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Revisiting rice transformation for a fail-safe protocol and its application for various gene functional and molecular studies

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Abstract

An efficient transformation and regeneration system is prerequisite for gene functional studies in rice, the staple food crop of the Asian subcontinent. Despite the availability of a large number of rice transformation protocols, it is hard to find a simple and minimum input based, but fail-safe protocol that ensures zero number of escapes (or the non-transformants). The fear of obtaining an unpredictable percentage of escapes at the end of rice tissue culture prompts carrying out multiple batches of transformation which not only consumes costly resources and time but also burdens one with screening of a large number of tissue culture derived plants. In the present study, we have described a simple but fail-safe rice transformation protocol for functional validation of the genes by overexpression and CRISPR-Cas9 mediated gene knockout systems. By taking the advantage of high transformability of japonica rice tissues, we have also revealed that the present rice transformation protocol can be effectively employed to visualize protein localization in various subcellular compartments of rice root and callus tissues, an alternative to conventional tobacco/onion peel infiltration or protoplast transformation which are either time consuming or tricky. Additionally, the paper also discusses the importance of Southern blotting in gene overexpression studies, utility of non-conventional antibiotic selection approaches and significance of sgRNA designing for gene knockout studies in rice. Various troubleshooting advice are also being presented. Overall, the present protocol might serve as an excellent guide for functional validation of several genes present in the rice genome waiting to be explored.

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Acknowledgements

MM gratefully acknowledges CSIR, New Delhi, India for awarding Senior Research Fellowship during PhD. MM also gratefully acknowledges SERB, DST, Government of India for awarding National Post‐Doctoral Fellowship (NPDF; File number: PDF/2020/000511) and Department of Biotechnology (DBT) for awarding BioCARe Fellowship (Sanction Order No. BT/PR51354/BIC/101/1344/2023) for post-doctoral research. MKR acknowledges research grant from National Agricultural Science Fund, ICAR. AKS acknowledges Sir J.C. Bose National Fellowship Award from SERB, Government of India. Authors also thank the Confocal Microscopy Facility, GFAPC Facility and the Central Instrumentation Facility of NIPGR, New Delhi, India. The authors are thankful to DBT-eLibrary Consortium (DeLCON) for providing access to e-resources.

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Authors and Affiliations

  1. National Institute of Plant Genome Research, Aruna Asaf Ali Marg, New Delhi, 110067, India

    Mrinalini Manna, Balakrishnan Rengasamy & Alok Krishna Sinha

  2. International Centre for Genetic Engineering and Biotechnology, Aruna Asaf Ali Marg, New Delhi, 110067, India

    Mrinalini Manna & Malireddy K. Reddy

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  1. Mrinalini Manna
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  2. Balakrishnan Rengasamy
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  3. Malireddy K. Reddy
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  4. Alok Krishna Sinha
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Contributions

Conceived and designed the experiments: MM, MKR, AKS. Performed the experiments: MM, RB. Analyzed the data: MM. Wrote the paper: MM. Proofreading: MKR, AKS.

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Correspondence to Mrinalini Manna or Alok Krishna Sinha.

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Manna, M., Rengasamy, B., Reddy, M.K. et al. Revisiting rice transformation for a fail-safe protocol and its application for various gene functional and molecular studies. J Plant Growth Regul (2024). https://doi.org/10.1007/s00344-024-11486-6

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