【作者】 吴姗；

【导师】 梁月荣；

【作者基本信息】 浙江大学 ， 茶学， 2004， 博士

【摘要】 茶树（Camellia sinensis（L．）O．Kuntze）作为我国一种重要的经济作物，其优良品种的培育和推广，是科技在茶叶生产中发挥效益的重要体现。仅仅依靠传统的育种方法已不能满足现代社会需求的迅猛发展。因而必须吸收和借鉴现代科学技术成果，利用这些新的方法和手段，培育出更优秀的茶树品种。植物转基因技术作为植物基因工程的一个重要组成部分，不仅为基因表达调控和遗传研究提供了一个理想的实验体系，更为植物（尤其是农作物）的定向改良和分子育种提供了一个有效的途径和方法。 本研究在前人的基础上，优化了茶树的遗传转化系统。主要内容如下： 进一步调整了茶树常用的遗传转化体系—农杆菌体系。在用农杆菌浸染前，将茶树外植体预培养在含有PVP（16g／L）的预培养基上2-3d可提高转化频率。但在预培养基中添加CaCl₂反而使转化频率降低。 对基因枪介导的茶树转化体系的有关参数进行优化。比较了不同的预培养条件，结果表明：AgNO₃对转化频率影响不大；此外，茶树外植体若在含有AgNO₃的培养基上培养时间超过30d，会发生褐化。当AgNO₃和CaCl₂与PVP一起用于预培养时，得到的转化率最高。即使在没有AgNO₃和CaCl₂的条件下，在愈伤诱导培养基（1／2MS+2，4-D+KT）中添加PVP作为预培养培养基，也能得到较好的转化效果。渗透处理不仅不能提高GUS的表达率，反而使愈伤再生困难。优化后的基因枪转化制弹程序为：60mg／ml钨粉悬浮液10μl中加入1.6μl质粒DNA（1μg／μl），再分别加入0.1M的亚精胺4μl，2.5M的CaCl₂ 15μl，最后定容至48μl；每次轰击上样量为8-10μl。在轰击压力7MPa，射程5cm，真空度-0.095MPa的条件下轰击一次，不论对GUS表达还是愈伤的再生都是较为适合的轰击条件。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％。 比较了农杆菌法（AGR），基因枪法（BOM）及农杆菌与基因枪结合（BTA，BPA和BOA）等方法对茶树遗传转化效率的影响。瞬间表达的结果及抗性愈伤筛选的结果都显示，农杆菌与基因枪结合使用比两种方法单独使用更有助于提高茶树转化的效率。 本实验中三个茶树品种一乌牛早、劲峰和紫笋得到的转化频率相似。根据本实验的结果，品种对转化的影响，不存在于品种间细胞感受能力的差异;然而，可能不同品种间再生能力的强弱是实验成败的另一个关键。更多还原

【Abstract】 Tea plant (Camellia sinensis (L.) O. Kuntze) is one of the most important economic plants in China. To breed and popularize the improved tea cultivars is a very important aspect of science and technology to show their power on tea production. Nowadays, the traditional breeding methods can not meet the great demand of the modern tea production any more. It becomes urgent to take advantage of the findings and productivity of the modern science and technology to breed tea cultivars with high quality. Plant transformation technology, as an important part of plant engineering, not only offers a perfect platform for research on gene regulation and expression but also provide an effective way for plant directional upgrade and plant breeding on a molecular level.In this research, tea plant genetic transformation systems, including Agrobacterium-mediated system and particle bombardment-mediated system, have been optimized.In Agrobacterium-mediated system, if tea explants were pre-cultured on medium containing PVP 16g/L for 2-3d before infiltrated by Agrobacterium tumefaciens, transformation frequency was increased. However, pre-cultured on the medium containing CaCl2 can only make the transformation frequency lower.Furthermore, a micro-projection protocol using particle delivery system and GUS report gene was developed for tea plant callus. Result of the comparison among several pre-bombardment culture media showed that AgNO3 had no significant effect on the tea plant transformation. In addition, if tea calli were cultured on the AgNO3-containing medium more than 30 d, they would become browned. When AgNO3 and CaCl2 were used in integration with PVP, the media always yielded the highest GUS gene activity. Even pre-cultured on the callus inducing medium (1/2MS plus 2, 4-D and KT) without AgNO3 and CaCl2, the bombarded targets could also show relatively higher transient GUS expression rates, only if PVP was added. Instead of making contribution to improving GUS expression, osmotic pre-treatment wouldbombardment micro-particle were that: 10# 1 tungsten suspension (60mg/ml), 1.6 # 1 plasmid DNA (1 # g/# 1), 4# 1 spermidin (0.1M) and 15 # 1 CaCl2 (12.5M). Ethanol was used to dilute the final volume up to 48 # 1. The loading volume for each bombardment was 8-10# 1. Bombarding tea callus one time, at 7MPa with a target distance of 5cm and vacuum at -0.095MPa was suitable for both GUS activity and regeneration. The survival rate of resistant calli was 5.0%-12.1%, when the bombarded explants were cultured on selection media with kanamycin or hygromycin.Besides Agrobacterium-mediated transformation ( AGR ) system and particle bombardment (BOM) system, several combinations of the two systems (BTA, BPA and BOA), have also been tested and compared with each other. Based on the results of GUS expression and survival rates of the resistant calli, the method combining Agrobacterium and particle bombardment systems is suitable to apply for tea plant transformationIn terms of the transformation frequency, there were no significant differences among Wuniuzao, Jinfeng and Zisun, three tea cultivars employed in the experiment. According to this, the differences of cell competent ability among different cultivars have no great influence on tea transformation. However, the regeneration capability of the cultivar would play a more critical role in tea plant transformation.更多还原