茶树农杆菌转化系统和基因枪转化系统的优化

Optimization of Agrobacterium-mediated and Particle Bombardment-mediated Transformation Systems in Tea Plant (Camellia sinensis)

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:60 mg/1ml钨粉悬浮液10 μl中加入1.6 μl质粒DNA(1 μg/μl),再分别加入0.1 mol/L的亚精胺4 μl,2.5 mol/L 的CaCl2 15 μl,最后定容至48 μl;每次轰击上样量为8-10 μl.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%.

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: 10 l tungsten suspension (60 mg/1ml), 1.6 l plasmid DNA (1 g/ l), 4 l spermidin (0.1 mol/L) and 15 l CaCl2 (l2.5 mol/L). Ethanol was used to dilute the final volume up to 48 l. The loading volume for each bombardment was 8-10 l. The survival rate of resistant calli was 5.0%-12.1%, when the bombarded explants were cultured on selection media with kanamycin or hygromycin.