74. Cryopreservation of in vitro shoot tips of potato varieties by V- Cryo-plate method

• Published in: Cryobiology 2012-12, Vol.65 (3), p.363-363
• Main Authors: Yamamoto, Shin-ichi, Fukui, Kuniaki, Hirai, Dai, Niino, Takao
• Format: Article
• Language: English
• ISSN: 0011-2240; 1090-2392
• DOI: 10.1016/j.cryobiol.2012.07.075

Cryopreservation procedure using aluminium cryo-plates (V-Cryo-plate method) was successfully applied to in vitro-grown potato (Solanum tuberosum L.) shoot tips. The shoots were cultured at 25°C on MS medium containing 3 % (w/v) sucrose and 2.9mM calcium over a period of 14d after subculture. The shoot tip (1–1.5×1mm) was dissected from the shoot and precultured at 25°C for 1d on MS medium containing 0.3M sucrose. The precultured shoot tips were placed on aluminium cryo-plates (7mm×37mm×0.5mm) with ten wells (diameter 1.5mm, depth 0.75mm) and embedded with alginate gel. Osmoprotection was performed by immersing the cryo-plates for 30min at 25°C in 25ml pipetting reservoirs filled with loading solution (2M glycerol+0.8M sucrose). For dehydration, the cryo-plates were transferred and immersed in another 25ml pipetting reservoirs filled with PVS2 for 30min at 25°C. Then, the cryo-plate was transferred in an uncapped 2ml cryotube and directly plunged into liquid nitrogen. After storage in LN2, shoot tips attached to the cryo-plate were directly rewarmed by immersion into a cryotube containing 2ml 1M sucrose solution. Using this procedure, the regrowth rate of cryopreserved shoot tips of cultivar ‘Sayaka’ reached more than 95%. This protocol was successfully applied to 12 different cultivars, which had quite high regrowth levels ranging from 93% to 100% and the average regrowth level of those cultivars reached 99%. This new method has many advantages and will facilitate the cryostorage of varieties of potato germplasms.