Improvement of asymbiotic seed germination and seedling development of Cypripedium macranthos Sw. with organic additives

  • Huh, Yoon Sun ;
  • Lee, Joung Kwan ;
  • Nam, Sang Young ;
  • Paek, Kee Yoeup ;
  • Suh, Gang Uk
  • Received : 2016.02.11
  • Accepted : 2016.03.15
  • Published : 2016.03.31


To find the optimal propagation condition for endangered Cypripedium macranthos Sw., also known as lady's slipper orchid, the effect of various organic additives on in vitro germination, protocorm formation and seedling growth was investigated during asymbiotic seed culture. When $100ml{\cdot}L^{-1}$ coconut water was added to the basal medium, the highest germination rate and protocorm formation rate were achieved, with 70.8% and 74.2% respectively. Supplementation of phloem sap from birch tree or maple tree also showed a facilitating effect to improve the germination and protocorm development. With $100ml{\cdot}L^{-1}$ birch sap or maple sap, both the germination and protocorm formation rates were roughly more than 65% and 68%. The roots and buds of the seedlings grew vigorously in the medium containing $100ml{\cdot}L^{-1}$ coconut water or phloem sap, in particular, their bud formation rates increased by more than 70%. Addition of banana powder and peptone could not create a more significantly favorable culture condition, and non-addition had the worst results. Our results demonstrated that proper organic amendments such as coconut water and phloem sap might be preferred to in vitro germination and the growth of seedlings developed from the protocorm of C. macranthos Sw. during asymbiotic seed culture.


Lady's slipper orchid;Asymbiotic seed culture;Organic amendments;Germination;Seedling growth


  1. Agampodi VA, Jayawardena B (2009) Effect of coconut (Cocos nucifera L.) water extracts on adventitious root development in vegetative propagation of Dracaena purplecompacta L. Acta Physiol Plant 31:279-284
  2. Akter S, Nasiruddin KM, Khaldun ABM (2007) Organogenesis of Dendrobium orchid using traditional media and organic extracts. J Agric Rural Dev 5:30-35
  3. Al-Khateeb AA (2008) Regulation of in vitro bud formation of date palm (Phoenix dactylifera L.) cv. Khanezi by different carbon sources. Bioresource Technol 99:6550-6555
  4. Amin M, Reusch J (1987) High-performance liquid chromatography of water-soluble vitamins: II. Simultaneous determinations of vitamins B1, B2, B6 and B12 in pharmaceutical preparations. J Chrom 390:448-453
  5. Arditti J (1967) Factors affecting the germination of orchid seeds. Bot Rev 33:1-97
  6. Arditti J (1977) Orchid biology, reviews and perspectives I. Cornell University Press, Ithaca, New York, USA
  7. Arditti J, Ernst R, Yam TW, Glabe C (1990) The contribution of orchid mycorrhizal fungi to seed germination: a speculative review. Lindleyana 5:249-255
  8. Baque MA, Shin YK, Elshmari T, Lee EJ, Paek KY (2011) Effect of light quality, sucrose and coconut water concentration on the micropropagation of Calanthe hybrids ('Bukduseong' x 'Hyesung') Aust J Crop Sci 5:1247-1254
  9. Chu CC, Mudge KW (1994) Effects of prechilling and liquid suspension culture on seed germination of the Yellow Lady's Slipper orchid, Cypripedium calceolus var. pubescens. Lindleyana 9:153-159
  10. Chung MJ, Lee SJ, Shin JH, Jo JS, Sung NJ (1995) The components of the sap from birches, bamboos and darae. J Kor Soc Food Nutr 24:727-733
  11. Colli S, Kerbauy GB (1993) Direct root tip conversion of Catasetum into protocorm-like bodies: Effect of auxin and cytokinin. Plant Cell Tiss Org Cult. 33:39-44
  12. Cribb P (1997) The genus Cypripedium, p. 301. Timber Press, Portland, USA
  13. Cribb P, Sandison M (1998) A preliminary assessment of the conservation status of species in the wild. Bot J Linnean Soc 126:183-190
  14. DeMarie E, Weimer M, Mudge W (1991) In vitro germination and development of Showy Lady's Slipper orchid (Cypripedium reginae Walt.) seeds. HortScience 26:727
  15. Deng L, Zhang Y, Wang MM, Zhao SW (2012) Study on nonasymbiotic germination of Cypripedium macranthos seed. Seed 31:31-39
  16. De Pauw MA, Remphery WR (1993) In vitro germination of three Cypripedium species in relation to time of collection, media and cold treatment. Can J Bot 71:879-885
  17. De Pauw MA, Remphery WR and Palmer CE (1995) The cytokinin preference for in vitro germination and protocorm growth of Cypripedium candidum. Ann Bot 75:267-275
  18. Fonnesbech M (1972) Organic nutrients in the media for propagation of Cymbidium in vitro. Physiologia Plantarum 27:360-364
  19. Gnasekaran P, Xavier R, Uma Rani S, Sreeramanan S (2010) A study on the use of organic additives on the protocorm-like bodies (PLBs) growth of Phalaenopsis violacea orchid. J Phytol 2:29-33
  20. Harvais G (1982) An improved culture medium for growing the orchid Cypripedium reginae axenically. Can J Bot 60:2547-2555
  21. Homa Y, Asahira TM (1985) New means of Phalaenopsis propagation with intermodal sections of flower stalk. J Jpn Soc Hortc Sci 54:379-387
  22. Huang JL, Hu H (2001). Seed germination requirements of Cypripedium flavum in axenic culture. Acta Bot Yunn 23:105-108
  23. Islam MO, Rahman ARMM, Matsui S, Prodhan AKMA (2003) Effects of complex organic extracts on callus growth and PLB regeneration through embryogenesis in the Doritaenopsis orchid. Jpn Agric Res Quart 4:229-235
  24. Kitsaki CK, Zygouraki S, Ziobora M, Kintzios S (2004) In vitro germination, protocorm formation and platlet development of mature versus immature seeds from several Ophrys species (Orchidaceae). Plant Cell Reports 23:284-290
  25. Kuka M, Cakste I, Gersebeka E (2013) Determination of bioactive compounds and mineral substances in latvian birch and maple saps. In Proceedings of the Latvian Academy of Sciences, Section B, Natural, Exact, and Applied Sciences 67:437-441
  26. Lee YI, Lu CF, Chung MC, Yeung EC, Lee N (2007) Developmental changes in endogenous abscisic acid concentrations and asymbiotic seed germination of a terrestrial orchid, Calanthe tricarinata Lindle. J Am Soc Hortic Sci 132:246-252
  27. Leroux G, Barabe D, Vieth J (1995) Comparative morphogenesis of Cypripedium acaule (Orchidaceae) protocorms cultivated in vitro with or without sugar. Can J Bot 73:1391-1406
  28. Masanori T, Tomita M (1997) Effect of culture media and cold treatment on germination in asymbiotic culture of Cypripedium macranthos and Cypripedium japonicum. Lindleyana 12:208-213
  29. Mclntyre DK, Veitch GJ, Wrigley JW (1974) Australian terrestrial orchids from seed. II. Improvements in techniques and further successes. Amer Orchid Soc Bull 43:52-53
  30. Miyoshi K, Mii M (1998) Stimulatory effects of sodium and calcium hypochlorite, pre-chilling and cytokinins on the germination of Cypripedium macranthos seed in vitro. Physiol Plant 102:481-486
  31. Murashige T, Skoog F (1962) A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiologia plantarum 15:473-497
  32. Murdad R. Latip MA, Aziz ZA, Ripin R (2010) Effects of carbon source and potato homogenate on in vitro growth and development of Sabah's endangered orchid: Phalaenopsis gigantea. Asia-Pac J Mol Biol 18:199-202
  33. Nakurte I, Keisa A, Rostoks N (2012) Development and validation of a reversed-phase liquid chromatography method for the simultaneous determination of indole-3-acetic acid, indole-3-pyruvic acid, and abscisic acid in barley (Hordeum vulgare L.). J Anal Methods Chem doi:10.1155/2012/103575
  34. 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 5:10-18
  35. Piao RZ, Wang YL, Zhao HY (2011) Preliminary exploration in in vitro seed culture conditions of Cypripedium macrnathos Sw. J Anhui Agri Sci 39:18428-18429, 18445
  36. Pieper W, Zimmer K (1974) Blutungssafte als Zusatz zu Nahrmedien fur Gewebekulturen. Gartnwelt 74:461-462
  37. Pyati AN, Murthy HN, Hahn EJ, Paek KY (2002) In vitro propagation of Dendrobiummacrostachyum Lindle.- A threatened orchid. Indian J Exp Biol 40:620-623
  38. Rasmussen HN (1992) Seed dormancy patterns in Epipactis palustris (Orchidaceae): requirements for germination and establishment of mycorrhiza. Physiol Plant 86:161-167
  39. Rasmussen HN (1995) Terrestrial Orchids: From Seed to Mycotrophic Plant. Cambridge University Press, New York, USA. pp. 444
  40. St-Arnaud M, Lauzer D, Barabe D (1992) In vitro germination and early growth of seedling of Cypripedium acaule (Orchidaceae). Lindleyana 7:22-27
  41. Steele WK (1995) Growing Cypripedium reginae from seed. Amer Orchid Soc Bull 64:383-391
  42. Tawaro S, Suraninpong P, Chanprame S (2008) Germination and regeneration of Cymbidum findlaysonianum Lindl. on a medium supplemented with some organic sources. Walailak J Sci and Tech 5:125-135
  43. Texeira da Silva JA, Chan MT, Chai ML, Tanaka M (2006) Priming abiotic factors for optimal hybrid Cymbidium (Orchidaceae) PLB and callus induction, plantlet formation, and their subsequent cytogenetic stability analysis. Sci Hortic 109:368-378
  44. Van der Kinderen G (1987) Abscisic acid in terrestrial orchid seeds: a possible impact on their germination. Lindleyana 2:84-87
  45. Van Waes JM, Debergh PC (1986) In vitro germination of some Western European orchids. Physiologia Plantarum 67:253-261
  46. Vujanovic V, St-Arnaud M, Barabe D, Thibeault G (2000) Viability testing of orchid seed and the promotion of colouration and germination. Ann Bot 86:79-86
  47. Yan N, Hu H, Huang JL, Xu K, Wang H, Zhou ZK (2006) Micropropagation of Cypripedium flavum through multiple shoots of seedlings derived from mature seeds. Plant Cell Tiss Org Cult 84:113-117
  48. Yoon YJ, Murthy HN, Hahn EJ, Paek KY (2007) Biomass production of Anoectichilus formosanus Hayata in a bioreactor system. J Plant Biol 50:573-576
  49. Zelmer CD, Currah RS (1997) Symbiotic germination of Spiranthes lacera (Orchidaceae) with a naturally occurring endophyte. Lindleyana 12:142-148
  50. Zettler LW, McInnis TM Jr. (1994) Light enhancement of symbiotic seed germination and development of an endangered terrestrial orchid (Platanthera intergrilabia). Plant Sci 7:154-161
  51. Zimmer K, Pieper W (1975) Weitere Untersuchungen zur Kultur in vitro con Aechmea. Gartenbauwissenschaft 40:129-132
  52. Zimmer K, Pieper W (1976) Zur Wirkung con Blutungssaft der Weissbirke auf das Wachstum von Geweben in vitro. Die Orchidee 27:267-269
  53. Zimmer K, Pieper W (1978) Clonal propagation of Phalaenopsis by excised buds. The Orchid Review 86:223-226

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