Cryopreservation technique of Dioscorea opposita Thunb. germplasm resources by vitrification

中国人. 本文对怀山药种质资源的玻璃化法超低温保存技术进行了研究。结果表明，怀山药带芽茎段玻璃化法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4℃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）在室温下处理60min，再用100％的PVS1在0℃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5％蔗糖的MS培养液洗涤4次，每次停留10min；转至再生培养基（MS＋KT 2mg／L＋NAA 0．02mg／L）再培养，成活率可达77.14％，再生苗与常温苗形态指标差异不大。

英语. Because of its unique medical and edible value, plantation of Dioscorea opposita Thunb. is being taken into account. In the traditional production method, it is universal to use nutrition organs to propagate, which is apt to cause its qualities to degradate and its output to drop. And with development of modern agriculture, a few high-yield foreign species replace gradually the low-yield, having important value and meeting local circumstance Dioscorea opposita Thunb. species. Thus the genuine Dioscorea opposita Thunb. are abandoned and close to extinction, which cause genuine Dioscorea opposita Thunb. germplasm resources to lapse. At present, Dioscorea opposita Thunb. germplasm resources were mainly preserved in the field. Because it not only needed a large number of manpower, material resources and financial resources, but also was affected unavoidably by various kinds of natural calamity（ such as aridity, flood, hail, plant diseases and pests）and human error（ such as negligent management, hanging label by mistake, etc. ）, Dioscorea opposita Thunb. germplasm resources were lost or mixed finally. Cryopreservation technology by vitrification based on cryobiology offered the technological assurance for preservation in vitro of Dioscorea opposita Thunb. germplasm resources, which could keep a large number of germplasm resources in the relatively narrow and small space, avoid invasion and attack of plant diseases and pests, keep genuine gene of Dioscorea opposita Thunb., and be favorable to international exchange of Dioscorea opposita Thunb. germplasm resources. The essay studied on the cryopreservation technique of Dioscorea opposita Thunb. germplasm by vitrification. Results were as follows： A best procedure of cryopresevation by vitrification was reported using the stem with a bud as materials. At first,the plantlets of Dioscorea opposita Thunb. cultured in vitro for 60 days were treated at 4℃ for 7 days. Stems of Dioscorea opposita Thunb. about 1-1.5 cm in length were cut and precultured for 2 days at 4℃ in 5% sucrose and 3% Mannose, they were dehydrated with 60% virtrification solution（PVS1 ： 22% glycerol ＋ 13% glycol ＋ 13% polyethyleneglycol ＋ 10% dimethyl sulfoxide）for 60 minutes at room temperature and then dehydrated with 100% virtrification solution for 60 min at 0℃ . At last, 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 4 times with MS medium supplemented with 5 % sucrose, then transferred and re-cuhured on the MS medium supplemented with KT 2 mg/L and NAA 0.02 mg/L. The survival rate was about 77.14%. The regenerated plantlets of Dioscorea opposita Thunb. were as same as the normal plantlets in morphology. This research sets up successfully a cryopresevation system by vitrification technology for Dioscorea opposita Thunb. germplasm, offering theoretical foundation for cultivation universally and genuineness-keeping of good-quality Dioscorea opposita Thunb. species（such as Dioscorea opposita Thunb. Tiegun）in production practices and international exchange of Dioscorea opposita Thunb. germplasm resources. This experimental results also provide an effective way for conservation in vitro of Dioscorea opposita Thunb. germplasm resources, which is very significant for conservation, exploitation, production and effective utilization of Dioscorea opposita Thunb.