关键词: 江西铅山, 红芽芋, 胚性愈伤组织, 小滴玻璃化法, 超低温保存

Abstract: To explore the cryopreservation of Jiangxi Yanshan red bud taro (Colocasia esculenta L. Schott var. cormosus CV. Hongyayu) embryogenic calli by droplet vitrification, which will provide the technical and theoretical basis for the cryopreservation of its germplasm resources. Plant tissue culture and single factor test were applied. A cryopreservation system of Jiangxi Yanshan red bud taro embryogenic calli by droplet-vitrification was established: About 0.2 g embryogenic calli was pre-cultured in liquid MS medium supplemented with 2 mg·L^-1 TDZ, 1 mg·L^-1 NAA and 0.5 mmol·L^-1 sucrose and maintained under a 14 h photoperiod at 25℃ for 3 d. Pre-cultured embryogenic calli was loaded with MS+2 mmol·L^-1 Gly+0.4 mmol·L^-1 sucrose for 20 min at 25℃ and then dehydrated with PVS2 at 0℃ for 40 min. Five drops of PVS2 were absorbed and dropped to aluminum foil strip, the dehydrated red bud taro embryogenic callus pieces were transferred to the PVS2 droplet on the aluminum foil strip. The aluminum foil strips were dipped in the liquid nitrogen using tweezers, then quickly transferred to 2 mL freezing tubes filled with liquid nitrogen. After keeping for 1 d, the aluminum foil strips were removed from freezing tubes using tweezers and immersed into the preheated 40℃ washing solution (MS+TDZ 2 mg·L^-1+NAA 1 mg·L^-1+1.2 mmol·L^-1 sucrose), the embryogenic callus blocks dropped from the aluminum foil strips, and then washed three times with liquid MS medium supplemented with 2 mg·L^-1 TDZ, 1 mg·L^-1 NAA and 1.2 mmol·L^-1 sucrose and each kept for 10 min and then post-cultured on solidified MS medium supplemented with 2 mg·L^-1 TDZ, 1 mg·L^-1 NAA in the dark for 7 d and then transferred to a 14 h photoperiod. After 30 d, the differentiated embryoids were again transferred onto fresh solidified MS medium supplemented with 2 mg·L^-1 TDZ, 1 mg·L^-1 NAA under 14 h photoperiod to developed into the whole plantlets. The average survival rate of embryogenic calli after cryopreservation by droplet-vitrification amounted to about 80%. No significant difference was observed in the morphological, physiological and cytological index of plantlets coming from control and cryopreserved embryogenic calli. Red bud taro embryogenic calli cryopreservation by droplet-vitrification could guarantee the stability of its genetic resources, which provided a new way for in vitro conservation of Jiangxi Yanshan red bud taro germplasm resources.

Key words: Jiangxi Yanshan, red bud taro(Colocasia esculenta L. Schott var. cormosus CV. Hongyayu), embryogenic callus, droplet-vitrification, cryopreservation