Abstract
Crop-improvement efforts for Chakhao black rice (Oryza sativa L.) through genetic engineering have not made much progress due to the absence of a protocol for callus induction and regeneration. We developed a tissue culture protocol for the Agrobacterium-mediated transformation of Chakhao Amubi and Chakhao Poireiton cultivars of rice using mature seeds as the explants. The transformation protocol is simple and can be carried out in laboratories with basic equipment for tissue culture. The effect of temperature, growth regulators, gelling agents, and the duration of infection on different stages of the protocol is also described. Transgenic rice seedlings can be obtained in 69–80 days with > 30% transformation efficiency using the optimized method for each genotype.
Key message
This article describes a time-efficient and high transformation efficiency protocol for Agrobacterium tumefaciens-mediated transformation of aromatic black rice cultivars, Chakhao Amubi and Chakhao Poireiton, for their genetic improvement.
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Acknowledgements
Authors would like to thank Science and Engineering Board (SERB), Government of India; Department of Biotechnology (DBT), Government of India; and Department of Science and Technology (DST), Government of India for funding. RCN would like to thank the Science and Engineering Board (SERB), Government of India for the Award of National Post-doctoral Fellowship (NPDF). AA would like to thank DBT State Level Biotech Hub Project for financial assistance received as Junior Research fellowship. The authors would like to acknowledge Professor Suman Kumaria, Rebati Sinam and Hanjabam Pritam Sharma.
Funding
Financial support received from Department of Biotechnology, Government of India, New Delhi for work vide Grant. No. BT/PR5397/PBD/16/974/2012 and BT/o4/NE/2009 to NKC. Financial support received from National Post-Doctoral Fellowship Scheme (NPDF), Science and Engineering Research Board, Government of India, New Delhi for work vide Sanction No. PDF/2019/001269.
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This research was designed by RCN and NKC. RCN and AA conducted the experiments and analysed the data. The manuscript was written by all three authors. All authors read and approved the final manuscript. RCN and AA contributed equally to this research and preparation of this manuscript.
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Supplementary file1 (PDF 182 kb) Statistical test for significance was carried out using one-way ANOVA, all pairwise multiple comparison procedures (Holm–Sidak method) using SigmaPlot 12.0 (Systat) and represented as superscript letters. Values with different letters are significantly different at p ≤ 0.05 (n = 3) and values with at least one letter in common have no significant differences
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Ali, A., Nongpiur, R.C. & Chrungoo, N.K. An efficient Agrobacterium-mediated transformation and regeneration protocol for aromatic black rice, Oryza sativa L. (cv. Chakhao Poireiton and Chakhao Amubi). Plant Cell Tiss Organ Cult 152, 619–633 (2023). https://doi.org/10.1007/s11240-022-02435-w
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DOI: https://doi.org/10.1007/s11240-022-02435-w