Abstract
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 T0 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 T2 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.
Highlights
Among the cloned salt-tolerant genes in rice, OsRR22 is one of the most important negative regulatory genes for salt tolerance in rice; this gene encodes the B-type response regulatory protein composed of 696 amino acids, which is located on chromosome 1 of rice and is involved in cytokine signal transduction and metabolism
The information of the constructed vector and specific insertion position is shown in Figure 1
This study indicated that the salt tolerance of the novel OsRR22 ment of major crops such as rice, maize, oilseed rape, and soybeans [18]
Summary
As the first selected grain crop for improving coastal and saline-alkali land, has tremendous potential for comprehensive utilization. Salt stress has detrimental effects on rice molecules, cells, as well as physiological and biochemical levels, resulting in a reduction in rice yield or death [4] It is one of the significant actions to guarantee world food security via screening and innovating salinealkali tolerant rice germplasm resources, studying the genetic and molecular mechanism of salt-alkali tolerance, cultivating novel rice varieties with saline-alkali-tolerant capacity, and conducting promotion and application in large areas [5,6]. Among the cloned salt-tolerant genes in rice, OsRR22 is one of the most important negative regulatory genes for salt tolerance in rice; this gene encodes the B-type response regulatory protein composed of 696 amino acids, which is located on chromosome 1 of rice and is involved in cytokine signal transduction and metabolism. The existing elite parents with excellent comprehensive traits were improved to create a novel salt-tolerant rice germplasm by using up-to-date gene-editing technology, which accelerates the process of salt-tolerant rice breeding
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