关键词: 怀山药, 种质资源, 玻璃化法, 超低温保存, 存活率

Abstract:

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 modern agriculture’s development, 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 affcted 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 hours. 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-cultured 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.

Key words: Dioscorea opposita Thunb., Germplasm resources, Vitrification, Vryopreservation, Survival rate