Phenotypic, hydroponic performance assessment, and expression profiling of triple knockout negative regulator genes in rice (Oryza sativa L.)

Rice is one of the most significant crops consumed daily by individuals. Enhancing and biofortifying rice to augment its nutritional value is a promising strategy for improving public health and tackling the widespread issue of micronutrient deficiencies. This study primarily attempted to evaluate the developed Cas9-free edited lines grown hydroponically with a triple knockout of the negative metal sensor regulator uptake ( OsHRZ1, OsHRZ2 , and OsLCT1 ). This result shows that the Cas9-free edited lines’ performance was excellent, with no significant effect on the plant's agronomic performance or yield penalty due to multiplex knockout of genes, in addition to growing under cadmium stress conditions. The protein content of seeds was higher in the Cas9-free edited lines than the protein concentrations observed in the wild type (control/treated), where the protein concentration ranged from 17 to18 mg g −1 FW protein compared to the wild type (normal/treated) (7.24, 7.08) mg g −1 FW, respectively. The concentration varies based on the growth condition under deficient or sufficient Fe/Zn. Photosynthetic rates were increased in Cas9-free edited plants in comparison to wild rice plants, correlating with enhanced agronomic yield parameters and tolerance of cadmium conditions. Rice pollen grain viability and fertility were examined to check the effect of excess iron/zinc on pollen grain viability, fertility, and germination. The expression of genes closely linked to iron, zinc, and cadmium uptake and translocation in rice endosperm was studied. Developed rice lines hold a huge promise to overcome micronutrient malnutrition worldwide.