The 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 efficiencies which necessities screening of large number of transgenic plants to find the suitable edits. In the present study, we have simplified 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 efficiency for both Japonica and Indica cultivars. The transformation efficiency 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 efficiency by transforming rice cultivars with CRISPR/Cas9 construct harbouring sgRNA against OsWRKY24 gene and found about 90% editing efficiency 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.

最新的CRISPR-Cas9介导的基因组编辑技术有望给水稻产量和品质改善带来革命，因此使用CRISPR-Cas9-gRNA构建体验证水稻转化方案是当前的需要。此外，更多数量的转基因水稻植株的再生是开发基因组编辑水稻品系的先决条件，因为顽抗的水稻品种被证明具有较低的编辑效率，这需要筛选大量的转基因植株以找到合适的编辑。在本研究中，我们使用 CRISPR/Cas9 空载体构建体简化了农杆菌介导的籼稻和粳稻品种的水稻转化方案，并对方案进行了适当优化，以便在最短的时间内获得大量再生植株。粳稻转基因品系在 65 天内获得，而籼稻品种则需要约 76-78 天。我们还获得了粳稻和籼稻品种约 90% 的再生效率。粳稻的转化效率约为 97%，籼稻品种的转化效率约为 69-83% 。此外，我们通过使用带有针对OsWRKY24基因的 sgRNA 的 CRISPR/Cas9 构建体转化水稻品种来筛选OsWRKY24基因编辑效率，发现粳稻品种的编辑效率约为 90% ，而转化的籼稻品种的编辑效率为 30%。这表明籼稻品种中存在强大的修复机制。