Development of Novel Rice Germplasm for Salt-Tolerance at Seedling Stage Using CRISPR-Cas9

Saline-alkali tolerant rice, as the first selected grain crop for improving coastal tidal land and saline-alkali land, has great potential for comprehensive utilization. In this study, an elite three-line restorer in breeding, R192, was taken as the receptor, and CRISPR/Cas9 technology was used to perform the directional editing of OsRR22, the main effect gene, which controls salt tolerance in rice. Eight transgenic plants of the T-0 generation with the OsRR22 gene knockout were obtained, and the transgenic seedlings were screened by using PCR amplification and sequence comparison. The homozygous mutant lines, M16 and M18, with OsRR22 knockout, which did not contain a transgenic vector skeleton, were identified in the T-2 generation. There were +1 bp and -20 bp in the exon regions of M16 and M18, respectively. At the three-leaf and one-heart stage, the seedlings were treated with 0.4% and 0.8% NaCl solution, and then their salt tolerance during the seedling stage was identified. The results showed that, without the salt treatment, no significant differences were found in plant height, the number of green leaves, total dry weight, and total fresh weight between the mutant lines M16 and M18 and the wildtype (WT) R192. However, after treatment with two different salt concentrations, the M16 and M18 mutants showed extremely significant differences in comparison with WT in plant height, the number of green leaves, total dry weight, and total fresh weight; between mutants and WT, there were significant differences in the number of green leaves, total fresh weight, and total dry weight after two salt treatments. Our results indicate the new germplasm with the OsRR22 mutation induced by CRISPR/Cas9 technology could improve the salt tolerance of rice, providing a reference for the improvement of salt tolerance of rice.