A simplified and improved protocol of rice transformation to cater wide range of rice cultivars.

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.