在用农杆菌浸染前,将茶树外植体预培养在含有PVP（polyvinylpyrrolidone,16g/L）的预培养基上2-3d可提高转化频率。优化后的茶树基因枪转化体系,即制弹程序：60βmg/1ml钨粉悬浮液10βµl中加入1.6βµl质粒DNA（1βµg/µl）,再分别加入0.1βmol/L的亚精胺4βµl,2.5βmol/L 的CaCl2 15βµl,最后定容至48βµl;每次轰击上样量为8-10βµl。基因枪转化后抗性筛选2个月,抗性愈伤组织存活率为5.0%~12.1%。
关键词：
茶学; 茶树; 转化; 农杆菌; 基因枪
When tea explants were pre-cultured on medium containing PVP 16g/L for 2-3d before infiltrated by Agrobacterium tumefaciens the transformation frequency was increased. Optimum conditions for preparing bombardment micro-particle were as follows: 10βµl tungsten suspension (60βmg/1ml), 1.6βµl plasmid DNA (1βµg/βµl), 4βµl spermidin (0.1βmol/L) and 15βµl CaCl2 (l2.5βmol/L). Ethanol was used to dilute the final volume up to 48βµl. The loading volume for each bombardment was 8-10βµl. The survival rate of resistant calli was 5.0%-12.1%, when the bombarded explants were cultured on selection media with kanamycin or hygromycin.
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