Authors : Toshikazu Matsumoto
Disciplines : Plant Genetics
Keywords : cryo-plate, cryopreservation, genetic stability, shoot tips,vitrification
Full Text: : HTML PDF


Plant genetic resources (PGR) are important for agriculture and are mainly conserved in seed and field genebanks. Cryopreservation is an in vitro conservation method, which has become an important tool for the long-term storage of PGR. Within this method, the techniques of vitrification and encapsulation/dehydration are commonly used worldwide. Recently, new cryogenic methods using cryo-plates (the V cryo-plate and D cryo-plate) have been developed. The cryo-plate methods have advantages, such as ease of handling during the procedure and high regrowth rates after cryopreservation. It is important to research genetic stability in long-term tissue-cultured plants and cryopreserved plants for successful conservation of plant germplasm. The present paper reviews current cryopreservation techniques. Thus far, studies have reported no or small differences between cryopreserved and non-cryopreserved samples under optimized conditions according to various morphological, biochemical and molecular analyses (e.g., RAPD-PCR, AFLP, and MSAP).


Castillo NRF, Bassil NV, Wada S and Reed BM (2010) Genetic stability of cryopreserved shoot tips of Rubus germplasm. In Vitro Cell. Dev. Biol. Plant 46: 246-256.

Charoensub R, Hirai D and Sakai A (2004) Cryopreservation of in vitro-grown shoot tips of cassava by encapsulation-vitrification method. CryoLett. 25: 51-58.

Dixit-Sharma S, Ahuja-Ghosh S, Mandel BB and Srivastava PS (2005) Metabolic stability of plants regenerated from cryopreserved shoot tips of Dioscorea deltoidea - An endangered medicinal plant. Sci. Hort. 105: 513-517.

Dhungana SA, Kunitake H, Niino T, Yamamoto S, Fukui K and Matsumoto T (2015) Cryopreservation of blueberry (Vaccinium L.) shoot tips by D cryo-plate method. Hort. Res. (Japan) 14 (suppl. 2): 363.

Dereuddre J, Blandis S and Hassen N (1991a) Resistance of alginate-coated somatic embryos of carrot (Daucus carota L.) to desiccation and freezing in liquid nitrogen: 1. Effects of preculture, CryoLett. 12: 125-134.

Engelmann F (2004) Plant Cryopreservation: Progress and Prospects. In Vitro Cell. & Devel. Biol. Plant 40 (5): 427-433.

Engelmann F, Gonzalez MTY, Wu Y and Escobar R (2008) Development of Encapsulation Dehydration, In: Plant Cryopreservation: A Practical Guide. (Barbara MR, ed). pp.59-75. Springer New York.

Febre J, and Dereuddre J (1990) Encapsulation-dehydration: A new approach to cryopreservation of solanum shoot-tips, CryoLett., 11: 413-426.

Fukai S and Oe M (1990) Morphological observations of chriysanthemum shoot tips cultured after cryopreservation and freezing. J Japan. Soc. Hort. Sci. 59: 383-387.

Harding K and Staines H (2001) Biometric analysis of phenotypic characters of tomato shoot tips recovered from tissue culture. dimethyle sulphoxide treatment and cryopreservation. CryoLett. 22: 255-262.

Hirai D, Shirai K, Shirai S and Sakai A (1998) Cryopreservation of in vitro-grown meristems of strawberry (Fragaria × ananassa Duch.) by encapsulation-vitrification. Euphytica 101: 109-115.

Hirai D and Sakai A (2001) Recovery growth of plants cryopreservation by encapsulation-vitrification, Bul. Hokkaido Pref. Agri. Exp. Sta. 80: 55-64.

Kartha KK, Leung NL and Mroginski LA (1982) In vitro growth responses and plant regeneration from cryopreserved meristems of cassava (Manihot esculenta Crantz). Zeitschrift für Pflanzenphysiologie 107, 133-140.

Kaviani B (2011) Conservation of plant genetic resources by cryopreservation. Australian J. Crop Sci. 5: 778-800.

Keller ERJ and Dreiling M (2002) Potato cryopreservation in Germany-using the droplet method for the establishment of a new large collection. In: Proc. Int. Hort. Con. Toronto, Canada.

Kim HH, Lee YG, Park SU, Lee SC, Baek HJ, Cho EG and Engelmann F (2009) Development of alternative loading solutions in droplet-vitrification procedures. CryoLett. 30: 291-299.

Kumu Y, Harada T and Yakuwa T (1983) Development of a whole plant from a shoot tip of asparagus officinalis L. frozen down to -196 °C. Journal of the Faculty of Agriculture, Hokkaido University 61: 285-394.

Leunufna S and Keller ERJ (2003) Investigating a new cryopreservation protocol for yam (Discorea spp.). Plant Cell Reports, 21: 1159-1166.

Maki S, Hirai Y, Niino T and Matsumoto T (2015) Assessment of molecular genetic stability between long-term cryopreserved and tissue cultured wasabi (Wasabia japonica) plants, CryoLett. 36:318-324.

Martin C and Gonzalez-Benito ME (2005) Survival and genetic stability of Dendranthema grandiflora Tzvelev shoot apices after cryopreservation by vitrification and encapsulation-dehydration, Cryobiol. 51: 281-289.

Matsumoto T, Sakai A and 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, Takahashi C and Yamada K (1995a) Cryopreservation of in vitro-grown meristems of wasabi (Wasabia japonica) by encapsulation-vitrification method. CryoLett. 16: 189-196.

Matsumoto T, Sakai A, and Yamada K (1995b) Cryopreservation of in vitro-grown apical meristems of lily (Lilium japonicum) by vitrification, Plant Cell Tiss. Org. Cul. 41: 231-241.

Matsumoto T, Takahashi C, Sakai A and Nako Y (1998) Cryopreservation of in vitro-grown apical meristems of hybrid statice by three different procedures. Sci. Hort. 76: 105-114.

Matsumoto T, T. Akihiro, Maki S, Mochida K, Kitagawa M, Tanaka D, Yamamoto S and Niino T (2013) Genetic stability assessment of Wasabi plants regenerated from long-term cryopreserved shoot tips using morphological, biochemical and molecular analysis, CryoLett. 34: 128-136.

Matsumoto T. and T. Niino (2014) The development of plant vitrification solution 2 and recent PVS2-based vitrification protocols. In: Reed, B.M. (ed.) Proceedings of the Second International Symposium on Plant Cryopreservation. Acta Hort. 1039: 21-28.

Matsumoto T, Yamamoto S, Fukui K and Niino T (2014a) Cryopreservation of blueberry dormant shoot tips using V cryo-plate method. 2014 Annu. confe. Ameri. soc. horti. sci.: S337.

Matsumoto T, Yoshimatsu K, Kawahara N, Yamamoto S and Niino T (2014b) Development of in vitro propagation by node culture and cryopreservation by V Cryo-plate method for Perilla frutescens, Adv. Hort. Sci., 28: 79-83.

Matsumoto T, Yamamoto S, Fukui K, Rafique T, Engelmann F and Niino T (2015) Cryopreservation of persimmon shoot tips from dormant buds using the D cryo-plate technique, The Hort. J. 84: 106-110.

Niina R, Castillo F, Bassil NV, Wada S and Reed BM (2010) Genetic stability of cryopreserved shoot tips of Rubus germplasm, In Vitro Cell. Dev. Biol. Plant 46: 246-256.

Niino T (2006) Developments in plant genetic resources cryopreservation technologies. In proceedings of the APEC workshop on effective gene bank management in APEC member economies. Suwon, Korea, pp. 197-217.

Niino T, Yamamoto s, Fukui K, Martínez CRC, Arizaga MV, Matsumoto T and Engelmann F (2013) Dehydration improves cryopreservation of mat rush (Juncus decipiens Nakai) basal stem buds on cryo-plate, CryoLett. 34: 549-560.

Niino T, Wunna, Watanabe K, Nohara N, Rafique T, Yamamoto S, Fukui K, Arizaga MV, Martinez CRC, Matsumoto T and Engelmann F (2014) Cryopreservation of mat rush lateral buds by air dehydration using aluminum cryo-plate, Plant Biot. 31: 281-287.

Panis B (1995) Cryopreservation of Banana (Musa spp.) Germplasm. Doctoral Paper (No. 272), Katholieke Universiteit Leuven, Belgium.

Pennycooke JC and Towill LE (2001) Medium alterations improve regrowth of sweet potato (Ipomoea batatas (L.) Lam.) shoot tips cryopreserved by vitrification and encapsulation-dehydration. CryoLett. 22: 381-389.

Peredo EL, Arroyo-Garcia R, Reed BM and Revilla MA (2008) Genetic and epigenetic stability of cryopreserved and cold-stored hops (Humulus lupulus L.) Cryobiol. 57: 234-241.

Phunchindawan M, Hirata K, Miyamoto K and Sakai A (1997) Cryopreservation of encapsulated shoot primordia induced in horseradish (Armoracia rusticana) hairy root cultures. Plant Cell Rep. 16: 469-473.

Reed BM (2008) Cryopreservation-Practical considerations, In: Plant Cryopreservation: A Practical Guide (Reed BM, ed), Springer, New York, pp.3-13.

Rey NA, Papacchioli V, Tavazza R and Pagnotta MA (2013) Gauging the genetic changes occurring across globe artichoke micropropagation towards an appropriate variety registration and nursery production. Sci. Hort. 156: 121-126.

Rafique T, Yamamoto S, Fukui K, Mahmood Z and Niino T (2015) Cryopreservation of sugarcane using the V cryo-plate technique. CryoLett. 36: 51-59.

Sakai A and Yoshida S (1967) Survival of plant tissue at super-low temperature VI. Effects of cooling and rewarming rates on survival. Plant Physiol. 42: 1695-1701.

Sakai A, Kobayashi S and 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 (1995) Cryopreservation of germplasm of woody plants, in Biotechnology in Agriculture and Forestry 32, Cryopreservation of Plant Germplasm I, (Bajaj YPS, ed), Springer-Verlag, Berlin, Heidelberg, pp. 53-69.

Sakai A (1997) Conservation of Plant Genetic Resources In Vitro, (eds) Razdan MK and Cocking EC, Science Publishers, New Hampshire, pp.53-66.

Sakai A and Engelmann F (2007) Vitrification, encapsulation-vitrification and droplet-vitrification: A review. CryoLett. 28: 151-172.

Sakai A (2008) Development of PVS-based vitrification and encapsulation-vitrification protocols, In: Plant cryopreservation A practical guide (Reed MB, ed), Springer, New York, pp.33-57.

Salma M, Fki L, Engelmann-Sylvestre I, Niino T and Engelmann F (2014) Comparison of droplet-vitrification and D-cryoplate for cryopreservation of date palm (Phoenix dactylifera L.) polyembryonic masses, Sci. Hort. 179: 91-97.

Schafer-Menuher A, Schumacher HM and Mix-Wagner (1997) Cryopreservation of potato cultivars-design of a method for routine application in genebanks. In: Proc. III int. Sym. In vitro cul. hort. Breed.

Sekizawa K, Yamamoto S, Rafique T, Fukui K and Niino T (2011) Cryopreservation of in vitro-grown shoot tips of carnation (Dianthus caryophyllus L.) by vitrification method using aluminium cryo-plates, Plant Biot. 28: 401-405.

Skyba M, Urbanova M, Toteva VK, Kosuth J, Harding K and Ellarova E (2010) Physiological, biochemical and molecular characteristics of cryopreserved Hypericum Perforatum L. shoot tips. CryoLett. 31: 249-260.

Urbanova M, Kosuth J and Cellarova E (2006) Genetic and biochemical analysis of Hypericum perforatum L. plants regenerated after cryopreservation. Plant Cell Rep. 25: 140-147.

Vasanth K and Vivier MA (2011) Improved cryopreservation procedure for long term storage of synchronised culture of grapevine, Biologia Plantarum 55: 365-369.

Withers LA (1980) The cryopreservation of higher plant tissue and cell cultures - an overview with some current observations and future thoughts. CryoLett. 1: 239-250.

Yamamoto S, Rafique T, Priyantha WS, Fukui K, Matsumoto T and Niino T (2011) Development of a cryopreservation procedure using aluminium cryo-plates, CryoLett. 32: 256-265.

Yamamoto S, Rafique T, Fukui K, Sekizawa K and Niino T (2012a) V-Cryo-plate procedure as an effective protocol for cryobanks: Case study of mint cryopreservation, CryoLett. 33: 12-23.

Yamamoto S, Fukui K, Rafique T, Khan NI, Martinez CRC, Sekizawa K, Matsumoto T and Niino T (2012b) Cryopreservation of in vitro-grown shoot tips of strawberry by the vitrification method using aluminium cryo-plates, Plant Gen. Res.: Charac. and Uti. 10: 14-19.

Yamamoto S, Rafique T, Sekizawa K, Koyama A, Ichihshi T and Niino T (2012c) Development of an effective cryopreservation protocol using alminium cryo-plates for in vitro-grown shoot tips of mulberries (Morus spp.) originated from the tropics and subtropics. Sanshi-Konchu Biotec, 81: 57-62. (in Japanese)

Yamamoto S, Rafique, Arizaga MV, Fukui K, Gutierrez EJC, Martinez CRC, Watanabe K and Niino T (2015) The aluminum cryo-plate increases efficiency of cryopreservation protocols for potato shoot tips, Amer. J. Potato Res. 92: 250-257.

Yoshimatsu K, Yamaguchi H & Shimomura K (1996) Traits of Panax ginseng hairy roots after cold storage and cryopreservation. Plant Cell Reports 15: 555-560.