作 者 ：洪森荣;尹明华*;王艾平
期 刊 ：植物研究 2014年 34卷 03期 页码：333-338
Keywords:Jiangxi Yanshan, red bud taro(Colocasia esculenta L. Schott var. cormosus CV. Hongyayu), embryogenic callus, droplet-vitrification, cryopreservation,
摘 要 ：探索江西铅山红芽芋胚性愈伤组织的小滴玻璃化法超低温保存，为其种质资源的超低温保存提供技术基础和理论依据。植物组织培养和单因子试验的方法。江西铅山红芽芋胚性愈伤组织小滴玻璃化法超低温保存的较佳程序为：约0.2 g胚性愈伤组织在25℃下转入MS+2 mg·L-1 TDZ+1 mg·L-1 NAA+0.5 mmol·L-1蔗糖的培养基中，于14 h·d-1光周期下预培养3 d后用MS+2 mmol·L-1甘油+0.4 mmol·L-1蔗糖在25℃下装载20 min，然后用PVS2在0℃下脱水40 min，吸取5滴PVS2到铝箔条上，将脱水后的红芽芋胚性愈伤组织块转到铝箔条上的PVS2液滴里。附有胚性愈伤组织块的铝箔条在液氮里蘸一下，然后迅速将其转入2 mL装满液氮的冷冻管中，再投入液氮保存1 d。从液氮中取出冷冻管中的铝箔条，浸入用40℃温水预热过的洗涤液（MS+2 mg·L-1 TDZ+1 mg·L-1 NAA+1.2 mmol·L-1蔗糖）中，使胚性愈伤组织块从铝箔条上脱落下来，然后再将胚性愈伤组织块转入新鲜的洗涤液中洗涤3次，每次10 min。洗涤后转入MS+2 mg·L-1 TDZ+1 mg·L-1 NAA固体培养基上，先暗培养7 d再转到14 h·d-1光周期中培养。30 d后将分化出的胚状体再次转入MS+2 mg·L-1 TDZ+1 mg·L-1 NAA固体培养基上可再生完整植株。红芽芋胚性愈伤组织包埋玻璃化超低温保存后的平均成活率约为80%。红芽芋胚性愈伤组织冻后再生苗没有发生形态学、生理学和细胞学的变异。红芽芋胚性愈伤组织小滴玻璃化法超低温保存可以保证其遗传资源的稳定性，为江西铅山红芽芋种质资源的离体保存提供了一条新的途径。
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.