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

Abstract

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 2.9 mM calcium over a period of 14 d after subculture. The shoot tip (1–1.5 × 1 mm) was dissected from the shoot and precultured at 25 °C for 1 d on MS medium containing 0.3 M sucrose. The precultured shoot tips were placed on aluminium cryo-plates (7 mm × 37 mm × 0.5 mm) with ten wells (diameter 1.5 mm, depth 0.75 mm) and embedded with alginate gel. Osmoprotection was performed by immersing the cryo-plates for 30 min at 25 °C in 25 ml pipetting reservoirs filled with loading solution (2 M glycerol + 0.8 M sucrose). For dehydration, the cryo-plates were transferred and immersed in another 25 ml pipetting reservoirs filled with PVS2 for 30 min at 25 °C. Then, the cryo-plate was transferred in an uncapped 2 ml cryotube and directly plunged into liquid nitrogen. After storage in LN 2 , shoot tips attached to the cryo-plate were directly rewarmed by immersion into a cryotube containing 2 ml 1 M 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.