Skip to main content
SpringerLink
Log in

Asymbiotic seed germination of Cymbidium bicolor Lindl. (Orchidaceae) and the influence of mycorrhizal fungus on seedling development

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

Abstract

An in vitro plant regeneration protocol was successfully established for Cymbidium bicolor an epiphytic orchid by culturing seeds from green pods. Immature seeds were germinated on four basal media viz., Murashige and Skoog (MS) medium, Knudson C (KC) orchid medium, Knudson C modified Morel (KCM) medium and Lindemann orchid (LO) medium. Seed germination and protocorm development was significantly higher in LO medium (96.6 %) followed by KCM (89 %), MS (77.5 %) and KC (62.7 %) media after 56 days. For multiple shoot induction the protocorms were transferred to B5 medium supplemented with cytokinin. Among the various cytokinins tested, BAP (4.42 μM) induced maximum (27.59) number of multiple shoots per explant. IBA was effective in inducing healthy roots. Tissue-cultured protocorms and seedlings of C. bicolor were inoculated with AC-01 fungal strain (Moniliopsis sp.) isolated from the mycorrizal roots of an epiphytic orchid Aerides crispum. Mycorrhizal fungi significantly enhanced number of roots, root length and shoot number.

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

Similar content being viewed by others

Abbreviations

BAP:

Benzyl amino purine

KIN:

Kinetin

TDZ:

Thidazuron –N phenyl-N′-1,2,3-thadiazol-5-ylurea

LO:

Lindemann orchid medium

KCM:

Knunson C modified Morel medium

NAA:

α-Napthalene acetic acid

MS:

Murashige and Skoog medium

KC:

Knudson C orchid medium

a.s.l:

Above sea level

References

  • Abadie JC, Puttsepp U, Bonfante P, Selosse MA (2006) Cephalanthera longifolia (Neottieae, Orchidaceae) is mixotrophic: acomparative study between green and non photosynthetic individuals. Can J Bot 84:1462–1477

    Article  CAS  Google Scholar 

  • Andronova EV, Batygina TB, Vasilyeava VE (2000) Protocorm. In: Batygina T (ed) Embryology of flowering plants. Terminology and concepts 3: 329–334

  • Begum AA, Tamaki M, Kako S (1994a) Formation of protocorm-like bodies (PLBs) and shoot development through in vitro cultures of outer tissue of Cymbidium PLB. Jpn Soc Hortic Sci 63:663–673

    Article  CAS  Google Scholar 

  • Begum AA, Tamaki M, Tahara M, Kako S (1994b) Somatic embryogenesis in Cymbidium through in vitro culture of inner tissue of protocorm-like bodies. J Jpn Soc Hortic Sci 63:419–427

    Article  CAS  Google Scholar 

  • Bhadra SK, Bhattacherjee B, Barua AK, Hossain MM (2002) Micropropagation of Dendrobium aphyllum (Roxb.) G.E.C.Fisher. Bang J Gent Biotech 3:47–50

    Google Scholar 

  • Chang C, Chang WC (1998) Plant regeneration from callus cultures of Cymbidium ensifolium var misericors. Plant Cell Report 17:251–255

    Article  CAS  Google Scholar 

  • Chang DCN, Chou LC (2007) Growth responses, enzyme activities and component changes as influenced by Rhizoctonia orchid mycorrhiza on Anoectochilus formosanus Hayata. Bot Stud 48:445–451

    CAS  Google Scholar 

  • Chang DCN, Chou LC, Lee GC (2007) New cultivation methods for Anoectochilus formosanus Hayata. Orchid Sci Biotech 1:56–60

    Google Scholar 

  • Chugh S, Guha S, Usha Rao I (2009) Micropropagation of orchids: a review on the potential of different explants. Sci Hort 22:507–520

    Google Scholar 

  • Chung JD, Chun CK, Choi SO (1985) Asymbiotic germination of Cymbidium ensifolium. II. Effects of several supplements to the medium, pH values and light and/or dark culture periods on growth of rhizome and organogenesis for rhizome. J Korean Soc Hortic Sci 26:86–92

    Google Scholar 

  • Currah RS, Zelmer C (1992) A key and notes for the genera of fungi mycorrhizal with orchids and a new species of the genus Epuhorhiza. Rep Tottori Mycol Inst 30:43–59

    Google Scholar 

  • Currah RS, Zelmer CD, Hambleton S, Richardson KA (1997) Fungi from orchids mycorhizas. In: Arditti J, Pridycon AM (eds) Orchid Biology: Reviews and Prespectives. VII. Kluwer Academic publishers, Great Britan, pp 117–170

    Google Scholar 

  • Dave N, Purohit SD (2004) In vitro growth shoot multiplition of Achras sapota a controlled carbon dioxide environment. Biol Plant 48:621–624

    Article  Google Scholar 

  • Deb CR, Pongener A (2011) Asymbiotic seed germination and in vitro seedling development of Cymbidium aloifolium (L.) Sw.: a multipurpose orchid. J Plant Biochem Biotechnol 20:90–95

    Article  Google Scholar 

  • Deb CR, Temjensangba (2006) In vitro propagation of threatened terrestrial orchid. Malaxis khasiana Soland ex. Swartz through immature seed culture. Indian J Exp Biol 44:762–766

    PubMed  CAS  Google Scholar 

  • Arditti J, Ernst, R (1993) Micropropagation of orchids. Wiley, New York

  • Fan L, Guo SX, Xiao PG (1999) Study on the structure and localization of acid phospatase of mycorrhiza root of Cymbidium sinense (Orchidaceae). Acta Bot Yunnan 21:197–201

    CAS  Google Scholar 

  • Fang D, Hongxia LIU, Hui JIN, Yibo LUO (2008) Symbiosis between fungi and the hybrid Cymbidium and its mycorrhizal microstructures. For Stud China 10:41–44

    Article  Google Scholar 

  • Godo T, Komori M, Nakaoki E, Yukawa T, Miyoshi K (2010) Germination of mature seeds of Calanthe tricarinata Lindl. an endangered terrestrial orchid, by asymbiotic culture in vitro. In Vitro Cell Dev Biol Plant 46:323–328

    Article  CAS  Google Scholar 

  • Hartman H, Kester D, Davis F, Geneve R (1997) Plant propagation; Principles and Practices, 6th edn. Prentice-hall, New Jersey, pp 125–144

    Google Scholar 

  • Harvais G (1982) An improved culture medium for growing the orchid Cypripedium reginae axienically. Can J Bot 51:327–332

    Article  Google Scholar 

  • Hoque MI, Roy AR, Sarker RH, Haque MM (1994) Micropropagation of Cymbidium bicolor through in vitro culture. Plant Tissue Cult 4:45–51

    Google Scholar 

  • Hossain MM, Sharma M, Teixeira da Silva JA, Pathak P (2010) Seed germination and tissue culture of Cymbidium giganteum Wall. ex Lindl. Sci Hortic 123:479–487

    Article  CAS  Google Scholar 

  • Hou XQ, Guo SX (2009) Interaction between a dark septate entophytic isolate from Dendrobium sp. and roots of D. nobile seedlings. J Integ Plant Biol 51:374–381

    Article  CAS  Google Scholar 

  • Huan LVT, Tanaka M (2004) Callus induction from protocorm-like body segments and plant regeneration in Cymbidium (Orchidaceae). J Hortic Sci Biotechnol 79:406–410

    CAS  Google Scholar 

  • Johnson TR, Stewart SL, Daniela D, Kane ME, Richardson L (2007) A symbiotic and symbiotic seed germination of Eulophia alta (orchidaceae)—Preliminary evidence for the symbiotic culture advantage. Plant Cell Tiss Org Cult 90:313–323

    Article  Google Scholar 

  • Julou T, Burghardt B, Gebauer G, Berveiller D, Damesin C, Selosse M (2005) Mixotrophy in orchids: insights from a comparative study of green individuals and non-photosynthetic individuals of Cephalanthera damasonium. New Phytol 166:639–653

    Article  PubMed  CAS  Google Scholar 

  • Kapoor R, Sharma D, Bhatnagar AK (2008) Arbuscular mycorrhizae in micropropation system and their potential applications. Sci Hortic 116:227–239

    Article  Google Scholar 

  • Kauth PJ, Vendrame WA, Kane ME (2006) In vitro seed culture and seedling development of Calopogon tuberosus. Plant Cell Tiss Org Cult 85:91–102

    Article  Google Scholar 

  • Kauth PJ, Dutra D, Johnson TR, Stewart SL, Kane ME, Vendrame W (2008) Techniques and applications of in vitro orchid seed germination. In: Teixeira da Silva JA (ed) Floriculture, Ornamental and Plant Biotechnology: Advances and Topical Issues, vol V, 1st edn. Global Science Books Ltd., Isleworth, pp 375–391

    Google Scholar 

  • Kishor R, Sunitibala Devi H (2009) Induction of multiple shoots in a monopodial orchid hybrid (Aerides vandarum Reichb.f x Vanda stangeana Reichb.f) using thidiazuron and analysis of their genetic stability. Plant Cell Tiss Organ Cult 97:121–129

    Article  Google Scholar 

  • Knudson L (1946) A new nutrient solution for the germination of orchid seed. Am Orchid Soc Bull 15:214–217

    CAS  Google Scholar 

  • Lauzer D, St- Arnaud M, Barabe D (1994) Teterazolium straining and in vitro germination of mature seeds of Cypripedium calceolus (orchidaceae). Lindeleyana 9:197–204

    Google Scholar 

  • Lee SS, Oh CH, Paek KY, Lee TS (1998) Isolations of the orchid mycorrhizal fungi from the roots of the Korean native orchids and inoculations of the isolates to four different orchids. Korean J Plant Pathol 14:536–542

    Google Scholar 

  • Leonerd JH (1979) Practical nuclear staining procedures for Rhizoctonia like fungi. Phytopathol 69:958–961

    Article  Google Scholar 

  • Lindemann EGP, Gunckel JE, Davidson OW (1970) Meristem culture of Cattleya. Am Orchid Soc Bull 39:1002–1004

    Google Scholar 

  • 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 76:241–262

    Article  Google Scholar 

  • Mahendran G, Narmatha Bai V (2009) Mass propagation of Satyrium nepalense D.Don- A medicinal orchid via seed germination. Sci Hort 119:203–207

    Article  CAS  Google Scholar 

  • Mahendran G, Narmatha Bai V (2012) Direct somatic embryogenesis and plant regeneration from seed derived protocorms of Cymbidium bicolor Lindl. Sci Hort 135:40–44

    Article  CAS  Google Scholar 

  • Malabadi RB, Teixeira da Silva JA, Nataraja K, Mulgund GS (2008) Shoot tip transverse thin cell layers and 2, 4-epibrassinolide in the micropropagation of Cymbidium bicolor Lindl. Floricult Ornamental Biotechnol 2:44–48

    Google Scholar 

  • Mariat F (1949) Action de l’acide nicotinique sur la germination et al. developmentdes embryous de Cattleya. Compt Rend Acad Sci 229:1355–1357

    CAS  Google Scholar 

  • Massey EE, Zettler L (2007) An expanded role for in vitro symbiotic seed germination as a conservation tool: two case studies in North America (Platanthera leucophaea and Epidendrum noturnum). Lankesteriana 7:303–308

    Google Scholar 

  • Masuhara G, Katsuya K (1994) In situ and in vitro specificity between Rhizoctonia sp. and Spiranthes sinensis (Persson) Ames. var.amoena (M. Bieberstein) Hara (Orchidaceae). New Phytol 127:711–718

    Article  Google Scholar 

  • Mead JW, Bulard C (1979) Vitamins and nitrogen requirements of Orchis laxiflora Lamk. New Phytol 83:129–136

    Article  CAS  Google Scholar 

  • Mohapatra A, Rout GR (2005) In vitro micropropagation of Geoderum purpureum R.Br. Indian J Biotech 4:568–570

    CAS  Google Scholar 

  • Morel G (1964) Tissue culture—a new means of clonal propagation of orchids. Am Orchid Soc Bull 33:437–478

    Google Scholar 

  • Morel GM (1965) Clonal propagation of orchids by meristem culture. Cymbidium Soc News 20:3–11

    Google Scholar 

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

    Article  CAS  Google Scholar 

  • Nayak NR, Rath SP, Patnaik S (1997) In vitro propagation of three epiphytic orchids, Cymbidium aloifolium (L.) Sw., Dendrobium aphyllum (Roxb.) Fisch. and Dendrobium moschatum (Buch-Ham) Sw. through thidiazuron-induced high frequency shoot proliferation. Sci Hortic 71:243–250

    Article  Google Scholar 

  • Nhut DT, Tien TNT, Huong MTN, Hien NTH, Huyen PX, Luan VQ, Le BV, Teixeira da Silva JA (2005) Artificial seeds for preservation and propagation of Cymbidium spp. Prop Ornamental Plants 5:67–73

    Google Scholar 

  • Nontachiyapoom S, Sasirat S, Manoch L (2010) Isolation and identification of Rhizoctonia-like fungi from roots of three orchids genera, Paphiopedium, Dentrobium and Cymbidium collected in Chiang Rai and Chiang Mai provinces of Thailand. Mycorrhiza 20:459–471

    Article  Google Scholar 

  • Nowak J (1998) Benefits of in vitro biotization of plant tissue cultures with microbial inoculants. In Vitro Cell Dev Biol Plant 34:122–130

    Article  Google Scholar 

  • Panwar D, Ram K, Harish, Shekhawat NS (2012) In vitro propagation of Eulophia nuda Lindl., an endangered orchid. Sci Hort 139:46–52

    Article  CAS  Google Scholar 

  • Pathak P, Mahant KC, Gupta A (2001) In vitro propagation as an aid to conservation and commercialization of Indian orchids: seed culture. In: Pathak P, Sehgal RN, Shekhar N, Sharma M, Sood A (eds) Orchids: Science and Commerce. Bishen Singh Mahendra Pal Singh, India, pp 319–362

    Google Scholar 

  • Rasmussen H (1995) Terrestrial orchids from seed to mycotrophic plant. Cambridge University Press, Cambridge

    Book  Google Scholar 

  • Rasmussen HN (2002) Recent developments in the study of orchid mycorrhiza. Plant Soil 244:149–163

    Article  CAS  Google Scholar 

  • Rasmussen HN, Rasmussen FN (2007) Trophic relationships in orchid mycorrhiza diversity and implications for conservation. Lankesteriana 7:334–341

    Google Scholar 

  • Roy AR, Patel RS, Patel VV, Sajeev S, Deka BC (2011) Asymbiotic seed germination, mass propagation and seedling development of Vanda coerulea Griff ex. Lindl. (BlueVanda):an in vitro protocol for an endangered orchid. Sci Hort 128:325–331

    Article  CAS  Google Scholar 

  • Sharma SK, Tandon P, Mishra RR (1991) Vitamins as related to axenic seed germination and seedling growth of Cymbidium elegans Lindl. and Coelogyne punctulata Lindl. J Orchid Soc India 5:25–28

    Google Scholar 

  • Sharrock S (2007) Botanic gardens and their role in national plant conservation strategies. In: Ishida G, Iwashinam T, Konishi T, Kurashige Y, Oikawa J, Yukawa T (eds) Conservation of plant diversity in Japanese botanic gardens. Japan Association of Botanical Gardens, Tsukuba Botanical Gardens, National Science Museum and Botanic Gardens Conservation International, Tokyo, pp 2–8

    Google Scholar 

  • Sheelavantmath SS, Murthy HN, Pyati AN, Ashok Kumar HG, Ravishanker BV (2000) In vitro propagation of the endangered orchid Geodorum densiflorum (Lam.) Schltr. through rhizome section culture. Plant Cell Tiss Org Cult 60:151–154

    Article  CAS  Google Scholar 

  • Shimasaki K, Uemoto S (1990) Micropropagation of a terrestrial Cymbidium species using rhizomes developed from seeds and pseudo bulbs. Plant Cell Tiss Org Cult 22:237–244

    Article  CAS  Google Scholar 

  • Somiya K (2007) Role of botanical gardens from the standpoint of the national biodiversity strategy of Japan. In: Ishida G, Iwashinam T, Konishi T, Kurashige Y, Oikawa J, Yukawa T (eds) Conservation of plant diversity in Japanese botanic gardens. Japan Association of Botanical Gardens, Tsukuba Botanical Gardens, National Science Museum and Botanic Gardens Conservation International, Tokyo, pp 9–23

    Google Scholar 

  • Stewart SL, Kane ME (2006) Symbiotic germination of Habenaria macroceratitis (Orchidaceae), a rare Florida terrestrial orchid. Plant Cell Tiss Org Cult 86:159–167

    Article  CAS  Google Scholar 

  • Stoutamire WP (1974) Terrestrial orchid seedlings. In: Withner CL (ed) The Orchids Scientific Studies. John Wily and Son, New York, pp 101–128

    Google Scholar 

  • Sungkumlong, Deb CR (2008) Effects of different factors on immature embryo culture, PLBs differentiation and rapid mass multiplication of Coelogyne suaveolens (Lindl.) Hook. Indian J Exp Biol 46:243–248

    PubMed  CAS  Google Scholar 

  • Sunitibala H, Kishor R (2009) Micropropagation of Dendrobium transparens L. from axenic pseudobulb segments. Indian J Biotech 8:448–452

    Google Scholar 

  • Swarts ND, Dixon KW (2009) Terrestrial orchid conservation in the age of extinction. Ann Bot 104:543–556

    Article  PubMed  Google Scholar 

  • Tatarenko IV, Vakhrameeva MG (1998) On vegetative propagation in orchids. Byulleten’ Botanicheskogo Sada I S Kosenko 7:155–157 (in Russian)

    Google Scholar 

  • Teixeira da Silva JA, Giang DTT, Chan MT, Sanjaya Norikane A, Chai ML, Chico-Ruı′z J, Penna S, Granstrom T, Tanaka M (2007) The influence of different carbon sources, photohetero-, photoauto- and photo mixotrophic conditions on protocorm-like body organogenesis and callus formation in thin cell layer culture of hybrid Cymbidium (Orchidaceae). Orchid Sci Biotechnol 1:15–23

    Google Scholar 

  • Vellupillai M, Swaru PS, Goh CJ (1997) Histological and protein changes during early stage of seed germination in the orchid Dendrobium curmenatum. J Hortic Sci 76:941–948

    Google Scholar 

  • Vujanovic V, St-Arnaud M, Barabe D, Thibeault G (2000) Viability testing of orchid seed and the promotion of coloration and germination. Ann Bot 86:79–86

    Article  Google Scholar 

  • Vyas S, Purohit SD (2003) In vitro growth and shoot multiplication of Wrightia tomentosa (Roxb.) Roem et Shult in controlled carbon dioxide environment. Plant Cell Tiss Org Cult 75:283–286

    Article  CAS  Google Scholar 

  • Wang X (1988) Tissue culture of Cymbidium: plant and flower induction in vitro. Lindleyana 3:184–189

    Google Scholar 

  • Wu JR, Ham SF, Zhu YY, Lv M, Wang HP, Guo W (2005) Ultra-structure of symbiosis mycorrhizal between Cymbidium goeringii and Rhizoctonia.sp. Nanjing for Univ Nat Sci Ed 29:105–108

    Google Scholar 

  • Yam TW, Arditti J (2009) History of orchid propagation: a mirror of the history of biotechnology. Plant Biotechnology Reports 3:1–56

    Article  Google Scholar 

  • Zettler LW, Poulter SB, McDonald KI, Stewart SL (2007) Conservation-driven propagation of an epiphytic orchid (Epidendrum nocturnum) with a mycorrhizal fungus. Hort Sci 42:135–136

    Google Scholar 

  • Zhu YG, Miller RM (2003) Carbon cycling by arbuscular mycorrhizal fungi in soil-plant systems. Trends Plant Sci 8:407–409

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgments

The financial support by University Grants Commission (UGC), 37-97/2009 (SR) Dated: 19.12.2009. New Delhi is gratefully acknowledged.

Author information

Authors and Affiliations

  1. Department of Botany, School of Life Sciences, Bharathiar University, Coimbatore, 641046, Tamilnadu, India

    V. Muniappan, M. Ashwini, T. Muthukumar & V. Narmatha Bai

  2. Department of Botany, Plant Tissue culture lab, Bharathiar University, Coimbatore, 641046, India

    G. Mahendran

Authors
  1. G. Mahendran
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. V. Muniappan
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. M. Ashwini
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. T. Muthukumar
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. V. Narmatha Bai
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to G. Mahendran.

Additional information

Communicated by J. van Staden.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Mahendran, G., Muniappan, V., Ashwini, M. et al. Asymbiotic seed germination of Cymbidium bicolor Lindl. (Orchidaceae) and the influence of mycorrhizal fungus on seedling development. Acta Physiol Plant 35, 829–840 (2013). https://doi.org/10.1007/s11738-012-1127-3

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s11738-012-1127-3

Keywords

Search

Navigation