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An efficient method for Agrobacterium-mediated genetic transformation of chilli pepper (Capsicum annuum L.)

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Abstract

In this study, we have describe a highly efficient transformation protocol for two elite Indian cultivars of chilli pepper (Capsicum annuum L.), Pusa Sadabahar and Pusa Jwala. We used hypocotyls and cotyledons derived from young seedlings as explants, standardized regeneration medium for enhanced regeneration, and optimized parameters for promoting efficient transformation in these chilli cultivars. The optimized regeneration medium consisting of MS medium augmented with 1 mg l−1 indole-3-acetic acid and 5 mg l−1 6-benzyl amino purine induced profuse shoot regeneration from the hypocotyl and cotyledon explants, with a regeneration frequency of 81% in Pusa Sadabahar and 78% in Pusa Jwala. The transformation protocol was standardized using Agrobacterium tumefaciens strain LBA4404 harbouring pCAMBIA2301 construct with GUS reporter and NPT-II marker genes. The results demonstrated that the hypocotyl explants are more amenable to transformation than cotyledonary explants in both the cultivars studied. Further, co-cultivation of the hypocotyl explants with agrobacterial cells (OD600 = 0.2–0.5) for the duration of 72 h was found to be optimum for obtaining high transformation efficiency of about 30% in both the cultivars. The transgenic status of chilli transformants recovered in the selection medium having 30 mg l−1 kanamycin was confirmed by GUS activity, PCR, Southern and RT-PCR analyses. About 85–90% of rooted transgenic plants survived hardening and acclimatization processes. Using this protocol we produced several independent transgenic lines of each chilli cultivar.

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Acknowledgements

The research work was financed by the Department of Biotechnology (Grant No. BT/PR5399/AGR/36/722/2012), Government of India, to SD and MVR. BKM gratefully acknowledges Department of Science and Technology-INSPIRE (DST-INSPIRE), Government of India, for providing fellowship.

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Authors and Affiliations

  1. TERI School of Advanced Studies, Institutional Area, Vasant Kunj, New Delhi, 110070, India

    Binod K. Mahto

  2. Division of Sustainable Agriculture, The Energy and Resources Institute (TERI), India Habitat Centre, Lodi Road, New Delhi, 110003, India

    Binod K. Mahto, Poonam Sharma, Pallavolu M. Reddy & Swatismita Dhar-Ray

  3. Department of Genetics, University of Delhi South Campus, Benito Juarez Road, New Delhi, 110021, India

    M. V. Rajam

Authors
  1. Binod K. Mahto
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  2. Poonam Sharma
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  3. M. V. Rajam
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  4. Pallavolu M. Reddy
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  5. Swatismita Dhar-Ray
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Correspondence to Pallavolu M. Reddy or Swatismita Dhar-Ray.

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Poonam Sharma and Swatismita Dhar-Ray: Formerly associated with TERI.

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Mahto, B.K., Sharma, P., Rajam, M.V. et al. An efficient method for Agrobacterium-mediated genetic transformation of chilli pepper (Capsicum annuum L.). Ind J Plant Physiol. 23, 573–581 (2018). https://doi.org/10.1007/s40502-018-0389-1

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  • DOI: https://doi.org/10.1007/s40502-018-0389-1

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