Skip to main content
Log in

Plant cryopreservation: Progress and prospects

  • Published:
In Vitro Cellular & Developmental Biology - Plant Aims and scope Submit manuscript

Summary

Cryopreservation (liquid nitrogen, −196°C) represents the only safe and cost-effective option for long-term conservation of germplasm of non-orthodox seed species, vegetatively propagated species, and of biotechnology products. Classical cryopreservation techniques, which are based on freeze-induced dehydration, are mainly employed for freezing undifferentiated cultures and apices of cold-tolerant species. New cryopreservation techniques, which are based on vitrification of internal solutes, are successfully employed with all explant types, including cells suspensions and calluses, apices, and somatic and zygotic embryos of temperate and tropical species. The development of cryopreservation protocols is significantly more advanced for vegetatively propagated species than for recalcitrant seed species. Even though its routine use is still limited, there are a growing number of examples where cryopreservation is employed on a large scale for different types of materials, including seeds with orthodox and intermediate storage behaviour, dormant buds, pollen, biotechnology products, and apices sampled from in vitro plantlets of vegetatively propagated species. Cryopreservation can also be employed for uses other than germplasm conservation, such as cryoselection, i.e., the selection through freezing of samples with special properties, or cryotherapy, i.e., the elimination of viruses from infected plants through apex cryopreservation. Because of its high potential, it is expected that cryopreservation will become more frequently employed for long-term conservation of plant genetic resources.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Similar content being viewed by others

References

  • Assy-Bah, B.; Engelmann, F. Cryopreservation of mature embryos of coconut (Cocos nucifera L.) and subsequent regeneration of plantlets. CryoLetters 13:117–126; 1992.

    Google Scholar 

  • Bercetche, J.; Galerne, M.; Dereuddre, J. Efficient regeneration of plantlets from embryogenic callus of Picea abies (L.) Karst after freezing in liquid nitrogen. C. R. Acad. Sci. Paris Sér. 3 310:357–363; 1990.

    Google Scholar 

  • Berjak, P.; Farrant, J. M.; Mycock, D. J.; Pammernter, N. W. Homoiohydrous (recalcitrant) seeds: the enigma of their desiccation sensitivity and the state of water in axes of Landolphia kirkii Dyer. Planta 186:249–261; 1989.

    Google Scholar 

  • Brison, M.; de Boucaud, M. T.; Pierronnet, A.; Dosba, F. Effect of cryopreservation on the sanitary state of a cv. of Prumus rootstock experimentally infected with Plum Pox Potyvirus. Plant Sci. 123:189–196; 1997.

    Article  CAS  Google Scholar 

  • Chandel, K. P. S.; Chaudhury, R.; Radhamani, J.; Malik, S. K. Desiccation and freezing sensitivity in recalcitrant seeds of tea, cocoa and jackfruit. Ann. Bot. 76:443–450; 1995.

    Article  Google Scholar 

  • Côte, F. X.; Goue, O.; Domergue, R.; Panis, B.; Jenny, C. In-field behavior of banana plants (Musa AA sp.) obtained after regeneration of cryopreserved embryogenic cell suspensions. CryoLetters 21:19–24; 2000.

    PubMed  Google Scholar 

  • Cyr, D. R. Cryopreservation: roles in clonal propagation and germplasm conservation of conifers. In: Engelmann, F.; Takagi, H., eds. Cryopreservation of tropical plant germplasm—current research progress and application. Tsukuba: JIRCAS; Rome: IPGRI; 2000:261–268.

    Google Scholar 

  • Dereuddre, J.; Hassen, M.; Blandin, S.; Kaminski, M. Resistance of alginatecoated somatic embryos of carrot (Daucus carotaL.) to desiccation and freezing in liquid nitrogen: 2. thermal analysis. CryoLetters 12:135–148; 1991.

    Google Scholar 

  • Dixit, S.; Mandal, B. B.; Ahuja, S.; Srivastava, P. S. Genetic stability assessment of plants regenerated from cryopreserved embryogenic tissues of Dioscorea bulbifera L. using RAPD, biochemical and morphological analysis. CryoLetters 24:77–84; 2003.

    PubMed  CAS  Google Scholar 

  • Dumet, D. Cryoconservation des massifs d'embryons somatiques de palmier à huile (Elaeis guineensis Jacq.) par déshydratation-vitrification. Etude du rôle du saccharose pendant le prétraitement. PhD thesis, Université Paris 6, France; 1994.

    Google Scholar 

  • Dumet, D.; Engelmann, F.; Chabrillange, N.; Duval, Y. Cryopreservation of oil palm (Elaeis guineensis Jacq.) somatic embryos involving a desiccation step. Plant Cell Rep. 12:352–355; 1993.

    Article  CAS  Google Scholar 

  • Dussert, S.; Chabrillange, N.; Engelmann, F.; Anthony, F.; Hamon, S. Cryopreservation of coffee (Coffea arabica L.) seeds: importance of the precooling temperature. CryoLetters 18:269–276; 1997.

    Google Scholar 

  • Dussert, S.: Chabrillange, N.; Vasquez, N. Cryopreservation of seeds for long-term conservation of coffee germplasm and elite varieties: successful application at CATIE. In: Proc. 18th Int. Conf. on Coffee Science (ASIC'99), Helsinki, August 2–6, 1999.

  • Dussert, S.; Engelmann, F.; Noirot, M. Development of probalistic tools to assist in the establishment and management of cryopreserved plant germplasm collections. CryoLetters 24:149–160; 2003.

    PubMed  Google Scholar 

  • Ellis, R. E.; Hong, T.; Roberts, E. H. An intermediate category of seed storage behaviour?, I. coffee. J. Exp. Bot. 41:1167–1174; 1990.

    Article  Google Scholar 

  • Engelmann, F.. In vitro conservation of tropical plant germplasm—a review. Euphytica 57:227–243; 1991.

    Article  Google Scholar 

  • Engelmann, F. Cryopreservation of embryos. In: Dattée, Y.; Dumas, C.; Gallais, A., eds. Reproductive biology and plant breeding. Berlin: Springer Verlag; 1992:281–290.

    Google Scholar 

  • Engelmann, F. Importance of desiccation for the cryopreservation of recalcitrant seed and vegetatively propagated species. Plant Genet. Res. Newslett. 112:9–18; 1997a.

    Google Scholar 

  • Engelmann, F. In vitro conservation methods. In: Ford-Lloyd, B. V.; Newburry, J. H.; Callow, J. A., eds. Biotechnology and plant genetic resources: conservation and use. Wallingford: CABI; 1997b:119–162.

    Google Scholar 

  • Engelmann, F. Alternative methods for the storage of recalcitrant seeds—an update. In: Marzalina, M.; Khoo, K. C.; Tsan, F. Y.; Krishnapillay, B. eds. IUFRO Seed Symposium 1998 ‘Recalcitrant Seeds’, Kuala Lumpur, Malaysia, October 12–15, 1998, Kuala Lumpur: FRIM; 1999:159–170.

    Google Scholar 

  • Engelmann, F. 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. Tsukuba: JIRCAS. Rome: IPGRI; 2000:8–20.

    Google Scholar 

  • Engelmann, F.; Dumet, D.; Chabrillange, N.; Abdelnour-Esquivel, A.; Assy-Bah, B.; Dereuddre, J.; Duval, Y. Factors affecting the cryopreservation of coffee, coconut and oil palm embryos. Plant Genet. Res. Newslett. 103:27–31; 1995.

    Google Scholar 

  • Engelmann, F.; Takagi, H. Cryopreservation of tropical plant germplasm—current research progress and applications. Tsukuba: JIRCAS; Rome: IPGRI; 2000.

    Google Scholar 

  • Fahy, G. M.; MacFarlane, D. R.; Angell, C. A.; Meryman, H. T. Vitrification as an approach to cryopreservation. Cryobiology 21:407–426; 1984.

    Article  PubMed  CAS  Google Scholar 

  • Florin, B.; Brulard, E.; Lepage, B. Establishment of a cryopreserved coffee germplasm bank. In: Abstracts Cryo'99, World Congress of Cryobiology, Marseilles, France, July 12–15, 1999:167.

  • Forsline, P. L.; McFerson, J. R.; Lamboy, W. F.; Towill, L. E. Development of base and active collections of Malus germplasm with cryopreserved dormant buds. Acta Hort. 484:75–78; 1999.

    Google Scholar 

  • Gagliardi, R. F.; Pacheco, G. P.; Carneiro, L. A.; Valls, J. F. M.; Vieira, M. L. C.; Mansur, E. Cryopreservation of Arachis species by vitrification of in vitro grown shoot apices and genetic stability of recovered plants. CryoLetters 24:103–110; 2003.

    PubMed  CAS  Google Scholar 

  • Ganeshan, S.; Rajashekaran, P. E. Current status of pollen cryopreservation research: relevance to tropical agriculture. In: Engelmann, F.; Takagi, H., eds. Cryopreservation of tropical plant germplasm—current research progress and applications. Tsukuba: JIRCAS:Rome:IPGRI; 2000:360–365.

    Google Scholar 

  • Golmirzaie, A.; Panta, A. Advances in potato cryopreservation at the International Potato Center, Peru. In: Engelmann, F.; Takagi, H., eds. Cryopreservation of tropical plant germplasm—current research progress and applications. Tsukuba: JIRCAS; Rome: IPGRI; 2000:250–254.

    Google Scholar 

  • Gonzalez Arnao, M. T. Desarollo de una tecnica para la crioconservacion de meristemos apicales de caña de azucar. Tesis presentada en opcion al grado de Doctor en ciencias tecnicas. Centro Nacional de Investigaciones Cientificas. La Habana, Cuba; 1996.

    Google Scholar 

  • Gonzalez-Benito, M. E.; Martin, C.; Iriondo, J. M. Conservation of rare and endangered plants endemic to Spain. In: Benson, E. E., ed. Plant conservation biotechnology. London: Taylor & Francis; 1999:251–264.

    Google Scholar 

  • Helliot, B.; Panis, B.; Poumay, Y.; Swennen, R.; Lepoivre, P.; Frison, E. Cryopreservation for the elimination of cucumber mosaic and banana streak viruses from banana (Musa spp.). Plant Cell Rep. 20:1117–1122; 2002.

    Article  CAS  Google Scholar 

  • Hummer, K. E.; Reed, B. M. Establishment and operation of a temperate clonal field genebank. In: Engelmann, F. Management of field and in vitro germplasm collections. Rome: IPGRI; 2000:29–31.

    Google Scholar 

  • Kartha, K. K.; Engelmann, F. Cryopreservation and germplasm storage. In: Vasil, I. K.; Thorpe, T. A., eds. Plant cell and tissue culture. Dordrecht: Kluwer; 1994:195–230.

    Google Scholar 

  • Kendall, E. J.; Qureshi, J. A.; Kartha, K. K.; Leung, N.; Chevrier, N.; Caswell, K.; Chen, T. H. H. Regeneration of freezing tolerant spring wheat (Triticum aestivum L.) plants from cryoselected callus. Plant Physiol. 94:1756–1762; 1990.

    Article  PubMed  CAS  Google Scholar 

  • Mandal, B. B. Cryopreservation research in India: current status and future perspectives. In: Engelmann, F.; Takagi, H. eds. Cryopreservation of tropical plant germplasm—current research progress and applications Tsukuba: JIRCAS: Rome: IPGRI; 2000:282–286.

    Google Scholar 

  • Matsumoto, T.; Sakai, A.: Cryopreservation of axillary shoots of in vitro grown grape (Vitis) by a two-step vitrification protocol. Euphytica 131:299–304; 2003.

    Article  CAS  Google Scholar 

  • Matsumoto, T.; Sakai, A.; Yamada, K. Cryopreservation of in vitro grown apical meristems of wasabi (Wasabia japonica) by vitrification and subsequent high plant regeneration. Plant Cell Rep. 13:442–446; 1994.

    Article  Google Scholar 

  • Mazur, P. Freezing of living cells: mechanisms and applications. Am. J. Physiol. Cell Physiol. 247:C125-C142; 1984.

    CAS  Google Scholar 

  • Meryman, H. T.; Williams, R. J.; Douglas, M. S. J. Freezing injury from solution effects and its prevention by natural or artificial cryoprotection. Cryobiology 14:287–302; 1977.

    Article  PubMed  CAS  Google Scholar 

  • Mix-Wagner, G.; Conner, A. J.; Cross, R. J. Survival and recovery of asparagus shoot tips after cryopreservation using the ‘droplet’ method. NZ J. Crop Hort. Sci. 28:283–287; 2000.

    Google Scholar 

  • Mix-Wagner, G.; Schumacher, H. M.; Cross, R. J. Recovery of potato apices after several years of storage in liquid nitrogen. CryoLetters 24:33–41; 2003.

    PubMed  CAS  Google Scholar 

  • Niino, T. Cryopreservation of germplasm of mulberry. In: Bajaj, Y. P. S., ed. Biotechnology in agriculture and forestry, vol. 32. Cryopreservation of plant germplasm I. Berlin: Springer Verlag: 1995:102–113.

    Google Scholar 

  • Panis, B.; Strosse, H.; Van den Henda, S.; Swennen, R. Sucrose preculture to simplify cryopreservation of banana meristem cultures. CryoLetters 23:375–384; 2002.

    PubMed  CAS  Google Scholar 

  • Pence, V. C. Cryopreservation of seeds of Ohio native plants and related species. Seed Sci. Technol. 19:235–251; 1991.

    Google Scholar 

  • Pence, V. C. Cryopreservation of recalcitrant seeds. In: Bajaj, Y. P. S., ed. Biotechnology in agriculture and forestry, vol. 32. Cryopreservation of plant germplasm I. Berlin: Springer Verlag; 1995:29–52.

    Google Scholar 

  • Pence, V. C. The application of biotechnology for the conservation of endangered plants. In: Benson, E. E., ed. Plant conservation biotechnology. London: Taylor & Francis; 1999:227–250.

    Google Scholar 

  • Pritchard, H. W. Cryopreservation of seeds. In: Day, J. G.; McLellan, M. R., eds. Methods in molecular biology, vol. 38. Cryopreservation and freeze-drying protocols. Totowa, NJ.: Humana Press; 1995:133–144.

    Google Scholar 

  • Reed, B. M.; Chang, Y. Medium- and long-term storage of in vitro cultures of temperature fruit and nut crops. In: Razdan, M. K.; Cocking, E. C., eds. Conservation of plant genetic resources in vitro, vol. 1. General aspects. Enfield: Science Publishers; 1997:67–105.

    Google Scholar 

  • Reed, B. M.; DeNoma, J.; Chang, Y. Application of cryopreservation protocols at a clonal genebank. In: Engelmann, F.; Takagi, H., eds. Cryopreservation of tropical plant germplasm—current research progress and applications. Tsukuba: JIRCAS; Rome: IPGRI; 2000:246–249.

    Google Scholar 

  • Roberts, H. F. Predicting the viability of seeds. Seed Sci. Technol. 1:499–514; 1973.

    Google Scholar 

  • Roca, W.; Debouck, D.; Escobar, R.; Mafla, G. Cryopreservation and cassava germplasm conservation at CIAT. In: Engelmann, F.; Takagi, H., eds. Cryopreservation of tropical plant germplasm—current research progress and applications. Tsukuba: JIRCAS: Rome: IPGRI; 2000:273–279.

    Google Scholar 

  • Sakai, A. Development of cryopreservation techniques. In: Engelmann, F.; Takagi, H., eds. Cryopreservation of tropical plant germplasm—current research progress and applications. Tsukuba: JIRCAS; Rome. IPGRI; 2000:1–7.

    Google Scholar 

  • Sakai, A.; Kobayashi, S.; Oiyama, I. Cryopreservation of nucellar cells of navel orange (Citrus sinensis Osb. var. brasiliensis Tanaka) by vitrification. Plant Cell Rep. 9:30–33; 1990.

    Article  Google Scholar 

  • Sakai, A.; Matsumoto, T.; Hirai, D.; Charoensub, R. Survival of tropical apices cooled to −196°C by vitrification. In: Li, P.H.; Palva, E. T., eds. Plant cold hardiness, gene regulation and genetic engineering. New York: Kluwer Academic/Plenum Publishers; 2002:109–119.

    Google Scholar 

  • Sakai, A.; Matsumoto, T.; Hirai, D.; Niino, T. Newly developed encapsulation-dehydration protocol for plant cryopreservation. CryoLetters 21:53–62; 2000.

    PubMed  Google Scholar 

  • Schäfer-Menuhr, A. Refinement of cryopreservation techniques for potato. Final Report for the period 1 Sept. 1991–31 Aug. 1996; Rome: IPGRI; 1996.

    Google Scholar 

  • Spencer, M. The challenges of developing cryopreservation strategies to suit the requirements of a large industrial in vitro plant cell collection. In: Abstract Cryo'99, World Congress of Cryobiology, Marseilles, France, July 12–15, 1999:245.

  • Touchell, D. H.; Dixon, K. W. Cryopreservation for seedbanking of Australian species. Ann. Bot. 40:541–546; 1994.

    Article  Google Scholar 

  • Towill, L. E.; Eberhart, S.; Roos, E. Cryopreservation at the USDA-ARS national seed storage laboratory. In: Abstracts Int. Workshop on Cryopreservation of Tropical Plant Germplasm—current research progress and applications, Tsukuba, Japan, October 20–23, 1998.

  • Towill, L. E.; Walters, C. Cryopreservation of pollen. In: Engelmann, F.; Takagi, H., eds. Cryopreservation of tropical plant germplasm—current research progress and applications. Tsukuba: JIRCAS; Rome: IPGRI; 2000:115–129.

    Google Scholar 

  • Uragami, A.; Sakai, A.; Magai, M. Cryopreservation of dried axillary buds from plantlets of Asparagus officinalis L. grown in vitro. Plant Cell Rep. 9:328–331; 1990.

    Article  Google Scholar 

  • Wang, Q.; Mawassi, M., Li, P.; Gafny, R.; Sela, I.; Tanne, E. Elimination of grapevine virus A (GVA) by cryopreservation of in vitro-grown shoot tips of Vitis vinifera L. Plant Sci. 165:321–327; 2003.

    Article  CAS  Google Scholar 

  • Watanabe, K.; Yamada, Y.; Ueno, S.; Mitsuda, H. Change of freezing resistance and retention of metabolic and differentiation potentials in cultured green Lavandula vera cells which survived repeated freeze-thaw procedures. Agr. Biol. Chem. Tokyo 49:1727–1731; 1985.

    CAS  Google Scholar 

  • Wesley-Smith, J.; Vertucci, C. W.; Berjak, P.; Pammenter, N. W.; Crane, J. Cryopreservation of desiccation-sensitive axes of Camellia sinensis in relation to dehydration, freezing rate and the thermal properties of tissue water. J. Plant Physiol. 140:596–604; 1992.

    Google Scholar 

  • Withers, L. A.; Engelmann, F. In vitro conservation of plant genetic resources. In: Altman, A., ed. Biotechnology in agriculture. New York: Marcel Dekker Inc.; 1998:57–88.

    Google Scholar 

  • Zhai, Z.; Wu, Y.; Huang, X.; Chen, R.; Zhao, Y.; Engelmann, F. Assessments of genetic stability of grape and kiwi in vitro plantlets regenerated from cryopresorved shoot tips using RAPD. CryLetters 24:315–322; 2003.

    Google Scholar 

  • Zhao, Y.; Wu, Y.; Engelmann, F.; Zhou, M.; Chen, S. Cryopreservaton of apple in vitro shoot tips by the droplet freezing method. CryoLetters 20:109–112; 1999.

    Google Scholar 

Download references

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Florent Engelmann.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Engelmann, F. Plant cryopreservation: Progress and prospects. In Vitro Cell.Dev.Biol.-Plant 40, 427–433 (2004). https://doi.org/10.1079/IVP2004541

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1079/IVP2004541

Key words

Navigation