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An improved Agrobacterium-mediated transformation of recalcitrant indica rice (Oryza sativa L.) cultivars

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Abstract

Agrobacterium-mediated transformation of indica rice varieties has been quite difficult as these are recalcitrant to in vitro responses. In the present study, we established a high-efficiency Agrobacterium tumefaciens-mediated transformation system of rice (Oryza sativa L. ssp. indica) cv. IR-64, Lalat, and IET-4786. Agrobacterium strain EHA-101 harboring binary vector pIG121-Hm, containing a gene encoding for β-glucuronidase (GUS) and hygromycin resistance, was used in the transformation experiments. Manipulation of different concentrations of acetosyringone, days of co-culture period, bacterial suspension of different optical densities (ODs), and the concentrations of l-cysteine in liquid followed by solid co-culture medium was done for establishing the protocol. Among the different co-culture periods, 5 days of co-culture with bacterial cells (OD600 nm = 0.5–0.8) promoted the highest frequency of transformation (83.04 %) in medium containing l-cysteine (400 mg l−1). Putative transformed plants were analyzed for the presence of a transgene through genomic PCR and GUS histochemical analyses. Our results also suggest that different cultural conditions and the addition of l-cysteine in the co-culture medium improve the Agrobacterium-mediated transformation frequencies from an average of 12.82 % to 33.33 % in different indica rice cultivars.

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Acknowledgments

This work was supported by OLP-31. MS, SD, AR, and PKV acknowledge the financial support from CSIR. NG acknowledges the financial support from ICMR.

Conflict of interest

The authors declare that they have no conflict of interest associated with this work.

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Authors and Affiliations

  1. CSIR-National Botanical Research Institute, Rana Pratap Marg, Lucknow, India

    Manju Shri, Arti Rai, Pankaj Kumar Verma, Prashant Misra, Sonali Dubey, Smita Kumar, Sikha Verma, Neelam Gautam, Rudra Deo Tripathi, Prabodh Kumar Trivedi & Debasis Chakrabarty

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  1. Manju Shri
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  2. Arti Rai
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  3. Pankaj Kumar Verma
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  4. Prashant Misra
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  5. Sonali Dubey
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  6. Smita Kumar
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  7. Sikha Verma
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  8. Neelam Gautam
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  9. Rudra Deo Tripathi
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  10. Prabodh Kumar Trivedi
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  11. Debasis Chakrabarty
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Correspondence to Debasis Chakrabarty.

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Handling Editor: Peter Nick

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Shri, M., Rai, A., Verma, P.K. et al. An improved Agrobacterium-mediated transformation of recalcitrant indica rice (Oryza sativa L.) cultivars. Protoplasma 250, 631–636 (2013). https://doi.org/10.1007/s00709-012-0439-x

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  • DOI: https://doi.org/10.1007/s00709-012-0439-x

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