Cryopreservation of Jiangxi Yanshan Red Bud Taro Embryogenic Calli by Encapsulation-vitrification
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摘要: 为长期安全保存江西铅山红芽芋种质资源，本文以江西铅山红芽芋的胚性愈伤组织为对象，研究了包埋玻璃化冻存过程中各因素对细胞活力和愈伤组织成活率的影响，优化建立了江西铅山红芽芋胚性愈伤组织包埋玻璃化超低温保存体系。将约0.2 g胚性愈伤组织块包埋成海藻酸钙凝胶珠后，在25℃下转入MS + 2 mg/L TDZ +1 mg/L NAA + 0.75 mol/L 蔗糖的培养基中于14 h/d光周期下预培养1 d；预培养后的胚性愈伤组织块用 2 mol/L甘油和0.4 mol/L蔗糖的混合物在25℃下装载40 min；采用PVS2在25℃下脱水30 min，更换PVS2后直接投入液氮保存1 d；再将胚性愈伤组织块置于37℃恒温水浴中化冻3 min，然后用MS + 2 mg/L TDZ + 1 mg/L NAA + 1.2 mol/L蔗糖的液体培养基洗涤3次，每次10 min；洗涤后的胚性愈伤组块转入MS+2 mg/L TDZ+1 mg/L NAA固体培养基上先暗培养7 d再转到14 h/d光周期中培养。7 d后胚性愈伤组织块开始恢复生长，并且在30 d 内分化出胚状体；将胚状体再次转入MS + 2 mg/L TDZ + 1 mg/L NAA固体培养基上，60 d后形成完整的植株。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60%，并且红芽芋胚性愈伤组织冻后再生苗没有发生形态性状和染色体数目的变异，此结果为长期安全保存江西铅山红芽芋种质资源奠定了良好的基础。Abstract: 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.
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