Generic placeholder image

Recent Patents on Biotechnology


ISSN (Print): 1872-2083
ISSN (Online): 2212-4012

Review Article

Cryopreservation of Orchids – A Review

Author(s): Saranjeet Kaur *

Volume 13 , Issue 2 , 2019

Page: [114 - 123] Pages: 10

DOI: 10.2174/1872208313666181127143058

Price: $65


Background: The orchids are one of the beautiful creations of nature which stand apart from any other assemblage of flowering plants. They are highly evolutionary and ecologically significant group of plants that have effectively occupied almost every habitat on the earth. Indiscriminate collections and extermination of their natural habitats have threatened many species of orchids with extinction, resulting in a severe reduction of their genetic resources in nature according to recent patents. It is necessary to adopt sound scientific protocols for the preservation of orchid species.

Method: This cost-effective technique provides large storage time for the conservation of germplasm. Presently, efforts have been made to explore various cryopreservation techniques utilized so far and factors affecting the longevity of the propagules (in vivo and in vitro) while cryopreserving them. The sample to be cryopreserved is freeze-preserved in two ways, a) stepwise at two different subzero temperatures and b) in the rapid method, the samples are placed directly in the liquid nitrogen.

Results: The orchid seeds and pollen are the most suitable propagules for cryopreservation of orchids due to their minute size and less space requirement.

Conclusion: Among the tissues (such as seeds, pollen, protocorms etc.) seeds are the most reliable. The present article reviews the cryopreservation techniques and factors effecting the cryopreservation, for in vitro conservation of orchid gene pool.

Keywords: Conservation, cryoprotectant, endangered, in vitro, seeds, orchids.

Graphical Abstract
Vendrame W, Tadeu de Faria R, Sorace M, Sahyun AS. Orchid cryopreservation. Ciênc Agrotec Lavras 2014; 38: 213-29.
Arditti J. Fundamentals of orchid biology. New York: John Wiley and Sons 1992.
Chauhan NS. Medicinal orchids of Himachal Pradesh. J Orchid Soc India 1990; 4: 99-105.
Rastogi RP, Dhawan BN. Anticancer and antiviral activities in Indian medicinal plants: a review. Drug Dev Res 1990; 19: 1-12.
Chen Y, Qing C, Zhang Y, et al. 1,4,5-Trihydroxy-7-methoxy-9H-fluoren-9- one, a new cytotoxic compound from Dendrobium chrysotoxum. Food Chem 2008; 108: 973-6.
De Clercq E. Trends in drug development for the treatment of AIDS: compounds interfering with the initial stages of HIV replicate cycle. Eur J Pharm Sci 1994; 2: 4-6.
De CL, Pathak P, Rao AN, Rajeevan PK. Medicinal and aromatic orchids.In: Golachowska M, Monu E,Eds Commercial Orchids Warsaw, Berlin: De Gruter Open Ltd . 2014; 243-9.
Goodenough DR. Vanilla, vanillin and vanillin derivatives. Bakers Digest 1982; 56: 8-10.
Edible orchids. Available at:
The Orchid Gifts Madeirahttp. Available at:
Captured beauty in yellow - resin dipped yellow real miniature rose dangle earrings. Available at: purple-orchid/
Zanenga-Godoy R, Costa CG. Foliar anatomy of four species of genus Cattleya Lindl. (Orchidaceae) of the Brazilian Central Planalt. Acta Bot Bras 2003; 17: 101-18.
Tikhonova VL. Long term storage of seed Fiziol. Rast, Moscow. Russ J Plant Physiol 1999; 46: 467-76.
Hu WH, Yang YH, Liaw SI, et al. Cryopreservation the seeds of a Taiwanese terrestrial orchid, Bletilla formosana (Hayata) Schltr. by vitrification. Bot Stud 2013; 54: 33-40.
Hay FR, Merritt DJ, Soanes JA, Dixon KW. Comparative longevity of Australian orchid (Orchidaceae) seeds under experimental and low temperature storage conditions. Bot J Linn Soc 2010; 164: 26-41.
Pritchard HW. Liquid nitrogen preservation of terrestrial and epiphytic orchid seeds. Cryo Lett 1984; 5: 295-300.
Thammasiri K. Cryopreservation of seeds of a Thai orchid (Doritis pulcherrima lindl.) by vitrification. Cryo Lett 2000; 21: 237-44.
Thammasiri K. Cryopreservation of some Thai orchid species. Acta Hortic 2008; (788): 53-62.
Wu RY, Chang SY, Hsieh TF, et al. Cryopreservation of Bletilla formosana seeds (Orchidaceae) by desiccation. Sci Hortic 2013; 157: 108-12.
Popova EV, Nikishina TV. The effect of seed cryopreservation on the development of protocorms by the hybrid orchid Bratonia. Russ J Plant Physiol 2003; 50: 672-7.
Merritt DJ, Hay FR, Swarts ND, et al. Ex situ Conservation and cryopreservation of orchid germplasm. Int J Plant Sci 2014; 175: 46-58.
Hoque MM, Khaleda L. A-l Forkan M. Development of cryopreservation protocols for five indigenous medicinal orchids of Bangladesh. J Orchid Soc India 2016; 30: 105-10.
Engelmann F. Use of biotechnologies for the conservation of plant biodiversity. In Vitro Cell Dev Biol Plant 2011; 47: 5-46.
Thammasiri K, Soamkul L. Cryopreservation of Vanda coerulea Griff. ex Lindl. Sci Asia 2007; 33: 223-7.
Hirano T, Godo T, Miyoshi K, et al. Cryopreservation and low-temperature storage of seeds of Phaius tankervilleae. Plant Biotechnol Rep 2009; 3: 103-9.
Vendrame WA, Carvalho VS, Dias JMM. In vitro germination and seedling development of cryopreserved Dendrobium hybrid mature seeds. Sci Hortic 2007; 114: 188-93.
Pegg DE. Principles of cryopreservation. Methods Mol Biol 2007; 368: 39-57.
Benelli C. Carlo de-A, Engelmann F. Recent advances in the cryopreservation of shoot-derived germplasm of economically important fruit trees of Actinidia, Diospyros, Malus, Olea, Prunus, Pyrus and Vitis. Biotechnol Adv 2013; 31: 175-85.
Wang B, Wang RR, Cui ZH, et al. Potential applications of cryogenic technologies to plant genetic improvement and pathogen eradication. Biotechnol Adv 2014; 32: 583-95.
Engelmann F. Plant cryopreservation: progress and prospects. In Vitro Cell Div. Biol Plant 2004; 40: 427-33.
Pritchard HW. Cryopreservation of desiccation-tolerant seeds. Methods Mol Biol 1995; 38: 133-44.
Pritchard HW. Cryopreservation of desiccation-tolerant seeds.In: Day JG, McLellan MR, Eds Methods in molecular biology cryopreservation and freeze-drying protocols. New Jersey: Humana Press Inc. 2007; pp. 185-201.
Walters C, Wheeler L, Stanwood PC. Longevity of cryogenically stored seeds. Cryobiol 2004; 48: 229-44.
Gonzalez-Arnao MT, Panta A, Roca WM, et al. Development and large scale application of cryopreservation techniques for shoot and somatic embryo cultures of tropical. Plant Cell Tissue Organ Cult 2008; 92: 1-13.
Na HY, Kondo K. Cryopreservation of tissue-cultured shoot primordia from shoot apices of cultured protocorms in Vanda pumila following ABA preculture and desiccation. Plant Sci 1996; 118: 195-201.
Matsumoto T, Mochida K, Itamura H, et al. Cryopreservation of persimmon (Diospyros kaki Thunb.) by vitrifiation of dormant shoot tips. Plant Cell Rep 2001; 20: 398-402.
Engelmann F. Importance of desiccation for the cryopreservation of recalcitrant seed and vegetatively propagated species. Plant Genet Resour Newsl 1997; 112: 9-18.
Cripps RF, McGregor K. Determination of the optimal dehydration period for the protocorms of Paralophia epipyhtica (Orchidacae) using differential scanning calorimetry. Cryobiol 2006; 53: 424.
Sopalun K, Kanchit K, Ishikawa K. Vitrification-based cryopreservation of Grammatophyllum speciosum protocorms. Cryo Lett 2010; 31: 347-57.
Jitsopakul N, Thammasiri K, Yukawa T, et al. Effect of cryopreservation on seed germination and protocorm development of Vanda tricolor. Sci Asia 2012; 38: 244-9.
Hirano T, Ishikawa K, Mii M. Advances in orchid cryopreservation. In: Teixeira da-Silva JA, Ed Floriculture ornamental and plant biotechnology: advances and topical issues. UK: Global Science Books 2006; pp. 410-4.
Luza JG, Polito VS. Cryopreservation of english walnut (Juglans regia L.) pollen. Euphytica 1988; 37: 141-8.
Popov AS, Popova EV, Nikishina TV, et al. Cryobank of plant genetic resources in Russian academy of sciences. Int J Refrig 2006; 29: 403-10.
Nikishina TV, Popov AS, Kolomeitseva GL, et al. Effect of cryoconservation on seed germination of rare tropical orchids. Russ J Plant Physiol 2001; 48: 810-5.
Nikishina TV, Popova EV, Vakhrameeva MG, et al. Cryopreservation of seeds and protocorms of rare temperate orchids. Russ J Plant Physiol 2007; 54: 121-7.
Tsai SF, Yeh SD, Chan CF, Liaw SI. High-efficiency vitrification protocols for cryopreservation of in vitro grown shoot tips of transgenic papaya lines. Plant Cell Tissue Organ Cult 2009; 98: 157-64.
Sakai A. Development of cryopreservation techniques.In: Engellmann F, Takagi H, EdsCryopreservation of Tropical Plant Germplasm. Tsukuba: Japan International Research Center for Agricultural Sciences 2000; pp. 1-7.
Popova E, Kim HH, Saxena PK, Engelmann F. Frozen beauty: the cryobiotechnology of orchid diversity. Biotechnol Adv 2016; 34: 380-403.
Finkle BJ, Zavala ME, Ulrich JM. Cryoprotective compounds in the viable freezing of plant tissues.In:Kartha KK, EdsCryopreservation of Plant Cells and Organs Boca Raton, Florida: CRC Press Inc.. 1985; 75-113.
Fuller BJ. Cryoprotectants: the essential antifreezes to protect life in the frozen state. Cryo Lett 2004; 25: 375-88.
Volk GM, Walters C. Plant vitrification solution 2 lowers water content and alters freezing behavior in shoot tips during cryoprotection. Cryobiol 2006; 52: 48-61.
Galdiano RF Jr, Lemos EGM, Faria RT, Vendrame WA. Cryopreservation of Dendrobium hybrid seeds and protocorms as affected by phloroglucinol and supercool X1000. Sci Hortic 2012; 148: 154-60.
Galdiano RF Jr, Lemos EGM, Vendrame WA. Cryopreservation, early seedling development, and genetic stability of Oncidium flexuosum Sims. Plant Cell Tissue Organ Cult 2013; 114: 139-48.
Galdiano RF Jr, Lemos EGM, de-Faria RT, et al. Seedling development and evaluation of genetic stability of cryopreserved Dendrobium hybrid mature seeds. Appl Biochem Biotechnol 2014; 172: 2521-9.
Ishikawa K, Harata K, Mii M, et al. Cryopreservation of zygotic embryos of a Japanese terrestrial orchid (Bletilla striata) by vitrification. Plant Cell Rep 1997; 16: 754-7.
Thammasiri K. Cryopreservation of seeds of a Thai orchid (Doritis pulcherrima Lindl.) by vitrification. Cryo Lett 2000; 21: 237-44.
Zhang W, Luzhen W, Guanqun C, Li R. Method for optimizing vitrification ultra-low temperature preservation effect of protocorm of Cymbidium hydridum CN104255708A, 2016.
Batty AL, Dixon KW, Brundrett M, Sivasithamparam K. Long-term storage of mycorrhizal fungi and seed as a tool for the conservation of endangered Western Australian terrestrial orchids. Aust J Bot 2001; 49: 619-28.
Pritchard HW, Poynter ALC, Seaton PT. Interspecific variation in orchid seed longevity in relation to ultra-dry storage and cryopreservation. Lindleyana 1999; 14: 92-101.
Pritchard HW. Liquid nitrogen preservation of terrestrial and epiphytic orchid seeds. Cryo Lett 1984; 5: 295-300.
Fabre J, Dereuddre J. Encapsulation-dehydration: a new approach to cryopreservation of Solanum shoot tips. Cryo Lett 1990; 11: 413-26.
Surenciski MR, Flachsland EA, Terada G, et al. Cryopreservation of Cyrtopodium hatschbachii Pabst (Orchidaceae) immature seeds by encapsulation-dehydration. Biocell 2012; 36: 31-6.
Flachsland E, Terada G, Scocchi A, et al. Cryopreservation of seeds and in vitro-cultured protocorms of Oncidium bifolium sims.(Orchidaceae) by encapsulation-dehydration. Cryo Lett 2006; 27: 235-42.
Martín C, Regalado JJ, Perán-Quesada R, et al. Cryopreservation of rhizome buds of Asparagus officinalis L. (cv. Morado de Huétor) and evaluation of their genetic stability. Plant Cell Tissue Organ Cult 2018; 133: 395-403.
Zhou Y, Zhang J, Chen M. . A method of increasing Anoectochilus PLB preservative effect.CN104304239B 2016.
Ajeeshkumar S, Decruse SW. Fertilizing ability of cryopreserved pollinia of Luisia macrantha, an endemic orchid of Western Ghats. Cryo Lett 2013; 34: 20-9.
Seaton PT, Hailes SJ. Effect of temperature and moisture content on the viability of Cattleya aurantiaca seed.In: Pritchard HW, EdModern methods in orchid conservation: the role of physiology, ecology and management. UK: Cambridge University 1989; pp. 17-29.
Miyoshi K, Mii M. Stimulatory effects of sodium and calcium hypochlorite, pre-chilling and cytokinins on the germination of Cypripedium macranthos seed in vitro. Physiol Plant 1998; 102: 481-6.
Seaton P, Kendon JP, Pritchard HW, et al. Orchid conservation: the next ten years. Lankestriana 2013; 13: 93-101.
Li DZ, Pritchard HW. The science and economics of ex situ plant conservation. Trends Plant Sci 2009; 14: 614-21.
Bowling JC, Thompson PA. On storing orchid seeds. Orchid Rev 1972; 80: 120-1.
Pritchard HW. Orchid seed storage at the Royal Botanic Gardens, Kew, England. 2. Physiology Unit, Wakehurst Place. Orchid Res News 1986; l: 7-18.
Hirano T, Ishikawa K, Mii M. Cryopreservation of immature seeds of Ponerorchis graminifolia var. suzukiana by vitrification. Cryo Lett 2005; 26: 139-46.
Manning JC, Van Staden J. The development and mobilization of seed reserves in some African orchids. Aust J Bot 1987; 35: 343-53.
Yeung EC, Law SK. Embryology of Calypso bulbosa. II. Embryo development. Can J Bot 1992; 70: 461-8.
Cerna M, Valdivieso P, Cella R, et al. Cryopreservation of orchid seeds through rapid and step freezing methods. F1000 Res 2018; 7: 209.
Lee YG, Popova E, Cui HY, et al. Improved cryopreservation of chrysanthemum (Chrysanthemum morifolium) using droplet-vitrification. Cryo Lett 2011; 32: 487-97.
Shin DJ, Kong HJ, Popova EV, et al. Cryopreservation of Kalopanax septemlobus embryogenic callus using vitrification and droplet-vitrification. Cryo Lett 2012; 33: 402-10.
Kim HH, Popova EV, Shin DJ, et al. Development of a droplet-vitrification protocol for cryopreservation of Rubia akane (Nakai) hairy roots using a systematic approach. Cryo Lett 2012; 33: 506-17.
Vujović T, Chatelet P, Ružić Ð, et al. Cryopreservation of Prunus spp. using aluminium cryo-plates. Sci Hortic 2015; 195: 173-82.
Yamamoto S, Wunna T, Rafique T, et al. The aluminium cryo-plate increases efficiency of cryopreservation protocols for potato shoot tips. Am J Potato Res 2015; 92: 250-7.
Nipawan J, Kanchit T, Keiko I. Cryopreservation of Bletilla striata Mature Seeds, 3-Day germinating seeds and protocorms by droplet-vitrification. Cryo Lett 2008; 29: 517-26.
Rahmah S, Mubbarakh SA, Ping KS, et al. Effects of droplet-vitrification cryopreservation based on physiological and antioxidant enzyme activities of Brassidium shooting star orchid. The Sci World J 2015; 38: 1-10.
Ping KS, Poobathy R, Rahmad Z, et al. Development of a PVS2 droplet-vitrification cryopreservation technique for Aranda Broga blue orchid protocorm-like bodies (PLBs). Cryo Lett 2017; 38: 290-8.
Kondo K, Tatarenko IV, Varghese SB, Iwai Y, Matsumoto KI. Orchid cryopreservation by using induced shoot primordia and protocorm-like bodies of tissue culture. In: Pathak P, Sehgal RN, Shekhar N,Sharma N, Sood A, EdsOrchids: science and commerce. Dehra Dun: Bishen Singh Mahendra Pal Singh 2001; pp. 397-412.
Paek KY, Murthy HN. Temperate oriental cymbidium species. In: Kull T, Arditti J EdsOrchid biology reviews and perspectives. Netherlands: Springer 2002; pp. 235-86.

Rights & Permissions Print Export Cite as
© 2022 Bentham Science Publishers | Privacy Policy