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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。
The tissue culture method was used to optimize the cryopreservation procedure of Guangfeng medicinal tuberosclerosis stem cells by embedding vitrification method. The AFLP molecular markers and flow cytometry Genetic stability of the test, at the same time the application of ultra-low temperature program to other parts of Jiangxi Yam, aims to lay the theoretical foundation for the long-term preservation of the genus Dioscorea. The results showed that the best pre-culture time of shoot tip of Guangfeng was 5 days, the best sucrose concentration was 0.75 mol·L-1, the best loading time was 40 min, the best temperature of dehydration was 0 ℃, The best time was 60 min; the darkness after freezing for 7 days could significantly improve the survival rate. The AFLP molecular markers and flow cytometry were used to detect the genetic stability of the shoots after freezing, and no abnormal bands and ploidy changes were observed. Stomatal observation showed no significant variation of leaf stomatal parameters. This cryopreservation program for other genotypes of yam in Jiangxi, the survival rate of about 40% to 85%. The cryopreservation program of embedding vitrification established in this study can ensure the genetic stability of Jiangxi yam and provide some technical support for the establishment of cryopreservation reservoir of Jiangxi yam germplasm resources.