An efficient Agrobacterium rhizogenes-mediated transformation protocol of Withania somnifera
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- 作者：Sayed Mehdi Dehdashti ; Sumita Acharjee…
- 关键词：Agrobacterium rhizogenes mediated ; GusA transgene expression ; Hairy roots ; Transformation efficacy ; Withania somnifera
- 刊名：Plant Cell, Tissue and Organ Culture (PCTOC)
- 出版年：2017
- 出版时间：January 2017
- 年：2017
- 卷：128
- 期：1
- 页码：55-65
- 全文大小：
- 刊物类别：Biomedical and Life Sciences
- 刊物主题：Plant Sciences; Plant Physiology; Plant Genetics & Genomics; Plant Pathology;
- 出版者：Springer Netherlands
- ISSN：1573-5044
- 卷排序：128
文摘
This is the first report on Agrobacterium rhizogenes-mediated transformation of Withania somnifera for expression of a foreign gene in hairy roots. We transformed leaf and shoot tip explants using binary vector having gusA as a reporter gene and nptII as a selectable marker gene. To improve the transformation efficiency, acetosyringone (AS) was added in three stages, Agrobacterium liquid culture, Agrobacterium infection and co-culture of explants with Agrobacterium. The addition of 75 μM AS to Agrobacterium liquid culture was found to be optimum for induction of vir genes. Moreover, the gusA gene expression in hairy roots was found to be best when the leaves and shoot tips were sonicated for 10 and 20s, respectively. Based on transformation efficiency, the Agrobacterium infection for 60 and 120 min was found to be suitable for leaves and shoot tips, respectively. Amongst the various culture media tested, MS basal medium was found to be best in hairy roots. The transformation efficiency of the improved protocol was recorded 66.5 and 59.5 % in the case of leaf and shoot tip explants, respectively. When compared with other protocols the transformation efficiency of this improved protocol was found to be 2.5 fold higher for leaves and 3.7 fold more for shoot tips. Southern blot analyses confirmed 1–2 copies of the gusA transgene in the lines W1-W4, while 1–4 transgene copies were detected in the line W5 generated by the improved protocol. Thus, we have established a robust and efficient A. rhizogenes mediated expression of transgene (s) in hairy roots of W. somnifera.