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Item Revisiting rice transformation for a fail-safe protocol and its application for various gene functional and molecular studies(Springer Nature Publishing AG, 2025) Manna, Mrinalini; Rengasamy, Balakrishnan; Reddy, Malireddy K.; Sinha, Alok KrishnaAn efcient 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 fnd 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 efectively employed to visualize protein localization in various subcellular compartments of rice root and callus tissues, an alternative to conventional tobacco/onion peel infltration 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 signifcance 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.Item Breeding rice for yield improvement through CRISPR/Cas9 genome editing method: current technologies and examples(Springer Nature Publishing AG, 2024) Rengasamy, Balakrishnan; Manna, Mrinalini; Thajuddin, Nargis Begum; Sathiyabama, Muthukrishnan; Sinha, Alok KrishnaThe impending climate change is threatening the rice productivity of the Asian subcontinent as instances of crop failures due to adverse abiotic and biotic stress factors are becoming common occurrences. CRISPR-Cas9 mediated genome editing ofers a potential solution for improving rice yield as well as its stress adaptation. This technology allows modifcation of plant’s genetic elements and is not dependent on foreign DNA/gene insertion for incorporating a particular trait. In this review, we have discussed various CRISPR-Cas9 mediated genome editing tools for gene knockout, gene knock-in, simultaneously disrupting multiple genes by multiplexing, base editing and prime editing the genes. The review here also presents how these genome editing technologies have been employed to improve rice productivity by directly targeting the yield related genes or by indirectly manipulating various abiotic and biotic stress responsive genes. Lately, many countries treat genome-edited crops as non-GMOs because of the absence of foreign DNA in the fnal product. Thus, genome edited rice plants with improved yield attributes and stress resilience are expected to be accepted by the public and solve food crisis of a major portion of the globe.Item A simplified and improved protocol of rice transformation to cater wide range of rice cultivars(Springer Nature Publishing AG, 2024) Rengasamy, Balakrishnan; Manna, Mrinalini; Jonwal, Sarvesh; Sathiyabama, Muthukrishnan; Thajuddin, Nargis Begum; Sinha, Alok KrishnaThe latest CRISPR-Cas9-mediated genome editing technology is expected to bring about revolution in rice yield and quality improvement, and thus validation of rice transformation protocols using CRISPR-Cas9-gRNA constructs is the need of the hour. Moreover, regeneration of more number of transgenic rice plants is prerequisite for developing genome-edited rice lines, as recalcitrant rice varieties were shown to have lower editing efciencies which necessities screening of large number of transgenic plants to fnd the suitable edits. In the present study, we have simplifed the Agrobacterium-mediated rice transformation protocol for both Indica and Japonica rice cultivars using CRISPR/Cas9 empty vector construct, and the protocols have been suitably optimized for getting large numbers of the regenerated plantlets within the shortest possible time. The Japonica transgenic lines were obtained within 65 days and for the Indica cultivars, it took about 76–78 days. We also obtained about 90% regeneration efciency for both Japonica and Indica cultivars. The transformation efciency was about 97% in the case of Japonica and 69–83% in the case of Indica rice cultivars. Furthermore, we screened the OsWRKY24 gene editing efciency by transforming rice cultivars with CRISPR/Cas9 construct harbouring sgRNA against OsWRKY24 gene and found about 90% editing efciency in Japonica rice cultivars, while 30% of the transformed Indica cultivars were found to be edited. This implicated the presence of a robust repair mechanism in the Indica rice cultivars.