Rice Genomics and Genetics 2025, Vol.16, No.3, 140-149 http://cropscipublisher.com/index.php/rgg 144 additional performances may not have been planned at first, but later proved to be quite valuable, especially in broadening the diversity of agronomic traits. In general, these mutants have not only improved in ear shape and grain size, but also enhanced yield-related traits. It seems that multiple editing can indeed bring a lot of usable changes in one generation of plants. Figure 2 CRISPR/Cas9-induced gs3 mutant plants and phenotype analysis. (A) Schematic map of the genomic region of GS3 and the sgRNA target site; arrows show the positions of PCR primers used for mutation detection; The PAM motif (NGG) is shown in red; (B) Gel electrophoresis of PCR products amplified from the mutated region digested with CEL I; WT and 1-9 are DNA samples from wild type and different transgenic plants. Arrows show the expected band sizes after CEL I digestion. (C) Sequence alignment of the sgRNA target region showing altered bases in different mutant lines; (D) Representative pictures showing the morphology of the main panicle; (E) Phenotype of the mutant plants grown in a greenhouse; (F) Comparison of the grain size of mutant gs3-5 plants with that of WT plants; statistics for the average grain length (G) and average grain weight (H) of representative mutant plants. Data were collected from 10 to 15 plants per mutant line. * indicates a significant difference (P<0.05) in comparison to WT counterparts (Adopted from Li et al., 2016) 5.3 Implications for breeding and scalability This study shows that through multiple CRISPR editing, multiple important yield-related traits can be improved in one variety at the same time, making the breeding process simpler. Stacking multiple beneficial genes in the same genetic background also makes it easier to figure out the regulatory relationship between genes, which is very helpful for accelerating the breeding of superior varieties. Because these edited traits can be efficiently expressed and stably inherited, this method has great potential in actual rice breeding and is suitable for large-scale promotion and application.
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