Professional Documents
Culture Documents
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.
Orchids Phytochemistry,
Biology and Horticulture
Fundamentals and Applications
This Springer imprint is published by the registered company Springer Nature Switzerland AG.
The registered company address is: Gewerbestrasse 11, 6330 Cham, Switzerland
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:
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.
v
Contents
vii
viii Contents
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
Djah François Malan UFR des Sciences de la Nature (SN), Université NANGUI
ABROGOUA, Abidjan, Ivory Coast
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
NANGUI ABROGOUA, Abidjan, Ivory Coast
Potshangbam Nongdam Department of Biotechnology, Manipur University,
Canchipur, Manipur, India
Noufou Doudjo Ouattara 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
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
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
ABROGOUA, Abidjan, Ivory Coast
Centre Suisse de Recherches Scientifiques en Côte d’Ivoire, Abidjan, Ivory Coast
Part I
Biogeography, Biodiversity, and
Environmental Factors
The Role of Ecological Factors in
Distribution and Abundance of Terrestrial 1
Orchids
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
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
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)
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.
2.1 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]
8 V. Djordjević and S. Tsiftsis
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,
1 The Role of Ecological Factors in Distribution and Abundance of Terrestrial. . . 9
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].
10 V. Djordjević and S. Tsiftsis
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
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
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
1 The Role of Ecological Factors in Distribution and Abundance of Terrestrial. . . 15
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
1 The Role of Ecological Factors in Distribution and Abundance of Terrestrial. . . 17
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)
18 V. Djordjević and S. Tsiftsis
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
20 V. Djordjević and S. Tsiftsis
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].
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].
1 The Role of Ecological Factors in Distribution and Abundance of Terrestrial. . . 21
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].
22 V. Djordjević and S. Tsiftsis
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)
1 The Role of Ecological Factors in Distribution and Abundance of Terrestrial. . . 23
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
1 The Role of Ecological Factors in Distribution and Abundance of Terrestrial. . . 25
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
26 V. Djordjević and S. Tsiftsis
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].
Table 1 (continued)
The soil
Orchid taxon pH range Soil pH values/literature sources
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
Epipactis leptochila subsp. 6.63–7.36 6.63–7.36 [25]
nauosaensis
Epipactis leptochila subsp. 5.1–7.5 5.1–7.5 [126]
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)
1 The Role of Ecological Factors in Distribution and Abundance of Terrestrial. . . 29
Table 1 (continued)
The soil
Orchid taxon pH range Soil pH values/literature sources
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)
30 V. Djordjević and S. Tsiftsis
Table 1 (continued)
The soil
Orchid taxon pH range Soil pH values/literature sources
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)
1 The Role of Ecological Factors in Distribution and Abundance of Terrestrial. . . 31
Table 1 (continued)
The soil
Orchid taxon pH range Soil pH values/literature sources
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]
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]
V. Djordjević and S. Tsiftsis
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
34 V. Djordjević and S. Tsiftsis
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
1 The Role of Ecological Factors in Distribution and Abundance of Terrestrial. . . 35
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].
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
1 The Role of Ecological Factors in Distribution and Abundance of Terrestrial. . . 37
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.
40 V. Djordjević and S. Tsiftsis
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].
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
42 V. Djordjević and S. Tsiftsis
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.
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
44 V. Djordjević and S. Tsiftsis
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].
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].
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)
V. Djordjević and S. Tsiftsis
1 The Role of Ecological Factors in Distribution and Abundance of Terrestrial. . . 51
6 Effects of Disturbance
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
56 V. Djordjević and S. Tsiftsis
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].
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
1 The Role of Ecological Factors in Distribution and Abundance of Terrestrial. . . 57
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
1 The Role of Ecological Factors in Distribution and Abundance of Terrestrial. . . 59
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
60 V. Djordjević and S. Tsiftsis
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
1 The Role of Ecological Factors in Distribution and Abundance of Terrestrial. . . 61
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
62 V. Djordjević and S. Tsiftsis
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.
References
1. Chase M, Christenhusz M, Mirenda T (2017) The book of orchids: a life-size guide to six
hundred species from around the world. Ivy Press, London
2. Dressler RL (1981) The orchids: natural history and classification. Harvard University Press,
Cambridge, MA
3. Swarts ND, Dixon KW (2009) Terrestrial orchid conservation in the age of extinction. Ann Bot
104:543–556
4. Chase MW, Cameron KM, Freudenstein JV, Pridgeon AM, Salazar G, van den Berg C,
Schuiteman A (2015) An updated classification of Orchidaceae. Bot J Linn Soc 177:151–174
5. Hágsater E, Dumont V (eds) (1996) Orchids: status, survey and conservation action plan.
IUCN, Gland/Cambridge, UK
6. Whigham DF, Willems JH (2003) Demographic studies and life-history strategies of temperate
terrestrial orchids as a basis for conservation. In: Dixon KW, Kell SP, Barrett RL, Cribb PJ
(eds) Orchid conservation. Natural History Publications, Kota Kinabalu
1 The Role of Ecological Factors in Distribution and Abundance of Terrestrial. . . 63
7. Waterman RJ, Bidartondo MI (2008) Deception above, deception below: linking pollination
and mycorrhizal biology of orchids. J Exp Bot 59:1085–1096
8. Averyanov LV (1990) A review of the genus Dactylorhiza. In: Arditti J (ed) Orchid biology:
reviews and perspectives, vol V. Timber Press, Oregon
9. Delforge P (2006) Orchids of Europe, North Africa and the Middle East. A & C Black, London
10. Tatarenko I (2007) Growth habits of temperate terrestrial orchids. In: Cameron KM, Arditti J,
Kull T (eds) Orchid biology – reviews and perspectives, vol IX. The New York Botanical
Garden Press, Bronx/New York
11. Tsiftsis S, Štípková Z, Kindlmann P (2019) Role of way of life, latitude, elevation and climate
on the richness and distribution of orchid species. Biodivers Conserv 28:75–96
12. Buttler PK (1991) Field guide to orchids of Britain and Europe. The Crowood Press, Swindon
13. Tsiftsis S, Antonopoulos Z (2017) Atlas of the Greek orchids, vol I. Mediterraneo Editions,
Rethymno
14. Antonopoulos Z, Tsiftsis S (2017) Atlas of the Greek orchids, vol II. Mediterraneo Editions,
Rethymno
15. Nikolić T (2019) Flora Croatica Vol. 4: vascular flora of the Republic of Croatia. Excursion
flora, Alfa d.d., Zagreb
16. GIROS (2009) Orchidee d’Italia – Guida alle orchidee spontanee. Il Castello, Cornaredo (Milano)
17. Kreutz CAJ, Çolak AH (2009) Türkiye Orkideleri. Rota Yayınlari, Istanbul
18. Vogt-Schilb H, Pradel R, Geniez P, Hugot L, Delage A, Richard F, Schatz B (2016) Responses
of orchids to habitat change in Corsica over 27 years. Ann Bot 118:115–123
19. Bernardos S, García-Barriuso M, Sánchez-Anta MA, Amich F (2007) Composition, geograph-
ical affinities and endemism of the Iberian Peninsula orchid flora. Nord J Bot 25:227–237
20. Baumann H, Künkele S, Lorenz R (2006) Die Orchideen Europas. Mit angrenzenden
Gebieten. Eugen Ulmer KG, Stuttgart
21. WCSP (2019) World checklist of selected plant families. Royal Botanic Gardens, Kew. http://
apps.kew.org/wcsp/
22. Tranchida-Lombardo V, Cafasso D, Cristaudo A, Cozzolino S (2011) Phylogeographic pat-
terns, genetic affinities and morphological differentiation between Epipactis helleborine and
related lineages in a Mediterranean glacial refugium. Ann Bot 107:427–436
23. Kull T, Hutchings MJ (2006) A comparative analysis in decline in the distribution ranges of
orchid species in Estonia and the United Kingdom. Biol Conserv 129:31–39
24. Djordjević V, Tsiftsis S, Lakušić D, Stevanović V (2016) Niche analysis of orchids of
serpentine and non-serpentine areas: implications for conservation. Plant Biosyst 150:710–719
25. Tsiftsis S, Tsiripidis I, Karagiannakidou V, Alifragis D (2008) Niche analysis and conservation
of the orchids of east Macedonia (NE Greece). Acta Oecol 33:27–35
26. Djordjević V, Tsiftsis S, Lakušić D, Jovanović S, Stevanović V (2016) Factors affecting the
distribution and abundance of orchids in grasslands and herbaceous wetlands. Syst Biodivers
14:355–370
27. Acharya KP, Vetaas OR, Birks HJB (2011) Orchid species richness along Himalayan
elevational gradients. J Biogeogr 38:1821–1833
28. Zhang SB, Chen WY, Huang JL, Bi YF, Yang XF (2015) Orchid species richness along
elevational and environmental gradients in Yunnan, China. PLoS One 10:e0142621
29. Zhang Z, Yan Y, Tianb Y, Lib J, Hea JS, Tanga Z (2015) Distribution and conservation of
orchid species richness in China. Biol Conserv 181:64–72
30. Jacquemyn H, Micheneau C, Roberts DL, Pailler T (2005) Elevational gradients of species
diversity, breeding system and floral traits of orchid species on Réunion Island. J Biogeogr
32:1751–1761
31. Ackerman JD, Trejo-Torres JC, Crespo-Chuy Y (2007) Orchids of the West Indies: predict-
ability of diversity and endemism. J Biogeogr 34:779–786
32. Štípková Z, Traxmandlová I, Kindlmann P (2016) Determinants of orchid species diversity in
Latin America. Lankesteriana 16:293–297
33. Barbaro L, Dutoit T, Grossi JL (2003) Influence des facteurs agro-écologiques sur les
assemblages d’orchidées dans les pelouses calcicoles du Vercors (Préalpes, France). Bot
Helv 113:63–79
64 V. Djordjević and S. Tsiftsis
34. Djordjević V (2018) Spatial distribution and ecology of orchids (Orchidaceae) of western
Serbia. Dissertation, University of Belgrade (in Serbian with English abstract)
35. Pellissier L, Vittoz P, Internicola AI, Gigord LDB (2010) Generalized food-deceptive orchid
species flower earlier and occur at lower altitudes than rewarding ones. J Plant Ecol
3:243–250
36. Körner C (2007) The use of ‘altitude’ in ecological research. Trends Ecol Evol 22:569–574
37. Chen YK, Yang XB, Yang Q, Li DH, Long WX, Luo WQ (2014) Factors affecting the
distribution pattern of wild plants with extremely small populations in Hainan Island, China.
PLoS One 9:e97751
38. Colwell RK, Lees DC (2000) The mid-domain effect: geometric constraints on the geography
of species diversity. Trends Ecol Evol 15:70–76
39. Traxmandlová I, Ackerman JD, Tremblay RL, Roberts DL, Štípková Z, Kindlmann P (2018)
Determinants of orchid species diversity in world islands. New Phytol 217:12–15
40. Schödelbauerová I, Roberts DL, Kindlmann P (2009) Size of protected areas is the main
determinant of species diversity in orchids. Biol Conserv 142:2329–2334
41. Blinova IV (2012) Intra-and interspecific morphological variation of some European terrestrial
orchids along a latitudinal gradient. Russ J Ecol 43:111–116
42. Wotavová K, Balounová Z, Kindlmann P (2004) Factors affecting persistence of terrestrial
orchids in wet meadows and implications for their conservation in a changing agricultural
landscape. Biol Conserv 118:271–279
43. Janečková P, Wotavová K, Schödelbauerová I, Jersáková J, Kindlmann P (2006) Relative
effects of management and environmental conditions on performance and survival of
populations of a terrestrial orchid, Dactylorhiza majalis. Biol Conserv 129:40–49
44. Blinova IV (2008) Populations of orchids at the northern limit of their distribution (Murmansk
Oblast): effect of climate. Russ J Ecol 39:26–33
45. Tsiftsis S, Djordjević V, Tsiripidis I (2019) Neottia cordata (Orchidaceae) at its southernmost
distribution border in Europe: threat status and effectiveness of Natura 2000 network for its
conservation. J Nat Conserv 48:27–35
46. Pfeifer M, Passalacqua NG, Bartram S, Schatz B, Croce A, Carey PD, Kraudelt H, Jeltsch F
(2010) Conservation priorities differ at opposing species borders of a European orchid. Biol
Conserv 143:2207–2220
47. Pillon Y, Fay MF, Shipunov AB, Chase MW (2006) Species diversity versus phylogenetic
diversity: a practical study in the taxonomically difficult genus Dactylorhiza (Orchidaceae).
Biol Conserv 129:4–13
48. Kotilínek M, Tatarenko I, Jersáková J (2018) Biological Flora of the British Isles: Neottia
cordata. J Ecol 106:444–460
49. Vakhrameeva MG, Denissova LV, Nikitina SV, Samsonov SK (1991) Orchids of our country.
Nauka, Moscow (in Russian)
50. Jermakowicz E, Brzosko E, Kotowicz J, Wróblewska A (2017) Genetic diversity of orchid
Malaxis monophyllos over European range as an effect of population properties and post-
glacial colonization. Pol J Ecol 65:69–86
51. Tamm CO (1991) Behaviour of some orchid populations in a changing environment. Obser-
vations on permanent plots, 1943–1990. In: Wells TCE, Willems JH (eds) Population ecology
of terrestrial orchids. SPB Academic Publishers, The Hague
52. Blinova I, Chmielewski FM (2008) Subarctic warming and its influence on the growth of
orchid populations in the extreme North-East of Europe Murmansk region. J Eur Orch
40:663–680. https://www.pabgi.ru/people/ilona_blinova/paper/t_orchids_08.pdf. Accessed
12 August 2019
53. Light MHS, MacConaill M (1998) Factors affecting germinable seed yield in Cypripedium
calceolus var. pubescens (Willd.) Correll and Epipactis helleborine (L.) Crantz (Orchidaceae).
Bot J Linn Soc 126:3–26
54. Øien DI, Moen A (2002) Flowering and survival of Dactylorhiza lapponica and Gymnadenia
conopsea in the Solendet nature reserve, Central Norway. In: Kindlmann P, Willems JH,
1 The Role of Ecological Factors in Distribution and Abundance of Terrestrial. . . 65
Whigham DF (eds) Trends and fluctuations and underlying mechanisms in terrestrial orchid
populations. Backhuys Publishers, Leiden
55. Inghe O, Tamm CO (1988) Survival and flowering of perennial herbs. V. Patterns of flowering.
Oikos 51:203–219
56. Rasmussen HN, Dixon KW, Jersáková J, Těšitelová T (2015) Germination and seedling
establishment in orchids: a complex of requirements. Ann Bot 116:391–402
57. Zhang S, Yang Y, Li J, Qin J, Zhang W, Huang W, Hu H (2018) Physiological diversity of
orchids. Plant Diversity 40:196–208
58. Carey PD, Farrell L (2002) Himantoglossum hircinum (L.) Sprengel (Biological flora of the
British Isles, 641.1). J Ecol 90:206–218
59. Wells TCE, Cox R (1989) Predicting the probability of the bee orchid (Ophrys apifera)
flowering or remaining vegetative from the size and number of leaves. In: Pritchard H (ed)
Modern methods in orchid conservation. Cambridge University Press, Cambridge
60. Štípková Z, Romportl D, Černocká V, Kindlmann P (2017) Factors associated with the
distributions of orchids in the Jeseníky Mountains, Czech Republic. Eur J Environ Sci
7:135–145. http://www.ejes.cz/index.php/ejes/article/view/318. Accessed 13 August 2019
61. Meekers T, Hutchings MJ, Honnay O, Jacquemyn H (2012) Biological Flora of the British
Isles: Gymnadenia conopsea s.l. J Ecol 100:1269–1288
62. Rasmussen HN (1995) Terrestrial orchids from seed to mycotrophic plant. Cambridge Uni-
versity Press, Cambridge
63. Wells TCE, Cox R (1991) Demographic and biological studies on Ophrys apifera: some
results from a 10 year study. In: Wells TCE, Willems JH (eds) Population ecology of terrestrial
orchids. SPB Academic Publishing, The Hague
64. Bódis J, Biró É, Nagy T, Takács A, Sramkó G, Bateman RM, Gilián L, Illyés Z, Tökölyi J,
Lukács BA, Csábi M, Molnár AV (2019) Biological flora of Central Europe Himantoglossum
adriaticum H. Baumann. Perspect Plant Ecol Evol Syst 125461. https://doi.org/10.1016/j.
ppees.2019.125461
65. Willems JH, Bik L (1991) Population biology of Orchis simia in the Netherlands. In: Wells
TCE, Willems JH (eds) Population ecology of terrestrial orchids. SPB Academic Publishing,
The Hague, pp 1972–1990
66. Brzosko E (2002) The dynamics of Listera ovata populations on mineral islands in the Biebrza
National Park. Acta Soc Bot Pol 71:243–251
67. Wells TCE (1981) Population ecology of terrestrial orchids. In: Synge H (ed) The biological
aspects of rare plant conservation. Wiley, Chichester
68. Jacquemyn H, Brys R, Hutchings MJ (2014) Biological flora of the British Isles: Epipactis
palustris. J Ecol 102:1341–1355
69. Tsiftsis S (2016) Morphological variability of Himantoglossum s.s. (Orchidaceae). Phytotaxa
245:17–30
70. Bailes C, Clements M, Cribb PJ, Muir H (1986) The cultivation of European orchids. Curtis’s
Bot Mag 3:8–13. JSTOR. http://www.jstor.org/stable/45066409 Accessed 12 August 2019
71. Abernethy A (2002) Light regimes as a control of terrestrial orchid distribution in New
Zealand. Dissertation, University of Canterbury
72. Diez JM, Pulliam HR (2007) Hierarchical analysis of species distribution and abundance
across environmental gradients. Ecology 88:3144–3152
73. Lõhmus A, Kull T (2011) Orchid abundance in hemiboreal forests: stand-scale effects of clear-
cutting, green-tree retention, and artificial drainage. Can J For Res 41:1352–1358
74. Shefferson RP, Kull T, Tali K (2005) Adult whole-plant dormancy induced by stress in long-
lived orchids. Ecology 86:3099–3104
75. Zhang SB, Hu H, Xu K, Li ZR, Yang YP (2007) Flexible and reversible responses to different
irradiance levels during photosynthetic acclimation of Cypripedium guttatum. J Plant Physiol
164:611–620
76. Jacquemyn H, Brys R, Jongejans E (2010) Size-dependent flowering and costs of reproduction
affect population dynamics in a tuberous perennial woodland orchid. J Ecol 98:1204–1215
66 V. Djordjević and S. Tsiftsis
77. Tsiftsis S, Djordjević V (2018) Habitat effects and differences in the reproductive success of
Orchis punctulata and Orchis purpurea (Orchidaceae). Turk J Bot 42:400–411
78. Jacquemyn H, Brys R, Honnay O, Hermy M (2008) Effects of coppicing on demographic
structure, fruit and seed set in Orchis mascula. Basic Appl Ecol 9:392–400
79. Jacquemyn H, Brys R, Honnay O, Hutchings MJ (2009) Biological flora of the British Isles:
Orchis mascula (L.) L. J Ecol 97:360–377
80. Jacquemyn H, Brys R (2010) Temporal and spatial variation in flower and fruit production in a
food-deceptive orchid: a five year study. Plant Biol 12:145–153
81. Selosse MA, Weiss M, Jany JL, Tillier A (2002) Communities and populations of sebacinoid
basidiomycetes associated with the achlorophyllous orchid Neottia nidus-avis (L.) L.C.M.
Rich. and neighbouring tree ectomycorrhizae. Mol Ecol 11:1831–1844
82. Taylor L, Roberts DL (2011) Biological flora of the British Isles: Epipogium aphyllum Sw. J
Ecol 99:878–890
83. Preiss K, Adam IKU, Gebauer G (2010) Irradiance governs exploitation of fungi: fine-tuning
of carbon gain by two partially myco-heterotrophic orchids. Proc R Soc B 277:1333–1336
84. Hornemann G, Michalski SG, Durka W (2012) Short-term fitness and long-term population
trends in the orchid Anacamptis morio. Plant Ecol 213:1583–1595
85. Jersáková J, Malinová T, Jeřábková K, Dötteri S (2011) Biological Flora of the British Isles:
Pseudorchis albida (L.) Á. & D. Löve. J Ecol 99:1282–1298
86. Tsiftsis S, Antonopoulos Z (2011) Pseudorchis albida: an enigmatic orchid for the Greek flora.
J Eur Orch 43:795–806
87. Jacquemyn H, Brys R, Adriaens D, Honnay O, Roldán-Ruiz I (2009) Effects of population size
and forest management ongenetic diversity and structure of the tuberous orchid Orchis
mascula. Conserv Genet 10:161–168
88. Cruz-Fernández QT, Alquicira-Arteaga ML, Flores-Palacios A (2011) Is orchid species rich-
ness and abundance related to the conservation status of oak forest? Plant Ecol 212:1091–1099
89. Hurskainen S, Jäkäläniemi A, Ramula S, Tuomi J (2017) Tree removal as a management
strategy for the lady’s slipper orchid, a flagship species for herb-rich forest conservation. Forest
Ecol Manag 406:12–18
90. Shefferson RP, Kull T, Tali K (2006) Demographic response to shading and defoliation in two
woodland orchids. Folia Geobot 41:95–106
91. van der Meer S, Jacquemyn H, Carey PD, Jongejans E (2016) Recent range expansion of a
terrestrial orchid corresponds with climate-driven variation in its population dynamics.
Oecologia 181:435–448
92. Shefferson RP, Mizuta R, Hutchings MJ (2017) Predicting evolution in response to climate
change: the example of sprouting probability in three dormancy prone orchid species. R Soc
Open Sci 4:160647
93. Liu H, Feng CL, Luo YB, Chen BS, Wang ZS, Gu HY (2010) Potential challenges of climate
change to orchid conservation in a wild orchid hotspot in southwestern China. Bot Rev
76:174–192
94. Reina-Rodríguez GA, Rubiano Mejía JE, Castro Llanos FA, Soriano I (2017) Orchid distri-
bution and bioclimatic niches as a strategy to climate change in areas of tropical dry forest in
Colombia. Lankesteriana 17:17–47
95. Ongaro S, Martellos S, Bacaro G, De Agostini A, Cogoni A, Cortis P (2018) Distributional
pattern of Sardinian orchids under a climate change scenario. Community Ecol 19:223–232
96. Gaskett AC, Gallagher RV (2018) Orchid diversity: spatial and climatic patterns from herbar-
ium records. Ecol Evol 8:11235–11245. https://doi.org/10.1002/ece3.4598
97. Molnár VA, Tökölyi J, Végvári Z, Sramkó G, Sulyok J, Barta Z (2012) Pollination mode
predicts phenological response to climate change in terrestrial orchids: a case study from
Central Europe. J Ecol 100:1141–1152
98. Hutchings MJ, Robbirt KM, Roberts DL, Davy AJ (2018) Vulnerability of a specialized
pollination mechanism to climate change revealed by a 356-year analysis. Bot J Linn Soc
186:498–509
1 The Role of Ecological Factors in Distribution and Abundance of Terrestrial. . . 67
99. Tsiftsis S, Tsiripidis I, Trigas P (2011) Identifying important areas for orchid conservation in
Crete. Eur J Environ Sci 1:28–37
100. Hijmans RJ, Cameron SE, Parra JL, Jones PG, Jarvis A (2005) Very high resolution interpo-
lated climate surfaces for global land areas. Int J Climatol 25:1965–1978
101. Fick SE, Hijmans RJ (2017) WorldClim 2: new 1-km spatial resolution climate surfaces for
global land areas. Int J Climatol 37:4302–4315
102. Bowles M, Zettler L, Bell T, Kelsey P (2005) Relationship between soil characteristics,
distribution and restoration potential of the federal threatened eastern prairie fringed orchid,
Platanthera leucophaea (Nutt.) Lindl. Am Midl Nat 154:273–285
103. Dijk E, Willems JH, van Andel J (1997) Nutrient responses as a key factor to the ecology of
orchid species. Acta Bot Neerl 46:339–363
104. Tsiftsis S, Tsiripidis I, Papaioannou A (2012) Ecology of orchid Goodyera repens in its
southern distribution limits. Plant Biosyst 146:857–866
105. Landi M, Frignani F, Lazzeri C, Angiolini C (2009) Abundance of orchids on calcareous
grasslands in relation to community species, environmental, and vegetation conditions. Russ J
Ecol 40:486–494
106. Lang D (2004) Britain’s orchids, a guide to the identification and ecology of the wild orchids of
Britain and Ireland. Wild Guides Ltd., Old Basing
107. Djordjević V, Tsiftsis S (2019) Patterns of orchid species richness and composition in relation
to geological substrates. Wulfenia 26:1–21
108. Tyler G (2003) Some ecophysiological and historical approaches to species richness and
calcicole/calcifuge behaviour – contribution to a debate. Folia Geobot 38:419–428
109. Farrell L (1985) Biological Flora of the British Isles: Orchis militaris L. J Ecol
73:1041–1053
110. Tali K, Foley MJY, Kull T (2004) Biological flora of the British Isles, 232. Orchis ustulata L. J
Ecol 92:174–184
111. Jersáková J, Traxmandlová I, Ipser Z, Matthias K, Pellegrino G, Schatz B, Djordjević V,
Kindlmann P, Renner SS (2015) Biological flora of Central Europe: Dactylorhiza sambucina
(L.) Soó. Perspect Plant Ecol Evol Syst 17:318–329
112. Kotilínek M, Těšitelová T, Jersáková J (2015) Biological Flora of the British Isles: Neottia
ovata. J Ecol 103:1354–1366
113. Petrova AS, Venkova DY (2006) Epipactis pontica (Orchidaceae): a new species for the
Bulgarian flora. Phytol Balcan 12:249–253. http://www.bio.bas.bg/~phytolbalcan/PDF/14_1/
14_1_11_Petrova_&_Venkova.pdf. Accessed 12 August 2019
114. van der Ent A, Wood JJ (2013) Orchids of extreme serpentinite (ultramafic) habitats in
Kinabalu Park. Malesian Orchid J 12:39–54
115. van der Ent A, van Vugt R, Wellinga S (2015) Ecology of Paphiopedilum rothschildianum at
the type locality in Kinabalu Park (Sabah, Malaysia). Biodivers Conserv 24:1641–1656
116. Filimonova E, Lukina N, Glazyrina M, Borisova G, Kumar A, Maleva M (2019) A compar-
ative study of Epipactis atrorubens in two different forest communities of the Middle Urals,
Russia. J For Res 1–10. https://doi.org/10.1007/s11676-019-01010-y
117. Stuckey I (1967) Environmental factors and the growth of native orchids. Am J Bot
54:232–241
118. Knudson MD, Vanlooy JA, Hill MJ (2015) A habitat suitability index (HSI) for the western
prairie fringed orchid (Platanthera praeclara) on the Sheyenne National Grassland, North
Dakota, USA. Ecol Indic 57:536–545
119. Wolken PM, Sieg CH, Williams SE (2001) Quantifying suitable habitat of the threatened
western prairie fringed orchid. J Range Manag 54:611–616
120. Sieg CH, King RM (1995) Influence of environmental factors and preliminary demographic
analysis of a threatened orchid, Platanthera praeclara. Am Midl Nat 134:307–323
121. Jacquemyn H, Waud M, Merckx VS, Lievens B, Brys R (2015) Mycorrhizal diversity, seed
germination and long-term changes in population size across nine populations of the terrestrial
orchid Neottia ovata. Mol Ecol 24:3269–3280
68 V. Djordjević and S. Tsiftsis
122. Illyés Z, Halász K, Rudnóy S, Ouanphanivanh N, Garay T, Bratek Z (2009) Changes in the
diversity of the mycorrhizal fungi of orchids as a function of the water supply of the habitat. J
Appl Bot Food Qual 83:28–36
123. Foley M, Clarke S (2005) Orchids of the British Isles. Griffin Press Publishing Limited,
Cheltenham
124. Danihelka J, Chrtek J Jr, Kaplan Z (2012) Checklist of vascular plants of the Czech Republic.
Preslia 84:647–811
125. Ståhlberg D (2009) Habitat differentiation, hybridization and gene flow patterns in mixed
populations of diploid and autotetraploid Dactylorhiza maculata s.l. (Orchidaceae). Evol Ecol
23:295–328
126. Molnár A (ed) (2011) Magyarország orchideáinak atlasza. Kossuth kiadó, Budapest
127. Hrivnák R, Hrivnák M, Slezák M, Vlčko J, Baltiarová J, Svitok M (2014) Distribution and
eco-coenotic patterns of the forest orchid Epipactis pontica in Slovakia. Ann For Res
57:55–69
128. Djordjević V, Jakovljević K, Stevanović V (2016) Three taxa of Epipactis (Orchidaceae-
Epidendroideae) new for the flora of Serbia. Phyton-Ann Rei Bot 56:77–89
129. Zilioli DM, Bini C, Wahsha M, Ciotoli G (2011) The pedological heritage of the Dolomites
(Northern Italy): features, distribution and evolution of the soils, with some implications for
land management. Geomorphology 135:232–247
130. Tsiftsis S (2009) The orchids (Orchidaceae) of E. Macedonia: distribution, ecology and high
conservation value areas. Dissertation, Aristotle University of Thessaloniki (in Greek with
English summary)
131. Sundermann H (1980) Europäische und mediterrane Orchideen. Brücke-Verlag Kurt
Schmersow, Hildesheim
132. Breiner R (1979) pH-Messungen an Orchideen-Standorten auf Kreta und Zypern. Mitt Bl Arb
Heim Orchid Baden-Wurttemberg 11:54–58. http://biolis.ub.uni-frankfurt.de/search/detail/
22028. Accessed 12 August 2019
133. Vakhrameeva MG, Tatarenko IV, Varlygina TI, Torosyan GK, Zagulski MN (2008) Orchids of
Russia and adjacent countries (within the borders of the former USSR). ARG Gantner Verlag,
Ruggell
134. Syska M (1995) Die Orchideenflora des westlichen Nestos-Deltas und des angrenzenden
Berglandes (Nordost-Griechenland): Verbreitung – Ökologie – Gefahrdung. J Eur Orch
27:339–552
135. Wallenwein F, Saad A (2000) Messungen des pH-Wertes an den Wuchsorten mediterraner
Orchideen. J Eur Orch 32:375–386
136. Möller O (1985) Die Mineralsalze der Standortböden der europäischer Orchideen. Die
Orchidee 36:118–121. https://orchidee.de/gesellschaft/die-orchidee/. Accessed 12 August
2019
137. Tsiripidis I (2001) Plant communities of beech forests in Rodopi mountain range and their
environmental assessment for reforestation. Dissertation, Aristotle University of Thessaloniki
(in Greek)
138. Kull T (1999) Biological Flora of the British Isles: Cypripedium calceolus L. J Ecol
87:913–924
139. Mróz L (1994) Ekologia Dactylorhiza sambucina (L.) Soó w Sudetach. Acta Univ Wratisl
Prace Botaniczne LXXVI:103–157
140. Molnár AV, Sramkó G (2012) Epipactis albensis (Orchidaceae): a new species in the flora of
Romania. Biologia 67:883–888
141. Parzych A, Sobisz Z (2013) Preliminary ecology research on Epipactis atrorubens (Hoffm.)
Besser on the Słowińskie coast (Northern Poland). Ecol Quest 18:21–32
142. Urban D (2013) Characteristics of the locality of Hammarbya paludosa (L.) O. Kuntze on the
Łęczna-Włodawa plain (West Polesie). Teka Komisji Ochrony i Kształtowania Środowiska
Przyrodniczego. OL PAN 10:448–454
143. Procházka F, Velísek V (1983) Orchideje naší přírody. Čekoslovenské Akademie Věd, Praha
1 The Role of Ecological Factors in Distribution and Abundance of Terrestrial. . . 69
165. Rewicz A, Jaskuła R, Rewicz T, Tończyk G (2017) Pollinator diversity and reproductive
success of Epipactis helleborine (L.) Crantz (Orchidaceae) in anthropogenic and natural
habitats. PeerJ 5:e3159
166. Fekete R, Nagy T, Bódis J, Biró É, Löki V, Süveges K, Takács A, Tökölyi J, Molnár VA (2017)
Roadside verges as habitats for endangered lizard-orchids (Himantoglossum spp.): ecological
traps or refuges? Sci Total Environ 607–608:1001–1008
167. Fekete R, Löki V, Urgyán R, Süveges K, Lovas Kiss Á, Vincze O, Molnár VA (2019) Roadside
verges and cemeteries: comparative analysis of anthropogenic orchid habitats in the eastern
Mediterranean. Ecol Evol. https://doi.org/10.1002/ece3.5245
168. Grant CD, Koch JM (2003) Orchid species succession in rehabilitated bauxite mines in
Western Australia. Aust J Bot 51:453–457
169. Norman MA, Koch JM, Grant CD, Morald TK, Ward SC (2006) Vegetation succession after
bauxite mining in Western Australia. Restor Ecol 14:278–288
170. Girlanda M, Selosse MA, Cafasso D, Brilli F, Delfine S, Fabian R, Ghignone S, Pinelli P,
Segreto R, Loreto F, Cozzolino S, Perotto S (2006) Inefficient photosynthesis in the Mediter-
ranean orchid Limodorum abortivum is mirrored by specific association to ectomycorrhizal
Russulaceae. Mol Ecol 15:491–504
171. Mucina L, Bültmann H, Dierßen K, Theurillat JP, Raus T, Čarni A, Šumberová K, Willner
W, Dengler J, Gavilán García R, Chytrý M, Hájek M, Di Pietro R, Iakushenko D, Pallas J,
Daniëls FJA, Bergmeier E, Santos Guerra A, Ermakov N, Valachovič M, Schaminée JHJ,
Lysenko T, Didukh YP, Pignatti S, Rodwell JS, Capelo J, Weber HE, Solomeshch A,
Dimopoulos P, Aguiar C, Freitag H, Hennekens SM, Tichý L (2016) Vegetation of Europe:
hierarchical floristic classification system of plant, lichen, and algal communities. Appl Veg
Sci 19:3–264
172. Djordjević V, Tomović G, Lakušić D (2010) Epipactis purpurata Sm. (Orchidaceae) – a new
species in the flora of Serbia. Arch Biol Sci 62:1175–1180
173. Kovalchuk A (2016) On the occurrence of Orchis pallens L. in the Ukrainian Carpathians. J
Eur Orch 48:29–36
174. Tsiftsis S, Karagiannakidou V, Tsiripidis I (2007) The orchid flora of East Macedonia (NE
Greece). J Eur Orch 39:489–526
175. Djordjević V (2016) Epipactis muelleri (Orchidaceae-Neottieae), a species new to the flora of
Serbia. Phyton-Ann Rei Bot 56:303–312
176. Djordjević V, Tsiftsis S, Lakušić D, Jovanović S, Stevanović V (2017) Distribution and
conservation status of some rare and threatened orchid taxa in the Central Balkans and the
southern part of the Pannonian plain. Wulfenia 24:143–162
177. Djordjević V, Jovanović S, Stevanović V (2014) Dactylorhiza fuchsii (Orchidaceae), a new
species in the flora of Serbia. Arch Biol Sci 66:1227–1232
178. Pierce S, Vagge I, Brusa G, Cerabolini BEL (2014) The intimacy between sexual traits and
Grime’s CSR strategies for orchids coexisting in semi-natural calcareous grassland at the Olive
Lawn. Plant Ecol 215:495–505
179. Slaviero A, Del Vecchio S, Pierce S, Fantinato E, Buffa G (2016) Plant community attributes
affect dry grassland orchid establishment. Plant Ecol 217:1533–1543
180. Oberdorfer E (1994) Pflanzensoziologische Exkursionsflora. Ulmer, Stuttgart
181. Krauss J, Klein AM, Steffan-Dewenter I, Tscharntke T (2004) Effects of habitat area, isolation,
and landscape diversity on plant species richness of calcareous grasslands. Biodivers Conserv
13:1427–1439
182. Leuschner C, Ellenberg H (2017) Ecology of central European non-Forest vegetation: coastal
to alpine, natural to man-made habitats: vegetation ecology of Central Europe. Springer
International Publishing, Cham
183. Haraštová-Sobotková M, Jarsáková J, Kindlmann P, Čurn L (2005) Morphometric and genetic
divergence among populations of Neotinea ustulata (Orchidaceae) with different flowering
phenologies. Folia Geobot 40:385–405
1 The Role of Ecological Factors in Distribution and Abundance of Terrestrial. . . 71
184. Budzhak VV, Chorney II, Tokariuk AI, Kuzemko AA (2016) Numeric syntaxonomical
analysis of the communities with participation of species from Molinia caerulea complex in
the southwest of Ukraine. Hacquetia 15:63–78
185. Schrautzer J, Fichtner A, Huckauf A, Rasran L, Jensen K (2011) Long-term population
dynamics of Dactylorhiza incarnata (L.) Soó after abandonment and re-introduction of
mowing. Flora 206:622–630
186. Blinova IV (2016) Spatial population structure of rare orchid species in rich fens in the central
part of Murmansk oblast. Russ J Ecol 47:234–240
187. Hrivnák R, Gömöry D, Cvachová A (2006) Inter-annual variability of the abundance and
morphology of Dactylorhiza majalis (Orchidaceae-Orchideae) in two permanent plots of a
mire in Slovakia. Phyton-Ann Rei Bot 46:27–44
188. Löki V, Tökölyi J, Süveges K, Lovas-Kiss A, Hürkan K, Gábor S, Molnár AV (2015) The
orchid flora of Turkish graveyards: a comprehensive field survey. Willdenowia 45:231–243
189. Molnár VA, Takács A, Mizsei E, Löki V, Barina Z, Sramkó G, Tökölyi J (2017) Religious
differences affect orchid diversity of Albanian graveyards. Pak J Bot 49:289–303
190. Rewicz A, Rewers M, Jędrzejczyk I, Rewicz T, Kołodziejek J, Jakubska-Busse A (2018)
Morphology and genome size of Epipactis helleborine (L.) Crantz (Orchidaceae) growing in
anthropogenic and natural habitats. PeerJ 6:e5992
191. Rewicz A, Kolodziejek J, Jakubska-Busse A (2016) The role of anthropogenic habitats as
substitutes for natural habitats: a case study on Epipactis helleborine (L.) Crantz (Orchidaceae,
Neottieae). Variations in size and nutrient composition of seeds. Turk J Bot 40:258–268
192. Scade A, Brundrett MC, Batty AL, Dixon KW, Sivasithamparam K (2006) Survival of
transplanted terrestrial orchid seedlings in urban bushland habitats with high or low weed
cover. Aust J Bot 54:383–389
193. Lim WH (2015) Aspects of the physiological ecology of the Western-Australian ruderal
orchid, Microtis media R. Br, with special reference to the functions of its mycorrhizal
fungi. Dissertation, the University of Western Australia
194. Grime JP (1979) Plant strategies and vegetation processes. Wiley, Chichester
195. Coates F, Lunt ID, Tremblay RL (2006) Effects of disturbance on population dynamics of
threatened orchid Prasophyllum correctum D. L. Jones and implications for grassland man-
agement in south-eastern Australia. Biol Conserv 129:59–69
196. Willems JH, Melser C (1998) Population dynamics and life-history of Coeloglossum viride
(L.) Hartm.: an endangered orchid species in The Netherlands. Bot J Linn Soc 126:83–93
197. Smith PH, Cross S (2016) Effect of mowing regime on abundance of green-winged orchid
Anacamptis morio on coastal grassland in Merseyside, England. Conserv Evid 13:79–81.
https://www.conservationevidence.com/individual-study/5910. Accessed 13 August 2019
198. Sletvold N, Øien DI, Moen A (2010) Long term influence of mowing on population dynamics
in the rare orchid Dactylorhiza lapponica: the importance of recruitment and seed production.
Biol Conserv 143:747–755
199. Humphrey JW, Coombs EL (1997) Effects of forest management on understorey vegetation in
a Pinus sylvestris L. plantation in NE Scotland. Bot J Scotl 49:479–488
200. Duncan M (2012) Response of orchids to bushfire: black Saturday Victoria 2009 – natural values
fire recovery program. Department of Sustainability and Environment. Victoria, Heidelberg
201. Fridley JD, Vandermast DB, Kuppinger DM, Manthey M, Peet RK (2007) Co-occurrence
based assessment of habitat generalists and specialists: a new approach for the measurement of
niche width. J Ecol 95:707–722
202. Boulangeat I, Lavergne S, Van Es J, Garraud L, Thuiller W (2012) Niche breadth, rarity and
ecological characteristics within a regional flora spanning large environmental gradients. J
Biogeogr 39:204–214
203. Schiebold JMI, Bidartondo MI, Lenhard F, Makiola A, Gebauer G (2018) Exploiting mycor-
rhizas in broad daylight: partial mycoheterotrophy is a common nutritional strategy in meadow
orchids. J Ecol 106:168–178
72 V. Djordjević and S. Tsiftsis
204. McCormick MK, Jacquemyn H (2014) What constrains the distribution of orchid populations?
New Phytol 202:392–400
205. McCormick MK, Whigham DF, Canchani-Viruet A (2018) Mycorrhizal fungi affect orchid
distribution and population dynamics. New Phytol 219:1207–1215
206. McCormick MK, Lee Taylor D, Whigham DF, Burnett RK (2016) Germination patterns in
three terrestrial orchids relate to abundance to mycorrhizal fungi. J Ecol 104:744–754
207. Dearnaley JDW (2007) Further advances in orchid mycorrhizal research. Mycorrhiza
17:475–486
208. Rasmussen HN, Rasmussen FN (2014) Seedling mycorrhiza: a discussion of origin and
evolution in Orchidaceae. Bot J Linn Soc 175:313–327
209. Selosse MA, Faccio A, Scappaticci G, Bonfante P (2004) Chlorophyllous and achlorophyllous
specimens of Epipactis microphylla (Neottieae, Orchidaceae) are associated with
ectomycorrhizal septomycetes, including truffles. Microb Ecol 47:416–426
210. Rasmussen HN, Rasmussen FN (2009) Orchid mycorrhiza: implications of a mycophagous
life circle. Oikos 118:334–345
211. Bailarote BC, Lievens B, Jacquemyn H (2012) Does mycorrhizal specificity affect orchid
decline and rarity? Am J Bot 99:1655–1665
212. Davis BJ, Phillips RD, Wright M, Linde CC, Dixon KW (2015) Continent-wide distribution in
mycorrhizal fungi: implications for the biogeography of specialized orchids. Ann Bot
116:413–421
213. Shefferson RP, Taylor DL, Weiß M, Garnica S, McCormick MK, Adams S, Gray HM,
McFarland JW, Kull T, Tali K, Yukawa T, Kawahara T, Miyoshi K, Lee YI (2007) The
evolutionary history of mycorrhizal specificity among lady’s slipper orchids. Evolution
61:1380–1390
214. Pellegrino G, Luca A, Bellusci F (2016) Relationships between orchid and fungal biodiversity:
mycorrhizal preferences in Mediterranean orchids. Plant Biosyst 150:1–10
215. McCormick MK, Lee Taylor D, Juhaszova K, Burnett RK, Whigham DF, O’Neill JP (2012)
Limitations on orchid recruitment: not a simple picture. Mol Ecol 21:1511–1523
216. Tremblay RL, Ackerman JD, Zimmerman JK, Calvo RN (2005) Variation in sexual reproduc-
tion in orchids and its evolutionary consequences: a spasmodic journey to diversification. Biol
J Linn Soc 84:1–54
217. van der Pijl L, Dodson CH (1966) Orchid flowers: their pollination and evolution. University
of Miami Press, Coral Gables
218. Jersáková J, Johnson SD, Kindlmann P (2006) Mechanisms and evolution of deceptive
pollination in orchids. Biol Rev 81:219–235
219. Vereecken NJ, Dafni A, Cozzolino S (2010) Pollination syndromes in Mediterranean orchids –
implications for speciation, taxonomy and conservation. Bot Rev 76:220–240
220. Neiland MRM, Wilcock CC (1998) Fruit set, nectar reward, and rarity in the Orchidaceae. Am
J Bot 85:1657–1671
221. Darwin C (1862) On the various contrivances by which British and foreign orchids are
fertilised by insects. John Murray, London
222. Jacquemyn H, Brys R, Hermy M, Willems JH (2005) Does nectar reward affect rarity and
extinction probabilities of orchid species? An assessment using historical records from Bel-
gium and the Netherlands. Biol Conserv 121:257–263
223. Catling P (1990) Auto-pollination in the Orchidaceae. In: Arditti JE (ed) Orchid biology:
reviews and perspectives, vol V. Timber Press, Portland
Which Environmental Factors Drive
Distribution of Orchids? A Case Study from 2
South Bohemia, Czech Republic
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
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.
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
76 Z. Štípková et al.
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
78 Z. Štípková et al.
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 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.
80 Z. Štípková et al.
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
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
2 Which Environmental Factors Drive Distribution of Orchids? A Case Study. . . 81
1 3 5 7 9 11 13 17 19 22 24 29 32 34 37 39
kves
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
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. 4 Potential distribution map of Anacamptis morio in the region of South Bohemia
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
Regularized training gain
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.
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
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
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. 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.
86 Z. Štípková et al.
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
Regularized training gain
With only variable Without variable With all variables
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
2 Which Environmental Factors Drive Distribution of Orchids? A Case Study. . . 87
1 3 5 7 9 11 13 17 19 22 24 29 32 34 37 39
kves
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
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. 10 Potential distribution map of Dactylorhiza fuchsii in the region of South Bohemia
solar_rad
summer_days
temp_1
temp_2
trop_days
veg_season
Total gain
0 0,2 0,4 0,6 0,8 1
Regularized training gain
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
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
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
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.
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
2 Which Environmental Factors Drive Distribution of Orchids? A Case Study. . . 91
Fig. 13 Potential distribution map of Epipactis palustris in the South Bohemian region
solar_rad
summer_days
temp_1
temp_2
trop_days
veg_season
Total gain
0 0,2 0,4 0,6 0,8 1
Regularized training gain
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
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
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
2 Which Environmental Factors Drive Distribution of Orchids? A Case Study. . . 93
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
94 Z. Štípková et al.
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.
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.
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
Regularized training gain
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
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
0.60
0.55
0.50
0.45
0.40
0.35
0.30
0.25
Fig. 18 Responses of Neottia ovata to (a) consolidated layer of ecosystems (KVES), (b) slope of
a terrain (slope), (c) solar radiation
96 Z. Štípková et al.
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.
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
2 Which Environmental Factors Drive Distribution of Orchids? A Case Study. . . 97
solar_rad
summer_days
temp_1
temp_2
trop_days
veg_season
Total gain
0 0,5 1 1,5 2 2,5
Regularized training gain
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.
98 Z. Štípková et al.
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
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
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
2 Which Environmental Factors Drive Distribution of Orchids? A Case Study. . . 99
Fig. 22 Potential distribution map of Platanthera chlorantha in the South Bohemian region
100 Z. Štípková et al.
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]
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.
2 Which Environmental Factors Drive Distribution of Orchids? A Case Study. . . 103
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.
References
1. Dirzo R, Raven PH (2003) Global state of biodiversity and loss. Annu Rev Environ Resour
28:137–167. https://doi.org/10.1146/annurev.energy.28.050302.105532
2. Possingham HP, Wilson KA (2005) Biodiversity – turning up the heat on hotspots. Nature
436:919–920. https://doi.org/10.1038/436919a
3. Tsiftsis S, Tsiripidis I, Trigas R (2011) Identifying important areas for orchid conservation in
Crete. Eur J Environ Sci 1(2):28–37
4. Margules CR, Pressey RL (2000) Systematic conservation planning. Nature 405:243–253.
https://doi.org/10.1038/35012251
5. Gaston KJ, Pressey RL, Margules CR (2002) Persistence and vulnerability: retaining bio-
diversity in the landscape and in protected areas. J Biosci 27:361–384. https://doi.org/
10.1007/BF02704966
6. Williams PH, Margules CR, Hilbert DW (2002) Data requirements and data sources for
biodiversity priority area selection. J Biosci 27:327–338. https://doi.org/10.1007/BF02704963
7. Heywood VH, Iriondo JM (2003) Plant conservation: old problems, new perspectives. Biol
Conserv 113:321–335. https://doi.org/10.1016/S0006-3207(03)00121-6
8. Efimov PG (2011) Revealing the decline and expansion of orchids of NW European Russia.
Eur J Environ Sci 1(2):7–17
9. Feldman D, Prat D (2011) Conservation recommendations from a large survey of French
orchids. Eur J Environ Sci 1(2):8–27
10. Dressler RL (1993) Phylogeny and classification of the orchid family. Cambridge University
Press, Cambridge
11. Chase MW, Cameron KM, Barrett RL, Freudebstein JV (2003) DNA data and Orchidaceae
systematics: a new phylogenetic classification. In: Dixon KW, Kell SP, Barrett RL, Cribb PJ
(eds) Orchid conservation. Natural History Publications, Kota Kinabalu
12. Cribb PJ, Kell SP, Dixon KW, Barrett RL (2003) Orchid conservation: a global perspective.
In: Dixon KW, Kell SP, Barrett RL, Cribb PJ (eds) Orchid conservation. Natural History
Publications, Kota Kinabalu
13. Pillon Y, Chase M (2006) Taxonomic exaggeration and its effects on orchid conservation.
Conserv Biol 21:263–265
14. Swarts ND, Dixon KW (2009) Terrestrial orchid conservation in the age of extinction. Ann Bot
104:543–556
15. Zhang ZJ, Yan YJ, Tian Y, Li JS, He JS, Tang ZY (2015) Distribution and conservation
of orchid species richness in China. Biol Conserv 181:64–72. https://doi.org/10.1016/j.biocon.
2014.10.026
16. Vakhrameeva MG, Tatarenko IV, Varlygina TI, Torosyan GK, Zagulski MN (2008) Orchids of
Russia and adjacent countries (within the borders of the former USSR). ARG Gantner Verlag
KG, Ruggell
17. Hutchings MJ (1989) Population biology and conservation of Ophrys sphegodes. In:
Pritchard HW (ed) Modern methods in orchid conservation: the role of physiology, ecology
and management. Cambridge University Press, Cambridge
18. Kull T, Kindlmann P, Hutchings M, Primack B (2006) Conservation biology of orchids:
introduction to the special issue. Biol Conserv 129:1–3
19. Wotavová K, Balounová Z, Kindlmann P (2004) Factors affecting persistence of terrestrial
orchids in wet meadows and implications for their conservation in a changing agricultural
landscape. Biol Conserv 118:271–279
104 Z. Štípková et al.
40. Czech National Phytosociological Database. Vegetation Science Group, Department of Botany
and Zoology, Faculty of Science, Masaryk University (2005) http://www.sci.muni.cz/botany/
vegsci/dbase.php?lang¼cz. Accessed 21 Feb 2014
41. South Bohemian Branch. Czech Botanical Society (2017) https://botanospol.cz/cs/node/42.
Accessed 03 Mar 2014
42. Pearson RG, Thuiller W, Araújo MB et al (2006) Model-based uncertainty in species range
prediction. J Biogeogr 33:1704–1711
43. Wisz MS, Hijmans RJ, Li J, Peterson AT, Graham CH, Guisan A, NCEAS Predicting Species
Distributions Working Group (2008) Effect of sample size on the performance of species
distribution models. Divers Distrib 14:763–773
44. Štípková Z, Kosánová K, Romportl D, Kindlmann P (2018) Chapter 8, Determinants of orchid
occurrence: a Czech example. In: Şen B, Grillo O (eds) Selected studies in biodiversity.
InTechOpen, London
45. Kosánová K (2017) Dynamika výskytu orchidejí ve vybraném modelovém území v jižních
Čechách. Mgr. Thesis, Charles University
46. Dykyjová D (2003) Ekologie středoevropských orchidejí. KOPP, České Budějovice
47. Jersáková J, Kindlmann P (2004) Zásady péče o orchidejová stanoviště. KOPP, České
Budějovice
48. Průša D (2005) Orchideje České republiky. Computer Press, Brno
49. Lepší P, Lepší M, Boublík K, Stech M, Hans V (2013) Červená kniha květeny jižní části Čech.
Jihočeské muzeum v Českých Budějovicích, České Budějovice
50. AOPK ČR (2013) Konsolidovaná vrstva ekosystémů. Agentura ochrany přírody a krajiny ČR.
[Electronic geographical data]
51. ČÚZK (2010) Digitální model reliéfu České republiky 4. generace (DMR4G). http://geoportal.
cuzk.cz/(S(wbdeojptgceogdtyhisrjzjf))/Default.aspx?head_tab¼sekce-00-gp&mode¼TextMeta&
text¼uvod_uvod&menu¼01&news¼yes&UvodniStrana¼yes
52. Kindlmann P, Balounová Z (1999) Flowering regimes of terrestrial orchids: chaos or regularity?
J Veg Sci 10:269–273
53. Jersáková J, Kindlmann P, Stříteský M (2002) Population dynamics of Orchis morio in the
Czech Republic under human influence. In: Kindlmann P, Willems JH, Whigham DF (eds)
Trends and fluctuations and underlying mechanisms in terrestrial orchid populations. Backhuys
Publishers, Leiden
54. Chán V (1999) Komentovaný červený seznam květeny jižní části Čech. AOPK ČR, Praha
55. Štípková Z, Kindlmann P (2015) Extent and reasons for meadows in South Bohemia becoming
unsuitable for orchids. Eur J Environ Sci 5:142–147
56. Baumann H, Künkele S, Lorenz R (2009) Orchideje Evropy a přilehlých oblastí.
Academia, Praha
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
Keywords
Mascarenes · Biodiversity · Invasive alien species · Island · Extinction
1 Introduction
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
110 C. Baider and F. B. V. Florens
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.
3 Diversity, Ecology, and Conservation of Mauritius Orchids 111
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].
112 C. Baider and F. B. V. Florens
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
3 Diversity, Ecology, and Conservation of Mauritius Orchids 113
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])
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
114 C. Baider and F. B. V. Florens
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
3 Diversity, Ecology, and Conservation of Mauritius Orchids 115
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
116 C. Baider and F. B. V. Florens
Fig. 5 Number of species of native orchids per bracket of elevation range, in terms of both
minimum and maximum known elevation per species
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
3 Diversity, Ecology, and Conservation of Mauritius Orchids 117
4.1 Deforestation
4.2 Harvesting
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
120 C. Baider and F. B. V. Florens
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)
3 Diversity, Ecology, and Conservation of Mauritius Orchids 121
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.
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)
122 C. Baider and F. B. V. Florens
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
3 Diversity, Ecology, and Conservation of Mauritius Orchids 123
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.
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].
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
124 C. Baider and F. B. V. Florens
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)
3 Diversity, Ecology, and Conservation of Mauritius Orchids 125
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,
126 C. Baider and F. B. V. Florens
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.
3 Diversity, Ecology, and Conservation of Mauritius Orchids 127
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
128 C. Baider and F. B. V. Florens
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.
References
1. Ripple WJ, Wolf C, Newsome TM et al (2017) World scientists’ warning to humanity: a
second notice. Bioscience 67:1026–1028
2. Ceballos G, Ehrlich PR, Raven PH (2020) Vertebrates on the brink as indicators of biological
annihilation and the sixth mass extinction. Proc Natl Acad Sci 117:13596–13602
3. Cardinale BJ, Duffy JE, Gonzalez A et al (2012) Biodiversity loss and its impact on humanity.
Nature 486:59–67
4. Mace GM, Norris K, Fitter AH (2012) Biodiversity and ecosystem services: a multilayered
relationship. Trends Ecol Evol 27:19–26
5. Ripple WJ, Wolf C, Newsome TM et al (2019) World scientists’ warning of a climate
emergency. Bioscience 70:8–12
6. Dinerstein E, Joshi AR, Vynne C et al (2020) A “Global Safety Net” to reverse biodiversity
loss and stabilize Earth’s climate. Sci Adv 6:eabb2824
7. Griscom BW, Adams J, Ellis PW et al (2017) Natural climate solutions. Proc Natl Acad Sci
114:11645–11650
8. Gillson L, Biggs H, Smit IPJ et al (2019) Finding common ground between adaptive
management and evidence-based approaches to biodiversity conservation. Trends Ecol Evol
34:31–44
9. Kueffer C, Kinney K (2017) What is the importance of islands to environmental conservation?
Environ Conserv 44:311–322
10. Whittaker RJ, Fernandez-Palacios JM (2007) Island biogeography: ecology, evolution, and
conservation. OUP, Oxford
11. Darwin C (1859) On the origin of species by means of natural selection, or, the preservation of
favoured races in the struggle for life. Murray, London
12. Fridriksson S (2013) Surtsey: evolution of life on a volcanic island. Elsevier, Kent
13. Ibanez T, Keppel G, Baider C et al (2018) Regional forcing explains local species diversity and
turnover on tropical islands. Glob Ecol Biogeogr 27:474–486
14. MacArthur RH, Wilson EO (1967) The theory of island biogeography. Princeton University
Press, Princeton
3 Diversity, Ecology, and Conservation of Mauritius Orchids 129
15. Norder SJ, Proios K, Whittaker RJ et al (2019) Beyond the last glacial maximum: island
endemism is best explained by long-lasting archipelago configurations. Glob Ecol Biogeogr
28:184–197
16. Kier G, Kreft H, Lee TM et al (2009) A global assessment of endemism and species richness
across island and mainland regions. Proc Natl Acad Sci 106:9322–9327
17. Jenkins CN, Pimm SL, Joppa LN (2013) Global patterns of terrestrial vertebrate diversity and
conservation. Proc Natl Acad Sci 110:E2602–E2610
18. Myers N, Mittermeier RA, Mittermeier CG et al (2000) Biodiversity hotspots for conservation
priorities. Nature 403:853–858
19. Keegan WF, Diamond JM (1987) Colonization of islands by humans: a biogeographical
perspective. In: Schiffer MB (ed) Advances in archaeological method and theory. Academic
Press, San Diego, pp 49–92
20. Rick TC, Kirch PV, Erlandson JM et al (2013) Archeology, deep history, and the human
transformation of island ecosystems. Anthropocene 4:33–45
21. COL (2021) Catalogue of life, 2021. Available from: https://www.catalogueoflife.org/data/
taxon/DPL
22. Taylor A, Weigelt P, König C et al (2019) Island disharmony revisited using orchids as a model
group. New Phytol 223:597–606
23. Hermans J, Rajaovelona L, Cribb P et al (2020) New species and nomenclatural changes in
Cynorkis (Orchidaceae) from Madagascar, the Comoros and the Mascarenes. Kew Bull 75:50
24. Pailler T (2020) Un nouveau Cynorkis (Orchidaceae) pour la flore des Mascareignes. Plant
Ecol Evol 153:492–497
25. Pailler T, Verlynde S, Bytebier B et al (2020) Revision of Angraecum sect. Perrierangraecum
(Orchidaceae; Epidendroideae; Vandeae) for the Mascarenes, with a description of a new
endemic species for Mauritius. Phytotaxa 442:183–195
26. Pailler T, Andilyat M, Andrianarivo C et al (2018) Guide des orchidées des îles de l’Océan
Indien: espèces indigènes et endémiques. Université de La Réunion, St Denis, La Réunion
27. Pailler T, Baider C (2020) Bulbophyllum mascarenense Pailler and Baider sp. nov.: a new
endemic orchid species from the Mascarenes. Bot Lett 167:417–423
28. Pailler T, Henze F (2020) Orchidées de La Réunion. Editions Orphie, St Denis, La Réunion
29. Chase MW, Cameron K, Barrett RL et al (2003) DNA data and Orchidaceae systematics: a
new phylogenetic classification. In: Dixon KW, Kell SP, Barret RL, Cribb PJ (eds) Orchid
conservation. Natural History Publications (Borneo), Kota Kinabalu, pp 69–89
30. Fay MF, Chase MW (2009) Orchid biology: from Linnaeus via Darwin to the 21st century.
Ann Bot 104:359–364
31. Gesner C (1751) Opera botanica. Mich. Seligmanni, Nuremberg
32. Kumbaric A, Savo V, Caneva G (2013) Orchids in the Roman culture and iconography:
evidence for the first representations in antiquity. J Cult Herit 14:311–316
33. Cohen IM, Ackerman JD (2009) Oeceoclades maculata, an alien tropical orchid in a Carib-
bean rainforest. Ann Bot 104:557–563
34. Swarts ND, Dixon KW (2009) Terrestrial orchid conservation in the age of extinction. Ann Bot
104:543–556
35. Montaggioni L, Nativel P (1988) La Réunion, Ile Maurice: Géologie et aperçus biologiques.
Masson, Paris
36. Padya BM (1989) Weather and climate of Mauritius. Mahatma Gandhi Institute, Moka
37. Cheke A, Hume J (2008) Lost land of the Dodo: an ecological history of the Mascarene
Islands. T and AD Poyser, London
38. de Boer EJ, Hooghiemstra H, Florens FBV et al (2013) Rapid succession of plant associations
on the small ocean island of Mauritius at the onset of the Holocene. Quat Sci Rev 68:114–125
39. Hammond DS, Gond V, Baider C et al (2015) Threats to environmentally sensitive areas from
peri-urban expansion in Mauritius. Environ Conserv 42:256–267
40. Page W, D’Argent GA (1997) A vegetation survey of Mauritius (Indian Ocean) to identify
priority rainforest areas for conservation management. IUCN/MWF report, Port Louis,
Mauritius
130 C. Baider and F. B. V. Florens
41. Florens FBV (2013) Conservation in Mauritius and Rodrigues: challenges and achievements
from two ecologically devastated oceanic islands. In: Sodhi NS, Gibson L, Raven PH (eds)
Conservation biology: voices from the tropics. Wiley-Blackwell, Chichester, pp 40–50
42. Florens FBV, Baider C, Martin GM et al (2016) Invasive alien plants progress to dominate
protected and best-preserved wet forests of an oceanic island. J Nat Conserv 34:93–100
43. Vaughan RE, Wiehe PO (1937) Studies on the vegetation of Mauritius: I. A preliminary survey
of the plant communities. J Ecol 25:289–343
44. Du Petit-Thouars AA (1804) Cahier de six planches, tirées en couleur, grand in-folio. The
author, Paris
45. Du Petit-Thouars AA (1809) Extrait de trois mémoires lus à la première classe de l’Institut, sur
l’histoire des plantes orchidées des îles australes d’Afrique. Tableau des espèces des plantes
orchidées qui composent le genre Angorkis. Nouv Bull Sci Philom Paris 1:314–319
46. Du Petit-Thouars AA (1822) Histoire particulière des plantes orchidées recueillies sur les trois
îles Australes d’Afrique, de France, de Bourbon et de Madagascar. The author, Paris, 32 pp +
109 plates
47. Vermeulen JJ, Schuiteman A, de Vogel EF (2018) New taxa in Bulbophyllum (Orchidaceae),
Epidendroideae: Malaxideae from New Guinea, lifting a mega-genus over the 2000-species
mark. Malesian Orchid J 21:31–68
48. Vaughan RE (1958) Wenceslaus Bojer 1795–1856. Proc R Soc Arts Sci Mauritius 2:73–98
49. Bojer W (1837) Hortus Mauritianus: ou énumération des plantes, exotiques et indigènes, qui
croissent à l’Ile Maurice, disposées d’après la méthode naturelle. Imprimerie d’Aimé Mamarot
et Compagnie, Mauritius
50. Roberts DL, Florens FBV, Baider C et al (2004) Taeniophyllum coxii (Summerh.) Summerh.
(Orchidaceae): a new record for Mauritius, Indian Ocean. Kew Bull 59:493
51. Baider C, Florens FBV, Rakotoarivelo F et al (2012) Two new records of Jumellea
(Orchidaceae) for Mauritius (Mascarene Islands) and their conservation status. Phytotaxa
52:21–28
52. Pailler T, Baider C (2012) Polystachya jubaultii Pailler (Orchidaceae), une espèce nouvelle
endémique de Mascareignes. L’Orchidophile 195:285–289
53. Fournel J, Micheneau C, Baider C (2015) A new critically endangered species of Angraecum
(Orchidaceae) endemic to the island of Mauritius, Indian Ocean. Phytotaxa 222:211–220
54. Pailler T, Rakotoarivelo FP, Razafimandimbison SG et al (2020) Taxonomic revision of
Jumellea (Orchidaceae, Angraecinae) in the Mascarenes. Phytotaxa 477:1–34
55. Ryan WBF, Carbotte SM, Coplan JO et al (2009) Global multi-resolution topography synthe-
sis. Geochem Geophys Geosyst 10:Q03014
56. Bosser J, Cadet T, Guého J et al (1976) Flore des Mascareignes: La Réunion, Maurice,
Rodrigues. MSIRI/KEW/IRD, Port Louis
57. Baider C (2009) MEL Joseph Guého 1937–2008: in memoriam. Taxon 58:679
58. Roberts DL (2001) Reproductive biology and conservation of the orchids of Mauritius. PhD
thesis, University of Aberdeen, 290 p
59. Mallet B, Pailler T, Blambert L et al (2012) Différences morphologiques et identification de
Jumellea rossii et Jumellea fragrans (Orchidaceae) à l’Ile de la Réunion: Implications pour la
conservation. Rev Ecol (Terre Vie) 11(Suppl):73–83
60. Bosser J (1965) Contribution à l’étude des Orchidaceae de Madagascar V. A. Révision de
quelques sections du genre Bulbophyllum à Madagascar. B. Autres espèces nouvelles de
Bulbophyllum. C. Espèces nouvelles des genres Cryptopus et Angraecum. Adansonia 5:375–410
61. Fay MF (2018) Orchid conservation: how can we meet the challenges in the twenty-first
century? Bot Stud 59:16
62. Isaac NJB, Mallet J, Mace GM (2004) Taxonomic inflation: its influence on macroecology and
conservation. Trends Ecol Evol 19:464–469
63. Givnish TJ, Spalink D, Ames M et al (2015) Orchid phylogenomics and multiple drivers of
their extraordinary diversification. Proc R Soc B Biol Sci 282:20151553
3 Diversity, Ecology, and Conservation of Mauritius Orchids 131
64. Taylor A, Keppel G, Weigelt P et al (2021) Functional traits are key to understanding orchid
diversity on islands. Ecography 4:703–714
65. Martos F, Dulormne M, Pailler T et al (2009) Independent recruitment of saprotrophic fungi as
mycorrhizal partners by tropical achlorophyllous orchids. New Phytol 184:668–681
66. Bosser J (2006) Contribution à l’étude des Orchidaceae de Madagascar, des Comores et des
Mascareignes. XXXV. Description d’un Oeceoclades nouveau de Madagascar, et notes sur
trois genres nouveaux pour les Mascareignes. Adansonia 28:45–54
67. Jacquemyn H, Micheneau C, Roberts DL et al (2005) Elevational gradients of species
diversity, breeding system and floral traits of orchid species on Réunion Island. J Biogeogr
32:1751–1761
68. Ackerman JD, Trejo-Torres JC, Crespo-Chuy Y (2007) Orchids of the West Indies: predict-
ability of diversity and endemism. J Biogeogr 34:779–786
69. Ding Y, Liu G, Zang R et al (2016) Distribution of vascular epiphytes along a tropical
elevational gradient: disentangling abiotic and biotic determinants. Sci Rep 6:19706
70. Keppel G, Gillespie TW, Ormerod P et al (2016) Habitat diversity predicts orchid diversity in
the tropical south-west Pacific. J Biogeogr 43:2332–2342
71. Roberts DL, Dixon KW (2008) Orchids. Curr Biol 18:R325–R329
72. Vollering J, Schuiteman A, de Vogel E et al (2016) Phytogeography of New Guinean orchids:
patterns of species richness and turnover. J Biogeogr 43:204–214
73. Micheneau C, Fournel J, Humeau L et al (2008) Orchid–bird interactions: a case study from
Angraecum (Vandeae, Angraecinae) and Zosterops (white-eyes, Zosteropidae) on Reunion
Island. Botany 86(10):1143–1151
74. Micheneau C, Fournel J, Pailler T (2006) Bird pollination in an angraecoid orchid on Reunion
Island (Mascarene Archipelago, Indian Ocean). Ann Bot 97:965–974
75. Micheneau C, Fournel J, Warren BH et al (2010) Orthoptera, a new order of pollinator. Ann
Bot 105:355–364
76. Safford RJ (1997) A survey of the occurrence of native vegetation remnants on Mauritius in
1993. Biol Conserv 80:181–188
77. FAO (2020) Global forest resources assessment 2020, report Mauritius. FAO, Rome
78. Angulo E, Deves A-L, Saint Jalmes M et al (2009) Fatal attraction: rare species in the spotlight.
Proc R Soc B Biol Sci 276:1331–1337
79. Courchamp F, Angulo E, Rivalan P et al (2006) Rarity value and species extinction: the
anthropogenic Allee effect. PLoS Biol 4:e415
80. Baider C (2017) Fireside Science, The smallest orchid of Mauritius, 2017. Available from:
https://scifundchallenge.org/firesidescience/2017/03/08/the-smallest-orchid-of-mauritius/
81. IUCN SSC (2001) IUCN Red List categories and criteria: version 3.1. IUCN Species Survival
Commission, Gland/Cambridge
82. Thormann B (2010) Threat-analysis of endemic tree fern species of Mauritius – role of
invasive plant species. BSc thesis, University of Applied Sciences, Eberswalde
83. Rouillard G, Guého J (1999) Les Plantes et leur histoire à l’île Maurice. MSM Ltd, Mauritius
84. Mawlah U (2007) Ecology of native Mauritian orchids and response to conservation manage-
ment. BSc thesis, University of Mauritius, Réduit
85. Florens FBV, Baider C (2013) Ecological restoration in a developing island nation: how useful
is the science? Restor Ecol 21:1–5
86. Vaughan RE, Wiehe PO (1941) Studies on the vegetation of Mauritius: III. The structure and
development of the upland climax forest. J Ecol 29:127–160
87. Florens FBV, Baider C, Seegoolam NB et al (2017) Long-term declines of native trees in an
oceanic island’s forests invaded by alien plants. Appl Veg Sci 20:94–105
88. Lorence DH, Sussman RW (1986) Exotic species invasion into Mauritius wet forest remnants.
J Trop Ecol 2:147–162
89. Florens FBV (2008) Ecologie des forêts tropicales de l’Ile Maurice et impact des espèces
introduites envahissantes. PhD thesis, Université de La Réunion
132 C. Baider and F. B. V. Florens
90. Baider C, Florens FBV (2006) Current decline of the ‘Dodo-tree’: a case of broken-down
interactions with extinct species or the result of new interactions with alien invaders? In:
Laurance W, Peres C (eds) Emerging threats to tropical forests. Chicago University Press,
Chicago, pp 199–214
91. Rasmussen HN, Rasmussen FN (2018) The epiphytic habitat on a living host: reflections on
the orchid–tree relationship. Bot J Linn Soc 186:456–472
92. Zarate-García AM, Noguera-Savelli E, Andrade-Canto SB et al (2020) Bark water storage
capacity influences epiphytic orchid preference for host trees. Am J Bot 107:726–734
93. Baider C, Florens FBV (2011) Control of invasive alien weeds averts imminent plant extinc-
tion. Biol Invasions 13:2641–2646
94. Monty MF, Florens FBV, Baider C (2013) Invasive alien plants elicit reduced production of
flowers and fruits in various native forest species on the tropical island of Mauritius
(Mascarenes, Indian Ocean). Trop Conser Sci 6:35–49
95. Krivek G, Florens FBV, Baider C et al (2020) Invasive alien plant control improves foraging
habitat quality of a threatened island flying fox. J Nat Conserv 54:125805
96. Bindewald A (2011) Threat analysis of the large epiphytic ferns Asplenium nidus L. and
Microsorum punctatum (L.) Copel. in the wet lowland forests of Mauritius: impact of invasive
alien plants. BSc thesis, University of Mauritius, Réduit
97. Hugel S (2012) Impact of native forest restoration on endemic crickets and katydids density in
Rodrigues island. J Insect Conserv 16:473–477
98. Florens FBV, Mauremootoo JR, Fowler SV et al (2010) Recovery of indigenous butterfly
community following control of invasive alien plants in a tropical island’s wet forests.
Biodivers Conserv 19:3835–3848
99. Bernet P (2010) Les Orchidées de La Réunion, MSM, Mauritius
100. de Cordemoy EJ (1895) Flore de l’île de la Réunion:(phanérogames, cryptogames, vasculaires,
muscinées) avec l’indication des propriétés économiques & industrielles des plantes,
P. Klinsksieck, Paris
101. Szelengowicz M, Tamon JM (2013) Les orchidées des Mascareignes. Printec, Seychelles
102. Reinegger RD, Oleksy RZ, Bissessur P et al (2021) First come, first served: fruit availability to
keystone bat species is potentially reduced by invasive macaques. J Mammal. 102:428–439
103. Florens FBV, Baider C, Martin GM et al (2012) Surviving 370 years of human impact: what
remains of tree diversity and structure of the lowland wet forests of oceanic island Mauritius?
Biodivers Conserv 21:2139–2167
104. Baider C, Florens FBV (2013) Eugenia alletiana (Myrtaceae), a new critically endangered
endemic species to the island of Mauritius. Phytotaxa 94:1–12
105. Singer FJ, Swank WT, Clebsch EEC (1984) Effects of wild pig rooting in a deciduous forest.
J Wildl Manag 48:464–473
106. Hone J (2002) Feral pigs in Namadgi National Park, Australia: dynamics, impacts and
management. Biol Conserv 105:231–242
107. Harrison RD, Tan S, Plotkin JB et al (2013) Consequences of defaunation for a tropical tree
community. Ecol Lett 16:687–694
108. Terborgh J, Nuñez-Iturri G, Pitman NCA et al (2008) Tree recruitment in an empty forest.
Ecology 89:1757–1768
109. Kingston T, Florens FBV, Oleksy RZ, et al. (2018) Pteropus niger, The IUCN Red List of
Threatened Species 2018: https://doi.org/10.1109/e.T18743A86475525
110. Florens FBV, Baider C, Marday V et al (2017) Disproportionately large ecological role of a
recently mass-culled flying fox in native forests of an oceanic island. J Nat Conserv 40:85–93
111. Jones CG, Lawton JH, Shachak M (1997) Positive and negative effects of organisms as
physical ecosystem engineers. Ecology 78:1946–1957
112. Florens FBV (2015) Flying foxes face cull despite evidence. Science 350:1325
113. Florens FBV (2012) Going to bat for an endangered species. Science 336:1102
114. Florens FBV (2016) Mauritius culls threatened fruit bats. Nature 530:33
3 Diversity, Ecology, and Conservation of Mauritius Orchids 133
115. Florens FBV, Baider C (2019) Mass-culling of a threatened island flying fox species failed to
increase fruit growers’ profits and revealed gaps to be addressed for effective conservation. J
Nat Conserv 47:58–64
116. Vincenot CE, Florens FBV, Kingston T (2017) Can we protect island flying foxes? Science
355:1368–1370
117. McConkey KR, Drake DR (2006) Flying foxes cease to function as seed dispersers long before
they become rare. Ecology 87:271–276
118. Albert S, Flores O, Baider C et al (2021) Differing severity of frugivore loss contrasts the fate
of native forests on the land of the Dodo (Mascarene archipelago). Biol Conserv 257:109131
119. Albert S, Flores O, Strasberg D (2020) Collapse of dispersal trait diversity across a long-term
chronosequence reveals a strong negative impact of frugivore extinctions on forest resilience. J
Ecol 108:1386–1397
120. Gouvenain RC d, Kobe RK, Silander JA (2007) Partitioning of understorey light and
dry-season soil moisture gradients among seedlings of four rain-forest tree species in Mada-
gascar. J Trop Ecol 23:569–579
121. Sodhi NS, Butler R, Laurance WF et al (2011) Conservation successes at micro-, meso-and
macroscales. Trends Ecol Evol 26:585–594
122. Florens FBV (2012) National parks: Mauritius is putting conservation at risk. Nature 481:29–29
123. Florens FBV (2013) Research safeguards protected areas: the important role of governments.
Trends Ecol Evol 28:504
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,
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.
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.
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].
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.
138 A. Bakayoko et al.
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].
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
140 A. Bakayoko et al.
(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,
4 Diversity of Orchids from Continental Sub-Saharan Africa 141
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.
4 Diversity of Orchids from Continental Sub-Saharan Africa 145
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.
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
146 A. Bakayoko et al.
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
148 A. Bakayoko et al.
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
4 Diversity of Orchids from Continental Sub-Saharan Africa 149
Fig. 8 Image of
Diaphananthe bidens
(Afzel. ex Sw.) Schltr.
(From Swiss Center for
Scientific Research)
Fig. 9 Image of
Listrostachys pertusa. (From
the Parc National des Île
Ehotilés)
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.
4 Diversity of Orchids from Continental Sub-Saharan Africa 151
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.
References
1. White F (1986) La végétation de l’Afrique. ORSTOM-UNESCO, Paris, p 391
2. Govaerts R, Cribb PJ, Wood J (2003) Monocot checklist. Royal Botanic Gardens, London
3. Geerinck D (1992) Orchidaceae (seconde partie). In: Bamps P (ed) Flore d’Afrique centrale
(Zaïre, Rwanda, Burundi). Spermatophyte. Jardin Botanique National de Belgique, Meise
4. Perez-Vera F (2003) Les Orchidées de Côte d’Ivoire. Collection Parthénope. Biotope, Mèze,
p 576
5. Lebrun JP, Stork AL (1995) Enumération des plantes à fleurs d’Afrique tropicale. Mono-
cotylédones: Limnocharitaceae à Poaceae, vol 3. Conservatoire et Jardin Botaniques de la
Ville de Genève, Geneva, p 341
6. Lebrun JP, Stork AL (1997) Enumération des plantes à fleurs d’Afrique tropicale. Gamopétales:
Clethraceae à Lamiaceae, vol 4. Conservatoire et Jardin Botaniques de la Ville de Genève,
Geneva, p 342
7. Hamisy WC (2007) Development of conservation strategies for the wild edible orchid in
Tanzania. Progress report for the Rufford Small Grants Foundation. The Rufford Foundation,
London
8. Owen J (2011) Kew scientists lead fight to save orchids from extinction. The Independent 15(4):
509–514
9. Lebrun JP, Stork AL (2015) Tropical African flowering plants. Ecology and distribution.
Orchidaceae, part one (Genera A–G). Conservatoire et Jardin Botaniques de la Ville de Genève,
Geneva
10. Lebrun JP, Stork AL (2017) Tropical African flowering plants. Ecology and distribution.
Orchidaceae, part two (Genera H–Z), vol 10. Conservatoire et Jardin Botaniques de la Ville
de Genève, Geneva, p 352
11. Schatz B, Geniez P (2001) Les orchidées, un patrimoine naturel à conserver. In: Le génie de la
nature. Biotope, Mèze, p 47
12. Johansson D (1974) Ecology of vascular epiphytes in West African rain forest. Acta Phytogeogr
Suec 59:1–136
13. Freudenstein JV, Chase MW (2015) Phylogenetic relationships in Epidendroideae
(Orchidaceae), one of the great flowering plant radiations: progressive specialization and
diversification. Ann Bot 115:665–681
14. Simo-Droissart M, Plunkett GM, Droissart V, Edwards MB, Farminhão JNM, Jecmenica V,
D’Haijère T, Lowry PP II, Sonké B, Micheneau C, Carlsward BS, Azandi L, Verlynde S, Hardy
OJ, Martos F, Bytebier B, Fischer E, Stévart T (2018) New phylogenetic insights toward
developing a natural generic classification of African Angraecoid orchids (Vandeae,
Orchidaceae). Mol Phylogenet Evol 126:241–249
15. Cribb PJ, Thomas S, Rasmussen FN (2001) The orchids of Ethiopia and Eritrea. Biol Skr 54:
75–84
16. Zhou Y, Liu B, Mbuni Y, Yan X, Mwachala G, Hu G, Wang Q (2017) Vascular flora of Kenya,
based on the Flora of Tropical East Africa. PhytoKeys 90:113–126
17. Droissart V, Sonké B, Stévart T (2006) Les Orchidaceae endémiques d’Afrique centrale
atlantique présentes au Cameroun. Syst Geogr Plants 76(1):3–84
18. Borokini TI (2014) A systematic compilation of endemic flora in Nigeria for conservation
management. J Threat Taxa 6(11):6406–6426
152 A. Bakayoko et al.
19. Cribb PJ, Pérez-Vera F (1975) Bulbophyllum ivorense P. J.Cribb & Pérez-Vera. Asansonia
15:209
20. Figueiredo E, Smith GF (2008) Plants of Angola. Strelitzia 22:292
21. Morris B (1982) The orchids of Malawi. Soc Malawi J 35(2):7–23
22. White F, Dowsett-Lemaire F, Chapman JD (2001) Evergreen forest flora of Malawi. Royal
Botanic Gardens, Kew, p 708
23. Darbyshire I, Timberlake J, Osborne J, Rokni S, Matimele H, Langa C, Datizua C, de Sousa C,
Alves T, Massingue A, Hadj-Hammou J, Dhanda S, Shah T, Wursten B (2019) The endemic
plants of Mozambique: diversity and conservation status. PhytoKeys 136:45–96
24. Mapaura A (2002) Endemic plant species of Zimbabwe. Nat Herb Bot Gard 18(1):117–149
25. Fonge BA, Essomo SE, Bechem TE, Tabot PT, Arrey BD, Afanga Y, Assoua EM (2019) Market
trends and ethnobotany of orchids of Mount Cameroon. J Ethnobiol Ethnomed 15(29):1–11
26. Menzepoh SB (2011) Les orchidées comestibles chez le peuple Bagam au Cameroun.
Biotechnologie. Agron Soc Environ 15(4):509–514
27. Kasulo V, Mwabumba L, Cry M (2009) A review of edible orchids in Malawi. J Hortic For 1(7):
133–139
28. Mapunda LND (2007), Edible orchids in Makete district, the Southern Highlands of Tanzania:
distribution, population and status. Master thesis. Swedish Biodiversity 553 Centre, Uppsala
Universitet, Uppsala, p 68
29. Assédé ESP, Djagoun CAMS, Azihou AF, Kouton MD, Gogan YSC, Geldenhuys CJ, Chirwa
PW, Sinsin BA (2017) Folk perceptions and patterns of use of orchid species in Benin, West
Africa. Flora et Vegetatio Sudano-Sambesica 20:26–36
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
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
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, 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].
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
5 Orchid Biodiversity and Genetics 159
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.
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
5 Orchid Biodiversity and Genetics 161
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
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].
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
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.
References
1. Mondragon-Palomino M, Theissen G (2009) Why are orchid flowers so diverse? Reduction of
evolutionary constraints by paralogues of class B floral homeotic genes. Ann Bot (Lond) 104:
583–594
2. Schiestl FP, Schluter PM (2009) Floral isolation, specialized pollination, and pollinator behav-
ior in orchids. Annu Rev Entomol 54:425–446
3. Wang SL, Viswanath KK, Tong CG, An HR, Jang S, Chen FC (2019) Floral induction and
flower development of orchids. Front Plant Sci 10:1258
4. Blanchard MG, Runkle ES (2006) Temperature during the day, but not during the night,
controls flowering of Phalaenopsis orchids. J Exp Bot 57(15):4043–4049
5. Campos KO, Kerbauy GB (2004) Thermoperiodic effect on flowering and endogenous hor-
monal status in Dendrobium (Orchidaceae). J Plant Physiol 161(12):1385–1387
6. Zhang Y, Zhao S, Liu D, Zhang Q, Cheng J (2014) Flowering phenology and reproductive
characteristics of Cypripedium macranthos (Orchidaceae) in China and their implication in
conservation. Pak J Bot 46(4):1303–1308
7. Wang WY, Chen WS, Huang KL, Huang L-S, Chen WH, Su WR (2003) The effects of day
length on protein synthesis and flowering in Doritis pulcherrima. Sci Hortic 97:49–56
8. Blanchard MG, Runkle ES (2008) Benzyladenine promotes flowering in Doritaenopsis and
Phalaenopsis orchids. J Plant Growth Regul 27(2):141–150
9. Wickland DP, Hanzawa Y (2015) The FLOWERING LOCUS T/TERMINAL FLOWER 1 gene
family: functional evolution and molecular mechanisms. Mol Plant 8:983–997
10. Hanzawa Y, Money T, Bradley D (2005) A single amino acid converts a repressor to an activator
of flowering. Proc Natl Acad Sci U S A 102:7748–7753
11. Ahn JH, Miller D, Winter VJ, Banfield MJ, Lee JH, Yoo SY, Henz SR, Brady RL, Weigel D
(2006) A divergent external loop confers antagonistic activity on floral regulators FT and TFL1.
EMBOJ 25:605–614
12. Suarez-Lopez P, Wheatley K, Robson F, Onouchi H, Valverde F, Coupland G (2001)
CONSTANS mediates between the circadian clock and the control of flowering in Arabidopsis.
Nature 410:1116–1120
13. Lee JH, Yoo SJ, Park SH, Hwang I, Lee JS, Ahn JH (2007) Role of SVP in the control of
flowering time by ambient temperature in Arabidopsis. Genes Dev 21:397–402
14. Kumar SV, Lucyshyn D, Jaeger KE, Alo’s E, Alvey E, Harberd NP, Wigge PA (2012)
Transcription factor PIF4 controls the thermos-sensory activation of flowering. Nature 484:
242–245
15. Adrian J, Farrona S, Reimer JJ, Albani MC, Coupland G, Turck F (2010) Cis-regulatory
elements and chromatin state coordinately control temporal and spatial expression of
FLOWERING LOCUS T in Arabidopsis. Plant Cell 22:1425–1440
16. Searle I, He Y, Turck F, Vincent C, Fornara F, Krober S, Amasino RA, Coupland G (2006) The
transcription factor FLC confers a flowering response to vernalization by repressing meristem
competence and systemic signaling in Arabidopsis. Genes Dev 20:898–912
17. Abe M, Kobayashi Y, Yamamoto S, Daimon Y, Yamaguchi A, Ikeda Y, Ichinoki H,
Notaguchi M, Goto K, Araki T (2005) FD, a bZIP protein mediating signals from the floral
pathway integrator FT at the shoot apex. Science 309:1052–1056
168 S. G and S. S
18. Wigge PA, Kim MC, Jaeger KE, Busch W, Schmid M, Lohmann JU, Weigel D (2005)
Integration of spatial and temporal information during floral induction in Arabidopsis. Science
309:1056–1059
19. Amasino R (2010) Seasonal and developmental timing of flowering. Plant J 61:1001–1013
20. Simon R, Igeno MI, Coupland G (1996) Activation of floral meristem identity genes in
Arabidopsis. Nature 384:59–62
21. Hanano S, Goto K (2011) Arabidopsis TERMINAL FLOWER1 is involved in the regulation of
flowering time and inflorescence development through transcriptional repression. Plant Cell 23:
3172–3184
22. Jang S, Choi SC, Li HY, An G, Schmelzer E (2015) Functional characterization of Phalaenopsis
aphrodite flowering genes PaFT1 and PaFD. PLoS One 10(8):e0134987
23. Li R, Wang A, Sun S, Liang S, Wang X, Ye Q et al (2012) Functional characterization of FT and
MFT ortholog genes in orchid (Dendrobium nobile Lindl.) that regulate the vegetative to
reproductive transition in Arabidopsis. Plant Cell Tissue Organ Cult 111(2):143–151
24. Ding L, Wang Y, Yu H (2013) Overexpression of DOSOC1, an ortholog of Arabidopsis
SOC1, promotes flowering in the orchid Dendrobium Chao Parya Smile. Plant Cell Physiol
54(4):595–608
25. Sawettalake N, Bunnag S, Wang Y, Shen L, Yu H (2017) DOAP1 promotes flowering in the
orchid Dendrobium Chao Praya Smile. Front Plant Sci 8:400
26. Yu H, Yang SH, Goh CJ (2000) DOH1, a class 1 knox gene, is required for maintenance of the
basic plant architecture and floral transition in orchid. Plant Cell 12(11):2143–2160
27. Hou CJ, Yang CH (2009) Functional analysis of FT and TFL1 orthologs from orchid (Oncidium
Gower Ramsey) that regulate the vegetative to reproductive transition. Plant Cell Physiol 50(8):
1544–1557
28. Hsu HF, Huang CH, Chou LT, Yang CH (2003) Ectopic expression of an orchid (Oncidium
Gower Ramsey) AGL6-like gene promotes flowering by activating flowering time genes in
Arabidopsis thaliana. Plant Cell Physiol 44(8):783–794
29. Chin DC, Hsieh CC, Lin HY, Yeh KW (2016) A low glutathione redox state couples with a
decreased ascorbate redox ratio to accelerate flowering in Oncidium orchid. Plant Cell Physiol
57(2):423–436
30. Sun X, Qin Q, Zhang J, Zhang C, Zhou M, Paek KY et al (2012) Isolation and characterization
of the FVE gene of a Doritaenopsis hybrid involved in the regulation of flowering. Plant
Growth Regul 68(1):77–86
31. Chen W, Qin Q, Zhang C, Zheng Y, Wang C, Zhou M et al (2015) DhEFL2, 3 and 4, the three
EARLY FLOWERING4-like genes in a Doritaenopsis hybrid regulate floral transition. Plant
Cell Rep 34(12):2027–2041
32. Li X, Luo J, Yan T, Xiang L, Jin F, Qin D et al (2013) Deep sequencing-based analysis of the
Cymbidium ensifolium floral transcriptome. PLoS ONE 8(12):e85480. https://doi.org/10.1371/
journal.pone.0085480
33. Lin CS, Hsu CT, Liao DC, Chang WJ, Chou ML, Huang YT et al (2016) Transcriptome-wide
analysis of the MADS-box gene family in the orchid Erycina pusilla. Plant Biotechnol J 14(1):
284–298. https://doi.org/10.1111/pbi.12383
34. Aceto S, Gaudio L (2011) The MADS and the beauty: genes involved in the development of
orchid flowers. Curr Genomics 12(5):342–356
35. Schwarz-Sommer Z, Huijser P, Nacken W, Saedler H, Sommer H (1990) Genetic control of
flower development by homeotic genes in Antirrhinum majus. Science 250:931–936
36. Coen ES, Meyerowitz EM (1991) The war of the whorls: genetic interactions controlling flower
development. Nature 353:31–37
37. Theißen G, Melzer R, Rümpler F (2016) MADS-domain transcription factors and the floral
quartet model of flower development: linking plant development and evolution. Development
143(18):3259–3271
38. Yanofsky MF, Ma H, Bowman JL, Drews GN, Feldmann KA et al (1990) The protein encoded
by the Arabidopsis homeotic gene agamous resembles transcription factors. Nature 346:35–39
5 Orchid Biodiversity and Genetics 169
39. Ó’Maoiléidigh DS, Graciet E, Wellmer F (2014) Gene networks controlling Arabidopsis
thaliana flower development. New Phytol 201:16–30
40. Irish VF (2010) The flowering of Arabidopsis flower development. Plant J 61:1014–1028
41. Angenent GC, Colombo L (1996) Molecular control of ovule development. Trends Plant Sci 1:
228–232
42. Favaro R, Pinyopich A, Battaglia R, Kooiker M, Borghi L, Ditta G, Yanofsky MF, Kater MM,
Colombo L (2003) MADS-box protein complexes control carpel and ovule development in
Arabidopsis. Plant Cell 15:2603–2611
43. Pinyopich A, Ditta GS, Savidge B, Liljegren SJ, Baumann E, Wisman E, Yanofsky MF (2003)
Assessing the redundancy of MADS-box genes during carpel and ovule development. Nature
424:85–88
44. Theißen G (2001) Development of floral organ identity: stories from the MADS house. Curr
Opin Plant Biol 4:75–85
45. Yu H, Goh C (2000) Differential gene expression during floral transition in an orchid hybrid
Dendrobium Madame Thong-In. Plant Cell Rep 19:926–931
46. Yu H, Goh CJ (2002) Molecular genetics of reproductive biology in orchids. Plant Physiol 127:
1390–1393
47. Lu ZX, Wu M, Loh CS, Yeong CY, Goh CJ (1993) Nucleotide sequence of a flower-specific
MADS box cDNA clone from orchid. Plant Mol Biol 23:901–904
48. Mondragon-Palomino M, Theissen G (2008) MADS about the evolution of orchid flowers.
Trends Plant Sci 13:51–59
49. Mondragon-Palomino M, Theissen G (2011) Conserved differential expression of paralogous
DEFICIENS- and GLOBOSA-like MADS-box genes in the flowers of Orchidaceae: refining
the ‘orchid code’. Plant J 66:1008–1019
50. Yu H, Goh CJ (2000) Identification and characterization of three orchid MADS-box genes of the
AP1/AGL9 subfamily during floral transition. Plant Physiol 123(4):1325–1336
51. Nadeau JA, Zhang XS, Li J, O’Neill SD (1996) Ovule development: identification of stage-
specific and tissue-specific cDNAs. Plant Cell 8:213–239
52. O’Neill SD, Nadeau JA, Zhang XS, Bui AQ, Halevy AH (1993) Interorgan regulation of
ethylene biosynthetic genes by pollination. Plant Cell 5:419–432
53. Porat R, Halevy AH, Serek M, Borochov A (1995) An increase in ethylene sensitivity following
pollination is the initial event triggering an increase in ethylene production and enhanced
senescence of Phalaenopsis orchid flower. Physiol Plant 93:778–784
54. Porat R, Borochov A, Halevy AH (1994) Pollination induced senescence in Phalaenopsis
petals. Relationship of ethylene sensitivity to activity of GTP-binding proteins and protein
phosphorylation. Physiol Plant 90:679–684
55. Halevy AH, Porat R, Spiegelstein H, Borochov A, Botha L, Whitehead CS (1996) Short chain
saturated fatty acid in the regulation of pollination-induced ethylene sensitivity of Phalaenopsis
flowers. Physiol Plant 97:469–474
56. Zhang XS, O’Neill SD (1993) Ovary and gametophyte development are coordinately regulated
by auxin and ethylene following pollination. Plant Cell 5:403–418
57. Yu H, Goh CJ (2001) Molecular genetics of reproductive biology in orchids. Plant Physiol 127:
1390–1393
58. Neelam SS, Jyoti SJ, Sandhya T, Bhawana M, Lokesh KN, Rajender SS (2018) Plant metabolic
engineering. In: Barh D, Azevedo V (eds) Omics technologies and bio-engineering towards
improving quality of life, volume 2: microbial, plant, environmental and industrial technologies,
Academic, Elsevier, London, pp 143–175. https://doi.org/10.1016/B978-0-12-815870-8.00009-7
59. Singer AC, Crowley DE, Thompson IP (2003) Secondary plant metabolites in
phytoremediation and biotransformation. TRENDS Biotechnol 21(3):123–130
60. Verpoorte R, Contin A, Memelink J (2002) Biotechnology for the production of plant secondary
metabolites. Phytochem Rev 1:13–25
61. Edreva A, Velikova V, Tsonev T et al (2008) Stress-protective role of secondary metabolites:
diversity of functions and mechanisms. Gen Appl Plant Physiol 34(1–2):67–78
170 S. G and S. S
62. Isah T (2019) Stress and defense responses in plant secondary metabolites production. Biol Res
52:39
63. Xu S, Schlüter PM, Grossniklaus U, Schiestl FP (2012) The genetic basis of pollinator
adaptation in a sexually deceptive orchid. PLoS Genet 8(8):e1002889. https://doi.org/10.
1371/journal.pgen.1002889
64. Mashilo J, Odindo AO, Shimelis HA, Musenge P, Tesfay SZ, Magwaza LS (2017) Drought
tolerance of selected bottle gourd [Lagenaria siceraria (Molina) Standl.] landraces assessed by
leaf gas exchange and photosynthetic efficiency. Plant Physiol Biochem 120:75–87
65. de Matos NJ, Bertodo LOO, Da Rosa LMG, Von Poser GL, Rech SB (2014) Stress induction of
valuable secondary metabolites in Hypericum polyanthemum acclimatized plants. South Afr J
Bot 94:182–189
66. Niinemets Ü (2015) Uncovering the hidden facets of drought stress: secondary metabolites
make the difference. Tree Physiol 36(2):129–132
67. Quan NT, Anh LH, Khang DT et al (2016) Involvement of secondary metabolites in response to
drought stress of rice (Oryza sativa L.). Agriculture 6(2):23
68. Afzal SF, Yar AK, Ullah RH et al (2017) Impact of drought stress on active secondary
metabolite production in Cichorium intybus roots. J Appl Environ Biol Sci 7(7):39–43
69. Piasecka A, Sawikowska A, Kuczyńska A et al (2017) Drought-related secondary metabolites
of barley (Hordeum vulgare L.) leaves and their metabolomic quantitative trait loci. Plant J 89
(5):898–913
70. Johnson M, Janakiraman N (2013) Phytochemical and TLC studies on stem and leaves of the
orchid dendrobium panduratum subsp. villosum Gopalan & AN Henry. Indian J Nat Prod
Resour 4:250–254
71. Banerjee J, Chauhan N, Dey BK (2018) Pharmacognostical, physiochemical and phytochemical
evaluation of leaf, stem and root of orchid Dendrobium ochreatum. J Appl Pharma Res 6(1):16–25
72. Minh TN, Khang DT, Tuyen PT, Minh LT, Anh LH, Quan NV, Ha PT, Quan NT, Toan NP,
Elzaawely AA, Xuan TD (2016) Phenolic compounds and antioxidant activity of Phalaenopsis
orchid hybrids. Antioxidants (Basel) 5(3):31. https://doi.org/10.3390/antiox5030031
73. Minh TN, Tuyen PT, Khang DT, Quan NV, Ha PTT, Quan NT, Andriana Y, Xinyan F, Van TM,
Khanh TD, Xuan TD (2017) Potential use of plant waste from the moth orchid (Phalaenopsis
Sogo Yukidian “V3”) as an antioxidant source. Foods 27(6–10):85
74. Sunita S, Suman K, Deepti G, Mayuresh J, Prasenjit P, Nasheman K (2015) Variation in the
marker content of five different Dendrobium species: comparative evaluation using validated
HPTLC technique. J App Pharm Sci 5(10):32–38
75. Lam Y, Tzi Bun NG, Yao RM, Shi J, Xu K, Sze SCW, Zhang KY (2015) Evaluation of chemical
constituents and important mechanism of pharmacological biology in Dendrobium plants. Evid
Based Complement Alternat Med. https://doi.org/10.1155/2015/841752. Accessed 10 July 2021
76. Williams (1979) The leaf flavonoids of the Orchidaceae. Phytochemistry 18(5):803–813
77. Li BJ, Zheng BQ, Wang JY, Tsai WC, Lu HC, Zou LH et al (2020) New insight into the
molecular mechanism of colour differentiation among floral segments in orchids. Commun Biol
3(89):1–13. https://doi.org/10.1038/s42003-020-0821-8
78. Brockington SF, Walker RH, Glover BJ, Soltis PS, Soltis DE (2011) Complex pigment
evolution in the Caryophyllales. New Phytol 190(4):854–864
79. Panche AN, Diwan AD, Chandra SR (2016) Flavonoids: an overview. J Nutr Sci 5(e47):1–15.
https://doi.org/10.1017/jns.2016.41
80. Zhao D, Tao J (2015) Recent advances on the development and regulation of flower color in
ornamental plants. Front Plant Sci 6:261. https://doi.org/10.3389/fpls.2015.00261
81. Martin C, Gerats T (1993) Control of flower coloration. In: Jordan BR (ed) The molecular
biology of flowering. CAB International, Wallingford
82. Glover BJ (2011) The diversity of flower color: how and why?. WIT Transactions on State of
the Art in Science and Engineering, WI T Press
83. Tan SH, Manap SA, Karim MR, Rashid SS, Mahmood M, Ling Ma N (2014) Comparative
flower pigment study of orchid plants. Adv Environ Biol 8(20):20–24
5 Orchid Biodiversity and Genetics 171
84. Vagas FD, Jimenez AR, Paredes-Lopez O (2000) Natural pigments: carotenoids, anthocyanins
and betalains-characteristics, biosynthesis, processing and stability. Crit Rev Food Sci 40:
173–289
85. Matsui S, Nakamura M (1988) Distribution of flower pigments in perianth of Cattleya and
genera I. species. J Japan Soc Hort Sci 57(2):222–232
86. Honda K, Ômura H, Hori M, Kainoh Y (2010) Allelochemicals in plant-insect interactions. In:
Comprehensive natural products II: chemistry and biology, vol 4. Elsevier Ltd, Boston, pp
563–594
87. Liew CF, LOH CS, Goh CJ, Lim SH (1998) The isolation, molecular characterization and
expression of dihydroflavonol 4-reductase cDNA in the orchid, Bromheadia finlaysoniana.
Plant Sci 135(2):161–169
88. Johnson ET, Yi H, Shin B, Oh BJ, Cheong H, Choi G (1999) Cymbidium hybrida
dihydroflavonol 4-reductase does not efficiently reduce dihydrokaempferol to produce orange
pelargonidin-type anthocyanins. Plant J 19(10):81–85
89. Wang LM, Zhang J, Dong XY, Fu ZZ, Jiang H, Zhang HC (2018) Identification and functional
analysis of anthocyanin biosynthesis genes in Phalaenopsis hybrids. Biol Plant 62(1):45–54
90. Li Y, Liu X, Cai X, Shan X, Gao R, Yang S, Han T, Wang S, Wang L, Gao X (2017)
Dihydroflavonol 4-reductase genes from Freesia hybrida play important and partially over-
lapping roles in the biosynthesis of flavonoids. Front Plant Sci 8:428. https://doi.org/10.3389/
fpls.2017.00428
91. Hieber AD, Mudalige-Jayawickrama RG, Kuehnle AR (2006) Color genes in the orchid
Oncidium Gower Ramsey: identification, expression and potential genetic instability in an
interspecific cross. Planta 223:521–531
92. Mudalige-Jayawickrama RG, Champagne MM, Hieber AD, Kuehnle AR (2005) Cloning and
characterization of two anthocyanin biosynthetic genes from Dendrobium orchid. J Am Soc
Hortic Sci 130:611–618
93. Pitakdantham W, Sutabutra T, Chiemsombat P, Pitaksutheepong C (2011) Isolation and char-
acterization of dihydroflavonol 4-reductase gene in Dendrobium flowers. J Plant Sci 6:88–94
94. Anggraini R, Febriani AL, Mazieda MN, Al-Yamini TH, Listyorini DD (2017) Isolation of
dihydroflavonol-4-reductase (DFR) gene in Dendrobium helix cv. Pomeo Brown. KNE Life Sci
3(4):213–218. https://doi.org/10.18502/kls.v3i4.707
95. Whang SS, Um WS, Song IJ, Lim PO, Choi K, Park KW, Kang KW, Choi MS, Koo JC (2011)
Molecular analysis of anthocyanin biosynthetic genes and control of flower coloration by
flavonoid 30 ,50 -hydroxylase (F30 50 H) in Dendrobium moniliforme. J Plant Biol 54:209–218
96. Khumkarjorn N, Thanonkeo S, Yamada M, Thanonkeo P (2017) Cloning and expression
analysis of a flavanone 3-hydroxylase gene in Ascocenda orchid. J Plant Biochem Biotechnol
26(2):179–190
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
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.
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
178 H. Herrera et al.
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
6 Orchid-Associated Bacteria and Their Plant Growth Promotion Capabilities 179
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].
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
180 H. Herrera et al.
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].
6 Orchid-Associated Bacteria and Their Plant Growth Promotion Capabilities 181
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
182 H. Herrera et al.
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
6 Orchid-Associated Bacteria and Their Plant Growth Promotion Capabilities 183
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).
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
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
188 H. Herrera et al.
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.
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.
190 H. Herrera et al.
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.).
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.
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
192 H. Herrera et al.
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.
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.
6 Orchid-Associated Bacteria and Their Plant Growth Promotion Capabilities 193
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.
References
1. Roberts DL, Dixon KW (2008) Orchids. Curr Biol 18(8):R325–R329
2. Herrera H et al (2019) Orchid mycorrhizal interactions on the Pacific side of the Andes from
Chile. A review. J Soil Sci Plant Nutr 19(1):187–202
3. Clements M et al (1999) Genera Orchidacearum Vol. 1: General introduction, Apostasioideae,
Cypripedioideae. Oxford University Press, Oxford
4. Herrera H et al (2017) Mycorrhizal compatibility and symbiotic seed germination of orchids
from the coastal range and Andes in south Central Chile. Mycorrhiza 27(3):175–188
5. Merckx V et al (2013) Mycoheterotrophy, vol 10. Springer, New York, pp 978–971
6. Herrera H et al (2018) Adaptation and tolerance mechanisms developed by mycorrhizal
Bipinnula fimbriata plantlets (Orchidaceae) in a heavy metal-polluted ecosystem. Mycorrhiza
28(7):651–663
7. Selosse M-A, Roy M (2009) Green plants that feed on fungi: facts and questions about
mixotrophy. Trends Plant Sci 14(2):64–70
8. Valadares RB et al (2012) Narrow fungal mycorrhizal diversity in a population of the orchid
Coppensia doniana. Biotropica 44(1):114–122
9. Fan X-L et al (2020) Transitions between the terrestrial and epiphytic habit drove the evolution
of seed-aerodynamic traits in orchids. Am Nat 195(2):275–283
10. Liu H, Luo Y, Liu H (2010) Studies of mycorrhizal fungi of Chinese orchids and their role in
orchid conservation in China—a review. Bot Rev 76(2):241–262
11. Fuji M et al (2020) Relative effectiveness of Tulasnella fungal strains in orchid mycorrhizal
symbioses between germination and subsequent seedling growth. Symbiosis 81:53–63
12. Yeung EC (2017) A perspective on orchid seed and protocorm development. Bot Stud 58(1):
1–14
13. Valadares R et al (2014) Proteome changes in Oncidium sphacelatum (Orchidaceae) at
different trophic stages of symbiotic germination. Mycorrhiza 24(5):349–360
194 H. Herrera et al.
14. Herrera H et al (2020) Isolation and identification of endophytic bacteria from mycorrhizal
tissues of terrestrial orchids from southern Chile. Diversity 12(2):55
15. Gontijo JB et al (2018) Bioprospecting and selection of growth-promoting bacteria for
Cymbidium sp. orchids. Sci Agric 75(5):368–374
16. Sisti LS et al (2019) The role of non-mycorrhizal fungi in germination of the mycoheter-
otrophic orchid Pogoniopsis schenckii Cogn. Front Plant Sci 10:1589
17. Dearnaley J, Perotto S, Selosse MA (2016) Structure and development of orchid mycorrhizas.
Mol Mycorrhizal Symbiosis 2016:63–86
18. Peterson RL et al (1996) The interface between fungal hyphae and orchid protocorm cells. Can
J Bot 74(12):1861–1870
19. Kuga Y, Sakamoto N, Yurimoto H (2014) Stable isotope cellular imaging reveals that both live
and degenerating fungal pelotons transfer carbon and nitrogen to orchid protocorms. New
Phytol 202(2):594–605
20. Herrera H et al (2020) Mycorrhizal Fungi isolated from native terrestrial orchids from region
of La Araucanía, Southern Chile. Microorganisms 8(8):1120
21. Weiß M et al (2016) Sebacinales–one thousand and one interactions with land plants. New
Phytol 211(1):20–40
22. Jiang J et al (2019) Fusarium oxysporum KB-3 from Bletilla striata: an orchid mycorrhizal
fungus. Mycorrhiza 29(5):531–540
23. Meng Y-Y et al (2019) Are fungi from adult orchid roots the best symbionts at germination? A
case study. Mycorrhiza 29(5):541–547
24. Valadares R et al (2020) Proteomic and transcriptomic analyses indicate metabolic changes
and reduced defense responses in mycorrhizal roots of Oeceoclades maculata (Orchidaceae)
collected in nature. Journal of Fungi 6(3):148
25. Dearnaley JD, Cameron DD (2016) Nitrogen transport in the orchid mycorrhizal symbiosis-
further evidence for a mutualistic association. New Phytol 213(1):10–12
26. Sathiyadash K et al (2020) Orchid mycorrhizal fungi: structure, function, and diversity. In:
Orchid biology: recent trends & challenges. Springer, Singapore, pp 239–280
27. Gnanamanickam SS (2006) Plant-associated bacteria, vol 1. Springer, Dordrecht
28. Yu AO, Leveau JH, Marco ML (2020) Abundance, diversity and plant-specific adaptations of
plant-associated lactic acid bacteria. Environ Microbiol Rep 12(1):16–29
29. Eida AA et al (2018) Desert plant bacteria reveal host influence and beneficial plant growth
properties. PLoS One 13(12):e0208223
30. Dellagi A, Quillere I, Hirel B (2020) Beneficial soil-borne bacteria and fungi: a promising way
to improve plant nitrogen acquisition. J Exp Bot 71(15):4469–4479
31. Basu S, Rabara R, Negi S (2017) Towards a better greener future-an alternative strategy using
biofertilizers. I: plant growth promoting bacteria. Plant Gene 12:43–49
32. Soto J et al (2019) Enhanced arsenic tolerance in Triticum aestivum inoculated with arsenic-
resistant and plant growth promoter microorganisms from a heavy metal-polluted soil. Micro-
organisms 7(9):348
33. Olanrewaju OS, Glick BR, Babalola OO (2017) Mechanisms of action of plant growth
promoting bacteria. World J Microbiol Biotechnol 33(11):1–16
34. Glick BR (1995) The enhancement of plant growth by free-living bacteria. Can J Microbiol 41
(2):109–117
35. Bhattacharyya PN, Jha DK (2012) Plant growth-promoting rhizobacteria (PGPR): emergence
in agriculture. World J Microbiol Biotechnol 28(4):1327–1350
36. Remigi P et al (2016) Symbiosis within symbiosis: evolving nitrogen-fixing legume symbi-
onts. Trends Microbiol 24(1):63–75
37. Ejaz S et al (2020) Effects of inoculation of root-associative Azospirillum and Agrobacterium
strains on growth, yield and quality of pea (Pisum sativum L.) grown under different nitrogen
and phosphorus regimes. Sci Hortic 270:109401
38. Sumbul A et al (2020) Azotobacter: a potential bio-fertilizer for soil and plant health manage-
ment. Saudi J Biol Sci 27(12):3634
6 Orchid-Associated Bacteria and Their Plant Growth Promotion Capabilities 195
39. Madigan MT, Martinko JM, Parker J (1997) Brock biology of microorganisms, vol 11.
Prentice hall, Upper Saddle River
40. Din M et al (2019) Production of nitrogen fixing Azotobacter (SR-4) and phosphorus solubi-
lizing Aspergillus niger and their evaluation on Lagenaria siceraria and Abelmoschus
esculentus. Biotechnol Rep 22:e00323
41. You M et al (2020) Isolation and characterization of Burkholderia cenocepacia CR318, a
phosphate solubilizing bacterium promoting corn growth. Microbiol Res 233:126395
42. Ludueña LM et al (2017) Effects of P limitation and molecules from peanut root exudates on
pqqE gene expression and pqq promoter activity in the phosphate-solubilizing strain Serratia
sp. S119. Res Microbiol 168(8):710–721
43. Zeng X et al (2012) Characterization and potassium-solubilizing ability of Bacillus Circulans
Z 1–3. Adv Sci Lett 10(1):173–176
44. Rajawat MVS et al (2016) A modified plate assay for rapid screening of potassium-solubiliz-
ing bacteria. Pedosphere 26(5):768–773
45. Etesami H, Emami S, Alikhani HA (2017) Potassium solubilizing bacteria (KSB):: mecha-
nisms, promotion of plant growth, and future prospects a review. J Soil Sci Plant Nutr 17(4):
897–911
46. Lian B et al (2002) A comprehensive review of the mechanism of potassium releasing by
silicate bacteria. Acta Mineral Sin 22(2):179–183
47. Sugumaran P, Janarthanam B (2007) Solubilization of potassium containing minerals by
bacteria and their effect on plant growth. World J Agr Sci 3(3):350–355
48. Ahmed E, Holmström SJ (2014) Siderophores in environmental research: roles and applica-
tions. Microb Biotechnol 7(3):196–208
49. Thomine S, Lanquar V (2011) Iron transport and signaling in plants. In: Transporters and
pumps in plant signaling. Springer, Berlin, pp 99–131
50. Braud A et al (2009) Enhanced phytoextraction of an agricultural Cr-and Pb-contami-
nated soil by bioaugmentation with siderophore-producing bacteria. Chemosphere 74(2):
280–286
51. Sabet H, Mortazaeinezhad F (2018) Yield, growth and Fe uptake of cumin (Cuminum
cyminum L.) affected by Fe-nano, Fe-chelated and Fe-siderophore fertilization in the calcar-
eous soils. J Trace Elem Med Biol 50:154–160
52. Kumar P et al (2018) Inoculation of siderophore producing rhizobacteria and their consortium
for growth enhancement of wheat plant. Biocatal Agric Biotechnol 15:264–269
53. El Attar I et al (2019) Screening of stress tolerant bacterial strains possessing interesting multi-
plant growth promoting traits isolated from root nodules of Phaseolus vulgaris L. Biocatal
Agric Biotechnol 20:101225
54. Novais SV et al (2016) Phosphorus-zinc interaction and iron and manganese uptake in the
growth and nutrition of phalaenopsis (Orchidaceae). Rev Bras Ciênc Solo 40:1–10
55. Zuo Y, Zhang F (2011) Soil and crop management strategies to prevent iron deficiency in
crops. Plant Soil 339(1):83–95
56. Kim J, Rees D (1992) Structural models for the metal centers in the nitrogenase molybdenum-
iron protein. Science 257(5077):1677–1682
57. Reichard P (1993) From RNA to DNA, why so many ribonucleotide reductases? Science 260
(5115):1773–1777
58. Camprubi E et al (2017) Iron catalysis at the origin of life. IUBMB Life 69(6):373–381
59. Li J, Li C, Smith SM (2017) Hormone metabolism and signaling in plants. Academic
60. Duca D et al (2014) Indole-3-acetic acid in plant–microbe interactions. Antonie Van Leeu-
wenhoek 106(1):85–125
61. Großkinsky DK et al (2016) Cytokinin production by Pseudomonas fluorescens G20-18
determines biocontrol activity against Pseudomonas syringae in Arabidopsis. Sci Rep 6(1):
1–11
62. Akhtar SS et al (2020) Role of cytokinins for interactions of plants with microbial pathogens
and pest insects. Front Plant Sci 10:1777
196 H. Herrera et al.
63. Dubois M, Van den Broeck L, Inzé D (2018) The pivotal role of ethylene in plant growth.
Trends Plant Sci 23(4):311–323
64. Desbrosses G et al (2009) PGPR-Arabidopsis interactions is a useful system to study signaling
pathways involved in plant developmental control. Plant Signal Behav 4(4):319–321
65. Gowtham H et al (2020) Induction of drought tolerance in tomato upon the application of ACC
deaminase producing plant growth promoting rhizobacterium Bacillus subtilis Rhizo SF 48.
Microbiol Res 234:126422
66. Win KT et al (2018) The ACC deaminase expressing endophyte Pseudomonas spp. enhances
NaCl stress tolerance by reducing stress-related ethylene production, resulting in improved
growth, photosynthetic performance, and ionic balance in tomato plants. Plant Physiol
Biochem 127:599–607
67. Pandey S, Gupta S (2020) Diversity analysis of ACC deaminase producing bacteria associated
with rhizosphere of coconut tree (Cocos nucifera L.) grown in Lakshadweep islands of India
and their ability to promote plant growth under saline conditions. J Biotechnol 324:183–197
68. Glick BR (2014) Bacteria with ACC deaminase can promote plant growth and help to feed the
world. Microbiol Res 169(1):30–39
69. del Carmen Orozco-Mosqueda M, Glick BR, Santoyo G (2020) ACC deaminase in plant
growth-promoting bacteria (PGPB): an efficient mechanism to counter salt stress in crops.
Microbiol Res 235:126439
70. Belimov AA et al (2019) Rhizobial ACC deaminase contributes to efficient symbiosis with pea
(Pisum sativum L.) under single and combined cadmium and water deficit stress. Environ Exp
Bot 167:103859
71. Berg G et al (2016) The plant microbiome explored: implications for experimental botany. J
Exp Bot 67(4):995–1002
72. Backer R et al (2018) Plant growth-promoting rhizobacteria: context, mechanisms of action,
and roadmap to commercialization of biostimulants for sustainable agriculture. Front Plant Sci
9:1473
73. Zhang R, Vivanco JM, Shen Q (2017) The unseen rhizosphere root–soil–microbe interactions
for crop production. Curr Opin Microbiol 37:8–14
74. Zhang Y et al (2017) Soil bacterial and fungal diversity differently correlated with soil
biochemistry in alpine grassland ecosystems in response to environmental changes. Sci Rep
7(1):1–10
75. Prashar P, Kapoor N, Sachdeva S (2014) Rhizosphere: its structure, bacterial diversity and
significance. Rev Environ Sci Biotechnol 13(1):63–77
76. Pinton R (2001) The rhizosphere as a site of biochemical interactions among soil components,
plants, and microorganisms. In: Willig S, Varanini Z, Nannipieri P (Eds) The rhizosphere:
Biochemistry and Organic Substance at the Soil-Plant Interface, 1st ed. CRC Press, Boca
Raton
77. Gobat J-M, Aragno M, Matthey W (2004) The living soil: fundamentals of soil science and
soil biology. Science Publishers, Enfield
78. Grayston S, Vaughan D, Jones D (1997) Rhizosphere carbon flow in trees, in comparison with
annual plants: the importance of root exudation and its impact on microbial activity and
nutrient availability. Appl Soil Ecol 5(1):29–56
79. Beckers B et al (2017) Structural variability and niche differentiation in the rhizosphere and
endosphere bacterial microbiome of field-grown poplar trees. Microbiome 5(1):1–17
80. Massalha H et al (2017) Small molecules below-ground: the role of specialized metabolites in
the rhizosphere. Wiley Online Library
81. Cesco S et al (2012) Plant-borne flavonoids released into the rhizosphere: impact on soil bio-
activities related to plant nutrition. A review. Biol Fertil Soils 48(2):123–149
82. Sugiyama A, Yazaki K (2014) Flavonoids in plant rhizospheres: secretion, fate and their
effects on biological communication. Plant Biotechnol 31(5):431–443
83. Edwards J et al (2015) Structure, variation, and assembly of the root-associated microbiomes
of rice. Proc Natl Acad Sci 112(8):E911–E920
6 Orchid-Associated Bacteria and Their Plant Growth Promotion Capabilities 197
84. Bulgarelli D et al (2012) Revealing structure and assembly cues for Arabidopsis root-
inhabiting bacterial microbiota. Nature 488(7409):91–95
85. Petrini O (1991) Fungal endophytes of tree leaves. In: Microbial ecology of leaves. Springer,
pp 179–197
86. Hardoim PR et al (2015) The hidden world within plants: ecological and evolutionary
considerations for defining functioning of microbial endophytes. Microbiol Mol Biol Rev 79
(3):293–320
87. Papik J et al (2020) The invisible life inside plants: deciphering the riddles of endophytic
bacterial diversity. Biotechnol Adv 44:107614
88. Hardoim PR, van Overbeek LS, van Elsas JD (2008) Properties of bacterial endophytes and
their proposed role in plant growth. Trends Microbiol 16(10):463–471
89. Reinhold-Hurek B, Hurek T (2011) Living inside plants: bacterial endophytes. Curr Opin Plant
Biol 14(4):435–443
90. Yu X et al (2015) Effects of growth stage and fulvic acid on the diversity and dynamics of
endophytic bacterial community in Stevia rebaudiana Bertoni leaves. Front Microbiol 6:867
91. Yandigeri MS et al (2012) Drought-tolerant endophytic actinobacteria promote growth of
wheat (Triticum aestivum) under water stress conditions. Plant Growth Regul 68(3):411–420
92. van der Heijden MGA et al (2015) Mycorrhizal ecology and evolution: the past, the present,
and the future. New Phytol 205(4):1406–1423
93. Prasad R et al (2017) Introduction to mycorrhiza: historical development. In: Varma A, Prasad
R, Tuteja N (eds) Mycorrhiza – function, diversity, state of the art. Springer International
Publishing, Cham, pp 1–7
94. Malhi GS et al (2020) Arbuscular mycorrhiza in combating abiotic stresses in vegetables: an
eco-friendly approach. Saudi J Biol Sci 28(2):1465
95. Bonfante P, Genre A (2010) Mechanisms underlying beneficial plant–fungus interactions in
mycorrhizal symbiosis. Nat Commun 1(1):1–11
96. Martin F et al (2016) Unearthing the roots of ectomycorrhizal symbioses. Nat Rev Microbiol
14(12):760–773
97. Teotia P et al (2017) Mobilization of micronutrients by mycorrhizal fungi. In: Varma A, Prasad
R, Tuteja N (eds) Mycorrhiza – function, diversity, state of the art. Springer International
Publishing, Cham, pp 9–26
98. Peterson RL, Massicotte HB, Melville LH (2004) Mycorrhizas: anatomy and cell biology.
NRC Research Press, Ottawa
99. Garbaye J (1994) Tansley review no. 76 helper bacteria: a new dimension to the mycorrhizal
symbiosis. New Phytol 128(2):197–210
100. Frey-Klett P, Garbaye J, Tarkka M (2007) The mycorrhiza helper bacteria revisited. New
Phytol 176(1):22–36
101. Deveau A, Labbé J (2016) Mycorrhiza helper bacteria. In: Martin F (ed) Molecular Mycor-
rhizal Symbiosis. Wiley, Hoboken
102. Rigamonte TA, Pylro VS, Duarte GF (2010) The role of mycorrhization helper bacteria in the
establishment and action of ectomycorrhizae associations. Brazilian J Microbiol 41(4):832–
840
103. Singh R et al (2015) Biochemistry and genetics of ACC deaminase: a weapon to “stress
ethylene” produced in plants. Front Microbiol 6:937
104. Barnawal D et al (2014) ACC deaminase-containing Arthrobacter protophormiae induces NaCl
stress tolerance through reduced ACC oxidase activity and ethylene production resulting in
improved nodulation and mycorrhization in Pisum sativum. J Plant Physiol 171(11):884–894
105. Lies A et al (2018) Using mycorrhiza helper microorganisms (MHM) to improve the mycor-
rhizal efficiency on plant growth. In: Meena VS (ed) Role of rhizospheric microbes in soil:
volume 1: stress management and agricultural sustainability. Springer Singapore, Singapore,
pp 277–298
106. Myo EM et al (2019) Indole-3-acetic acid production by Streptomyces fradiae NKZ-259 and
its formulation to enhance plant growth. BMC Microbiol 19(1):155
198 H. Herrera et al.
107. Krause K et al (2015) Biosynthesis and secretion of Indole-3-acetic acid and its morphological
effects on Tricholoma vaccinum-spruce ectomycorrhiza. Appl Environ Microbiol 81(20):
7003–7011
108. Pettipher GL, Jay JM, Wang HH (2005) Microbiological techniques. In: Worsfold P,
Townshend A, Poole C (eds) Encyclopedia of analytical science, 2nd edn. Elsevier, Oxford,
pp 16–25
109. Fuentes A et al (2020) Fungal and bacterial microbiome associated with the rhizosphere of
native plants from the Atacama Desert. Microorganisms 8(2):209
110. Fouda AH et al (2015) Biotechnological applications of fungal endophytes associated with
medicinal plant Asclepias sinaica (Bioss.). Ann Agric Sci 60(1):95–104
111. Verma VC et al (2009) Endophytic actinomycetes from Azadirachta indica a. Juss.: isolation,
diversity, and anti-microbial activity. Microb Ecol 57(4):749–756
112. Herrera H et al (2019) Root-associated fungal communities in two populations of the fully
mycoheterotrophic plant arachnitis uniflora phil.(corsiaceae) in Southern Chile. Microorgan-
isms 7(12):586
113. Bodor A et al (2020) Challenges of unculturable bacteria: environmental perspectives. Rev
Environ Sci Biotechnol 19(1):1–22
114. Emerson D et al (2008) Identifying and characterizing bacteria in an era of genomics and
proteomics. Bioscience 58(10):925–936
115. Gürtler V, Stanisich VA (1996) New approaches to typing and identification of bacteria using
the 16S-23S rDNA spacer region. Microbiology (Reading) 142(Pt 1):3–16
116. Hunt DE et al (2006) Evaluation of 23S rRNA PCR primers for use in phylogenetic studies of
bacterial diversity. Appl Environ Microbiol 72(3):2221–2225
117. Karah N et al (2011) Species identification and molecular characterization of Acinetobacter
spp. blood culture isolates from Norway. J Antimicrob Chemother 66(4):738–744
118. Lau SKP et al (2015) Chapter 12 – Gene amplification and sequencing for bacterial identifi-
cation. In: Sails A, Tang Y-W (eds) Methods in microbiology. Academic Press, pp 433–464
119. Welker M (2011) Proteomics for routine identification of microorganisms. Proteomics 11(15):
3143–3153
120. Risch M et al (2010) Comparison of MALDI TOF with conventional identification of
clinically relevant bacteria. Swiss Med Wkly 140:w13095
121. Rahi P, Prakash O, Shouche YS (2016) Matrix-assisted laser desorption/ionization time-of-
flight mass-spectrometry (MALDI-TOF MS) based microbial identifications: challenges and
scopes for microbial ecologists. Front Microbiol 7:1359–1359
122. Stewart EJ (2012) Growing unculturable bacteria. J Bacteriol 194(16):4151–4160
123. Pham VHT, Kim J (2012) Cultivation of unculturable soil bacteria. Trends Biotechnol 30(9):
475–484
124. Muyzer G, Smalla K (1998) Application of denaturing gradient gel electrophoresis (DGGE)
and temperature gradient gel electrophoresis (TGGE) in microbial ecology. Antonie Van
Leeuwenhoek 73(1):127–141
125. Al-Mailem DM, Kansour MK, Radwan SS (2017) Capabilities and limitations of DGGE for
the analysis of hydrocarbonoclastic prokaryotic communities directly in environmental sam-
ples. Microbiology 6(5):e00495
126. Ravin NV, Mardanov AV, Skryabin KG (2015) Metagenomics as a tool for the investigation of
uncultured microorganisms. Russ J Genet 51(5):431–439
127. Baker GC, Smith JJ, Cowan DA (2003) Review and re-analysis of domain-specific 16S
primers. J Microbiol Methods 55(3):541–555
128. Muhamad Rizal NS et al (2020) Advantages and limitations of 16S rRNA next-generation
sequencing for pathogen identification in the diagnostic microbiology laboratory: perspectives
from a middle-income country. Diagnostics 10(10):816
129. Kaul S, Sharma T, Dhar MK (2016) “Omics” Tools for better understanding the plant-
endophyte interactions. Front Plant Sci 7:955–955
6 Orchid-Associated Bacteria and Their Plant Growth Promotion Capabilities 199
130. Marchesi JR, Ravel J (2015) The vocabulary of microbiome research: a proposal. Microbiome
3(1):31
131. Alibrandi P et al (2020) Diversity and structure of the endophytic bacterial communities
associated with three terrestrial orchid species as revealed by 16S rRNA gene metabarcoding.
Front Microbiol 11:604964
132. Li O et al (2017) Bacterial and diazotrophic diversities of endophytes in Dendrobium
catenatum determined through barcoded pyrosequencing. PLoS One 12(9):e0184717
133. Yu J et al (2013) Design and application of specific 16S rDNA-targeted primers for assessing
endophytic diversity in Dendrobium officinale using nested PCR-DGGE. Appl Microbiol
Biotechnol 97(22):9825–9836
134. Pei C et al (2017) Diversity of endophytic bacteria of Dendrobium officinale based on culture-
dependent and culture-independent methods. Biotechnol Biotechnol Equip 31(1):112–119
135. Lin M et al (2020) The effect of plant geographical location and developmental stage on root-
associated microbiomes of Gymnadenia conopsea. Front Microbiol 11:1257
136. Pavlova A et al (2017) Colonization strategy of the endophytic plant growth-promoting strains
of Pseudomonas fluorescens and Klebsiella oxytoca on the seeds, seedlings and roots of the
epiphytic orchid, Dendrobium nobile Lindl. J Appl Microbiol 123(1):217–232
137. Tsavkelova EA et al (2007) Orchid-associated bacteria produce indole-3-acetic acid, promote
seed germination, and increase their microbial yield in response to exogenous auxin. Arch
Microbiol 188(6):655–664
138. Tsavkelova EA et al (2016) Dendrobium nobile Lindl. Seed germination in co-cultures with
diverse associated bacteria. Plant Growth Regul 80(1):79–91
139. Vorholt JA (2012) Microbial life in the phyllosphere. Nat Rev Microbiol 10(12):828–840
140. Alibrandi P et al (2020) Plant growth promoting potential of bacterial endophytes from three
terrestrial mediterranean orchid species. Plant Biosyst https://doi.org/10.1080/11263504.2020.
1829731
141. White JF et al (2014) Occurrence of B acillus amyloliquefaciens as a systemic endophyte of
vanilla orchids. Microsc Res Tech 77(11):874–885
142. Faria DC et al (2013) Endophytic bacteria isolated from orchid and their potential to promote
plant growth. World J Microbiol Biotechnol 29(2):217–221
143. Altinkaynak H, Ozkoc I (2020) Isolation and molecular characterization of plant growth
promoting bacteria from the rhizosphere of orchids in Turkey. Rhizosphere 16:100280
144. Tsavkelova E, Cherdyntseva T, Netrusov A (2005) Auxin production by bacteria associated
with orchid roots. Microbiology 74(1):46–53
145. Tsavkelova EA et al (2007) Bacteria associated with orchid roots and microbial production of
auxin. Microbiol Res 162(1):69–76
146. Aguilar Díaz T et al (2018) Rizobacterias promotoras de crecimiento en Guarianthe skinneri
(Orchidaceae). Rev Biol Trop 66(3):953–968
147. Ortiz J et al (2019) The endophytic fungus Chaetomium cupreum regulates expression of
genes involved in the tolerance to metals and plant growth promotion in Eucalyptus globulus
roots. Microorganisms 7(11):490
148. Srivastava S, Kadooka C, Uchida JY (2018) Fusarium species as pathogen on orchids.
Microbiol Res 207:188–195
149. Hou XQ, Guo SX (2009) Interaction between a dark septate endophytic isolate from
Dendrobium sp. and roots of D. nobile seedlings. J Integr Plant Biol 51(4):374–381
150. Herrera H et al (2020) Isolation and identification of plant growth-promoting bacteria from
rhizomes of Arachnitis uniflora, a fully mycoheterotrophic plant in southern Chile. Appl Soil
Ecol 149:103512
151. Adamo M et al (2020) The dark side of orchid Symbiosis: can Tulasnella calospora decompose
host tissues? Int J Mol Sci 21(9):3139
152. Tsavkelova E, Cherdyntseva T, Netrusov A (2004) Bacteria associated with the roots of
epiphytic orchids. Microbiology 73(6):710–715
200 H. Herrera et al.
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
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.
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
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].
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. 3 Pelotons
interconnected with the
pelotons of neighboring cells
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].
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].
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
7 Mycorrhiza in Orchids 207
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].
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].
10 Transport of Phosphorus
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].
7 Mycorrhiza in Orchids 211
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.
References
1. Christenhauz MJM, Byng JW (2016) The number of known plants species in the world and its
annual increase. Phytotaxa 261:201–217
2. Pecoraro L, Girlanda M, Kull T, Perini C, Perotto S (2012) Molecular identification of root
fungal associates in Orchis pauciflora Tenore. Plant Biosys 146:985–991
212 S. Kaur
3. McCormick MK, Whigham Dennis F, Sloan DM, Hodkinson K, Brendan H (2006) Orchid–
fungus Fidelity: a marriage meant to last? Ecol 87:903–911
4. Zhang J, Jia W, Yang J, Ismail AM (2006) Role of ABA in integrating plant responses to
drought and salt stresses. Field Crop Res 97:111–119
5. Hartley SE, Gange AC (2009) Impacts of plant symbiotic Fungi on insect herbivores: mutual-
ism in a multitrophic context. Ann Rev Ento 54:323–342
6. Guo SX, Wang QY (2001) Character and action of good strain on stimulating seed germination
of Gastrodia elata. Mycosystema 3:408–412
7. Tang MJ, Meng ZX, Guo SX (2008) Effect of endophytic fungi on the content of sugars
and inorganic elements of cultivated seedling of Anoectochilus. Chin Tradit Herb Drugs
39:1565–1568
8. Yu XM, Guo SM (2000) Establishment of symbiotic system for Anoectochilus roxburghii
(wall.) Lindl. And endophytic fungi. J Chin Materia Med 25:81–82
9. Chen X, Wu CH, Tang JJ, Hu SJ (2005) Arbuscular mycorrhizae enhance metal lead uptake and
growth of host plants under a sand culture experiment. Chemosphere 60:665–671
10. Zhang X, Lund AA, Sarath G, Cerny RL, Roberts DM, Chollet R (1999) Soybean nodule
sucrose synthase (Nodulin-100): further analysis of its phosphorylation using recombinant and
authentic root-nodule enzymes. Arch Biochem Biophys 371:70–82
11. X-m C, Guo S-X (2005) Effects of four species of endophytic fungi on the growth and
polysaccharide and alkaloid contents of Dendrobium nobile. China Materia Med 30:253–257
12. Chutima R, Dell B, Suyanee V, Boonsom B (2011) Endophytic fungi from Pecteilis susannae
(L.) Rafin (Orchidaceae), a threatened terrestrial orchid in Thailand. Mycorrhiza 21:221–229
13. Taylor D, Lee B, Thomas D, Hodges SA (2004) Evidence for mycorrhizal races in a cheating
orchid. Proceedings of biological sciences. R Soc Pub 271(1534):35–43
14. Jacquemyn H, Honnay O, Cammue Bruno PA, Brys R, Lievens B (2010) Low specificity and
nested subset structure characterize mycorrhizal associations in five closely related species of
the genus Orchis. Mol Ecol 19:4086–4095
15. Pereira G, Romero C, Suz LM, Atala C (2014) Essential mycorrhizal partners of the endemic
Chilean orchids Chloraea collicensis and C. gavilu. Flora Morph Dist Funct Ecol Plants
209:95–99
16. Peterson RL, Currah RS (1990) Synthesis of mycorrhizae between protocorms of Goodyera
repens (Orchidaceae) and Ceratobasidium cereale. Can J Bot 68:1117–1125
17. Leake JR (1994) The biology of myco-heterotrophic (‘saprophytic’) plants. New Phytol
127:171–216
18. Pecoraro L, Girlanda M, Kull T, Perini C, Perotto S (2012) Molecular identification of root
fungal associates in Orchis pauciflora. Plant Biosys 146:985–991
19. Smith SE, Read DJ, Harley JL (1997) Mycorrhizal symbiosis, 2nd edn. Academic Press, San
Diego
20. Rasmussen HN (2002) Recent developments in the study of orchid mycorrhiza. Plant Soil
244:149–163
21. Hadley G, Williamson B (1971) Analysis of the post-infection growth stimulus in orchid
mycorrhiza. New Phytol 70:445–455
22. Selosse M-A, Minasiewicz J, Boullard B (2017) On the germination of Neottia nidus-avis.
Mycorrhiza 27:611–618
23. Peterson RL, Uetake Y, Bonfante P, Faccio A (1996) The interface between fungal hyphae and
orchid protocorm cells. C J Botany 74:1861–1870
24. Alvarez MR (1968) Quantitative changes in nuclear DNA accompanying post germination
embryonic development in Vanda (Orchidaceae). Am J Bot 55:1036–1041
25. Rasmussen HN (2009) Orchid mycorrhiza: implications of a mycophagous life style. Oikos
118:334–345
26. Shefferson RP, WeiB M, Kull T, Taylor DL (2005) High specificity generally characterizes
mycorrhizal association in rare lady’s slipper orchids, genus Cypripedium. Mol Ecol 14:613–626
27. Alghamdi SA (2017) Influence of mycorrhizal fungi on seed germination and growth in
terrestrial and epiphytic orchids. Saudi J Biol Sci 26:495–502
7 Mycorrhiza in Orchids 213
28. Bidartondo MI, Burghardt B, Gebauer G, Bruns TD, Read DJ (2004) Changing partners in the
dark: isotopic and molecular evidence of ectomycorrhizal liaisons between forest orchids and
trees. Proc Biol Sci 271(1550):1799–1806. The Royal Society Publishers
29. McKendrick SL, Leake JR, Read DJ (2000) Symbiotic germination and development of myco-
heterotrophic plants in nature: transfer of carbon from ectomycorrhizal Salix repens and Betula
pendula to the orchid Corallorhiza trifida through shared hyphal connections. New Phytol
145:539–548
30. Martos F, Munoz F, Pailler T, Kottke I, Gonneau C, Selosse M-A (2012) The role of epiphytism
in architecture and evolutionary constraint within mycorrhizal networks of tropical orchids. Mol
Ecol 21:5098–5109
31. Zi X-M, Sheng C-L, Goodale UM, Shao S-C, Gao J-Y (2014) In situ seed baiting to isolate
germination-enhancing fungi for an epiphytic orchid, Dendrobium aphyllum (Orchidaceae).
Mycorrhiza 24:487–499
32. McKendrick SL, Leake JR, Taylor DL, Read DJ (2002) Symbiotic germination and develop-
ment of the myco-heterotrophic orchid Neottia nidus-avis in nature and its requirement for
locally distributed Sebacina spp. New Phytol 154:233–247
33. Sakamoto Y, Yokoyama J, Maki M (2015) Mycorrhizal diversity of the orchid Cephalanthera
longibracteata in Japan. Myco Sci 56:183–189
34. Kuga Y, Sakamoto N, Yurimoto H (2014) Stable isotope cellular imaging reveals that both live
and degenerating fungal pelotons transfer carbon and nitrogen to orchid protocorms. New
Phytol 202:594–605
35. Dearnaley JDW, Martos F, Selosse MA (2012) Orchid mycorrhizas: molecular ecology, phys-
iology, evolution and conservation aspects. In: Esser K (ed) The mycota, vol IX – fungal
associations, 2nd edn. Springer, Berlin, pp 207–230
36. Barroso J, Casimiro A, Carrapico F, Pais M, Salome S (1988) Localization of uricase in
mycorrhizas of Ophrys lutea. New Phytol 108:335–340
37. Selosse M-A, Weiss M, Jany J-L, Tillier A (2002) Communities and populations of sebacinoid
basidiomycetes associated with the achlorophyllous orchid Neottia nidus-avis (L.) L.C.M.
Richb. And neighbouring tree ectomycorrhizae. Mol Ecol 11:1831–1844
38. Girlanda M, Segreto R, Cafasso D, Liebel HT, Rodda M, Ercole E, Cozzolino S, Gebauer G,
Perotto S (2011) Photosynthetic mediterranean meadow orchids feature partial
mycoheterotrophy and specific mycorrhizal associations. Am J Bot 98:1148–1163
39. Alexander C, Hadley G (1985) Carbon movement between host and mycorrhizal endophyte
during the development of the orchid Goodyera repens. New Phytol 101:657–665
40. Zimmer K, Hynson NA, Gebauer G, Allen EB, Allen MF, Read DJ (2007) Wide geographical
and ecological distribution of nitrogen and carbon gains from fungi in pyroloids and mono-
tropoids (Ericaceae) and in orchids. New Phytol 175:166–175
41. Smith SE, Gianinazzi PV, Koide R, Cairney JWG (1994) Nutrient transport in mycorrhizas:
structure, physiology and consequences for efficiency of the symbiosis. Plant Soil 159:103–113
42. Balestrini R, Nerva L, Sillo F, Girlanda M, Perotto S (2014) Plant and fungal gene expression in
mycorrhizal protocorms of the orchid Serapias vomeracea colonized by Tulasnella calospora.
Plant Signal Behav 9:e977707
43. Fochi V, Falla N, Girlanda M, Perotto S, Balestrini R (2017) Cell-specific expression of plant
nutrient transporter genes in orchid mycorrhizae. Plant Sci 263:39–45
44. Imhof S (1998) Subterranean structures and mycotrophy of the achlorophyllous Triuris hyalina
(Triuridaceae). Can J Bot 76:2011–2019
45. Imhof S (1999) Anatomy and mycotrophy of the achlorophyllous Afrothismia winkleri. New
Phytol 144:533–540
46. Fochi V, Chitarra W, Kohler A, Voyron S, Singan VR, Lindquist EA, Barry KW, Girlanda M,
Grigoriev IV, Martin F, Balestrini R, Perotto S (2017) Fungal and plant gene expression in the
- symbiosis provides clues about nitrogen pathways in orchid mycorrhizas. New Phytol
213:365–379
47. Cameron DD, Johnson I, Leake JR, Read DJ (2007) Mycorrhizal acquisition of inorganic
phosphorus by the green-leaved terrestrial orchid Goodyera repens. Ann Bot 99:831–834
214 S. 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
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
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
8 Phytoalexins in Orchids 219
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].
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].
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
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
8 Phytoalexins in Orchids 221
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.
References
1. Braga MR et al (1991) Phytoalexins induction in Rubiacea. J Chem Ecol 17:1079–1090
2. Grayer RJ, Kokubun T (2015) Plant-fungal interactions: the search for phytoalexins and other
antifungal compounds from higher plants. Phytochemistry 56:253–263
3. Arruda RL, Santana Paz AT, Freitas Bara MT, Côrtes MVCB, Filippi MCC, Conceição EC
(2016) An approach on phytoalexins: function, characterization and biosynthesis in plants of the
family Poaceae. Ciência Rural 46(7):1206–1216. https://doi.org/10.1590/0103-
8478cr20151164
4. Cavalcanti LS et al (2005) Aspectos bioquímicos e moleculares da resistência induzida. In:
Cavalcanti LS et al (eds) Indução de resistência em plantas a patógenos e insetos, vol 81.
FEALQ, Piracicaba, p 124
5. Boller AH, Corrodi F, Gaumann E et al (1957) Uber induzierte Abwehrstoffe bei Orchideen
Pt. 1. Helv Chim Acta 40:1062–1066
222 S. Kaur
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
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
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].
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.
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),
228 K. Thammasiri et al.
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.
2.2.2 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
9 Micropropagation of Some Orchids and the Use of Cryopreservation 229
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.
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
232 K. Thammasiri et al.
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
9 Micropropagation of Some Orchids and the Use of Cryopreservation 233
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].
2.2.9 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
9 Micropropagation of Some Orchids and the Use of Cryopreservation 235
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].
Table 1 (continued)
Orchid species and
hybrids Explant Regenerants Medium composition References
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)
9 Micropropagation of Some Orchids and the Use of Cryopreservation 239
Table 1 (continued)
Orchid species and
hybrids Explant Regenerants Medium composition References
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
3 Orchid Cryopreservation
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
242 K. Thammasiri et al.
apple [132–134], cherry [135], blueberry [136], etc. There is no research on orchid
dormant bud 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.
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
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].
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
9 Micropropagation of Some Orchids and the Use of Cryopreservation 247
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).
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]
248 K. Thammasiri et al.
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
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)
250 K. Thammasiri et al.
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.
9 Micropropagation of Some Orchids and the Use of Cryopreservation 251
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.
References
1. Knudson L (1922) Symbiosis and asymbiosis relative to orchids. New Phytol 26:328–336
2. Arditti J (1984) An history of orchid hybridization, seed germination and tissue culture. Bot
J Linn Soc 89:359–381
3. Arditti J (1967) Factors affecting the germination of orchid seeds. Bot Rev 33:1–96
4. Sheehan TJ (1983) Recent advances in botany, propagation and physiology of orchids.
In: Janick J (ed) Horticultural reviews, vol 5. AVI Publishing Company, Westport, pp 279–315
5. Stephen R, Jayakoalen N (2000) Artificail seed production in coriander (Coriandrum sativum
L.). Plant Tissue 10(1):45–49
6. Arditti A, Ernst R (1993) History. In: Micropropagation of orchids. Wiley, New York, pp 3–23
252 K. Thammasiri et al.
7. Arditti J, Ghani AKA (2000) Numerical and physical properties of orchid seeds and their
biological implications. New Phytol 145:367–421
8. Seaton P, Ramsay M (2005) Growing orchids from seed. Mondadori Printing, Surrey
9. Kalimuthu K, Senthilkumar R, Vijayakumar S (2007) In vitro micropropagation of orchid,
Oncidium sp. (Dancing Dolls). Afr J Biotechnol 6(10):1171–1174
10. Manning JC, Staden J (1987) The development and mobilization of seed reserves in some
African orchids. Aust J Bot 35:343–353
11. Rasmussen HN, Anderson TF, Johansen B (1990) Temperature sensitivity of in vitro germi-
nation and seedling development of Dactylorhiza majalis (Orchidaceae) with and without
a mycorrhizal fungus. Plant Cell Environ 13:171–177
12. Knudson L (1946) A nutrient for germination of orchid seeds. Am Orchid Soc Bull
15:214–217
13. Lee YI, Yeung EC, Chung MC (2007) Embryo development of orchids. In: Orchid biotech-
nology. Stallion Press, Singapore, pp 23–44
14. Champagnat M, Morel G (1972) La culture in vitro des tissues tubercules d’Ophrys. Comptes
Rendus de I’Academie de Sciences, Paris 274:3379–3380
15. Norstog K (1979) Embryo culture as a tool in the study of comparative and developmental
morphology. In: Plant cell and tissue culture: principles and applications. Ohio State
University, Columbus, pp 179–202
16. Georage EF, Hall MA, Klerk GJD (2008) Plant propagation by tissue culture, 3rd edn.
Springer, Dordrecht
17. Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco
tissue cultures. Physiol Plant 15:473–497
18. Vacin EF, Went FW (1949) Some pH changes in nutrient solutions. Bot Gaz 110:605–613
19. Puchooa D (2004) Comparison of different culture media for the in vitro culture of
Dendrobium (Orchidaceae). Int J Agric Biol 6:884–888
20. Naing AH, Chung JD, Lim KB (2011) Plant regeneration through indirect somatic
embryogenesis in Coelogyne cristata orchid. Am J Plant Sci 2:262–267
21. Sopalun K, Thammasiri K, Ishikawa K (2010) Micropropagation of the Thai orchid
Grammatophyllum speciosum Blume. Plant Cell Tissue Organ Cult 101:143–150
22. Kishor R, Sharma GJ (2009) Intergeneric hybrid of two rare and endangered orchids,
Renanthera imschootiana Rolfe and Vanda coerulea Griff. ex L. (Orchidaceae): synthesis
and characterization. Euphytica 165(2):247–256
23. Kishor R, Devi HS, Jeyaram K et al (2008) Molecular characterization of reciprocal
crosses of Aerides vandarum and Vanda stangeana (Orchidaceae). Plant Biotechnol Rep
2:145–152
24. Wattanawikkit P, Bunn E, Chayanarit K, Tantiwiwat S (2011) Effect of cytokinins (BAP and
TDZ) and auxin (2,4-D) on growth and development of Paphiopedilum callosum. Kasetsart J
(Nat Sci) 45:12–19
25. Jaime ATS (2012) New basal media for protocorm like body and callus induction of hybrid
Cymbidium. J Fruit Ornam Plant Res 20(2):127–133
26. Long B, Niemiera AX, Cheng ZY et al (2010) In vitro propagation of four threatened
Paphiopedilum species (Orchidaceae). Plant Cell Tissue Organ Cult 101:151–162
27. Schenk RU, Hildebrant AC (1972) Medium and techniques for induction and growth of
monocotyledonous and dicotyledonous plant cell cultures. Can J Bot 50:199–204
28. Luo JP, Zha XQ, Fan XJ (2006) Effects of carbon and nitrogen sources on protocorm-
like bodies proliferation of Dendrobium huoshanense by liquid culture. Acta Hortic 725(1):
197–202
29. Gamborg OL, Miller RA, Ojima K (1968) Nutrient requirements of suspension cultures of
soybean root cells. Exp Cell Res 50(1):151–158
30. Chu CC (1978) The N6 medium and its applications to another culture of cereal crops.
In: Proceedings of symposium on plant tissue culture. Science Press, Peking, pp 43–50
9 Micropropagation of Some Orchids and the Use of Cryopreservation 253
31. Silva JAT, Yam TW, Fukai S et al (2005) Establishment of optimum nutrient media for in vitro
propagation of Cymbidium sw. (Orchidaceae) using protocorm-like body segments. Propag
Ornam Plants 5(3):129–136
32. Pierik RLM (1988) In vitro culture of higher plants as a tool in the propagation of horticultural
crops. Acta Hortic 226:25–40
33. Pierik RLM, Sprenkels PA, Harst BV et al (1988) Seed germination and further development
of plantlets of Paphiopedilum ciliolare Pfitz. in vitro. Sci Hortic 34:139–153
34. Hong PI, Chen JT, Chang WC (2008) Promotion of direct somatic embryogenesis of Oncidium
by adjusting carbon sources. Biol Plant 52(3):597–600
35. Lou JP, Wawrosch C, Kopp B (2009) Enhanced micropropagation of Dendrobium
huoshanense C.Z. Tang et S.J. Cheng through protocorm-like bodies: the effects of cytokinins,
carbohydrate sources and cold pretreatment. Sci Hortic 123:258–262
36. Ferreira WM, Suzuki RM, Pescador R et al (2011) Propagation, growth, and carbohydrates
of Dendrobium Second Love (Orchidaceae) in vitro as affected by sucrose, light, and dark.
In Vitro Cell Dev Biol 47:420–427
37. Chen TY, Chen JT, Chang WC (2004) Plant regeneration through direct shoot bud formation
from leaf cultures of Paphiopedilum orchids. Plant Cell Tissue Org Cult 76:11–15
38. Wu IF, Chen JT, Chang WC (2004) Effects of auxins and cytokinins on embryo formation
from callus of Oncidium ‘Gower Ramsey’. Plant Cell Tissue Org Cult 77:107–109
39. Steward FC, Mapes MO, Mears K (1958) Growth and organised development of cultured
cells: II. Organisation in cultured grown from freely suspended cells. Am J Bot 45:705–707
40. Chen JT, Chang WC (2000) Efficient plant regeneration through somatic embryogenesis from
callus culture of Oncidium (Orchidaceae). Plant Sci 160:87–93
41. Jheng FY, Do YY, Liauh YW et al (2006) Enhancement of growth and regeneration efficiency
from embryogenic callus cultures of Oncidium ‘Gower Ramsey’ by adjusting carbohydrate
sources. Plant Sci 170:1133–1140
42. Nayak NR, Sahoo S, Patnaik S, Rath SP (2002) Establishment of thin cross section (TCS)
culture method for rapid micropropagation of Cymbidium aloifolium (L.) Sw. and Dendrobium
nobile Lindl. (Orchidaceae). Sci Hortic 94:107–116
43. Tokuhara K, Mii M (1993) Micropropagation of Phalaenopsis and Doritaenopsis by culturing
shoot tips of flower stalk buds. Plant Cell Rep 13:7–11
44. Tokuhara K, Mii M (2001) Induction of embryogenic callus and cell suspension culture from
shoot tips excised from lower flower stalk buds of Phalaenopsis (Orchidaceae). In Vitro Cell
Dev Biol Plant 37:457–461
45. Chen Y, Piluek C (1995) Effect of thidiazuron and N6-benzylaminopurine on shoot regener-
ation of Phalaenopsis. Plant Growth Regul 16:99–101
46. Roy J, Banerjee N (2003) Induction of callus and plant regeneration from shoot-tip explants of
Dendrobium fimbriatum Lind. var oculatum Hk.f. Sci Hortic 97:333–340
47. Huetteman CA, Preece JE (1993) Thidiazuron: a potent cytokinin for woody plant tissue
culture. Plant Cell Tissue Org Cult 33:105–119
48. Ernst R (1994) Effect of thidiazuron on in vitro propagation of Phalaenopsis and
Doritaenopsis (Orchidaceae). Plant Cell Tissue Org Cult 39:273–275
49. Wasiksiri W, Thammasiri K, Chuenboonngarm N et al (2010) Medium development for
micropropagation of Vanda coerulea Griff. ex Lindl. from lateral buds. In: Abstract of 36th
Congress on science and technology of Thailand
50. Wu K, Zeng S, Silva JAT et al (2012) Efficient regeneration of Renanthera Tom Thumb ‘Qilin’
from leaf explants. Sci Hortic 135:194–201
51. Vyas S, Guha S, Bhattacharya M et al (2009) Rapid regeneration of plants of Dendrobium
lituiflorum Lindl. (Orchidaceae) by using banana extract. Sci Hortic 121:32–37
52. Lo SF, Narawade SM, Kou CL et al (2004) Asymbiotic germination of immature seeds,
plantlet, development and ex vitro establishment of plant of Dendrobium tosaense Makino –
a medicinally important orchid. In Vitro Cell Dev Biol Plant 40:528–535
254 K. Thammasiri et al.
53. Nambiar N, Tee CS, Maziah M (2012) Effects of organic additives and different carbohydrate
sources on proliferation of protocorm-like bodies in Dendrobium Alya Pink. Plant Omics J
5(1):10–18
54. Sinha P, Jahan MAA, Munshi JL et al (2010) High frequency regeneration of Phalaenopsis
amabilis (L.) Bl. Cv. Lovely through in vitro culture. Plant Tissue Cult Biotechnol 20(2):
185–193
55. Gnasekaran P, Rathinam X, Sinniah UR et al (2010) A study on the use of organic additives
on the protocorm-like bodies (PLBS) growth of Phalaenopsis violacea orchid. J Phytology
2:29–33
56. Li Z, Xu L (2009) In vitro propagation of white-flower mutant of Rhynchostylis gigantea
(Lindl.) Ridl. through immature seed-derived protocorm-like bodies. J Hortic For 1(6):93–97
57. Vyas S, Kapoor-Pandey P, Guha S et al (2011) Synchronous plantlet formation by using
banana extract and in vitro hardening in orchid, Dendrobium lituiflorum Lindl. J Ornam Hortic
Plants 1(3):175–184
58. Zeng S, Wu K, Silva JAT et al (2012) Asymbiotic seed germination, seedling development and
reintroduction of Paphiopedilum wardii Sumerh., an endangered terrestrial orchid. Sci Hortic
138:198–209
59. Gnasekaran P, Poobathy R, Mahmood M et al (2012) Effects of complex organic additives on
improving the growth of PLBs of Vanda Kasem’s delight. Aust J Crop Sci 6:1245–1248
60. Ali MJ, Murdad R, Latip MA et al (2011) In vitro seed germination of Bornean endemic
orchids Dendrobium tetrachromum and Dendrobium hamaticalcar. In: Empowering science,
pp 770–777
61. Lee YI (2010) Micropropagation of Cypripedium formosanum Hayata through axillary buds
from mature plants. HortScience 45(9):1369–1372
62. Dix L, Van SJ (1982) Auxin and gibberellins-like substances in coconut milk and malt extract.
Plant Cell Tissue Org Cult 1:239–245
63. Staden JV, Zazimalova E, George EF (2008) Plant growth regulators II: cytokinins,
their analogues and antagonists. In: Plant propagation by tissue culture, 3rd edn. Springer,
Dordrecht, pp 206–217
64. Baque MA, Shin YK, Elshmari T et al (2011) Effect of light quality, sucrose and coconut water
concentration on the microporpagation of Calanthe hybrids (‘Bukduseong’ ‘Hyesung’ and
‘Chunkwang’ ‘Hyesung’). Aust J Crop Sci 5(10):1247–1254
65. Ng CY, Saleh NM (2011) In vitro propagation of Paphiopedilum orchid through formation of
protocorm-like bodies. Plant Cell Tissue Organ Cult 105:193–202
66. Asghar S, Ahmad T, Hafiz IA et al (2011) In vitro propagation of orchid (Dendrobium nobile)
var. Emma white. Afr J Biotechnol 10(16):3097–3103
67. Parab G, Krishnan S (2012) Rapid in vitro mass multiplication of Aerides maculosa and
Rhynchostylis retusa from immature seeds. Indian J Biotechnol 11:288–294
68. Nge KL, Nwe N, Chandrkrachang S et al (2006) Chitosan as a growth stimulator in orchid
tissue culture. Plant Sci 170:1185–1190
69. Chanrkrachange S (2002) The applications of chitin and chitosan in agriculture in Thailand, In:
Suchiva K, Chanrkrachange S, Methacanon P and Peter MG. (Eds). Advances in Chitin
Science Volume 5. Bangkok, Thailand:458–462
70. Thomus TD (2008) The role of activated charcoal in plant tissue culture. Biotechnol Adv
26:631–618
71. Pan MJ, Staden JV (1998) The use of charcoal in vitro culture – a review. Plant Growth Regul
26:155–163
72. Znaniecka J, Królicka A, Sidwa-Gorycka M et al (2005) Asymbiotic germination, seedling
development and plantlet propagation of Encyclia aff. oncidioides – an endangered orchid.
Acta Soc Bot Pol 74(3):193–198
73. Shin YK, Baque AM, Elghamedi S et al (2011) Effects of activated charcoal, plant growth
regulators and ultrasonic pre-treatments on in vitro germination and protocorm formation of
Calanthe hybrids. Aust J Crop Sci 5(5):582–588
9 Micropropagation of Some Orchids and the Use of Cryopreservation 255
74. Ket NV, Hahn EJ, Park SY et al (2004) Micropropagation of an endangered orchid
Anoectochilus formosanus. Biol Plant 48(3):339–344
75. Thomus TD, Michael A (2007) High-frequency plantlet regeneration and multiple shoot
induction from cultured immature seeds of Rhynchostylis retusa Blume., an exquisite orchid.
Plant Biotechnol Rep 1:243–249
76. Te-chato S, Kongruk S, Khaimuk W (2010) Micropropagation of Chang Daeng (Rhynchostylis
rubrum) by embryogenic callus. J Agric Technol 6(3):589–597
77. Roy AR, Patel RS, Patel VV et al (2011) Asymbiotic seed germination, mass propagation and
seedling development of Vanda coerulea Griff ex. Lindl. (Blue Vanda): an in vitro protocol for
an endangered orchid. Sci Hortic 128:325–331
78. Arenmongla T, Deb CR (2012) Germination of immature embryos and multiplication of
Malaxis acuminata D. Don, an endangered therapeutically important orchid, by asymbiotic
culture in vitro. Indian J Biotechnol 11:464–469
79. Rittirat S, Thammasiri K, Te-chato S (2012) Effect of media and sucrose concentrations with
or without activated charcoal on the plantlet growth of P. cornu-cervi (Breda) Blume & Rchb.
f. J Agric Technol 8(6):2077–3087
80. Khatun MM, Khatun H, Khanam D et al (2010) In vitro root formation and plantlet develop-
ment in Dendrobium orchid. Bangladesh J Agric Res 35(2):257–265
81. Prizão EC, Gonçalves LM, Gutierre MAM et al (2012) Activated charcoal and graphite for the
micropropagation of Cattleya bicolor Lindl. and an orchid double-hybrid ‘BLC Pastoral
Innocence’. Acta Sci 34(2):157–161
82. Arditti J (1977) Clonal propagation of orchids by means of tissue culture – a manual.
In: Arditti J (ed) Orchid biology: reviews and perspectives, vol I. Cornell University Press,
Ithaca, pp 203–293
83. Malmgren S (1996) Orchid propagation: theory and practice. In: Allen C (eds) North Amer-
ican native orchids: propagation and production. North American Native Terrestrial Orchid
Conference, Germantown, Maryland, pp 63–71
84. Barbery R, Morales I (2011) Manual para el cultivo in vitro de la orquídea Cattleya nobilior.
Flor símbolo de Concepción. CEPAD, Editorial El País, Santa Cruz, 46 pp
85. Mitra GC, Prasad RN, Roychowdhary A (1976) Inorganic salts and differentiation of pro-
tocorms in seed-callus of an orchid and correlated changes in its free amino acid content.
Indian J Exp Biol 14:350–351
86. Rasmussen HN, Dixon KW, Jersáková J et al (2015) Germination and seedling establishment
in orchids: a complex of requirements. Ann Bot 116:391–402
87. Bajquz A, Piotrowska A (2009) Conjugates of auxin and cytokinin. Phytochemistry
70(8):957–969
88. Thammasiri K (1997) Orchids in Thailand: a success story. APAARI, FAO, Angkor Publishers
(P.) Ltd., New Delhi
89. Utami ESW, Hariyanto S (2019) In vitro seed germination and seedling development of a rare
Indonesian native orchid Phalaenopsis amboinensis J.J Sm. Hindawi Scientifica 2019:1–6
90. Jainol JE, Jualang AG (2015) In vitro shoot multiplication and rooting of shoot tip explants of
Dimorphorchis lowii: an endemic orchid of Borneo. Plant Physiol 7:14–25
91. Utami ESW, Hariyanto S, Sri Y et al (2017) In vitro propagation of the endangered medicinal
orchid, Dendrobium lasianthera J.J. Sm through mature seed culture. Asian Pac J Trop
Biomed 7(5):406–410
92. Pant B, Swar S (2011) Micropropagation of Cymbidium iridioides. Nepal J Sci Technol
12:91–96
93. Pant B, Gurung R (2005) In vitro seed germination and seedling development in Aerides
odorata Lour. J Orchid Soc India 19:51–55
94. Pant B, Swar S, Karanjeet A (2008) Micropropagation of Coelogyne cristata Lindl. J Orchid
Soc India 22(1–2):45–48
95. Pradhan S, Pant B (2009) In vitro seed germination in Cymbidium elegans Lindl. and
Dendrobium densiflorum Lindl. ex Wall (Orchidaceae). Bot Orient J Plant Sci 6:100–102
256 K. Thammasiri et al.
96. Pant B, Pradhan S (2010) Micropropagation of Cymbidium elegans Lindl. through protocorm
and shoot tip culture. Role of biotechnology in food security and climate change. In:
Proceedings of 6th international plant tissue culture and biotechnology conference, Dhaka,
Bangladesh, December, pp 3–5
97. Pant B, Shrestha S, Pradhan S (2011) In vitro seed germination and seedling development of
Phaius tancarvilleae (L’Her.) Blume. Sci World 9(9):50–52
98. Rajkarnikar KM (2011) Propagation of Cymbidium aloifolium (L.) Sw. in vitro by seeds. Bull
Dept Plant Res 33:27–30
99. Koirala D, Pradhan S, Pant B (2013) Asymbiotic seed germination and plantlet development
of Coelogyne fuscescens Lindl., a medicinal orchid of Nepal. Sci World 11(11):97–100
100. Pradhan S, Regmi T, Parmar G et al (2013b) Effect of different media on in vitro seed
germination and seedling development of Cymbidium aloifolium (L.) Sw. Nepal J Sci Technol
14(1):51–56
101. Parmar G (2014) In vitro seed germination and seedling development of Cymbidium
devonianum Paxton (Orchidaceae). Bull Dept Plant Res 36:61–64
102. Pant B, Shrestha S (2011) In vitro mass propagation of a ground orchid – Phaius tancarvilleae
(L’Her.) Blume through shoot tip culture. Plant Tissue Cult Biotechnol 21(2):181–188
103. Prasongsom S, Thammasiri K, Chuenboonngarm N (2016) Efficient adventitious shoot regen-
eration from shoot tip culture of Rhynchostylis gigantea (Lindl.) Ridl. (amethyst-purple), a rare
Thai orchid species. Walailak J Sci Technol 13(9):757–767
104. Sangdum S, Thammasiri K, Chuenboonngarm N et al (2017) Micropropagation of
Dendrobium cruentum Rchb. f., a rare Thai orchid species. Acta Hortic 1167:69–74
105. Prasongsom S, Thammasiri K, Narangajavana J et al (2020) Cryopreservation of Dendrobium
cruentum Rchb. f. seeds by D cryo-plate and V cryo-plate techniques. Walailak Journal of
Science and Technology 17(3):181–191
106. Jitsopakul N, Thammasiri K, Ishikawa K (2013) Efficient adventitious shoot regeneration from
shoot tip culture of Vanda coerulea, a Thai orchid. ScienceAsia 39:449–455
107. Pornchuti W, Thammasiri K, Chuenboonngarm N et al (2017) Micropropagation of
Spathoglottis eburnea Gagnep, a Thai orchid species, through shoot tips. Acta Hortic 1167:
87–94
108. Dewir YH, El-Mahrouk EM, Murthy HN et al (2015) Micropropagation of Cattleya: improved
in vitro rooting and acclimatization. Hortic Environ Biotechnol 56(1):89–93
109. Morel G (1970) Neres ouf dem Gebiet der Meristem-Forchung. Die Orchidee 20:433–443
110. Nagaraju V, Parthasarathy VS (1995) In vitro propagation of Phaius and bamboo orchid by
shoot tip culture. Ann Plant Physiol 9:102–104
111. Devi JB, Borthakur B, Deka PC (1997) Clonal propagation of Dendrobium moschatum and
Cymbidium aloifolium through shoot tip culture. J Orchid Soc India 11:19–21
112. Subramanium G, Taha RM (2003) Morphogenesis of Cymbidium atropurpureum in vitro.
Malays J Sci 22:1–5
113. Sharma A, Tandon P (1992) In vitro culture of Dendrobium wardianum Warner: morphoge-
netic effects of some heterogenous adjuvants. Indian J Plant Physiol 35:80–85
114. Sheela VL, Rajmohan K, Anita S et al (2004) Effect of growth regulators on development and
multiplication of protocorm like bodies in Dendrobium cv Sonia. J Orchid Soc India 18:21–23
115. Sharon M, Vasundhara G (1990) Micropropagation of Dendrobium Joannie Ostenhault.
J Orchid Soc India 4:145–148
116. Kalimuthu K, Senthikumar R, Murugalatha N (2006) Regeneration and mass multiplication of
Vanilla planifolia Andr. – a tropical orchid. Curr Sci 91:1401–1403
117. Sheelavanthmath SS, Murthy HN, Hema BP et al (2005) High frequency of protocorm like
bodies (PLBs) induction and plant regeneration from protocorm and leaf sections of Aerides
crispum. Sci Hortic 106:395–401
118. Vij SP, Aggarwal S, Pathak P (2004) Regeneration competence of Cymbidium Great Waltz
Valley flower roots: a study in vitro. J Orchid Soc India 18:109–115
9 Micropropagation of Some Orchids and the Use of Cryopreservation 257
119. Vij SP, Sembi JK, Verma J et al (2004) In vitro rapid mass multiplication of Aerides multiflora,
a floriculturally significant species. J Orchid Soc India 17:63–68
120. Chung HH, Chen JT, Chang WC (2005) Cytokinins induce direct somatic embryogenesis of
Dendrobium Chiengmai Pink and subsequent plant regeneration. In Vitro Cell Dev Biol Plant
41:765–769
121. Martin KP, Madassery JP (2006) Rapid in vitro propagation of Dendrobium hybrids through
direct shoot formation from foliar explants and protocorm like bodies. Sci Hortic 108:95–99
122. Bhadra SK, Hossain MM (2004) Induction of embryogenesis and direct organogenesis in
Micropera pallida Lindl., an epiphytic orchid of Bangladesh. J Orchid Soc India 18:5–9
123. Kuo HL, Chen JT, Chang WC (2005) Efficient plant regeneration through direct somatic
embryogenesis from leaf explants of Phalaenopsis ‘Little Steve’. In Vitro Cell Dev Biol Plant
41:453–456
124. Janarthanam B, Seshadri S (2008) Plantlet regeneration from leaf derived callus of Vanilla
planifolia Andr. In Vitro Cell Dev Biol Plant 44:84–89
125. Balilashaki K, Ghehsareh MG (2016) Micropropagation of Phalaenopsis amabilis var.
‘Manila’ by leaves obtained from in vitro culturing the nodes of flower stalks. Not Sci Biol
8(2):164–169
126. Goh CJ, Wong PF (1990) Micropropagation of the monopodial orchid hybrid Aranda Deborah
using inflorescence explants. Sci Hortic 44:315–321
127. Chen LR, Chen JT, Chang WC (2002) Efficient production of protocorm like bodies and plant
regeneration from flower stalk explants of the sympodial orchid Epidendrum radicans.
In Vitro Cell Dev Biol Plant 38:441–445
128. Chen JT, Chang WC (2002) Effects of tissue culture characteristics on direct somatic embryo-
genesis in Oncidium ‘Gower Ramsey’. Plant Cell Tissue Org Cult 69:41–44
129. Mitsukuri K, Mori G, Johkan M et al (2009) Effects of explant position and dark treatment on
bud formation in floret culture of Ponerorchis graminiflolia Rchb. f. Sci Hortic 121:243–247
130. Baby R, Valsala PA, Doddamani MB (2019) In vitro micropropagation protocol for Vanda
hybrid ‘Dr. Anek’. Int J Curr Microbiol App Sci 8(4):2073–2084
131. Sakai A (1960) Survival of the twig of woody plants at 196 °C. Nature 185:393–394
132. Sakai A, Nishiyama Y (1978) Cryopreservations of winter vegetative buds of hardy fruit trees
in liquid nitrogen. HortScience 13:225–227
133. Forsline PL, Stushnoff C, Towill LE et al (1998) Recovery and longevity of cryopreserved
apple buds. J Am Soc Hortic Sci 123:365–370
134. Towill LE, Bonnart R (2003) Cracking in a vitrification solution during cooling or warming
does not affect growth of cryopreserved mint shoot tips. CryoLetters 24:341–346
135. Towill LE, Forsline PL (1999) Cryopreservation of sour cherry (Prunus cerasus L.) using
a dormant vegetative bud method. Cryo-Letters 20:215–222
136. Jenderek MM, Reed BM (2017) Cryopreserved storage of clonal germplasm in the USDA
national plant germplasm system. In Vitro Cell Dev Biol Plant 53:299–308
137. Panis B, Lambardi M (2005) Status of cryopreservation technologies in plants (crops and
forest trees). In: The role of biotechnology. Villa Gualino, Turin, Italy, 5–7 March
138. Uemura M, Sakai A (1980) Survival of carnation (Dianthus caryophyllus L.) shoot to the
temperature of liquid nitrogen. Plant Cell Physiol 21:85
139. Uragami A, Sakai A, Nagai M et al (1989) Survival of cultured cells and somatic embryos of
Asparagus officinalis cryopreserved by vitrification. Plant Cell Rep 8:418–421
140. Sakai A, Kobayashi S, Oiyama I (1990) Cryopreservation of nucellar cells of navel orange
(Citrus sinensis Osb. var brasiliensis Tanaka) by vitrification. Plant Cell Rep 9:30–33
141. Nishizawa S, Sakai A, Amano Y et al (1993) Cryopreservation of asparagus (Asparagus
officinalis L.) embryogenic suspension cells and subsequent plant regeneration by vitrification.
Plant Sci 88:67–73
142. Sakai A, Matsumoto T, Hirai D et al (2000) Newly developed encapsulation-dehydration
protocol for plant cryopreservation. CryoLetters 21:53–62
258 K. Thammasiri et al.
143. Niino T, Tanaka D, Tantely RR et al (2007) Cryopreservation of basal stem buds of in vitro
growth mat rush (Juncus spp.) by vitrification. CryoLetters 28:197–206
144. Chen Y, Wang JH (2002) Cryopreservation of carrot (Daucus carota L.) cell suspensions and
protoplasts by vitrification. CryoLetters 24:57–64
145. Thammasiri K (2000) Cryopreservation of seeds of Thai orchid (Doritis pulcherrima Lindl.)
by vitrification. CryoLetters 21:237–244
146. Matsumoto T, Sakai A (1995) An approach to enhance dehydration tolerance of alginate-
coated dried meristems cooled to 196 °C. CryoLetters 16:299–306
147. Hirai D, Shirai K, Shirai S et al (1998) Cryopreservation of in-vitro grown meristems
of strawberry (Fragaria X ananassa Duch.) by encapsulation- vitrification. Euphytica 101:
109–115
148. Niino T, Sakai A (1992) Cryopreservation of alginate – coated in-vitro grown shoot tips of
apple, pear and mulberry. Plant Sci 87:199–206
149. Fabre J, Dereuddre J (1990) Encapsulation-dehydration: a new approach to cryopreservation
of Solanum shoot-tips. CryoLetters 11:413–426
150. Jitsopakul N, Thammasiri K, Ishikawa K (2008) Cryopreservation of Vanda coerulea
protocorms by encapsulation-dehydration. CryoLetters 29:253–260
151. Hirai D, Sakai A (1999) Cryopreservation of in vitro-grown axillary shoot-tip meristems of
mint (Mentha spicata L.) by encapsulation vitrification. Plant Cell Rep 19(2):150–155
152. Sakai A, Hirai D, Niino T (2008) Development of PVS-based vitrification and encapsulation-
vitrification protocols. In: Reed BM (ed) Plant cryopreservation: a practical guide. Springer,
New York, pp 33–58
153. Matsumoto T (2017) Cryopreservation of plant genetic resources: conventional and new
methods. Rev Agric Sci 5:13–20
154. Thammasiri K, Prasongsom S, Kongsawadworakul P et al (2019) Cryopreservation
of Arundina graminifolia (D. Don) hochr. seeds using D cryo-plate method. Acta Hortic
1234:301–308
155. Sakai A, Englemann F (2007) Vitrification, encapsulation-vitrification and droplet-
vitrification: a review. CryoLetters 28:151–172
156. Kim HH, Yoon JW, Park YE et al (2006) Cryopreservation of potato cultivated varieties and
wild species: critical factors in droplet vitrification. CryoLetters 27(4):223–234
157. Agrawal A, Swennen R, Panis B (2004) A comparision of four methods for cryopreservation
of meristems in banana (Musa spp.). CryoLetters 25:101–110
158. Yoon JW, Kim HH, Ko HC et al (2006) Cryopreservation of cultivated and wild potato
varieties by droplet- vitrification: effect of subculture of mother-plants and of preculture of
shoot tips. CryoLetters 27:211–222
159. Halmagyi A, Deliu C, Coste A (2005) Plant regrowth from potato shoots tips cryopreserved by
a combinated vitrification-droplet method. CryoLetters 26:313–322
160. Pennycooke JC, Towill LE (2000) Cryopreservation of shoot tips in vitro plants of sweet
potato [Ipomoea batatas (L.) Lam.] by vitrification. Plant Cell Rep 19:733–737
161. Ashmore S, Azimi M, Drew RA (2001) Cryopreservation trials in Carica papaya. Acta Hortic
(ISHS) 560:117–120
162. De Boucaud MT, Brison M, Helliot B et al (2002) Cryopreservation of Prunus. In: Towill LE,
YPS B (eds) Biotechnology in agriculture and forestry 50. Cryo-preservation of plant
germplasm II. Springer, Berlin/Heidelberg, pp 287–311
163. Leunufna S, Keller ERJ (2003) Investigating a new cryo-preservation protocol for yams
(Dioscorea spp.). Plant Cell Rep 21:1159–1166
164. Halmagyi A, Fischer-Kluver G, Mix-Wagner G et al (2004) Cryopreservation of
Chrysanthemum morifolium (Dendranthema grandiflora Ramat.) using different approaches.
Plant Cell Rep 22:371–375
165. Panis B, Piette B, Swennen R (2005) Droplet vitrification of apical meristem: a cryopreserva-
tion protocol applicable to all Musaceae. Plant Sci 168:45–55
9 Micropropagation of Some Orchids and the Use of Cryopreservation 259
166. Halmagyi A, Pinker I (2006) Plant regeneration from Rosa shoot tips cryopreserved by
a combined droplet vitrification method. Plant Cell Tissue Org Cult 84:145–153
167. Jitsopakul N, Thammasiri K, Ishikawa K (2008) Cryopreservation of Bletilla striata mature seeds,
3-day germination seeds and protocorms by droplet-vitrification. CryoLetters 29:517–526
168. Sopalun K, Thammasiri K, Ishikawa K (2010) Vitrifcation-based cryopreservation of
Grammatophyllum speciosum protocorms. CryoLetters 31:347–357
169. Barraco G, Sylvestre I, Engelmann F (2011) Comparing encapsulation-dehydration and
droplet-vitrification for cryopreservation of sugarcane (Saccharum spp.) shoot tips. Sci Hortic
130:320–324
170. Gonzalez-Arnao MT, Lazaro-Vallejo CE, Engelmann F et al (2009) Multiplication and
cryopreservation of vanilla (Vanilla planifolia ‘Andrews’). In Vitro Cell Dev Biol Plant
45:574–582
171. Hernandez-Ramírez F, Gonzalez-Arnao MT, Cruz-Cruz CA et al (2014) Comparison of
different preconditioning and loading treatments with vanilla (Vanilla planifolia Jack.) apices
cryopreserved using the droplet-vitrification procedure. Acta Hortic 1039:173–180
172. Gantait S, Sinniah UR, Suranthran P et al (2015) Improved cryopreservation of oil palm
(Elaeis guineensis Jacq.) polyembryoids using droplet-vitrification approach and assessment
of genetic fidelity. Protoplasma 252:89–101
173. Pathirana R, McLachlan A, Hedderley D et al (2015) Removal of leafroll viruses from infected
grapevine plants by droplet vitrification. Acta Hortic 1083:491–498
174. Volk G, Bonnart R, Krueger R et al (2012) Cryopreservation of citrus shoot tips using
micrografting for recovery. CryoLetters 33:418–426
175. Yamamoto S, Rafique T, Priyantha WS et al (2011) Development of a cryopreservation
procedure using aluminum cryo-plates. CryoLetters 32:256–265
176. Jitsopakul N, Sangyojarn P, Homchan P et al (2019) Efficiency of aluminum cryo-plates for
cryopreservation of Dendrobium signatum Rchb. F. pollinia. Acta Hortic 1234:279–286
177. Imsomboon T, Thammasiri K, Kosiyachinda P et al (2020) Cryopreservation of protocorm-
like bodies of Vanda lilacina Teijsm. & Binn., a Thai Orchid species, by V-cryo-plate and
D-cryo-plate methods. Walailak J Sci Technol 17(4):369–379
178. Niino T, Yamamoto S, Fukui K et al (2013) Dehydration improves cryopreservation of mat
rush (Juncus decipiens Nakai) basal stem buds on cryo-plates. CryoLetters 34:549–560
179. Niino T, Watanabe K, Nohara N et al (2014) Cryopreservation of mat rush lateral buds by air
dehydration using aluminum cryo-plate. Plant Biotechnol 31(3):281–287
180. Cordova LB II, Thammasiri K (2016) Cryopreservation on a cryo-plate of Arundina
graminifolia protocorms, dehydrated with silica gel and drying beads. CryoLetters 37:68–76
181. Ishikawa K, Harata K, Mii M et al (1997) Cryopreservation of zygotic embryos of a Japanese
terrestrial orchid (Bletilla striata) by vitrification. Plant Cell Rep 16:754–757
182. Wang JH, Ge JG, Liu F et al (1998) Cryopreservation of seeds and protocorms of Dendrobium
candidum. CryoLetters 19:123–128
183. Hirano T, Godo T, Mii M et al (2005) Cryopreservation of immature seeds of Bletilla striata by
vitrification. Plant Cell Rep 23:534–539
184. Hirano T, Ishikawa K, Mii M (2005) Cryopreservation of immature seeds Ponerochis
graminifolia var. suzukiana by vitrification. CryoLetters 26:139–146
185. Thammasiri K, Soamkul L (2007) Cryopreservation of Vanda coerulea Griff. ex Lindl. seeds
by vitrification. ScienceAsia 33:223–227
186. Jitsopakul N, Thammasiri K, Yukawa T et al (2012) Effect of cryopreservation on seed
germination and protocorm development of Vanda tricolor. ScienceAsia 38:244–249
187. Flachsland E, Terada G, Scocchi A et al (2006) Cryopreservation of seeds and in vitro-cultured
protocorms of Oncidium bifolium Sims. (RCHIDACEAE) by encapsulation-dehydration.
CryoLetters 27:235–242
188. Tsukazaki H, Mii M, Tokuhara K et al (2000) Cryopreservation of Doritaenopsis suspension
culture by vitrification. Plant Cell Rep 19:1160–1164
260 K. Thammasiri et al.
189. Nikishina TV, Popov AS, Vakhrameeva MG et al (2007) Cryopreservation of seed and
protocorms of rare temperate orchids. Russ J Plant Physiol 54:121–127
190. Pornchuti W, Thammasiri K (2008) Cryopreservation of protocorms of Dendrobium
cariniferum Rchb. f. Acta Hortic 788:63–68
191. Yin M, Hong S (2009) Cryopreservation of Dendrobium candidum Wall. ex Lindl. protocorm
like-bodies by encapsulation-vitrification. Plant Cell Tissue Org Cult 98(2):179–185
192. Na HY, Kando K (1996) Cryopreservation of tissue-cultured shoot primordial from shoot
apices of cultured protocorms in Vanda pumila following ABA preculture and desiccation.
Plant Sci 118:195–201
193. Lurswijidjarus W, Thammasiri K (2004) Cryopreservation of shoot tips of Dendrobium Walter
Oumae by encapsulation/dehydration. ScienceAsia 30:293–299
194. Vendrame WA, Carvalho V, Dias MM et al (2008) Pollination of Dendrobium hybrids using
cryopreserved pollen. HortScience 43(1):264–267
195. Ajeeshkumar S, Decruse SW (2013) Fertilizing ability of cryopreserved pollinia of Luisia
macrantha, an endemic orchid of Western Ghats. CryoLetters 34:20–29
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
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
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
10 Cymbidium: Botany, Production, and Uses 265
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
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
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.
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.)
6 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.
the growing medium. Always use sterilized cutting tools as CymMV and ORSV
spread through mechanical means [23].
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
10 Cymbidium: Botany, Production, and Uses 271
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.
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.
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
10 Cymbidium: Botany, Production, and Uses 273
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.
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.
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
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].
10 Cymbidium: Botany, Production, and Uses 277
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.
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.
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.
10 Cymbidium: Botany, Production, and Uses 279
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.
8.1.1 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.
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.
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.
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%.
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.
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.
10 Cymbidium: Botany, Production, and Uses 285
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.
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.
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.
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.
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.
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.
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.
10 Cymbidium: Botany, Production, and Uses 291
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.
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.
References
1. Hew CS (2001) Ancient Chinese orchid cultivation: a fresh look at an age-old practice. Sci
Hortic 87:1–10
2. Puy D, Cribb DJP (2007) The genus Cymbidium. Kew Publishing, Kew
3. Du Puy DJ, P Cribb (1988) The classification of Cymbidium. In: Du Puy D, Cribb P (eds) The
genus Cymbidium. Kew Publishing, Royal Botanic Garden, Kew, United kingdom, pp 50–194
4. Seth CJ, Cribb PJ (1984) An assessment of sectional limits in the genus Cymbidium Swartz.
In: Arditti J (ed) Orchid biology reviews and perspectives, vol 3. Cornell University Press,
Ithaca, pp 411–483
5. Obara-Okeyo P, Fujii K, Kako S (1998) Isozyme variation in Cymbidium species
(Orchidaceae). Hortic Sci 33:133–135
6. Choi SH, Kim MJ, Lee JS, Ryu KH (2006) Genetic diversity and phylogenetic relationships
among and within species of oriental cymbidiums based on RAPD analysis. Sci Hortic
108:79–85
7. Wang H-Z, Lu J-J, Hu X, Liu J-J (2011) Genetic variation and cultivar identification in
Cymbidium ensifolium. Plant Syst Evol 293:101–110
8. Sharma SK, Kumaria S, Tandon P, Rama Rao S (2013) Assessment of genetic variation and
identification of species-specific ISSR markers in five species of Cymbidium (Orchidaceae).
J Plant Biochem Biotechnol 22:250–255
9. Chung MY, Chung GM, Chung MG, Epperson B (1998) Spatial genetic structure in
populations of Cymbidium goeringii. Genes Genet Syst 73:281–285
10. Aoyama M (1989) Karyomorphological studies in Cymbidium and its allied genera,
Orchidaceae. Bull Hiroshima Botanical Garden 11:1–121
11. Leoonhardt KW (1977) Chromosome numbers and cross compatibility in the genus cymbid-
ium and some related tropical genera (Orchidaceae). Dissertation, University of Hawaii
12. Stort MNS, Morales MAM (1987) Sterility barriers in some artificial F1 orchid hybrids: female
sterility. Rev Brasil Genet 10:109–118
13. Stort MNS (1984) Sterility barriers of some artificial hybrids Male sterility I. Microsporogen-
esis and pollen germination. Am J Bot 71:309–319
14. Wimber DE (1956) Cytogenetic studies in the genus Cymbidium dissertation, Claremont
Graduate University
15. Du Puy DJ (1986) A taxonomic revision on of the genus Cymbidium Sw. (Orchidaceae). PhD
thesis, University of Birmingham and Royal Botanic Gardens, Kew
16. Govaerts R (2019) World checklist of Orchidaceae. Facilitated by the Royal Botanic Gardens,
Kew. Published on the Internet. http://wcsp.science.kew.org/. Retrieved
17. King G, Pantling R (1898) The Orchids of Sikkim Himalaya. Ann Roy Bot Gard, Calcutta
18. Menninger ED (1963) Diary of a colchicine induced tetraploid Cymbidium. Am Orchid Soc
Bull 32:885–887
19. Wimber DE, Van Cott A (1967) Artificially induced polyploidy in Cymbidium. In: Proceed-
ings of 5th World Orchid Conference, pp 27-32
20. Kim MS, Kim JY, Eun JS (2003) Chromosome doubling of a Cymbidium hybrid with
colchicine treatment in meristem culture. In: Proceedings of NIOC2003, Nagoya, Japan
21. Hwang SH, Kim MS, Park PH, Park SY (2016) Scent analysis using an electronic nose and
flowering period of potted diploid and Tetraploid Cymbidium. Korean J Hortic Sci Technol
34:163–171
10 Cymbidium: Botany, Production, and Uses 293
22. Fitch CM (2004) Propagating your Orchids: vegetative propagation. In: The gardener’s guide
to growing of orchids. Botanic Garden, Brooklyn
23. Bag TK (2006) Orchid disease and their management. Tech Bull, ICAR-NRC Orchid Sikkim
3:15–17
24. Soon TE (2005) Orchid Asia. Marshall Cavendish International (Asia) Private Ltd., Singapore
25. Tibs, M. 2007. Orchids: A practical guide to care and cultivation, New Holland, pp. 52-63.
26. Pal R, Medhi RP (2011) Technoguide for propagation of Cymbidiums through backbulbs.
National Research Centre for Orchids, Sikkim
27. Morel GM (1960) Producing viruses free Cymbidium. Am Orchid Soc Bull 29:495–497
28. Wimber DE (1963) Clonal multiplication of Cymbidium through tissue culture of the shoot
meristem. Am Orchid Soc Bull 32:105–107
29. Gu Z, Higaki PC, Nishidha MM, Arditti J, Nyman LP (1987) The effects of benzyladenine, 2,
4-dichlorophenoxyacetic acid and indole acetic acid on shoot-tip culture of Cymbidium.
Lindleyana 2:88–90
30. Begum AA, Tamaki M, Kako S (1994) Formation of protocorm-like-bodies (PLB) and shoot
development in vitro culture of outer tissue of Cymbidium PLB. J Jpn Soc Hortic Sci
63:663–673
31. Nayak NR, Sahoo S, Pathak S, Rath SP, Pathak S (2002) Establishment of thin section (TCS)
method for rapid micropropagation of Cymbidium aloifolium (L) Sw. and Dendrobium nobile
Lindl. (Orchidaceae). Sci Hortic 94:107–116
32. Singh F (1992) In vitro propagation of orchids ‘state of the art’. J Orchid Soc India 6:11–14
33. Bernard N (1899) Sur la germination du Neottia nidusavis. Compt Rend Acad Sci Paris
128(1253):1255
34. Bernard N (1909) Levolution dans la symbiose, les orchidees et leurs champignons
commensaux. Ann Sci Nat Bot 9:1–196
35. Knudson L (1922) Non symbiotic germination of orchid seeds. Bot Gaz 73:1–25
36. Ballion G, Ballion M (1924) The non-symbiotic germination of orchid seed in Belgium. Orch
Rev 32:305–308
37. Ballion G, Ballion M (1928) A symbiotic germination of orchid seed. Orch Rev 36:103–112
38. Clement E (1925) A resume of the non-symbiotic germination of orchid seed. Orch Rev
33:199–200
39. Smith F (1932) Raising orchid seedlings asymbiotically. Gard Chron 91:9–11
40. Arditti J, Clements MA, Fast G, Hadley G, Nashimura G, Ernst R (1982) Orchid seed
germination and seedling culture -A manual. In: Arditti J (ed) Orchid biology: reviews and
perspectives, vol II. Cornell University, London
41. Sharma J (1996) Orchids of India commercialization and conservation. Daya Publishing
House, New Delhi
42. Xiang Y, Yu FA, Peng ZH (2003) Tissue culture of Cymbidium sinense. Forest Res Beijing
16:434–438
43. Chang C, Chen YC, Hsu YH, Wu JT, Hu CC, Chang WC, Lin NS (2005) Transgenic resistance
to Cymbidium mosaic virus in Dendrobium expressing the viral capsid protein gene. Trans-
genic Res 14:41–46
44. Mitra GC, Prasad RN, Roychowdhury A (1976) Inorganic salt and differentiation of pro-
tocorms in seed callus of orchid and correlative changes in its free amino acid content. Indian J
Exp Biol 14:350–351
45. Hossain M, Sharma M, DaSilva JAT, Pathak P (2010) Seed germination and tissue culture of
Cymbidium giganteum Wall ex. Lindl. Sci Hortic 123:479–487
46. Arditti J, Ernst R (1993) Micro propagation of Orchids. Willey, New York
47. Sagawa Y, Shoji T, Shoji T (1966) Clonal propagation of Cymbidiums through shoot meristem
culture. Am Orchid Soc Bull 35:118–122
48. Kim KW, Kako S (1984) Morphological and histological studies on the formation of pro-
tocorm-like bodies and explants development in Cymbidium shoot apex culture in vitro. J
Korean Soc Hortic Sci 25:156–163
49. Subramaniam G, Taha RM (2003) Micro propagation of Cymbidium atropurpureum in vitro
Malaysian. J Sci 22:310–316
294 R. Pal et al.
73. Ueda H, Torikata H (1968) Organogenesis in meristem culture Cymbidium: I studies on the
effect of growth substances added to culture media under continuous illumination. J Jpn Soc
Hortic Sci 37:240–248
74. Conner LN, Conner AJ (1984) Comparative water loss from leaves of Solanum lancilatum
plants cultured in vitro. Plant Sci Lett 36:241–246
75. Ticha IB, Rodociiova B, Kadlecek P (1999) Stomatal morphology during acclimatization of
tobacco plantlets to ex vitro condition. Biol Plant 42:469–474
76. Barinerd KE, Fuchigami LH (1981) Acclimatization of aseptically cultured apple plants to low
relative humidity. J Am Soc Hortic Sci 106:515–518
77. Fujiwara K, Kozai T (1995) Physical microenvironment and its effects. In: Aitken-Christie J.,
Kozai T., Smith M.A.L. (eds) Automation and environmental control in plant tissue culture.
Springer, Dordrecht, pp 319–369
78. Pospisilova J, Solanova M, Ondej T, Opatryny Z (1988) The photosynthetic characteristics
during the micropropagation of tobacco and potato plants. Photosynthetica 22:205–213
79. De Proft MPMML, Debergh P (1985) Carbon dioxide and ethylene evolution in the culture
atmosphere of Magnolia cultured in vitro. Plant Physiol 65:375–379
80. Kozai T, Kubota C (2005) In vitro aerial environments and their effects. In: Kozai T, Afreen F,
Zobayed SMA (eds) Photoautotrophic (sugar-free medium) micropropagation as a new micro-
propagation and transplant production system. Springer, Dordrecht, pp 31–52
81. Pospisilova J, Solavova J, Catasky J (1992) Photosynthetic response to stress during in vitro
cultivation. Photosynthetica 26:3–18
82. Mani SK, Nagaraju V (2004) Influence of potting media on acclimatization and growth of in
vitro plantlets of Cymbidium hybrids. J Orn Hort 7:23–70
83. Powell CL, Caldwell KI, Littler RA, Warrington I (1988) Effect of temperature regime and
nitrogen fertilizer level on vegetative and reproductive bud development in Cymbidium
orchids. J Am Soc Hortic Sci 113:552–556
84. Went FW (1957) The experimental control of plant growth. Cron Bot 17:148–152
85. Li J, Zhao X, Matsui S (2001) Effect of temperature on rate of nutrient concentrations in
pseudobulbs and leaves and flowering in a Cattleya and a Cymbidium hybrid. J Soc High Tech
Agric 13:85–90
86. Ichihashi S (1997) Orchid production and research in Japan. In: Arditti J, Pridgeon AM (eds)
Orchid biology: reviews and perspectives, vol 7. Kluwer Academic Publishers, Dordrecht
87. Komori T, Yoneda K (2002) Effects of autumn and winter light and temperature management
on lead emergence in mericloned Cymbidium lovely angel ‘The Two Virgins’. Environ
Control Biol 40:383–387
88. Lee N, Lee CZ (1993) Growth and flowering of Cymbidium ensifolium var. misericors as
influenced by temperature. Acta Hortic 30:223–231
89. Komori T, Niitsu A, Murakami T (1990) Effects of light intensity on the growth, flowering and
carbohydrate contents of Cymbidium orchids. (Japanese text with English abstract) Bull.
Yamanashi Agric Res Cent 4:17–26
90. Kim YJ, Lee HJ, Kim KS (2011) Night interruption promotes vegetative growth and flowering
of Cymbidium. Sci Hortic 130:887–893
91. Hatamzadeh A, Shafyii Masouleh SS (2011) The influence of vermicompost on the growth and
productivity of cymbidiums. Caspian J Env Sci 9:125–132
92. Yamane M, Sakuamoto (1993) Effect of a hydrophilic polymer and granular soil containing it
on the growth and flowering at first potting of mericlones young cymbidium growing bark
compost. Bull Fac Agric Tottori Univ 46:7–11
93. Naik SK, Barman D, Medhi RP (2010) Response of Cymbidium Pine Clash Moon Venus to
major nutrient at vegetative growth stage. J Ornam Hortic 13:182–188
94. Poole HA, Seeley JG (1978) Nitrogen, potassium and magnesium nutrition of three orchid
genera. J Am Soc Hortic Sci 103:485–488
95. Bik AR, van den Berg TJM (1984) Effect of substrate and nitrogen supply on yield and quality
of mini-cymbidium. Acta Hortic 150:289–296
296 R. Pal et al.
96. De Pascale S, Maturi T, Paradiso R, Barbieri G (2001) Comparison of water and nutrients use
of three cultivars of cymbidium in a soilless culture system. Acta Hortic 559:535–542
97. de Kreij C, van den Berg TJM (1990) Effect of electrical conductivity of the nutrient solution
and fertilization regime on spike production and quality of Cymbidium. Sci Hortic 44:293–300
98. Song S-J, Boo C-H, Zhang-Kual U (1999) Mineral absorption by Cymbidium Jungfrau in the
solution culture. Korean J Exp Agric 18:35–40
99. Naik SK, Barman D, Devadas R, Ushabharathi T (2013) Evaluation of Panchgavya as source
of nutrient for Cymbidium orchids. Afr J Agric Res 8:5728–5732
100. Carver M (1978) The black citrus aphid, Toxoptera citricidus (Kirkaldy) and T. aurantii
(Boyer de Fonscolombe) (Homoptera: Aphididae). J Aust Entomol Soc 17:263–270
101. Dekle GW (1965) Arthropods of Florida and neighboring land areas: Florida armored scale
insects, vol 3. Florida Department of Agriculture & Consumer Services, Division of Plant
Industry, Gainesville
102. Nagrare VS, Pal R, Barman D (2009) Pests associated with orchid Dendrobium nobile under
mid-Altitude of Sikkim. Environ Ecol 27:560–562
103. Zimmerman EC (1948) Insects of Hawaii. vol 5. Homoptera: Sternorhyncha. Honolulu, Univ.
Hawaii Press p 464
104. Miller DR, Devidson JA (2005) Armored scale insect pests of trees and shrubs (Hemiptera:
Diaspididae). Cornell University Press, Ithaca
105. Meena NK, Pal R, Barman D, Medhi RP (2013) Biology and seasonal abundance of two-
spotted spider mite, Tetranychus urticae on orchids and rose. Phytoparasitica 41:597–609
106. Nagrare VS (2001) Pests of orchids and their management in Sikkim- a survey. J Orchid Soc
India 15:65–68
107. Pant RP, Das M, Khan MR, Pun KB, Medhi RP (2012) Association of an ecto-parasite
nematode- Helicotylenchus microcephalus Sher, with poor growth of Cymbidium hybrids in
Sikkim. Indian Phytopath 65:196–197
108. Johnson M, Babu DVN, Latha S, Muthaiyan MC (1993) Interception from imported orchid
plants. Plant Prot Bull 45:26–27
109. Latha S, Babu DVN, Sathyanarayana N, Reddy OR, Sharma R (1999) Plant parasitic nema-
todes intercepted from imported ornamental plants. Indian Phytopath 52:283–284
110. Lin YY, Wang KM, Tsay TT (1992) The occurrence of Aphelenchoides besseyi Christie on
Dendrobium Lady Fay. Plant Prot Bull- Taipei 34:202–215
111. Magee CJ (1943) Orchid mosaic. Aust Orchid Rev 8:51–52
112. Jenson DD (1950) Mosaic of Cymbidium orchids. Phytopathology 40:966–967
113. Jenson DD, Gold HA (1951) A virus ringspot of Odontoglossum orchid: symptoms, transmis-
sion and electron microscopy. Phytopathology 41:648
114. Sherpa AR, Hallan V, Pathak P, Zaidi AA (2006a) Coat protein gene of Cymbidium mosaic
virus: characterization of geographical isolates from India. J Phytopathol 154:275–280
115. Pant RP, Das M, Pun KB, Ramachandran P, Medhi RP (2010) Occurrence of Cymbidium
mosaic and Odontoglossum ringspot viruses in orchid germplasm of Sikkim and Darjeeling
hills, their identification and diagnosis. Indian Phytopath 63:326–332
116. Singh MK, Sherpa AR, Hallan V, Zaidi AA (2007) A potyvirus in Cymbidium spp. in Northern
India. Aust Plant Dis Notes 2:11–13
117. Doi Y, Chang MU, Yora K (1977) Orchid fleck virus. In: CMIIAAB descriptions of plant
viruses, vol. 183. Commonwealth Mycological Institute
118. Blanchfield AL, Mackenzie AM, Gibbs A, Kondo H, Tamada T, Wilson CR (2001) Identifi-
cation of orchid fleck virus by reverse transcriptase-polymerase chain reaction and analysis of
isolate relationships. J Phytopathol 149:713–718
119. Gibbs A, Mackenzie A, Cross P, Wilson C, Kitajima EW, Nightingale M, Clements M (2000)
Viruses of orchids in Australia; their identification, biology and control. Aust Orchid Rev
65:10–21
120. Kitazima EW, Kondo H, Mackenzie A, Rezende JAM, Gioria R, Gibbs A, Tamada T (2001)
Comparative cytopathology and immunochemistry of Japanese, Australian and Brazillian
isolates of orchid fleck virus. J Gen Plant Pathol 67:231–237
10 Cymbidium: Botany, Production, and Uses 297
121. Chang MU, Arai K, Doi Y, Yora K (1976) Morphology and intracellular appearance of orchid
fleck virus. Ann Phytopathol Soc Jpn 42:156–167
122. Rani P, Pant RP, Jain RK (2010) Serological detection of Cymbidium mosaic and
Odontoglossum ringspot viruses in orchids with polyclonal antibodies produced against
their recombinant coat proteins. J Phytopathol 158:542–545
123. Morel GM (1964) A new means of clonal propagation of orchids. Am Orchid Soc Bull
31:437–477
124. Retheesh ST, Bhat AI (2010) Simultaneous elimination of Cucumber mosaic virus and
Cymbidium mosaic virus infecting Vanilla planifolia through meristem culture. Crop Prot
29:1214–1217
125. Loi JS, Lee SM, Can Lam LT, Fan S, Yiang WH (1991) Eradication of orchid viruses in
Dendrobium Sonia using virazole. Singap J Prim Ind 19:16–22
126. Toussaint A, Kummert J, Maroquin C, Lebrun A, Roggemans J (1993) Use of Virazole to
eradicate ORSV from in vitro cultures of Cymbidium Sw. Plant Cell Tissue Organ Cult
32:303–309
127. Watanabe K, Tanaka R, Sakurai H, Iguchi K, Yamada Y, Hsu CS, Sakuma C, Kikuchi H,
Shibayama H, Kawai T (2007) Structure of Cymbidine A, a monomeric peptidoglycan-related
compound with hypotensive and diuretic activities, isolated from a higher plant, Cymbidium
goeringii (Orchidaceae). Chem Pharm Bull 55:780–783
128. Kim SM, Jang EJ, Hong JW, Song SH, Pak CH (2016) A comparison of functional fragrant
components of Cymbidium (Oriental orchid) species. Korean J Hortic Sci Technol 34:331–341
129. Shengji P, Zhiwei Y (2018) Orchids and its uses in Chinese medicine and health care products.
Med Res Innov 2:1–3
Biotechnology Approaches
on Characterization, Mass Propagation, 11
and Breeding of Indonesian Orchids
Dendrobium lineale (Rolfe.) and Vanda
tricolor (Lindl.) with Its Phytochemistry
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
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
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.
302 E. Semiarti et al.
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.
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
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:
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.
304 E. Semiarti et al.
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]
Genetic transformation by using KNAT1 gene
Dendrobium lineale In vitro embryogenesis (zygotic and somatic) [14, 15]
Genetic transformation by using AtRKD4 gene
Dendrobium phalaenopsis In vitro embryogenesis (zygotic and somatic) [16, 17]
Genetic transformation by using AtRKD4 gene
Coelogyne pandurata In vitro embryogenesis (zygotic and somatic) [18, 19]
Genetic transformation by using KNAT1 gene
Dendrobium capra In vitro embryogenesis (zygotic and somatic) [20, 21]
Genetic transformation by using AtRKD4 gene
11 Biotechnology Approaches on Characterization, Mass Propagation, and. . . 305
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.
306 E. Semiarti et al.
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
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
308 E. Semiarti et al.
7 Conclusions
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.
References
1. Irawati (2002) Pelestarian jenis anggrek Indonesia [En: Preservation of Indonesian orchids].
Buku panduan Seminar Anggrek Indonesia 2002:34–45
2. Schuiteman A, Vermeulen JJ, de Vogel EF (2010) Flora Malesiana: orchids of New Guinea,
vol VI; genus Bulbophyllum. (CD-ROM). ETI/National Herbarium Nederland,
Amsterdam/Leiden
310 E. Semiarti et al.
22. Mudalige RG, Kuehnle AR (2004) Orchid biotechnology in production and improvement.
HortScience 39:11–17
23. Dwiyani R, Purwantoro A, Indrianto A, Semiarti E (2010) Improvement of genetic transfor-
mation technique in Vanda tricolor orchid using acetosyringone. Ann Bogoriense 14(2):27–32
24. Kossel A (1891) Ueber die chemische Zusammensetzung der Zelle. Arch Phys 4:181–186
25. Aditama TY (2014) Jamu dan Kesehatan. Lembaga Penerbit Balitbangkes, Jakarta
26. Kirtiker KR, Basu BD (1975) Indian medicinal plants, IV. Bishen Singh Mohendra Pal Singh,
Dehradun
27. Das SP, Bhattacherjee SK (2006) Orchids. In: Bhattacherjee SK (ed) Herbaceous perennials and
shade loving foliage plants. Pointer Publishers, Jaipur
28. Williams CA (1979) The leaf flavonoids of the Orchidaceae. Phytochemistry 18(5):803–813.
https://doi.org/10.1016/0031-9422(79)80019-9
29. Lam Y, Ng TB, Yao RM, Shi J, Xu K, Sze SCW, Zhang KY (2015) Evaluation of chemical
constituents and important mechanism of pharmacological biology in Dendrobium Plants. Evid
Based Complement Alternat Med 2015:841752. https://doi.org/10.1155/2015/841752.
Published online 2015 Apr 6
30. Khan H, Tarun Belwal M, Tariq M, Atanasov AG, Devkota HP (2019) Genus Vanda: A review
on traditional uses, bioactive chemical constituents and pharmacological activities. J
Ethnopharmacol 229:46–53
31. Abbas B, Dailami M, Listyorini FH, Munarti (2017) Genetic variations and relationships of
Papua’s endemic orchids based on RAPD markers. Nat Sci 9(11):377–385. https://doi.org/
10.4236/ns.2017.911035377
32. Kasutjianingati K, Firgiyanto R (2018) Characterization of Morphology from Orchid Vanda sp.
as a Genetic Information Source for Preservation and Agribusiness of Orchids in Indonesia. In
IOP Conference Series: Earth and Environmental Science (207(1):012006). IOP Publishing
33. Lim SH, Teng PC, Lee YH, Goh CJ (1999) RAPD analysis of some species in the genus Vanda
(Orchidaceae). Ann Bot 83:193–196
34. Lo SF, Nalawade SM, Mulabagal V et al (2004) In vitro propagation by asymbiotic seed
germination and 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity studies of
tissue culture raised plants of three medicinally important species of Dendrobium. Biol Pharm
Bull 27(5):731–735. https://doi.org/10.1248/bpb.27.731
35. Luo AX, He XJ, Zhou SD, Fan YJ, He T, Chun Z (2009) In vitro antioxidant activities of a
water-soluble polysaccharide derived from Dendrobium nobile Lindl. extracts. Int J Biol
Macromol 45(4):359–363. https://doi.org/10.1016/j.ijbiomac.2009.07.008
36. Wang J-H, Luo J-P, Zha X-Q, Feng B-J (2010) Comparison of antitumor activities of different
polysaccharide fractions from the stems of Dendrobium nobile Lindl. Carbohydr Polym
79(1):114–118. https://doi.org/10.1016/j.carbpol.2009.07.032
37. Yang L, Qin LH, Bligh SWA et al (2006) A new phenanthrene with a spirolactone from
Dendrobium chrysanthum and its anti-inflammatory activities. Bioorg Med Chem
14(10):3496–3501. https://doi.org/10.1016/j.bmc.2006.01.004
38. Zhang Y, Liu S, Che Y, Liu X (2007) Epicoccins A-D, epipolythiodioxopiperazines from a
Cordyceps-colonizing isolate of Epicoccum nigrum. J Nat Prod 70(9):1522–1525. https://doi.
org/10.1021/np070239u
39. Chen RM, Chen TG, Chen TL et al (2005) Anti-inflammatory and antioxidative effects of
propofol on lipopolysaccharide activated macrophages. Ann N Y Acad Sci 1042:262–271
40. Song JI, Kang YJ, Yong HY, Kim YC, Moon A (2012) Denbinobin, a phenanthrene from
Dendrobium nobile, inhibits invasion and induces apoptosis in SNU-484 human gastric cancer
cells. Oncol Rep 27(3):813–818
41. Ye Q, Qin G, Zhao W (2002) Immunomodulatory sesquiterpene glycosides from Dendrobium
nobile. Phytochemistry 61(8):885–890. https://doi.org/10.1016/s0031-9422(02)00484-3
42. Charoenrungruang S, Chanvorachote P, Sritularak B, Pongrakhananon V (2014) Gigantol, a
bibenzyl from Dendrobium draconis, inhibits the migratory behavior of non-small cell lung
cancer cells. J Nat Prod 77(6):1359–1366. https://doi.org/10.1021/np500015v
312 E. Semiarti et al.
43. Chen C-C, Huang Y-L, Teng C-M (2000) Antiplatelet aggregation principles from
Ephemerantha lonchophylla. Planta Med 66(4):372–374. https://doi.org/10.1055/s-2000-8553
44. Okada Y, Miyauchi N, Suzuki K et al (1995) Search for naturally occurring substances to
prevent the complications of diabetes. II. Inhibitory effect of coumarin and flavonoid
derivatives on bovine lens aldose reductase and rabbit platelet aggregation. Chem Pharm Bull
43(8):1385–1387. https://doi.org/10.1248/cpb.43.1385
45. Pan L-H, Li X-F, Wang M-N et al (2014) Comparison of hypoglycemic and antioxidative
effects of polysaccharides from four different Dendrobium species. Int J Biol Macromol
64:420–427
46. Gong CY, Yu ZY, Lu B et al (2014) Ethanol extract of Dendrobium chrysotoxum Lindl
ameliorates diabetic retinopathy and its mechanism. Vasc Pharmacol 62(3):134–142. https://
doi.org/10.1016/j.vph.2014.04.007
47. Simmler C, Antheaume C, André P, Bonté F, Lobstein A (2011) Glucosyloxybenzyl eucomate
derivatives from Vanda teres stimulate HaCaT cytochrome c oxidase. J Nat Prod 74:949–955
48. Vijaykumar K (2013) In vitro anti-oxidant activity of pet-ether extract of Vanda tessellata Roxb.
Int Ayur Med J 1:1–4
49. Thaakur SR, Pokkula S (2013) Ameliorative effects of Vanda testacea in sciatic nerve
transection-induced neuropathy in rats. Int J Pharm Bio Sci 4:271–284
50. Andre P, Archambault JC, Renimel I (2011) Use of an extract of the orchid Vanda coerulea as a
skin hydrating agent. US Patent 8,039,028
51. Bonté F, Simmler C, Lobstein A, Pellicier F, Cauchard JH (2011) Action d0 un extrait de Vanda
coerulea sur la sénescence de fibroblastescutanés. In Annales pharmaceutiques francaises (69
(3):177–181). Elsevier Masson
52. Dasari R, Sathyavathi D, Belide SK, Soumy BR (2013) Pharmacological evaluation for
antidepressant activity of Vanda spathulata in mice. Int J Pharm Bio Sci 4:866–872
53. Anwar M, Kumar SN, Mahendran B (2013) Hepatoprotective activity of pet-ether extract of
Vanda tessellata Roxb. Int Ayur Med J 1:1–4
54. Kumar PS, Subramoniam A, Pushpangadan P (2000) Aphrodisiac activity of Vanda tessellata
(Roxb.) Hook exdon extract in male mice. Indian J Pharm 32:300–304
55. Khan H, Marya, Belwal T, Tariq M, Atanasov AG, Devkota HP (2019) Genus Vanda: a review
on traditional uses, bioactive chemical constituents and pharmacological activities.
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
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?
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)
Annual frequency of buying orchids
(average standard deviation)
Own use For gift
Evaluated Total of Average standard Average standard
criterion respondents deviation deviation
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)
12 Preferences of Orchid Consumers and the Substitute Products’ Influences 317
Table 3 Comparison by level of education 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
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
Kruskal–Wallis Test p ¼ 0,032 p ¼ 0,025
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)
Annual frequency of buying orchids
(average standard deviation)
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
Kruskal–Wallis Test p ¼ 0,080 p ¼ 0,005
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
318 A. Anacleto and L. Scheuer
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
Multiple choice questions
Preferences of Orchid Consumers and the Substitute Products’ Influences
319
320 A. Anacleto and L. Scheuer
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
Multiple choice questions
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
12 Preferences of Orchid Consumers and the Substitute Products’ Influences 321
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
322 A. Anacleto and L. Scheuer
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
Multiple choice questions
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,
12 Preferences of Orchid Consumers and the Substitute Products’ Influences 323
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.
324 A. Anacleto and L. Scheuer
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.
References
1. Anacleto A, Negrelle RRB, Cuquel FL, Muraro D (2017) Profile and behavior of flower
consumer: subsidies for marketing actions. Ceres 64(6):557–566. https://doi.org/10.1590/
0034-737x201764060001
2. Tathe S (2019) Global floriculture market research gain impetus due to the growing demand
over 2019–2028 with a CAGR of 5.00%. Relatório Técnico
3. Bornancin AAP, Anacleto A (2018) Perfil e comportamento do consumidor de
bromélias: orientação a produção rural. Revista Brasileira de Planejamento e Desenvolvimento
7(1):51–66. https://doi.org/10.3895/rbpd.v7n1.7055
4. ITC, International Trade Centre (2019) Market dynamics, annual report. Disponível em
http://www.intracen.org. Acesso em 05/11/2019
5. Hirschman E, Holbrook M (1982) Hedonic consumption: emerging concepts, methods and
propositions. J Mark 26 (4 ed):138–149. https://doi.org/10.1177/002224298204600314
6. Kotler P, Keller KL, Ancarani F, Costabile M (2014) Marketing management, 14th edn.
Pearson, Upper Saddle River
7. Solomon MO (2002) Comportamento do consumidor: comprando, possuindo, sendo, 5th edn.
Porto Alegre, Bookman
8. Churchill GA, Peter JP (1995) Marketing: creating value for customers. Irwin, Boston
9. Malhotra NK (2010) Marketing research: an applied orientation. Pearson Education Australia,
Boston
10. Hair JF, Black WC, Babin BJ, Anderson RE, Tatham RL (2009) Análise multivariada de dados.
Bookman, Porto Alegre
12 Preferences of Orchid Consumers and the Substitute Products’ Influences 325
11. Porter ME (2008) Competitive strategy: techniques for analyzing industries and competitors.
Simon and Schuster, New York
12. Muraro D, Negrelle RR, Cuquel FL, AnacletoA (2016) Market management: the impact on the
development of an ornamental plants supply chain in Curitiba, Brazil. Ciencia e Investigación
Agraria 42(3):453–460. https://doi.org/10.4067/S0718-16202015000300013
13. Schewe CD, Smith RM (1979) Marketing: concepts and applications. McGraw-Hill, New York
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
Qualisud, Université de La Réunion, Université Montpellier, CIRAD, Institut Agro, Avignon
Université, Montpellier, France
e-mail: hippolyte.kodja@univ-reunion.fr
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
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)
332 K. Mahadeo et al.
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.
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,
334 K. Mahadeo et al.
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
13 Vanilla: Culture, Reproduction, Phytochemistry, Curing, Pest, and Diseases 335
“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].
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].
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
336 K. Mahadeo et al.
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
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].
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.
References
1. Bouriquet G (1954) Le vanillier et la vanille dans le monde. In: Lechevalier P (ed) Encyclopédie
biologique, vol XLVI. Editions Paul Lechevalier, Paris VI, p 748
2. Purseglove JW, Brown EG, Green CI, Robbins SRJ (1981) Vanilla. In: Spices, vol 2. Longman
Inc., New York
3. Palama TL (2010) NMR-based metabolomic characterization of Vanilla planifolia. Doctoral
Thesis. Leiden University, Netherlands
4. Bory S, Grisoni M, Duval M-F, Besse P (2008) Biodiversity and preservation of vanilla: present
state of knowledge. Genet Resour Crop Evol 55:551–571
5. Soto Arenas MA (2003) Vanilla. In: Pridgeon AM, Cribb PJ, Chase MW, Rasmussen FN (eds)
Genera Orchidacearum, vol 3. Oxford University Press, USA, pp 321–334
6. Arditti J, Rao AN, Nair H (2009) Hand-pollination of vanilla: how many discoverers? Orchid
Biol: Rev Perspect X:233–249
7. Kanisawa T (1993) Flavor development in Vanilla beans. Kouryou 180:113–123
8. Kanisawa T, Tokoro K, Kawahara S (1994) Flavor development in the beans of Vanilla
planifolia. In: Kurihara K, Suzuki N, Ogawa H (eds) Proceedings of the International Sympo-
sium Tokyo, Japan, pp 268–270
9. Tokoro K, Kawahara S, Amano A, Kanisawa T, Indo M (1990) Glucosides in vanilla beans and
changes of their contents during maturation. In: Bessiere Y, Thomas AF (eds) Flavour science
and technology. Wiley, Chichester, New York, Brisbane, Toronto, Singapore, pp 73–76
10. Palama TL, Fock I, Choi YH, Verpoorte R, Kodja H (2010) Biological Variation of Vanilla
planifolia leaves metabolome. Phytochemistry 71(5):567–573
11. Odoux E, Brillouet JM (2009) Anatomy, histochemistry and biochemistry of glucovanillin,
oleoresin and mucilage accumulation sites in green mature vanilla pod (Vanilla planifolia;
Orchidaceae): a comprehensive and critical reexamination. Fruits 64:221–241
12. Palama TL, Khatib A, Choi YH, Côme B, Fock I, Verpoorte R, Kodja H (2011) Metabolic
characterization of green pods from Vanilla planifolia accessions grown in La Réunion.
Metabolic characterization of green pods from Vanilla planifolia accessions grown in La
Réunion. Environ Exp Bot 72(2):258–265
13 Vanilla: Culture, Reproduction, Phytochemistry, Curing, Pest, and Diseases 339
13. Garros-Patin J, Hahn J (1954) La chimie de la vanille. In: Le Vanillier et la Vanille dans le
Monde
14. Balls AK, Arana FE (1941) The curing of vanilla. Ind Eng Chem 33:1073–1075
15. Dignum MJW, Kerler J, Verpoorte R (2002) Vanilla curing under laboratory conditions. Food
Chem 79:165–171
16. Havkin-Frenkel D, French JC, Graft N, Joel DM, Pak FE, Frenkel C (2004) Interrelation of
Curing and Botany in Vanilla (Vanilla planifolia) Bean. Acta Hortic 629:93–102
17. Odoux E (2000) Changes in vanillin and glucovanillin concentrations during the various stages
of the process traditionally used for curing Vanilla fragrans beans in Réunion. Fruits 55:119–
125
18. Pérez-Silva A, Odoux E, Brat P, Ribeyre F, Rodriguez-Jimenes G, Robles-Olvera V, García-
Alvarado MA, Günata Z (2006) GC-MS and GC-olfactometry analysis of aroma compounds in
a representative organic aroma extract from cured vanilla (Vanilla planifolia G. Jackson) beans.
Food Chem 99:728–735
19. Koyyappurath S, Conéjéro G, Dijoux JB, Lapeyre-Montès F, Jade K, Chiroleu F, Gatineau F,
Verdeil JL, Besse P, Grisoni M (2015) Differential responses of Vanilla accessions to root rot
and colonization by Fusarium oxysporum f. sp. radicis-vanillae. Front Plant Sci 6:1125
20. Tombe M, Liew ECY (2010) Fungal diseases of Vanilla. In: Odoux E, Grisoni M (eds) Vanilla.
CRC Press, Boca Raton, FL
21. Fravel D, Olivain C, Alabouvette C (2003) Research review: Fusarium oxysporum and its
biocontrol. New Phytol 157:493–502
22. Havkin-Frenkel D, Belanger FC (2018) Vanilla diseases. In: Handbook of vanilla science and
technology. Wiley, Oxford, pp 27–40
23. Charron C, Hubert J, Minatchy J, Wilson V, Chrysot F, Gerville S, Loos R, Jeandel C, Grisoni M
(2018) Characterization of Colletotrichum orchidophilum, the agent of black spot disease of
vanilla. J Phytopathol 166(7–8):525–531
24. Zettler FW, Ko NJ, Wisler GC, Elliott MS, Wong SM (1990) Viruses of orchids and their
control. Plant Dis 74:621–626
25. Leclercq-Le Quillec F, Rivière C, Lagorce A (2001) Spread of cymbidium mosaic potexvirus
and potyviruses in vanilla plants grown in shade houses in Reunion Island. Fruits 56:249–260
26. Hu JS, Ferreira S, Xu MQ, Lu M, Iha M, Pflum E, Wang M (1994) Transmission, movement
and inactivation of cymbidium mosaic and odontoglossum ringspot viruses. Plant Dis 78:
633–636
27. Farreyrol K, Pearson MN, Grisoni M, Leclercq-Le Quillec F (2001) Severe stunting of Vanilla
tahitensis in French polynesia caused by Cucumber mosaic virus (CMV), and the detection of
the virus in V. fragrans in Reunion Island. Plant Pathol 50:414–414
28. Grisoni M, Pearson M, Farreyrol K (2010) Virus diseases of vanilla. In: Odoux E, Grisoni M
(eds) Vanilla. CRC Press Taylor & Francis Group, USA, pp 97–123
29. Madhubala R, Bhadramurthy V, Bhat AI, Hareesh PS, Retheesh ST, Bhai RS (2005)
Occurencre of Cucumber mosaic virus on vanilla (Vanilla planifolia Andrews) in India. J
Biosci 30:339–350
30. Farreyrol K, Pearson MN, Grisoni M, Cohen D, Beck D (2006) Vanilla mosaic virus isolates
from French polynesia and the Cook Islands are Dasheen mosaic virus strains that exclusively
infect vanilla. Arch Virol 151:905–919
31. Wisler GC, Zettler FW, Mu L (1987) Virus infections of vanilla and other orchids in French
Polynesia. Plant Dis 71:1125–1129
32. Grisoni M, Davidson F, Hyrondelle C, Farreyrol K, Caruana ML, Pearson M (2004) Nature,
incidence, and symptomatology of viruses infecting Vanilla tahitensis in French polynesia.
Plant Dis 88:119–124
33. Grisoni M, Moles M, Farreyrol K, Rassaby L, Davis R, Pearson M (2006) Identification of
potyviruses infecting vanilla by direct sequencing of a short RT-PCR amplicon. Plant Pathol 55:
523–529
34. Pearson MN, Brunt AA, Pone SP (1990) Some hosts and properties of a potyvirus infecting
Vanilla fragrans (Orchidaceae) in the kingdom of Tonga. J Phytopathol 128:46–54
340 K. Mahadeo et al.
35. Wang YY, Beck DL, Gardner RC, Pearson MN (1993) Nucleotide sequence, serology and
symptomatology suggest that vanilla necrosis potyvirus is a strain of watermelon mosaic virus
II. Arch Virol 129:93–103
36. Wang YY, Pearson MN (1992) Some characteristics of potyvirus isolates from Vanilla tahitensis
in French Polynesia and the Cook Islands. J Phytopathol 135:71–76
37. Bhat AI, Bhadramurthy V, Siju S, Hareesh PS (2006) Detection and identification of Cymbid-
ium mosaic virus infecting vanilla (Vanilla planifolia Andrews) in India based on coat protein
gene sequence relationships. J Plant Biochem Biotechnol 15:33–37
38. Richard A, Farreyrol K, Rodier B, Leoce-Mouk-San K, Wong M, Pearson MN, Grisoni M
(2009) Control of virus diseases in intensively cultivated vanilla plots of French Polynesia. Crop
Prot 28:870–877
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
Qualisud, Université de La Réunion, Université Montpellier, CIRAD, Institut Agro, Avignon
Université, Montpellier, France
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
Qualisud, Université de La Réunion, Université Montpellier, CIRAD, Institut Agro, Avignon
Université, Montpellier, France
e-mail: hippolyte.kodja@univ-reunion.fr
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
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
344 S. Khoyratty et al.
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.
14 Vanillin: Biosynthesis, Biotechnology, and Bioproduction 345
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
346 S. Khoyratty et al.
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.
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
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
348 S. Khoyratty et al.
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
14 Vanillin: Biosynthesis, Biotechnology, and Bioproduction 349
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.
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.
350 S. Khoyratty et al.
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
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
14 Vanillin: Biosynthesis, Biotechnology, and Bioproduction 351
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.
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).
352 S. Khoyratty et al.
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.
14 Vanillin: Biosynthesis, Biotechnology, and Bioproduction 353
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.
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
354 S. Khoyratty et al.
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.
References
1. Firmenich (2017) Firmenich enriches Vanilla palette with sustainable and cost-effective natural
tonalities. http://www.firmenich.com/en_INT/company/news/FIRMENICH-ENRICHES-
VANILLA-PALETTE.html. Accessed 09 Sept 2017
2. Givaudan (2016) Anything but Plain Vanilla. Givaudan Taste Essentials Vanilla. https://www.
givaudan.com/flavours/world-flavours/tasteessentials/vanilla. Accessed 29 July 2016
3. Ni J, Tao F, Du H, Xu P (2015) Mimicking a natural pathway for de novo biosynthesis: natural
vanillin production from accessible carbon sources. Sci Rep 5:13670. https://doi.org/10.1038/
srep13670
14 Vanillin: Biosynthesis, Biotechnology, and Bioproduction 355
4. Perfumer and Flavorist (2014) Givaudan submits patent application for Vanilla fermentation
process. http://www.perfumerflavorist.com/networking/news/company/Givaudan-Submits-Pat
ent-Application-For-Vanilla-Fermentation-Process-238896611.html. Accessed 29 July 2016
5. Kaur B, Chakraborty D (2013) Biotechnological and molecular approaches for vanillin
production: a review. Appl Biochem Biotechnol 169:1353–1372
6. Hansen EH, Møller BL, Kock GR, Bünner CM, Kristensen C, Jensen OR, Okkels FT,
Olsen CE, Motawia MS, Hansen J (2009) De novo biosynthesis of vanillin in fission yeast
(Schizosaccharomycespombe) and Baker’s yeast (Saccharomyces cerevisiae). Appl Environ
Microbiol 75:2765–2774
7. Zheng L, Zheng P, Sun Z, Bai Y, Wang J, Guo X (2007) Production of vanillin from waste
residue of rice bran oil by Aspergillusniger and Pycnoporuscinnabarinus. Bioresour Technol
98:1115–1119
8. Lesage-Meessen L, Lomascolo A, Bonnin E, Thibault JF, Buleon A, Roller M, Asther M,
Record E, Ceccaldi BC, Asther M (2002) A biotechnological process involving filamentous
fungi to produce natural crystalline vanillin from maize bran. Appl Biochem Biotechnol
103:141–153
9. Thibault FJ, Asther M, Colonna-Ceccaldi B, Couteau D, Delattre M, Duarte JC, Craig F, Heldt-
Hansen PH, Kroon PA, Lesage-Meessen L, Micard VC, Renard MT, Van Hulle S, Williamson G
(1998) Fungal bioconversion of agricultural by-products to vanillin. Lebenson Wiss Technol
31:530–536
10. Rasoanaivo P (1998) Essential oils of economic value in Madagascar: present state of knowl-
edge. HerbalGram 43:31–59
11. Toms A, Wood JM (1970) Degradation of trans-ferulic acid by Pseudomonas acidovorans.
Biochemist 9:337–343
12. Sinha AK, Sharma UK, Sharma N (2008) A comprehensive review on Vanilla flavor:
extraction, isolation and quantification of vanillin and others constituents. Int J Food Sci Nutr
59:299–326
13. Ranadive AS (2011) Quality control of Vanilla beans and extracts. In: Havkin-Frenkel D,
Belanger FC (eds) Handbook of Vanilla science and technology. Wiley-Blackwell, Oxford,
pp 141–160
14. Gleason-Allured J (2011) Vanilla: anything but plain. Perfumer and Flavorist. http://www.
perfumerflavorist.com/flavor/application/vanilla/132347233.html. Accessed 30 July 2016
15. Dunphy P, Bala K (2012a) The role of microorganisms in Vanilla curing. Part 1: evidence for
microbial involvement. Perfum Flavor 37:24–29
16. Cameron KM (2011) Vanilla phylogeny and classification. In: Havkin-Frenkel D, Belanger FC
(eds) Handbook of Vanilla science and technology. Wiley-Blackwell, Oxford, p 243
17. Alwahti AY (2003) A taste of Vanilla. The TED case studies. Number 686. http://www1.
american.edu/TED/vanilla.htm. Accessed 30 July 2016
18. Hoffman PG, Zapf CM (2011) Flavor, quality, and authentication. In: Havkin-Frenkel D,
Belanger FC (eds) Handbook of Vanilla science and technology. Wiley-Blackwell, Oxford,
pp 162–179
19. Kennedy CR (2015) The flavor rundown: natural vs. artificial flavors: What’s in a flavor?
Harvard University. http://sitn.hms.harvard.edu/flash/2015/the-flavor-rundown-natural-vs-artifi
cial-flavors/. Accessed 29 July 2016
20. Zhang S, Mueller C (2012) Comparative analysis of volatiles in traditionally cured Bourbon and
Ugandan Vanilla bean (Vanilla planifolia) extracts. J Agric Food Chem 60:10433–10444
21. Toth S, Lee KJ, Havkin-Frenkel D, Belanger FC, Hartman TG (2011) Volatile compounds in
Vanilla. In: Havkin-Frenkel D, Belanger FC (eds) Handbook of Vanilla science and technology.
Wiley-Blackwell, Oxford, pp 183–218
22. Sigma-Aldrich (2014) Ingredients catalog. Flavour Frag J
23. Burdock GA (2010) Fenaroli’s handbook of flavor ingredients, 6th edn. CRC Press, New York
24. Dignum M, Kerler J, Verpoorte R (2001) Vanilla production: technological, chemical and
biosynthetic aspects. Int Food Res J 17:199–219
356 S. Khoyratty et al.
25. Dignum MJW, Kerler J, Verpoorte R (2002) Vanilla curing under laboratory conditions. Food
Chem 79:165–171
26. Royal Society of Chemistry (2016) Chemistry in its element – vanillin. http://www.rsc.org/
chemistryworld/podcast/CIIEcompounds/transcripts/vanillin.asp. Accessed 29 July 2016
27. Labuda I (2011) Biotechnology of vanillin: vanillin from microbial sources. In: Havkin-
Frenkel D, Belanger FC (eds) Handbook of Vanilla science and technology. Wiley-Blackwell,
Oxford, pp 301–327
28. Dionisio AP, Molina G, Carvalho DS, Pastore GM (2012) Natural flavourings from biotech-
nology for foods and beverages. In: Baines D, Seal R (eds) Natural food additives, ingredients
and flavourings. Woodhead Publishing, Cambridge, pp 231–259
29. Gimenez C, Cabrera R, Reina M, Gonzalez-Coloma A (2007) Fungal endophytes and their role
in plant protection. Curr Org Chem 11:707–720
30. Kucht S, Groß J, Hussein Y, Grothe T, Keller U, Basar S, König W, Steiner U, Leistner E (2004)
Elimination of ergoline alkaloids following treatment of Ipomoea asarifolia (Convolvulaceae)
with fungicides. Planta 219:619–625
31. Schardl CL, Young CA, Hesse U, Amyotte SG, Andreeva K, Calie PJ, Fleetwood DJ,
Haws DC, Moore N, Oeser B, Panaccione DG, Schweri KK, Voisey CR, Farman ML,
Jaromczyk JW, Roe BA, O'Sullivan DM, Scott B, Tudzynski P, An Z, Arnaoudova EG,
Bullock CT, Charlton ND, Chen L, Cox M, Dinkins RD, Florea S, Glenn AE, Gordon A,
Güldener U, Harris DR, Hollin W, Jaromczyk J, Johnson RD, Khan AK, Leistner E,
Leuchtmann A, Li C, Liu J, Liu J, Liu M, Mace W, Machado C, Nagabhyru P, Pan J,
Schmid J, Sugawara K, Steiner U, Takach JE, Tanaka E, Webb JS, Wilson EV, Wiseman JL,
Yoshida R, Zeng Z (2013) Plant-symbiotic Fungi as chemical engineers: multi-genome analysis
of the Clavicipitaceae reveals dynamics of alkaloid loci. PLoS Genet 9:e1003323
32. Steiner U, Hellwig S, Leistner E (2008) Specificity in the interaction between an epibioticcla-
vicipitalean fungus and its convolvulaceous host in a fungus/plant symbiotum. Plant Signal
Behav 3:704–706
33. Steiner U, Leibner S, Schardl CL, Leuchtmann A, Leistner E (2011) Periglandula, a new fungal
genus within the Clavicipitaceae and its association with Convolvulaceae. Mycologia
103:1133–1145
34. Stierle A, Strobel G, Stierle D (1993) Taxol and taxane production by Taxomyces andreanae, an
endophytic fungus of Pacific yew. Science 260:214–216
35. Strobel GA, Hess WM, Li JY, Ford E, Sears J, Sidhu RS, Summerell B (1997) Pestalotiopsis
guepinii, a Taxol-producing endophyte of the Wollemi pine, Wollemia nobilis. Aust J Bot
45:1073–1082
36. Souza JJD, Vieira IJC, Rodrigues-Filho E, Braz-Filho R (2011) Terpenoids from endophytic
Fungi. Molecules 16:10604–10618
37. Hardoim PR, Overbeek LSV, Berg G, Pirttila AM, Compant S, Campisano A, Doring M,
Sessitsch A (2015) The hidden world within plants: ecological and evolutionary considerations
for defining functioning of microbial endophytes. Microbiol Mol Biol R 79:293–320
38. Bálint M, Tiffin P, Hallström B, O’Hara RB, Olson MS, Fankhauser JD, Piepenbring M,
Schmitt I (2013) Host genotype shapes the foliar fungal microbiome of balsam poplar
(Populusbalsamifera). PLoS One 8:e53987
39. Álvarez-Loayza P, White JF, Torres MS, Balslev H, Kristiansen T, Svenning JC, Gil N (2011)
Light converts endosymbiotic fungus to pathogen, influencing seedling survival and niche-
space filling of a common tropical tree, Iriarteadeltoidea. PLoS One 6:e16386
40. Arnold AE, Mejía LC, Kyllo D, Rojas EI, Maynard Z, Robbins N, Herre EA (2003) Fungal
endophytes limit pathogen damage in a tropical tree. Proc Natl Acad Sci USA 100:15649–15654
41. Herre EA, Mejía LC, Kyllo DA, Rojas E, Maynard Z, Butler A, Van Bael SA (2007) Ecological
implications of anti-pathogen effects of tropical fungal endophytes andmycorrhizae. Ecology
88:550–558
42. Bayat F, Mirlohi A, Khodambashi M (2009) Effects of endophytic fungi on some drought
tolerance mechanisms of tall fescue in a hydroponics culture. Russ J Plant Physiol 56:510–516
14 Vanillin: Biosynthesis, Biotechnology, and Bioproduction 357
43. De Sassi C, Müller CB, Krauss J (2006) Fungal plant endosymbionts alter life history and
reproductive success of aphid predators. Proc R Soc Biol Sci 273:1301–1306
44. Rudgers JA, Afkhami ME, Rúa MA, Davitt AJ, Hammer S, Huguet VM (2009) A fungus
among us: broad patterns of endophyte distribution in the grasses. Ecology 90:1531–1539
45. Saari S, Sundell J, Huitu O, Helander M, Ketoja E, Ylönen H, Saikkonen K (2010) Fungal-
mediated multitrophic interactions – do grass endophytes in diet protect voles from predators?
PLoS One 5:e9845
46. Havkin-Frenkel D, Belanger FC (2016) Biotechnological production of vanillin. In: Havkin-
Frenkel D, Dudai N (eds) Biotechnology in flavor production, 2nd edn. Wiley, West Sussex,
p 168
47. Korthout H, Verpoorte R (2007) Vanilla. In: Berger RG (ed) Flavours and fragrances: chemistry,
bioprocessing and sustainability. Springer Science + Business Media, Leipzig, pp 203–217
48. Dixon RA (2011) Vanillin biosynthesis – not as simple as it seems? In: Havkin-Frenkel D,
Belanger FC (eds) Handbook of Vanilla science and technology. Wiley-Blackwell, Oxford,
pp 292–298
49. Kundu A (2017) Vanillin biosynthetic pathways in plants. Planta 245:1069–1078
50. Yang H, Barros-Rios J, Kourteva G, Rao X, Chen F, Shen H, Liu C, Podstolski A, Belanger F,
Havkin-Frenkel D, Dixon RA (2017) A re-evaluation of the final step of vanillin biosynthesis in
the orchid Vanilla planifolia. Phytochemistry 139:33–46
51. Funk C, Brodelius PE (1990) Phenylpropanoid metabolism in suspension cultures of Vanilla
planifolia. Andr Plant Physiol 94:95–101
52. Havkin-Frenkel D, Belanger FC (2007) Application of metabolic engineering to vanillin
biosynthetic pathways in Vanilla planifolia. In: Verpoorte R, Alfermann AW, Johnson TS
(eds) Applications of plant metabolic engineering. Springer, Dordrecht, pp 175–196
53. Gallage NJ, Hansen EH, Kannangara R, Olsen CE, Motawia MS, Jørgensen K, Holme I,
Hebelstrup K, Grisoni M, Møller BL (2014) Vanillin formation from ferulic acid in Vanilla
planifolia is catalysed by a single enzyme. Nat Commun 5:4037
54. Dewick PM (2002) The shikimate pathway: aromatic amino acids and phenylpropanoids. In:
Dewick PM (ed) Medicinal natural products. Wiley, West Sussex, pp 121–166
55. Havkin-frenkel D, Podstolski A, Dixon RA (2003) Vanillin biosynthetic pathway enzyme from
Vanilla planifolia. US Patent US20030070188 A1 (US Patent Office: United States, 2003)
56. Mathew S, Abraha TE (2004) Ferulic acid: an antioxidant found naturally in plant cell walls and
feruloylesterases involved in its release and their applications. Crit Rev Biotechnol 24:59–83
57. Lesage-Meessen L, Stentelaire C, Lomascolo A, Couteau D, Asther M, Moukha S, Record E,
Sigoillot J, Asther M (1999) Fungal transformation of ferulic acid from sugar beet pulp to
natural vanillin. J Sci Food Agric 79:487–490
58. Roling WFM, Kerler J, Braster M, Apriyantono A, Stam H, Verseveld HWV (2001)
Microorganisms with a taste for Vanilla: microbial ecology of traditional Indonesian Vanilla
curing. Appl Environ Microbiol 67:1995–2003
59. Pinaria AG, Liew ECY, Burgess LW (2010) Fusariumspecies associated with Vanilla stem rot in
Indonesia. Australas Plant Pathol 39:176–183
60. Porras-Alfaro A, Bayman P (2007) Mycorrhizal fungi of Vanilla: diversity, specificity and
effects on seed germination and plant growth. Mycologia 99:510–525
61. Dunphy P, Bala K (2012b) The role of microorganisms in Vanilla curing. Part 2: Microbial
transformation of phenols and other compounds. Perfum Flavor 37:22–27
62. Gu F, Chen Y, Fang Y, Wu G, Tan L (2015) Contribution of Bacillus isolates to the flavor
profiles of Vanilla beans assessed through aroma analysis and chemometrics. Molecules
20:18422–18436
63. Mäkelä MR, Marinović M, Nousiainen P, Liwanag AJ, Benoit I, Sipilä J, Hatakka A, de Vries
RP, Hildén KS (2015) Aromatic metabolism of filamentous fungi in relation to the presence of
aromatic compounds in plant biomass. Adv Appl Microbiol 91:63–137
64. Reineccius G (1998) Natural flavoring materials. In: Reineccius G (ed) Source book of flavors,
2nd edn. Chapman & Hall, New York, p 357
358 S. Khoyratty et al.
65. Takahashi M, Inai Y, Miyazawa N, Kurobayashi Y, Fujita A (2013) Key odorants in cured
Madagascar Vanilla beans (Vanilla planifolia) of differing bean quality. Biosci Biotech Bioch
77:606–611
66. Leffingwell & Associates (2017) Odor & flavor detection thresholds in water (in parts per
billion). http://www.leffingwell.com/odorthre.htm. Accessed 5 Mar 2017
67. Buccellato F (2011) Vanilla in perfumery and beverage flavors. In: Havkin-Frenkel D, Belanger
FC (eds) Handbook of Vanilla science and technology. Wiley-Blackwell, Oxford, pp 235–240
68. Grice K, Lu H, Atahan P, Asif M, Hallmann C, Greenwood P, Maslen E, Tulipani S,
Williford K, Dodson J (2009) New insights into the origin of perylene in geological samples.
Geochim Cosmochim Acta 73:6531–6543
69. David AS, Seabloom EW (2016) Plant host species and geographic distance affect the structure
of aboveground fungal symbiont communities, and environmental filtering affects belowground
communities in a coastal dune ecosystem. Microb Ecol 71:912–926
70. Zarraonaindia I, Owens SM, Weisenhorn P, West K, Hampton-Marcell J, Lax S, Bokulich NA,
Mills DA, Martin G, Taghavi S, van der Lelie D, Gilbert JA (2015) The soil microbiome
influences grapevine-associated microbiota. Microb Cell 2:171–173
71. Varma VC, Gange AC (2014) Advances in endophytic research. Springer, New Delhi
72. Faeth SH, Fagan WF (2002) Fungal endophytes: common host plant symbionts but uncommon
mutualists. Integr Comp Biol 42:360–368
73. Pusztahelyi T, Holb IJ, Pócsi I (2015) Secondary metabolites in fungus-plant interactions. Front
Plant Sci 6:573. https://doi.org/10.3389/fpls.2015.00573
74. Ludwig-Müller J (2015) Plants and endophytes: equal partners in secondary metabolite pro-
duction? Biotechnol Lett 37. https://doi.org/10.1007/s10529-015-1814-4
75. Hongsheng Y, Zhang L, Li L, Zheng C, Guo L, Li W, Sun P, Qin L (2010) Recent developments
and future prospects of antimicrobial metabolites produced by endophytes. Microbiol Res
165:437–449
76. Gallage NJ, Møller BL (2015) Vanillin–Bioconversion and Bioengineering of the Most Popular
Plant Flavor and Its De Novo Biosynthesis in the Vanilla Orchid. Mol Plant 8:40–57
77. Khoyratty S, Kodja H, Verpoorte R (2018) Vanilla flavor production methods: a review. Ind
Crops Prod 125:433–442
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
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].
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
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
2.3 Unani
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.
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.
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.
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.
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.
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.
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.
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.
over-exploitation for medicinal purpose and lack of cultivation, the plant has become
endangered, and IUCN has categorized the plant as critically endangered.
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.
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.
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.
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].
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.
15 Ethnobotany and Recent Advances in Indian Medicinal Orchids 381
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].
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.
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.
References
1. Jeffrey D, White JD, O’Keefe BR, Sharma J, Javed G, Nukala V, Ganguly A, Khan IA,
Kumar NB, Mukhtar HF, Pauli GF, Walker L, Sivaram Rajaraman SP, Trimble EL (2016)
India-United States dialogue on traditional medicine: toward collaborative research and
generation of an evidence base. J Glob Oncol. https://doi.org/10.1200/JGO.17.00099.
Published online November 16, 2017
15 Ethnobotany and Recent Advances in Indian Medicinal Orchids 383
28. Anuradha V, Rao P (1994) Praemorsin, a new phenanthropyran from Acampae praemorsa.
Phytochemistry 37(3):909–910
29. Kala S, Senthilkumar S (2010) Antimicrobial activity of Acanthephippium bicolor, Lindley,
Malaysian Journal of Microbiology 6(2):140–148
30. Joshi GC, Tewari LM, Lohani N, Upreti K, Jalal JS, Tewari G (2009) Diversity of orchids
in Uttarakhand and their conservation strategy with special reference to their medicinal
importance. Rep Opin 1:47–52
31. Anuradha V, Prakash NS (1998) A phenanthropyran from Aerides crispum. Phytochemistry
48:185–186
32. Linthoingambi L, Das AK, Singh PK, Ghosh SK (2013) Medicinal uses of orchids by tribes in
India: a review. Int J Curr Res 5:2796–2798
33. Ghanaksh A, Kaushik P (1999) Antibacterial effect of Aerides multiflora Roxb. A study in
vitro. J Orchid Soc India 13:65–68
34. Majumder PL, Banerjee S, Sen S (1995) Stilbenoids from the orchids Agrostophyllum
callosum and Coelogyne flaccida. Phytochemistry 39:649–653
35. Majumder PL, Banerjee S, Sen S (1996) Three stilbenoids from the orchid Agrostophyllum
callosum. Phytochemistry 42:847–853
36. Majumder PL, Banderjee S, Lahari S, Mukhoti N, Sen S (1998) Dimeric phenanthrenes from
two Agrostophyllum species. Phytochemistry 47:855–860
37. Majumder PL, Sen S, Banerjee S (1999) Agrostophyllol and isoagrostophyllol, two novel
diastereomeric 9,10-dihydrophenanthropyran derivatives from the orchid Agrostophyllum
callosum. Tetrahedron 55:6691–6702
38. Majumdar PL, Majumder S, Sen S (2003) Triterpenoids from the orchids Agrostophyllum
breirps and Agrostophyllum callosum. Phytochemistry 62:591–596
39. Biswas A, Bari MA, Roy M, Bhadra SK (2010) Inherited folk pharmaceutical knowledge of
tribal people in the Chittagong Hill tracts Bangladesh. Indian J Tradit Knowl 9:77–89
40. Singh A, Duggal S (2009) Medicinal orchids: an overview. Ethnobot Leafl 13:351–363
41. Majumder PL, Ghosal S (1991) Arundinol, a new triterpene from the orchid Arundina
bambusifolia. J Indian Chem Soc 68:88–91
42. Majumder PL, Ghosal S (1993) Two stilbenoids from the orchid Arundina bambusifolia.
Phytochemistry 32:439–444
43. Majumder PL, Ghosal S (1994) Two stilbenoids from the orchid Arundina bambusifolia.
Phytochemistry 35:205–208
44. Ahmad N (1997) Wild flowers of Bangladesh. UPL, Dhaka
45. Liu MF, Ding Y, Zhang DM (2005) Phenanthrene constituents from rhizome of Arundina
graminifolia. Zhongguo Zhong Yao Za Zhi 30:353–356
46. Liu MF, Han Y, Xing DM, Shi Y, Xu LZ, Du LJ, Ding YI (2004) A new stilbenoid from
Arundina graminifolia. Asian Nat Prod Res 6:229–232
47. Liu MF, Han Y, Xing DM, Wang W, Xu LZ, Du LJ, Ding Y (2005) One new benzyldihy-
drophenanthrene from Arundina graminifolia. Asian Nat Prod Res 7:767–770
48. Liu M, Lv H, Ding Y (2012) Antitumoral bibenzyl derivatives from tuber of Arundina
graminifolia. Zhongguo Zhong Yao Za Zhi 37(1):66–70
49. Lalitharani S, Mohan VR, Maruthupandian A (2011) Pharmacognostic investigations on
Bulbophyllum albidum (Wight) Hook. f. Int J PharmTech Res 3:556–562
50. Saranya B, Gopalan R, Narmatha bai V, Mahendran G (2012) Rare and threatened orchid
species used for various diseases by Irulars, Vellingiri hills, Coimbatore. Int J Sci Res Publ
2:1–2
51. Kalaiarasan A, Kumar P, Ahmed John S (2012) Bio-chemical investigation of Bulbophyllum
kaitense rechib root by GC-MS. Eastern Ghats of India. Nat Sci 10:29–31
52. Kalaiarasan PK, Ahmed John S (2012) Antimicrobial activity of Bulbophyllum kaitense.
Rechib. Stem eastern penisular flora in India. J Nat Sci 10:41–44
53. Kalaiarasan A, Ahmed John S (2012) In-vitro screening for anti-inflammatory activity of
Bulbophyllum kaitense Rechib. Pseudobulb extract by HRBC method, eastern peninsular flora
in South India. Int J Sci Res Publ 2:1–5
15 Ethnobotany and Recent Advances in Indian Medicinal Orchids 385
54. Rajendran AN, Rao R, Ravi Kumar K, Henry AN (1997) Some medicinal orchids of Southern
India. Anc Sci Life 17:10–14
55. Barthel M (2008) Pharmaceutical compositions containing bulbophyllum and their use for
treating illnesses. Patent grant No. 7939112; publication date Sep 4 2008
56. Kumari P, Joshi GC, Tewari LM (2011) Diversity and status of ethno-medicinal plants of
Almora district in Uttarakhand, India. Int J Biodivers Conserv 3:298–326
57. Sharma C, Kumari T, Arya KR (2014) Ethnopharmacological survey on bone healing plants
with special references to Pholidota articulata and Coelogyne cristata (Orchidaceae) used in
folk tradition of Kumaon, Uttarakhand, India. Int J Pharma Res Health Sci 2:185–190
58. Majumder PL, Laha S, Datta N (1982) Coelonin, a 9.10-dihydrophenanthrene from the orchids
Coelogyne ochracea and Coelogyne elata. Phytochemistry 21:478–480
59. Majumder PL, Sen S, Majumder S (2001) Phenanthrene derivatives from the orchid
Coelogyne cristata. Phytochemistry 58(4):581–586
60. Lyudmyla B, Halyna T, Zbigniew O, Lyudmyla K, Oleksand G (2016) Antimicrobial activity
screening of extrants from leaves and pseudobulbs of Coelogyne cristata Lindl. (Orchidaceae).
In: The scientific proceedings of the international network AgroBioNet, agrobiodiversity for
improving nutrition, health and life quality, pp 40–44
61. Pramanick DD (2016) Pharmacognostic studies on the pseudobulb of Coelogynecristata Lindl.
(Orchidaceae)-an epiphytic orchid of ethno-medicinal importance. J Pharmacogn Phytochem
5:120–123
62. Jain SK (1991) Dictionary of Indian folk medicine and ethnobotany. Deep Publications, Lucknow
63. Agrawal DP (2005) Himalayan medicine system and its materia medica. http://www.
indianscience.org/essays.pdf
64. Dhiman AK (2004) Medicinal plants of Uttaranchal state. Chaukhambha Sanskrit Series
Office, Varanasi
65. Trivedi VP, Dixit RS, Lal VK (1980) Orchids in the drug markets of Bareilly, Kanpur and
nearby districts. Nagarjun (Calcutta) 23(8):157–163
66. Moin S, Shibu BS, Wesley PS, Devi BC (2012) Bioactive potential of Coelogyne stricta
(D. Don) Schltr: an ornamental and medicinally important orchid. J Pharm Res 5:2191–2196
67. Joseph J (1977) Quest of medicinal plants and re-establishment of their medicinal virtues. In:
Atal C, Kapur BM (eds) Cultivation and utilization of medicinal and aromatic plants. Regional
Research Laboratory, Jammu Tawi
68. Kovacs A, Vasas A, Hohmann J (2007) Natural phenanthrenes and their biological activity.
Phytochemistry 69:1084–1110
69. Howlader MA, Alam M (2011) Central nervous system depressant effects of the ethanolic
extract of Cymbidium aloifolium (L.). J Appl Pharm Sci 1:60–62
70. Juneja RK, Sharma SC, Tandon JS (1987) Two substituted bibenzyls and a
dihydrophenanthrene from Cymbidium aloifolium. Phytochemistry 26:1123–1125
71. Barua AK, Ghosh BB, Ray S, Patra A (1990) Cymbinobin-A, a phenanthraquinone from
Cymbidium aloifolium. Phytochemistry 29:3046–3047
72. Ghosh BB, Ray S, Bhattacharyya P et al (1992) Cymbinodin B, a phenanthraquinone from
Cymbidium aloifolium. Indian J Chem 31B:557–558
73. Howlader MA, Alam M, Ahmad KT, Khatun F, Apu AS (2011) Antinociceptive and anti-
inflammatory activity of the ethanolic extract of Cymbidium aloifolium (L.). Pak J Biol Sci
14:909–911
74. Radhika B, Murthy JVVSN, Grace DN (2013) Preliminary phytochemical analysis & anti-
bacterial activity against clinical pathogen of medicinally important orchid Cymbidium
aloifolium (L.) Sw. Int J Pharm Sci Res 4:3925–3931
75. Vij SP, Srivastav RC, Mainra AK (1992) On the occurrence of Dactylorhiza hatagirea
(D.Don) Soo in Sikkim. Orchid News 8–9:14–15
76. Lal B, Negi HR, Singh RD, Ahuja PS (2004) Medicinal uses of Dactylorhiza hatagirea among
the natives of higher altitudes in Western Himalaya. J Orchid Soc India 18:97–100
77. Bulpitt CJ (2005) Occasional paper: the uses and misuses of orchids in medicine. Q J Med
98:625–631
386 R. Pal et al.
78. Kala CP (2005) Indigenous uses, population density, and conservation of threatened medicinal
plants in protected areas of the Indian Himalayas. Conserv Biol 19:368–378
79. Thakur M, Dixit VK (2007) Effect of some vajikaran herbs on pendiculation activities and in
vitro sperm count in male. Sex Disabil 25:203–207
80. Ranpal S (2009) An assessment of status and antibacterial properties of Dactylorhiza
hatagirea in Annapurna Conservation Area (A case study of Paplekharka, Lete VDC,
Mustang). B. Sc. Forestry Research thesis submitted to Tribhuvan University, Institute of
Forestry, Pokhara
81. Izu HK, Aneko EK, Omimori TT (1999) Studies on Nepalese crude drugs, chemical constit-
uents of panch aunle, the roots of Dactylorhiza hatagirea D. Chem Pharm Bull 47:1618–1625
82. Rao TA, Sridhar S (2007) Wild orchids in Karnataka. A pictorial compendium, Institute of
Natural Resources Conservation, Education, Research and Training (INCERT), Bangalore
83. Nagananda GS, Satishchandra N (2013) Antimicrobial activity of cold and hot successive
pseudobulb extracts of Flickingeria nodosa (Dalz.) Seidenf. Pak J Biol Sci 16:1189–1193
84. Chakrabarty M, Datta GK, Ghosh S, Debnath PK (2001) Induction of antioxidative enzyme by
the ayurvedic herb Desmotrichum fimbriatum Bl. in mice. Indian J Exp Biol 39:485–486
85. Sudhanshu, Rao N, Mittal S, Vishal, Menghani E (2012) Antioxidant agents alternative source
for malaria disease. Indian J Appl Pharm 4:14–16
86. Medhi RP, Chakrabarti S (2009) Traditional knowledge of NE people on conservation of wild
orchids. Indian J Tradit Knowl 8:11–16
87. Maridass M, Thangavel K, Raju G (2008) Antidiabetic activity of tuber extract of Eulophia
epidendraea (Retz.) Fisher (Orchidaceae) in alloxan diabetic rats. Pharmacologyonline
3:606–617
88. Maridass M, Ramesh U (2010) Investigation of phytochemical constituents from Eulophia
epidendraea. Int J Biol Technol 1:1–7
89. Maridass M (2011) Anti diarrhoeal activity of rare orchid Eulophia epidendraea (Retz.) Fisher.
Nat Pharm Technol 1:5–10
90. Anonymous (2003) The wealth of India: a dictionary of India raw material and industrial
products. Council of Scientific and Industrial Research, New Delhi
91. Tatiya A, Surana S, Bhavsar S, Patil D, Patil Y (2012) Pharmacognostic and preliminary
phytochemical investigation of Eulophia herbacea Lindl. tubers (Orchidaceae). Asian Pac
J Trop Dis 2:S50–S55
92. Sikarwar RLS, Phathak B, Jaiswal A (2008) Some unique ethnomedical properties of tribal
communities of Chitrakoot, Madya Pradesh. Indian J Tradit Knowl 7:613–617
93. Jagdale SP, Shimpi SD, Chachad D (2009) Pharmacological studies of salep. J Herb Med
Toxicol 3:153–156
94. Ruchi KS, Upadhyaya R, Trivedi US, Tiwari S (2012) Qualitative phytochemical analysis of
Eulophia nuda Lindl. an endangered orchid. Int J Pharm Res Bio-Sci 1:456–462
95. Aberoumand A (2010) Evaluating potential values of phytate, trypsin inhibitors and
polyphenols in plants based diets. Pak J Food Sci 20:50–53
96. Nadkarni N (1976) Indian natural medical, vol 1. Popular Prakashan, Bombay
97. Meena AK, Rao MM (2009) Folk herbal medicines used by the Meena community in
Ali Aberoumand and S.S. Deokule proximate and mineral composition of wild coco (Eulophia
ochreata L.) tubers in Iran. J Food Agro-Ind 2:203–209
98. Jagtap S, Gilda S, Bhondave P, Paradkar A, Pawar P, Harsulkar A (2009) Validation of the
potential of Eulophia ochreata L. tubers for its anti-inflammatory and antioxidant activity.
Pharmacologyonline 2:307–316
99. Maridass M, Victor B, Ramesh U (2005) Ethnobotanical information of Eulophia epidendraea
(Retz) Fischer (Orchidaceae) in the Kambli Malaikovil Forest, Tirunelveli district, Tamil
Nadu. Bombay Nat Hist Soc 102:255–257
100. Jain A, Katewa S, Galav P, Ambika N (2007) Unrecorded ethnomedicinal uses of biodiversity
from Tadgarh-Raoli wild life sanctuary, Rajasthan, India. Acta Bot Yunnanic 29:337–344
15 Ethnobotany and Recent Advances in Indian Medicinal Orchids 387
101. Aswal BS (1992) Less known medicinal uses of three plants from Kumaun Himalaya (India)
Indian. J For 15:76–77
102. Chauhan NS (1990) Medicinal orchids of Himachal Pradesh. J Orchid Soc India 4:99–105
103. Dey AC (1982) Indian medicinal plants used in Ayurvedic preparations. Bishan Singh
Mahender Pal Singh, Dehradun
104. Habbu PV, Smita DM, Mahadevan KM, Shastry RA, Biradar SM (2012) Protective effect of
Habenaria intermedia tubers against acute and chronic physical and psychological stress
paradigs in rats. Braz J Pharmacogn 22:568–579
105. Pérez Gutiérrez RM (2010) Orchids: a review of uses in traditional medicine, its phytochem-
istry and pharmacology. J Med Plants Res 4:592–638
106. Chinmay R, Kumari S, Bishnupriya D, Mohanty RC, Dixit R, Padhi MM, Babu R (2011)
Phyto-pharmacognostical studies of two endangered species of Malaxis (Jeevak and
Rishibhak). Pharmacogn J 3:77–85
107. Bhatnagar JK, Handa SS, Duggal SC (1970) Chemical investigation on Microstylis wallichii.
Planta Med 20:157–161
108. Chauhan RS, Nautiyal MO, Prasad P, Purohit H (2008) Ecological features of an endangered
medicinal orchid – Malaxis muscifera (Lindley) Kuntze in Western Himalaya. McAllen Int
Orchid Soc J 9:8–12
109. Sarkar PK, Agrawal VV (1978) Notes on Pholidota pallida Lindl. and its use in Ranchi district
Bihar. Bull Bot Surv India 20:182–183
110. Nadkarni AK (1954) Dr. K.M. Nadkarni’s Indian materia medica, vol 2, 3rd edn. Popular Book
Depot, Bombay
111. Bi ZM, Wang ZT, Xu LS, Xu GJ (2004) Studies on chemical constituents of Pholidota
yunnanensis. Zhongguo Zhong Yao Za Zhi 29:47–49
112. Guo XY, Wang J, Wang NL, Kitanaka S, Liu HW, Yao XS (2006) New stilbenoids from
Pholidota yunnanensis and their inhibitory effects on nitric oxide production. Chem Pharm
Bull (Tokyo) 54:21–25
113. Al-Amin M, Sultana GNN, Hossain CF (2012) Analgesic and anti-inflammatory activities of
Rhynchostylis retusa. Biol Med 3:55–59
114. Kirtikar PK, Basu BD (1975) Indian medicinal plants, 2nd edn. (Reprint Ed. 1975). M/
s Bishen Singh MahendraPal Singh, Dehradun
115. Usman MRB, Chhaya G, Surekha CG, Lalit S, Samadhan B (2012) Pharmacognostical
Evaluations of Some Herbal Plant. IJARPB 2(3):302–310
116. Kumar PKS, Subramoniam A, Pushpangadan P (2000) Aphrodisiac activity of Vanda
tessellata (Roxb.) Hook.Ex Don extract in male mice Indian. J Pharmacol 32:300–304
117. Ahmad F, Sayeed A, Islam A, Abdus Salam SM, Sadik G, Sattar MA, Astaq Mohal
Khan GRM (2002) Antimicrobial activity of extracts a glycoside from Vanda roxburghii R
Br. Pak J Biol Sci 5:189–191
118. Kumar S (2002) The medicinal plants of North-East India. Scientific Publishers, Jodhpur
119. Singh MP, Dey S (2005) Indian medicinal plants. Satish Serial Publishing House, Delhi
120. Reddy CS, Pattanaik C, Murthy MSR, Reddy KN (2005) Orchids of Eastern Ghats, India.
EPTRI-ENVIS Newslett 11:6–12
Traditionally Used Medicinal Dendrobium:
A Promising Source of Active Anticancer 16
Constituents
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.
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
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.
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.
Fig. 2 (continued)
394 M. R. Paudel et al.
Growth Inhibition ð%Þ ¼ 100 ðTotal cells Dead cellsÞ=Total cells 100
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.
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.
to day first – 0 weight, (ii) median survival time and increase in lifespan (%ILS), and
(iii) hematological parameters [12, 26].
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.
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)
Compound 28 A549, HL-60 cell lines 97.3%, 55.7%
(104 mol L1)
M. R. Paudel et al.
16
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
404 M. R. Paudel et al.
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
16 Traditionally Used Medicinal Dendrobium: A Promising Source of. . . 405
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].
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
408 M. R. Paudel et al.
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)
16 Traditionally Used Medicinal Dendrobium: A Promising Source of. . . 409
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.
References
1. Hossain MM (2011) Therapeutic orchids: traditional uses and recent advances – an overview.
Fitoterapia 82:102–140
2. Bulpitt CJ, Li Y, Bulpitt PF, Wang J (2007) The use of orchids in Chinese medicine. J R Soc
Med 100:558–563
3. Gutiérrez RMP (2010) Orchids: a review of uses in traditional medicine, its phytochemistry
and pharmacology. J Med Plants Res 4:592–638
4. Bulpitt CJ (2005) The uses and misuses of orchids in medicine. Q J Med 98:625–631
5. Ng TB, Liu J, Wong JH, Ye X, Sze SCW, Tong Y, Zhang KY (2012) Review of research on
Dendrobium, a prized folk medicine. Appl Microbiol Biotechnol 93:1795–1803
6. Xu J, Han QB, Li SL, Chen XJ, Wang XN, Zhao ZZ, Chen HB (2013) Chemistry, bioactivity
and quality control of Dendrobium, a commonly used tonic herb in traditional Chinese
medicine. Phytochem Rev 12:341–367
7. da Silva JAT, Ng TB (2017) The medicinal and pharmaceutical importance of Dendrobium
species. Appl Microbiol Biotechnol 101:2227–2239
8. Lam Y, Ng TB, Yao RM, Shi J, Xu K, Sze SCW, Zhang KY (2015) Evaluation of chemical
constituents and important mechanism of pharmacological biology in Dendrobium plants.
Evid Based Complement Alternat Med 2015:841752
410 M. R. Paudel et al.
9. Stanaway JD, Afshin A, Gakidou E, Lim SS, Abate D, Abate KH, Abbafati C, Abbasi N,
Abbastabar H, Abd-Allah F (2018) Global, regional, and national comparative risk assessment
of 84 behavioural, environmental and occupational, and metabolic risks or clusters of risks for
195 countries and territories, 1990–2017: a systematic analysis for the Global Burden of
Disease Study 2017. Lancet 392:1923–1994
10. Siegel RL, Miller KD, Jemal A (2019) Cancer statistics, 2019. CA Cancer J Clin 69:7–34
11. Millimouno FM, Dong J, Yang L, Li J, Li X (2014) Targeting apoptosis pathways in cancer
and perspectives with natural compounds from mother nature. Cancer Prev Res 7:1081–1107
12. Gordon JL, Brown MA, Reynolds MM (2018) Cell-based methods for determination of
efficacy for candidate therapeutics in the clinical Management of Cancer. Diseases 6:85
13. Bray F, Ferlay J, Soerjomataram I, Siegel RL, Torre LA, Jemal A (2018) Global cancer
statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers
in 185 countries. CA Cancer J Clin 68:394–424
14. Guilbaud N, Kraus-Berthier L, Meyer-Losic F, Malivet V, Chacun C, Jan M, Tillequin F,
Michel S, Koch M, Pfeiffer B (2001) Marked antitumor activity of a new potent acronycine
derivative in orthotopic models of human solid tumors. Clin Cancer Res 7:2573–2580
15. Greenwell M, Rahman P (2015) Medicinal plants: their use in anticancer treatment. Int
J Pharm Sci Res 6:4103–4112
16. Moraes DFC, de Mesquita LSS, do Amaral FMM, de Sousa Ribeiro MN, Malik S (2017)
Anticancer drugs from plants. In: Malik S (ed) Biotechnology and production of anti-cancer
compounds. Springer, Cham, pp 121–142
17. Hossain M, Akter S, Uddin S (2020) Screening of bioactive phytochemicals in some indig-
enous epiphytic orchids of Bangladesh. In: Khasim S, Hegde S, Gonzalez-Arnao M,
Thammasiri K (eds) Orchid biology: recent trends & challenges. Springer, Singapore,
pp 481–506
18. Cakova V, Bonte F, Lobstein A (2017) Dendrobium: sources of active ingredients to treat
age-related pathologies. Aging Dis 8:827–849
19. Kierkegaard P, Pilotto AM, Leander K (1970) The constitution and relative configuration of
crepidine, an alkaloid from Dendrobium crepidatum Lindl. Acta Chem Scand 24:3757–3759
20. Majumder PL, Guha S, Sen S (1999) Bibenzyl derivatives from the orchid Dendrobium
amoenum. Phytochemistry 52:1365–1369
21. Li Y, Wang CL, Wang YJ, Wang FF, Guo SX, Yang JS, Xiao PG (2009) Four new bibenzyl
derivatives from Dendrobium candidum. Chem Pharm Bull 57:997–999
22. Ren G, Deng WZ, Xie YF, Wu CH, Li WY, Xiao CY, Chen YL (2020) Bibenzyl derivatives
from leaves of Dendrobium officinale. Nat Prod Commun 15:1–5
23. Nam B, Ryu SM, Lee D, Jung CH, Jin CH, Kim JB, Lee IS, Han AR (2019) Identification of
two new phenanthrenes from Dendrobii Herba and their cytotoxicity towards human hypo-
pharynx squamous carcinoma cell (FaDu). Molecules 24:2339
24. Tang H, Zhao T, Sheng Y, Zheng T, Fu L, Zhang Y (2017) Dendrobium officinale Kimura et
Migo: a review on its ethnopharmacology, phytochemistry, pharmacology, and industrializa-
tion. Evid Based Complement Alternat Med 2017:7436259
25. Sut S, Maggi F, Dall’Acqua S (2017) Bioactive secondary metabolites from orchids
(Orchidaceae). Chem Biodivers 14:e1700172
26. Ediriweera MK, Tennekoon KH, Samarakoon SR (2019) In vitro assays and techniques
utilized in anticancer drug discovery. J Appl Toxicol 39:38–71
27. Galluzzi L, Bravo-San Pedro JM, Vitale I, Aaronson SA, Abrams JM, Adam D, Alnemri ES,
Altucci L, Andrews D, Annicchiarico-Petruzzelli M et al (2015) Essential versus accessory
aspects of cell death: recommendations of the NCCD 2015. Cell Death Differ 22:58–73
28. Gali-Muhtasib H, Hmadi R, Kareh M, Tohme R, Darwiche N (2015) Cell death mechanisms of
plant-derived anticancer drugs: beyond apoptosis. Apoptosis 20:1531–1562
29. Montero J, Sarosiek KA, DeAngelo JD, Maertens O, Ryan J, Ercan D, Piao H, Horowitz NS,
Berkowitz RS, Matulonis U et al (2015) Drug-induced death signaling strategy rapidly redicts
cancer response to chemotherapy. Cell 160:977–989
16 Traditionally Used Medicinal Dendrobium: A Promising Source of. . . 411
30. Liu Y, Levine B (2015) Autosis and autophagic cell death: the dark side of autophagy. Cell
Death Differ 22:367–376
31. Nimse SB, Pal D (2015) Free radicals, natural antioxidants, and their reaction mechanisms.
RSC Adv 5:27986
32. Paudel MR, Pant B (2017) Cytotoxic activity of crude extracts of Dendrobium amoenum and
detection of bioactive compounds by GC-MS. Botanica Orientalis: J Plant Sci 11:38–42
33. Klongkumnuankarn P, Busaranon K, Chanvorachote P, Sritularak B, Jongbunprasert V,
Likhitwitayawuid K (2015) Cytotoxic and antimigratory activities of phenolic compounds
from Dendrobium brymerianum. Evid Based Complement Alternat Med 2015:350410
34. Zheng Q, Qiu D, Liu X, Zhang L, Cai S, Zhang X (2015) Antiproliferative effect of
Dendrobium catenatum Lindley polypeptides against human liver, gastric and breast cancer
cell lines. Food Funct 6:1489–1495
35. Paudel MR, Chand MB, Pant B, Pant B (2019) Assessment of antioxidant and cytotoxic
activities of extracts of Dendrobium crepidatum. Biomol 9:478
36. Prasad R, Koch B (2016) In vitro anticancer activities of ethanolic extracts of Dendrobium
crepidatum and Dendrobium chrysanthum against T-cell lymphoma. J Cytol Histol 7:432
37. Chen Y, Li Y, Qing C, Zhang Y, Wang L, Liu Y (2008) 1,4,5-Trihydroxy-7-methoxy-9H-
fluoren-9-one, a new cytotoxic compound from Dendrobium chrysotoxum. Food Chem
108:973–976
38. Hu J, Fan W, Dong F, Miao Z, Zhou J (2012) Chemical components of Dendrobium
chrysotoxum. Chin J Chem 30:1327–1330
39. Li M, He Y, Peng C, Xie X, Hu G (2018) Erianin inhibits human cervical cancer cell through
regulation of tumor protein p53 via the extracellular signal-regulated kinase signaling pathway.
Oncol Lett 16:5006–5012
40. Wang H, Zhang T, Sun W, Wang Z, Zuo D, Zhou Z, Li S, Xu J, Yin F, Hua Y (2016) Erianin
induces G2/M-phase arrest, apoptosis, and autophagy via the ROS/JNK signaling pathway in
human osteosarcoma cells in vitro and in vivo. Cell Death Dis 7:e2247
41. Sun J, Fu X, Wang Y, Liu Y, Zhang Y, Hao T, Hu X (2016) Erianin inhibits the proliferation of
T47D cells by inhibiting cell cycles, inducing apoptosis and suppressing migration. Am
J Transl Res 8:3077–3086
42. Fan YJ, Luo AX (2011) Evaluation of anti-tumor activity of water-soluble polysaccharides
from Dendrobium denneanum. Afr J Pharm Pharmacol 5:415–420
43. Wattanathamsan O, Treesuwan S, Sritularak B, Pongrakhananon V (2018) Cypripedin, a
phenanthrenequinone from Dendrobium densiflorum, sensitizes non-small cell lung cancer
H460 cells to cisplatin-mediated apoptosis. J Nat Med 72:503–513
44. Charoenrungruang S, Chanvorachote P, Sritularak B, Pongrakhananon V (2014) Gigantol, a
bibenzyl from Dendrobium draconis, inhibits the migratory behavior of non-small cell lung
cancer cells. J Nat Prod 77:1359–1366
45. Charoenrungruang S, Chanvorachote P, Sritularak B, Pongrakhananon V (2014) Gigantol-
induced apoptosis in lung cancer cell through mitochondrial-dependent pathway. Thai J Pharm
Sci 38:67–73
46. Unahabhokha T, Chanvorachote P, Sritularak B, Kitsongsermthon J, Pongrakhananon V
(2016) Gigantol inhibits epithelial to mesenchymal process in human lung cancer cells. Evid
Based Complement Alternat Med 2016:4561674
47. Unahabhokha T, Chanvorachote P, Pongrakhananon V (2016) The attenuation of epithelial to
mesenchymal transition and induction of anoikis by gigantol in human lung cancer H460 cells.
Tumor Biol 37:8633–8641
48. Hlosrichok A, Sumkhemthong S, Sritularak B, Chanvorachote P, Chaotham C (2018) A
bibenzyl from Dendrobium ellipsophyllum induces apoptosis in human lung cancer cells.
J Nat Med 72:615–625
49. Chaotham C, Pongrakhananon V, Sritularak B, Chanvorachote P (2014) A bibenzyl from
Dendrobium ellipsophyllum inhibits epithelial-to-mesenchymal transition and sensitizes lung
cancer cells to anoikis. Anticancer Res 34:1931–1938
412 M. R. Paudel et al.
70. Hsu JL, Lee YJ, Leu WJ, Dong YS, Pan SL, Uang BJ, Guh JH (2014) Moniliformediquinone
induces in vitro and in vivo antitumor activity through glutathione involved DNA damage
response and mitochondrial stress in human hormone refractory prostate cancer. J Urol
191:1429–1438
71. Tseng TH, Lin WL, Chen ZH, Lee YJ, Shie MS, Lee KF, Shen CH, Kuo HC (2016)
Moniliformediquinone as a potential therapeutic agent, inactivation of hepatic stellate cell
and inhibition of liver fibrosis in vivo. J Transl Med 14:263
72. Lu TL, Han CK, Chang YS, Lu TJ, Huang HC, Bao BY, Wu HY, Huang CH, Li CY, Wu TS
(2014) Denbinobin, a phenanthrene from Dendrobium nobile, impairs prostate cancer migra-
tion by inhibiting Rac1 activity. Am J Chin Med 42:1539–1554
73. Song JI, Kang YJ, Yong HY, Kim YC, Moon A (2012) Denbinobin, a phenanthrene from
Dendrobium nobile, inhibits invasion and induces apoptosis in SNU-484 human gastric cancer
cells. Oncol Rep 27:813–818
74. Luo AX, Fan YJ (2011) Immune stimulating activity of water-soluble polysaccharide fractions
from Dendrobium nobile Lindl. Afr J Pharm Pharmacol 5:625–631
75. Wang JH, Luo JP, Zha XQ, Feng BJ (2010) Comparison of antitumor activities of different
polysaccharide fractions from the stems of Dendrobium nobile Lindl. Carbohydr Polym
79:114–118
76. Zhou XM, Zheng CJ, Wu JT, Chen GY, Chen J, Sun CG (2016) Five new lactone derivatives
from the stems of Dendrobium nobile. Fitoterapia 115:96–100
77. Zhou XM, Zheng CJ, Gan LS, Chen GY, Zhang XP, Song XP, Li GN, Sun CG (2016)
Bioactive phenanthrene and bibenzyl derivatives from the stems of Dendrobium nobile.
J Nat Prod 79:1791–1797
78. Zhang L, Wang F, Ren X (2018) Inhibitory effect of Dendrobium officinale polysaccharide on
human gastric cancer cell xenografts in nude mice. Food Sci Technol 38:78–83
79. Zhao Y, Liu Y, Lan XM, Xu GL, Sun YZ, Li F, Liu HN (2016) Effect of Dendrobium officinale
extraction on gastric carcinogenesis in rats. Evid Based Complement Alternat Med 2016:1213090
80. Hu JM, Zhao YX, Miao ZH, Zhou J (2009) Chemical components of Dendrobium poly-
anthum. Bull Kor Chem Soc 30:2098–2100
81. Bhummaphan N, Pongrakhananon V, Sritularak B, Chanvorachote P (2018) Cancer stem cell–
suppressing activity of chrysotoxine, a bibenzyl from Dendrobium pulchellum. J Pharmacol
Exp Ther 364:332–346
82. Chanvorachote P, Kowitdamrong A, Ruanghirun T, Sritularak B, Mungmee C, Likhitwi-
tayawuid K (2013) Anti-metastatic activities of bibenzyls from Dendrobium pulchellum. Nat
Prod Commun 8:115–118
83. Mittraphab A, Muangnoi C, Likhitwitayawuid K, Rojsitthisak P, Sritularak B (2016) A new
bibenzyl-phenanthrene derivative from Dendrobium signatum and its cytotoxic activity. Nat
Prod Commun 11:657–659
84. Chimsook T (2016) Phytochemical screening, total phenolic content, antioxidant activities and
cytotoxicity of Dendrobium signatum Leaves. MATEC Web Conf 62:03005
85. Chen XJ, Mei WL, Cai CH, Guo ZK, Song XQ, Dai HF (2014) Four new bibenzyl derivatives
from Dendrobium sinense. Phytochem Lett 9:107–112
86. Weng HY, Hsu MJ, Chen CC, Chen BC, Hong CY, Teng CM, Pan SL, Chiu WT, Lin CH
(2013) Denbinobin induces human glioblastoma multiforme cell apoptosis through the IKKα–
Akt–FKHR signaling cascade. Eur J Pharmacol 698:103–109
87. Kuo CT, Hsu MJ, Chen BC, Chen CC, Teng CM, Pan SL, Lin CH (2008) Denbinobin induces
apoptosis in human lung adenocarcinoma cells via Akt inactivation, Bad activation, and
mitochondrial dysfunction. Toxicol Lett 177:48–58
88. Chen TH, Pan SL, Guh JH, Chen CC, Huang YT, Pai HC, Teng CM (2008) Denbinobin
induces apoptosis by apoptosis-inducing factor releasing and DNA damage in human colo-
rectal cancer HCT-116 cells. Naunyn Schmiedeberg's Arch Pharmacol 378:447–457
89. Chanvorachote P, Chamni S, Ninsontia C, Phiboonchaiyanan PP (2016) Potential anti-
metastasis natural compounds for lung cancer. Anticancer Res 36:5707–5717
414 M. R. Paudel et al.
90. Hassan M, Watari H, AbuAlmaaty A, Obha Y, Sakuragi N (2014) Apoptosis and molecular
targeting therapy in cancer. Biomed Res Int 2014:150845
91. Pai HC, Chang LH, Peng CY, Chang YL, Chen CC, Shen CC, Teng CM, Pan SL (2013)
Moscatilin inhibits migration and metastasis of human breast cancer MDA-MB-231 cells
through inhibition of Akt and Twist signaling pathway. J Mol Med 91:347–356
92. Nishida N, Yano H, Nishida T, Kamura T, Kojiro M (2006) Angiogenesis in cancer. Vasc
Health Risk Manag 2:213–219
93. Guan L, Zhou J, Lin Q, Zhu H, Liu W, Liu B, Zhang Y, Zhang J, Gao J, Feng F (2019) Design,
synthesis and antitumour and anti-angiogenesis evaluation of 22 moscatilin derivatives.
Bioorg Med Chem 27:2657–2665
94. Su C, Zhang P, Liu J, Cao Y (2017) Erianin inhibits indoleamine 2, 3-dioxygenase–induced
tumor angiogenesis. Biomed Pharmacother 88:521–528
95. Gong YQ, Fan Y, Wu DZ, Yang H, Hu ZB, Wang ZT (2004) In vivo and in vitro evaluation of
erianin, a novel anti-angiogenic agent. Eur J Cancer 40:1554–1565
96. Yu Z, Zhang T, Gong C, Sheng Y, Lu B, Zhou L, Ji L, Wang Z (2016) Erianin inhibits high
glucose-induced retinal angiogenesis via blocking ERK1/2-regulated HIF-1α-VEGF/
VEGFR2 signaling pathway. Sci Rep 6:34306
97. da Silva JAT, Cardoso JC, Dobránszki J, Zeng SJ (2015) Dendrobium micropropagation: a
review. Plant Cell Rep 34:671–704
98. Pant B, Paudel MR, Chand MB, Pradhan S, Malla BB, Raskoti BB (2018) Orchid diversity in
two community forests of Makawanpur District, Central Nepal. J Threat Taxa 10:12523–12530
99. Pant B (2013) Medicinal orchids and their uses: tissue culture a potential alternative for
conservation. African J Plant Sci 7:448–467
100. Pant B, Pradhan S, Paudel MR, Shah S, Pandey S, Joshi PR (2019) Various culture techniques
for the mass propagation of medicinal orchids from Nepal. Acta Hortic 1262:109–124
101. Pant B (2014) Application of plant cell and tissue culture for the production of phytochemicals
in medicinal plants. In: Adhikari R, Thapa S (eds) Infectious diseases and nanomedicine
II. Springer, India, pp 25–39
102. Espinosa-Leal CA, Puente-Garza CA, García-Lara S (2018) In vitro plant tissue culture: means
for production of biological active compounds. Planta 248:1–18
103. Wang HQ, Jin MY, Paek KY, Piao XC, Lian ML (2016) An efficient strategy for enhancement
of bioactive compounds by protocorm-like body culture of Dendrobium candidum. Ind Crop
Prod 84:121–130
104. Cui HY, Murthy HN, Moh SH, Cui YY, Paek KY (2015) Establishment of protocorm
suspension cultures of Dendrobium candidum for the production of bioactive compounds.
Hortic Environ Biotechnol 56:114–122
105. Cui HY, Murthy HN, Moh SH, Cui YY, Lee EJ, Paek KY (2014) Production of biomass and
bioactive compounds in protocorm cultures of Dendrobium candidum Wall ex Lindl. using
balloon type bubble bioreactors. Ind Crop Prod 53:28–33
106. Bhattacharyya P, Kumaria S, Diengdoh R, Tandon P (2014) Genetic stability and phytochem-
ical analysis of the in vitro regenerated plants of Dendrobium nobile Lindl., an endangered
medicinal orchid. Meta Gene 2:489–504
107. Zheng YP, Jiang W, Liao FL, Lu HF (2012) Optimization of light quality for production of
alkaloid and polysaccharide in Dendrobium candidum Wall. ex Lindl. J Med Plants Res
6:560–565
108. Park SY, Ho TT, Paek KY (2020) Medicinal orchids: production of bioactive compounds and
biomass. In: Khasim S, Hegde S, Gonzalez-Arnao M, Thammasiri K (eds) Orchid biology:
recent trends & challenges. Springer, Singapore, pp 439–450
109. Murthy HN, Paek KY, Park SY (2018) Micropropagation of orchids by using bioreactor
technology. In: Lee YI, Yeung ECT (eds) Orchid propagation: from laboratories to green-
houses – methods and protocols. Springer, Germany, pp 195–208
110. Paudel MR (2019) Biological activities of in vivo and in vitro plant materials of some
Dendrobium species. PhD thesis, Tribhuvan University, Nepal
A Review on Phytochemistry, Nutritional
Potential, Pharmacology, and Conservation 17
of Malaxis acuminata: An Orchid with
Rejuvenating and Vitality Strengthening
Properties
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
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
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.
hairs [9]. Anatomical similarity between rhizomes and pseudobulbs indicates that
species can be propagated from its rhizomes as well as pseudobulbs (Fig. 1).
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
17 A Review on Phytochemistry, Nutritional Potential, Pharmacology, and. . . 419
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.
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
17 A Review on Phytochemistry, Nutritional Potential, Pharmacology, and. . . 421
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.
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).
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].
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).
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].
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.
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,
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.
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.
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).
References
1. Shukla PK, Chaubey OP (2008) Threatened wild medicine plants, assessment, conservation and
managemet. Anmol Publications Pvt Ltd, New Delhi
2. Bose B, Choudhury H, Tandon P, Kumaria S (2017) Studies on secondary metabolite profiling,
anti-inflammatory potential, in vitro photoprotective and skin-aging related enzyme inhibitory
activities of Malaxis acuminata, a threatened orchid of nutraceutical importance. J Photochem
Photobiol B Biol 173:686–695
3. Polunin O, Stainton A (1977) Flowers of the Himalaya. Oxford University Press, Calcutta
4. Balkrishna A, Srivastava A, Mishra RK, Patel SP, Vashistha RK, Singh A, Jadon V, Saxena P
(2012) Astavarga plants-threatened medicinal herbs of the North-West Himalaya. Int J Med
Arom Plants 2:661–676
5. Yonzone R, Lama D, Bhujel RB, Rai S (2013) Diversity, distribution and present availability
status of Malaxis Soland. ex Sw. (Orchidaceae) in Darjeeling Himalaya of WB, India. Lifesci
Leaflet 9:18–28
6. Govindarajan R, Singh DP, Rawat AKS (2007) High performance liquid chromatographic
method for the quantification of phenolics in ‘Chyavanprash’, a potent Ayurvedic drug.
J Pharma Biomed Anal 43:527–532
430 R. Suyal et al.
7. Lohani N, Tewari LM, Joshi GC, Kumar R, Kishor K, Upreti BM (2013) Population assessment
and threat categorization of endangered medicinal orchid Malaxis acuminata D. Don. from
North-West Himalaya. Int J Conserv Sci 4:493–502
8. Chowdhary HJ, Wadhwa BM (1984) Flora of Himachal Pradesh. Botanical Survey of India,
Calcutta
9. Sharma YP, Rani J, Raina R, Bandana K (2014) New insights into the morphology of Malaxis
acuminata D Don. Int J Farm Sci 4:136–146
10. Chinmay R, Suman K, Bishnupriya D, Mohanty RC, Dixit R, Padhi MM, Babu R (2011) Phyto-
Pharmacognostical studies of two endangered species of Malaxis (Jeevak and Rishibhak).
Pharmacog J 3:77–85
11. Uma E, Rajendran R, Muthukumar T (2015) Morphology, anatomy and mycotrophy of
pseudobulb and subterranean organs in Eulophia epidendraea and Malaxis acuminata
(Epidendroideae, Orchidaceae). Flora 217:14–23
12. Bose TK, SK Bhattacharjee, P Das, Basak UC (1980) Orchids of India. Naya Prokash, Calcutta
13. Reddy CS, Pattanaik C, Murthy MSR, Reddy KN (2006) Floristic census of orchids of Eastern
Ghats, India. The Botanica 56:79–76
14. Suyal R, Bhatt ID, Rawal RS, Tewari LM (2019) Status of two threatened astavarga herbs,
Polygonatum cirrhifolium and Malaxis muscifera, in West Himalaya: conservation implica-
tions. Proc Natl Acad Sci, India Sect B: Biol Sci 2019. https://doi.org/10.1007/s40011-019-
01144-3
15. Kant R, Verma J, Thakur K (2012) Distribution pattern, survival threats and conservation of
‘Astavarga’ orchids in Himachal Pradesh, Northwest Himalaya. Plant Arch 12:165–168
16. Jalal JS, Rawat GS (2009) Habitat studies for conservation of medicinal orchids of Uttarakhand,
Western Himalaya. African J Plant Sci 3:200–204
17. Dobhal A (2016) Diversity of medicinal plants in Arakot-Khadikal forest area of district Tehri
Garhwal, Uttarakhand, India. Environ Conserv J 17:47–66
18. Bhardwaj A, Verma RK, Rana JC (2017) Phytosociology of terrestrial orchid species in
Cedrusdeodara (Roxb. ex D. Don) G. Don forest of western Himalaya, India. Environ Ecol
35:3373–3377
19. Sharma R, Martins N, Kuca K, Chaudhary A, Kabra A, Rao MM, Prajapati PK (2019)
Chyawanprash: a traditional Indian bioactive health supplement. Biomol Ther 9:161
20. Singh AP, Sandhu AS (2005) A dictionary of medicinal plants. Singhal S. Sundeep Publishers,
New Delhi
21. Chakarvarty HL (1976) Plant wealth of Iraq. A dictionary of economic plants, vol 1. Botany
Directorate, Ministry of Agriculture and Agrarian Reform (Iraq), Baghdad
22. Jalal JS, Kumar P, Pangtey YPS (2008) Ethnomedicinal orchids of Uttarakhand, western
Himalaya. Ethnobot Leaflets 12:1227–1230
23. Gaur RD (1999) Flora of district Garhwal North West Himalaya. Transmedia, Srinagar
24. Jadhav D (2008) Medicinal plants of Madhya Pradesh and Chhattisgarh. Daya Publishing
House, Delhi
25. Rai V, Agarwal M, Khatoon S, Rawat AKS, Mehrotra S (2001) Estimation of Co and Mn in
some medicinal plants. Bull Environ Contaminat Toxicol 66:427–432
26. Aggarwal S, Zettler LW (2010) Reintroduction of an endangered terrestrial orchid, Dactylorhiza
hatagirea (D. Don) Soo, assisted by symbiotic seed germination: first report from the Indian
subcontinent. Nat Sci 8:139–145
27. Arditti J, Ernst R (1984) Physiology of germinating orchid seeds. In: Arditti J (ed) Orchid
biology: reviews and perspectives III. Cornell University Press, New York, pp 177–222
28. Subedi A, Kunwar B, Choi Y, Dai Y, van Andel T, Chaudhary RP, de Boer HJ, Gravendeel B
(2013) Collection and trade of wild-harvested orchids in Nepal. J Ethnobiol Ethnomed 9:64
29. Warrier PK, Nambiar VPK, Ramankutty C (1997) Indian medicinal plants: a compendium of
500 medicinal plants. Arya Vaidya Sala, Oriental Longman, Chennai
30. Khajuria AK, Kumar G, Bisht NS (2017) Diversity with ethnomedicinal notes on orchids: a case
study of Nagdev forest range, Pauri Garhwal, Uttarakhand, India. J Med Plant Stud 5:171–174
31. Chauhan NS (1990) Medicinal orchids of Himachal Pradesh. J Orchid Soc India 4:99–105
17 A Review on Phytochemistry, Nutritional Potential, Pharmacology, and. . . 431
32. Arora M, Singh S, Mahajan A, Sembi JK (2017) Propagation and phytochemical analysis of
Crepidium acuminatum (D. Don) Szlach. IOSR J Pharm Biol Sci 12:14–20
33. Lohani N, Tewari LM, Joshi GC, Kishor K, Kumar S, Tewari G, Joshi N (2013) Chemical
composition of Microstylis wallichii Lindl. from Western Himalaya. J Med Plant Plants Res
7:2289–2292
34. Giri L, Belwal T, Bahukhandi A, Suyal R, Bhatt ID, Rawal RS, Nandi SK (2017) Oxidative
DNA damage protective activity and anti-oxidant potential of Ashtvarga species growing in the
Indian Himalayan region. Ind Crop Prod 102:173–179
35. Rawat S, Jugran A, Giri L, Bhatt ID, Rawal RS (2011) Assessment of antioxidants properties in
fruits of Myrica esculenta: a popular wild edible species in Indian Himalayan region. Evid
Based Compl Alt Med 2011:512787
36. Bhatt ID, Dauthal P, Rawat S, Gaira K, Jugran A, Rawal RS, Dhar U (2012) Characterization of
essential oil composition, phenolic content and antioxidant properties in wild and planted
individuals of Valeriana jatamansi Jones. Sci Hort 136:61–68
37. Bahukhandi A, Rawat S, Bhatt ID, Rawal RS (2013) Influence of solvent types and source of
collection on total phenolic content and antioxidant activities of Acorus calamus L. Natl Acad
Sci Lett 36:93–99
38. Suyal R, Rawat S, Rawal RS, Bhatt ID (2019) Variability in morphology, phytochemicals and
antioxidants in Polygonatum verticillatum (L.) All. populations under different ecological
habitats in Himalaya. Environ Monit Assess 191-S:783
39. Arora M, Kaur G, Singh S, Mahajan A, Sembi JK (2019) Quantification of phytochemicals in
the pseudobulbs of Crepidium acuminatum (D. Don) Szlach-a critically endangered medicinal
plant. Curr Trend Biotech Pharm 13:366–375
40. Whitney E, Rolfes SR (2008) Understanding nutrition, 11th edn. Thomson Wadsworth,
California
41. Calder PC (2004) n 3 Fatty acids, inflammation, and immunity—relevance to postsurgical and
critically III patients. Lipids 39:1147–1161
42. Finkemeier I, König AC, Heard W, Nunes-Nesi A, Pham PA, Leister D et al (2013) Trans-
criptomic analysis of the role of carboxylic acids in metabolite signaling in Arabidopsis leaves.
Plant Physiol 162:239–253
43. De Angeli A, Zhang J, Meyer S, Martinoia E (2013) AtALMT9 is a malate-activated vacuolar
chloride channel required for stomatal opening in Arabidopsis. Nat Commun 4:1804
44. Dyson BC, Miller MA, Feil R, Rattray N, Bowsher C, Goodacre R et al (2016) FUM2, a
cytosolic fumarase, is essential for acclimation to low temperature in Arabidopsis thaliana. Plant
Physiol 172:118–127
45. Drincovich MF, Voll LM, Maurino VG (2016) On the diversity of roles of organic acids. Front
Plant Sci 7:1592
46. Rajurkar NS, Gaikwad KN (2014) Identification and quantification of amino acids from
medicinally important plants by using high-performance thin-layer chromatogramphy. J Liq
Chromat Relat Technol 37:2197–2205
47. Singh D, Kumar S, Pandey R, Hasanain M, Sarkar J, Kumar B (2017) Bioguided chemical
characterization of the antiproliferative fraction of edible pseudo bulbs of Malaxis acuminata D.
Don by HPLC-ESI-QTOF-MS. Med Chem Res 26:3307–3314
48. Raval SS, Vaghela PG, Mandavia MK, Golakiya BA (2016) Phytochemical analysis of Malaxis
acuminata D. Don (Jeevak), an ingredient of Jeevaniya Group of Ashtavarga. Indian J Agric
Biochem 29:155–160
49. Rawat S, Bhatt ID, Rawal RS (2011) Total phenolic compounds and antioxidant potential of
Hedychium spicatum Buch. Ham. ex D. Don in west Himalaya, India. J Food Compos Anal
24:574–579
50. Bhatt ID, Rawat S, Rawal RS (2013) Antioxidants in medicinal plants. In: Chandra S, Lata H,
Verma A (eds) Biotechnology for medicinal plants. Springer-Verlag, Berlin/Heidelberg, pp
296–326
51. Garg P, Aggarwal P, Sharma P, Sharma S (2012) Antioxidant activity of the butanol extract of
Malaxis acuminata (Jeevak). J Pharm Res 5:2888–2288
432 R. Suyal et al.
52. Bose B, Kumaria S, Choudhury H, Tandon P (2017) Insights into nuclear DNA content,
hydrogen peroxide and antioxidative enzyme activities during transverse thin cell layer organ-
ogenesis and ex vitro acclimatization of Malaxis wallichii, a threatened medicinal orchid.
Physiol Mol Biol Plants 23:955–968
53. Oikarinen A (2004) Connective tissue and aging. Int J Cosm Sci 26:107–107
54. Amaro-Ortiz A, Yan BD, Orazio JA (2014) Ultraviolet radiation, aging and the skin: prevention
of damage by topical cAMP manipulation. Molecules 19:6202–6219
55. Chung HY, Kim DH, Lee EK, Chung KW, Chung S, Lee B et al (2019) Redefining chronic
inflammation in aging and age-related diseases: proposal of the Seno-inflammation concept.
Aging Dis 10:367–382
56. Sunitha K, Suresh P, Santhosh MS, Hemshekhar M, Thushara RM, Marathe GK,
Thirunavukkarasu C, Kemparaju K, Kumar SM, Girish KS (2013) Inhibition of hyaluronidase
by N-acetyl cysteine and glutathione: role of thiol group in hyaluronan protection. Int J Biol
Macromol 55:39–46
57. Bharal A, Kashyap M, Sohpal VK, Sembi JK (2014) Evaluation of antimicrobial properties of
terrestrial orchids (collected from northern Himalayas) against certain human pathogens. Int J
Bioassays 3:3036–3039
58. Arora M, Kaur G, Kahlon PS, Mahajan A, Sembi JK (2017) Pharmacognostic evaluation and
antimicrobial activity of endangered ethnomedicinal plant Crepidium acuminatum (D. Don)
Szlach. Pharmacog J 9:S56–S63
59. Sharma P, Nipun M, Pankaj G, Gurukirpal S, Sumit D, Sakshi S (2011) Malaxis acuminata: a
review. Int J Res Ayurveda Pharm 2:422–425
60. Tamta BP, Sharma AK, Puni L, Singh A (2015) Propagation and conservation of endangered
orchid Microstylis wallichii Syn Malaxis acuminata (Jeevak) in its natural habitats of
Uttarakhand Himalayas. Int J Sci Technol 4:424–432
61. Arenmongla T, Deb CR (2012) Germination of immature embryos and multiplication of
Malaxis acuminata D. Don, an endangered therapeutically important orchid, by asymbiotic
culture in vitro. Indian J Biotechnol 11:464–469
62. Cheruvathur MK, Abraham J, Mani B, Thomas TD (2010) Adventitious shoot induction from
cultured internodal explants of Malaxis acuminata D. Don, a valuable terrestrial medicinal
orchid. Plant Cell Tissue Org Cult 101:163–170
63. Kaur S, Bhutani KK (2010) Micropropagation of Malaxis acuminata D. Don: a rare orchid of
high therapeutic value. J Med Arom Plants 1:29
64. Arenmongla T, Deb CR (2012) Studies on in vitro morphogenetic potential of nodal segments
of Malaxis acuminata D. Don. Appl Biol Res 14:156–163
65. Giri L, Jugran A, Rawat S, Dhyani P, Andola H, Bhatt ID, Rawal RS, Dhar U (2012) In vitro
propagation, genetic and phytochemical assessment of Habenaria edgeworthii: an important
Astavarga plant. Acta Physiol Plant 34:869–875
66. Mishra J, Bhandari H, Singh M, Rawat S, Agnihotri RK, Mishra S, Purohit S (2011) Hairy root
culture of Picrorhiza kurroa Royle ex Benth.: a promising approach for the production of
picrotin and picrotoxinin. Acta Physiol Plant 33:1841–1846
67. Giri L, Dhyani P, Rawat S, Bhatt ID, Nandi SK, Rawal RS, Pande V (2012) In vitro production
of phenolic compounds and antioxidant activity in callus suspension cultures of Habenaria
edgeworthii: a rare Himalayan medicinal orchid. Ind Crop Prod 39:1–6
68. Badhani A, Rawat S, Bhatt ID, Rawal RS (2015) Variation in chemical constituents and
antioxidant activity in yellow Himalayan (Rubus ellipticus smith) and hill raspberry (Rubus
niveus Thunb.). J Food Biochem 39:663–372
69. Rawat S, Bhatt ID, Rawal RS (2017) Geographical and environmental variation in chemical
constituents and antioxidant properties in Roscoea procera wall. J Food Biochem 41:e12302
70. Jugran AK, Bhatt ID, Rawal RS, Rawat S (2020) Essential oil composition, phenolics and
antioxidant activities of Valeriana jatamasi Jones (Caprifoliaceae) at different phenological
stages. Plant Biosyst. (in press)
17 A Review on Phytochemistry, Nutritional Potential, Pharmacology, and. . . 433
71. Sharma H, Kumar P, Singh A, Aggarwal K, Roy J, Sharma V, Rawat S (2020) Development of
polymorphic EST-SSR markers and their applicability in genetic diversity evaluation in Rho-
dodendron arboreum. Mol Biol Rep 47:2447–2457
72. Sharma H, Bhandawat A, Rawat S (2020) Cross-transferability of SSR markers developed in
Rhododendron species of Himalaya. Mol Biol Rep 47.: (in press). https://doi.org/10.1007/
s11033-020-05606-0
73. Dhyani P, Sharma B, Singh P, Masand M, Seth R, Sharma RK (2020) Genome-wide discovery
of microsatellite markers and, population genetic diversity inferences revealed high anthropo-
genic pressure on endemic populations of Trillium govanianum. Ind Crop Prod 154:112698
Phytochemistry, Pharmacology,
and Conservation of Ansellia africana: 18
A Vulnerable Medicinal Orchid of Africa
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
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
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
438 P. Bhattacharyya et al.
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.
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.
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
18 Phytochemistry, Pharmacology, and Conservation of Ansellia. . . 439
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
18 Phytochemistry, Pharmacology, and Conservation of Ansellia. . . 441
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).
442 P. Bhattacharyya et al.
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.
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
18 Phytochemistry, Pharmacology, and Conservation of Ansellia. . . 443
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).
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.
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
18 Phytochemistry, Pharmacology, and Conservation of Ansellia. . . 445
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].
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.
18 Phytochemistry, Pharmacology, and Conservation of Ansellia. . . 449
References
1. Bhattacharyya P, Van Staden J (2016) Ansellia africana (Leopard orchid): a medicinal orchid
species with untapped reserves of important biomolecules—a mini review. S Afr J Bot
106:181–185
2. Chinsamy M, Finnie JF, Van Staden J (2011) The ethnobotany of South African medicinal
orchids. S Afr J Bot 77:2–9
3. Hutchings A, Scott AH, Lewis G, Cunningham A (1996) Zulu medicinal plants: an inventory.
University of Natal Press, Scottsville
4. Chinsamy M, Finnie JF, Van Staden J (2014) Anti-inflammatory, antioxidant, anti-
cholinesterase activity and mutagenicity of South African medicinal orchids. S Afr J Bot
91:88–98
5. Hossain MM (2011) Therapeutic orchids: traditional uses and recent advances—an overview.
Fitoterapia 82:102–140
6. Germishuizen G, Meyer NL (2003) Plants of southern Africa: an annotated checklist. National
Botanical Institute, Pretoria
7. Vasudevan R, Van Staden J (2011) Cytokinin and explant types influence in vitro plant
regeneration of leopard orchid (Ansellia africana Lindl.). Plant Cell Tissue Organ Cult
107:123–129
8. Gelfand M (1985) The traditional medical practitioner in Zimbabwe: his principles of practice
and pharmacopoeia. Mambo Press, Gweru
9. Vasudevan R, Van Staden J (2010) In vitro asymbiotic seed germination and seedling growth of
Ansellia africana Lindl. Sci Hortic (Amsterdam) 123:496–504
10. Tandon P, Sharma M, Swarup R (2005) Biodiversity: status and prospects. Narosa Publishing
House, New Delhi
11. Deb CR, Tandon P (2004) Establishment of an embryogenic suspension culture of Pinus kesiya
(Khasi pine) from various explants. Indian J Biotechnol 3:445–448
12. Tandon P, Kumaria S (1998) Threats to plant diversity in high altitude of North-East India and
conservation of rare and endangered plants using biotechnological approaches. In: Science at
High Altitude. Proceedings of the National Symposium, Allied Publishers Ltd, India, pp 140–147
13. Paul S, Kumaria S, Tandon P (2012) An effective nutrient medium for asymbiotic seed
germination and large-scale in vitro regeneration of Dendrobium hookerianum, a threatened
orchid of northeast India:1–7. https://doi.org/10.1093/aobpla/plr032
14. Papenfus HB, Naidoo D, Pošta M et al (2015) The effects of smoke derivatives on in vitro seed
germination and development of the leopard orchid Ansellia africana. Plant Biol 18:289–294
15. Ngoroyemoto N, Gupta S, Kulkarni MG et al (2019) Effect of organic biostimulants on the
growth and biochemical composition of Amaranthus hybridus L. S Afr J Bot 124:87–93
16. Gupta S, Chaturvedi P, Kulkarni MG, Van Staden J (2020) A critical review on exploiting the
pharmaceutical potential of plant endophytic fungi. Biotechnol Adv 39:107462
17. Bhattacharyya P, Kumar V, Van Staden J (2017) Assessment of genetic stability amongst
micropropagated Ansellia africana, a vulnerable medicinal orchid species of Africa using
SCoT markers. S Afr J Bot 108:294–302
18. Ara H, Jaiswal U, Jaiswal VS (2000) Synthetic seed: prospects and limitations. 78:1438–1444
19. Gantait S, Bustam S, Sinniah UR (2012) Alginate-encapsulation, short-term storage and plant
regeneration from protocorm-like bodies of Aranda Wan Chark Kuan ‘Blue’ ?? Vanda coerulea
Grifft. ex. Lindl. (Orchidaceae). Plant Growth Regul 68:303–311
20. Bhattacharyya P, Kumar V, Van Staden J (2018) In vitro encapsulation based short term storage
and assessment of genetic homogeneity in regenerated Ansellia africana (leopard orchid) using
gene targeted molecular markers. Plant Cell Tissue Organ Cult 133:299–310
21. Nadeem M, Kumar A, Nandi SK, Palni LMS (1998) Tissue culture of medicinal plants with
particular reference to Kumaun Himalaya. In: Proceedings of the workshop on Himalayan
medicinal plants-potential and prospects. Kosi-Katarmal, Almora, pp 5–7
22. Rout GR, Samantaray S, Das P (2000) In vitro manipulation and propagation of medicinal
plants. Biotechnol Adv 18:91–120
450 P. Bhattacharyya et al.
23. Verpoorte R, Contin A, Memelink J (2002) Biotechnology for the production of plant secondary
metabolites. Phytochem Rev 1:13–25
24. Yesil-Celiktas O, Nartop P, Gurel A et al (2007) Determination of phenolic content and
antioxidant activity of extracts obtained from Rosmarinus officinalis ’calli. J Plant Physiol
164:1536–1542
25. Shinde AN, Malpathak N, Fulzele DP (2010) Determination of isoflavone content and antiox-
idant activity in Psoralea corylifolia L. callus cultures. Food Chem 118:128–132
26. Deikman J, Hammer PE (1995) Induction of anthocyanin accumulation by cytokinins in
Arabidopsis thaliana. Plant Physiol 108:47–57
27. Szopa A, Ekiert H (2014) Schisandra chinensis (Turcz.) Baill. (Chinese magnolia vine) in vitro
cultures. In: Govil JN (ed) Recent progress in medicinal plants. Biotechnology and genetic
engineering II, vol 39. Studium Press LLC, Houston, pp 405–434
28. Amoo SO, Aremu AO, Van Staden J (2013) Shoot proliferation and rooting treatments
influence secondary metabolite production and antioxidant activity in tissue culture-derived
Aloe arborescens grown ex vitro. Plant Growth Regul 70:115–122
29. Tsay HS, Gau TG, Chen CC (1989) Rapid clonal propagation of Pinellia ternata by tissue
culture. Plant Cell Rep 8:450–454
30. Roy J, Banerjee N (2003) Induction of callus and plant regeneration from shoot-tip explants of
Dendrobium fimbriatum Lindl. var. oculatum Hk. f. Sci Hortic (Amsterdam) 97:333–340
31. Martin KP, Madassery J (2006) Rapid in vitro propagation of Dendrobium hybrids through
direct shoot formation from foliar explants, and protocorm-like bodies. Sci Hortic (Amsterdam)
108:95–99
32. Bhattacharyya P, Kumar V, Grúz J, Doležal K, Van Staden J (2019) Deciphering the phenolic
acid reserves and antioxidant activity within the protocorm like bodies of Ansellia africana: a
vulnerable medicinal orchid. Ind Crop Prod 135:21–29
33. Mesfin YM, Hailemariam D, Biadglign S, Kibret KT (2014) Association between HIV/AIDS
and multi-drug resistance tuberculosis: a systematic review and meta-analysis. PLoS One 9:
e82235
34. Bandeira SO, Gaspar F, Pagula FP (2001) African ethnobotany and healthcare: emphasis on
Mozambique. Pharm Biol 39:70–73
35. Luo X, Pires D, Aínsa JA et al (2011) Antimycobacterial evaluation and preliminary phyto-
chemical investigation of selected medicinal plants traditionally used in Mozambique.
J Ethnopharmacol 137:114–120
36. Ndawonde BG, Zobolo AM, Dlamini ET, Siebert SJ (2007) A survey of plants sold by traders at
Zululand muthi markets, with a view to selecting popular plant species for propagation in
communal gardens. Afr J Range Forage Sci 24:103–107
37. Penduka D, Mthembu W, Cele KH et al (2018) Extracts of Ansellia africana and Platycarpha
glomerata exhibit antibacterial activities against some respiratory tract, skin and soft tissue
infections implicated bacteria. S Afr J Bot 116:116–122
38. He Z, De Buck J (2010) Cell wall proteome analysis of Mycobacterium smegmatis strain MC2
155. BMC Microbiol 10:121
39. Masondo NA, Stafford GI, Aremu AO, Makunga NP (2019) Acetylcholinesterase inhibitors
from southern African plants: an overview of ethnobotanical, pharmacological potential and
phytochemical research including and beyond Alzheimer’s disease treatment. S Afr J Bot
120:39–64
40. Bohlin L, Göransson U, Alsmark C, Weden C, Backlund A (2010) Natural products in modern
life science. Phytochem Rev 9:279–301
41. Hedrick PW (2001) Conservation genetics: where are we now? Trends Ecol Evol 16:629–636
42. Braun AC (1959) A demonstration of the recovery of the crown-gall tumor cell with the use of
complex tumors of single-cell origin. Proc Natl Acad Sci U S A 45:932
43. Feyissa T, Welander M, Negash L (2007) Genetic stability, ex vitro rooting and gene expression
studies in Hagenia abyssinica. Biol Plant 51:15–21
44. Peyvandi M, Noormohammadi Z, Banihashemi O et al (2009) Molecular analysis of genetic
stability in long-term micropropagated shoots of Olea europaea L.(cv. Dezful). Asian J Plant
Sci 8:146–152
18 Phytochemistry, Pharmacology, and Conservation of Ansellia. . . 451
45. Salvi ND, George L, Eapen S (2001) Plant regeneration from leaf base callus of turmeric and
random amplified polymorphic DNA analysis of regenerated plants. Plant Cell Tissue Organ
Cult 66:113–119
46. Larkin PJ, Scowcroft WR (1981) Somaclonal variation—a novel source of variability from cell
cultures for plant improvement. Theor Appl Genet 60:197–214
47. Gantait S, Kundu S, Ali N, Sahu NC (2015) Synthetic seed production of medicinal plants: a
review on influence of explants, encapsulation agent and matrix. Acta Physiol Plant 37:1–12
48. Balunas MJ, Kinghorn AD (2005) Drug discovery from medicinal plants. Life Sci 78:431–441
49. Egan AN, Schlueter J, Spooner DM (2012) Applications of next-generation sequencing in plant
biology. Am. J. Bot. 99:175–185
50. Yan L, Wang X, Liu H, Tian Y, Lian J, Yang R et al (2015) The genome of Dendrobium
officinale illuminates the biology of the important traditional Chinese orchid herb. Mol Plant
8:922–934
51. Hardoim PR, Van Overbeek LS, Berg G et al (2015) The hidden world within plants: ecological
and evolutionary considerations for defining functioning of microbial endophytes. Microbiol
Mol Biol Rev 79:293–320
52. Bacon CW, White J (2000) Microbial endophytes. CRC Press
53. Larriba E, Jaime MDLA, Nislow C et al (2015) Endophytic colonization of barley (Hordeum
vulgare) roots by the nematophagous fungus Pochonia chlamydosporia reveals plant growth
promotion and a general defense and stress transcriptomic response. J Plant Res 128:665–678
54. Nassimi Z, Taheri P (2017) Endophytic fungus Piriformospora indica induced systemic
resistance against rice sheath blight via affecting hydrogen peroxide and antioxidants. Biocon-
trol Sci Tech 27:252–267
55. Tan RX, Zou WX (2001) Endophytes: a rich source of functional metabolites. Nat Prod Rep
18:448–459
56. Gupta S, Chaturvedi P (2015) Phytochemical screening and extracellular enzymatic enumera-
tion of foliar endophytic fungal isolates of Centella asiatica (L.) urban. Int J Pharm Sci Rev Res
35:21–24
57. Gupta S, Bhatt P, Chaturvedi P (2018) Determination and quantification of asiaticoside in
endophytic fungus from Centella asiatica (L.) urban. World J Microbiol Biotechnol 34:111
58. Kumara PM, Zuehlke S, Priti V et al (2012) Fusarium proliferatum, an endophytic fungus from
Dysoxylum binectariferum Hook. f, produces rohitukine, a chromane alkaloid possessing anti-
cancer activity. Antonie Van Leeuwenhoek 101:323–329
59. Mousa WK, Raizada MN (2013) The diversity of anti-microbial secondary metabolites pro-
duced by fungal endophytes: an interdisciplinary perspective. Front Microbiol 4:65
60. Tao G, Liu ZY, Hyde KD et al (2008) Whole rDNA analysis reveals novel and endophytic fungi
in Bletilla ochracea (Orchidaceae). Fungal Divers 33:101–112
61. Chen XM, Dong HL, Hu KX, Sun ZR, Chen J, Guo SX (2010) Diversity and antimicrobial and
plant-growth-promoting activities of endophytic fungi in Dendrobium loddigesii Rolfe. J Plant
Growth Regul 29:328–337
62. Chutima R, Dell B, Vessabutr S, Bussaban B, Lumyong S (2011) Endophytic fungi from
Pecteilis susannae (L.) Rafin (Orchidaceae), a threatened terrestrial orchid in Thailand. Mycor-
rhiza 21:221–229
63. Aggarwal S, Nirmala C, Beri S et al (2012) In vitro symbiotic seed germination and molecular
characterization of associated endophytic fungi in a commercially important and endangered
Indian orchid Vanda coerulea Griff. Ex Lindl. Eur J Environ Sci 2:33–42
64. Parthibhan S, Rao MV, Kumar TS (2017) Culturable fungal endophytes in shoots of
Dendrobium aqueum Lindley–an imperiled orchid. Ecol Genet Genom 3:18–24
65. Yuan Z, Chen Y, Yang Y (2009) Diverse non-mycorrhizal fungal endophytes inhabiting an
epiphytic, medicinal orchid (Dendrobium nobile): estimation and characterization. World
J Microbiol Biotechnol 25:295
66. Chen S, Yao H, Han J, Liu C, Song J, Shi L, Zhu Y, Ma X, Gao T, Pang X (2010) Validation of
the ITS2 region as a novel DNA barcode for identifying medicinal plant species. PloS One.
https://doi.org/10.1371/journal.pone.0008613
Dendrobium sp.: In vitro Propagation of
Genetically Stable Plants and 19
Ethnomedicinal Uses
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
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
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
456 L. Tikendra et al.
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
458 L. Tikendra et al.
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].
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
19 Dendrobium sp.: In vitro Propagation of Genetically Stable Plants. . . 459
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.
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.
460 L. Tikendra et al.
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
19 Dendrobium sp.: In vitro Propagation of Genetically Stable Plants. . . 463
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.
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)
19 Dendrobium sp.: In vitro Propagation of Genetically Stable Plants. . . 465
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.
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.
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)
19 Dendrobium sp.: In vitro Propagation of Genetically Stable Plants. . . 469
Table 1 (continued)
Culture medium having optimal
Species Explant used growth response References
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)
470 L. Tikendra et al.
Table 1 (continued)
Culture medium having optimal
Species Explant used growth response References
• 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)
19 Dendrobium sp.: In vitro Propagation of Genetically Stable Plants. . . 471
Table 1 (continued)
Culture medium having optimal
Species Explant used growth response References
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)
472 L. Tikendra et al.
Table 1 (continued)
Culture medium having optimal
Species Explant used growth response References
+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)
19 Dendrobium sp.: In vitro Propagation of Genetically Stable Plants. . . 473
Table 1 (continued)
Culture medium having optimal
Species Explant used growth response References
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)
474 L. Tikendra et al.
Table 1 (continued)
Culture medium having optimal
Species Explant used growth response References
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
19 Dendrobium sp.: In vitro Propagation of Genetically Stable Plants. . . 475
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.
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
19 Dendrobium sp.: In vitro Propagation of Genetically Stable Plants. . . 477
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%
478 L. Tikendra et al.
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
19 Dendrobium sp.: In vitro Propagation of Genetically Stable Plants. . . 481
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,
482 L. Tikendra et al.
P3
a
P4
P2
P1
MP
P5
P6
P7
P8
P9
P7
b P8
P5
P4
P3
P2
P1
MP
P6
P9
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
Acknowledgments LT and PN are thankful to UGC (University Grant Commission), New Delhi,
India, for financial support.
References
1. Lam Y, Ng TB, Yao RM, Shi J, Xu K, Sze SCW, Zhang KY (2015) Evaluation of chemical
constituent and important mechanism of pharmacological biology in Dendrobium plants. Evid
Based Complementary Altern Med 2015:841752
2. Nongdam P, Nirmala C (2011) In vitro rapid propagation of Cymbidium aloifolium (L.) Sw.: a
medicinally important orchid via seed culture. J Biol Sci 11:254–260
3. Wraith J, Norman P, Pickering C (2020) Orchid conservation and research: an analysis of gaps
and priorities for globally red listed species. Ambio 49:1601–1611
4. Nongdam P, Nirmala C (2012) In vitro seed germination and mass propagation of Cymbidium
dayanum Reichb: an important ornamental orchid of North-East India. Trends Hortic Res 2
(2):28–37
5. Xiaohua J, Singchi C, Yibo L (2008) Taxonomic revision of Dendrobium monifolium complex
(orchidaceae). Sci Hort 120:143–145
6. Paudel MR, Bhattarai HD, Pant B (2020) Traditionally used medicinal Dendrobium: a
promising source of active anticancer constituents. In: Orchids phytochemistry, Biology
horticulture: fundamentals and applications. Springer, Cham. pp 1–26. https://doi.org/10.
1007/978-3-030-11257-8_16-1.
7. Wood HP (2006) The dendrobiums. Timber Press, Portland
8. Moudi M, Go R, Yien CYS, Saleh MN (2013) A review on molecular systematic of the Genus
Dendrobium Sw. Acta Biol Malays 2:71–78
9. Nongdam P, Nirmala C, Tiwari R (2006) In vitro multiplication of Cymbidium pendulum
orchids via embryo culture. Plant Cell Biotech Mol Biol 7:145–150
10. Hinsley A, Boer HJD, Fay MF, Gale SW, Gardiner LM, Gunasekara RS, Kumar P, Masters S,
Metusala D, Roberts D, Veldman S, Wong S, Phelps J (2018) A review of the trade in orchids
and implications for conservation. Bot J Linn Soc 186:435–455
11. Tikendra L, Amom T, Nongdam P (2018) Effect of phytohormones on rapid in vitro propa-
gation of Dendrobium thyrsiflorum Rchb.f.: an endangered medicinal orchid. Pharmacogn
Mag 14:495–500
12. Senthilkumar S (2001) Problems and prospects of orchid mycorrhizal research. J Orchid Soc
India 15:23–32
13. Wraith J, Pickering C (2018) Tourism and recreation of global threat to orchids. Biodivers
Conserv 26:3407–3420
14. Tee CS, Wong CQ, Lam XL, Maziah M (2010) A preliminary study of protocorm-like bodies
(PLBs) induction using leaf explants of Vanda and Dendrobium orchids. Asia Pac J Mol Biol
Biotechnol 18:189–191
484 L. Tikendra et al.
15. Zhao XB, Wu WJ, Pang L, Cai GX (2012) Study on key technology of rapid culture and
propagation for Dendrobium candidum as rare and endangered medicinal materials. J TCM
Univ Hunan 32(3):27–30. (in Chinese with English abstract)
16. Winarto B, Rachmawati F (2013) In vitro propagation protocol of Dendrobium ‘Gradita 31’
via protocorm like bodies. Thammasat Int J Sci Technol 18(2):54–68
17. Bhattacharyya P, Kumaria S, Tandon P (2016) High frequency regeneration protocol for
Dendrobium nobile: a model tissue culture approach for propagation of medicinally important
orchid species. S Afr J Bot 104:232–243
18. Paul P, Joshi M, Gurjar D, Shailajan S, Kumaria S (2017) In vitro organogenesis and
estimation of β-sitosterol in Dendrobium fimbriatum Hook.: an orchid of biopharmaceutical
importance. S Afr J Bot 113:248–252
19. Lin W, Wang J, Xu X, Wu Y, Qiu D, He B, Sarsaiya S, Ma X, Chen J (2020) Rapid propagation
in vitro and accumulation of active substances of endangered Dendrobium cariniferum Rchb.
F. Bioengineered 11:386–396
20. Rawat JM, Rawat B, Agnihotri RK, Chandra A, Nautiyal S (2013) In vitro propagation,
genetic and secondary metabolite analysis of Aconitum violaceum Jacq.: a threatened medic-
inal herb. Acta Physiol Plant 35:2589–2599
21. Krishna H, Alizadeh M, Singh D, Singh U, Chauhan N, Eftekhari M, Sadh RK (2016)
Somaclonal variations and their applications in horticultural crops improvement. 3. Biotech
6:54
22. Antony JJJ, Shamshir RA, Poobathy R, Chew BL, Subramaniam S (2015) Somaclonal
variations were not induced by the cryopreservation: levels of somaclonal variations of in
vitro and thawed protocorms of Dendrobium bobby Messina analysed by SCoT and TRAP
DNA markers. S Afr J Bot 100:148–157
23. Chin CK, Lee ZH, Mubbarakh SA, Antony JJJ, Chew BL, Subramaniam S (2019) Effects of
plant growth regulators and activated charcoal on somaclonal variations of protocorm-like
bodies (PLBs) of Dendrobium sabin blue orchid. Biocatal Agric Biotechnol 22:101426
24. Tikendra L, Amom T, Nongdam P (2019) Molecular genetic homogeneity assessment of
micropropagated Dendrobium moschatum Sw. - a rare medicinal orchid, using RAPD and
ISSR markers. Plant Gene 19:100196
25. Tikendra L, Koijam AS, Nongdam P (2019) Molecular markers based genetic fidelity assess-
ment of micropropagated Dendrobium chrysotoxum Lindl. Meta Gene. https://doi.org/10.
1016/j.mgene.2019.100562
26. Chandra S, Rawat DS (2015) Medicinal plants of the family Caryophyllaceae: a review of
ethno medicinal uses and pharmacological properties. Integr Med Res 4:123–131
27. Schultes RE (1962) The role of ethnobotanist in search for new medicinal plants. Lloydia 25
(4):257–266
28. Quinlan MB (2011) Ethnomedicine. Wiley online library https://doi.org/10.1002/
9781444395303.ch19
29. Pohle P (1990) Useful plants of Manang district: a contribution to the ethnobotany of the
Nepal. Himalaya, Franz Steiner Verlag Wiesbaden GmbH, Stuttgart
30. Withner CL (1959) The orchid: a scientific survey. Ronald Press Company, New York
31. Hossain MM (2011) Therapeutic orchids: tradicinal uses and recent advances - an overview.
Fitoterapia 82:102–104
32. Bulpitt CJ (2007) The use of orchids in Chinese medicine. J R Soc Med 100:558–563
33. Vaidya B, Shrestha M, Joshee N (2000) Report on Nepalese orchid species with medicinal
properties. In: Watanabe T, Takano A, Bista MS, Bista, Siju HK (eds) The Himalayas plants,
can they save us? Proceeding of Nepal-Japan joint symposium on conservation and utilization
of Himalayan medicinal resources, Society for the conservation and development of Himala-
yan Medicinal Resources (SCDHMR), Japan, pp 146–152
34. Joshi KK, Joshi SD (2000) Genetic heritage of medicinal and aromatic plants of Nepal
Himalayas. Buddha Academy Publisher and Distributors, Pvt, Kathmandu
35. Bulpitt CJ (2005) The uses and misuses of orchids in medicine. QJM 98:625–631
19 Dendrobium sp.: In vitro Propagation of Genetically Stable Plants. . . 485
36. Baral SR, Kurmi PP (2006) A compendium of medicinal plants of Nepal. Publisher Rachana
Baral, Mass Printing Press, Kathmandu
37. Subedi A (2011) New species, pollinator interactions and pharmaceutical potential of Hima-
layan orchids. Ph. D. Thesis, Leiden University, The Netherlands
38. Joshi G, Tewari LM, Lohani N, Upreti K, Jalal JS, Tewari G (2009) Diversity of orchids on
Uttarakhand and their conservation strategy with special reference to their medicinal impor-
tance. Rep Opin 1:47–52
39. Meitei A, Pamarthi RK, Kumar R, Bhutia NT, Rai D, Kiran BP, Singh AK, Gazmer R, Singh
DR (2019) Dendrobium nobile Lindl. in Indian traditional medicine, a phytochemical
approach. Indian J Horticul 76(3):557–560
40. Pant B, Raskoti BB (2013) Medicinal orchids of Nepal. Himalayan Map House, Pvt Ltd,
Kathmandu
41. Nongdam P (2014) Ethno-medicinal uses of some orchids of Nagaland, North-East India. Res
J Med Plant 8(3):126–139
42. Matsuda H, Morikawa T, Xie H, Yoshikawa M (2004) Antiallergic phenanthrenes and
stilbenes from the tubers of Gymnadenia conopsea. Planta Med 70:847–855
43. Arditti J (1992) Fundamentals of orchid biology. Wiley, New York, pp 243–260
44. Arora M, Mahajan A, Sembi JK (2007) A review on phytochemical and pharmacological
potential of family orchidaceae. Int Res J Pharm 8(10):9–24
45. Guteirrez RMP (2010) Orchids: a review of uses in traditional medicine, its phytochemistry
and pharmacology. J Med Plant Res 4(8):592–638
46. Khasim S, Rao PRM (1999) Medicinal importance of orchids. The Botanica 49:86–91
47. Li Y, Wang CL, Guo SX, Yang JS (2008) Two new compounds from Dendrobium candidum.
Chem Pharm Bull 56:1477–1479
48. Fan C, Wang W, Wang Y, Qin G, Zhao W (2001) Chemical constituents from Dendrobium
densiflorum. Phytochemistry 57:1255–1258
49. Venkateswarlu S, Raju MS, Subbarahu GV (2002) Synthesis and biological activity of
isamoenylin, a metabolite of Dendrobium amoenum. Biosci Biotechnol Biochem 66:2236–
2238
50. Miyazawa M, Shimamura H, Nakamura S, Kameoka H (1997) Antimutagenic activity of
gigantol from Dendrobium nobile. J Agric Food Chem 45:2849–2853
51. Vaidya BN (2019) Nepal global hotspot for medicinal orchids. In: Medicinal plants. pp 35–80.
https://doi.org/10.1007/978-3-030-31269-5_3
52. Arditti J, Ernst R (1984) Physiology of orchid seed germination. In: Arditi J (ed) Orchid
biology reviews and perspectives. Cornell University Press, New York
53. Hossain MM (2009) Traditional therapeutic uses of some indigenous orchids of Bangladesh.
Med Aromat Plant Sci Biotechnol 42:101–106
54. Singh DK (2001) Morphological diversity of the orchids of Orissa/Sarat Misra. In: Pathak P,
Sehgal RN, Shkhar N, Sharma M, Sood ABSMPS (eds) Orchids: science and commerce. New
Delhi, Bishen Singh Mahendra Pal Singh, pp 35
55. Yang L, Wang Z, Xu L (2006) Simultaneous determination of phenols (bibenzyl phenanthrene
and fluorenone) in Dendrobium species by high performance liquid chromatography with
diode array detection. J Chromatograp 1104:230–237
56. Zhao P, Wang W, Feng FS, Wu F, Yang ZQ, Wang WJ (2007) High frequency shoot
regeneration through transverse thin cell layer culture in Dendrobium candidum Wall Ex
Lindl. Plant Cell Tissue Organ Cult 90:131–139
57. Wu HS, Xu JH, Chen LZ, Sun JJ (2004) Studies on antihyperglycemic effect and its
mechanism of Dendrobium candidum. Zhongguo Zhong Yao Za Zhi 29:160–163
58. Zheng Y (2005) Shihuin China pharmacopoeia. Chemical Technology Press, Beijing, pp 61–63
59. Ding YP, Wu LS, Yu LW (1998) Optimized harvesting period for Dendrobium candidum.
China J Chinese Materia Medica 23:458–460
60. Bensky D, Gemble A (1993) A Chinese herbal medicine Materia Medica. Eastland Press,
Seattle
486 L. Tikendra et al.
61. Xu J, Han BQ, Li SL, Chen XJ, Wang XN, Zhao ZZ et al (2013) Chemistry, bioactivity and
quality control of Dendrobium, a commonly used tonic herbin traditional Chinese medicine.
Phytochem Rev 12:341–367
62. Pant B (2013) Medicinal orchids and their uses: tissue culture a potential alternative for
conservation. Afric J Plant Sci 7(10):448–467
63. Bi ZM, Wang ZT, Xu LS, XU GJ (2003) Studies on the chemical constituents of Dendrobium
fimbriatum. Yao Xue Xue Bao 38: 526–529
64. Jin H, Xu ZX, Chen JH, Han SF, Ge S, Luo YB (2009) Interaction between tissue-cultured
seedlings of Dendrobium officinale and mycorrhizal fungus (Epulorhiza sp.) during symbiotic
culture. Chin J Plant Ecol 33:433–441
65. Ho CK, Chen CC (2003) Moscatilin from the orchid Dendrobium loddigesii is a potential
anticancer agent. Cancer Investig 21:729–736
66. Roy J, Naha S, Majumdar M, Banerjee N (2007) Direct and callus mediated protocorm-like
body induction from shoot-tips of Dendrobium chrysotoxum Lindl. (Orchidaceae). Plant Cell
Tissue Organ Cult 90:31–39
67. Nimisha PS, Hiranmai Yadav R (2012) Proximate and phytochemical analysis of Dendrobium
macrostachyum Lindl. Int J Pharmaceutic Sci 4(1):385–386
68. Arditti J, Clementsm MA, Fast G, Handley G, Nishimura (1982) Orchid seed germination and
seedling culture -a manual. In: Arditti J (ed) Orchid biology -reviews and perspectives, vol II.
Cornell University Press, Ithaca, pp 243–370
69. Dressler RL (1993) Phylogeny and classification of the orchid family. Cambridge University
Press, New York
70. Rama Rao V (2002) Distributional status survey of threatened plants of Gujarat. PhD Thesis,
Sardar Patel University, Vallabh Vidyanagar
71. Gavali D, Sharma D (2004) Traditional knowledge and biodiversity conservation in Gujarat.
Indian J Trad Knowledge 3:51–58
72. Kirtikar KR, Bashu BD (2001) Orchidaceae Dendrobium. In: Indian medicinal plants, 2nd
edn. Oriental Press, India, pp 3316–3317
73. Kovacs A, Vasas A, Hohmann J (2008) Natural phenanthrenes and their biological activity.
Phytochemistry 69:1084–1110
74. Balzarini J, Neyts J, Schols D, Hosoya M, Van Damme E, Peumans W, De Clercq E (1992)
The mannose-specific plant lectins from Cymbidium hybrid and Epipactis helleborine and the
(N-acetylglucosamine) N-specific plant lectin from Urtica dioca are potent and selective
inhibitors of human immunodeficiency virus and cytomegalovirus replication in vitro. Antivir
Res 18:191–207
75. Wu ZG, Jiang W, Chen SL, Mantri N, Tao ZM, Jiang CX (2016) Insights from the cold
transcriptome and metabolome of Dendrobium officinale: global reprogramming of metabolic
and gene regulation networks during cold acclimation. Front Plant Sci 7:1653
76. Chen KK, Chen AL (1935) The alkaloid of Chin-Shih-Hu. J Biol Chem 111:653–658
77. Liu QF, Zhao WM (2009) A new dedonbrine type alkaloid from Dendrobium nobile. Chin
Chem Lett 14:278–279
78. Chauhan NS (1999) Medicinal and aromatic plants of Himachal Pradesh. Indus Publishing,
New Delhi, p 632
79. Devi PU, Selvi S, Devipriya D, Murugan S, Suja S (2009) Antitumor and antimicrobial
activities and inhibition of in-vitro lipid peroxidation by Dendrobium nobile. Afr J Biotechnol.
https://doi.org/10.5897/AJB2009.000-9292
80. Zhu S, Niu Z, Xue Q, Hui W, Xie X, Ding X (2018) Accurate authentication of Dendrobium
officinale and its closely related species by comparative analysis of complete plastomes. Acta
Pharm Sin B 8(6):969–980
81. Lin Y, Li J, Li B, He T, Chun Z (2010) Effects of light quality on growth and development of
protocorm-like bodies of Dendrobium officinale in vitro. Plant Cell Tissue Organ Cult 105
(3):329–335
19 Dendrobium sp.: In vitro Propagation of Genetically Stable Plants. . . 487
82. Zhou Y, Honda M, Zhu H, Zhang Z, Guo X, Li T, Li Z, Peng X, Nakajima K, Duan L (2015)
Spatiotemporal sequestration of miR165/166 by Arabidopsis argonaute 10 promotes shoot
apical meristem maintenance. Cell Rep 10:1819–1827
83. Zhang GN, Zhang CF, Wang ZT, Xu LS (2004) Chemical constituents of Dendrobium
thyrsiflorum Rchb f (II). Chinese J Natural Med 2(2):78–81
84. Zhang D, Zhang JJ (2005) Effect of Coeloglossum viride var. bracteatum extract on oxidation
injury in sub-acute senescent model mice. Zhongguo Yi Xue Ke Xue Bao 27:729–733
85. Song C, Kang J, Ji X, Fu C, Wen X, Yu L, Qiao G (2007) Efficient plant regeneration and
genetic fidelity assessment of in vitro-derived plants of Dendrobium nobile- an endangered
medicinal and ornamental herb. Orchid Sci Biotechnol 1:51–55
86. Yan L, Wang CL, Wang YJ, Gua SX, Yang JS, Chen XM, Xiao PG (2009) Three new bibenzyl
derivatives from Dendrobium candidum. Chem Pharm Bull 57:218–219
87. Wu JW, Hu CY, Shahid MQ et al (2013) Analysis on genetic diversification and heterosis in
autotetraploid rice. Springer Plus 2:439
88. You HL, Park JD, Baek NI, Kim S, Ahn BZ (1995) In vitro and in vivo antimural phenan-
threnes from the aerial parts of Dendrobium nobile. Planta Med 61:178–180
89. Manandhar NP (1995) A survey of medicinal plants of Jajarkot district, Nepal. J
Ethnopharmacol 48(1):1–6
90. Pant B, Thapa D (2012) In vitro mass propagation of an epiphytic orchid, Dendrobium
primulinum Lindl. through shoot tip culture. Afr J Biotech 11(42):9970–9974
91. Yuan Y, Yu M, Jia Z, Song X, Liang Y, Zhang J (2018) Analysis of Dendrobium huoshanense
transcriptome unveils putative gene associated with active ingredients synthesis. BMC Geno-
mics 19:978
92. Kartikar KR, Basu BD (1981) Indian medicinal plants, vol 2. International Book Distribution,
Dehradun
93. Mk A, Rashid MH, Hossain MS, Salam MA, Rouf MA (2002) In vitro seed propagation of
Dendrobium (Dendrobium transparens) orchid as influenced by different media. Biotechnol-
ogy 1:111–115
94. Teixeira da Silva JA (2013) The role of thin cell layers in regeneration and transformation in
orchids. Plant Cell, Tissue and Organ Cult 113:149–161
95. Zahara M, Datta A, Boonkorkaew P (2016) Effects of sucrose, carrot juice and culture
mediaon growth and net CO2 exchange rate in Phalaenopsis hybrid ‘pink’. Sci Hortic
205:17–24
96. Utami ESW, Hriyanto S (2020) Organic compounds: contents and their role in improving seed
germination and protocorm development in orchids. Int J Agronomy 2020:2795108
97. Zahara M, Datta A, Boonkorkaew P, Mishra A (2017) The effects of different media, sucrose
concentrations and natural additives on plantlet growth of Phalaenopsis hybrid ‘pink’. Braz
Arch Biol Technol 60:e160149
98. Anderson AB (1996) The reintroduction of Plantanthera ciliaris in Canada. In: Allen C (ed)
North American native terrestrial orchids propagation and production. North American Native
Terrestrail Orchid Conference, Germantown, pp 73–76
99. Stewart SL, Kane ME (2006) Asymbiotic seed germination and in vitro seedling development
of Habenaria macroceratitis (Orchidaceae), a rare Florida terrestrial orchid. Plant Cell Tissue
Organ Cult 86:147–158
100. Dohling S, Kumaria S, Tandon P (2012) Multiple shoot induction from axillary bud cultures of
the medicinal orchid, Dendrobium longicornu. AoB Plants pls032. https://doi.org/10.1093/
aobpla/pls032
101. Murashige T, Skoog F (1962) A revised medium for rapid growth and bio assays with tobacco
tissue culture. Physiologia Planturam 15(3):473–497
102. Mitra GC, Prasad RN, Roychowdhury A (1976) Inorganic salts and differentiation of pro-
tocorms in seed callus of orchid and correlative changes in its free amino acid content. Indian J
Exp Biol 14:350–351
488 L. Tikendra et al.
103. Knudson L (1946) A new nutrient solution for germination of orchid seed. Amer Orchid Soc
Bull 15:214–217
104. Vacin E, Went FW (1949) Some pH changes in nutrient solutions. Bot Gaz 110:605–613
105. Gamborg OL, Miller RA, Ojima K (1968) Nutrient requirements of suspension culture of
soybean root cells. Exp Cell Res 50(1):151–158
106. Kramer PJ, Kozlowski TT (1979) Physiology of woody plants. Academic Press, New York, p
811
107. Suzuki RM, Moreira VC, Pescador R, Melo FW (2012) Asymbiotic seed germination and in
vitro seedling development of the threatened orchid Hoffmennseggella cinnaberina. In Vitro
Cell Dev Biol 48:500–511
108. Yildiz M (2012) The prerequisite of the success in plant tissue culture: high frequency shoot
regeneration. In: Leva A, Rinaldi LMR (eds) Recent advances in plant in vitro culture, 1st edn.
InTech Rijeka, London, UK pp 63–90. https://doi.org/10.5772/51097
109. Teixeira da Silva JA, Dobranszki J, Cardoso JC, Zeng SJ (2015) Mocropropagation of
Dendrobium: a review. Plant Cell Rep 34:671–704
110. Kong Q, Yuan SY, Végvári GY (2007) Micropropogation of an orchid Dendrobium
strongylanthum Rchb.f. Int J Hortic Sci 13:61–64
111. Kaewduangta W, Reamkatog P (2011) Effect of modification medium on growth development
of Dendrobium parishii in vitro. Am Eurasian J Agric Environ Sci 11:117–121
112. Nongdam P, Tikendra L (2014) Establishment of an efficient in vitro regeneration protocol for
rapid and mass propagation of Dendrobium chrysotoxum lindl. using seed culture. Sci World J.
https://doi.org/10.1155/2014/740150
113. Bhadra SK, Barua AK, Bhattacharjee B, Hossain MM (2002) In vitro micropropagation of
Dendrobium aphyllum (Roxb.) G. E. C. Fisher. Bangladesh J Genet Biotechnol 3:47–50
114. Bai MF, Wu TL, Huang M, Zhang TG (2004) Rapid propagation of Dendrobium loddigesii
Rolfe by tissue culture. Seed 23:44–46. (in Chinese with English abstract)
115. Huang WC, Yin LQ, Hu YH, Wang XQ, Zhao XF, Li XF (2008) In vitro rapid propagation of
Dendrobium fimbriatum. J Shanghai Jiaotong Univ (Agric Sci) 26:584–587. (in Chinese with
English abstract)
116. Guan P, Shi JM (2009) Tissue culture of stem segment and induction of floral buds of
Dendrobium denndanum. Lishizhen Med Materia Med Res 20:205–206. (in Chinese with
English abstract)
117. Pradhan S, Paudel YP, Pant B (2013) Efficient regeneration of plants from shoot tip explants of
Dendrobium densiflorum Lindl., a medicinal orchid. Afr J Biotechnol 12:1378–1383
118. Yasugi S, Shinto H (1994) Formation of multiple shoots and regenerated plantlets by culture of
pseudobulb segment in nobile type Dendrobium. Plant Tissue Cult Lett 11(2):153–156
119. Sharma U, Rao VR, Nohan JSS, Reddy AS (2007) In vitro propagation of Dendrobium
microbulbon A. Rich—a rare ethnomedicinal herb. Indian J Biotechnol 6:381–384
120. Ferreira WDM, Kerbauy GB, Costa APP (2006) Micropropagation and genetic stability of a
Dendrobium hybrid (Orchidaceae). In Vitr Cell Dev Biol Plant 42:568–571
121. Webster S, Mitchell SA, Ahmad MH (2003) A novel surface sterilization method for reducing
fungal and bacterial contamination of field grown medicinal explants intended for in vitro
culture. In Proceedings of 17th SRC conference entitled “Science and technology for economic
development: Technology driven agriculture and agro processing SRC, Jamaica
122. Oyebanji OBO, Nweke O, Odebunmi NB, Galadima MS, Idris UN, Nnodi A, Afolabi S,
Ogbadu GH (2009) Simple, effective and economical explant-surface sterilization protocol for
cowpea, rice and sorghum seeds. Afri J Biotechnol 8(20):5395–5399
123. Hossain MM, Sharma M, Pathak P (2013) In vitro propagation of Dendrobium aphyllum
(Orchidaceae)—seed germination to flowering. J Plant Biochem Biotechnol 22:157–167
124. Zhang X, Gao J (2020) In vitro tetraploid induction from multigenotype protocorms and
tetraploid regeneration in Dendrobium officinale. Plant Cell Tissue Organ Cult 141:289–298
125. Tomita M, Tomita M (1997) Plant regeneration from immature seed -derived callus of
Cypripedium macranthos Swartz var. taiwanianum (Masamune) F. Maekawa. Breed Sci
47:279281
19 Dendrobium sp.: In vitro Propagation of Genetically Stable Plants. . . 489
126. Hynson NA, Madesen TP, Selosse MA, Adam IKU, Ogura-Tsujita Y, Roy M et al (2013) The
physiological ecology of mycoheterotropy. In: Merckx VSFT (ed) Mycoheterotropy: the
biology of plants living on fungi. Springer, Berlin, pp 297–342
127. Parthibhan S, Senthil Kumar T, Rao MV (2015) Phenology and reintroduction strategies for
Dendrobium aqueum Lindley – an endemic, near threatened orchid. J Nat Conserv 24:68–71
128. Nontachaiyapoon S, Sasirat S, Manoch L (2011) Symbiotic seed germination of
Grammatophyllum speciosum Blume and Dendrobium draconis Rchb. f., native orchids of
Thailand. Sci Hortic 130:303–308
129. Paul S, Kumaria S, Tandon P (2012) An effective nutrient medium for asymbiotic seed
germination and large-scale in vitro regeneration of Dendrobium hookerianum, a threatened
orchid of northeast India. AoB Plants plr032. https://doi.org/10.1093/aobpla/plr032
130. Utami ESW, Hariyanto S, Manuhara YSW (2017) In vitro propagation of the endangered
medicinal orchid, Dendrobium lasianthera JJ Sm through mature seed culture. Asian Pac J
Trop Biomed 7:406–410
131. Phillip VJ, Nainar SAZ (1986) Clonal propagation of Vanilla planifolia (Salisb.) Ames using
tissue culture. J Plant Physiol 122:211–215
132. Morel G (1966) Meristem culture clonal propagation of orchids. Orchid Dig 30:45–49
133. Dohling S, Das MC, Kumaria S, Tandon P (2007) Conservation of splendid orchids of North-
East India. Biodivers. Its significance. IK International Publishers, New Delhi, pp 354–365
134. Sharma A, Tandon P (1992) In vitro culture of Dendrobium wardianum Warner: morphoge-
netic effects of some nitrogenous adjuvants. Indian J Plant Physiol 35(1):80–85
135. Kanjilal B, DeSarkar D, Mitra J, Dutta BK (1999) Stem disc culture: development of a rapid
mass propagation method of Dendrobium moschatum (Buch-Ham) Swartz—an endangered
orchid. Curr Sci 77:497–500
136. Roy J, Banerjee N (2003) Induction of callus and plant regeneration from shoot-tip explant of
Dendrobium fimbriatum Lindl. var. oculatum Hk. f. Sci Hortic 97:333–340
137. Malabadi RB, Mulgund GS, Kallappa N (2005) Micropropagation of Dendrobium nobile from
shoot tip sections. J Plant Physiol 162:473–478
138. Pornpienpakdee P, Singhasurasak R, Chaiyasap P, Pichyangkura R, Bunjongrat R,
Chadchawan S, Limpanavech P (2010) Improving the micropropagation efficiency of hybrid
Dendrobium orchids with chitosan. Sci Hortic 124:490–499
139. Asghar S, Ahmad T, Ahmad I, Yaseen M (2011) In vitro propagation of orchid (Dendrobium
nobile) var. Emma white. Afr J Biotechnol 10:3097–3103
140. Vij SP, Pathak P (1989) Micropropagation of Dendrobium chrysanthum Wall. through
pseudobulb segments. J Orchid Soc India 3:25–28
141. Gao PY, Chen JM, Gan Q (2002) Tissue culture of stem segment and plantlet regeneration of
Dendrobium nobile. Chin Tradit Herbal Drug 33:1031–1033. (in Chinese with English
abstract)
142. Bai Y, Bao YH, Wang WQ, Yan YN (2006) Tissue culture and rapid propagation of
Dendrobium crepidatum Lindl. ex Paxt. Plant Physiol Commun 42:R28–S68. (in Chinese)
143. Bhattacharyya P, Paul P, Kumaria S, Tandon P (2018) Transverse thin cell layer (t-TCL)
mediated improvised micropropagation protocol for endangered medicinal orchid
Dendrobium aphyllum Roxb: an integrated phytomolecular approach. Acta Physiol Plant
40:137
144. Pant B, Sha HS, Shrestha R, Pandey S, Joshi PR (2017) An overview on orchid endophytes.
In: Varma A, Prasad R, Tuteia N (eds) Mycorrhiza-nutrient uptake biocontrol ecorestoration.
Springer, Berlin, pp 503–524
145. Martin KP, Geevarghese J, Jodheph D, Madassery J (2005) In vitro propagation of
Debdrobium hybrids using flower stalk node explants. Indian J Exp Biol 43:280–285
146. Nugroho JD, Arobaya AYS, Tanur EA (2019) Propagation of Dendrobium antennatum Lindl
via seed culture in vitro using simple medium: fertilizer and complex organic based medium.
HIYATI J Biosci 26(3):133–138
147. Ma NL, Khoo SK, Lee JX, Soon CF, Shukor NAAB (2020) Efficient micropropagation of
Dendrobium aurantiacum from shoot explant. Plant Sci Today 7(3):476–482
490 L. Tikendra et al.
148. Parthibhan S, Kumar TS, Rao MV (2015) In vitro regeneration from protocorms in
Dendrobium aqueum Lindley- an imperilled orchid. J Nat Conserv 24:68–71
149. Partibhan S, Rao MV, da Silva T, Kumar TS (2018) Somatic embryogenesis from stem thin
cell layers of Dendrobium aqueum. Biol Plant 62(3):439–450
150. Riva SS, Islam A, Hoque ME (2016) In vitro regeneration and rapid multiplication of
Dendrobium bensoniae, an indigenous ornamental orchid. The Agriculturists 14(2):24–31
151. Refish NMR, Wang L, Fu C, Xu X, Jin W, Li M, Yu L (2016) Establishment and optimization
of high efficiency embryogenic callus induction system in Dendrobium candidum. African J
Plant Sci 10(4):77–83
152. Maharajan S, Thakur LS, Thapal BB, Pradhan S, Pant KK, Joshi GP, Pant B (2020) In vitro
propagation of the endangered orchid Dendrobium chryseum Rolfe from protocorms culture.
Nepal J Sci Technol 19(1):39–47
153. Kaur S (2017) In vitro regeneration of shoots from nodal explants of Dendrobium chrysotoxum
LINDL. J Hortic Res 25(1):27–34
154. Hapsoro D, Septiana VT, Ramadiana S, Yusnita Y (2018) A medium containing commercial
foliar fertilizer and some organic additives could substitute MS medium for in vitro growth of
Dendrobium hybrid seedlings. J Floratek 13(1):11–22
155. Kaur S, Bhandari P, Bhutani KK (2015) Chracterization of bioactive compounds at seedling
stage and optimization of seed germination, culture multiplication and Dendrobium nobile
Lindl. - a study in vitro. Int J Adv Res 3(4):1041–1052
156. Priyanka S, Verma LS, Satyanarayana E, Subhankar (2018) In vitro regeneration and rapid
multiplication of Dendrobium nobile. Int J Chem Studies 6(6):1286–1288
157. Shetty V, Thomas A, Pujari I, Babu VS (2015) Asymbiotic hypergeneration of protocorm like
bodies -an efficient and simple micropropagation strategy for conserving the therapeutic
ornamental Dendrobium ovatum. Int J Recent Sci Res 6(12):8009–8015
158. Bhowmik TK, Rahman MM (2020) Micropropagation of commercially important orchid
Dendrobium palpebrae Lindl. through in vitro developed psudobulb culture. J Adv Biotechnol
Exp Ther 3(3):225–232
159. Kunagorn N, Roopkam C, Aumroong P, Anukul N (2017) Meristem tip culture of Dendrobium
orchid for boosting efficiency of hygienic large scale micropropagation. Acta Hortic 1155:
419–424
160. Adhikari H, Pant B (2019) In vitro seed germination and seedling growth of the orchid
Dendrobium primulinum Lindl. African J Plant Sci 13(12):324–331
161. Mamun AA, Islam MM, Ahmed M, Ghose GC (2018) In vitro mass propagation from shoot
tip of Dendrobium Red Bull-an endangered epiphytic orchid. Plant Tissue Cult Biotech 28
(2):161–169
162. Rattana K, Sangchanjiradet S (2017) Micropropagation of Dendrobium signatum Rchb.F.
Pertanika J Trop Agric Sci 40(4):577–586
163. Billore V, Jain M, Suprasanna P (2017) Monochromic radiation through light-emitting diode
(LED) positively augments in vitro shoot regeneration in orchid (Dendrobium Sonia). Can J
Biotech 1(2):50–58
164. Sahagun J, Kongbangkerd A, Ratannasut K (2018) Organogenic potential of Dendrobium
floral tissues for stable transformation applications. Philippine J Sci 147(4):667–676
165. Obsuwan K, Serayheap K, Thepsithar C (2019) Effects of calcium silicate and proline-induced
salt tolerance on the in vitro propagation of Dendrobium Sonia ‘Red jo’. Acta Hortic 1262:87–
91
166. Bing HYN, Tham DT, Vinh TT, Hoi QV, Cong VK, Duy NV (2018) In vitro propagation of the
new orchid Dendrobium trankimianum T. Yukawa. J Biotechnol 16(4):649–657
167. Larkin P, Scowcroft W (1981) Somaclonal variation—a novel source of variability from cell
cultures for plant improvement. Theor Appl Genet 60:197–214
168. Lakshmanan V, Venkataramareddy SR, Neelwarne B (2007) Molecular analysis of genetic
stability in long-term micropropagated shoots of banana using RAPD and ISSR markers.
Electron J Biotechnol. https://doi.org/10.2225/vol10-issue1-fulltext-12
19 Dendrobium sp.: In vitro Propagation of Genetically Stable Plants. . . 491
169. Sahijram L, Soneji JR, Bollamma KT (2003) Analyzing somaclonal variation in micro-
propagated bananas (Musa spp). In Vitro Cell Dev Biol 39:551–556
170. Saravanan S, Sarvesan R, Vinod MS (2011) Identification of DNA elements involved in
somaclonal variants of Rauvolfia serpentine (L.) arising from indirect organogenesis as
evaluated by ISSR analysis. Indian J Sci Technol 4:1241–1245
171. Vázquez AM (2001) Insight into somaclonal variation. Plant Biosyst 135:57–62
172. Kunitake H, Nakashima T, Mori K, Tanaka M, Mii M (1995) Plant regeneration from
mesophyll protoplasts of Lisianthus (Eustoma grandiflorum) by adding activated charcoal
into protoplast culture medium. Plant Cell Tissue Organ Cult 43:59–65
173. Zayova E, Vassilevska IR, Kraptchev B, Stoeva D (2010) Somaclonal variations through
indirect organogenesis in eggplant (Solanum melongena L.). Biol Divers Conserv 3:1–5
174. Farahani F, Yari R, Masoud S (2011) Somaclonal variation in Dezful cultivar of olive (Olea
europaea subsp. europaea). Gene Conserve 10:216–221
175. Siragusa M, Carra A, Salvia L, Puglia A, De Pasquale F, Carimi F (2007) Genetic instability in
calamondin (Citrus madurensis Lour.) plants derived from somatic embryogenesis induced by
diphenylurea derivatives. Plant Cell Rep 26:1289–1296
176. Sun S, Zhong J, Li S, Wang X (2013) Tissue culture-induced somaclonal variation of
decreased pollen viability in torenia (Torenia fournieri Lind.). Bot Stud 54(1):36
177. De Carvalho SR, Luis ZG, Scherwinski-Pereira JE (2014) The histodifferentiation events
involved during the acquisition and development of somatic embryogenesis in oil palm (Elesis
guineensis Jacq.). Plant Growth Regul 72:67–80
178. Arnhold-Schmitt B (1993) Rapid changes in amplification and methylation pattern of genomic
DNA in cultured carrot root explants (Daucus carota L.). Theor Appl Genet 85:793–800
179. Eeuwens CJ, Lord S, Donough CR, Rao V, Vallejo G, Nelson S (2002) Effects of tissue culture
conditions during embryoid multiplication on the incidence of “mantled” flowering in clonally
propagated oil palm. Plant Cell Tissue Organ Cult 70:311–323
180. Kaeppler SM, Kaeppler HF, Rhee Y (2000) Epigenetic aspects of somaclonal variation in
plants. Plant Mol Biol 43:179–188
181. Ngezahayo F, Dong Y, Liu B (2007) Somaclonal variation at the nucleotide sequence level in
rice (Oryza sativa) as revealed by RAPD and ISSR markers, and by pairwise sequence
analysis. J Appl Genet 48:329–336
182. Leva AR, Petruccelli R, Rinaldi LMR (2012) Somaclonal variation in tissue culture: a case
study with olive. In: Leva AR, Rinaldi LMR (eds) Recent advances in plant in vitro culture.
INTECH Open Access Publisher, Croatia, pp 123–150
183. Neelankandan AK, Wang K (2012) Recent progress in the understanding of tissue culture-
induced genome level changes in plants and potential applications. Plant Cell Rep 31:597–620
184. Tiwari JK, Chandel P, Gupta S, Gopal J, Singh BP, Bhardwaj V (2013) Analysis of genetic
stability of in vitro propagated potato microtubers using DNA markers. Physiol Mol Biol
Plants 19:587–595
185. Azizi P, Hanafi MM, Sahebi M, Harikrishna JA, Taheri S, Yassoralipour A, Nasehi A (2020)
Epigenetic changes and their relationship to somaclonal variation: a need to monitor the
micropropagation of plantation crops. Funct Plant Biol. https://doi.org/10.1071/fp19077
186. Sabir A, Newbury HJ, Todd G, Catty J, Ford-Lloyd BV (1992) Determination of genetic
stability using isozymes and RFLPs in beet plants generated in vitro. Theor Appl Genet
84:113–117
187. Dixit S, Mandal BB, Sangeeta A, Srivastava PS (2003) Genetic stability assessment of plants
regenerated from cryopreserved embryogenic tissues of Dioscorea bulbifera l. using RAPD,
biochemical and morphological analysis. Cryo Letters 24:77–84
188. Mallon R, Rodriguez-Oubina J, Gonzalez M (2010) In vitro propagation of the endangered
plant Centaurea ultreiae: assessment of genetic stability by cytological studies, flow cytometry
and RAPD analysis. Plant Cell Tissue Organ Cult 101:31–39
189. Loureiro J, Rodriguez E, Dolezel J, Santos C (2006) Comparison of four nuclear isolation
buffers for plant DNA cytometry. Ann Bot 98:679–689
492 L. Tikendra et al.
190. Bairu MW, Aremu AO, van Staden J (2011) Somaclonal variation in plants: cause and
detection methods. Plant Growth Regul 63:147–173
191. Butiuc-Keu A, Farkas A, Cristea V (2016) Genetic stability assessment of in vitro plants by
molecular markers. Studia Univeritatis Babes - Bolyai Biologia 61(1):107–114
192. Singh SR, Dalal S, Singh R, Dhawan AK, Kalia RK (2013) Ascertaining clonal fidelity of
micropropagated plants of Dendrocalamus hamiltonii Nees et Arn. ex Munro using molecular
markers. In Vitro Cell Dev Biol 49:572–583
193. Bandupriya HDD, Iroshini WWMA, Perera SACN, Vidhanaarachchi VRM, Fernando SC,
Santha ES, Gunathilake TR (2017) Genetic fidelity testing using SSR marker assay confirms
trueness to type of micropropagated coconut (Cocos nucifera L) plantlets derived from
unfertilized ovaries. The Open Plant Sci J 10:46–54
194. Raynalta E, Elina J, Sudarsono SD (2018) Clonal fidelity of micro propagated Phalaenopsis
plantlets based on assessment using eighteen Ph-Pto SNAP marker loci. Agrivita J Agri Sci 40
(3):390–402
195. Rohela GK, Jogam P, Bylla P, Reuben C (2019) Indirect regeneration and assessment of
genetic fidelity of acclimated plantlets by SCoT, ISSR and RAPD markers in Rauwolfia
tetraphylla L.: an endangered medicinal plant. Biomed Res Int 2019:3698742
196. Dey A, Nongdam P, Nandy S, Mukherjee S, Tikendra L, Hazra AK, Pandey DK (2020)
Polyamine elicited aristolochic acid production in in vitro clonally fidel Aristolochia indica L.:
an ISSR and RAPD markers and HPTLC based study. S Afr J Bot https://doi.org/10.1016/j.
sajb.2020.06.018
197. Dey A, Nandy S, Nongdam P, Tikendra L, Mukherjee A, Mukherjee S, Pandey DK (2020)
Methyl jasmonate and salicylic acid elicit indole alkaloid production and modulate antioxidant
defence and biocidal properties in Rauvolfia serpentina Benth. ex Kurz. in vitro culture. S Afri
J Bot 135:1–17
198. Muralidharan K, Wakeland EK (1993) Concentration of primer and template qualitatively
affects products in random- amplified polymorphic DNA PCR. BioTechniques 14:362–364
199. Amom T, Tikendra L, Rahaman H, Potshangbam A, Nongdam P (2018) Evaluation of genetic
relationship between 15 bamboo species of North-East India based on ISSR marker analysis.
Mol Biol Res Commun 7:7–15
200. Zerini HN, Jafari H, Ramandi HD, Bolandi AR, Karimishahri (2019) A comparative assess-
ment of DNA fingerprinting assays of ISSR and RAPD markers for molecular diversity of
Saffron and other Crocus spp. in Iran. Nucleus 62:39–50
201. Gianchino RRA (2020) Investigation of the genetic variation of anise (Pimpinella anisum L.)
using RAPD and ISSR markers. Genet Resour Crop Evol 67:763–780
202. Zietkiewicz E, Rafalski A, Labuda D (1994) Genome fingerprinting by simple sequence repeat
(SSR) anchored polymerase chain reaction amplification. Genomics 20:176–183
203. Collard BCY, Mackill DJ (2009) Start codon targeted (SCoT) polymorphism: a simple, novel
DNA marker technique for generating gene-targeted markers in plants. Plant Mol Biol Rep
27:86–93
204. Amom T, Tikendra L, Apana N, Goutam M, Sonia P, Koijam AS, Potshangbam AM, Rahaman
H, Nongdam P (2020) Efficiency of RAPD, ISSR, iPBS, SCoT and phytochemical markers in
the genetic relationship study of five native and economical important bamboos of North-East
India. Phytochemistry 174:112330
205. Amom T, Nongdam P (2017) The use of molecular marker methods in plants: a review. Int J
Cur Res Rev 9:1–9
206. Hu J, Vick BA (2003) Target region amplification polymorphism: a novel marker technique for
plant genotyping. Plant Mol Biol Rep 21:289–294
207. Liu Q, Chen J, Corlett RT, Fan X, Yu D, Yang H, Gao J (2015) Orchid conservation in the
biodiversity hotspot of Southwestern China. Conserv Biol 29:1563–1572
208. Smulders M, de Klerk G (2011) Epigenetics in plant tissue culture. Plant Growth Regul
63:137–146
19 Dendrobium sp.: In vitro Propagation of Genetically Stable Plants. . . 493
209. Hao YJ, Wen XP, Deng XX (2004) Genetic and epigenetic evaluations of citrus calluses
recovered from slow-growth culture. J Plant Physiol 161:479–484
210. Baurens FC, Causse S, Lagavre T (2008) Methylation-sensitive amplification polymorphism
(MSAP) protocol to assess CpG and CpNpG methylation in the banana genome. Fruits
63:117–123
211. Morcello F, Gagneur C, Adam H, Richaud F, Singh R, Cheah SC, Rival A, Duval Y, Trebear
JW (2006) Somaclonal variation in micropropagated oil palm. Characterization of two novel
genes with enhanced expression in epigenetically abnormal cell lines and in response to auxin.
Tree Physiol 26:585–594
212. Wang D, Zhao J, Bai Y, Ao Y, Guo C (2017) The variation analysis of DNA methylation in
wheat carrying gametocidal chromosome 3c from Aegilops triuncialis. Int J Mol Sci 18:1738
213. Nadeem MA, Nawaz MA, Shahid MQ, Dogan Y, Comertpay G, Yildiz M, Hatipoglu R,
Ahmed F, Alsaleh A, Labhane N et al (2018) DNA molecular markers in plant breeding:
current status and recent advancement in genomic selection and genome editing. Biotechnol
Biotechnol Equip 32:261–285
214. Dey A, Hazra AK, Nongdam P, Nandy S, Tikendra L, Mukharjee A, Banerjee S, Mukherjee S,
Pandey DK (2019) Enhanced bacoside content in polyamine treated in vitro raised Bacopa
monnieri (L.) Wettst. S Afr J Bot 123:259–269
215. Gantait S, Mandai N, Bhattacharyya S, Kanti Das P (2010) Determination of genetic integrity
in long-term micropropagated plantlets of Allium ampeloprasum L. using ISSR markers.
Biotechnol 9:218–223
216. Rathore NNS, Rai MK, Phulwaria M, Rathore NNS, Shekhawat NS (2014) Genetic stability in
micropogated Cleome gynandra revealed by SCoT analysis. Acta Physiol Plant 36:555–559
217. Bhattacharyya P, Kumaria S, Diengdoh R, Tandon P (2014) Genetic stability and phytochem-
ical analysis of the in vitro regenerated plants of Dendrobium nobile Lindl., an endangered
medicinal orchid. Meta Gene 2:489–504
218. Rani V, Raina SN (2000) Genetic fidelity of organized meristem-derived micropropagated
plants: a critical reappraisal. In Vitro Cell Dev Biol Plant 36:319–330
219. Bhattacharyya P, Kumaria S, Job N, Tandon P (2015) Phyto-molecular profiling and assess-
ment of antioxidant activity within micropropagated plants of Dendrobium thyrsiflorum: a
threatened, medicinal orchid. Plant Cell Tissue Organ Cult 122:535–550
220. Wannajindaporn A, Poolsawat O, Chaowiset W, Tantasawat PA (2015) Evaluation of genetic
variability in in vitro sodium azide-induced Dendrobium ‘Earsakul’ mutants. Genet Mol Res
13(3):5333–5342
221. Bhattachryya P, van Staden J (2018) Molecular insights into genetic diversity and population
dynamics of five medicinal Eulophia species: a threatened orchid texa of Africa. Physiol Mol
Biol Plants 24:631–641
222. Poczai P, Varga I, Laos M et al (2013) Advances in plant gene targeted and functional markers:
a review. Plant Methods 9:6
223. Doyle JJ, Doyle JL (1990) Isolation of plant DNA from fresh tissue. Focus 12:13–15
224. Galvan MZ, Bornet B, Balatti PA, Balachandra M (2003) Inter simple sequence repeat (ISSR)
markers as a tool for the assessment of both genetic diversity and gene pool origin in common
bean (Phaseolus vulgaris L.). Euphytica 132:297–301
225. Ajibade SR, Weeden NF, Chite SM (2000) Inter simple sequence repeat analysis of genetic
relationships in the genus Vigna. Euphytica 111:47–55
Eulophia spp.: In Vitro Generation, Chemical
Constituents, and Pharmacological 20
Activities
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
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
498 V. Shriram and V. Kumar
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
20 Eulophia spp.: In Vitro Generation, Chemical Constituents, and. . . 499
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
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
4 Pharmacological Activities
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],
502 V. Shriram and V. Kumar
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].
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.
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. . .
(continued)
506
Table 2 (continued)
Plant
Eulophia species part Extract/Pure molecule Ethnopharmacological/Pharmacological activities References
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. . .
(continued)
508
Table 2 (continued)
Plant
Eulophia species part Extract/Pure molecule Ethnopharmacological/Pharmacological activities References
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
20 Eulophia spp.: In Vitro Generation, Chemical Constituents, and. . . 509
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
510 V. Shriram and V. Kumar
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
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
512 V. Shriram and V. Kumar
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)].
References
1. Abhyankar RK, Upadhyay R (2011) Ethnomedicinal studies of tubers of Hoshangabad
M.P. Bull Environ Pharmcol Life Sci 1:57–59
2. Ahirrao YA, Patil MV, Patil DA (2008) On identities and ethnomedicinal claims of some common
botanicals sold by vendors in North Maharashtra, India. Indian J Tradit Knowl 2:9–13
20 Eulophia spp.: In Vitro Generation, Chemical Constituents, and. . . 513
3. Alam MK, Rashid MH, Hossain MS, Salam MA, Rouf MA (2002) In vitro seed propagation of
Dendrobium (Dendrobium transparens) orchid as influenced by different Media. Biotechnol
1:111–115
4. Arenmongla T, Deb CR (2012) Germination of immature embryos and multiplication of
Malaxis acuminate D. Don, an endangered therapeutically important orchid, by asymbiotic
culture in vitro. Indian J Biotechnol 11:464–469
5. Bhagaonkar PY, Kadam VN (2006) Ethnopharmacology of Bajara tribe of Umarkhed taluka,
district Yavatmal, Maharashtra for reproductive disorders. Indian J Tradit Knowl 5:336–341
6. Bhandari SR, Kapadi AH (1983) A 9, 10-dihydrophenanthrene from tubers of Eulophia nuda.
Phytochemistry 22:747–748
7. Bhandari SR, Kapadi AH, Majumder PL, Joardar M, Shoolery JN (1985) Nudol, a phenanthrene
of the orchids Eulophia nuda, Eria carinata and Eria stricta. Phytochemistry 24:801–804
8. Bhatt D, Jethva K, Zaveri M (2020) Phytopharmacognostical study of tubers of Eulophia nuda
Lindl. Int J Pharm Sci Res 49:3483–3488
9. Bhurat MR, Kawatikwar PS, Sanghavi RS, Patil PP, Salunke PA, Kapure SV (2012) Evalu-
ation of Eulophia herbacea tubers mucilage as an innovative suspending agent. J Pharm Res
5:321–323
10. Bulpitt CJ (2005) The uses and misuses of orchids in medicine. QJM: An Int J Med 98:625–631
11. Chanda R, Mohanty JP, Bhuyan NR, Kar PK, Nath LK (2007) Medicinal plants used against
gastrointestinal tract disorders by the traditional healers of Sikkim Himalayas. Indian J Tradit
Knowl 6:606–610
12. Chang C, Hu WH, Chen YC, Su YL, Chiu YT (2010) In vitro flowering and mating system of
Eulophia graminea Lindl. Bot Stud 51:357–362
13. Chase MW, Cameron KM, Freudenstein JV, Pridgeon AM, Salazar G, Van den Berg C,
Schuiteman A (2008) An updated classification of Orchidaceae. Bot J Linn Soc 177(2):151–174
14. Das S, Dash SK, Padhy SN (2003) Ethno-medicinal informations from Orissa State, India, A
review. J Hum Ecol 14:165–227
15. Datla P, Kalluri MD, Basha K, Bellary A, Kshirsagar R, Kanekar Y et al (2010) 9,10-Dihydro-
2,5- dimethoxyphenanthrene-1,7-diol, from Eulophia ochreata, inhibits inflammatory signalling
mediated by Toll like receptors. Br J Pharmacol 160:1158–1170
16. Dawande V, Gurav R (2015) In-vitro seed germination and effect of growth regulators on
subsequent development of protocorms of Eulophia nuda Lindl. Int J Appl Sci Biotechnol 3:
243–247
17. Decruse SW, Reny N, Shylajakumari S, Krishnan PN (2013) In vitro propagation and field
establishment of Eulophia cullenii (Wight) Bl., a critically endangered orchid of Western Ghats,
India through culture of seeds and axenic seedling-derived rhizomes. In Vitro Cell Dev Biol-
Plant 49:520–528
18. Devkar S, Jagtap SD, Kale Y, Kasote D (2009) Antibacterial activity of Eulophia ochreata L.
tubers. J Herb Med Toxicol 3:31–33
19. Dey A, Nath De J (2010) A survey of ethnomedicinal plants used by the tribals of Ajoydha hill
region, Purulia District, India. Am-Eurasian J Sustain Agric 4:280–290
20. Dodson CH (2020) Orchid. Encyclopædia Britannica. Available at https://www.britannica.com/
plant/orchid. Accessed 20 Aug 2020
21. Fay MF (2018) Orchid conservation: how can we meet the challenges in the twenty-first
century? Bot Stud 59:16
22. Ghule BV, Darwhekar GD, Jain DK, Yeole PG (2006) Evaluation of binding properties of
Eulophia campestris Wall. Mucilage. Indian J Pharm Sci 68:566–569
23. Gledhill D (2002) The names of plants, 3rd edn. Cambridge University Press. https://doi.org/10.
1017/CBO9780511754951
24. Gupta VK, Kaushik A, Chauhan DS, Ahirwar RK, Sharma S, Bisht D (2018) Anti-
mycobacterial activity of some medicinal plants used traditionally by tribes from Madhya
Pradesh, India for treating tuberculosis related symptoms. J Ethnopharmacol 227:113–120
25. Gutierrez RMP (2010) Orchids: A review of uses in traditional medicine, its phytochemistry
and pharmacology. J Med Plants Res 4:592–638
514 V. Shriram and V. Kumar
26. Hew CS (2001) Ancient Chinese orchid cultivation: a fresh look at an age-old practice. Scientia
Horticulturae 87(1–2):1–10
27. Hossain MM (2011) Therapeutic orchids: traditional uses and recent advances – an overview.
Fitoterapia 82(2):102–140
28. Hossain MM (2015) Ex vitro seedling development from in vitro rhizome-like bodies in
Eulophia promensis Lindl.: a new technique for orchid propagation. J Bot:207694. https://
doi.org/10.1155/2015/207694
29. Jagtap SD, Deokule SS, Bhosle SV (2008) Ethanobotanical uses of endemic and RET plants by
Pawra tribe of Nandurbar district, Maharashtra. Indian J Tradit Knowl 7:311–315
30. Jagdale SP, Shimpi S, Chachad D (2009) Pharmacological studies of Salep. Herb Med Toxicol
3:153–156
31. Jagtap SD, Deokule SS, Pawar PK, Harsulkar AM (2009) Traditional ethnomedicinal knowl-
edge confined to the Pawra tribe of Satpura Hills, Maharashtra, India. Ethnobot Lealf 13:98–115
32. Jain A, Katewa SS, Galav PK, Sharma P (2005a) Medicinal plant diversity of wildlife sanctuary,
Rajasthan, India. J Ethnopharmacol 102:143–157
33. Jain A, Katewa SS, Galav PK (2005b) Some phytotherapeutic claims by tribals of southern
Rajasthan. Indian J Tradit Knowl 4:291–297
34. Jain B, Kumane SC, Bhattacharya S (2006) Medicinal flora of Madhya Pradesh and Chhattis-
garh – a review. Indian J Tradit Knowl 5:237–242
35. Jalal JS, Kumar P, Pangtey YPS (2008) Ethnomedicinal Orchids of Uttarakhand, Western
Himalaya. Ethnobot Leafl 1:164
36. Jansakul C, Yorsin S, Naphatthalung J, Tachanaparugse K, Changwichai K, Chootip K,
Ingkaninan K (2019) Relaxant mechanism of Eulophia macrobulbon ethanolic extract and
1-(4’-hydroxybenzyl)-4, 8-dimethoxyphenanthrene-2, 7-diol on human corpus cavernosum.
Funct Foods Health Dis 9:328–340
37. Johnson TR, Stewart SL, Dutra D, Kane ME, Richardson L (2007) Asymbiotic and symbiotic
seed germination of Eulophia alta (Orchidaceae)-preliminary evidence for the symbiotic culture
advantage. Plant Cell Tissue Organ Cult 90:313–323
38. Jones DL (2006) A complete guide to native orchids of Australia including the island territories.
Frenchs Forest, N.S.W, New Holland, p 358
39. Joshi GC, Tewari LM, Lohani N, Upreti K, Jalal JS, Tewari G (2009) Diversity of orchids in
Uttarakhand and their conservation strategy with special reference to their medicinal impor-
tance. Rep Opin 1:47–52
40. Kapale R (2012) Ethnobotany of Baiga tribals with reference to utilization of forest resources in
Achanakmar biosphere reserve (India). Bull Env Pharm Life Sci 1(6):73–76
41. Karuppusamy S (2007) Medicinal plants used by Paliyan tribes of Sirumalai hills of southern
India. Nat Prod Radiance 6:436–442
42. Kayalvizhi K, Divya K, Sankari A (2020) Medicinal orchids – an overview. Biotica Res Today
2:1084–1087
43. Kirtikar KR, Basu BD (1918) Indian medicinal plants. Springer, Allahabad, pp 1242–1243
44. Kong JM, Goh NK, Chia LS, Chia TF (2003) Recent advances in traditional plant drugs and
orchids. Acta Pharmacol Sin 24:7–21
45. Kovács A, Vasas A, Hohmann J (2008) Natural phenanthrenes and their biological activity.
Phytochemistry 69:1084–1110
46. Kshirsagar RD, Kanekar YB, Jagtap SD, Upadhyay SN, Rao R, Bhujbal SP et al (2010)
Phenanthrenes of Eulophia ochreata Lindl. Int J Green Pharm 4:147–152
47. Kumar V, Lemos M, Sharma M, Shriram V (2013) Antioxidant and DNA damage protecting
activities of Eulophia nuda Lindl. Free Radicals Antioxid 3:55–60
48. Mahendran G, Muniappan V, Ashwini M, Muthukumar T, NarmathaBai V (2013) Asymbiotic
seed germination of Cymbidium bicolor Lindl. (Orchidaceae) and the influence of mycorrhizal
fungus on seedling development. Acta Physiol Plant 35:829–840
49. Mali PY, Bhadane VV (2008) Some rare plants of ethnomedicinal properties from Jalgaon
district of Maharashtra. Int J Green Pharm 2:76–78
50. Maridass M (2011) Anti diarrhoeal activity of rare orchid Eulophia epidendraea (Retz.) Fisher.
Nat Pharm Technol 1:5–10
20 Eulophia spp.: In Vitro Generation, Chemical Constituents, and. . . 515
75. Rajendran A, Rao NR, Kumar KR, Henry AN (1997) Some medicinal orchids of Southern
India. Anc Sci Life 17:1–4
76. Sastri BN (1952) The wealth of India, A dictionary of Indian raw materials and industrial
products: raw materials, vol 3. Council of Scientific Industrial Research, New Delhi, p 221
77. Schuster R, Zeindl L, Holzer W, Khumpirapang N, Okonogi S, Viernstein H, Mueller M (2017)
Eulophia macrobulbon – an orchid with significant anti-inflammatory and antioxidant effect and
anticancerogenic potential exerted by its root extract. Phytomedicine 24:157–165
78. Sharma M, Vij SR (1986) Micropropagation of Eulophia dabia (D. Don) Hochr. Orchid
News, 2(11)
79. Shriram V, Kumar V, Kishor PB, Suryawanshi SB, Upadhyay AK, Bhat MK (2010) Cytotoxic
activity of 9,10-dihydro-2,5-dimethoxyphenanthrene-1,7-diol from Eulophia nuda against
human cancer cells. J Ethnopharmacol 128:251–253
80. Shriram V, Nanekar V, Kumar V, Kishor PB (2014) In vitro regeneration and ploidy level
analysis of Eulophia ochreata Lindl. Indian J Exp Biol 52:1112–1121
81. Shroti RK, Upadhyay R (2014) In vitro micropropagation of Eulophia nuda Lind an endangered
terrestrial orchid through PLB (protocorm like bodies). Int J Plant Animal Environ Sci 4:172–176
82. Sikarwar RL, Pathak B, Jaiswal A (2008) Some unique ethnomedicinal perceptions of tribal
communities of Chitrakoot, Madhya Pradesh. Indian J Tradit Knowl 7:613–617
83. Singh A, Duggal S (2009) Medicinal orchids: an overview. Ethnobot Leaflets 13:351–363
84. Swarts ND, Dixon KW (2009) Terrestrial orchid conservation in the age of extinction. Ann Bot
104:543–556
85. Tatiya AU, Puranik PM, Surana SJ, Patil YS, Mutha RE (2013) Evaluation of hypolipidemic,
antidiabetic and anti-oxidant activity of Eulophia herbacea tubers. Bangladesh J Pharmacol 8:
269–275
86. Tatiya AU, Bari N, Surana SJ, Kalaskar MG (2014) Effect of bioassay guided isolation of
1-phenanthrene carboxylic acid from Eulophia herbacea Lindl. Tubers on human cancer cell
lines. Res J Phytochem 8:155–161
87. Tayade SK, Patil DA (2005) Hitherto untapped plantlore from Nandurbar district (Maharashtra).
Nat Prod Radiance 4:46–50
88. Temkitthawon P, Changwichit K, Khorana N, Viyoch J, Suwanborirux K, Ingkaninan K (2017)
Phenanthrenes from Eulophia macrobulbon as novel phosphodiesterase-5 inhibitors. Nat Prod
Commun 12:1934578X1701200121
89. Teoh ES (2016) Medicinal orchids of Asia. Springer International Publishers, Basel
90. Thomas S (1998) Note on nomenclature and typification of Eulophia hians Spreng.
(Orchidaceae). Kew Bull 1005–1007
91. Tuchinda P, Udchachon J, Khumtaveeporn K, Taylor WC, Engelhardt LM, White AH (1988)
Phenanthrenes of Eulophia nuda. Phytochemistry 27:3267–3271
92. Tuchinda P, Udchachon J, Khumtayeeporn K, Taylor WC (1989) Benzylated phenanthrenes
from Eulophi nuda. Phytochemistry 28:2463–2466
93. Upadhyay S, Kanetkar Y, Kshirsagar R, Rajgopal V, Datla P, Bellary A, Singh SP (2009) Toll
like receptor (TLR) signaling antagonist. US Patent 20090215908
94. Vij SP, Sood A, Pathak P (1989) On the utility of rhizome segments in micropropagating
Eulophia hormusjii Duth. J Orchid Soc India 3:41–45
95. Wisutthathum S, Demougeot C, Totoson P, Adthapanyawanich K, Ingkaninan K,
Temkitthawon P, Chootip K (2018) Eulophia macrobulbon extract relaxes rat isolated pulmo-
nary artery and protects against monocrotaline-induced pulmonary arterial hypertension.
Phytomedicine 50:157–165
96. Yadav DP, Chhipa RC, Singh B (2012) Anti-glycation effect of whitton root (eulophia nuda) in-
vitro condition. Int J Pharm Sci Res 3:3502
97. Yan-Qiu Z, Rong-Yi Z, Rui M, Zhi-Qiong T, Yu-Jie W (2009) Endophytic fungi of roots of
Eulophia flava. Southwest China J Agric Sci 22:675–680
Cyrtopodium glutiniferum, an Example of
Orchid Used in Folk Medicine: 21
Phytochemical and Biological Aspects
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
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
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.
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],
520 C. F. Araujo-Lima et al.
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].
21 Cyrtopodium glutiniferum, an Example of Orchid Used in Folk. . . 521
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].
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.
21 Cyrtopodium glutiniferum, an Example of Orchid Used in Folk. . . 523
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
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])
526 C. F. Araujo-Lima et al.
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.
5 Conclusions
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.
References
1. Batista JAN, Bianchetti LB (2004) Three new taxa in Cyrtopodium (Orchidaceae) from central
and southeastern Brazil. Brittonia 56(3):260–274. https://doi.org/10.1663/0007-196X(2004)
056[0260:TNTICO]2.0.CO;2
2. Barreto DW, Parente JP (2006) Chemical properties and biological activity of a polysaccharide
from Cyrtopodium cardiochilum. Carbohydr Polym 64(2):287–291. https://doi.org/10.1016/j.
carbpol.2005.11.038
3. Dutra D, Kane ME, Richardson L (2009) Asymbiotic seed germination and in vitro seedling
development of Cyrtopodium punctatum: a propagation protocol for an endangered Florida
native orchid. Plant Cell Tissue Organ Cult 96(3):235–243. https://doi.org/10.1007/s11240-
008-9480-z
4. Chase MW, Cameron KM, Freudenstein JV et al (2015) An updated classification of
Orchidaceae. Bot J Linn Soc 177(2):151–174. https://doi.org/10.1111/boj.12234
5. Romero-González GA, Carnevali G, Duno R et al (2019) A new species of Cyrtopodium
(Orchidaceae: Cyrtopodiinae) from Belize. Phytotaxa 424(2):71–86. https://doi.org/10.11646/
phytotaxa.424.2.1
6. Maciel AA, Cardoso JCF, Oliveira PE (2020) On the low reproductive success of two
Cyrtopodium species (Orchidaceae: Cyrtopodiinae): the relative roles of biotic and abiotic
pollination. Plant Species Biol 35(1):49–58. https://doi.org/10.1111/1442-1984.12260
7. Vogel IN, Macedo AF (2011) Influence of IAA, TDZ, and light quality on asymbiotic germi-
nation, protocorm formation, and plantlet development of Cyrtopodium glutiniferum Raddi., a
21 Cyrtopodium glutiniferum, an Example of Orchid Used in Folk. . . 529
26. Guimarães FAR, Pereira MC, Felício C d S et al (2013) Symbiotic propagation of seedlings of
Cyrtopodium glutiniferum Raddi (Orchidaceae). Acta Bot Bras 27(3):590–596. https://doi.org/
10.1590/S0102-33062013000300016
27. Pereira MC, Rocha DI, Veloso TGR et al (2015) Characterization of seed germination and
protocorm development of Cyrtopodium glutiniferum (Orchidaceae) promoted by mycorrhizal
fungi Epulorhiza spp. Acta Bot Bras 29(4):567–574. https://doi.org/10.1590/0102-
33062015abb0078
28. Sousa KCI, De Araújo LG, De Sousa Silva C et al (2019) Seed germination and development of
orchid seedlings (Cyrtopodium saintlegerianum) with fungi. Rodriguesia 70. https://doi.org/10.
1590/2175-7860201970004
29. Rodrigues LA, de Paiva Neto VB, Boaretto AG et al (2015) In vitro propagation of
Cyrtopodium saintlegerianum Rchb. f. (Orchidaceae), a native orchid of the Brazilian savan-
nah. Crop Breed Appl Biotechnol 15(1):10–17. https://doi.org/10.1590/1984-
70332015v15n1a2
30. Couto DR, Francisco TM, Manhães VC, Dias HM., Pereira MCA (2017) Floristic composition
of a Neotropical inselberg from Espírito Santo state, Brazil: an important area for conserva-
tion. Check List 13(1):2043–2054
31. do Brasil F. 2020 Flora do Brasil. Jardim Botânico do Rio de Janeiro. http://floradobrasil.jbrj.
gov.br/. Acessed 24 June 2020
32. Silva AG, Boldrini RF, Kuster RM (2013) Os sumarés cicatrizantes da medicina tradicional
brasileira, ou, as surpresas químicas ativas do desconhecido gênero Cyrtopodium
(Orchidaceae). Nat Line 11:152–154
33. Corrêa MP, de Azeredo Penna L (1984) Dicionário Das Plantas Úteis Do Brasil e Das Exóticas
Cultivadas: HL (Vol. 4). Ministério da Agricultura, Instituto Brasileiro de Desenvolvimento
Florestal
34. Gonçalves Brasileiro B, Ramos Pizziolo V, Soares Raslan D, Mashrouah Jamal C, Silveira D
(2006) Antimicrobial and cytotoxic activities screening of some Brazilian medicinal plants used
in Governador Valadares district. Rev Bras Cienc Farm 42(2):195–202. https://doi.org/10.1590/
s1516-93322006000200004
35. Pereira CEDB, Felcman J (1998) Correlation between five minerals and the healing effect of
Brazilian medicinal plants. Biol Trace Elem Res 65(3):251–259. https://doi.org/10.1007/
bf02789100
36. Boscolo OH, De Senna Valle L (2008) Plantas de uso medicinal em Quissamã, Rio de Janeiro,
Brasil. Iheringia Ser Bot 63(2):263–272
37. Boscolo OH, Lucia RRMV, Fernandes, De L (2010) An ethnobotanical survey as subsidy for
the generation of researches related to biotechnology. Int Res J Biotechnol 1:1–6
38. Parente JP, Adão CR, Da Silva BP, Tinoco LW (2014) Structural characterization of an
acetylated glucomannan with antiinflammatory activity and gastroprotective property from
Cyrtopodium andersonii. Carbohydr Res 391(1):16–21. https://doi.org/10.1016/j.carres.2014.
03.021
39. Morales-Sánchez V, Rivero-Cruz I, Laguna-Hernández G, Salazar-Chávez G, Mata R (2014)
Chemical composition, potential toxicity, and quality control procedures of the crude drug of
Cyrtopodium macrobulbon. J Ethnopharmacol 154(3):790–797. https://doi.org/10.1016/j.jep.
2014.05.006
40. Auberon F, Olatunji OJ, Herbette G et al (2016) Chemical constituents from the aerial parts of
Cyrtopodium paniculatum. Molecules 21(10):1–18. https://doi.org/10.3390/
molecules21101418
41. Auberon F, Olatunji OJ, Raminoson D et al (2017) Isolation of novel stilbenoids from the roots
of Cyrtopodium paniculatum (Orchidaceae). Fitoterapia 116:99–105. https://doi.org/10.1016/j.
fitote.2016.11.015
42. Teoh ES (2019) Orchids as aphrodisiac, medicine or food. Springer, Singapore
43. Magalhães K do N, Guarniz WAS, Sá KM et al (2019) Medicinal plants of the Caatinga,
northeastern Brazil: ethnopharmacopeia (1980–1990) of the late professor Francisco José de
Abreu Matos. J Ethnopharmacol 237:314–353. https://doi.org/10.1016/j.jep.2019.03.032
21 Cyrtopodium glutiniferum, an Example of Orchid Used in Folk. . . 531
44. Martínez GJ, Barboza GE (2010) Natural pharmacopoeia used in traditional Toba medicine for
the treatment of parasitosis and skin disorders (Central Chaco, Argentina). J Ethnopharmacol
132(1):86–100. https://doi.org/10.1016/j.jep.2010.07.049
45. Araújo-Lima CF, Paula da Silva Oliveira J, Coscarella IL et al (2020) Metabolomic analysis of
Cyrtopodium glutiniferum extract by UHPLC-MS/MS and in vitro antiproliferative and
genotoxicity assessment. J Ethnopharmacol 253:112607. https://doi.org/10.1016/j.jep.2020.
112607
46. Sponchiado G, Adam ML, Silva CD et al (2016) Quantitative genotoxicity assays for analysis
of medicinal plants: a systematic review. J Ethnopharmacol 178:289–296. https://doi.org/10.
1016/j.jep.2015.10.026
47. Dvorakova M, Landa P (2017) Anti-inflammatory activity of natural stilbenoids: a review.
Pharmacol Res 124:126–145. https://doi.org/10.1016/j.phrs.2017.08.002
48. Oliviero F, Scanu A, Zamudio-Cuevas Y, Punzi L, Spinella P (2018) Anti-inflammatory effects
of polyphenols in arthritis. J Sci Food Agric 98(5):1653–1659. https://doi.org/10.1002/jsfa.
8664
49. Russo GL, Tedesco I, Spagnuolo C, Russo M (2017) Antioxidant polyphenols in cancer
treatment: friend, foe or foil? Semin Cancer Biol 46:1–13. https://doi.org/10.1016/j.
semcancer.2017.05.005
50. Malaguarnera L (2019) Influence of resveratrol on the immune response. Nutrients 11(5):1–24.
https://doi.org/10.3390/nu11050946
51. Yahfoufi N, Alsadi N, Jambi M, Matar C (2018) The immunomodulatory and anti-inflammatory
role of polyphenols. Nutrients 10(11):1–23. https://doi.org/10.3390/nu10111618
52. Atanaskova N, Tomecki KJ (2010) Innovative management of recurrent furunculosis. Dermatol
Clin 28(3):479–487. https://doi.org/10.1016/j.det.2010.03.013
53. Pinkus H (2009) Short communication. Clin Chem Lab Med 26(9):517–518. https://doi.org/10.
1515/cclm.1988.26.9.579
54. Roje Z, Roje Ž, Matić D, Librenjak D, Dokuzović S, Varvodić J (2011) Necrotizing fasciitis:
literature review of contemporary strategies for diagnosing and management with three case
reports: torso, abdominal wall, upper and lower limbs. World J Emerg Surg 6(1):1–17. https://
doi.org/10.1186/1749-7922-6-46
55. Chambers HF (2008) Clinical decisions management of skin and soft-tissue infection incision
and drainage plus anti-MSSA therapy:1063–1067
56. Dornelas MT, Correa M d PD, Barra FML et al (2012) Fournier’s syndrome: a 10-year
evaluation study. Rev Bras Cir Plást 27(4):600–604
57. Clinical and Laboratory Standards Institute (CLSI) (2015) M07-A10: methods for dilution
antimicrobial susceptibility tests for bacteria that grow aerobically; approved standard – tenth
edition. Clin Infect Dis 35(2). www.clsi.org
58. Lei Z, Zhang D, Lu B, Zhou W, Wang D (2018) Activation of mast cells in skin abscess induced
by Staphylococcus aureus (S. aureus) infection in mice. Res Vet Sci 118(8):66–71
59. Kobayashi SD, Malachowa N, Deleo FR (2015) Pathogenesis of Staphylococcus aureus
abscesses. Am J Pathol 185(6):1518–1527. https://doi.org/10.1016/j.ajpath.2014.11.030
60. Natsis NE, Cohen PR (2018) Coagulase-negative Staphylococcus skin and soft tissue infec-
tions. Am J Clin Dermatol 19(5):671–677. https://doi.org/10.1007/s40257-018-0362-9
61. Żbikowska B, Franiczek R, Sowa A, Połukord G, Krzyzanowska B, Sroka Z (2017) Antimi-
crobial and antiradical activity of extracts obtained from leaves of five species of the genus
Bergenia: identification of antimicrobial compounds. Microb Drug Resist 23(6):771–780.
https://doi.org/10.1089/mdr.2016.0251
62. Moghrovyan A, Sahakyan N, Babayan A, Chichoyan N, Petrosyan M, Trchounian A (2019)
Essential oil and ethanol extract of oregano (Origanum vulgare L.) from Armenian flora as a
natural source of terpenes, flavonoids and other phytochemicals with antiradical, antioxidant,
metal chelating, tyrosinase inhibitory and antibacterial activity. Curr Pharm Des 25(16):1809–
1816. https://doi.org/10.2174/1381612825666190702095612
63. Ma C, He N, Zhao Y, Xia D, Wei J, Kang W (2019) Antimicrobial mechanism of hydroquinone.
Appl Biochem Biotechnol 189(4):1291–1303. https://doi.org/10.1007/s12010-019-03067-1
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
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.
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 (continued)
Species Compound Ref.
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)
Species Compound Ref.
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)
22 Phenanthrenes from Orchidaceae and Their Biological Activities 539
Table 1 (continued)
Species Compound Ref.
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
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
22 Phenanthrenes from Orchidaceae and Their Biological Activities 541
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.
22 Phenanthrenes from Orchidaceae and Their Biological Activities 543
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].
22 Phenanthrenes from Orchidaceae and Their Biological Activities 545
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].
22 Phenanthrenes from Orchidaceae and Their Biological Activities 549
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].
22 Phenanthrenes from Orchidaceae and Their Biological Activities 551
3 Chemotaxonomical Significance
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].
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].
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].
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
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
References
1. The Plant List:Orchidaceae http://www.theplantlist.org/1.1/browse/A/Orchidaceae/ Retrieved
18 November 2020
2. Dressler RL (1993) Phylogeny and classification of the orchid family. Dioscorides Press,
Portland
3. Dressler RL (1981) The orchids: natual history and classification. Harvard University Press
4. Gutiérrez RMP (2010) Orchids: a review of uses in traditional medicine, its phytochemistry
and pharmacology. J Med Plant Res 4:592–638
5. Tóth B, Hohmann J, Vasas A (2018) Phenanthrenes: a promising group of plant secondary
metabolites. J Nat Prod 81:661–678
6. Chinsamy M, Finnie JF, Van Staden J (2014) Anti-inflammatory, antioxidant, anti-
cholinesterase activity and mutagenicity of South African medicinal orchids. South Afr J
Bot 91:88–98
7. Kovács A, Vasas A, Hohmann J (2008) Natural phenanthrenes and their biological activity.
Phytochemistry 69:1084–1110
22 Phenanthrenes from Orchidaceae and Their Biological Activities 563
30. Lee CL, Chang FR, Yen MH, Yu D, Liu YN, Bastow KF, Morris-Natschke SL, Wu YC, Lee
KH (2009) Cytotoxic phenanthrenequinones and 9,10- dihydrophenanthrenes from Calanthe
arisanensis. J Nat Prod 72:210–213
31. Lee CL, Nakagawa-Goto K, Yu D, Liu YN, Bastow KF, Morris-Natschke SL, Chang FR, Wu
YC, Lee KH (2008) Cytotoxic calanquinone A from Calanthe arisanensis and its first total
synthesis. Bioorg Med Chem Lett 18:4275–4277
32. Chang CF, Hsu YL, Lee CY, Wu CH, Wu YC, Chuang TH (2015) Isolation and cytotoxicity
evaluation of the chemical constituents from Cephalantheropsis gracilis. Int J Mol Sci 16:
3980–3989
33. Yoshikawa K, Ito T, Iseki K, Baba C, Imagawa H, Yagi Y, Morita H, Asakawa Y, Kawano S,
Hashimoto T (2012) Phenanthrene derivatives from Cymbidium great flower Marie Laurencin
and their biological activities. J Nat Prod 75:605–609
34. Yoshikawa K, Baba C, Iseki K, Ito T, Asakawa Y, Kawano S, Hashimoto T (2014) Phenan-
threne and phenylpropanoid constituents from the roots of Cymbidium great flower
‘Marylaurencin’ and their antimicrobial activity. J Nat Med 68:743–747
35. Zhao N, Yang G, Zhang Y, Chen L, Chen Y (2016) A new 9,10-dihydrophenanthrene from
Dendrobium moniliforme. Nat Prod Res 30:174–179
36. Kuo W, Huang YL, Shen CC, Shieh BJ, Chen CC (2007) Prenylated benzoic acids and
phenanthrenes from Liparis nakaharai. J Chin Chem Soc 54:1359–1362
37. Yamaki M, Kato T, Bai L, Inoue K, Takagi S (1991) Methylated stilbenoids from Bletilla
striata. Phytochemistry 30:2759–2760
38. Majumder PL, Banerjee S, Sen S (1996) Three stilbenoids from the orchid Agrostophyllum
callosum. Phytochemistry 42:847–852
39. Wu YP, Liu WJ, Zhong WJ, Chen YJ, Chen DN, He F, Jiang L (2017) Phenolic compounds
from the stems of Flickingeria fimbrata. Nat Prod Res 31:1518–1522
40. Majumder PL, Banerjee S (1988) Structure of flavanthrin, the first dimeric 9,10-
dihydrophenanthrene derivative from the orchid Eria flava. Tetrahedron 44:7303–7308
41. Apel C, Dumontet V, Lozach O, Meijer L, Guéritte F, Litaudon M (2012) Phenanthrene
derivatives from Appendicula reflexa as new CDK1/cyclin B inhibitors. Phytochem Lett 5:
814–818
42. Yan X, Tang B, Liu M (2018) Phenanthrenes from Arundina graminifolia and in vitro
evaluation of their antibacterial and anti-haemolytic properties. Nat Prod Res 32:707–710
43. Lin CW, Hwang TL, Chen FA, Wu TS, Huang CH, Hung HY, Wu TS (2016) Chemical
constituents of the rhizomes of Bletilla formosana and their potential anti-inflammatory
activity. J Nat Prod 79:1911–1921
44. Zhou D, Chen G, Ma YP, Wang CG, Lin B, Yang YQ, Li W, Koike K, Hou Y, Li N (2019)
Isolation, structure elucidation, optical resolution, and antineuroinflammatory activity of
phenanthrene and 9,10-dihydrophenanthrene derivatives from Bletilla striata. J Nat Prod 82:
2238–2245
45. Chen Y, Xu J, Yu H, Qing C, Zhang Y, Wang L, Liu Y, Wang J (2008) Cytotoxic phenolics
from Bulbophyllum odoratissimum. Food Chem 107:169–173
46. Leong YW, Kang CC, Harrison LJ, Powell AD (1997) Phenanthrenes, dihydrophenanthrenes
and bibenzyls from the orchid Bulbophyllum vaginatum. Phytochemistry 44:157–165
47. Majumder P, Laha S, Datta N (1982) Coelonin, a 9,10-dihydrophenanthrene from the orchids
Coelogyne ochracea and Coelogyne elata. Phytochemistry 21:478–480
48. Tu Y, Huang J, Li Y (2018) Anticholinesterase, antioxidant, and beta-amyloid aggregation
inhibitory constituents from Cremastra appendiculata. Med Chem Res 27:857–863
49. Juneja RK, Sharma SC, Tandon JS (1987) Two substituted bibenzyls and a
dihydrophenanthrene from Cymbidium aloifolium. Phytochemistry 26:1123–1125
50. Lertnitikul N, Pattamadilok C, Chansriniyom C, Suttisri R (2020) A new dihydrophenanthrene
from Cymbidium finlaysonianum and structure revision of cymbinodin-A. J Asian Nat Prod
Res 22:83–90
22 Phenanthrenes from Orchidaceae and Their Biological Activities 565
51. Auberon F, Olatunji OJ, Raminoson D, Muller CD, Soengas B, Bonte F, Lobstein A (2017)
Isolation of novel stilbenoids from the roots of Cyrtopodium paniculatum (Orchidaceae).
Fitoterapia 116:99–105
52. Inthongkaew P, Chatsumpun N, Supasuteekul C, Kitisripany T, Putalun W,
Likhitwitayawuid K, Sritularak B (2017) α-Glucosidase and pancreatic lipase inhibitory
activities and glucose uptake stimulatory effect of phenolic compounds from Dendrobium
formosum. Rev Bras Farmacogn 27:480–487
53. Chen DN, Wang YY, Liu WJ, Chen YJ, Wu YP, Wang JX, He F, Jiang L (2020) Stilbenoids
from aerial parts of Dendrobium plicatile. Nat Prod Res 34:323–328
54. Sarakulwattana C, Mekboonsonglarp W, Likhitwitayawuid K, Rojsitthisak P, Sritularak B
(2020) New bisbibenzyl and phenanthrene derivatives from Dendrobium scabrilingue and
their α-glucosidase inhibitory activity. Nat Prod Res 34:1694–1701
55. Majumder PL, Banerjee S (1990) Two stilbenoids from the orchid Eria flava. Phytochemistry
29:3052–3055
56. Schuster R, Zeindl L, Holzer W, Khumpirapang N, Okongi S, Viernstein H, Mueller M (2017)
Eulophia macrobulbon – an orchid with significant anti-inflammatory and antioxidant effect
and anticancerogenic potential exerted by its root extract. Phytomedicine 24:157–165
57. Tuchinda P, Udchachon J, Khumtaveeporn K, Taylor WC, Engelhardt LM, White AH (1988)
Phenanthrenes of Eulophia nuda. Phytochemistry 27:3267–3271
58. Blitzke T, Masaoud M, Schmidt J (2000) Constituents of Eulophia petersii. Fitoterapia 71:
593–594
59. Matsuda H, Morikawa T, Xie H, Yoshikawa M (2004) Antiallergic phenanthrenes and
stilbenes from the tubers of Gymnadenia conopsea. Planta Med 70:847–855
60. Morikawa T, Xie H, Matsuda H, Wang T, Yoshikawa M (2006) Bioactive constituents from
Chinese natural medicines. XVII. Constituents with radical scavenging effect and new
glucosyloxybenzyl 2-isobutylmalates from Gymnadenia conopsea. Chem Pharm Bull 54:506–513
61. Chen Y, Cai S, Deng L, Xia Q, Du LF, Cui GZ, Li J, Zhou XM, Ye Q, Zhou Y et al (2015)
Separation and purification of 9,10-dihydrophenanthrenes and bibenzyls from Pholidota
chinenesis by high-speed countercurrent chromatography. J Sep Sci 38:453–459
62. Wang J, Wang L, Kitanaka S (2007) Stilbene and dihydrophenanthrene derivatives from
Pholidota chinensis and their nitric oxide inhibitory and radical-scavenging activities. J Nat
Med 61:381–386
63. Bai L, Yamaki M, Takagi S (1996) Stilbenoids from Pleione bulbocodioides. Phytochemistry
42:853–856
64. Li Y, Zeng KW, Zhao MB, Jiang Y, Li J, Tu PF, Wu ZH (2017) A new prenylated flavone from
Pleione bulbocodioides. J Asian Nat Prod Res 19:738–743
65. Tezuka Y, Ueda M, Kikuchi T (1989) Studies on the constituents of Orchidaceae plants. VIII.
Constituents of Spiranthes sinensis (Pers.) Ames var. amoena (M. Biebersin) Hara. (1).
Isolation and structure elucidation of spiranthol-A, spiranthol-B, and spirasineol-A, new
isopentenyldihydrophenanthrenes. Chem Pharm Bull 37:3195–3199
66. Majumder PL, Banerjee S, Maiti DC, Sen S (1995) Stilbenoids from the orchids
Agrostophyllum callosum and Coelogyne flaccida. Phytochemistry 39:649–653
67. Auberon F, Olatunji OJ, Krisa S, Antheaume C, Herbette G, Bonte F, Merillon JM, Lobstein A
(2016) Two new stilbenoids from the aerial parts of Arundina graminifolia (Orchidaceae).
Molecules 21:1430
68. Baller A, Corrodi H, Gaumann E, Hardegger E, Kern H, Winterhalter-Wild N (1957) Wilting
agents and antibiotics. XX. Induced defensive substances in the Orchidaceae. 1. Helv Chim
Acta 40:1062–1066
69. Fisch MH, Flick BH, Arditti J (1973) Structure and antifungal activity of hircinol, loroglossol
and orchinol. Phytochemistry 12:437–441
70. Liu MF, Han Y, Xing DM, Wang W, Xu LZ, Du LJ, Ding Y (2005) One new benzyldihy-
drophenanthrene from Arundina graminifolia. J Asian Nat Prod Re.:767–770
566 A. Vasas
71. Takagi S, Yamaki M, Inoue K (1983) Antimicrobial agents from Bletilla striata. Phytochem-
istry 22:1011–1015
72. Klongkumnuankarn P, Busaranon K, Chanvorachote P, Sritularak B, Jongbunprasert V,
Likhitwitayawuid K (2015) Cytotoxic and antimigratory activities of phenolic compounds
from Dendrobium brymerianum. Evid Based Complement Alternat Med 350410
73. Fan C, Wang W, Wang Y, Qin G, Zhao W (2001) Chemical constituents from Dendrobium
densiflorum. Phytochemistry 57:1255–1258
74. Lee YH, Park JD, Baek NI, Kim SI, Ahn BZ (1995) In vitro and in vivo antitumoral
phenanthrenes from the aerial parts of Dendrobium nobile. Planta Med 61:178–180
75. Zhou XM, Zheng CJ, Gan LS, Chen LS, Chen GY, Zhang XP, Song XP, Li GN, Sun CG
(2016) Bioactive phenanthrene and bibenzyl derivatives from the stems of Dendrobium nobile.
J Nat Prod 79:1791–1797
76. Kyokong N, Muangnoi C, Thaweesest W, Kongkatitham V, Likhitwitayawuid K,
Rojsitthisak P, Sritularak B (2019) A new phenanthrene dimer from Dendrobium palpebrae.
J Asian Nat Prod Res 21:391–397
77. Yamaki M, Honda C (1996) The stilbenoids from Dendrobium plicatile. Phytochemistry 43:
207–208
78. Bhummaphan N, Petpiroon N, Prakhongeheep O, Sritularak B, Chanvorachote P (2019)
Lusianthridin targeting of lung cancer stem cells via Src-STAT3 suppression. Phytomedicine
62:152932
79. Majumder PL, Lahiri S (1990) Lusianthrin and lusianthridin, two stilbenoids from the orchid
Lusia indivisa. Phytochemistry 29:621–624
80. Hernandez-Romero Y, Rojas JI, Castillo R, Rojas A, Mata R (2004) Spasmolytic effects, mode
of action, and structure-activity relationships of stilbenoids from Nidema boothii. J Nat Prod
67:160–167
81. Li B, Ali Z, Chan M, Li J, Wang M, Abe N, Wu CR, Khan IA, Wang W, Li SX (2017)
Chemical constituents of Pholidota cantonensis. Phytochemistry 137:132–138
82. Estrada S, Rojas A, Mathison Y, Israel A, Mata R (1999) Nitric oxide/cGMP mediates the
spasmolytic action of 3,4’-dihydroxy-5,5’-dimethoxybibenzyl from Scaphyglottis livida.
Planta Med 65:109–114
83. Yang J, Jiang J, Huang G, Liu B, Liu Y, Zhan R, Chen Y (2014) Two new flavanone glycosides
from Sunipia scariosa. Biochem Syst Ecol 57:317–321
84. Majumder PL, Roychowdhury M, Chakraborty S (1998) Thunalbene, a stilbene derivative
from the orchid Thunia alba. Phytochemistry 49:2375–2378
85. Yang L, Qin LH, Bligh SWA, Bashall A, Zhang CF, Zhang M, Wang ZT, Xu LS (2006) A new
phenanthrene with a spirolactone from Dendrobium chrysanthum and its anti-inflammatory
activities. Bioorg Med Chem 14:3496–3501
86. Majumder PL, Pal S (1992) Rotundatin, a new 9,10-dihydrophenanthrene derivative from
Dendrobium rotundatum. Phytochemistry 31:3225–3228
87. Majumder PL, Sen RC (1987) Structure of moscatin – a new phenanthrene derivate from
Dendrobium rotundatum. Indian J Chem 26B:18–20
88. Chen CC, Wu LG, Ko FN, Teng CM (1994) Antiplatelet aggregation principles of
Dendrobium loddigesii. J Nat Prod 57:1271–1274
89. Chen Y, Shi X, Liu Y, Li Y, Zhang Y (2013) Aromatic compounds from Coelogyne longipes.
Biochem Syst Ecol 50:72–74
90. Wang Y, Guan SH, Meng YH, Zhang YB, Cheng CR, Shi YY, Feng RH, Zeng F, Wu ZY,
Zhang JX, Yang M, Liu X, Li Q, Chen XH, Bi KS, Guo DA (2013) Phenanthrenes, 9,10-
dihydrophenanthrenes, bibenzyls with their derivatives, and malate or tartrate benzyl ester
glucosides from tubers of Cremastra appendiculata. Phytochemistry 94:268–276
91. Ren J, Qian XP, Guo YG, Li T, Yan SK, Jin HZ, Zhang WD (2016) Two new phenanthrene
glycosides from Liparis regnieri Finet and their antibacterial activities. Phytochem Lett 18:64–67
92. Liu XQ, Li XP, Yuan QY (2016) Two new phenanthrene glucosides from Cremastra
appendiculata. Chem Nat Compd 52:23–25
22 Phenanthrenes from Orchidaceae and Their Biological Activities 567
93. Ye QH, Zhao WM, Qin GW (2003) New fluorenone and phenanthrene derivatives from
Dendrobium chrysanthum. Nat Prod. Res 17:201–205
94. Tezuka Y, Ji L, Hirano H, Ueda M, Nagashima K, Kikuchi T (1990) Studies on the constituents
of orchidaceous plants. IX. Constituents of Spiranthes sinensis (Pers.) Ames var. amoena
(M. Bieberson) Hara. (2). Structures of spiranthesol, spiranthoquinone, spiranthol-C, and
spirasineol-B, new isopentenyldihydrophenanthrenes. Chem Pharm Bull 38:629–635
95. Liu L, Yin QM, Yan X, Hu C, Wang W, Wang RK, Luo X, Zhang XW (2019) Bioactivity-
guided isolation of cytotoxic phenanthrenes from Spiranthes sinensis. J Agric Food Chem 67:
7274–7280
96. Anuradha V, Rao NSP, Bhaskar MU (1994) Ochrolic acid, a precursor to phenanthropyrones,
from Coelogyne ochracea. Phytochemistry 36:1515–1517
97. Yao S, Tang CP, Ye Y, Kurtán T, Kiss-Szikszai A, Antus S, Pescitelli G, Salvadori P, Krohn K
(2008) Stereochemistry of atropisomeric 9,10-dihydrophenanthrene dimers from Pholidota
chinensis. Tetrahedron: Asymmetry 19:2007–2014
98. Connolly JD, Rycroft DS, Srivastava DL, Cole WJ, Ifeadike P, Kimbu SF, Singh J, Hughes M,
Thom C, Gerhard U, Organ AJ, Smith RJ, Harrison LJ (1999) Aromatic compounds from the
liverwort Plagiochila spinulosa. Phytochemistry 50:1159–1165
99. Liu XQ, Guo YQ, Gao WY, Zhang TJ, Yan LL (2008) Two new phenanthrofurans from
Pleione bulbocodioides. J Asian Nat Prod Res 10:453–457
100. Guo XY, Wang J, Wang NL, Kitanaka S, Yao XS (2007) 9, 10-Dihydrophenanthrene deriv-
atives from Pholidota yunnanensis and scavenging activity on DPPH free radical. J Asian Nat
Prod Res 9:165–174
101. Yang M, Cai L, Fang H, Yang S, Fang Y, Ding Z (2014) Phenolic compounds from
Monomeria barbata. Chem Nat Compd 50:88–92
102. Guo XY, Wang J, Wang NL, Kitanaka S, Liu HW, Yao XS (2006) New stilbenoids from
Pholidota yunnanensis and their inhibitory effects on nitric oxide production. Chem Pharm
Bull 54:21–25
103. Yamaki M, Bai L, Inoue K, Takagi S (1990) Benzylphenanthrenes from Bletilla striata.
Phytochemistry 29:2285–2287
104. Lin YL, Chen WP, Macabalang AD (2005) Dihydrophenanthrenes from Bletilla formosana.
Chem Pharm Bull 53:1111–1113
105. Bai L, Kato T, Inoue K, Yamaki M, Takagi S (1993) Stilbenoids from Bletilla striata.
Phytochemistry 33:1481–1483
106. Woo KW, Park JE, Choi SU, Kim KH, Lee KR (2014) Phytochemical constituents of Bletilla
striata and their cytotoxic activity. Nat Prod Sci 20:91–94
107. Bai L, Kato T, Inoue K, Yamaki M, Takagi S (1991) Blestrianol A, B and C, biphenanthrenes
from Bletilla striata. Phytochemistry 30:2733–2735
108. Liu L, Li J, Zeng KW, Li P, Tu PF (2013) Three new phenanthrenes from Cremastra
appendiculata (D. Don) Makino. Chin Chem Lett 24:737–739
109. Tuchinda P, Udchachon J, Khumtaveeporn K, Taylor WC (1989) Benzylated phenanthrenes
from Eulophia nuda. Phytochemistry 28:2463–2466
110. Yang MH, Cai L, Li MH, Zeng XH, Yang YB, Ding ZT (2010) Three new phenanthrenes from
Monomeria barbata Lindl. Chin Chem Lett 21:325–328
111. Dong HL, Liang HQ, Wang CL, Guo SX, Yang JS (2013) Shancigusins E-I, five new
glucosides from the tubers of Pleione yunnanensis. Magn Reson Chem 51:371–377
112. Lin YL, Huang RL, Don MJ, Kuo YH (2000) Dihydrophenanthrenes from Spiranthes sinensis.
J Nat Prod 63:1608–1610
113. Lin YL, Wang WY, Kuo YH, Liu YH (2001) Homocyclotirucallane and two
dihydrophenanthrenes from Spiranthes sinensis. Chem Pharm Bull 49:1098–1101
114. Hu J, Fan W, Dong F, Miao Z, Zhou J (2012) Chemical components of Dendrobium
crepidatum and their neurite outgrowth enhancing activities. Chin J Chem 30:1327–1330
115. Yao S, Tang CP, Li XQ, Ye Y (2008) Phochinenins A-F, Dimeric 9,10-dihydrophenanthrene
derivatives, from Pholidota chinensis. Helv Chim Acta 91:2122–2129
568 A. Vasas
116. Liu XQ, Yuan QY, Guo YQ (2009) Two new stilbenoids from Pleione bulbocodioides. J Asian
Nat Prod Res 11:116–121
117. Yamaki M, Bai L, Kato T, Inoue K, Takagi S (1993) Three dihydrophenanthropyrans from
Bletilla striata. Phytochemistry 32:427–430
118. Bai L, Yamaki M, Yamagata Y, Takagi S (1996) Shanciol, a dihydrophenanthropyran from
Pleione bulbocodioides. Phytochemistry 41:625–628
119. Bai L, Yamaki M, Takagi S (1998) Flavan-3-ols and dihydrophenanthropyrans from Pleione
bulbocodioides. Phytochemistry 47:1125–1129
120. Jiang S, Chen CF, Ma XP, Wang MY, Wang W, Xia Y, Zhang N, Pan WD, Wu MK (2019)
Antibacterial stilbenes from the tubers of Bletilla striata. Fitoterapia 138104350
121. Bai L, Masukawa N, Yamaki M, Takagi S (1982) Four stilbenoids from Pleione
bulbocodioides. Phytochemistry 48:327–331
122. Dong H, Wang C, Li Y, Guo S, Yang J (2010) Complete assignments 1H and 13C NMR data of
three new dihydrophenanthrofurans from Pleione yunnanensis. Magn Reson Chem 48:256–260
123. Leong YW, Harrison LJ (2004) A biphenanthrene and a phenanthro[4,3-b]furan from the
orchid Bulbophyllum vaginatum. J Nat Prod 67:1601–1603
124. Wang L, Zhang CF, Wang ZT, Zhang M, Xu LS (2009) Five new compounds from
Dendrobium crystallinum. J Asian Nat Prod Res 11:903–911
125. Dong FW, Fan WW, Xu FQ, Wan QL, Su J, Li Y, Zhou L, Zhou J, Hu JM (2013) Inhibitory
activities on nitric oxide production of stilbenoids from Pholidota yunnanensis. J Asian Nat
Prod Res 15:1256–1264
126. Sharma C, Dixit M, Singh R, Agrawal M, Mansoori MN, Kureel J, Singh D, Narender T, Arya
KR (2015) Potential osteogenic activity of ethanolic extract and oxoflavidin isolated from
Pholidota articulata Lindley. J Ethnopharmacol 170:57–65
127. Simmler C, Antheaume C, Lobstein A (2010) Antioxidant biomarkers from Vanda coerulea
stems reduce irradiated HaCaT PGE-2 production as a result of COX-2 inhibition. PLoS One
5:1–9
128. Majumder PL, Datta N, Sarkar AK, Chakraborti J (1982) Flavidin. A novel 9,10-
dihydrophenanthrene derivative of the orchids Coelogyne flavida, Pholidota articulata and
Otochilus fusca. J Nat Prod 45:730–732
129. Majumder P, Bandyopadhyay D, Joardar S (1982) Coelogin and Coeloginin: Two Novel 9,l0-
dihydrophenanthrene derivatives from the orchid Coelogyne cristata. J Chem Soc Perkin
Trans I:1131–1136
130. Sharma C, Kumari T, Mansoori MN, Dixit M, Shukla P, Singh D, Bhandari SPS, Narender T,
Arya KR (2014) Ethanolic extract of Coelogyne cristata Lindley (Orchidaceae) and its
compound coelogin promote osteoprotective activity in overiectomized estrogen deficient
mice. Phytomedicine 21:1702–1707
131. Veerraju P, Rao NSP, Rao LJ, Rao KVJ Rao PRMn(1989) Amoenumin, a 9,10-dihydro-5H-
phenanthro-(4,5-b,c,d)-pyran from Dendrobium amoenum. Phytochemistry 28:950951
132. Majumder PL, Basak M (1991) Two stilbenoids from the orchid Cirrhopetalum andersonii.
Phytochemistry 30:3429–3432
133. Majumder PL, Basak M (1990) Cirrhopetalin, a phenanthrene derivative from Cirrhopetalum
andersonii. Phytockemistry 29:1002
134. Sun J, Zhan R, Chen Y, Zhang Y, Chen L (2018) A new phenanthrene and a new
9,1dihydrophenanthrene from Bulbophyllum retusiusculum. Nat Prod Res 32:2447–2451
135. Yamaki M, Bai L, Kato T, Inoue K, Takagi S, Yamagata Y, Tomita KI (1993) Blespirol, a
phenanthrene with a spirolactone ring from Bletilla striata. Phytochemistry 33:1497–1498
136. Alder AC, Rüedi P, Eugster CH (1984) Plectranthone A, B, C und D. Diterpenoide
Phenanthren-1,4-dione aus Blattdrüsen einer Plectranthuus sp. (Labiatae). Helv Chim Acta
67:1003–1011
137. Krohn K, Loock U, Paavilainen K, Hausen BM, Schmalle HW, Kiesele H (2001) Synthesis
and electrochemistry of annoquinone-A, cypripedin methyl ether, denbinobin and related
1,4-phenanthrenequinones. Arkivoc 1:88–130
22 Phenanthrenes from Orchidaceae and Their Biological Activities 569
138. Xiao S, Xu D, Zhang M, Linhu L, Ding L, Zhou S, Zhou Y (2016) A novel phenanthrene-1,2-
dione from Bletilla striata. Chin J Org Chem 36:638–641
139. Sun A, Pang S, Liu J, Lin J, Xu R (2016) Two novel phenanthrenequinones with anti-cancer
activity isolated from Bletilla striata. Bioorg Med Chem Lett 26:2375–2379
140. Bhaskar MU, Rao LJM, Rao NSP, Rao PRM (1991) Ochrone A, a novel 9,10-dihydro-1,4-
phenanthraquinone from Coelogyne ochracea. J Nat Prod 54:386–389
141. Barua AK, Ghosh BB, Ray S, Patra A (1990) Cymbinodin-A, a phenanthraquinone from
Cymbidium aloifolium. Phytochemistry 29:3046–3047
142. Ghosh BB, Ray S, Bhattacharyya P, Datta PK, Mukherjee BB, Patra A, Banerjee AK, Barua
AK (1992) Cymbinodin B, a phenanthraquinone from Cymbidium aloifolium. Indian J Chem
31B:557–558
143. Liu D, Ju J, Zou Z, Lin G, Yang J (2005) Isolation and structure determination of
cypritibetquinone A and B, two new phenanthraquinones from Cypripedium tibeticum. Acta
Pharm Sin 40:255–257
144. Hu JM, Chen JJ, Yu H, Zhao YX, Zhou J (2008) Five new compounds from Dendrobium
longicornu. Planta Med 74:535–539
145. Sritularak B, Anuwat M, Likhitwitayawuid K (2011) A new phenanthrenequinone from
Dendrobium draconis. J Asian Nat Prod Res 13:251–255
146. Chen XJ, Mei WL, Zuo WJ, Zeng YB, Guo ZK, Song XQ, Dai HF (2013) A new antibacterial
phenanthrenequinone from Dendrobium sinense. J Asian Nat Prod Res 15:67–70
147. Lin TH, Chang SJ, Chen CC, Wang JP, Tsao LT (2001) Two phenanthraquinones from
Dendrobium moniliforme. J Nat Prod 64:1084–1086
148. Yang D, Liu LY, Cheng ZQ, Xu FQ, Fan WW, Zi CT, Dong FW, Zhou J, Ding ZT, Hu JM
(2015) Five new phenolic compounds from Dendrobium aphyllum. Fitoterapia 100:11–18
149. Tezuka Y, Hirano H, Kikuchi T, Xu GJ (1991) Constituents of Ephemerantha lonchophylla;
isolation and structure elucidation of new phenolic compounds, ephemeranthol-A,
ephemeranthol-B, and ephemeranthoquinone, and of a new diterpene glucoside,
ephemeranthoside. Chem Pharm Bull 39:593–598
150. Chen DN, Wu YP, Chen YJ, Liu WJ, Wang JX, He F, Jiang L (2017) Two new stilbenoids from
aerial parts of Flickingeria fimbriata. J Asian Nat Prod Res 212:117–122
151. Williams RB, Martin SM, Hu JF, Garo E, Rice SM, Norman VL, Lawrence JA, Hough GW,
Goering MG, O’Neil-Johnson M, Eldridge GR, Starks CM (2012) Isolation of apoptosis-
inducing stilbenoids from four members of the Orchidaceae family. Planta Med 78:160–165
152. Masuda Y, Suzuki R, Sakagami H, Umemura N, Shirataki Y (2012) Novel cytotoxic
phenanthrenequinone from Odontioda Marie Noel 'Velano'. Chem Pharm Bull 60:1216–1219
153. Xue Z, Li S, Wang S, Wang Y, Yang Y, Shi J, He L (2006) Mono-, bi-, and triphenanthrenes
from the tubers of Cremastra appendiculata. J Nat Prod 69:907–913
154. Shi Y, Zhang B, Lu Y, Qian C, Feng Y, Fang L, Ding Z, Cheng D (2017) Antiviral activity of
phenanthrenes from the medicinal plant Bletilla striata against influenza A virus. BMC Compl
Altern Med 17:273
155. Majumder PL, Pal A, Joardar M (1990) Cirrhopetalanthrin, a dimeric phenanthrene derivative
from the orchid Cirrhopetalum maculosum. Phytochemistry 29:271–274
156. Li CY, Liu J, Su XH, Yuan ZP, Zhong YJ, Li YF, Liang B (2013) New dimeric phenanthrene
and flavone from Spiranthes sinensis. J Asian Nat Prod Res 15:417–421
157. Liu L, Yin QM, Zhang XW, Wang W, Dong XY, Yan X, Hu R (2016) Bioactivity-guided
isolation of biphenanthrenes from Liparis nervosa. Fitoterapia 115:15–18
158. Yang X, Yang J, Xu C, Lv J, Wang C, Song P (2016) Antimicrobial stilbenoids from Bletilla
yunnanensis. Chem Nat Compd 52:19–22
159. Shi X, Li Y, Liu Y, Jiang J, Wang L, Zhang Y, Chen Y (2010) A new 9,10-
dihydrophenanthropyran dimer and a new natural 9,10-dihydrophenanthropyran from
Otochilus porrectus. Biochem Syst Ecol 38:842–845
160. Yamaki M, Bai L, Inoue K, Takagi S (1989) Biphenanthrenes from Bletilla striata. Phyto-
chemistry 28:3503–3505
570 A. Vasas
161. Yang M, Cai L, Tai Z, Zeng X, Ding Z (2010) Four new phenanthrenes from Monomeria
barbata Lindl. Fitoterapia 81:992–997
162. Xu J, Yu H, Qing C, Zhang Y, Liu Y, Chen Y (2009) Two new biphenanthrenes with cytotoxic
activity from Bulbophyllum odoratissimum. Fitoterapia 80:381–384
163. Liu L, Li J, Zeng KW, Jiang Y, Tu PF (2016) Five new biphenanthrenes from Cremastra
appendiculata. Molecules 21:1089/1–10.
164. Qian CD, Jiang FS, Yu HS, Shen Y, Fu YH, Cheng DQ, Ding ZS, Gan LS (2015) Antibacterial
biphenanthrenes from the fibrous roots of Bletilla striata. J Nat Prod 78:939–943
165. Bai L, Yamaki M, Inoue K, Takagi S (1990) Blestrin A and B, bis(dihydrophenanthrene)ethers
from Bletilla striata. Phytochemistry 29:1259–1260
166. Phiboonchaiyanan PP, Petpiroon N, Sritularak B, Chanvorachote P (2018) Phoyunnanin E
induces apoptosis of non-small cell lung cancer cells via p53 activation and down-regulation
of survivin. Anticancer Res 38:6281–6290
167. Yamaki M, Bai L, Kato T, Inoue K, Takagi S, Yamagata Y, Tomita KI (1992) Bisphenanthrene
ethers from Bletilla striata. Phytochemistry 31:3985–3987
168. Cakova V, Urbain A, Antheaume C, Rimlinger N, Wehrung P, Bonté F, Lobstein A (2015)
Identification of phenanthrene derivatives in Aerides rosea (Orchidaceae) using the combined
systems HPLC-ESI-HRMS/MS and HPLC-DAD-MS-SPE-UV-NMR. Phytochem Anal 26:
34–39
169. Majumder PL, Banerjee S, Lahiri S, Mukhoti N, Sen S (1998) Dimeric phenanthrenes from
two Agrostophyllum species. Phytochemistry 47:855–860
170. Li JY, Hou B, Ren FC, Yang XB, Lv YF, Kuang MT, Yang L, Zhou J, Hu JM (2018) Poly
p-hydroxybenzyl substituted bibenzyls and phenanthrenes from Bletilla ochracea Schltr with
anti-inflammatory and cytotoxic activity. Fitoterapia 129:214–248
171. Xiao SJ, Yuan FM, Zhang MS, Yu SY, Li JD, Yi XD, Xu DL (2017) Three new
1-(p-hydroxybenzyl)phenanthrenes from Bletilla striata. J Asian Nat Prod Res 19:140–144
172. Xiao SJ, Yuan FM, Zhang MS, Yu SY, Li JD, Yi XD, Xu DL (2016) Three new
1-(p-hydroxybenzyl)phenanthrenes from Bletilla striata. J Asian Nat Prod Res 31:1–5
173. Waratchareeyakul W, Fusi F, Durante M, Ahmed A, Knirsch W, Mas-Claret E (2020)
Vasorelaxing activity of stilbenoid and phenanthrene derivatives from Brasiliorchis
porphyrostele: involvment of smooth muscle CaV1.2 channels. Planta Med 86:631–642
174. Majumder PL, Banerjee S (1988) A ring-B oxygenated phenanthrene derivative from the
orchid Bulbophyllum gymnopus. Phytochemistry 27:245–248
175. Majumder PL, Kar A, Shoolery JN (1985) Bulbophyllanthrin, a phenanthrene of the orchid
Bulbophyllum leopardium. Phytochemistry 24:2083–2087
176. Chen YG, Xu JJ, Yu H, Qing C, Zhang YL, Liu Y, Wang JH (2007) 3,7-Dihydroxy-2,4,6-
trimethoxyphenanthrene, a new phenanthrene from Bulbophyllum odoratissimum. J Korean
Chem Soc 51:352–355
177. Majumder PL, Bandyopadhyay S, Pal S (2008) Rigidanthrin, a new dimeric phenanthrene
derivative of the orchid Bulbophyllum rigidum. J Indian Chem Soc 85:1116–1123
178. Leong YW, Harrison LJ, Powell AD (1999) Phenanthrene and other aromatic constituents of
Bulbophyllum vaginatum. Phytochemistry 50:1237–1241
179. Xue Z, Li S, Wang SJ, Yang YC, He DX, Ran GL, Kong LZ, Shi JG (2005) Studies on
chemical constituents from the corm of Cremastra appendiculata. China Journal of Chinese
Materia Medica 30:511–513
180. Majumder PL, Sen S, Majumder S (2001) Phenanthrene derivatives from the orchid
Coelogyne cristata. Phytochemistry 58:581–586
181. Majumder PL, Sen RC (1991) Pendulin, a polyoxygenated phenanthrene derivative from the
orchid Cymbidium pendulum. Phytochemistry 30:2432–2434
182. Li YP, Wang YJ, Chen LL (2016) Chemical constituents of Dendrobium chrysotoxum. Chem
Nat Compd 52:90–92
183. Treesuwan S, Chanvorachote P, Pongrakhananon V, Treesuwan S, Chanvorachote P,
Pongrakhananon V, Sritularak B (2018) Cypripedin diminishes an epithelial-to mesenchymal
22 Phenanthrenes from Orchidaceae and Their Biological Activities 571
transition in non-small cell lung cancer cells through suppression of Akt/GSK-3β signalling.
Sci Rep 8:8009
184. Zhang YY, Wang P, Wang H, Chen LL, Mei WL, Dai HF, Zhang YY, Song XQ, Zuo WJ (2019)
Chemical constituents from Dendrobium hainanense. J Asian Nat Prod Res 21:873–880
185. Hwang JS, Lee SA, Hong SS, Han XH, Lee C, Kang SJ, Lee D, Kim Y, Hong JT, Lee MK,
Hwang BY (2010) Phenanthrenes from Dendrobium nobile and their inhibition of the
LPS-induced production of nitric oxide in macrophage RAW 264.7 cells. Bioorg Med Chem
Lett 20:3785–3787
186. Zhang X, Xu JK, Wang NL, Kurihara H, Yao XS (2008) Antioxidant phenanthrenes and
lignans from Dendrobium nobile. J Chin Pharm Sci 17:314–318
187. Kim JH, Oh SY, Han SB, Uddin GM, Kim CY, Lee JK (2015) Anti-inflammatory effects of
Dendrobium nobile derived phenanthrenes in LPS-stimulated murine macrophages. Arch
Pharm Res 38:1117–1126
188. Zhang GN, Zhong LY, Bligh SWA, Guo YL, Zhang CF, Zhang M, Wang ZT, Xu LS (2005)
Bi-bicyclic and bi-tricyclic compounds from Dendrobium thyrsiflorum. Phytochemistry 66:
1113–1120
189. Chen CC, Huang YL, Teng CM (2000) Antiplatelet aggregation principles from
Ephemerantha lonchophylla. Planta Med 66:372–374
190. Hernandez-Romero Y, Acevedo L, de Sanchez M, Shier WT, Abbas HK, Mata R (2005)
Phytotoxic activity of bibenzyl derivatives from the orchid Epidendrum rigidum. J Agric Food
Chem 53:6276–6280
191. Bhandari SR, Kapadi AH, Majumder PL, Joardar M, Shoolery JN (1985) Nudol, a phenanthrene
of the orchids Eulophia nuda, Eria carinata and Eria stricta. Phytochemistry 24:801–804
192. Majumder PL, Kar A (1987) Confusarin and confusaridin, two phenanthrene derivatives of the
orchid Eria confusa. Phytochemistry 26:1127–1129
193. Tatiya AU, Bari N, Surana SJ, Kalaskar MG (2014) Effect of bioassay guided isolation of
1-phenanthrene carboxylic acid from Eulophia herbacea Lindi. tubers on human cancer cell
lines. Res J Phytochem 8:155–161
194. Wisutthathum S, Demougeot C, Totoson P, Adthapanyawanich K, Ingkaninan K,
Temkitthawon P, Chootip K (2018) Eulophia macrobulbon extract relaxes rat isolated pulmo-
nary artery and protects against monocrotaline-induced pulmonary arterial hypertension.
Phytomedicine 50:157–165
195. Temkifthawon P, Changwichit K, Khorana N, Viyoch J, Suwanborirux K, Ingkaninan K
(2017) Phenanthrenes from Eulophia macrobulbon as novel phosphodiesterase-5 inhibitors.
Nat Prod Commun 12:79–82
196. Shriram V, Kumar V, Kishor PB, Suryawanshi SB, Upadhyay AK, Bhat MK (2010) Cytotoxic
activity of 9,10-dihydro-2,5-dimethoxyphenanthrene-1,7-diol from Eulophia nuda against
human cancer cells. J Ethnopharmacol 128:251–253
197. Kshirsagar RD, Kanekar YB, Jagtap SD, Upadhyay SN, Rao R, Bhujbal SP, Kedia JN (2010)
Phenanthrenes of Eulophia ochreata Lindl. Int J Green Pharm:147–152
198. Cretton S, Righi D, Sahib L, Christen P, Oyarzun A, Fajardo V, Kaiser M (2018)
A new antifungal and antiprotozoal bibenzyl derivative from Gavilea lutea. Nat Prod Res
32:695–701
199. Savaris CR, Ghiraldi DMB, Chiavelli LUR, de Oliveira Santin SM, Milaneze-Gutierre MA,
Kischkel B, Negri M, Scariot DB, Garcia FP, Nakamura CV, Pomini AM (2018) Phytochem-
ical and biological studies of Gomesa recurva R. Br. (Orchidaceae): Chemotaxonomic signif-
icance of the presence of phenanthrenoids. Biochem Syst Ecol 80:11–13
200. Majumder PL, Lahiri S (1990) Volucrin, a new dimeric phenanthrene derivative from the
orchid Luisia volucris. Tetrahedron 46:3621–3626
201. Estrada S, Toscano RA, Mata R (1999b) New phenanthrene derivatives from Maxillaria
densa. J Nat Prod 62:1175–1178
202. Estrada S, López-Guerrero JJ, Villalobos-Molina R, Mata R (2004) Spasmolytic stilbenoids
from Maxillaria densa. Fitoterapia 75:690–695
572 A. Vasas
203. Valencia-Islas NA, Paul RN, Shier WT, Mata R, Abbas HK (2002) Phytotoxicity and ultra-
structural effects of gymnopusin from the orchid Maxillaria densa on duckweed (Lemna
pausicostata) frond and root tissues. Phytochemistry 61:141–148
204. Suzuki R, Tanaka T, Yamamoto M, Sakagami H, Tomomura M, Tomomura A, Satoh K,
Shirataki Y (2012) In search of new biological activities of isolates from Odontoglossum
Harvengtense ‘Tutu’. In Vivo 26:993–1000
205. Estrada-Soto S, López-Guerrero JJ, Villalobos-Molina R, Mata R (2006) Endothelium-
independent relaxation of aorta rings by two stilbenoids from the orchids Scaphyglottis livida.
Fitoterapia 77:236–239
206. Yang KC, Uen YH, Suk FM, Liang YC, Wang YJ, Ho YS, Li IH, Lin SY (2005) Molecular
mechanisms of denbinobin-induced anti-tumorigenesis effect in colon cancer cells. World J
Gastroenterol 11:3040–3045
207. Song JI, Kang YJ, Yong HY, Kim YC, Moon A (2012) Denbinobin, a phenanthrene from
Dendrobium nobile, inhibits invasion and induces apoptosis in SNU-484 human gastric cancer
cells. Oncol Rep 27:813–818
208. Sánchez-Duffhues G, Calzado MA, de Vinuesa AG, Appendino G, Fiebich BL, Loock U,
Lefarth-Risse A, Krohn K, Muñoz E (2009) Denbinobin inhibits nuclear factor-κB and induces
apoptosis via reactive oxygen species generation in human leukemic cells. Biochem
Pharmacol 77:1401–1409
209. Huang YC, Guh JH, Teng CM (2005) Denbinobin-mediated anticancer effect in human K562
leukemia cells: role in tubulin polymerization and Bcr-Abl activity. J Biomed Sci 12:113–121
210. Lu TL, Han CK, Chang YS, Lu TJ, Huang HC, Bao BY, Wu HY, Huang CH, Li CY, Wu TS
(2014) Denbinobin, a phenanthrene from Dendrobium nobile, impairs prostate cancer migra-
tion by inhibiting Rac1 activity. Am J Chin Med 42:1539–1554
211. Datla P, Kalluri MD, Basha K, Bellary A, Kshirsagar R, Kanekar Y, Upadhyay S, Singh S,
Rajagopal V (2010) 9,10-Dihydro-2,5-dimethoxyphenanthrene-1,7-diol, from Eulophia
ochreata, inhibits inflammatory signalling mediated by Toll-like receptors. Br J Pharmacol
160:1158–1170
212. Yang H, Lee PJ, Jeong EJ, Kim HP, Kim YC (2012) Selective apoptosis in hepatic stellate cells
mediates the antifibrotic effect of phenanthrenes from Dendrobium nobile. Phytother Res 26:
974–980
Orchids of Genus Bletilla: Traditional Uses,
Phytochemistry, Bioactivities, 23
and Commercial Importance
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
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
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
23 Orchids of Genus Bletilla: Traditional Uses, Phytochemistry,. . . 577
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
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
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].
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
23 Orchids of Genus Bletilla: Traditional Uses, Phytochemistry,. . . 581
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
582 H. P. Devkota et al.
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].
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].
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].
584 H. P. Devkota et al.
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].
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].
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].
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].
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
23 Orchids of Genus Bletilla: Traditional Uses, Phytochemistry,. . . 585
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].
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].
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].
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
586 H. P. Devkota et al.
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.
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
23 Orchids of Genus Bletilla: Traditional Uses, Phytochemistry,. . . 587
years has resulted in the decline of natural habitats, which needs immediate concern
from the scientific community and other stakeholders.
References
1. Cakova V, Urbain A, Le Quéméner C et al (2015) Purification of vandaterosides from Vanda
teres (Orchidaceae) by stepwise gradient centrifugal partition chromatography. J Sep Sci 38:
3006–3013. https://doi.org/10.1002/jssc.201500349
2. Khan H, Marya BT et al (2019) Genus Vanda: a review on traditional uses, bioactive chemical
constituents and pharmacological activities. J Ethnopharmacol 229:46–53
3. De Lakshman C, Pathak P, Rao AN, Rajeevan PK (2015) Commercial orchids. De Gruyter
Open, Warsaw/Berlin
4. Bulpitt CJ, Li Y, Bulpitt PF, Wang J (2007) The use of orchids in Chinese medicine. J R Soc
Med 100:558–563. https://doi.org/10.1258/jrsm.100.12.558
5. Gutiérrez RMP (2010) Orchids: a review of uses in traditional medicine, its phytochemistry and
pharmacology. J Med Plants Res 4:592–638. https://doi.org/10.5897/JMPR10.012
6. Pant B (2013) Medicinal orchids and their uses: tissue culture a potential alternative for
conservation. African J Plant Sci 7:448–467. https://doi.org/10.5897/ajps2013.1031
7. Hadi H, Razali SNS, Awadh AI (2015) A comprehensive review of the cosmeceutical benefits
of vanda species (orchidaceae). Nat Prod Commun 10:1483–1488
8. Teoh ES (2016) Medicinal orchids of Asia. Springer, Cham. https://doi.org/10.1007/978-3-319-
24274-3
9. Teoh ES (2019) Orchids as aphrodisiac, medicine or food. Springer Nature Switzerland AG,
Cham
10. Chen X, Gale SW, Cribb PJ (2009) BLETILLA H. G. Reichenbach, Fl. Serres Jard. Eur. 8: 246.
1853, nom. cons. In: Wu ZY, Raven PH (eds) Flora of China, Flagellariaceae through
Marantaceae, vol 24. Science Press/Missouri Botanical Garden Press, Beijing/St. Louis, pp
209–210
11. Govaerts R (2020) World checklist of orchidaceae. http://wcsp.science.kew.org/. Accessed
21 Feb 2020
12. Li JY, Kuang MT, Yang L et al (2018) Stilbenes with anti-inflammatory and cytotoxic activity
from the rhizomes of Bletilla ochracea Schltr. Fitoterapia 127:74–80. https://doi.org/10.1016/j.
fitote.2018.02.007
13. Cai JY, Zhao L, Zhang DZ (2007) Chemical constituents from Bletilla ochracea Schltr. Chem
Res Chinese Univ 23:705–707. https://doi.org/10.1016/S1005-9040(07)60153-6
14. Yu H, Dai B, Qian C et al (2016) Antibacterial activity of chemical constituents isolated from
fibrous roots of Bletilla striata. Zhong Yao Cai 39:544–547
15. He X, Wang X, Fang J et al (2017) Bletilla striata: medicinal uses, phytochemistry and
pharmacological activities. J Ethnopharmacol 195:20–38
16. Watanabe M, Devkota HP, Sugimura K, Watanabe T (2018) A guidebook of medicinal plant
park. School of Pharmacy, Kumamoto University, Kumamoto
17. Guo Y, Zhai L, Long H et al (2018) Genetic diversity of Bletilla striata assessed by SCoT and
IRAP markers. Hereditas 155:35. https://doi.org/10.1186/s41065-018-0074-4
18. Zeng X, Zhang S, Zhang L et al (2006) A study of the neuroprotective effect of the phenolic
glucoside gastrodin during cerebral ischemia in vivo and in vitro. Planta Med 72:1359–1365.
https://doi.org/10.1055/s-2006-951709
19. Yang L, Wang Z, Xu L (2006) Simultaneous determination of phenols (bibenzyl, phenanthrene,
and fluorenone) in Dendrobium species by high-performance liquid chromatography with diode
array detection. J Chromatogr A 1104:230–237. https://doi.org/10.1016/j.chroma.2005.12.012
20. Williams CA (1979) The leaf flavonoids of the orchidaceae. Phytochemistry 18:803–813.
https://doi.org/10.1016/0031-9422(79)80019-9
588 H. P. Devkota et al.
21. Lin YL, Chen WP, Macabalang AD (2005) Dihydrophenanthrenes from Bletilla formosana.
Chem Pharm Bull 53:1111–1113. https://doi.org/10.1248/cpb.53.1111
22. Lin CW, Hwang TL, Chen FA et al (2016) Chemical constituents of the rhizomes of Bletilla
formosana and their potential anti-inflammatory activity. J Nat Prod 79:1911–1921. https://doi.
org/10.1021/acs.jnatprod.6b00118
23. Yang X, Tang C, Zhao P et al (2012) Antimicrobial constituents from the tubers of Bletilla
ochracea. Planta Med 78(6):606–610. https://doi.org/10.1055/s-0031-1298264
24. Li JY, Yang L, Hou B et al (2018) Poly p-hydroxybenzyl substituted bibenzyls and phenan-
threnes from Bletilla ochracea Schltr with anti-inflammatory and cytotoxic activity. Fitoterapia
129:241–248. https://doi.org/10.1016/j.fitote.2018.07.007
25. Wang B, Xu Y, Chen L et al (2020) Optimizing the extraction of polysaccharides from bletilla
ochracea schltr. Using response surface methodology (RSM) and evaluating their antioxidant
activity. Processes 8:341. https://doi.org/10.3390/pr8030341
26. Shena X, Jiang W, Shena X, Jiang W (2020) Polysaccharides from Bletilla striata: extraction,
optimization and their antioxidant activities in vitro. Med Res 4:200010. https://doi.org/10.
21127/yaoyimr20200010
27. Qu Y, Li C, Zhang C et al (2016) Optimization of infrared-assisted extraction of Bletilla striata
polysaccharides based on response surface methodology and their antioxidant activities.
Carbohydr Polym 148:345–353. https://doi.org/10.1016/j.carbpol.2016.04.081
28. Jiang F, Li M, Wang H et al (2019) Coelonin, an anti-inflammation active component of Bletilla
striata and its potential mechanism. Int J Mol Sci 20(18):4422. https://doi.org/10.3390/
ijms20184422
29. Zu YY, Liu QF, Tian SX et al (2019) Effective fraction of Bletilla striata reduces the
inflammatory cytokine production induced by water and organic extracts of airborne fine
particulate matter (PM2.5) in vitro. BMC Complement Altern Med 19:369. https://doi.org/10.
1186/s12906-019-2790-3
30. Wang W, Meng H (2015) Cytotoxic, anti-inflammatory and hemostatic spirostane-steroidal
saponins from the ethanol extract of the roots of Bletilla striata. Fitoterapia 101:12–18. https://
doi.org/10.1016/j.fitote.2014.11.005
31. Dong J, Zhao L, Cai L et al (2014) Antioxidant activities and phenolics of fermented Bletilla
formosana with eight plant pathogen fungi. J Biosci Bioeng 118:396–399. https://doi.org/10.
1016/j.jbiosc.2014.03.003
32. Chen Z, Zhao Y, Zhang M et al (2020) Structural characterization and antioxidant activity of a
new polysaccharide from Bletilla striata fibrous roots. Carbohydr Polym 227:115362. https://
doi.org/10.1016/j.carbpol.2019.115362
33. Song Y, Zeng R, Hu L et al (2017) In vivo wound healing and in vitro antioxidant activities of
Bletilla striata phenolic extracts. Biomed Pharmacother 93:451–461. https://doi.org/10.1016/j.
biopha.2017.06.079
34. Zhang C, Gao F, Gan S et al (2019) Chemical characterization and gastroprotective effect of an
isolated polysaccharide fraction from Bletilla striata against ethanol-induced acute gastric ulcer.
Food Chem Toxicol 131:110539. https://doi.org/10.1016/j.fct.2019.05.047
35. Jiang F, Li W, Huang Y et al (2013) Antioxidant, Antityrosinase and antitumor activity
comparison: the potential utilization of fibrous root part of Bletilla striata (Thunb.) Reichb.f.
PLoS One 8(2):e58004. https://doi.org/10.1371/journal.pone.0058004
36. Niu J, Wang S, Wang B et al (2020) Structure and anti-tumor activity of a polysaccharide from
Bletilla ochracea Schltr. Int J Biol Macromol 154:1548–1555. https://doi.org/10.1016/j.
ijbiomac.2019.11.039
37. Sun A, Liu J, Pang S et al (2016) Two novel phenanthraquinones with anti-cancer activity
isolated from Bletilla striata. Bioorganic Med Chem Lett. 26(9):2375–2379. https://doi.org/10.
1016/j.bmcl.2016.01.076
38. Guo JJ, Dai BL, Chen NP et al (2016) The anti-Staphylococcus aureus activity of the
phenanthrene fraction from fibrous roots of Bletilla striata. BMC Complement Altern Med
16(1):491. https://doi.org/10.1186/s12906-016-1488-z
23 Orchids of Genus Bletilla: Traditional Uses, Phytochemistry,. . . 589
39. Jiang S, Chen CF, Ma XP et al (2019) Antibacterial stilbenes from the tubers of Bletilla striata.
Fitoterapia 138:104350. https://doi.org/10.1016/j.fitote.2019.104350
40. Jiang S, Wan K, Lou HY et al (2019) Antibacterial bibenzyl derivatives from the tubers
of Bletilla striata. Phytochemistry 162:216–223. https://doi.org/10.1016/j.phytochem.2019.
03.022
41. Qian CD, Jiang FS, Yu HS et al (2015) Antibacterial biphenanthrenes from the fibrous roots of
Bletilla striata. J Nat Prod 78(4):939–943. https://doi.org/10.1021/np501012n
42. Dong L, Liu X-X, Wu S-X et al (2020) Rhizoma Bletillae polysaccharide elicits hemostatic
effects in platelet-rich plasma by activating adenosine diphosphate receptor signaling pathway.
Biomed Pharmacother 130:110537
43. Yang C, Xia T, Wang C et al (2019) Using the UPLC–ESI–Q-TOF–MSE method and intestinal
bacteria for metabolite identification in the nonpolysaccharide fraction from Bletilla striata.
Biomed Chromatogr 33:e4637. https://doi.org/10.1002/bmc.4637
44. Lu B, Xu YM, Zhang HM, Li TJQY (2005) Effects of different extracts from Bletilla colloid on
rabbit platelet aggregation. Pharm J PLA 21:330–331
45. Wang W, Cheng MH, Gao JZX (2016) Study on hemostasis of Bletilla striata hemostatic
sponge. J Pharm Pract 34:32–35
46. Wang Y, Han S, Li R et al (2019) Structural characterization and immunological activity of
polysaccharides from the tuber of Bletilla striata. Int J Biol Macromol 122:628–635. https://doi.
org/10.1016/j.ijbiomac.2018.10.201
47. Peng Q, Li M, Xue F, Liu H (2014) Structure and immunobiological activity of a new
polysaccharide from Bletilla striata. Carbohydr Polym 107:119–123. https://doi.org/10.1016/
j.carbpol.2014.02.042
48. Wang Y, Liu D, Chen S et al (2014) A new glucomannan from Bletilla striata: structural and
anti-fibrosis effects. Fitoterapia 92:72–78. https://doi.org/10.1016/j.fitote.2013.10.008
49. Shi Y, Zhang B, Lu Y et al (2017) Antiviral activity of phenanthrenes from the medicinal plant
Bletilla striata against influenza a virus. BMC Complement Altern Med 17:273. https://doi.org/
10.1186/s12906-017-1780-6
50. Chen Z, Cheng L, He Y, Wei X (2018) Extraction, characterization, utilization as wound
dressing and drug delivery of Bletilla striata polysaccharide: a review. Int J Biol Macromol
120:2076–2085
51. Diao H, Li X, Chen J et al (2008) Bletilla striata polysaccharide stimulates inducible nitric
oxide synthase and Proinflammatory cytokine expression in macrophages. J Biosci Bioeng 105:
85–89. https://doi.org/10.1263/jbb.105.85
52. Zhou D, Chen G, Ma YP et al (2019) Isolation, structural elucidation, optical resolution, and
Antineuroinflammatory activity of Phenanthrene and 9,10-Dihydrophenanthrene derivatives from
Bletilla striata. J Nat Prod 82(8):2238–2245. https://doi.org/10.1021/acs.jnatprod.9b00291
53. Morita H, Koyama K, Sugimoto Y, Kobayashi J (2005) Antimitotic activity and reversal of
breast cancer resistance protein-mediated drug resistance by stilbenoids from Bletilla striata.
Bioorganic Med Chem Lett 15(4):1051–1054. https://doi.org/10.1016/j.bmcl.2004.12.026
54. Shi S, Huang Z, Luo Y et al (2012) Bletilla striata polysaccharide for the treatment of ulcerative
colitis in mice. J China Pharm Univ 43(6):535–540
55. Ke CYZC (2011) Effects of Bletilla striata polysaccharides on ulcerative colitis. Chin Pharm
22:2132–2134
56. Yu L, Nie X, Pan H et al (2011) Diabetes mellitus ulcers treatment with Bletilla striata
polysaccharide. Zhongguo Zhongyao Zazhi 36(11):1487–1491. https://doi.org/10.4268/
cjcmm20111118
57. Li Q, Li K, Huang SS et al (2014) Optimization of extraction process and antibacterial activity
of bletilla striata polysaccharides. Asian J Chem 26:3574–3580. https://doi.org/10.14233/
ajchem.2014.16500
58. Wu RY, Chang SY, Hsieh TF, Sen CY (2013) Cryopreservation of Bletilla formosana seeds
(Orchidaceae) by desiccation. Sci Hortic 157:108–112. https://doi.org/10.1016/j.scienta.2013.
03.033
590 H. P. Devkota et al.
59. Hsiao YY, Pan ZJ, Hsu CC et al (2011) Research on orchid biology and biotechnology. Plant
Cell Physiol 52:1467–1486. https://doi.org/10.1093/pcp/pcr100
60. Hirano T, Godo T, Mii M, Ishikawa K (2005) Cryopreservation of immature seeds of Bletilla
striata by vitrification. Plant Cell Rep 23:534–539. https://doi.org/10.1007/s00299-004-0893-9
61. Hu WH, Yang YH, Liaw SI, Chang C (2013) Cryopreservation the seeds of a Taiwanese
terrestrial orchid, Bletilla formosana (Hayata) Schltr. by vitrification. Bot Stud 54:33. https://
doi.org/10.1186/1999-3110-54-33
62. Wu R-Y, Chang S-Y, Hsieh T-F et al (2016) Cryopreservation of orchid genetic resources by
desiccation: a case study of Bletilla formosana. In: Marco-Jiménez F, Akdemir H (eds)
Cryopreservation in Eukaryotes. IntechOpen. https://doi.org/10.5772/65302
63. Ishikawa K, Harata K, Mii M et al (1997) Cryopreservation of zygotic embryos of a Japanese
terrestrial orchid (Bletilla striata) by vitrification. Plant Cell Rep 16:754–757. https://doi.org/
10.1007/s002990050314
Orchids of Genus Vanda: Traditional Uses,
Phytochemistry, Bioactivities, 24
and Commercial Importance
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
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
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
594 H. P. Devkota et al.
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].
24 Orchids of Genus Vanda: Traditional Uses, Phytochemistry,. . . 595
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].
596 H. P. Devkota et al.
4 Phytochemistry
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.
Fig. 6 Structures of eucomic acid derivatives, bibenzyl derivative, and phenolic compounds
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].
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
600 H. P. Devkota et al.
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.
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.
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].
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.
602 H. P. Devkota et al.
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.
References
1. Cakova V, Urbain A, Le Quéméner C et al (2015) Purification of vandaterosides from Vanda
teres (Orchidaceae) by stepwise gradient centrifugal partition chromatography. J Sep Sci 38:
3006–3013. https://doi.org/10.1002/jssc.201500349
2. Khan H, Marya, Belwal T et al (2019) Genus Vanda: a review on traditional uses, bioactive
chemical constituents and pharmacological activities. J Ethnopharmacol 229:46–53
3. Bulpitt CJ, Li Y, Bulpitt PF, Wang J (2007) The use of orchids in Chinese medicine. J R Soc
Med 100:558–563. https://doi.org/10.1258/jrsm.100.12.558
4. Gutiérrez RMP (2010) Orchids: a review of uses in traditional medicine, its phytochemistry and
pharmacology. J Med Plants Res 4:592–638. https://doi.org/10.5897/JMPR10.012
5. Pant B (2013) Medicinal orchids and their uses: tissue culture a potential alternative for
conservation. African J Plant Sci 7:448–467. https://doi.org/10.5897/ajps2013.1031
6. Hadi H, Razali SNS, Awadh AI (2015) A comprehensive review of the cosmeceutical benefits
of vanda species (orchidaceae). Nat Prod Commun 10:1483–1488
7. Gardiner LM, Kocyan A, Motes M et al (2013) Molecular phylogenetics of Vanda and related
genera (Orchidaceae). Bot J Linn Soc 173:549–572. https://doi.org/10.1111/boj.12102
8. The Plant List (2013) The Plant List. Version 1.1. In: Internet. http://www.theplantlist.org/1.1/
browse/A/Orchidaceae/Vanda/. Retrived on 15 Dec 2020
9. Musharof Hossain M (2011) Therapeutic orchids: traditional uses and recent advances – an
overview. Fitoterapia 82:102–140
10. Hossain MM (2009) Traditional therapeutic uses of some indigenous orchids of Bangladesh.
Med Aromat Plant Sci Biotechnol 3:100–106
11. Pant B, Raskoti BB (2013) Medicinal orchids of Nepal. Himalayan Map House, Kathmandu
12. De LC, Rao AN, Rajeevan PK et al (2015) Morphological characterization in Vanda spp. Int J
Sci Res 4:26–32
13. De Lakshman C, Pathak P, Rao AN, Rajeevan PK (2015) Commercial orchids. De Gruyter
Open Ltd, Warsaw/Berlin
14. Motes MR (1997) Vandas: their botany, history and culture. Timber Press, Portland/Cambridge
15. Panda A, Mandal D (2013) The folklore medicinal orchids of Sikkim. Anc Sci Life 33:90.
https://doi.org/10.4103/0257-7941.139043
16. Deb CR, Deb MS, Jamir NS, Imchen T (2009) Orchids in indigenous system of medicine in
Nagaland, India. Pleione 3:209–211
17. Manandhar NP (2002) Plants and people of Nepal. Timber Press, Inc., Portland
18. Subedi A, Kunwar B, Choi Y et al (2013) Collection and trade of wild-harvested orchids in
Nepal. J Ethnobiol Ethnomed:9. https://doi.org/10.1186/1746-4269-9-64
19. Singh MP, Dey S (2005) Indian medicinal plants. Satish Serial Publishing House, New Delhi
24 Orchids of Genus Vanda: Traditional Uses, Phytochemistry,. . . 603
20. Rao TA (1998) Conservation of wild orchids of Kodagu in the Western Ghats. Agricultural
Technologies and Services Pvt., Bangalore
21. Dasari R, Sathyavati D, Belide SK et al (2013) Evaluation of antioxidant activity of two
important memory enhancing medicinal plants Celtis timorensis and Vanda spathulata. Asian
J Pharm Clin Res 6:153–155
22. Vaidya B, Shrestha M, Joshee N (2000) Report on Nepalese orchid species with medicincal
properties. In: Watanabe T, Takano A, Bista MS, Saiju HK (eds) Proceeding of Nepal-Japan
joint symposium on conservation and utilization of Himalayan medicinal resources
23. Shengji P, Zhiwei Y (2018) Orchids and its uses in chinese Chinese medicine and health care
products. Med Res Innov 2. https://doi.org/10.15761/mri.1000133
24. Rajendran A, Rao NR, Kumar KR, Henry AN (1997) Some medicinal orchids of southern India.
Anc Sci Life 17:10–104
25. Zeng X, Zhang S, Zhang L et al (2006) A study of the neuroprotective effect of the phenolic
glucoside gastrodin during cerebral ischemia in vivo and in vitro. Planta Med 72:1359–1365.
https://doi.org/10.1055/s-2006-951709
26. Yang L, Wang Z, Xu L (2006) Simultaneous determination of phenols (bibenzyl, phenanthrene,
and fluorenone) in Dendrobium species by high-performance liquid chromatography with diode
array detection. J Chromatogr A 1104:230–237. https://doi.org/10.1016/j.chroma.2005.12.012
27. Williams CA (1979) The leaf flavonoids of the orchidaceae. Phytochemistry 18:803–813.
https://doi.org/10.1016/0031-9422(79)80019-9
28. Simmler C, Antheaume C, Lobstein A (2010) Antioxidant biomarkers from Vanda coerulea
stems reduce irradiated HaCaT PGE-2 production as a result of COX-2 inhibition. PLoS One
5. https://doi.org/10.1371/journal.pone.0013713
29. Anuradha V, Prakasa Rao NS (1998) Parviflorin a phenanthropyran from Vanda parviflora.
Phytochemistry 48:181–182. https://doi.org/10.1016/S0031-9422(97)00610-9
30. Anuradha V, Prakasa Rao NS (1998) Tessallatin a phenanthropyran from Vanda tessalata.
Phytochemistry 48:183–184. https://doi.org/10.1016/S0031-9422(97)00611-0
31. Anuradha V, Basaveswara Rao MV, Aswar AS (2008) Oxo-tessallatin, a novel
phenanthrapyrone isolated from Vanda tessalata. Orient J Chem 24:1119–1122
32. Uddin MN, Afrin R, Uddin MJ et al (2015) Vanda roxburghii chloroform extract as a potential
source of polyphenols with antioxidant and cholinesterase inhibitory activities: identification of
a strong phenolic antioxidant. BMC Complement Altern Med 15. https://doi.org/10.1186/
s12906-015-0728-y
33. Junka N, Kanlayanarat S, Buanong M, Wongs-Aree C (2012) Characterisation of floral antho-
cyanins and their antioxidant activity in Vanda hybrid (V. teres x V. hookeriana). J Food Agric
Environ 10:221–226
34. Junka N, Kanlayanarat S, Buanong M et al (2011) Analysis of anthocyanins and the expression
patterns of genes involved in biosynthesis in two Vanda hybrids. Int J Agric Biol 13:873–880
35. Tatsuzawa F, Saito N, Seki H et al (2004) Acylated anthocyanins in the flowers of Vanda
(Orchidaceae). Biochem Syst Ecol 32:651–664. https://doi.org/10.1016/j.bse.2004.02.004
36. Brandänge S, Granelli I, Johanson R et al (1973) Studies on Orchidaceae alkaloids. XXXVI.
Alkaloids from some Vanda and Vandopsis species. Acta Chem Scand 27:1096–1097. https://
doi.org/10.3891/acta.chem.scand.27-1096
37. Subramoniam A, Gangaprasad A, Sureshkumar PK et al (2013) A novel aphrodisiac compound
from an orchid that activates nitric oxide synthases. Int J Impot Res 25:212–216. https://doi.org/
10.1038/ijir.2013.18
38. Chawla AS, Sharma AK, Handa SS, Dhar KL (1992) Chemical studies and antiinflammatory
activity of Vanda roxburghii roots. Indian J Pharm Sci 54(4):159–161
39. Das S, Bhattacharya A, Bhattacharya AK (1967) Active constituents of Vanda roxburghi.
J Indian Chem Soc 44:804–805
40. Joshi PR, Paudel MR, Chand MB et al (2020) Cytotoxic effect of selected wild orchids on two
different human cancer cell lines. Heliyon 6:e03991. https://doi.org/10.1016/j.heliyon.2020.
e03991
604 H. P. Devkota et al.
41. Dahmén J, Leander K (1976) The structure of parishin, a glucoside from Vanda parishii.
Phytochemistry 15:1986–1987. https://doi.org/10.1016/S0031-9422(00)88865-2
42. Prakash B, Bias RT (2016) A novel chemical from the leaf extract of Vanda tesselata (Roxb.)
hook. ex. G. Don. World J Pharceutical Res 5:1049–1058
43. Begum Y, Sen P, Bulbul I, Nasrin F (2018) Evaluation of analgesic and anti-inflammatory
potentials of the leaf and root extracts of Vanda roxburghii (Roxb). J Complement Altern Med
Res. https://doi.org/10.9734/jocamr/2018/40002
44. Vijaykumar K (2013) In vitro anti-oxidant activity of pet-ether extract of Vanda tessellata Roxb.
Int Ayur Med J 1:1–4
45. Thaakur SR, Pokkula S (2013) Ameliorative effects of Vanda testacea in sciatic nerve
transection-induced neuropathy in rats. Int J Pharma Bio Sci 4(1):271–284
46. Islam SS, Sayeed H, Shahriyar SA, Ferdous AIA (2016) Antioxident, analgesic and cytotoxic
activity of methanolic extract of Vanda roxburghii root. Aging (Albany NY) 1:3
47. Chowdhury MA, Rahman MM, Chowdhury HRH et al (2014) Antinociceptive and cytotoxic
activities of an epiphytic medicinal orchid: Vanda tessellata Roxb. BMC Complement Altern
Med 14. https://doi.org/10.1186/1472-6882-14-464
48. Prakash B, Bais RT, Sahu RK (2018) Screening of antioxidant and antidepressant activity of
Vanda tessellata leaves extract. Int J Green Pharm 12(4):S829–S834. https://doi.org/10.22377/
ijgp.v12i04.2262
49. Anwar M, Indro S, Wisnu RW et al (2013) Hepatoprotective activity of pet-ether extract of
Vanda tessellata Roxb. Proc 2013 J Int Conf Rural Inf Commun Technol Electr Technol rICT
ICEV-T 2013:3–6
50. Bhattacharjee B, Islam T, Rahman MZ, Shahinul Islam SM (2015) Antimicrobial activity and
phytochemical screening of whole plant extracts of Vanda tessellata (Roxb.) Hook.ex.D.Don.
World J Pharm Pharm Sci 4(1):72–83
51. Andre P, Archambault JC, Renimel I (2011) Use of an extract of the orchid Vanda coerulea as a
skin hydrating agent. US Patent US8039028B2
52. Bonté F, Simmler C, Lobstein A et al (2011) Action d’un extrait de Vanda coerulea sur la
sénescence de fibroblastes cutanés. Ann Pharm Fr 69:177–181. https://doi.org/10.1016/j.
pharma.2011.02.001
53. Cauchard JH, Pellicier F, Archambault JC, et al (2011) Orchid vanda coerulea as a cosmetic
active agent. US Patent 0091588 A1
54. Mundugaru R, Sharma S, Sivanesan S et al (2020) Neuroprotective effect of vanda roxburghii
extract in endothelin-1 (Et-1) induced hippocampal ischemic damage and ameliorate cognitive
deficit. Indian J Pharm Educ Res 54(3):740–749. https://doi.org/10.5530/ijper.54.3.125
55. Uddin MJ, Rahman MM, Abdullah-Al-Mamun M, Sadik G (2015) Vanda roxburghii: an
experimental evaluation of antinociceptive properties of a traditional epiphytic medicinal orchid
in animal models. BMC Complement Altern Med 15:305. https://doi.org/10.1186/s12906-015-
0833-y
56. Pathan D, Ambavade S (2014) Investigation of anticonvulsant activity of Vanda roxburghii.
J Pharmacog Phytochem 2:95–99
57. Suresh KPK, Subramoniam A, Pushpangadan P (2000) Aphrodisiac activity of Vanda tessellata
(Roxb.) Hook. ex Don extract in male mice. Indian J Pharmacol 32:300–304
58. Nayak BS, Suresh R, Rao AVC et al (2005) Evaluation of wound healing activity of Vanda
roxburghii R.Br (Orchidacea): a preclinical study in a rat model. Int J Low Extrem Wounds 4:
200–204. https://doi.org/10.1177/1534734605282994
59. Hsiao YY, Pan ZJ, Hsu CC et al (2011) Research on orchid biology and biotechnology. Plant
Cell Physiol 52:1467–1486. https://doi.org/10.1093/pcp/pcr100
60. Teoh ES (2016) Medicinal orchids of Asia. Springer, Cham. https://doi.org/10.1007/978-3-319-
24274-3
61. Sripotar D (2008) Non-detrimental finding of Vanda coerulea. NDF WORKSHOP WG
4 – Geophytes and Epiphytes case study 4 summary
24 Orchids of Genus Vanda: Traditional Uses, Phytochemistry,. . . 605
62. Hinsley A, De Boer HJ, Fay MF et al (2018) A review of the trade in orchids and its implications
for conservation. Bot J Linn Soc 186:435–455. https://doi.org/10.1093/botlinnean/box083
63. Pant B, Pradhan S, Paudel MR et al (2019) Various culture techniques for the mass propagation
of medicinal orchids from Nepal. Acta Horticulturae 1262:109–124
64. Kaur S, Bhutani KK (2009) In vitro propagation of Vanda testacea (Lindl.) Reichb. f. – a rare
orchid of high medicinal value. Plant Tissue Cult Biotechnol 19:1–7. https://doi.org/10.3329/
ptcb.v19i1.4077
65. Chand K, Shah S, Sharma J et al (2020) Isolation, characterization, and plant growth-promoting
activities of endophytic fungi from a wild orchid Vanda cristata. Plant Signal Behav 15. https://
doi.org/10.1080/15592324.2020.1744294
66. Aggarwal S, Nirmala C, Beri S et al (2012) In vitro symbiotic seed germination and molecular
characterization of associated endophytic fungi in a commercially important and endangered
Indian orchid Vanda coerulea Griff. Ex Lindl. Eur J Environ Sci 2:33–42. https://doi.org/10.
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
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.
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].
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
612 M. Kanlayavattanakul and N. Lourith
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].
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].
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].
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
614 M. Kanlayavattanakul and N. Lourith
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].
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
25 Orchid Extracts and Cosmetic Benefits 615
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].
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].
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
616 M. Kanlayavattanakul and N. Lourith
(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].
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].
25 Orchid Extracts and Cosmetic Benefits 617
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].
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].
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].
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
618 M. Kanlayavattanakul and N. Lourith
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].
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].
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
Orchid Extracts and Cosmetic Benefits
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
25 Orchid Extracts and Cosmetic Benefits 621
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
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
Orchid Extracts and Cosmetic Benefits
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
624 M. Kanlayavattanakul and N. Lourith
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.
References
1. Bulpitt CJ, Li Y, Bulpitt PF, Wang J (2007) The use of orchids in Chinese medicine. J R Soc
Med 100:558–563
2. Singh A, Duggal S (2009) Medicinal orchids-an overview. Ethnobot Leafl 13:399–412
3. Kong JM, Goh NK, Chia LS, Chia TF (2003) Recent advances in traditional plant drugs and
orchids. Acta Pharmacol Sin 24:7–21
4. Hossain MM (2011) Therapeutic orchids: traditional uses and recent advances – an overview.
Fitoterapia 82:102–140
5. Ng TB, Liu J, Wong JH, Ye X, Sze SCW, Tong Y, Zhang KY (2012) Review of research on
Dendrobium, a prized folk medicine. Appl Microbiol Biotechnol 93:1795–1803
6. Kanlayavattanakul M, Lourith N (2015) An update on cutaneous aging treatment using herbs.
J Cosmet Laser Ther 17:343–352
7. Lourith N, Kanlayavattanakul M (2016) Biopolymeric agents for skin wrinkle treatment.
J Cosmet Laser Ther 18:301–310
8. Kammeyer A, Luiten RM (2015) Oxidation events and skin aging. Ageing Res Rev 21:16–29
9. Pradhan S, Madke B, Kabra P, Singh AL (2016) Anti-inflammatory and immunomodulatory
effects of antibiotics and their used in dermatology. Indian J Dermatol 61:469–481
10. Kanlayavattanakul M, Lourith N (2015) Biopolysaccharides for skin hydrating cosmetics.
In: Ramawat KG, Mérillon JM (eds) Polysaccharides. Springer, Switzerland
11. Kanlayavattanakul M, Lourith N (2018) Plants and natural products for the treatment of skin
hyperpigmentation – a review. Planta Med 84:988–1006
12. Kanlayavattanakul M, Lourith N (2018) Skin hyperpigmentation treatment using herbs:
a review of clinical evidences. J Cosmet Laser Ther 20:123–131
13. Bhattacharyya P, Van Staden J (2016) Ansellia africana (leopard orchid): a medicinal orchid
species with untapped reserves of important biomolecules – a mini review. S Afr J Bot
106:181–185
25 Orchid Extracts and Cosmetic Benefits 625
14. Chinsamy M, Finnie JF, Van Staden J (2014) Anti-inflammatory, antioxidant, anti-cholinester-
ase activity and mutagenicity of South African medicinal orchids. S Afr J Bot 91:88–98
15. Athipornchi A, Jullapo N (2018) Tyrosinase inhibitory and antioxidant activities of orchid
(Dendrobium spp.). S Afr J Bot 119:188–192
16. Kanlayavattanakul M, Lourith N, Chaikul P (2018) Biological activity and phytochemical
profiles of Dendrobium: a new source for specialty cosmetic materials. Ind Crop Prod
120:61–70
17. Wang Q, Sun P, Li G, Zhu K, Wang C, Zhao X (2014) Inhibitory effects of Dendrobium
candidum Wall ex Lindl. on azoxymethane- and dextran sulfate sodium-induced colon
carcinogenesis in C57BL/6 mice. Oncol Lett 7:493–498
18. Dong FW, Luo HR, Wan QL, Xu FQ, Fan WW, Wang KJ, Li N, Hu J-M (2012) Two new
bibenzyl glucosides from Dendrobium chrysotoxum. Bull Kor Chem Soc 33:2247–2250
19. Lin Y, Wang F, Yang L-J, Chun Z, Bao J-K, Zhang G-I (2013) Anti-inflammatory phenanthrene
derivatives from stems of Dendrobium denneanum. Phytochemistry 95:242–251
20. Ge J-C, Zha X-Q, Nie C-Y, Yu N-J, Li Q-M, Peng D-Y, Duan J, Pan L-H, Luo J-P (2018)
Polysaccharides from Dendrobium huoshanense stems alleviates lungs inflammation in
cigarette smoke-induced mice. Carbohydr Polym 189:289–295
21. Li Y, Li F, Gong Q, Wu Q, Shi J (2011) Inhibitory effects of Dendrobium alkaloids on memory
impairment induced by lipopolysaccharide in rats. Planta Med 77:117–121
22. Kim JH, Oh S-Y, Han S-B, Uddin GM, Kim CY, Lee JK (2015) Anti-inflammatory effects
of Dendrobium nobile derived phenanthrenes in LPS-stimulated murine macrophages.
Arch Pharm Res 38:1117–1126
23. Zeng Q, Ko C-H, Siu W-S, Li L-F, Han X-Q, Yang L, Lau CB-S, Hu J-M, Leung P-C (2017)
Polysaccharides of Dendrobium officinale Kimura & Migo protect gastric mucosal cell against
oxidative damage-induced apoptosis in vitro and in vivo. J Ethnopharmacol 208:214–224
24. Liang J, Chen S, Chen J, Lin J, Xiong Q, Yang Y, Yuan J, Zhou L, He L, Hou S, Li S, Hong S,
Lai X (2018) Therapeutic roles of polysaccharides from Dendrobium officinale on colitis and its
underlying mechanisms. Carbohydr Polym 185:159–168
25. Wu Y-Y, Liang C-Y, Liu T-T, Liang Y-M, Li S-J, Lu Y-Y, Liang J, Yuan X, Li C-J, Hou S-Z,
Lai X-P (2018) Protective roles and mechanisms of polysaccharides from Dendrobium officinal
on natural aging-induced premature ovarian failure. Biomed Pharmacother 101:953–960
26. Zhang Z, Zhang D, Dou M, Li Z, Zhang J, Zhao X (2016) Dendrobium officinale Kimura et
Migo attenuates diabetic cardiomyopathy through inhibiting oxidative stress, inflammation and
fibrosis in steptozotocin-induced mice. Biomed Pharmacother 84:1350–1358
27. Huang X, Nie S, Cai H, Zhang G, Cui SW, Xie M, Philips GO (2015) Study on Dendrobium
officinale O-acetyl-glucomannan (Dendronan ®): part VI. Protective effect against oxidative
stress in immunosuppressed mice. Food Res Int 72:168–173
28. Cai H-L, Huang H-J, Nie S-P, Xie M-Y, Philips GO, Cui SW (2015) Study on Dendrobium
officinale O-acetyl-glucomannan (Dendronan ®): part III – immunomodulatory activity in vitro.
Bioact Carbohydr Diet Fibre 5:99–105
29. Huang X, Nie S, Cai H, Zhang C, Cui SW, Xie M, Philipes GO (2015) Study on Dendrobium
officinale O-acetyl-glucomannan (Dendronan): part IV. Immunomodulatory activity in vivo.
J Funct Foods 15:525–532
30. Zhang Y, Zhang L, Liu J, Liang J, Si J, Wu S (2017) Dendrobium officinale leaves as a new
antioxidant source. J Funct Foods 37:400–415
31. Wu C-T, Huang K-S, Yang C-H, Chen Y-C, Liao J-W, Kuo C-L, Chen C-L, Lo S-F, Hsieh C-C,
Tsay H-S (2014) Inhibitory effects of cultured Dendrobium tosaense on atopic dermatitis
murine model. Int J Pharm 463:193–200
32. Schuster R, Zeindl L, Holzer W, Khumpirapang N, Okonogi S, Viernstein H, Mueller M (2017)
Eulophia macrobulbon – an orchid with significant anti-inflammatory and antioxidant effect
and anticancerogenic potential exerted by its root extract. Phytomedicine 24:157–165
33. Tatsuzawa F, Saita N, Seki H, Yokoi M, Yukawa T, Shinoda K, Honda T (2004) Acylated
anthocyanins in the flowers of Vanda (Orchidaceae). Biochem Syst Ecol 32:651–664
626 M. Kanlayavattanakul and N. Lourith
34. Simmler C, Antheaume C, Lobstein A (2010) Antioxidant biomarkers from Vanda coerulea
stems reduces irradiated HaCaT PGE-2 production as a result of COX-2 inhibition. PLoS One
5:e13713
35. Bonté F, Simmler C, Lobstein A, Pellicier F, Cauchard J-H (2011) Action of an extract of Vanda
coerulea on the senescence of skin fibroblasts. Ann Pharm Fr 69:177–181
36. Nayak B, Suresh R, Rao AVC, Pilai GK, Davis EM, Ramkisson V, McRae A (2005) Evaluation
of wound healing activity of Vanda roxburghii R. Br (Orchidaceae): a preclinical study in a rat
model. Int J Low Extrem Wounds 4:200–204
37. Uddin MN, Afrin R, Uddin MJ, Uddin MJ, Alam AHMK, Rahman AA, Sadik G (2015)
Vanda roxburghii chloroform extract as a potential source of polyphenols with antioxidant
and cholinesterase inhibitory activities: identification of a strong phenolic antioxidant. BMC
Complement Altern Med 15:195
38. Simmler C, Antheaume C, André P, Bonté F, Lobstein A (2011) Glucosyloxybenzyl eucomate
derivatives from Vanda teres stimulate HaCaT cytochrome c oxidase. J Nat Prod 74:949–955
39. Singh DK (2001) Orchid diversity in India: an overview. In: Patak P, Sehgal RN, Shekhar N,
Sharma M, Sood A (eds) Orchid: science and commerce. Bishen Singh Mahendra Pal Singh,
Dehradun
40. Lin CC, Huang PC, Lin JM (2000) Antioxidant and hepatoprotective effects of Anoectochilus
formosanus and Gynostemma pentaphyllum. Am J Chin Med 28:87–96
41. Shih CC, Wu YW, Lin WC (2002) Antihyperglycaemic and anti-oxidant properties of
Anoectochilus formosanus in diabetic rats. Clin Exp Pharmacol Physiol 29:684–688
42. Kumar S (2002) The medicinal plants of North-East India. Scientific Publishers, Jodhpur
43. Maridass M, Hussain MIZ, Raju G (2008) Phytochemical survey of orchids in the Tirunelveli
hills of South India. Ethnobot Leafl 12:705–712
44. Jalal JS, Kumar P, Pangtey YPS (2008) Ethnomedicinal orchids of Uttarakhand, Western
Himalaya. Ethnobot Leaft 12:1227–1230
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.
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
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
Orchids from Basilicata: The Scent
OH CO2Me
CHO CHO
O O
H H
3 4
2
1
5 6 7
CH2OH CH2OH
O O OH
H H
8 9 10
O OH O
O O
O
11 12 13
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
26 Orchids from Basilicata: The Scent 633
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
634 M. D’Auria et al.
14 15
16
17 18
CO2Et
19 20
CHO
21 22
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
26 Orchids from Basilicata: The Scent 635
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
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.
26 Orchids from Basilicata: The Scent 637
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
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),
26 Orchids from Basilicata: The Scent 639
Monoterpenes
OH
OH
OH
Sesquiterpenes
OH
OH
H H H
OHC
H H H H
Terpenoids
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
Orchids from Basilicata: The Scent
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.
26 Orchids from Basilicata: The Scent 643
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
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.
References
1. Pawliszyn J (1997) Solid-phase microextraction: theory and practice. Wiley, New York
2. Bentivenga G, D’Auria M, De Luca E, De Bona A, Mauriello G (2001) The use of SPME-GC-
MS in the analysis of flavor of virgin olive oil. Riv Ital Sostanze Gr 78:157–162
3. Bentivenga G, D’Auria M, De Bona A, Mauriello G (2002) On the flavor of virgin olive oil. Riv
Ital Sostanze Gr 79:101–105
4. Bentivenga G, D’Auria M, Mauriello G, Racioppi R, Viggiani L (2004) Characterization of
virgin olive oils from Basilicata by using 1H-NMR and SPME-GC-MS. Riv Ital Sostanze Gr
81:86–89
5. D’Auria M, Emanuele L, Mauriello G, Racioppi R (2003) On the origin of “Goût de Lumiere”
in champagne. J Photochem Photobiol A Chem 158:21–26
6. Mauriello G, Capece A, D’Auria M, Garde-Cerdán T, Romano P (2009) SPME-GC method as a
tool to differentiate VOC profiles in Saccharomyces cerevisiae wine yeasts. Food Microbiol
26:246–252
7. D’Auria M, Emanuele L, Racioppi R (2009) The effect of heat and light on the composition of
some volatile compounds in wine. Food Chem 117:9–14
8. Cellamare L, D’Auria M, Emanuele L, Racioppi R (2009) The effect of light on the composition
of some volatile compounds in wine: an HS-SPME-GC-MS study. Int J Food Sci Technol
44:2377–2384
9. Acquaviva V, D’Auria M, Racioppi R (2014) Changes in aliphatic ester composition in white
wines during exposition to light. An HS-SPME-GC-MS study. J Wine Res 25:63–74
10. D’Auria M, Mauriello G, Rana GL (2004) Volatile organic compounds from saffron. Flav Fragr
J 19:17–23
11. D’Auria M, Mauriello G, Racioppi R, Rana GL (2006) Use of SPME-GC-MS in the study of
time evolution of the constituents of saffron aroma: modifications of the composition during
storage. J Chromatogr Sci 44:18–21
12. Mauriello G, Marino R, D’Auria M, Cerone G, Rana GL (2004) Determination of volatile
organic compounds from truffles via SPME-GC-MS. J Chromatogr Sci 42:299–305
13. D’Auria M, Rana GL, Racioppi R, Laurita A (2012) Studies on volatile organic compounds of
Tuber borchii and T. asa-foetida. J Chromatogr Sci 50:775–778
14. D’Auria M, Racioppi R, Rana GL (2013) Volatile organic compounds of Schenella pityophilus.
Nat Prod Res 27:41–44
15. D’Auria M, Racioppi R, Rana GL, Laurita A (2014) Studies on volatile organic compounds of
some truffles and false truffles. Nat Prod Res 28:1709–1717
16. Mang SM, Racioppi R, Camele I, Rana GL, D’Auria M (2015) Use of volatile metabolite
profiles to distinguish three Monilinia species. J Plant Pathol 97:55–59
17. Mang SM, Zotta T, Camele I, Racioppi R, D’Auria M, Rana GL (2017) Morphological,
physico-chemical and molecular investigations on Tuber bellonae from Basilicata – Italy.
J Anim Plant Sci 27:528–541
18. Bentivenga G, D’Auria M, Fedeli P, Mauriello G, Racioppi R (2004) SPME-GC-MS analysis of
volatile organic compounds in honey from Basilicata. Evidence for the presence of pollutants
from anthropogenic activities. Int J Food Sci Technol 39:1079–1086
19. D’Auria M, Mauriello G, Racioppi R (2004) SPME-GC-MS analysis of horseradish
(Armoracia rusticana). Ital J Food Sci 16:501–504
26 Orchids from Basilicata: The Scent 647
20. D’Auria M, Mauriello G, Marino R, Racioppi R (2005) Composition of volatile fraction from
Thymus, Origanum, Lavandula and Acinos species. J Essent Oil Bear Plants 8:36–51
21. D’Auria M, Racioppi R (2015) The effect of drying on the composition of volatile organic
compounds in Rosmarinus officinalis, Laurus nobilis, Salvia officinalis and Thymus serpyllum.
A HS-SPME-GC-MS study. J Essent Oil Bear Plants 18:1209–1223
22. Celano G, D’Auria M, Xiloyannis C, Mauriello G, Baldassarre M (2006) Composition and
seasonal variation of soluble cuticolar waxes in Actinidia deliciosa leaves. Nat Prod Res
20:701–709
23. D’Auria M, Racioppi R, Velluzzi V (2008) A comparison of results obtained using liquid
injection and headspace solid-phase microextraction for crude oil analysis by GC with mass
spectrometer detection. J Chromatogr Sci 46:332–338
24. D’Auria M, Racioppi R (2017) HS-SPME-GC-MS analysis of onion (Allium cepa L.) and
shallot (Allium ascalonicum L.). Food Res 1:161–165
25. D’Auria M, Emanuele L, Racioppi R, Stefanizzi N (2020) Determination of simple carbohy-
drates in beer using SPME, on fiber derivatization and gas chromatography mass spectrometry.
Food Res 4:1156–1161
26. Rosati L, Romano VA, Cerone L, Fascetti S, Potenza G, Bazzato E, Cillo D, Mecca M, Racioppi
R, D’Auria M, Farris E (2020) Pollination features and floral volatiles of Gymnospermium
scipetarum (Barberidaceae). J Plant Res 132:49–56
27. Kaiser R (1993) Von Duft der Orchideen. Editiones Roche, Basel
28. Tollsten L, Bergström LG (1993) Fragrance chemotypes of Platanthera (Orchidaceae) – the
result of adaptation to pollinating moths? Nord J Bot 13:607–613
29. Peplys D, Ibarra F, Francke W, Löfstedt C (2002) Odour-mediated nectar foraging in the silver
Y moth, Autographa gamma (Lepidoptera: Noctuidae): behavioural and electrophysiological
responses to floral volatiles. Oikos 99:75–82
30. Esposito F, Vereecken NJ, Gammella M, Rinaldi R, Laurent P, Tyteca D (2018) Characterization
of sympatric Platanthera bifolia and Platanthera chlorantha (Orchidaceae) populations with
intermediate plants. PeerJ 6:e4256
31. D’Auria M, Lorenz R, Racioppi R, Romano VA (2017) Fragrance components of Platanthera
bifolia subsp. osca. Nat Prod Res 31:1612–1619
32. D’Auria M, Lorenz R, Mecca M, Racioppi R, Romano VA, Viggiani L (2020) Fragrance
components of Platanthera bifolia subsp. osca and Platanthera chlorantha collected in several
sites in Italy. Nat Prod Res. https://doi.org/10.1080/14786419.2019.1593166
33. Knudsen JT, Tollsten L, Bergström LG (1993) Floral scent – a checklist of volatile compounds
isolated by head-space techniques. Phytochemistry 33:253–280
34. Williams NH, Written WM (1983) Orchid floral fragrance and male euglossine bees: methods
and advances in the fast sesquidecade. Biol Bull 163:355–395
35. Claessens J, Kleynen J (2013) The pollination of European orchids: part 2: Cypripedium and
Cephalanthera. J Hardy Orch Soc 10:114–120
36. D’Auria M, Lorenz R, Mecca M, Racioppi R, Romano VA (2020) Aroma components of
Cephalanthera orchids. Nat Prod Res. https://doi.org/10.1080/14786419.2019.1616724
37. Nilsson LA (1983) Mimesis of bellflower (Campanula) by the red helleborine orchid
Cephalathera rubra. Nature 305:799–800
38. Claessens J, Beentjes KK, Heijrman T, Miller J, Gravandeel B (2015) Beobachtungen von
Miarus campanulae als Bestäuber von Cephalanthera rubra. J Eur Orch 47:77–87
39. Milet-Pinheiro P, Ayasse M, Dötterl S (2015) Visual and olfactory cues of Campanula
(Campanulaceae) and their significance for host recognition by an oligolectic bee pollinator.
PLoS One 10:e0128577
40. Lorenz R, Romano VA (2014) Beiträge über die morphologische Differenzierung in der
Gattung Serapias: 1. Serapias cordigera in Süditalien. J Eur Orch 46:616–660
41. Pellegrino G, Luca A, Bellusci F, Musacchio A (2012) Comparative analysis of floral scents in
four sympatric species of Serapias L. (Orchidaceae): clues on their pollination strategies. Plant
Syst Evol 298:1837–1843
648 M. D’Auria et al.
42. Schiestl FP, Cozzolino S (2008) Evolution of sexual mimicry in the orchid subtribe orchidinae:
the role of preadaptations in the attraction of male bees as pollinators. BMC Evol Biol 8:27
43. Vereecken NJ, Wilson CA, Hötling S, Schulz S, Banketov SA, Mardulyn P (2012) Pre-
adaptations and the evolution of pollination by sexual deception: Cope’s rule of specialization
revisited. Proc R Soc B 279:4786–4794
44. D’Auria M, Lorenz R, Mecca M, Racioppi R, Romano VA (2020) The composition of the
aroma of Serapias orchids in Basilicata (southern Italy). Nat Prod Res. https://doi.org/10.1080/
14786419.2020.1713127
45. Delforge P (2016) Orchidées d’Europe, d’Afrique du Nord et du Proche-Orient: la bible des
orchidophiles, plus de 600 espèces et de nombreuses variétés et illustrées, 4th edn, revue et
augmentée. Delachaux et Niestlé, Paris
46. Bartolucci F, Peruzzi L, Galasso G, Albano A, Alessandrini A, Ardenghi NMG, Astuti G,
Bacchetta G, Ballelli S, Banfi E, Barberis G, Bernardo L, Bouvet D, Bovio M, Cecchi L, Di
Pietro R, Domina G, Fascetti S, Fenu G, Festi F, Foggi B, Gallo L, Gottschlich G, Gubellini L,
Iamonico D, Iberite M, Jiménez-Mejías P, Lattanzi E, Marchetti D, Martinetto E, Masin RR,
Medagli P, Passalacqua NG, Peccenini S, Pennesi R, Pierini B, Poldini L, Prosser F, Raimondo
FM, Roma-Marzio F, Rosati L, Santangelo A, Scoppola A, Scortegagna S, Selvaggi A, Selvi F,
Soldano A, Stinca A, Wagensommer RP, Wilhalm T, Conti F (2018) An updated checklist of the
vascular flora native to Italy. Plant Biosyst 152:179–303
47. Vereecken NJ, Dafni A, Cozzolino S (2010) Pollination syndromes in Mediterranean orchids –
implications for speciation, taxonomy and conservation. Bot Rev 76:220–240
48. Doneddu M (2015) Osservazioni sull’impollinazione di Barlia robertiana ad opera dei
coleotteri Tropinota squalida e Oxythyrea funesta (Cetoniidae) in Sardegna. GIROS Orch
Spont Eur 58:262–265
49. Smithson A, Gigord LDB (2001) Are there fitness advantages in being a rewardless orchid?
Reward supplementation experiments with Barlia robertiana. Proc R Soc Lond B Biol Sci
268:1435–1441
50. McBrien HL, Millar JG, Rice RE, McElfresh JS, Cullen E, Zalom FG (2002) Sex attractant
pheromone of the red-shouldered stink bug Thyanta pallidovirens: a pheromone blend with
multiple redundant components. J Chem Ecol 28:1797–1818
51. Tulp M, Bohlin L (2005) Rediscovery of known natural compounds: nuisance or goldmine?
Bioorg Med Chem 13:5274–5282
52. Gallego E, Gelabert A, Roca FJ, Perales JF, Guardino X (2012) Identification of volatile organic
compounds (VOC) emitted from three European orchid species with different pollination
strategies: two deceptive orchids (Himantoglossum robertianum and Ophrys apifera) and a
rewarding (Gymnadenia conopsea). J Biodivers Environ Sci 2:18–29
53. Salzmann CC, Cozzolino S, Schiestl FP (2007) Floral scent in food-deceptive orchids: species
specificity and sources of variability. Plant Biol 9:720–729
54. Jersáková J, Johnson SD, Kindlmann P (2006) Mechanisms and evolution of deceptive
pollination in orchids. Biol Rev 81:219–235
55. Moya S, Ackerman JD (1993) Variation in the floral fragrance of Epidendrum ciliare
(Orchidaceae). Nord J Bot 13:41–47
56. Smithson A, Macnair MR (1997) Negative frequency-dependent selection by pollinators on
artificial flowers without rewards. Evolution 51:715–723
57. Ackerman JD (1986) Mechanisms and evolution of food-deceptive pollination systems in
orchids. Lindleyana 1:108–113
58. Johnson SD, Nilsson LA (1999) Pollen carryover, geitonogamy, and the evolution of deceptive
pollination systems in orchids. Ecology 80:2607–2619
Index
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