Plant Cell, Tissue and Organ Culture

, Volume 78, Issue 3, pp 273–276 | Cite as

Micropropagation of Cypripedium macranthos var. rebunense through Protocorm-like Bodies Derived from Mature Seeds

  • Hanako Shimura
  • Yasunori KodaEmail author


Cypripedium macranthos var. rebunense is the most famous terrestrial orchid in Japan, since the variety has large beautiful yellowish-white flowers and is threatened with extinction. Establishment of an efficient method for micropropagation is urgently needed. When imbibed mature seeds of the orchid, that had been pre-chilled at 4°C for 3 months, were sown onto Hyponex-peptone medium that contained both α-naphthaleneacetic acid (NAA) and cytokinin, protocorm-like bodies (PLBs) were formed from germinated seeds. Although the growth of PLBs was very slow, plantlets were easily regenerated from the PLBs on hormone-free medium. The PLBs were subcultured eight times along 2 years without loss of ability to regenerate plantlets, and one aggregate of PLBs (ca. 5 mm in diameter) produced ca. 10 plants within a year. A reduction of commercial value through a large-scale micropropagation by this method will be able to prevent illegal collection from the wild populations.

asymbiotic germination conservation of threatened orchid Cypripedium macranthos var. rebunense micropropagation protocorm-like bodies 


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. Arditti J (1977) Clonal propagation of orchids by means of tissue culture: a manual. In: Arditti J (ed) Orchid Biology: Reviews and Perspectives, I (pp. 203–293). Cornell University Press, Ithaca, New YorkGoogle Scholar
  2. Colli S & Kerbauy GB (1993) Direct root tip conversion of Catasetum into protocorm-like bodies. Effects of auxin and cytokinin. Plant Cell Tiss. Org. Cult. 33: 39–44CrossRefGoogle Scholar
  3. De Pauw MA & Remphrey WR (1993) In vitro germination of three Cypripedium species in relation to time of seed collection, media, and cold treatment. Can. J. Bot. 71: 879–885Google Scholar
  4. Lauzer D, St-Arnaud M & Barabe D (1994) Tetrazolium staining and in vitro germination of mature seeds of Cypripedium acaule (Orchidaceae). Lindleyana 9(3): 197–204Google Scholar
  5. 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: 481–486CrossRefGoogle Scholar
  6. Murashige T & Skoog F (1962) A revised medium for rapid growth and bioassays with tabacco tissue cultures. Physiol. Plant 15: 473–497Google Scholar
  7. Rasmussen HN (1992) Seed dormancy patterns in Epipactis palustris (Orchidaceae): requirements for germination and establishment of mycorrhiza. Physiol. Plant 86: 161–167CrossRefGoogle Scholar
  8. Tomita M (1996) Effects of media and time of seed collection on seed germination of Cypripedium macranthum var. rebunense. Combined Proc. Int. Plant Propagator's Soc. 46: 730–734Google Scholar
  9. Tomita M & Tomita M (1997) Plant regeneration from immature seed-derived callus of Cypripedium macranthos Swartz var. taiwanianum (Masamune) F. Maekawa. Breed. Sci. 47: 279–281Google Scholar
  10. Van Waes JM & Debergh PC (1986) In vitro germination of some Western European orchids. Physiol. Plant 67: 253–261Google Scholar

Copyright information

© Kluwer Academic Publishers 2004

Authors and Affiliations

  1. 1.Department of Botany, Graduate School of AgricultureHokkaido UniversitySapporoJapan

Personalised recommendations