简单高效的根癌农杆菌介导的水稻基因转化方法

本文在研究影响农杆菌介导的水稻转化的主要因素基础上,建立了一套简单、高效的水稻转基因系统。将水稻成熟胚来源的愈伤组织用农杆菌EHA101/pHQ9,EHA 101/pHQ 10,EHA 101/pHQ T3感染后,筛选抗性愈伤,经分化获得转化株。抗性愈伤的平均得率为约100个愈伤/g愈伤外植体,抗性愈伤的分化频率平均高达85%。转基因植株的GUS染色、Southern杂交结果表明,T-DNA上的外源基因已整合进转基因植物的基因组中。转基因植株T1代对潮霉素的抗性表明,多数转基因株系符合孟德尔分离比3∶1。该系统的建立将有助于应用T-DNA标签法和基因打靶法进行水稻功能基因组的研究。

A SIMPLE AND HIGHLY EFFICIENT AGROBACTERIUM -MEDIATED RICE TRANSFORMATION SYSTEM

A simple and highly efficient rice transformation system was established based on the studying of lactors influencing the Agrobacteritnn -mediated rice transformation. Embryogenic calli derived from mature embryos were infected and cocultivated with A . tumejaciens EHA101 harboring binary vectors:pHQ9,pHQ10,pHQT3. The highest transformation frequency was about JOO bypromycin resistance calli per gram of calli explants,and above 85% of these calli could be regenerated into plants. The putative transgenic plants were confirmed by GUS assay and southern-blot analysis. Hygromycin resistance tests indicated that the segregation of transgene in T1 progeny corresponded to the Mendelian ratio,3:1. This system will benefit the functional genomic study of rice by using T-DNA insertion mutagenesis and gene targeting.