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An efficient Agrobacterium rhizogenes-mediated transformation protocol of Withania somnifera

Plant Cell, Tissue and Organ Culture (PCTOC) volume 128, pages 55–65 (2017)Cite this article

Abstract

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.

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Acknowledgments

We acknowledge the Iranian Ministry of Science, Research and Technology (MSRT) Scholarships for Iranian students abroad from the Iranian Government received by SMD. We thank the Department of Agricultural Biotechnology, Assam Agricultural University for providing laboratory facilities to do gene cloning, Agrobacterium transformation works. We would also like to thank Institute of Microbial Technology (IMTECH), Chandigarh for the bacterial strains.

Author contributions

SMD and SA performed bacterial transformations. Under the guidance of SA, SMD conducted the experiments for hairy roots transformation. SK helped SMD with southern blots. SMD and MD were responsible for data analyses. SMD and MD wrote the manuscript. All the authors agree with the contents of the manuscript.

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Authors and Affiliations

  1. Department of Bioengineering and Technology, Faculty of Technology, Gauhati University, Guwahati-14, Assam, India

    Sayed Mehdi Dehdashti & Manab Deka

  2. Department of Agricultural Biotechnology, Assam Agriculture University, Jorhat, Assam, India

    Sumita Acharjee

  3. Agricultural Biotechnology Research Institute of Iran (ABRI), Karaj, Iran

    Shahla Kianamiri

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  1. Sayed Mehdi Dehdashti
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  2. Sumita Acharjee
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  4. Manab Deka
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Correspondence to Manab Deka.

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Dehdashti, S.M., Acharjee, S., Kianamiri, S. et al. An efficient Agrobacterium rhizogenes-mediated transformation protocol of Withania somnifera . Plant Cell Tiss Organ Cult 128, 55–65 (2017). https://doi.org/10.1007/s11240-016-1081-7

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