Plant Biotechnology Reports

, Volume 3, Issue 1, pp 103–109 | Cite as

Cryopreservation and low-temperature storage of seeds of Phaius tankervilleae

  • Tomonari Hirano
  • Toshinari Godo
  • Kazumitsu Miyoshi
  • Keiko Ishikawa
  • Masaya Ishikawa
  • Masahiro Mii
Original Article


In this study we established reliable methods for conservation of seeds of Phaius tankervilleae as an orchid genetic resource. The seeds, which were dehydrated to 5% water content and preserved at 4°C, showed no decrease in viability and germinability after three months. After storage for six months, however, the seeds showed a drastic decrease in germinability, even though survival rate was high. For long-term preservation of seeds of P. tankervilleae, cryopreservation is applied to the freshly harvested seeds. When the seeds were cryopreserved by the vitrification method for up to 12 months there was no apparent deterioration effect of storage time. These results indicate that cryopreservation by the vitrification method is useful for long-term conservation of P. tankervilleae seeds, which are difficult to preserve for more than three months under dry and low-temperature conditions.


Cryopreservation Orchid Phaius tankervilleae Seed storage Vitrification 


  1. Hirano T, Godo T, Mii M, Ishikawa K (2005a) Cryopreservation of immature seeds of Bletilla striata by vitrification. Plant Cell Rep 23:534–539PubMedCrossRefGoogle Scholar
  2. Hirano T, Ishikawa K, Mii M (2005b) Cryopreservation of immature seeds of Ponerorchis graminifolia var. suzukiana by vitrification. CryoLetters 26:139–146PubMedGoogle Scholar
  3. Hirano T, Ishikawa K, Mii M (2006) Advances in orchid cryopreservation. In: Teixeira da Silva JA (ed) Floriculture ornamental and plant biotechnology: advances and topical issues, vol 2, 1st edn. Global Science Book, London, pp 410–414Google Scholar
  4. Ishikawa K, Harata K, Mii M, Sakai A, Yoshimatsu K, Shimomura K (1997) Cryopreservation of zygotic embryos of Japanese terrestrial orchid (Bletilla striata) by vitrification. Plant Cell Rep 16:754–757CrossRefGoogle Scholar
  5. Koopowitz H (1992) A stochastic model for the extinction of tropical orchids. Selbyana 13:115–122Google Scholar
  6. Koopowitz H, Thornhill A, Anderson M (1993) Species distribution profiles of the neotropical orchids Masdevallia and Dracula (Pleurothallidinae, Orchidaceae); implications for conservation. Biodivers Conserv 2:681–690CrossRefGoogle Scholar
  7. Langis R, Steponkus PL (1990) Cryopreservation of rye protoplasts by vitrification. Plant Physiol 92:666–671PubMedCrossRefGoogle Scholar
  8. Lauzer D, St-arnaud M, Barabé D (1994) Tetrazolium staining and in vitro germination of mature seeds of Cypropedium acaule (Orchidaceae). Lindleyana 9:197–204Google Scholar
  9. Nikishina TV, Popov AS, Kolomeitseva GL, Golovkin BN (2001) Effect of cryopreservation on seed germination of rare tropical orchids. Russ J Plant Physiol 48:810–815CrossRefGoogle Scholar
  10. Popov AS, Popova EV, Nikishina TV, Kolomeytseva GL (2004) The development of juvenile plants of the hybrid orchid Bratonia after seed cryopreservation. CryoLetters 25:205–212PubMedGoogle Scholar
  11. Pritchard HW (1984) Liquid nitrogen preservation of terrestrial and epiphytic orchid seed. CryoLetters 5:295–300Google Scholar
  12. Pritchard HW (1985) Determination of orchid seed viability using fluorescein diacetate. Plant Cell Environ 8:727–730Google Scholar
  13. Pritchard HW, Poyner ALC, Seaton PT (1999) Interspecific variation in orchid seed longevity in relation to ultra-dry storage and cryopreservation. Lindleyana 14:92–101Google Scholar
  14. Rasmussen HN (1995) Seed survival. In: Rasmussen HN (ed) Terrestrial orchids from seed to mycotrophyic plant. Cambridge University Press, Cambridge, pp 28–38Google Scholar
  15. Roberts EH, Ellis RH (1989) Water and seed survival. Ann Bot 63:39–52Google Scholar
  16. Roos EE, Davidson DA (1992) Record longevities of vegetable seeds in storage. HortScience 27:393–396Google Scholar
  17. Sakai A, Kobayashi S, Oiyama I (1990) Cryopreservation of nucellar cells of navel orange (Citrus sinensis Osb. var. brasiliensis Tanaka) by vitrification. Plant Cell Rep 9:30–33CrossRefGoogle Scholar
  18. Seaton PT, Hailes NSJ (1989) Effect of temperature and moisture content on the viability of Cattleya aurantiaca seed. In: Pritchard HW (ed) Modern methods in orchid conservation: the role of physiology, ecology and management. Cambridge University Press, Cambridge, pp 17–29Google Scholar
  19. Shoushtari BD, Heydari R, Johnson GL, Arditti J (1994) Germination and viability staining of orchid seeds following prolonged storage. Lindleyana 9:77–84Google Scholar
  20. Singh F (1981) Differential staining of orchid seeds for viability testing. Am Orchid Soc Bull 50:416–418Google Scholar
  21. Thammasiri K (2000) Cryopreservation of seeds of a Thai orchid (Doritis pulcherrima Lindl.) by vitrification. CryoLetters 21:237–244PubMedGoogle Scholar
  22. Tokuhara K, Mii M (1993) Micropropagation of Phalaenopsis and Doritaenopsis by culturing shoot tips of flower stalk buds. Plant Cell Rep 13:7–11CrossRefGoogle Scholar
  23. Towill LE (1990) Cryopreservation of isolated mint shoot tips by vitrification. Plant Cell Rep 9:178–180CrossRefGoogle Scholar
  24. Van Waes JM, Debergh PC (1986a) Adaptation of the tetrazolium method for testing the seed viability, and scanning electroscopy study of some Western European orchid. Physiol Plant 66:435–442CrossRefGoogle Scholar
  25. Van Waes JM, Debergh PC (1986b) In vitro germination of some Western European orchid. Physiol Plant 67:253–261CrossRefGoogle Scholar
  26. Volk GM, Walters C (2003) Preservation of genetic resources in the national plant germplasm clonal collections. Plant Breed Rev 23:291–344Google Scholar
  27. Wang JH, Ge JG, Liu F, Bian HW, Huang CN (1998) Cryopreservation of seeds and protocorms of Dendrobium candidum. CryoLetters 19:123–128Google Scholar

Copyright information

© Korean Society for Plant Biotechnology and Springer 2008

Authors and Affiliations

  • Tomonari Hirano
    • 1
    • 6
  • Toshinari Godo
    • 2
  • Kazumitsu Miyoshi
    • 3
  • Keiko Ishikawa
    • 4
  • Masaya Ishikawa
    • 5
  • Masahiro Mii
    • 1
  1. 1.Laboratory of Plant Cell Technology, Faculty of HorticultureChiba UniversityMatsudoJapan
  2. 2.Botanic Gardens of ToyamaToyamaJapan
  3. 3.Crop Science Laboratory, Faculty of BioresourcesAkita Prefectural UniversityAkitaJapan
  4. 4.Japan Horticultural Production and Research InstituteMatsudoJapan
  5. 5.Environmental Stress Research Unit, Division of Plant SciencesNational Institute of Agrobiological SciencesTsukubaJapan
  6. 6.Division of Innovative ResearchCreative Research Initiative “Sousei” (CRIS), Hokkaido UniversitySapporoJapan

Personalised recommendations