Cryopreservation

Protocol
Part of the Springer Protocols Handbooks book series (SPH)

Abstract

The diversity and beauty of orchids place them among the top flowering plants in the international market. They are commercialized either as cut flowers or potted plants. With over 25,000 estimated known species and over 100,000 hybrids developed, they represent about 10% of all flowering plants. The development of feasible techniques for the conservation of orchid material with low maintenance costs, greater storage time, and good phytosanitary conditions is essential for the conservation of species as well as commercial use of hybrids. Cryopreservation is a technique that involves the conservation of biological material at ultra-low temperatures, generally at −196 °C in liquid nitrogen or at −150 °C in nitrogen’s vapor phase. Currently, this is the only feasible technique for the long-term preservation of genetic material from plants that are vegetatively propagated, or with unviable or recalcitrant seeds. Due to the large diversity of orchids, this is a developing and relevant field that requires continued research and development of improved techniques according to the species and/or hybrid. In this chapter, we present simple methods and their applications for the cryopreservation of orchid seeds, protocorms, and pollen.

Key words

Conservation Germplasm storage Liquid nitrogen Orchidaceae Pollinia Protocorm Seed Ultra-low temperature 

References

  1. 1.
    Gonzalez-Benito ME, Perez C (1997) Cryopreservation of nodal explants of an endangered plant species (Centaurium rigualii Esteve) using the encapsulation–dehydration method. Biodivers Conserv 6:583–590CrossRefGoogle Scholar
  2. 2.
    Reed BM (2008) Plant cryopreservation: a practical guide. Springer, New YorkCrossRefGoogle Scholar
  3. 3.
    Harvengt L, Meier-Dinkel A, Dumas E, Collin E (2004) Establishment of a cryopreserved gene bank of European elms. Can J For Res 34:43–55CrossRefGoogle Scholar
  4. 4.
    Engelmann F (2004) Plant cryopreservation: progress and prospects. In Vitro Cell Dev Biol Plant 40:427–433CrossRefGoogle Scholar
  5. 5.
    Mazur P (1984) Freezing of living cells: mechanisms and applications. Amer J Physiol Cell Physiol 247:125–142CrossRefGoogle Scholar
  6. 6.
    Kartha KK, Engelmann F (1994) Cryopreservation and germplasm storage. In: Vasil IK, Thorpe TA (eds) Plant cell and tissue culture. Kluwer, Dordrecht, pp 195–230Google Scholar
  7. 7.
    Engelmann F (1997) In vitro conservation methods. In: Callow J, Ford-Lloyd B, Newbury HJ (eds) Biotechnology and plant genetic resources: conservation and use. CAB Intl., Wallingford, pp 119–161Google Scholar
  8. 8.
    Rao NK (2004) Plant genetic resources: advancing conservation and use through biotechnology. Afr J Biotechnol 3:136–145Google Scholar
  9. 9.
    Hirai D, Sakai A (2003) Simplified cryopreservation of sweet potato [Ipomoea batatas (L.) Lam.] by optimizing conditions for osmoprotection. Plant Cell Rep 21:961–966CrossRefGoogle Scholar
  10. 10.
    Engelmann F (2000) Importance of cryopreservation for the conservation of plant genetic resources. In: Engelmann F, Takagi H (eds) Cryopreservation of tropical plant germplasm: current research progress and applications. JIRCAS, Tsukuba, pp 8–20Google Scholar
  11. 11.
    Panis B, Piette B, Swennen R (2005) Droplet vitrification of apical meristems: a cryopreservation protocol applicable to all Musaceae. Plant Sci 168:45–55CrossRefGoogle Scholar
  12. 12.
    Redenbaugh K (1993) Synseeds: applications of synthetic seeds to crop improvement. CRC Press, Boca Raton, FLGoogle Scholar
  13. 13.
    Engelmann F (2011) Use of biotechnologies for the conservation of plant biodiversity. In Vitro Cell Dev Biol Plant 47:5–16CrossRefGoogle Scholar
  14. 14.
    Fabre J, Dereuddre J (1990) Encapsulation-dehydration: a new approach to cryopreservation of Solanum shoot-tips. CryoLetters 11:413–426Google Scholar
  15. 15.
    Dereuddre J, Blandin S, Hassen N (1991) Resistance of alginate-coated somatic embryos of carrot (Daucus carota L.) to desiccation and freezing in liquid nitrogen: 1. Effects of preculture. CryoLetters 12:125–134Google Scholar
  16. 16.
    Engelmann F, Takagi H (2000) Cryopreservation of tropical plant germplasm-current research progress and applications. JIRCAS, TsukubaGoogle Scholar
  17. 17.
    Gonzalez-Arnao MT, Engelmann F (2006) Cryopreservation of plant germplasm using the encapsulation–dehydration technique: review and case study on sugarcane. CryoLetters 27:155–168PubMedGoogle Scholar
  18. 18.
    Sakai A, Engelmann F (2007) Vitrification, encapsulation–vitrification and droplet–vitrification: a review. CryoLetters 28:151–172PubMedGoogle Scholar
  19. 19.
    Engelmann F, Gonzalez-Arnao MT, Wu WJ, Escobar RE (2008) Development of encapsulation–dehydration. In: Reed BM (ed) Plant cryopreservation: a practical guide. Springer, Berlin, pp 59–76CrossRefGoogle Scholar
  20. 20.
    Shibli RA, Haagenson DM, Cunningham SM, Berg WK, Volenec JJ (2001) Cryopreservation of alfalfa (Medicago sativa L.) cells by encapsulation-dehydration. Plant Cell Rep 20:445–450CrossRefGoogle Scholar
  21. 21.
    Dumet D, Grapin A, Bailly C, Dorion N (2002) Revisiting crucial of an encapsulation/desiccation based cryopreservation process: importance of thawing method in the case of pelargonium meristems. Plant Sci 163:1121–1127CrossRefGoogle Scholar
  22. 22.
    Gonzalez-Arnao MT, Juaréz J, Navarro L, Duran-Vila N (2003) Cryopreservation of ovules and somatic embryos of citrus using the encapsulation-dehydration technique. CryoLetters 24(2):85–94PubMedGoogle Scholar
  23. 23.
    Wang QC, Mawassi M, Sahar N, Li P, Violeta CT, Gafny R, Sela I, Tanne E, Perl A (2004) Cryopreservation of grapevine (Vitis spp.) embryogenic cell suspensions by encapsulation–vitrification. Plant Cell Tiss Org Cult 77:267–275CrossRefGoogle Scholar
  24. 24.
    Fang JY, Wetten A, Johnston J (2008) Headspace volatile markers for sensitivity of cocoa (Theobroma cacao L.) somatic embryos to cryopreservation. Plant Cell Rep 27:453–461CrossRefGoogle Scholar
  25. 25.
    Scocchi A, Faloci M, Medina R, Olmos S, Mroginski L (2004) Plant recovery of cryopreserved apical meristem-tips of Melia azedarach L. using encapsulation/dehydration and assessment of their genetic stability. Euphytica 135:29–38CrossRefGoogle Scholar
  26. 26.
    Suzuki M, Akihama T, Ishikawa M (2005) Cryopreservation of encapsulated gentian axillary buds following 2 step-preculture with sucrose and desiccation. Plant Cell Tiss Org Cult 83:115–121CrossRefGoogle Scholar
  27. 27.
    Wang Q, Laamanen J, Uosukainen M, Valkonen JPT (2005) Cryopreservation of in vitro-grown shoot tips of raspberry (Rubus idaeus L.) by encapsulation–vitrification and encapsulation–dehydration. Plant Cell Rep 24:280–288CrossRefGoogle Scholar
  28. 28.
    Ishikawa K, Harata K, Mii M, Sakai A, Yoshimatsu K, Shimomura K (1997) Cryopreservation of zygotic embryos of a Japanese terrestrial orchid (Bletilla striata) by vitrification. Plant Cell Rep 16:754–757CrossRefGoogle Scholar
  29. 29.
    Hirano T, Godo T, Mii M, Ishikawa K (2005) Cryopreservation of immature seeds of Bletilla striata by vitrification. Plant Cell Rep 23:534–539CrossRefGoogle Scholar
  30. 30.
    Jitsopakul N, Thammasiri K, Ishikawa K (2008) Cryopreservation of Bletilla striata mature seeds, 3-day germinating seeds and protocorms by droplet-vitrification. CryoLetters 29:517–526PubMedGoogle Scholar
  31. 31.
    Lurswijidjarus W, Thammasiri K (2004) Cryopreservation of shoot tips of Dendrobium Walter Oumae by encapsulation/dehydration. Sci Asia 30:293–299CrossRefGoogle Scholar
  32. 32.
    Vendrame WA, Carvalho VS, Maguire I (2007) In vitro propagation and plantlet regeneration from Doritaenopsis purple gem ‘Ching Hua’ flower explants. HortScience 42(5):1256–1258Google Scholar
  33. 33.
    Galdiano RF Jr, Lemos EGM, Faria RT, Vendrame W (2012) Cryopreservation of Dendrobium hybrid seeds and protocorms as affected by phloroglucinol and supercool X1000. Sci Hort 148:154–160CrossRefGoogle Scholar
  34. 34.
    Galdiano RF Jr, Lemos E, Vendrame W (2014) Seedling development and evaluation of genetic stability of cryopreserved Dendrobium hybrid mature seeds. Appl Biochem Biotechnol 172:2521–2529CrossRefGoogle Scholar
  35. 35.
    Galdiano RF Jr, Lemos EGM, Vendrame W (2013) Cryopreservation, early seedling development, and genetic stability of Oncidium flexuosum Sims. Plant Cell Tiss Org Cult 114:139–148CrossRefGoogle Scholar
  36. 36.
    Tsukazaki H, Mii M, Tokuhara K, Ishikawa K (2000) Cryopreservation of Doritaenopsis suspension culture by vitrification. Plant Cell Rep 19:1160–1164CrossRefGoogle Scholar
  37. 37.
    Na HY, Kondo K (1996) Cryopreservation of tissue-cultured shoot primordia from shoot apices of cultured protocorms in Vanda pumila following ABA preculture and desiccation. Plant Sci 118(2):195–201CrossRefGoogle Scholar
  38. 38.
    Vendrame W, Faria RT, Sorace M, Sahyun SA (2014) Orchid cryopreservation. Ciênc Agrotec 38(3):213–229CrossRefGoogle Scholar
  39. 39.
    Vendrame WA, Khoddamzadeh AA (2017) Orchid biotechnology. Hort Rev 44:173–228Google Scholar
  40. 40.
    Vendrame WA, Carvalho VS, Maguire I, Dias JMM (2007) In vitro germination and plant regeneration of cryopreserved Dendrobium hybrid mature seeds. Sci Hort 114:188–193CrossRefGoogle Scholar
  41. 41.
    Pritchard HW, Seaton PT (1993) Orchid seed storage: historical perspective, current status, and future prospects for long-term conservation. Selbyana 14:89–104Google Scholar
  42. 42.
    Merritt DJ, Hay FR, Swarts ND, Sommerville KD, Dixon KW (2014) Ex situ conservation and cryopreservation of orchid germplasm. Int J Plant Sci 175:46–58CrossRefGoogle Scholar
  43. 43.
    Bowling JC, Thompson PA (1972) On storing orchid seed. Orchid Rev 80:120–121Google Scholar
  44. 44.
    Pritchard HW (1985) Growth and storage of orchid seeds. In: Tan KW (ed.) Proc. eleventh world orchid conference. 11th W.O.C., Miami, pp 290–293Google Scholar
  45. 45.
    Ito I (1965) Ultra-low temperature storage of orchid pollinia and seeds. Jpn Orchid Soc Bull 11:4–15Google Scholar
  46. 46.
    Pritchard HW (1984) Liquid nitrogen preservation of terrestrial and epiphytic orchid seed. CryoLetters 5:295–300Google Scholar
  47. 47.
    Seaton PT, Hailes SJ (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 17–29CrossRefGoogle Scholar
  48. 48.
    Thornhill A, Koopowitz H (1992) Viability of Disa uniflora Berg (Orchidaceae) seeds under variable storage conditions: is orchid gene-banking possible? Biol Conserv 62:21–27CrossRefGoogle Scholar
  49. 49.
    Surenciski MR, Gonzales EA, Terada G, Mroginski LA, Rey HY (2012) Cryopreservation of Cyrtopodium hatschbachii Pabst (Orchidaceae) immature seeds by encapsulation-dehydration. Biocell 36:31–36PubMedGoogle Scholar
  50. 50.
    Harding K (2004) Genetic integrity of cryopreserved plant cells: a review. CryoLetters 25:3–22PubMedGoogle Scholar
  51. 51.
    Panis B, Lambardi M (2006) Status of cryopreservation technology in plants (crops and forest trees). In: Ruane J, Sonnino A (eds) The role of biotechnology in exploring and protecting agricultural genetic resources. Food and Agriculture Organization, Rome, pp 61–78Google Scholar
  52. 52.
    Popova EV, Nikishina TV, Kolomeitseva GL, Popov AS (2003) The effect of seed cryopreservation on the development of protocorms by the hybrid orchid Bratonia. Russian. J Plant Physiol 50:672–677Google Scholar
  53. 53.
    Morel G (1960) Producing virus free Cymbidium. Amer Orchid Soc Bull 29:459–497Google Scholar
  54. 54.
    Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plant 15:473–497CrossRefGoogle Scholar
  55. 55.
    Vendrame WA, Faria RT (2011) Phloroglucinol enhances recovery and survival of cryopreserved Dendrobium nobile protocorms. Sci Hort 128:131–135CrossRefGoogle Scholar
  56. 56.
    Gogoi K, Kumaria S, Tandon P (2012) A comparative study of vitrification and encapsulation-vitrification for cryopreservation of protocorms of Cymbidium eburneum L., a threatened and vulnerable orchid of India. CryoLetters 33:443–452PubMedGoogle Scholar
  57. 57.
    Mata-Rosas M, Lastre-Puertos E (2015) Long-term conservation of protocorms of Brassavola nodosa (L) Lind. (Orchidaceae): effect of ABA and a range of cryopreservation techniques. CryoLetters 36(5):289–298PubMedGoogle Scholar
  58. 58.
    Connor KF, Towill LE (1993) Pollen-handling protocol and hydration/dehydration characteristics of pollen for application to long-term storage. Euphytica 68:77–84CrossRefGoogle Scholar
  59. 59.
    Grout BWW, Roberts AV (1995) Storage of free pollen, pollen embryos and the zygotic embryos of seed by cryopreservation and freeze drying. In: Grout BWW (ed) Genetic preservation of plant cells in vitro. Springer, Berlin, pp 63–74CrossRefGoogle Scholar
  60. 60.
    Sacks EJ, St Clair DA (1996) Cryogenic storage of tomato pollen: effect on fecundity. HortScience 31:447–448Google Scholar
  61. 61.
    Towill LE (2002) Cryopreservation of plant germplasm: introduction and some observations. In: Towill LE, Bajaj YPS (eds) Cryopreservation of plant germplasm II. Springer, Berlin, pp 4–21CrossRefGoogle Scholar
  62. 62.
    Pritchard HW, Prendergast FG (1989) Factors influencing the germination and storage characteristics of orchid pollen. In: Pritchard HW (ed) Modern methods in orchid conservation: the role of physiology, ecology and management. Cambridge University Press, Cambridge, pp 1–16CrossRefGoogle Scholar
  63. 63.
    Ganeshan S, Rajasekharan PE, Shashikumar S, Decruze W (2008) Cryopreservation of pollen. In: Reed BM (ed) Plant cryopreservation: a practical guide. Springer, Berlin, pp 443–464CrossRefGoogle Scholar
  64. 64.
    Pritchard HW, Poynter ALC, Seaton PT (1999) Interspecific variation in orchid seed longevity in relation to ultra-dry storage and cryopreservation. Lindleyana 14(2):92–101Google Scholar
  65. 65.
    Vendrame WA, Carvalho VS, Dias JMM, Maguire I (2008) Pollination of Dendrobium hybrids using cryopreserved pollen. HortScience 43:264–267Google Scholar
  66. 66.
    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
  67. 67.
    Hosomi ST, Custódio CC, Seaton PT, Marks TR, Machado-Neto NB (2012) Improved assessment of viability and germination of Cattleya (Orchidaceae) seeds following storage. In Vitro Cell Dev Biol Plant 48:127–136CrossRefGoogle Scholar
  68. 68.
    Nishizawa S, Sakai A, Amano Y, Matsuzawa T (1993) Cryopreservation of asparagus (Asparagus officinalis L.) embryogenic suspension cells and subsequent plant regeneration by vitrification. Plant Sci 91:67–73CrossRefGoogle Scholar
  69. 69.
    International Seed Testing Association (1993) International rules for seed testing 1993. Seed Sci Technol:21Google Scholar
  70. 70.
    Singh F (1999) Differential staining of orchid seeds for viability testing. Amer Orchid Soc Bull 50:416–418Google Scholar
  71. 71.
    Pritchard HW (1985) Determination of orchid seed viability using fluorescein diacetate. Plant Cell Environ 8:727–730Google Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  1. 1.Environmental Horticulture DepartmentUniversity of Florida, Tropical Research and Education CenterHomesteadUSA

Personalised recommendations