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Micropropagation of Cypripedium flavum through multiple shoots of seedlings derived from mature seeds

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Cypripedium flavum, known as the rare lady’s slipper orchid, is one of the endemics with a yellow flower in China. Due to its conservation and commercial requirement, establishment of an efficient method for micropropogation is urgently needed. Multiple shoots were obtained by placing seedlings from seeds of C. flavum on Harvais media supplemented with two cytokinins (BAP or KIN) used alone or in addition to different concentration of potato homogenate. The effect of BAP was better than that of KIN on shoot multiplication. The Havais media supplemented with BAP (2.22 μM) and potato homogenate (20 g l−1) was the most effective, providing high shoot multiplication frequencies (95%) associated with a high number of shoots per explant (2.55 shoots/plant). For root formation, high rooting and survival were achieved using 1/2 Harvais media supplemented with 0.6 g l−1activated charcoals. High-level activated charcoal increased the number and the length of roots because the activated charcoal could absorb BAP in the media. This study demonstrated that C. flavum could be micropropagated by using multiple shoots of seedlings derived from mature seeds.

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  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 Cornell University Press, Ithaca, New York 203–293

  2. Case AM, Mlodozeniec HT, Wallace LE, Weldy TW, (1998) Conservation genetics and taxonomic status of the rare Kentucky Lady’s slipper: Cypripedium Kentuckiense (Orchidaceae) Am. J. Bot. 85: 1779–1786

  3. Chen XC, Zhu GH, Ji CH, Lang KY, Luo YB, Cribb P, (2005) Orchidaceae. In: Wu ZY, Raven PH., (eds) Flora of China: Vol. 25 Science Press, Beijing, and Missouri Botanical Garden Press, St. Louis

  4. Cribb P, Sandison MS, (1998) A preliminary assessment of the conservation status of Cypripedium species in the wild Bot. J. Linn. Soc. 126: 183–190

  5. Cribb P, (1997) The Genus Cypripedium. Timber Press, Portland, Oregon

  6. De Pauw MA, Remphrey WR, Palmer CE, (1995) The cytokinin preference for in vitro germination and protocorm growth of Cypripedium candidum Ann. Bot. 75: 267–275

  7. Harvais G, (1973) Growth requirements and development of Cypripedium reginae in axenic culture Can. J. Bot. 51: 327–332

  8. Huang JL, Hu H, (2001) Seed germination requirements of Cypripedium flavum in axenic culture (Chinese) Acta Bot. Yunn. 23: 105–108

  9. Lee YI, Lee N, (2003) Plant regeneration from protocorm-derived callus of Cypripedium formosanum In Vitro Cell. Dev. Biol., Plant 39: 475–479

  10. Shimura H, Koda Y, (2004) Micropropagation of Cypripedium macranthos var rebunense through protocorm-like bodies derived from mature seeds Plant Cell Tiss. Org. Cult. 78: 273–276

  11. Tomita M, Tomita M, (1997) Plant Regeneration from immature seed-derived callus of Cypripedium macranthos Swartz var taiwanianum (Masamune) F. Maekawa Breed. Sci. 47: 279–281

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The authors thank Cun-Xin Lee, Shi-bao Zhang, Can-Kun Xiong, Hong-Mei Yang, Da-Eng Shi and Yi-bo Luo for their technical assistance. The authors are also grateful to Mr Roger Brennan for his helpful advice on expression of the manuscript. This work was supported by the National Natural Science Foundation of China (30270151, 30470182).

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Correspondence to Hong Hu.

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Yan, N., Hu, H., Huang, J. et al. Micropropagation of Cypripedium flavum through multiple shoots of seedlings derived from mature seeds. Plant Cell Tiss Organ Cult 84, 114–118 (2006).

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  • Cypripedium flavum
  • conservation
  • induction of multiple shoots
  • micropropagation
  • root formation