The Development of a Transformation System for Four Local Rice Varieties and CRISPR/Cas9-Mediated Editing of the OsCCD7 Gene

Agrobacterium-mediated transformation systems are extensively applied in japonica rice varieties. However, the adaptability of local rice varieties to existing transformation systems remains limited, owing to their complex genotypes, posing a substantial challenge to transformation. In this study, four local rice varieties were selected to optimize the effects of different culture media on callus induction, browning resistance, contamination resistance, callus tolerance, differentiation, regeneration, and root development, and then two varieties were selected to improve plant architecture and tiller development by CRISPR/Cas9-mediated gene editing, based on constructive transformation systems. The goal was to enhance the transformation efficiency of local varieties and innovate germplasms. The results demonstrated that japonica rice varieties XG293 and WD68 exhibited higher induction rates under the treatment of 2 mg/L 2,4-D (2,4-Dichlorophenoxyacetic acid) + 1 mg/L NAA (Naphthaleneacetic acid), whereas indica rice varieties H128 and E33 performed the best under 3 mg/L 2,4-D + 1 mg/L NAA. Severe browning in H128 was effectively mitigated by a carbon source of 20 g/L maltose supplemented with 40 mg/L ascorbic acid. Contamination after Agrobacterium infection was controlled by 300 mg/L Tmt (Timentin). Under a treatment of 200 µM/L acetosyringone +10 min infection duration, XG293 and WD68 exhibited higher callus tolerance, differentiation rates, and GUS staining rates, achieving transformation efficiencies of 43.24% and 52.38%, respectively. In contrast, H128 and E33 performed better under the treatment of 200 µM/L Acetosyringone + 5 min, with transformation efficiencies of 40.00% and 40.74%, respectively. The mutants after OsCCD7 gene editing in WD68 and H128 displayed a dwarfness of plant height, a significant increase in tiller numbers, and compact architecture. These findings demonstrate that an optimized combination of plant growth regulators and infection durations effectively improves transformation efficiency for local varieties, and the OsCCD7 gene regulates plant architecture and tiller development with variable effects, depending on the rice complex genotypes. This study provides a theoretical basis for the efficient transformation of local rice varieties and germplasm innovation.