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茶叶科学 ›› 2003, Vol. 23 ›› Issue (1): 6-10.doi: 10.13305/j.cnki.jts.2003.01.002

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茶树农杆菌转化系统和基因枪转化系统的优化

吴姗, 梁月荣, 陆建良, 金惠淑, 吴颖   

  1. 浙江大学茶叶研究所,浙江 杭州 310029
  • 收稿日期:2002-10-12 修回日期:2003-01-06 出版日期:2003-05-25 发布日期:2019-09-16
  • 作者简介:吴姗(1976-)女,浙江杭州人,浙江大学茶叶研究所博士生,主要从事茶树生物技术与资源利用研究。
  • 基金资助:
    国家自然科学基金(30170591)和国家博士点基金(200033539)资助项目

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

WU Shan, LIANG Yue-rong, LU Jian-liang, KIM Hyesuk, WU Ying   

  1. Tea Research Institute, Zhejiang University;Hangzhou 310029, China
  • Received:2002-10-12 Revised:2003-01-06 Online:2003-05-25 Published:2019-09-16

摘要: 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

Abstract: 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.

Key words: Tea science, Tea plant, Transformation, Agrobacterium tumefaciens, Particle bombardment

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