National Botanical Research Institute, Council of Scientific and Industrial Research, Rana Pratap Marg, Lucknow 226001, UP, India.
Plant Cell Rep. 2010 Feb;29(2):133-41. doi: 10.1007/s00299-009-0805-0. Epub 2009 Dec 11.
This report describes Agrobacterium tumefaciens-mediated transformation of Withania somnifera--an important Indian medicinal plant. A. tumefaciens strain LBA4404, containing the binary vector pIG121Hm was used for transformation, along with the gusA reporter gene with intron under the transcriptional control of the Cauliflower Mosaic Virus (CaMV) 35S promoter. The leaf segments from two-and-a-half-month-old green house-grown seedlings were more efficient in transformation, as compared to those from the in vitro-grown shoots. Second expanded leaf from the shoot tip gave the highest transient transformation efficiency. Selection of transgenic shoots was done in the presence of 50 mg l(-1) kanamycin. Polymerase chain reaction analysis of T(0) transgenic plants showed the presence of gusA and nptII genes. The expression of these transgenes in T(1) progeny was confirmed by RT-PCR. The integration of gusA gene was confirmed by Southern blot analysis. The transformation efficiency was found to be 1.67%.
本报告描述了根癌农杆菌介导的印度药用植物睡茄的遗传转化。使用携带二元载体 pIG121Hm 的根癌农杆菌菌株 LBA4404 以及在花椰菜花叶病毒（CaMV）35S 启动子转录控制下带有内含子的 gusA 报告基因进行转化。与来自体外生长芽的叶片相比，来自 2.5 个月大的温室生长幼苗的叶片在转化中更有效。从芽尖的第二片展开叶获得了最高的瞬时转化效率。在 50mg l(-1)卡那霉素存在下选择转基因芽。对 T(0)转基因植物的聚合酶链反应分析显示存在 gusA 和 nptII 基因。通过 RT-PCR 证实了这些转基因在 T(1)后代中的表达。通过 Southern blot 分析证实了 gusA 基因的整合。转化效率为 1.67%。
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