应用农杆菌介导的生长点转化方法建立甜瓜遗传转化技术  

Establishment of Transformation Method for Melon Using Agrobacterium-mediated Plant Genetic Transformation Via Plant Apical Meristem

作　　者：郝金凤[1] 荆培培[1] 张丽[1] 哈斯阿古拉[1] 

机构地区：[1]内蒙古大学生命科学学院,内蒙古自治区牧草与特色作物生物技术重点实验室,内蒙古呼和浩特010021

出　　处：《华北农学报》2014年第2期116-120,共5页Acta Agriculturae Boreali-Sinica

基　　金：国家自然科学基金资助项目(31101559;31360486);教育部"春晖计划"科研合作项目(Z2005-2-01003)

摘　　要：为探讨甜瓜的遗传转化技术,以甜瓜品种河套蜜瓜腋芽生长点做外植体,利用庆大霉素基因作为筛选基因,对影响农杆菌转化的菌液浓度、转化时间、共培养时间进行了正交试验。通过优化转化条件,建立了农杆菌介导的甜瓜腋芽生长点遗传转化体系。结果表明,采用培养20 d的无菌苗,以腋芽作为外植体,预培养1 d;用OD600=0.7的菌液转化10 min后,黑暗中共培养2 d,然后用40 mg/L庆大霉素进行筛选;移栽后在叶片上喷洒20 mg/L庆大霉素筛选转基因植株,效果最好。通过对庆大霉素抗性苗进行PCR检测,初步验证外源基因已经整合进甜瓜基因组,转化率达到76%,幼苗移栽成活率为76.3%。A high-frequency transformation system has been established by Agrobacterium tumefaciens transformation technology and using meristems of axillary buds as transformation receptor. External factors that affect the efficiency of genetic transformation on the base of gentamicin were studied and optimized,such as the bacterial concentration and the times of transformation and co-cultivation. The major results were as follows： seedling was grown for 20 days,then axillary buds excised and stabbed were infected with Agrobacterium tumefaciens which OD600was 0. 7 for 10 min. Then after co-cultivation for 2 days,transformed plants were selected by 20 mg / L and 40 mg / L gentamicin on the leaves and rooting respectively. It was originally proved by the PCR that expression cassettes had been transferred into the genome. The positive transformation rate was 76% by the analysis of PCR. And the living rate of the transplanted transgenic plants was 76. 3%.

关 键 词：甜瓜 植物生长点 遗传转化 农杆菌 

分 类 号：S652[农业科学—果树学]