Improvement of Rice Salt Tolerance by Pyramiding Two Genes in Xian and Geng Backgrounds Through CRISPR-Cas9 System

Salinity is one of the main environmental factors influencing rice production. Many genes affecting salt tolerance (ST) have been cloned in rice so far. In the present study, four genes negatively regulating ST, including HST1, LRRK1, STRK2, and PC1, were edited by CRISPR-Cas9 technology in six rice varieties (three in indica (xian) and three in japonica (geng) backgrounds), and three two-gene editing combinations, including hst1-lrrk1, hst1-strk2, and hst1-pc1, were created. All combinations of hst1-pc1, hst1-lrrk1, and hst1-strk2 significantly improved the ST of all the tested materials in both xian and geng backgrounds and had much better ST than single-gene editing lines. The combination of hst1-pc1 had the poorest ST in CH70 and 8TX23 backgrounds but showed almost the same level of ST as the combinations of hst1-strk2 and hst1-lrrk1 in the C199S background for 17 days after salinization, which clearly brought out the background effect on ST and its utilization in ST breeding. As a comparison of the recipient varieties, almost all gene-edited lines except hst1-pc1 in the CH70 background showed significantly reduced grain weight owing to reduced seed setting rate in normal conditions. The hst1-strk2 showed the highest level of ST at the seedling stage and a relatively higher grain yield among all the lines; thus, it is feasible to enhance the ST of high-yielding rice varieties by simultaneously gene-editing against the two loci or pyramiding these two alleles with the other major ST genes of rice. Our results provide valuable gene resources and germplasms for improving rice salt tolerance and high yield.