农杆菌介导的苜蓿次级体细胞胚的遗传转化

摘要

采用农杆菌菌株GV3101感染子叶期苜蓿体细胞胚来研究苜蓿次级体细胞胚的遗传转化方法.农杆菌菌株GV3101双相载体pCAMBIA2301,此双相载体具有gus报告基因和npt Ⅱ抗卡那霉素筛选基因.感染的子叶期苜蓿体细胞在75 mg/L卡那霉素筛选压下,经过一系列诱导培养,最终获得转基因植株.然后,通过GUS组织化学定位分析来检测转基因植株不同器官中的GUS表达,并进一步通过PCR和Southern杂交确定转基因的稳定整合和转化率.结果表明转基因植株不同器官均有GUS表达,整合的nptⅡ基因的拷贝数是1～4,获得的转基因植株的转化率是65.82％.%We describe a genetic transformation method of secondary somatic embryogenesis in alfalfa through cotyledon-stage somatic embryos of alfalfa infected by Agrobacterium strain GV3101. The Agrobacterium strain GV3101 contained binary vector pCAMBIA2301 that had gus gene as reporter and nptll gene as selectable marker. The infected primaryembryos were induced through series of medium under 75 mg/L kanamycin selection. We obtained the transgenic alfalfa plants. Then, GUS expression in different tissue of transgenic alfalfa was tested by GUS histochemical analysis. Further, the stable integration and transformation efficiency were tested by polymerase chain reaction and Southern blotting hybridization. The result showed that GUS expression was different in different organs of transgenic alfalfa; the copy number of integrated npt I/gene was from 1 to 4; the transformation efficiency via primary somatic embryogenesis was 65.82%.