Abstract
Significant increases in rice yield and stress resistance are constant demands for breeders. However, high yield and high stress resistance are often antagonistic to each other. Here, we report several new rice mutants with high yield and excellent cold tolerance that were generated by simultaneously editing three genes, OsPIN5b (a panicle length gene), GS3 (a grain size gene) and OsMYB30 (a cold tolerance gene) with the CRISPR–Cas9 (clustered regularly interspaced short palindromic repeats-associated protein 9) system. We edited two target sites of each gene with high efficiency: 53% for OsPIN5b-site1, 42% for OsPIN5b-site2, 66% for GS3-site1, 63% for GS3-site2, 63% for OsMYB30-site1, and 58% for OsMYB30-site2. Consequently, the ospin5b mutants, the gs3 mutants, and the osmyb30 mutants exhibited increased panicle length, enlarged grain size and increased cold tolerance, respectively. Then nine transgenic lines of the ospin5b/gs3, six lines of ospin5b/osmyb30 and six lines of gs3/osmyb30 were also acquired, and their yield related traits and cold tolerance corresponded to the genes being edited. Additionally, we obtained eight ospin5b/gs3/osmyb30 triple mutants by editing all three genes simultaneously. Aside from the ospin5b/gs3/osmyb30-4 and ospin5b/gs3/osmyb30-25 mutants, the remaining six mutants had off-target events at the putative off-target site of OsMYB30-site1. The results also showed that the T2 generations of these two mutants exhibited higher yield and better cold tolerance compared with the wild type. Together, these results demonstrated that new and excellent rice varieties with improved yield and abiotic stress resistance can be generated through gene editing techniques and may be applied to rice breeding. Furthermore, our study proved that the comprehensive agronomic traits of rice can be improved with the CRISPR–Cas9 system.
Highlights
Rice is one of the most important cereal crops in the world and feeds more than half of the world’s population (Khush, 1999)
The results showed that the two triple mutants, ospin5b/gs3/ osmyb30-4 and ospin5b/gs3/osmyb30-25, in which all three genes were edited simultaneously without off-target events, exhibited higher yield and better cold tolerance compared to the wild type
The results showed that the CRISPR/Cas9 system had a high editing efficiency in the T0 generation: 53% for OsPIN5b-site1, 42% for OsPIN5b-site2, 66% for GS3-site1, 63% for GS3-site2, 63% for OsMYB30-site1, and 58% for OsMYB30-site2 (Table 1)
Summary
Rice is one of the most important cereal crops in the world and feeds more than half of the world’s population (Khush, 1999). Precise gene engineering technologies, such as the transcription activator-like effector nucleases (TALENs) (Boch and Bonas, 2010), zinc finger nucleases (ZFNs) (Kim et al, 1996) and CRISPR–Cas (Jinek et al, 2012), have become fashionable strategies for developing new elite plant varieties to alleviate food insecurity owing to their high efficiency, speed and accuracy (Li et al, 2016; Li et al, 2017; Shen et al, 2018) Among those fashionable strategies, the CRISPR–Cas system is the most popular method to solve the outstanding issues currently because of its well-researched and constant improvement (Cong et al, 2013; Li et al, 2013; Mali et al, 2013)
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