玻璃化法超低温保存怀山药种质的技术研究

Cryopreservation technique of Dioscorea opposita germplasm by vitrification

目的 研究怀山药种质资源的玻璃化超低温保存技术。方法 以B号怀山药带芽茎段为材料进行玻璃化超低温保存，并采用培养法检测保存后材料的成活率，从而筛选出最佳的玻璃化超低温保存技术体系。结果 B号怀山药带芽茎段玻璃化超低温保存较佳的技术体系是继代生长60d的怀山药无菌苗置4C冰箱低温锻炼7d；在无菌条件下切取1～1．5cm的带芽茎段，转至含5％蔗糖＋3％甘露糖的培养基内，置4（？冰箱预培养2d；用60％的PVS1（22％甘油＋13％乙二醇＋13％聚乙二醇＋10％二甲基亚砜）在0℃处理60rain，再用100％的PVS。在0C条件下处理60min，随即迅速投入液氮；保存24h后，在37℃水浴中快速化冻，用含7％蔗糖的MS培养液洗涤4次，每次停留10min；转至再生培养基（MS＋KT2mg／L＋NAA0．02mg／L）再培养，成活率可达75％以上，再生苗与常温苗形态指标差异不大。结论 本试验成功地建立了怀山药种质资源玻璃化法超低温保存的技术体系，为怀山药种质资源的长期保存提供了一条有效途径。

Objective To study the cryopreservation technique of Dioscorea opposita germplasm resources by vitrification. Methods Stems with buds of B^# D. opposita germplasm were used as materials for cryopreservation by vitrification and the survival rates were examined by culture method in order to optimize a cryopresevation by vitrification technical system for D. opposita germplasm. Results A best procedure of cryopresevation by vitrification was as below： At first, the aseptic plantlets of stems with buds of B^# D. opposita subcuhured in vitro for 60 d were treated at 4 ℃ for 7 d. The stems with buds of D opposita about 1--1.5 em in length were cut and preeultured at 4℃ for 2 d in 5 M sucrose and 3M mannose media, they were dehydrated with 60% vitrification solution （PVS1 ： 22% glycerol＋ 13% glycol ＋ 13 % polyethyleneglycol＋10% dimethyl sulfoxide） at 0 ℃ for 60 min and then dehydrated with 100M vitrification solution at 0 ℃ for 60 min. At last, the stems were immersed immediately into liquid nitrogen directly and conserved for 24 h. After rapidly thawing in a water bath at 37 ℃, the stems were washed four times with MS medium supplemented with 7 % sucrose and 10 min each time, then transferred and re-cultured on the MS medium supplemented with KT 2 mg/L and NAA 0.02 mg/L. The survival rate was up to 75%. The regenerated plantlets of D. opposita showed no more difference than the normal plantlets in morphology. Conclusion This test sets up successfully a eryopresevation by vitrification system technique for D. opposita germplasm, which provides an effective way for long-lasting conservation in vitro of D. opposita germplasm resources.