Climate change has caused high salinity in many fields, particularly in the mud flats in coastal regions. The resulting salinity has become one of the most significant abiotic stresses affecting the world’s rice crop productivity. Developing elite cultivars with novel salinity-tolerance traits is regarded as the most cost-effective and environmentally friendly approach for utilizing saline-alkali land. To develop a highly efficient green strategy and create novel rice germplasms for salt-tolerant rice breeding, this study aimed to improve rice salinity tolerance by combining targeted CRISPR/Cas9-mediated editing of the OsRR22 gene with heterosis utilization. The novel alleles of the genic male-sterility (GMS) and elite restorer line (733S^rr22-T1447-1 and HZ^rr22-T1349-3) produced 110 and 1 bp deletions at the third exon of OsRR22 and conferred a high level of salinity tolerance. Homozygous transgene-free progeny were identified via segregation in the T2 generation, with osrr22 showing similar agronomic performance to wild-type (733S and HZ). Furthermore, these two osrr22 lines were used to develop a new promising third-generation hybrid rice line with novel salinity tolerance. Overall, the results demonstrate that combining CRISPR/Cas9 targeted gene editing with the “third-generation hybrid rice system” approach allows for the efficient development of novel hybrid rice varieties that exhibit a high level of salinity tolerance, thereby ensuring improved cultivar stability and enhanced rice productivity.

MicroRNAs (miRNAs) are small molecule RNAs widely involved in responses to plant abiotic stresses. We performed small RNA sequencing of cotton anthers at four developmental stages under normal and high temperature (NT and HT, respectively) conditions to investigate the stress response characteristics of miRNA to HT. A total of 77 miRNAs, including 33 known miRNAs and 44 novel miRNAs, were identified, and 41 and 28 miRNAs were differentially expressed under NT and HT stress conditions, respectively. The sporogenous cell proliferation (SCP), meiotic phase (MP), microspore release period (MRP), and pollen maturity (PM) stages had 10 (including 12 miRNAs), four (including six miRNAs), four (including five miRNAs), and seven (including 11 miRNAs) HT stress-responsive miRNA families, respectively, which were identified after removing the changes in genotype-specific miRNAs under NT condition. Seven miRNA families (miR2949, miR167, and miR160 at the SCP stage; miR156 and miR172 at the MP stage; miR156 at the MRP stage; and miR393 and miR3476 at the PM stage), which had expression abundance of more than 10% of the total expression abundance, served as the main regulators responding to HT stress with positive or negative regulation patterns. These miRNAs orchestrated the expression of the corresponding target genes and led to different responses in the HT-tolerant and the HT-sensitive lines. The results revealed that the HT stress response of miRNAs in cotton anthers were stage-specific and differed with the development of anthers. Our study may enhance the understanding of the response of miRNAs to HT stress in cotton anthers and may clarify the mechanism of plant tolerance to HT stress.