytophthora parasitica
Symptoms
Black rot is the most destructive disease of orchids. The disease appears as water-
soaked small brown patches on the aerial parts of plants. Black necrotic lesions
develop on pseudobulbs and roots which later spread upward, resulting in complete
defoliation of the plant. The disease later migrates to other potted plants kept within
the vicinity of the disease beds. New shoots also show black rot symptoms,
which starts from the portion attached with the mother plants/pseudobulbs. Several
Cymbidium hybrids in pots also get infected with black rot causing initial water-
soaked small leaf spot, which later get transformed into blight symptoms covering
larger leaf area. Black rot symptoms on leaves might be initiated by secondary
airborne inoculum. These pathogens also cause damping-off of seedlings. The
disease appears from last week of May or first week of June and continues to
occur up to September under Darjeeling and Sikkim conditions when temperature
is around 30 C and humidity is above 90%.
Epidemiology
The disease spreads through contaminated potting media or water splash from
adjacent infected plants or even through irrigation water. Plants grown in soil are
more infected than raised bed. Soil bed over saturated with more than 90% water
holding capacity favors the disease. Rotting occurs mostly on the Cymbidium plants
grown in clayey soil beds, which are practically observed lower in elevation
and easily get moistened with excessive water by rain or overhead irrigation.
Besides, where rainwater was continuously dropped on the plants make the plant
surface wet for longer period. Temperature in the range of 24–30 C and relative
humidity of 80–95% and continuous rainfall coupled with misty foggy weather favor
the disease.
Management
Use of unsterilized potting media and pots should be avoided. Remove the infected
plants and also destroy infected parts to check further spread of the disease. A good
aeration in the nursery is essential. Reduce watering when the disease is expected to
286 R. Pal et al.
occur (June–September). It is advisable to keep the orchid plants on benches

90–120 cm above the ground level to avoid contamination through water splash.
In terrestrial orchids proper drainage should be provided. For effective control,
Matco MZ or metalaxyl @ 1 g/l or mancozeb @ 2 g/l can be used as spray or soil
drenching. Application of contact fungicide, e.g., Captain, Thiram, or mancozeb,
alternately with a systemic base fungicide metalaxyl is recommended.
8.2.3 Bacterial Soft Rot
Causal Pathogen: Erwinia sp.

The disease is caused by Erwinia sp. Severe infestation of bacterial soft rot has been
reported on some cymbidium hybrids and Eria pubescens during rainy season. The
infected plants initially showed water-soaking lesions and grayish-green lesions that
rapidly enlarged. The affected areas are soft, decayed, and brown in color. With
increasing severity, the internal tissues disintegrate and produce foul smell, and
subsequently the bulb gets completely rotted and the entire plant collapses. When
the disease portion is surface sterilized and plated on nutrient agar, bacterial colonies
are formed after 48 h. Bacterial colonies on nutrient agar are creamish white, about
1.0 mm in diameter, circular, and raised with entire edge.
Management: Proper sanitation and sterilization of cutting tools with alcohol is
recommended. Only disease-free plant material should be planted. Watering fre-
quency needs to be less to minimize leaf wetness. Application of copper-based
fungicides is effective against the bacterium.
8.2.4 Bacterial Brown Rot

Causal Pathogen: Pseudomonas cattleya
Symptoms: Small soft, water-soaked sunken spots are found on leaves that later
become black/brown. The disease advances rapidly, resulting in immediate death of
plants.
Epidemiology: High temperature and high humidity favor the disease. The bacte-
rium spreads very fast by rain splash and overhead irrigation water.
Management
Acquire disease-free plants and isolate new stocks for at least 4 weeks before
integration with existing stock. It is advised to reduce prolonged wetness by increas-
ing air circulation and the water retention capability of the growing medium.
The infected leaves may be cut off to check further spread of the disease. Overhead
irrigation should be avoided. Copper-based fungicides are effective against the
bacteria. The infected plants should be drenched or sprayed with 8-
hydroxyquinoline at a dilution of 1:2000 in water.
8.2.5 Nematode Disease

Causal Organism: Helicotylenchus microcephalus
An ectoparasitic nematode has been reported from Cymbidium hybrids in differ-

ent localities in Sikkim [107]. The disease was observed mostly on imported
cymbidium hybrids.
Symptoms
Roots of infected Cymbidium hybrids develop severe necrosis, swelling, and fluffy
root system. The leaves of infected plants exhibited typical bending, twisting,
and abnormal growth. The associated nematode, Helicotylenchus microcephalus,
is spiral and the most frequent plant parasitic nematode found worldwide in tem-
perate and tropical countries. The species is considered to be migratory ectoparasitic
feeders and feed from outside the roots by inserting their stylet into the epidermis
of young succulent roots. Eggs are laid in the soil close to the roots or on the root
surface and hatch in 2 or 3 days under favorable temperature conditions.
There is no evidence of plant parasitic nematode on orchids from India except few
interceptions at the plant quarantine check posts. In India, four nematode species
were intercepted from Madras Airport during 1989–1991 from the orchid consign-
ment imported from East Asian countries [108]. Again, five nematode species,
namely, Aphelenchoides bicaudatus, A. besseyi, Helicotylenchus dihystera,
Rotylenchulus reniformis, and Xiphinema elongatum, were intercepted from
Oncidium, Cattleya, Dendrobium, and Vanda species imported from Thailand and
Singapore [109]. Species of Helicotylenchus pseudorobustus, Aphelenchoides
besseyi, A. composticola, and A. aligarhensis have been reported on Phalaenopsis
and Cattleya sp. imported from Taiwan to Shanghai [110]. Aphelenchoides besseyi
has also been reported from Dendrobium “Lady Fay.”
Management
Control of plant parasitic nematode is achieved with nematode-free planting mate-
rial. Tissue-cultured plants are the best option. Proper cultural practices limit the
spread of nematode. Hot water treatment of propagative material has limited
the survival of plant parasitic nematode. Mustard oil cake and neem oil cake can
be incorporated with the planting media. For heavy infestation, carbofuran 3G can be
used.
8.2.6 Viral Diseases of Orchids

Virus diseases are serious threat to orchid industry. They not only reduce the general
vigor of the plant but also lower the flower quality, thereby reducing the marketabil-
ity of orchids and incurring serious economic losses. The first evidence of virus
disease on orchid was described by [111] from Australia as flexuous or rigid rod-
shaped particles associated with mosaic disease of orchids.
Cymbidium Mosaic Virus (CymMV)

The virus was first reported by [112] from California on Cymbidium sp. The virus
produces symptoms like mosaic, necrosis, chlorotic flecks, water-soaked lesions,
and flower necrosis on different orchid hosts. Cymbidium mosaic virus is filamen-
tous with a model length of 480 nm and width of 13 nm. The axial canal is obscure.
288 R. Pal et al.
The basic helix is obvious and the pitch of the basic helix is 2.8 nm. The sedimen-
tation coefficient of CymMV is 121 S. The virus contains 5.6% nucleic acid and 94%
protein. Genome of CymMV is unipartite and consists of linear ss RNA with the total
genome size of 8.1 Kb.
Odontoglossum Ringspot Virus (ORSV)

The virus was first reported by [113] on Odontoglossum grande from the USA.
The virus produces ringspot on O. grande and diamond mottle on Cymbidium spp.
It produces color breaking, chlorotic streaking, mosaic, and necrosis. Some orchid
cultivars are also symptomless. The virus particles are rod-shaped, not enveloped,
and straight having a model length of about 300 nm and width of 18 nm. Axial canal
is obvious. The purified preparations have two sedimentation components: 212 S
and 119 S. The virions contain 5% nucleic acid, and the molecular weight of the coat
protein is 17.5 kDa. In ultrathin sections, virions are found in the mesophyll,
epidermis, and vascular parenchyma. The infected cell has crystal inclusion in
the cytoplasm. The major ORSV hosts are Cattleya, Cymbidium, Epidendrum,
Odontoglossum, and Oncidium, but it infects many orchid species and hybrids.
CymMV and ORSV are generally found in mixed infections causing severe damage
to the orchid species. Such plants showed severe necrosis, large sunken patches, and
cracking of leaves. In India, the incidence of Odontoglossum ringspot virus (ORSV)
has been reported in 42 different species of orchids from Sikkim by slot-blot
hybridization method [114] and by ELISA on 71 species [115].
Calanthe Mild Mosaic Virus (CalMMV)

Calanthe mild mosaic virus has been reported on Cymbidium pendulum and C.
tigrinum from Sikkim [116]. The infected samples have been tested by ELISA, RT-
PCR, and Northern blot analysis and confirmed the presence of potyvirus.
The sequencing of an RT-PCR-amplified amplicon using potyvirus-specific primers
revealed that the virus is closely related to Calanthe mild mosaic virus.
Orchid Fleck Virus (OFV)

Orchid fleck virus was first reported in Japan on Cymbidium sp. showing chlorotic
and necrotic fleck symptoms [117]. Later on, it was reported from Australia, Brazil,
Denmark, Germany, Korea, and the USA producing chlorotic and necrotic spots and
rings in many genera of Orchidaceae [118–120]. It is an important virus of orchids
and reported on many orchid species from different parts of the world. The virus is
sap transmitted to Dendrobium sp. and few species of family of Chenopodiaceae,
Solanaceae, Leguminosae, and Aizoaceae [117, 121]. This virus has been reported
from Sikkim and Kalimpong area of Darjeeling districts on Coelogyne elata, C.
flaccida, and many Cymbidium species. Electron microscopy of negatively stained
preparations showed bacilliform particles measuring 32–40 nm in diameter and
100–150 nm in length.
8.2.7 Detection and Management

During the last two decades, there has been great advancement in the field of
detection technology. There are a number of diagnostic techniques available for
the detection of orchid viruses which are accurate, reliable, and less time-consuming
and can detect up to attogram (ag) level. Some of the diagnostic techniques like
enzyme-linked immunosorbent assay (ELISA), immunosorbent electron microscopy
(ISEM), dot immunobinding assay (DIBA), tissue blot immunoassay (TBIA),
reverse transcription-polymerase chain reaction (RT-PCR), immune capture PCR,
multiplex PCR, DIG-labeled cRNA probes, matrix-assisted laser desorption-ioniza-
tion (MALDI), TaqMan real-time PCR, and molecular beacons are some of the
highly sensitive and specific tests used for the detection of orchid viruses. A number
of companies like Mukoyama Orchids in Japan, Forsite Diagnostics Ltd in Britain,
and Agdia Incorporation in the USA have developed easy and rapid techniques for
diagnosis of orchid viruses which can be performed in the field and provide quick
results within 5–10 min. Extensive surveys of orchid nurseries in Sikkim and
Darjeeling hills and detection of samples by ELISA revealed that most of the
commercial hybrids and species grown in this region are contaminated
with CymMV and ORSV. These two viruses are often found as mixed infec-
tions [115]. Recently, polyclonal antibodies against CymMV and ORSV have been
developed using bacterially expressed recombinant coat protein as immuno-
gen [122]. The antisera are being used for the detection of both viruses from the
planting materials.
Orchids are vegetatively propagated crops, and cultural practices play a very
significant role particularly as orchid viruses generally spread by inadvertent
propagation. Hence, strict sanitation practices are essential for the control of
CymMV and ORSV. Clean cultivation by avoiding the sources of inoculums is
the best option requiring minimum inputs, but unfortunately this is not practiced
by orchid growers. Morel [27] first developed revolutionary techniques of meri-
stem culture to produce virus-free cymbidiums through meristem culture which
established the techniques as a clonal propagation procedure. He standardized
meristem tip culture to obtain virus-free clonal material of Cymbidium based on
the concept that majority of plant viruses do not infect the meristematic dome
despite severe systematic infection of the plants, and therefore, this region can be
excised and regenerated to plantlet [123]. Meristem culture is an important method
to produce virus-free plants, and it should be attempted only with valuable
breeding plant or valuable unique clone [124].
Chemotherapy using antiviral compounds like ribavirin (Virazole) and
dithiouracil is being used in tissue culture media to get virus-free plants. Loi et al.
[125] reported that Virazole was found effective in obtaining virus-free
Dendrobium shoot tips and callus from parent plants infected by CymMV and
ORSV from in vitro cultures of Cymbidium [126]. Genetic engineering coupled
with tissue culture techniques offers a useful way to introduce specific genes into
plants. The development of a suitable gene transfer system for Dendrobium orchids
would provide the breeder with greater opportunity to produce commercially
desirable hybrids.
290 R. Pal et al.
9 Other Uses
Cymbidiums are primarily cultivated for cut flower and pot plant production, but
they also possess value for medicine, food, and other uses. Many cymbidium species
are used in herbal medicine for curing various kinds of body ailments. Isolation of
phytochemicals in a few species has shown encouraging results and can lead to
development of useful drugs.
9.1 Medicinal Use
In herbal medicine roots, leaves or whole plant cymbidiums are used for curing
various body ailments particularly in India, China, and Australia. Herbalist uses the
plant parts fresh, dried, or in powdered form. The choice of herbal plant for curing
the disease depends upon its availability, effectiveness, and other substitutes avail-
able in the surrounding locality. Cymbidium aloifolium is most abundantly used
India, Nepal, and Bangladesh, whereas C. ensifolium, C. goeringii, and C. faberi are
amply used cymbidiums in Chinese medicine system. The survey of published
reports indicates that cymbidiums can cure from minor ailments, viz., cut, boils,
and earache, to severe illnesses like paralysis, traumatic injuries, and lung- and
kidney-related problems. C. aloifolium, C. hookerianum, C. devonianum, C.
lancifolium, and C. bicolor are used for treating cuts and boils, curing cracks in
feet, and healing fractures. The roots of species such as C. aloifolium, C.
devonianum, C. ensifolium, C. faberi, C. goeringii, C. kanran, C. sinense, and C.
wilsonii cure reparatory-related diseases like cough, asthma, and bronchitis. C.
ensifolium and C. lancifolium improve the blood circulation in the body. The roots
of C. faberi, C. goeringii, and C. kanran are used to clear out stomach worms. C.
aloifolium and C. ensifolium are used in treating menstrual-related disorders in
women. C. aloifolium, C. wilsonii, and C. goeringii are used for treating body
weaknesses. Cymbidium species are also reported useful in treating for dysentery
(C. madidum), gastroenteritis (C. kanran), tumor (C. aloifolium), rheumatism (C.
lancifolium), inflammation (C. ensifolium), headache (C. faberi), fever (C. macro-
rhizon), and diarrhea (C. iridioides). The seeds of C. madidum are used as oral
contraceptive.
The activity of the species against a particular disease is due to the presence of
phytochemicals in the plant. A few species have been surveyed for phytochemical
present in them. C. aloifolium contains several phenanthrene aloifol I, alifol II,
coelolin, 6-methoxycoelonin, pendulin, and denthyrsinin. Watanabe et al. [127]
isolated cymbidine, a monomeric peptidoglycan-related compound with hypoten-
sive and diuretic activities, from a C. goeringii. Gigantol was isolated from the
whole plant C. goeringii. Kim et al. [128] reported that fragrant compounds isolated
from C. goeringii have either cytotoxic or antibacterial properties. α-Bergamotene
has a cytotoxic effect on cancerous cells, while nerolidol has antibacterial property.
Nerolidol and β-bisabolene were isolated from C. forestii. β-Bisabolene is cytotoxic.
α-Pinene inhibits the growth of glioblastoma cells, and 1,8-cineole that functions as
pheromone is found in C. faberi.
9.2 Cosmetics
Cymbidium flowers are used in perfume, skin cream, and antiaging cosmetics [129].
9.3 Food
Olatshe is a popular Bhutanese dish made from the flower buds of Cymbidium
hookerianum. People harvest the flower buds from the inflorescence just before
opening and then wash and boil them in water until they become soft. After draining
the water, they add a mixture of spices, melted cheese, and salt to it and cook further
for 5 min, and the dish is ready. Olatshe is served with rice and noodles or used
as a dip. The orchid flowers add bitterness, and the addition of spices offset the
bitter taste. In China, the flowers of cymbidium orchids are used as herbal tea and
drinks [129]. Aboriginals in Australia eat pseudobulbs of C. canaliculatum and C.
madidum as mucilaginous food [2]. In Darjeeling, India monkeys often take out and
eat the tender part of stem base of leaves of C. lowianum, C. tracyanum, and
Cymbidium hybrids.
9.4 Other Uses
The juice of the pseudobulbs of C. canaliculatum is used for making glue. In western
Jawa, roasted pseudobulbs of C. lancifolium are grounded to stick substance, and the
sticky substance is made into handle to fasten Sudanese knives [2]. In India
the senesced leaves of cymbidium are collected from the field and woven into
baskets.
10 Conclusion
Cymbidiums rank on the top among all the orchids cultivated for cut flower
production because of heavy substance and high keeping quality of the flowers.
Only a few species of cymbidium are utilized for evolving about 16,000 hybrids
from a gene pool of 52 species. Thus, a broad scope still exists for using the other
suitable species in the breeding program. Cymbidium breeders mostly use hybrid-
ization and selection in the improvement of the cultivars. However, the different
breeding methods, such as mutation breeding, are rarely applied in the varietal
improvement of cymbidiums, despite promising results in other ornamental crops.
Two mechanically viral diseases of cymbidium, CyMV and ORSV, cause severe
limitation in the cultivation of cymbidium; no source of resistance is reported so far,
292 R. Pal et al.
and the results have shown that transgenic breeding can help in developing the
resistant cultivars. Since cultivars of cymbidium are highly heterozygous, induction
and use of haploid in the breeding program would be useful in developing new
varieties.
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Biotechnology Approaches
on Characterization, Mass Propagation, 11
and Breeding of Indonesian Orchids
Dendrobium lineale (Rolfe.) and Vanda
tricolor (Lindl.) with Its Phytochemistry
Endang Semiarti, Aziz Purwantoro, and Ika Puspita Sari
Contents
1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 300
2 Breeding Programs on Orchids Revealed by Biotechnology Approaches . . . . . . . . . . . . . . . . . 302
3 Phytochemical Compounds of Dendrobium lineale and Vanda tricolor . . . . . . . . . . . . . . . . . . . 305
4 Dendrobium Chemical Compounds . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 305
5 Vanda Compounds . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 306
6 Pharmacological Activities . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 307
7 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 309
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 309
Abstract
Mass propagation of orchids is very important to do extensively due to their
slower reproduction and growth naturally. Besides this, many growers do hunting
expansionally on orchids especially orchid species which is having uniqueness
and exclusivity characters. Moreover, orchids such as Dendrobium and Vanda
also have phytochemistry such as sesquiterpenoid, alkaloid, bibenzyl, and phe-
nolic that could be used as medicinal properties. Mass propagation could be
conducted through biotechnology approaches using either conventional in vitro
technique with adding plant growth regulator or orchid breeding using genetic
engineering revealed by Agrobacterium-mediated transformation techniques. All
of these methods are proposed in high embryogenesis of orchids. The
E. Semiarti (*)
Faculty of Biology, Universitas Gadjah Mada, Yogyakarta, Indonesia
e-mail: endsemi@ugm.ac.id
A. Purwantoro
Faculty of Agriculture, Universitas Gadjah Mada, Yogyakarta, Indonesia
e-mail: azizp@ugm.ac.id
I. Puspita Sari
Faculty of Pharmacy, Universitas Gadjah Mada, Yogyakarta, Indonesia

300 E. Semiarti et al.
combination of 2,4-D and ascorbic acid was absolutely required in the

Agrobacterium-mediated transformation. Insertion of KNAT1 gene produced
more buds in micropropagation compared to the wild-type plants. The efficiency
of transformation was increased caused by acetosyringone application.
Keywords
Mass propagation · Orchid breeding · Orchid biotechnology · Dendrobium ·
Vanda
1 Introduction
Orchid is a kind of an ornamental plant. There are more than 5000 species of orchids
that could be found in Indonesia [1]. Indonesia is rich with natural orchid species
because of its complete agroclimate from the lowlands to the highlands for tropical
climate. As [2] stated that approximately 5000 orchid species in the major divisions
of Indonesia (Table 1). Of the main islands, only the orchid flora of Java is probably
almost completely catalogued. It is well known that orchids are valuable in many
ways as follows.
Orchid is preferred because of the beauty of the flowers that were used for
decoration in offices and receptions in certain places and as a collection plant in
the garden. Some of them have a specific character which gives a special value due to
their uniqueness and exclusivity. A common feature of orchids is due to their beauty
of the flowers including the last long a time without wilting. In addition to the beauty
of the flowers, it turns out that the body parts of orchids also contain phytochemicals,
in the form of flavonoids, polysaccharides, bibenzyls, phenanthrenes, coumarins,
sesquiterpenoids, alkaloids, and steroids. In general plants produce various com-
pounds for survival by producing organic compounds called primary and secondary
metabolites. Primary metabolites are compounds produced by plants that function
directly in the process of photosynthesis, respiration, and growth. Secondary metab-
olites are intermediate or side compounds of primary metabolites. Secondary metab-
olites are specific, only produced by plants in certain families because they have
different functions. Therefore, the secondary metabolites which are produced by
Table 1 The richness of orchid flora of the major divisions of Indonesiaa

Island (group) Number of orchid species Percentage of endemic orchids
Sumatra 1185 42
Java 799 29
Lesser Sunda Islands 208 26
Borneo 1571 56
Sulawesi 585 66
Moluccas 390 46
New Guinea 2824 86
a
Taken from [2]
11 Biotechnology Approaches on Characterization, Mass Propagation, and. . . 301
orchids could be used in medicinal purposes. Some orchids that are known to have
medicinal properties include Spathoglottis plicata seeds for itching, Dendrobium
crumenatum for anticancer medicine, Dendrobium lineale for anticancer drugs, and
Vanda tricolor fragrance for aroma therapy, while the roots contain metabolites for
anticancer drugs (Fig. 1). Therefore, these orchids are now being hunted, taken from
the forest to be traded. This will certainly threaten the existence of these orchids in
nature, besides the threat of natural disasters and conversion of forest land to
settlements or roads.
Most of the orchids are endemic in certain areas in Indonesia, and because of their
uniqueness and exclusivity, many growers do hunting expansionally. On the other
hand, the reproduction and growth of an orchid are very slow due to its natural
biology. Seeds of the orchid were difficult to germinate since it has no endosperm.
Naturally the seeds will germinate after getting source of nutrients which is supplied
by mycorrhiza in the form of symbiosis mutualism. This causes the population of
orchids in nature not too much. Air humidity, sufficient light, wind, and environ-
mental conditions are natural mechanisms for regulating the abundance of orchids in
their natural habitat. The destruction of habitat and difficulties in cultivation, how-
ever, are threatening these orchid species [3]. Rare and threatened orchid species are
propagated by seeds rather than by vegetative methods [4]. Therefore, it is necessary
to develop a technique for sowing the seed of orchid in order to reach the highest
germination rate to be a protocorm. Since the orchid seeds are having no endosperm,
the germination of those seeds was established on the artificial medium. On the other
hand, micropropagation efforts are an important way to maintain the population of
Fig. 1 Potential Indonesian orchids for phytochemistry. (a and b) Spathoglottis plicata Blume.;
(c–d) Vanda tricolor Lindley; (e–f) Dendrobium crumenatum Sw.; (g–h) Dendrobium lineale Rolfe.
these orchids in nature and the existence of these orchids for biopharmaceutical
stock. Thus, a biotechnological approach can be applied to conserve them both in
situ and ex situ by using micropropagation methods.
2 Breeding Programs on Orchids Revealed by Biotechnology

Approaches
Plant breeding is often considered as an interaction of art and science to alter the
natural variation of crop plants in the direction of human needs [5]. However, at
present plant breeding is not only included on art and science but also considered on
the technology. Therefore, plant breeding is the art, science, and technology of
improving important agricultural plants for the benefit of humankind.
In Fig. 2, it can be explained that basically the plant breeding programs include
three kinds of activities which could be started from any point of view as given
below.
In general, the main purpose of plant breeding with agricultural crops is to
improve both qualities and quantities of traits with the profitable values such as
yields, nutritional qualities, and other characters. The qualitative characters, or traits,
are the easiest to deal with since most of them are discontinuous traits that are
governed by one or a few major genes. On the contrary, quantitative characters are
much more difficult for the breeder to control since these traits are governed by many
genes, each having a small effect. Unfortunately, many traits of economic
Fig. 2 Basic activities on the

plant breeding program
importance are of this type, e.g., height, cold and drought tolerance, time to maturity,
and, in particular, yield. However, plant breeding is a rapidly advancing science. It is
able to make use of genetic and biotechnological innovations to efficiently develop
better crop varieties.
Orchids belong to the ornamental plants which are highly priced in the interna-
tional market due to their designed spectacular flowers; brilliant colors; delightful
appearances; myriad sizes, shapes, and forms; and long-lasting qualities. In breeding
ornamental plants, attention is paid to such factors as longer blooming periods,
improved keeping qualities of flowers, general thriftiness, and other features that
contribute to usefulness and aesthetic appeal. Novelty itself is often a virtue in
ornamentals, and the spectacular, even the bizarre, is often sought.
Breeding orchids can be a highly challenging endeavor. Most of the purposes on
orchid breeding are to produce commercially important hybrids that have a market
demand and are liked by the consumers. The concept behind the development of
hybrids in orchids may vary according to the genus and species. The generalized
objectives as stated by Bhattacharjee and Das [6] are given below:
• To breed for better color, size, and substance of the flower

• To introduce a perfect blending of colors in sepals, petals, and lips
• To create round and full form of sepals and petals with minimum fenestrations
and twists
• To increase the length of inflorescence
• To increase the number of flowers/inflorescence
• To achieve compactness in flower facing on the spike
• To develop hybrids showing a correct mode of display
• To extend the blooming period
• To produce miniature forms
• To produce fragrant varieties
• To produce flowers with longer vase life
• To develop types suitable as pot plants
• To develop hybrids insensitive to strict climatic regime
• To develop hybrids resistant to biotic stress like diseases particularly to viruses
Orchid breeding programs can be divided into two groups including classical and
modern plant breeding. In the first one, plants are selected with desirable characters,
and elimination of undesirable characters occurs. Otherwise, the modern plant
breeding programs called biotechnology have used molecular biology techniques
and omics technologies [7]. Biotechnology approaches are more efficient compared
to the classical one. It could be due to its most rapid, easiest, cheapest approach, and
the important things are not influenced by the environment. Furthermore, biotech-
nological developments are helping breeders make the desired genetic changes with
much greater precision. Therefore, applications of biotechnology on orchid breeding
have significantly shortened the time. Another purpose using biotechnology
method on orchid breeding is the production of a huge number of plantlets.
Therefore, improvement in orchid breeding is aimed to get stable production of high-

quality clones by micropropagation.
Purposing of mass propagation of orchid plants in addition to in vitro seed
germination, it can also be done with various in vitro techniques, namely, organ
culture, cell culture, protoplast culture, callus culture, and somatic embryogenesis.
Even the current genetic engineering approach is not only used to improve the
specific trait of the orchid; it is also used to increase its mass propagation by embryo
gene insertion, bud formation genes, etc. Embryogenesis is the one method which
could be applied on mass propagation of orchid either zygotic embryogenesis or
somatic embryogenesis (Table 2).
The addition of 2 g/L peptone in all culture media accelerated the growth and
increased the percentage of seed germination of up to 100%, enlarged the size, and
greenish the protocorms and shoot initiation [14].
The most suitable method of biotechnology conducted on orchid breeding is
genetic engineering. In this case the modern genetic techniques can insert desirable
traits into plants, and the new plants are called transgenic or genetically modified
organisms (GMOs). Whatever the purpose on orchid breeding, the method of
Agrobacterium-mediated transformation system is the popular method.
There are several transformation methods which have been employed for the
transfer of exogenous genes into orchid tissues. The most widely used method is
particle bombardment, followed by Agrobacterium-mediated transformation
[22]. Semiarti et al. [10] used Agrobacterium tumefaciens for doing the transforma-
tion of the wild orchid species Phalaenopsis amabilis. Moreover, [22] mentioned
that both cells of Vanda tricolor and P. amabilis transformant carrying KNAT1 gene
were more meristematic compared to the wild-type plants as the organ produced
more buds in micropropagation. During working on orchid transformation, much
effort has been spent on testing different chemical compounds and selectable
markers and their corresponding selection agents for the efficacy and efficiency of
Table 2 Some techniques that have been used for mass propagation of Indonesian orchid species
through in vitro embryogenesis and Agrobacterium-mediated transformation
Orchid species Techniques conducted References
Phalaenopsis amabilis In vitro embryogenesis (zygotic and somatic) [8–11]
Genetic transformation by using KNAT1 gene
Genetic transformation by using AtRKD4 gene
Vanda tricolor In vitro embryogenesis (zygotic and somatic) [12, 13]
Dendrobium lineale In vitro embryogenesis (zygotic and somatic) [14, 15]
Dendrobium phalaenopsis In vitro embryogenesis (zygotic and somatic) [16, 17]
Coelogyne pandurata In vitro embryogenesis (zygotic and somatic) [18, 19]
Dendrobium capra In vitro embryogenesis (zygotic and somatic) [20, 21]
transgenic orchids. Pre-culture treatment and application of ascorbic acid were

absolutely required in the Agrobacterium-mediated transformation of V. tricolor.
Moreover, acetosyringone had been added in order to improve the efficiency of
Vanda transformation [12]. Finally, the choice of an appropriate method, a chemical
compound, and also a selectable marker is vital to the success of orchid transforma-
tion protocol.
3 Phytochemical Compounds of Dendrobium lineale

and Vanda tricolor
In general, secondary metabolites are produced by plants for various purposes,

including:
1. As a plant protector against the threat of herbivore animals or microbial infec-

tions, fungi, or pathogenic viruses
2. As an attraction for pollinators and seed-dispersing animals
3. As a reproductive hormone
Generally, secondary metabolites are classified based on their biosynthetic path-

ways, namely, phenolics, terpenes and steroids, alkaloids, and flavonoids. It has been
clearly demonstrated that secondary metabolites are the plant adaptation agents to
their environment [24]. Secondary metabolites are unique sources for pharmaceuti-
cals. Since the ancient times in Indonesia, the oldest references regarding the use of
medicinal herbs are found in temple relief. While the term Jamu (Jampi Oesada) may
also be traced to the relics of ancient writing, some say it might be in the manuscripts
of Madhawapura, Ghatotkacasraya (MPU Panuluh), Serat Centhini, and Serat
Kawruh Bab Jampi-Jampi Jawi. In 1775, Rumphius, a Dutch botanist, conducted a
research on herbal medicine in Indonesia. He published a book called Herbarium
Amboinense possibly in which there are parts of the orchid plant as herbal medicine
[25]. In India and China, Dendrobium and Vanda are already used in Ayurveda and
Chinese pharmacopoeia [26, 27]. Pharmacopoeia is a drug standard book issued by a
government official body that outlines the ingredients of a drug, the chemicals in a
drug and its properties, the medicinal properties, and the usual dosage. In Indonesia
there is an Indonesian Herbal Pharmacopoeia since 2008, but unfortunately until
now there has been no description of the herbs that come from orchids while
Indonesia’s wealth in the form of orchids is very abundant.
4 Dendrobium Chemical Compounds
The use of plant parts from Dendrobium as herbal medicine has been going on for a
long time such as tonic, astringent, analgesic, and anti-inflammatory agents. With the
development of content analysis techniques in herbal medicine, chemical compound
research has been carried out from various materials derived from Dendrobium.
Table 3 Dendrobium compounds

No Group Compounds Dendrobium species
1 Polysaccharide Polysaccharide D. nobile, D. fimbriatum
2 Bibenzyl Gigantol, dendrocandin D. amoenum, D. candidum,
D. aphyllum, D. densiflorum,
D. nobile
3 Phenanthrene Phenanthrenedione, D. chrysotoxum,
phenanthrene, moscatin, D. densiflorum, D. nobile
moscatilin, denbinodin
4 Coumarin Ayapin D. densiflorum
5 Sesquiterpenoid Dendronobilin D. nobile
6 Alkaloid Scopoletin D. densiflorum
7 Steroid Sitosterol D. thyrsiflorum
The results of research from [28] found that constituents that are commonly found in
Dendrobium are flavone C-glycosides and flavonols. This research was conducted
on 142 species. A few years later, many researchers found about 100 compounds
from 42 Dendrobium species including 32 alkaloids, 6 coumarins, 15 bibenzyls,
4 fluorenones, 22 phenanthrenes, and 7 sesquiterpenoids (Table 3).
Nearly all of the Dendrobium studied were found to be compounds that emerged
from almost all of them, polysaccharide, gigantol, dendrocandin, and moscatin/
moscatilin. Some appear dendronophenol [29]. Given the abundance of content in
Dendrobium species, research has also been carried out on many pharmacological
activities of all of these compounds.
5 Vanda Compounds
Compounds commonly found in Vanda include bibenzyl derivatives (gigantols),

phenanthrene derivatives, phenolic compounds, anthocyanins, alkaloids, steroids,
and triterpenoids [30] (Table 4).
Research on compound and pharmacological activities of D. lineale and
V. tricolor has never been done. However, there are several ways to estimate the
compound and pharmacological activities of these two orchids, one of which is by
understanding genetic similarity and chemotaxonomy studies. In chemotaxonomy it
is known that plants that are taxonomically close also have genetic similarities. This
genetic similarity will give rise to the suspicion that similar compounds will be found
(although in varying degrees). By knowing the possibility that the compounds
owned by D. lineale and Dendrobium species have similarities, it will be able to
be used to estimate the pharmacological activities that are also not far away
(although there are variations in intensity). This also applies to V. tricolor with
close Vanda species [23].
Research conducted by [31] on Papua’s endemic orchid including D. lineale
found several similarities among Dendrobium. The study was conducted by isolation
and extraction of genomic DNA from Papua’s dried leaf orchids (26 types). In this
Table 4 Vanda compound

No Group Compounds Vanda species
1 Bibenzyl Gigantol V. coerulea,
V. roxburghii
2 Phenanthrene Tessalatin, oxo-tessalatin, parviflorin, imbricatin, V. tessellate,
methoxycoe lonin, coelonin V. parviflora,
V. coerulea
3 Phenolic Parishin, gallic acid, tetracosylferulate V. parishii,
V. tessellata,
V. roxburghii
4 Anthocyanins Delphinidin and cyanidin derivatives Vanda hybrid
5 Steroid and β-Sitosterol-D-glucoside, β-sitosterol, V. roxburghii
triterpenoids Υ-sitosterol, stigmasterol
6 Alkaloid Laburnine acetate V. hindsii
study, markers that are commonly used in researching genetic variation are random
amplified polymorphism DNA (RAPD). From the analysis using the unweighted
pair-group method arithmetic average (UPGMA) with the sequential agglomerative
hierarchical nested cluster analysis (SAHN-clustering) and TREEE programs, it is
known that D. lineale has similarities with D. mirbelianum and Dendrobium species
(variation score 0.23–0.30). Based on chemotaxonomy, it can be assumed that
D. lineale compound includes polysaccharide, gigantol, dendrocandin, moscatin/
moscatilin, and dendronophenol [29].
V. tricolor has similarities with several Vanda from research results based on
morphological characters (V. tricolor is similar to V. limbata, V. celebica, V. retusa,
V. scanen, and V. foetida [32]. Based on the RAPD analysis, V. tricolor has genetic
similarities with V. lamellata, V. limbata, V. luzonica, V. sumatrana, and V. merrillii
with percentage similarity of all >50% [33].
6 Pharmacological Activities
Polysaccharides isolated from Dendrobium have antioxidant and free radical scav-
enging activities. Environmental stress on Dendrobium will result in increased nitric
oxide (NO) radicals, namely, catalase (CAT), peroxidase (POD), ascorbate peroxi-
dase (APX), 2,2-azinobis (3-ethylbenzothiazoline-6-sulphonic acid) or ABTS,
hydroxyl, and 1,1-diphenyl-2-picrylhydrazyl (DPPH) radicals, and enzymatic anti-
oxidant superoxide dismutase (SOD). Stress-fighting activity is shown in D. nobile
polysaccharide (scavenging 20–40% of DPPH and 40-0.60% of ABTS).
D. fimbriatum polysaccharide has the highest scavenging activity of ABTS among
Dendrobium species at 90% [34, 35]. Furthermore, other studies confirm the anti-
tumor action of Dendrobium stem fraction in inhibiting sarcoma 180 in vivo and
HL-60 cells in vitro [36].
Phenanthrene and dihydrophenanthrene from Dendrobium species can inhibit
tumor necrosis factor alpha (TNFa), interleukin 8 (IL-8), and IL-10 during
lipopolysaccharide (LPS) stimulation [37, 38]. Upon lipopolysaccharide (LPS)

stimulation, macrophages produce a large amount of inflammatory factors, such as
tumor necrosis factor α (TNF-α), interleukin-1 beta (IL-1β), interleukin 6 (IL-6),
interferon (IFN), and other cytokines [39]. In addition, denbinobin is a major
phenanthrene isolated from the stems of D. nobile. The antitumor action of
denbinobin in SNU-484 cells decreased the expressions of matrix
metalloproteinase-2 and matrix metalloproteinase-9 [40].
Sesquiterpene glycosides (dendrosides) significantly promoted cell proliferation
and stimulation of mouse B lymphocytes [41]. B lymphocytes are important to
immune cells and play a key role for responses in the immune system. Gigantol
inhibits lung cancer cell line due to downregulating caveolin-1 (Cav-1), activating
ATP-dependent tyrosine kinase, and regulating cell division cycle 42 (Cdc42) [42].
Moscatilin, moscatin, and moscatin diacetate inhibit arachidonic acid
(AA) platelet aggregation-induced in rabbit and collagen induced (50 μg/mL and
100 μg/mL) [43]. Studies on the aggregation of platelets are conducted through
thrombin, arachidonic acid (AA), thrombin, or collagen induction method [44]. Poly-
saccharide from D. officinale, D. nobile, and D. chrysotoxum showed a hypoglyce-
mia activity in diabetic rats [45]. Besides D. chrysotoxum is also able to inhibit the
occurrence of diabetic retinopathy [46] (Table 5).
The ethanolic extract of stem V. coerulea exhibited significant antioxidant and
anti-inflammatory activities due to reduction in the production of COX-2 enzymes
and PGE2 in irradiated HaCaT cells [47]. The petroleum ether extract of leaves of
V. tessellata showed potential DPPH and nitric oxide (NO) radical scavenging
activities [48], while the hydro-alcoholic extracts of leaves of V. testacea reduced
axotomy-induced peripheral neuropathy in rats due to antioxidant activity [49]. The
extracts of V. coerulea showed a skin-hydrating property [50]. V. teres stem extract
exhibited antiaging effects in immortalized keratinocyte cell lines of human origin
(HaCaT) [51]. The methanolic extract of the flowers of V. spathulata exhibited an
antidepressant activity in mice using the forced swim test and the tail suspension test
[52]. Anwar et al. [53] had reported that the petroleum ether extract of leaves
of V. tessellata showed a dose-dependent hepato-protective activity in rats.
Table 5 Pharmacological activity of Dendrobium species

Pharmacological
No activity Compounds Orchid species
1 Antioxidant Polysaccharides D. nobile, D. fimbriatum,
D. huoshanense, and D. chrysotoxum
2 Antitumor Polysaccharides D. nobile
3 Anti-inflammatory Phenanthrene D. nobile and D. chrysanthum
4 Immunomodulatory Dendrosides D. nobile, D. moniliforme
5 Anticancer Gigantol D. draconis
6 Antiplatelet Moscatilin, moscatin, and D. chrysotoxum
aggregation moscatin diacetate
7 Antidiabetes Polysaccharides D. officinale, D. nobile, and
D. chrysotoxum
Table 6 Pharmacological activity of Vanda species

Pharmacological
No activity Compounds Orchid species
1 Anti- Imbricatin, gigantol, and methoxycoe lonin V. coerulea
inflammatory
2 Antiaging Vandateroside and eucomic acid; imbricatin, V. coerulea
methoxycoe lonin, and gigantol and V. teres
3 Antidepressant Phenol compounds V. spathulata
4 Antioxidant Phenanthrene V. coerulea
5 Hepatoprotective Phenanthrene V. tessellata
6 Aphrodisiac Phenanthrene V. tessellata
Alcoholic extracts of flowers of V. tessellata showed an aphrodisiac activity in male

mice [54] (Table 6).
Khan et al. [55] reported that using in vitro and in vivo models, some members of
Vanda species have bioactive chemical constituents and pharmacological activities
from the aerial parts of the plants and often used as a traditional medicine for
ethnopharmacology in Asian countries. The stem and aerial parts of V. tessellata
and V. coerulea have potential anti-inflammatory and anti-microbial activities. Based
on the review, it is necessary to prove the pharmacology activity of D. lineale and
V. tricolor by using a guided bioassay of the pharmacological effects that have been
done on Dendrobium and Vanda.
7 Conclusions
Orchids have distinguishable, unique, and exclusive characters. Phytochemical on

orchids could be useful for medicinal purposes, such as antitumor, anticancer, anti-
inflammatory, etc. At present, the achievement of orchid breeding is mass propaga-
tion or micropropagation through biotechnology approaches, namely, either in vitro
techniques or genetic engineering by using Agrobacterium-mediated transformation.
Acknowledgments We thank Mrs. Hj. Sri Suprih Lestari, the owner of “TITI” Orchid Nursery;
Pakem-Yogyakarta and Mrs. Titik Handayani, the owner of “Keboen Kita” Orchid Nursery; and
Godean, Sleman-Yogyakarta for the permission taking the photograph of Dendrobium lineale, and
special thanks are given to Mr. Aries Bagus Sasongko for his assistance to take the photo of
Dendrobium crumenatum.
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J Ethnopharmacol 229:46–53
Preferences of Orchid Consumers and the
Substitute Products’ Influences 12
Adilson Anacleto and Luciane Scheuer
Contents
1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 314
2 Material and Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 315
3 Results and Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 316
4 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 323
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 324
Abstract
The global floriculture market is very varied, but it is worth mentioning the orchid
trade, being among the most important species of this market. Despite the
economic relevance of the orchids, the consumer preferences about these flowers
are unknown. Thus, an exploratory-descriptive survey was conducted between
August and December 2019 with 150 consumers, in order to investigate the
profile and behavior of this kind of consumer. The study found that women had
a higher average purchase per year than men. As consumers advanced their
education, there was a tendency to increase the consumption. Roses were the
main choice of the consumers when they did not buy orchids, and the main
substitute products for orchids were perfumes and chocolates. Further studies on
the use of the orchid as a means of seduction are recommended, given the
consumer’s availability in these cases to pay for the desired orchid up to 50%
more than the market price.
Keywords
Flowers · Floriculture · Orchidaceae · Marketing · Relationship marketing ·
Gardens · Ornamental plants · Home decor · Orchidists · Flower retail market
A. Anacleto (*) · L. Scheuer

State University of Paraná, Paranaguá, Brazil
e-mail: adilson.anacleto@unespar.edu.br; luciane.scheuer@unespar.edu.br

314 A. Anacleto and L. Scheuer
1 Introduction
The floriculture market in the world has revenues of approximately US $ 67.3 billion
per year and is expected to grow by an average around 5% in countries where this
market is already consolidated, and up to 12% per year in developing countries,
especially in Latin America and Asia [1, 2].
The flowers trade other than traditional ones has revealed a new market config-
uration, which is expected to boost the market over the next decade, with Europe
being the main destination, representing the largest share (n ¼ 30%) of the global
floriculture market [2, 3].
The global floriculture market is very varied, but it is important to mention the
orchid trade, which generates a marketed amount close to US$ 20 billion per year,
being among the most important species of this market [4].
Despite the economic relevance of orchids to the global floriculture market,
Anacleto et al. [1] report that consumer preferences are unknown about the most
flower species, and for orchids this lack of information is even greater given the
economic importance of this group of flower species.
According to Anacleto et al. [1] and Bornancin et al. [3] in a globalized scale of
trade experienced by the market today, it is essential to know the factors that
influence consumer preference, and then delimit strategic actions aimed at the
market positioning, resulting in greater trading capacity, expanding the presence of
the product, and consequently increasing the consumption.
According to Hirschman [5], the consumption is a continuous process and goes
far beyond exchanging a financial amount for a good. This process involves issues
that influence the consumer before, during, and after the purchase. According to
Kotler et al. [6], the consumption is linked to the marketing, which is a social and
managerial process whereby individuals and groups get what they need and want by
creating, offering, and freely exchanging valuable products and services with other
individuals. The domain of the consumption is when people and objects are brought
into contact, acquiring sense, producing social meanings and distinctions.
Solomon [7] explains that when talking about consumption, not only tangible
products are cited but also intangible experiences, ideas, and characteristics, many
consumer experiences, such as fantasies, feelings, and fun, are behind the buying
decisions and are important considerations for the consumption phenomenon.
For Churchill et al. [8], consumers are people who buy goods and services for
themselves or others, and every buying process must first have passed for the
recognition of a need. The needs may have different stimuli as well as the inner
sensations, which are characterized by desires. When the need arises inside the
consumer, the impulse to meet it is called motivation. Anacleto et al. [1] describes
that the motivation in the flower consumption is a relevant factor to the success of
this type of product.
The flower trade has been growing all over the world, but the demand for flowers
is still quite irregular and concentrated on the holidays, so it is strongly related to
seasonality. So, the growth of flower consumption is highly dependent on the
country economic development and the cultural increment, and according to
12 Preferences of Orchid Consumers and the Substitute Products’ Influences 315
Churchill et al. [8], it is essential to know the consumers, identify their profile, their
preferences and the factors that influence the decision at the time of purchase, as well
as understand the perceptions of consumers in front of many possibilities available
[8].
Thus, given this context, the research results are presented, which aimed to
answer the following questions:
(i) What were the factors that determined the profile and behavior of orchid
consumers?
(ii) What products were the main competitors of the orchids in the market?
(iii) What were the main reasons for buying orchids?
(iv) What was the client’s willingness to pay more when they found the orchid they
wanted?
2 Material and Methods
An exploratory-descriptive survey was conducted between August and December

2019 with 150 consumers who traditionally promoted the purchase of orchids for
consumption or for gift giving. The research met the marketing research guidelines
related to the consumer preferences, considering also when the population is
unknown as proposed by Malhotra [9], and the sample was directed to 150
consumers.
The sampling as proposed by Anacleto et al. [1] required that the consumer had
bought orchids at least once in the last 6 months and had agreed in participating in an
unidentified research.
The data analysis sought to identify the existence of correlation between the
annual consumption averages of orchids among the investigated class considering
the following variables: gender, education, income, age, and marital status that were
considered as the explanatory factors [1, 9]. The qualitative variables were charac-
terized through absolute and relative frequencies (%) and quantitative variables
through the average and standard deviation.
The evaluation of the influence of gender, age, education, and economic condi-
tion on the orchid consumption levels was evaluated according to Anacleto et al. [1].
For this, the nonparametric Mann–Whitney and Kruskal–Wallis tests were applied,
followed by the multiple comparison test of Dunn averages, at a significance level of
5% ( p <0.05), in order to identify the pairs of groups with statistically significant
differences [10].
The measurement of the most important species, the factors that limited the
increase of orchid consumption, and the influence of substitute products were
obtained by calculating the relevance index, obtained from the sum resulting from
the hierarchical categorization that allowed each interviewee to issue three indica-
tions categorizing the most important with 3 (three) and the least important with 1
(one) among the range of alternatives which were highlighted as the most important
ones.
3 Results and Discussion
The research related to the orchid consumption revealed that most consumers were
women (n ¼ 58%), and when compared to males, they had higher buying frequency
for both their own consumption and gift giving (Mann–Whitney Test ¼ p <0.001)
(Table 1).
The predominant age group of purchases (70.1%) was between 20 and 40 years
old, and no statistically significant correlation was observed between age and orchid
consumption, as well as no significant variations related to marital status of the
consumers were observed. In both cases, there were no significant differences in the
frequency of orchids purchase for their own use or for gift giving (Table 2).
Regarding education, as schooling progressed, there was a slight tendency to
increase consumption, both in purchases for own use and for gift (Table 3).
Related to the monthly family income, significant statistical differences were
observed in the number of orchids purchased per year, both for use ( p ¼ 0.080) and
for gift giving ( p ¼ 0.005), having a correlated relation to the increase in the number
Table 1 Gender comparison on the number of times consumers have bought orchids in the last
12 months (N ¼ 150)
Annual frequency of buying orchids
(average standard deviation)
Own use For gift
Evaluated Total of Average standard Average standard
criterion respondents deviation deviation
Female 87 2,72 3,46a 2,37 2,14a
Male 63 1,29 2,36 b
2,43 1,77b
Mann-Whitney Test p <0,001 p ¼ 0,513
p – Significance value
Averages followed by the same letter do not differ statistically
equal letters do not differ statistically from each other (p > 0.05 in the Mann-Whitney Test)
a,b,c
Table 2 Comparison by marital status on the number of times consumers have bought orchids in
the last 12 months (N ¼ 150)
Own use For gift
Married 73 2,11 3,31ª 2,09 1,79ª
Single 56 2,20 2,38ª 2,57 2,14ª
Divorced 9 2,22 3,42ª 2,14 1,91ª
Widow(er) 8 2,29 2,37ª 1,82 1,99ª
Kruskal–Wallis Test p ¼ 0,028 p ¼ 0,070
A average, SD standard deviation, p significance value, a groups with no significant differences
( p >0.05 in the multiple comparison test by Dunn)
Table 3 Comparison by level of education on the number of times consumers have bought orchids
in the last 12 months (N ¼ 150)
Own use For gift
Elementary 17 1,61 1,99ª 1,72 1,08ª
school
High school 68 1,73 1,91ª 1,79 1,08ª
University 50 2,36 2,69b 2,02 1,69b
graduation
Postgraduate 15 3,55 2,99c 3,23 1,70c
p significance value of Kruskal–Wallis Test
Equal letters do not differ statistically from each other ( p >0.05 in the multiple comparison test
a,b,c
by Dunn)
Table 4 Comparison by the degree of monthly family income on the number of times consumers
have bought in the last 12 months (N ¼ 150)
Own use For gift
Total of Average standard Average standard
Evaluated criterion respondents deviation deviation
Up to US$ 329 22 1,74 1,87ª 1,69 1,34ª
From US$ 330 to US$ 47 2,01 1,90ª 1,99 1,54ª
549
From US$ 550 to US$ 50 2,55 2,34b 2,34 1,72b
975
From US$ 976 to US$ 20 4,31 2,88c 3,54 1,70c
1902
Over US$ 1903 11 5,30 2,60c 4,59 1,88c
p significance value of Kruskal–Wallis Test
Equal letters do not differ statistically from each other ( p >0.05 in the multiple comparison test
a,b,c
by Dunn)
of orchids purchased per year according to the increase in monthly family income
(Table 4).
Mother’s Day and Valentine’s Day were the main dates of the year (Table 5) when
there was greater demand for orchids, similar to data reported in other studies [1], but
the study revealed that the use of flowers in the seduction processes and birthdays are
also periods that motivate orchid consumers to make new purchases.
Regarding the main flowers found in the market and which competed in the
flower shops with the orchids (Table 6), it was observed that roses were the main
Table 5 Top dates when consumers used to buy orchids

Classification Dates Relevance index
1 Mother’s Day 236
2 Valentine’s Day 122
3 Seduce a person 82
4 Celebrate birthdays 49
5 Woman’s Day 47
6 Own use (decoration) 47
7 All Soul’s Day 26
8 Graduation Tribute 16
9 Wedding 15
10 Gifts for various special dates 10
Multiple choice questions
flowers in the consumer’s choice. However, studies developed by Anacleto et al. [1]
analyzing the flower sales on the shelves of the flower shops, they found that orchids
are the flower group favored by consumers for their own use and they are the second
option for gift.
The consumer’s decision about the flower to be purchased, and whether or not to
buy it, depends on factors such as value, species, color, size, payment terms,
durability, discounts obtained and especially the easy access to the product, where
and when buying the orchids. The orchid consumption tends to be made easier if this
set of factors gives the consumer the sense of a routine decision.
The main substitute products for orchids were perfumes and chocolates (Table 7)
but other flower species were also considered by consumers as substitutes. The
absolute majority of the respondents (n ¼ 83%) admitted that when they did not find
the desired orchid for his own use, he/she searched nearby and considered buying a
replacement product. However, when the acquisition was for gift, the interviewees
(n ¼ 54%) admitted that they searched in up to three different flower shops or did
previous research on the Internet in order to facilitate the purchase.
Although the study on the orchid consumer profile reveals data similar to that
already reported by Anacleto et al. [1] related to the consumption levels by age,
marital status, and education, this study also revealed a greater influence of the
substitute products in relation to the orchids, as well as a high willingness of the
customers in replacing the orchids if they could not find the desired flower when it
was for their own use.
The substitute products for flowers have presented better performance in terms of
global market in relation to the flowers. Anacleto et al. [1] describe that the perfume
and chocolate industries are better structured and promote massive investment in
marketing, especially on seasonal dates when gift giving is a habit. The clothes have
a high level of ability to act as a substitute product, in part due to the fact that in the
daily life, clothes are more easily perceived by the consumer, and it is easier to get
indications from the people close to person that will receive the gift, getting easier to
please the receiver [6], as well as the clothes come in a variety of types, shapes, and
with wide price range.
12
Table 6 Flower species preferred by consumers (N ¼ 150) when they do not buy orchids
Classification (for gift) Flower species Relevance index Classification (own use) Flower species Relevance index
1 Rose 232 1 Rose 172
2 Violet 53 2 Tulip 44
3 Lisianthus 26 3 Cyclamen 28
4 Cyclamen 20 4 Lisianthus 24
5 Astromeliad 15 5 Violet 18
6 Tulip 15 6 Bromeliad 16
7 Bromeliad 10 7 Astromeliad 15
8 Begonia 9 8 Begonia 12
9 Daisy 9 9 Gerbera daisy 11
10 Carnation flower 8 10 Dahlia 8
Preferences of Orchid Consumers and the Substitute Products’ Influences
319
Table 7 Main products to replace orchids when the consumer did not find the desired orchid
(N ¼ 150)
Classification Substitute products Relevance index
1 Perfumes 131
2 Chocolates 114
3 Other flower species 103
4 Clothes 26
5 Wines 25
6 Books 23
7 Cosmetics/makeup 22
8 Stuffed animals 14
9 Semi jewelry or imitation jewelry 14
10 Decoration items/ornaments 12
The trade of orchids must consider two factors, if the level of desire and will of
the customer in having the product is high and if the accessibility to the product is
facilitated, because if the consumers can easily purchase the product of their need,
naturally occurs the predisposition to pay the values asked when he/she finds the
flower object of his/her desire, otherwise the space of the substitute products
increases.
The substitute products, according to Porter [11], can be classified as those that
can replace the desired product targeted by the consumer under free market condi-
tions. The biggest part of the consumers, facing a great desire of having a product,
have a price limit or purchase effort that they are willing to pay in order to access that
product. The substitute products reduce the potential returns of a good to a producer
or manufacturer, because the more attractive the price in relation to the performance
of the substitute product, the greater the pressure on the product profits that can
replace them in the case of orchids, as well as in the consumer decision-making
process.
Most customers, when faced with conditions that make it difficult to buy any
product, tend to consider replacing it, and according to Kotler et al. [6], this pre-
purchase tension depends intrinsically on the main reasons that drive the consumer
to buy. The process of buying orchids depends basically on the internal or external
motivational factors that lead the consumer to search for the product. After this phase
of the buying process, there is a decision whether or not to purchase the product.
A product can be classified as a substitute for another when both can perform the
same function, similar function or even cause a similar effect as desired by the
consumer, such as orchids as internal decoration in homes, if they have high cost,
other lower priced species may have consumer preference as it would perform the
same function in the residence decorating. The similar function would occur if the
consumer in this case made the purchase of plastic flowers, and the similar effect
could be obtained by the consumer with the purchase of another product, as the
consumer wishes to provide the residence decoration. All commercial products, in
general, have a substitute product, which are manufactured or produced by other
companies. The ability of a substitute product for orchids and their efficiency are
linked to satisfaction and the sense of pleasure or disappointment that results of the
expected performance of the replacement product in relation to the expectations of
the orchid consumer, which will impact in the decision process of the consumer in
the future.
Most of the time, the decision-making process is imperceptible to the consumer,
who first feels the desire to have the flower due to the visual, aesthetic, and/or smell
interest, and then recognizes the need to own it.
The need to buy flowers in a general context according to Muraro et al. [12] and
Anacleto et al. [1] originate from several motivational factors but they point out that
the consumer is inserted in which people tend to seek the behavior of repetition of
the collective habit in the case of decoration with the use of flowers in their homes or
workplaces; however, Kotler et al. [6] describes that the need and desire for
consumption may be linked to situation came from childhood memories, loved
ones, psychological needs in relation to the environment, or factors such as social
and cultural influence.
People make routine decisions every day, and they become consumers as they
acquire customary things without having to think and analyze the purchase process
more deeply. But the orchid consumption is classified as an extensive decision which
degree of difficulty requires the consumer take a longer time to make the decision,
which can go from minutes to hours; in a general context, the more relevant the
extensive decision, the longer the analysis time. The orchids are classified in this
group of purchases as they are not essential products, so the consumer should
consider whether they can afford those resources to buy or not.
Orchid consumers, in a general context, showed high price sensitivity when used
for their own use; the predisposition in this case to pay more expensive averaged an
increase of 5.94% of the market value of the flower, and if the cost was higher for the
desired flower, this would be replaced by other products. However, the consumers
were less sensitive to the price when buying for the purpose of seducing a person or
when orchids were the chosen form of gift for Valentine’s Day; in both the cases,
they were more likely to spend up to eight times higher than those compared to
flowers purchased for their own use (Table 8).
Specifically, in the case of orchids that if the importance of nonmonetary values is
increased, the wide range of substitute products disposed to trade tend to have less
power of influence for the consumer, and this perception is more pronounced when
buying as a gift when cost is less relevant to the consumer.
The preference for consumption of an orchid type can be determined by two sets
of factors, first of all by the intrinsic factors of each individual that are able to make
the purchase decision; in this respect, it is highlighted the set of feelings, thoughts,
and factors that the individual who will give the gift wants to provoke in the receiver,
either by remembering colors or smells, which tied to the various types of arrange-
ments, exposure, price, and others which can influence the internal decision-making
process and result in an easier behavior of buying. In this case, orchids may have a
strong appeal over competing products or even other competing flowers since the
orchids have a strong relationship to two relevant organoleptic properties in the
Table 8 Value that customers are predisposed to overpay for the desired orchid according to
motivation, need and desire (N ¼ 150)
Classification Dates How much would pay more for the desired orchid (%)
1 Loving seduction 52.5
2 Valentine’s Day 34.8
3 Celebrate Birthday 32.0
4 Mother’s Day 20.1
5 Woman’ Day 20.0
6 All Soul’s Day 10.1
7 Graduation Tribute 7.02
8 Wedding 6.60
9 End of the year 6.23
10 Own use (decoration) 5.94
buying process, especially smell and color, which may arouse the consumer’s
unconscious to the consumption preference.
Effectively a person’s buying action depends on the motivation he/she receives,
as well as the perception of a possible internal situation, and whether this process
will make him/her take action to seek this satisfaction of desire. The perception
combines sensation with the meaning that a previous experience attributes to each
individual, yet it depends on one’s memory and thought, and the symbolic values
that permeate it. According to Schewe and Smith [13], the symbolic perception is a
process by which we value what individuals feel, and that value influences and adds
to our sensory impressions bringing our past experiences to influence them by giving
meaning of completeness and continuity, which were constructed from fragmentary
stimuli collected by the sense organs. The sensation depends on the stimulus and the
ability of the each individual to record it in previous events that involved the same
stimulus and that will affect the interpretation of the sensation by the brain, and thus
influencing the consumer’s behavior in a situation of a revived sensation, and it is in
this context that occurs the motivation for buying which is the inner impulse to fulfill
the need.
The substitute products may have reduced strength, and one way to achieve this is
by building sales relationships between sellers and buyers. The flower merchants,
especially those who have direct contact with the end consumers, develop relation-
ships that bring many benefits to the companies, and then the buyers believe in the
sellers, considered partners by establishing a lasting relationship that generates a
competitive advantage. Such partnerships between establishments and consumers
generate win-win relationship, resulting in what both parties seek; for Kotler et al.
[6], the key to reduce the power of the substitute products, especially in the case of
nonessentials such as orchids, is the relationship marketing that is a developmental
process that must happen in these cases. First of all, the company has to identify the
potential customers who are all people able to consume its products. Once identified,
the next step is to identify potential customers by analyzing their purchase profile,
the interest by the product and whether they can afford it and if they can promote
repeated purchases.
Identifying regular and prospective customers is important in the process, because
precisely this class of customers can make purchases of substitute products, so the
differentiated service can reduce this impact then these regular customers can
become preferred customers, who are those who have identification with the product
and recommend them to other ones [6].
In this context, the more intensive the relationship marketing, the greater the
number of loyal orchids customers and, therefore, the higher the consumption.
Companies typically have in their records the market share and the number of
potential customers. Within the market, there is a subgroup of clients that have
different needs, and for the company to know these clients better, it is necessary to
collect more data, through the these information collected, it is possible to identify
the profile of the clients, which can be separated by: gender, education, marital state,
and with this information, it is possible to identify which type of orchid will be
targeted to meet the wants and needs of each individual or each client group.
Fulfilling the consumers’ desires in relation to the product is an important factor
in the loyalty of a product consumption, Kotler et al. [6] describe that the satisfaction
of the consumer desires can be understood as the feeling of pleasure or disappoint-
ment resulting from the comparison of the expected performance of the product (or
result) in relation to the expectations of the person.
The consumers when looking for a product to buy mentally form an expectation
about the quality of this product. The expectations are based on the needs and desires
of each individual, his/her past personal experiences, and the influence of others.
Then after consumption, the consumer makes a comparison between what he/she
wanted and what he/she actually got after purchase, as also described by Bornancin
et al. [3], if the performance exceeds expectations, the consumer will be highly
satisfied, leading to the possibility of new consumption and the customer loyalty in
the orchid consumption.
The relationship marketing actions point to a very viable form of customer
loyalty, it also points to organizations the advantages of retaining their current
customers over gaining new customers, due to it is usually cheaper to keep cus-
tomers on track then to conquer new ones. In addition, customer loss can be
disastrous, so customer loyalty based on genuine and continuing satisfaction of
desires and needs is one of the greatest assets that a flower-trading company can
acquire.
4 Conclusion
The study revealed that women, in relation to orchid consumption, had a higher
average purchase per year than men.
The predominant age group of purchases (70.1%) was between 20 and 40 years
old, but with no difference in consumption between age groups.
It was also observed that as consumers advanced their educations level, there was
a slight tendency towards increasing the consumption.
Roses were the main flowers in the choice of consumers when they did not buy
orchids, and the main substitute products for the orchids were perfumes and
chocolates.
The study showed that there is an increase in the number of orchids purchased per
year according to the increase in monthly family income. In this context, orchids,
due to the high cost, when compared to the decision-making factors for purchase
apparently fits into nonmonetary valuation decision-making processes, so this con-
cept can be realized as, with the exception of roses, all other flowers classified as
competitor of the orchids on the shelves of the flower shops have much lower
purchase costs to the consumer. Thus, considering that in some cases, this cost
reaches less than 50% of the value of an orchid, apparently the nonmonetary
appreciation of orchids may be constituting one of the most important factors in
the consumer’s perception for the motivation to buy. This tendency should be used as
a consumer loyalty alternative by relationship marketing to be developed by mer-
chants with their consumers.
Finally, it is recommended to carry out further studies regarding the use of the
orchid as a means of seduction between people. It is important to mention that in the
seduction processes, the extensive decision has less influence, and the price ceases to
be the main issue in the consumption preference and consumer willingness to pay for
the orchid he/she wants can reach 50% of the market price.
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Part IV
Agri-food Applications
Vanilla: Culture, Reproduction,
Phytochemistry, Curing, Pest, and Diseases 13
Keshika Mahadeo, Tony L. Palama, Bertrand Côme, and
Hippolyte Kodja
Contents
1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 330
2 Cultivation Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 331
3 Flowering . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 332
4 Phytochemistry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 333
5 Curing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 334
6 Pest and Diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 335
6.1 Fungal Diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 335
6.2 Viral Diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 337
7 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 338
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 338
K. Mahadeo (*)
Qualisud, Université de La Réunion, Université Montpellier, CIRAD, Institut Agro, Avignon
Université, Montpellier, France
Laboratoire de Chimie des Produits Naturels, Université de la Réunion, Faculté des Sciences et
Technologies, 15 Avenue René Cassin, CS 92 003, 97 744 St Denis Cedex 9, La Réunion, France
e-mail: keshika.mahadeo@univ-reunion.fr
T. L. Palama
Université Sorbonne Paris Nord, Laboratoire de Chimie, Structures, Propriétés de Biomatériaux et
d’Agents Thérapeutiques, CSPBAT, CNRS, UMR 7244, Villetaneuse, France
e-mail: tony.palama@univ-paris13.fr
B. Côme
La Vanilleraie, Sainte-Suzanne, La Réunion, France
e-mail: la.vanilleraie@orange.fr
H. Kodja
e-mail: hippolyte.kodja@univ-reunion.fr

330 K. Mahadeo et al.
Abstract
This chapter describes relevant information on vanilla (Vanilla planifolia) includ-
ing the plant description, flowering, and phytochemistry of green pods and leaves.
The harvesting and post-harvesting processes are also described along with the
cultivation methods. The chapter closes by looking at fungal and viral diseases of
vanilla plants.
Keywords
Vanilla · Vanilla planifolia · Vanilla cultivation · Curing process · Vanilla diseases
1 Introduction
Vanilla is considered to be the best contribution of the Americas to the world of

flavors. The orchid Vanilla planifolia is indigenous to Central America. In Mexico,
vanilla fruit was called “tlilxochitl,” which comes from the words “tlilli” and
“xochitl” and means “black” and “pod,” respectively [1, 2]. When the Spaniards
discovered America, they called it “vanilla” which comes from the Spanish word
“vaina” meaning “pod.” The Latin word “planifolia” described the flatness (“plani”)
of the leaves (“folia”) [3].
The genus Vanilla belongs to the largest family of flowering plants, the
Orchidaceae. Among the 800 genera and the 25,000 species within the family, the
genus Vanilla contains by itself 110 species [4]. Among the 110 species, only 15
species develop an aromatic fruit. V. planifolia, V. tahitensis, and V. pompona (Fig. 1)
are the only three species cultivated and commercialized for their relevant flavors
Fig. 1 Cultivation of V. tahitensis (left), V. planifolia (middle), and V. pompona (right). V.

tahitensis: thin and elongated leaves; V. planifolia: flat and large leaves; V. pompona: the leaves
exhibit dimorphism (large and small leaves)
13 Vanilla: Culture, Reproduction, Phytochemistry, Curing, Pest, and Diseases 331
and aroma compounds. However, the vanilla market is dominated by Bourbon like
vanilla (V. planifolia). It represents more than 95% of the world production of
vanilla.
2 Cultivation Methods
Vanilla is a tropical plant that grows best in warm and moist climate. Therefore,
natural growth is obtained between latitude 15° north and 27° south on all continents
except Australia [1, 3, 5]. The optimal conditions for Vanilla plants growth are
temperature comprised between 21–32 °C, while precipitation required falls between
2000 and 2500 mm per year [1].
Vanilla is a hemi-epiphytic orchid that needs a tree to provide physical support,
shade, and organic material. The cultivation is performed through three cultural
practices: in forest-type land, intensively in deforested land, and in shade houses
(Fig. 2).
In forest-type land, trees are used as tutors that support the vanilla plants. The
aerial roots help the vines to climb on the trees to reach the canopy in order to
accumulate the maximum of sunlight before flowering. The aerial roots also con-
tribute to absorb water from the air humidity. Ideal soil for vanilla is light, rich in
humus and porous allowing the roots to spread without molds development [1, 3].
The plant nutrition comes from the roots found in the soil that feed on decaying
organic matter (decomposition of dead leaves from the tutor’s trees). A relatively
low density of vanilla plants is cultivated in forest-type land leading to low yields of
green pod/ha.
In contrast, the production of vanilla in deforested land is an intensive system.
This cultural practice consists of planting support trees with sufficient distance for
ventilation between the trees in order to avoid molds development. After a year,
Fig. 2 Cultivation of vanilla in forest-type land (left), deforested land (middle), and in shade
houses (right)
when there is sufficient shade (60%), the vanilla is planted. The plant feeding is
ensured by input of sugar cane mulch, coconut fiber, and organic compost.
The cultivation in shade houses is another intensive system of vanilla production.
It is performed in semi-controlled conditions with shade accumulation of 60%. In
this intensive system, dead wood trunks are used as tutors and the plant feeding is
provided by natural organic compost.
Those two last systems of vanilla cultivation have the advantage of relatively high
yields.
Vanilla vines grow around 10 m/year. Crops are established from cuttings of
vigorous vines. Higher temperatures or insufficient shading can cause the death of
the plant. On the contrary, excessive humidity and shading can induce susceptibility
of the plant to diseases (mildew and root rot) [1]. A water stress of about 50 days and
sunlight exposure are necessary to induce flowering.
3 Flowering
In general, the first flowers appear 3 years following planting. The physiological cue
to flower is promoted by climatic (dry season) or mechanical stress. In Reunion
Island, the flowering season is September to December. The flowers (8–10 flowers)
are grouped into an inflorescence located underneath the leaf. During the blooming
season, the flowers open a few at a time and lasts a single day (Fig. 3). Vanilla
flowers are hermaphroditic. The rostellum, a cap like structure, hangs exactly in
between the anther sac (male organ) and the stigma (female organ) and acts as a
physical barrier to prevent from self-fertilization. Thus, vanilla flowers are either
naturally pollinated by the Melipona bee (found in Mexico), or by hand pollination.
V. planifolia plants were introduced to Reunion Island in 1822. However, without the
specific pollinators on the island, no vanilla pods were produced. The biggest
advancement in vanilla growth and pollination happened in 1841, when Edmond
Albius, a slave on Reunion Island, discovered a practical method of vanilla hand
pollination [6]. Albius discovered that the rostellum could be lifted out of the way
(using a small stick), so that the anther sac can hang down unimpeded over the
stigma lobes. Now practically all vanilla is produced by hand pollination.
Fig. 3 Flower of V. planifolia

Fig. 4 Mature fruit of

V. planifolia
Immediately after hand pollination, pollen tubes begin their germination and fertil-
ization of the ovules. The ovary begins to enlarge and develops a dark green pod.
The maximum length (approximately 20 cm) and diameter of the fruit is achieved
within 2 months after pollination. Afterward, the fruit enters into a long period of
maturation of 7–8 months. When the fruit is mature, the distal tip of the bean changes
color from green to yellow (Fig. 4). This senescence zone is the starting point of the
fruit dehiscence that can continue after harvesting. At this stage, the vanilla pod
starts to lose their aromatic compounds and flavors. Therefore, all mature fruits are
harvested 9 months after pollination before the dehiscence appears.
4 Phytochemistry
The leaves and green pods of V. planifolia contain various glucosides including the
glucoside A (bis[4-(β-D-glucopyranosyloxy)-benzyl]-2-isopropyltartrate) and the
glucoside B (bis[4-(β-D-glucopyranosyloxy)-benzyl]-2-(2-butyl) tartrate) [3, 7–9].
These two glucosides were reported in vanilla leaves and may contribute to discrim-
inate the development stage of the leaves. Young leaves were found to have higher
level of glucose, glucoside A, and glucoside B whereas older leaves have more
sucrose, acetic acid, homocitric acid, and malic acid [10]. As glucosides A and B are
thought to be precursors of glucovanillin, there is maybe a connection between their
accumulation in green pods and young leaves.
The 1H NMR metabolomic analysis of developing green pods (between 3 and
8 months after pollination) had showed that younger pods contain more glucose,
malic acid, homocitric acid, glucosides A, and glucoside B. In the green mature
beans, vanillin, the main flavoring constituent of vanilla is present only in very low
trace amounts. However, it is mainly present in the uncured pods in the form of a
glucoside called glucovanillin. In a recent study, Odoux and Brillouet [11] unam-
biguously proved that glucovanillin is stored in the placental laminae (92%) of
mature green pods whereas only a small amount of glucovanillin (7%) is present
in the trichomes [3]. Compared to younger pods, older pods contain more sucrose,
glucovanillin, vanillin, p-hydroxybenzaldehyde glucoside, and p-hydroxyben-
zaldehyde [12]. In green mature pods, free vanillin can reach 10–20% of the total
vanillin content. Besides, the lipid content of vanilla mature beans have also been
investigated and the authors reported various fatty acids such as lauric acid (12:0),
myristic acid (14:0), pentadecanoic acid (15:0), palmitic acid (16:0), stearic acid
(18:0), oleic acid (18:1 n-9), and linoleic acid (18:2 n-6) [13]. The major fatty acids
were found to be oleic acid, palmitic acid, and linoleic acid.
Various other minor glycosides were identified in mature green pods after enzy-
matic hydrolysis including glucosides of vanillic acid, vanilly alcohol, 4-vinyl-
guaiacol, acetovanillon, caffeic acid, ferulic acid, methyl-3,4-dihydroxycinnamate,
2-methoxy-4-cresol, homovanillyl alcohol, 3,4-dihydroxybenzoic acid, ethyl-4-
hydroxy-3-methoxyphenylacetate, p-cresol, 4-vinylphenol, methylsalicylate, p-
hydroxybenzylethyl ether, p-hydroxybenzaldehyde, p-hydroxycinnamic acid,
cinnamic alcohol, cinnamic acid, phenethyl alcohol, 3-phenylpropanol [3, 8].
The phenolic compounds detected in mature pods were mostly present as gluco-
sides, underlining the need of the curing process to ensure the release of the
aglycones to increase the vanilla flavor.
5 Curing
After harvesting, mature green pods of V. planifolia are almost odorless and they
develop their characteristic flavor and aroma after a “curing” process. Several pro-
cedures have been developed for curing vanilla. Every vanilla growing country has
developed its own curing process, but they all generally involve four common steps:
“killing,” “sweating,” “drying,” and “conditioning” [3, 14–18]. The whole process
takes about 9–12 months. In course of the “killing” process the natural physiological
processes in the harvested beans are stopped and thus avoid any dehiscence of the
fruit. This can be carried out by hot water scalding, sun or oven heating, and
scarification. Killing methods allow cell structure disruption and thus various
enzymes can come into contact with their substrates like glucovanillin. During the
killing process, the glucovanillin is hydrolyzed to form vanillin and glucose through
the action of a β-glucosidase [17]. The “sweating” step allows the moisture to escape
rapidly to reach a level which will avoid microbial spoilage during the subsequent
operations. This step is also crucial because it lets indigenous enzymes taking effect
to develop the characteristic vanilla aroma and flavor. During the “sweating” step the
beans get their characteristic brown color and develop a proper texture and flexibil-
ity. They are wrapped up in sheets and put in a container overnight. During the
Fig. 5 Curing steps of vanilla
“drying” step, decrease of the moisture content reduces undesirable enzyme activ-
ities and biochemical changes. This step usually lasts for 2–4 weeks and is
performed indoor and outdoor. The final step of vanilla curing process is the
“conditioning” step. Vanilla pods are stored in closed boxes for several months
(6 months) to refine its flavor and develop new compounds from Maillard reactions.
The full curing process last at least 6 months but can go for 1 year or more
(Fig. 5) [3].
6 Pest and Diseases
Vanilla cultivation tends to develop into a more intensive practice. Thus, occurrence
of pest and diseases affecting Vanilla is more and more increasing. In the last
decades, vanilla growers had to face several fungal and/or virus diseases [3].
6.1 Fungal Diseases
Favorable conditions for the development of fungal diseases are drought, deficient
soil drainage, poor ventilation, excessive watering of the roots and over-pollination.
Fungal diseases are frequently opportunistic infections caused by poor management
practices.
Among them Fusarium, a cryptogamic infection, is the most damaging disease
of vanilla. The root and stem rot (RSR) of vanilla is a disease caused by Fusarium
oxysporum f. sp. radicis-vanillae (Forv) [19]. It is a soil-borne fungus found in all
vanilla producing countries causing severe damage and yield losses. In the early
stage of the disease, there is browning and death of underground roots, followed
by the death of aerial roots. Subsequently, the plant stops its shoot growth
suggesting a lack of nutrients and the leaves and stems begin to shrivel [3].
When the fungus infects the plant, it is difficult to eradicate the disease. Chemical
fungicides, essential oils (clove and cinnamon oil), or biological control such as
Pseudomonas and Trichoderma are used as control methods for RSR [20].
However, none of these methods were efficient enough to restore productivity
of vanilla plots. The best management of the disease can be an approach in which
the biological control can play a major role in complement of the varietal
selection for resistance. However, cultural practices should be the first methods
to be used to prevent the development of the pathogen (equipment in good
sanitary conditions, managing water in an appropriate manner and use of land
with good drainage, keeping plant well-nourished, avoid overcrowding, and
excess shade). Besides, identifying genotypes resistant to Fusarium can also be
considered as an alternative [21]. Indeed, several species including V. pompona,
V. phaeantha, and V. bahiana showed resistance to the pathogen [2]. Most of the
V. planifolia accessions, V. tahitensis and V. odorata were susceptible to RSR.
However, Koyyappurath et al. [19] identified new accessions of V. planifolia
resistant to Fusarium.
Anthracnose, caused by Colletotrichum sp., is another fungal pathogen that
attacks leaves, fruits, stems, and flowers of vanilla plant. Characteristic of the disease
are irregular brown spots on the leaves, shoots, and fruits [22]. The spots develop
afterward into elliptic necrotic brown spots (Fig. 6). In general, the symptoms appear
on the first young leaves of the apical part of the plant followed by fruit damage
during the humid and warm months. Infected fruits fall prematurely before reaching
their maturity. Anthracnose occurs on poorly maintained plantations with proper
Fig. 6 Brown spots on

vanilla pods caused by
Colletotrichum sp [23].
Reprinted with permission
from John Wiley & Sons
shading control. Indeed, an excess of shade and high density of plants are favorable
conditions for anthracnose development. In 2011, Colletotrichum orchidophilum has
been identified as the causal agent of anthracnose in Reunion Island. The annual pod
production was reduced by 10–30% due to this pathogen [23].
Vanilla stems and pods can also be infected by mildew. It generally occurs after a
significant rainfall event. The propagation of mildew can be rapid in the field
resulting in important loss of mature pods.
In these conditions, infected plants have to be eliminated before spread of the
disease to the entire plot. The causal agent of mildew is either Plasmopara
palmivora, or Phytophtora. Phytophthora jatrophae is one of the sources of vanilla
mildew with direct economic impact since it attacks the developing fruits [1, 2]. The
disease starts to one extremity and then spreads to the whole pod. Affected parts of
the pods turn into a brown chocolate color. Diseased pods lose their swelling and
rapidly fall to the ground [1, 3].
6.2 Viral Diseases
Vanilla is also affected by viral diseases due to intensification of cultivation. The

damage caused by viruses can be difficult to distinguish: some plants do not show
clear symptoms or are asymptomatic. Cymbidium Mosaic Virus (CymMV,
Potexvirus) and Odontoglossum RingSpot Virus (ORSV, Tobamovirus) are
reported as the most prevalent viruses having economical incidences [3, 24].
Plants infected by CymMV and ORSV are generally asymptomatic, but occa-
sionally they exhibit mild chlorosis or small spots on the leaves and stems of V.
planifolia and V. tahitensis [25]. These two viruses are transmitted through the
sap and are dispersed through contaminated cutting tools. They may also be
transmitted by contaminated pollen [26]. Potential destructive impact and rapid
spread of this virus made it of interest for vanilla growers. The virus was first
reported in South Pacific in French Polynesia. Farreyrol et al. [27] reported for
the first time infection of Vanilla by Cucumber Mosaic Virus (CMV,
Cucumovirus) in the Indian Ocean in La Réunion. Although the authors did not
observe severe symptoms, leaves of CMV-infected plants were slightly elon-
gated. It has since been found in vanilla vines growing in other countries in the
Indian Ocean, such as Madagascar and India [3, 28, 29]. Another virus, the
Vanilla Mosaic Virus (VanMV, Potyvirus) occurs mainly in the islands of French
Polynesia. The virus causes leaf distortion and mosaic lesions in V. planifolia,
V. pompana and V. tahitensis vines [30, 31]. This virus may be transmitted by
aphids [22]. Other viruses have also been reported to infect vanilla vines: the
Watermelon Mosaic Virus (WMV, Potyvirus), Bean Common Mosaic Virus
(BCMV, Potyvirus), Bean Yellow Mosaic Virus (BYMV, Potyvirus), Cowpea
Aphid-Borne Mosaic Virus (CABMV, Potyvirus), Ornithogalum Mosaic Virus
(OrMV, Potyvirus) and Wisteria Vein Mosaic Virus (WVMV, Potyvirus) [3,
32–36].
Nowadays, efficient techniques have been developed to detect virus [33, 35,
37], and virus spread can be control with good practice cultivation [3]. In
prevention of virus diseases, it is important to use healthy certified cuttings, to
install insect-proof netting around the shade houses to avoid the insect vectors
(aphids), to eliminate crops around the plantation that can be reservoirs of
the virus and to eliminate diseased vanilla vines as soon as they are detected
[22, 38].
7 Conclusion
Vanilla is the principal source of natural vanilla of commerce and it is mainly used in
food, perfumery, and pharmaceutical preparations. The quality of the bean depends
on several aspects including the species used, the volatile constituents, the curing
process adopted, and the vanillin content. Hence, this chapter is elaborated on the
cultivation methods of vanilla vines, the curing process, and how to avoid the main
pest and diseases of vanilla cultivation.
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Vanillin: Biosynthesis, Biotechnology,
and Bioproduction 14
Shahnoo Khoyratty, Rob Verpoorte, and Hippolyte Kodja
Contents
1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 342
1.1 Vanilla Species as Source of Vanillin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 346
1.2 Vanillin Sources: Plants and Microorganisms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 349
1.3 Volatiles Related to Vanilla Flavor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 350
1.4 Endophyte Species Occurrences Depending on Geographical Location . . . . . . . . . . . . 351
1.5 Plant and Endophyte Cohabitation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 353
2 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 354
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 354
Abstract
Further understanding of Vanilla flavor compounds synthesis, in Vanilla plants, is
lacking in literature. This can aid in better production of flavor compounds.
Additionally, commercial importance of Vanilla pods can then be improved.
Although vanillin amounts in pod, predominates among flavor compounds, the
natural flavor is made of over 200 chemicals. Despite being low cost to produce,
synthetic pure vanillin alone does not match the much sought complex notes of
the natural product, used in products with high-quality attributes. This, despite the
S. Khoyratty (*)
Natural Products Laboratory, Institute of Biology, Leiden University, Leiden, The Netherlands
e-mail: shahnookhoyratty@gmail.com
R. Verpoorte
Natural Products Laboratory, Institute of Biology, Leiden University, Leiden, The Netherlands
e-mail: verpoort@chem.leidenuniv.nl
H. Kodja
e-mail: hippolyte.kodja@univ-reunion.fr

342 S. Khoyratty et al.
natural product being more than 200 times the price of the synthetic version,
fungal biotransformation reactions of vanillin precursors yield “natural” vanillin.
Endophytic fungi form symbiotic relationships with asymptomatic plants, where
they produce secondary metabolites. In some cases, such metabolites were
previously thought to be produced by the plant, but later revised to originate
from the fungi. In this view, Vanilla flavor metabolites may be due to fungal
participation, within the plant. Those fungi may either participate partially or
completely, on the vanillin biosynthetic pathway, within the plant. This potential
participation requires to be elucidated, to gain a better understanding of the still
debatable vanillin biosynthesis, within the plant. The occurrence of different
fungal endophyte species, across plant culture regions, may contribute to the
observed terroir effect on pod flavor. The study of fungal endophytes within
Vanilla plants, and their biosynthetic potential, is thus warranted.
Keywords
Vanilla · Vanillin · Flavor · Endophyte · Biosynthesis
1 Introduction
Vanilla is a member of the Orchidaceae family, itself comprising 110 species. Within
this family, Vanilla planifolia Jacks. ex Andrews, Vanilla pompona Schiede., and
Vanilla tahitensis J.W. Moore, all originating from Central America, are important in
terms of their flavors. For this reason, the three species have commercial values and thus
cultivated. Vanilla spp. were introduced in Europe by the Spanish, about the year 1520,
whereas Reunion Island initiated its cultivation around 1819 (Table 1).
Given substrate specificity is lacking during vanillin synthesis, the process is
inefficient and expensive as more substrates are then required [5]. Additionally, this
synthesis is not environmental friendly [77]. There is also the issue of slow growth of
Vanilla plants produced through tissue culture, and a low expression of vanillin
biosynthesis. As such, vanillin production from those tissues is also inefficient and
expensive. Better alternatives, which are more efficient and more cost-effective, are
then preferred. Such an alternative is in the synthesis of vanillin by microorganisms.
Among different microorganisms, some fungi have the highest amounts of vanillin
synthesis (Table 1). A possible characteristic, for a microorganism to achieve higher
amounts of vanillin synthesis, is in the ability to not be affected, from toxicity due to
vanillin. Generally, this characteristic can be attributed to the ability to convert vanillin
into chemicals of lower toxicity. These chemicals include vanillyl alcohol and vanillic
acid. The latter conversion process would reduce the total amount of vanillin obtained,
which is not desirable [5]. There is, however, one method to decrease vanillin toxicity,
which does not reduce vanillin amount, by the glycosylation of vanillin. With this
view, yeast has been genetically modified to obtain this glycosylation ability [5]. It is
worth noting that vanillin occurs as glucovanillin, a glycosylated form, in green pods
of Vanilla. Of interest, fungal endophytes may reside within pods, a Vanilla plant
organ, and may be involved with vanillin synthesis.
14 Vanillin: Biosynthesis, Biotechnology, and Bioproduction 343
Table 1 Selected landmark events in relation to Vanilla flavor

Year Events
Ca. 1520 Vanilla plants imported to Europe. Given absence of required pollinators from
Mexico, no pods formed in Europe
1819 Vanilla plants imported to Reunion Island, where artificial pollination method
developed. This method opened possibilities of commercialization
1874 Nicholas-Theodore Gobley isolates and purifies vanillin from Vanilla pods
After From lignin, vanillin synthesized. However, procedure is not environmental friendly
1920s
1970 Green vanillin biotransformation: Vanillin synthesized by Pseudomonas
acidovorans (bacteria), from ferulic acid precursors
1998 Production of green vanillin: Rice bran oil and wheat bran are inexpensive
2002 agricultural by-products. These are sources of ferulic acid, as precursors towards
2007 vanillin synthesis, by Aspergillusniger (fungus) combined with
Pycnoporuscinnabarinus (fungus). The later yields a maximum vanillin of 300–
2800 mg L1 of liquid media. This amount depends on the fungal strains, addition
of glucose, and adsorbent resinsa, concentrating ferulic acid amount. Synthetic
vanillin was shown to differ from this green vanillin, through isotope ratio δ13CPDB
analysis
2009 Production of de novo green vanillin: Genetically modified yeasts
Schizosaccharomycespombe and Saccharomyces cerevisiae convert glucose to
vanillin. This is only possible after a modification that introduced a novel
biosynthetic pathway. The yield of vanillin is 45–65 mg L1 of liquid media
2011 Supply cannot match demand, for Vanilla flavoring from Vanilla pods. This
prompted flavor companies, like International Flavors and Fragrances, Inc. and
Givaudan, like to find alternative methods to produce substitutes
2014 Green ripeVanilla beans incubated with microorganism yields vanillin. This process
is part of a patent application by Givaudan (flavor company). Vanillin amounts equal
to, or higher than in cured pods, and has additional consistent complex sensory
profiles, devoid of off-notes
2015 Production of de novo green vanillin: Genetically modified bacteria Escherichia coli
convert glucose to vanillin.This is only possible after a modification that introduced
a novel biosynthetic pathway. The yield of vanillin is 97.2 mg L1 from l-tyrosine
precursor, 19.3 mg L1 from glucose precursor, 13.3 mg L1 from xylose
precursor, and 24.7 mg L1 from glycerol precursor
2017 Novel natural Vanilla products, by Firmenich. These are less expensive and come
from sustainable production practices
Adapted from [1–11]
a
Vanillin synthesized by microorganisms is removed from media by adsorbent resins. This allows
for more vanillin production [5]. In high amounts, vanillin is toxic, prompting the microorganism to
degrade vanillin. The latter reduces the yield of vanillin
A literature search shows that de novo vanillin synthesis has not been reported for
natural microorganisms. The ability towards de novo synthesis has only been
possible after genetically engineering microorganisms e.g. biosynthesis of vanillin
from glucose in recombinant yeast [76]. The process has several associated prob-
lems, e.g., genetically modified E. coli production of vanillin suffers from isovanillin
synthesis, a contaminating chemical that reduces vanillin yield [5]. To prevent a
decrease in vanillin yield, gene knockout technology was applied to genetically
modified yeasts [5]. The gene knockout targeted a gene, for which enzyme (alcohol
dehydrogenase ADH6) is responsible for the conversion of vanillin to vanillyl
alcohol. Despite this, another issue occurred in accumulation of vanillin, and
hence toxicity, as well as decrease in solubility, within the culture medium.
Flavors within cured Vanilla pods are of commercial importance. A flavor
component of Vanilla cured pods is vanillin, generally occurring at 1–2% w/w
[12]. In terms of amount, vanillin is thus the major flavored component. Still,
other minor components present in lesser amounts than vanillin in the pods also
contribute to Vanilla flavor. Those minor components are responsible for specific
flavors, which give value to natural Vanilla flavor, over synthetic vanillin. Flavor
descriptors for natural Vanilla may range across floral, woody, and spicy [13].
When vanillin and Vanilla flavor are compared in terms of organoleptic proper-
ties, the former is one-dimensional, whereas the latter is multidimensional [14]. This
makes Vanilla flavor complex, although subtle as well [14, 15]. Vanilla pods thus
qualify as spice [16]. One parameter that influences Vanilla flavor, is the conditions
under which plants were grown [15]. The types of combination of flavor-related
chemicals within the pods confer specific flavors to those pods [17]. This is a quality
feature which affects Vanilla trade and prices.
The type and amount of metabolite, as well as the ratio at which these occur,
affect the final flavor of the pods. This, then, constitutes the organoleptic properties
of the pod. Given the contribution of minor components (in terms of quantity), to
flavor, it is essential to find the minimum amount of such flavor components that can
be detected in terms of sensory attributes, by humans. Vanillyl alcohol is an example
of this, given olfactory-GC analysis shows the latter registers as intense as vanillin,
despite being at an amount 1000 times less than vanillin, in cured pods [18]. Given
over 250 components contribute to flavor in cured pods, vanillin accounts to only
less than 12% of variability in organoleptic qualities, when different pods, with
varying flavors, are compared [18]. Some flavor components are volatiles detected as
aromas. These evaporate the moment pods are heated. Thus, baking can lead to
flavor alteration [19].
Other minor flavor components include vanillin putative precursors, given these
also contribute to flavor qualities of pods. A nonexhaustive list includes volatiles
such as monoterpenes, sesquiterpenes, and phenolics [20, 21]. Nonvolatiles include
vanillic acid with creamy flavors [22], vanillyl alcohol with balsamic flavors [23],
p-hydroxybenzoic acid having phenolic tones [22], and p-hydroxybenzaldehyde
with a sweet flavor [22]. Volatiles are detected with the nose, whereas nonvolatiles
with the mouth, i.e., both have differing contributions to flavor.
Vanilla production requires significant labor intervention, compared to other
cultivated crops [24, 25]. The cost of labor involved in its production thus makes
the pod expensive. Labor availability is also dependent on political stability, which
then affects the price of the pods. Climatic conditions also affect the pod supply,
leading to price fluctuations. The volatility of pod price, on the international market,
has encouraged the production of cheaper synthetic vanillin and nonnatural Vanilla
flavors, which are then incorporated in food and beverages. The latter production
is also more economical than attempting to extract Vanilla flavor from pods.
As a comparison, natural extracts are at least 200 times more expensive than
synthetic vanillin. This price difference has sometimes led to imitation natural
flavors being sold, thus requiring quality control, to prevent this practice [26].
Global demand to supply ratio for Vanilla cured pods is at least at 10:1 [2]. Due to
this, synthetic vanillin competes with natural Vanilla flavor, despite the flavor
attributes of natural flavor being better. With time, 95% of Vanilla flavor world
consumption is that of synthetic vanillin. Through habit, consumers have got used to
vanillin over natural Vanilla [26].
Still, market trends are shifting towards natural vanillin, synthesized by green
chemical reactions, instead of the synthetic version. Even so, natural vanillin is not
set to substitute natural Vanilla flavor, rather it will take the place of synthetic
vanillin at a more accessible price [27]. High-quality products, e.g., fine chocolates,
will always contain real Vanilla flavor though. Other products using Vanilla flavor
are Cola beverages, ice cream, and baked foods [26].
As consumer trend shift towards a greater demand for natural products, demand
for biotechnology-derived natural flavors are also increasing [28]. Two main
methods of flavor compound synthesis are either through biotransformation or
through de novo synthesis. The difference between both production methods is
with regards to the number of steps in reactions, from precursor, leading to the
same vanillin [28]. In biotransformation reactions, single reactions would
biotransform precursor into vanillin, whereas in de novo synthesis a precursor is
biotransformed into vanillin after going through complex metabolic pathways.
The search for microorganisms capable to produce “natural” vanillin de novo is
sought after. Finding such microorganisms could partially address the problem of not
having enough natural Vanilla flavor extract. Some fungi are good candidates
towards this end, given their ability to synthesize vanillin from ferulic acid
precursors.
Endophytes are microorganisms that produce secondary metabolites and are
sometimes responsible for the source of those metabolites, and in several cases,
previously thought to be produced by the plants [29]. Additionally, metabolites from
the same endophytes can participate in biosynthetic pathways of the plants, to yield
novel secondary metabolites. These include pharmacological metabolites ergoline
alkaloids [30–33] and taxol [34, 35].
Endophytes (bacteria and fungi) form symbiotic relationships with plant organs
while, unlike pathogens, the plant shows no visible disease symptoms [36]. Still, the
definition of an endophyte is not clear-cut. For instance, if a plant pathogen loses
its virulence, this microorganism can then be termed as an endophyte [37]. The
presence of the endophytes in the plant is due to the metabolome of the plant
[38]. Endophyte function within the plant is varied and so far is reported as
participating in conferring to the host, survival tools, within the environment in
which the plant grows. Endophyte assistance to the plant encompasses stress condi-
tions, e.g., pathogenic attack [39–41], under low water regimes [42] and consump-
tion by herbivores [43–45].
In a similar way, it is reasonable to assume fragrances and flavors traditionally
thought to be produced by plants, may either be produced only, or partially, by
endophytes instead. For this reason, an in-depth study into endophytes residing in
plants, those important for their flavors, is warranted. From there, to study the
biosynthetic abilities or biotransformation reactions of those endophytes, on pre-
cursors and towards flavor metabolites. Given endophyte composition generally
varies across environments, it may also be possible that such differences are respon-
sible for the terroir effects of Vanilla.
1.1 Vanilla Species as Source of Vanillin
Flavor metabolites are organ specific within Vanilla plants. Specifically, mature
pods, but not the leaves, contain flavors. The age of the pod is important, given
immature pod are devoid of vanillin. During the maturation period, vanillin content
increases within the pod, as glucovanillin. The highest yield of vanillin is reached at
the end of maturation. Vanilla pods take about 8 to 9 months after pollination to
mature. Most of the glucovanillin within the mature pods would then be hydrolyzed
with the enzyme glucosidase, during the curing process, into vanillin [24, 25].
Although Vanilla pods harbor the highest vanillin content, the presence of vanillin
is not limited to Vanilla species. Other plants also contain vanillin (Table 2).
The synthesis of vanillin, in terms of all steps on the biosynthetic pathway, and
the identity of participating vanillin precursors are still uncertain. One issue is that
studies conducted to elucidate this pathway, or part of it, used varying biological
materials, e.g., Vanilla pods, cell culture, and plants [47]. Each of these materials
may have different vanillin synthesis pathways which increase uncertainty, in any
Table 2 Vanillin presence and amounts in different plants

Vanillin percentage with respect to plant dry
Plant species weight
Syzygium aromaticum (L.) Merr. and L.M. Trace
Perry)
Common name: Clove
Solanum tuberosum L. 0.01
Common name: Potato
Proboscidea louisianica (Mill.) Thell. 0.01
Common name: Ram’s horn
Narcissus tazetta L. 0.01–0.60
Common name: Narcissus
Hyacinthus orientalis L. 0.20–0.50
Common name: Hyacinth
Vanilla pomponaSchiede 0.01–2.00
Common name: Vanilla
Vanilla tahitensis J.W. Moore 0.50–2.00
Vanilla planifolia Jacks. ex Andrews* 2.00–3.00 (Highest vanillin content)
Adapted from [46]
direct comparison between results for different biological materials. Given the
simplicity of vanillin structure, enzyme promiscuity on phenolic pathways, it is
hypothesized that synthesis of vanillin can thus occur through several potential
pathways, which is a further complication [48]. Due to these issues, it is not quite
possible to reconstruct a single reliable pathway for vanillin synthesis, based on data
from different sources.
Figure 1 illustrates vanillin putative synthesis network. Precursors include tyro-
sine and phenylalanine.
Vanillin can be converted into vanillic acid and vanillyl alcohol, both of which
also contribute to Vanilla flavor as well. As a result, vanillin can either be the product
of the vanillin biosynthesis pathway or a precursor to other intermediates.
Through the shikimic acid pathway, tyrosine and phenylalanine precursors
(phenylpropanoids) lead to the synthesis of vanillin [52], which is generally
accepted. Despite this, the steps through which this conversion occurs vary based
on two hypotheses, i.e., either the ferulate or benzoate pathways. C6C3 methylation
and hydroxylation yield ferulic acid [52], followed by chain shortening, to give
vanillin. The latter reaction describes the ferulate pathway hypothesis. In the alter-
native benzoate pathway, phenylalanine or tyrosine go through a chain shortening
reaction [52], then the aromatic ring undergoes methylation or hydroxylation to yield
vanillin. It is also possible for another vanillin precursor, such asp-hydroxybenzoic
acid, to originate directly from the shikimate pathway as well. In this case, vanillin
synthesis is not related to phenylalanine or to tyrosine, making this possibility,
different from the ferulate and benzoate pathway hypotheses, for vanillin synthesis.
Enzymes tyrosine or phenylalanine ammonia lyase are two enzymes that inter-
vene at the entry point of the phenylpropanoid pathway. Cinnamic acid derivatives
are synthesized through the reactions catalyzed by both aforementioned enzymes.
The precursor p-hydroxybenzaldehyde can lead to vanillin synthesis. Metabolites
from the latter pathway are present in Vanilla pods, which suggests that this pathway
may be possible towards vanillin synthesis. Such metabolites include p-coumaric
acid, vanillyl alcohol [18], p-hydroxybenzaldehyde, protocatechuic aldehyde, and p-
hydroxybenzoic acid [13]. And yet, there is the contradictory result that radiolabeled
p-hydroxybenzaldehyde, after feeding within pod material, is not integrated into
glucovanillin [53]. Still, this is not a conclusive result, since highly reactive alde-
hydes, like the radiolabeled p-hydroxybenzaldehyde, may already be used in other
reactions before being able to participate within vanillin synthesis. As such, this still
opens the possibility that other nonradiolabeled p-hydroxybenzaldehyde, already
present in the pods, may have been used in vanillin synthesis.
Another controversy is with regards to the function of enzymes that intervene on
pathways, leading to vanillin synthesis. An enzyme reported by a research work to
participate in vanillin synthesis from ferulic acid as precursor (ferulate pathway) [53]
is instead reported by another research work to participate in p-hydroxyben-
zaldehyde synthesis from p-coumaric acid (US patent application) [55]. It is possible
that an enzyme has broad substrate specificities, as hypothesized by Dixon. For
instance, cinnamic aldehydes may be synthesized from the related acid derivatives
by an enzyme. Also, aldehyde reduction may occur through enzymes that react
Fig. 1 Scheme of potential pathways to vanillin in relation to confirmed pathways in the plant
Vanilla planifolia (Bold, Dark lines). Vanilla planifolia reactions adapted from [47–53]. The
hydroxylation reaction marked with # has not been found in plants and fungi, as phenylalanine
and tyrosine are synthesized separately from different precursors [54]. The system (cell culture, pod,
and callus) in which the step was shown experimentally to occur is indicated but only for those
confirmed steps and not those proposed to occur by the authors without experimental results
similarly to aromatic ring substituents on several substrates. Both vanillin and one of
its precursor (ferulic acid, a cinnamic acid derivative) have comparable aromatic ring
substitution patterns. Additionally, ferulic acid occurs in large amounts either freely
or linked in plant cell walls [56, 57], making it a good candidate towards vanillin
synthesis.
1.2 Vanillin Sources: Plants and Microorganisms
Variation in the microbial species within Vanilla pods depends on the treatment
conditions in the curing process of pods post-harvest. In this way, microbial species
within Indonesian pods differed significantly after being subjected to scalding (part
of the curing process) [58]. The exact temperature and length of time, through which
pods are treated during scalding, vary depending on the region of the world. In
Indonesia, the treatment is to put pods in water at 65–70 °C for a period of 2 min.
With a decrease in the number of microbial species was associated the proliferation
of fungi. Furthermore, Fusarium spp. were also identified within Indonesian pods
[59]. The latter were identified through sequencing of elongation factor genes,
combined with morphological similarities against Fusarium spp. references.
Other fungi are also reported to occur within V. planifolia. In this way, Tulasnella
spp., Ceratobasidium spp., and Thanatephorus spp. are all mycorrhizal fungi, found
in roots, while not causing any plant symptoms [60].
Other research work focused, instead, on actinomycetes and bacterial microor-
ganisms, and the species of these present during the curing process [15, 61]. Some
bacterial endophytes, living inside Vanilla pods, were found to raise vanillin
amounts after curing. Those bacteria are Bacillus subtilis and Bacillus vanillea
[62]. It may also be possible that other Bacillus species would have similar
properties.
The world demand towards natural products at the expense of synthetic ones has
pushed for a higher consumption of natural vanillin [57]. To satisfy this increase in
demand, a production of natural vanillin by making use of microorganisms has
gained favor. As early as the 1940s through the 1990s, some research work was
already conducted, focused on producing vanillin, by fungal microorganisms
[63]. However, this type of research was not seen as important over the years and
is thus lacking. Instead, work concerned with biotransformation of cinnamic acid
and ferulic acid was promoted. For this reason, more work is now warranted in the
usage of fungi towards vanillin synthesis. In fact, fungal species are known to
synthesize vanillin through ferulic acid as precursor [47, 57, 63] (Fig. 2).
What is also lacking is that no work is reported on Vanilla pod fungal endophytes
in connection to synthesis of Vanilla flavor metabolites, whether through biotrans-
formation reaction of precursors or through de novo synthesis. By knowing which
fungal species occur within pods that can intervene in the elaboration of Vanilla
flavors, it may be possible to gain greater control on fine-tuning towards the best
Vanilla flavor in pods, while these are still growing on the plant. This can be done by
encouraging the growth, on the plant, of those specific fungal species.
Fig. 2 Scheme of potential pathways to vanillin in relation to confirmed pathways in fungi (Bold,
Dark lines). Fungus F1: Pycnoporuscinnabarinus, F2: Trametes sp., F3: Aspergillusniger, F4:
Botrytis sp., F5: Cephalosporium sp., F6: Penicillium sp., F7: Trichoderma sp., F8: Verticillium sp.,
F9: Schizophyllum commune, F10: Paecilomycesvariotii, F11: Fusariumsolani, F12: Sporotrichum
thermophile, F13: Debaromyceshansenii. Fungal reactions adapted from [63]. R ¼ H
1.3 Volatiles Related to Vanilla Flavor
Despite the importance of vanillin to Vanilla flavor, the desirability of pods does not
depend only on vanillin amount within the pod [64]. The problem is that vanillin has
been given too much importance at the detriment of other nonvanillin Vanilla flavor
metabolites. A contributing factor to this is that vanillin is the first metabolite related
to Vanilla flavor which was isolated (Table 1). Aroma detected by the nose is also an
essential component that adds value to pods. For this reason, and since research is
lacking in this direction, more attention should be dedicated to volatiles related to
flavor in the pods. A technique to detect odor-active volatiles is through the use of
GC-olfactory analysis. There are, however, complications given it is not easy to
relate single metabolites from complex combinations to specific flavor components.
Generally, nonvanillin Vanilla flavor metabolites occur in amounts much lower
than vanillin, within the pods. And yet, the former can be appreciated by the senses
as a flavor component, at the same title as vanillin. As such, detection of amounts
through analytical methods has never been an issue with vanillin but can be difficult
with other Vanilla flavor metabolites. As a comparison, the Vanilla flavor metabolite
guaiacol typically occurs 20 times lower in amounts than vanillin [65]. Still, sensory
organs can detect guaiacol at 50 times a lower threshold than that of vanillin
(detection threshold guaiacolis 13 ppb, vanillin is 680 ppb [66]).
Despite research generally lacking on this theme, some research work has been
previously conducted on finding volatile metabolites present in Vanilla pods. In this
way, a number of pod-derived volatiles are known [20, 21, 62]. Of interest, volatiles
detected in pods can be grouped into phenols, sesquiterpenes, monoterpenes, lac-
tones, arenes, and ethers.
Within the group of volatiles from pods, a further step was done in some work,
identifying those that can be detected by olfactory senses [20]. Within the context of
Vanilla flavor, this is still not enough, given not all volatiles from pods detected by
olfactory senses are relevant to Vanilla flavor. For this reason, it is also essential to do
research work, to find which volatiles from pods are related to Vanilla flavor.
Sesquiterpenes volatiles, for instance, found in pods may contribute to woody
notes, which also define Vanilla flavor [67]. References such as [22] are available
that describe organoleptic tones produced by specific metabolites. These references
are then an important first step for this type of work of associating volatile metab-
olites in pods to Vanilla flavors.
Some fungi are reported in literature to produce terpenoids with flavor-related
properties [63, 68]. The same could be the case with pod-derived fungal endophytes,
producing volatiles with Vanilla flavor–related properties. As yet, literature is absent
on potential participation of endophytes, in volatile Vanilla flavor synthesis. This,
then, represents a research avenue of interest.
1.4 Endophyte Species Occurrences Depending on Geographical

Location
Depending on the cultivation site of a plant, fungal endophyte species tend to differ
for the same plant species [69]. In conjecture, a similar occurrence is possible for
fungal endophytes in Vanilla pods, depending on the cultivation sites across Reunion
Island. Due to this diversity, it is essential to obtain samples which reflect the general
fungal endophyte community, in Vanilla and across Reunion Island. Thus, the step
forward is to sample plants from different cultivation regions in Reunion Island, in
view of isolating and identifying endophytes from these (Fig. 3).
Fig. 3 East and southeastern regions in Reunion Island where Vanilla cultivation is performed
This difference in Vanilla flavor across cultivation sites is not limited to Reunion
Island. As such, for the same plant species (Vanilla planifolia), flavor also varies
across different cultivation zones in the world [13] (Table 3). This type of difference
in flavor is known as the terroir effect, which is also popular in grapes used in wine
making. Diversity in microorganisms in grapes has been observed, based on the
culture region of the plant [70]. Additionally, it was also shown for those grapes that
the metabolome differs due to microorganism species present within the plant. This
then contributes to the terroir effect of the final wine product. Additionally, from
their results, the authors speculate wine terroir of a location may be obtained across
any other locations, by inoculating the same fungal species from that location onto
any grape plants. That fungal endophytes may contribute to Vanilla flavor, while the
fungal species may vary across regions may at least partially explain the observed
terroir effect. This then forms a plausible hypothesis to investigate.
Table 3 Region-specific flavors depending on cultivation region

Common
Cultivation region name Species Region-specific flavor properties
Indian ocean islands Bourbon Vanilla Deeply balsamic notes
including Reunion planifolia
Island
India Indian Vanilla Lacks balsamic tones, creamy, sweetness but
planifolia more body compared to Bourbon Vanilla.
Mildly pungent sour flavor
Mexico Mexican Vanilla Lesser body than Bourbon Vanilla
planifolia
Indonesian Islands Indonesian Vanilla Lacks creamy and sweet notes compared to
planifolia Bourbon Vanilla. Pencil tones, intense
woody, and mildly smoky
Adapted from: [13]
Even if all cultivated Vanilla planifolia plants are genetically similar clones in
Reunion Island, pod flavor differences have still been observed depending on
cultivation zones, in the same Island. Additionally, the same curing process is used
for all pods (Bertrand Côme – La Vanilleraie, pers. comm.) (Fig. 3). As such,
observed differences in flavor notes cannot be attributed to plant genetic factors,
nor to pod post-harvest processing methods. This opens the possibility to other
factors that need to be elucidated and that contribute to differences in pod flavor
properties. One such possibility may be fungal endophyte diversity within Vanilla
planifolia, depending on culture regions for the plant.
Vanilla flavor does not occur in all organs of Vanilla plants. For instance, this
flavor occurs in the pods but not the leaves. For this reason, it may be informative to
find fungal endophytes species occurring within the pods, and to compare against
those occurring in the leaves. It is only through such a comparison that fungal
endophyte species specific to the pods only, but absent from the leaves, can be
found. These endophytes then are likely to be associated more to flavor metabolite
synthesis. By performing such an experiment, but including a further parameter, i.e.,
for plants coming from different cultivation regions in Reunion Island, it may be
possible to make a correlation between fungal species and the terroir effect. Also of
interest is to find the capacity of fungal endophyte species to synthesize Vanilla
flavor metabolites and the associated precursors.
1.5 Plant and Endophyte Cohabitation
Interest in endophytes has been growing over time [71]. What was found is that
every plant species harbors endophytes [72]. The number of endophytic species
varies depending on the plant host. On some hosts, only one species was found, and
in other cases, hundreds of species were isolated. While the endophytes reside within
the plant host, a physiological, metabolic interaction is formed between both types of
organisms. This interaction differs to that, when a pathogen infects a plant, where the
plant mounts a defense reaction. The defense reaction translates in the synthesis of
phytoalexins, whereas for endophytes, the plant does not mount a defense reaction.
Rather, a constitutive synthesis of metabolites is obtained. Another possible reaction
within this interaction is the endophyte that biotransforms plant-derived precursors.
In fact, the resulting secondary metabolites from endophyte interaction with the
plant can protect the plant against pathogens, through constitutive defense. Overall,
the metabolic implication of a plant–endophyte interaction is quite complex, given
there are many endophyte species, each with its own interaction, within the plant
[73]. This complexity might add to terroir effects, within the context of Vanilla
flavor.
2 Conclusions
The review here elaborates on vanillin and Vanilla flavor synthesis, as well as points
to the research direction taken, so far, on the same themes. A discussion of the
research direction to follow in the future themes on those flavors where literature as
research work is lacking is also mentioned. The vanillin biosynthetic network occurs
in plants, especially Vanilla spp., and in different microorganisms. That some
microorganisms participate in vanillin synthesis has led to their usage for this
synthesis. By introducing selected genes in some microorganisms, it has even
been possible for those to synthesize vanillin de novo, when this was not possible
before. It is now well documented that endophytes contribute to the synthesis of
economically valuable secondary metabolites [73–75]. It may be possible that
microorganisms present inside Vanilla plants assist in Vanilla flavor synthesis,
during pod maturing or during the curing process. Fungal endophyte variability,
across plant culture regions, may also contribute to the observed terroir effect in
Vanilla pods. Given variability in endophyte species within a plant, this variability
may, at least partially, help to understand the conflicting studies about vanillin
synthesis, within Vanilla pods. More studies on bacterial species within Vanilla
plants are warranted.
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Part V
Phytochemistry and Medicinal Properties
Ethnobotany and Recent Advances in
Indian Medicinal Orchids 15
Ram Pal, N. K. Meena, M. Dayamma, and D. R. Singh
Contents
1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 362
2 Orchids in Indian System of Medicine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 363
2.1 Ayurveda . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 363
2.2 Siddha . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 365
2.3 Unani . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 365
2.4 Tribal Medicine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 366
3 Orchids in Indian Medicine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 366
3.1 Acampe Lindley . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 366
3.2 Acanthephippium Lindl. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 367
3.3 Aerides Lour. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 368
3.4 Agrostophyllum Blume . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 368
3.5 Arundina Blume . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 369
3.6 Bulbophyllum Thouars . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 369
3.7 Coelogyne Lindl. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 371
3.8 Cymbidium Swartz. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 372
3.9 Dactylorhiza Necker ex Nevski . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 373
3.10 Dendrobium Sw. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 374
3.11 Eulophia R. Br. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 375
3.12 Habenaria R. Br. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 377
3.13 Malaxis Sol ex. Sw. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 379
3.14 Pholidota Lindl. ex Hook. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 380
3.15 Rhynchostylis Blume . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 381
3.16 Vanda Jones ex R Br. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 381
4 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 382
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 382
R. Pal (*) · M. Dayamma

ICAR-National Research Centre for Orchids, Darjeeling Campus, Darjeeling, West Bengal, India
e-mail: rampal_nrco@yahoo.com
N. K. Meena
ICAR-National Research Center on Seed and Spices, Tabiji, Ajmer, India
D. R. Singh
ICAR-National Research Centre for Orchids, Pakyong, East Sikkim, Sikkim, India

362 R. Pal et al.
Abstract
India is very rich in orchid genetic resource. It is estimated that about 1300
species occur within the political boundaries of India, of which nearly 150 species
are used in Ayurvedic, Siddha, Unani, and tribal system of medicine. The country
has been paying attention to ornamental orchids for the use in floriculture
industry, but the orchid therapeutics did not catch such attention which also has
similar potential to contribute in economy and health of the people. The present
chapter takes a look on uses of orchids in traditional medicine system as well
as progress made for their utilization in healthcare system.
Keywords
Orchids · Ayurveda · Medicinal · Phytochemicals
1 Introduction
Traditional medicines have a long history of catering the healthcare needs of people
all over the world. In India, traditional medicine system includes Ayurveda, Siddha,
Unani and Homeopathy, Yoga, and Naturopathy. Of these, the first four are heavily
dependent on plant-base formulations for curing various kinds of illnesses. The two
famous treatises on Ayurveda, Charaka Samhita and Sushruta Samhita, were com-
posed during 600 B.C., the former listed 341 types of plants and plant products
and the latter described 1120 types of illnesses, 700 medicinal plants, and 121
preparations. India possesses 47,000 species of plants distributed in 15 agroclimatic
zones: 15,000 of them have medicinal value. Of these, 7000 used in Ayurveda, 700
in Unani, 600 in Siddha, and 30 in modern medicine [1]. Ashtavarga is a group of
eight plant species and have prodigious role in Ayurvedic system of medicine.
In Sanskrit they are called as Kakoli, Kshirakakoli, Meda, Mahameda, Jeevaka,
Rishbhaka, Riddhi, and Vriddhi [2]. The plants in Ashtavarga are called Jeevaniya
(strengthens vitality and immunity), Brhnayiya [activates cell regeneration system],
and Vayasthapan (activates metabolic and anabolic process leading to youthfulness)
[3]. The scientific identity of Ashtavarga plants has been established Kakoli as
Roscoea procera Wall. (Roscoea purpurea Sm), Kshirakakoli as Lilium polyphyllum
D.Don, Meda as Polygonatum verticillatum (L.) All., Mahameda as Polygonatum
cirrhifolium (Wall.) Royle Jeevak as Malaxis acuminate D. Don. (Crepidium
acuminatum), Rishbhaka as Microstylis muscifera Ridl. (Malaxis muscifera), Riddhi
as Habenaria edgeworthii Hook f. ex Colt (Platanthera edgeworthii), and Vriddhi as
Habenaria intermedia (H. arietina). The last four are terrestrial orchids found in the
Himalayas. The other orchids mentioned in Ayurvedic literature are Jivanti,
Flickingeria macraei (Dendrobium alpestre), shwethuli, and rasna (Acampe
papillosa and Vanda tessellata). Additionally, the tribal medicine system is practiced
by 645 tribes who live in isolation and practice their system of medicine. The tribal
have passed down their knowledge and practices of curing illnesses generation after
15 Ethnobotany and Recent Advances in Indian Medicinal Orchids 363
generation by word of mouth. With other plants, tribal also use several species
of orchids in their healthcare system.
Ethnobotanical studies indicate about 150 species of orchids are used in tradi-
tional medicinal system in India. Despite their known potential value in medicine
and perennial use, a very few species have been subjected to phytochemical analysis
and clinical evaluation. Orchids produce a large number of phytochemical
like alkaloids, flavonoids, carotenoids, anthocyanins, and sterols. Aeridin was
isolated from Aerides crispum Lindl: Agrostophyllin, Agrostophyllinone, and
Agrostophyllinol from Agrostophyllum brevipes King & Pantl.; Arundinan from
Arundina graminifolia (D. Don) Hochr; Bulbophythrin A and Bulbophythrin B from
Bulbophyllum odoratissimum (Sm.); and Coeloginanthrin and Combretastatin C-1
from Coelogyne cristata Lindl. The study of phytochemical constituents is essential
to validate their usage against a specific disease. The clinically tested biomolecules
would have a usage in the medical science. The integration of traditional medicines
with western medicine would not only bring down the cost of medication but also
increase the safety from side effects of western medicines. The People’s Republic of
China has successfully incorporated traditional herbal medicine into a modern
healthcare system. The unique blend herbal medicine, acupuncture, and western
medicine cater the healthcare needs of its people [4].
2 Orchids in Indian System of Medicine
India has six, Ayurveda, Siddha, Unani, Yoga, Naturopathy, and Homeopathy, well-
acknowledged systems of medicine. Ayurveda, Siddha, Yoga, and Naturopathy
originated in India. Unani system of medicine has its roots in Greece and introduced
by Arabs in India. It spread, enriched, and assimilated with Indian culture during the
Mughal period. Homeopathy was introduced in India during the eighteenth century
and became part of the Indian medicine system. Thus, the systems of medicine
that originated in India and the systems of medicine that came from outside into the
country got assimilated with Indian culture are called Indian System of Medicine [5].
Though not officially recognized, there are tribal or folk medicines in indigenous
healthcare which plays a vital role in meeting the healthcare needs of tribal in India.
2.1 Ayurveda
Ayurveda is one of the most acknowledged holistic healthcare systems based on

varying medicinal uses of plants to prevent and cure a variety of illnesses. Charaka
Samhita and Sushruta Samhita, the two compilations are dating back to 600 BC, the
primary source of knowledge in healthcare in ancient India. The Charaka Samhita
consists of 8 sections and 120 chapters dealing with various aspects of medicine and
associated subjects. Around 600 drugs of plant, animal, and mineral origin are
mentioned in this treatise [6]. Sushruta Samhita, a treatise on surgery, describes
364 R. Pal et al.
surgical instruments, operative procedures, and presents a description of 650 drugs.

The medical books were written during medieval India (seventh to the eleventh
century) mention Ashtavarga which consists of eight plant species with their
Sanskrit names as Kakoli, Kshirakakoli, Jeevaka, Rishbhaka, Meda, Mahameda,
Riddhi, and Vriddhi [2]. The plants in Ashtavarga are called as Jeevaniya
(strengthens vitality and immunity), Brhnayiya (activates cell regeneration system),
and Vayasthapan (activates metabolic and anabolic process leading to youthfulness)
[3]. There had been a problem of correct identification of plants mentioned in
Ashtavarga. However, the researches carried out after the independence of the
country, the majority of Indian scientists agree with the plants mentioned in the
Ashtavarga. The Jeevak is Malaxis acuminate D. Don. (Crepidium acuminatum),
Rishbhaka is Microstylis muscifera Ridl. (Malaxis muscifera), Riddhi is Habenaria
edgeworthii Hook f. ex Colt (Platanthera edgeworthii), and Vriddhi is Habenaria
intermedia (H. arietina), respectively. The plants mentioned in Ashtavarga occur in
the Himalayas between 1200 and 4000 m. In Himachal Pradesh, an Indian state in
Western Himalayas, all the four orchids occur between 1800 and 2800 m altitude and
flower during monsoon [7].
Chyavanprash is formulation made up of about 50 herbs, sugar, and honey and
very popular in the northern part of India. It maintains physique, vigor, and vitality
and also delays the process of aging. Approximately 15,000 tons of Chyavanprash
produced annually in India. Adults and children consume it as a remedy for cold,
cough, and respiratory infections. Ashtavarga plants were indispensable constituents
of Chyavanprash and collectively increased the antioxidant activity of Amla
(Phyllanthus emblica), one of the principal constituent of the Chyavanprash. The
unregulated use of these plants might have led to decline, and replacement of these
herbs was proposed in Ayurvedic text Bhavprakash (sixteenth century),
Yogaratnakara (seventeenth century), and Vaidyachintamani (eighteenth century).
The Ayurvedic Pharmacopoeia of India published by Ministry of AYUSH, Govern-
ment of India, permits replacement of Malaxis acuminata (Crepidium acuminatat)
and Malaxis muscifera with Pueraria tuberosa and Habenaria edgeworthii
(Platanthera edgeworthii) and Habenaria intermedia (Habenaria arietina) with
Dioscorea bulbifera. Malaxis acuminata used for the preparation of Chyavanprash
and contains Beta-sitosterol, piperitone, citronellal, eugenol, Limonene, 1,8-cineole,
p-cymene, O-Methylbatatasin, and cetyl alcohol [8]. The ethyl acetate extract of
Chyavanprash exhibited a higher level of scavenging activity, i.e., close to ascorbic
acid (IC50 20.693 μg/ml) and thus inhibits formation free radicals in the body [9].
The other orchids used in Ayurvedic system of medicine include Munjataka
(Dactylorhiza hatagirea), Jeevanti (Flickingeria macraei) (Dendrobium macraei),
and Rasna (Vanda tessellata). In India, medicinal orchids are under threat due to
various human-made and natural calamities. All four orchids of Ashtavarga group
have been placed under the Red Data Book of Indian Plants. The causes of the
rarity of medicinal orchids vary with the region. In Himachal Pradesh, Ashtavarga
orchids are threatened due construction of houses and hydroelectric projects,
grazing, and collection fodder from natural habitats [7]. In Niyamgiri hills of
Odisha, they are facing endanger because of mining operations [10]. Shifting
cultivation, upcoming industries, use of forests for human settlement, and felling
of trees are a major threat for the survival of the orchids in other parts of Odisha [11].
Acampe praemorsa, Geodorum densiflorum, and Habenaria marginata are endan-
gered in Harda district of Madhya Pradesh [12]. Unregulated collection of orchids
for various purposes including medicines is endangering orchids in their natural
habitats. Therefore, it is necessary to ease the pressure on natural habitats by
cultivating orchids propagated through tissue culture rather than collecting from
natural habitats.
2.2 Siddha
Siddha system of medicine is one of the ancient traditional healthcare system

practiced mostly in Tamil-speaking areas of India. Ayurveda system of medicine is
relatively more popular than the Siddha system of medicine. Unlike Ayurveda, in
Siddha system of medicine, orchids do not find much favor for medicines. However,
tribal living in Tamil Nadu uses orchids to cure the illnesses.
2.3 Unani
Unani system of medicine originated in Greece and introduced in India by Arabs

and Persians in the eleventh century. It flourished under the patronage Mughal
rulers and spread all over the country. The Unani system medicine suffered a
setback during British rule in India, but the efforts of Nizam of Hyderabad, Azizi
family of Lucknow, and Sharifi family of Delhi brought it to life in a short time
[13]. There are several institutions to undertake research and education in the
country: the Central Council for Research in Unani Medicine (CCRUM) to pro-
mote research; the National Institute of Unani Medicine (NIUM) for postgraduate,
teaching, training, and research; and the Central Research Institute on Unani
Medicine for research and education in Unani medicine. In Turkey, the salep is
prepared by grinding of tuberous orchids to a fine powder to use as food and drug.
It is used as a thickening agent in Turkish ice cream and also mixed with milk to
make the hot drink known as salep served sprinkled with cinnamon [14]. In Unani
system of medicine Salam Panja (palm-like), Salam lahsunia (garlic type), Salam
Mishri (translucent and globular), and Salam Badshah (like a king) are common.
These names were given to the appearance of tubers used in the preparation of
salep. In India, several species of orchids such as Dactylorhiza hatagirea,
Eulophia nuda, Habenaria commelinifolia, and Satyrium nepalense are used in
preparation of salep.
366 R. Pal et al.
2.4 Tribal Medicine
The tribal system of medicine in India developed based on the experiences and
experimentation of tribal people living in isolation in the want of healthcare facili-
ties. This tradition of knowledge passed down from one generation to other. Tribal
in India use many plant species, including orchids to get rid of many diseases.
Dongaria Kondha tribes of Niyamgiri hills in southwest Odisha, India, use 16
species of orchids to treat 33 kinds of diseases [10]; in another study from Odisha,
it was found that Bonda, Dongaria Kondha, Juang, and Saora tribes use 26 species
orchids to cure various kinds of body ailments [11]. Tribals in the neighboring state,
Andhra Pradesh, in the Eastern Ghats use 23 species of orchids to cure various kinds
of diseases. Pragada et al. [15] surveyed 53 families of the tribals in Andhra Pradesh
and reported that Geodorum densiflorum is used to treat ephemeral fever.
Hill-Korwa tribe of Chhattisgarh uses 30 medicinal herbs belonging to 18 families.
Among them, there are two orchids, Saccolabium papillosum (Acampe praemorsa)
used for bone fracture and body ache and Bulbophyllum leopardinum used against
sunstroke and diabetes [16]. Northeastern Himalayan state, Nagaland, is rich in
orchid genetic resources; 396 species belonging to 92 genera inhabit in this state.
There are 15 species of orchids used by local practitioners to treat various diseases
like rheumatism, cholera, nervous disorder, and tuberculosis that are also used as
antimicrobial agent and antidotes to snake and insect bites [17]. According a survey
of 198 respondents consisting of 57 females and 141 males in Arunachal Pradesh,
101 plant species belonging to 50 families are used in ethnomedicine [18]. It is quite
surprising that a state with the highest species of orchids in India did not document
orchids species in part of their ethnobotanic medicines, whereas in neighboring state,
Nagaland, 15 species of orchids have been recorded for medicinal uses. In the
Kashmir Himalayas, 7 species of orchid are used to cure 24 types of different
diseases [19]. Not all the orchids with medicinal value have been documented in
the country, and those need to be documented earliest. Most of the studies do not
focus on the complete preparation and use of herbal medicines. For realizing the
benefits of herbal-based medicines used in tribal or folk medicine, a complete
formulation and its use should be documented.
3 Orchids in Indian Medicine
3.1 Acampe Lindley
Epiphytic or lithophytic, monopodial perennial herb, stem erect or branched, leaves

fleshy thick, leathery, bilobulate at apex. Inflorescence axillary, erect sometimes
branched, small-medium sized, non-resupinating flowers. The genus is consisting of
about 12 species distributed in India, Southwest China, Malaysia, and Africa. In
India, this genus is represented by six species, and two of them, viz., A. praemorsa
and A. carinata, are of medicinal interest. A. praemorsa is the most widely and
intensively used species of this genus.
3.1.1 Acampe carinata (Griff.) Panigrahi

Found in India, Nepal, Bhutan, Thailand, Cambodia, and Vietnam. Monopodial
epiphyte, plants robust; thick coriaceous leaves; inflorescence, umbel with 5–12
small flowers.
Roots [10] and leaf paste [20] are used to alleviate abdominal pain. Root paste of
A. carnata is applied locally on scorpion sting and snakebite [10].
3.1.2 Acampe praemorsa (Roxb.) Blatt & McCann

Found in northeastern states of India, Nepal, Sri Lanka, and Myanmar at an elevation
ranging from 600 to 1600 m. Plants are robust monopodial epiphyte; leaves thick,
fleshy, leathery oblong, notched at tip; inflorescence umbel; flowers small, fragrant.
Ethnobotanical surveys conducted in the country showed that roots, leaves, whole
plant, seed capsule rind, and seeds are used for curing various body ailments. The
paste made from the roots of A. praemorsa and Asparagus racemosus is taken orally
on an empty stomach for 15 days to alleviate arthritis [20]. The decoction made from
the fresh roots consumed orally (2 tsp) along with honey (5 tsp) taken twice a day for
5 days for curing cough [21]. Hossain [20] recorded that roots are used for treating
asthma and have expectorant properties, and the root paste is also applied externally
on scorpion and snakebite. Shanavaskhan et al. [22] recorded that tribes of Kerala
use whole plant to treat rheumatism. The root paste is applied to fractured organ of
the cattle [23]. Leaf paste along with a piece of garlic is taken daily for 7 days to get
relief from chest pain and stomach disorder caused by hyper acidity [10]. The juice
from leaves is applied over nipple for relief from abdominal pain, and dispensing
two to three drops in the ear relieves earache [22]. The fractured bones can also be
treated by applying leaf paste [24, 25]. The fibers from the capsule are used to treat
the wounds. The capsules fibers are placed on the wound and tied with a piece of
cloth. The old fibers and cloth are replaced with new one every day till wound is
healed. The seeds are directly applied on the old wound as an antibiotic. The plant is
used as tonic [26] and in rheumatism [26]. The leaves contain flavonoids and
cyanogenic glycosides [27]. Anuradha and Rao [28] isolated a new phenanthropyran
derivative [1,7-dihydroxy-3-methoxy-9,10-dihydrophenanthropyran] from the
whole plant.
3.2 Acanthephippium Lindl.
Terrestrial, medium- to small-sized herbs; pseudobulb ovoid, ovoid cylindric, cylin-

dric pseudobulb with a few nodes and internodes and usually clasped with mem-
branous sheath at base. The genus consists of about 12 species and is distributed in
tropical Asia, Malaysia, to Fiji Islands. Three species inhabited in India, only one
used in medicine.
3.2.1 Acanthephippium bicolor Lindl.

Hot- to warm-growing, small-sized, terrestrial herb found in southern India and
Sri Lanka at an elevation of 650 m. Pseudobulbs clustered, large, smooth, ovate,
368 R. Pal et al.
deeply sulcate tapering above into thin stem; flowers during winter on a short,
erect 3.0–4 cm long, up to six-flowered inflorescence. Flowers are pale purple
with 9–11 veins.
Tribes in Kolli Hills in Namakkal district of Tamil Nadu (Eastern Ghats), use leaf
extract A. bicolor for treating urinary tract infections mainly caused by Escherichia
coli, Staphylococcus saprophyticus, Klebsiella, Enterococci bacteria, and Proteus
mirabilis. Kala and Senthilkumar [29] tested antimicrobial activity of leaf extract of
A. bicolor against 35 microbial agents and found Gram-negative bacterium, like
Staphylococcus aureus, Streptococcus faecalis, and Bacillus cereus, among Gram-
positive bacteria. Proteus vulgaris, Proteus mirabilis, Enterobacter aerogenes,
Shigella dysenteriae, Klebsiella pneumoniae, and Escherichia coli and pathogenic
fungi, viz., Microsporum audouinii, Microsporum fulvum, Candida albicans, and
Trichophyton rubrum, are sensitive to leaf extract. This might be the reason for using
this plant in treating the urinary tract infection. Further study may lead to develop-
ment of drug suitable for combating urinary tract infections.
3.3 Aerides Lour.
Epiphytic medium-sized monopodial epiphytic herbs with many thick roots; stem
with many nodes, short and enclosed by leaf sheaths. Leaves are distichous leathery,
fleshy, and apex bilobed. Inflorescence arises from the axils of leaves densely
flowered. The genus consists of about 20 species distributed from Sri Lanka,
India, Nepal, Bhutan, Myanmar, China, Thailand, Indochina, and Malaysia to the
Philippines and Indonesia. Eight species under this genus have been reported from
India. Three species, namely, A. crispum, A. emericii, and A. maculosa are endemic.
A. rosea, A. odorata, A. multiflora, and A. crispum, have been grouped as medicinal
orchids. The paste made from the leaves of A. multiflorum is applied as poultice on
cuts and wound [30]. The root infusion of A. maculosa is given once a day for
1–2 months to a patient suffering from tuberculosis. Anuradha and Prakash [31]
isolated Aeridin (2,7-dihydroxy-1,3-dimethoxy-9,10-dihydrophenanthropyran),
a derivative of phenanthropyran from whole plant part of A. crispum. Powder of
A. crispum is boiled in neem oil and filtered, and 2–3 drops is poured into ears once
during night for 3 days to cure pain and ear deafness [32]. The premedical studies
conducted by Ghanakash and Kaushik [33] have shown that leaf extract of A.
multiflora has antibacterial property.
3.4 Agrostophyllum Blume
3.4.1 Agrostophyllum callosum Rchb. f.

Epiphytic herbs, pseudostems are clustered bilaterally flattened with many
internodes; leaves are twisted and almost lie in the same plane; inflorescence
terminal, globular, densely flowered; flowers usually white, resupinating, often
self-pollinating. Not much known about ethnobotanical use of this species. Two
stilbenoids (agrostophyllol and isoagrostophyllol) and two diastereomeric 9,10-

dihydrophenanthropyran derivatives from A. callosum Rchb. f. [34–37] and two
terpenoids (agrostophyllinol and agrostophylline) from A. brevipes Ridley and A.
callosum Rchb. f. [38].
3.5 Arundina Blume
3.5.1 Arundina graminifolia (D. Don) Hochr.

A. graminifolia is commonly known as a bamboo orchid. It is herbaceous terrestrial
plant found in India, Nepal, Thailand, Malaysia, Singapore, South China, through
the Pacific Islands. Terrestrial, thin and tall stem, leaves alternating acuminate,
distributed from sea level to 1200 m, flowers similar to Cattleya and short-lived
usually 5–6 days.
Flower stalk paste and rhizome are used for treating ear pain and rheumat-
ism, whereas the roots are used in snakebite and intestinal biliary colic in Bangladesh
[39].
It is used for healing the cracks on the skin [40]. A. graminifolia contains numerous
stilbenoids, namely, arundin and its analogues: triterpenoid, Arundinol, and hydroxy-
benzaldehyde [41–43]. The plant contains tannins, saponin, and heptacosane [44].
Benzyldihydrophenanthrene, arundinaol (7-hydroxy-1-( p-hydroxybenzyl)-
2,4-dimethoxy-9,10-dihydrophenanthrene), and five phenanthrene constituents
(7-hydroxy-2,4-dimethoxy-9,10-dihydrophenanthrene(1),4,7-dihydroxy-2-methoxy-
9,10-dihydrophenanthrene (2),2,7-dihydroxy-4-methoxy-9,10-dihydrophenanthrene
(3),7-hydroxy-2-methoxyphenanthrene-1,4-dione (4),7-hydroxy-2-methoxy-9,10-
dihydrophenanthrene-1,4-dione (5) have also been isolated from the rhizomes of
this plant [45–47]. It is believed, this genus is effective for detoxification, is
antiarthritis, and used as an antidote and demulcent. Liu et al. [48] isolated six
bibenzyl derivatives, namely, 2,7-dihydroxy-1-( p-hydroxybenzyl)-4-methoxy-9,10-
dihydrophenanthrene (1); 4,7-dihydroxy-1- ( p-hydroxybenzyl)-2-methoxy-9,10-
dihydrophenanthrene (2); 3, 30 -dihydroxy-5-methoxybibenzyl (3); (2E)-2-propenoic
acid-3-(4-hydroxy-3-methoxyphenyl)-tetracosyl ester (4); (2E)-2-propenoic acid-3-
(4-hydroxy-3-methoxyphenyl)-pentacosyl ester (5); and pentadecyl acid (6), from
the tuber of this species and evaluated for antitumor activity. The compound 3(3,30 -
dihydroxy-5-methoxybibenzyl) has exhibited stronger antitumor activity than other
compounds.
3.6 Bulbophyllum Thouars
Largest genus, plants epiphytic or lithophytic, small to large in size; one- to two-
leaved, arising from apex of pseudobulb. Inflorescence arises from base of pseudo-
bulb or from rhizome, flowers small to large, flowers with the foot of the column is
hinged attached to the column, petals shorter than the dorsal sepal.
370 R. Pal et al.
3.6.1 Bulbophyllum acutiflorum A.Rich. (Syn: Bulbophyllum albidum

(Wight) Hook.f.)
Found in the Eastern Himalayas at an elevation ranging from 900 to 1800 m, warm-
to cool-growing epiphyte, pseudobulbs globose-ovoid; leaves lanceolate, bilobed at
apex; inflorescence, umbel, six- to eight-flowered; flowers during spring to winter.
Kanikkar tribe of Agasthyamalai Hills uses leaves and pseudobulbs of B. albidum
(B. acutiflorum) for strengthening weak uterus for conception. A preliminary study
conducted to analyze the phytochemicals has shown that it contains saponins,
steroids, and flavonoids along with other phytochemicals [49].
3.6.2 Bulbophyllum cariniflorum Rchb.

Found in the Northeastern Himalayas and eastern Southeast Asia at an elevation
ranging from 1100 to 2100 m, cool-growing epiphyte; pseudobulbs five-angled,
deeply grooved; leaves 2–3 elliptic pointed at apex; inflorescence 10- to 20-flow-
ered, flowers with a fetid smell.
Paste made by mixing 2 g of dried root, 1 g of black pepper, and 5 ml of cow milk
is taken half spoon orally with a cup of water by women for 5–10 days to induce
abortion during first trimester of pregnancy in the districts of Mondanala and
Sutanguni in the Niyamgiri Hill Ranges of Odisha, India [10].
3.6.3 Bulbophyllum fusco-purpureum Wight

Found in southern Western Ghats of India; pseudobulbs ovoid taper toward apex,
one-leaved; leaves oblong, obtuse; inflorescence semi-erect, four- to five-flowered;
flowers yellowish pink; flowering April–May.
Tribals living in Reserve Forest of Nilgiri use paste of pseudobulb to cure skin
diseases. The pseudobulbs are washed and made into paste. The paste is applied on
affected parts [50].
3.6.4 Bulbophyllum kaitiense Rchb.f.

B. kaitiense is an epiphytic, endemic orchid found in South India at an elevation
ranging from 2000 to 2800 m in Kolli hills of Eastern Ghats, India. It grows trees or
rocks, forms a dense mat-like structure; leaves 9–13 cm long, inflorescence umbel,
five- to six-flowered, mentum in flowers absent, flowering winters to late spring.
B. kaitiense is used to cure several diseases and likely to have anticancer,
antioxidant, anti-inflammatory, and antimicrobial activity [51]. Terpenoids, flavo-
noids, saponins, tannins, coumarin, and quinine carbohydrates are present in the
pseudobulbs of B. kaitiense. The ethanolic extract of roots has antibacterial activity
against 12 human pathogenic microorganisms. The ethanolic extract activity was
more pronounced for fungi than bacteria [52]. Ethanolic extract of pseudobulbs also
has anti-inflammatory activity [53].
3.6.5 Bulbophyllum sterile (Lam.) Suresh Syn: Bulbophyllum

nilgherrense Wight
Warm- to cool-growing creeping epiphyte; found in India, Nepal, Bangladesh,
and Myanmar at an elevation ranging from 1300 to 1700 m. Pseudobulbs fleshy,
yellowish green, four-angled; leaves erect, oblong-elliptic, obtuse at apex; inflores-

cence many-flowered; flowering December to January.
The crushed whole plant is used to cure pimples and skin allergy by the tribal
living in Reserve Forests of Nilgiri, Tamil Nadu, India. The whole plant is washed
and crushed. The plant paste is applied externally on affected parts [50]. Pseudo-
bulbs and leaves of B. nilgherrense are grounded into fine paste and mixed with
cow’s milk and administered orally for curing leucoderma [54]. Barthel [55] derived
some active compound from the pseudobulbs of B. nilgherrense (=B. sterile) and
claimed that these are effective in treating various disorders of cardiovascular
system. The invention claimed that it has the only 1/6th of the cost of conventional
treatment and has no side effects. Chopped pseudobulbs are cooked in coconut oil,
and oil is applied to cure rheumatism, and the paste made from whole plant is applied
on swellings [22].
3.7 Coelogyne Lindl.
Genus Coelogyne consists of nearly 200 species distributed in the Northeastern

Himalayas and South India (Western Ghats), Nepal, Bhutan, Upper Myanmar,
China, and Vietnam. Plants are epiphytic or lithophytic. Pseudobulbs ovoid, conical
or cylindrical; two-leaved (rarely one), arise from the apex of pseudobulb, leathery;
inflorescence racemose or panicled; flowers small to large. Thirty-four species occur
in India. The two C. cristata and C. stricta (C. elata) are medicinally important.
3.7.1 Coelogyne cristata Lindl.

Found in northeastern India, West Bengal, Bhutan, and Nepal at an elevation ranging
from 800 to 1800 m. Plants epiphytic, pendulous; pseudobulbs shiny, partially
covered with sheath; leaves linear-lanceolate, pointed at tip; inflorescence one
or two, arising from the base of the pseudobulb, up to 30 cm long; flowers lightly
scented, white with a yellow spot on the lip; flowering March–April.
The fruits resin of C. cristata is used to heal the bone fractures of domestic
animals. Some people also apply its resin externally on the injured portion of the
body for decocting the blood [56]. C. cristata and Pholidota imbricata are the most
frequently used plants in treating bone fractures in Kumaon Hills of Uttarakhand
[57]. Ethanolic extract of C. cristata restored trabecular bone (both in femoral and
tibial bones) without producing uterine estrogenicity changes when fed to ovariec-
tomized mice. Coelogin, a compound isolated from C. cristata, enhanced the
markers of osteoblastic differentiation and activity in vitro in the mice. The findings
supported the claim of folk tradition of Kumaon region for using C. cristata in the
treatment of fractured bones. They proposed that ethanolic extract of C. cristata and
its pure compound coelogin has potential in the management of postmenopausal
osteoporosis [57]. Majumder et al. [58, 59] isolated coelogin and coeloginin and two
novel 9,10-dihydrophenanthrene derivatives, coeloginanthridin and coeloginanthrin,
from the whole plant of C. cristata. The ethanol extracts of leaves and pseudobulbs
of C. cristata exhibit potential antimicrobial properties against Staphylococcus
372 R. Pal et al.
aureus [60]. This bacterium commonly acts as a commensal of the human micro-
biota but can become an assertive pathogen and can cause skin infections including
abscesses, respiratory infections such as sinusitis, and food poisoning. Pramanik [61]
reported pharmacognostic characters along with physicochemical and fluorescence
values for C. cristata pseudobulbs as a diagnostic tool for the standardization of the
medicinal plant product.
3.7.2 Coelogyne stricta (D. Don) Schltr.

Found in the Northeastern Himalayas, Bhutan, and Nepal at an elevation ranging
from 1400 to 1600 m. Plants are epiphyte or lithophytes. Pseudobulbs long, shiny,
compressed, sheathed at base; leaves elliptic oblong, acute leathery; inflorescence
21–40 cm long, erect; flowers yellowish white to pinkish white, yellow to orange
spot on the lip; flowering April–June.
The pseudobulbs are used to treat fever and headache [62]. In tribal and folk
medicine, it is also used to cure bone fractures, fever, and headache [63]. A hot drink
made from pseudobulbs of C. cristata relieves from constipation and also acts an
aphrodisiac [64]. Pseudobulbs are applied externally to promote bone healing of
fractured limbs in northeastern India [65]. The ethyl acetate extract is exhibit
potential antibacterial activity against Pseudomonas aeruginosa and Salmonella
enteric, Enterococcus faecalis and Bacillus subtilis. The MIC (minimum inhibitory
concentration) value was 0.250 mg/ml and The MIC value for Salmonella enterica,
Corynebacterium sp. and Candia albicans was 0.750 mg/ml, 0.500 mg/ml and
0.500 mg/ml, respectively. Ethyl acetate extract of C. stricta leaves inhibits human
cervical cell lines (HeLA) cell growth at concentration 50 μg/ml [66]. Majumdar
et al. [58] isolated 9,10dihydrophenanthropyrone (Coelonin) from C. elata (= C.
stricta).
3.8 Cymbidium Swartz.
The widely distributed genus consists of about 52 species. Plants are epiphytic or
terrestrial. Pseudobulbs short to long covered with leaf bases, older are without
leaves; leaves clasping the pseudobulbs; inflorescence erect, arching, or pendulous
with a few- to many-flowered; flowers showy, spreading small to large. Several
species of this genus are medicinally important, but C. aloifolium is widely used
in India.
3.8.1 Cymbidium aloifolium (L.) Sw.

In India, this species occurs in the Northeastern Himalayas and Southern India at
an elevation ranging from sea level to 1100 m. Hot- to warm-growing; pseudobulbs
small clasped with leaf bases; leaves fleshy, linear-oblong, notched at apex.
Inflorescence one or two arising from the base of pseudobulb, pendulous; flowers
3–4 cm across; flowering April–July.
Leaves, roots, as well as whole plant are used to cure various body ailments.
The paste made from the aerial roots is used to cure fractured bones [20, 54]. Dried
powder C. aloifolium roots (2 g), dried ginger (2 g), and black pepper (1 g) are taken
with a cup of cow’s milk twice a day for 2 months to reduce paralysis [10]. The juice
from the pod used against earache [22, 67]. The leaf juice alone or mixed with salt is
used to treat earache [20], otitis and inflammatory conditions [68], and boils and
fever [69]. The plant is emetic and purgative [54]. The salep made from the
pseudobulbs is used as a tonic and in treating weakness of eyes, chronic illness,
vertigo, burns, and sores [69].
Several phenanthrenes aloifol I and II, coelonin, 6-O-methoxycoelonin, batatasin
III, and gigantol [70], cymbinodin A [71], and cymbinodin B [72] have been isolated
from C. aloifolium. In a test on Swiss albino mice, ethanolic extract of C. aloifolium
at 200 and 400 mg kg1 body weight showed central nervous system (CNS)
depressant effects [69]. Ethanolic leaf extract of C. aloifolium leaves has analgesic
and anti-inflammatory activities in mice [73]. The leaves of C. aloifolium contain
flavonoids, reducing sugars, cyanogenic glycosides, terpenoids, and tannins [27, 74].
The analgesic and anti-inflammatory effect may be due to the presence of tannins
and flavonoids in the leaf extract.
3.9 Dactylorhiza Necker ex Nevski
The genus is consists of about 111 species and has pan-global distribution.
Cold-growing terrestrials characterized by two to three flat tubers; leaves linear-
lanceolate. In India, Dactylorhiza hatagirea Soo is used as medicine.
3.9.1 Dactylorhiza hatagirea Soo

Found in Pakistan, Afghanistan, Nepal, Bhutan, and Tibet at an elevation
of 2500–5000 m. Plants are terrestrial perennials up to a height of 70 cm. Tubers
are five lobbed; leaves broadly lanceolate; dense flowering inflorescence; flowers
usually purple but rarely white; flowers during summer.
D. hatagirea is an important aphrodisiac in Ayurvedic and Unani system of
medicine and used to increase vigor and vitality. The salep made from the tubers
of D. hatagirea is extensively used in local medicine as a nervine tonic for its
astringent and aphrodisiac properties [75, 76]. In Turkey, root powder is also used
to make ice-cream and beverages [77]. The tubers are used to cure diabetes, diarrhea,
dysentery, paralysis, convalescence, impotence, and malnutrition [40] and to cure
kidney complaints and aphrodisiac [78]. In Kashmir, root tuber extract is commonly
used for curing fever, whereas leaf extract is used for dysentery.
Administering root extract of D. hatagirea daily 200 mg kg1 body weight
for 28 days to male rats increased testosterone concentrations from 2.33 ng ml1
to 9 ng ml1. Increased libido was evident by an increase in sexual parameters like
mount, intromission, and ejaculatory frequency as well as latency [79]. The tuber
extract has antibacterial activity against Gram-positive and Gram-negative bacteria,
including Escherichia coli [80]. Izu et al. [81] isolated five new compounds
(Dctylorhin A-E) and two natural compounds named dactyloses A and B. Due to
374 R. Pal et al.
over-exploitation for medicinal purpose and lack of cultivation, the plant has become
endangered, and IUCN has categorized the plant as critically endangered.
3.10 Dendrobium Sw.
The genus Dendrobium is the largest genus of family Orchidaceae consisting about
900 species. The species is distributed throughout Asia, Australia, and Europe.
Nearly 100 species found in India. There are 32–40 species of this genus used
for the preparation of Shinhu, a popular tonic. In India a few species such as
D. cruentum, D. ovatum, D. nobile, D. moschatum, etc. are used to treat minor
ailments; however, Dendrobium nodosum (Flickingeria nodosa) and Dendrobium
plicate (Flickingeria fimbriata) and Dendrobium macraei Lindl. (Flickingeria
macraei) are widely used in Ayurvedic healthcare system in India.
3.10.1 Dendrobium nodosum Dalzell (Syn. Flickingeria nodosa (Dalzell)

Seidenf.)
Plants are creeping epiphytes. Rhizomes branched terminating into pseudobulbs;
pseudobulbs grooved compressed, fusiform; leaves sessile arise at the apex
of pseudobulb, oblong-elliptic; flowers small (about 1 cm), white distributed in
Southeast Asia at an elevation ranging from sea level to 2100 m. The plant is a
source of Jivanti mentioned in Charak Samhita.
In folk medicine of Karnataka, halva is prepared from Flickingeria nodosa
(D. nodosa), and it is consumed as astringent, aphrodisiac, and expectorant. Besides,
it is also useful for curing asthma, bronchitis, throat infections, and dermatological
infections and also acts as a blood purifier [82]. Juice of the pseudobulb and leaves
is administered for asthma. The plant is also used for the treatment of asthma,
bronchitis, consumption, fever, burning sensation, biliousness, and diseases of the
blood [22]. The cold chloroform extract of pseudobulbs shows good antifungal
activity against Trichophyton mentagrophytes, a fungus that causes athlete’s foot,
inflammation of skin, and infection of face, scalp, and body [83].
3.10.2 Dendrobium plicatile Lindl. (Syn. Flickingeria fimbriata (Blume)

A.D. Hawkes; Dendrobium macraei Lindl.)
The species is widely distributed in the Himalayas, China, India, Sri Lanka to
Southeast Asia, and Papua New Guinea at an elevation ranging from 700 to
1700 m. Plants are epiphyte with creeping and branched rhizome. The branched
rhizomes terminating into yellowish green pseudobulbs; leaves oblong-lanceolate,
leathery; inflorescence one- to three-flowered.
The plant cures from disorders of disorders of the bile, blood, and phlegm. It is
generally used in decoctions with other plants having similar properties and some-
times used alone as a stimulant and tonic, the latter to treat debility associated with
seminal loss [2]. In West Bengal, India, it is used in Rasayana therapy and sold as
Jibanti [84]. It is used for treating asthma, bronchitis, sore throat, stomach ache,
biliousness, and fever in Uttar Pradesh [65]. For curing skin allergies, hill tribes of
Odisha consume one spoonful of root paste of Flickingeria macraei (Dendrobium

plicatile) with 1 g of black pepper powder on an empty stomach for 21 days. They
also apply it directly on eczematous lesions [10].
3.11 Eulophia R. Br.
The genus consists of nearly 210 species distributed in the tropical and sub-tropical
region of Africa, Asia, India, Australia, and America. Primarily, eulophias
are terrestrial, but a few species have also adapted to epiphytic and lithophytic
mode of living. About 28 species are found in India. These are distributed in the
Northeastern Himalayas and Peninsular regions of the country. Eulophia species
have gained prominence due to nutraceutical ability and rejuvenating and their
effects, anti-fatigue, anticancer, and aphrodisiac properties. The eulophias are exten-
sively utilized as tribal food and medicine. The eulophias may lead to newer sources
of drugs in modern society.
3.11.1 Eulophia dabia (D.Don) Hochr (Syn. Eulophia campestris Wall.

Ex Stapf.)
Terrestrial herb, hot- to cool-growing small- to medium-sized plants, height
20–40 cm; pseudobulb, cylindric, globose, leafless during flowering; leaves two,
linear-lanceolate, acute; inflorescence erect, emerges, raceme; flowers 2.0–2.5 cm,
pink; found in India, Bangladesh, Sri Lanka and Western Himalayas; flowering
April–May.
Sudhanshu et al. [85] screened Eulophia camprestis (Eulophia dabia) for the
presence phytochemicals, viz., alkaloids, flavonoids, tannins, saponins, glycosides,
etc., and noticed that phytochemicals have considerable antioxidant activity against
radical scavenging assay and could be used in malaria therapy. Eulophia camprestis
(Eulophia dabia) rhizomes are used as a tonic and effective in curing stomach
problems, cough, paralysis, and also used as aphrodisiac [86], for worm infestation,
and for scrofula [40].
3.11.2 Eulophia epidendraea (J.Koenig ex Retz.) C.E.C.Fisch

Terrestrial medium-sized; pseudobulbs subterranean; leaves ovoid, linear, two
to three; inflorescence one- to two-flowered; flowers fragrant; flowering
December–January, found in India, Bangladesh, Sri Lanka, and Western Himalayas.
The leaves and tubers of E. epidendraea are traditionally used for the treatment
of tumor, abscess, and wound healing of animals [87]. Maridass and Ramesh [88]
isolated four phytochemicals (β-sitosterol, β-sitosterolglucoside, β-amyrin, and
β-lupeol) from the tubers and four flavonoids (apigenin, luteolin, kaempferol, and
quercetin) from the leaves. The ethanolic extract of tuber (500 mg kg1) of
E. epidendraea has antidiarrheal activity [89]. Maridass [27] reported significant
healing response in rats treated with 100 mg ml1 E. epidendraea tuber extract. The
tuber extract of E. epidendraea has a stimulating effect on collagen
synthesis. Maridass et al. [87] observed a reduction of blood glucose levels when
376 R. Pal et al.
alloxan-induced rats were induced with 200 mg ethanolic tuber extract

E. epidendraea. Besides antidiabetic ethanolic extract of tubers also has hypoglycemic
and protective effects in the liver and kidney, a significant weight gain was observed in
rats when they were administered with 100 and 300 mg kg1 body weight.
3.11.3 Eulophia herbacea Lindl.

Terrestrial herb, small to medium in size, cool-growing; pseudobulb above the
ground, ovoid; leaves two to three, linear-lanceolate; inflorescence erect six- to
ten-flowered; flowers fragrant; flowering June; found in China, Bangladesh, India,
Western Himalayas, Laos, Myanmar, and Thailand at an elevation ranging from
1000 to 2000 m.
E. herbacea is used in the treatment of tumor of the scrofulous gland of the neck
[90]. It is also used to make salep and nutritious beverages from dried tubers
of various species of orchids including Eulophia. It shows multiple activities such
as anticancer, nutritional, antihyperlipidemic, antioxidant, antiarthritic, anti-inflam-
matory, antimicrobial, and immunomodulator [90]. Phytochemical analysis revealed
the presence of acidic compounds, carbohydrates, amino acids, mucilage, tannins,
steroids and triterpenoid [91]. Crushed bulbs are fried in mustard oil, and the residue
is applied on rheumatism thrice a day till cure [92]. 10 g dried tuber of E. herbacea,
5 g dried leaves of Withania somnifera, 5 g dried leaves of Curculigo orchioides, and
5 g black pepper are grounded into powder taken orally with a cup of water for
20 days against aphrodisiac by Dongria Kandha tribe of Niyamgiri Hills in Odisha,
India. The leaf decoction is also used against vermifuge [10].
3.11.4 Eulophia nuda Lindl. (Syn. Eulophia spectabilis Suresh)

Terrestrial, large-sized, hot- to warm-growing herb. Pseudobulbs subterranean,
round, leafless at flowering; leaves 3–4 elliptic-lanceolate, acuminate appear after
flowering; inflorescence erect, 20–30 long, many-flowered (12–20); flowers green-
ish purple.
The tubers of E. nuda are used to treat a tumor, scrofulous infection, anthelmintic,
bronchitis, and juice from the roots is used to treat the snakebite [92]. Salep or Salam
Mishri prepared from the roots of E. nuda and Orchids latifolia and said to be
aphrodisiac addition to other medicinal properties [24]. Jagdale et al. [93] tested
aphrodisiac claims of these two orchids and found that found crude drugs are non-
toxic up to a level of 2 g kg1 body weight in adult rats. Administering the crude
extract of E. nuda and O. latifolia to adult rats increased mounting behavior but
Orchis fed rats had higher mounts than the Eulophia fed. The remarkable increase in
the organ weights, as well as sperm counts, a significant increase in the protein,
hemoglobin, and testosterone content, was observed over the control. E. nuda
contains phytochemically active compounds such as alkaloids, flavonoids, saponins,
cardiac glycosides, terpenoids, and steroids having significance in medicine [94].
3.11.5 Eulophia ochreata Lindl.

Terrestrial plant 20–30 cm tall, warm- to cool-growing herb; pseudobulb conical
ovoid; leaves two to five, oblong-lanceolate to ovate, plicate; inflorescence erect,
many-flowered; flowers golden yellow; flowering in June; endemic, found in the

Western Ghats, India, at an elevation of 1000 m.
Tubers of E. ochreata are rich source of plant fiber, protein, and carbohydrates
which can be consumed as nutrition for healthy growth and adequate protection
against diseases arising from malnutrition; however, a study on anti-nutritional/
toxicological factors and biological evaluation of nutrient contents is necessary
before recommending them as source of food. E. ochracea tubers contain anti-
nutrients (alkaloids, saponins, and steroids) and need adequate processing before
they are consumed or fed to animals [95]. Tubers of E. ochreata are used for the
treatment of stomachache and as an astringent, anti-fatigue, aphrodisiac, and
anthelminthic and act as a blood purifier. The tubers are also used in cough,
cold, and heart troubles [96]. In Rajasthan, E. ochreata is locally known as Mishri,
and the crushed bulbs are used to cure diarrhea [97]. Tubers of E. ochreata are a
potential source of natural antioxidant and can be exploited for its nutraceutical
and medicinal properties [98]. The tubers contain a large quantity of white muci-
lage; they are astringent, used as a nutritive tonic, aphrodisiac, anthelmintic, and
blood purifier [96, 99, 100]. They are also used to cure cough, cold, and heart
troubles [26, 101, 102]. It is mainly used as a tonic by the local tribes in the
study area.
3.12 Habenaria R. Br.
Terrestrial herbaceous plants bear ellipsoid, fusiform, or ovoid underground tubers;

the leaves fall off during winters, and new leaves emerge during early summer or
monsoon. The genus consists of about 600 species distributed in America, Asia, and
Africa. In India, the genus is represented by 61 species, occurring in Himalayan,
Central, and Peninsular India. Riddhi (Habenaria intermedia) and Vriddhi
(Habenaria edgeworthii) are the critical ingredients of Ashtaverga in Ayurvedic
system of medicine.
3.12.1 Habenaria commelinifolia (Roxb.) Wall. ex Lindl.

Terrestrial, warm- to cool-growing herb found in the Himalayas, India, Nepal,
Myanmar, Thailand, and Vietnam at an elevation ranging from 375 to 2000 m.
Plants are 90 cm tall; tubers ellipsoidal; leaves oblong, lanceolate; inflorescence
raceme many-flowered (10–15), species flowers during the end of the monsoon.
The tubers of this species have long been utilized to cure spermatorrhea among
tribal of Orissa. To cure spermatorrhea, equal quantity of dried tubers of Habenaria
commelinifolia and roots of Saraca indica are boiled in one liter of water till the
volume is reduced to 100 ml. The decoction (6–8 drops) is administered to a patient
orally on an empty stomach for 10 days [10]. The tubers of this plant can cure
snakebite and snakebite wounds, roots cure fever, and the whole plant can cure nose
bleeding [62].
378 R. Pal et al.
3.12.2 Habenaria edgeworthii (Hook f. ex Colett) R.K.Gupta (Syn:

Habenaria acuminata Lindl. syn Platanthera edgeworthii (Hook.
f. ex Collett) R.K. Gupta; Herminium edgeworthii (Hook.f. ex
Collett) X.H.Jin.)
The species has been placed under genus Herminium as Herminium edgeworthii
(Hook.f. ex Collett) X.H.Jin. In India, this species is popularly known as Habenaria
edgeworthii, hence described under Habenaria. The species is found in Western
Himalayas on open grassy slopes in association with Satyrium nepalense at altitude
ranging from 2500 to 3000 m. The plants are 40–80 cm tall and bear many small
flowers with 15–25-cm-long inflorescence; it flowers during the month of
September.
The dried tubers are commonly known as Vriddhi and important constituent of
Ashtavarga. The tubers of this species are used to prepare Chyavanprash and also
used as health tonic, blood purifier, and rejuvenator [103].
3.12.3 Habenaria intermedia D.Don.

The species is found in moist open grasslands of the Western and Eastern Himalayan
regions of the country at an elevation ranging from 2000 to 2500 m. Plants are up
to 50 cm tall and bear 10–15-cm-long plicate ovate-oblong, acuminate leaves.
Inflorescence four- to six-flowered; flowers are large 5–6 cm across. The species
flowers from July to September.
The underground tubers of this plant are steamed or boiled and dried (Ridhi)
before marketing. Tubers are used in Asokaghrta, Amrtaprasa Ghrta,
Dashmularishta, and Chagaladya Ghrita, and it cures fever, e.g., Ksaya,
Raktavikara, and Murchha (http://www.ayurveda.hu/api/API-Vol-5.pdf). It is a con-
stituent of Chyavanprash and also used as a tonic, expectorant, rejuvenator, and life-
span promoter [30, 102]. The tubers are known to promote intellect, aphrodisiac,
depurative, anthelmintic, rejuvenating, and tonic. Habu et al. [104] reported that
tubers of H. intermedia have anti-stress/adaptogenic activity due to presence of
scopoletin and gallic acid or their synergistic properties and the activity might be
mediating through an antioxidant mechanism.
3.12.4 Habenaria longicorniculata Graham

Terrestrial, medium-sized warm- to cool-growing herb distributed in India and
Sri Lanka at an elevation of 800–1000 m. Plants up to 80 cm tall; tubers unequal
ellipsoid; leaves five to seven clustering at base, lanceolate, acute at tip; inflores-
cence with one to four scented flowers. The species is distributed in central and
peninsular regions of the country.
The tuber paste is mixed with an equal amount of turmeric powder and applied
externally on affected parts for 2 weeks to cure leucoderma [50].
3.12.5 Habenaria marginata Colebr.

Terrestrial, hot- to cool-growing herb found in China, India, Nepal, Bangladesh,
Myanmar, and Thailand at an elevation of 100–1200 m. Tubers cylindrical; leaves
sessile linear-oblong, margined with yellow; inflorescence many-flowered; blooms
during summer. The plant can be easily identified by the presence of a cluster of
marginated leaves at the base, 6–8 small greenish yellow flowers on 10–25-cm-tall
plants during monsoon.
The tubers (250 g) are boiled in 1 l of water until the volume is reduced to 250 ml,
and then decoction is mixed with 5 ml of honey and taken daily on an empty stomach
for 14 days to cure malignant ulcer. According to Joshi et al. [30], thoroughly boiled
plant extract is useful in curing flatulence.
3.12.6 Habenaria roxburghii Nicolson

Terrestrial, hot- to warm-growing herb with two to three orbicular, elliptic leaves
lying on the ground. Plants are up to 30 cm tall; inflorescence 20–25-cm-long
bearing 10–20 white fragrant flowers. The species is endemic to the Western Ghats
and found in shady locations of forest floors. It flowers from July to September.
The tubers’ extract of this species eaten with sugar gives a cooling effect to the
body [54]. Eating tubers before breakfast controls burning micturition. Tuber also
cures from diabetes [105]. The other species of Habenaria also used in herbal
medicine. The crushed leaves of Habenaria pectinata can cure snakebite and
snakebite wounds, whereas tubers relieve arthritis [20]. The mixture of Habenaria
plantaginea tubers, black pepper (Piper nigrum), and garlic (Allium sativum) is
useful for curing chest and stomach pain [54]; Habenaria susannae are used to
cure blebs or bullae occurring on the palm [20].
3.13 Malaxis Sol ex. Sw.
The genus Malaxis consists of nearly 300 species distributed from tropical
to temperate regions of the world. Nineteen species are found in India. They
are distributed in temperate to tropical regions of the country. Two species, namely,
M. acuminata (Rishabhaka) (Now known as Crepidium acuminatum) and M.
muscifera (Jeevika), are components of Ayurvedic formulation Ashtaverga. The
dried pseudobulbs are used for the preparation of herbal tonic (Chyawanprash) and
to cure tuberculosis. Lack of proper production supply system and increasing
demands of herbal drugs are promoting the practice of adulteration and substitution,
causing the degradation of the desired therapeutic effect of plant species used in
Ayurveda [106].
3.13.1 Malaxis acuminata D. Don (Syn Microstylis wallichii Lindl.)

(Currently known as Crepidium acuminatum (D. Don) Szlach.)
Terrestrial rarely lithophytic herbaceous perennial found under shady locations of
forest floors. It is distributed in India (Himachal Pradesh to Arunachal Pradesh,
Madhya Pradesh), Nepal, Bhutan, China, Thailand, and Myanmar. M. acuminate
(Crepidium acuminatum) plant up to 25 cm height, distributed all over India,
2000–3000 m altitude. Pseudobulbs are conical fleshy smooth, shiny 2–9 cm long,
and mucilaginous.
380 R. Pal et al.
Powder made from dried pseudobulbs is used in Chyavanprash, a nutritive tonic.

It is reported to cure tuberculosis, bleeding diathesis, fever, phthisis, and bronchitis,
soothe burning sensation, and enhance sperm production (spermatogenesis) [30, 56,
103]. M. auminata pseudobulbs contain piperitone, citronellal, eugenol, Limonene,
1,8-cineole, p-cymene, O-Methylbatatasin, and cetyl alcohol and two sugars,
namely, glucose and rhamnose [107]. Recently, one sterol, β-sitosterol, has also
been isolated from the ethyl acetate fraction of pseudobulbs. The plant has become
vulnerable in its natural habitat. The loss of forest cover and an extensive collection
of rhizomes from the wild are major causes of its threats in natural habitat.
3.13.2 Malaxis muscifera (Lindl.) Ktze.

Terrestrial, cool-growing herb, pseudobulbs long-sheathed and many-nerved; leaves
two not equal, ovate-lanceolate; inflorescence ribbed, erect, many-flowered; flowers
small; flowering time June to August. The species is distributed in India, Bhutan, and
Nepal at an elevation ranging from 2600 to 4300 m.
The herb is an important ingredient of Chyavanprash. Additionally, it is used to
cure disorders related to blood, inflammation, male sterility, fever, dysentery, exter-
nal and internal hemorrhage, and general weakness. It is an aphrodisiac. It is also
used for insect bite and rheumatism [102, 108].
3.14 Pholidota Lindl. ex Hook.
Plants are lithophyte or epiphyte with creeping rhizome; often the leaves are in pairs
and sometimes solitary; inflorescence are drooping and densely flowered. It consists
of 27 species distributed from India to South China, Malaysia, Indonesia, and New
Guinea. Seven species are found in India. Pholidota chinensis and Pholidota pallida
are medicinally important.
Pseudobulbs are crushed to expel the spines and applied on the body [22]. For
curing inflammation, freshly collected pseudobulbs are made into a paste including
the juice from the coconut kernel, and the paste is applied on the inflamed areas until
the swelling or inflammation subsides [22]. Rhizome paste is applied for finger
abscess [54]. Finely macerated pseudobulbs are made into a paste with mustard oil
and applied to joints to remove rheumatic pains [109]. Pseudobulbs of P. chinensis
are used to cure duodenal ulcer, scrofulous glands of neck, and toothache, and
tincture made from pseudobulb are used to treat bronchitis [110]. Pseudobulb of P.
imbricata extract can cure abdominal pain, and rheumatism and leaf and root paste
when applied externally heals fractures [105]. P. pallida pseudobulbs are used for
getting rid of intestinal worms and abdominal pain, and roots are used for rheuma-
tism [20]. Bi et al. [111] isolated n-nonacosane, cyclopholidone, n-dotriacontanoic
acid, n-octacostyl ferulate, cyclopholidonol, cycloneolitsol, and beta-sitosterol,
respectively, from P. chinensis. Guo et al. [112] isolated six stilbenoids,
(bibenzyldihydrophenanthrene) ether, from P. yunnanensis, and all the isolated
compound were found to inhibit nitric oxide production in a murine macrophage-
like cell line activated by lipopolysaccharide and interferon gamma.
3.15 Rhynchostylis Blume
The genus consists of three to four species distributed from India to the Philippines.
Two species are found in India. Only Rhynchostylis retusa (Linn.) Bl. is used for
medicinal purpose. A single leaf is made into a paste without using water and applied
externally to cure throat inflammation [22]. Fresh roots 3–4 g and 2 g leaf bud of
Pisum sativum are made into a paste; about 1 g paste is administered to the patient
orally with water on an empty stomach two times a day for 7 days to cure blood
dysentery. The plant is also used for softening the skin, and the leaf paste is applied
externally for healing wounds [10]. The whole plant preparation is used to treat
asthma, tuberculosis, infantile epilepsy, kidney stone, and menstrual disorders [105].
Methanol leaf extract of R. retusa has analgesic and anti-inflammatory activity.
Methanolic leaf extract inhibited acetic acid-induced writhing and carrageenan-
induced paw edema in mice when they were administered with 200 and 400 mg kg1
and 100 and 200 mg kg1 extract, respectively [113]. For treating malarial fever,
decoction of the fresh roots is made and stored. 5 g paste of young shoot of
Andrographis paniculata (Burm. f.) Wall. ex Nees along with 100 ml of this
decoction is taken orally twice a day for 5 days day till it is cured [21].
3.16 Vanda Jones ex R Br.
Vanda is the most important genus known for its horticultural as well as therapeutic
value. The name Vanda owes its origin from Sanskrit name for Vanda tessellata. This
genus consists of 70 plus species distributed in India, Indonesia, Malaysia, Thailand,
China, the Philippines, China, and Australia. There are 13 species known to occur
within the political boundaries of India, among which V. spathulata and V. tessellata
are medicinally important. Most of the species under this genus are epiphytic or
lithophytic with monopodial growth habit and strap-shaped leaves arranged in
a fishbone pattern. Flowers are large, and color ranges from green to blue.
3.16.1 Vanda spathulata (L.) Spreng.

The golden yellow flowers of V. spathulata are being eaten raw to reduce asthma or
powdered and 0.5–1 g powder mixed with honey and taken twice a day orally to get
rid of asthma. Flowers are also consumed against asthma and mania [26]. Flowers
are taken to cure tuberculosis, asthma, and mania. Plant juice is given to temper bile
and to abate frenzy [22].
3.16.2 Vanda tessellata (Lindl.) Rchb. f. (Syn. Vanda roxburghii R. Br.)

This perennial herb is generally called a “Rasna” in Ayurvedic system of medicine.
Roots, leaves, or whole plant are used to treat various kinds of ailments. The roots
are used to treat rheumatism and similar disorders [54]. The aerial roots and leaves
are grounded with a tender bud of Phoenix loureirii, and the paste is plastered over
fractured bone, and the extract of the same five spoonful is served twice a day orally
till cure by Konda Reddis and Koyas tribe in Andhra Pradesh [54]. The roots of this
382 R. Pal et al.
plant found to have alexiteric and antipyretic properties. Root enters into the
composition of various medicated oils for external application in nervous disorders
and rheumatism [26]. The paste made from the leaves when applied on body
alleviates fever. Shanavasakhan et al. [22] carried out an extensive survey for the
medicinal use of this plant in Kerala and noticed that purpose and method of its use
varied with the region. In Palakkad, leaf juice is applied for earache; in Kollam, leaf
poultice is applied to relieve sprains, lumbago, and back pain, and Waynad juice
from the leaves and aerial roots mixed with neem oil and garlic is used for treating
earache. It has also been used to treat bronchitis inflammation, hiccup, piles, and
boils on the scalp [114]. Usman et al. [115] studied pharmacognostical properties of
“Rasna” and found dried roots are brown, fragrant, bitter, and longitudinally wrin-
kled, whereas the powder is muddy brown in color. Kumar et al. [116] reported
aphrodisiac property of alcoholic extract of V. tessellata flowers. Alcoholic extract of
flowers of V. tessellata at doses of 50 and 200 mg kg1 increased mating perfor-
mance and also increased male and female ratio of resulting offspring without any
toxicity. Methanol and acetate extract of V. tessellata is found to have antibacterial
activity against a number of pathogenic and fungi. Melanin (VR1), a compound that
was isolated, has a very strong activity against bacteria [117].
Some other Vanda species like V. coerulea, V. teres (now known as Vandopsis
undulata), and V. cristata (Syn. Trudellia cristata) also used for medicinal purpose.
The flower juice of V. coerulea is used as an eye drop solution to cure glaucoma and
blindness [118]. Leaves of V. cristata are used to cure cough, whereas the extract
from leaves is used to inhibit a number of foodborne pathogens like Klebsiella
pneumoniae, E. coli, and Salmonella typhi [119, 120].
4 Conclusion
India has a long tradition of using orchids and other medicinal plants in traditional
medicine system such as Ayurveda, Siddha, Unani, and Homeopathy. However,
overexploitation and apathy for use modern techniques for commercial use have
pushed many medicinal orchids to rarity. A very few orchids in India have been
subjected to search for active principle compound and clinical evaluation despite the
fact that a plethora of information is available on their use in traditional as well
as tribal medicine system. Further, more emphasis has to be laid down on mass
propagation, cultivation practices, and post-harvest handling practices along with
breeding of medicinal orchids for a better quality of products.
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EPTRI-ENVIS Newslett 11:6–12
Traditionally Used Medicinal Dendrobium:
A Promising Source of Active Anticancer 16
Constituents
Mukti Ram Paudel, Hari Datta Bhattarai, and Bijaya Pant
Contents
1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 390
2 Anticancer Compounds Isolated from Dendrobium Species . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 392
3 Methods of Screening Anticancer Effect . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 392
3.1 In Vitro Assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 396
3.2 In Vivo Assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 398
4 Anticancer Effects of Dendrobium Species . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 399
4.1 Cytotoxicity Effect . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 399
4.2 Anti-metastasis Effect . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 406
4.3 Antiangiogenesis Effect . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 407
5 Production of Anticancer Compounds Through In vitro Culture of Dendrobium Species . . . 408
6 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 409
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 409
Abstract
Dendrobium represents one of the most important genera of the Orchidaceae
family, having medicinal and ornamental value. Dendrobium species have been
traditionally used as first-rate medicinal herbs in the treatment of a variety of
disorders, such as nourishing the stomach and enhancing the production of body
fluids. Many species of this genus are the sources of tonic for astringent, analge-
sic, anti-pyretic, antioxidant, antimicrobial, antidiabetic, anticancer, anti-
inflammatory, anti-metastasis, and antiangiogenesis because they have alkaloids,
aromatic compounds, sesquiterpenoids, and polysaccharides as main compo-
nents. This chapter includes the active constituents, extract and pure isolate,
from 23 Dendrobium species and their effect in the anticancer, anti-metastasis,
and antiangiogenesis.
M. R. Paudel (*) · H. D. Bhattarai · B. Pant

Central Department of Botany, Tribhuvan University, Kathmandu, Nepal
e-mail: mr.paudel@cdbtu.edu.np; muktiram.paudel@cdb.tu.edu.np

390 M. R. Paudel et al.
Keywords
Antiangiogenesis · Anticancer · Anti-metastasis · Bibenzyl · Dendrobium ·
Fluorenone · Phenanthrene
1 Introduction
Dendrobium species represent the most important plants, ornamentally and medic-
inally. They have been used for a thousand years as first-rate herbs and prized folk
medicine in China, India, Australia, and other countries of the world [1]. In tradi-
tional Chinese medicine (TCM), they are the source of tonic, nourishing the stomach
and enhancing the production of body fluids, astringent, analgesic, and anti-
inflammatory substances [2]. A common name “Shi-Hu” is used in TCM for
30 species of Dendrobium under two monographs, Dendrobii Caulis (Shi-Hu) and
Dendrobii Officinalis Caulis (Tie-Pi Shi-Hu) [1, 2]. The Chinese consider
Dendrobium as one of the 50 fundamental herbs used to treat all kinds of ailments
and use Dendrobium tonic for longevity [3]. In Indian Ayurvedic system of medi-
cine, the commonly used orchids are “Salem” (Orchis latifolia and Eulophia
latifolia), “Jewanti” (Dendrobium alpestre), “Shwethuli” (Acampe papillosa), and
“Rasna” (Vanda tessellata) [4]. Dendrobium teretifolium and D. discolor have been
used for treating different ailments as dysentery, relieving pain, and controlling
ringworm, and Dendrobium speciosum has been employed as emergency bush
food in Australian traditional medicine [1].
In light of traditional importance as a medicinal plant, knowledge on
the constituents of various Dendrobium species and their pharmacological activi-
ties has been growing, and methodologies have been developed for effective
propagation [5, 6]. Indeed, a large number of pharmacological activities have
been assigned to different Dendrobium species, such as anti-inflammatory, anti-
platelet aggregation, hepatoprotective, anti-fibrotic, antiviral, antifungal, antimi-
crobial, antioxidant, antidiabetic, neuroprotective, immunomodulatory, and
anticancer [7, 8].
Cancer is the second biggest health problem after cardiovascular diseases
accounting for an estimated 9.56 million deaths worldwide in 2017. The number
of cancer deaths increased between 1990 and 2017 by 66% [9, 10]. It is caused by
dysfunction of gene coding for proteins such as growth factors, growth factor
receptors, anti-apoptotic proteins, transcription factors, and tumor suppressors
[11]. Treatment of cancer currently includes the surgical removal of cancerous tissue,
radiotherapy, chemotherapy, and a combination of chemotherapy and radiotherapy.
The use of anticancer drugs (chemotherapy), while often more beneficial when used
in conjugation with radiation therapy or surgery, is nonetheless a key line of
treatment [10, 12, 13]. Because anticancer drugs are the mainstay of chemotherapy,
it is important to discover novel anticancer drugs with diverse activity, a novel
mechanism of action, and minimal issues of toxicity [14–16]. In parallel, there is
increasing evidence for the potential compounds from different Dendrobium species
16 Traditionally Used Medicinal Dendrobium: A Promising Source of. . . 391
Fig. 1 Some Dendrobium species rich in anticancer compounds: D. chrysanthum (a),

D. fimbriatum (b), D. moniliforme (c), and D. polyanthum (d). (Photo credit: B.B. Raskoti for
D. chrysanthum and D. polyanthum)
(Fig. 1) as inhibitors of various stages of tumorigenesis and associated processes,

underlining the importance of these products in cancer prevention and therapy
[7, 17]. Cancer chemoprevention by anticancer drugs derived from Dendrobium
species has shown promising results against various malignancies [18]. Therefore,
they potentially represent an inexhaustible source of chemicals for the discovery of
new anticancer drugs.
Knowing that Dendrobium species have been traditionally used for the prepara-
tion of natural remedies, they are worth investigating by scientists who are looking
for new natural ingredients, which could become the main feature of a novel
mechanism of action in cancer. This book chapter is mainly focused on the constit-
uents of Dendrobium species which have been subjected to investigation of cancer.
2 Anticancer Compounds Isolated from Dendrobium Species
As the Dendrobium species have been used in traditional medicines [2, 6], their
misidentification and adulteration led to a loss of therapeutic potency and potential
intoxication [5]. For decades, fast-developing molecular techniques using DNA
fingerprinting, DNA sequencing, and DNA microarray have been applied exten-
sively to authenticate medicinal materials, including various Dendrobium species
[5]. Chemical studies on Dendrobium species have been conducted since the 1930s,
while alkaloids, aromatic compounds, sesquiterpenoids, and polysaccharides have
been identified as the main components [19–22]. To date, various Dendrobium
species are known to produce a variety of secondary metabolites. The biological
activities and pharmacological actions of all the isolated compounds were investi-
gated [23]. More than 100 compounds from 42 Dendrobium species including
32 alkaloids, 6 coumarins, 30 bibenzyls, 4 fluorenones, 22 phenanthrenes,
and 7 sesquiterpenoids have been identified and discussed [6, 8, 18, 24,
25]. The active anticancer bibenzyls, 3,4,30 -trimethoxy-5,40 -dihydroxybibenzyl (1),
3,4-dihydroxy-30 ,40 -dimethoxybibenzyl (2), 4-(3-hydroxy-4-methoxyphenethyl)-2,6-
dimethoxylphenol (3), 4,40 -dihydroxy-3,5-dimethoxybibenzyl (4), 4,5,40 -trihydroxy-
3,30 -dimethoxybibenzyl (5), 5,30 -dihydroxy-3,4-dimethoxy-bibenzyl (6), aloifoll (7),
chrysotoxine (8), dendrocandin B (9), dendrocandin I (10), dendrocandin U (11),
dendrofalconerol A (12), dendrosignatol (13), dengraols A and B (14) and (15),
denofficin (16), erianin (17), fimbriadimerbibenzyl A, B, E, F, and G (18–22), giga-
ntol (23), longicornuol A (24), moscatilin (25), and tristin (26) (Fig. 2);
phenanthrenes, chrysotoxol B (27), confusarin (28), cypripedin (29), denbinobin (30),
epheranthol B (31), lusianthrindin (32), moniliformediquinone (33), and moscatin (34)
(Fig. 3); and fluorenones, 1,4,5-trihydroxy-7-methoxy-9H-fluoren-9-one (35) and
dendroflorin (36) (Fig. 4), have been isolated from different Dendrobium species.
3 Methods of Screening Anticancer Effect
Cell proliferation, inducing apoptosis, growth suppressors, activating invasion,

metastasis, and angiogenesis are the major hallmarks of cancer [26]. They acquired
during the development of human cancers. Several in vitro and in vivo assays have
been developed to evaluate each hallmark feature of cancer, and the selection of
particular assay mainly depends on the specific research question(s) to be examined.
Nowadays, a wide range of in vitro and in vivo assays (some of them are described
here) are available for cytotoxicity, apoptosis, cell migration, and invasion; angio-
genesis in cancer cells is commonly used in cancer drug discovery.
Fig. 2 (continued)
Fig. 2 Structure of anticancer bibenzyl derivatives (1–26)

Fig. 3 Structure of anticancer phenanthrene derivatives (27–34)

Fig. 4 Structure of anticancer fluorenone derivatives (35 and 36)
3.1 In Vitro Assay
A number of in vitro assays are available to investigate the anticancer potential

of plant-derived extract or drug such as trypan blue dye exclusion assay, resazurin
cell growth inhibition assay, LDH (lactic acid dehydrogenase) assay, SRB
(sulforhodamine B) assay, MTT (3-[4,5-dimethylthiazole-2-yl]-2,5-
diphenyltetrazolium bromide) assay, MTS (3-(4,5-dimethylthiazol-2-yl)-5-
(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) assay, and XTT
(2,3-bis[2-methoxy-4-nitro-5-sulfophenyl]-2H tetrazolium-5-carboxyanilide inner
salt) assay [12, 26].
3.1.1 Trypan Blue Dye Exclusion (TBDE) Assay

TBDE assay is the most commonly performed test to assess the viability of cells. In
this assay, the cells are washed with HBSS (Hank’s buffered salt solution) and
centrifuged for 10–15 min at 10,000 rpm, and this entire step is repeated thrice.
The cells are suspended in a known quantity of HBSS, and the cell count is adjusted
to 2 106 cells mL1. The cell suspension is distributed into Eppendorf tubes. The
cells are exposed to drug dilutions and incubated at 37 C for 3 h. Thereafter, in the
dye exclusion test, i.e., the equal quantity of the drug-treated cells are mixed with
trypan blue (0.4%) and incubated for 1 min. It is then loaded in a hemacytometer,
and viable and nonviable cells are counted. Viable cells do not take up color, whereas
dead cells take up color. However, if taken longer, live cells also generate and take up
color. The percentage of growth inhibition is calculated using the following formula:
Growth Inhibition ð%Þ ¼ 100 ðTotal cells Dead cellsÞ=Total cells 100
3.1.2 Resazurin Cell Growth Inhibition (RCGI) Assay

RCGI (also called as alamar blue) assay measures the cellular viability as well as the
function of the mitochondria. In this assay, cells are treated with trypsin (0.025%)
and EDTA (0.25 mM) for 5 min. Subsequently, cells are washed with PBS, counted
and seeded in 96-well plate containing 5 103 cells per well, and incubated for
overnight growth. Then, cells are treated with test samples and kept for 48 h
incubation, followed by the addition of 20 μL of resazurin (0.01%) and further
incubation for 1–2 h at 37 C. Fluorescence of 96-well plate is measured by a multi-
well plate reader at excitation and an emission wavelength of 540 and 590 nm,
respectively, and inhibitory concentration (IC50) values are calculated. The IC50
value is the amount of anticancer drug needed to inhibit 50% of cell proliferation.
3.1.3 Lactic Dehydrogenase (LDH) Assay

Lactic dehydrogenase activity spectrophotometrically measured the cellular lysates
at 340 nm by analyzing NADH reduction during the pyruvate-lactate transformation.
Cells are lysed with 50 mM Tris-HCl buffer with 20 mM EDTA and sodium dodecyl
sulfate (SDS, 0.5%) at pH 7.4. Further cells are disrupted by sonication and
centrifuged at 13,000 rpm for 15 min. The assay mixture for the enzymatic analysis
consists of 33 μL of a sample in 48 mM PBS with 1 mM pyruvate and 0.2 mM
NADH at pH 7.5. The percentage of LDH released is calculated as a percentage of
the total amount, considered as the sum of the enzymatic activity present in the
cellular lysate and that in the culture medium.
3.1.4 Sulforhodamine B (SRB) Assay

SRB is a bright pink aminoxanthene dye that binds to the basic amino acids in mild
acidic condition and dissociates under basic condition. Cells are seeded in 96-well
plate at 5000–10000 cells per well. The difference in cell number seeded adjusts for
differences in the growth rate of the various cell lines. Cells are allowed to adhere to
the wells overnight, and then the test samples are added in wells. Water is added to
the control well at 1:10 dilution in a medium. The plate is incubated under 5% CO2 at
37 C for 3 days. The cells are fixed by the addition of cold 50% trichloroacetic acid
to a final concentration of 10%. After 1 h incubation at 4 C, the cells are washed five
times with deionized water. The cells are then stained with 0.4% SRB dissolved in
1% acetic acid for 15–30 min and subsequently washed five times with 1% acetic
acid to remove an unbound stain. After the plate is air-dried at room temperature, the
bound dye is solubilized with 10 mM Tris base, and the plate is read on a multi-well
plate reader at 595 nm.
3.1.5 3-(4,5-Dimethylthiazole-2-yl)-2,5-Diphenyltetrazolium Bromide

(MTT) Assay
MTT assay, based on the conversion of the purple tetrazolium salt to yellow
tetrazolium crystals of formazan of metabolically active cells, provides a quantitative
determination of viable cells. Cells are seeded in the 96-well plate at a cell density of
2 105 cells per well in 100 μL of cell-cultured medium and allow to grow in a CO2
incubator for 24 h at 37 C. The medium is then replaced by fresh medium
containing different concentrations of the test sample and allow for 48-h incubation.
Thereafter, 20 μL MTT (5 mg mL1 in PBS) is added to each well and incubate for
4 h. The medium is removed, and 200 μL DMSO is added to each well to dissolve
the MTT metabolic product. The plate is shaken at 150 rpm for 5 min, and optical
density is measured at 560 nm.
3.1.6 3-(4,5-Dimethylthiazol-2-yl)-5-(3-Carboxymethoxyphenyl)-2-
(4-Sulfophenyl)-2H-Tetrazolium (MTS) Assay
Like MTT, it is also a reliable, convenient, and economical method. This method is
performed in a similar way like MTT, but MTS is used to perform this assay.
3.1.7 2,3-Bis(2-Methoxy-4-Nitro-5-Sulfophenyl)-2H Tetrazolium-5-

Carboxyanilide Inner Salt (XTT) Assay
In order to measure the proliferation response, the XTT assay is used. The tetrazo-
lium salt, XTT, is especially useful in quantifying viable cells. This assay is designed
for the spectrophotometric quantification of cell growth and viability without the use
of radioactive isotopes and is based on the cleavage of yellow tetrazolium salt, XTT,
to form an orange formazan dye by metabolically active cells. XTT cleavages into an
orange formazan dye by the mitochondrial enzyme, dehydrogenase, occur exclu-
sively in living cells. Cells are grown in a growth medium with 10% FBS in 96-well
plate. They are treated with appropriate test sample for 24 h. An XTT assay is
performed at the end of the incubation. Briefly, 50 mL of XTT labeling mixture
solution is added to each well, and the cells are incubated at 37 C for 4 h. The
formazan crystals formed is soluble in aqueous solution, and optical density at
450 nm is compared with that of control wells with a screening multi-well plate
reader. The reference wavelength is 650 nm.
3.2 In Vivo Assay
3.2.1 Induction of Ehrlich Ascites Carcinoma

Antitumor activity of the test compounds is determined using Ehrlich ascites carci-
noma (EAC) tumor model in mice. The ascitic carcinoma-bearing mice (donor) are
used for the study, 15 days after tumor transplantation. The animals are divided into
groups of normal mice (a), tumor-bearing mice (b), tumor-bearing mice treated with
the standard drug (c), and tumor-bearing mice treated with test drug (d). The ascitic
fluid is drawn using an 18-gauge needle with a sterile syringe. A small amount is
tested for microbial contamination. Tumor viability is determined by trypan blue
exclusion test, and cells are counted by hemacytometer. The ascitic fluid is suitably
diluted in normal saline to get a concentration of 106 cells mL1 of tumor cell
suspension. This is injected intraperitoneally to obtain ascitic tumor. The mice are
weighed on the day of tumor inoculation and then once in 3 days thereafter.
Treatment is started on the tenth day of tumor inoculation. Standard (one dose) is
injected on the tenth day intraperitoneally. The drug is administered from the tenth
day for 5 days intraperitoneally, after the administration of the last dose followed by
18-h fasting. The animals in each group are kept to check the mean survival time
(MST) of the tumor-bearing hosts. Antitumor effects of a drug are assessed by
observation of following parameters: (i) percentage increase in weight as compared
to day first – 0 weight, (ii) median survival time and increase in lifespan (%ILS), and
(iii) hematological parameters [12, 26].
4 Anticancer Effects of Dendrobium Species
The death of the cells can be either accidental or regulated. Regulated cell death is
genetically strictly operated and is an essential biological phenomenon for multi-
cellular organisms [27]. In healthy cells, it is involved in several biological pro-
cesses, such as development, homeostasis, embryogenesis, maturation of the
immune system, and protecting an organism from microbial attacks. The activity
of regulated cell death is not limited to functions in healthy cells – it plays a
significant role in several pathological conditions, such as cancer, chronic inflam-
mation, and neurodegenerative diseases [28]. In such conditions, the mechanisms of
death are impaired by increasing or decreasing cells’ ability to self-destruct. Acci-
dental or necrotic cell death is not controlled, and it occurs as a result of physical
(e.g., temperature), chemical (e.g., pH), or mechanical (e.g., shearing) stimuli
[29, 30].
Cancer is the uncontrolled virtually autonomous growth of abnormal cells that
can arise in any organ or tissue of the body. It is a heterogeneous disease that
develops through the transformation of healthy cells to rapidly and uncontrollably
dividing cancer cells as a result of, for example, oxidative stress, gene oncogenes, or
viruses [30]. The immune system usually isolates and destroys the abnormal cells
before they proliferate enough to be noticeable as a tumor. Free radicals can
compromise immune cell function reducing immune responses which can allow
the abnormal cells to continue growing [31].
Extracts of many Dendrobium species as well as their isolate of pure compounds
have been studied as a valuable natural source of promising anticancer agents
(Table 1). Indeed, cell proliferation is the most important characteristic of cancer/
tumor cells and could be indicated by the viability of the cells. Due to the high
diversity of the types of cancer, specific mechanisms of action have been explored to
assign to cytotoxic, antitumor, antiproliferative, anti-metastatic, antimigratory, or
apoptotic properties in vitro or in vivo and suggest their potential anticancer effects.
4.1 Cytotoxicity Effect
The extracts and isolate of pure compounds of different Dendrobium species have
capacities to kill the growth of various types of cancer cells. Even if some researches
have shown preliminary anticancer effects of Dendrobium, the specific mechanisms
have not been fully clarified. The various solvent extracts of D. amoenum,
D. chrysanthum, D. crepidatum, D. formosum, D. longicornu, D. moniliforme
(synonym, D. candidum), and D. signatum have in vitro anticancer effect on
different types of human cancer cell lines, cervical cancer (HeLa) and brain tumor
(U-251) [32, 35, 62, 63], colon cancer (HCT-116) [66], breast cancer (MCF-7) [65],
400
Table 1 Isolates of different Dendrobium species and their anticancer effect on different cancer cell lines
Dendrobium Extract/active
species compound isolate Biological target IC50 value Activity Ref.
D. amoenum Methanol extract HeLa cell line 110.22 μgmL1 Anticancer [32]
U-251 cell line 550.55 μgmL1
D. brymerianum Compound 25 H460 cell line 196.7 μgmL1 Cytotoxic, [33]
Compound 23 23.4 μgmL1 antimigratory
Compound 32 65.0 μgmL1
Compound 36 125.8 μgmL1
D. catenatum Protein extract HepG2, SGC-7901, MCF-7 cell lines 73.38%, 78.91%, 86.8% Cytotoxic, [34]
Peptides 21.72–33.42% antiproliferative
27.81–33.94%
30.02–41.80%
D. crepidatum Methanol extract HeLa cell line 194.14 μgmL1 Anticancer [35, 36]
U-251 cell line 301.99 μgmL1
Ethanol extract T-cell lymphoma 325 μgmL1
D. chrysanthum Ethanol extract T-cell lymphoma 400 μgmL1 Anticancer [23, 36]
Compound 25 FaDu cell line 2.55 μM
D. chrysotoxum Compound 17 T47D, 143B, HUVECs, HeLa, MG63.2 cell 80–160 nM, 58.19 nM, 34.2 nM, Antiproliferative, [37–41]
lines 8.3 μM, 88.69 nM apoptosis,
Compound 35 K562, HL-60, A549, BEL-7402, SGC-7901 cell 32.18, 10.39, 18.40, 1.49, 15.48 cytotoxic,
lines μgmL1 antimigratory
Compound 36 K562, HL-60, A549, BEL-7402, SGC-7901 cell 26.65, 10, 9.03, 0.97, 5.53
lines μgmL1
Compound 27 A549, HL-60 cell lines 81.1%, 65.0%
(104 mol L1)
(104 mol L1)
M. R. Paudel et al.
16

(104 mol L1)
(104 mol L1)
(104 mol L1)
(104 mol L1)
(104 mol L1)
D. denneanum Polysaccharides S180 induced mice 72.04% Antitumor [42]
D. densiflorum Compound 29 H460 cell line >50 μM Anticancer, [43]
apoptosis
D. draconis Compound 23 H460 cell line >20 μM Antimigratory, [44–47]
apoptosis
D. ellipsophyllum Compound 5 H460, H292, H23 cell lines 100, 100, 188.89 μM Apoptosis, [48–50]
antimigratory
D. falconeri Compound 12 H460 cell line >10 μM Antimigratory [51, 52]
D. fimbriatum Compound 3, 18– HL-60, SMMC-7721, A-549, SW480, MCF-7 2–21.23 μM Cytotoxic [53]
22, and 25 cell lines
D. formosum Ethanolic extract T-cell lymphoma 350 μgmL1 Antitumor [54]
D. gratiosissimum Compound 14, 15, HL-60 2.1, 6.4, 0.082, 10.6 μM Cytotoxic [55]
23, 25
Traditionally Used Medicinal Dendrobium: A Promising Source of. . .
D. loddigesii Compound 25 HUVECs, A-549, H23, MDA-MB-231, 4.5–20 μM Antiproliferative, [56–61]

HCT-116, SCC, A375 cell lines antiangiogenesis,
antimigratory,
apoptosis
D. longicornu Acetone extract U-251 cell line 620.56 μgmL1 Anticancer [62]
Ethanol extract HeLa cell line 294.70 μgmL1
(continued)
401
402
Table 1 (continued)
Dendrobium Extract/active
species compound isolate Biological target IC50 value Activity Ref.
D. moniliforme Methanol extract HeLa, HCT-116, MCF-7 cell lines, 26-M3.1 155.80 μgmL1, 1–2 mgmL1 Anticancer, [63–71]
induced mice cytotoxic,
Ethanol extract U-251 cell line 772.50 μgmL1 anti-metastasis,
Compound 30 K562 cell line 1.84 μM antiproliferative,
Apoptosis
Compound 33 HSC-T6, BNL CL.2, PC-3 and DU-145 cell 0.5–3.0 μM
lines
D. nobile Compound 30 K562, PC-3, SNU-484, SK-Hep-1, HeLa cell 1.84, 7.5, 7.9, 16.4, 22.3 μM Cytotoxic, [69,
lines antimigratory 72–77]
Denobilone A HeLa, MCF-7, A549 cell lines 9.8, 9.4, 9.9 μM
Lactone derivatives HeLa, MCF-7, A549 cell lines 18.2–25.3, 15.3–30.0, 19.2–
28.2 μM
Polysaccharides S180 induced mice, HL-60, HepG2 cell lines 2.5 mgmL1, 200 μgmL1
D. officinale Aqueous extract MNNG-induced gastric tumorigenesis in rats 2.4–4.8 g Apoptosis, [22, 78,
Compound 4, 9, 11, HeLa cell line 8.0–92.4 μM anticancer 79]
16, 23, 25
Polysaccharides SGC-7901 xenograft mice <1.0 g
D. polyanthum Compound 25 A549, HL-60 cell lines 67.5%, 53.0% Cytotoxic [80]
(104 mol L1)
M. R. Paudel et al.
16
D. pulchellum Compound 8 H460, H23 cell lines 127.34, 145.47 μM Anti-metastasis [59, 81,
Compound 25 H23 cell line 33.41 μM 82]
D. signatum Ethanol extract MCF-7, NCI-H187 cell lines – Cytotoxic [83, 84]
Compound 13 MDA-231, HepG2, HT-29 cell lines 25.2, 51.3, 30.4 μM
Compound 2 80.5, 137.8, 88.2 μM
Compound 9 25.9, 49.6, 29.3 μM
Compound 10 42.5, 89.7, 52.0 μM
Compound 12 30.3, 50.3, 35.7 μM
D. sinense Compound 1 SGC-7901, BEL-7402, K562 cell lines 16.7, >30, >30 μM Cytotoxic [85]
Compound 7 12.8, >30, >30 μM
Compound 6 7.8, 11.7, 15.7 μM
Compound 24 >30, 10.0, 10.3 μM
Traditionally Used Medicinal Dendrobium: A Promising Source of. . .
403
T-cell (Dalton’s) lymphoma [36, 54], and MCF-7 and lung cancer (NCI-H187)
[84]. The extract has induced the apoptosis on HCT-116 cell line by chromatin
condensation and formation of apoptotic bodies. During the apoptosis, the expres-
sion of caspase 3, caspase 9, and Bax was made elevated, whereas the expression of
Bcl-2 and pro-inflammatory COX-2, iNOS, and NF-κB was made low [66]. The
growth inhibition mechanism of the MCF-7 cell by the extract was induced by
enhancing the cell cycle arrest in G2/M phase and regulating the key biomarkers
(tumor growth-associated biomarkers including Erα, IGPBP2, IGFBP4, and GATA3
and apoptosis-associated biomarkers including ELF5, p53, p21, p18, CDH1, CDH2,
and p12) [65]. Also, treatment with D. candidum at any concentration and any time
point caused no inhibitory effect on cell proliferation of normal breast epithelial
(MCF10A) cell line [65].
The majority of the anticancer effect of extracts deal with in vitro evaluations.
Only a few of them were deepened in vivo. D. candidum extract is effective in the
prevention of chemically induced colon carcinogenesis in C57BL/6 mice by increas-
ing the serum SOD level and decreasing the levels of pro-inflammatory cytokines
IL-6, IL-12, TNF-α, and IFN-γ [67]. The ethanolic extract of D. formosum has
cytotoxic activity on T-cell (Dalton’s) lymphoma bearing mice. It has significant
increase in apoptotic cell death in a dose- and time-dependent manner by arresting
the cells in the G2/M phase of the cell cycle with a treatment of 150 mg/kg body
weight [54]. Similarly, the gastric carcinogenesis in rats was inhibited by oral
administration of D. officinale extract (4.8 and 2.4 g/kg). The extract could down-
regulate the expression of malondialdehyde (MDA) and 8-hydroxy-2-
deoxyguanosine and upregulate the activity of glutathione peroxidase as well as
IL-2 during N-methyl-N0 -nitro-N-nitrosoguanidine (MNNG)-induced gastric tumor-
igenesis in rats. It also reduced the level of inflammatory cytokines including
activin A, Agrin, IL-1α, ICAM-1 (intracellular adhesion molecule-1), and TIMP-1
(tissue inhibitor of matrix metalloproteinase-1) and increased the level of IL-10.
Additionally, it increases the protein level of Bax and caspase-3 and decreases the
expression of Bcl-2 [79]. D. officinale polysaccharide can inhibit the growth of
human gastric cancer cell (SGC-7901) xenograft in nude mice [78].
Mainly, the bibenzyl (see Fig. 1), phenanthrene (see Fig. 2), and fluorenone (see
Fig. 3) derivatives isolated from different Dendrobium species seem to be very active
in promising anticancer compounds. There are more than 25 bibenzyl derivatives
have been isolated from Dendrobium species which have a potential anticancer
effect on different types of cancer cell lines. Compound 17, a promising anticancer
bibenzyl compound, has been isolated from the stems of D. chrysotoxum. It has an
antitumor effect in estrogen receptor (ER) positive breast cancer cells (T47D), and its
effect has been evaluated on multiple cancer-associated pathways. Besides, com-
pound 17 induced apoptosis in T47D cells by reducing Bcl-2 expression and
activating caspase signaling and also suppressed the expression of CDKs and caused
cell cycle arrest. Meanwhile, compound 17 did not affect the proliferation of
MCF10A cell line [41]. Compound 17 inhibited the growth of HeLa cells and
induced apoptosis in a dose- and time-dependent manner, inducing cell cycle arrest
at the G2/M phase. Its treatment increased the expression of Bax and caspase-3 but
decreased levels of Bcl-2 and phosphorylated-ERK1/2 [39]. The effect of compound

17 on osteosarcoma (OS) cell lines (143B and MG63.2) was also explored and
further elucidated the underlying molecular mechanisms of action. It inhibits cell
proliferation and induces cell cycle G2/M arrest by regulating cell cycle-related
proteins. Furthermore, it induced cell apoptosis by activating both extrinsic (expres-
sion of downstream apoptotic protein caspase-8) and intrinsic (caspase-9) pathways
[40]. Bibenzyl derivatives like compounds 4, 9, 11, 14, 15, 16, 23, and 25 obtained
from D. brymerianum, D. gratiosissimum, and D. officinale possessed significant
cytotoxicity against human lung cancer (H460), HL-60 cells, and HeLa cell lines
[22, 33, 55]. Similarly, compounds 1, 6, 7, and 24, isolated from D. sinense, have
possessed different degrees of cytotoxicity on human gastric carcinoma
(SGC-7901), human hepatocellular carcinoma (BEL-7402), and chronic myeloge-
nous leukemia (K562) cell lines [85]. Bibenzyls derivatives from D. fimbriatum such
as compound 18–22 exhibited broad spectrum and cytotoxicity with five human
cancer cell lines: promyelocytic leukemia (HL-60), hepatocellular carcinoma
(SMMC-7721), lung carcinoma (A-549), breast cancer (MCF-7), and colorectal
adenocarcinoma (SW480) [53]. Furthermore, compounds 3 and 25 also have the
most cytotoxic effect from the same plant [53]. Additionally, bibenzyl derivatives,
compounds 2, 9, 10, 12, and 13, isolated from the whole plant of D. signatum, have
appreciable cytotoxic activity against three human cancer cell lines, including breast
cancer (MDA-23), liver hepatocellular carcinoma (HepG2), and colorectal adeno-
carcinoma (HT-29) cells [83]. Compound 5 from D. ellipsophyllum had demon-
strated high cytotoxicity on lung cancer cells (H23, H460 and H292) and revealed a
significant increase of early and late apoptosis with an absence of necrosis cell death.
The upregulation of a tumor repressor protein p53 was elucidated in lung cancer
cells [48].
Compound 30, promising phenanthrene, isolated from D. nobile and
D. moniliforme, has anticancer effect in many cancer cell lines, including pancreatic
adenocarcinoma, leukemia, and glioblastoma [64, 73]. It induces human glioblas-
toma multiforme (GBM) cell apoptosis through IκB kinase inactivation, followed by
Akt and fork head in rhabdomyosarcoma dephosphorylation and caspase-3 activa-
tion signaling cascade [86]. Furthermore, it induces apoptosis in lung and colorectal
cancer cell via Akt inactivation, Bad activation, mitochondrial dysfunction,
apoptosis-inducing factor releasing, and DNA damage [87, 88]. The combination
of compound 30 with Fas ligand reduces the concentration of Fas ligand needed to
activate caspases and cell apoptosis. This compound inhibits nuclear factor-κB and
induces apoptosis via ROS generation, and this effect takes place in a MAPK-
independent pathway [64]. Also, compound 30 has been reported to increase the
levels of tubulin polymerization and deregulation of Bcr-Abl signaling to inhibit
human leukemia (K562) cell proliferation. Furthermore, it significantly suppressed
the expression of Bcr-Abl and phosphorylation of CrkL, a crucial tyrosinase kinase
and an adaptor protein in chronic myeloid leukemia, respectively [69]. Similarly,
phenanthrene compound 27–29, 31–34 obtained from D. chrysotoxum,
D. densiflorum, D. moniliforme, and D. brymerianum possessed significant cytotox-
icity against several human cancer cell lines [33, 38, 43, 71].
Compound 35 and 36, fluorenone derivatives obtained from D. chrysotoxum and

D. brymerianum, possessed significant cytotoxicity against different human cancer
cell lines: lung (H460), leukemia (K562 and HL-60), lung (A549), hepatoma
(BEL-7402), and stomach (SGC-7901) [33, 37].
4.2 Anti-metastasis Effect
Metastasis is dissemination of cancer cells from primary tumors to secondary sites

through the blood and lymphatic system and forms new tumors on other parts. It
consists of several steps involving detachment of malignant transformed cells from
the primary tumor site, attachment to the extracellular matrix (ECM), degradation of
the ECM and basement membrane (invasion), cell migration, and establishment of
secondary tumors [89]. It is the leading cause of mortality among cancer patients.
Epithelial to mesenchymal transition (EMT) is the hallmark of cancer metastasis
[46]. EMT is a process during which epithelial cells acquire migratory and invasion
ability [59]. The change of cancer cells from epithelial to mesenchymal phenotypes
facilitates the aggressiveness of cancer [90]. Several signaling proteins are known to
be responsible for metastasis, such as a protein tyrosine kinase called focal adhesion
kinase (FAK), ATP-dependent tyrosine kinase (Akt), cell division cycle 42 (Cdc-42),
metalloproteinases (MMPs), Ras-related C3 botulinum toxin substrate 1 (Rac-1),
E-cadherin, N-cadherin, vimentin, slug, and caveolin-1 (Cav-1) [12, 26]. EMT was
suppressed by compound 12 in expression from E-cadherin to N-cadherin and a
decrease in the protein expression level of slug and vimentin [51]. It also suppressed
Cav-1, which is a protein implicated in aggressiveness, and downregulated Akt in
lung cancer (H460) cells. The expression of migration-related integrin, including
integrin β-1 and integrin α-4, was significantly reduced in response to compound 12
[52]. The migration and invasion of lung cancer (H292) cells by compound 5 were
reduced by decreasing migration-regulating proteins, including integrins αv, α4, β1,
β3, and β5, as well as FAK, Rac-1, and Cdc-42 [49, 50]. Compound 25 can attenuate
migration and invasion in human lung cancer cells (H23) associated with an atten-
uation of endogenous reactive oxygen species (ROS), in which hydroxyl radical
(OH•) was identified as a dominant species [59]. It also suppresses the migration and
metastasis of human breast cancer (MDA-MB-231) and lung cancer (H460) cells by
inhibiting mRNA and protein expression of Twist, Akt phosphorylation, N-cadherin,
and Cav-1 [33, 91]. Compound 23 has the inhibitory effect on lung cancer (H292 and
H460) cell migration, downregulation of Cav-1, and activation of Akt and Cdc-42,
thereby suppressing filopodia formation. Besides, it greatly decreases EMT markers,
including N-cadherin, vimentin, and slug, leading to significant suppression of
protein kinase B, extracellular signal-regulated kinase, and Cav-1 survival pathways
during the detached condition [33, 44–47]. Compound 30 suppresses invasion and
metastasis in human gastric cancer (SNU-484) and prostate cancer (PC-3) cells.
Tumor invasion and metastasis are often associated with the enhanced synthesis
and/or activation of matrix-degrading enzymes Rac-1 and matrix metalloproteinases
(MMPs), among which MMP-2 and MMP-9 are of central importance [72, 73,
86]. Increased FAK activation is tightly associated with enhanced migratory behav-
ior and cancer metastasis, and FAK signaling regulates the formation and turnover of
focal adhesions in cells in a moving state [44]. Likewise, increased Akt phosphor-
ylation is associated with metastasis behavior in some cancer cells and has been
shown, in certain cases, to be the downstream effector of FAK [44]. Recent evidence
suggests that Cav-1 plays an essential role in cancer aggressiveness and metastasis,
and its overexpression is closely associated with increased cancer migration. High
level of Cav-1 is found in many cancer types, and overexpression of Cav-1 results in
increased motility of human lung cancer (H460) cells, while knockdown of the
protein causes the opposite effect [47, 51].
Only two isolated compounds from Dendrobium species were evaluated in vivo:
the antimigratory and anti-metastatic effects of compound 25 on human breast
cancer in an MDA-MB-231 metastatic model. Compound 25 (100 mg/kg) signifi-
cantly suppresses breast cancer metastasis to the lungs and reduced the number of
metastatic lung nodules and lung weight without causing any toxicity [91]. An
in vivo orthotopic osteosarcoma (OS) model was established by intra-tibial injection
of OS 143B cells to confirm the antitumor effect of compound 17. The mice were
injected with 5% DMSO intraperitoneally every other day for seven times in total.
Compound 17 markedly inhibited the growth of OS with no major organ-related
toxicity [40]. Besides the in vitro evaluation of D. candidum extract, it has anti-
metastatic effect in BALB/c mice with tumors propagated by the injection of colon
carcinoma cells (26-M3.1). It has reduced the serum cytokine levels of IL-6, IL-12,
TNF-alpha, and IFN-gamma to a greater extent. The most prominent anti-metastatic
effect of extract has been associated with the marked decrease in expression of
MMP-2 and MMP-9, together with a marked increase in expression of TIMP-1 and
TIMP-2 [66, 68].
4.3 Antiangiogenesis Effect
Angiogenesis is a process that is known as the formation of new blood vessels with
the help of existing blood vessels and to play a major role in cancer growth and
metastasis [92]. Therefore, angiogenesis is an important factor in the progression of
cancer. Angiogenesis is stimulated when tumor tissues require nutrients and oxygen.
New blood vessels can feed growing tumors with nutrients and oxygen, allowing
cancer cells to spread (metastasis). Angiogenesis has a four-step process: (i) the
basement membrane in tissue is injured locally, (ii) endothelial cells activated by
angiogenic factors migrate, (iii) endothelial cells proliferate and stabilize, and
(iv) angiogenic factors continue to influence the angiogenic process [26]. It is
regulated by both activator and inhibitor molecules. However, upregulation of the
activity of angiogenic factors is itself not sufficient for angiogenesis. Many different
proteins have been identified as an angiogenic factor, including vascular endothelial
growth factor (VEGF), basic fibroblast growth factor (bFGF), platelet-derived
growth factor (PGF), angiogenin, transforming growth factor-α (TGF-α), TGF-β,
and tumor necrosis factor-α (TNF-α) [12, 26, 92]. Among these, VEGF and its
receptors play an important role in angiogenesis. For in vitro angiogenesis assays,

human umbilical vein endothelial cells (HUVECs) and human dermal microvascular
endothelial cells have commonly been used as a model system. The effects of
compound 25 were assessed on VEGF- and bFGF-induced angiogenesis in cultured
HUVECs in vitro and in vivo [56]. It significantly inhibited the growth of lung
cancer cell line (A549) and suppressed growth factor-induced neovascularization.
Moreover, VEGF- and bFGF-induced cell proliferation, migration, and tube forma-
tion of HUVECs were markedly inhibited by compound 25 [57]. Compound 25
impeded angiogenesis by suppressing the activation of VEGF receptor 2 (Flk-1/
KDR) and c-Raf-MEK1/2-ERK1/2 signals [93]. Compound 17 downregulates the
expression of inflammation factors through the regulation of IDO-induced tumor cell
angiogenesis mimicry and endothelial cell-dependent angiogenesis by targeting
JAK2/STAT3 pathway [94]. Compound 17 induced depolymerization of F-actin
and β-tubulin more prominently in proliferating endothelial cells. It also inhibits
high glucose-induced retinal angiogenesis by blocking ERK1/2-mediated HIF-1α
and suppressing VEFG-induced activation of VEGFR2 [95, 96].
5 Production of Anticancer Compounds Through In vitro

Culture of Dendrobium Species
Many Dendrobium species are highly demanded in traditional herbal medicine, and
they are the sources of modern anticancer compounds as described earlier. The wild
resources of them have been depleted by overexploitation to meet their demand for
medicinal use and health products [97, 98]. In general, plant cell and tissue culture
techniques provide an alternative way to produce clonal plants for mass production
as well as to conserve germplasm for future uses [99, 100]. Also, this is an excellent
way to produce quality plant materials for agriculture, forestry and horticulture, and
bioactive secondary metabolites for pharmaceutical industries [101]. Recently, this
technique has received greater attention for producing plant-specific bioactive com-
pounds which have applications in pharmaceutical, cosmetic, and nutraceutical
industries [101, 102]. Efforts have been made for the establishment of cell, callus,
protocorm-like bodies (PLBs), and organ cultures of some Dendrobium species for
the production of value-added compounds [103–107]. The use of bioreactors for
large-scale cultivation of cells, calluses, and PLBs of some Dendrobium species has
become feasible for the production of bioactive secondary metabolites [108,
109]. By contrast, biomass production for harvesting bioactive compounds is
aimed at maximizing the growth of callus, PLBs, and tissues containing high
amounts of bioactive metabolites [108]. Some stress signaling substances such as
salicylic acid, methyl jasmonate, and thidiazuron is added to the culture medium for
increasing the content of useful secondary metabolites like alkaloids, polysaccha-
rides, and aromatic compounds [103, 108, 109]. However, pure compounds have not
been isolated so far from the in vitro culture of Dendrobium species. Preliminary
research showed anticancer activity of in vitro culture of D. amoenum,
D. crepidatum, and D. longicornu toward cervical (HeLa) and brain tumor (U251)
cell lines [110]. This paves the pathways for the future potential of promising
anticancer compounds from in vitro raised tissue of Dendrobium species. Thus,
plant cell and tissue culture could be an alternative and suitable tool for improve-
ment, enhancement, and production of desirable bioactive compounds which also
help to minimize the pressure on the natural population of Dendrobium species and
result in their sustainable utilization.
6 Conclusion
Organic extracts and isolated compounds, with various chemical structures from
different Dendrobium species, explored to have an anticancer effect toward different
cancer cells in vitro and/or in tumor-bearing mice in vivo. Altogether 36 anticancer
compounds under three groups, bibenzyl, phenanthrene, and fluorenone, have been
isolated from Dendrobium species, among them, 26 compounds of bibenzyl, 8 com-
pounds of phenanthrene, and 2 compounds of fluorenone derivatives. Inhibition of
the cancer cell proliferation, induction of apoptosis, suppression of metastasis, and
angiogenesis have been discussed in detail of the aforementioned compounds. Out
the 36 compounds, 5, 17, 23, 25, and 30 have been largely discussed in vitro and
in vivo anticancer effect on the different cancer cell lines. The application of tissue
culture technique has proven as an alternative strategy for the production of anti-
cancer compounds, especially when the plant resources are overexploited. Impor-
tantly, in some cases, the anticancer principles are devoid of cytotoxicity toward
normal cells, and their potencies relative to anticancer drugs currently in use may not
all be known, but hopefully, some of these anticancer principles can be developed
into chemopreventive therapeutics.
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A Review on Phytochemistry, Nutritional
Potential, Pharmacology, and Conservation 17
of Malaxis acuminata: An Orchid with
Rejuvenating and Vitality Strengthening
Properties
Renu Suyal, Sandeep Rawat, R. S. Rawal, and Indra D. Bhatt
Contents
1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 416
2 Botanical Description of the Species . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 417
3 Habitat, Distribution, and Ecology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 418
4 Medicinal and Other Uses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 419
5 Nutritional Composition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 420
6 Bioactive Compounds Isolated from M. acuminata . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 422
7 Biological and Pharmacological Activities . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 423
7.1 Antioxidant Activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 423
7.2 Antiaging Activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 424
7.3 Sun Protection Factor (SPF) and UV-A Blocking Activity . . . . . . . . . . . . . . . . . . . . . . . . . . 424
7.4 Anti-Inflammatory Activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 425
7.5 Antiproliferative Activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 425
7.6 Antimicrobial Activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 425
8 Propagation and Cultivation Effort . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 426
8.1 Vegetative Propagation Techniques . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 426
8.2 In Vitro Propagation Techniques . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 427
9 Future Prospective and Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 428
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 429
Abstract
Malaxis acuminata is one among the 300 species of the genus with medicinal
properties and hence used in traditional Indian medicine system. The species is a
perennial, monopodial, threatened terrestrial orchid distributed in moist ground
and in rocks laden with mosses in south Asia including, Himalaya and southern
R. Suyal · R. S. Rawal · I. D. Bhatt

G.B. Pant National Institute of Himalayan Environment, Almora, Uttarakhand, India
e-mail: idbhatt@gbpihed.nic.in
S. Rawat (*)
G.B. Pant National Institute of Himalayan Environment, Sikkim Regional Centre, Gangtok,
Sikkim, India
e-mail: sandeep_rawat15@rediffmail.com

416 R. Suyal et al.
Indian hills, Australia, and western region of South America. Medicinally, the
species is used in Ayurvedic formulations in the preparation of energetic tonic
with adaptogenic, immunomodulating, rejuvenating, and other health benefits.
Various essential nutrients and pharmacological compounds are identified and
detected in the pseudobulb of the species. The species has been successfully
validated for antioxidant, antiaging, UV-A blocking, anti-inflammatory, anti-
proliferative, and antimicrobial activities, which supported its traditional use
also. Propagation methods for large-scale multiplication of the species are avail-
able but need further refining for robustness for farming purposes. Various
research gap areas and possible research areas for harnessing the potential of
the species have been highlighted in the end of the chapter.
Keywords
Astavarga · Anti-aging · Malaxis · Himalaya · Medicinal plant · Orchid · Vitality
strengthening
Abbreviations
A/F Abundance and frequency ration
AAE Ascorbic acid equivalent
ABTS 2,2-Azinobis (3-ethylbenzoline-6-sulfonic acid) radical scavenging
assay
BA 6-Benzylaminopurine
CITES The Convention on International Trade in Endangered Species of Wild
Fauna and Flora
DPPH 1,1-Diphenyl-2-picrylhydrazyl radical scavenging assay
IBA Indole-3-butyric acid
IC50% Inhibitory concentration.
LOX Lipoxygenase
NAA 1-Naphthaleneacetic acid
NCBI National Center for Biotechnological Information
ppm Parts per million
ROS Reactive oxygen species
SPF Sun Protection Factor
TDZ Thidiazuron
1 Introduction
Malaxis is a genus of orchids that prefer terrestrial, epiphytic, or occasionally

holomycotrophic habitats in mountainous region. The genus is distributed world-
wide and consists of more than 300 species, but its diversity is highly concentrated in
southeast Asia. Among the 19 species found in India, Malaxis acuminata D. Don.
(Syn. Crepidium acuminatum D. Don. Szlach.) is a perennial, monopodial,
17 A Review on Phytochemistry, Nutritional Potential, Pharmacology, and. . . 417
medicinally important threatened terrestrial orchid [1]. The herb is found in temper-
ate and subtropical region on moist, shady places in the pine, oak forests, moist
ground, and in rocks laden with mosses within an altitude between 1200 and 2200 m
asl [2]. The species is found in mountain grasslands of south Asia, Australia, and
western region of South America [3–5]. Pseudobulbs of the species are important
ingredients of the traditional Indian Ayurvedic formulation “Chyavanprash” [4],
which is considered as a polyherbal energetic tonic with adaptogenic, immunomo-
dulating, rejuvenating, and other health benefits [6].
Traditionally, in India, pseudobulbs of M. acuminata are used in the treatment of
several diseases such as seminal debility, internal and external hemorrhages, dysentery,
fever, emaciation and weakness, forcing its collection from wild habitats to be utilized
in pharmaceutical industries. Generally, due to rapid loss of specific habitats and
overexploitation, several orchids are listed in Appendix II of the Convention on
International Trade in Endangered Species of Wild Fauna and Flora (CITES) [7].
The objective of this chapter is to summarize all the research findings available on
various aspects, such as botanical description and distribution, ethnopharmacology,
phytochemistry, propagation, and conservation measures of M. acuminata. Also, a
systematic approach for conservation and sustainable utilization of the species has
been suggested. Based on all the compiled information, research gap has also been
discussed. This chapter provides the basis for further studies on conservation and
development of identifying better therapeutic agents and health products from the
species.
2 Botanical Description of the Species
M. acuminata is a perennial, medium-sized terrestrial orchid with a height of 30 cm

and consists of pseudobulbs and fibrous roots at the base. The plant is erect and has a
small stem composed of aerial flowering axis and basal swollen stem (pseudobulbs),
bearing nodes and internodes arising from the base of mother rhizome. Several
adventitious roots arise below this rhizomatous structure, firmly anchoring the
plant into the soil. Rhizomatous structure and pseudobulbs are covered by sheathing
leaf bases, which appear marginally on nodes. The leaves are simple, three to four in
number, alternate, ovate to lanceolate, membranous, 5–15-cm long, and has an acute
apex with a sheathing leaf base [8]. Leaf constants recorded as, palisade ratio as ~0.8,
stomatal index ~8.4, stomata number ~70 nos., stomata size ~0.03 0.03 mm, and
anomocytic type of stomata [9]. The flowers are terminal racemes, yellowish green
in color with a purple tinge, and 3 mm in diameter [10, 11]. Fruit is capsule, seeds
minute and powdery, and ovoid in shape. Pseudobulbs are 3–9 cm long, fleshy,
smooth, shining, greenish, covered with membranous sheath, and slightly mucilag-
inous [4, 8]. The plant flowers from July to August and fruiting takes place from
September to October [10]. Anatomical and histochemical studies revealed the
presence of endophytic mycorrhizal fungus in the root and protocorm, and the
endophytic bacterium (probably Corynebacterium) detected in the leaves and root
418 R. Suyal et al.
Fig. 1 Plant of Malaxis acuminata along with inflorescence and pseudobulbs
hairs [9]. Anatomical similarity between rhizomes and pseudobulbs indicates that
species can be propagated from its rhizomes as well as pseudobulbs (Fig. 1).
3 Habitat, Distribution, and Ecology
M. acuminata grows in group that contains 5–25 individuals in moist, shady, and
humus-rich forest floors and forms symbiotic relationship with mycorrhizal fungi
that nourishes this species [7]. In India, this species is distributed in Himalaya from
Jammu Kashmir to Arunachal Pradesh, including states such as Himachal Pradesh,
Uttarakhand, Assam, Nagaland, Manipur, Mizoram, and Tripura [4]. In India, the
species also been reported in Andaman Islands, Kerala, Anaimalai Hills, east
Godavari district of Andhra Pradesh, and Madhya Pradesh [3, 4, 12, 13]. Outside
India, the species is found in Myanmar, Thailand, Malaysia, Laos, Cambodia,
Southern China, the Philippines, Australia, Peru, and Ecuador in mountain grass-
lands [3, 5].
Studies on quantitative assessment and ecologocal studies of the threatened
medicinal plant along with the assessment of availability, growth preferences,
distribution pattern and habitat preference are vital steps for development of conser-
vation measures [14]. However, only few studies are available on ecological aspect
confined to Indian Himalayan region (Table 1), which shows low population density
across the surveyed populations and indicates poor availability of the species in
the wild. Loss of habitat and illegal collection puts this species at higher risk
of endangerment [15]. Dominant associates reported for M. acuminata at most
sites are Roscoea procera, Thalictrum foliolosum, Valeriana jatamansi, Rumex
Table 1 Quantitative assessment of M. acuminata in different regions

Density
(plant/ Frequency
Region Habitat types m2) (%) A/F References
Uttarakhand Banj Oak, mixed 1.9–2.4 13–23 – [16]
forest and oak-pine
Kumaun Himalaya Quercus 1.70– 50–90 0.03– [7]
leucotrichophora and 15.30 0.24
Cedrus deodara
Arakot-Khadikal Moist, shady, and 0.2 20 – [17]
forest, Tehri humus-rich forest
floor
Chail Wildlife Different type 2.0 10 0.2 [18]
Sanctuary,
Himachal Pradesh
nepalensis, Oxalis corniculata, Asparagus curillus, Potentilla fulgense, and Polyg-

onum species [7, 16–18]. However, other regions of the world remain unexplored in
this context, which is needed to formulate a global strategy for conservation of the
species.
4 Medicinal and Other Uses
M. acuminata has been used in traditional Indian medicine system in the preparation of
various polyherbal formulations and tonics and in folklore medicines. Pseudobulbs of
the species are used in the preparation of Chyavanprash and other Ayurvedic formu-
lations like Astavarga, Astavarga-churna, Chitrakadi-taila, Vachadi-taila, Mahakalyan-
taila, Jivaniya-ghrita, Mahamayura-ghrita, Vajikarma-taila, Brahini-gutika, and
Himvana agada [4]. Among these, Chyavanprash is a well-recognized formulation
that is used as immunomodulator, health promoter, rejuvenator, and brain tonic due to
its antiaging, antioxidant, cardioprotective, and adaptogenic properties [6, 19]. Ayur-
vedic properties of the plant species has been described as, sweetness in taste, cold in
potency, pacifies vata and aggravates kapha [20]. Pseudobulbs of M. acuminata are
considered as sweet, refrigerant, and febrifuge [4].
Traditionally, paste of pseudobulbs is applied topically for insect bites and is used
in the treatment of rheumatism along with other herbal plants [21]. Its swollen stem
is considered as refrigerant, aphrodisiac, styptic, anti-dysenteric, febrifuge, and
tonic. Decoction of bulb is used to increase the quantity of semen or to stimulate
the production of semen [22]. Pseudobulbs are used in the treatment of skin diseases,
piles, and burning sensation [7]. Pseudobulbs and rhizomes are used for bronchitis
and is used as a tonic by local people of Uttarakhand [23]. It is used to treat sterility,
vitiated condition of pitta and vata, seminal weakness, internal and external hemor-
rhages, dysentery, fever, emaciation, and general debility [24]. Rhizome and pseudo-
bulbs of the species are edible and are consumed in northeastern India [9, 25].
Various medicinal uses of the species described in the literature are given in Table 2.
420 R. Suyal et al.
Table 2 Medicinal uses of M. acuminata reported in different parts of the world

Medicinal uses Parts used Utilized form Reference
Burning sensation, bronchitis, fever, Rhizome, root, Dry powder [26–28]
tuberculosis, and weakness pseudobulb
Tonic and bleeding diathesis Pseudobulbs Fresh [5]
Bronchitis and tonic Pseudobulbs Fresh [23]
Refrigerant and febrifuge Pseudobulb – [29]
Cooling, spermopiotic, analgesic, internal Pseudobulb [30]
and external hemorrhages
Rheumatism Pseudobulbs Mixture with [21]
other plants
Aphrodisiac Pseudobulbs Powder [31]
Styptic, anti-dysenteric, emaciation Swollen stem – [24]
5 Nutritional Composition
The edible pseudobulbs of the species are rich in essential nutrients, minerals,
vitamins, and other metabolites (Table 3). Analysis of pseudobulbs shows the
following: total ash content between 1.49 and 6.9 (% w/w), moisture content
6.8%, total fat content 1.45%, and carbohydrate content 112 μg/ml [10, 32]. Pseudo-
bulbs are also rich in valuable minerals such as copper (6.48 ppm), zinc (43 ppm),
manganese (35 ppm), iron (331 ppm), potassium (21,600 ppm), calcium
(9000 ppm), magnesium (2800 ppm), and aluminum (198 ppm) [33]. High levels
of important vitamins are also reported in the pseudobulbs, specifically α-tocopherol
(12.00–9.80 mg/100 dw) and γ-tocopherol (695.00–786.7 mg/100 g dw) [33].
Likewise, valuable classes of secondary metabolites also reported in the species.
Total phenolic contents (1.72 mg/g), total tannins (1.69 mg/g), total flavonoid
(1.71 mg/g), and total flavonol (1.81 mg/g) have also been reported in significant
quantity in the pseudobulbs [34], and these metabolites are known for their antiox-
idant, anticancer, antidiabetic, and various medicinal properties [35–38] (Table 3).
Among these important metabolites, some are thoroughly analyzed. Among the
different fatty acids, linoleic acid (18:2ω6: 61.20–65.23%), α-linolenic acid (18:3ω3,
15.50–18.10%), and oleic acid (18:1ω9, 12.00–14.87) were the major constituents;
however, palmitic acid (16:0, 6.00–5.90%) stearic acid (18:0, 2.10–2.50%), γ-linolenic
acid (18:3ω6, 2.20–1.87%), eicosanoic acid (20:0, 0.81–0.69%), eicosenoic acid
(20:1, 0.42–0.52%) and eicosadienoic acid (20:2, 0.04–0.07%) were also present in
trace amount [33]. Among these, α-linolenic acid and γ-linolenic acid are considered
as essential fatty acids, which cannot be synthesized by the human body [40]. Besides
their structural role, these essential fatty acids play a significant role in cellular
signaling and activating or inhibiting transcription factors such as NF-κB, which is
linked to pro-inflammatory cytokine production [41].
A significant amount of organic acids were also found in the pseudobulbs, and
among them. Acetic acid, propenoic acid, malonic acid, succinic acid, propanoic
acid, fumaric acid, itaconic acid, and pipecolic acid were majorly found [2]. These
Table 3 Chemical composition of pseudobulbs and leaf and stem extract of M. acuminata
S. no. Nutritional content Available content References
Nutrients
1 Total ash content (%w/w) 1.49–6.90 [9, 32]
2 pH (10% solution) 6.80 [32]
3 Moisture content (%) 53.00 [32]
4 Total fat content (%) 1.45 [39]
5 Alkaloid content (%) 5.00 [39]
6 Resin content (%) 0.90 [39]
7 Crude fiber content (%) 5.10 [39]
8 Saponin content (%) 2.00 [39]
9 Carbohydrate content(μg/g) 112.00 [39]
Minerals (ppm)
10 Cu 6.48 [33]
11 Zn 43.00 [33]
12 Mn 35.00 [33]
13 Fe 331.00 [33]
14 K 21600.00 [33]
15 Ca 9000.00 [33]
16 Mg 2800.00 [33]
17 Al 198.00 [33]
18 Ba 26.70 [33]
19 B 55.60 [33]
20 Mo 0.30 [33]
21 Cl 156.00 [33]
22 Co 1.41 [25]
Vitamins and other secondary metabolites
23 α-Tocopherol (mg/100 g) 9.80–12.00 [33]
24 γ-Tocopherol (mg/100 g) 695.00–786.70 [33]
25 Total phenolic content (mg/g) 1.72 [34]
26 Total tannins (mg/g) 1.69 [34]
27 Total flavonoid content (mg/g) 1.71 [34]
28 Total flavonol content (mg/g) 1.81 [34]
metabolites not only act as a reserve energy resource for the plants but also regulate
physiological activities of the species by adjusting pH level of the cell, modulate
cellular transport through membrane and secondary messenger of cell signaling, and
play a vital role during cold stress [42–45]. Similarly, various essential (L-valine, L-
leucine, L-threonine, L-phenylalanine, and L-methionine) and non-essential amino
acids were also recorded in pseudobulbs [2, 46]. Thus, consumption of pseudobulbs
fulfills various basic dietary requirements. However, compositional variation noted
in different studies emphasized the requirement of agro-climatic, agronomic, post-
harvesting management techniques for obtaining quality produce from the species.
In addition, variability in such qualitative and quantitative traits is always desirable
for varietal improvement through breeding programs (Table 4).
422 R. Suyal et al.
Table 4 Essential nutrients detected in the rhizome of M. acuminata

Class Compound References
Fatty acid Linoleic acid, γ-linolenic acid, oleic acid, stearic acid, [33]
hexadecanoic acid, heptadecanoic acid, palmitic acid;
eicosanoic acid, and eicosadienoic acid
Organic acids Acetic acid, propenoic acid, malonic acid, succinic acid, [2]
propanoic acid, fumaric acid, itaconic acid, pipecolic acid,
hydroxybutyric acid, malic acid, ribonic acid, hydrocinnamic
acid, shikimic acid, D-xylonolactone, and gluconolactone
Sugars and Galactose, D-glucose, D(+)-sucrose, D-mannose, fructose, β- [2]
glycosides maltose, α-D-glucopyranose, levoglucosan, methyl beta-
glucofuranoside, 15,β-galacto-pyranose, and α-D-(+)-
mannopyranose
Amino acids L-alanine, L-valine, L-leucine, L-glycine, L-serine, L- [2, 46]
and amines threonine, L-proline, L-aspartic acid, L-phenylalanine, L
glutamic acid, L-asparagine, ornithine, ethanolamine, and L-
valine
6 Bioactive Compounds Isolated from M. acuminata
Phytochemical analysis of methanolic extract of pseudobulbs, leaf, and stem showed

that they are rich in flavonoids, phenolic acid, sterols, and alcohols (Table 5). Among
the different flavonoids and phenolic acids, catechin, phloridzin, rutin, caffeic acid,
chlorogenic acid, 3-hydroxy benzoic acid, 4-hydroxy benzoic acid, protocatechuic
acid, 3-hydroxy cinnamic acid, and p-coumaric acid are found in the species [2, 34].
Also, GC-MS analysis showed the presence of different volatile compounds such as
sibutramine, limonene, diethylene glycol, p-cymene, eugenol, benzene, piperitone,
and uridine the methanolic extracts of pseudobulbs. More recently, Singh et al. (2017)
identified and characterized isorhamnetin O-glycoside, bulbophythrin A, gigantol,
batatasin III, lusianthrin, 2,3-dimethoxy-9,10-dihydrophenanthrene-4,7-diol, lipar
acid C, and 30 -O-methylbatatasin from ethyl acetate fraction obtained from methanolic
extract of the rhizomes of M. acuminate using HPLC-ESI-QTOF-MS/MS analysis.
This fraction showed strong antiproliferative activity in comparison with standard
doxorubicin against cancer cell lines, such as A549 (70.29%), DLD1 (73.12%), MCF-
7 (79.10%), and DU145 (68.65%) [47].
Similarly, dry root powder of M. acuminata extracted with 9:1 methanol/water
solution yielded several fractions and 67 compounds including diethyl phthalate,
heptadecanoic acid, methyl ester, cyclohexadecanolide, octadecanoic, diethyl
phthalate, heneicosane, hexadecane, docosane, pentadecanoic acid, and others
were detected by gas chromatography/mass spectrometry (GC-MS) analysis. Few
of them have been reported as pharmacologically active molecules [48]. Although
various compounds have been identified in the species, their quantitative assessment
and other biological activity remain unexplored. However, quantitative information
on these compounds and their hereditability, variability, and bioactivity are manda-
tory for commercialization of this product.
Table 5 Bioactive compounds isolated and identified in M. acuminata

Class Compound References
Flavonoid Catechin, phloridzin, and rutin [34]
Phenolic Caffeic acid, chlorogenic acid, ellagic acid, 3-hydroxy benzoic, 4- [2, 34]
acid hydroxy benzoic, protocatechuic acid, 3-hydroxy cinnamic acid,
and p-coumaric acid
Sterols Stigmasterol and β-sitosterol [2]
Volatile Sibutramine, limonene, diethylene glycol, p-cymene, eugenol, [2]
compounds benzene, and piperitone
Other 1,2-Benzenedicarboxylic acid, 11-hexadecenoic acid, 13- [47, 48]
compounds octadecenal, 1-butanol, 3-methyl-, acetate, 2,3-dimethoxy-9,10-
dihydrophenanthrene-4,7-diol, 2,6-diisopropylnaphthalene, 30 -o-
methylbatatasin, 6-octadecenoic acid, 8-octadecenoic acid, 9-
octadecenal, batatasin III, bulbophythrin A, butyl oleate, cerasynt,
cis-oleic acid, cyclopentadecanolide, diethyl phthalate,
cyclopentanetridecanoic acid, e-8-hexadecen-1-ol acetate,
gigantol, heneicosane, heneicosanoic acid, hydrofol acid,
Isorhamnetin o-glycoside, lignoceric acid, lipar acid C,
lusianthrin, margaric acid, methyl (6e)-6-octadecenoate, methyl
margarate, methyl ricinoleate, methyl tetradecanoate, n-
octadecanoic acid, octadec-9-enoic acid, octadecanoic acid, octyl-
thiirane, oxacyclododecan-2-one, tert-hexadecanethiol,
tetradecenal, tetradecyl-oxirane, thiirane, and triarachine
Alcohols Glycerol, ribitol, and myo-inositol [2]
7 Biological and Pharmacological Activities
Biological and pharmacological properties of isolated compounds, solvent extracts

or solvent fractions reported in the pseudobulb of M. acuminata has been described
under following sections and a summary of these properties is given in Table 6.
7.1 Antioxidant Activity
In the human body, various physiological processes are continuously mediated by

production and exchange of free radicals. During stress, higher production of these
oxidative agents is escaped by antioxidant system of the body and these oxidants
create oxidative stress through degeneration of macromolecules, resulting in aging
process along with chronic degenerative diseases such as diabetes, neurodegenera-
tive disease, cancer, Parkinson’s disease, and Alzheimer’s disease. Diet
supplemented with adequate amount of antioxidants can prevent degradation of
macromolecules and related chronic disease [49, 50]. Giri et al. [33] reported that
the pseudobulbs of the species have strong antioxidant potential, as evaluated by
various in vitro free radical scavanging and reducing assays such as, 2,2-azinobis(3-
ethylbenzoline-6-sulfonic acid) radical assay (ABTS: 4.02 mM AAE/100 g dry
weight) and 1,1-diphenyl-2-picrylhydrazyl radical assay (DPPH: 1.10 mM AAE/
424 R. Suyal et al.
Table 6 Biological and pharmacological activity of M. acuminata

Extract/
S. Pharmacological/biological Plant part experimental
No. properties used model References
1 Antioxidant activity Pseudobulbs, Methanolic [32, 34,
leaf and stem extract 39]
2 Antiaging activity Leaf and stem Methanolic [52]
extract
3 Anti-inflammatory activity Leaf and stem Methanolic [52]
extract
4 Sun protection factor (SPF) and UV- Leaf and stem Methanolic [52]
A blocking activity extract
5 Antiproliferative activity Pseudobulbs Ethanolic [47]
extract/human
cancer cell lines
6 Antimicrobial activity (against E. Pseudobulb Solvent extracts [58, 59]
coli, S. aureus, P. aeruginosa, B.
subtilis, K. aerogenes, P. mirabilis, C.
albicans
100 g dry weight), ferric reducing anti-oxidant properties (FRAPS: 1.18 mM AAE/
100 g dry weight) and superoxide scavenging assays (0.16 unit/mg dry weight).
Similarly, Garg et al. [51] reported DPPH scavenging properties and ferric ion
reducing properties of butanol extract of pseudobulbs of the species in dose-depen-
dent manner. Also, Bose et al. [52] reported that methanolic leaf extract of in vitro-
derived plants showed significant antioxidant activity (IC50: 42.66 μg/mg) compared
to standard ascorbic acid (IC50: 38.24 μg/mg), and aqueous stem extracts of wild
plant showed moderate DPPH activity(178.56 μg/mg).
7.2 Antiaging Activity
Pseudobulbs of M. acuminata are used in the preparation of vitality strengthening

and rejuvenating formulation due to its antiaging properties. Antioxidants have
ability to maintain the structural integrity of skin and thus, its use has been adopted
as an important strategy for skin glowing mechanism in order to develop anti-aging
products. Bose et al. [52] reported that methanolic leaf extract showed anti-collage-
nase activity with IC50 of 32.52 μg/ml [standard EDTA (IC50: 35.45 μg/ml)] and
very strong elastase inhibitory activity with IC50 of 32.24 μg/ml [standard oleanolic
acid (IC50: 30.56 μg/ml)], respectively. Hence, collagen and elastin are known to
maintain skin structural integrity and elasticity of skin [53]; therefore, M. acuminata
could be used as one of the potential rejuvenators and antiaging agents.
7.3 Sun Protection Factor (SPF) and UV-A Blocking Activity
Various environmental exposures such as harmful radiations (e.g., sun-light and

ultraviolet radiation) lead to oxidation of the lipids, proteins, and DNA of the
outer surface of the body. Skin as a protective covering of the body also gets
damaged by these reactive radicals or reactive oxygen species (ROS). UV-A (400–
315 nm) and UV-B (315–280 nm) radiations contribute predominantly to extrinsic
premature photoaging [54]. Plant-based formulations have been reported to absorb
ultraviolet radiations (UV-A and UV-B) and act as potential solar filters in develop-
ing new sunscreen formulations. Methanolic extracts of both wild and in vitro-
derived plants of M. acuminata showed promising UV-A blocking potentials,
whereas leaf and stem extracts at a concentration of 250 μg/ml showed 21.68 and
27.64 μg/ml in vitro SPF values. These values can be attributed to the presence of
various polyphenolic and flavonoid compounds present in the plant extracts [52].
7.4 Anti-Inflammatory Activity
In aging process, immune response with the influence of oxidative stress is chron-
ically started to degenerate, leads to chronic systemic inflammation. Various pro-
inflammatory mediators such as, cytokines and chemokines are involved in the
development of chronic inflammation and the immune-senescence process [55].
Regulation of such inflammation can be prevented at a certain level by consumption
of plant-based formulations. Among the enzymatic machineries involved in the
prevention of chronic systemic inflammation process, hyaluronidase is the key
enzyme responsible for increased inflammation, angiogenesis, fibrosis, and collagen
deposition in wound healing [56]. Bose et al. [52] showed that methanolic leaf (IC50:
14.32 μg/ml) and stem extracts (IC50: 16.2 μg/ml) of tissue culture-derived plants
exhibited promising anti-5-LOX activity [known inhibitor of 5-LOX (IC50:
2.56 0.4 μg/ml)] and strongest anti-hyaluronidase activity (IC50: 60.36 μg/ml) as
compared to oleanolic acid (IC50: 32.45 μg/ml).
7.5 Antiproliferative Activity
Sulphorhodamine B assay showed that ethanol extract and its fractions exhibited
antiproliferative activity against four human cancer cell lines, i.e., A549 (non-small
cell lung cancer cells), DU145 (human prostate carcinoma), DLD1 (human colorectal
adenocarcinoma), and MCF-7 (human breast adenocarcinoma). The ethyl acetate
fraction obtained from methanolic extract showed a potent antiproliferative activity
(A549: 70.29%), DLD1: 73.12%, MCF-7: 79.10%, and DU145: 68.65% inhibition) in
comparison with standard doxorubicin against cancer cell lines (A549: 80.13%),
DLD1: 64.45%, MCF-7: 79.82%, and DU145: 89.26% inhibition). However, ethanol
extract and its n-butanol fraction produced a moderate antiproliferative activity [47].
7.6 Antimicrobial Activity
Minimal microbial static concentration (MIC) assay showed that the ethanol and
methanol extracts of M. acuminata were found to be highly active against both P.
426 R. Suyal et al.
aeruginosa and S. aureus strains. The plants demonstrated antimicrobial properties

against Gram-negative bacterial strains [57]. Also, different extracts (hexane, chlo-
roform, ethyl acetate, ethanol, and residual aqueous) of pseudobulbs of M.
acuminata exhibited antibacterial activity against four bacterial strains: two Gram-
positive strains (Staphylococcus aureus MTCC 87 and Bacillus subtilis MTCC 121)
and two Gram-negative strains (Escherichia coli MTCC 40 and Pseudomonas
aeruginosa MTCC 424). Among these strains, E. coli (20 mm) and B. subtilis
(15.33 mm) showed maximum zone of inhibition (ZOI) in chloroform extract [58].
Similarly, Sharma et al. [59] analyzed antifungal (against Candida albicans) and
antibacterial activities of butanol extracts against Escherichia coli, Klebsiella
aerogenes, Pseudomonas aeruginosa, Proteus mirabilis, Staphylococcus aureus
and reported strong antifungal activity against Candida albicans strain with
32 mm inhibition zone at a concentration of 50 mg/mg as compared to 28 mm
inhibition zone of standard antifungal compound fluconazole in 50 μg/mg concen-
tration [59]. This antimicrobial property of the species can be utilized in the
preparation of preservative agent formulations and food products.
8 Propagation and Cultivation Effort
Propagation and cultivation efforts of M. acuminata are at preliminary level. A

summary of the multiplication and propagation techniques is given in Table 7.
8.1 Vegetative Propagation Techniques
In order to conserve and multiplicate M. acuminata in its natural habitat, Tamta et al.
[60] propagated M. acuminata by vegetative methods through nodal cuttings,
Table 7 Various research and results of in vitro propagation conducted in M. acuminata

Explant Culture media Response References
Adventitious MS+ 3 mg/L TDZ+ 0.5 mg/L 96% organogenesis; 100% [62]
shoot buds NAA; MS+ 3 mg/LTDZ shoot induction with 14.6
+0.5 mg/L NAA+ 0.4 mM shoots per explant; 96%
spermidine; MS+ 4 mg/L rooting with 3.3 roots per
IBA + 1.5 mg/L activated shoots
charcoal (AC)
Pseudobulbs MS + 1 mg/L BAP+ 1 mg/L 65% explant responded with [63]
segment NAA+ 2 g/L activated charcoal proliferation of protocorm-like
(AC) body
Seeds MS+ sucrose (3%, w/ 85% germination after [64]
v) + 4 μM NAA 135 days
Nodal MS+ sucrose (3% w/v) + 3 μM 75% survival rate with [58]
segment NAA+ 3 μM BA maximum plant height, and
number of leaves, shoots, and
roots
i.e. using tip, middle, and bottom parts and whole pseudobulb (control) in experi-
mental plots at Deodar and Oak Forests at Chakrata, Dhanolti and Mussoorie Forest
areas of Uttarakhand Himalaya. Maximum survival (up to 85%) and optimum
growth were observed in whole pseudobulb as compared to other propagules.
8.2 In Vitro Propagation Techniques
Seed germination and micropropagation studies are available for the species for
mass propagation. Arenmongla and Deb [61] used immature seeds of 7–8 weeks
after pollination to study the effect of culture condition on asymbiotic seed germi-
nation. Immature seeds cultured on MS medium containing sucrose (3%) and 4 μM
α- naphthalene acetic acid under diffused light condition (20 μ mol/m2/s) showed
85% seed germination after 135 days as compared to full light condition (40 μ mol/
m2/s). Germinated seeds were converted into protocorm-like bodies, which further
differentiated into young plantlets. Rooted plantlets with well-expanded leaves and
distinct bulbs were obtained in medium supplemented with sucrose (3%), activated
charcoal (0.3%), and NAA and BA (3 μM, each), and up to 15 shoots and protocorm-
like body were achieved. After hardening, about 75% survival was achieved after
2 months of transfer into the field.
In a study conducted by Cheruvathur et al. (2010) [62], adventitious shoot buds
were induced from internodal explants grown on Murashige and Skoog (MS)
medium supplemented with different concentrations of 6-benzyladenine (BA), kine-
tin (Kn), and thidiazuron (TDZ). TDZ at 3 mg/l induced the highest frequency (82%)
of organogenic explants. In the presence of 3 mg/l TDZ and 0.5 mg/l NAA, the
frequency of organogenesis was 96% with a mean number of 6.1 shoots per explant.
Highest frequency of shoot induction (100%) and mean shoot number per explant
(14.6) were observed on MS medium with 3 mg/l TDZ, 0.5 mg/l NAA, and 0.4 mM
spermidine, and highest frequency of rooting (96%) and mean number of roots per
shoot (3.3) were observed on MS medium with 4 mg/l indole-3-butyric acid (IBA)
and 1.5 mg/l activated charcoal (AC). In another study, using pseudobulb segments
of M. acuminata as explant on MS media, supplemented with 1 mg/L BAP, 1 mg/L
NAA, and 2 g/L activated charcoal (AC), 65% explant response was obtained with
proliferation of protocorm-like bodies [63]. Similarly, in vitro propagation of M.
acuminata using nodal segment on MS medium fortified with sucrose (3% w/v),
3 μM NAA, and 3 μM BA showed bud formation after 3 weeks of inoculation with
the following: development of 18 shoot buds; plant height, 2.4 cm; number of leaves,
4.5; and roots, 4.0 [58]. The hardened plants that were transferred to community
potting mix containing mixture of charcoal pieces, chopped forest litter, coconut
husk, sand and black soil showed 75% survival rate.
Propagation protocols using seed germination as well as micropropagation tech-
niques are well developed for M. acuminata. However, genetic fidelity and quality of
plants raised by tissue culture have not been analyzed. During micropropagation
protocol development studies, small pseudobulbs were obtained, but the accumula-
tion of medicinally important ingredients was not analyzed; however, such in vitro
428 R. Suyal et al.
raised tissues may be very important for direct production of secondary metabolites
[65]. Many different in vitro approaches have been used in plant system for
increased biosynthesis and the accumulation of bioactive metabolites. These
methods includes, suspension culture, biotransformation, and Agrobacterium-medi-
ated transformation [65–67]. These new technologies served to enhance the contin-
ued produtivity of phytochemicals, especially medicinal compounds from higher
plants as a renewable source. It is expected that continuous and intensified efforts in
this field will lead to controllable and successful biotechnological production of
specific, valuable, and as yet unknown plant chemicals.
9 Future Prospective and Conclusion
M. acuminata is well recognized for its vitality strengthening and rejuvenating

properties and thus used in traditional Ayurvedic formulations. Pharmacologically,
this species has been evaluated for antiaging, antioxidant, antiinflammatory, sun
protection and UV-A blocking, antiproliferative, and anti-microbial activities [2, 32,
34, 47, 52, 58–59,]. All these assays were based on in vitro experiments; however, to
reach any conclusive remark, these activities need further evidences at molecular and
clinical levels. Further, before development of any pharmacological products, clin-
ical efficacy needs to be analyzed. Also, various molecules extracted from the
species using various solvents and fractions need to be examined for these pharma-
cological activities in deeper manner for supporting its ethno-pharmacological
significance. Generally, a particular compound-rich fraction or extract of the species
is considered to be responsible for a particular specific biological activity, but actual
and reasonable mechanism of action at physiological level needs exact information
on active molecule. Similarly, statistical difference among phytochemicals with any
activity is required for efficacy testing during the drug development.
Market success of medicinal plant as a drug depends upon the content of active
ingredient present in the species. However, drug derived from plant required con-
sistent and continuous supply of the active ingredient overcomming the issue of
seasonality. Content of secondary metabolites is highly influenced by different
factors such as growing conditions, seasons, climatic conditions, sun light exposure,
altitude, along with genetic makeup [36, 38, 68–70]. Therefore, selection of elite
genotype and climatic conditions are essentially required for obtaining higher
phytochemical content and better pharmacological activity. Identification of suitable
growing conditions, agronomic practices, and inheritance of the active metabolite
need more scientific exploration.
Genetic information of bioactive compound can improve the quality traits. In
NCBI database, only 38 nucleotide sequences are available (retrieved on
July 12, 2020), and most of them belong to genomic region of DNA. Thus, gene
related markers, such as, ESTs, or ant other functional markers are scared in the
species. In the recent past, modern biotechnology tools such as high throughput
sequencing technology has become a reliable tool for the generation of large set data
of expression part at sequence level in shorter time and cost effective manner.
However, in mordern arena it bacome a very useful tool for trait-specific marker
development for molecular breeding, genetic diversity analysis or population genet-
ics studies [71, 72]. Currently, whole-genome and transcriptome sequencing are also
preferred method to identify novel genome-wide polymorphic SSR markers, and is
cost-effective irrespective of model and non-model plants [73]. Such information
can be useful for rapid genotyping, molecular breeding, and functional marker
identification for trait improvement.
Propagation protocols such as tissue culture, seed germination, and clonal propaga-
tion for rapid multiplication are available in the species [58, 62–64], but a more robust
technique needs to be developed for the benefit of farmers. With the product develop-
ment, demand of the species will increase consequently, which will create more
pressure in the species on its wild stock. Further, robust and rapid propagation method
for quality-related traits can facilitate farmers to enhance yield and productivity.
Overall, on the basis of available information on M. acuminata, it can be
concluded it has proven vitality strengthening and rejuvenating properties. Various
pharmacological activities has been carried out in support of its traditional uses.
However, most of these phytochemical and pharmacological studies are in its
preliminary stage and need comprehensive scientific evidences. Today, M.
acuminata is collected from wild populations for commercial supply, and therefore
robust propagation protocols and quality planting material are needed to farmers to
cultivate this species for sustainability and conservation.
Acknowledgments The authors thank the Director of G.B. Pant National Institute of Himalayan
Environment for the support and encouragement.
Funding information:
This study was partially funded by the Botanical Garden Scheme of Ministry of Environment,
Forest & Climate Change (MoEF&CC), Government of India, New Delhi (F.N. BSI-290/6/2013-
Tech; September 29, 2013).
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Phytochemistry, Pharmacology,
and Conservation of Ansellia africana: 18
A Vulnerable Medicinal Orchid of Africa
Paromik Bhattacharyya, Shubhpriya Gupta, and

Johannes Van Staden
Contents
1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 436
2 Ansellia africana as a Species . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 437
3 Ethnomedicinal, Horticultural, and Traditional Uses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 438
4 Ex Situ Conservation Using In Vitro Technologies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 438
5 Phytoconstituents and Biological Activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 442
5.1 Antimicrobial and Membrane Damaging Activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 442
5.2 Acetylcholinesterase Inhibitory, Antiinflammatory, and Antioxidant Activity . . . . . . 444
6 Molecular Biology Approaches . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 444
7 Future Research Prospects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 445
7.1 Bioassays and In Vivo Model-Based Studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 445
7.2 Drug Discovery . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 446
7.3 Next-Generation Sequencing and Transcriptome Data Mining . . . . . . . . . . . . . . . . . . . . . . 446
7.4 Endophyte Mapping and Metabolite Production . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 446
7.5 Phylogeography and DNA Barcoding . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 447
8 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 448
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 449
Abstract
Medicinal plants are natural reserves of various therapeutic biomolecules.
Orchids occupy a significant position among these. Ansellia africana is one of
the most important orchids used in various pharmacopeias worldwide, especially
in Traditional African Pharmacopeia (TAP). South Africa, in particular, houses
approximately 494 species of orchids with a 75% rate of endemism. A. africana is
a wonder orchid having large reserves of prized biomolecules that provide
remedies to chronic ailments such as Alzheimer’s disease. Apart from its
P. Bhattacharyya · S. Gupta · J. Van Staden (*)

Research Centre for Plant Growth and Development, School of Life Sciences, University of
KwaZulu-Natal Pietermaritzburg, Scottsville, South Africa
e-mail: rcpgd@ukzn.ac.za

436 P. Bhattacharyya et al.
pharmaceutical importance, the plant is of high importance in the horticultural

industry, primarily because of its flowers. However, as with many orchid
species, the wild populations of this fascinating orchid species are under severe
threat. Presently A. africana has been categorized as “vulnerable” and mandates
systematic as well as scientific approaches to conserve as well as sustainably
utilize its economic potentials. Keeping into consideration the huge potentiality
of A. africana as a medicinal herb, the present chapter documents all the
major research aspects with a focused aim to provide holistic information
about the pharmaco-horticultural importance of this prized orchid species of
Africa.
Keywords
Medicinal orchids · Alzheimer’s disease · Traditional African pharmacopeia
(TAP) · Phytomedicine
1 Introduction
Global climate change, along with an increased rate of deforestation, has adversely
affected the distribution of flora and fauna on a rapid scale stressing the need to
conserve natural habitats on a priority basis. Africa as a continent is one of the main
nuclei of the few remaining biodiversity reserves of the world. Among the various
parts, the southern part of Africa is a rich reserve for various flora and fauna [1,
2]. Being a part of the African mega biodiversity hotspot, South Africa holds a rich
reserve of various herbaceous plant species including rare, endangered, and threat-
ened (RET) – medicinal aromatic plants (MAP) [2, 3, 4]. In Africa, a large popula-
tion of people depends a lot on traditional healers for the treatment of various chronic
ailments. Orchids are a vital part of African traditional medicine as in other tradi-
tional pharmacopoeias of the world. However, the exact time of inclusion of orchids
(for medicinal purposes) in African traditional medicine is not known [2, 5]. In
South African traditional pharmacopeias, approximately 49 orchid species are being
used [3, 6]) of which Ansellia africana Lindl stands out prominently. Traditionally
various parts of A. africana have been used in the Traditional African Pharmacopeia
(TAP) for centuries [2, 3]. It is reported that the smoke generated after burning the
stem and roots of A. africana has exhibited a vital role in the treatment of ailments
involving the central nervous system (CNS) [1]. The stem and root infusions
obtained by A. africana have been reported to possess aphrodisiac properties.
Along with its medicinal usage, A. africana is highly valued for its beautiful flowers
which are reported to have a high shelf life in comparison to other orchid species.
The medicinal orchids of Southern Africa have not been extensively studied for
taxonomical aspects and very little research has been done on the phenotypic and
genotypic diversity of wild populations of A. africana and its associated species.
These lacunae can be filled by performing research focused on the systematic studies
along with their conservation and ethnopharmacological aspects.
18 Phytochemistry, Pharmacology, and Conservation of Ansellia. . . 437
2 Ansellia africana as a Species
Orchids are one of the most diversified forms of plant taxa. Being one of the mega
biodiversity hotspots in the world, the orchid biodiversity in southern Africa is
tremendous of which A. africana deserves special mention [1]. A. africana is an
epiphyte and generally grows in clusters on trees with a predominant presence in the
semitropical areas (Fig. 1a, b). The roots are specially adapted to provide better
attachment to the substratum on which it grows along with the absorption of
nutrients and moisture for its survival in harsh and rugged climatic conditions.
Unlike other epiphytes, the roots of A. africana are of needle shape pointing upward
which later on multiply exponentially and form a dense clump around the pseudo-
bulb which gathers the senescing leaves and tissue debris upon which the orchid
survives (Fig. 1c). It blooms generally in the dry winters and produces a surplus of
Fig. 1 (a) Ansellia africana plants in the wild (b) Plants growing in the wild with attached capsules
after pollination (c) Clustered needle like pointed roots of A. africana
yellow or greenish-yellow flowers that are marked with light or deep brown spots
from which it has got its popular name “leopard orchid.” It has a reported distribution
in tropical Africa along with Namibia, Botswana, and Swaziland, along with the
Northern Cape in South Africa. The orchid has a specific habitat specificity and is
found to grow in hot and dry river valleys [1, 7]. The natural populations of
A. africana are under tremendous anthropogenic pressure and are facing the risk
of extinction. Keeping into consideration its present threatened status, IUCN has
categorized A. africana as “vulnerable.” Like most orchid species A. africana is also
enlisted within Appendix 1 of CITES.
3 Ethnomedicinal, Horticultural, and Traditional Uses
A. africana is one of the most prized medicinal orchid species, not only in Africa but
also globally. In TAP, it is one of the most important ingredients primarily because of
its huge reserves of prized biomolecules [1–3]. Traditionally, stem infusions and
smoke from A. africana are used by Zulu traditional healers as antidotes for bad
dreams [3], whereas leaf and stem extracts were used by the Mpika tribes of Zambia
in controlling madness and related mental disorders [8]. The orchid extract has also
found important usage as an aphrodisiac along with imparting various protective
charms [8]. Apart from its ethnomedicinal usage, the showy flowers of A. africana
make it an important candidate taxa for the horticultural industry along with a high
shelf life. In short, A. africana is one of the few orchid species which has both
horticultural and medicinal usage, making it a prized species for collectors and
export.
4 Ex Situ Conservation Using In Vitro Technologies
The rapid depletion of orchid bio-resources from their natural habitats demands
urgent strategies for their conservation. Due to various developmental activities and
other anthropogenic pressures in the African mega biodiversity hotspot, the wild
populations of various orchids including A. africana are facing the risk of fragmen-
tation as well as extinction. The attractive flowers and medicinal properties of
A. africana have made it a highly traded orchid species leading to indiscriminate
collection from the wild, thus rendering the species to become threatened in the wild
[1, 2, 9]. Lack of comprehensive annual data on annual trade of A. africana is also a
major concern.
Habitat destruction has various far-reaching impacts, which are not only the loss
of precious gene pools useful in plant development or biosynthesis of new com-
pounds but also the loss of a pharmaceutically important source of various vital
compounds. The modern tools of biotechnology can be utilized for the propagation
and conservation of plant genetic resources [10]. In general, these could be accom-
plished both by in situ and ex situ methods. These techniques were initially intro-
duced for plant species having agricultural and horticultural importance, but are now
rapidly being applied to the collection, propagation, preservation, and evolution of

rare and endangered plant germplasm. In situ conservation, which is an ideal and
dynamic approach that allows plants to interact and coevolve, includes the protection
of genetic resources in the natural environment through the protection of the
environment itself [11]. It is the most suitable option to ensure the natural growth,
proliferation, and perpetuation of the species. Globally, to promote the cause of in
situ conservation, many forested areas have been categorized as national parks,
wildlife sanctuaries, and biosphere reserves. However, it is costly to maintain and
is highly susceptible to natural calamities. On the other hand, ex situ conservation
programs have played an important role in acclimatization, rehabilitation, multipli-
cation, and judicious exploitation. Biotechnological approaches of conservation are
complementary to conventional methods. These can directly assist plant conserva-
tion programs through molecular marker technology, molecular diagnostics, in vitro
technologies, and cryopreservation [10, 12].
One of the major reasons behind the depletion of natural reserves of orchids is
their extremely low rates of seed germination in nature, along with dependence on
symbiotic fungal strains which augments the process of seed germination in nature
[13]. Like other orchids, A. africana also has a very low rate of seed germination in
nature which is less than 3%, and efforts are being made to supplement the process
using asymbiotic seed germination techniques [9]. It was observed that the growth
medium on which the seeds were germinated impacts the process rather than the
sterilization process. In addition, dark preconditioning after inoculation significantly
increased the rate of seedling growth particularly the rhizoids in the protocorms
[9]. However, recently Papenfus et al. [14], reported a positive synergy of smoke-
water on the germination of A. africana. Smoke is generally reported to have a
conducive effect in enhancing plant vigor and influences pollen growth [15, 16]. The
research done by Papenfus et al. [14] provided the first insights on the impact of the
two smoke-derived chemical compounds, i.e., karrikinolide (KAR1) and tri-
methylbutenolide (TMB) on the in vitro seed germination and seedling growth of
A. africana.
It was found that the half-strength MS medium supplemented with 1:250 (v:v)
smoke-water (SW) significantly improved the germination rate index (GRI) and the
development rate index (DRI) of A. africana seeds. The SW-treated seeds in MS
medium significantly enhanced the production of large protocorm-like bodies
(PLBs). However, the KAR1-treated seeds showed no significant effect on the
germination and development of seeds, whereas, TMB-treated seeds significantly
reduced the GRI and DRI of A. africana seeds [14].
Along with the development of symbiotic seed germination modules, the improve-
ment of micropropagation procedures can assure sufficient supply of plant material
along with the probability of producing elite genotypes with a subsequent reduction
in over-collection of the wild germplasm (Fig. 2a, b). There exist a few reports on the
high frequency in vitro propagation, acclimatization, and clonal fidelity assessment of
the microclones of A. africana [7, 14]. The media supplemented with meta-Topolin
Riboside (mTR) enhanced the shoot and root length, leaf number, frequency of root
organogenesis, and fresh weight of A. africana [17].
440
Fig. 2 (a) Asymbiotic germination of seeds (b) Stages of germinating seed (c) Initiation of plantlet from nodal explant (d) Proliferation of multiple shoots (e)
Synthetic seed encapsulated in sodium alginate beads (f) Proliferated roots with prominent white needle-like structures (g) Hardened micropropagated plants of
P. Bhattacharyya et al.
A. africana
The report also demonstrated the efficacy of mTR which is a derivative of meta-
Topolin over the conventional cytokinin-based PGRs like BA and TDZ. Working in
similar lines, Bhattacharyya et al. [7] demonstrated the comparative efficacy of
aromatic cytokinin mT in terms of ascertaining genetic stability and regeneration
potential over conventional cytokinin BA. The research also provided vital insights
into the role of phenolic elicitors like phloroglucinol singly and also in combination
with conventional auxins like indole Butyric Acid (IBA) in induction, proliferation,
and branching of A. africana roots which are highly valued for their medicinal
properties [17] (Fig. 1b and 2c, d).
Being a recalcitrant species, the development of artificial seed technology in
A. africana deserves special mention. Synthetic seed technology has played a pivotal
role in orchid biotechnology as it offers tremendous potential for easy handling,
micropropagation, and long- and short-term storage of plant germplasm through
cryopreservation techniques [18–20]. Furthermore, the successful development of
synthetic seed technology is largely dependent on the formation and production of
viable artificial seeds that would be able to transform into complete plantlets
[18]. Recently, Bhattacharyya et al. [20] reported the synthetic seed production
and short-term storage of A. africana using protocorm-like body (PLB) segments.
The developed propagule provides vital insights into the role of aromatic cytokinin
meta-Topolin and its derivatives (mTR, mTTHP, and memTTHP) in the short-term
storage of A. africana synthetic seeds. The MS media supplemented with 7.5 μM
mem-TTHP showed the highest response percentage of encapsulated PLBs which
were successfully stored for 75 days at 8 °C. The in vitro regeneration of A. africana
will be helpful in maintaining its population in the wild and can be utilized in the
conservation of various other orchids that are endangered (Fig. 2e).
Apart from the formulation of conservation strategies, in vitro propagation
techniques have contributed significantly to the growth of the pharmaceutical indus-
try over the past several decades in a multidisciplinary manner, including varietal
improvement and development of elite cultivars and production of secondary
metabolites [21–25]. PGRs such as auxins and cytokinins (CK) regulates the activ-
ities of phenylalanine ammonia-lyase and chalcone synthase enzymes, thereby
affecting the biochemical synthesis of phenolic acids [26, 27]. Amoo et al. [28]
reported that CK supplemented MS medium significantly enhanced the various
bioactive metabolites including phenolics and iridoids in Aloe arborescens. The
orchids have distinctive somatic embryos which are known as protocorm-like bodies
or PLBs and are reported to contain crucial bioactive metabolites that are present in
wild plants [29–31]. Therefore, these PLBs can be propagated on a large scale as
they are highly differentiated and can be used as a substitute for wild medicinal
sources for exploiting the therapeutic potential. Bhattacharyya et al. [32] reported
that A. africana PLBs are a potential source of biologically important phenolic acids
such as hydroxybenzoic and hydroxycinnamic acid derivatives. They discovered
that the treatment of topolins (mT, mTR, Mem T, and MemTR) and TDZ significantly
influenced the biosynthesis and accumulation of various phenolic acids (including
hydroxybenzoic and hydroxycinnamic acid derivatives) and exponentially enhanced
the biomass, FW, and DW of A. africana PLBs (Fig. 2f, g).
Also, natural antioxidants, which are present in fruits, vegetables, and medicinal
plants, have received much attention and have been studied extensively since they
are effective free radical scavengers and are assumed to be less toxic than synthetic
antioxidants. Rapid multiplication rate, higher genetic stability, and significantly
higher antioxidant activity reported in the study ensure the utility of this micro-
propagation protocol developed for further utilization in the ex-situ conservation and
commercial utilization of A. africana [17, 32]. In short, micropropagation is an
important biotechnological tool, which largely assists in combating the various
biodiversity conservation issues arising primarily due to unplanned over-collection
and habitat destruction. Proper use of plant tissue culture modules can further
facilitate the production of elite cultivars with significantly higher yields of second-
ary metabolites on a mass scale, which will cater to both medicinal as well as
horticultural industries.
5 Phytoconstituents and Biological Activity
5.1 Antimicrobial and Membrane Damaging Activity
The ethnomedicinal applications of medicinal herbs are useful not only in conser-
vation but also in cultural tradition and biodiversity at community levels [33]. The
whole plant of A. africana has been reported to be used in the treatment of
respiratory disorders such as asthma [34, 35], while its shoots are being used in
the treatment of lice [36]. However, studies on the biological activities of A. africana
are rare. Penduka et al. [37] provided some vital insights into the antimicrobial
activities of A. africana by testing its extract against Moraxella catarrhalis (clinical
isolate), Klebsiella pneumoniae (ATCC 4352), Staphylococcus aureus (ATCC
25925), and Mycobacterium smegmatis (ATCC 14468). The exhibited minimum
inhibitory concentration (MIC) value of A. africana extracts was significantly low
ranging from 2.5 to 10 mg/l. One of the most significant findings of the research is
the efficacy of A. africana extracts against the M. tuberculosis strains. The African
subcontinent is severely engulfed by the phenomenon of malnutrition which further
magnifies the risk of tuberculosis in the region. The situation gets more aggravated
due to the lack of modern healthcare systems. The potential activity of A. africana
extracts against M. tuberculosis strains provides a new prospect in the development
of indigenous medicines and drugs at an affordable rate [37]. Comparative antimi-
crobial activity of root and stem extracts of A. africana was also estimated, which
closely supports the traditional use of the whole plant in treating respiratory prob-
lems [34, 35]. Furthermore, the studies revealed no antagonistic influence of the
plant extract with antibiotic ciprofloxacin, which provides baseline information on
the utilization of A. africana in multiple drug therapy (MDT) after in vivo confir-
mation [37]. Interestingly, plant extracts exhibited a low membrane-damaging activ-
ity against M. smegmatis strains which can be due to the thick and waxy cell wall of
Mycobacteria which makes it have a highly impermeable outer surface, enabling
Mycobacteria strains to withstand extreme environmental conditions also in the
presence of antibacterial agents [38]. The findings of Penduka et al. [37] validates the
traditional use of A. africana by traditional healers in the treatment of skin, respira-
tory, and soft tissue infections. The research findings also envisage the fact that the
strategies for the treatment of infectious diseases are multidimensional and more
dedicated investigations are required using in vivo models along with compound
isolation (Table 1).
Table 1 Biological activities attributed to Ansellia africana

Name of the Plant parts
biological activity used Salient findings References
COX-1 and COX-2 Leaves, 1. Roots of A. africana showed high Chinsamy
assay stems, and COX-1 and COX-2 inhibition activity et al. [4]
roots 2. The dicholoromethane (DCM) root
extract of A. africana exhibited the
highest EC50 activity among seven
south African medicinal orchid species
tested, i.e., 0.25 0.10 mg/ml
Acetylcholinisterase Leaves, Highest acetylcholinesterase inhibitory
inhibitory activity stems, and activity exhibited among the seven
roots most potential medicinal orchid
species tested. The most potent extract
was the ethanolic root extract
β-Carotene Leaves, Exhibited moderate β-carotene
bleaching activity stems, and bleaching activity
roots
Mutagenic activity Leaves, DCM root extract and ethanolic leaf,
stems, and stem, and root extract exhibited
roots mutagenic effects
Antioxidant power Leaves, High antioxidant levels determined by
stems, and FRAP (Fluorescence Recovery After
roots Photobleaching) assay
Antimicrobial Leaves, 1. Demonstrated high efficacy against Penduka et al.
activity stem, and both gram-positive and gram-negative [37]
roots strains of bacteria.
2. In extremely low concentration (2.5-
10 mg/l) root and stem extracts showed
high efficacy.
3. Potentiality to combat pathogenicity
of Mycobacterium tuberculosis and
Staphylococcus aureus causing
chronic respiratory track ailments in
humans.
Phenolic acid Protocorm- Modulating the production of Bhattacharyya
activity like bodies biopharmaceutically important et al. [32]
(PLB) phenolic acids, i.e., hydroxybenzoic
acid and hydroxycinnamic acid
derivatives in protocorm-like bodies
(PLB)
5.2 Acetylcholinesterase Inhibitory, Antiinflammatory,

and Antioxidant Activity
In the recent past, a lot of medicinal plant bioprospection has been carried out based
on their activities related to the CNS [39]. Reports on the traditional use of
A. africana in the treatment of mental disorders like insanity, hyper fits, nervous
disorders have been confirmed to have prominent acetylcholinesterase (AChE)
inhibitory activity [2, 4]. A total of 64 African plant species are reported to exhibit
potent AChE activity among which A. africana figures prominently [2, 39]. The
findings of Chinsamy et al. [4] have revealed that the ethanolic root extracts of
A. africana have a promising AChE activity which is in close synchrony to its
traditional usages [1].
Traditionally, A. africana is also reported to possess potent anti-inflammatory
activity and is being used by traditional healers as an aphrodisiac [1, 3, 8]. Recent
studies on the phytochemical constitution of A. africana revealed the presence of
high levels of gallotanin in A. africana plant parts [4]. Gallotanin is a prized
molecule and is reported to possess various biological activities such as anti-
inflammatory activity [37]. In recent times, Alzheimer’s disease has become a
major concern for society. Inflammatory responses, cholinergic system, and oxida-
tive stress often collectively account for the various symptoms prevalent in aged
persons and Alzheimer-affected patients [37].
One of the primary enzymes which are involved in anti-inflammatory responses is
cyclooxygenase (COX). In general, COX enzymes are being classified into COX-1
and COX-2. According to Bohlin et al. [40], flavonoids, naphthoquinones,
alkylamides, phenolic phenyl-propane derivatives are some of the compounds
involved in COX enzyme inhibition. Chinsamy et al. [4] reported that the extracts
from the roots of A. africana showed high COX-1 and COX-2 inhibition activity.
The highest EC50 activity (0.25 0.10 mg/ml) was shown by dichloromethane
(DCM) root extract of A. africana. Also, the aqueous concoctions of A. africana
plant parts exhibited high levels of COX-1 and COX-2 activity which explains the
practice of Mpika tribes and other tribes of Africa to administer warm aqueous
concoctions of A. africana plants in the treatment of bad dreams or madness
[1]. Apart from AChE activity and anti-inflammatory activity the A. africana
extracts exhibited a potent antioxidant and antimutagenic activity justifying the
importance and significance of A. africana in TAP.
6 Molecular Biology Approaches
The use of DNA fingerprinting has a wide range of applications. It has been used in
forensic science to solve criminal cases and settle parental disputes. It has a wide
spectrum of applications in the field of plant sciences; it is used to identify genetic
diversity within breeding populations, to positively identify and differentiate acces-
sions, cultivars, and species that might be challenging to illustrate due to related
phenotypic characters or indistinct traits, and to identify plants encompassing genes
of interest (such as the confirmation of transformation events). As the natural

populations are exposed to several genetic factors or forces affecting the amount
and kind of genetic variation such as mutation, chance events like genetic drift and
founder events, selection, migration, and a species mating system [41], a careful
assessment of these factors and their role in the formulation of conservation strate-
gies is essential.
The sustainability of micropropagation techniques depends upon the production
of true-to-type plants and maintenance of the genetic integrity of the in vitro-raised
plants so that the advantage in the use of elite genotypes over natural seedlings is
maintained. However, in vitro techniques are known to induce clonal variations.
Since the first observation and report of clonal variations [42], it remains an issue of
great concern for tissue culture-raised plants as there are reports of genetic disparities
in micropropagated plants [43, 44]. The occurrence of these variations depends on
several factors such as the source of the explants, media composition, and cultural
conditions [45]. Thus, monitoring the degree of genetic variability within the
in vitro-raised plants is of prime consideration for the effective commercial utiliza-
tion of the technique and also for large-scale production of true-to-type plants of the
desired genotype [46]. In the recent past, various high-frequency regeneration pro-
tocols have been developed in orchids including A. africana with high rates of
genetic stability [17, 20, 47].
7 Future Research Prospects
7.1 Bioassays and In Vivo Model-Based Studies
A. africana offers various promising leads both in terms of floriculture and

biopharmaceuticals. This orchid is highly prized for its showy flowers along with
its therapeutic properties, primarily due to the reserves of various bio entities in its
aerial parts [1]. This chapter summarized the existing ethnobotanical and horticul-
tural uses, phytochemistry, pharmacological activities along with conservation and
molecular insights on A. africana [1, 32].
To make this prized ethnomedicinal herb more acceptable, systematic clinical
trials evaluating its in-depth biological activities using in vivo models is needed [1].
Also, more systematic studies on various toxicological as well as mutagenic prop-
erties must be taken up. In short, there is an urgent need for systematic clinical trials
to establish the efficacy of A. africana in medicine. Along with clinical trials,
attempts should also be made to mass propagate elite cultivars of A. africana
using various biotechnological tools [1, 17, 19]. The PLBs can serve as reserves of
important medicinal compounds like phenolic acids which can be further enhanced
by the use of suitable elicitors [32]. Fast regeneration protocols, which will be cheap
and reproducible, must be developed to further facilitate the sustainable utilization of
A. africana germplasm. To facilitate such endeavors, interventions of innovative
biotechnological tools such as cryopreservation methods and bioreactors are
required [1, 17].
7.2 Drug Discovery
The current trend of research in drug discovery from traditionally reported medicinal
herbs includes a multidirectional approach comprising of botanical, biological,
phytochemical, and molecular approaches. Discovery of new biological molecules
provides significantly important research leads against chronic disorders such as
cancer, malaria, and various neurodegenerative disorders [4, 5]. In recent years,
various plant-based drugs of immense medicinal importance like arteether,
dendrobine, gigantol, moscatillin, galantamine, nitisinone, etc. have made a major
impact in the areas of drug discovery from traditional ethnomedicinal herbs [48]. Var-
ious research studies on African medicinal herbs with special reference to
A. africana have revealed that it houses various phytochemical entities that are
largely unexplored, requiring thorough clinical evaluation. The results exhibited
by various fractions of A. africana plant extract in the treatment of neurodegenera-
tive disorders provides evidence that it houses molecules that may play a substantial
role in the treatment of CNS disorders [1, 4].
7.3 Next-Generation Sequencing and Transcriptome Data Mining
Development of next-generation sequencing (NGS) has opened new gateways for

exploring the genomes of various nonmodel plants which might play a pivotal role in
deciphering the medicinal properties in various traditional medicinal herbs [49]. To
date, several molecular and analytical methods have been applied in the identifica-
tion and genotyping of various medicinally important orchid species, mostly
dendrobiums. However, none to date have taken genome sequencing of the orchids
into account. So far, only the genome of D. officinale has been reported [50].
Development of NGS-based approach attempting to sequence the selected medicinal
orchid transcriptomes like A. africana to identify and characterize transcripts poten-
tially contributing to their observed medicinal properties will largely help in answer-
ing various unanswered questions in plant therapeutics [1]. In addition to it,
understanding the medicinal potential of A. africana might illuminate the basic
understanding of the genes involved in the biosynthesis and channelization of
bioactive secondary metabolites such as phenylpropanoids, alkaloids, and terpe-
noids. In addition to that, knowledge of NGS provides a better understanding of
the various biological interactions such as stress tolerance and mycorrhizal associ-
ation. Utilizing this advanced biotechnological tool, the important role played by
symbiotic association with fungi in nature and how it controls the germination
pathways can be deciphered for A. africana.
7.4 Endophyte Mapping and Metabolite Production
Microorganisms are known to play an important role in most ecosystems including

soil and plant habitats. The microorganisms associated with plants maintain their
biological diversity in terrestrial ecosystems through various biological processes

[51]. The endophytic microorganisms (including bacteria, fungi, virus, and pro-
tozoa) reside in intercellular spaces of stems, petioles, roots, and leaves of plants
without causing any disease [16, 52]. Endophytes are well known to promote plant
growth, plant productivity, and assist their hosts to withstand biotic and abiotic
stresses by the production of countless biologically active metabolites [53,
54]. Besides this, endophytes are known to produce a larger number of metabolites
and enzymes as validated by a number of endophyte culture studies [16, 55–
57]. Many of these metabolites are the same as produced by their respective hosts
and may have potential benefits in the pharmaceutical industry [58, 59]. Various
orchids (i.e., Bletilla ochracea, Dendrobium nobile, Dendrobium loddigesii,
Dendrobium aqueum, Pecteilis Susannae, Vanda coerulea, etc.) have been explored
for their endophytes and their secondary metabolites [60–64]. However, the natu-
rally occurring endophytic microorganisms present in A. africana are not yet known.
There is a need to investigate and map the endophytic diversity (particularly bacterial
and fungal endophytes) of A. africana which might be a source of vital metabolites
including phytohormones which may have a positive effect on the development of
this endangered species. The associated endophytic flora of A. africana may serve as
beneficial microorganisms that could be used in its propagation and improving
acclimatization and vigor [65].
7.5 Phylogeography and DNA Barcoding
Scientific analysis of the phytogeography that is mainly influenced by the occurrence

of concerted evolution which plays a significant role in conservation schemes of
RET plants particularly in case of orchids. In concerted evolution, hundreds to
thousands of tandemly repeated copies of DNA such as ribosomal DNA (rDNA)
evolve in a concerted way. Therefore, the copies of related genes have more
similarities within the species as compared to between the species. The homogeni-
zation of these multiple-copy genes through unequal crossing-over and high fre-
quency of gene conversion is the key factor in influencing the phenomenon of
concerted evolution. On the other hand, under distinct circumstances, the vital nature
of the tandem repeats of such genes gets disturbed and incomplete intra-genomic
deviation appears under such conditions. Incomplete concerted evolution for r-DNA
has been reported in several plant species including orchids. A. africana is one of the
species from South Africa that is highly endangered due to high rates of deforesta-
tion and habitat destruction, therefore, studies on phylogeography and existing
genetic variations using molecular markers can be of great implication. The studies
related to the genetic variability of several orchid species have demonstrated an
irregular distribution of genetic diversity among different geographic zones. Fur-
thermore, being an endangered orchid, DNA barcoding of A. africana and other
related South African orchid species is of relevance. DNA barcodes such as rbcL
(ribulose-bisphosphate carboxylase), matK (Megakaryocyte-Associated Tyrosine
Kinase), psbA-trnH, rpoC1, and ITS2 (internal transcribed spacer 2) are popular
worldwide. Few studies have employed psbA-trnH barcode for identifying species
of medicinal pteridophytes and within the genus Dendrobium. Chen et al. [66] have
shown that ITS2 is a universal barcode in the identification of plants, as 92.7% cases
from 6600 samples in seven phyla (Angiosperms, Gymnosperms, Ferns, Mosses,
Liverworts, Algae, and Fungi) has been correctly identified by ITS2. Consequently,
ITS2 region has been useful in differentiating plants from various families including
Orchidaceae. Therefore, these potential barcodes can be used for authentication of
orchids including A. africana.
8 Conclusions
The orchid A. africana holds intriguing prospects, not only in the field of medicinal
plant research but also in the horticultural industry because of its showy flowers and
it can be one of the most desired orchid species for commercial growers. Moreover,
research insights providing its potent activity on the CNS make it an important plant
species for bioprospecting of drugs against Alzheimer’s disease. It also possesses
potent antibacterial activity which can further strengthen Africa’s indigenous med-
icine production. Efforts are required for further studies, especially evaluating its
in vivo biological activities along with toxicological and mutagenic properties to
better validate the safety of these different plant-derived compounds. Also, to
establish its efficacy, there is a need for preclinical and clinical trials. However, the
high demand for A. africana in national and international markets has led to its over-
exploitation and habitat destruction. This has resulted in the loss of the wild
population of A. africana. Therefore, for the successful commercialization of this
threatened taxon wide research is needed that includes conservation practices and a
sustainable supply of plants. This can be achieved by utilizing biotechnological
techniques such as micropropagation, cryopreservation, and bioreactors. Detailed
research on synthetic seed technology (artificially encapsulated somatic embryos) is
required for the improvement in germination frequency of A. africana synthetic
seeds and subsequent plantlet growth in the soil such that it can be used on a
commercial scale. Furthermore, hairy root culture can be used as a model system
to improve the valuable phytochemicals of A. africana. To prevent misidentification
and possible adulteration of A. africana, quality control protocols are needed. In the
future, new research findings may increase the present therapeutic importance of
A. africana and its future use in modern medicine. In short, A. africana is one of the
most prized orchid species occurring in Africa, which has the potential to boost the
African bio-economy by promoting phyto-horticultural ventures.
Acknowledgments PB and SG thank the University of KwaZulu-Natal, South Africa for financial
support in the form of postdoctoral fellowships. The authors are grateful to the Microscopy and
Microanalysis Unit (MMU), UKZN, Pietermaritzburg for microscopic assistance. We are thankful
to Dr. Heino B. Papenfus, Kelp Products International (Pty) Ltd., Simon’s Town, South Africa, Mrs.
Louise Van Staden, Pietermaritzburg, South Africa and Mrs. Lee Warren, Senior Administrative
Assistant, Research Centre for Plant Growth and Development, University of KwaZulu-Natal,
Pietermaritzburg, South Africa for providing the photographs of wild plants as well as seed
germination photographs of A. africana.
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https://doi.org/10.1371/journal.pone.0008613
Dendrobium sp.: In vitro Propagation of
Genetically Stable Plants and 19
Ethnomedicinal Uses
Leimapokpam Tikendra, Nandeibam Apana,

Angamba Meetei Potshangbam, Thoungamba Amom,
Ravish Choudhary, Rajkumari Sanayaima, Abhijit Dey, and
Potshangbam Nongdam
Contents
1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 454
2 Ethnomedicinal Uses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 455
3 In Vitro Propagation of Dendrobiums . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 458
3.1 Culture Media and Plant Growth Regulators . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 458
3.2 Explants (Selection and Surface Sterilization) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 459
3.3 In Vitro Propagation of Dendrobiums Using Different Explants . . . . . . . . . . . . . . . . . . . . 460
4 Genetic Stability of In Vitro Propagated Dendrobiums . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 467
4.1 Somaclonal Variation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 467
4.2 Genetic Stability Assessment Using DNA Markers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 475
5 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 482
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 483
Abstract
Plants belonging to the Dendrobium genus occupy a dominant position among
the orchids because of their high ornamental and therapeutic values. They are
widely popular in the international floriculture trade as they bear stunning flowers
with diverse coloration, varied forms, and patterns. The ethnomedicinal uses of
these orchids are also prominently found due to the possession of immense
medicinal properties. Excessive exploitation through rampant unregulated
L. Tikendra · N. Apana · A. M. Potshangbam · T. Amom · P. Nongdam (*)

Department of Biotechnology, Manipur University, Canchipur, Manipur, India
e-mail: nongpuren@gmail.com
R. Choudhary
DSST, Indian Agricultural Research Institute, New Delhi, India
R. Sanayaima
DDU College, University of Delhi, New Delhi, India
A. Dey
Department of Life Sciences, Presidency University, Kolkata, India
e-mail: abhijit.dbs@presiuniv.ac.in

454 L. Tikendra et al.
collection and widespread habitat destruction have dwindled the Dendrobium

natural populations at an alarming rate. The fast and reliable micropropagation
techniques offer an alternative to the slow conventional methods of Dendrobium
propagation. The rapid in vitro regeneration of genetically stable Dendrobiums is
essential for effective germplasm conservation and large-scale orchid commer-
cialization. Several genetic stable Dendrobiums have been successfully propa-
gated by ascertaining the clonal fidelity of the regenerants using different
molecular markers. This chapter focuses on the uses of Dendrobiums as important
ethnomedicine, their in vitro propagation, and clonal assessment for producing
genetically stable orchids using DNA markers.
Keywords
Dendrobiums · Phytochemicals · Alkaloids · Micropropagation · In vitro
propagation · Genetic variation · Somaclonal variation · Genetic stability ·
DNA markers
1 Introduction
Orchids, the incredible flowering plants which belong to the Orchidaceae family,
comprise about 1000 genera and 35,000 species [1]. They have captivating floristic
characters and color patterns with diverse forms and growth habits and are distrib-
uted worldwide from tropics to high alpine [2, 3]. They are considered as luxurious
plants because of their exquisite beauty and fragrance of flowers, brilliance in
coloration, and remarkable range in sizes and manifold shapes [4]. The genus
Dendrobium, composed of about 1400 species, is significant among the orchids
because of their high ornamental and medicinal values [5, 6]. They exhibit tremen-
dous diversity with numerous interspecific hybrids and are widely distributed geo-
graphically in most parts of Asia, Australia, and Europe [7, 8]. Many Dendrobiums
possess extraordinarily beautiful flowers with varied floral patterns and forms,
making them one of the most sought after ornamental plants in the International
floricultural market. The large part of contemporary orchid trade is mostly domi-
nated by artificially propagated plants and hybrids of Dendrobium, Cymbidium, and
Phalaenopsis orchids [9, 10]. Dendrobiums are also in huge demand for the phar-
maceutical industry due to the rich content of diverse useful phytochemicals.
The natural populations of these multiutility orchids have witnessed drastic reduc-
tion due to excessive unregulated collection for illegal trade and rampant habitat
destruction [11]. The whole Orchidaceae is listed in the Red Data Book of Interna-
tional Union of Conservation of Nature (IUCN), and the entire Dendrobiums are
included in Appendix ΙΙ of threatened species of plants and animals under CITES [12,
13]. Rapid large-scale propagation of the orchids is the need of the hour to meet the
increasing commercial demands and for effective germplasm conservation. But con-
ventional propagation methods are slow, labor-intensive, and extremely time-consum-
ing. Plant tissue culture techniques may substitute the conventional approaches for
19 Dendrobium sp.: In vitro Propagation of Genetically Stable Plants. . . 455
effective conservation and commercialization of Dendrobiums. Many workers have

successfully micropropagated several Dendrobiums using different explants [11, 14–
19]. But there is a possibility of the emergence of somaclonal variation among the
regenerated orchids as plant tissues are routinely exposed to different stress conditions
during in vitro culture. Incorporation of plant growth regulators, long culture cycle,
and differential explant regeneration patterns might change the genetic makeup of the
regenerants leading to somaclonal variation [20, 21]. Genetic variation among the in
vitro propagated plants is unwanted if the primary regenerants are the desired end
products. Production of genetically stable plants similar to the elite mother plants is
beneficial to orchid cut-flower industry as it assists in uniform blooming during
predictable periods fulfilling the market demands. The genetically stable Dendrobiums
can be propagated by ascertaining the clonal fidelity of the regenerants using different
DNA molecular markers. Many investigators have successfully used several DNA
markers for genetic homogeneity assessment of different micropropagated
Dendrobiums [17, 22–25]. In this chapter, we highlight the ethnomedicinal uses of
Dendrobiums for therapeutic control of ailments and recent works on the in vitro
propagation of genetically stable plants using different DNA markers.
2 Ethnomedicinal Uses
The study of how people of a particular culture and area use indigenous plants in
their lives for daily health management and other requirements is called ethnobotany
[26]. Schultes [27] had termed it as “The study of the relationship which existed
between people of ancient societies and their environment.” Every society harbors a
specific medical culture or “Ethnomedicine,” which is concerned with the cultural
interpretations of health, illness, disease prevention, and local healing practices [28].
Since time immemorial, the traditional healers in any ethnic community use wild
plants for making indigenous medicines. These local medicine men also used
Dendrobiums for folk medicine preparation in the form of herbal paste or medicinal
concoctions to treat different ailments [29]. The ethnomedicinal uses of
Dendrobiums date back to twenty-eighth century B.C when their therapeutic appli-
cation was mentioned in “Material medica” during the time of Emperor “Shen-
Nung” [1, 30]. They are prominently employed in the traditional Chinese medicine
(TCM) by utilizing about 30 different Dendrobium species under Dendrobii Caulis
(Shi-Hu) and Dendrobii officinalis Caulis (Tie-Pi Shi-Hu) [6, 31]. They are used in
Chinese folk medicines as a tonic, analgesic, fluid body enhancer, and anti-inflam-
matory substances [32]. Dendrobiums are also applied in Indian Ayurveda medicine
with D. alpestre as a source of “Jewanti” and D. teretifolium, D. macraei,
D. densiflorum, D. fimbriatum, and D. discolor in the management of dysentery,
pain, pimples, skin eruption, liver upset, nervous debility, asthma, bronchitis, throat
trouble, and fever and is used as an aphrodisiac [33–41]. Many Dendrobiums have
rich contents of phytochemical compounds such as gigantol, moscatilin, dendrobinae,
mucilage, dendrobine, denbinobine, dendroside derivatives, nobilin D, nobilin E and
nobilone in D. nobile [31, 42, 43]; dendromoniliside derivatives, moniliformin,

4-phenanthrenequinone, and daucosterol in D. monoliforme [44, 45]; dibutyl phthal-
ate, ethyl haematommate, methyl B-orcinol carboxylate, N-docosyl trans-ferulate, and
ferulaldehyde in D. longicornu [1]; jebantine and jibantic acid in D. macraei [1, 46];
flavanthrin, coelonin, iusianthridin, moscatin, gigantol, dibutyl phthalate, and
P-hydroxyphenylpropionic methyl ester in D. aphyllum [1]; dendrocandin derivatives,
amotin, amoenin, flaccidin, and 3,4-dihydroxy-5,4 dimethoxybibenzyl in
D. candidum [1, 47]; crepidine, crepidamine, and dendrocrepine in D. crepidatum
[43]; homoeridictyol, scoparone, bibenzyl, densiflorol, cypripedin, gigantol,
moscatilin, tristin, naringenin, homoeriodictyol, moscatin, and scoparone in
D. densiflorum [1, 48]; isoamoenylin, amoenylin and moscatilin in D. amoenum
[1, 49]; rotundatin, moscatin, moscatilin, and scopoletin in D. moschatum [50]. The
presence of the diverse alkaloids, flavonoids, and glycosides in Dendrobiums makes
them highly important medicinal herbs with antimicrobial, anti-inflammatory, anti-
cancer, antioxidative, and antiviral properties [51]. The ethnomedicinal uses of some
important Dendrobiums are described below.
D. amoenum Wall. Ex. Lindl.: It is an important epiphytic orchid with high
floricultural and medicinal values. The dried stems are powdered to prepare a
decoction, which can be used as a tonic [40]. The fresh paste obtained from
grounded pseudobulbs may be applied to treat burnt skin and dislocated bones [52].
D. aphyllum (Roxb.) C.E.C. Fisch.: The leaves can be grounded after drying
completely and made into a fine paste with water. The paste obtained from the leaves
can be put on abnormal or deformed parts of the head of a newly born baby to get
into normal shape [53]. The leaves poultice is also used in the treatment of boils and
pimples in the skin [54].
D. aurantiacum Rchb. F: The dried stems of the orchid are utilized as traditional
or folk medicines in China for their antipyretic, eyesight improving, immune-
modulatory, anti-oxidant, and antiaging properties [45]. The infusion or decoction
obtained from the leaves is used in diabetic treatment [55].
D. candidum Wall ex. Lindl.: It is considered a crucial herbal medicine in South
and Southeast Asia [56]. The decoction obtained from the leaves is used to treat
diabetes in China as it showed stimulation of insulin secretion from the beta cells
while inhibiting glucagon production [57]. They may also be used in maintaining the
tonicity of the stomach and promoting the secretion of body fluid [58]. There are
reports of employing them in relieving the symptoms of throat inflammation,
gastritis, dehydration, and blurred vision [59].
D. chrysotoxum Lindl.: The medicinal properties of the plant are bestowed by the
presence of diverse polysaccharides and phenanthrenes derivatives in leaves and
pseudobulbs. The anti-inflammatory activities of the plant are due to the presence of
erianthridin [55]. The liquid extract, which is prepared by boiling the leaves, can be
used as tonic and antipyretic [41].
D.chrysanthum Wall: The dried and grounded leaves are used for anti-pyretic,
eyes-benefitting, and immune-regulatory purposes. They are also applied for skin
improvement and treatment of some skin diseases [60].
D. crepidatum Griff: Stems and pseudobulbs are plant parts used for making
herbal medicines. The dried stems of the plant are employed for managing cancer
and diabetes and in the treatment of cataracts and fever [1, 61]. The grounded
pseudobulbs are made into a paste with some water used to treat fractured and
dislocated bones [52, 62].
D. densiflorum Lindl.: The plant is used in traditional or folk medicines as a yin
tonic in China. The dried and grounded pseudobulbs are made into a paste which is
then used to promote body fluid secretion and strengthen the stomach and relieve
fatigue and pain [48]. They are also employed as body immune booster and in the
treatment of boils, pimples, and other skin rashes [40, 43].
D. fimbriatum Hook: The infusion or decoction of the leaves is consumed as a
tonic as it promotes body fluid secretion. The paste prepared from the dried leaves
can be put on the surface of the fractured part of the body for setting the cracked
bones [63]. The whole plant can be used to treat liver upset and controlling debility
and nervous breakdown [36].
D. loddigesii Rolfe: This is one of the most prominent medicinal orchids in
Southern China [64]. The infusion or decoction made from the leaves is used as a
tonic for nourishing the stomach and stimulating the secretion of body fluid. It is also
utilized for lowering body temperature during fever and also acts as an anticancer
agent [65].
D. macrostachyum Lindl.: Juice can be extracted from the juvenile leaves and
tender shoot tips of the orchid. The extracted juice may be used as effective ear drops
for curing ear pain and treating boils, pimples, and other skin rashes [31, 66, 67].
D. macraei Lindl.: The whole plant is useful for treating bronchitis, throat
problem, fever, asthma, and as an aphrodisiac [68]. It can also be utilized as a
tonic for general debility [69]. The dried tubers can be made into powder by grinding
and used as a stimulant for lowering blood pressure and curing skin allergy [62].
D. microbulbon A.Rich: This is a small rare epiphytic orchid having useful
medicinal properties [70]. The leaves are crushed to make a paste that can be applied
on the stomach for curing stomach pain [71]. The bulbs are also eaten by the tribal
people of Gujarat, India, as a source of food [72].
D. moschatum Lindl.: The plant is used in traditional medicines for its antimi-
crobial, anticancer, antiallergic, and anti-inflammatory activities due to the high
content of phenanthrenes [73]. The liquid extract from the leaves is utilized as ear
drops for curing earaches. The pastes prepared from the dried and powdered
pseudobulbs are used to treat dislocated and fractured bones [62, 74].
D.moniliforme (L.) Sw.: It is an important medicinal orchid which is widely
distributed in China [75]. The pseudobulbs, after drying, can be boiled or soaked
in hot water to get infusion or decoction. The infusion from the dried stems is used as
an aphrodisiac and for antipyretic, analgesic, and tonic purposes [76].
D. nobile Lindl.: The dried pseudobulbs are ground to powder and mixed with
water to form liquid extract, which can be used as a tonic for nourishing the stomach,
promoting the body fluid secretion, and reducing fever [77]. It is also utilized in
managing tuberculosis, general debility, lowering salivation, night sweats, and
anorexia [78]. Fresh dried stems can also be employed in the preparation of the drugs
that work as an aphrodisiac, analgesic, and life longevity [79].
D. officinale Kimura et Migo: It is a crucial herbal medicine in many Asian
countries for hundreds of years due to its high content of polysaccharides [80]. The
plant can be employed as a nourishing yin tonic for promoting body fluid production
[81]. They are also reportedly used in improving immunity, strengthening memory,
preventing and combating cancers, and prohibiting thrombokinesis [82].
D.thyrsiflorum Rchb.f: This is the most widely used orchid in Chinese herbal
preparation after D. nobile due to its widespread distribution and strong reproductive
ability [83]. The high content of scoparone and coumarins in its system produces
effects of relaxing smooth muscles, expanding vessels, and anticoagulating blood
[84]. The orchid has been used as a good immune-modulator due to the rich presence
of diverse polysaccharides [85]. Apart from managing many chronic disorders, it is
also employed as a stimulant and commercial dye [86].
D. tosaense Makino: This is a medicinal orchid that locals consume as a quality
health food in Taiwan [87]. The infusion or decoction can be prepared by either
soaking the leaves in hot water or boiling them. The decoction made from the leaves
is used to treat anxiety and panic attacks in China [88].
Other Dendrobiums: The whole plant of D. longicornu is used to treat fever and
coughs [89]. The dried pseudobulbs of D. primulinum are grounded to make into a
pulp, which is used as an immune enhancer [90]. The paste made from the pseudo-
bulbs of D. transparens is applied for curing fractured and dislocated bones [62].
The stem parts of D. huoshanense are also utilized for promoting body immunity and
fluid secretion and also to treat throat inflammation, stomach pain, and ophthalmic
disorders [91]. The paste prepared from the dried pseudobulb of D. heterocarpum is
used to treat fractured and dislocated bones [52]. The juice extracted from the fresh
plant of D. ovatum can be given internally for controlling stomach pain. It also
stimulates the bile and acts as a laxative to the intestines [54, 92]. The pulp made
from the pseudobulbs of D. monticola is used in the treatment of boils, pimples, and
other skin rashes [54]. The pseudobulbs of D. tokai on the other hand, are used as
oral contraceptives in India [93].
3 In Vitro Propagation of Dendrobiums
3.1 Culture Media and Plant Growth Regulators
The success of orchid in vitro propagation depends mainly on the choice of culture
media and plant growth regulators (PGRs) used. This is because varied quantities of
inorganic and organic nutrients are provided to the growing tissues depending on the
culture media type employed to propagate orchids [94]. A defined culture medium
contains minor and major inorganic salts, carbon source, amino acids, and several
vitamins. However, as per requirement, the culture media can be incorporated with
organic acids, organic nitrogenous compounds, and other plant extracts [95, 96].
Normal media consist of few mineral salts with 30 mM each of inorganic nitrogen
and potassium, and ammonium salts at the range of 2–20 mM. Low concentration
(1–3 mM) of calcium, sulfate, phosphate, and magnesium salts is sufficient to impart
in vitro tissue growth. Sucrose is integrated into the medium as only carbon source,
but glucose, fructose, and other sugars like mannose and galactose may also be
included as the carbon source to assist cell growth [97]. Vitamins (thiamine HCl,
nicotinic acid, pyridoxine HCl, riboflavin, biotin, folic acid), an amino acid (glycine)
may be incorporated at varying concentrations depending on the kind of medium and
specific culture requirement. Nitrogen in the culture medium was known to affect in
vitro seed germination in several orchids [98, 99]. The nitrogen requirement for seed
germination is provided by NH4+ and NO3 [100].
Ammonium nitrate is the source of nitrogen in Murashige and Skoog (MS)
medium [101], while ammonium sulfate provides nitrogen in Mitra, Knudson C
(KC), Vacin, and Went (VW), and B5 media [102–105]. The high seed germination
and subsequent development for many Dendrobiums in MS medium may be attrib-
uted to the presence of ammonium nitrate and rich content of macro- and micro-
nutrients [92, 106]. The seed germination rate and culture growth on nutrient media
vary for different orchids as the nutritional requirement is species-specific [9, 107].
The in vitro propagation of Dendrobiums is generally performed on MS, Mitra, VW,
KC, and B5 media. The media are appended with different growth hormones to
enhance culture growth and differentiation. Cytokinins like benzyl amino purine
(BAP), kinetic (KN), isopentenyl adenine (2ip), and thidiazuron (TDZ) are used for
shoot initiation, multiplication, and plant regeneration. Auxins such as indole-3-
acetic acid (IAA), indole-3- butyric acid (IBA), 1- naphthalene acetic acid (NAA),
and 2,4-Dichlorophenoxyacetic acid (2,4-D) are essential for rooting induction and
multiplication apart from inducing cell division and cell expansion. The cytokinin
and auxin are often employed in combination at different concentrations to promote
shoot and root development leading to complete orchid regeneration.
3.2 Explants (Selection and Surface Sterilization)
The selection of the right explant is one of the key steps for effective micro-
propagation of Dendrobiums. The wrong choice of explant seriously undermines
the success of in vitro orchid propagation. The explant culture response is affected
by several factors such as genotypes, physiological stage of mother plants and
explant source, age, size, density, and its portion in donor/mother plant [108]. The
choice of the explant is dependent on plant material availability, seasonal abundance,
medium type and culture environment, age of the tissue explant, and other physio-
logical factors [109]. Several workers gave explant preference to immature seeds
[110–112], nodal part [113–116], shoot tips [90, 117], pseudobulb segment, and
axillary buds [118–120] for in vitro propagation of Dendrobiums. Juvenile tissues
must be chosen as they have more regeneration capability compared to differentiated
ones. Explants from the in vitro derived plants are favorable due to their high
regeneration potential, less exudation of phenolic compounds, and non-requirement
of disinfection before starting a tissue culture process.
The surface sterilization of explants is crucial as it removes the microbial con-

taminants from its surface before being inoculated to the nutrient medium. The
failure to eliminate the infectious agents during the process leads to unsuccessful
orchid micropropagation. Culture contamination with microbes is undesirable as it
compromises the normal in vitro culture development [121]. Active competition
exists between the microbes and growing tissues for nutrients, and the presence of
contaminants often leads to increase culture mortality, variable growth, tissue necro-
sis, and failure in shoot and root multiplication [122]. Different chemical agents are
employed for explant sterilization of Dendrobiums. Choosing proper concentration
and treatment duration is vital for minimizing explant tissue injury due to the toxic
nature of the disinfectants. Complete decontamination of explants with sterile
distilled water is required after every chemical treatment. Mostly mercuric chloride
(1–2%) and sodium hypochlorite (4–8%) have been used for explant surface steril-
ization to initiate culture in several Dendrobiums [11, 17–19, 123, 124].
3.3 In Vitro Propagation of Dendrobiums Using Different Explants
3.3.1 Seed Culture

Dendrobiums are epiphytic orchids whose seeds do not have thick seed coat.
Compared to the heavily lignified seed coat of terrestrial orchids, lignin and cuticular
materials are absent in the seed coats of Dendrobiums. The seeds are simpler and
easier to germinate, unlike terrestrial orchid seeds, as they can easily absorb water
and nutrient from the culture environment [109]. The mature seeds as explants are
preferred as they produce a higher germination percentage than the immature seeds
[107]. The disinfectant concentration for surface sterilization of the mature seeds in
dehisced capsules should be low as the thin seed coat of Dendrobiums may not
tolerate the harsh chemical effect of the sterilizing agent [125]. The seeds, when
inoculated on to appropriate nutrient medium, germinate by enlarging the embryos
and transforming into a highly meristematic spherical shape structure called pro-
tocorms [126]. The protocorms, under appropriate growth conditions, subsequently
develop into complete seedlings with well-formed leaves and roots.
Parthibhan et al. [127] cultured seeds of D. aqueum on half-strength MS basal
medium to produce protocorms. The protocorms were used to initiate in vitro
propagation of D. aqueum on half-strength MS medium augmented with different
cytokinins (BAP, 2ip, KN, and TDZ) and auxins (IBA, NAA, 2, 4-D) at varying
concentrations (1.0, 3.0, 5.0, 7.0, and 10.0 mgL1) along with other natural additives
of banana extract (BE) and coconut water (CW) (1%, 3%, 5%, 7%, and 10%). The
highest shoot number (9.3) per explants was obtained on 3.0 mgL1 NAA enriched
medium followed by the production of seven shoots per explants on medium
incorporated with 3% of BE. Rooting of the shoots was best observed with 8.75
roots per shoot on half MS medium supplemented with 5.0 mgL1 IBA. Root
elongation was maximum with root length measuring at 1.48 cm in 7.0 mgL1
NAA incorporated medium. Well-developed plantlets were transferred to small pots
with brick pieces and charcoal mixture (1:1) along with layers of mosses for better
acclimatization before they were hardened and transferred to a greenhouse. Non-

tachaiyapoon et al. [128] studied the effectiveness of eight putative orchid mycor-
rhizal fungi from three orchid genera in promoting in vitro seed germination and
protocorm development of D. draconis. The seed and protocorm developmental
stages were examined weekly by culturing on MS medium and Oat meal agar
(OMA), which was inoculated with one of the eight fungal isolates (PV-QS-0-2,
C1-Dt-TC-1, CS-QS-0.1, Da-KP-0-1, PV-PC-1-1, C3-DT-TC-2, Pch-Qs-0-3, PV-
QS-0-1, and C1-QS-0-1). The seeds of D. draconis germinated successfully (100%)
in all the treatments tested in 2 weeks, but further seed development varied consid-
erably with different treatment types. Three fungal isolates of different anamorphic
species of Tulasnella (C1-DT-TC-1, PV-PC-1-1, and 3-DT-TC-2) produced signif-
icant protocorm development, but none of the fungal isolates performed better than
the normal MS medium in regard to seed germination, protocorm formation, and
seedling development.
Paul et al. [129] studied the seed germination response and in vitro propagation of
D. hookerianum on MS, Mitra, KC, and B5 media. The seed germination was fastest
in MS medium with the highest seed germination percentage (95.27%) achieved in
3–4 weeks. The longest time in seed germination (7–8 weeks) with the lowest
germination percentage (51.38%) was observed in B5 medium. The protocorm
development, leaf, and root organogenesis and subsequent seedling development
were superior in MS medium as compared to the other three media. The study
suggested that growth hormone incorporation into the media was not essential for the
stimulation of orchid growth. Nongdam and Tikendra [112] adopted seed culture to
in vitro propagate D. chrysotoxum on Mitra medium enriched with different combi-
nations and concentrations of auxins and cytokinins. Maximum seed germination
was attained in 2 weeks when seeds were grown on medium supplemented with
2.0 mgL1 BAP, 2.0 mgL1 IAA, and 0.4% activated charcoal (AC). A higher
concentration of cytokinins (BAP or KN) with a low level of auxin (NAA or IBA)
promoted shoot and leaf multiplication. But the reduction in shoot response was
noticed when the medium was incorporated with the only cytokinin, thereby
suggesting the synergetic effect of auxin and cytokinin in shoot and leaf develop-
ment. IBA was better than NAA in inducing rooting, but root formation was more
pronounced when auxin was incorporated with a low concentration of cytokinin.
Utami et al. [130] germinated seeds of D. lasianthera on VW medium fortified
with varying concentrations of peptones (1.0, 2.0, 3.0 gL1). Maximum seed
germination (100%) and shoot formation (84%) were accomplished with medium
enriched with 2.0 mgL1 peptone. The role of organic additives on subsequent shoot
development was also examined by inoculating the nascent seedlings with 1–2
leaves on a medium incorporated with different organic nutrients. The shoot and
root growth in the seedlings were much improved in medium containing 15%
coconut water. Tikendra et al. [11] reported the in vitro propagation of D.
thyrsiflorum using seeds obtained from unripe capsules. Multiple shoot formation
was observed when either BAP or KN was present in the Mitra medium. But raising
the concentration of cytokinin increased shoot production and shoot length in the
culture. Medium containing 2.0 mgL1 KN generated more shoots (3.16 0.47)
compared to BAP (2.18 0.54) at a similar concentration. The presence of both

cytokinin (BAP or KN) and auxin (IAA, IBA, or NAA) in the medium gave more
shoots than the medium supplemented with only cytokinin. Shoot formation was the
highest (3.83 0.48) when the medium was appended with 1.0 mgL1 KN and
2.5 mgL1 IAA. Among the auxins tested, IAA produced the best root initiation with
maximum root formation (6.32 0.37) attained in 2.0 mgL1 IAA-enriched
medium. Tikendra et al. [24] also accomplished effective seed germination and
subsequent protocorm formation of D. moschatum in Mitra medium supplemented
with different concentrations of BAP (Figs. 1a & b). Shooting initiation was
Fig. 1 In vitro propagation of D. moschatum by seed culture. (a) Swelling of seeds indication
successful germination in M + 2.4 mgL1 BAP. (b) Protocorm formation witnessed in
M + 2.4 mgL1BAP. (c) In vitro shoot initiation after protocorm development in
M + 0.6 mgL1TDZ. (d) Shoot multiplication and leaf formation in M + 1.2 mgL1TDZ + 1.2 mgL1
NAA. (e) High root multiplication observed in M + 1.2 mgL1IBA + 1.2 mgL1 TDZ. (f)
Hardening of healthy and well-developed plantlets
prominent when TDZ and BAP were incorporated singly or in combination with
auxin in the medium (Fig. 1c). High shoot multiplication and leaf formation were
noticed in medium supplemented either with 2.4 mgL1 BAP or1.2 mgL1
TDZ + 1.2 mgL1 NAA (Fig. 1d). IAA was the least responsive to in vitro rooting
as compared to IBA and NAA. Root formation was high in the medium
supplemented either with 1.2 mgL1IBA + 1.2 mgL1 TDZ or 1.2 mgL1 NAA
+1.2 mgL1 TDZ (Fig. 1e). The well-developed seedlings were hardened after
acclimatizing them in the greenhouse condition using bricks, charcoal, and coconut
husk (1:1:1) as a potting mixture with 95% survival rate (Fig. 1F).
Lin et al. [19] germinated the seeds of D. cariniferum on half-strength MS and
MS basic medium. The seeds showed faster germination in half-strength MS com-
pared to full- strength MS medium after 30 days of culture. They noticed the
influence of pH of the medium on the protocorm multiplication rate with the highest
proliferation recorded in the medium with pH 5.7 and the lowest observed under
pH 5.9. Different concentrations of NAA and BAP were incorporated into the
medium to test their influence on protocorm differentiation. Protocorm proliferation
and differentiation were best noticed in medium with 0.1 mgL1 NAA and
1.0 mgL1 BAP compared to other hormonal combinations. Protocorm differentia-
tion to seedlings was influenced by the level of peptone concentration. Medium
integrated with 1.5 mgL1 peptone produced the fast-growing thickest seedlings
with the best root development.
3.3.2 Shoot Tips

Different explants like shoot tip, nodal and pseudobulb segment, floral stalk, and
inflorescence were employed apart from the seeds to in vitro propagate several
Dendrobiums. While the seeds differentiated into protocorms, other explants gave
rise to protocorm-like bodies (PLBs) before developing into plantlets [131]. Morel
[132] first coined the term “Protocorm like bodies,” which were the structures similar
to the seed-derived meristematic protocorms obtained directly from tissue explants
and or/callus under in vitro condition. The PLBs differentiate into multiple shoot
buds, but growth hormone concentration and combination are the keys to successful
PLB formation and multiplication and subsequent development into seedlings [133].
Sharma and Tandon [134] employed excised shoot apices to grow D. wardianum
on MS medium incorporated with various inorganic and organic sources like
calcium nitrate, ammonium sulfate, urea, and amino acids. Direct multiple shoot
formation was observed in the medium supplemented with 2.5 mgL1calcium
nitrate and 0.5 mgL1 urea, apart from generating PLBs, which subsequently
developed into complete plantlets. Kanjilal et al. [135] used a transverse section of
shoots obtained from 8 weeks old in vitro grown seedlings to propagate D.
moschatum on KC medium enriched with 15% coconut water (CW) and varied
concentration of growth hormones. Medium supplemented with 15% CW and
1.0 mgL1 2,4-D prompted PLB formation but PLB proliferation was more prom-
inent in medium augmented with 15% CW, 3.0 mgL1 IBA, and 2.0 mgL1 NAA.
The explant survival rate was 93%, with the formation of 7.6 PLBs per explant.
Rooting development was best with 1.0 mgL1 NAA supplemented in the potting
mixture consisting of bricks, charcoal chips, sand, and soil in 1:1:1:1 ratio. Roy and
Banerjee [136] used shoot tips for in vitro propagation of D. fimbriatum on KC
medium supplemented with different concentrations of NAA and BAP. Callus
formation was observed in NAA and BAP supplemented medium with 66.67% of
the shoot tip explants responding to callus formation. However, the hormone-free
medium produced PLBs instead of callus formation, which subsequently developed
into shoots. The culture upon transferred to medium enriched with 4.0 mgL1 BA
and 0.5 mgL1 NAA induced shoot proliferation through axillary branching.
Malabadi et al. [137] inoculated a thin transverse section (1 mm) from the shoot
tips of in vitro cultured D. nobile on Mitra medium appended with different
concentrations of triacontanol (TRIA). Medium supplemented with 4.0 mgL1
TRIA proved effective for PLB initiation from shoot tips and further shoot bud
proliferation with 93% of the explants producing many PLBs and shot buds in the
culture. Roy et al. [66] tested the growth potential of shoot tips of D. chrysotoxum on
KC medium augmented with different concentrations of TDZ, BAP, and NAA.
Medium incorporated with either 2.0 μM TDZ or BAP recorded the highest
callusing, while the presence of 0.5 μM NAA in the medium produced 69-fold
increase in callus weight in 3 months. High PLBs formation through the intervening
callus phase was noticed with 1.0 μM NAA-enriched medium. The direct PLB
formation from the explant was also witnessed depending upon the type of cytokinin
used and its dosage. Medium augmented either with 1.0 μM TDZ or 8.0 μM BAP
produced similar PLBs yield in the culture.
Pornpienpakdee et al. [138] found the development of PLBS from shoot tips of
Dendrobium Eiskul hybrid on VW medium appended with six different chitosan
molecules. Medium with either 10 mgL1 P-70 or 20 mgL1 P-90 chitosan pro-
duced optimal PLB initiation and multiplication. But the presence of 20 mgL1 O-80
chitosan in the medium was essential for shoot induction in PLBs. Medium incor-
porated with 10 mgL1 O-80 chitosan promoted plantlet regeneration after shoot
induction of the PLBs. The increased concentration of P-70 chitosan from 10 mgL1
to 16 mgL1 in the medium enhanced the in vitro to ex vitro transplanting efficiency
from 95 to 100%. Asghar et al. [139] employed lateral shoots (8.0 cm) as explants to
propagate D. nobile in phytotechnology medium (O753) supplemented with BAP
and KN along with CW as additives. Medium with 2.0 mgL1 BAP produced a
maximum number of shoots (4.33), while the most extended shoots (4.18 cm) were
obtained in medium augmented with 1.5 mgL1 KN. Incorporation of higher
concentrations of BAP, KN (3.0 mgL1), and CW (300 mlL1) led to necrosis,
low growth, and yellowing of shoots, thus hampering shoot growth and develop-
ment. High rooting percentage (97.5%), root number (4.70), and root length (3.4 cm)
were noticed in IBA supplemented medium. Rooting development was more pro-
nounced with IBA than with NAA though the increased concentration of both auxins
(3.0 mgL1) resulted in an inadequate rooting response.
Pant and Thapa [90] initiated shooting of shoot tips (0.3–0.5 mm) derived from in
vitro propagated seedlings of D. primulinum, on MS medium fortified with different
growth hormones. Maximum shoots (4.5 shoots per explant) was recorded in MS
medium enriched with 1.5 mgL1 of IBA. The root formation (three roots per shoot)
was best observed 3 weeks after the in vitro regenerated shoots were transplanted to
a rooting medium with 0.5 mgL1 IBA. Pradhan et al. [117] grew D. densiflorum
using shoot tips on MS medium appended with varied combinations of cytokinins
and auxins. Shooting initiation was noticed in medium supplemented with
0.5 mgL1 NAA and BAP (0.5–2.0 mgL1). Maximum shooting (four shoots per
explants) was witnessed in MS medium incorporated with 2.0 mgL1 BAP and
0.5 mgL1 NAA. Rooting of the in vitro propagated shoots was accomplished on a
medium integrated with auxins (NAA, IAA, or IBA) at varying concentrations
(0.5–-2.0 mgL1). IBA proved more effective in rooting compared to IAA and
NAA. The best rooting response (4.5 roots per shoot) was produced in MS medium
with 1.5 mgL1 IBA. Winarto et al. [16] initiated shoot tip culture of Dendrobium
‘Zahra FR 62’ leading to PLB formation and multiplication in half-strength MS
medium augmented with 1.0 mgL1 TDZ and 0.5 mgL1 IBA. The initial PLB
production was high, with 5–10 new PLBs generated from 3–5 PLBs in 4–5 months.
PLB proliferation and plantlet conversion took place with a medium incorporated
with 2% sucrose and 0.05 mgL1 BA.
3.3.3 Pseudobulb Segments

Vij and Pathak [140] employed a pseudobulb segment (0.5–1.0 cm) from a 40-week old
axenic culture to grow D. chrysanthum in MS medium supplemented with different
PGRs. Among the growth hormone tested, IAA, gibberellic acid (GA3), or BAP did not
elicit any culture response. Medium enriched with 1.0 mgL1 NAA and 1.0 mgL1 KN
along with 2.0 gmL1 yeast extract and 25 ml urea evoked organogenic response.
Thirty-seven percent of the explant responded, leading to the development of complete
plants with well-developed leaves and roots in 18–25 weeks.
Yasugi and Shinto [118] also observed positive shooting responses from the
pseudobulb segment on MS medium supplemented with 0.1 mgL1 NAA and
0.1 mgL1 BAP. Shoot generation was the highest in BAP- and NAA-enriched
medium with the formation of 2.3–2.5 shoots per pseudobulb segment in 8 weeks of
culture. The robust rooting response was also noticed in the same hormonal combi-
nations with the production of 5.8–9.0 roots per shoot and corresponding shoot
length ranging from 5.3–7.3 mm. Sharma et al. [119] successfully induced axillary
bud development from pseudobulb explants on MS medium augmented with
7.5 mgL1IAA and 20 mgL1 BAP. Medium supplemented with 2.0 mgL1 BAP
generated maximum shoot number (39) with increased shoot length and pronounced
bulblet formation. Incorporation of IAA in the medium induced rooting of in vitro
shoots with 90% of them successfully rooted. Hossain et al. [123] utilized pseudo-
bulb sections derived from in vitro grown seedlings of D. aggregatum to assess
culture morphogenetic response. MS medium augmented with 1.0 mgL1 BAP and
0.5 mgL1 picloram produced multiple shootings with the highest shoot formation
of 7.75 shoots per explant in about 35 days. The presence of 0.5 mgL1 IAA in the
medium induced rooting, and well-rooted plantlets were successfully hardened with
80% survival rate by transferring them to community pots filled with sterilized bricks
pieces, charcoal, and peat moss (1:1:1).
3.3.4 Nodal Segment

Nodal segments were used by Gao et al. [141] to grow D. nobile in different culture
media viz., MS, B5, and KC supplemented with different concentrations of BAP and
NAA. The best shoot induction was observed in MS + 0.5 mgL1 BAP + 0.2 mgL1
NAA, while maximum shoot multiplication was noticed in MS + 3.0 mgL1
BAP + 0.5 mgL1 NAA. IBA promoted root initiation, but the highest rooting
response was found in MS medium fortified with 0.1 mgL1 NAA. Bai et al. [142]
tested in vitro culture response of axenic nodal segments on either half or full-
strength MS medium appended with BAP, NAA, and IBA. Induction of adventitious
bud was witnessed in either hormone-free half or full-strength MS medium. BAP
promoted shooting and in vitro shoots were rooted completely on half-strength MS
medium enriched with IBA at a concentration range of 0.2–0.4 mgL1 and 0.1%
activated charcoal (AC). Bhattacharyya et al. [143] propagated D. aphyllum by
implanting a thin cell layer obtained from nodal segments on MS medium aug-
mented with varied combinations of growth regulators and meta-topolin. Shoot
proliferation was best with an average production of 39.4% shoots per explant in
medium with 15 mM meta-topolin, 10 μM TDZ, and 10 μM AgNO3. The rooting
frequency was maximum (82.34%) in half-strength MS medium incorporated with
15 μM IBA. Dohling et al. [100] employed the nodal segment to propagate D.
longicornu on MS medium with 3% sucrose, 0.8% agar, and growth hormones
(NAA, 2,4-D, and BAP) having a concentration in the range 1–50 μM. NAA
produced the highest shoot bud number without PLBs formation, and the maximum
shoot number was generated in 30 μM NAA-enriched medium. The presence of 2, 4-
D, on the other hand, gave a variable response by producing both shoots and PLBs.
When 15 or 20 μM of 2, 4-D was associated with 15 μM BAP, only PLB formation
took place with the occurrence of the highest PLB conversion (41.48%).
Bhattacharyya et al. [17] tested the efficiency of nodal segments of D. crepidatum
for PLB and shoot formation on MS medium containing diverse concentrations of
either TDZ or KN with 0.5 mgL1 NAA. Incorporation of TDZ singly at 3 mgL1
produced a shooting response frequency of 55%, which was enhanced to 97% by
adding 0.5 mgL1NAA in the medium. Influence of different polyamine concentra-
tion on shoot multiplication was also examined for explants subcultured in MS+
2.0 mgL1 TDZ + 0.5 mgL1 NAA. Medium supplemented with 0.8 mM putres-
cine, 2.0 mgL1 TDZ, and 0.5 mgL1 NAA generated maximum shoots (11.8 per
explant), while the highest rooting response was achieved in 2.0 mgL1 IBA
incorporated medium.
Pant et al. [144] documented in vitro organogenesis from the nodal segment in D.
fimbriatum. Medium supplemented either with BAP or KN singly did not produce
any effective shoot organogenesis. But medium with BAP and NAA at a similar
concentration of 17.76 μM showed shoot bud initiation, proliferation, and shoot
multiplication after 10 weeks of culture. However, enhancement of hormone con-
centration level in the medium lowered the shoot formation. The regenerated shoots
were best rooted in a rooting medium with 8.88 μM of NAA, producing maximum
root number (9.20 0.24) and longer root length (2.68 0.01 cm). The well-
developed seedlings were initially acclimatized in small pots with vermiculite for
about 2 weeks. Axenic stem nodal segments were used by Zhang and Gao [124] to
induce PLB formation in D. officinale on solid half-strength MS medium containing
2.0 mgL1 BAP, 0.5 mgL1 NAA, and 30 gL1 sugar. The axillary buds developed
from the stem nodes in 14 days gave rise to PLBs, which differentiated into shoot
tips. The induced PLBs with emerging shoot tips, when shifted to H16 media,
differentiated into cluster buds, which on further subculturing subsequently devel-
oped into well-rooted seedlings. The final hardening was performed by transferring
the partially acclimatized seedlings to thermocol pots filled with brick pieces,
charcoal, and mosses (1:1:1) with 84% survival rate.
3.3.5 Flower Stalk Node

Flower stalk nodes were utilized to in vitro propagate Dendrobium hybrid Sonia 17
and 28 by Martin et al. [145]. Bud break was initiated when the flower stalk node
was cultured on half-strength MS medium enriched with either 6.97 μM KN and
15% coconut water (CW) or 13 μM BAP alone. KN in the medium improved shoot
bud formation as five shoots per shoot bud were formed when the excised shoots
were transferred to KN-enriched medium. BAP, on the other hand, promoted PLB
formation with shoot buds developing into multiple PLBs on 44.4 μM BAP incor-
porated medium. Further conversion of PLBs into shoots was accomplished when
they were moved to half-strength MS medium augmented with 67 μM KN. The
rooting of the shoots was best on half-strength MS medium incorporated with 2 gL1
AC, and the complete seedlings with leaves and roots were acclimatized and
hardened with 80% survival rate.
The recent in vitro propagation works on Dendrobiums performed by several
researchers using different explants in the last 5 years are listed in Table 1.
½ MS ¼ half strength Murashige and Skoog medium; 2,4-D ¼ 2,4-dichlorophenoxy
acetic acid; CW ¼ coconut water; BAP ¼ 6-benzylamino purine; FT ¼ foliar fertilizer;
GA3 ¼ gibberellic acid; IAA ¼ indole-3- acetic acid; IBA ¼ indole-3- butyric acid;
KC ¼ Knudson C medium; KN ¼ kinetin; MS ¼ Murashige and Skoog medium;
M ¼ Mitra medium; NAA ¼ 1- napthaleneacetic acid; Pic ¼ picloram; PLB(s) ¼ pro-
tocorm-like bodies; PM ¼ phytamax medium; TDZ ¼ thidiazuron; VW ¼ Vacin and
Went; ZN ¼ zeatin
4 Genetic Stability of In Vitro Propagated Dendrobiums
4.1 Somaclonal Variation
The primary objective of micropropagation is the production of identical clones

genetically similar to the elite mother plants. Genetic differences may appear among
the propagated plants as the cells and tissues are continuously confronted with
various stress conditions during in vitro culture. The genetic variation detected in
the plants derived from any in vitro cultured cells or tissues was termed “Somaclonal
variation” by Larkin and Scowkraft [167]. Somaclonal variation in the regenerated
plants is not only detrimental for elite genotype conservation but also undesirable for
Table 1 In vitro propagation of different Dendrobiums from various explants in the last 5 years
Culture medium having optimal
Species Explant used growth response References
D. antennatum Lindl Seeds • 100% seed germination was [146]
observed in three different
combinations: MS + 10% CW; MS+
growmore (1.0 mgL1) + 10% CW;
MS+ growmore
(1.0 mgL1) + 50 mgL1 spring
onion
• Plantlet height (58.0 5.0 mm)
was maximum in MS + growmore
(1.0 mgL1) + 10% CW, while the
root number (5.9 2.1) and root
length (33.3 7.8 mm) were
superior in MS+ growmore
(1.0 mgL1) + 50 mgL1 spring
onion
• Shoot number was maximum
(1.4 0.5) in MS + 10% CW
D. aurantiacum Rchb.f Shoot tips • Callusing was 100% successful in [147]
all the combinations tested but
MS + 10.0 mgL1 2,4-D took the
shortest period (3 days) along with
the highest cell concentration (43.73
x 106)
D. aqueum Lindl Seeds-derived • ½ MS + 3.0 mgL1 NAA induced [148]
protocorms the maximum shoot number
(9.4 1.81) per explant while the
shoot length was highest
(1.52 0.20 cm) in ½
MS + 7.0 mgL1 NAA
• ½ MS + 5.0 mgL1 IBA recorded
the highest root number
(8.75 1.18) per explant while the
longest root length (1.48 0.13 cm)
was witnessed in ½ MS + 7.0 mgL1
NAA
Stem • Highest (42.67 0.58) globular [149]
transverse somatic embryogenesis (SE) per
thin cell tTCL was observed in ½ MS + 1.5
layers mgdm3 2iP
(tTCLs) • Indirect SE callus response was
best (53.33%) in ½ MS + 1.5
mgdm3 2iP + 1.0 mgdm3 IBA
• Direct SE response of 25.0% was
observed in ½ MS + 0.5 mgdm3
BAP + 1.5 mgdm3 2iP
D. bensoniae Rchb.f Shoot nodes • Shoot induction (80%), shoot [150]
multiplication (4.66 0.57 per
explants), and leaves per explant
(9.33 1.15) were highest in
(continued)
Table 1 (continued)
MS + 2.0 mgL1 BAP
• Root induction (90%) was
maximum in MS + 1.0 mgL1
BAP + 1.5 mgL1 IBA
• Root multiplication was highest
(10.35 0.07 per explant) in
MS + 0.5 mgL1 BAP + 1.0 mgL1
IBA
D. candidum wall. Ex Shoot derived • Earliest (7 days) callus formation [151]
Lindl PLBs with 100% rate of callus induction in
MS + 1.0 mgL1 2,4-D + 0.5 mgL1
KN, but the re-differentiation rate
was lower than 30%
• Re-differentiation rates were
superior (about 50%) in
MS + 10.0 mgL1
NAA + 0.25 mgL1 BAP;
MS + 10.0 mgL1
NAA + 0.5 mgL1 BAP;
MS + 10.0 mgL1
NAA + 0.25 mgL1 KN;
MS + 10.0 mgL1
NAA + 0.5 mgL1 KN
D. cariniferum Rchb. f Seeds • Seed germination was faster in ½ [19]
MS basal medium
• Medium at pH 5.7 witnessed high
rate (5.8 0.92) of protocorm
proliferation, with temperature
(23 2 °C) and light intensity (1000
lux) optimal for protocorm
multiplication
• Protocorm proliferation was highest
(10.27 0.52) in MS + 0.1 mgL1
NAA + 1.0 mgL1 BAP
• Seedling growth with best
(10.01 0.28) rooting response was
observed in MS + 1.5 mgL1
peptone
• Culture period (60 days),
temperature (23 2 °C), and light
intensity (1500 ~ 2000 lux) were the
optimal conditions for bioactive
compounds accumulation
D. chryseum Rolfe Protocorms • Shoot number (18.75 0.48 shoots [152]
per culture) was highest in ½
MS + 2.0 mgL1 KN + 10% CW,
while the shoot length was longest
(2.0 0.20 cm) in ½
MS + 1.0 mgL1 GA3 + 10% CW
(continued)
Table 1 (continued)
• Rooting (5.0 roots; 1.7 0.17 cm)
was best achieved in ½
MS + 1.5 mgL1 IAA
D. chrysotoxum Lindl In vitro- • Highest regeneration response [153]
derived nodal (100%) with 20.70 0.0 PLBs
segments observed in MS (liquid) + 5.37 μM
NAA. The plantlet development was
also earliest (9 weeks) in this
hormonal combination
D. fimbriatum hook Nodal • Shoot multiplication (14.00 0.47) [18]
segments and shoot length (1.50 0.02) were
superior in MS + 17.76 μM
BAP + 17.76 μM NAA
• Maximum root number
(9.20 0.24) and root length
(2.68 0.01 cm) were witnessed in
MS + 8.88 μM NAA
D. Hybrid In vitro- • FT NPK (32:10:10) had [154]
derived significantly higher growth response
seedlings than ½ MS medium
• FT + tomato extract produced the
highest fresh weight (1.8 g), and
height (6.78 cm) of the seedlings
• Leaf number (5.86), and root
number (9.58) were highest in
FT + mungbean sprout extract, while
the root length (3.72 cm) was
maximum in the medium containing
potato extract
D. lasianthera J.J.Sm Seeds • MVW + 3.0 gL1 peptone recorded [130]
the best seed germination (84%)
• MS + 15% CW regenerated the
highest length of plantlet
(3.4 1.7 cm), leaves
(1.9 0.5 cm), roots (1.8 0.4 cm),
and maximum number of leaves
(5.2 2.1) and roots (6.0 3.6) after
16 weeks of culture
D. moschatum Sw. Seeds • Overall shoot multiplication was [24]
superior in MT medium appended
with TDZ
• Root formation with high
(7.46 0.64 roots) in
M + 1.2 mgL1 TDZ + 1.2 mgL1
IBA
• Maximum shoot length
(6.41 0.54 cm) was noticed in
MT + 0.6 mgL1 TDZ, while the
(continued)
Table 1 (continued)
highest root length (6.92 0.60 cm)
was witnessed in
MT + 1.2 mgL1TDZ + 1.2 mgL1
IBA
D. nobile Lindl Seeds, PLBs • M + 1.0 mgL1 NAA recorded the [155]
earliest seed germination
(2.05 0.05 weeks), protocorm
development (4.40 0.14 weeks),
and seedlings formation
(13.25 0.50 weeks) along with the
highest frequency of seed
germination (98.50 1.3%)
• The highest PLB formation per
explant (10.0 0.4) and maximum
shoot per explant (14.0 0.0) were
observed in M + 20% CW along with
the earliest development of plantlets
(14.0 0.40 weeks)
Nodal • Shoot proliferation was highest [17]
segments (21.8 0.5) in MS+ 1.0 mgL1
meta-topolin +0.8 mgL-1 putrescine
• Rooting frequency was maximum
(10.1 0.4) in ½ MS+ 2.0 mgL-1
IBA + 0.5 mgL-1 phloroglucinol
• The highest shoot induction (80%) [156]
and shoot multiplication
(4.32 1.05 per explant) were
achieved in MS + 2.5 mgL1 BAP
• Rooting was best (90%) in
MS + 1.0 mgL1 IBA + AC
D. ovatum (Willd.) PLBs • Spherule formation from micro [157]
Kranzl seeds was highest (94.7 1.01%;
95.5 0.97%) in modified ½ MS
incorporated separately with
0.1 mgL1 ZN or 10% CW
• Maximum (348.6 6.90) PLB
development was observed in ½
MS + 0.1 mgL1 ZN
• PLB conversion to plantlets was
most efficient (93.0 1.65%) in ½
MS + 10% CW
D. palpebrae Lindl Seeds • Seed germination (80%) was best in [158]
PM + 2.0% sucrose than other media
(KC, MS, MVW) experimented
• In vitro flowering occurred only in
MS + 0.8% agar +3.0% sucrose
+0.5 mgL1 pic +1.0 mgL1 BAP,
and MS+ PM + 2.0% sucrose
(continued)
Table 1 (continued)
+0.5 mgL1 NAA + 1.0 mgL1 BAP
• Root length (4.63 0.22 cm) and
root number (2.53 0.14) were
highest in MS + 0.5 mgL1 IBA
D. “pompadour” Meristem tips • Highest survival rate (64%) and [159]
(hybrid of D. Louis occurrence of the green and visible
Bleriot X D. development (3.2) from the
Phalaenopsis) meristem-tips were noticed in VW
(liquid) + 0.1 mgL1 NAA
• A frequency of 87.7% clones or
49.5% of the total explants were
virus free
D. primulinum Lindl Seeds • Seed germination started in 2 weeks [160]
in MS + BAP and the maximum
capability of seedling growth
(4.5 1.29) was obtained in
MS + 1.5 mgL1 BAP
• Protocorm formation was earliest in
basal MS medium but enhanced
when the medium was incorporated
with 0.5 mgL1 each of BAP and
NAA
D. Red bull Shoot tips • After 150 days of culture, [161]
maximum number of shoots (7.66)
was observed in MS + 3.0 mgL1
BAP + 1.0 mgL1 NAA
• Shoot length was highest
(21.19 cm) in MS +3.0 mgL1
BAP + 1.5 mgL1 NAA
• Root multiplication was maximum
(4.67) in MS + 4.0 mgL1
BAP + 1.5 mgL1 NAA
D. signatum Rchb.f Seeds • MS + 10% potato extract; ½ [162]
MS + 10% potato extract;
MS + 5.0% mashed banana recorded
100% seed germination
• ½ MS basal and ½ MS + 2.0 mgL1
TDZ +0.5 mgL1 NAA produced the
highest shoot proliferation
(67.0 8.33%), while the root
multiplication was best (5.3 2.02)
in ½ MS + 2.0 mgL1
BAP + 0.5 mgL1 NAA
D. Sonia Rhizome • Shoot induction was best in [163]
buds MS + 11 μM BAP
• Shoot multiplication, PLB
formation, and rooting was
maximum in MS + 11 μM
(continued)
Table 1 (continued)
BAP + 11.42 μM IAA
• The monochromatic light spectra
best suited for shoot initiation and
proliferation was yellow light (at an
intensity of 24.6 μmol/m2/s and
590 nm wavelength). High shoot
proliferation rate (98%) with 290
shoots per ten explants was recorded
under yellow light influence
• Although the leaf area
(32.1 1.22 mm2), and fresh weight
(5.5 0.40gm) were highest in
yellow monochromatic light, the
shoot length (5.4 0.39 cm) and
root length (2.1 0.33 cm) were
superior under blue light (at intensity
of 22.5 μmol/m2/s and 470 nm
wavelength)
D. Sonia “Earsakul” Petals and • ½ MS + 1.0 mgL1 [164]
sepals BAP + 0.5 mgL1 NAA produced
maximum (75.0 11.2%)
meristemoid induction of the petal
tissues transiently transformed by
Agrobacterium tumefaciens strain
EHA105 harboring pCAMBIA-1301
that infiltrated the petal tissues
• Larger (0.5–2.0 mm) and highest
(4.8 1.8) mean meristemoids tissue
per explant was observed in ½ MS
(liquid) +1.0 mgL1
BAP + 1.0 mgL1 NAA
• The maximum (90 10.0%)
survival rate with 0.0% bacterial
contamination was observed in the
agroinfiltrated petal tissues treated
with 20 mgL1 meropenem
D. Sonia “red jo” PLBs • Seedlings grew better in [165]
VW + NaCl (5.0–40 mM) than
medium without NaCl or with
CaSiO3 or proline
• Maximum shoot (0.61 0.2 cm)
and root length (0.81 0.36 cm)
were evident in VW + 5.0 mM NaCl
• Fresh weight (0.11 0.04) was
maximum in VW + 40 mM NaCl
D. thyrsiflorum Rchb.f Seeds • Successful seed germination [11]
occurred in 2–3 weeks of culture
• Highest (3.49 0.96) shoot
multiplication was observed in
(continued)
Table 1 (continued)
M + 1.0 mgL1 KN + 1.0 mgL1
IAA
• Maximum root multiplication
(6.52 0.37) was recorded in
M + 2.0 mgL1 IAA
• Transferring of plantlets from
M + 1.0 mgL1 KN + 1.0 mgL1
IAA to new combination of
M + 1.0 mgL1 KN + 2.5 mgL1
IAA further increased the shoot
number (3.83 0.48)
D. trankimianum T Latent buds • With 92.06% of PLBs formation, [166]
Yukawa MS (modified) + 1.5 mgL1
TDZ + 0.5 mgL1 NAA produced
the highest (14.11) PLB regeneration
per explant
• Maximum shoot number (22.35
shoots/explant) and shoot length
(1.96 cm) were recorded in
MS + 1.5 mgL1 BAP
• Among the various effects of mash,
medium incorporated with 60 g ripe
banana per liter of medium
regenerated the highest shoots/
explant (25.11), and shoot length
(2.12 cm)
• With 98.51% rooting, ½
MS + 0.5 mgL1 NAA produced the
highest root number (7.91) and root
length (4.01 cm)
economic profits if the primary regenerants are the desired end products [21, 168].
Therefore, it is imperative to ascertain clonal fidelity to propagate only the geneti-
cally stable plants.
Choosing proper explant for culture initiation is critical for preserving the genetic
stability of the regenerated plants. Genetic variation is anticipated among in vitro
clones propagated through seed explants as they are derived from the fusion product
of gametes from genetically different parents. Utilization of meristematic tissues like
shoot tips, axillary,, and stem nodes as starting materials for micropropagation is
likely to generate genetically identical plants by reducing variation [169]. The
mature and differentiated tissues like roots, leaves, and stems produce more variation
than juvenile explants with preexisting meristems due to the intervening callus phase
[119, 170]. The callus-mediated plant propagation is involved with the risk of
genetic aberration as callus formation is associated with the dedifferentiation
phase, which is generally followed by abnormal cell divisions [171]. As explant
preparation from the same donor plant escalates the chance of variant production, it
is important to select a donor plant with inherent genetic composition and genome
uniformity [172]. Plants generated through somatic embryogenesis are more genet-
ically stable than those propagated through organogenic differentiation, as DNA
methylation is less in early embryogenesis [171].
Factors like longer explant disinfection in sterilants, high hormone concentration,
long culture duration/ subculture cycle, the callus transition phase, explant cell/tissue
heterogeneity, and other spontaneous mutations maybe the reasons for the occur-
rence of a genetic aberration among the in vitro clones [20, 21]. The chances of
somaclone production in prolong culture increase as there is a possibility of greater
accumulation of genetic variation during successive subcultures [173]. The rise in
subculture frequency may also intensify the rate of somaclonal variation produced
by nucleotide sequence alternation rather than quantitative changes as shown by
constant C-value even after the seventh subculture cycle of olive genotypes [174].
Some growth hormones at specific concentrations/in different combinations may
incite mutation producing genetic variation among the regenerants [175, 176].
Carvalho et al. [177] observed the increased generation of somaclonal variants
with 2,4-D in prolonged culture. DNA methylation rate was raised in the presence
of 2, 4-D, which changed the DNA ploidy level resulting in the generation of variant
clones. Arnhold-Schmitt [178] observed changes in chromosome arrangement and
DNA methylation in carrot callus culture when IAA was present with inositol in the
medium. The plant growth regulator ratio also affected the in vitro genetic changes
as low and high incidence of the variant “mantled” flowering was witnessed in high
auxin/cytokinins and high cytokinins/ auxin ratio, respectively, by Eeuwens et al.
[179] in oil palm.
4.2 Genetic Stability Assessment Using DNA Markers
The long-term benefit of Dendrobium micropropagation lies in the production of

genetically stable plants identical to elite mother plants. Genetically uniform plant
production confers a great advantage to the cut-flower industry as it helps in the
production of uniform blooming during predictable periods to meet the market
demands. If the genetic uniformity can be retained for a longer duration, the overall
production cost will be reduced significantly, and the whole process would be highly
profitable. The genetic variation is generally due to alteration in chromosome
numbers (aneuploidy and polyploidy), chromosome structure (due to deletion,
duplication, insertion, or translocation), DNA base mutations, and gene amplifica-
tion or methylation, as in the case of epigenetics [180–185]. Different strategies that
may be employed to assess the genetic stability include morphological characters
based phenotypic identification, cytological analysis of chromosomal alteration, and
isozyme-based variation study [186–188]. Flow cytometry analysis can also be
performed to complement the traditional cytological studies to give a correct assess-
ment of any change in ploidy [189, 190]. Since these methods are associated with
limitations, more effective PCR-based DNA markers have been used for genetic
stability testing of the micropropagated orchids.
DNA markers have become a versatile tool in plant genotyping in the last three
decades. The two-classes of DNA markers, viz., the codominant markers such as
simple sequence repeat (SSR), restriction fragment length polymorphism (RFLP),
amplified fragment length polymorphism (AFLP), and single nucleotide polymor-
phism (SNP); and the dominant markers like random amplified polymorphic DNA
(RAPD), inter-simple sequence repeat (ISSR), inter-retrotransposon targeted ampli-
fied region (IRAP) and start codon targeted polymorphism (SCoT), have allowed the
researchers to study the somaclonal variation present either in a single known locus
or at different loci in the plant genome, respectively [191–197]. RAPD, the simplest
and inexpensive DNA marker, is extensively used to detect somaclonal variation but
is coupled with drawbacks of low reliability and reproducibility [198]. ISSR markers
are more effective and reliable with longer primers (15–30 mers) and higher
annealing temperature, giving greater stringency than RAPD [199]. But both the
markers are simple, fast, and cost-effective and use only a single primer to amplify
the genomic DNA [200, 201]. They are also easier technically than RFLP, SSR, and
AFLP markers as no prior sequence information is required for generating DNA
amplification products [201, 202]. With the introduction of gene-targeted markers
such as SCoT and TRAP (targeted region amplified polymorphism), the polymor-
phism can be detected with reproducible bands and accurate results [22]. SCoT is an
emerging marker that gains popularity over other dominant markers such as RAPD
and ISSR. This marker system requires a single primer during amplification and is
based on the short-ranged conserved region that flanked the “ATG” start codon in the
plant genes [203, 204]. Arbitrary markers like RAPD and ISSR may fail to detect
variations as their genetic information is based on the noncoding regions of DNA not
linked to functional traits. SCoT markers are derived either from the gene itself or its
immediate flanking regions and are associated with functional genes and their
corresponding traits [205]. TRAP technique employs a public express sequence
tag (EST) database to design primers against the annotated EST sequences for the
detection of polymorphic markers to link the EST sequences with its respective
phenotypes [206]. TRAP markers may yield more accurate estimates of genetic
variation than other markers [207].
The epigenetic nature of somaclonal variation is caused by methylation that does
not change the DNA sequence, affecting the cell’s ability to read the genes, thereby
the gene activity [208]. In such cases, specific markers like methylation-sensitive
amplified polymorphism (MSAP), RFLP, or identification via gene expression can
be employed for the detection of somaclonal variants [209–213]. The use of a single
marker system for genetic stability testing of the micropropagated plants does not
always guarantee accurate results [214]. So, utilization of two or more marker types
is crucial for genetic clonal assessment as this will help validate the outcomes of
variability analysis by different markers [196, 215, 216].
Ferreira et al. [120] carried out RAPD analysis to check for possible genetic
alterations in Dendrobium Second Love plants which were originated from six
consecutive subcultures. Twenty RAPD primers produced 172 bands with band
number ranging from 5 to 12 per primer. Two phenotypic alterations of fasciated and
elongated leaves were detected during the culture, but the RAPD analysis did not
reveal any genetic polymorphism among the phenotypically variant plants. This
showed that variation might not necessarily correspond to alteration in the DNA
sequence. The plants with fasciated or elongated leaves generated normal shoots on
further subculturing under similar culture conditions, which affirmed the notion that
phenotypical variations did not ensure changes in the DNA sequence. RAPD
analysis did not divulge any genetic polymorphism in the micropropagated
Dendrobium Second Love in six consecutive subcultures (540 days). The study
presented the possibility of direct in vitro propagation of genetically stable plants
under the influence of TDZ as a sole promoter for multiple shoot initiation and rapid
proliferation. Song et al. [85] tested the genetic stability of callus-derived plants of
D.nobile using ISSR markers. Thirty eight ISSR primers were used, which generated
141 scorable bands with an average of 3.7 bands per primer. Monomorphic banding
patterns were observed for shoots obtained from the callus subcultured for less than
15 cycles, but a low degree of polymorphism was detected for those derived from
callus with more than 16 culture cycles. The genetic uniformity was maintained
among the 20 randomly selected plants generated from shoots obtained from
15 cycles of callus subculture. The finding illustrated the genetic stability of D.
nobile regenerants obtained from callus-derived shoots, which had been subcultured
for 15 cycles on MS medium enriched with 4.0 mM NAA. Bhattacharyya et al. [217]
used two markers, viz., RAPD and SCoT, to evaluate the genetic stability of
micropropagated D. nobile. Seven RAPD and 15 SCoT primers, which gave repro-
ducible and scorable bands, were selected after screening 80 RAPD and 35 SCoT
primers. The band number for RAPD ranged from 2 to 6, while it varied from 4 to12
bands for SCoT markers. The PIC values recorded for RAPD and ISSR markers
were 0.92 and 0.76, respectively, while resolving power (RP) ranged from 3.66 to 10
for RAPD and 4 to 12 for SCoT markers. The cumulative RAPD and SCoT data
showed only five polymorphic bands, indicating a high level of genetic monomor-
phism (97%) between the in vitro clones and mother plant. The observation of high
Rp and PIC values for both the marker systems indicated greater marker efficiency in
detecting the genetic stability of the in vitro propagated orchids. The Mantel test also
showed a high correlation between RAPD and SCoT markers (r ¼ 0.51), portraying
similar efficacy of the two markers in identifying variability between the regenerants.
Investigation on the genetic stability of regenerated Dendrobium Bobby Messina
(DBM) following cryopreservation procedure was performed by Antony et al. [22]
using RAPD markers. Many RAPD primers were screened, but only ten primers
generated distinct and reproducible bands were chosen for the study. The percentage
of polymorphic bands found for ten primers examined in the three cryopreserved
DBM viz., DBVG2P1, DBVG2P2, and DBVG2P3 was between 20 and 39.9%.
Variation in the RAPD banding profiles indicated the existence of somaclonal
variation within the regenerated plants. The clonal variability may appear due to
DMSO toxicity- (PVS2), freezing-, or thawing-induced injury or the regeneration
phase [218]. The study disclosed a high polymorphism in cryopreserved plantlets
18 months post-cryopreservation compared to the earlier report of only 10%
polymorphism in DBM cryopreserved for 3 months. Antony et al. [22] further

performed a somaclonal variation detection study of cryopreserved PLBs of
Dendrobium Bobby Messina using targeted region amplification polymorphism
(TRAP) and SCoT markers for producing genetically stable plants. PLBs, non-
cryopreserved PLBs, and thawed cryopreserved PLBs were examined for genetic
uniformity using the molecular markers. Eight pairs of TRAP primers yielded
distinct, scorable, and reproducible bands with size varying from 100 to 2000 bp.
The TRAP primers disclosed absolute polymorphism in the cryopreserved PLBs. In
non-cryopreserved PLBs, only primer TRAP 20-6B generated monomorphism,
while the remaining seven TRAP primers displayed both complete and partial
polymorphism. The four SCoT primers produced specific and reproducible amplifi-
cation fragments with band size ranging from 500 to 3000 bp. four SCoT primers
(S26, S32, S33, and S36) generated polymorphism for both the cryopreserved and
non-cryopreserved PLBs. The investigation revealed that both TRAP and SCoT
markers could be effectively used to detect somaclonal variation in regenerated
cryopreserved PLBs, which will assist in the in vitro propagation of genetically
stable Dendrobium Bobby Messina.
Bhattacharyya et al. [219] assessed the genetic stability of ISO (indirect shoot
organogenesis) and DSO (direct shoot organogenesis) regenerated D. thrysiflorum
using SCoT and ISSR markers. Thirty six SCoT and Twenty five ISSR primers were
screened to select 8 SCoT and 5 ISSR primers, which yielded reproducible and
unambiguous bands. SCoT markers identified a small degree of polymorphism with
3.22 and 8% clonal variability among the DSO-and ISO-derived plants, respectively.
However, ISSR markers detected low polymorphism of 4.76% in the ISO-derived
plants, while no genetic variation was witnessed between the DSO-derived plants.
Pooled SCoT and ISSR data approach showed a very low variability of 1.88% in
the DSO generated plants and 6.52% polymorphism within ISO-derived plants. The
study showed the effectiveness of SCoT to reveal more variability among the
regenerated plants than the ISSR markers. Molecular analysis disclosed the DSO
regenerated plants to be more genetically stable than ISO propagated plants of D.
thyrsiflorum, which might be due to hormone-induced stress during PLB formation,
shoot induction, and long culture cycle. Wannajindaporn et al. [220] executed a
genetic variation assessment of 25 Dendrobium “Earsakul” mutants obtained from
NaN3 treatment along with ten untreated control plants using ISSR markers. Marker
analysis of the putative mutants using 11 ISSR primers produced 173 fragments
across all genotypes with 39 polymorphic fragments generating 22.5% polymor-
phism. The ISSR fragments size varied from 140 bp (ISSR 835) to 5000 bp (ISSR
835) with ISSR 827 giving the highest polymorphism (47.8%), and the lowest of
39.1% by ISSR 811. But the untreated ten controls exhibited similar DNA banding
profiles with no existence of polymorphism among them. The study demonstrated
the effectiveness of ISSR markers to identify Dendrobium mutants derived from
NaN3-induced PLBs, allowing earlier selection and producing greater genetic sta-
bility among the clones by reducing mutant population size. Bhattacharyya et al. [17]
assessed the genetic stability of acclimatized plants of D. crepidatum using SCoT
and ISSR markers. Eight SCoT yielded 30 amplified bands with 3 polymorphic
bands generating 10% polymorphism, while 5 ISSR primers produced 18 reproduc-

ible bands with 100% monomorphism among the regenerants. Combined RAPD and
ISSR data revealed low clonal variability of 6.38% with a Jaccard’s coefficient
varying from 0.94 to 1.00. The genetically stable D. crepidatum was successfully
propagated with the two markers detecting a very low level of genetic polymorphism
among the regenerants.
To produce genetically stable plants of Dendrobium Sabin Blue, Ching et al. [23]
examined the presence of somaclonal variation in PLBs treated at different concen-
trations of NAA, kinetin, TDZ, and AC using ISSR and direct amplification of
minisatellite DNA region (DAMD) markers. Nine ISSR and eleven DAMD primers
were utilized to evaluate the genetic differentiation of PLBs subcultured under the
influence of different additives for 2 years. PLBs under the treatment of 1.5 mgL1
kinetin harbored the highest genetic variation while the protocorms grown on
medium supplemented with either 4 mgL1 TDZ or 0.5gL1 AC exhibited the
maximum genetic stability. The study observed that genetic stability assessment of
PLBs for long-term culture maintenance should be conducted with molecular
markers so that only the genetically uniform plants were propagated. Bhattacharyya
et al. [221] employed IRAP and ISSR markers to check the genetic consistency of D.
aphyllum micropropagated through a transverse thin cell layer approach. Five IRAP
and nine ISSR primers, which produced scorable reproducible bands, were chosen
for the genetic stability test of in vitro propagated orchids. The IRAP produced 26
scorable bands with an average of 5.20 bands per primer, while ISSR generated 50
amplifiable bands with 5.55 average bands per primer. Both the markers produced
two polymorphic bands each because of which IRAP and ISSR detected 7.69 and
4% polymorphism, respectively. The cumulative IRAP and ISSR data analysis
revealed a polymorphism of 5.26% among the regenerants and mother plants.
IRAP markers were more effective than ISSR in polymorphism detection as ISSR
targeted a particular region of the genome, which might not display the genetic
variation among the clones. But IRAP, which was a retrotransposon-targeted molec-
ular marker, affirmed the results of ISSR marker analysis. IRAP markers are appro-
priate for somaclonal variability detection as the movement of transposable elements
in the genome is an important contributing factor for the emergence of somaclonal
variation [167, 222]. The investigation revealed high genetic stability within the
plantlets of D. aphyllum propagated through the use of t-TCL as an explant source.
Tikendra et al. [24, 25] successfully in vitro propagated genetically stable D.
moschatum and D. chrysotoxum by assessing the clonal fidelity using RAPD and
ISSR markers. The two markers are dominant markers that require no prior infor-
mation of the targeted sequence of the plant genome. Genetic stability assessment
conducted in these Dendrobiums using DNA markers revealed a high degree of
monomorphism among the regenerants and mother plants. Experimentally, the leaf
samples from the mother plants and randomly selected micropropagated plants were
used for genomic DNA extraction using CTAB (Cetyltrimethylammonium bromide)
method [223]. The quantity and purity of the extracted genomic DNA were checked
using a UV spectrophotometer and 0.8% agarose gel electrophoresis, respectively.
The genomic DNA was subjected to PCR amplification using different RAPD and
ISSR primers. The amplified DNA fragments were separated on 2.0% agarose gel
electrophoresis in 1X Tris-acetate EDTA buffer and stained with 0.5μgL1 ethidium
bromide (EtBr). A 1 kb DNA ladder was used to ascertain the size of the separated
unknown DNA fragments. The agarose gel containing the separated bands was
visualized and photographed using a gel documentation system. The experimental
steps involved in the molecular genetic stability assessment of in vitro propagated D.
chrysotoxum and D. moschatum are illustrated in Fig. 2.
In the genetic stability assessment of the micropropagated D. chrysotoxum, 12
RAPD and 11 ISSR primers were selected after screening based on the production of
reproducible and scorable bands. Twelve RAPD primers generated 74 scorable
bands, out of which 73 bands were monomorphic, ensuing 98.81% of monomor-
phism. Similarly, 11 ISSR primers produced 76 reproducible bands, from which 73
bands were monomorphic, producing 97.47% of monomorphism. In D. moschatum,
genetic homogeneity analysis using 10 primers each of RAPD and ISSR markers
yielded 48 and 54 scorable bands out of which 45 and 53 bands were monomorphic,
resulting in 95.2% and 98.0% of monomorphism, respectively. The combined use of
two marker systems ensured that the outcome of RAPD analysis was validated by
the follow-up results of the ISSR markers. The ISSR generated a higher average
number of bands per primer than RAPD markers indicating its effectiveness in
analyzing genetic polymorphism. The banding profiles of OPF-14 (Fig. 3A) and
UBC-814 (Fig. 3B) in D. chrysotoxum, and so also the amplification profiles of
OPC-08 (Fig. 3C) and UBC-868 (Fig. 3D) in D. moschatum, showed total similarity
in their respective banding pattern between the regenerants and mother plants.
Fig. 2 Experimental steps involved in the genetic stability assessment of in vitro propagated
Dendrobiums using RAPD and ISSR molecular markers
Fig. 3 Banding profiles of OPF-14 RAPD primer (a) and UBC-814 ISSR primer (b) in D.
chrysotoxum; OPC-08 RAPD primer (c) and UBC-868 ISSR primer (d) in D. moschatum
The genetic association and closeness of mother plant (MP) and in vitro clones
(P1-P9) can be portrayed by constructing UPGMA dendrograms. The dendrograms
showed the clustering of MP with P1, P2, P3, and P4 in D. chrysotoxum, while MP
was associated with P1, P2, P3, P4, P5, P6, P7, and P8 in a major cluster in D.
moschatum (Fig. 4).
The consistency of genetic similarity defined by the cluster analysis was checked
by performing PCoA (Principal coordinate analysis). PCoA revealed the grouping of
the regenerants and the mother plants similar to the clustering patterns exhibited by
UPGMA dendrograms. The correlation between the different markers employed in
the analysis was checked by performing the Mantel test. The genetic matrices of
RAPD and pooled RAPD+ISSR showed no significant correlation for D.
chrysotoxum (r ¼ 0.620; p ¼ 0.02) and D. moschatum (r ¼ 0.966; p ¼ 0.10). But,
significant correlation was observed between ISSR and RAPD-ISSR pooled matri-
ces in D. chrysotoxum (r ¼ 0.936, p ¼ 0.002), and D. moschatum (r ¼ 0.753,
P3
a
P4
P2
P1
MP
P5
P6
P7
P8
P9
0.015 0.010 0.005 0.000
P7
b P8
P5
P4
P3
P2
P1
MP
P6
P9
0.015 0.010 0.005 0.000
Fig. 4 Dendrograms depicting the clustering pattern between the mother plants and in vitro
regenerants of (a) D. chrysotoxum and (b) D. moschatum
p ¼ 0.010). In both the investigations, the ISSR markers were more effective than
RAPD in determining the genetic stability of in vitro propagated Dendrobiums.
Galvan et al. [224] and Ajibade et al. [225] also demonstrated the effectiveness of
ISSR over RAPD markers in clonal fidelity assessment of regenerants. The ISSR
markers are distributed in the entire genome allowing amplification of genomic
DNA in a greater number of fragments per primer than RAPD markers.
5 Conclusions
Dendrobiums are extensively used in traditional medicines to treat diverse ailments

due to the inherent therapeutic properties they acquired from numerous phytochem-
ical contents. The in vitro propagation of different Dendrobiums is successfully
accomplished utilizing mainly seeds, shoot tips, pseudobulb, and nodal segments as
reliable explants. Clonal fidelity assessment using DNA markers is performed to in

vitro propagate genetically stable Dendrobiums. Of the several DNA markers
employed for clonal evaluation, mostly ISSR, IRAP, and SCoT are preferred to the
less reliable and inconsistent RAPD markers. The application of two markers system
is more appropriate than using a single marker in genetic stability assessment as the
result of variability analysis of one marker type can be validated by that of the others.
The rapid and mass propagation of genetically stable orchids will ensure the
effective conservation and commercialization of Dendrobiums, narrowing down
the existing gap between demand and supply.
Acknowledgments LT and PN are thankful to UGC (University Grant Commission), New Delhi,
India, for financial support.
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Eulophia spp.: In Vitro Generation, Chemical
Constituents, and Pharmacological 20
Activities
Varsha Shriram and Vinay Kumar
Contents
1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 496
2 Distribution and Botanical Description . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 498
3 Phytochemical Constituents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 499
4 Pharmacological Activities . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 501
4.1 Ethnopharmacological Uses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 501
4.2 Evidence-Based Pharmacological Activities . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 502
5 In vitro Regeneration, Phytochemical Production, and Conservation . . . . . . . . . . . . . . . . . . . . . 504
6 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 512
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 512
Abstract
Eulophia R.Br. ex Lindl represents one of the largest, wide-spread, and important
genera of the family Orchidaceae. Eulophia shows an extraordinary kind of
morphological diversity and occupies a wide variety of habitats. Current records
testify that it encompasses around 230 species, out of which 203 are the accepted
ones. This genus is of prime importance because of its distinctive ecology and
broad-spectrum ornamental and therapeutic properties. Crude solvent extracts
and phytochemicals have been assessed from the members of this genus and
found to possess potent pharmacological activities including anticancer, anti-
diabetic, anti-inflammatory, and DNA protection among others. Keeping this in
view, we are presenting herein an account on the distribution and botanical
description of the genus Eulophia, chemical constituents reported from the
V. Shriram (*)
Department of Botany, Prof. Ramkrishna More Arts, Commerce and Science College, Savitribai
Phule Pune University, Pune, India
e-mail: varshashriram76@gmail.com
V. Kumar
Department of Biotechnology, Modern College of Arts, Science and Commerce, Savitribai Phule
Pune University, Pune, India

496 V. Shriram and V. Kumar
Eulophia species along with their ethno/pharmacological activities/claims

besides their evidenced-based therapeutic activities. Since many species of
Eulophia are reportedly threatened, their conservation is required on urgent
basis. However, low germination and survival rate in natural conditions along
with requirement for specific fungal associations for germination and growth, in
situ conservation approaches have met with limited success. Plant tissue culture
technologies as emerging platforms for in vitro regeneration, phytochemical
production and ex situ conservation for the members of Eulophia are discussed
with a perspective approach.
Keywords
Anticancer · Asymbiotic germination · Eulophia · In vitro regeneration ·
Orchidaceae · Phenanthrene · Phytomolecules · Protocorms · Protocorm-like
bodies
1 Introduction
The family Orchidaceae comprises highly evolved monocot plants, representing sec-
ond largest and most diversified group, which boasts a worldwide distribution of an
estimated 28,000 species and 736 genera [13, 21]. Family Orchidaceae belongs to the
Asparagales (order) of class monocotyledonae of Angiospermae (Sub-division) in the
kingdom Plantae. The word orchid was coined by Theophrastus, originated from Greek
word Orchis as the tubers’ shape in many species from the genus resembling to the
testicles. Orchids were the symbol of virility for the Greeks, whereas for Chinese people
these were the plants of the kings’ fragrance [26]. Orchids hold a peak position in plant
evolution, often used for ornamental purpose because of their incredible diversity and
beautiful flowers besides their great value in horticulture and plant-based medicines
[4]. They exhibit broad diversity in morphology, growth form, life history, and habitat.
These are herbaceous, perennial plants, either terrestrial or epiphytic. The epiphytes can
absorb water from the surrounding air. Most of the members are autotrophs while some
are saprophytic, being helped to obtain nourishment by their rhizospheric fungi. Orchid
flowers show tremendous variations in size (2 mm diameter in genus Pleurothallism
and more than 30 cm in Brassia). Further, the diversity is observed in the methods of
pollination as well as types of pollinators. Orchid species have evolved to imitate
several other organisms like bee, fly, etc. among other creatures they try to attract. Some
have developed elaborate systems of water traps and tunnels, hinged petals, and sticky
packets of pollen devices often described as devious or deceitful.
Orchids are distributed through wide ecological conditions. They occur in all the
terrestrial ecosystems except pole and desert regions. This family primarily found in
tropical regions, whereas many species are spread in the northern and southern
temperate zones. Utmost diversity is observed in tropical regions where rainforest,
temperature lush, and high humidity conditions prevail. Many species in the north-
ern temperate zones are found in bogs, prairies, grasslands, and hardwood forests.
20 Eulophia spp.: In Vitro Generation, Chemical Constituents, and. . . 497
Orchids often blossom in high altitude regions (4500 m above mean sea level). A
large number of orchids are found in cloud forests in tropical regions on mountain-
sides, often covered with mosses and lichens. This is a favorable habitat for epiphytic
orchids. Few orchid species flourish in desert conditions. A few species like
Brassavola are known to grow on mangrove roots, whereas some species grow on
bare rocks [20]. Some orchid species are widespread (e.g., Ionopsis utricularioides);
however, some are restricted to single mountain [20].
Orchids are historically used as food, medicines, adhesives, perfumes, and
flavoring agents. For instance, Vanilla (a product obtained from the orchid Vanilla
planifolia) is commercially important flavoring agent derived from orchid. They
comprise one of the topmost horticultural plant families and their cultivation is
highly significant for global nursery industry [3]. The orchids have taken an impor-
tant place in the flower industry because of their charm, shelf-life, productivity, and
ease with packing and/or shipping. Orchids account for a large share of global
floriculture trade both as cut flowers and as potted plants. They have been used
across the world in various traditional medicinal systems [67].
The record of orchids might begin with their uses in the therapeutic purpose.
Chinese were the first to describe and cultivate various orchids [35]. These plants
first received recognition in the traditional herbal writings of China and Japan, and
they were the first to describe medicinal use of orchids [10]. In recent times, more
species belonging to different genera have been reported for their medicinal proper-
ties and this list is expected to expand in future [25, 64]. As reported in the literature,
various species of orchids are used as antimicrobial, anti-inflammatory, antioxidant,
anticancer, anti-pyretic, anti-mutagenic, anti-convulsive, anti-helmintic, anti-
hepatotoxic, wound healing, anti-platelet, anti-diabetic, anti-allergic, immunomod-
ulatory, anti-aging, pain reliever, antiviral, and herbicidal agents [39, 42, 83,
89]. Phytomolecules of orchids have been investigated and several constituents
have been reported [44]. The phytochemicals from these plants are mainly alkaloids,
stilbenoids and phenanthrenes, flavonoids, terpenoids, essential oils, glycosides, and
coumarins. Phenanthrene derivatives are the common phyto-constituent in orchids
[27]. Hydroxylated and 9,10-dihydroxy phenanthrenes are most commonly found in
rhizome/ bulbs/ tubers, roots of these plants besides other derivatives. Phenanthrene
derivatives are synthesized from stilbenes that originate from cinnamic acid deriv-
atives. Biosynthesis of 9, 10 dihydroxy phenanthrenes is via phenylpropionic acid
derivatives.
Eulophia R.Br. ex Lindl is an important and largest terrestrial genus of the
Orchidaceae. According to current records, it encompasses around 230 species
[90] and out of that 203 are the accepted ones. It belongs to the subtribe Eulophiinae
(subfamily Epidendroideae; tribe Cymbidieae) [74]. The center of diversity of the
genus is Africa, mostly found in the palaeotropics, although six species extend into
the neotropics, and Eulophia shows an extraordinary morphological diversity and
occupies a surprisingly wide variety of habitats. This genus is of prime importance
due to its distinctive ecology and broad-spectrum ornamental and therapeutic prop-
erties. Keeping this in view, we are presenting herein an account on the distribution
and botanical description of Eulophia, chemical constituents reported from the
Eulophia species along with their ethno/pharmacological activities/claims besides

their evidenced-based therapeutic activities. Further, many species of Eulophia are
under IUCN threatened categories and hence conservation of the genus is the focus
for the conservation biologist. However, low germination and survival rate in natural
conditions along with requirement for specific fungal associations for germination
and growth, in situ conservation approaches have met with limited success. Plant
tissue culture technologies as emerging platforms for in vitro regeneration, phyto-
chemical production, and ex situ conservation for the members of Eulophia are
discussed with a perspective approach.
2 Distribution and Botanical Description
Eulophia belongs to the family Orchidaceae and its members are known as “cordu-
roy orchids” [38], while in India they are known as “Amarkand.” The name
Eulophia is of Greek origin (eu-lopos), describing the “beautiful crest,” denoting
lip-crests [23]. This genus name was coined by Robert Brown, and was established
by Lindley in 1821. Eulophia are predominantly terrestrial, rarely lithophytic and
sympodial herbs whereas 2 are epiphytic as found on Madagascar. It shows wide
distribution across the world and frequently found in Africa and Asia; some species
are deciduous while few are evergreen. Africa is considered as a center of diversity
of this genus with an extensive diversity of habitats comprising the desert-margins,
marshes, and dry savanna woodlands besides wet tropical forests. It is large
pan-tropical genus distributed in shady rainforests with grass or shrubs or in open
scrub or woodland in the tropics and subtropics of Africa, India, Asia, Queensland,
and the Americas, while some of its species (such as E. petersii) are habituated to
arid climates (desert species). Some species have shown broad latitudinal spread as
in E. cucullata, which can be correlated to the polyploidy.
Phylogenetic studies reveal that genus Eulophia is paraphyletic. It is either
autotrophic or at times chlorophyll deficient. Root system is adventitious, slender
to stout, produced from the tubers’/ pseudobulbs’ base often with a well-defined
white velamen. Pseudo-bulbs/ tubers are the perennating organ either above the
ground and rhizomatous or tuberous if subterranean. They are cylindrical/ fusiform/
conical/ ovoid/ irregular, with several nodes, homoblastic, and grow into a chain of
tubers. Leaves appear at or after anthesis. Green Leaves usually present, may be
highly reduced in some species, scale-like and brown or even buff. Green leaves
thin-textured or fleshy or coriaceous, petiolate, long and narrow, linear/ lanceolate/
ovate/ elliptic/ sometimes pleated, with or without prominent longitudinal veins,
acute to acuminate, articulate or not to a sheathing or petiole-like leaf base, some-
times overlapping and forming a false stem. Flowering stalk (scape) produced with
leaves from the current year’s growth that are enclosed with subscarious tubular
sheaths.
Inflorescence is erect, terminal/ lateral/ basal laxly to sub-densely, many flowered
or rarely reduced to a solitary flower, usually racemose or rarely paniculate, simple or
rarely branching; bracts are persistent. Flowers are small to large, green/ brown/ showy
and brightly colored at times bicolored, resupinate/ non-resupinate. Sepals and petals
either similar or petals are much broader. Dorsal sepal free, oblong/ elliptic/ lanceolate/
oblanceolate, reflexed, erect/ porrect; lateral sepals oblique at base and decurrent on
column foot. Labellum / Lip flat, sessile, 3-lobed, free to base or fused to base of the
column, lacking a claw, usually with a callus 2 or 3 ridged or papillose on upper
surface, either spurred at the base or without spurred or saccate. If spur is present, it
may be obscure and sac-like. Labellum disk-like/ entirely tuberculate/ nerves tuber-
culate/ verrucose/ keeled; mid-vein present on side lobes often raised into a ridge at the
base, lateral lobes free / fused to base of column, mid-lobe flat or convex. Columns are
short to long, with or without a column-foot. Ovary is cylindrical, grooved. This genus
includes 203 accepted species and 2 are the cross (hybrid species), for the complete list
of species and the description refer to http://www.plantsoftheworldonline.org/taxon/
urn:lsid:ipni.org:names:325750-2).
3 Phytochemical Constituents
Owing to the wide-spectrum uses of Eulophia genus, several researchers have

focused on investigation of its phytomolecules and their bioactivities in the recent
past, refer Table 1. Key attention was to isolate and to identify their respective phyto-
constituents and then exploring them for broad-spectrum bioactivities. Several
important biomolecules with tremendous therapeutic values have been reported
from a number of Eulophia species. Kovács et al. [45] investigated the phytochem-
ical constituents of E. ochreata and E. nuda and confirmed the presence of essential
minerals, polyphenols, saponins, alkaloids, phytic acid among others in these spe-
cies. Several phenanthrenes have been reported from E. nuda till date. Diverse
Phenanthrene derivatives are most commonly occurring phytomolecules in this
genus, for instance Bhandari and Kapadi [6] isolated crystalline molecule and
characterized it as 9,10-Dihydrophenanthrene from ethanol extract of tubers of
E. nuda and named it as eulophiol with molecular formula C6H604. Later in 1985,
Nudol, a phenanthrene from E. nuda with molecular formula C16H1404 and mol.
Mass 270) was reported by Bhandari et al. [7]. A research group from Thailand and
Australia took the interest and described six phenanthrene derivatives from E. nuda
9,10-Dihydro-2,5-dimethoxyphenanthrene-1,7-diol (mol. Formula C16H1604; mol.
Mass 272), 9,10-dihydro-4-methoxyphenanthrene-2,7-diol (mol. Formula
C15H14O3; mol. Mass 242.0941), l,5-Dimethoxyphenanthrene-2,7-diol (mol. For-
mula C16H14O4; mol. Mass 270), 1,5,7,-trimethoryphenanthrene-2,6-diol (mol. For-
mula C17H16,O5; mol. Mass 300.0098), 5,7-dimethoxyphenanthrene-2,6-diol (mol.
Formula C16H14O4; mol. Mass 270.28), and 4,40 ,8,80 -tetramethoxy [1,10 -
biphenanthrenel-2,20 ,7,70 -tetrol (mol. Formula C32H26O8; mol. Mass 538)]. Besides,
4-Hydroxybenzaldehyde and 4-hydroxybenzyl alcohol were reported by this group
[91]. Further, by the same research group in 1989 two more phenanthrene deriva-
tives, viz., 9,10-dihydro-l-(40 -hydroxybenzyl) -4,7-dimethoxyphenanthrene-2,8-diol
(C23H22O5) with the molecular mass 378, 1-(40 -hydroxybenzyl)-4,8
dimethoxyphenanthrene- 2,7-diol (C23H20O5) with molecular mass 376 were
Table 1 List of phytochemicals (pure compounds) reported from Eulophia species

Plant
Eulophia species part Pure Compound reported References
Eulophia epidendrea (JKoen) Schltr. Leaves Apigenin, luteolin, Maridass
kaempferol, and quercetin and
Tuber fractions β-sitosterol, Ramesh
β-sitosterolglucoside, β- [51]
amyrin and lupeol
Eulophia nuda Lindl. Tuber n-hexacosyl alcohol and Merchant
lupeol et al. [56]
1,5-dihydroxy-2,7- Bhandari
dimethoxy-9,10- and
dihydrophenanthrene Kapadi [6]
(Eulophiol)
2,7-dihydroxy-3,4- Bhandari
dimethoxyphenanthrene et al. [7]
(Nudol)
9,10-dihydro-2,5- Tuchinda
dimethoxyphenanthrene-1,7- et al. [91]
diol
9,10-dihydro-4- Tuchinda
methoxyphenanthrene-2,7- et al. [91]
diol
1,5-dimethoxyphenanthrene- Tuchinda
2,7-diol et al. [91]
1,5,7,- Tuchinda
trimethoxyphenanthrene-2,6- et al. [91]
diol
5,7-dimethoxyphenanthrene- Tuchinda
2,6-diol et al. [91]
4,40 ,8,80 -tetramethoxy Tuchinda
[1,10 -biphenanthrene]- et al. [91]
2,20 ,7,70 tetrol.
4-hydroxybenzaldehyde Tuchinda
et al. [91]
4-hydroxybenzyl alcohol Tuchinda
et al. [91]
9,10-dihydro-1- Tuchinda
(40 -hydroxybenzyl)-4,7 et al. [92]
dimethoxyphenanthrene-
2,8-diol
1-(40 -hydroxybenzyl)-4,8- Tuchinda
dimethoxyphenanthrene- et al. [92]
2,7-diol
3,40 -dihydroxy- Tuchinda
30 ,5,50 -trimethoxybibenzyl et al. [92]
bis(4-hydroxybenzyl) ether
Tubers: Alkaloids, flavonoids, Bhatt et al.
water saponins, carbohydrates, [8]
extract steroids, triterpenoids,
(continued)
Table 1 (continued)
Plant
Eulophia species part Pure Compound reported References
tannins, phenolics, coumarins
and anthraquinones
Eulophia ochreata Lindl. Fresh 9, 10-Dihydro-2, Datla et al.
tubers 5-Dimethoxyphenanthrene-1, [15]
7-diol
5, 7-Dimethoxyphenanthrene-
2, 6-diol
described from same species [92]. Moreover, 3,40 -dihydroxy-30 ,5,50 -

trimethoxybibenzyl with molecular weight 304 and bis(4-hydroxybenzyl) ether
with molecular weight 230 were found [92].
Various important classes of secondary metabolites including flavonoids, reduc-
ing sugars, cyanogenic glycosides terpenoids, and tannins were reported from
methanolic extracts of E. epidendrea of tubers [53]. The same group further reported
the presence of flavonoids, sterols, and terpenoids in the fractions collected from leaf
and root extracts of E. epidendrea [51]. Major phytochemicals reported from species
of Eulophia are presented in Table 1.
4 Pharmacological Activities
4.1 Ethnopharmacological Uses
Rhizomes or tubers of many Eulophia species are regularly consumed by the tribes,
especially in India, both as food and for therapeutic materials for maintenance of
health and longevity. In traditional medical practices like the Ayurveda, it is fre-
quently recommended as an expectorant, tonic, diuretic, astringent, and a soft
purgative [64]. The tubers of several Eulophia species have traditional medicinal
usages against scrofulous glands of the neck, bronchitis, and rheumatoid arthritis,
besides being used as a vermifuge, appetizer, anti-fatigue, anti-helminthic, anti-
bellyache, anti-scrofulous blood purifier, anti-helminthic, anti-tumor, wound
healing, and an aphrodisiac agent. For instance, fresh juice of E. campestris tubers
is used in traditional systems for treating diarrhea, and dysentery, besides its use as a
laxative and an appetizer. The rhizomes of E. campestris have tonic properties and
are also used as aphrodisiac [11]. The tubers are useful against worm infestation and
scrofula [83]. E. epidendrea tubers paste is useful against boils and is used as breast
pain reliever for feeding mothers [75]. The tubers of E. epidendrea are used
traditionally for curing diarrhea and tumors [69]. The tubers of this orchid are also
used as appetizer and as blood purifier and are particularly helpful during heart
problems [52]. Similarly, E. herbacea tubers are used for reducing liver swelling [2],
for increasing the sperm-count [2], for treating pimples [70], while fried tubers in
mustard oil are externally applied for rheumatism [82].
E. nuda Lindl., is an important and heavily explored species of this genus for its
tremendous therapeutic potentials. Its tubers are used for treating worm infestation
and scrofula [83], skin rashs, acidity, piles, anorexia, anthrax, and stomach com-
plaints [79], rheumatoid arthritis [49], cancer, asthma, and bronchitis [34]. The
whole tubers of E. nuda are used to get relief from abdominal pain [14], while the
root juices are used against snakebites [82]. The “Salep” of E. ochreata tubers is used
for the treatment of sexual impotency and male sterility [27, 43], the tubers paste is
used against asthma and acute bronchitis [32, 33], whereas the tuber-decoction
works as antidote for snakebite and to cure leukemia [49].
4.2 Evidence-Based Pharmacological Activities
Investigations have been carried out to scientifically validate the ethnopharma-

cological activities and therapeutic claims of some important species of Eulophia,
including E. nuda, E. campestris, E. herbacea, and E. epidendrea, among others.
Most of the investigated species have wide-spectrum and strong therapeutic activ-
ities, thus confirming their ethnopharmacological importance. The E. campestris
tubers have tremendous binding properties [97], and thus their mucilage is heavily
used for their binding attributes in drug tablet formulations, which ultimately
regulate the drug-release rate [22]. Yadav et al. [96] evaluated the effects of
“Salep” of E. campestris on glycation inhibitory activities, and the authors reported
that the application of “Salep” reduced the formation of glycated products/ advanced
glycation end-products. Similarly, mucilage of the tubers of E. herbacea has been
reported as an excellent suspending agent, besides its properties like less sedimen-
tation rates, high viscosity, acidic pH, and abilities to re-disperse make it a good
pharmaceutical adjuvant [9]. The methanolic extracts from the tubers of
E. epidendrea exhibited anti-diarrheal potencies in experimental rats, as confirmed
by the abilities of the extract in inhibiting the intestinal fluid accumulation and the
propulsive movements of the intestinal contents [50]. These investigations hold
significance for validating the folk medicines claims. Jagdale et al. [30] validated
the traditional uses of E. nuda tubers as an aphrodisiac on mice models and reported
significant aphrodisiac potentials of this plant. Jagtap et al. [31] reported anti-
inflammatory and antioxidant activities from the methanol extracts of E. ochreata.
Besides, solvent extracts of E. ochreata showed noteworthy antibacterial activities
against Bacillus subtilis, Staphylococcus Aureus, and Escherichia coli [18]. The
tubers of E. ochreata have also been reported to possess antioxidant, antiglycan, and
amylase inhibitory activities and thus might prove a potential source for identifying
anti-diabetic molecules [63]. E. ochreata have also shown high antioxidative activ-
ities [18, 62, 63]. In a comprehensive in vitro assessment, Kumar et al. [47] studied
phytochemical profiling, and antioxidant and free-radical scavenging activities of
different solvent extracts of E. nuda and the authors attributed these potencies to
high amounts of phenols, flavonoids, vitamins, and carotenoids. All the extracts
showed a broad-spectrum antioxidant properties including DNA protection from

hydroxyl-radical-induced damage [47]. The results provided the scientific basis for
the traditional uses of this orchid as a natural antioxidant and phytotherapeutic agent.
Our group further reported antioxidant, free-radical scavenging, and cytotoxicity
activities of E. nuda tuber extracts [61]. The Reversed Phase High Performance Liquid
Chromatography-Electrospray Ionization/Mass Spectrometry-based phytochemical
profiling of ethyl acetate extract confirmed the presence of a total of 37 compounds
including some known antioxidants like catechin, tocopherol, and trigallic among
others, and the antioxidants were reported to act via induction of nuclear transcription
factor-erythroid-2 related factor (Nrf2) and hemeoxygenase-1 (HO-1) pathways [61].
Further, Preedapirom et al. [73] reported aphrodisiac activities of E. macrobulbon
crude extract on erectile dysfunction in male aged rats. Jansakul [36] studied potential
applications of ethanolic extract of E. macrobulbon tubers, and an isolated constituent,
1-(40 -hydroxybenzyl)-4,8-dimethoxyphenanthrene-2,7-diol on human erectile dys-
function. Authors in an attempt to study the underlying mechanism of action, they
reported the relaxant mechanism of on human corpus cavernosum.
In similar vein, several therapeutically potent, pure molecules have been reported
from the members of Eulophia. For instance, our group isolated a phenanthrene
derivative 9,10-dihydro-2,5-dimethoxyphenanthrene-1,7-diol from E. nuda which
showed excellent cytotoxic activity (better efficacy than the standard anticancer
molecule carboplatin) against human cancer cells [79]. This compound was also
isolated from E. ochreata and it effectively inhibited lipopolysaccharide-induced and
Toll-like receptors-mediated, nuclear factor κB-activated inflammatory genes; how-
ever, it reduced both lipopolysaccharide-induced tumor necrosis factor (TNF-α)
release and carrageenan-induced paw edema in rats [15]. Overall, these results
confirmed the anti-inflammatory potencies of this plant, besides highlighting the
underlying mechanism targeted by this compound. Tatiya et al. [86] successfully
attempted the bioassay-guided isolation of 1-phenanthrenecarboxylic acid 1, 2, 3, 4,
4a, 9, 10, 10a-octahydro-1, 4a-dimethyl-, methyl ester from E. herbacea and this
compound showed strong anti-proliferative activities against human cancer cell
lines, thus confirming the traditional anticancer uses of the tuber in the folklores.
Upadhyay et al. [93] isolated phenanthrene derivative Eulophiol from Eulophia
species, and examined its potential application in inhibition of immune stimulation
involving Toll-like receptor ligands, especially TLR-4, the authors labeled it as a
potent Toll-like receptor signaling antagonist. Temkitthawon et al. [88] isolated a
new phenanthrene, 9,10-dihydro-4-(40 -hydroxybenzyl)-2,5-dimethoxyphenanthrene-
1,7-diol, besides three known phenanthrenes, 1-(40 -hydroxybenzyl)-4,8-
dimethoxyphenanthrene-2,7-diol, 9,10-dihydro-2,5-dimethoxyphenanthrene-1,7-diol,
and 1,5,7-trimethoxyphenanthrene-2,6-diol from the tubers of E. macrobulbon, and
the phytomolecules were found to be potent phosphodiesterase inhibitors.
Wisutthathum et al. [95] undertook pharmacological characterization of the vascular
actions of the E. macrobulbon ethanolic extract and its active compound, 1-(40 -
hydroxybenzyl)-4,8-dimethoxyphenanthrene-2,7-diol using isolated pulmonary arter-
ies from rats having pulmonary arterial hypertension induced by monocrotaline. The
pulmonary arterial hypertension was reported to be improved by the tuber extract via
pulmonary arteries relaxation mediated through endothelial nitric oxide, reduced Ca2+-
mobilization, besides reduced arteries wall thickness and the right ventricular hyper-
trophy [95]. Ethnopharmacological uses and evidenced-based pharmacological activ-
ities of different Eulophia species are listed in Table 2.
5 In vitro Regeneration, Phytochemical Production,

and Conservation
A large number of orchid species including Eulophia are under threat due to their
over-exploitation, beside loss of habitats and the pollinators [3, 84]. This genus
mostly in marginal situations is exposed to several challenges like unsuitable
environmental conditions besides predation, parasitism, or competition are also
major threats to the population of its species. Physical alterations, habitat destruction
and degradation, climatic changes and challenges coupled with the over-
exploitation, and the introduction of non-native species have worsen the situation
and consequently contributed to the decline in numbers and distribution of Eulophia
genus [65], it has further resulted into an increasing number of its species in IUCN
Red Lists.
Eulophia shows vegetative propagation by tubers and sexual reproduction by
seeds. They can also be propagated by stem and rhizome cuttings. These vegetative
methods of propagation are helpful because they produce exact clones unlike sexual
reproduction. However, asexual reproduction is exceptionally slow and usually
produces 2–4 plants in a year. This difficulty in natural population drives many
medicinal as well as horticultural orchids including few Eulophia species to be
endangered and some are even reached the extinction [67]. Seed germination is
another method of propagation but this is not genetically identical to the parent.
Besides, Eulophia flowers only after they are 4–5 years old. In nature, only around
5% flowers get pollinated, leaving the ovules of 95% flowers as unfertilized, a major
hindrance in capsule formation. Further, though each capsule bears many seeds but
only 5% seeds germinate in their natural environment besides their slow growth
characteristics [57]. Suppressed endosperm and lack of nutrients also hamper the
germination and early vegetative growth, and thus these plants require highly
specialized symbiotic fungal associations towards fulfilling their nutritional require-
ments [57, 72].
In situ conservation of orchids under threat is thus a difficult task and necessitate
viable ex situ alternatives for their multiplication and conservation [48]. Plant tissue
culture-based technologies have emerged in recent years as sound platforms for mass
multiplication and germplasm conservation of orchids, besides in vitro production
platforms for high-value secondary metabolites. Plant tissue culture techniques have
made significant contributions in multiplication of many threatened orchid species.
As per the recent reports, seeds can be germinated asymbiotically without the
association of any fungal partner in a nutrient medium for best results [67].
In initial attempts, the micropropagation of E. dabia was successfully achieved
described by Sharma and Vij [78] followed by the micropropagation of E. hormusjii
20
Table 2 List of plant species from Eulophia genus, their ethnopharmacological uses, and evidence-based pharmacological activities of their crude extracts
and/or pure molecules
Plant
Eulophia species part Extract/Pure molecule Ethnopharmacological/Pharmacological activities References
A. Ethnopharmacological Uses
Eulophia Tuber Fresh Juice Gastro-intestinal disorders (diarrhea, dysentery, Chanda et al. [11]
campestris Wall. stomach pain, laxative), appetizer
Eulophia Rhizome – Tonic, stomach problem, aphrodisiac, cough, cold Medhi and Chakrabarti [55]
campestris Wall.
Eulophia Tuber Mucilage Worm infestation, scrofula Singh and Duggal [83]
campestris Wall.
Eulophia dabia Tuber – Cough, cold Joshi et al. [39]
(D. Don.) Hochr.]
Eulophia epidendrea Tuber Paste Ease the pain due to milk clotting Rajendran et al. [75]
(JKoen) Schltr.
Eulophia epidendrea Tuber – Tumor, diarrhea Patil and Mahajan [69]
(JKoen) Schltr.
Eulophia epidendrea Tuber – Appetizer, anthelmintic, aphrodisiac, stomachic, Maridass et al. [52]
(JKoen) Schltr. worm infestation, blood purifier, heart troubles
Eulophia epidendrea Tuber Narkhede et al. [64]
(JKoen) Schltr.
Eulophia spp.: In Vitro Generation, Chemical Constituents, and. . .
Eulophia graminae Tuber Extract Earache Karuppusamy [41]

Lindl.
Eulophia herbacea Tuber Extract Reduces the liver swelling Ahirrao et al. [2]
Lindl
Eulophia herbacea Tuber Roasted Increases the sperm count Ahirrao et al. [2]
Lindl
Eulophia herbacea Tuber Crushed; fried in mustered oil Rheumatism Sikarwar et al. [82]
Lindl
505
(continued)
506
Table 2 (continued)
Plant
Eulophia herbacea Tuber – Bellyache Dey and Nath [19]
Lindl
Eulophia herbacea Tuber Paste Treatment for pimples Patil and Patil [70]
Lindl
Eulophia herbacea Seed Powder Weakness (Fatigue) Tayade and Patil [87]
Lindl
Eulophia herbacea Hypolipidemic, antidiabetic and anti-oxidant Tatiya et al. [85]
Lindl activity
Eulophia herbacea Anabolic and Reproductive Activity Patil et al. [71]
Lindl
Eulophia macrobulbon Root Extract inflammatory and antioxidant effect and Schuster et al. [77]
anticancerogenic potential exerted
Eulophia mannii Tuber Extract Antioxidant activity Narkhede et al. [64]
(Rchb. f.)
Hook. f. (EM)
Eulophia nuda Lindl. Tuber Extract Worm infestation and scrofula Singh and Duggal [83]
Eulophia nuda Lindl. Tuber Raw material without Skin rash, acidity, piles, anorexia, anthrax, stomach Shriram et al. [79]
processing complaints
Eulophia nuda Lindl. Raw Raw material without Rheumatoid arthritis Mali and Bhadane [49]
tuber processing
Eulophia nuda Lindl. Tubers Extract Anticancer, anti-asthmatic, anti-bronchitis Jain et al. [34]
Eulophia nuda Lindl. Whole Paste Boils, abscesses Hossain [27]
plant
Eulophia nuda Lindl. Root Juice Treatment for snakebite Sikarwar et al. [82]
Eulophia nuda Lindl. Tubers Extract Anti-inflammatory activity Abhyankar and Upadhyay
[1]
V. Shriram and V. Kumar
20
Eulophia nuda Lindl. Whole Raw material without Abdominal pain due to non-menstruation, Das et al. [14]
tuber processing Spermatorrhea, Leucorrhea
Eulophia nuda Lindl. Tuber Extract Vermifuge, blood purifier Sastri [76]
Eulophia nuda Lindl. – – Appetizer, tonic Patil and Mahajan [69]
Eulophia nuda Lindl. – – Rheumatoid arthritis Mali and Bhadane [49]
Eulophia nuda Lindl. – – Anthelmintic, bronchitis Merchant et al. [56]
Eulophia nuda Lindl. – – Snake bite Sikarwar et al. [82],
Abhyankar and Upadhyay
[1]
Eulophia nuda Lindl. – – Worm infestation and scrofula Singh and Duggal [83]
Eulophia nuda Lindl. – – Gastric problems Kapale [40]
Eulophia nuda Lindl. Tuber Aquous methanol extract DNA damage protecting activity Kumar et al. [47]
Eulophia nuda Lindl. – – Anti-glycation effect Yadav et al. [96]
Eulophia nuda Lindl. – – Aphrodisiac activities of Salep in adult Swiss male Jagdale et al. [30]
mice
Eulophia nuda Lindl. – – Anti-inflammatory activity Tuchinda et al. [92]
Eulophia nuda Lindl. – Acetone extract Antibacterial activity against Escherichia coli, Nagulwar et al. [59]
Pseudomonas aeruginosa, Staphylococcus aureus
Eulophia nuda Lindl. – – Antifungal Activity against Aspergillus niger, Nagulwar et al. [59]
A. flavus, Candida albicans
Eulophia nuda Lindl. – – Hepatoprotective activity Nagulwar et al. [59]
Eulophia spp.: In Vitro Generation, Chemical Constituents, and. . .
(Acute ccl4 induced hepatotoxicity in rats)

Eulophia nuda Lindl. Tuber Ethanol extract Anti-mycobacterial activity Gupta et al. [24]
Eulophia ochreata Tuber Salep Treatment of sexual impotency and male sterility Hossain [27], Das et al. [14]
Lindl
Eulophia ochreata Tuber Paste Asthma, acute bronchitis Jain et al. [32], Jain et al.
Lindl [33]
Eulophia ochreata Tuber Powder Increase the stamina for physical activities Jagtap et al. [29]
Lindl
507
(continued)
508
Table 2 (continued)
Plant
Eulophia ochreata Tuber Extract For restoring general health, strength, vigor Jagtap et al. [31]
Lindl
Eulophia ochreata Tuber Decoction Antinode in snakebite, cure leukemia Mali and Bhadane [49]
Lindl
Eulophia pratensis Tuber Paste To remove scrofulous gland in the neck Hossain [27]
Lindl.
Eulophia ramentaceae Tuber – Impotency related problems Bhagaonkar and Kadam [5]
Lindl Ex. Wight
B. Pure Molecules and/or their Pharmacological Activities
Eulophia ochreata Tuber 9, 10-Dihydro-2, Inhibits inflammatory signaling mediated by Toll- Upadhyay et al. [93]
Lindl 5-Dimethoxyphenanthrene-1, like receptors in human THP1 cells Datla et al. [15]
7-diol
Eulophia ochreata Tuber 9, 10-Dihydro-2, Antioxidant activity Kshirsagar et al. [46]
Lindl 5-Dimethoxyphenanthrene-1,
7-diol
5, 7-Dimethoxyphenanthrene-
2, 6-diol
Eulophia nuda Lindl. Tuber 9, 10-Dihydro-2, Anti-proliferative activity against Human cancer Shriram et al. [79]
5-Dimethoxyphenanthrene-1, cells (MCF 7)
7-diol
V. Shriram and V. Kumar
using rhizome segments [94]. McAlister and Van Staden [54] reported in vitro seed
germination of E. cucullata, E. streptopetala, and E. petersii on MS medium [58],
medium supplemented with 3% sucrose and 0.01% myoinositol. Chang et al. [12]
reported in vitro seed germination of the orchid E. graminea, a species native to
Taiwan, and was successfully developed into rhizomes. The in vitro plants could
flower and produce the fruits (capsule) through the autogamous mating system.
Obtained seeds were sown in vitro and in this manner four generations were cultured
over a period of 4 year [12].
Panwar et al. [68] reported a standardized method for in vitro propagation and
tuberization of E. nuda, using tuber explants on MS medium supplemented with
6-benzyladenine (BA) and additives (ascorbic acid, adenine sulfate, arginine, and
citric acid). Multiplication of shoots was achieved successfully up to three sub-
cultures [68]. The rate of in vitro tuber formation was comparably higher than in
the natural conditions. Rooting of shoots was achieved and the in vitro generated
plantlets were acclimatized to the greenhouse conditions [68]. It was thus an
efficient method for regeneration, multiplication, and production of large number
of tuberous plantlets of E. nuda. Similarly, an optimized protocol for micro-
propagation of E. nuda from axillary bud segments was reported by Shroti and
Upadhyay [81] using MS medium supplemented 2,4-D. The explants developed
protocorm like bodies (PLBs) within 6–8 weeks of inoculation on the growth
medium, and the subculture PLBs on basal MS medium differentiated plantlets
into tubers.
A method for asymbiotic seed germination, seedling development, and establish-
ment of in vitro generated plantlets of E. nuda was reported by our group [60]. The
authors optimized several parameters such as most suitable seed age, culture medium
compositions, type and concentrations of phytohormones, additives and/or supple-
ments, and reported a standardized and reproducible method for asymbiotic seed
germination and plantlet regeneration of E. nuda (Figs. 1 and 2; [60]). These findings
hold significance and may help in mass multiplication and conservation of this and
other closely related species. Further, ploidy analysis of in vitro raised plants, first
work of this kind in this plant, revealed that these plantlets kept their genome stable
and true-to-typeness with mother plants (Fig. 3), a key consideration for conserva-
tion of any species without genetic alteration.
We conducted a study on another species of the genus, E. ochreata [80], and
described a first report on direct as well as indirect in vitro plant regeneration of this
important orchid. A number of parameters comprising type of explant, artificial
growth medium types and compositions, and phytohormones were standardized for
optimal plant regeneration. Among the explants, the PLBs were found to be the best
choice for induction, proliferation of shoots, as well as for callus production
[80]. The MS medium supplemented with 2.5 mg L1 BAP and 1.0 mg L1 Kin
proved best for shoot multiplication with synchronized growth. The number of
shoots was further enhanced with subcultures on same media composition; achiev-
ing up to 40 shoots per explant after 3 such cycles of 30 days each. The shoots were
successfully rooted in vitro on ¼ strength MS fortified with activated charcoal and
additives; rooted plantlets were acclimatized greenhouse conditions. The similar
Fig. 1 Asymbiotic germination of Eulophia nuda seeds obtained from capsules. (a) Capsules of
plants growing in wild conditions, (b) Micrograph of seed with embryo (arrow) at pre-germination
stage, (c) Scanning electron micrographs of E. nuda seed (arrow), (d) Scanning electron micrograph
showing testa-breaking leading to protocorm formation (arrows), (e) Shoot emergence from PLBs
(arrow). Reprinted with permission from Springer, Nanekar et al. [60]. https://doi.org/10.1007/
s40011-014-0353-4; https://link.springer.com/article/10.1007/s40011-014-0353-4
ploidy levels of in vitro regenerated plants in comparison with the field-grown

mother plants confirmed the true-to-typeness of tissue culture generated plantlets.
Similarly, in vitro germination of seeds, differentiation of embryos, as well as ex
vitro seedling production was reported from in vitro rhizome-like bodies of
E. promensis by Hossain [28]. Through the subcultures, profuse proliferation of
protocorms was observed which later developed rhizome-like bodies (RLBs). These
RLBs further produced in between one to three seedlings per RLB. Authors thus
developed a fast and cost-effective method for the micropropagation of E. promensis
which can potentially be used for other orchids as well for their propagation and
conservation. Symbiotic germination of Eulophia species was established success-
fully by Ochora et al. [66] using oats medium and germination medium
supplemented with banana homogenate and activated charcoal. Further, in vitro
asymbiotic germination and clonal propagation of E. cullenii was reported by
Fig. 2 Asymbiotic seed germination and plant developmental stages of Eulophia nuda, (a) Seed
germination and PLB formation after 60 days of inoculation on BM1 + CW, (b) Protocorm-derived
seedling formation on BM1 + CW containing 1 mg/L each of NAA and BAP after 120 days of seed
inoculation, (c and d) Stages of shoot multiplication on BM1 + CW containing 2.5 mg/L BAP and
1.5 mg/L Kin, 60 days after seedling inoculation and further shoot proliferation after two sub-
cultures of 30 days each, (e) In vitro rooting of microshoots on MS medium supplemented with
200 mg/L activated charcoal and 2 mg/L IBA, (f) Acclimatized plants of E. nuda in greenhouse,
inset: tuber of E. nuda. Bar ¼ 1 cm. Reprinted with permission from Springer, Nanekar et al.
[60]. https://doi.org/10.1007/s40011-014-0353-4; https://link.springer.com/article/10.1007/s40011-
014-0353-4
a b 100 NC
300
90
NC 80
250
70
200 60
Count
Count
50
150
40
100 30
20
50
10
0 0
100 200 300 400 500 100 200 300 400 500
BL2-A (10^3) BL2-A (10^3)
Fig. 3 Histograms of flow cytometric analysis of field-grown (a) and in vitro raised plants (b) of
E. nuda. Reprinted with permission from Springer, Nanekar et al. [60] https://doi.org/10.1007/
s40011-014-0353-4; https://link.springer.com/article/10.1007/s40011-014-0353-4
Decruse et al. [17]. These authors like previous reports used organic supplements
like coconut water, peptone, yeast extract, and casein hydrolysate for enhancing the
protocorm growth followed by shoot development and then linear mini-rhizomes
formation. These findings thus hold significance from eco-restoration perspectives of
orchids as well.
In vitro asymbiotic seed germination and plantlet development in E. nuda on
Knudson-C medium was also reported by Dawande and Gurav [16]. Symbiotic seed
culture of E. alta was well attempted by Johnson [37] using fungal isolates collected
from its roots, and authors found that seedlings co-cultured with fungal isolate (Ealt-
396) grew more rapidly than the asymbiotic seedlings. Authors advocated that the
symbiotically grown seedlings to be more appropriate for reintroduction to natural
areas than their asymbiotic counterparts.
6 Conclusion
Orchids represent highly evolved and valuable plants, extensively utilized for
ornamental and therapeutic purposes. They have remarkable ethnopharmacological
applications in traditional medicinal systems and many of these claims have been
scientifically validated through pharmacological assessments of crude extracts as
well as pure molecules from these plants. Eulophia represents a diverse group of
orchids with tremendous potentials, commercially, ecologically, and therapeutically;
however, owing to these potentials many of the species of this genus are over-
exploited and thus belong to threatened taxa. Considering the low germination and
survival rate in natural conditions along with requirement for specific fungal asso-
ciations for germination and growth, in situ conservation approaches have met with
limited success. Plant tissue culture technologies have emerged as potent means for
large-scale production and conservation of important members of Eulophia genus.
There are some important reports in recent years describing micropropagation of a
number of species of Eulophia; however, most the species are remained to be
explored, and thus more of such investigations are required. Besides, though many
of the Eulophia species are known to biosynthesize potent bioactive
phytomolecules, there are very few or no reports on in vitro production of these
phytomolecules using plant cell and tissue cultures of these plants, future investiga-
tions need to be conducted to fill this big gap.
Acknowledgments VS wish to acknowledge the financial assistance from the University Grants
Commission (UGC), Government of India [No.: F 39-426/2010 (SR)].
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Cyrtopodium glutiniferum, an Example of
Orchid Used in Folk Medicine: 21
Phytochemical and Biological Aspects
Carlos Fernando Araujo-Lima, Israel Felzenszwalb, and

Andrea Furtado Macedo
Contents
1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 518
2 The Genus Cyrtopodium R. Br. (Orchidaceae) and Cyrtopodium glutiniferum Raddi:
Biological, Cultivation, and Phytochemical Aspects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 519
3 Ethnopharmacological Aspects of Cyrtopodium glutiniferum . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 521
4 Novel Evidences of C. glutiniferum Efficacy on Skin Lesions Treatment . . . . . . . . . . . . . . . . . 526
5 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 528
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 528
Abstract
The center of Cyrtopodium spp. diversity is in a typical Brazilian savanna-like
formation. C. glutiniferum has thick big exposed pseudobulbs and yellow high
paniculate inflorescences. This species has been taxonomically confused with
many other correlates, which also have yellow flowers. C. glutiniferum is located
in hotspots in Brazil and is relevant ethnopharmacologically. The plant’s bulb
traditional uses include the treatment of abscesses and wound healing. In previous
work, phenanthrene was the most abundant subclass in metabolomic analysis,
however the most abundant molecules were dihydroformononetin, caffeic acid
Carlos Fernando Araujo-Lima and Israel Felzenszwalb contributed equally with all other
contributors.
C. F. Araujo-Lima · I. Felzenszwalb
Laboratory of Environmental Mutagenesis, Department of Biophysics and Biometry, Rio de Janeiro
State University, Rio de Janeiro, Brazil
Roberto Alcantara Gomes Institute of Biology, Universidade do Estado do Rio de Janeiro, UERJ,
Rio de Janeiro, Brazil
A. F. Macedo (*)
Integrated Laboratory of Plant Biology, Department of Botany, Institute of Biosciences, Federal
University of Rio de Janeiro State, UNIRIO, Rio de Janeiro, Brazil
e-mail: andrea.macedo@unirio.br

518 C. F. Araujo-Lima et al.
4-O-glucoside (glucocaffeic acid), and arbutin. As C. glutiniferum is traditionally

used to treat skin lesions, our research group has verified the positive anti-
inflammatory and antiproliferative effects of the extract against activated mono-
nuclear cells. However, probably the therapeutic effect of this orchid is not
directly related to the elimination of the pathogen but to the control of tissue
lesions mediated by acute inflammation, acting against the cells of the immune
response, involved in the occurrence of abscesses.
Keywords
Ethnopharmacology · Ethnobotany · Cyrtopodium · Antimicrobial ·
Antioxidant · Anti-inflammatory · Plant tissue culture
Abbreviations
ATCC American Type Culture Collection
CFU Colony-forming unit
FDA United States Food and Drug Administration Agency
IAA Indole-3-acetic acid
OA Oatmeal agar
PLBs Protocorm-like bodies
UHPLC-MS/MS Ultra-high-performance liquid chromatography tandem mass
spectrometry
UV Ultraviolet radiation
1 Introduction
The Cyrtopodium genus (Epidendroideae: Cymbidieae: Cyrtopodiinae) has approx-

imately 50 species of neotropical scope, from southern Florida to northern Argen-
tina, mostly terrestrial, with few epiphytes [1–6]. Most species are found in Brazil,
about 40, followed by Bolivia and Venezuela with 9 species each [7]. Only two
species have been registered in the USA (C. polyphyllum and C. punctatum),
however, C. polyphyllum has been naturalized [8]. In fact, the center of genus
diversity is in the “cerrado,” a typical Brazilian savanna-like formation, whose
physiognomies include grassy field (“campo limpo”), grass-herb-sub-shrub field
(“campo sujo”), and semideciduous xeromorphic medium tall forest (“cerrado”
sensustricto) [9]. Despite the great importance of the genus object of this work, it
is relevant to state that the classification and, therefore, the identification of
Cyrtopodium species in Brazil have been revised due to innumerable problems
concerning the material deposited in the herbariums. Many holotypes that were
deposited consisted of incomplete plant material, mainly without a flower. Due to
these problems, epitypes have been used for the correct identification of
Cyrtopodium species. Terrestrial species with large pseudobulbs and yellow flowers,
mainly, have been the subject of a major revision due to their taxonomic complexity.
Species of this group, which were collected: (a) in the northern part of South
America, in the Amazon basin, and, probably, along the north and northeast coast of
21 Cyrtopodium glutiniferum, an Example of Orchid Used in Folk. . . 519
Brazil are C. andersonii (Lamb. Ex Andrews) R. Br. and (b) in southeastern Brazil
are C. glutiniferum Raddi or C. cardiochilum Lindl [9]. The last two species are
considered co-specific [10]. Consequently, older articles published, with plant mate-
rial collected in Brazil, may present identification errors. For this reason, we have
chosen to make a more general overview of the genus Cyrtopodium, with special
attention to Cyrtopodium glutiniferum.
2 The Genus Cyrtopodium R. Br. (Orchidaceae) and

Cyrtopodium glutiniferum Raddi: Biological, Cultivation,
and Phytochemical Aspects
Brazilian Cyrtopodium species occur according to the soil and the surrounding
vegetation, most of which are terrestrial and bloom at the end of the dry season
and at the beginning of the rainy season [1]. Few studies on pollination have been
carried out on this taxon which can occur through optional self-pollination, with
rain-assisted autogamy, as well as cross-pollination, by the action of large bees by
food deceit or not [6, 11–15]. There are reports that some species produce fewer
fruits and seeds due to the low pollination rate [3, 6].
The most common Brazilian biomes occurrence of these species are:
(a) “cerrado”; (b) rocky field vegetation (“campo rupestre”), a vegetation with
more or less continuous herbaceous stratum intermixed by small shrubs or subshrubs
on sandy soil with pebbles or graves; (c) “restinga” vegetation, a lowland coastal
plain vegetation on lacustrine and marine sands that occur discontinuously along the
Brazilian coast within the Atlantic Forest – which is considered one of the global
biodiversity hotspots; and (d) along the coast on rocks [9, 11, 16, 17].
Some Cyrtopodium species are endemic to Brazil and are classified as “vulnerable”
(Cyrtopodiumtriste Rchb. f. and Warm.; Cyrtopodium palmifrons Rchb. f. and Warm.),
“endangered” (Cyrtopodium poecilum var. roseum Bianch. and J. A. N. Bat.;
Cyrtopodium lissochiloides Hoehne and Schltr.), “critically endangered” (Cyrtopodium
lamellaticallosum J. A. N. Bat. and Bianch.; Cyrtopodium latifolium Bianch. and J. A.
N. Bat.; Cyrtopodium linearifolium J. A. N. Batista and Bianchetti), and “data defi-
cient” (Eulophia ruwenzoriensis Rendle) [12]. Notably, Cyrtopodium species from
southeastern Brazil, including C. glutiniferum, are found growing in the remnants of the
Atlantic rain forest and in sandy coastal plain habitats [18–21]. Precisely, populations in
lowland areas where land is desirable for real estate development and habitat exploi-
tation are vanishing [22]. Despite the considerable economic importance of their showy
flowers and the medicinal value of the pseudobulbs, most species of the genus
Cyrtopodium are unknown to horticulturists and are rarely seen in cultivation. Large-
scale propagation is a prerequisite to meet future pharmaceutical and ornamental
requirements, and to prevent eradication of this highly valuable plant [7].
Therefore, due to issues of endemism, ethnopharmacological importance, orna-
mental value, original habitat destruction, modification and fragmentation, low seed
production, and pressure on the preservation of the group, since many species have
been overcollected and only exist in areas of environmental preservation [3, 23],
studies have been developed to establish cultivation protocols. Among these publi-
cations, some stand out and will be commented below.
In vitro asymbiotic seed propagation was carried out for C. punctatum, where a
higher germination rate was registered in the P723 medium, in the dark and greater
seedling development in the same medium, but with a photoperiod of 16 h [3]. Guo et al.
[24] established a protocol for the production of C. paranaense seedlings, from in vitro
root tips cultivation, with the formation of protocorm-like body (PLB). In this work, it
was verified that the PLBs were formed from the tips of the roots or the stele of the root of
the mother plant, maintaining an initial dependence on its vascular tissue [24]. Picolotto
et al. also established a C. paludicolum cultivation protocol from root tips [25]. Vogel
and Macedo [7] produced the only work on in vitro production of C. glutiniferum. In this
work it was verified that germination was faster under white and blue light and highest
under green light. The three light conditions also induced the development of pro-
tocorms. Indole-3-acetic acid (IAA) positively affected protocorm-like bodies (PLBs),
shoots, and roots multiplication from protocorms (Fig. 1). Later, Guimarães et al. [26]
published work on the symbiotic propagation of C. glutiniferum with positive results for
germination and growth on oatmeal agar (OA) medium inoculated with the mycorrhizal
fungus Epulorhiza sp. C. glutiniferum seems to have a preference for strains of
Epulorhiza and that fungus digestion is essential to protocorm development [27].
Similarly, in studies carried out with C. paludicolum and C. saintlegerianum it was
observed that symbiotic seed germination is more beneficial than asymbiotic germina-
tion [23, 28]. Environmental restoration models may require basic biotechnology
techniques such as in vitro germination, since this technique has an advantage of not
producing individuals genetically identical to the matrix, generating variability. This is
the exact opposite of micropropagation, another tissue culture technique. As for
Cyrtopodium species, which represent a valuable germplasm, in vitro cultivation tech-
niques for germplasm propagation and conservation are essential [29].
Cyrtopodium glutiniferum Raddi occurs only in Brazil (endemic), in the Atlantic
forest, in the states of Espírito Santo, Rio de Janeiro, and Minas Gerais, common,
especially, in rock outcrops, granite (inselbergs) but also possible to be observed
developing in sandy soil. There are three worldwide hotspots of inselberg plant
diversity. Southeastern Brazil, where C. glutiniferum is located, is one of them [17].
As described previously, C. glutiniferum is subjected to conditions of exposure to
full sun, high UV incidence, lack of soil, constant winds, water and nutrient scarcity,
difficulty in affixing roots, and high temperatures (Fig. 2a), including the group of
succulent plants, which are tolerant to dissection [17, 30].
C. glutiniferum has thick big exposed (not buried) fusiform pseudobulbs (30–
90 cm high) (Fig. 2a) and yellow high (90–240 cm long) paniculate axillary
inflorescences, with simple or 1–4 ramifications that rise from the developing
shoot (Fig. 2b). Its large flowers (expanded lip with 1.6–2.3 cm long), completely
or predominantly yellow, appear at the end of the dry season and beginning of the
rainy season (August–October) (Fig. 2c). The ovate sepals may be greenish-yellow
or yellow-brown in color and have brown spots at the apex, while the callus may
have orange spots that occasionally extend to the surrounding area and the base of
the lateral lobes (Fig. 2c) [7, 31]. In C. glutiniferum pollination occurs through
sexual propagation (i.e., gene recombination) [7].
Fig. 1 Representative photographs of the sequence of events and stages of in vitro germination of
C. glutiniferum seed: (a) seed germination after 50 days, (b) protocorm formation after 50 days, (c)
4-weeks-old PLBs (arrow – PLB mass), and (d) plantlets and PLBs after 4 weeks of culture on IAA
medium (black arrow – plantlet; blue arrow – root with velamen; yellow arrows – bud protuber-
ance). Scale bars ¼ 1 mm. (Source: Vogel and Macedo [7])
As previously explained, many species of the genus have already been confused
with each other because of their close resemblance [5]. C. glutiniferum has already
been mistakenly identified as C. cardiochilum, C. andersonii, or C. paranaenses
Schltr. Due to its similarity with C withneri L. C. Menezes has not yet enabled a
taxonomic solution [7, 31].
3 Ethnopharmacological Aspects of Cyrtopodium

glutiniferum
Popularly known as “Sumaré,” the genus Cyrtopodium is used on the production of

ointments in Brazil, with no or poor distinction of the species utilized [32]. The
genus has ethnopharmacological relevance, being used for treatment of chest colds,
Fig. 2 Cyrtopodium glutiniferum in inselbergh at Morro da Urca, Rio de Janeiro. (a) Whole plant
with pseudobulbs, leaves, and inflorescence; (b) detail of flower buds and flowers [7]; (c) flower
detail [31]
tuberculosis, and hemoptysis [33], as a topic antibiotic [34] and to treat perforation
wounds [24]. Species of the genus are also used in the form of ointments for the
treatment of lesions on the eyelids; in the form of juice to treat abscesses, folliculitis,
or in the form of syrups to treat cough and pertussis [10, 32]. Researchers reported
that extracts from pseudobulbs of C. paranaenses Schltr. and C. andersonii R. Br.
are useful for healing wounds [7, 35–37]. C. punctatum is part of some phytophar-
maceutical formulations for treating abscesses and burn [38]. The C. andersonii
pseudobulb is used in traditional medicine to treat inflammatory symptoms, with
healing properties and antihemorrhagic activity [38]. Previous studies have already
reported the presence of glucomannan with possible immunomodulatory, anti-
inflammatory, and gastroprotective activity [2, 38]. In an article published with
C. macrobulbon, a species that is traditionally used to treat urinary infections, it
was found that its extract and stilbenoids have antinociceptive, anti-inflammatory
properties [39, 40]. Subsequently, it was also found that extracts of C. paniculatum,
which are rich in stilbenoids, had a moderate toxic effect on cancer cells [41].
Stilbenoids appear to be a chemical characteristic of several species of Orchidaceae,
some of which are more frequent in the Cyrtopodium genus [41] (Table 1).
Stilbenoids are phytoalexins that protect the plant against herbivory. This chemical
group has a great structural diversity that goes through stilbenes, bibenzyls,
9,10-dihydrophenanthrenes, phenanthrenes, (dihydro)-phenanthrenequinones,
p-hydroxybenzyl-phenanthrenes, 9,10-dihydrophenanthrofurans, and phenanthrene
dimers. These molecules have anti-inflammatory, antifibrotic, and antimicrobial prop-
erties, which may justify the traditional use of several species of the genus [41].
The genus Cyrtopodium has an ethnopharmacological basis, such as the folk use
of C. cardiochilum Lindl. and C. andersonni R. Br. on gastric inflammatory diseases,
mainly associated to polysaccharidic contents [2, 38, 39]. C. macrobulbon (La Llave
and Lex.) G. A. Romero and Carnevali and C. paniculatum (Ruiz and Pav.) Garay
are traditionally used for treating urinary infections [39] and their extracts are rich in
stilbenoids and phenanthrenes derivatives, as cyrtopodinone, cyrtopodinol, and
cyrtopodin. Some of these molecules showed moderate cytotoxicity activity towards
a cancer cell line [40, 41]. C. macrobulbon is also employed in the treatment of
abscesses, and as a balsamic agent [39]. C. punctatum (L.) Lindl. is traditionally used
as an expectorant in the recovery of dry cough, and amelioration of the inflammatory
symptoms of bronchitis and asthma as syrup [5, 32]. C. punctatum is also used as
emetic, on blood pressure control [42], to treat rheumatism [43] and also for treating
boils and abscesses [44].
Recently, we described a phenolic content emphasized metabolomic analysis of
C. glutiniferums by UHPLC-MS/MS, since, as previously reported, some stilbenoids
appear to be characteristic of the genus. This was the first work on metabolomics of
the genus Cyrtopodium [45]. In a survey made from publications on the phytochem-
istry of the species of Cyrtopodium genus, it was found that some molecules, mostly
stilbenoids, were reported in more than one species: glucomnann, confusarin,
gigantol, potatoesin III, denthyrsinin, and shancidin (Table 1).
In our work it was found that phenanthrenes was the most abundant subclass,
despite the fact that the most abundant molecules were dihydroformononetin, caffeic
acid 4-O-glucoside (glucocaffeic acid), and arbutin. In this study, we also proceed an
in vitro genotoxicity assessment, recommended by FDA for any chemical used in
human therapeutics and also some aspects of antiproliferative, anti-inflammatory,
and antioxidant activity of C. glutiniferum aqueous extract [45]. The genotoxicity
assessment is fundamental for any natural product intended by human use. Despite
524
Table 1 Survey of common published molecules of Cyrtopodium spp.

Confusarin Gigantol Batatasin III Denthyrsinin Shancidin Glucomannan
C. paniculatum + + + + +
Pseudobulbs [40]
C. macrobulbon + + +
Pseudobulbs [39]
C. andersonii +
Pseudobulbs [38]
C. cardiochilum +
Pseudobulbs [2]
C. paniculatum + + + +
Roots [41]
C. glutiniferum + +
Pseudobulbs [45]
C. F. Araujo-Lima et al.
the popular belief that the use of plants as an alternative for the treatment of several
diseases is free of hazards, the chemical composition of the vegetal extracts can
imply in the occurrence of the most diverse lesions to living matter and, above all, to
DNA [46]. In our investigation, C. glutiniferum extract induced mutagenicity only
on TA100 strain at the highest tested dose (5.0 mg) on Ames Test and did not
increase micronucleated RAW264.7 macrophages. Because of the facts presented,
the extract cannot be considered genotoxic [45].
Our recent findings about C. glutiniferum extract efficacy suggest extract can be
considered a good direct antioxidant, reducing the DPPH+ radicals, acting as a free
radical scavenger, presenting an EC50 of 132.6 6.2 μg/mL. We also observed a
dose-dependent and time-dependent anti-inflammatory response in LPS-activated
macrophages (Fig. 3) by the decrease of both stimulation index (calculated by the
variation in mitochondrial function using WST-1 reagent – Fig. 3a) and viable cells
counts (through trypan blue method – Fig. 3b), suggesting an immunomodulatory
effect. In fact, the secondary metabolites detected on C. glutiniferum extract are
described as antiproliferative and anti-inflammatory agents [47–49].
Fig. 3 Effects of
Cyrtopodium glutiniferum
extract on RAW 264.7
macrophages activation and
proliferation status. After
offering 1 μg/mL of E. coli
LPS as a phlogistic agent to
RAW 264.7 cells and treat
them with C. glutiniferum
aqueous extract (from 0 to
50,000 ng mL1) for 24 or
48 h, WST-1 (a) and trypan
blue (b) cell counting were
performed to evaluate
activation and proliferation.
(Source: Araújo-Lima et al.
[45])
Stilbenoids and other polyphenols are described as regulators of transcriptional

factors, as NF-κB, IKK and mTOR, acting directly on cell activation responses [50,
51]. In comparison to other orchids from the same genus, the pseudobulbs of
C. paranaense are used for wound healing, but no biological activity was established
scientifically. The pseudobulbs of C. flavum, which are popularly used to heal skin
lesions in Brazilian folk medicine, have been studied as an analgesic and anti-
inflammatory agent. Its 20% pseudobulb aqueous extract and a polysaccharide
extracted from it, cyrtopodine, were described as anti-inflammatories, besides the
absence of analgesic activity [32].
4 Novel Evidences of C. glutiniferum Efficacy on Skin Lesions

Treatment
Regarding the ethnotherapeutical perspective, Cyrtopodium ointments are widely

used in Brazilian traditional medicine to treat boils and abscesses, as mentioned
above. Besides that, they can be considered as the most common treatment to skin
and soft tissue diseases [52], furuncles, and abscesses since they have a complex
pathophysiology and are mediated by both infectious and inflammatory processes
[53]. A skin abscess is essentially a suppurative sequela of folliculitis, and when
infection involves several adjacent follicles, producing a coalescent inflammatory
mass with pus draining from multiple orifices, the larger nodule is then termed a
carbuncle [52]. The tissular homeostasis misbalance caused by bacterial infection
can produce pyogenic abscesses, which can culminate in hemodynamic alterations,
as ischemia and subcutaneous thrombosis, resulting in tissue necrosis, decurrent of
inflammation [54].
Skin abscesses are commonly caused by infectious pathogens, and the bacterial
milieu can consist both in Gram-positive and Gram-negative bacteria, such as
Staphylococcus aureus infection in boils [55] and Escherichia coli infection in
Fournier’s syndrome [56].
Trying to demystify some aspects concerning the mechanism of action of
C. glutiniferum as therapeutic alternative for treatment of skin and soft tissue
diseases, we decided to investigate the efficacy of the aqueous extract against S.
aureus (ATCC 25923 strain) and E. coli (ATCC 25922 strain). The minimal inhib-
itory concentration (MIC) tests in flat bottom microtiter 96 wells plate were
performed according to Antimicrobial Susceptibility Testing guidelines from the
Institute of Clinical and Laboratory Standards Institute [57].
For antimicrobial activity experiments, the tested concentrations of
C. glutiniferum extract were 3.00, 1.00, 0.33, 0.11, 0.04, and 0.01 mg/mL. The
maximum concentration of C. glutiniferum extract was defined as 1/1000 of the mass
found in topical ointments (3%). So, an aliquot of each strain was thawed and diluted
in Mueller Hinton Broth – MHB in the proportion of 1:500, generating an inoculum
of ATCC 25923 from S. aureus and another of ATCC 25922 from E. coli, both with
concentration of approximately 2.0 105 CFU/mL. From these suspensions, 100 μL
were pipetted into each well of the microtiter plates, one strain on each plate. In the
first row of each, another 100 μL of MH broth without inoculum was pipetted to
control the test sterility (white). The antimicrobial tetracycline (Sigma Aldrich;
St. Louis, MO, USA) was used in all experiments as a positive control, in concen-
trations from 1.8 to 445 μg/mL.
With the addition of the inoculum, the concentration of the tested compound/
extract was adjusted to the desired value (dilution 1:2) and the final concentrations of
the inocula were approximately 1.0 105 CFU.
Microtiter plates were incubated for 24 h at 35 °C and the growth of the samples
was evaluated both visually and spectrophotometrically using the SpectraMax Plus
384 Microplate Reader spectrophotometer, measuring the absorbance of the cultures
(Optical Growth Dosage) at the wavelength of 620 nm. The experiments were
carried out in triplicate and repeated three times.
According to Table 2, the results of the above-mentioned extract’s antimicrobial
activity were not encouraging. The extract was only able to cause partial inhibition
of E. coli 25922 in the highest concentration tested. At the other concentrations of
the assay, both E. coli 25922 and S. aureus 25923 did not suffer any inhibition effect
after being exposed to C. glutiniferum extract. These findings interpose the results of
the bacterial mutagenesis model, in which death of Salmonella enterica serovar
Typhimurium was detected in concentrations greater than 50 μg.
Considering the complexity of pathophysiological aspects on skin lesions, and
correlating these data to our recent findings about the anti-inflammatory and anti-
proliferative effects of C. glutiniferum against activated mononuclear cells, probably
the therapeutic effect of this orchid is not directly related to eliminate the pathogen
but in controlling the tissue injuries mediated by the acute inflammation, acting
against immune response cells, involved in abscess occurrence [58–60].
Despite our negative results about antimicrobial efficacy, one of the most abun-
dant compounds detected by metabolomics in our C. glutiniferum extract, arbutin, is
associated to the bactericidal effect of other plant extracts from Bergenia genus [61],
and also in essential oils [62]. The chemical nature of arbutin (derivative of hydro-
quinone) and its tyrosinase inhibitor effect can be related to its efficacy against
different bacteria, interfering in protein scaffolds and resulting in cell wall damages,
even when these effects being observed in high doses of this polyphenol [62, 63].
The other two compounds (glucocaffeic acid and dihydroformononetin) have no
evidences on literature about its microbicidalefficacy.
Table 2 Antimicrobial activity of Cyrtopodium glutiniferum extract (in mg/mL) against

Escherichia coli (25922 strain) and Staphylococcus aureus (25923 strain)
Strain Blank Negative control 3.00 1.00 0.33 0.11 0.04 0.01
E. coli (25922) + / + + + + +
S. aureus (25923) + + + + + + +
Categories: growth without inhibition: + (80% of growth, in comparison to negative control);
partial inhibition: / (between 10% and 80% of growth, in comparison to negative control; total
inhibition: (<10% of growth, in comparison to negative control)
5 Conclusions
Cyrtopodium species from southeastern Brazil, including Cyrtopodium glutiniferum

Raddi, are found growing in biodiversity hotspots, remnants of the Atlantic rain
forest and in sandy coastal plain habitats and, besides that, C. glutiniferum is relevant
for biotechnological and pharmacognosy purposes. The orchid is widely prescribed
in Brazilian folk medicine and some of its medicinal properties had to be scientif-
ically validated. In our previous metabolomic analysis, the most abundant molecules
detected on aqueous extract were dihydroformononetin, caffeic acid 4-O-glucoside
(glucocaffeic acid), and arbutin. As C. glutiniferum is traditionally used to treat skin
lesions, as abscesses, furunculosis, and wound healing, our research group has
verified the positive anti-inflammatory and antiproliferative effects of the extract
against activated mononuclear cells. However, the therapeutic effect of this orchid
probably is most directly related to the modulation of host immune responses and
that about the elimination of the bacteria on inflammation site. The effects of
C. glutiniferum on inflammation-mediated tissue damage can favor host cellular
and tissue repair and resolutive response phase in cutaneous infections.
Acknowledgments Funding: This work was supported by the National Council of P&D of Brazil
(CNPq) and Coordination of Superior Level Staff Improvement (CAPES), which granted fellow-
ships; and the Foundation for Research of Rio de Janeiro State (FAPERJ), UERJ, UNIRIO, and
CNPq, which gave financial support.
Technical support: The authors thank AykeAdnet de Lima and Renato Geraldo da Silva Filho,
from the Department of Microbiology and Parasitology of Federal University of Rio de Janeiro
State (UNIRIO) by technical support on antimicrobial experiments.
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Phenanthrenes from Orchidaceae and Their
Biological Activities 22
Andrea Vasas
Contents
1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 534
2 Occurrence of Phenanthrenes in Orchidaceae Species . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 535
2.1 Monophenanthrenes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 539
2.2 Di- and Triphenanthrenes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 547
2.3 Occurrence of phenanthrenes in Orchidaceae . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 551
3 Chemotaxonomical Significance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 551
4 Pharmacological Activities of Orchidaceae Phenanthrenes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 552
4.1 Traditional Uses of Orchids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 552
4.2 Biological Activities of Orchidaceae Phenanthrenes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 553
5 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 561
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 562
Abstract
Orchidaceae is one of the largest plant family. Its members are distributed world-
wide, and many of them are used in traditional medicines for the treatment of
different diseases. In the last few decades, phytoconstituents with great chemical
diversity were isolated from several orchids, and a wide range of biological activities
were detected either for crude extracts or for pure compounds. Among the isolated
compounds, phenolic derivatives, especially stilbenoids and phenanthrenes, are the
most important ones. In the plant kingdom, orchids are the best sources of phenan-
threnes – around 400 components were isolated from 100 species. Investigation of
the phenanthrene content of orchids can be contributed to place taxonomically
questionable or rather controversial species into the proper genera. Pharmacological
investigation of Orchidaceae phenanthrenes revealed that several compounds
possess promising activities – mainly antiproliferative, antimicrobial, anti-
inflammatory, and antioxidant effects. This chapter summarizes the isolated
A. Vasas (*)
Department of Pharmacognosy, University of Szeged, Szeged, Hungary
e-mail: vasasa@pharmacognosy.hu

534 A. Vasas
phenanthrenoids, their occurrence in Orchidaceae species, chemotaxonomical

significances, and biological activities of the most promising representatives.
Keywords
Orchidaceae · Bletilla · Dendrobium · Phenanthrenes · Phenanthroquinones ·
Chemotaxonomy · Antiproliferative
1 Introduction
Orchidaceae, one of the largest and most evolved family of the flowering plants,
comprises approximately 27,000 species in 900 genera [1]. Orchids are widely
distributed, and the diversity of the plants increases toward the tropic and
especially tropical mountains. Approximately 73% of orchids are epiphytic.
The most represented genera are Bulbophyllum (n ¼ 1000), Dendrobium (n ¼
900), Eria (n ¼ 500), Maxillaria (n ¼ 420), Oncidium (n ¼ 420), Liparis (n ¼
350), Eulophia (n ¼ 200), and Calanthe (n ¼ 150) [2, 3]. Many of the orchids
are used in traditional medicines for the treatment of different diseases, e.g.,
tumors, infectious diseases, pain and fever, diabetes, skin diseases, problems
concerning circulation, digestive, respiratory, and reproduction organs [4, 5]. In
the last decades, several studies have been performed to justify the medicinal use
of various plants in the treatment of diseases. As a result of these investigations,
a wide range of chemical compounds, including alkaloids, bibenzyl derivatives
(stilbenes and phenanthrenes), flavonoids, and terpenoids, were identified from
orchids, and diuretic, anti-inflammatory, antipyretic, antirheumatic, myorelaxant,
antitumor, antimicrobial, hypoglycemic, and neuroprotective activities of the
extracts and pure compounds were detected [6, 7]. Moreover, phenanthrenes
are important phytoalexins and act as defense against diverse biotic and abiotic
factors [8, 9].
Phenanthrenes and stilbenoids are the most relevant components present in
Orchidaceae species. Phenanthrenes are aromatic secondary metabolites, occur-
ring relatively rare in nature. To date, only a few phenanthrene-containing plant
families (n ¼ 17) have been identified, and less than 500 phenanthrenes were
isolated [5]. Most of these metabolites were described from Orchidaceae,
Juncaceae, Combretaceae, and Dioscoreaceae species [5]. However, the best
sources of phenanthrenes are orchids; approximately 75% of the compounds
were isolated from Orchidaceae species. As phenanthrenes are derived from
stilbenes by oxidative coupling of the aromatic rings of stilbene precursors, it
is not surprising that they occur together in orchids. The phenanthrene core could
also be generated from stilbenes by UV irradiation in the presence of oxidants
[10, 11], and fungal infection also increases the formation of phenanthrenes
[9]. After the biosynthesis of the phenanthrene scaffold, further modifications
are catalyzed by different oxidases. Most of the phenanthrenes are stable, as it
22 Phenanthrenes from Orchidaceae and Their Biological Activities 535
was confirmed by an investigation proving that heat processing did not affect sig-
nificantly the anti-inflammatory activity of bibenzyls and phenanthrenes isolated
from Bletilla striata tuber [12]. In case of phenanthrenes, certain substituents
seem to be specific to certain families, e.g., p-hydroxybenzyl- and stilbene-
substituted compounds are almost exclusively found in Orchidaceae species.
Therefore, these components are considered by some as chemotaxonomic
markers.
Although phenanthrenes comprise a relatively small group of natural products,
discovering new phenanthrene derivatives and evaluating their prospective biolog-
ical activities have become of great interest to many research groups worldwide.
Several phenanthrenoids possess promising antitumor, antimicrobial, and anti-
inflammatory activities. For some of them, studies have been conducted to elucidate
their possible mode of action and to deduce structure–activity relationships. Based
on 213 references, this review covers the 376 phenanthrenes that have been isolated
from orchids, their occurrence in Orchidaceae species, chemotaxonomical signifi-
cances, and biological activities of the most promising compounds.
2 Occurrence of Phenanthrenes in Orchidaceae Species
Phenanthrenes isolated so far from natural sources may be classified into three
major groups: mono-, di-, and triphenanthrenes. The diversity of mono-
phenanthrenes is due to the number and type of the connecting substituents and
the saturation of the bond between C-9 and C-10. Compounds with saturated C-9–
C-10 bond are named as dihydrophenanthrenes, while 9,10-dehydro derivatives are
the phenanthrenes. 9,10-Dehydro and dihydro derivatives occur almost equally in
monophenanthrenes; about 60% of the compounds are dihydrophenanthrenes
[7]. Another group of monophenanthrenes is the phenanthrenequinones. Di- and
triphenanthrenes can be classified by the type of connecting monomers, and the
location of the linkage. Up to now, only a few compounds of the triphenanthrene
group have been described [5].
According to the literature data, the best sources of phenanthrenes are
Orchidaceae species, approximately 75% of the identified compounds isolated
from Orchidaceae species (Table 1). Mono-, di-, and triphenanthrenes were equally
isolated from orchids. Interestingly, up to now, phenanthrene trimers were isolated
only from orchids.
In many cases, depictions of the compounds are not correct, as rings A and C
are usually replaced. During biosynthesis, incorporation of dihydro-m-coumaric
acid into a 9,10-dihydrophenanthrene (hircinol), through a bibenzyl, was con-
firmed by Fritzemeier and Kindl [8]; therefore, most of the Orchidaceae phen-
anthrenes, substituted with hydroxy and methoxy groups at C-2 and C-4 on one of
their rings, have to be depicted as ring A. In many cases, the hyroxy group is
changed to methoxy group at C-2. In most cases, ring C is substituted with a
hydroxy group at C-7.
536 A. Vasas
Table 1 Phenanthrenes in orchids

Species Compound Ref.
1 Aerides rosea 28, 191 [168]
2 Agrostophyllum 4, 17, 18, 20, 187, 342, 343 [38, 66, 169]
callosum
3 A. khasiyanum 342, 343 [169]
4 Appendicula 1, 17, 174, 177, 193, 339 [41]
reflexa
5 Arundina 1, 4, 5, 15, 83, 88, 258, 272, 275, [42, 67, 70]
graminifolia 289, 290
6 Bletilla formosana 1, 24, 30, 33, 72–79, 107, 110, 170, [43, 104]
174, 177, 226, 228, 229, 241, 244,
258, 290, 293–295, 297, 313, 315,
324, 342, 349–351, 353
7 Bletilla ochracea 229, 231, 242, 243, 245 [170]
8 Bletilla striata 1, 3, 5, 8, 9, 11, 16, 24, 25, 29, 69, [12, 37, 44, 71, 106, 107,
72–75, 81, 82, 88, 93–95, 110, 122, 138, 139, 117, 120, 135,
129, 134, 140–144, 147, 148, 165, 154, 164, 171, 172]
170–175, 179, 180, 192, 226, 227,
229–231, 244, 253, 284, 285, 289,
290, 294, 297, 300, 306, 307, 313,
318, 319, 322, 324–326, 331–335,
344–348, 350, 352, 353, 356, 373
9 B. yunnanensis 81, 291, 313, 353 [158]
10 Brasiliorchis 17, 181 [173]
prophyrostele
11 Bulbophyllum 35, 208, 211, 216 [174]
gymnopus
12 B. leapardium 185 [175]
13 B. odoratissimum 1, 43, 166, 220, 272, 301, 302 [45, 176, 162, 176]
14 B. reptans 1, 35, 172, 208, 211, 216, 289, 353, [24]
364
15 B. retusiusculum 103, 224 [134]
16 B. rigidum 358 [177]
17 B. vaginatum 1, 17, 19, 24, 35–37, 136, 172, 174, [46, 123, 178]
176, 178, 184, 186, 190, 208–210,
357, 359
18 Calanthe 45–48, 268, 278, 279 [30, 31]
arisanensis
19 Cephalantheropsis 49, 50 [32]
gracilis
20 Cremastra 1, 4, 9, 12–14, 92, 120, 127, 169, [26, 26, 26, 48, 90, 92,
appendiculata 172, 194, 195, 221, 222, 232–239, 108, 153, 163, 179]
286, 287, 290, 321, 324, 337, 340,
341, 353–355, 360–363, 376
21 Cirrhopetalum 102, 223 [132, 133]
andersonii
(continued)
Table 1 (continued)
22 C. maculosum 353 [155]
23 Coelogyne cristata 41, 160, 162, 215 [129, 130, 180]
24 C. elata 1 [47]
25 C. flaccida 20 [66]
26 C. flavida 157 [128]
27 C. longipes 157, 158, 160, 166 [89]
28 C. ochracea 1, 252, 256, 271 [22]
29 Cymbidium 1, 17, 276, 277 [49, 141, 142]
aloifolium
30 C. finlaysonianum 1, 5, 17, 25, 80, 170, 177, 269, 276 [50]
31 Cymbidium Great 51, 52, 53, 269 [34]
32 Flower Marie 54, 281 [34]
Laurencin
33 C. pendulum 212, 218 [181]
34 Cypripedium 272, 274 [143]
tibeticum
35 Cyrtopodium 1, 5, 24, 33, 39, 55, 121, 123, 132, [17, 51]
paniculatum 170, 174, 196, 211, 212, 217, 240,
258, 267, 290, 297, 313, 316, 323,
324, 327, 328, 336, 353
36 Dendrobium 159 [131]
amoenum
37 D. aphyllum 283 [148]
38 D. brymerianum 5, 172 [72]
39 D. chrysanthum 166, 190, 248, 254 [85, 93]
40 D. chrysotoxum 122, 145, 166, 170, 190, 272 [114, 182]
41 D. crystallinum 247 [124]
42 D. denneanum 2, 8, 56–60, 166, 197–199 [13]
43 D. densiflorum 5, 166, 212, 259, 272 [73, 183]
44 D. draconis 8, 32, 261 [145]
45 D. fimbriatum 62 [15]
46 D. formosum 1, 5, 8, 24, 32, 174, 211, 261 [52]
47 D. hainanense 43, 44 [184]
48 D. loddigesii 5, 31, 166, 168, 200, 246 [14, 88]
49 D. longicornu 32, 260 [144]
50 D. moniliforme 8, 42, 63, 136, 270, 282 [35, 147]
51 D. moscatum 166 [87]
52 D. nobile 1, 5, 8, 10, 23–26, 64, 66, 170, 172, [18, 74, 74, 101, 185–187]
176, 184, 190, 201–204, 211, 270,
292
53 D. palpebrae 5, 190, 212, 338 [76]
54 D. plicatile 1, 5, 8, 23–25, 31, 46, 48, 69, 166, [23, 53, 77, 159]
170, 174, 212, 225, 258, 288
(continued)
538 A. Vasas
Table 1 (continued)
55 D. primulinum 67 [16]
56 D. rotundatum 31, 35, 166, 174, 208 [86, 87]
57 D. scabrilingue 1, 5, 38 [54]
58 D. sinense 262 [146]
59 D. thyrsiflorum 5, 8, 9, 23–25, 100, 101, 166, 167, [75, 188]
174, 211, 212, 255, 263, 288, 303,
310, 329, 330, 369, 370, 372
60 D. venustum 5, 308 [78, 166]
61 Ephemerantha 26, 184, 214 [20]
fimbriata
62 E. lonchophylla 23–25, 176, 182, 258, 270 [149, 189]
63 Epidendrum 23, 214 [190]
rigidum
64 Eria carinata 174 [191]
65 E. confusa 211, 217 [192]
66 E. flava 1, 25, 172, 176, 289 [40, 55]
67 E. stricta 174 [191]
68 Eulophia herbacea 164 [193]
69 E. macrobulbon 1, 16, 71, 172, 180, 188, 212, 229 [56, 194, 195]
70 E. nuda 1, 21, 22, 68, 82, 174, 176, 188, 189, [57, 109, 191, 196]
212, 356
71 E. ochreata 16, 170 [197]
72 E. petersii 1, 5, 21, 167, 188 [58]
73 Flickingeria 1, 5, 8, 23–25, 30, 34, 170, 174, 211, [39, 150]
fimbrata 212, 266, 273
74 Gavilea lutea 9 [198]
75 Gomesa recurva 185 [199]
76 Gymnadenia 1, 9, 75, 90, 91, 226, 289, 297 [59, 60]
conopsea
77 Liparis nakaharai 205 [36]
78 L. nervosa 353–355, 367, 368, 374 [157]
79 L. regnieri 206, 207 [91]
80 Loroglossum 7, 8 [69]
hircinum
81 Luisia indivisa 5, 167 [79]
82 L. volucris 371 [200]
83 Maxillaria densa 24, 27, 174, 183, 214, 216 [201–203]
84 Monomeria 69, 81, 96, 106, 245, 288, 314, 323, [101, 110, 161]
barbata 346, 375
85 Nidema boothii 5, 25, 40, 212, 258 [80]
86 Odontioda Marie 269, 280 [152]
Noel “Velano”
87 Odontoglossum 190 [204]
harvengtense
“Tutu”
(continued)
Table 1 (continued)
88 Oncidium isthmi 264 [151]
89 Orchis militaris 4 [68, 69]
90 Otochilus fusca 157 [128]
91 O. porrectus 312 [159]
92 Pholidota 157, 159, 161 [126]
articulata
93 P. cantonensis 5, 155, 265 [81]
94 P. chinensis 1, 2, 4–6, 8, 9, 24, 33, 65, 108–110, [61, 62, 97, 115]
113, 124, 177, 296, 299, 303, 309–
311, 317
95 P. imbricata 219, 246 [124]
96 P. yunnanensis 105, 114, 115, 156, 298, 308 [100, 102, 125]
97 Pleione 1, 5, 88, 104, 111, 116–119, 125, [21, 25, 63, 64, 99, 116,
bulbocodioides 126, 128, 132, 140–144, 146–148, 118, 119, 121]
226, 294, 320, 365, 366
98 P. yunnanensis 1, 5, 11, 75, 81, 133–135, 226, 353 [111, 122
99 Scaphyglottis 1, 5, 176, 213 [82, 205]
livida
100 Spiranthes sinensis 70, 83–86, 89, 98, 99, 131, 137– [95, 112, 113, 156]
139, 149–153, 252, 257, 258, 304
101 S. sinensis var. 4, 87, 98, 99, 130, 154, 257, 305 [65, 94]
amoena
102 Sunipia scariosa 5, 157, 158 [83]
103 Thunia alba 5, 176, 212, 289, 353 [84]
104 Vanda coerulea 1, 17, 156, 157 [127]
2.1 Monophenanthrenes
Around 80% of the phenanthrenes reported from orchids are monophenanthrenes.

All of them are substituted, most frequently at C-2, C-5, and C-7, but substitution is
freasible at every other place. Substitution at positions C-9 and C-10 are quite rare.
Five dihydrophenanthrenes, substituted with hydroxy (56–60) group at C-9, were
isolated from Dendrobium denneanum [13]. Loddigesiinol A (200), identified from
Dendrobium loddigesii [14], 62 from D. fimbriatum [15], 67 from D. primulinum
[16], 55 from Cyrtopodium paniculatum [17], 202 from D. nobile [18], and
phoimbrtol (219) from Pholidota imbricata are also substituted with a hydroxy
group at C-9 [19]. Fimbriol A (214) from E. fimbriata [20], 190 from several orchids,
and 202 from D. nobile are substituted with a methoxy group at C-9 [18]. There are
some examples, e.g., bulbocodioidins A–D (116–119) [21], ochrolone (163) [22],
plicatol A (225) [23], reptanthrin (364) [24], bulbocodioidins E (365), and F (366) of
9,10-disubstituted phenanthrenes [25].
540 A. Vasas
Almost all of the monophenanthrenes are substituted with hydroxy group(s); those
O-glycosides [e.g., cremaphenanthrene M (236), 250, 360, and 361] that do not bear this
functional group are suggesting to be biogenetically also hydroxylated [5, 26–29]. The
hydroxy group is usually linked at C-2 and/or C-7. There are only few compounds (e.g.,
2, 62) substituted with only hydroxy groups [13, 15]. The methoxy group is the second
most common substituent, linking mainly at C-2, C-4, C-5, and C-6. Most of the
Orchidaceae phenanthrenes are methoxylated, e.g., calanphenanthrene A (45),
calanhydroquinones A–C (46–48), calanquinones A–C (268, 278, 279) [30, 31],
cephanthrenes A (49) and B (50) [32], marylaurencinols A–C (52–54) [33], and
ephemeranthoquinone C (281) [34], 42, 63, 64 [18, 35] and 205 [36], identified from
Calanthe arisanensis, Cephalantheropsis gracilis, Cymbidium Great Flower Marie
Laurencin, Dendrobium moniliforme, D. nobile, and Liparis nakaharai. Compounds
3, 171, and 179, isolated from Bletilla striata [37], the 9,10-dihydrophenanthrene
callosumin (18) and its phenanthrene pair callosuminin (187) from A. callosum [38], and
34 from Flickingeria fimbrata, substituted with only methoxy groups [39].
The most common monophenanthrenes are the 9,10-dihydrophenanthrene coelonin
(1) and its phenanthrene pair flavanthrinin (172) [40], hircinol (8), and its unsaturated
pair plicatol-B (moscatin, 166), lusianthridin (5) and lusianthrin (167), and erianthridin
(24) and nudol (174). Coelonin (1) was isolated from almost every investigated
Orchidaceae species [Appendicula reflexa [41], Arundina graminifolia [42],
B. formosana [43], B. striata [12, 37, 44], Bulbophyllum reptans [24],
B. odoratissimum [45], B. vaginatum [46], Coelogyne elata [47], C. ochracea [47],
Cremastra appendiculata [26, 48], Cymbidium aloifolium [49], C. finlaysonianum
[50], Cyrtopodium paniculatum [51], D. formosum [52], D. plicatile [53],
D. scabrilingue [54], Eria flava [55], Eulophia macrobulbon [56], E. nuda [57],
E. petersii [58], Flickingeria fimbrata [39], G. conopsea [59, 60], Pholidota chinensis
[61, 62], Pleione bulbocodioides [63, 64], and Spiranthes sinensis var. amonea [65],
and it is a common constituent of several dimers. Orchinol (4) is also a common 9,10-
dihydrophenanthrene identified from A. callosum [38, 66], A. graminifolia [67],
C. appendiculata [48], O. militaris [68, 69], Pholidota chinensis [61], and
S. sinensis var. amoena [65]. Lusianthridin (5) was isolated from several orchids
such as Arundia graminifolia [70], B. striata [44, 71], Cymbidium finlaysonianum
[50], Cyrtopodium paniculatum [51], D. brymerianum [72], D. densiflorum [73],
D. formosum [52], D. nobile [74, 75], D. palpebrae [76], D. plicatile [53, 77],
D. scabrilingue [54], D. venustum [78], E. petersii [58], Flickingeria fimbrata [39],
L. indivisa [79], N. boothii [80], P. cantonensis [81], P. bulbocodioides [63, 64],
S. livida [82], S. scariosa [83], and T. alba [84]. Plicatol-B (166) was identified not
only from Dendrobium species D. chrysanthum [85], D. plicatile [23], D. rotundatum
[86, 87], D. densiflorum [73], D. moscatum [87], and D. loddigesii [88], but also from
B. odoratissimum [45],and Coelogyne longipes [89].
In Orchidaceae phenanthrenes, mono- (glucose) or disaccharide units (evolved
from glucose, apiose, and rhamnose) can also be joined to the skeleton. Mono-
glucosides were isolated from Bletilla striata (11, 93–95, 249, 251), Cremastra
appendiculata (12, 13) [90], Cymbidium Great Flower Marie Laurencin (51) [33],
Dendrobium primulinum (67) [16], and Liparis regnieri (206, 207) [91]. Moreover,
disaccharide-substituted phenanthrenes, such as phenanthrene-glucoapioside (198),
glucorhamnoside (199) [13], and diglucosides (14, 221, 222, 248, 250, 360, 361),
were also reported from orchids (B. striata, C. appendiculata, Dendrobium
chrysanthum, and Dendrobium denneanum) [26, 90, 92, 93].
The presence of prenyl-substituted phenanthrenes is relatively uncommon in
nature. However, seven prenylated compounds, namely sinensols B (84), C (85)
and F (86), spirasineol A (87), sinensol G (spiranthol-A, 98), spiranthol-B (99), and
spiranthesol (305) were isolated from Spiranthes sinensis and S. sinensis var.
amonea, one of them (305) is a dimer substituted with two prenyl groups [65, 94,
95, 112, 113]. A formyl group when present occurs at C-8
(spiranthesphenanthrene D, 70), while carboxyl group at C-4 (252) in the case of
compounds isolated from Coelogyne ochracea and S. sinensis [95, 96].
Considering the stilbene origin of almost all of the phenanthrenes, it is axiomatical
that stilbene-substituted phenanthrenes also occur in orchids. Surprisingly, this type of
542 A. Vasas
phenanthrenes is quite rare in plants. Most of the compounds (n ¼ 13) were isolated
from orchids. In cases of phochinenins I (108), J (109), and K (110) [97], isolated from
Pholidota chinensis, lusianthridin (5) is connected with batatasin III (dihydrostilbene),
and in phochinenin L (113) [97] a dihydrophenanthrene, isolated previously from a
liverwort (Plagiochila spinulosa), is linked to the stilbene thunalbene [98]. Other
Orchidaceae phenanthrenes, e.g., bleformin C (107), phochinenin K (110),
bulbocodioidin H (111), bletilladin D (112), and phoyunnanins A (114) and B (115)
are also derived from 9,10-dihydrophenanthrenes, substituted with stilbenes [25, 43,
44, 97, 99, 100]. Contrary to these phenanthrenes, where stilbenes and phenanthrenes
are connected via C-C coupling, monbarbatain E (106) is joined to stilbostemin E
(a dihydrostilbene moiety) through an O atom [101]. Similar compounds [shancilin
(104), and phoyunnanin (105)] were isolated from Pleione bulbocodioides and
Pholidota yunnanensis [63, 102]. Interestingly, up to now, only stilbene-substituted
mono-9,10-dihydrophenanthrenes were identified from orchids; stilbene-substituted
mono-, di-, and tri-9,10-dehydrophenanthrenes are not published in the literature.
Hydroxybenzyl-substituted monophenantheres are quite usual in Orchidaceae

species. Such compounds were isolated from the members of Arundia (83, 88)
[67, 70], Bletilla [e.g., 72–79, 82, 88, 241–244, bleochrin G (245)] [12, 43, 44, 71,
103–107, 170], Cremastra (e.g., 92) [108], Cymbidium (80) [50], Eulophia (82)
[109], Gymnadena [75, gymconopins A (90) and B (91)] [59, 60], Monomeria
(245) [110], Pleione (78, 81, 88) [63, 111], and Spiranthes [sinensols A (83),
B (84), C (85), F (86), spirasineol-A (87), and sinensol H (89)] genera
[65, 112, 113].
Another group of monophenanthrenes in orchids involves phenanthrofurans and

phenanthropyrans. Furan and pyran rings are generally linked to the phenanthrene
skeleton at C-1–C-2 (120–127, 129, 140–146) [17, 44, 90, 99, 114–121], but other
connections [e.g., C-2–C-3 (131–134) [17, 112, 122], C-5–C-6 (136) [35, 123], C-6–
C-7 (135, 148, 149) [112, 119, 120, 122], and C-7–C-8 (138, 139, 150–153)] also
544 A. Vasas
occur [95]. Interestingly, all of the furan ring-containing derivatives are

dihydrophenanthrenes, while loddigesiinol B (246) is the only phenanthropyran,
which has an unsaturated phenanthrene skeleton [19, 22]. Phenanthrenelactones
were reported from Dendrobium crystallinum (247) [124].
In case of 155–163, a ring closure is performed between C-4 and C-5 forming a
five- (155) or six-membered (156–162) oxygen-heterocyclic ring or a cyclohexane
ring (163). These compounds were isolated from Pholidota [81, 125, 126], Vanda
[127], Coelogyne [22, 89, 128–130], Otochilus [128], Sunipia [83], and Dendrobium
species [131]. Some monophenanthrenes have additional different substituents, e.g.,
in case of cirrhopetalanthridin (102) and bobulretin B (103), and their 9,10-
unsaturated pairs cirrhopetalin (223) and bobulretin A (224) a methylenedioxy
group is connected at C-2–C-3 [132–134]. Two spirolactone-substituted phenan-
threnes (253, 254) were isolated from Bletilla striata and D. chrysanthum [85, 135].
Phenanthroquinones are supposed to be the oxidative derivatives of phenan-

threnes or dihydrophenanthrenes; however, the detailed biosynthetic pathway of
these components needs further investigations. Phenanthrenequinones and
alkylated 1,4-phenanthrenequinones can be formed from the analogous oxygen-
ated phenanthrenes and abietane-type diterpenoids, respectively [5, 136, 137]. In
the past two decades, several phenanthroquinones were identified from orchids
belonging to different genera, e.g., Arundia (258, 272, 275) [42, 67], Bletilla (258)
[43, 138, 139], Bulbophyllum (272) [45], Calanthe (268, 278, 279) [30, 31],
Coelogyne (256, 271) [140], Cymbidium (276, 277, 269, 281) [33, 34, 50, 141,
142], Cyrtopodium (258, 267) [51], Cypripedin (272, 274) [143], Dendrobium
(258–262, 270, 272, 282) [52, 74, 77, 144–148], Eulophia (258) [149],
Flickingeria (266, 273) [150], Nidema (258) [80], Oncidium (264) [151],
Odontioda (280) [152], Pholidota (265) [81], and Spiranthes (257, 258)
[94, 95]. These compounds were found to be diversely substituted – hydroxy,
methyl, and methoxy groups being the most frequent moieties attached to the main
skeleton.
546 A. Vasas
Although phenanthrene-1,4-quinones are the most common phenanthrene-

quinones, the carbonyl group can also be attached at C-2, C-3, C-5, and/or C-8
(e.g., 282–287) [138, 139, 148, 153]. The saturation level of phenanthrenequinones
can vary depending on the type of the phenanthrene unit.
Interestingly, the most common 9,10-dihydrophenanthrenes have their own phen-
anthrene pairs, e.g., coelonin (1) and flavanthrinin (172), orchinol (4) and
dehydroorchinol (169), lusianthridin (5) and lusianthrin (167), hircinol (8) and
plicatol-B/moscatin (166), 3 and 171, and loroglossol (7) and 168.
2.2 Di- and Triphenanthrenes
More than 85 dimers and 2 trimers have been described from orchids. Mono-
phenanthrenes can connect through their functional groups or directly via C–C
coupling to form di- or triphenanthrenes. The monomers can be linked together at
many different positions (1-1’, 1-3’, 1-6’, 1-8’, 2-7’, 3-6’, 6-6’, 8-6’, and 8-8’);
usually, C-1 is involved in the connection. The linking monomers have usually been
isolated previously from the same plant, or the same genus. Di- and triphenanthrenes
are even more rare than monophenanthrenes.
548 A. Vasas
Most dimeric compounds isolated from orchids are supposed to be formed by the
coupling of prevalent monomeric units, e.g., lusianthridin (288, 298, 303, 346)
[99, 100, 115, 154], coelonin (289, 294, 295, 297, 300) [43, 51, 67, 107], flavanthrinin
(353) [43, 84, 155], orchinol (304) [95, 156], nudol (368) [157], 2,4,7-trimethoxy-
9,10-dihydrophenanthrene (291) [158], and 2-hydroxy-4,7-dimethoxyphenathrene
(355) [153, 157]. Interestingly, a prenylated dimer [spiranthesol (305)] is also identi-
fied from Spiranthes sinesis which is composed of two 3-6’ connected sinensol G (98)
monomers [94]. Among the identified compounds, symmetrical dimers also occur,
e.g., in case of 291, two 2,4,7-trimethoxy-9,10-dihydrophenanthrene (3) monomers
are attached through C-1 and C-1’ [158].
The C–C coupling of two flavidin monomers (157) resulted in 8,8’-biflavidin

(312) [159]. Different monomers, 9,10-dihydro and -dehydro derivatives, can also
be connected producing a great variety of dimers. In blestriarene A (290), two
9,10-dihydro derivatives, namely coelonin (1) and 6-methoxy coelonin (17), are
joining through their 1-1’ [160], while in blestriarene B (313) a moscatin (166) unit
connects to coelonin (1) [104]. In bleformin I (315), a nudol (174) and an
erianthridin (24), and in coeludol A (316), a coelonin (1) and a nudol (174) are
connected [43, 51].
550 A. Vasas
The coupling of lusianthridin (5) with lusianthrin (167) resulted in the formation
of monbarbatains B (314) and C (323), differing only in the positions of the linkages
[5, 161]. In bulbophythrin B (301), a lusianthridin (5) and a cannabidihyrophe-
nanthrene (6) monomer are connecting, through their C-1 and C-7’ [162]. In many
cases, 173 is one of the members of dimers, e.g., cremaphenanthrene C (321) [163],
318, 319, 322, 332 [164], 335 [154], and 354 [153]. In case of liparisphenanthrene C
(374), two 173 monomers are joining through an ether bridge [157]. Similarly, in
blestrins A (306) and B (307), the two monomers [two coelonin (1) in case of A, and
a coelonin (1) and a lusianthridin (5) in case of B] connect forming an ether bridge
[165]. Phoyunnanin E (308), phochinenis C–E (309–311), and blestrins C (333) and
D (334) are also dimers, in which the two monomers are connecting through a
hydroxy group, resulted in an ether bridge [115, 166, 167]. Dendropalpebrone (338),
isolated from D. palpebrae, is the only dimer containing a phenanthrene and a
phenanthroquinone moiety [76].
Until now, compounds 360 and 361, isolated from C. appendiculata, are the only
glycosylated phenanthrene dimers that have been reported from nature [26]. Presum-
ably, these diphenanthrenes are formed by the coupling of a flavanthrinin (172) and a
flavanthrinin-diglucoside. Several diphenanthrenes are formed by the coupling of
2,5-dihydroxy-6-methoxy-9,10-dihydrophenanthrene, identified from a liverwort
species Plagiochila spinulosa with other, more common monomers, e.g.,
lusianthridin (296, 310) and coelonin (299, 309), described from orchids [115]. In
case of phochinenin B (317), the same liverwort dihydrophenanthrene connects with
its previously unknown unsaturated form [115]. Compound 292, a constituent of
D. nobile [18], is a homodimer of two 2-hydroxy-3,4,7-trimethoxy-9,10-
dihydrophenanthrene, identified previously from P. spinulosa [98].
Up to now, only two triphenanthrenes are described from orchids; monbarbatain
D (375) is most likely derived from the coupling of three 9,10-dihydrophenanthrene
lusianthridin (5) units through C-1–C-8–C-6 [161], while in 376, three phenanthrene
[two flavanthrinin (172) and a 2-hydroxy-4,7-dimethoxyphenanthrene (173)] mono-
mers are connected [153].
2.3 Occurrence of phenanthrenes in Orchidaceae
3 Chemotaxonomical Significance
Orchidaceae phenanthrenes have chemotaxonomical significance, as prenyl-,

p-hydroxybenzyl-, and stilbene-substituted phenanthrenes were isolated almost
exclusively from orchids. Prenyl-substituted phenanthrenes were isolated only
from Spiranthes sinensis, and these compounds are substituted also with
552 A. Vasas
p-hydroxybenzyl group(s). Stilbene-substituted phenanthrenes were identified from

Pleione, Pholidota, Monomeria, Bletilla, and Cremastra species. Either prenyl- or
stilbene-substituted phenanthrenes belong to the 9,10-dihydrophenanthrene group,
and with the exception of spiranthesol (305), all of them are monophenanthrenes.
p-Hydroxybenzyl-substituted phenanthrenes were isolated from species belonging
to the genera Arundia, Bletilla, Eulophia, Cremastra, Cymbidium, Dendrobium,
Gymnadena, Pleione, and Spiranthes. Moreover, almost all of the Orchidaceae
phenanthrenes are substituted with hydroxy and/or methoxy groups at C-2, C-4,
and C-7.
The phenanthrene-containing species belong to the subfamilies Cypripedioideae,
Epidendroideae, Higher Epidendorideae, and Orchidoideae. Among them,
Epidendroideae is the best source of these compounds; members of 23 genera
(e.g., Arundia, Bletilla, Cremastra, Coelogyne, Pleione, Pholidota, Bulbophyllum,
Monomeria, and Dendrobium) have been reported to contain phenanthrenes. Several
genera (n ¼ 11) of the subfamily Higher Epidendroideae, especially members of
subtribe Cyrtopodiinae (e.g., Eulophia, Cyrtopodium, and Cymbidium), contain
phenanthrenes, and their structure is very close to that isolated from Epidendroideae
species. Spiranthes and Gymnadenia species belonging to the subfamily
Orchidoideae contain p-hydroxybenzyl- and/or prenyl-substituted compounds. Cyp-
ripedium tibeticum belonging to the subfamily Cypripedioideae is the only member
of this subfamily from which phenanthrenes (272, 274) have been reported [143].
Bletilla, Cremastra, Pleione, and Pholidota genera belonging to the subfamily
Epidendroideae contain the widest variety of phenanthrenes (n ¼ 80, 39, 33, and
36, respectively). From the members (Bulbophyllum and Cirrhopetalum species) of
the subtribe Bulbophyllinae (tribe Podochileae), methylenedioxy-substituted phen-
anthrenes (102, 103, 223, 224) were isolated. Dendrobium and Flickingeria species
belonging to the subtribe Dendrobiinae, tribe Podochileae, are rich in
phenanthroquinones.
4 Pharmacological Activities of Orchidaceae Phenanthrenes
4.1 Traditional Uses of Orchids
Several phenanthrene-containing plants are traditionally applied for treating differ-

ent diseases mainly in Asia, Africa, and South America, e.g., the tubers of
C. appendiculata are one of the main constituents of a popular traditional Chinese
medicine (TCM) product, “Shan-Ci-Gu,” which has been used for the treatment of
different types of cancer in Asia [26]. Pseudobulbs of B. striata and M. barbata are
used to cure pulmonary disorders in China [41, 110, 164]. The tubers of Bletilla
yunnanensis have long been used in TCM (“Baiji”) in southwest China to treat
tuberculosis, pneumonorrhagia, and pneumonophthisis [158]. A traditional medi-
cine, namely “Xiao Huang Cao Shi-Hu,” is prepared from the stems of different
Dendrobium species, e.g., D. aphyllum, D. chrysotoxum, D. denneanum, D. nobile,
and D. officinale, and applied to alleviate stomach disorders and fever [13, 14, 114,
148]. P. chinensis has been used in TCM for the treatment of high blood pressure and
headache [115]. S. sinensis is also a well-known member of TCM, and it is applied to
cure inflammatory diseases and different types of cancer [156]. The whole plant or
the pseudobulbs of Pholidota yunnanensis are applied to alleviate cough, trauma,
and stomachache [125]. Eulophia species are used in different parts of India for their
anticancer, immunomodulatory, aphrodisiac, and anthelminthic activities [196]. In
Indian folk medicine, Eulophia ochreata is used as an antiphlogistic agent. It also
used for its rejuvenating and aphrodisiac properties, and tuber sap is applied
externally for curing rheumatism [197].
4.2 Biological Activities of Orchidaceae Phenanthrenes
The pharmacological activities (e.g., antimicrobial, antioxidant, anti-inflammatory,

cytotoxic, and neuroprotective effects) of almost all newly isolated compounds have
been investigated, and several compounds showed multiple bioactivities. Based on
the accumulated data, denbinobin (270) can be considered one of the most promising
phenanthrene, and because of its novel mechanisms of action it could be a potential
anticancer lead compound for further development.
4.2.1 Antiproliferative Activity

Many Orchidaceae phenanthrenes showed significant cytotoxic activities against
different cancer cell lines. Among them, only compounds with IC50 values lower
than 10 μM are presented in this section. Ddenbinobin (270) has shown noteworthy
activities in different test systems. Denbinobin (270) causes cytotoxicity in several
different mechanisms of actions, involving 1) apoptosis-induction through caspase-
dependent and -independent ways [206], and by the activation of proapoptotic Bax
protein, and inactivation of the antiapoptotic Bcl-2 [207]; 2) blocking of NF-κB
activation [208]; 3) enhancing the synthesis of reactive oxygen species (ROS) [208];
4) downregulation of DcR3 which sensitized the cells toward programmed cell death
[209, 210]; and 5) antimigratory effects through reducing Src kinase activity,
inhibition of calcium-binding cell migration protein (S100A8), and by down-
regulation of matrix MMP-2 and MMP-9 metalloproteinases [207]. A structure-
activity relationship (SAR) study revealed that the quinone substruction present in
this compound is essential for the antiproliferative effect [208].
The D. nobile metabolites lusianthridin (5) and denbinobin (270) exerted cyto-
toxic effects against A549 human lung carcinoma [ED50: 7.7 μM (5); 1.3 μM (270)],
SK-OV-3 human ovary adenocarcinoma [ED50: 9.4 μM (5); 3.5 μM (270)], and
HL-60 human promyelocytic leukemia [ED50: 9.8 μM (5); 0.11 μM (270)] cell lines.
Denbinobin (270) proved to be more effective than lusianthridin (5). The methylated
derivatives of these compounds did not exert cytotoxic effect; therefore, it can be
concluded that the presence of a free phenolic hydroxy group in the molecule is
essential for the inhibitory activity. Moreover, lusianthridin (5) possessed antitumor
activity in ICR mice, implanted intraperitoneally with sarcoma 180 at a dose of
20 mg/kg [74]. In another investigation, lusianthridin (5) suppressed cancer stem
554 A. Vasas
cells in lung cancer cells through downregulation of Src-STAT3-c-Myc pathways.

The compound inhibited the formation of tumor spheres of primary lung cancer
cells. Moreover, after cancer stem cell attenuation by 5, the lung cancer cells
exhibited significantly higher susceptibility to chemotherapeutic drugs.
Lusianthridin (5) strongly suppressed CSC phenotypes of H460 and H292 cells [78].
Two dimeric phenanthrenes [denthyrsinol (369) and denthyrsinone (372)] and the
monophenanthrene denthirsinin (212), isolated from D. thyrsiflorum, showed sig-
nificant cytotoxicity against HeLa, K-562 and MCF-7 cells with IC50 values 9.3 μM
(HeLa), 1.6 μM (K-562) for denthyrsinol (369), 9.9 μM (HeLa), 6.0 μM (K-562),
and 3.5 μM (MCF-7) for denthyrsinone (372), and 2.7 μM (HeLa), 2.3 μM (K-562),
and 4.8 μM (MCF-7) for denthirsinin (212), respectively, using the MTT assay. SAR
investigations displayed that the dimerization of phenanthrenes is a very important
factor for the inhibition of cancer cell growth [188]. The D. chrysotoxum metabolite
4,9-dimethoxyphenanthrene-2,5-diol possessed cytotoxic activity against human
hepatoma (BEL-7402) (IC50 1.8 μM) and gastric cancer (SGC-7901) (IC50 2.9 μM)
cell lines [182]. Densiflorol B (272) was proved to be active against human leukemia
HL-60 (IC50 value 4.6 μM), human lung adenocarcinoma A549 (IC50 value 8.7 μM),
and human hepatoma BEL-7402 (IC50 value 7.2 μM) tumor cell lines. Cisplatin was
used as a positive control (IC50 values 0.7 μM on HL-60, 0.4 μM on A549, and 0.18
μM on BEL-7402, respectively) [45]. Compound 220, isolated from
B. odoratissimum, possessed cytotoxic activity against A549 and SGC-7901 tumor
cell lines with IC50 values 3.4 and 1.1 μM [176].
Calanquinone A (278) was proved to be cytotoxic against different [MCF-7
breast (IC50 0.03 μM), PC-3 prostate (IC50 0.16 μM), A549 lung (IC50 0.19 μM),
HCT-8 colon (IC50 0.20 μM), KB nasopharyngeal (IC50 0.32 μM), DU145 prostate
(IC50 0.34 μM), and KB-VIN vincristine-resistant nasopharyngeal (IC50 0.45 μM),
respectively] cancer cell lines. Moreover, it showed an improved drug resistance
profile compared to the positive control paclitaxel [31]. The phenanthrene dimers
bulbophythrins A (302) and B (301) possessed significant cytotoxic activity against
the HL-60 (IC50 1.3 and 4.0 nM), K562 (IC50 6.1 and 2.8 nM), SGC-7901 (IC50 6.1 nM
for 301), and A549 (IC50 9.2 and 1.2 nM) cell lines, respectively. These inhibition
values were comparable to that of the positive control cisplatin [IC50 2.3 nM (HL-60),
IC50 0.27 nM (K562), IC50 0.7 nM (SGC-7901), and IC50 1.5 nM (A549)] [162]. In a
WST-8 assay, ephemeranthoquinone B (269), a secondary metabolite of Cymbidium
Great Flower Marie Laurencin, possessed cytotoxic activity (IC50 2.8 μM) which was
comparable to that of the positive control mitomycin C (IC50 0.1 μM)
[33]. Monbarbatain B (314) inhibited the proliferation of HL-60 cells (IC50 7.3 μM),
and this activity was comparable with that of cisplatin (IC50 value 7.7 μM)
[161]. Cymbinodin A (276) possessed significant cytotoxic activity against
NCI-H187 cells with IC50 3.7 μM. Doxorubicin was used as a positive control
(IC50 0.24 μM) [50]. By using the MTT assay, ephemeranthoquinone B (269) and
5-hydroxy-2,3-dimethoxy-1,4-phenanthrenequinone (280), isolated from Odontioda
Marie Noel “Velano,” were found to possess specific cytotoxic effect against HL-60
cells (IC50 3.0 μM for 269, and 4.7 μM for 280). However, none of the compounds
caused apoptosis according to a DNA fragmentation assay [152]. A
phenanthrenequinone (264) of Oncidium isthmi inhibited the M14 (IC50 1.5 μM) and
NCI-H460 (IC50 5.0 μM) cell lines. Moreover, it induced apoptosis through caspase
3/7 activation and in an LDH release assay [151]. Compounds 177 and 193 signif-
icantly inhibited the effect of CDK1/cyclin B kinase (IC50s 0.07 and 0.2 μM,
respectively) [41].
Compound 82, isolated from B. striata, was proved to be cytotoxic against
HCT-15 cells (IC50 value 2.2 μM) using the SRB assay. Doxorubicin was used as
a positive control (IC50 value 0.02 μM) [106]. Cypripedin (259), a phenanthre-
nequinone from D. densiflourm, attenuated typical mesenchymal phenotypes,
including migratory behavior of nonsmall cell lung cancer H460 cells, with a
significant reduction of actin stress fibers and focal adhesion and with weakened
anchorage-independent growth. The negative activity of the compound on epithelial-
to-mesenchymal transition was due to ATP-dependent tyrosine kinase (Akt) inacti-
vation. The observation was also supported by a similar result on another lung cancer
H23 cell line, indicating that cypripedin (259) possesses a promising pharmacolog-
ical effect on lung cancer metastasis [183]. Coelonin (1), lusianthridin (5), 69, and
calanhydroquinone C (48) showed potent cytotoxic activity against MDA-MB231
cells with the IC50 values 5.4, 8,0, 7.7, and 2.0 μM, respectively. Erianthridin (24),
1, 5, 69, 170, and nudol (174) were effective against HepG2 cells with the IC50
values 8.4, 9.4, 8.7, 8.8, 6.1, and 4.2 μM, respectively. Finally, compounds 1, 5, 69,
48, and 174 proved to be active against A549 cells with the IC50 values 5.8, 8.6, 8.9,
4.9, and 9.1 μM, respectively [53].
Liparisphenanthrene A (367), 354, and 355 showed cytotoxic activity on
HGC-27 cell line (IC50 values 9.6, 8.2, and 10.0 μM, respectively) and on HT-29
cell line (IC50 values 9.3 μM for 367, and 8.5 μM for 355) [157]. P-configuration of
bulbocodioidin H (111) was cytotoxic against colon cancer (HCT-116), liver cancer
(HepG2), and breast cancer (MCF-7) cell lines with IC50 values of 7.6, 3.8, and 3.4
μM, respectively. Cyrtonesin A (132) showed cytotoxic activity on MCF-7 (IC50
value 5.4 μM). Taxol was used as a positive control (IC50 values 0.02 μM for
HCT-116, 0.03 μM for HepG2, and 0.01 μM for MCF-7) [25]. Bulbocodioidin A
(9R) (116) was cytotoxic against HepG2 (IC50 value 8.1 μM) and BGC-823 (IC50
value 8.4 μM), while bulbocodioidin D (10S) (119) against HCT-116 (IC50 value 8.3
μM), HepG2 (IC50 value 2.3 μM), and MCF-7 (IC50 value 2.5 μM) cells [21].
4.2.2 Antimicrobial Activity

The antimicrobial effect of numerous phenanthrenes was tested against various
(resistant and nonresistant) bacteria and fungi, and several of them inhibited the
growth of the microorganisms, which might be due to their phytoalexin peculiarities.
Orchinol (4) from O. militaris and hircinol (8) from L. hircinum are typical phyto-
alexins of orchids. Orchinol (4) inhibited completely the growth of Candida
lipolytica at 100 ppm for the first 6 days, whereas hircinol (8) or the control did so
for only 3 days [69].
The tubers of B. striata have been used in TCM for the treatment of
pneumonorrhagia and pneumonophthisis. Therefore, its constituents lusianthridin
(5) and 81 were tested against Staphylococcus aureus. Both compounds inhibited the
556 A. Vasas
growth of this gram-positive bacterium [MIC values 50 μg/mL (5) and 25 μg/mL
(81), respectively] [71]. The biphenanthrenes blestriarene A-C (289, 313, 353) were
effective against S. aureus and Streptococcus mutans. Blestriarene B (313) pos-
sessed the highest activity (MIC values 12.5 μg/mL and 6.25 μg/mL, respectively)
against both test organisms [160]. The antimicrobial activities of 318, 319, 322, 332,
353 (blestriarene C), and 356, isolated also from B. striata, were evaluated against
gram-positive and gram-negative bacterial strains using the microdilution method.
Compounds 318, 319, 322, 353, and 356 showed potent antibacterial activities
against methicillin-resistant S. aureus ATCC 43300 and ampicillin-resistant
S. aureus ATCC 29213, which were comparable in potency to the positive control
ampicillin. Among them, compound 318 possessed the highest inhibitory activities
against ampicillin-resistant S. aureus ATCC 29213 and methicillin-resistant
S. aureus ATCC 43300 (MIC values of 2 and 4 μg/mL, respectively)
[164]. Blestriarenes B (313) and C (353) were effective against S. aureus (MIC ¼
6.25 and 25 μg/mL, respectively) and Streptococcus epidermidis (MIC ¼ 25 μg/mL
for both) [158]. Coelonin (1) (26 μg/mL), flavanthridin (25) (53 μg/mL), shanciol C
(142) (26 μg/mL), shanciol F (143) (52 μg/mL), shanciol D (148) (52 μg/mL), and
blestriarene A (290) (6 μg/mL) from B. striata showed potent antibacterial activity,
with MICs of 6–52 μg/mL against S. aureus ATCC 6538 [120].
Ephemeranthoquinone B (269), the metabolite of Cymbidium Great Flower Marie
Laurencin, was active against B. subtilis (MIC ¼ 4.9 μg/mL) [33]. Another phen-
anthrene of the plant, marylaurencinol C (54), had an antimycotic activity against
Trichophyton rubrum (inhibition zone of 12.7 mm at 10 μg/disk) which was
comparable to that of ketoconazole (inhibition zone of 15.7 mm at 5 μg/disk)
[34]. Phenanthrenes (88, 272, and 290) from A. graminifolia were tested for anti-
bacterial and antihemolytic activities. Blestriarene A (290) and shancidin (88)
inhibited the growth of S. aureus and E. coli (MIC value 20 μg/mL for both), and
B. subtilis (MIC value 40 μg/mL), by breaking the integrity of membranes and the
cell wall. Meanwhile, 88, 290, and densiflorol B (272) showed antihemolytic activity
(50% inhibition rate at 16 μg/mL for 88, and 290, and 128 μg/mL for 272) on
erythrocytes hemolysis caused by bacteria [42].
4.2.3 Anti-inflammatory Activity

The inhibitory activity of compounds, isolated from B. formosana (72, 110, 295,
342), were tested on superoxide anion generation and elastase release by human
neutrophils in response to N-formyl-L-methionyl-L-leucyl-L-phenylalanine/cytocha-
lasin B (FMLP/CB) by using idelalisib (IC50 values of 0.1 μM in case of superoxide
anion generation, and 0.3 μM in case of elastase release, respectively) as a positive
control. The 3-p-hydroxybenzyl-substituted 9,10-dihydrophenanthene (72) exerted
the highest inhibitory activity against superoxide anion generation (IC50 value 0.2
μM). In case of 110, in which a stilbene unit (batatasin III) and a 9,10-
dihydrophenanthene connect through C-1–C-60 , high inhibitory activity was
detected for elastase release (IC50 value of 0.6 μM). Among the phenanthene dimers,
295, evolved by 1,30 connection of two 9,10-dihydrophenanthene units, one of them
substituted with a 10 -p-hydroxybenzyl group possessed the most potent inhibitory
activity against superoxide anion generation (IC50 value 0.2 μM). Compound 342
showed significant inhibitory activity against elastase release (IC50 value 0.3 μM). In
this compound, two 9,10-phenanthenes, containing methoxy groups at C-6, are
connected through their C-1 [43].
Compounds 59 and 195, isolated from D. denneanum, significantly inhibited the
phosphorylation of IκBα, which prevents the degradation of IκBα and subsequent
NF-κB activation. Therefore, these compounds may decrease the expression of
proinflammatory cytokines by inhibiting the activation of NF-κB [13]. Compound
16, isolated from Eulophia ochreata, inhibited the production of inflammatory
cytokine TNF-α from LPS-stimulated THP-1, RAW264.7 cells and PBMCs. More-
over, it blocked TLR4-induced signaling via the NF-kB pathway by inhibiting
MyD88-mediated signaling mechanisms. Treatment with 16 inhibited the expression
of IL-8 and IL-β in a dose-dependent manner [211].
The secondary metabolites of D. loddigesii, loddigesiinols A (200) and B (246),
moscatin (166), 5-hydroxy-2,4-dimethoxyphenanthrene (168), and lusianthridin (5),
inhibited the LPS-induced NO production in RAW 264.7 macrophage cells (IC50
values 2.6 μM for 200, and 10.9 μM for 246, 6.4 μM for 166, 5.3 μM for 168, and 4.6
μM for 5), compared to the positive control aminoguanidine (IC50 17.5 μM), while
rotundatin (31) and hircinol (8) were less active [14]. In an experiment, coelonin
(1, IC50 10.2 μM), lusianthridin (5) (IC50 9.6 μM), hircinol (8, IC50 26.4 μM),
ephemeranthol A (23, IC50 12.0 μM), erianthridin (24, IC50 19.5 μM), flavanthridin
(25, IC50 34.1 μM), ephemeranthol C (26, IC50 17.6 μM),
5,7-dimethoxyphenanthrene-2,6-diol (176, IC50 35.7 μM), fimbriol B (184, IC50
28.9 μM), and 203 (IC50 20.4 μM), the constituents of D. nobile, showed anti-
inflammatory activity which was comparable to the positive control aminoguanidine
(IC50 17.5 μM) [185]. The inhibitory activity of ephemeranthol A (23) mediated
through NF-κB and MAPK pathways. At a concentration of 25 μg/mL, compounds
23 and 204 decreased the production of TNF-α, IL-1β, and IL-6 [187].
Compound 190 inhibited RANKL-induced formation of osteoclasts, and the
lipopolysaccharide (LPS)-stimulated NO production (IC50 24.1 μM) in RAW
264.7 cells. Moreover, due to its inhibitory effect on the iNOS protein expression,
compound 190 scavenged the NO radical production in micromolar concentrations
[204]. The LPS-induced NO-production of RAW 264.7 cells was also inhibited by a
metabolite of Pholidota yunnanensis, 1-(40 -hydroxybenzy)-imbricatin (156) (IC50
20.8 μM) [125]. Besides the COX-2 inhibitory activity of compound 156 and the
Vanda coerulea metabolite 6-methoxy-coelonin (17) (IC50 values 12.0 and 5.8 μM,
respectively), a dose-dependent suppression of the PGE2 production in HaCaT cells
(IC50 values of 12.2 and 19.3 μM, respectively) was also observed. Moreover, 17
decreased COX-2 expression induced by UVB irradiation [127].
Phenanthrenes of B. striata were investigated for their antineuroinflammatory
activity. The dimeric phenanthrene 352 showed the most potent anti-
neuroinflammatory activity, with an IC50 value of 1.0 μM, followed by bleformin
F (350, IC50 5.0 μM). The heterodimer phochinenin K (110) also showed potent
activity (IC50 1.9 μM). Minocycline was used as a positive control (IC50 27.2
μM) [44].
558 A. Vasas
Dendrochrysanene (254) concentration-dependently inhibited the production of

proinflammatory cytokines and iNOS. At 1.1 μg/mL concentration, the compound
partially suppressed the mRNA level of TNF-α and IL-8 from LPS-activated mac-
rophages, respectively, and at 11.2 μg/mL it significantly inhibited the amount of
TNF-α and IL-8 mRNAs. Moreover, it suppressed IL-10 and iNOS mRNAs at 0.11
and 1.1 μg/mL, and at 11.2 μg/mL 254 significantly inhibited IL-10 and iNOS at the
mRNA level [85].
The anti-inflammatory activity of 188 and denthyrsinin (212) was tested by
measuring the reduction of anti-inflammatory (IL-6, TNF-α) and proinflammatory
(IL-10) cytokines in the LPS-stimulated macrophage model in vitro. The IC50 values
in case of the reduction of IL-6 were 23 μM for 188, and 25 μM for 212. Cortisol
(IC50 value 0.5 μM) was used as a positive control. TNF-α secretion was reduced by
about 50% by incubating the cells with coelonin (1), and about 40% after treatment
of 212. Coelonin (1), 212, and the positive control dexamethason completely
inhibited iNOS expression [56]. Coelonin (1) showed potent anti-inflammatory
activity on LPS-stimulated NO production in BV-2 microglial cells, with IC50
value of 5.4 μM, which was stronger than the positive control quercetin (IC50
value 10.5 μM) [63].
Nishidono et al. investigated the effect of heat processing on the chemical
constituents and NO-suppressing activity of Bletilla tuber. The Chinese Pharmaco-
poeia describes the heat-processing methods used on raw tubers of B. striata to
produce the herbal medicine Bletilla tuber. The comparison of the chemical profiles
of the extracts indicated that the relative content of phenanthrenes had not changed.
The extracts of the tuber with (BT1) or without (BT2) heat processing showed
similar IC50 values (120 μg/mL for BT1 and 140 μg/mL for BT2) on NO production
suppressing activity in IL-1β-treated hepatocytes. Furthermore, besides bibenzyls,
phenanthrenes [coelonin (1), flavanthrinin (172), 75, and blestriarenes A (290) and
C (353)] were identified as the compounds, responsible for suppressing the NO
activity (30.8 μM for 1, and 2.2 μM for 75). The results suggested that the biological
activities, such as the anti-inflammatory and hemostatic effects of Bletilla tuber, are
not affected by heat processing [12].
The inhibitory effect of phoimbrtol (219, IC50 value 11.6 μM) and loddigesiinol
B (246, IC50 value 10.9 μM) on nitric oxide (NO) production was stronger than those
of the positive controls quercetin (17.5 μM) and resveratrol (22.0 μM) [124].
4.2.4 Antioxidant Activity

The D. draconis metabolite, compound 32, exerted the same activity (IC50 10.2 μM)
in the DPPH radical-scavenging assay as the positive control trolox (IC50 11.7 μM)
[145]. Loddigesiinol A (200, IC50 26.1 μM)), moscatin (166, IC50 59.8 μM), and
lusianthridin (5, IC50 62.2 μM) possessed antioxidant effect in the DPPH-scavenging
assay which was comparable with the positive control resveratrol (IC50 28.7 μM)
[14]. Confusarin (211), flavanthrinin (172), and compounds 190 and 170 were
investigated by the DPPH assay for their antioxidant activity using ascorbic acid
(IC50 18.0 μM) as a positive control. Among the investigated compounds, 211
displayed comparable activity (IC50 value of 12.9 μM) to that of ascorbic acid.
According to the SAR investigations, a more pronounced antioxidant activity could

be detected when hydroxy and methoxy groups are presented at ortho position [186].
In the DPPH radical-scavenging assay, compound 190 and moscatin 166 from
D. chrysotoxum revealed antioxidant activity (IC50 6.8 μM for 190, and 9.2 μM for
166) of similar potencies to ascorbic acid (IC50 8.3 μM) [182]. In the same test
system, monbarbatains A–D (346, 314, 323, 375) (IC50 values 15.1, 12.9, 9.4, and
4.8 μM, respectively), 288 (IC50 7.7 μM), and 81 (IC50 12.2 μM) possessed
antioxidant activity. BHA and rutin were used as positive controls (IC50 values 9.3
and 5.2 μM, respectively) [101, 161]. The phenanthrenes of Odontoglossum
Harvengtense “Tutu,” compound 190 and flavanthrinin (172), exerted NO scaveng-
ing activity (IC50 values 19 and 2 μM) [204]. Free radical-scavenging activity of
16 and 170 was determined by DPPH method (IC50 values 29.5 and 40.0 μM,
respectively). The IC50 value of the positive control curcumin was determined as
45.0 μM [197].
Several dihydrophenanthrenes from Pholidota chinensis [coelonin (1), 2,4,7-
trihydroxy-9,10-dihydrophenanthrene (2), lusianthridin (5), cannabidihydrophe-
nanthrene (6), hircinol (8), isohircinol (9), erianthridin (24), and eulophiol (65)]
possessed antioxidant activity, with IC50 values of 16.7 (1), 16.2 μM (2), 22.3 (5),
24.7 (6), 28.9 (8), 21.4 (9), 14.9 (24), and 27.7 (65), respectively, in the DPPH
radical-scavenging assay [62]. Among the constituents of another Pholidota species
(P. yunnanensis), imbricatin (156) was the most active (EC50 8.8 μM) in the DPPH
radical-scavenging assay. The EC50 values of phoyunnanin C (298, 26.7 μM), 288
(15.6 μM), lusianthridin (5, 22.3 μM), eulophiol (65, 27.7 μM), and 2 (10.0 μM)
were comparable to that of the positive control resveratrol (21.2 μM). In case of
phoyunnanins A (114) and B (115), lower activities (EC50 values of 55.9 and 47.3
μM, respectively) were detected [100].
In vitro free radical-scavenging assays (DPPH, ·OH) were used to confirm the
antioxidant effect of coelonin (1), methoxycoelonin (17), imbricatin (156), and
flavidin (157) (DPPH assay: IC50 values of 8.5 (1), 9.0 (17), 8.4 (156), and 6.6
(157) μM; ·OH assay: IC50 values of 0.03 (1), 0.04 (17), 0.08 (156), and 0.08 (157)
μM, respectively). Furthermore, compounds 17 and 156 neutralized dose-
dependently (IC50 values of 9.4 and 8.8 μM, respectively) the ROS production in
H2O2-induced HaCaT cells [127]. Coelonin (1) and ochinol (4) showed DPPH and
ABTS radical-scavenging activities (EC50s 10.8 μM for both on DPPH, and 7.0 and
5.0 μM on ABTS). Blestriarene A (290) was the most effective in radical-scavenging
measurement (IC50 7.7 μM for DPPH and 4.1 μM for ABTS). Gallic acid and
ascorbic acid served as positive controls for antioxidant investigations (IC50 5.8
and 22.5 μM for DPPH, and 2.4 and 32.5 μM for ABTS) [48]. The DPPH radical-
scavenging activity (IC50 value of 18.1 μM) of phoimbrtol (219), isolated from
P. imbricata, was comparable to that of resveratrol (IC50 ¼ 24.5 μM), and quercetin
(IC50 ¼ 21.7 μM) [124].
4.2.5 Other Activities

In case of confusarin (211)-treated (10 μM) PC12 cells, an increase of the neurite
outgrowth (7.1%) was detected, after 72 h of incubation, proving the neuroprotective
560 A. Vasas
activity of the compound. NGF was used as a positive control (11.9% at

50 ng/mL) [114].
Coelonin (1, IC50 13.4 μM), compound 202 (IC50 9.0 μM), fimbriol B (184, IC50
11.0 μM), and denbinobin (270, IC50 15.2 μM), yielded from D. nobile, showed
inhibitory activity against hepatic stellate (HSC-T6) cell line, which was comparable
to that of (–)-epigallocatechin-3-gallate (IC50 9.9 μM) [18]. Compounds 184, 202,
and 270 decreased selectively, and dose- and time-dependently the viability of
HSC-T6 cells. At a 10 μM concentration, apoptotic cell transformations, generated
by the compounds, were observed, while at a concentration of 30 μM, necrotic
changes of the cells were performed. Moreover, fimbriol B (184) and 202 caused
caspase-dependent apoptosis in the caspase-3/7 activity assay [212].
Oxoflavidin (161) displayed remarkable osteogenic activity in the osteoblast ALP
assay on mice calvarial osteoblasts at concentrations of 1 pM, 100 pM, and 1 nM.
Cell mineralization was the most pronounced after using 100 pM of 161. At the same
concentration, applying real-time quantitative PCR measurements, compound 161
enhanced the transcription of osteogenic proteins (e.g., BMP-2, OCN) after 24 and
48 h. This observation supported the folk medicinal use of Pholidota articulata in
bone healing [126]. Coelogin (160) treatment to calvarial osteoblasts led to enhanced
ALP activity (a marker of osteoblast differentiation) and increased calcium nodule
formation compared to control untreated cells. Moreover, coelogin (160) also
increased the transcript levels of osteogenic gene markers such as BMP-2, Type I
Col, and RUNX-2 [130].
Compound 17 was shown to possess vasorelaxant activity on rat aorta rings
precontracted by phenylephrine (an α1 receptor agonist), the CaV1.2 stimulator
(S)-()-Bay K 8644, or depolarized with high K+ concentrations. Compound 181
was active solely on rings stimulated by 25 mM K+. The spasmolytic activity of 17
was significantly affected by the presence of an intact endothelium. The KATP
channel blocker glibenclamide and the KV channel blocker 4-aminopyridine signif-
icantly antagonized the vasorelaxant activity of 17. In patch-clamp experiments, 17
inhibited Ba2+ current through CaV1.2 channels in a concentration-dependent man-
ner, whereas it did not affect K+ currents through KATP and KV channels [173]. The
antihypertensive activity of EtOH extract of E. macrobulbon and its constituent 229
was investigated on pulmonary arteries. It is in accordance to specific expression of
PDE5 in pulmonary vasculature. Pulmonary arterial hypertension is improved by the
extract acting through pulmonary artery relaxation mediated through endothelial
NO, reduced Ca2+-mobilization, and reduced pulmonary artery wall thickness and
right ventricular hypertrophy [194]. In another investigation, compound 229 pos-
sessed potent PDE5 and PDE6 inhibitory activities (IC50 ¼ 1.7 and 1.8 μM,
respectively) evaluated by the [3H]cGMP radioassay method. Sildenafil was used
as a positive control (IC50 values 0.03 and 0.05 μM, respectively) [195].
Selective BChE (butyrylcholinesterase) inhibitory effects were observed for
coelonin (1, IC50 value 19.7 μM), orchinol (4, IC50 value 32.8 μM), blestriarene A
(290, IC50 value 88.5 μM), and dehydroorchinol (169, IC50 value 37.8 μM). The
most potent anti-BChE activity was exhibited by coelonin (1), which was compara-
ble to that of galantamine (IC50 ¼ 13.2 μM). Dimerization of monomeric 9,10-
dihydrophenanthrene significantly decreased the BChE activity [48]. Ochinol (4)

and blestriarene A (290) inhibited the β-amyloid peptide aggregation (64.5 and
29.5% at 20 μM, respectively), suggesting that they could serve as potential agents
for Alzheimer’s disease drug developments [48].
Compound 261 showed significant α-glucosidase and pancreatic lipase inhibitory
effects with IC50 values of 126.9 and 69.5 μM, respectively. A kinetic study, conducted
by the Lineweaver-Burk plot method, revealed that 32 was a noncompetitive inhibitor of
α-glucosidase and pancreatic lipase enzymes. Moreover, lusianthridin (5) showed
glucose uptake stimulatory effect without toxicity on L6 myotubes at concentrations
of 1 and 10 μg/mL [52].
5 Conclusions
Phenanthrenes are formed a group of natural products. To date, approx. 500 phen-
anthrene-type plant secondary metabolites have been identified from various
sources. Phenanthrenes have received much attention in the literature over the past
two decades, and a variety of potential beneficial effects have been elucidated.
Discovery of new phenanthrene derivatives, either from natural sources or by
synthetic routes, with promising pharmacological activities is a great challenge to
many research groups worldwide.
The occurrence of phenanthrenes is not restricted to one organ, they are found in
different plant parts (stem, rhizome, root, tuber bulb, and leaf); mainly, the whole
plants were investigated. Whereas the biosynthetic pathways of hircinol (8) and
orchinol (4) have been very well characterized [8], those of other phenanthrene
derivatives are unknown.
It can be concluded that orchids are the most abundant sources of such com-
pounds. Previously, more than 370 phenanthrenes have been isolated from approx-
imately 100 species of 40 genera of the Orchidaceae. Bletilla, Bulbophyllum,
Cremastra, Cymbidium, Cyrtopodium, Dendrobium, Pholidota, Pleione, and
Spiranthes species are the best sources of phenanthrenes. These species are belong-
ing to the subfamilies Epidendroideae, higher Epidendroideae, and Orchidoideae.
The most investigated genus in Orchidaceae is Dendrobium; 26 species of this genus
were studied for phenanthrene content, and 136 compounds were isolated from
them. However, Bletilla striata is the richest source of phenanthrenes as to date
80 compounds were isolated from this plant. The most common monophenanthrenes
are coelonin (1), lusianthridin (5), hircinol (8), erianthridin (24), plicatol-B
(moscatin, 166), lusianthrin (167), flavanthrinin (172), and nudol (174).
Because of their limited occurrence, phenanthrenes can be used as chemotaxo-
nomic markers. Besides the plant-specific enzymes requiring for the biosynthesis of
such compounds, certain phenanthrene substituents (e.g., hydroxybenzyl, prenyl,
vinyl, stilbene/dihydrostilbene, and mono-/disaccharide units) are also restricted to
specific families. Prenylated derivatives and stilbene/dihydrostilbene-connected
phenanthrenes have been identified solely from orchids, especially from the mem-
bers of the genus Pholidota. p-Hydroxybenzyl-substituted and glycosylated
562 A. Vasas
compounds seem to be also characteristic to orchids. These species are rich in

phenanthroquinones, too. To date, only two trimeric phenanthrenes are known
from natural source; both were reported from Orchidaceae species. It is also unique
in orchid phenanthrenes that, because of the close coexistence of liverwort species,
some of the dimers contain a monophenanthrene unit that has been isolated earlier
only from Plagiochila species.
The identified phenanthrenes have been studied in almost all cases for their
pharmacological effects, and many of them possessed diverse activities. In several
cases, detailed mechanism of action investigations has also been performed. The
accumulated data in case of denbionobin (270) implied that it could be a potential
anticancer lead compound. Compound 318 showed promising inhibitory activities
against ampicillin-resistant S. aureus and methicillin-resistant S. aureus (MIC values
2 and 4 μg/mL, respectively). Anti-inflammatory activity of lusianthridin (5),
moscatin (166), 168, and loddigesiinols A (200) and B (246) (IC50 values of 4.6,
6.4, 5.3, 2.6, and 10.9 μM, respectively), mediated through inhibition of
LPS-induced NO production, could also be promising for further investigations.
Compound 352 showed strong antineuroinflammatory activity (IC50 value of 1.0
μM). However, despite the promising in vitro results, in vivo effects of the phenan-
threnes are rarely investigated. Semisynthetic modifications or synthesis of the
compounds could allow to widening their biological investigations. Finally,
although orchids are used widespread in traditional medicines, the toxicity and
mutagenic potential of these species are seldom verified.
In conclusion, phytochemical investigation of taxonomically questionable or
rather controversial orchids, focusing on their phenanthrene content, can contribute
to clarify the taxonomic position of these species, and the great diversity of phen-
anthrenes provided by Orchidaceae species offers unique opportunities either for
medicinal chemists or pharmacologists to discover new classes of therapeutics.
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Orchids of Genus Bletilla: Traditional Uses,
Phytochemistry, Bioactivities, 23
and Commercial Importance
Hari Prasad Devkota, Rajan Logesh, Anjana Adhikari-Devkota, and

Mukti Ram Paudel
Contents
1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 574
2 Botanical Description, Distribution, and Ecology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 575
3 Traditional Uses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 576
4 Bioactive Compounds . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 576
5 Biological Activities . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 579
5.1 Anti-inflammatory Activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 580
5.2 Antioxidant Activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 580
5.3 Cytotoxic, Antitumor, and Anticancer Activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 581
5.4 Antimicrobial Activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 582
5.5 Hemostatic Activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 583
5.6 Immunological Activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 584
5.7 Anti-Fibrosis Activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 584
5.8 Antiviral Activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 584
5.9 Wound Healing Activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 584
5.10 Antiulcer Activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 585
5.11 Anti-neuroinflammatory Activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 585
5.12 Anti-Mitotic Activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 585
5.13 Anti-Tyrosinase Activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 585
H. P. Devkota (*) · A. Adhikari-Devkota

Graduate School of Pharmaceutical Sciences, Kumamoto University, Kumamoto, Japan
e-mail: devkotah@kumamoto-u.ac.jp; anjana@kumamoto-u.ac.jp
R. Logesh
TIFAC-CORE in Herbal Drugs, Department of Pharmacognosy and Phytopharmacy, JSS College
of Pharmacy (JSS Academy of Higher Education and Research), Udhagamandalam, Tamil Nadu,
India
M. R. Paudel
e-mail: muktiram.paudel@cdb.tu.edu.np

574 H. P. Devkota et al.
5.14 Anti-Ulcer Activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 586

6 Commercial Importance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 586
7 Conclusions and Future Remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 586
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 587
Abstract
The genus Bletilla comprises of six species, that is, B. chartacea, B. foliosa,
B. formosana, B. guizhouensis, B. ochracea, and B. striata, mainly distributed in
China, Japan, Taiwan, Vietnam, Thailand, and Myanmar. Few species of this
genus are used for medicinal purposes and they have great importance for their
ornamental values. Bletilla species are used traditionally for the treatment of
respiratory diseases and as hemostatic agents. Chemical analysis has revealed
the presence of various compounds such as phenanthrenes, bibenzyl derivatives,
terpenoids, and saponins, among others. Similarly, pharmacological activity and
evaluation of the extracts and isolated compounds have reported potent anti-
inflammatory, antioxidant, and hemostatic activities. However, extensive
harvesting in recent years has resulted in destruction of natural habitats and
needs urgent focus for preservation. This book chapter focuses on the traditional
uses, phytochemistry, and biological activities of Bletilla species along with their
conservation practices and commercialization potential.
Keywords
Bletilla · Bletilla striata · Orchids · Orchidaceae · Traditional uses ·
Phytochemistry · Conservation · Commercialization
1 Introduction
Orchidaceae is one of the largest families of flowering plants which consists of more
than 800 genera and 30,000 species [1]. Orchid flowers are well-known for their
beautiful color and fragrances and cultivated worldwide for ornamental purposes
[2, 3]. Many orchid species are also used in traditional medicines for the prevention
and treatment of various diseases and also studied widely for their interesting
bioactive chemical constituents and pharmacological activities along with their
potential in drug discovery and development and use in food, cosmetic, and other
industries [4–9].
The genus Bletilla consists of six species, that is, B. chartacea (King & Pantl.)
Tang & F.T.Wang, B. foliosa (King & Pantl.) Tang & F.T.Wang, B. formosana
(Hayata) Schltr., B. guizhouensis Jie Huang & G.Z.Chen, B. ochracea Schltr., and
Bletilla striata (Thunb.) Rchb.f. (http://www.plantsoftheworldonline.org/taxon/urn:
lsid:ipni.org:names:331150-2, retrieved on December 4, 2020). Species of Bletilla
are native to China, Japan, Korea, Myanmar, Taiwan, Thailand, and Vietnam. They
are widely cultivated for their ornamental values. Furthermore, B. formosana,
B. ochracea, and B. striata are reported to be used in various traditional medicine
23 Orchids of Genus Bletilla: Traditional Uses, Phytochemistry,. . . 575
Fig. 1 Photographs of Bletilla striata: (a) plant with flowers, (b) flowers close-up, (c) rhizomes, (d)
rhizomes close-up
systems in various respiratory disorders, swelling, hemorrhage, ulcer, and as hemo-

static agents. Among these six species, B. striata (Fig. 1) is one of the most widely
used species and is studied in detail for its traditional medicinal value, bioactive
chemical constituents, and pharmacological activities. However, extensive
harvesting in recent years has resulted in destruction of natural habitats and needs
urgent focus for preservation. This book chapter focuses on the traditional uses,
phytochemistry, and biological activities of Bletilla species along with their conser-
vation practices and commercialization potential.
2 Botanical Description, Distribution, and Ecology
Bletilla, commonly known as urn orchid, is a temperate and terrestrial genus

consisting of six species (B. chartacea, B. foliosa, B. formosana, B. guizhouensis,
B. ochracea, and B. striata) distributed through China, Japan, Taiwan, Vietnam,
Thailand, and Myanmar [10, 11]. They are small- to medium-sized herbs with
subterranean, tuberous, and irregular shaped rhizome containing many long and
fibrous roots. The erect stem bears a few plicate, narrowly oblong-lanceolate to
linear-lanceolate deciduous leaves sheathing at the base. The inflorescence is
terminal and loose with several flowers. The flowers are usually medium-sized, star-
shaped with white, pink, purple, or yellow colored tepals [8].
Bletilla species vary on their size and the habitat; however, most of them are
reported from tropical or temperate regions. B. formosana is a perennial herb found
in sunny locations from 1200 to 3000 m, and B. ochracea is found in open forest
with altitude of 900–2400 m. B. striata is the most commonly cultivated species that
usually grows on grassland at 1100–3200 m [8].
3 Traditional Uses
Mainly three species of genus Bletilla are being used in traditional/folk medicine
system of China, India, and other Asian countries. In China, the tubers of
B. formosana, B. ochracea, and B. striata are biological sources for the crude drug
“Bai-ji” used in the treatment of bleeding, burns, esophagus inflammation, gastritis,
and ulcers [12]. The details of the medicinal uses are provided below:
B. formosana: In China, the stems are known for their properties to strengthen the
lungs and to reduce swelling and bleeding. They are used for the treatment of cough,
tuberculosis, pneumonia, bronchiectasis, peptic ulcers, and bleeding from nose. The
scrapings of the stems are applied to treat cracks of the heel in India [8].
B. ochracea: It is reported to be used as substitute for B. striata in Southwest China
and used in treatment of pulmonary tuberculosis, pneumonia, and ulcers [12–14].
B. striata: B. striata is well known for its stringent hemostatic property [15]. Tubers
are the mainly used parts which are used for the treatment of swelling, bleeding from
cuts, burns and trauma, cough, tuberculosis, etc. [8]. B. striata is used locally for the
treatment of chapped skin, sores, ulcers, and wounds which helps in reducing the
swelling and promoting the regeneration of tissues [3]. It has hemostasis and lung-
supplementing effects, and is taken for hematemesis due to pulmonary tuberculosis
and gastric ulcer. Powder or grated material is applied to swellings, cuts, cracks, and
burns. It is also cultivated in the garden for ornamental purposes. Although Orchids
are very difficult to cultivate, it show vigorous growth even if they are left alone
[16]. It is also used in drinks, medicated diets, and also in wines for its beneficial
purposes. Other uses include as cosmetic, insecticide, and coating agent [17].
4 Bioactive Compounds
Orchids are well known for their various bioactive constituents, and they are mainly
derivatives of chemical classes such as hydroxyl-benzyl derivatives [18],
fluorenones and stilbenoids [19], and flavones C-glycosides [20], among others.
Few species of genus Bletilla have also been studied for their bioactive chemical
constituents, and B. striata is the most studied among these species for the isolation/
analysis of chemical constituents and biological activities. Studies so far have
reported dihydrophenanthrene derivatives, phenanthrene derivatives, bibenzyl
derivatives, triterpenoids, and saponins as major chemical constituents. The detailed

chemical components of these species are explained below:
Bletilla formosana: Lin et al. [21] performed the chemical analysis of the whole
plants of B. formosana. Three new dihydrophenanthrene derivatives named as
4-methoxy-9,10-dihydrophenanthrene-1,2,7-triol, 1-(4-hydroxybenzyl)-4,7-dimethoxy-
9,10-dihydrophenanthrene-2-ol, and 1,3,6-tri(4-hydroxylbenzyl)-4-methoxydihydrophe-
nanthrene-2,7-diol (Fig. 2) were isolated and identified along with seven already known
phenanthrene derivatives namely 1-(4-hydroxybenzyl)-4-methoxy9,10-dihydrophe-
nanthrene-2,7-diol, 1,6-di(4-hydroxylbenzyl)-4-methoxy-9,10-dihydrophenanthrene-2,7-
diol, 1,3-di(4-hydroxybenzyl)-4-methoxy-9,10-dihydrophenanthrene-2,7-diol, 1-(4-hydr
oxybenzyl)-4-methoxyphenanthrene-2,7-diol, 1-(4-hydroxybenzyl)-4,8-di-methoxyphe-
nanthrene-2,7-diol, 1,8-bi(4-hydroxybenzyl)-4-methoxyphenanthrene-2,7-diol, and
blestriarene B; a known bibenzyl derivative, 20 ,60 -bis( p-hydroxybenzyl)-3,30 -dihydroxy-
5- methoxybibenzyl; six known flavonoids, apigenin, kaempferol, 6-methoxykaempferol,
kaempferol 7-O-glucoside, 8-C-p-hydoxybenzylkaempferol and isorhamnetin; and three
simplephenolic compounds, 3,4-dihydroxybenzaldehyde, protocatechuic acid, and
p-(hydroxymethyl)phenyl-β-D-glucoside.
Fig. 2 Structures of some main compounds from B. formosana

Lin et al. [22] performed the chemical analysis of the rhizomes of B. formosana.
Total nine new phenanthrene derivatives named as bleformins A-I (Fig. 2), a new
bibenzylglucoside named as bleformin J were isolated and identified along with
45 known compounds including several phenanthrenes, bibenzyl derivatives and
other chemical classes.
B. ochracea: Cai et al. [13] isolated a new phenanthrene derivative, 2,7,-bis
(allyloxy)-5-methoxy-3-methyl-9,10-dihydrophenanthrene along with gastrodin,
gastrodigenin, and 4-hydroxybenzaldehyde (Fig. 3) and three steroids namely
β-sitosterol, stigmasterol, and daucosterol from the tubers of B. ochracea. Yang
et al. [23] isolated a new 2(2-methylpropyl)butanedioic acid derivative named as
bletillin A and a new bibenzyl derivative named as bletillin B (Fig. 3)
along with 17 known compounds such as gymnoside V, gymnoside VI, militarine,
erianthridine, nudol, 1,5,7-trimethoxyphenanthrene-2,6-diol, 4,7-dihydroxy-2-methoxy-
9,10-dihydrophenanthrene, 4,7-dihydroxy-1-p-hydroxybenzyl-2-methoxy-9,10-dihydro
phenanthrene, 2,7-dihydroxy-1-p-hydroxybenzyl-4-methoxy-9,10-dihydrophenanthrene,
blestriarene A, blestriarene B, blestriarene C, 3,30 -dihydroxy-2,6-bis(p-hydroxybenzyl)-
5-methoxybibenzyl, 20 ,60 -bis(p-hydroxybenzyl)-30 ,5-dimethoxy-3-hydroxybibenzyl, 3,
30 -dihydroxy-4-(p-hydroxybenzyl)-5-methoxybibenzyl, 3,30 -dihydroxy-2-(p-hydroxy
benzyl)-5-methoxybibenzyl, and 30 ,5-dihydroxy-2-(p-hydroxybenzyl)-3-methoxy
bibenzyl from the tubers. Some of the phenanthrene derivatives were reported to
show antibacterial activity against Gram-positive bacteria. Li et al. [12] isolated an
identified four new dihydrophenanthrenofuran derivatives named as bleochranols A-D
along with 10 known phenanthrenes and 11 known bibenzylderivatives from the
rhizomes. These compounds were also evaluated for their anti-inflammatory and
Fig. 3 Structures of some main compounds from B. ochracea

Fig. 4 Structures of some main compounds from B. striata
cytotoxicity activities. Li et al. [24] further isolated and characterized six new bibenzyl
derivatives named as bleochrins A-F and four new phenanthrene derivatives named as
bleochrins G-J along with 11 previously known compounds from the rhizomes.
Recently, few studies related to the extraction, characterization and bioactive evalua-
tion of polysaccharides from the tubers of B. ochracea are also reported [25].
B. striata: As mentioned before, B. striata is the most extensively studied species in
this genus regarding chemical constituents and biological activities. In 2017, He
et al. [15] have compiled the chemical constituents isolated and identified from
Bletilla striata, where they have tabulated a total of 125 compounds belonging to
different classes such as phenanthrene derivatives, biphenanthrene derivatives,
dihydrophenanthrene derivatives, bibenzyls, phenanthraquinones, terpenoids, and
saponins, among others. The details of these compounds can be found in the
mentioned review [15], and thus they are not discussed in detail here in this chapter.
Structures of some representative compounds, that is, 2,4,7-trimethoxyphe-
nenthrene, 2,3,4,7-tetramethoxyphenanthrene, batastatin III, and 3’-O-methylba-
tastatin III are given in Fig. 4. In recent years, studies have also focused on the
advanced techniques for the extraction, analysis, and characterization of polysac-
charides from the rhizomes along with their bioactivities [26, 27].
5 Biological Activities
Owing to their extensive use in traditional medicines, and presence of interesting

bioactive chemical constituents, Bletilla species are also studied widely for their
pharmacological activities using in vitro and in vivo models. Some of these studies
are discussed in detail in the following sections.
5.1 Anti-inflammatory Activity
Lin et al. have evaluated the anti-inflammatory activity of the chemical constituents
of the rhizomes of B. formosana. The 43 compounds were isolated from the
ethanolic extract and were evaluated for superoxide anion scavenging and elastase
inhibitory activities. The results revealed that most of the compounds showed potent
inhibitory activity against superoxide anion and elastase activity with an IC50 value
ranging from 0.2 0.1 and 0.4 0.1 μM to >10 μM, which were compared with the
CAL-101 used as a standard that possessed an IC50 value of 0.1 0.1 and 0.3 0.1,
respectively [22].
Li et al. carried out anti-inflammatory activity evaluation of the extracts of rhizomes
of Bletilla ochracea against murine monocytic RAW 264.7 cells and the results
showed that the ethanol fraction possessed an IC50 value of 45.85 2.21 μM, whereas
the compounds 3-(4-hydroxybenzyl)-4-methoxy-9,10-dihydrophenanthrene-2,7-diol,
4-methoxy-9,10-dihydrophenanthrene-2,7-diol and 4-methoxyphenanthrene-2,7-diol
showed most potent activity with an IC50 value of 8.17 0.64, 8.81 0.46, and
2.86 0.17 μM, respectively [12].
Jiang et al. have carried out anti-inflammatory activity using coelonin an active
compound isolated from the ethanol extract of the tubers of B. striata. The results
showed that the compound coelonin significantly inhibited lipopolysaccharide
(LPS)-induced interleukin-1β (IL-1β), interleukin-6 (IL-6), and tumor necrosis fac-
tor-α (TNF-α) expression at 2.5 μg/mL. The phosphorylation levels of the key
inflammatory regulators such as nuclear factor-kappa B (NF-κB) and cyclin-
dependent kinase inhibitor 1B (p27Kip1) were also significantly reduced [28].
Zu et al. carried out the pulmonary anti-inflammatory activity of the extracts from
B. striata in RAW264.7 cells using PM2.5. The pretreatment with the extract of
B. striata significantly decreased the inflammatory cytokines in the macrophage. The
extract also attenuated PM2.5-induced proinflammatory protein expression and
downregulated the levels of phosphorylated NF-κBp65, inhibitor of kappa B
(IκB)-α, c-Jun N-terminal kinase (JNK), extracellular signal-regulated kinase
(ERK), and p38 [29].
Wang et al. carried out the evaluation of anti-inflammatory activity against COX-1
and COX-2 for the compounds isolated from B. striata. The results showed that the
compounds, (1α,3α)-1-O-[(β-D-xylopyranosyl-(1!2)-α-L-rhamnopyranosyl)]-3-O-
glucopyranosyl-5α-spirostan, (1α,3α)-1-O-[(β-D-xylopyranosyl-(1!2)-α-Lrhamno-
pyranosyl)oxy]-3-O-D-glucopyranosyl-25(27)-ene-5αspirostan, (1α,3α)-1-O-[(β-D-
xylopyranosyl-(1!2)-α-L-rhamnopyranosyl)oxy]-epiruscogenin, (1α,3α)-1-O-[(β-D-
xylopyranosyl-(1!2)-α-Lrhamnopyranosyl)oxy]-epineoruscogenin, and 3-O-β-D-
glucopyranosyl-3-epi-neoruscogenin exhibited significant anti-inflammatory activity
with an IC50 value ranging from 35.5 to 96.4 μM [30].
5.2 Antioxidant Activity
Dong et al. carried out the DPPH, ABTS, and FRAP assay on the tubers of acetone
fractions on the fermented and nonfermented B. formosana. The results revealed that
the fermented B. formosana showed higher antioxidant activity on DPPH, ABTS,

and FRAP assays with IC50 values of 0.417, 43.57 4.72, and 120.1 0.2 μg/ml,
respectively [31].
Chen et al. studied the antioxidant activity of a new polysaccharide from the
fibrous roots of B. striata against DPPH, ABTS, and superoxide anion radicals. The
results showed that both the polysaccharide as well as B. striata extract showed
potent activity. The percentage scavenging activities for polysaccharide and B. striata
against DPPH, superoxide, and ABTS were 64.47% and 23.35%, 72.27% and
32.82%, and 29.49% and 18.90%, respectively [32].
Song et al. have carried out the antioxidant activity against DPPH, ABTS,
hydroxyl, superoxide anion, and reducing power using ethanol extract of B. striata
pure and ointment mixture. The results showed that both pure and ointment mixture
showed potent antioxidant activities [33].
Zhang et al. evaluated the antioxidant activity against ABTS, and FRAP assay
using BSP fraction. The results showed that B. striata could achieve 76% of ABTS
scavenging activity, and 46.2% of FRAP ability at 10 mg/ml concentration [34].
Qu et al. have carried out the antioxidant activity against superoxide anion,
hydroxyl, DPPH and chelation of ferrous ions using ethanol extract of B. striata.
The results showed that on treating with B. striata it possessed significant antioxi-
dant properties [27].
Jiang et al. have carried out the antioxidant activity against DPPH and FRAP
using fractions from ethanol extract of tubers of B. striata. The results showed that
ethanolic extracts from both FRP and PSP had strong free radical scavenging with
IC50 values of FRP (6.2 mg/L) which was slightly lower than the positive control
(2.4 mg/L) but was significantly higher than PSP (68.0 mg/L) activity,
respectively [35].
Wang et al. carried out the antioxidant activity of B. ochracea polysaccharides
(BOP) against DPPH, ABTS, hydroxyl, superoxide anion, and ferrous ions (Fe2+)
free radicals assay. The results showed that the BOP showed to inhibit with an EC50
value of 692.16, 224.09, 542.22, 600.53, and 515.70 μg/mL, respectively [25].
5.3 Cytotoxic, Antitumor, and Anticancer Activity
Li et al. have carried out cytotoxic activity against HL-60, SMMC-7721, A-549,
MCF-7, and SW-480 from the ethanol extract of rhizomes of Bletilla ochracea and
the results showed that the compounds bleochrin E, pleiobibenzynin A, pleiobi-
benzynin B, and 2,4-bis(p-hydroxybenzyl)-3,30 -dihydroxy-5-methoxybibenzyl were the
most potent inhibitors with an IC50 values ranging from 0.79 to 6.57 μM [24].
Niu et al. have carried out the antitumor activity using CT26 cells from the
polysaccharide fraction of tubers of B. ochracea (BOP). The results showed that
BOP significantly decreased tumor growth in a dose-dependent manner [36].
Sun et al. have carried out the anticancer activity against MCF-7, HT-29, HUVEC,
and A549 cells on the tubers of Bletilla striata. The compounds 7-hydroxy-
2-methoxy-phenanthrene-3,4-dione and 30 ,70 ,7-trihydroxy-2,20 ,40 -trimethoxy-
0
[1,8 -biphenanthrene]-3,4-dione showed promising as an antiproliferative agent
against cancer with an IC50 value ranging from 12.64 2.17 to 48.35 3.87 μM,
respectively [37].
Zhang et al. have carried out the anti-cancer activity against GES-1 cells using
BSP fraction. The results showed that BSP at higher concentrations of polysaccha-
rides in culture medium could potentially increase permeability of cell membrane
and did not significantly affect cell viability of GES1 cells after 24 h or 48 h
treatment [34].
Jiang et al. have carried out the antitumor activity against HepG2 cells using
fractions from ethanol extract of tubers of B. striata. The results showed that both
FRP and PSP can dose dependently induce HepG2 cells apoptosis, which implied
tumor therapeutic effect [35].
Guo et al. have carried out the cytotoxicity activity against human red blood cells
using phenanthrene fraction (EF60) of B. striata. The results showed that the EF60
possessed at 160 μg/mL showed no cytotoxicity against human erythrocytes, and was
minimally toxic to human umbilical vein endothelial cells with an IC50 of 75 μg/mL [38].
Wang et al. have carried out cytotoxicity activity against A-549 cells, BGC-823 cells,
HepG2 cells, HL-60, MCF-7 cells, SMMC-7721, and W480 for the compounds isolated
from B. striata. The results showed that the compounds (1α,3α)-1-O-[(β-D-xylo-
pyranosyl-(1!2)-α-L-rhamnopyranosyl)]-3-OD-glucopyranosyl-5α-spirostan, (1α,3α)-
1-O-[(β-D-xylopyranosyl-(1!2)-α-Lrhamnopyranosyl)oxy]-3-O-D-glucopyranosyl-25
(27)-ene-5αspirostan, (1α,3α)-1-O-[(β-D-xylopyranosyl-(1!2)-α-L-rhamnopyranosyl)
oxy]-epiruscogenin, (1α,3α)-1-O-[(β-D-xylopyranosyl-(1!2)-α-Lrhamnopyranosyl)
oxy]-epineoruscogenin and 3-O-β-D-glucopyranosyl-3-epi-neoruscogenin exhibited
significant cytotoxicity against all tested tumor cell lines with IC50 values less than 30
μM for all the cell lines used, whereas the compound bletilnoside A did not show any
cytotoxicity activity [30].
Yang et al. studied the antimicrobial activity against Staphylococcus aureus,

S. epidermidis, Bacillus subtilis, Escherichia coli, Candida albicans, C. krusei,
and C. parapsilosis for the compounds from the ethanol extract of the tubers of
B. ochracea. The results showed that the compound blestriarene A possessed
potent activity against three strains with an IC50 value ranging from 12.5 to 50 μg/
mL, and the other compounds showed lesser activity against all strains [23].
Jiang et al. have performed the antibacterial activity against S. aureus, B. subtilis,
and E. coli for the compounds isolated from the tubers of B. striata. The results
showed the compounds 2,7-dihydroxy-3,4-dimethoxyphenanthrene and
2,7-dihydroxy4-methoxy-9,10-dihydrophenanthrene showed potent inhibition
against S. aureus and B. subtilis with an MIC ranging from 26 to 53 μg/ml;
and the other compounds namely 2,7-dihydroxy-3,4-dimethoxy-9,10-dihydro
phenanthrene, shanciol C, shanciol D, shanciol F, and blestriarene A showed inhi-
bition only against S. aureus with an MIC ranging from 6 to 53 μg/ml, whereas all
the compounds did not show any inhibition against E. coli [39].
Jiang et al. have carried out the antibacterial activity against S. aureus, B. subtilis,
and E. coli using ethanol extract of the dried tubers of Bletilla striata. The results
showed that the compounds namely bletistrin G, bletistrin J, bulbocol,
shancigusin C, shanciguol, and shancigusin B showed good antibacterial activities
against S. aureus and B. subtilis. Among them, compounds bulbocol and
shancigusin B showed potent inhibitory activities against S. aureus, with MICs of
9 and 3 μg/mL, respectively [40].
Qian et al. have carried out the antibacterial activity against Gram-positive bacteria:
S. aureus, S. epidermidis, Enterococcus faecalis, and B. subtilis and Gram-negative
bacteria: E. coli and Proteus vulgaris using compounds isolated from the ethanol
extract of the rhizomes of B. striata. The results showed that the compounds 4,7,
70 -trimethoxy90 ,100 -dihydro(1,30 -biphenanthrene)-2,20 ,50 -triol, 4,7,40 -trimethoxy-90 ,100 -
dihydro(1,10 -biphenanthrene)-2,20 ,70 -triol, 4,7,30 ,50 -tetramethoxy-90 ,100 -dihydro(1,
0 0 0
1 -biphenanthrene)-2,2 ,7 -triol, 4,8,4 ,8 -tetramethoxy(1,10 -biphenanthrene)-2,7,20 ,
0 0
0
7 -tetrol, and blestriarene C showed potent antibacterial activities against six Gram-
positive bacteria strains, including methicillin-resistant S. aureus ATCC 43300 and
ampicillin-resistant S. aureus ATCC 29213. Among them, 4,7,40 -trimethoxy-90 ,100 -
dihydro(1,10 -biphenanthrene)-2,20 ,70 -triol showed the most potent inhibitory activities,
with MICs of 2 and 4 μg/mL against ampicillin-resistant S. aureus ATCC 29213 and
methicillin-resistant S. aureus ATCC 43300, respectively [41].
Guo et al. have carried out the antibacterial activity against Staphylococcus
aureus using phenanthrene fraction (EF60) of B. striata. The results showed that
the EF60 possessed significant minimum inhibitory concentration (MIC) values
against these pathogens ranged from 8 to 64 μg/mL [38].
5.5 Hemostatic Activity
In traditional medicines, Bletilla species are well known for their hemostatic prop-
erties. Polysaccharides from the rhizomes of B. striata are reported to induce the
hemostatic activity by activating adenosine diphosphate (ADP) receptor signaling
pathway through P2Y1, p2y12, and PKC receptors [42].
Yang et al. have carried out the hemostatic activity in in vivo using compounds
isolated from the tubers of B. striata. The results showed the parent compounds
underwent various metabolic processes and their metabolites had various activities
which could possess hemostatic activity [43].
Wang et al. have carried out hemostatic activity of the compounds isolated from
B. striata. The results showed that the compounds (1α,3α)-1-O-[(β-D-xylopyranosyl-
(1!2)-α-L-rhamnopyranosyl)]-3-OD-glucopyranosyl-5α-spirostan, (1α,3α)-1-O-[(β-D-
xylopyranosyl-(1!2)-α-Lrhamnopyranosyl)oxy]-3-O-D-glucopyranosyl-25(27)-
ene-5αspirostan, (1α,3α)-1-O-[(β-D-xylopyranosyl-(1!2)-α-L-rhamnopyranosyl)
oxy]-epiruscogenin, (1α,3α)-1-O-[(β-D-xylopyranosyl-(1!2)-α-Lrhamnopyranosyl)
oxy]-epineoruscogenin, and 3-O-β-D-glucopyranosyl-3-epi-neoruscogenin exhibited
highest hemostatic activity [30].
Lu et al. have carried out the hemostatic activity using water, n-butyl alcohol,
petroleum ether, and ethyl acetate fractions obtained from the ethanol extract of
B. striata. The results showed that the water and n-butyl alcohol fractions signifi-
cantly increased the platelet aggregation rate induced by ADP [44].
Wang et al. have evaluated the hemostatic activity using the extracts of tubers of
B. striata. The results showed that B. striata inhibited the rat tail hemorrhage,
traumatic hemorrhage of liver, and spleen in rabbits as well as traumatic hemorrhage
of liver and abdominal aorta in Beagle dogs [45].
5.6 Immunological Activity
Wang et al. have carried out the immunological activity in ICR mice with the ethanol
extract of Bletilla striata polysaccharide (BSP). The results showed on the BSP-1,
BSP-2, and CBSP on thymus were 23.5%, 3.3%, and 4.1%, spleen index were
36.0%, 24.4%, and 10.4%, respectively [46].
Peng et al. have carried out the immunobiological activity using the acetone
fraction of BSPF2. The results showed that BSPF2 was found to stimulate spleen
cells proliferation. On the basis of this finding, it was suggested that BSPF2 may be a
good source for the development of immunomodulator [47].
5.7 Anti-Fibrosis Activity
Wang et al. have carried out anti-fibrosis activity against human mesangial cells
(HMCs) using aqueous extract of BSP at a concentration of 5, 10, 20, 40, 80, and 160
μg/ml. The results showed that BSPb exhibited significant anti-fibrosis activity by
downregulating TGF-β RI, TGF-β RII, and α-SMA production [48].
5.8 Antiviral Activity
Shi et al. have studied the influenza A virus activity using ethanol extract of the
rhizomes of B. striata. The results on the compounds against cytotoxicity to MDCK
and reduction of CPE in MDCK cells, hemagglutination, and neuraminidase inhibition
assay showed to inhibit with an IC50 value ranging from 0.9 0.2 to 42.3 3.9 μM,
whereas the neuraminidase inhibition assay showed to possess IC50 values ranging
from 16.8 1.6 to 87.5 10.1 μM, respectively [49].
5.9 Wound Healing Activity
Bletilla plants are also widely used in traditional medicines for wound healing
activities. Song et al. have carried out the wound healing activity in vivo using
ethanol extract of BSP pure and ointment mixture. The results showed that animals
treated with “mixed ointment” experienced inflammatory infiltration, which was
lower than that of other groups. Both “BSPG ointment” and “Bletilla phenolic
ointment” demonstrated superior tissue repair compared to the control. Therefore,
this study confirmed that the BSP and ointment mixture has excellent wound healing
activities [33].
Chen et al. have carried out the wound healing activity using alcohol extract of the
tubers of B. striata polysaccharide (BSP). The results showed that the BSP exhibited
excellent healing function mainly due to its modulation of macrophages throughout
inflammation and proliferation periods [50].
Diao et al. have carried out the wound healing activity using B. striata polysac-
charide (BSP) isolated from B. striata. The results showed that BSP enhanced
vascular endothelial cell (EC) proliferation and vascular endothelial growth factor
(VEGF) expression and also changed the nitric oxide synthase (iNOS), tumor
necrosis factor alpha (TNF-α), and interleukin 1 beta (IL-1β) mRNA levels and
enhanced the expression of these cytokines, but has no effect on interferon gamma
(IFN-γ) level, respectively [51].
5.10 Antiulcer Activity
Zhang et al. have carried out the gastric ulcer activity in vivo using BSP fraction. The
results showed that the BSP treatment led to a 92% reduction in ulcer area. The
histopathology reports showed that on BSP treatment the ulcer model group
exhibited a remarkably gradual decrease in PAS staining intensity, compared to the
normal group, respectively [34].
5.11 Anti-neuroinflammatory Activity
Zhou et al. have carried out the anti-neuroinflammatory activity against

LPS-activated BV-2 microglial cells using the compounds isolated from the ethanol
extract of dried tubers of B. striata. The results showed that the compound
phochinenin K exhibited the most potent activity with an IC50 value of 1.9 μM [52].
5.12 Anti-Mitotic Activity
Morita et al. have carried out the antimitotic activity against K562/BCRP cells using
compounds isolated from the methanol extract tubers of B. striata. The results
showed that the stilbenoids strongly enhanced the cytotoxicity of SN-38 in K562/
BCRP cells but not in K562 cells to possess antimitotic activity [53].
5.13 Anti-Tyrosinase Activity
Jiang et al. have carried out the tyrosinase inhibitory activity using fractions from
ethanol extract of tubers of B. striata. The results showed that the ethanolic extracts
from PSP showed strong tyrosinase inhibition activity in a dose-dependent manner

with IC50 ¼ 751.4 mg/L [35].
5.14 Anti-Ulcer Activity
Shi et al. have carried out the anti-ulcer activity of BSP against mice. The results
showed that on BSP treatment (2, 10, and 50 mg/kg) exhibited dose-dependent
inhibitory activity against the expression of Th2 type cytokines including IL-4, IL-5,
and IL-13 [54]. Ke and Zhao et al. have performed the ulcerative colitis activity in
mice using BSP. The results on BSP treatment showed to inhibit lymphocyte
activation and secretion of related cytokines via suppressing the activation of
macrophages [55]. Yu et al. have carried out the anti-ulcer activity of BSP against
streptozotocin-induced diabetic ulcers in rats and the results revealed that BSP
effectively stimulated inflammatory cell infiltration, promoted epithelial tissue for-
mation and fibroblast proliferation, and increased hydroxyproline content [56].
6 Commercial Importance
B. striata and few other species of the genus are commercially important orchids due
to their extensive use in traditional medicine in Asian countries and also for their
ornamental purposes. Recent research regarding their bioactive chemical constitu-
ents and pharmacological activities have promoted their wide application. Many
studies are also focusing on the polysaccharides from rhizomes/tubers [25–27,
57]. The mucilaginous roots of B. striata are also used for writing by mixing with
vermilion, as insecticides and also in cosmetics [17]. However, extensive harvesting
in recent years has resulted in destruction of natural habitats and need urgent focus
for preservation [58]. Various new biotechnological tools have been applied in the
conservation, propagation, and cultivation of commercially and medicinally impor-
tant orchids in recent years [6, 59]. Cryopreservation techniques have also been
utilized for B. formosana and B. striata seeds [58, 60–63]. However, there is a urgent
need for proper conservation and cultivation techniques and approaches for sustain-
able harvesting/utilization of Bletilla species to meet the increasing commercial
market demand.
7 Conclusions and Future Remarks
In this chapter, we compiled the available information about the traditional medicinal
uses, phytochemistry and pharmacological activities of orchids belonging to the
genus Bletilla. B. striata along with few other species are very important in tradi-
tional medicine systems in Asia. They are also important for their other non-
medicinal uses such as for foods and cosmetics. Extensive harvesting in recent
years has resulted in the decline of natural habitats, which needs immediate concern
from the scientific community and other stakeholders.
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Orchids of Genus Vanda: Traditional Uses,
Phytochemistry, Bioactivities, 24
and Commercial Importance
Hari Prasad Devkota, Anjana Adhikari-Devkota, Rajan Logesh,

Tarun Belwal, and Bijaya Pant
Contents
1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 592
2 Botanical Description, Distribution, and Ecology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 593
3 Traditional Uses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 593
4 Phytochemistry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 596
5 Biological Activities . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 597
5.1 Antioxidant and Anti-Inflammatory Activities . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 597
5.2 Cytotoxic Activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 599
5.3 Hepatoprotective Activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 599
5.4 Antimicrobial Activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 599
5.5 Antiaging Activity and Cosmetic Applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 599
5.6 Antidepressant Activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 600
5.7 Neuroprotective Activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 600
5.8 Antinociceptive and Analgesic Activities . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 600
5.9 Other Pharmacological Activities . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 600
H. P. Devkota (*) · A. Adhikari-Devkota

Graduate School of Pharmaceutical Sciences, Kumamoto University, Kumamoto, Japan
e-mail: devkotah@kumamoto-u.ac.jp; anjana@kumamoto-u.ac.jp
R. Logesh
TIFAC-CORE in Herbal Drugs, Department of Pharmacognosy and Phytopharmacy, JSS College
of Pharmacy (JSS Academy of Higher Education and Research), Udhagamandalam, Tamil Nadu,
India
T. Belwal
College of Biosystems Engineering and Food Science, Key Laboratory for Agro-Products
Postharvest Handling of Ministry of Agriculture and Rural Affairs, Zhejiang Key Laboratory for
Agro-Food Processing, Zhejiang University, Hangzhou, China
B. Pant
e-mail: b.pant@cdbtu.edu.np

6 Commercial Importance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 601

7 Conclusions and Future Recommendations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 602
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 602
Abstract
The genus Vanda comprises of about 74 species, mainly distributed in South
and Southeast Asia. Many Vanda species are being used in traditional
medicines in India, Nepal, Bangladesh, and other south Asian countries.
Among these species, only very few species are studied in detail for their
bioactive chemical constituents, pharmacological activities, and are consid-
ered for commercial product development as medicines and cosmetics. In this
chapter, we aim to provide an overview about the traditional uses, phyto-
chemistry, and pharmacological activities of various important Vanda species
along with their conservation and cultivation practices and commercial
importance.
Keywords
Vanda · Orchids · Orchidaceae · Traditional uses · Phytochemistry ·
Commercialization
1 Introduction
Orchidaceae is one of the largest families of flowering plants, and they consist of
more than 880 genera and over 30,000 species [1, 2]. They are one of the most
evolutionary advanced plants and inhabit almost every habitat on earth. Orchid
plants are well known for their beautiful flowers, having attractive colors, shapes,
fragrance, and are widely cultivated around the world for their ornamental values
[2]. Although orchids are primarily cultivated and used largely in floriculture
industry, many are also used in traditional medicine as herbal plants and products,
in foods, and in cosmetics around the world [3–6].
The genus Vanda comprises of about 74 species, distributed mainly in South
and Southeast Asia [7, 8]. Many species are used as crude drugs under the name
of “Rasna” in Ayurvedic formulations [9]. They are also used in other traditional
medicine systems in China, India, Nepal, Bangladesh, and other south Asian
countries [2, 10, 11]. Among these 74 species, only very few species are studied
in detail for their bioactive chemical constituents, pharmacological activities, and
are considered for commercial product development as medicines and cosmetics.
In this chapter, we aim to provide an overview about the traditional uses,
phytochemistry, and pharmacological activities of various important Vanda spe-
cies along with their conservation and cultivation practices and commercial
importance.
24 Orchids of Genus Vanda: Traditional Uses, Phytochemistry,. . . 593
2 Botanical Description, Distribution, and Ecology
The genus Vanda comprises approximately 74 species and is widespread through-

out South and Southeast Asia – India, Nepal, Bhutan, Bangladesh, Myanmar, Sri
Lanka, Thailand, and East Asia, through southern China, Taiwan to Korea and
Japan, down through Indonesia, Philippines to northern Australia, and New Guinea
and the Solomon Islands [7].
The genus has monopodial growth habit, mostly epiphytic but sometimes litho-
phytes and terrestrial too. Plants range from small to large. Vanda species can form
large plants with long sturdy stems with extensive aerial root systems on trees with
stiffly erect, premorse tipped leaves. Vanda species are epiphytic monopodial herbs
with ascending or rarely arching stems with short internodes and many distichous
leaves and thick roots in the lower part. The colorful and long-lasting flowers are
arranged in few to many-flowered axillary racemes. The thick roots serve as the
primary water storage organs which allow these species to withstand drought in
semidormant conditions (http://www.plantsoftheworldonline.org/taxon/urn:lsid:
ipni.org:names:30077641-2, accessed on December 17, 2020). They are grouped
into four categories on the basis of their leaf character, e.g., strap shaped, terete,
semiterete, and channeled [12]. Inflorescence is axillary, erect, and simple with often
brightly colored, sometimes fragrant, flowers. There is great diversity in floral shape
and color. The flowers are small to large, few to many fleshy, heavy textured, long
lasting, and yellow, brown, purple, magenta, blue, and lavender in color. The flower
size varies from 2.5 to 10 cm [13]. The genus is one of the five most horticulturally
important orchid genera in the world [7]. A number of species of genus Vanda are
vulnerable to extinction in the wild and are being rare and geographically restricted
in distribution [14].
3 Traditional Uses
Many Vanda species are being used in different systems of traditional medicines in
South Asian countries for the treatment of inflammation, wounds, bone fractures,
nervous disorders, and rheumatism [2, 9–11]. Among them, the uses of Vanda
coerulea Griff. ex Lindl. (Fig. 1), Vanda cristata Wall. ex Lindl. (Fig. 2), Vanda
parviflora Lindl. Vanda spathulata (L.) Spreng, Vanda tessellata (Roxb.) Hook.
ex. G.Don (Syn. Vanda roxburghii R.Br.) (Fig. 3), and Vanda testacea (Lindl.) Rchb.
f (Fig. 4) are commonly reported. Traditional uses of individual species of Vanda are
mentioned below:
Vanda coerulea: The juice of the leaves is used to treat diarrhea and indigestion
[15]. Decoction of the flowers is used as appetizer and tonic [16].
Vanda cristata: The paste prepared from whole plants or roots is used to treat cuts,
wounds, and boils and to treat dislocated bones [17]. The juice of the leaves is used
Fig. 1 Photographs of Vanda coerulea. (Photos by H.P. Devkota)
Fig. 2 Photographs of Vanda cristata. (Photos by B. Pant)
to treat bronchitis, cough, tonsillitis, and weakness [15]. Leaf powder is used as
expectorant and the leaf paste applied to cuts and wounds [18].
Vanda parviflora: It is used to treat rheumatism, disorders of nervous system, and
also as anticancer and antiviral agent [19, 20].
Vanda spathulata: Powder prepared from dry flowers is used to treat asthma, maniac
disorders, and neurological disorder [21].
Fig. 3 Photographs of Vanda tessellata. (Photos by B. Pant)
Fig. 4 Photographs of Vanda testacea. (Photo by B. Pant)
Vanda tessellata: Roots are commonly used for the treatment of inflammatory
diseases and rheumatism. Rhizome paste is applied in dislocated bones
[15]. Leaf juice or paste is used to treat bronchitis, earache, rheumatism, and fever
[16, 22, 23]. Paste obtained from the roots and leaves is applied for the treatment of
sprains, rheumatism, and also used as antidote for spider, scorpion, and snake bite
[16, 18]. Root decoction is used in the treatment of cholera [16]. Plant ash with
mustered oil is used to treat bone fracture [16]. Roots are used for the treatment of
rheumatism and bronchitis. Paste made from the leaves is used to treat fever
[18]. Leaf juice is used to treat ear infection and skin diseases [24].
Vanda testacea: Leaf extract is used to treat earache as eardrop. It is also used to treat
cuts, wounds, and for antiviral activities. Leaves are used in the treatment of viral
diseases and cancer. Leaf drops are used during earache [11]. Powder obtained from
dried flowers and leaves is used to treat rheumatism [11, 16].
4 Phytochemistry
Orchids are rich in various bioactive chemical compounds. Hydroxyl-benzyl deriv-

atives [25], fluorenones and stilbenoids [26], and flavones C-glycosides [27] are
some of the most common chemical classes in plants of the Orchidaceae family.
Only a few species of Vanda genus have been studied for their chemical constituents,
and they are reported to contain phenanthrene derivatives, bibenzyl derivative, and
other compounds including anthocyanins, simple phenolic compounds, and volatile
compounds (Table 1). Phenanthrene derivatives (Fig. 5) are reported from
V. tessellata, V. parviflora, and V. coerulea [28–31]. Simmer et al. isolated a bibenzyl
derivative, gigantol (8) (Fig. 6), from the stems of Vanda coerulea [28]. It was also
isolated and identified from the roots of V. tessellata [32]. Few other phenolic
compounds are isolated such as tetracosylferulate (9), parishin (10), 4-(β-D-
glucopyranosyloxy) benzyl alcohol (11), 2,5-dimethoxy-6,8-dihydroxy isoflavone
(12), and gallic acid (13) from various Vanda species (Table 1, Fig. 6). Anthocyanins
such as delphinidin and cyanidin derivatives were reported from the flowers of
various Vanda hybrids [33–35]. An alkaloid, laburine acetate, was isolated from
Vanda hindsii [36]. Some other compounds are 2,7,7-tri methyl bicyclo [2.2.1],
heptane [37], and steroids ([38, 39]. Joshi et al. [40] performed the GC-MS analysis
of the methanol extract of whole plant of Vanda cristata and reported the identifi-
cation of 9-methyl-octadecenoate as a major component (53.43%) followed by
palmitic acid (23.51%), 15-methyl-hexadecanoic acid methyl ester (4.86%),
10-nonadecenoic acid methyl ester (3.55%), 2-methyl-Z,Z-3,13-octa-
decadienol (2.95%), and 11-tridecene-1-ol (2.74%). Other minor constituents were
alpha-bisabolol, 14-methyl-pentadecanoic acid methyl ester, 10-octadecenoic acid
methyl ester, hexadecanoic acid, linolelaidoyl chloride, etc.
Table 1 Major bioactive compounds identified from Vanda species

Chemical class Compounds Plant source References
Phenanthrene Tessallatin (1) V. tessellata [30]
derivatives Oxo-tessallatin (2) V. tessellata [31]
Parviflorin (3) V. parviflora [29]
Flavidin (4) V. coerulea [28]
Imbricatin (5) V. coerulea [28]
Coelonin (6) V. coerulea [28]
Methoxycoelonin (7) V. coerulea [28]
Bibenzyl derivative Gigantol (8) V. coerulea, [28]
V. tessellata [32]
Phenolic compounds Tetracosylferulate (9) V. tessellata [38]
Parishin (10) V. parishii [41]
4-(β-D-Glucopyranosyloxy) benzyl V. parishii [41]
alcohol (11)
2,5-Dimethoxy-6,8-dihydroxy isoflavone V. tessellata [42]
(12)
Gallic acid (13) V. tessellata [42]
Fig. 5 Structures of phenanthrene derivatives
5 Biological Activities
Some of the medicinal species of Vanda genus are evaluated for their pharmacolog-
ical activities such as anti-inflammatory, antioxidant, cytotoxic, antiaging,
hepatoprotective, and anticonvulsant activities, among others.
5.1 Antioxidant and Anti-Inflammatory Activities
Simmler et al. [28] evaluated the antioxidant and anti-inflammatory activities of

extracts obtained from Vanda coerulea. Preliminary experiments showed that the
aqueous ethanol extract of stems exhibited strong 1–1-diphenyl-2-picrylhydrazyl
(DPPH) and hydroxyl radical (OH) scavenging activities as compared to extracts of
leaves and roots. Stem extract also showed potent in vitro inhibitory activity against
type 2 prostaglandin (PGE-2) release from ultraviolet (UVB) irradiated HaCaT
keratinocytes. Five compounds, flavidin (4), imbricatin (5), coelonin (6),
methoxycoelonin (7), and gigantol (8), were isolated from the extract by using
bioassay-guided isolation procedures, and imbarcatin (5) and methoxycoelonin (7)
Fig. 6 Structures of eucomic acid derivatives, bibenzyl derivative, and phenolic compounds
showed potent antioxidant activities. Compounds 5, 7, and 8 also showed potent

inhibitory activity against cyclooxygenase (COX)-2 expression as revealed by
western blot analysis.
Chawla et al. [38] reported that for their anti-inflammatory activity, different
extracts were obtained from the roots of Vanda tessellata. Petroleum ether, chlo-
roform, and methanol extracts exhibited 54.2%, 42.1%, and 21.9% antiedema
activity, respectively. Further chemical isolation afforded tetracosylferulate and
β-sitosterol-D-glucoside from the petroleum ether and chloroform extracts,
respectively [38].
Begum et al. reported the anti-inflammatory activity of methanol extracts of
leaves and roots of Vanda tessellata in carragenan-induced paw edema test and
also reported that the leaf extract (50 &100 mg/kg) reduced paw edema significantly
at the third and fourth hours of the treatment with maximum 67.14% of inhibition,
whereas root extract (100 mg/kg) also showed potential anti-inflammatory potential
at third hour with 61.37% of inhibition [43].
Vijaykumar et al. [44] reported the DPPH and nitric oxide (NO) radical-
scavenging activities of the petroleum ether extract of leaves of Vanda tessellata.
Similarly, Thaakur and Pokkula [45] reported the ameliorative effects of valuated
hydro-alcoholic extracts of leaves of Vanda testacea in axotomy-induced periph-
eral neuropathy in rats possibly by enhanced antioxidant activity, reduced
calcium level, and inhibition of PGE2. Islam et al. reported the antioxidant
activity of the methanol extract of roots of Vanda tessellata in DPPH and NO
assays [46].
5.2 Cytotoxic Activity
Cytotoxic effects of Vanda cristata have been reported by Joshi et al. [40] against
cervical cancer (HeLa) and glioblastoma (U251) cell lines. They reported that
methanol extracts of the whole plant of V. cristata were effective cell growth
inhibitors with significant percentage inhibition of 54.56% at the highest concentra-
tion (400 μg/ml) in Hela cell lines with IC50 values of 317.23 μg/ml. Similarly, in the
case of glioblastoma cells (U251), V. cristata was significantly effective in inhibiting
growth, with percentage inhibition of 61.86%, with IC50 of 163.66 μg/ml.
Chowdhury et al. reported on the aqueous and methanol leaf extracts of
V. tessellata for cytotoxic activity using brine shrimp (Artemia salina), and the
results showed very low cytotoxicity against brine shrimps [47].
Islam et al. have studied the cytotoxic activity using brine shrimp lethality
bioassay for the methanolic extract of Vanda tessellata root, and the results showed
that the extract showed significant toxicity of brine shrimp nauplii with the LC50
value of 25.190.98 μg/ml [46]. Similarly, Prakash et al. reported the potent
antioxidant activities of chloroform and ethanol extracts of the leaves of Vanda
tessellata [48].
5.3 Hepatoprotective Activity
Anwar et al. [49] reported the dose-dependent hepatoprotective activity of the

petroleum ether extract of leaves of Vanda tessellata in rats. The activity was
evaluated through the determination of serum markers such as cholesterol, triglyc-
erides, alanine amino transferase, etc.
Bhattacharjee et al. [50] evaluated the antibacterial and antifungal activities of

different extracts of the whole plant of Vanda tessellata. Among different extracts,
the chloroform extract showed potent antibacterial activity and antifungal activity.
5.5 Antiaging Activity and Cosmetic Applications
Andre et al. [51] reported the antiaging potentials of the extracts of Vanda coerulea
for skin-hydrating properties in cosmetics composition. The extracts showed
skin-hydrating property mainly by increasing the expression of aquaporin 3 and
lympho-epithelial Kazal type-related inhibitor (LEKTI) protein, which resulted in
the limitation of intercellular water evaporation and enhancement of transport of
water in the epidermis. Similarly, Bonte et al. [52] evaluated the effects of ethanolic
extracts of Vanda coerulea against the cutaneous aging. Various bioactive constitu-
ents were identified such as imbricatin, methoxycoelonin, and gigantol which
significantly decreased the number of S phase cells and led to reduction in cyclin E
and cyclin-dependent kinase 2. Similarly, Cauchard et al. [53] also evaluated the
antiaging potential of Vanda coerulea and reported that the extracts can be used as
active constituents in cosmetics to diminish the aging signs of the skin.
5.6 Antidepressant Activity
Dasari et al. [21] reported that the methanolic extract of the flowers of Vanda
spathulata showed antidepressant activities in mice using the forced swim test and
the tail suspension test. Similarly, Prakash et al. [48] reported the antidepressant
activity of chloroform and ethanol extracts of the leaves of Vanda tessellata in rats
using forced swimming test and tail suspension test.
5.7 Neuroprotective Activity
Mundugaru et al. (2020) evaluated the neuroprotective activity of the hydroalcoholic

extract of Vanda tessellata in experimental models of ischemic hippocampal injury
in rats, and the results suggested that the extract at a concentration of 200 and
400 mg/kg showed neuroprotective potentials in ischemic hippocampal injury [54].
5.8 Antinociceptive and Analgesic Activities
Chowdhury et al. reported the antinociceptive activity of the aqueous and methanol
extracts of leaves of Vanda tessellata in antinociceptive activity in mice using acetic
acid-induced writhing test, hot plate test, and tail immersion test. Extracts at 200 and
400 mg/kg showed antinociceptive activity in a dose-dependent manner [47].
Islam et al. studied the analgesic activity of the methanolic extract of the roots of
Vanda tessellata in an acetic acid-induced writhing model of pain in mice. The
extract at the dose of 200 and 400 mg/kg body weight exhibited significant reduction
in acetic acid-induced writhing in mice [46]. Similarly, Begum et al. reported the
analgesic activity of methanolic extract of leaves and roots in acetic acid-induced
writhing and formalin-induced paw-licking models in mice. The methanolic extract
of the leaves at the dose of 100 mg/kg body weight showed significant analgesic
activity [43].
5.9 Other Pharmacological Activities
Apart from above-mentioned activities, various other pharmacological activities are

also reported for the extracts obtained from different species of Vanda. For example,
Uddin et al. [55] reported the antiacetylcholinesterase and antibutyrylcholinesterase

activities of the chloroform extract of Vanda tessellata. The ethanolic extract of roots
of Vanda tessellata showed potent anticonvulsant activity against picrotoxin,
pentylenetetrazole, and maximal electroshock-induced convulsions in mice
[56]. Suresh et al. evaluated the aphrodisiac activity of the water and alcohol extracts
of different plant parts (root, flower, and leaf) of Vanda tessellata in male mice. The
evaluation was carried out by observing the mounting behavior, mating performance,
and reproductive performance. The alcohol extract of the flower showed most potent
activity [57]. The aqueous extract of Vanda tessellata showed potent wound-healing
activity in rats after topical administration at a dose of 150 mg/kg/day [58].
6 Commercial Importance
Vanda orchids have very high commercial potential for their ornamental values and
also for their traditional medicinal uses, bioactive constituents, and pharmacological
activities. Extracts of Vanda plants are also gaining attentions for the potential use in
cosmetics [6, 53].
Breeding technologies, basic research into orchid biology, and the application of
biotechnology for their improvement and production in the orchid industry have
developed everlasting interest on them, globally [5, 59]. It indicates there will be a
huge demand for orchids in the future including esthetically and medicinally impor-
tant Vanda. Because of their extraordinary floral diversification and deep color
combination, Vanda is regarded as a commercially important group of plant in the
orchids floriculture industry, both as cut flowers and as potted plants. Vanda has been
designated as the “Queen Orchid of the East” due to its robust and large rounded
flowers [60]. One of the species, V. coerulea or blue Vanda, has a magnificent flower,
with purplish blue color. This species got the honor from American Orchid Society
39 times and has been used as the parental stock to develop new hybrids for more
than 4000 hybrids [61]. Various verieties and hybrids are also available in India
[13]. However, the proper conservation and sustainable utilization of Vanda species
for commercial purposes is an urgent issue as the natural plant populations are being
decreased due to extensive harvesting. The natural populations of Vanda species are
decreasing, and some of them are now categorized as threatened species under IUCN
Red List (http://www.iucnredlist.org/) [2]. In addition to esthetic importance, due to
the high medicinal value of Vanda, their illegal collection for trade and consumption
has resulted in more species in the threatened category [62]. Thus, various
researchers are involved in their ex situ conservation by in vitro culture technique
[63, 64]. Besides, the beneficial endophytes in them add on more value in them
[65, 66], and this is another potential of research to investigate their role for
medicinal properties of Vanda. Thus, advanced biotechnological tools for the
lab-based culture and propagation are necessary which may in future provide
solutions for large-scale production and commercial uses.
7 Conclusions and Future Recommendations
The species of Vanda are distributed and cultivated in various Asian countries and
are widely used for ornamental and medicinal purposes. Only very few studies have
been performed regarding the chemical constituents in these species and their
respective biological activities. Further research is necessary in the detailed chemical
isolation of active constituents and their bioactivity analysis using animal models to
explore their potential for pharmacological and cosmetic applications. Extensive
harvesting practices have also resulted in the decrease of natural habitat for these
species; thus, sustainable harvesting practices are necessary along with the develop-
ment of advanced techniques for their cultivation and propagation.
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14712/23361964.2015.36
Part VI
Cosmetic Applications
Orchid Extracts and Cosmetic Benefits
25
Mayuree Kanlayavattanakul and Nattaya Lourith
Contents
1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 610
2 Causes and Treatment Strategies of Dryness, Greasiness, Wrinkle, and Aging of Skin . . . 610
3 Impacts of Radical, UV, and Extracellular Matrix in Firmness, Wrinkle,
and Aging of Skin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 611
4 Orchids and Cosmetic Benefits . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 612
4.1 Ansellia africana . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 613
4.2 Bulbophyllum scaberulum . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 613
4.3 Dendrobium spp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 613
4.4 Dendrobium candidum . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 614
4.5 Dendrobium chrysotoxum . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 614
4.6 Dendrobium denneanum . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 614
4.7 Dendrobium huoshanense . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 614
4.8 Dendrobium nobile . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 615
4.9 Dendrobium officinale . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 615
4.10 Dendrobium tosaense . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 616
4.11 Eulophia hereroensis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 616
4.12 Eulophia macrobulbon . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 617
4.13 Eulophia petersii . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 617
4.14 Tridactyle tridentata . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 617
4.15 Vanda coerulea . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 617
4.16 Vanda roxburghii . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 618
4.17 Vanda teres . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 618
5 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 621
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 624
M. Kanlayavattanakul (*) · N. Lourith

School of Cosmetic Science, Mae Fah Luang University, Chiang Rai, Thailand
e-mail: mayuree@mfu.ac.th; nattayal@mfu.ac.th

610 M. Kanlayavattanakul and N. Lourith
Abstract
Orchid has long been used in several traditional medicines all around the world.
This medicinal herb is evidenced for immunomodulatory activity and functions
as longevity recipe. The most commonly used orchids in complementary medi-
cine, Ayurvedic and traditional Chinese recipes, are Vanda and Dendrobium.
In addition to these genera, different orchids are potentially to be implied for
health promotion aspects including their cosmetic benefits. Orchids with scien-
tific supports for cosmetic properties relevant for skin dryness, skin wrinkle, and
aging of skin are therefore summarized in this chapter. Furthermore, traditional
uses relevant to cosmetic benefits are disclosed as well as those commercialized
orchid extracts in cosmetic industry. Thus, the beautiful floriculture orchids
and full of availability are appreciable to be used for skin aging protection and
treatment products, and flow in the stream of the consumers’ awareness and
preference on natural or bio-based products are presented in this context.
Keywords
Orchid · Cosmetics · Hydration · Moisturizer · Antiaging · Anti-wrinkle
1 Introduction
Orchid is evidenced as a therapeutic herb that positively affects human health. This
flowering family has variety of species according to its beautiful flower and could be
cultivated in all continents except Antarctica and deserts. The economic important of
orchid is therefore unlimited for therapeutic uses but included floriculture proposes.
That drives orchid into a huge business as a second most cut flowers. Regarding its
importance in potted floriculture, orchid bleedings have been continuously taken
worldwide resulting of more than 25,000 species majorly being developed in tropical
and subtropical regions.
Pharmacological activities of orchids liberating variety applications of the herbs
in different recipes [1–5] and the specific phytochemistry and biological activities
contributing on diseases would be addressed in different chapters of this book. In this
chapter, appraisal of orchids for skin treatments is objectively to be focused. The
adverse effects of oxidants, radical, inflammatory mediators, and enzymes causing
dryness, wrinkle, and aging of skin as well as hyperpigmentation are firstly summa-
rized to figure out on these correlations exacerbating aging.
2 Causes and Treatment Strategies of Dryness, Greasiness,

Wrinkle, and Aging of Skin
When an individual ages, the skin barrier is impaired, and this is known as chrono-
logic aging. The turnover rate of epidermal cells slows down, and the vascular
network between epidermal cells, which consists of keratinocytes, fibroblasts,
25 Orchid Extracts and Cosmetic Benefits 611
Langerhans cells, and melanocytes, and the skin elastic fibers and fluids are
disrupted. In addition, these cells are decreased resulting in skin thickness reduction.
Consequently, skin absorption, sensory perception, protection, secretion, and excre-
tion are reduced including thermoregulation. Epidermis, particularly the stratum
corneum, is thinner leading to skin dryness due to a reduction in water holding
capacity resulting in severe skin damage. These cutaneous impairments are caused
by a reduction in collagen, elastin, and hyaluronan which are synthesized by
epidermal cells [6].
3 Impacts of Radical, UV, and Extracellular Matrix in

Firmness, Wrinkle, and Aging of Skin
Skin aging is caused by several factors which damage cell membranes and compo-
nents including lipids, proteins, and DNA. Reactive molecules with unpaired elec-
trons or free radicals initiate cellular damage known as intrinsic, chronologic, and
extrinsic aging. Natural cellular metabolism generates free radicals in a self-defense
mechanism and efficiently scavenges these species and neutralizes the radicals;
however, these are decreased with age. Dermal damage is also induced by UV
exposure at the shorter wavelengths (UVB), which are absorbed by the epidermis
prior to irradiation of keratinocytes. Meanwhile, longer wavelengths (UVA) pene-
trate the skin and interact with epidermal and dermal cells. Proteolytic enzyme
activities are propagated resulting in degradation of collagen and elastin fibers
including glycosaminoglycan (GAG), hyaluronan, chondroitin, keratin, dermatan,
and heparin. They are linked to proteins such as collagen (28 types) and elastin
and act as lubricants associated with the elasticity and tensile strength of skin.
In addition, the matrix metalloproteinase (MMP) is a degradation enzyme of the
extracellular matrix (ECM), including collagen, elastin, and GAG. These enzymes
with 28 members (MMP-1 to MMP-28) are function and accelerated with age and
radicals including inflammatory mediators as well. Therefore, deactivation, inhibi-
tion, and suppression of MMP, especially collagenase, elastase, and hyaluronidase,
in addition to stimulation of hyaluronan synthase are regarded as the leading strategy
in the management of skin aging. In addition, cellular damage results in inflamma-
tory mediators generating free radicals and worsens intrinsic aging in turn as well
as an induction of MMPs activation [6, 7].
Therefore, antioxidative molecules (e.g., superoxide dismutase, catalase,
glutathione peroxidase) and nonenzymatic antioxidants (for instance, vitamin E,
vitamin C, ubiquinone) to prevent free radical damage which terminate the radicals,
protect against radical generation, increase self-defense mechanisms, and act as
topical sun protectors limiting radical generation are contributing to antiaging
products and have been extensively commercialized as over-the-counter (OTC)
products. Antioxidants are therefore accepted as major therapeutic ingredients
which decelerate skin aging. Consequently, commercial interest in the incorporation
of antioxidants in cosmetic products is increasing, particularly in naturally derived
products as they are thought to be milder, safer, and healthier. Topical OTC products
are the main source of interest in treating skin disorders, including wrinkles, and
protecting against aging, particularly those containing botanical ingredients.
Oxidative stresses induce inflammatory responses and activate MAPK pathway
as well as NF-κB, AP-1, and pro-inflammatory cytokines and other inflammatory
mediators that upregulate MMPs activities that severely propagate aging process of
skin, which later generate radicals in the systems, accumulating or worsening
aging of skin [8] including dryness of skin. Accordingly, anti-inflammatory and
immunomodulatory agents are used in dermatology [9] not only for combating
skin aging but also for allergic skin treatment and suppression of skin dryness. The
treatment of excessive skin dryness is the subject of many cosmetic formulations,
as this ailment can impact personal appearance and self-confidence and over time
can result in reductions in elasticity and promote the generation of wrinkles. The
application of skin-hydrating products thus allows for skin hydration and enhanced
aesthetic appearance. Moisturizers considered safe can cause allergic skin reactions
in some users, and public perceptions are shifting from synthetic products toward
the use of non-irritating, natural moisturizers. Of these, plant-derived polysaccha-
rides are actives gaining interest among consumers and researchers in the cosmetic
field [10].
Skin hyperpigmentation is caused by several factors, i.e., UV radiation, radicals,
inflammatory mediators, and hormones. Briefly, UV radiation causes skin hyper-
pigmentation by stimulating keratinocytes to secrete α-melanocyte-stimulating
hormone (α-MSH), a small peptide hormone derived from proopiomelanocortin
(POMC). Consequently, α-MSH binds to melanocortin 1 receptor (MC1R)
expressed on melanocyte surfaces and thereafter induces melanogenesis via multiple
signaling pathways resulting from cAMP, protein kinase A (PKA), cAMP response
element-binding protein (CREB), and microphthalmia-associated transcription fac-
tor (MITF) activity. MITF is a key transcription factor regulating the transcription
of melanogenic enzymes, i.e., tyrosinase, tyrosinase-related protein (TRP)-1, and
TRP-2. In addition, UV radiation modulates nuclear factor E2-related factor 2 (Nrf2)
and further activates mitogen-activated protein kinases (MAPKs). MAPKs consist
of three subtypes: stress-activated protein kinases (SAPKs)/c-Jun NH2-terminal
kinases (JNK), p38, and extracellular signal-regulated kinases (ERKs). JNK and
p38 kinases are stimulated by pro-inflammatory cytokines and environmentally
induced stresses such as exposure to UV irradiation, heat, and hydrogen peroxide,
resulting in DNA damage. Melanogenesis is controlled by MAPKs, with MITF
being activated by p38 phosphorylation. By contrast, ERK activation inhibits mel-
anin synthesis by downregulating MITF expression [11, 12].
4 Orchids and Cosmetic Benefits
The potential of each orchid with cosmetic benefits is disclosed alphabetically on

the basis of scientific evidences. The phytochemically active compounds will
be included together with biological activities in vitro, ex vivo, and in vivo if
appreciable.
4.1 Ansellia africana
Leopard orchid has long been regarded as the important source of pharmacologically
active biomolecules beneficial for health [13]. This orchid posed anti-inflammatory
effect by the inhibition against COX-1. Its crude root extract using CH2Cl2 inhibited
COX-1 at EC50 of 0.25 0.10 mg/ml. In addition, this extract showed acetylcho-
linesterase inhibition, although at a lesser degree than the ethanolic and ether extracts
(EC50 = 0.34 0.14 and 0.24 0.03 and 0.33 0.03 mg/ml, respectively). EC50 of
galantamine, the positive control, was 0.44 0.10 μM [14].
4.2 Bulbophyllum scaberulum
Ethanolic extract of the orchid root inhibited COX-2 and acetylcholinesterase with
EC50 of 0.44 0.32 and 0.26 0.00 mg/ml, while that of CH2Cl2 extract were
1.43 0.86 and 0.02 0.00 mg/ml, respectively [14].
4.3 Dendrobium spp.
Dendrobium is the second largest genus in the family Orchidaceae, and more than
1,100 species are cultivated in Thailand regarding continuous bleeding of the orchid
to give glamor color and shape varieties. This world’s major stakeholder cut orchids
are widely in red-purple, pink, and white flowers, of which Sonia, Sonia Pink,
Snow Rabbit, and Shavin White are the most common floriculture varieties. The
methanolic extracts of these varieties were screened on in vitro antioxidant and
tyrosinase inhibitory effect. The flower of Shavin White was best in DPPH radical
scavenging activity, followed by Sonia Pink, while Snow Rabbit and Sonia were
comparatively low (IC50 = 463.08 15.68, 492.83 15.73, and > 500 μg/ml)
once compared with the standard gallic acid, quercetin, and ascorbic acid
(IC50 = 0.64 0.01, 0.85 0.04, and 0.94 0.05 μg/ml). Although anti-DPPH
activities of the orchid flower extracts were weak, their inhibitory effects against
mushroom tyrosinase were strong especially Sonia, Sonia Pink, and Shavin White
(IC50 = 57.38 9.26, 83.21 3.53, and 111.67 2.88 μg/ml) that were more
potent than the standard kojic acid (IC50 = 151.73 2.06 μg/ml), while the extract
of Snow Rabbit was lower (IC50 = 167.82 2.63 μg/ml) than the standard. These
activities would be governed by the active principles in terms of phenolics and
flavonoids. Nevertheless, the actives profile of these floriculture Dendrobium was
not carried out in the study [15]. The red-purple Dendrobium Sonia was assessed on
biological activity and phytochemical profile in different research. The anthocyanin-
rich extracts were prepared from the orchid flower. Of which, chemical quality in
terms of total phenolics was additionally reported together with the biological
activities beneficial for cosmetics, i.e., astringency, in vitro radical scavenging, and
enzyme inhibitory activities. The 70% ethanolic and water extract at different times
of extraction were highlighted as the interesting source of antioxidants and inhibitors
against collagenase, elastase, and tyrosinase, which is promising for prevention of

collagen and elastin degradation as per skin dullness. Moreover, the extracts were
safe in NHF posed antioxidant and MMP-2 inhibitory effect activities. In addition to
the safe and efficient activities in NHF, the extracts were safe in B16F10 melanoma
and were proved to suppress cellular melanogenesis, in which the mechanism was
revealed to be by tyrosinase and TRP-2 inhibition at a higher degree than the
standard kojic acid. The biological activities of the Dendrobium Sonia flower were
governed by their ten phenolics and three anthocyanins constituents. Of which,
sinapic and ferulic acids were the major phenolics, and pelargonidin was the
principal anthocyanin followed by cyanidin and keracyanin [16].
4.4 Dendrobium candidum
D. candidum stem is used in traditional Chinese medicine as yin tonic with

inflammation treatment. This methanolic extract of medicinal herb (200, 400, and
800 mg/kg) was reported to increase serum superoxide dismutase (SOD level), while
pro-inflammatory cytokines, i.e., interleukin (IL)-6, IL-12, tumor necrosis factor
(TNF)-α, and interferon (IFN)-γ, were decreased as examined in an animal model for
2 weeks in a dose-dependent manner [17].
4.5 Dendrobium chrysotoxum
The 95% ethanolic extract of the stem was assessed upon its inhibitory effect against
acetylcholine (AChE) and butyrylcholine (BChE) esterases. However, the activity of
the isolated pure compounds was moderate to weak [18].
4.6 Dendrobium denneanum
The isolated pure compounds, 2,5-dihydroxy-4-methoxy-phenanthrene 2-O-β-D-

glucopyranoside and 5-methoxy-2,4,7,9S-tetra-hydroxy-9,10-dihydrophenanthrene,
from the stem exhibited potent anti-inflammation. iNOS was suppressed as per the
inhibition against p38, JNK, MAPK, and IκBα, which suggested their dual mecha-
nism inhibition in MPKs and NF-κB pathways [19].
4.7 Dendrobium huoshanense
Polysaccharides derived from Dendrobium orchids are found to have several health
benefits especially against inflammation including those from D. huoshanense. The
orchid stem rich in polysaccharides was prepared into the extract that majorly
consists of mannose and glucose in a molar ratio of 1.89:1. The polysaccharides
extract was intragastrically administrated at 100, 200, and 400 mg/kg/day for
4 weeks into cigarette smoke-induced mice. The orchid extract was shown to inhibit
TNF-α and IL-1β secretion in serum. The anti-inflammatory effect was studied to be
caused by NF-κB reduction as well as phosphorylation of IκB, p65, p38, and JNK.
Thus, anti-inflammatory activity of D. huoshanense polysaccharides extract was by
alleviating NF-κB and MAPK signaling pathways [20].
4.8 Dendrobium nobile
The stem methanolic extract of the orchid contains alkaloids (96.1%) and poly-
saccharides (1.2%) shown to prevent lipopolysaccharide (LPS)-induced elevation
in tumor necrosis factor receptor 1 (TNFR1) mRNA and protein levels. LPS-
induced activation of phosphorylated p38 mitogen-activated protein kinases (p38
MAPK) and nuclear factor kappa-B (NF-κB) pathway was also suppressed as per
injection of 40, 80, and 160 mg/kg/day into rats for 14 days. Of which, the activity
was pronounced at higher concentration [21]. The isolated pure compounds,
ephemeranthol A and dehydroorchinol, were later confirmed upon these anti-inflam-
matory activities. They inhibited cellular NO production as per pro-inflammatory
cytokines in a dose-dependent behavior at the cellular safety concentration ranging
from 6.25 to 50 μg/ml. The significantly efficient dose (25 μg/ml) of the compounds
was later on confirmed on their inhibitory effect against IL-1β and IL-6, while TNF-
α was significantly suppressed by ephemeranthol A but not dehydroorchinol. There-
after, the stronger anti-inflammatory active, ephemeranthol A, was examined upon
its function in inflammatory signaling pathway. Ephemeranthol A reduced the level
of phosphorylated p38 and inhibited NF-κB activation [22].
4.9 Dendrobium officinale
The stem of the orchid has long been used in traditional Chinese medicine claimed
to reduce fever and to have immunological function. This Dendrobium species is
therefore commercially cultivated not only in China mainland but also in Southeast
Asian countries especially Thailand.
The 132 kDa polysaccharides (mannose and glucose of 3.8:1.0) derived from the
orchid were shown to remarkably reduce cellular oxidative stress by the capability
to inhibit ROS production (at the dose of 62.5–500 μg/ml). Furthermore, the poly-
saccharides extract significantly decreased the p-NF-κBp65/NF-κBp65 level
induced by H2O2. In addition, the extract was also efficient as examined in an animal
model [23]. Potency of the orchid polysaccharides against inflammation was con-
firmed by different in vivo studies as IL-1β, IL-6, IL-18, TNF-α, and IFN-γ were
significantly decreased following 50, 100, and 200 mg/kg administrated into the
induced-mice group [24]. Polysaccharides derived from the stem of the orchid
that is mainly composed of mannose, glucose, and arabinose (molecular weight of
393.8 kDa) were orally administrated into female mice (70 mg/kg) for 10 weeks. The
Dendrobium polysaccharides were evidenced to reduce pro-inflammatory cytokines
(TNF-α and IL-6) and MDA levels while estradiol, SOD, GSH-x, and total antiox-
idant capacity in serum. Moreover, it significantly balanced pro-inflammatory/anti-
inflammatory cytokines ratio, the key mechanism maintaining body health and
resisting damage, to normal level and was able to improve function of mitochondria
by an inhibition of p53/Bcl-2 mediated mitochondrial apoptosis signaling pathway
in natural aging-induced mice. Taken together, the orchid may alleviate cellular
aging by inhibitory effect against NF-κB, and the orchid was suggested to be used
for natural aging treatment in female [25]. The 72.1% polysaccharides extract
(mannose and glucose – 19.51% and 14.03%) of the orchid stem by 80%
EtOH was orally administrated into the diabetic cardiomyopathy-induced mice for
8 weeks. The treatment groups at the dose of 150 and 300 mg/kg were shown to
be increased in SOD with the suppression of MDA activities. In addition, NF-κB,
TNF-α, and IL-1β were significantly suppressed in a comparison with the diabetic
cardiomyopathy-induced mice [26].
The pharmacological benefits of D. officinale stem are confirmed with the
traditionally used by the scientific evidences that continuously explored. The poly-
saccharides extract, Dendronan ®, has been therefore commercialized and proved to
have protective effects against oxidative stress including its capability to increase
CAT, SOD, and GSH-Px with the reduction of MDA in animal model [27].
The orchid polysaccharides are therefore potentially used for immunomodulatory
activity enhancement [28, 29] associated with longevity or aging protection and
treatment.
To widen its application, the different parts of the orchid are explored. Leaf is
obviously one part of the medicinal herb that is essential to be revealed for its new
potential uses. The leaves of 11 different strains of D. officinale were extracted by
80% MeOH. They exhibited anti-DPPH activity at an interesting capability, which
corresponds with total flavonoids content, and rutin was shown to be the biologically
active marker of the orchid leaf [30].
4.10 Dendrobium tosaense
Stem extract of D. tosaense containing quercetin was orally administrated into

allergic modeling mice at the dose of 30, 100, and 300 mg/kg in a comparison
with quercetin (1.6 mg/kg). Anti-allergic potential of the extract was evidenced by
the significant reduction of serum IgG1 and IgE as per IL-4, IFN-γ, and IL-6 level
except the low dose (30 mg/kg). Thus, the orchid was potentially to be used for
atopic dermatitis therapy or other allergic disorder [31].
4.11 Eulophia hereroensis
Tuber extract of this orchid with CH2Cl2 showed anti-activity against COX-1, COX-
2, and acetylcholinesterase activity (EC50 = 0.87 0.28, 1.17 0.15, and
0.23 0.16 mg/ml). However, its ether extract posed only COX-2 and acetylcho-
linesterase inhibitions (EC50 = 1.12 0.33 and 1.20 0.24 mg/ml) [14].
4.12 Eulophia macrobulbon
This tropical orchid is a major floriculture species in Thailand and in other Southeast
Asian countries. It has been used to treat insect bites in local Thai folk remedy. Its
root was therefore extracted, challenged on activity against DPPH radical. The crude
extract scavenged 9 0% of the radical, whereas that of the standard ascorbic acid
was 67 1%, at the same test concentration of 100 μg/ml. Fractionation of the crude
extract was undertaken to improve antioxidant activity to 51 3% and 44 2%,
respectively. The crude extract and these two potent fractions were revealed to have
cellular anti-inflammatory activities. The secretions of IL-6, IL-10, and TNF-α,
inflammatory mediators, were shown to be suppressed at the same test concentration
at 100 μg/ml. The suppression (%) of the crude extract were 30 7, 67 10, and
81 9. Meanwhile, the potent fractions were 12 1% and 24 14%, 60 10%
and 77 4%, and 106 11% and 81 14%, respectively. Of which, the capability
of the orchid and orchid active fractions were noted potent against IL-6. Thus, the
IC50 of each sample against these mediators were examined and were shown to be
54, 25, and 54 μg/ml, respectively [32].
4.13 Eulophia petersii
This medicinal orchid inhibited COX-1 especially its CH2Cl2 extracts of the stem,
pseudobulb, and roots (EC50 = 1.49 0.05, 0.87 0.12, and 1.41 0.64 mg/ml)
and posed acetylcholinesterase inhibitory effect (EC50 = 0.39 0.04, 0.51 0.14,
and 0.51 0.05 mg/ml) [14].
4.14 Tridactyle tridentata
South Africans traditionally employed the orchid root in the remedy recipes, in
which its CH2Cl2 extract was later on confirmed for its anti-inflammatory effects via
the capability against COX-1 and acetylcholinesterase activities (EC50 = 1.47 0.89
and 0.46 0.01 mg/ml) [14].
4.15 Vanda coerulea
This orchid is commonly called blue orchid with regard to its anthocyanin constit-
uents [33]. The hydroalcoholic stem extract displayed in vitro radical scavenging
activity. In addition, the isolated pure compounds posed inhibitory effect against
PGE-2 production in irradiated HaCaT cell line and UVB-induced COX-2 expres-
sion as well [34].
The ethanolic extract of the stem was isolated to give active pure compounds. The
orchid-derived stilbenoid, imbricatin, methoxycoelonin, and gigantol replicated
senescence of normal human skin fibroblasts and were able to restore the percentage
at a rate equivalent to that of young cells together with the recovery of the cyclin E
and cyclin-dependent kinase 2 (cdk2). These results highlight the potential of the
orchid as raw material to fight against the visible signs of skin aging [35].
4.16 Vanda roxburghii
The orchid leaf extract (aqueous) was evidenced to have wound healing prop-
erties as examined in an excision wound animal model. Topical application of
the extract at a dose of 150 mg/kg/day consecutively for 10 days was shown to
reduce the wound diameter by 60%, while the control group has 48% reduction.
The wounds were fully healed in 13 days, whereas those of the control group
were 20 days. Moreover, the significant increment ( p <0.005) in wet and dry
granulation tissue weights, hydroxyproline, and hexosamine contents were
detected. Of which, the pro-healing action was suggested to be by an attribution
to increase collagen deposition or to better alignment and maturation or both
[36]. Moreover, its root extracts were prepared and screened for antioxidant
activity, in which the chloroform extract was exhibited as the most potent
fraction. IC50 against DPPH and OH radicals were 5.76 0.42 and
7.96 0.61 μg/ml, while those of the standard catechin were 4.55 0.33
and 9.45 0.57 μg/ml, respectively. In addition, antioxidant activity of the
extract assessed by FRAP assay was comparable to catechin (1.34 0.04 and
1.48 0.03 at 100 μg/ml). The active principles in terms of total phenolics and
flavonoids contents of the extract were noted to be significantly higher than the
other extracts (85.9 1.03 mg GAE/g dried extract and 300.1 0.61 mg CE/g
dried extract). The extract additionally posed inhibitory effects against AChE
and BChE at IC50 of 221.13 and 82.51 μg/ml, while those of the standard
donepezil and galantamine were 5.21 and 8.91 μg/ml, respectively. The active
compound responsible for these activities was found to be gigantol [37].
4.17 Vanda teres
The methanolic extract of V. teres stem was isolated to yield the active compounds.
The isolated eucomic acid and vandateroside II were shown to increase cytochrome
c oxidase activity and/or expression, without enhancing cellular mitochondrial
content. In addition, they contributed to stimulate respiratory functions in
keratinocytes. Thus, these orchid isolated compounds were suggested to become
new natural ingredients for antiaging preparations to remedy age-related disorders
such as skin aging [38].
The orchids with cosmetic benefits presented above are therefore summarized
in Table 1. The therapeutic uses of orchids relevant to cosmetic benefits are shown
in Table 2. Furthermore, orchid extracts commercially available for cosmetic prep-
aration are disclosed in Table 3 together with INCI (International Nomenclature
of Cosmetic Ingredients) name and CAS (Chemical Abstracts Service) number
for further reference.
Table 1 Candidate orchid with cosmetic properties (anti-inflam., anti-inflammatory)
25
Biological activity
Botanical name Part uses In vitro Ex vivo In vivo Phytochemical active References
Ansellia Root Anti-inflam. [13]
africana Lindl.
Bulbophyllum Root Anti-inflam. [14]
scaberulum
(Rolfe) Bolus
Dendrobium Flower Antioxidant, anti- Phenolics and flavonoids [15]
spp. tyrosinase
Astringency, NHF; antioxidant and 10 phenolics; majorly sinapic [16]
antioxidant, anti- anti-MMP-2 and ferulic acid
collagenase, anti- B16F10 melanoma; 3 anthocyanins; pelargonidin,
elastase, anti-tyrosinase anti-melanogenesis, cyanidin, keracyanin
anti-tyrosinase, anti-
TRP-2
D. candidum Stem Anti-inflam. in [17]
Wall. ex Lindl. animal model
D. Stem Anti-inflam. [18]
chrysotoxum
Lindl.
D. denneanum Stem Anti-inflam. 2,5-dihydroxy-4-methoxy- [19]
Kerr phenanthrene 2-O-β-D-
glucopyranoside, 5-methoxy-
2,4,7,9S-tetra-hydroxy-9,10-
dihydrophenanthrene
D. Stem Anti-inflam. in Polysaccharides [20]
huoshanense animal model
D. nobile Stem Anti-inflam. Anti-inflam. Anti-inflam. in Alkaloids, polysaccharides, [21, 22]
Lindl. animal model ephemeranthol A,
dehydroorchinol
619
(continued)
Table 1 (continued)
620
Biological activity
Botanical name Part uses In vitro Ex vivo In vivo Phytochemical active References
D. officinale Stem Antioxidant, anti- Anti-inflam., Polysaccharides [23–29]
Kimura & inflam. immunomodulatory
Migo in animal models
Leaf Antioxidant Flavonoids [30]
D. tosaense Stem Anti-allergy, anti- Quercetin [31]
Makino inflam. in animal
model
Eulophia Tuber Anti-inflam. [14]
hereroensis
Schltr.
E. Root Antioxidant Anti-inflam. [32]
macrobulbon
(E.C.Parish &
Rchb.f.) Hook.
f.
E. petersii Stem, Anti-inflam. [14]
(Rchb. f.) pseudobulb,
Rchb. f. root
Tridactyle Root Anti-inflam. [14]
tridentata
(Harv.) Schltr.
Vanda Stem Antioxidant Anti-inflam. Anthocyanins, stilbenoid, [33–35]
coerulea Griff. imbricatin, methoxycoelonin,
ex Lindl. gigantol
V. roxburghii Leaf Wound healing in Phenolics, flavonoids, [36, 37]
R.Br. animal model gigantol
V. teres Stem Antioxidant Eucomic acid, [38]
M. Kanlayavattanakul and N. Lourith
vandateroside II
Table 2 Therapeutic uses of orchids relevant to cosmetic benefits

Botanical name Part uses Uses References
Acampe papillosa (Lindl.) Lindl. Root Cooling agent, astringent, [39]
asthma
Anocetochilus formosanus Tuber Antioxidant [40, 41]
Hayata
Arundina graminifolia (D. Don) Rhizome Antibacterial, body ache [2, 42]
Hochr. treatment
Bletilla striata (Thunb.) Rchb. f. Whole For cracked feet and hands, [3]
plant anti-inflam., emollient for
burn and skin diseases, tonic
Cleisostoma williamsonii (Rchb. Whole Astringent [39]
f.) Garay plant
Coelogyne corymbosa Lindl. Pseudobulb Wound healing [42]
C. punctulata Lindl. [36]
Cymbidium aloifolium (L.) Sw. Seed Wound healing [43]
Dendrobium alpestre Royle Pseudobulb Tonic, pimples and skin [41]
problems
D. crumenatum Sw. Leaf Pimples [36]
D. monticola Hunt & Summerh. Pseudobulb Pimples and skin eruptions
D. nobile Lindl. Pseudobulb Burn soothing
Seed Wound healing
Stem Longevity
D. ovatum (Wild.) Kranzl. Whole Emollient [39]
plant
Eulophia dabia (D. Don) Hochr. Tuber Astringent [2, 39]
Flickingeria macraei (Lindl.) Pseudobulb Tonic, skin allergy and [42]
Seidenf. eczema
Luisia tenuifolia Blume Rhizome, Emollient [39, 42]
leaf
Rhynchostylis retusa Blume Whole Emollient, wound, asthma [39, 42,
plant and skin diseases curing, 44]
inhibitory effect against
Bacillus subtilis and
Escherichia coli
Vanda tessellate (Roxb.) Hook. Whole Inflammation, tonic, scalp [2, 39, 44]
Ex G. Don plant boils
5 Conclusions
Orchid has long served as the therapeutic herb in several traditional recipes world-
wide. This medicinal herb does not only have health benefits, but their beautiful
flowers are also cultivated for floriculture purposes resulting in variety of the orchid
cultivars. The appreciable of orchid for cosmetic proposes especially for skin aging
protection and treatment are enclosed. Traditional uses of orchid for astringency or
tonic effects are later scientifically proved by its anti-inflammatory activities
622
Table 3 Orchid extracts commercially supplied for cosmetics

Use
CAS level
Commercial name INCI name number Orchid/part Claim/application Supplier (%)
Orchid Water – – – Anti-inflammatories Biogründl –
Anti-redness/anti-couperose
agents
Softening/texturing agents
Dermalab Orchid – – – Skin care (facial care, facial Dermalab 2–4
extract cleansing, body care, baby care)
Hyacinth orchid BLETILLA STRIATA ROOT Bletilla striata Regenerating/revitalizing agents 2–4
root extract EXTRACT root Anti-inflammatories
Oriental Orchid – Stem Anti-inflammatories The Garden of –
Extract-SG Antioxidants Natural Solution
Orchid Extract – – Flower Moisturizing agents Specialty Natural –
Liquid Lightening/whitening agents Product
Antioxidants
Herbal Extract VANILLA PLANIFOLIA 8024- Seed pods of Skin softening. Used in Peter Jarvis –
Orchid EG BEAN EXTRACT 06-4 Vanilla moisturizers
Herbal Extract planifolia –
Orchid EO
Herbal Extract – –
Vanilla EO
Herbal Extract
Vanilla EG
Orchid Complex CYMBIDIUM Cymbidium Emollients United-Guardian 2–10
WS GRANDIFLORUM FLOWER orchids Lubricants/slip agents
Orchid Complex EXTRACT 65381- Moisturizing
OS 09-1 Lubricious feeling
M. Kanlayavattanakul and N. Lourith
25
Orchid Extract ORCHIS MASCULA FLOWER 90082- Orchis mascula Anti-inflammatories Carrubba –
EXTRACT 24-9 flower Antioxidants
Moisturizing agents
Nourishing agents
Nourishing
Moisturizing
Akomilk ® Orchid Orchis macula Skin care (facial care, facial Akott –
cleansing, body care, baby care)
Orchid CYMBIDIUM 65381- – Emollients Ashland –
Complex™ OS GRANDIFLORUM FLOWER 09-1 Moisturizing Specialty
ester EXTRACT Chemical
ORCHID ORCHIS MASCULA FLOWER 90082- Orchid flowers Emollients Provital 0.5–5
EXTRACT H. EXTRACT 24-9 Moisturizing agents
GL.-M.S. Soothing agents
Antiaging agents
Antioxidants
Conditioning agents
Moisturizing
ORCHISTEM™ CALANTHE DISCOLOR 7732- Stem Antiaging agents
EXTRACT 18-5 Anti-wrinkle agents
Radiance promoters
Shine/radiance
Smoothness
623
associated with prevention and treatment of skin dryness as per allergic skin as well as
those of cellular inflammatory lesions that resulted from cellular oxidative stress.
These benefits are accumulating in antiaging of skin capability. In addition, antiox-
idant activities of orchid contribute on suppression or downregulate adverse effects of
oxidative stress in dermal cells surplus with diminishing overproduction of skin
melanin pigments. Accordingly, cosmetic benefits of orchid would be initiated
by the skin hydrating potency and delineated for antiaging of skin eventually. Some
of the orchid species are already commercialized in cosmetic industry. Thus, the
researchers both in academic and industrial sections are encourage to versatile those
of evidence-based ones into higher value cosmetic ingredient and finished products as
well. Despite some being already commercialized, the scientific-based information
seems inadequate. This presenting context will be therefore available for those who
are interested in cosmetic applications of orchids and for sufficient data study to
ensure efficacy and safety of orchids used in health promotion products including
cosmetics. Eventually, maximize benefits or orchid will be pursed and flow in the
stream of the consumers’ choice and preference on natural or bio-based products.
Acknowledgments Mae Fah Luang University is acknowledged for facility supports during the
manuscript preparation.
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Orchids from Basilicata: The Scent
26
Maurizio D’Auria, Simonetta Fascetti, Rocco Racioppi,
Vito Antonio Romano, and Leonardo Rosati
Contents
1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 628
2 Platanthera Orchids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 628
3 Cephalanthera Orchids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 630
4 Serapias Orchids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 632
5 Barlia robertiana . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 635
6 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 645
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 646
Abstract
HS-SPME-GC-MS analysis of the scent of some orchid species found in Basili-
cata (Southern Italy) has been performed. The analysis of Platanthera species
showed the presence of two different behaviors. The samples from Basilicata
showed the presence of lilac derivatives, while samples from Calabria or Abruzzo
had as main component benzyl benzoate. The analysis of Cephalanthera orchids
showed that, probably, scent does not have a relevant role on the pollination
strategy of the plants in comparison to the role of color. The analysis of Serapias
orchids showed the presence of alkanes and alkenes but with a lower molecular
weight than those reported in previous works on these species. Furthermore, α-
amorphene was found as main component in Serapias cordigera and Serapias
cordigera subsp. lucana. The analysis of Barlia robertiana showed a high
variability in the composition of the scent.
M. D’Auria (*) · R. Racioppi · V. A. Romano

Dipartimento di Scienze, Università della Basilicata, Potenza, Italy
e-mail: maurizio.dauria@unibas.it; rocco.racioppi@unibas.it
S. Fascetti · L. Rosati
School of Agricultural Forestry, Food, and Environmental Science, Università della Basilicata,
Potenza, Italy
e-mail: simonetta.fascetti@unibas.it; leonardo.rosati@unibas.it

628 M. D’Auria et al.
Keywords
Orchids · Platanthera · Cephalanthera · Serapias · Barlia · Volatile organic
compounds · Solid phase micrextraction · Gas chromatography – mass
spectrometry
1 Introduction
The determination of the volatile organic compounds emitted from a natural source
is smell and taste which are the oldest of our senses. They probably developed in
very primitive organisms as a means of obtaining information about chemical
changes in the organism’s environment. Animals use smell and taste to find food
and to assess its quality. The smell of food has a powerful effect on animals.
Living organisms use the chemical sense as a means of communications. If the
communication is between different parts of the same organism, the messenger is
referred to as a hormone. Chemicals used to carry signals from one organism to
another are known as semiochemicals. In the case of flowers, the aroma components
are mainly devoted to attract pollinator insects.
The main problem in the determination of the composition of the volatile organic
compounds is the correct methodology to be used in order to determine the presence of
chemical compound in so low concentration. Pawliszyn at the end of the last century
developed a new method known as solid phase microextraction (SPME) [1]. It consists
in a gas-chromatographic syringe, where the needle contains a little section of the fiber
where a stationary phase such as that present in gas-chromatographic column. The fiber
was exposed to the mixture of volatile organic compounds, and then desorbed in the
injection sector of the gas-chromatographic apparatus.
During this time, our research group used SPME in order to determine the
composition of volatile organic compounds in a lot of natural matrices. Thus, we
determined the composition of volatile compounds mixtures in olive oil [2–4], wine
[5–9], saffron [10, 11], fungi and truffles [12–17], honey [18], horseradish [19],
thymus [20, 21], origanum [20], lavandula [20], acinos [20], rosmarinus [21], laurus
[21], salvia [21], actinidia [22], crude oil [23], onion [24], shallot [24], and beer [25].
Furthermore, recently, we used this methodology in the determination of the scent of
a flower plant [26]. Based on this work, when we received the proposal for a study on
the scent of the spontaneous orchids of Basilicata (Southern Italy), we were ready to
accept the idea.
2 Platanthera Orchids
In Europe, the genus Platanthera shows the presence of 13 taxa; in Italy, six species
have been described (Platanthera algeriensis, Platanthera bifolia subsp. bifolia,
Platanthera bifolia subsp. osca, Platanthera bifolia subsp. subalpina, Platanthera
26 Orchids from Basilicata: The Scent 629
chlorantha, and Platanthera kuenkelei var. sardoa). In Basilicata, only P. bifolia

subsp. osca (Fig. 1a) and P. chlorantha (Fig. 1b) are present.
There are some differences between these two taxa. However, in P. chlorantha,
the pollinator insects (Sphingidae and Noctuidae) can get close to the spur, while, in
P. bifolia subsp. osca, this is not possible to reach nectar. Furthermore, P. bifolia
subsp. osca emitted an intense scent during the night, while P. chlorantha emitted an
aroma during all day.
Previous study on the aroma of these taxa showed some differences. In a study on
P. bifolia, Kaiser found, as main components of the aroma, methyl benzoate (58.0%)
and linalool (18.8%). A small quantity of benzyl benzoate has been determined [27].
Another study analyzed the aroma of the same orchid in different places in Pays-Bas
and Sweden and Switzerland. In the site of Sandby (Pays-Bas), linalool (75%), lilac
derivatives (44%), geraniol (35%), and methyl benzoate (32%) were found to be the
main components of the aroma [28]. In Lenstad (Pays-Bas), they found linalool
(77%) and geraniol (47%); in the site of Horn (Pays-Bas), lilac compounds (75%),
linalool (63%), and methyl benzoate (47%) were found in the scent [28]. In Sweden,
they found lilac compounds, linalool, and methyl benzoate [28]. Finally, in Swiss
plants, the main components of the aroma were methyl benzoate and linaool [28].
Another study performed in Sweden showed the presence in the aroma of methyl
benzoate, benzyl acetate, benzyl benzoate, benzyl salicylate, and lilac aldehydes
Fig. 1 (a) Platanthera bifolia subsp. osca; (b) Platanthera chlorantha

[29]. Finally, in a work performed in Belgium, 3,7-dimethyl-1,3,6-octatriene, and

santolinatriene were detected in the aroma [30].
The authors of these studies, by using different chromatographic techniques,
found different results for plants in different sites, showing an adaptive behavior.
The same behavior was observed in P. chlorantha. In Switzerland, linalool, methyl
benzoate, and geraniol were the components of the aroma [27, 28]. In Pays-Bas, lilac
aldehydes and linalool were found [28]; in Sweden, methyl benzoate and linalool
[28]; and in Belgium, the main components were 3,7-dimethyl-1,3,6-octatriene,
3-carene, and lilac aldehyde [30].
Considering the observed high variability of the results, we performed a study on
both P. bifolia subsp. osca and P. chlorantha collected in different sites [31, 32]. The
results of analyses are reported in Table 1. The structures of the compounds involved
are collected in Fig. 2.
There is not a significant variation in the composition of the aroma between
P. bifolia subsp. osca reported in Basilicata and P. chlorantha deriving from the same
region. We observed a significant variation between the composition of the aroma
(mainly formed by lilac compounds and linalool) in P. bifolia subsp. osca deriving
from sites in Basilicata and those deriving from Calabria and Abruzzo where benzyl
benzoate is the main component of the scent.
We can justify the observed behavior only admitting an adaptive modification of
the scent as reported in the previous work on these taxa in relation with possible
differences in the nature of pollinating agents. Lilac compounds are known to be
attractive compounds [29]. However, benzyl benzoate is known to be an attractive
compound present in some other Orchidaceae floral volatiles [33, 34].
3 Cephalanthera Orchids
Eight species of Cephalanthera orchids have been observed in Europe. Only three
species have been found in Basilicata. They are Cephalanthera longifolia (L.),
Cephalanthera rubra (L.), and Cephalanthera damasonium (Mill.) (Fig. 3). There
are some differences between these three species: in particular, while Cephalanthera
damasonium is autogamous, Cephalanthera logifolia and Cephalanthera rubra are
allogamous [35]. The analysis of the scent of these species has been performed, and
the results are reported in Table 2 and Fig. 4 [36].
Except for some terpenes and terpenoid compounds, cis-β-farnesene (found only
in C. longifolia), α-farnesene (found in C. rubra and C. damasonium), and pristane
(found in C. damasonium), the main components of the scent for all the species are
hydrocarbons (tetra-, penta-, hexa-, and heptadecane). It is interesting to note that,
considering that C. damasonium is a self-pollinating species, while the other two are
allogamous, there is no significant difference in the scent. Furthermore, it is known
that C. rubra mimics bellflower (Campanula) colors in order to receive pollination
service [37, 38].
At this purpose, it is noteworthy that the components of the scent of C. rubra are
completely different from those of Campanula species [39]. The mimic effect is then
26
Table 1 Volatile organic compounds from Platanthera. Only the compounds with area % higher than 3% are reported
Platanthera bifolia subsp. osca
Rifreddo Grisolia Palena
Compound (Basilicata) Pignola (Basilicata) Marsico Nuovo (Basilicata) (Calabria) (Abruzzo)
Sample Sample Sample Sample Sample Sample Sample
1 2 3 4 1 2 3
Area %
1 Linalool 8.83 10.00 17.77
2 Methyl benzoate 9.04
3 Lilac aldehyde A 6.16
4 Lilac aldehyde B 11.80 8.61 3.01 7.80 8.64 8.02
5 Lilac aldehyde C 3.11 1.39

6 Lilac alcohol A 10.73
7 Lilac alcohol B 11.48 7.41 9.90 13.08 13.52 15.10 15.86 12.99
8 Lilac alcohol C 11.39 10.10 14.96 10.13 18.45 9.08 9.75 13.99
9 Lilac alcohol D 11.43 7.50 21.02 32.45 34.28 32.80 25.07 34.53
10 Geraniol 4.05
11 Benzyl benzoate 55.83 74.72
12 Galaxolide 6.66
13 Benzyl 2- 3.19
hydroxybenzoate
Platanthera chloranta
Satriano
(Basilicata)
Area %
3 Lilac aldehyde A 15.13
4 Lilac aldehyde B 5.39
9 Lilac alcohol D 25.78
631
OH CO2Me
CHO CHO
O O
H H
3 4
2
1
CHO CH2OH CH2OH

O O O
H H H
5 6 7
CH2OH CH2OH
O O OH
H H
8 9 10
O OH O
O O
O
11 12 13
Fig. 2 Volatile compounds found in P. bifolia subsp. osca and P. chlorantha
due to a similar color and not to the presence of the similar attractive compounds in
the aroma.
The absence of differences between different species with different pollinating
mechanisms, and the absence of correlation between the scent of C. rubra and
Campanula species, induces us to consider that, probably, scent has not an important
role to attract insects.
4 Serapias Orchids
Five species of Serapias orchids can be observed in Basilicata. We can find allog-
amous diploid Serapias cordigera L. subsp. cordigera, Serapias cordigera subsp.
lucana [40], Serapias vomeracea subsp. longipetala (Ten.), autogamous diploid
Fig. 3 (a) Cephalanthera longifolia (L.); (b) Cephalanthera rubra (L.); and (c) Cephalanthera
damasonium (Mill)
Table 2 Volatile organic compounds of Cephalanthera orchids from Basilicata. Only the com-
pounds with area % higher than 3% are reported
C. longifolia C. rubra C. damasonium
Compound Area %
14 Tetradecane 3.49
15 cis-β-Farnesene 5.04
16 Pentadecane 48.94 5.52 7.41
17 α-Farnesene 3.60 4.35
18 Hexadecane 6.00 7.01
19 Ethyl dodecanoate 5.18
20 Heptadecane 3.83 5.15 5.92
21 Pristane 3.04
22 Hexadecanal 3.08
14 15
16
17 18
CO2Et
19 20
CHO
21 22
Fig. 4 Volatile organic compounds found in Cephalanthera orchids
Serapias parviflora Parl., and sexual deceptive tetraploid Serapias lingua L. (Fig. 5).
We analyzed the scent of all the species recovered in Basilicata.
Previous studies performed in Calabria and in Campania on this species have
been performed on the hexane extracts of the labellum [41, 42]. They found
longchain (with more than 21 carbon atoms) alkanes and alkenes in different ratio.
S. lingua showed 45% alkanes and 55% alkenes; in S. cordigera, they found 32%
alkanes and 68% alkenes; S. parviflora had 93% alkanes and 7% alkenes. In another
study, different ratios have been found [43]. In this case, in S. lingua, 40/60 (alkenes/
alkanes) was found, while S. cordigera showed a 30/70 ratio. The results of our
analyses are reported in Table 3 and Fig. 6 [44].
In S. cordigera subsp. Cordigera, the analysis did not find alkanes and alkenes
with high molecular weights as in the previous works on this species; on the
contrary, the main component was α-amorphene. The same main component was
found in S. cordigera subsp. lucana. In S. vomeracea, the main components were an
alkane (pentadecane) and some alkenes; however, in previous work [41], hepta-
cosane and tetracosane were the main components. We found only alkanes and
alkenes with lower molecular weight. The same behavior has been observed with the
other species.
The different molecular weight of the compounds we determined in comparison
with those found in the previous works can depend on intrinsic limits of SPME
Fig. 5 (a) Serapias cordigera L. 1753; (b) Serapias cordigera subsp. lucana R. Lorenz and V. A.
Romano 2014; (c) Serapias vomeracea subsp. longipetala (Ten.) H. Baumann and Künkele 1989;
(d) Serapias lingua L. 1753; and (e) Serapias parviflora Parl. 1837
determination. On the other hand, the compounds whose presence has been deter-
mined in the previous work in this field [41–43] are compounds with a high boiling
point and very low vapor pressure, and probably, they cannot represent the actual
components of the aroma, but only the precursors.
5 Barlia robertiana
Barlia robertiana (Loisel.) Greuter (Orchidaceae) (Fig. 7) is among the numerous

orchids which present nonrewarding flowers, and it is obligatorily insect pollinated.
It has a steno-mediterranean distribution [45] and can be found in open habitats (e.g.,
clearings in the scrublands and termophilous woods, dry grasslands, and roads
edges), usually on silty-sandy soils. In Italy, it is present in all the regions except
for some of the Alpine sector [46]. In the Basilicata region, (Southern Italy) it is
rather widespread from the coast to the inner mountainous area of Apennines.
Unlike most of the mediterranean orchids, which are pollinated by specialized
bees occupying narrow ecological niches, B. robertiana attracts many different
636
Table 3 Volatile organic compounds of Serapias orchids from Basilicata. Only the compounds with area % higher than 3% are reported
Compound S. cordigera S. lucana S. vomeracea S. lingua S. parviflora
23 2-Undecanone 3.31
24 Pentadecene 7.05
16 Pentadecane 11.13 25.43 7.41 11.03
25 α-Amorphene 28.85 38.06
18 Hexadecane 3.96
26 3-Heptadecene 3.02
20 Heptadecane 3.96 4.55 13.89 11.70 6.38
11 Benzyl benzoate 4.18 3.80
27 Octadecane 3.24 4.49
28 Isopropyl myristate 7.80
29 Farnesylacetaldehyde 3.15
30 5-Nonadecene 14.48
31 Nonadecene 5.16
32 Nonadecane 6.38 4.28
33 2-Heptadecanone 3.42
34 Isopropyl palmitate 4.86
35 Propyl undecanoate 5.15
36 Heneicosane 4.14 4.31
37 Ethyl elaidate 3.39 3.47
38 Ethyl oleate 15.33
39 Ethyl stearate 3.33
M. D’Auria et al.
H
O
H
24 24
25
26 27
O
CHO
O
28 29
30 31
32 33
O
O
O
O
34 35
36
O
O
O
O
38
37
39
Fig. 6 Volatile organic compounds found in Serapias orchids
species of pollinators [47] such as Apoidea (Hymenoptera) and Cetoniidae (Cole-

optera) [48, 49]. In this context, it is worth to note as an appropriate knowledge about
pollination and reproduction biology is not only important for scientific purpose, but
is also crucial to develop effective management and conservation actions of rare and
endangered plants [26] such as terrestrial orchids.
Overall, within the 11 samples, 68 compounds were identified (min 14; max 28;
and mean 19.2) with a high between samples variability both for composition and
Fig. 7 Barlia robertiana
abundance of VOCs (Figs. 8 and 9, Table 4). The most frequent compounds
(i.e., found in >60% of plants) were hexadecane, which was present in 9 samples,
β-bisabolene, β-sesquiphellandrene and ethyl dodecanoate found in 8 samples, and
caryophyllene, cis-α-bergamotene, δ-selinene, and octadecane which were detected
in 7 samples. As concern dominant compounds, each sample returned a different
result: We alternatively found as dominant in the samples: α-zingiberene, i-propyl
14-methyl-pentadecanoate, farnesol, p-Menth-8-en-1-ol, and pristane. An exception
was represented by β-sesquiphellandrene and verbenone which were found both
prevailing in 2 samples. β-Sesquiphellandrene was dominant in the samples Pisticci
2 and Vietri 2, whereas verbenone was dominant in Pisticci 1 and Sant’Arcangelo 2.
Remarkably, these 2 pairs of samples group together localities resulting rather
distant, geographically and ecologically.
The results of VOCs compositions for each sample are reported in Table 4. Two
samples from the lower course of Basento river, the closest to the Ionian coast
(Pisticci municipality, 10 m a.s.l.), were found and analyzed (Table 4). The first
one showed the presence of verbenone (45.22%), a monoterpene, α-zingiberene
(6.88%) (Fig. 9), a sesquiterpene, and ethyl tetradecanoate (15.96%). Verbenone
shows a camphor mentholic flavor and acts as a pheromone. Also α-zingiberene is
a pheromone [50]. The second sample showed a completely different composition
of the aroma (Table 4). The main products were caryophyllene (8.51%) (Fig. 9),
Fig. 8 Barlia robertianai

during the determination
β-sesquiphellandrene (29.06%), and farnesal (5.81%). All the compounds are

sesquiterpenes. β-Sesquiphellandrene has a herbal fruity flavor. It is interesting to
note that α-zingiberene and β-sesquiphellandrene are compounds with a very
similar structure (Fig. 9), both deriving from a possible cyclization of E,Z-farnesyl
cation (Fig. 10). On the contrary, caryophyllene derives from E,E-farnesyl cation
(Fig. 10).
Two samples were collected at Sant’Arcangelo (218 m amsl) (Table 4). The first
one showed the presence of α-zingiberene (17.14%), δ-selinene (9.06%), i-propyl
14-methylpentadecanoate (3.74%), β-curcumene (Fig. 9) (2.43%), nonadecane
(2.06%), longipinene (1.77%), ethyl dodecanoate (1.75%), and nerolidol (Fig. 9)
(1.69%). The second sample has as component of the scent verbenone (31.48%),
\δ-selinene (17.59%), pristane (9.61%), longipinene (4.49%), limonene (Fig. 9)
(1.65%), decanal (2.12%), and β-curcumene (1.56%). Longipinene and δ-selinene
are both sesquiterpenes deriving from E,E-farnesyl cation (Fig. 10). Pristane is
present in shark liver, fish oil, ambergris, plankton, and anise fruits [51].
The samples collected at Tolve (307 m) showed the presence of the following
compounds: sample 2, caryophyllene (10.11%), ethyl dodecanoate (8.29%), i-propyl
Monoterpenes
OH
OH
OH
Sesquiterpenes
OH
OH
H H H
OHC
H H H H
Terpenoids
Fig. 9 Volatile tepenes and terpenoids found in Barlia robertiana scent
14-methyl-pentadecanoate (12.26%), tetradecane (3.59%), Z-β-farnesene (1.49%)

(Fig. 9), β-sesquiphellandrene (2.94%), hexadecane (3.09%), and ethyl tetra-
decanoate (2.57%); sample 3, β-sesquiphellandrene (8.39%), farnesol (36.63%)
26
Table 4 Volatile organic compounds of Barlia robertiana from different sampled sites in Basilicata region (Italy). Only the compounds with area % higher than
3% are reported
Pisticci Pisticci S. S. Arcan. Tolve Tolve Vietri Vietri
1 2 Arcan.1 2 2 3 Pomarico 1 2 Potenza Savoia
Compound Area %
α-Pinene 7.87
β-Myrcene 3.26
β-Phellandrene 4.51
D-limonene 3.21
γ-Terpinene 5.24
p-Menth-8-en-1-ol 21.68
α-Terpineol 5.39
Verbenone 45.22 31.48
Citronellol 17.96
Methyl citronellate 3.01 3.61
Tetradecane 3.09
α-Zingiberene 6.88 17.14
Caryophyllene 8.51 10.11 3.68 3.07 5.21 6.75 17.96
Cis-α-bergamotene 14.04 4.15
E-β-farnesene 7.10
Longipinene 4.40
(continued)
641
642
Table 4 (continued)
Pisticci Pisticci S. S. Arcan. Tolve Tolve Vietri Vietri
1 2 Arcan.1 2 2 3 Pomarico 1 2 Potenza Savoia
Compound Area %
β-Bisabolene 4.61 8.98 3.17 5.32
δ-selinene 9.06 17.59
β-Sesquiphellandrene 29.06 8.39 17.59 43.08 15.14 8.97 25.05
Ethyl dodecanoate 8.29 3.76 2.60
Hexadecane 3.09 4.01 3.36
Pristane 9.61 22.57
Farnesol 36.63 19.36
Farnesal 5.81
Ethyl tetradecanoate 15.96
Dihydrofarnesol 22.23
i-Propyl 14-methyl- 3.74 12.26
pentadecanoate
M. D’Auria et al.
Fig. 10 Biosynthesis of some sequiterpenes

(Fig. 9), dihydrofarnesol (22.23%), E-β-farnesene (1.97%), and β-bisabolene

(2.13%).
In the sample collected at Pomarico (348 m amsl), several monotepenes were
found: β-myrcene (3.26%) (Fig. 2), β-phellandrene (4.51%), limonene (3.21%), γ-
terpinene (5.24%), p-menth-8-en-1-ol (21.68%), and α-terpineol (5.39%). Further-
more, some sesquiterpenes were found: caryophyllene (3.68%), β-bisabolene
(4.61%), and β-sesquiphellandrene (17.59%).
The site of Vietri (410 m) gave two samples. They contain almost the same
compounds in different amounts: citronellol (1.77% in sample 1 and 17.96% in
sample 2), methyl citronellate (3.01% and 3.61%), tridecane (2.35% and 1.29%),
tetradecane (3.09% and 1.39%), caryophillene (3.07% and 5.21%), cis-α-
bergamotene (Fig. 9) (14.04% and 4.15%), β-bisabolene (8.98% and 3.17%), δ-
selinene (2.49% and 1.16%), β-sesquiphellandrene (43.08% and 15.14%), ethyl
dodecanoate (3.76% and 2.63%), hexadecane (4.01% and 1.80%), and heptadecane
(2.24% and 1.49%).
A sample collected at Potenza (727 m msl) showed the following compounds: α-
pinene (7.87%), caryophillene (6.75%), β-bisabolene (2.00%), β-sesquiphellandrene
(8.97%), pristane (22.57%), and farnesol (19.36%).
Finally, the sample collected at Savoia di Lucania (760 m) showed the presence of
caryophillene (17.96%), E-β-farnesene (7.10%), β-bisabolene (5.32%), and β-
sesquiphellandrene (25.05%).
Considering only the main components, we observed this behavior: In Pisticci,
the main components were verbenone and β-sesquiphellandrene. In Sant’Arcangelo,
the main components were α-zingiberene and verbenone. Tolve showed the presence
of caryophyllene, i-propyl 14-methyl-pentadecanoate, and farnesol. The site of
Pomarico showed as main components p-menth-8-en-1-ol and sequiphellandrene.
The main components found at Vietri were β-sesquiphellandrene, and citronellol.
The analyses of the sample at Potenza showed as the main component pristane. The
main component of the sample collected at Savoia di Lucania was β-
sesquiphellandrene.
The high variability of Barlia robertiana floral scent detected in this study
involved both qualitative and quantitative aspects of VOCs composition. Composi-
tions of our samples were not in agreement with the volatile organic compounds
found in the scent of Barlia robertiana (sub Himantoglossum robertianum (Loisel.)
P. Delforge) in a previous work [52]. For example, concerning the main components,
Gallego and coworkers found as prevailing compounds: α-pinene, β-pinene, and
limonene, whereas our samples were characterized by β-sesquiphellandrene,
verbenone, zingiberene, i-propyl 14-methyl-pentadecanoate, farnesol, p-menth-8-
en-1-ol, and pristane. However, α-pinene, β-pinene, and limonene were often
detected in our samples too, but with lower abundance. The high variability of floral
scent detected between sampled sites from Basilicata does not seem at all related in
any way to environmental factors or to geographic locations. Therefore, these
findings suggest that there is no adaptation by the species to local conditions or
specific communities of pollinators. A high variation in floral signals such as color
and floral scent has been highlighted in several deceptive orchids [53]. Even if some
compounds detected in our study can act as pheromone (e.g., verbenone and α-
zingiberene), this specific function probably is not realized in Barlia robertiana. The
characteristic morphology and coloring of the flowers of Barlia robertiana seem to
exclude attraction mechanisms widely used by other deceptive orchids such as
shelter imitation, brood-site imitation, psudoantagonism, or sexual attraction. In
fact, B. robertiana strategy to attract pollinators involves together scent, appearance
of the flowers, and showiness of the plant during the flowering phase. This pollina-
tion system of rewardless species that does not imitate specific nectariferous plants is
defined as “generalized food deception” [54]. In this framework, it has been
suggested as in such species a huge variation in floral characters both within and
between populations may behave the effect to disrupt the associative learning of
visiting insects [55]. In fact, rewardlessness can be considered a dangerous strategy
because some insects can soon learn to avoid such flowers [56], having as a
consequence reduced fruit set via reduced pollinium removal and deposition. The
causes of the evolution of rewardlessness have been frequently discussed but are still
incompletely known [57, 58] because few experiments specifically addressed this
question. A specific study involving Barlia robertiana [49] demonstrated probably
for the first time an evolutionary advantage for rewardlessness in the Orchidaceae. In
particular, in this study, the experiments comparing flowers artificially supplied with
sucrose solution showed an increasing probability (approximately ten times) of
pollinia removing by pollinators for flowers without nectar (i.e., the natural condi-
tion), probably leading as a consequence to an higher seed paternity. The striking
results are due to the fact that bees visiting a rewardless flower were more vigorous
to seek a potential reward inside the corolla; therefore, pollinia were more likely
detached.
However, it must be stressed again that this (male) reproductive advantage can
only occur if pollinating insects do not learn to associate the floral signals of a
species with the lack of nectar inside the flowers.
6 Conclusion
HS-SPME-GC-MS analysis of the scent of some orchid species found in Basilicata

(Southern Italy) has been performed. The analysis of Platanthera species showed the
presence of two different behaviors. The samples from Basilicata showed the
presence of lilac derivatives, while samples from Calabria or Abruzzo had as main
component benzyl benzoate. Probably, it is the result of an adaptive behavior. The
analysis of Cephalanthera orchids showed that, probably, scent does not have a
relevant role on the pollination strategy of the plants. The scent has the same
composition both for allogamous and autogamous species. Alkanes are the main
components of the scent. The analysis of Serapias orchids showed the presence of
alkanes and alkenes but with a lower molecular weight than those reported in
previous works on these species. Furthermore, α-amorphene was found as a main
component in Serapias cordigera and Serapias cordigera subsp. lucana. The anal-
ysis of Barlia robertiana showed a high variability in the composition of the scent.
At this time, it is not possible to understand the reason for this high variety of
compounds in the scent. This situation leads us to think that we do not yet perfectly
know the real reasons that lead to an effective composition of the aroma. The study
of this phenomenology will have to be continued.
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Index
A Angolan flora, 144

Abruzzo, 645 Angraecopsis, 115, 138
Abscesses, 523 Angraecum, 111
Acampe carnata, 367 Ansellia africana, 613
Acampe praemorsa, 367 acetylcholinesterase (AChE) inhibitory
Acanthphippium bicolor, 368 activity, 444
ACC deaminase (ACCD), 178 anti-inflammatory activity, 444
Accidental/necrotic cell death, 399 antimicrobial and membrane damaging
Acclimatization, 18 activity, 442–443
Acclimatized, 509 bioassays and in-vivo model based
Acidic soils, 34 studies, 445
Activated charcoal (AC), 235–236 drug discovery, 446
Active photosynthetic tissues, 138 endophyte mapping and metabolite
Additives, 509 production, 446–447
Aeranthes, 115 ethno-medicinal, horticultural and
Aerial roots, 331 traditional uses, 438
Agrobacterium, 428 ex situ conservation, 438–442
Agrobacterium-mediated transformation, 304 micropropagation protocol, 442
Agrobacterium tumefaciens, 304 molecular biology approaches, 444–445
Agronomic practices, 428 natural populations, 438
Alamar blue assay, 396 next-generation sequencing, 446
Alien animals, 121–123 phytochemical constitution, 444
Alien plants, 119–121 roots of, 437
Alkaline soils, 34 short-term storage using protocorm like
Alluvial forests, 44 body, 441
Alps, 43 stem and root infusions, 436
Alternaria brassicicola, 219 Anthesis, 498
Altitude, 80 Anthocyanins-rich extracts, 613
Alzheimer’s disease, 444 Anthracnose, 336
Amino acids, 230 Anthroposols, 38–39
1-Aminocyclopropane-1-carboxylate Anti-aging, 424
(ACC), 177 Anti-angiogenesis effect, 407, 408
α-Amorphene, 634 Anticancer, 497, 503
Amplified fragment length polymorphism bibenzyl derivatives, 394
(AFLP), 476 drugs, 390
Anacamptis morio, 79–80 fluorenone derivatives, 396
Angiogenesis, 407, 425 phenanthrene derivatives, 395
Angiogenin, 407 Anti-collagenase, 424
Angiospermae, 496 Anti-hyaluronidase, 425

Reference Series in Phytochemistry, https://doi.org/10.1007/978-3-030-38392-3
650 Index
Anti-inflammation, 614 Bioactivities, 499

Anti-inflammatory, 502, 523, 525, 527, 528 Biodiversity, 108–110, 112, 117, 125–127, 154
activity, 425 hotspots, 109
effect, 615 Biotechnological tools, for Vanda sp. culture
Anti-metastasis effect, 406, 407 and propagation, 601
Antimicrobial properties, 523, 526, 527 Biotechnology, 428
Antioxidant, 502, 523, 617 Biotransformation, 349, 428
activity, 423–424 Birch, 42
Anti-proliferative activity, 425 Bird pollinated, 117
Apennines, 635 2,3-Bis(2-methoxy-4-nitro-5-sulfophenyl)-2H
Aphrodisiac, 501, 502 tetrazolium-5-carboxyanilide inner salt
Apoidea, 637 (XTT), 398
Arable land, 78 β-Bisabolene, 638, 644
Arachidonic acid (AA), 308 Black aphid, 279
Arginine, 210 Black River Gorges National Park, 115
Aroma, 334 Black rot, 285–286
Arundina gramminifolia, 369 Bleeding, 576
Ascomycetes, 57 Bletilla, 574
Ascorbic acid, 305 anti-fibrosis activity, 584
Asparagales, 496 anti-inflammatory activity of, 580
Astavarga, 419 anti-microbial activity, 582–583
Asymbiotic, 509, 512 anti-mitotic activity, 585
germination, 510 anti-neuroinflammatory activity, 585
Atopic dermatitis, 616 anti-oxidant activity, 580–581
ATP-dependent tyrosine kinase (Akt), 406 anti-tyrosinase activity, 585
Autogamous, 509 anti-ulcer activity, 585, 586
Autogamy, 60, 519 anti-viral activity, 584
Autotrophy, 16 bioactive compounds, 576–579
Auxins, 203, 229, 236 commercial importance, 586
Ayurveda, 363–365, 501 cytotoxic, antitumor and anticancer activity,
581–582
B hemostatic activity, 583–584
Bacteria, 527 immunological activity, 584
Bacterial soft rot, 286 terrestrial genus, 575
Balkan Peninsula, 46 traditional uses, 576
Balkans, 41 would healing activity, 584
Banana homogenate, 230–233 Bletilla striata, 220, 575, 579
Barlia robertiana, 635–645 Bletilla tuber, 558
Basic fibroblast growth factor (bFGF), 407 B lymphocytes, 308
Basidiomycetes, 57 Bogs, 48
Basilicata, 635, 645 Boisduval scale, 281
Batatasin, 422 Brazil, 518
Batatasin III, 524 Brazilian biomes, 519
Bean Common Mosaic Virus (BCMV), 337 Bronchitis, 419, 420
Beans, 334 Browning, 335
Bean Yellow Mosaic Virus (BYMV), 337 Bulbophyllum, 111
Bedrock types, 23 B. albidum, 370
Beech, 41 B. cariniflorum, 370
Benthamia, 115 B. nilgherrense, 370–371
Benzyl benzoate, 629, 630 B. scaberulum, 613
Bibenzyls, 404, 535 Bulbophythrin, 422, 423
Bidirectional nutrient flow, 208 Bulbophythrin A, 422
8,8’-Biflavidin, 549 Burns, 576
Index 651
C Colletotrichum sp., 336

Caffeic acid, 422, 423 Commercialization, 575
Calabria, 645 Competition, 23, 119
Calanquinone A, 554 Conditioning process, 335
Calanthe, 114 Confusarin, 524
Calanthe mild mosaic virus, 288 Coniferous forest, 42, 83
Calcareous geological substrates, 21 Conservation, 504, 586
Calcium carbonate, 37 biology, 74
Calcium oxalate, 37 management areas, 123
Cancer cell lines, 400 Consolidated layer of ecosystems (KVES), 76,
Candida albicans, 426 79, 83, 86, 92, 95, 100, 102
Capacity building, 126 Consumer preference, 314
Capsule, 202, 509 See also Orchid consumption
Carbon, 210 Consumers perceptions, 315
Cardiovascular diseases, 390 Corallorhiza maculata, 203
Caryophyllene, 638, 639, 644 Cortex, 202
Catechin, 422, 423 Cosmetic formulations, 612
Caveolin-1 (Cav-1), 406 Cowpea Aphid-Borne Mosaic Virus
Cell division cycle 42 (Cdc-42), 406 (CABMV), 337
Cellular aging, 616 Crepidium acuminatum, 416
Cellular anti-inflammation, 617 Cross-pollination, 60
Cellular damage, 611 Cryopreservation, orchid, 243–244
Cellular mitochondrial content, 618 D cryo-plate method, 250–251
Cellular oxidative stress, 615 dormant bud method, 241–242
Cephalanthera, 630–632, 645 droplet-vitrification method, 246, 247
C. damasonium, 630 encapsulation-dehydration method,
C. longifolia, 630 242–245
C. rubra, 80–85, 630 encapsulation-vitrification method,
Cervical cancer, 599 245–246
Cetoniidae, 637 slow freezing method, 242
Chalazal end, 204 V cryo-plate method, 247–248
Chalk, 21 vitrification method, 242
Chemokines, 425 Cryptopus, 118
Chemotaxonomy, 306 Cultivation of terrestrial orchids, 16
Chitinases, 207 Curing process, 334
Chitosan, 234–235 Cutaneous ageing, 599
Chlorogenic acid, 422, 423 Cyanidin, 596
Chronologic aging, 610 Cyclooxygenase (COX), 444
Chrysomphalus aonidum, 281 Cymbidium
cis-α-bergamotene, 638, 644 agroclimatic requirements, 275–277
cis-β-farnesene, 630 anthracnose, 284–285
CITES, 109 aphids, 279–280
Citronellol, 644 bacterial brown rot, 286
Climate, 90 bacterial soft rot, 286
Climatic data, 78 black rot, 285–286
CMV-infected plants, 337 breeding, 266–269
Coccus hesperidum, 281 chromosome number, 264
Coconut water (CW), 233–234 cosmetics, 291
Coelogyne cristata, 371 cultivation, 275–279
Coelogyne stricta, 372 food, 291
Coelonin, 541, 560, 597 genetic diversity, 263–264
Collagen, 424, 425 growing media for, 277
Collagenase, 614 medicinal use, 290–291
652 Index
Cymbidium (cont.) human cancer cell lines, 405

natural hybridization, 265–266 induced cell apoptosis, 405
nematode disease, 287 phenanthrene, 405
nutrient management, 277–278 solvent extracts, 399
plant health management, 279–289 T-cell (Dalton’s) lymphoma, 404
pre and post zygotic barriers, 264–265
propagation, 269–275
scale insects, 280–282
viral diseases of orchids, 287–289 D
watering, 279 Dactylorhiza fuchsii, 83–86
Cymbidium aloifolium, 372–373 Dactylorhiza hatagirea, 373
Cymbidium Mosaic Virus (CymMV), 287, 337 Databases, 76
Cynorkis, 112 D cryo-plate method, 250–251
Cypripedin, 555 Deciduous forest, 40
Cyrtopodium, 518 Deforestation, 110, 117
Cyrtopodium glutiniferum, 519 Deforested land, 331
Ames Test, 525 Delphinidin, 596
antimicrobial activity, 526 Dementia, 149
antinociceptive, 523 Denaturing gradient gel electrophoresis
arbutin, 527 (DGGE), 182
bacterial infection, 526 Denbinobin, 553
bactericidal effect, 527 Dendrobium, 307, 308, 613
cultivation, 519 agarose gel, 479
cyrtopodine, 526 alkaloids, 456
dihydroformononetin, 527 aneuploidy, 475
endangered, 519 anti-allergic, 457
ethnopharmacological, 519 anticancer compounds, 391, 392
ethnopharmacological aspects, 521–526 anticancer effect, 392, 408
extract, 527 anti-inflammatory, 457
flower detail, 522 anti-oxidative, 456
folliculitis, 526 anti-pyretic, 456
genotoxicity assessment, 523 auxins, 461
inflammation, 526 axillary, 474
microbicidal efficacy, 527 axillary branching, 464
mutagenesis model, 527 bacterial contamination, 473
ointments, 526 banding profiles, 478
polyphenols, 526, 527 basal MS medium, 472
seed propagation, 520 bud break, 467
on skin lesions treatment, 526–527 bulblet formation, 465
strain, 527 callus induction, 469
syrups, 522 callus phase, 464
taxonomic solution, 521 cataracts, 457
therapeutic alternative, 526 chitosan molecules, 464
traditional medicine, 523 clonal evaluation, 483
wounds, 522 clonal fidelity, 474
Cyrtopodium paludicolum, 520 coconut water, 460
Cytokines, 425 contaminants, 460
Cytokinins, 203, 229, 236 cost-effective, 476
Cytotoxic activity, 503 cut-flower industry, 475
Cytotoxicity effect cytokinins, 461
bibenzyls, 404 DNA markers, 455
biomarkers, 404 DNA methylation, 475
fluorenone, 406 dominant markers, 479
Index 653
elite mother plants, 467 plantlet conversion, 465

epiphytic, 457 PLB conversion, 471
ethidium bromide, 480 PLB formation, 471
ethnic community, 455 polyamine, 466
ethnomedicinal, 456 polymorphism, 476
explant disinfection, 475 polyploidy, 475
floral stalk, 463 polysaccharides, 458
functional traits, 476 post cryopreservation, 477
genomic DNA, 479 potato extract, 470, 472
genotypes, 478 potting mixture, 464
growmore, 468 pre-existing meristems, 474
growth hormone, 461 primers, 476
hardening, 467 principal coordinate analysis, 481
hormone-free, 466 proliferation, 463
hormone-induced stress, 478 propagate, 458
identical clones, 467 proteins, 390
immature seeds, 459 protocorm proliferation, 469
immunity, 458 protocorms, 460
infusion, 456, 457 pseudobulbs, 456
in vitro culture, 408 putrescine, 466
in vitro flowering, 471 re-differentiation, 469
in vitro propagation, 478 regenerants, 477
IRAP markers, 479 regenerated shoots, 465
ISSR markers, 482 regeneration, 455
kinetin, 479 reproducible bands, 479
latent buds, 474 resolving power, 477
Mantel test, 477 ripe banana, 474
mature seeds, 460 rooting, 459
meristematic, 463 rooting frequency, 471
meristemoid induction, 473 rooting response, 466
meristem-tips, 472 root initiation, 462
meta-topolin, 466 root length, 470
micropropagation, 459 scorable bands, 480
molecular markers, 479 seed germination, 461, 468
monochromatic light spectra, 473 seedlings, 463
monographs, 390 seeds derived protocorms, 468
monomorphic banding, 477 shoot buds, 463
MS medium, 467 shooting initiation, 462
mungbean sprout, 470 shoot multiplication, 463
natural remedies, 391 shoot nodes, 468
necrosis, 464 shoot tips, 459
nutrient, 460 skin rashes, 457
ophthalmic disorders, 458 somaclonal variation, 455, 467
organic nutrients, 458 somatic embryogenesis, 468
organogenesis, 466 spherule formation, 471
organogenic response, 465 subcultures, 476
ornamental, 454 sucrose, 465
petal tissues, 473 surface sterilization, 460
pharmacological activities, 390 therapeutic application, 455
phenotypical variations, 477 tonic, 458
phytochemical, 454, 455 traditional medicines, 457
picloram, 465 transposable elements, 479
plantlet, 470 unripe capsules, 461
654 Index
Dendrobium (cont.) Doxorubicin, 422, 425

UPGMA dendrograms, 481 DPPH, 598
vermiculite, 466 Droplet-vitrification method, 246, 247
vitamins, 459 Drought, 14
Dendrobium candidum, 614 Drying process, 335
Dendrobium chemical compound, 305, 306
Dendrobium chrysanthum, 216
Dendrobium chrysotoxum, 614 E
Dendrobium crumenatum, 301 E-cadherin, 406
Dendrobium huoshanense, 614 Ecological keystone, 123
Dendrobium lineale, 301, 306, 307 Ecological niche, 55
Dendrobium nobile, 615 Ecological plasticity, 21
Dendrobium nodosum, 374 Ecology, 108, 116–117
Dendrobium officinale, 615 Ecosystem restoration, 125
Dendrobium plicatile, 375 Ectomycorrhizae, 180
Dendrobium tosaense, 616 Ectorhizosphere, 179
Dendrochrysanene, 558 Ehrlich ascites carcinoma (EAC), 398
de novo green vanillin, 343 Eicosanoic acid, 420
Denthyrsinin, 524 Elastase, 614
Deodar, 427 Elastin, 424
Dermal damage, 611 Elevational gradient, 115
Development rate index (DRI), 439 Ellagic acid, 423
Diabetes, 423 Embryogenesis, 304
Diaphananthe, 138 Encapsulation-dehydration method, 242–245
Diaspis boisduvali, 281 Encapsulation-vitrification method, 245–246
Dichromothrips nakahari, 282 Endemic, 519
Diethylene glycol, 422, 423 Endemic species, 140, 141, 144
Dihydrofarnesol, 644 Endemism, 109
Dihydrophenanthrene, 307 Endomycorrhiza, 180, 208
9,10-Dihydrophenanthrenes, 499, 541, 546 Endophytes, 342, 349, 447
3-(4,5-Dimethylthiazol-2-yl)-5- Endophytic fungi, 203
(3-carboxymethoxyphenyl)-2- Endoplasmic reticulum, 207
(4-sulfophenyl)-2H-tetrazolium Endorhizosphere, 179
(MTS), 398 Endosphere, 179, 182, 191
3-(4,5-Dimethylthiazole-2-yl)-2,5- Environmental variables, 75, 76, 78, 79, 94
diphenyltetrazolium bromide Ephemeranthoquinone B, 554, 556
(MTT), 397 Epipactis palustris, 86–90
Disease and pest management, 262 Epiphytes, 137
Disperis, 114 Epiphytic, 113
Dispersal, 116 Epithelial to mesenchymal transition (EMT),
Distribution, 139, 140, 142, 143 406
Anacamptis morio, 79 Epulorhiza, 520
Cephalanthera rubra, 83 Escherichia coli, 426, 526
Dactylorhiza fuchsii, 83 Estrogen receptor (ER), 404
E. palustris, 90 Ethnopharmacological, 502
Neottia nidus-avis, 90 Ethyl dodecanoate, 638, 639
Neottia ovata, 94 Ethylene, 178
Platanthera chlorantha, 97 Eugenol, 422
Diversity, 74, 136, 138, 150 Eulophia, 497
DNA, 205 distribution and botanical description,
DNA barcoding of A. africana, 447, 448 498–499
DNA fingerprinting, 444 E. alta, 512
Dormant bud method, 241 E. campestris, 501
Index 655
E. cucullata, 509 Fungal-associated bacteria, 191–192

E. cullenii, 510 Fungal invasion, 202
E. dabia, 375, 504 Fusarium, 335
E. epidendrea, 375, 501
E. herbacea, 376, 501, 503
E. hereroensis, 616 G
E. hormusjii, 504 Garrigue, 44
E. macrobulbon, 503, 617 Gastric ulcer, 576
E. nuda, 376, 499, 502 Gastrodia, 115
E. ochreata, 377, 499 Genetic(s), 163
E. petersii, 509, 617 diversity, 429
E. promensis, 510 Genetically modified organisms (GMOs), 304
E. streptopetala, 509 Genotype, 428
ethnopharmacological uses, 501–502 Geographic distribution, 55
evidenced-based pharmacological activities, Geraniol, 629, 630
502–504 Germination, 14, 56, 504
in vitro regeneration, phytochemical Germination rate index (GRI), 439
production and conservation, 504–512 Gigantol, 422, 423, 524, 597
phytochemical constituents, 499–501 Glioblastoma, 599
Eulophiol, 499, 503 Glioblastoma multiforme (GBM), 405
Europe, 10 Global warming, 19
Evidence-based management, 126 Glucomannan, 524
Exocytosis, 207 Glucoside A, 333
ex situ conservation, Vanda sp., 601 Glucoside B, 333
Extinction, 108–110, 122, 123, 127 Glucosides, 334
Extracellular matrix (ECM), 406 Glucovanillin, 333
Glycosides, 334, 422
Goodyera pubescens, 203
GPS position, 76
F Grassland vegetation, 45
Farnesal, 639 Grazing, 53
α-Farnesene, 630
E-β-farnesene, 644
Farnesol, 644 H
Fatty acids, 420, 422 Habenaria, 122
Fens, 48 H. commelinifolia, 377
Feral pigs, 122 H. edgeworthii, 378
Ferulic acid, 343, 345, 349 H. intermedia, 378
Fibrosis, 425 H. longicorniculata, 378
Fir, 42 H. marginata, 379
Fire, 54 H. roxburghii, 379
Flavidin, 597 Habitat(s), 76, 80, 86, 94
Flavones C-glycosides, 576 destruction, 110, 438
Flavor, 334 heterogeneity, 9
Flore des Mascareignes, 112 Harvesting, 118
Floriculture, 497 Heavy metals, 39
Flowering, 14 Hemostatic agents, 575
Fluorenone, 406, 596 Herbaceous vegetation, 45–49
Focal adhesion kinase (FAK), 406 Herbaria Amboinesis, 305
Forest, 93, 125 Herbarium collections, 112
Forest-type land, 331 Hermaphroditic, 332
Fruiting, 14 Heterotrophy, 16
Fumaric acid, 420, 422 Hexadecane, 638
656 Index
Hircinol, 541 Island, 108, 110–113, 115–117, 119, 122,

Holomycotrophic habitats, 416 127, 128
Homeotic genes, 154, 158, 159 Isorhamnetin O-glycoside, 422, 423
Hornbeam, 41 Itaconic acid, 420, 422
Human dermal microvascular endothelial IUCN, 498
cells, 408 IUCN Red Lists, 504
Human umbilical vein endothelial cells
(HUVECs), 408
Humus, 38 J
Hyaluronidase, 425 Jackknife procedure
8-Hydroxy-2-deoxyguanosine, 404 Anacamptis morio, 80
Hydroxy benzoic acid, 422 Cephalanthera rubra, 82
Hydroxybenzyl-substituted Dactylorhiza fuchsii, 86
monophenantheres, 543 Epipactis palustris, 88
Hydroxy cinnamic acid, 422 Neottia nidus-avis, 91
Hydroxyl-benzyl derivatives, 576, 596 Neottia ovata, 94
Hyphae, 204 Platanthera chlorantha, 97
Hyphal coils, 210 Java deer, 123
Hyphal mass, 208 Jumellea, 111
K
Killing process, 334
I Knudson-C, 512
Igneous rock, 21
Imbricatin, 597
Immune system, 399 L
Immunology, 615 Lactic dehydrogenase (LDH), 397
Indigenous African ornamental orchids, 145 Lilac aldehyde, 630
Indole-3-acetic acid (IAA), 178, 180, 188, Lilac compounds, 629, 630
229, 520 Limestone, 21
Indole-3-butyric acid, 427 Limonene, 422, 423, 644
Indole butyric acid (IBA), 229 Linalool, 629, 630
Inflammatory cytokines, 404 Lineweaver-Burk plot method, 561
Inflammatory mediators, 611 Linoleic acid, 420
Inflorescence, 332, 520, 593 Linolenic acid, 420
International Union for the Conservation of Lipar acid C, 422, 423
Nature, 118 Liparisphenanthrene A, 555
Inter-retrotransposon targeted amplified region Lipopolysaccharide (LPS), 308
(IRAP), 476 Lithophytes, 114, 593
Inter-simple sequence repeat (ISSR), 476 L-leucine, 421, 422
Invasive, 109 L-Methionine, 421
Invasive alien species, 122, 127 Longipinene, 639
In vitro assays L-phenylalanine, 421, 422
LDH, 397 L-threonine, 421, 422
MTS, 398 Lusianthridin, 541
MTT, 397 Lusianthrin, 422, 423
RCGI, 396 L-valine, 421, 422
SRB, 397
TBDE, 396
XTT, 398 M
In vitro free radical-scavenging assays, 559 Macaca fascicularis, 117
In vivo assay, 398 Macaque, 122
Ipomeamarone, 218, 221 Macrophage, 580
Index 657
Macrosiphum luteum, 280 Microcoelia, 115

MADS box genes Micropropagation, orchid, 226–227, 237–241
class A, 160 activated charcoal, 235–236
class B, 160–161 amino acids, 230
class C and D, 161–162 banana homogenate, 230–233
and orchid flower development, 158–160 chitosan, 234–235
Magnesium, 37 coconut water, 233–234
Malaxis acuminate culture media, 228
anti-aging, 424 plant growth regulators, 229–230
anti-microbial properties, 426 potato extract, 233
anti-oxidant activity, 423–424 protocorm, 227
anti-proliferative activity, 425 solid/semi-solid supports, 236–239
bioactive compounds from, 422–423 sugar, 228–229
botanical description, 417–418 vitamins, 230
in medicinal uses, 419–420 Micropropagation, 427, 512, 520
nutritional composition, 420–422 Mid-domain effect (MDE), 8
propagation and cultivation efforts of, Mild chlorosis, 337
426–428 Mildew, 337
pseudobulbs of, 417 Minerals, 420, 421
sun protection factor, 424 Mires, 48
Malaxis acuminatum, 379 Molecular breeding, 429
Malaxis muscifera, 380 Molluscs, 123
Malondialdehyde (MDA), 404 Monocot, 202
Malonic acid, 420, 422 Monomorphic banding patterns, 477
Mantel test, 477 Monophenanthrenes, 539–546
Margelliantha, 138 Mother’s Day, orchid consumption, 317
Marsh vegetation, 49 Mountain reserves, 117
Mascarenes, 109, 111, 113, 117, 123 Mowing, 53
Mass extinction, 108, 109 Mutualistic interaction, 208
Matrix metalloproteinases (MMPs), 406 Mycellium, 209
Mauritius, 109–110 Mycoheterotrophic fungi, 211
Mauritius orchids, 111 Myco-heterotrophy, 16
alien animals, 121–123 Mycophagy, 207
alien plants, 119–121 Mycorrhiza helper bacteria (MHB), 179–181
deforestation, 117 Mycorrhizal fungi, 15, 56, 121
diversity and endemism, 112–114 Mycorrhizosphere, 180
ecology, 116–117 Myxotrophy, 38
harvesting, 118
indirect effects, 123
threats, 123–126 N
types and distribution, 113–116 Naphthalene acetic acid, 427
MaxEnt, 75, 78, 83, 90 Nature Reserves, 110
Meadows, 90, 93, 97 N-cadherin, 406
Mean survival time (MST), 398 Necrotic brown spots, 336
Medicinal aromatic plants (MAP), 436 Nematode disease, 287
Medicinal orchids, 436 Neotropical scope, 518
Mediterranean area, 5 Neottia ovata, 94–96
p-menth-8-en-1-ol, 644 Nepal, 592
Metabolomic analysis, 523 Next-generation sequencing (NGS), 446
Metalloproteinases (MMPs), 406 Nitrate, 210
Metamorphic rocks, 23 Nitric oxide (NO) radicals, 598
meta-Topolin Riboside (mTR), 439 Nitrogen, 210
Methoxycoelonin, 597 Non-rewarding pollination systems, 59
658 Index
Nuclear factor κB, 503 market configuration, 314

Nudol, 541 means of seduction, 324
Nutrients, 35 more attractive, 320
Mother’s Day, 317
O motivational factors, buy flowers, 321
Oak and oak-hornbeam forests, 41, 76 number of times consumers bought, 316
Oceanic islands, 108, 109 orchid type, 321
Octadecane, 638 overpay, 322
Octadecenoic acid, 423 own use, 316
Odontoglossum ringspot virus, 288 performance in global market, 318
Oligopeptide, 211 pre-purchase tension, main reasons, 320
Orchidaceae, 4, 207, 496–498, 574, 592 product accessibility, 320
African countries, uses of Orchidaceae profile, 315
species in, 145–150 relationship marketing, 322–324
botanical description and systematic, repeated purchases, 323
137–138 research related, 316
description, 534 residence decorating, 320
distribution of orchids, 137 vs. roses, 317
endemism of, 139–145 routine decisions, 321
phenanthrenes (see Phenanthrenes) satisfaction of desire, 322
Orchid-associated bacteria, 181–182 seasonal dates, 318
fungal-associated bacteria, 191–192 seduction processes, 317
phyllosphere-associated bacteria, 188–189 stimulus, 322
rhizosphere-associated bacteria, 189–190 substitute products, 315
root endosphere-associated bacteria, symbolic values, 322
190–191 tendency, 324
seed-associated bacteria, 183–188 Valentine’s Day, 317
Orchid consumption value, species, color, size, payment terms,
advantages, 323 durability, 318
age group of purchases, 316 Orchids of sub-Saharan Africa
average purchase, 323 data collection, 137
behavior of buying, 321 endemism of Orchidaceae species, 139–145
birthdays, 317 geographic areas, 138–139
colors/smells, 321 Organic acids, 420, 422
consumer's decision, 318 Ornamental, 496
consumer willingness, 324 Ornithogalum Mosaic Virus (OrMV), 337
continuing satisfaction, 323 Osteosarcoma (OS), 405, 407
decoration in homes, 320 Over-exploited, 512
desire, decision-making process, 321 Oxidative stress, 612, 616
desired flower cost, 321 Oxoflavidin, 560
economic development, 314 Oxygen-heterocyclic ring, 544
expectations of person, 323
and family income, 316 P
family income, 324 Palmitic acid, 420, 422
floriculture market, 314 Paraphyletic, 498
flower species, 314 Parenchyma cells, 202, 208
for gift, 316 Parishin, 596
hierarchical categorization, 315 Pastures, 46
important species, 314 p-Coumaric acid, 422, 423
inner impulse, 322 P-cymene, 422
lack of information, 314 Pelatons, 202, 204, 207
level of education, 317 Pentadecane, 634
loyalty, 323 Peroxisomes, 207
Index 659
Phaius, 114 genetic engineering, 304

Phalaenopsis amabilis, 304 hybrids, 303
Pharmacological benefits, 616 in vitro seed germination, 304
Pharmacopoeia, 305 mass propagation, 304
Phenanthrene(s), 307, 405, 497, 499, 503, 596 objectives, 303
anti-inflammatory activity, 556–558 qualitative and quantitative characters, 302
antimicrobial activity of, 555–556 Plant growth-promoting bacteria (PGPB),
antioxidant activity, 558–559 177–179, 192
antiproliferative activity, 553–555 Plant growth regulators (PGRs), 229–230
chemotaxonomical significance, 551–552 Plant vitrification solution (PVS), 242, 246
derivatives, 596, 597 Platanthera, 631, 645
description, 534 P. bifolia subsp. bifolia, 628
di-and triphenanthrenes, 547–550 P. bifolia subsp. osca, 628, 630
monophenanthrenes, 539–546 P. chlorantha, 629, 630
occurence in Orchidaceae species, 535 Platanthera chlorantha, 96–100
pharmacological activities, 552–561 Platelet aggregation, 584
sources, 534 Platelet-derived growth factor (PGF), 407
Phenanthrofurans, 543 Platylepis, 124
Phenanthropyrans, 543 Pleistocene, 116
Phenanthroquinones, 545, 552, 562 Plicatol-B, 541
Phloridzin, 422 Pods, 333, 334
Phorophytes, 115 Policy National Parks, 126
Phosphorus, 36, 209 Pollinating agents, 630
Photoaging, 425 Pollination, 332, 519
Photosynthetic activity, 14 systems, 116
Phyllosphere-associated bacteria, 188–189 Pollinator, 19
Phylogenetic studies, 113 insects, 628
Phylogeography, of A. africana, 447 Pollinium, 156
Phytoalexins Polyherbal energetic tonic, 417
accumulation, 216 Polyploidy, 498
bioactive chemical compounds, 216 Polysaccharides, 581, 612, 614, 615
biosynthesis, 217–218 Polystachya, 111
compounds, 218 Poplar plantations, 51
defence mechanism, 217 Population genetics, 429
gymnosperms, 220 Potassium, 36
human health, 220–221 Potential distribution map, 79
inhibitory compounds, 216 Cephalanthera rubra, 83
monocot family orchidaceae, 216–217 Dactylorhiza fuchsii, 86
orchids, 220 Epipactis palustris, 91
plant hormones, 219 Neottia nidus-avis, 93
types, 219 Neottia ovata, 96
Phytomolecules, 163, 497, 499, 503, 512, 575 Platanthera chlorantha, 99
Pigments, 165 Precipitation, 14–15, 80, 90, 93
Pine forests, 42 Prenyl-substituted phenanthrenes, 541
β-Pinene, 644 Primary metabolites, 300
α-Pinene, 644 Principal coordinate analysis (PCoA), 481
Pinnaspis buxi, 281 Pristane, 639, 644
Pinosylvin, 220 Pro-inflammatory cytokine, 420
Plant breeding Propanoic acid, 420
acetosyringone, 305 Propenoic acid, 420, 422
activities, 302 i-propyl 14-methyl-pentadecanoate, 640, 644
agricultural plants, 302 Protected areas, 116
biotechnology, 303 Proteus mirabilis, 426
660 Index
Protocatechuic acid, 422 Self-fertilization, 332

Protocorm, 36, 202, 204, 206, 227, 510, 521 Self-pollination, 60
Protocorm-like bodies (PLBs), 227, 233, 234, δ-Selinene, 638, 639
239, 248, 408, 509 Sensory profiles, 343
Pseudobulbs, 138, 417, 498, 518, 520 Sepals, 520
Pseudomaquis, 44 Seppe vegetation, 45
Pseudomonas aeruginosa, 426 Sequential agglomerative hierarchiacal nested
cluster analysis (SAHN-clustering), 307
R Serapias, 645
Random amplified polymorphic DNA (RAPD), S. cordigera, 634
307, 476 S. cordigera L. subsp. cordigera, 632
Rasna, 592 S. cordigera subsp. cordigera, 634
Ras-related C3 botulinum toxin substrate S. cordigera subsp. lucana, 632, 634
1 (Rac-1), 406 S. lingua, 634
Rats, 123 S. parviflora, 634
Reactive oxygen species (ROS), 406 S. vomeracea, 634
Red spider mite, 283–284 S. vomeracea subsp. longipetala, 632
Regulated cell death, 399 Serpentine, 23
Reintroduction, 512 β-Sesquiphellandrene, 638–640, 644
Reproductive biology, 156 Sesquiterpene glycosides, 308
Resazurin cell growth inhibition (RCGI), 396 Sexual deception, 59
Respiratory disorders, 575 Shade houses, 331
Restriction fragment length polymorphism Shan-Ci-Gu, 552
(RFLP), 476 Sibutramine, 422, 423
Resveratrol, 221 Siddha, 365
Rewarding pollination, 59 Siderophores, 209
Rheumatism, 419, 420 Silicate substrates, 21, 25
V. testacea treatment, 595 Simple sequence repeat (SSR), 476
Rhizoctonia fungi, 206 markers, 429
Rhizomatous structure, 417 Single nucleotide polymorphism (SNP), 476
Rhizome-like bodies (RLBs), 510 Skin aging, 611, 618
Rhizomes, 5 Skin hyperpigmentation, 612
Rhizosphere, 178–180, 182, 189 Slope, 79, 85, 90, 94
Rhizosphere-associated bacteria, 189–190 Slow freezing method, 242
Rhyncostylis retusa, 381 Slug, 406
River reserves, 117 Smoke-water (SW)-treated A. africana
Root cortex, 204–206 seeds, 439
Root endosphere-associated bacteria, 190–191 Sodium dodecyl sulfate (SDS), 397
Root system, 12 Solid phase microextraction (SPME), 628, 634
Rostellum, 332 South Bohemia, orchids species distribution
Ruderal, 52 Anacamptis morio, 79–82
Rutin, 422 Cephalanthera rubra, 80–85
Rwanda flora, 142 Dactylorhiza fuchsii, 83–86
Epipactis palustris, 86–91
S materials and methods, 75–79
Salep, 502 Neottia nidus-avis, 90–94
Scape, 498 Neottia ovata, 94–96
Scrub vegetation, 44 Platanthera chlorantha, 96–99
Secondary metabolites, 164, 166, 300, 305, Southeast Asia, 593
408, 420, 421, 428, 501 Southern Europe, 34
Sectarianism, 136 Spathoglottis plicata, 301
Seed(s), 116 Species, 97
germination, 206, 427 distribution models, 74–75
Seed-associated bacteria, 183–188 richness, 115
Index 661
Spermidine, 427 precipitation, 14–15

Spirolactone-substituted phenanthrenes, 544 root systems, 6
Spruce, 42 soil moisture, 26
Staphylococcus aureus, 426 soil organic matter, 37
Starch, 204 soil pH, 34–35
Start codon targeted polymorphism species-area relationship, 11
(SCoT), 476 temperature, 11–14
Stearic acid, 420, 422 Tetra-, penta-, hexa-and heptadecane, 630
Stilbenoids, 523, 534, 576, 596 Therapeutic, 497
Succinic acid, 420, 422 Thidiazuron, 427
Sugar, 228–229 Threatened medicinal plant, 418
Sulforhodamine B (SRB), 397 Thrips, 282
Sulphorhodamine B, 425 Ti scale, 281
Sun protection factor, 424 Tissue culture, 520
Superoxide anion, 581 Tocopherol, 420, 421
Superoxide dismutase (SOD), 307 Toll-like receptor, 503
Suppressed endosperm, 504 Toxicity, 390
Swards, 48 Toxoptera aurantii, 279
Sweating process, 334 Traditional African Pharmacopeia (TAP), 436,
Symbionts, 203, 206–207 438, 444
Symbiosis, 179, 180, 207, 209, 211 Traditional Chinese medicine (TCM), 390
Symbiotic fungal associations, 504 Transforming growth factor-α (TGF-α), 407
Symbiotic relation, 121 Transforming growth factor-β (TGF-β), 407
Synchronized growth, 509 Transporter proteins, 211
Synthetic seed technology, 441 Trans-resveratrol, 218
Synthetic vanillin, 344 Trehalose, 210
Tribal medicine, 366
Tridactyle tridentata, 617
T TRP-2 inhibition, 614
Taeniophyllum, 111 True-to-typeness, 509
Tail immersion test, 600 Tryphan blue dye exclusion (TBDE), 396
Tall-herb vegetation, 45 Tuberculosis, 576
Taxonomic inflation, 113 Tubers, 5
Temperature, 83, 85 Tumorigenesis, 391
Terrestrial, 496 Tumor necrosis factor-α (TNF-α), 407, 503
Terrestrial orchids, 4 Tyrosinase, 614
anthropogenic vegetation, 49–60 inhibitor effect, 527
anthroposols, 38–39
atmospheric humidity, 15
U
calcium and magnesium, soil, 37
Ultramafic rocks, 23
climate change, 19–20
Unani, 365
climatic factors, 11–20
Unweighted pair-group method arithmetic
ecology and rarity, 6
average (UPGMA), 307
elevation, 7–9
Uttarakhand, 418, 419, 427
forest and scrub vegetation, 40–45
generalists and specialists, 54–56
geological substrate, 20–25 V
herbaceous vegetation, 45–49 Valentine’s Day, orchid consumption, 317
high-altitude areas, 9 Vanda sp., 309, 592
latitude and longitude, 9–11 analgesic activity, 600
light regime, 16–19 anti-acetylcholinesterase activity, 601
nitrogen, phosphorus and potassium in soil, anti-aging activity and cosmetic
35–37 applications, 599–600
pollination system, 58–60 anti-bacterial and antifungal activity, 599
662 Index
Vanda sp. (cont.) odorless, 334

anti-butyrylcholinesterase activity, 601 orchidaceae, 330
antidepressant activities, 600 pathways, 347
antinociceptive activity, 600 phytochemistry, 333, 334
antioxidant and anti-inflammatory activities, pod post-harvest processing methods, 353
597–598 pods, 333, 346, 347
bioactive compounds, 306, 307, 596 pollination, 332
botanical description, distribution and production, 344
ecology, 593 quality, 345
commercialization, 601 species, 353
cosmetics, 601 substrate specificity, 342
cytotoxic activity, 599 synthesis, 342, 349
extinction, 593 terroir effect, 352
floriculture industry, 601 tropical plant, 331
hepatoprotective activity, 599 viral diseases, 337, 338
illegal collection for trade, 601 volatiles, 351
neuroprotective activity, 600 Vanilla Mosaic Virus (VanMV), 337
ornamental purpose, 602 Vanillic acid, 347
phytochemistry, 595–596 Vanillin, 334, 344, 346
traditional uses, 593–595 by fungal microorganisms, 349
V. coerulea, 593, 594, 617–618 Vanillyl alcohol, 347
V. cristata, 593, 594 Vascular endothelial growth factor (VEGF),
V. parviflora, 594 407
V. roxburghii, 618 V cryo-plate method, 247–248
V. spathulata, 381, 594 Verbenone, 638, 639, 644
V. teres, 618 Vernalization, 14
V. tessellata, 381–382, 595 Vimentin, 406
V. testacea, 595 Viral diseases, 337
V. tricolor, 301, 306, 307 V. testacea treatment, 595
wound-healing activity, 601 Vitality rejuvenating, 428
Vanilin, synthesis of, 346 Vitality strengthening, 428
Vanilla Vitamins, 230, 420, 421
aromatic, 330 Vitrification, 240, 242, 243, 246, 251
chain shortening, 347 Volatile organic compounds, 628, 631, 633,
characteristics, 342 634, 636, 637, 641, 644
commerce and food, 338 Volatiles, 351
cultivation, 331, 332, 335, 338
curing process, 334, 335, 346
description, 330 W
endophyte community, 351 Watermelon Mosaic Virus (WMV), 337
enzyme, 347 Weeding, 124
ferulate, 347 Wet meadows, 47
flavor metabolite guaiacol, 351 Wisteria Vein Mosaic Virus (WVMV), 337
flavors, 344, 345, 351, 353 Wound healing, 618
flavors and aroma compounds, 331 Writhing model, 600
flowering, 332, 333
fungal diseases, 335–337 Y
glucosides, 333 Yellow aphid, 280
hemi-epiphytic orchid, 331
hosts, 353
indigenous, 330 Z
mature fruit, 333 α-Zingiberene, 639
metabolites, 345 Zingiberene, 644

Orchids Phytochemistry, Biology and Horticulture: Jean-Michel Mérillon Hippolyte Kodja

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Reference Series in Phytochemistry

More information about this series at http://link.springer.com/series/13872

With 122 Figures and 50 Tables

ISSN 2511-834X ISSN 2511-8358 (electronic)

Part I: Biogeography, Biodiversity, and Environmental Factors

Villenave d’Ornon, France Jean-Michel Mérillon

Part I Biogeography, Biodiversity, and Environmental Factors ... 1

1 The Role of Ecological Factors in Distribution and Abundance of

2 Which Environmental Factors Drive Distribution of Orchids?

3 Diversity, Ecology, and Conservation of Mauritius Orchids ..... 107

4 Diversity of Orchids from Continental Sub-Saharan Africa . . . . . 135

5 Orchid Biodiversity and Genetics . . . . . . . . . . . . . . . . . . . . . . . . . . 153

Part II Biology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 173

6 Orchid-Associated Bacteria and Their Plant Growth

7 Mycorrhiza in Orchids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 201

8 Phytoalexins in Orchids ................................. 215

Part III Horticulture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 223

9 Micropropagation of Some Orchids and the Use of

10 Cymbidium: Botany, Production, and Uses . . . . . . . . . . . . . . . . . . . 261

11 Biotechnology Approaches on Characterization, Mass Propagation,

12 Preferences of Orchid Consumers and the Substitute

Part IV Agri-food Applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 327

13 Vanilla: Culture, Reproduction, Phytochemistry, Curing,

14 Vanillin: Biosynthesis, Biotechnology, and Bioproduction . . . . . . . 341

Part V Phytochemistry and Medicinal Properties ............. 359

15 Ethnobotany and Recent Advances in Indian Medicinal

16 Traditionally Used Medicinal Dendrobium: A Promising Source

17 A Review on Phytochemistry, Nutritional Potential, Pharmacology,

18 Phytochemistry, Pharmacology, and Conservation of

19 Dendrobium sp.: In vitro Propagation of Genetically Stable

Part VI Cosmetic Applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 607

25 Orchid Extracts and Cosmetic Beneﬁts . . . . . . . . . . . . . . . . . . . . . 609

Anjana Adhikari-Devkota Graduate School of Pharmaceutical Sciences, Kuma-

Thoungamba Amom Department of Biotechnology, Manipur University,

Nandeibam Apana Department of Biotechnology, Manipur University, Canchipur,

Cesar Arriagada Laboratorio de Biorremediación, Facultad de Ciencias

Cláudia Baider The Mauritius Herbarium, Agricultural Services, Ministry of

Paromik Bhattacharyya Research Centre for Plant Growth and Development,

Nipawan Jitsopakul Department of Plant Science, Textile and Design, Faculty of

Vito Antonio Romano Dipartimento di Scienze, Università della Basilicata,

Vladan Djordjević and Spyros Tsiftsis

© Springer Nature Switzerland AG 2022 3

8 Orchid Mycorrhizal Fungi . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 56

All representatives of the family Orchidaceae in Europe are terrestrial, belonging to

Epipactis includes a total of 79 species and subspecies, which are widespread in

2 Elevation, Latitude, Longitude, and the Orchid Species-

2.2 Latitude and Longitude

It should be emphasized that latitude is one of the most important determinants of

Attention should be called to a study of morphological variation in populations of

2.3 Orchid Species-Area Relationship

The inﬂuence of temperature on orchid populations varies depending on altitude and

umbilicata (a, f photos V. Djordjević; b–e, g–j photos S. Tsiftsis)

Air temperature affects germination and seedling establishment, photosynthetic

precipitation and its effects on orchid populations were also demonstrated by an

3.3 Atmospheric Humidity

The relationship between atmospheric humidity and abundance of orchid

previous growing season, whereas populations of Gymnadenia conopsea and

species inhabiting a forest ﬂoor can acclimate to reduced illumination by changing

of ﬂower production in the species Cephalanthera longifolia and to a decrease in the

3.5 Orchid Species Distribution and Its Patterns in a World

4 Geological Substrates and Soil Properties

4.1 Geological Substrates

A signiﬁcant number of terrestrial orchid species in the Balkans occur on