子房注射法转化‘紫宝石’蝴蝶兰的研究  

Genetic Transformation of Phalaenopsis ‘Purple Gem'(Orchidaceae)via Ovary-injection

作　　者：张莉 王佳[1,3] 郑枫 吴坤林[1,4] 房林[1] 李琳 曾宋君[1,4] ZHANG Li;WANG Jia;ZHENG Feng;WU Kunlin;FANG Lin;LI Lin;ZENG Songjun(Key Laboratory of South China Agricultural Plant Molecular Analysis and Gene Improvement, South China Botanical Garden, Chinese Academy of Sciences, Guangzhou, Guangdong 510650, China;University of Chinese Academy of Sciences, Beijing 100049, China;Shenzhen TiehanYifang Environmental Science and Technology Co. LTD, Shenzhen, Guangdong 518000, China;Guangdong Provincial Key Laboratory of Applied Botany, South China Botanical Garden, Chinese Academy of Sciences, Guangzhou, Guangdong 510650, China)

机构地区：[1]中国科学院华南植物园华南农业植物分子分析和遗传改良重点实验室,广东广州510650 [2]中国科学院大学,北京100049 [3]深圳市铁汉一方环境科技有限公司,广东深圳518000 [4]中国科学院华南植物园广东省应用植物学重点实验室,广东广州510650

出　　处：《热带作物学报》2018年第4期669-674,共6页Chinese Journal of Tropical Crops

基　　金：国家重点研发计划项目(No.2016YFC0503104);广东省现代农业产业技术体系花卉创新团队项目(No.2016LM1142);中国科学院战略生物资源服务网络计划植物种质资源创新项目(No.ZSZC-008);广东省科技计划项目(No.2017A020208036;No.2017B090901050;No.2015B020231005;No.2014B09091051)

摘　　要：采用子房注射法对‘紫宝石’蝴蝶兰进行外源DNA导入研究,结果表明:不同时期注射和不同注射处理方式对‘紫宝石’蝴蝶兰的落果率影响不同,对胚座组织和种子的GUS染色率的影响也不同,在授粉后65 d时,以2%DMSO为缓冲液,注射400μg/mL浓度质粒,种子的GUS染色率最高达6.14%。选取各注射时期无菌播种后获得的1 000个原球茎,经系列浓度的潮霉素筛选,结果发现授粉后65 d注射时获得的抗性植株最多,为31株,转化率为3.1%。切取转化植株幼叶及幼根进行GUS检测,均为阳性;随机挑选7株幼苗进行PCR检测,均能扩增出GUS基因和HPT基因条带,说明外源基因已经整合到植株的基因组内。此研究为兰科植物育种提供了一种简单高效的途径。The method of ovary injection was reported for Phalaenopsis ‘Purple Gem＇ transformation in this study. Results showed that different injection treatments in different development stage of P. Purple Gem had different effect on the percentage of fertile fruit and GUS staining rate of seeds and placentas. The highest GUS staining rate of seeds from the mature fruit by ovary-injection was 6.14%. The highest 3.1% hygromycin-resistance plants were gained after hygromycin（Hyg） screening by a series of concentration in 65 DAP fruit. Seven plants selected randomLy were detected by GUS assay and PCR analysis of GUS and HPT, and all of them were positive. The results showed the integration and expression of foreign gene were integrated into hygromycin-resistance plants. The successful application of ovary injection for P. Purple Gem transformation could provide a simple and efficient method for the Orchidaceae breeding.

关 键 词：'紫宝石’蝴蝶兰 子房注射法 遗传转化 PCR 

分 类 号：S682[农业科学—观赏园艺]