忍冬原生质体分离条件研究

Researches on Isolation Conditions of Protoplasts of Lonicera japonica

目的:探讨忍冬原生质体分离的最佳条件,为忍冬原生质体培养、细胞融合以及遗传转化提供大量优质的原生质体。方法:分别对忍冬原生质体分离过程中的若干影响因子如:酶液组成、酶解方式、酶解时间、酶解温度、渗透压进行比较分析,以确定最佳的分离条件。结果:确定了最优化的忍冬原生质体分离条件,即以生长旺盛的疏松的忍冬愈伤组织作为分离原生质体所需材料,以1.5%纤维素酶+0.25%果胶酶作为分离原生质体所用酶液,以含有0.7 mol.L-1甘露醇作为渗透保护剂的细胞-原生质体清洗液(CPW)溶液进行酶液的配制,于25℃静置条件下酶解12 h。结论:利用该实验所确定的忍冬原生质体最佳分离条件可获得大量优质的原生质体,为后续忍冬细胞融合以及遗传转化等实验奠定良好的实验基础。

Objective：To find out the best isolation conditions of protoplasts of Lonicera japonica.Method：Some influence factors on isolation of protoplasts,such as composition of enzyme solution,methods,hours and temperature of enzyme dissolving,osmotic pressure,and so on,were compared and analyzed to find out the best conditions of protoplasts isolation of L.japonica.Result：The callus flourishing well of L.japonica was used as materials for protoplasts isolation.0.7 mol · L-1 mannitol＋1.5% cellulose Onozuka R-10＋0.25% pectolase Yakult Y-23 was the best composition of enzyme solution,which was dissolved in cell protoplast wash medium（CPW） solution.12-hour enzyme dissolving at 25 ℃ was the best methods of protoplasts isolation.And standing was better than shaking.Conclusion：Abundant and superior protoplasts of L.japonica can be obtained under the best isolation conditions that this study has found out which will give the necessary help for protoplasts culture,cell fusion and genetic transformation of L.japonica.