Skip to main content
SpringerLink
Log in

Rapid and highly efficient callus induction and plant regeneration in the starch-rich duckweed strains of Landoltia punctata

  • Original Article
  • Published:
Acta Physiologiae Plantarum Aims and scope Submit manuscript

Abstract

The starch-rich duckweed Landoltia punctata is a valuable aquatic plant in wastewater purification, bioenergy production, and many other applications. A highly efficient callus induction and plant regeneration protocol is desirable so that biotechnology can be used to develop new varieties with added value and adaptation. We studied both known and unknown factors that influence callus induction in L. punctata and obtained almost 100 % induction rate in 30 days. The optimum medium for callus induction was MS basal medium supplemented with 1 % sorbitol, 15 mg/L 2,4-D, and 2 mg/L 6-BA. Green fragile callus was induced from the meristematic region in the budding pouches. The optimum photoperiod for callus induction was 16-h day, and the optimum explant orientation was dorsal side down on the medium. The optimum medium for callus subculture was WPM basal medium supplemented with 2 % sorbitol, 4 mg/L 2,4-D, and 0.5 mg/L TDZ. Green callus could be maintained by subculture once every 4 weeks. However, when the subculture cycle was prolonged to 6 weeks or longer, yellow fragile embryogenic callus was obtained. The optimum plant regeneration medium was MS medium supplemented with 0.5 % sucrose, 1 % sorbitol, and 1.0 mg/L 6-BA with frond regeneration rates of approximately 90 %. The regenerated fronds rooted in Hoagland’s liquid medium in 1 week. The callus induction and frond regeneration protocol was tested for its efficiency in geographically distinct strains 5502, 8721, and 9264. Thus, we obtained a rapid and efficient protocol for callus induction and frond regeneration of L. punctata, which takes only 9 weeks.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6

Similar content being viewed by others

Abbreviations

2,4-D:

2,4-Dichlorophenoxyacetic acid

NAA:

1-Naphthaleneacetic acid

6-BA:

6-Benzyladenine

2IP:

N6-[Δ2-Isopentyl] adenine

TDZ:

1-Phenyl-3-(1,2,3-thiadiazol-5-yl) urea

MS:

Murashige and Skoog medium

WPM:

Woody plant medium

SD:

Standard deviation

ANOVA:

Analysis of variance

References

  • Anderson KE, Lowman Z, Stomp AM, Chang J (2011) Duckweed as a feed ingredient in Laying Hen Diets and its effect on egg production and composition. Intern J Poultry Sci 10:4–7

    Article  CAS  Google Scholar 

  • Appenroth K-J, Borisjuk N, Lam E (2013) Telling duckweed apart: Genotyping technologies for the Lemnaceae. Chinese Journal of Applied Environmental Biology 19:1–10

    Article  CAS  Google Scholar 

  • Cantó-Pastor A et al (2015) Efficient transformation and artificial miRNA gene silencing in Lemna minor. Plant Biology 17:59–65

    Article  PubMed  PubMed Central  Google Scholar 

  • Chang WC, Chiu PL (1976) Induction of callus from fronds of duckweed (Lemna gibba L.). Bot Bull Acad Sinica 17:106–109

    Google Scholar 

  • Chang WC, Chiu PL (1978) Regeneration of Lemna gibba G3 through callus culture. Zeitschriftfschriftfür Pflanzenph 89:91–94

    Article  CAS  Google Scholar 

  • Chang WC, Hsing YI (1978) Callus formation and regeneration of frond-like structures in Lemna perpusilla 6746 on a defined medium. Plant Science Letters 13:133–136

    Article  Google Scholar 

  • Edelman M, Perl A, Flaishman M, Blumenthal A (1998) Transgenic Lemnaceae In: European Patent Specification EP 1021552B1

  • El-Shafai SA, El-Gohary FA, Nasr FA, van der Steen NP, Gijzen HJ (2007) Nutrient recovery from domestic wastewater using a UASB-duckweed ponds system. Bioresour Technol 98:798–807

    Article  CAS  PubMed  Google Scholar 

  • Ge X, Zhang N, Phillips GC, Xu J (2012) Growing Lemna minor in agricultural wastewater and converting the duckweed biomass to ethanol. Bioresour Technol 124:485–488

    Article  CAS  PubMed  Google Scholar 

  • Hanover J et al (1988) Nodule culture: a developmental pathway with high potential for regeneration, automated micropropagation, and plant metabolite production from woody plants. Genetic Manipulation of Woody Plants, vol 44. Basic Life Sciences. Springer, US, pp 149–166

    Chapter  Google Scholar 

  • Hoagland DR, Arnon DI (1950) The water-culture method for growing plants without soil. California Agricultural Experiment Station Circular 347:1–32

    Google Scholar 

  • Hossain MM, Kant R, Van PT, Winarto B, Zeng S, Teixeira da Silva JA (2013) The application of biotechnology to orchids. Critical Reviews in Plant Sciences 03/2013; 32(2) 32:69-139

  • Iqbal S (1999) Duckweed aquaculture: potentials, possibilities and limitations for combined wastewater treatment and animal feed production in developing countries. SANDEC Report No 6/99

  • Khvatkov P, Chernobrovkina M, Okuneva A, Shvedova A, Chaban I, Dolgov S (2015) Callus induction and regeneration in Wolffia arrhiza (L.) Horkel ex Wimm. Plant Cell Tiss Organ Cult 120:263–273

    Article  CAS  Google Scholar 

  • Landolt E (1986) Biosystematic investigations in the family of duckweeds (Lemnaceae) Vol. 2: The family of Lemnaceae: a monographic study. – Morphology, karyology, ecology, geographic distribution, nomenclature, descriptions. Eidgenössische Technische Hochschule Zürich, Zürich

  • Landolt E, Kandeler R (1987) The family of Lemnaceae - A monographic study. Vol. 4, Biosystematic investigations in the family of duckweeds (Lemnaceae) vol 4. Veröffentlichungen des Geobotanischen Institutes der ETH, Stiftung Rübel, Zürich

  • Lemon GD, Posluszny U (2000) Comparative shoot development and evolution in the Lemnaceae. Intern J Plant Sci 161:733–748

    Article  Google Scholar 

  • Lemon GD, Posluszny U, Husband BC (2001) Potential and realized rates of vegetative reproduction in Spirodela polyrhiza, Lemna minor, and Wolffia borealis. Aquat Bot 70:79–87

    Article  Google Scholar 

  • Leng RA, Stambolie JH, Bell R (1995) Duckweed - a potential high-protein feed resource for domestic animals and fish. Livestock Res Rural Dev 7:Article 5

  • Les DH, Crawford DJ (1999) Landoltia (Lemnaceae), a new genus of duckweeds. Novon 9:530–533

    Article  Google Scholar 

  • Li J et al (2004) Callus induction and regeneration in Spirodela and Lemna. Plant Cell Rep 22:457–464

    Article  CAS  PubMed  Google Scholar 

  • Li X, Jin Y, Gao X, Zhang G, Zhao H (2011) Fermentation method of high ratios of biobutanol with Landoltia punctata. China Brewing 31:85–88

    Google Scholar 

  • Lloyd G, McCown B (1980) Commercially feasible micropropagation of mountain laurel, Kalmia latifolia, by use of shoot-tip culture. Int Plant Propagation Soc Proc 30:421–427

    Google Scholar 

  • Moon HK, Stomp AM (1997) Effect of medium components and light on callus induction, growth and frond regeneration in Lemna gibba (duckweed) in vitro. In Vitro Cell Dev Biol Plant 33:21–25

    Article  Google Scholar 

  • Moon HK, Rajbhandari N, Stomp AM (1998) Effect of media components and phytohormones on in vitro frond proliferation of Lemna gibba G3 and 24 additional L. gibba strains. Plant Res 1:98–104

    Google Scholar 

  • Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plant 15:473–497

    Article  CAS  Google Scholar 

  • Oron G (1994) Duckweed culture for wastewater renovation and biomass production. Agric Water Manag 26:27–40

    Article  Google Scholar 

  • Pieterse AH (2013) Is flowering in Lemnaceae stress-induced? A review. Aquat Bot 104:1–4

    Article  CAS  Google Scholar 

  • Qian C, Jin Y, Zhang G, Fang Y, Xiao Y, Zhao H (2012) Improving production of bioethanol from duckweed (Landoltia punctata) by pectinase pretreatment Energies 5:3019-3032

  • Reinhold D, Handell L, Saunders FM (2011) Callus cultures for phytometabolism studies: phytometabolites of 3-trifluoromethylphenol in Lemnaceae plants and callus cultures. Int J Phytorem 13:642–656

    Article  CAS  Google Scholar 

  • Rival S et al (2008) Spirodela (duckweed) as an alternative production system for pharmaceuticals: a case study, aprotinin. Transgenic Res 17:503–513

    Article  CAS  PubMed  Google Scholar 

  • Sree KS, Maheshwari SC, Bokac K, Khuranad JP, Keresztesc A, Appenroth KJ (2015) The duckweed Wolffia microscopica: A unique aquatic monocot. Flora 210:31–39

    Article  Google Scholar 

  • Stefaniak B, Woźny A, Budna I (2002) Callus induction and plant regeneration in Lemna minor L. Biol Plant 45:469–472

    Article  Google Scholar 

  • Su H et al (2014) Use of duckweed (Landoltia punctata) as a fermentation substrate for the production of higher alcohols as biofuels. Energy Fuels 28:3206–3216

    Article  CAS  Google Scholar 

  • Vunsh R et al (2007) High expression of transgene protein in Spirodela. Plant Cell Rep 26:1511–1519

    Article  CAS  PubMed  Google Scholar 

  • Xu J, Shen G (2011) Growing duckweed in swine wastewater for nutrient recovery and biomass production. Bioresour Technol 102:848–853

    Article  CAS  PubMed  Google Scholar 

  • Xu J, Cui W, Cheng JJ, Stomp A-M (2011) Production of high-starch duckweed and its conversion to bioethanol. Biosyst Eng 110:67–72

    Article  Google Scholar 

  • Xu J, Cheng JJ, Stomp A-M (2012) Growing Spirodela polyrrhiza in swine wastewater for the production of animal feed and fuel ethanol: a pilot study. CLEAN – Soil. Air, Water 40:760–765

    Article  CAS  Google Scholar 

  • Xu Y et al (2015) Species distribution, genetic diversity and barcoding in the duckweed family (Lemnaceae). Hydrobiologia 743:75–87

    Article  CAS  Google Scholar 

  • Yamamoto Y, Rajbhandari N, Lin X, Bergmann B, Nishimura Y, Stomp A-M (2001) Genetic transformation of duckweed Lemna gibba and Lemna minor. In Vitro Cellular & Developmental Biology - Plant 37:349–353

    Article  CAS  Google Scholar 

  • Zhao Y et al (2015) Pilot-scale comparison of four duckweed strains from different genera for potential application in nutrient recovery from wastewater and valuable biomass production. Plant Biology 17:82–90

    Article  CAS  PubMed  Google Scholar 

  • Zheng MY, Konzak CF (1999) Effect of 2,4-dichlorophenoxyacetic acid on callus induction and plant regeneration in anther culture of wheat (Triticum aestivum L.). Plant Cell Rep 19:69–73

    Article  CAS  Google Scholar 

  • Ziegler P, Adelmann K, Zimmer S, Schmidt C, Appenroth KJ (2015) Relative in vitro growth rates of duckweeds (Lemnaceae) – the most rapidly growing higher plants. Plant Biology 17:33–41

    Article  PubMed  Google Scholar 

  • Ziv M, Shemesh D (1996) Propagation and tuberization of potato bud clusters from bioreactor culture. In vitro Cell Dev Biol Plant 32:31–36

    Article  CAS  Google Scholar 

  • Ziv M, Ronen G, Raviv M (1998) Proliferation of meristematic clusters in disposable presterilized plastic bioreactors for the large-scale micropropagation of plants. In Vitro Cellular & Developmental Biology - Plant 34:152–158

    Article  Google Scholar 

Download references

Acknowledgements

The authors would like express their gratitude to all the reviewers and editors for their comments that helped to improve the manuscript. The Rutgers Duckweed Stock Cooperative at, the State University of New Jersey, USA, provided the L. punctata strains 9264 and 8721. This research was supported by the International Science and Technology Cooperation Program of China (2014DFA30680), the Major Technology Project of Hainan (ZDZX2013023-1), and the National Nonprofit Institute Research Grant of ITBB (ITBB2015ZD07).

Author information

Author notes

    Authors and Affiliations

    1. Key Laboratory of Biology and Genetic Resources of Tropical Crops, Ministry of Agriculture; Hainan Bioenergy Center, Institute of Tropical Bioscience and Biotechnology, CATAS, Hainan, 571101, China

      Meng Huang, Lili Fu, Xuepiao Sun & Jiaming Zhang

    2. Rutgers, The State University of New Jersey, New Brunswick, NJ, 08901, USA

      Rong Di

    Authors
    1. Meng Huang
      View author publications

      You can also search for this author in PubMed Google Scholar

    2. Lili Fu
      View author publications

      You can also search for this author in PubMed Google Scholar

    3. Xuepiao Sun
      View author publications

      You can also search for this author in PubMed Google Scholar

    4. Rong Di
      View author publications

      You can also search for this author in PubMed Google Scholar

    5. Jiaming Zhang
      View author publications

      You can also search for this author in PubMed Google Scholar

    Corresponding author

    Correspondence to Jiaming Zhang.

    Additional information

    Communicated by S. Srivastava.

    M. Huang and L. Fu contributed equally to this work.

    Rights and permissions

    Reprints and permissions

    About this article

    Check for updates. Verify currency and authenticity via CrossMark

    Cite this article

    Huang, M., Fu, L., Sun, X. et al. Rapid and highly efficient callus induction and plant regeneration in the starch-rich duckweed strains of Landoltia punctata . Acta Physiol Plant 38, 122 (2016). https://doi.org/10.1007/s11738-016-2142-6

    Download citation

    • Received:

    • Revised:

    • Accepted:

    • Published:

    • DOI: https://doi.org/10.1007/s11738-016-2142-6

    Keywords

    Search

    Navigation