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Genotype independent and efficient Agrobacterium-mediated genetic transformation of the medicinal plant Withania somnifera Dunal

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Abstract

Withania somnifera one of the most reputed Indian medicinal plant has been extensively used in traditional and modern medicines as active constituents. A high frequency genotype and chemotype independent Agrobacterium-mediated transformation protocol has been developed for W. somnifera by optimizing several factors which influence T-DNA delivery. Leaf and node explants of Withania chemotype was transformed with A. tumefaciens strain GV3101 harboring pIG121Hm plasmid containing the gusA gene encoding β-glucuronidase (GUS) as a reporter gene and the hptII and the nptII gene as selection markers. Various factors affecting transformation efficiency were optimized; as 2 days preconditioning of explants on MS basal supplemented with TDZ 1 μM, Agrobacterium density at OD600 0.4 with inclusion of 100 μM acetosyringone (As) for 20 min co-inoculation duration with 48 h of co-cultivation period at 22 °C using node explants was found optimal to improved the number of GUS foci per responding explant from 36 ± 13.2 to 277.6 ± 22.0, as determined by histochemical GUS assay. The PCR and Southern blot results showed the genomic integration of transgene in Withania genome. On average basis 11 T0 transgenic plants were generated from 100 co-cultivated node explants, representing 10.6 % transformation frequency. Our results demonstrate high frequency, efficient and rapid transformation system for further genetic manipulation in Withania for producing engineered transgenic Withania shoots within very short duration of 3 months.

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Abbreviations

YEB:

Yeast Extract Broth

TDZ:

Thidiazuron

As:

Acetosyringone

MS:

Murashige and Skoog

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Acknowledgments

The authors are thankful to the NMITLI, New Delhi and Department of Biotechnology (DBT), New Delhi, for providing the financial support. The authors thank Dr. P. Misra, National Botanical Research Institute, Lucknow for providing constructs. Authors are also thankful to Director CIMAP for constant support and encouragement.

Conflicts of interest

The authors declare that they have no conflict of interest.

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Authors and Affiliations

  1. CSIR-Central Institute for Medicinal and Aromatic Plants (CSIR-CIMAP), Lucknow, 226015, UP, India

    Smrati Mishra, Shilpi Bansal, Rajender Singh Sangwan & Neelam S. Sangwan

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  1. Smrati Mishra
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  2. Shilpi Bansal
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  3. Rajender Singh Sangwan
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  4. Neelam S. Sangwan
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Correspondence to Neelam S. Sangwan.

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Mishra, S., Bansal, S., Sangwan, R.S. et al. Genotype independent and efficient Agrobacterium-mediated genetic transformation of the medicinal plant Withania somnifera Dunal. J. Plant Biochem. Biotechnol. 25, 191–198 (2016). https://doi.org/10.1007/s13562-015-0324-8

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  • DOI: https://doi.org/10.1007/s13562-015-0324-8

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