对金钗石斛 Dendrobium nobile 进行人工自花授粉，在授粉之后10、20、30、40、45、50和60 d分别将工程化根癌农杆菌 Agrobacterium tumefaciens EHA105(pCAMBIA1301)直接注射入子房内部.150 d后，金钗石斛蒴果成熟，摘取果实，进行种子β-葡萄糖苷酸酶（GUS）染色、无菌播种、潮霉素筛选以及PCR检测试验.结果表明：子房注射对果实发育具有较大影响，在授粉后10~30 d进行子房注射后，果实脱落；在授粉后45 d进行子房注射，成熟果实注射部位的种子GUS染色率最高，可达18%；无菌播种之后，经潮霉素筛选，获得11株幼苗，每株植株均表现出GUS染色反应；随机挑选4株幼苗进行PCR检测，发现均能扩增出预期条带，说明外源基因已经整合到植株基因组内.农杆菌子房注射法在兰科植物转基因的成功应用，可以为兰科植物育种提供一种简单高效的方法.
Dendrobium nobile was transformed by ovary-injection of Agrobacterium. After artificial pollination in 10, 20, 30, 40, 45, 50 and 60 d, EHA105(pCAMBIA1301) harboring both β-glucuronidase gene (GUS) and hpt was injected into the ovary. The seeds of split fruit of D. nobile were cultured on the N16 culture medium by aseptic processing and detected by GUS expression. Forty-five days after cultured, the protocorms were diverted to N16 medium with hygromycin to gain hygromycin-resistance protocorms, then four randomly selected hygromycin-resistance protocorms were detected by PCR analysis. The study showed that ovary-injection of agrobacterium had harmful effects on the fruit development and seed formation. Ovary-injection of agrobacterium after artificial pollination 45 d had the highest efficiency, 18% seeds from the injected position showing GUS expression. Eleven hygromycin-resistant plants were gained in this study, and four randomly selected hygromycin-resistant plants were detected by PCR analysis and all of them were positive. The results showed that ovary-injection of agrobacterium transformation had advantages in genetic transformation of orchids.
刘其府,董 会,曾宋君,陈之林,吴坤林,张建霞,段 俊.农杆菌子房注射法对金钗石斛的活体转化研究[J].华南农业大学学报,2013,34(3):378-382复制