农杆菌介导的地被菊遗传转化体系的优化_赵伶俐.pdfVIP

分子植物育种，2011 年，第9 卷，第1 期，第74-80 页 Molecular Plant Breeding, 2011, Vol.9, No.1, 74-80 研究报告 A Letter 农杆菌介导的地被菊遗传转化体系的优化 1 1 1, 2* 1 赵伶俐 石少川 张启翔 高亦珂 1 北京林业大学园林学院, 北京, 100083; 2 国家花卉工程研究中心, 北京, 100083 * 通讯作者, zqx@bjfu.edu.cn 摘 要 本研究以地被菊‘北林黄’无菌苗叶片外植体为受体材料，以卡那霉素抗性基因为选择标记基因，对 农杆菌介导的地被菊‘北林黄’的遗传转化体系进行优化。研究结果表明：选取植株中部叶片为转化受体，外植 体在经过1 d 的预培养，用活化的OD600 为0.6 的农杆菌侵染10 min，共培养2 d，再经过3 d 延迟培养后转移 到筛选培养基上培养，有利于提高遗传转化效率，外植体的愈伤组织获得数和抗性芽数均最高。在筛选后期， 不定芽生根阶段使用的Kanamycin (Kan)选择压力为25 mg/L。获得的部分Kan 抗性植株经PCR 检测，扩增出 特异条带，初步证明目的基因已整合到‘北林黄’基因组DNA 中。本研究将为进一步利用分子手段对地被菊进 行遗传改良，并获得综合抗逆性提高的优异种质奠定基础。 关键词 地被菊, 根癌农杆菌, 遗传转化体系 Optimization of Agrobacterium Tumfaciens-Mediated Transformation System for Chrysanthemum morifolium ‘Beilinhuang ’ Zhao Lingli 1 Shi Shaochuan 1 Zhang Qixiang 1, 2 * Gao Yike 1 1 College of Landscape Architecture, Beijing Forestry University, Beijing 100083; 2 China National Engineering Research Center for Floriculture, Beijing, 100083 * Corresponding author, zqx@bjfu.edu.cn DOI: 10.3969/mpb.009.000074 Abstract In this research ，we optimized an Agrobacterium tumfaciens-mediated genetic transformation system of Chrysanthemum ‘Beilinhuang ’, which in vitro cultured leaves were used as the explants for genetic transformation and the Kanamycin resistant gene as selective marker. The results showed that the percentages of resistant callus and resistant buds were increased with the procedures of the 1 d pre-culture, 10 min infection, 2 d co-cultivation and 3 d later-cultivation while selected the middle leaves of plant as transformed receptor. The optimal concentration of Kanamycin was 25 mg/L during the later rooting filtration stage. In this experiment we obtained some regenerat- ing plants with Kanamycin resistance, PCR validation might imply that the target gene was integrated into the genomes of Chrysanthemum ‘Beilinhuang ’. This study will lay the