Cryopreservation is a very important tool for the long-term storage of plant genetic resources for future generations, requiring only a minimum of space and maintenance. With increasing interest in the genetic engineering of plants, the preservation of cultured cells and somatic embryos with unique attributes is assuming greater importance. Recently, cryopreservation was reported to offer real hope for enhancing the preservation of endangered and rare plants (Touchell 1995; Touchell and Dixon 1996). This chapter describes protocols for successful vitrification using plant vitrification solution 2 (PVS2), and highlights some of the factors contributing to high levels of post-LN recovery. The development of a simple and reliable method for cryopreservation would allow the widespread storage of cultured cells, meristems, and somatic embryos. Vitrification involving vitrification solutions (Langis et al. 1990; Sakai et al. 1990; Yamada et al. 1991) and encapsulation-dehydration techniques (Fabre and Dereuddre 1990) were developed in the 1990s, and the number of cryopreserved species has increased markedly since then (Sakai 1995, 1997; Engelmann and Takagi 2000). A vitrification procedure using an ethylene glycol-based vitrification solution and French straws was presented by Steponkus and colleagues. They reported successful cryopreservation of Dianthus and Chrysanthemum (Langis et al. 1990; Schnabel-Preikstas et al. 1992), and potato (Lu and Steponkus 1994). This chapter will outline the development and uses of PVS2 developed by Sakai and associates.
Access provided by Autonomous University of Puebla. Download to read the full chapter text
Chapter PDF
Similar content being viewed by others
Keywords
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.
References
Agrawal A, Swennen R, Panis B (2004) A comparison of four methods for cryopreservation of meristems in banana (Musa spp.). CryoLetters 25: 101-110
Burke MJ (1986) The glassy state and survival of anhydrous biological systems. In: Leopold AC (ed) Membrane, Metabolism and Dry Organisms. Cornell Univ. Press, Ithaca, NY, pp 358-364
Charoensub R, Hirai D, Sakai A (2004) Cryopreservation of in vitro-grown shoot tips of cassava by encapsulation-vitrification method. CryoLetters 25: 51-58
Charoensub R, Phansiri S, Sakai A, Yongmenitchai W (1999) Cryopreservation of cassava in vitro grown shoot tips cooled to -196°C by vitrification. CryoLetters 20: 89-94
Chen Y, Wang JH (2003) Cryopreservation of carrot (Daucus carota L.) cell suspensions and protoplasts by vitrification. CryoLetters 24: 57-64
Cho EG, Hor YL, Kim HH, Rao VR, Engelmann F (2002) Cryopreservation of Citrus mandurensis zygotic embryonic axes by vitrification: Importance of loading and treatment with vitrification solution. CryoLetters 23: 317-324
Corredoira E, San-José MC, Ballester A, Vieitez AM (2004) Cryopreservation of zygotic embryo axes and somatic embryos of European chestnut. CryoLetters 25: 33-42
Crowe JH, Crowe LF, Chapman D (1984a) Preservation of membranes in anhydrobiotic organisms: The role of trehalose. Science 233: 701-703
Crowe JH, Crowe LF, Chapman D (1984b) Infrared spectroscopic studies on interactions of water and carbohydrates with a biological membrane. Arch Biochem Biophys 232: 400-407
De Carlo A, Benelli C, Lambardi M (2000) Development of a shoot-tip vitrification protocol and comparison with encapsulation-based procedures for plum (Prunus domestica L.) cryopreservation. CryoLetters 21: 215-222
Dereuddre J, Fabre J, Bassaglia C (1988) Resistance to freezing in liquid nitrogen of carnation (Dianthus caryophyllus L. var. Eolo) apical and axillary shoot tips excised from different aged in vitro plantlets. Plant Cell Rep 7: 170-173
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: 125-134
Dumet D, Engelmann F, Chabrillange N, Duval Y (1993a) Cryopreservation of oil palm (Elaeis guineensis Jacq.) somatic embryo involving a desiccation step. Plant Cell Rep 12: 352-355
Dumet D, Engelmann F, Chabrillange N, Duval Y, Dereuddre J (1993b) Importance of sucrose for the acquisition of tolerance to desiccation and cryopreservation of oil palm somatic embryos. CryoLetters 14: 243-250
Engelmann F (1991) In vitro conservation of tropical plant germplasm - a review. Euphytica 57: 227-243
Engelmann F, Takagi H (2000) Cryopreservation of Tropical Plant Germplasm: Current Research Progress and Applications. JIRCAS, Tsukuba and IPGRI, Rome
Fabre J, Dereuddre J (1990) Encapsulation-dehydration: A new approach to cryo-preservation of Solanum shoot tips. CryoLetters 11: 413-426
Fahy GM, MacFarlane DR, Angell CA, Meryman HT (1984) Vitrification as an approach to cryopreservation. Cryobiology 21: 407-426
Fukai S (1992) Studies on the cryopreservation of shoot tips of Dianthus and Chrysanthemum. Mem Fac Agric Kagawa Univ 56: 1-79
Gagliardi RF, Pacheco GP, Cameiro LA, Valls JFM, Vieira MLC, Mansur E (2003) Cryopreservation of Arachis species by vitrification of in vitro grown shoot apices and genetic stability of recovered plants. CryoLetters 24: 103-110
Gamez-Pastrana R, Martinez-Ocampo Y, Beristain CI, Gonzalez-Arnao MT (2004) An improved cryopreservation protocol for pineapple apices using encapsulation-vitrification. CryoLetters 25: 405-414
Golmirzaie AM, Panta A (2000) Advances in potato cryopreservation at CIP. In: Engelmann F, Takagi H (eds) Cryopreservation of Tropical Plant Germplasm: Current Research Progress and Applications. JIRCAS, Tsukuba and IPGRI, Rome. pp 250-254
Gonzalez-Arnao MT, Ravelo MM, Villavicencio CU, Montero MM, Engelmann F (1998) Cryopreservation of pineapple (Ananas comosus) apices. CryoLetters 19: 375-382
Hirai D, Sakai A (1999a) Cryopreservation of in vitro grown axillary shoot-tip meristems of mint (Mentha spicata) by encapsulation-vitrification. Plant Cell Rep 19: 150-155
Hirai D, Sakai A (1999b) Cryopreservation of in vitro grown meristems of potato (Solanum tuberosum L.) by encapsulation-vitrification. Potato Res 42: 153-160
Hirai D, Sakai A (2003) Simplified cryopreservation of sweet potato (Ipomoea batatas (L.) Lam.) by optimizing conditions for osmoprotection. Plant Cell Rep 21: 961-966
Hirai D, Shirai K, Shirai S, Sakai A (1998) Cryopreservation of in vitro-grown meristems of strawberry (Fragaria x ananassa Duch.) by encapsulationvitrification. Euphytica 101: 109-115
Huang CN, Wang JH, Yan QS, Zhang XQ, Yan QF (1995) Plant regeneration from rice (Oryza sativa L.) embryogenic suspension cells cryopreserved by vitrification. Plant Cell Rep 14: 730-734
Ishikawa K, Harada K, Mii M, Sakai A, Yoshimatsu K, Shimomura K (1997) Cryopreservation of protocorms of Japanese terrestrial orchid (Bletilla striata) by vitrification. Plant Cell Rep 16: 754-757
Jung DW, Sung CK, Touno K, Yoshimatsu K, Shimomura K (2001) Cryopreservation of Hyoscyamus niger adventitious roots by vitrification. J Plant Physiol 158: 801-805
Kamiya T, Sasaki K, Yoshimatsu K, Shimomura K (1995) Cryopreservation of hairy roots of Atropa belladona by vitrification. Proc Japan Soc Plant Tissue Cult., 14th Annual Meet Rep:112
Kim HH (2004) Analysis of factors affecting the cryopreservation of garlic shoot tips. Doctoral Paper, Graduate School, Seoul National University, Korea
Kim HH, Cho EG, Baek YJ, Kim CY, Engelmann F (2004) Cryopreservation of garlic shoot tips by vitrification: effect of dehydration, rewarming, unloading and regrowth conditions. CryoLetters 25: 59-70
Kohmura H, Ikeda Y, Sakai A (1994) Cryopreservation of apical meristems of Japanese shallot (Allium wakegi) by vitrification and subsequent high plant regeneration. CryoLetters 15: 289-298
Kohmura H, Sakai A, Chokyu S, Yakuwa T (1992) Cryopreservation of in vitrocultured multiple bud clusters of asparagus (Asparagus officinalis L. cv. Hiroshima green) by the technique of vitrification. Plant Cell Rep 11: 433-437
Kuranuki Y, Sakai A (1995) Cryopreservation of in vitro-grown shoot tips of tea (Camellia sinensis) by vitrification. CryoLetters 16: 345-352
Lambardi M, Fabbri A, Caccavale A (2000) Cryopreservation of white poplar (Populus alba L.) by vitrification of in vitro grown shoot tips. Plant Cell Rep 19: 213-218
Langis R, Schnabel B, Earle ED, Steponkus PL (1989) Cryopreservation of Brassica campestris cell suspensions by vitrification. CryoLetters 10: 421-428
Langis R, Schnabel-Preikstas BJ, Earle ED, Steponkus PL (1990) Cryopreservation of carnation shoot tips by vitrification. Cryobiology 276: 657-658
Leunufna S, Keller ERJ (2005) Cryopreservation of yams using vitrification modified by including droplet method: Effects of cold acclimation and sucrose. CryoLetters 26: 93-101
Langis R, Steponkus P (1991) Vitrification of isolated rye protoplasts: protection against dehydration injury by ethylene glycol. CryoLetters 12: 107-112
Lu S, Steponkus PL (1994) Cryopreservation of Solanum shoot-tips by vitrification. Cryobiology 31: 569
Luo J, Reed BM (1997) Abscisic acid-responsive protein, bovine serum albumin, and proline pretreatments improve recovery of in vitro currant shoot-tip meristems and callus cryopreserved by vitrification. Cryobiology 34: 240-250
Luyet BJ (1937) The vitrification of organic colloids and protoplasm. Biodinamica 29: 1-15
Makowska Z, Keller J, Engelmann F (1999) Cryopreservation of apices isolated from garlic (Allium sativum L.) bulbils and cloves. CryoLetters 20: 175-182
Martinez MT, Ballester A, Vieitez AM (2003) Cryopreservation of embryogenic cultures of Quercus robur using desiccation and vitrification procedures. Cryobiology 46: 182-189
Matsumoto T, Mochida K, Itamura H, Sakai A (2001) Cryopreservation of persimmon (Diopsyros kaki Thunb.) by vitrification of dormant shoot tips. Plant Cell Rep 20: 398-402
Matsumoto T, Sakai A (2003) Cryopreservation of axillary shoot tips of in vitrogrown grape (Vitis) by a two-step vitrification protocol. Euphytica 131: 299-304
Matsumoto T, Sakai A, Nako Y (1998) A novel preculturing for enhancing the survival of in vitro grown meristems of wasabi (Wasabia japonica) cooled to -196°C by vitrification. CryoLetters 19: 27-36
Matsumoto T, Sakai A, Takahashi C, Yamada K (1995a) Cryopreservation of in vitro-grown apical meristems of wasabi (Wasabia japonica) by encapsulation-vitrification method. CryoLetters 16: 189-196
Matsumoto T, Sakai A, Yamada K (1994) Cryopreservation of in vitro-grown apical meristems of wasabi (Wasabia Japonica) by vitrification and subsequent high plant regeneration. Plant Cell Rep 13: 442-446
Matsumoto T, Sakai A, Yamada K (1995b) Cryopreservation of in vitro-grown apical meristems of lily by vitrification. Plant Cell Tissue Organ Cult 41: 237-241
Matsumoto T, Takahashi C, Sakai A, Nako Y (1998) Cryopreservation of in vitrogrown apical meristems of hybrid statice by three different procedures. Scientia Hortic 76: 105-114
Murashige T, Skoog F (1962) A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiol Plant 15: 473-497
Niino T, Hettiarachchi A, Takahashi J, Samarajeewa PK (2000a) Cryopreservation of lateral buds of in vitro-grown plants of innala (Solemostemon rotundifolius) by vitrification. CryoLetters 21: 349-356
Niino T, Ivette S, Murayama T (2000b) Cryopreservation of vegetatively propagated species (mainly mulberry). In: Engelmann F, Takagi H (eds) Cryopreservation of Tropical Plant Germplasm: Current Research Progress and Applications. JIRCAS, Tsukuba and IPGRI, Rome, pp 75-80
Niino T, Sakai A, Yakuwa H, Nojiri K (1992a) Cryopreservation of in vitrogrown shoot tips of apple and pear by vitrification. Plant Cell Tissue Organ Cult 28: 261-266
Niino T, Sakai A, Enomoto S, Magoshi J, Kato S (1992b) Cryopreservation of in vitro-grown shoot tips of mulberry by vitrification. CryoLetters 13: 303-312
Niino T, Takano J, Saga T, Kobayashi M (2003a) Cryopreservation of in vitro grown Chinese leek “Nakamidori” shoot tips by vitrification. Hortic Res Jpn 2: 241-245
Niino T, Tanaka D, Ichikawa S, Takano J, Ivette S, Shirata K, Uemura M (2003b) Cryopreservation of in vitro-grown apical shoot tips of strawberry by vitrification. Plant Biotech 20: 75-80
Niino T, Tashiro K, Suzuki M, Ohuchi S, Magoshi J, Akihama T (1997) Cryopreservation of in vitro-grown shoot tips of cherry and sweet cherry by one-step vitrification. Scientia Hortic 70: 155-163
Nishizawa S, Sakai A, Amano Y, Matsuzawa T (1992) Cryopreservation of asparagus (Asparagus officinalis L.) embryogenic suspension cells and subsequent plant regeneration by a simple freezing method. CryoLetters 13: 379-388
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: 67-73
Niwata E (1995) Cryopreservation of apical meristems of garlic (Allium sativum L.) and high subsequent plant regeneration. CryoLetters 16: 102-107
Panis B (1995) Cryopreservation of banana (Musa spp.) germplasm. Doctoral paper (No. 272), Katholieke Universiteit, Leuven
Paul H, Daigny G, Sangwan-Noreel BS (2000) Cryopreservation of apple (Malus x domestica Borkh.) shoot tips following encapsulation or encapsulationvitrification. Plant Cell Rep 19: 768-774
Pennycooke JC, Towill LE (2001) Medium alterations improve regrowth of sweet potato (Ipomoea batatas (L.) Lam.) shoot tips by vitrification and encapsulation-dehydration. CryoLetters 22: 381-389
Phunchindawan M, Hirata K, Miyamoto K, Sakai A (1997) Cryopreservation of encapsulated shoot primordia induced by horseradish (Armoracia rusticana) hairy roots. Plant Cell Rep 16: 469-473
Reed BM (1992) Cryopreservation of Ribes apical meristems. Cryobiology 29: 740
Reinhoud P (1996) Cryopreservation of tobacco suspension cells by vitrification. Doctoral Paper, Rijks University, Leiden
Sakai A (1960) Survival of the twig of woody plants. Nature 185: 393-394
Sakai A (1995) Cryopreservation of germplasm of woody plants. In: Bajaj YPS (ed) Biotechnology in Agriculture and Forestry, Vol. 32, Cryopreservation of Plant Germplasm I , Springer-Verlag, Berlin, Heidelberg, pp 53-69
Sakai A (1997) Potentially valuable cryogenic procedures for cryopreservation of cultured plant meristems. In: Razdan MK, Cocking EC (eds) Conservation of Plant Genetic Resources In Vitro Science Publishers, USA, pp 53-66
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: 30-33
Sakai A, Kobayashi S, Oiyama I (1991a) Cryopreservation of nucellar cells of navel orange (Citrus sinensis Osb.) by a simple freezing method. Plant Sci 74: 243-248
Sakai A, Kobayashi S, Oiyama I (1991b) Survival by vitrification of nucellar cells of navel orange (Citrus sinensis var. brasiliensis Tanaka) cooled to -196°C. J. Plant Physiol 137: 465-470
Sakai A, Nishiyama Y (1978) Cryopreservation of winter vegetative buds of hardy fruit trees in liquid nitrogen. HortScience 13: 225-227
Sakai A, Yoshida S (1967) Survival of plant tissue at super-low temperatures. VI. Effects of cooling and rewarming rates on survival. Plant Physiol 42: 1695-1701
Schnabel-Preikstas B, Earle ED, Steponkus PL (1992) Cryopreservation of chrysanthemum shoot tips by vitrification. Cryobiology 29: 739
Sharma N, Sharma B (2003) Cryopreservation of shoot tips of Picrorhiza kurroa Royle ex Benth., an indigenous endangered medicinal plant, through vitrification. CryoLetters 24: 181-190
Shatnawi MA, Engelmann F, Frattarelli A, Damiano C (1999) Cryopreservation of apices of in vitro plantlets of almond (Prunus dulcis Mill). CryoLetters 20: 13-20
Shibli RA, Al-Juboory KH (2000) Cryopreservation of “nabali” olive (Olea europaea L.) somatic embryos by encapsulation-dehydration and encapsulationvitrification. CryoLetters 21: 357-366
Steponkus PL, Langis R, Fujikawa S (1992) Cryopreservation of plant tissues by vitrification. In: Steponkus PL (ed), Advances in Low Temperature Biology V.1. JAI Press, Hampton Hill, UK, pp 1-61
Tanaka D, Niino T, Isuzugawa K, Hikage T, Uemura M (2004) Cryopreservation of shoot apices of in vitro grown gentian plants: comparison of vitrification and encapsulation-vitrification protocols. CryoLetters 25: 167-176
Takagi H, Thinh NT, Kyesmu PM (1998) Cryopreservation of vegetatively propagated tropical crops by vitrification. Acta Hortic 461: 485-494
Takagi H, Thinh NT, Sakai A, Senboku T (1997) Cryopreservation of taro in vitrogrown shoot tips (Colocasia esculenta (L.) Schott) by vitrification. I. Investigation of basic vitrification conditions. Plant Cell Rep 16: 594-599
Thammasiri K (1999) Cryopreservation of embryonic axes of jackfruit. CryoLetters 20: 21-28
Thammasiri K (2000) Cryopreservation of seeds of a Thai orchid (Doritis pulcherrima Lindl.) by vitrification. CryoLetters 21: 237-244
Thinh NT (1997) Cryopreservation of germplasm of vegetatively propagated tropical monocots by vitrification. Doctoral Paper, Faculty of Agriculture, Kobe University, Japan
Thinh NT, Takagi H (2000) Cryopreservation of in vitro grown apical meristems of terrestrial orchids (Cymbidium spp.) by vitrification. In: Engelmann F, Takagi H (eds) Cryopreservation of Tropical Plant Germplasm: Current Research Progress and Applications. JIRCAS, Tsukuba and IPGRI, Rome, pp 441-443
Thinh NT, Takagi H, Sakai A (2000) Cryopreservation of in vitro grown apical meristems of some vegetatively propagated tropical monocots by vitrification. In: Engelmann F, Takagi H (eds) Cryopreservation of Tropical Plant Germplasm: Current Research Progress and Applications. JIRCAS, Tsukuba and IPGRI, Rome, pp 227-232
Thinh NT, Takagi H, Yashima S (1999) Cryopreservation of in vitro grown shoot tips of banana (Musa spp.) by vitrification method. CryoLetters 20: 163-175
Touchell D (1995) Principles of cryobiology for conservation of threatened Aus-tralian plants. Doctoral paper, Department of Botany, University of Western Australia, Perth.
Touchell DH, Dixon KW (1996) Cryopreservation for the conservation of Australian endangered plants. In: Normah MN, Narimah MK, Clyde MM (eds) International Workshop on the In Vitro Conservation of Plant Genetic Resources, Plant Bio-technology Lab., University Kebangsaan Malaysia, Kuala Lumpur, pp 169-180
Towill LE (1990) Cryopreservation of isolated mint shoot tips by vitrification. Plant Cell Rep 9: 178-180
Towill LE, Bonnart R (2003) Cracking in a vitrification solution during cooling or warming does not affect growth of cryopreserved mint shoot tips. CryoLetters 24: 341-346
Towill LE, Jarret RL (1992) Cryopreservation of sweet potato (Ipomoea batatas (L.) Lam.) shoot tips by vitrification. Plant Cell Rep 11: 175-178
Tsukazaki H, Mii M, Tokuhara K, Ishikawa K (2000) Cryopreservation of Doritaenopsis suspension culture by vitrification. Plant Cell Rep 19: 1160-1164
Turner SR, Tan B, Senaratna T, Bunn E, Dixon KW, Touchell DH (2000) Cryopreservation of the Australian species Macropidia fuliginosa (haemo-doraceae) by vitrification. CryoLetters 21: 379-388
Uragami A, Sakai A, Nagai M (1990) Cryopreservation of dried axillary buds from plantlets of Asparagus officinalis L. grown in vitro. Plant Cell Rep 9: 328-331
Valladares S, Toribio M, Celestino C, Vietiez A (2004) Cryopreservation of embryogenic cultures from mature Quercus suber trees using vitrification. CryoLetters 25: 177-186
Vandenbussche B, Weyens G, De Proft MP (2000) Cryopreservation of in vitro sugar beet (Beta vulgaris L.) shoot tips by a vitrification technique. Plant Cell Rep 19: 1064-1068
Volk GM, Maness N, Rotindo K (2004) Cryopreservation of garlic (Allium sativum L.) using plant vitrification solution 2. CryoLetters 25: 219-226
Wang Q, Batuman Ö, Li P, Bar-Joseph M, Gafny R (2002) A simple and efficient cryopreservation of in vitro-grown shoot tips of ‘Troyer’ citrange [Poncirus trifoliata (L.) Raf. x Citrus sinensis (L.) Osbeck.] by encapsulation-vitrification. Euphytica 128: 135-142
Wang ZC, Deng XX (2004) Cryopreservation of shoot-tips of citrus using vitrification: effect of reduced form of glutathione. CryoLetters 25: 43-50
Watanabe K, Steponkus PL (1995) Vitrification of Oryza sativa L. cell suspensions. CryoLetters 16: 255-262
Withers LA (1979) Freeze preservation of somatic embryos and clonal plantlets of carrot (Daucus carota). Plant Physiol 63: 460-467
Wu Y, Zhao Y, Engelmann F, Zhou M, Zang D, Chen S (2001) Cryopreservation of apple dormant buds and shoot tips. CryoLetters 22: 375-380
Wu Y, Engelmann F, Zhao Y, Zhou M, Chen S (1999) Cryopreservation of apple shoot tips: importance of cryopreservation technique and of conditioning of donor plants. CryoLetters 20: 121-130
Yamada T, Sakai A, Matsumura T, Higuchi S (1991) Cryopreservation of apical meristems of white clover (Trifolium repens L.) by vitrification. Plant Sci 78: 81-87
Yoshimatsu K, Yamaguchi H, Shimomura K (1996) Traits of Panax ginseng hairy roots after cold storage and cryopreservation. Plant Cell Rep 15: 555-560
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2008 Springer Science+Business Media, LLC
About this chapter
Cite this chapter
Sakai, A., Hirai, D., Niino, T. (2008). Development of PVS-Based Vitrification and Encapsulation–Vitrification Protocols. In: Reed, B.M. (eds) Plant Cryopreservation: A Practical Guide. Springer, New York, NY. https://doi.org/10.1007/978-0-387-72276-4_3
Download citation
DOI: https://doi.org/10.1007/978-0-387-72276-4_3
Publisher Name: Springer, New York, NY
Print ISBN: 978-0-387-72275-7
Online ISBN: 978-0-387-72276-4
eBook Packages: Biomedical and Life SciencesBiomedical and Life Sciences (R0)