Cryopreservation of Jiangxi Yanshan Red Bud Taro Embryogenic Calli by Encapsulation-vitrification

To ensure the long-term conservation of Jiangxi Yanshan red bud taro (Colocasia esculenta L. Schott var. cormosus ‘Hongyayu’) germplasm resource,the effect of the cryopreservation process by encapsulation-vitrification on cell viability and calli survival was studied using embryogenic calli,and the cryopreservation system was optimized. About 0.2 g of embryogenic calli were encapsulated into alginate-gel beads and then precultured in liquid MS medium supplemented with 2 mg/L TDZ,1 mg/L NAA and 0.75 mol/L sucrose and maintained under a 14 h photoperiod at 25℃ for 1 d. Precultured encapsulated embryogenic calli were loaded with a mixture of 2 mol/L glycerol plus 0.4 mol/L sucrose for 40 min at 25℃ and then dehydrated with PVS2 at 25℃ for 30 min. The encapsulated and dehydrated embryogenic calli were plunged directly into liquid nitrogen (LN) for 1 d. After rapidly thawing in a 37℃ water bath for 3 min,the embryogenic calli were washed three times with liquid MS medium supplemented with 2 mg/L TDZ,1 mg/L NAA and 1.2 mol/L sucrose for 10 min at 25℃,and were post-cultured on solidified MS medium supplemented with 2 mg/L TDZ and 1 mg/L NAA in the dark for 7 days and then transferred to a 14 h photoperiod. Successfully vitrified and warmed embryogenic calli resumed growth within a week and differentiated embryoids within 30 days. Embryoids were transferred onto fresh solidified MS medium supplemented with 2 mg/L TDZ and 1 mg/L NAA under a 14 h photoperiod to develop into whole plantlets within 2 months of culture. The average survival rate of the embryogenic calli after freezing was about 60%. Plantlets regenerated from cryopreserved embryogenic calli had no morphological and chromosomal variation,laying a good foundation for the long-term safe storage of Jiangxi Yanshan red bud taro germplasm resources.