江西山药茎尖包埋玻璃化法超低温保存及其遗传稳定性检测

采用植物组织培养法对广丰药薯茎尖的包埋玻璃化法超低温保存程序进行优化，并采用扩增片段长度多态性（AFLP）分子标记法和流式细胞术对其冻后再生苗的遗传稳定性进行检测，同时将超低温程序应用到江西山药其他地方品种，旨在为薯蓣属植物种质资源的长期保存奠定理论基础。结果表明，广丰药薯茎尖预培养的较佳时间为5 d，较佳的蔗糖浓度为0.75 mol·L-1；装载的较佳时间为40 min；脱水的较佳温度为0 ℃，较佳时间为60 min；冻后黑暗培养7 d可以显著提高其成活率。用AFLP分子标记法和流式细胞术对茎尖冻后再生植株的遗传稳定性进行检测，没有发现异常条带和染色体倍性变化，气孔观察也未发现叶下表皮气孔参数的显著变异。将这种超低温保存程序用于江西山药其他基因型，成活率约为40%～85%。该研究建立的包埋玻璃化法超低温保存程序能保证江西山药的遗传稳定性，可为建立江西山药种质资源超低温保存库提供一定的技术支撑。

Cryopreservation of Jiangxi yam shoot tip by encapsulation vitrification and its detection of genetic stability

In this paper， cryopreservation procedure of Guangfeng medicinal yam shoot tip by encapsulation vitrification would be optimized by the method of plant tissue culture， and the genetic stability of regenerated plantlets after cryopreservation would be tested with AFLP molecular markers and flow cytometry methods. At the same time， this cryopreservation procedure by encapsulation vitrification would be applied to other local varieties of Jiangxi yam， in order to lay the theoretical and technical foundation for long term conservation of genus Dioscorea germplasm. The results showed that: The better pre culture time of Guangfeng medicinal yam shoot tips was 5 d， the better sucrose concentration in pre culture medium was 075 mol·L-1; The better loading time was 40 min; The better dehydration temperature was 0 ℃， the better dehydration time was 60 min; 7 day dark culture could significantly improve the survival rate of shoot tips after cryopreservation. At last， the genetic stability of regenerated plants was detected by AFLP molecular marker and flow cytometry （FCM）， and no abnormal bands and chromosome ploidy changes were found. At the same time， no significant variation of stomatal parameters of leaves was observed. The cryopreservation technique was used in other genotypes of Jiangxi yam， the survival rate was about 40%-85%. Therefore， cryopreservation procedure by encapsulation vitrification could guarantee the genetic stability of Jiangxi yam germplasm resources. The results of this study provided a certain technical support for the establishment of cryopreservation bank of Jiangxi yam germplasm resources.