Plant Gene and Traits 2024, Vol.15, No.3, 141-151 http://genbreedpublisher.com/index.php/pgt 144 4.2 Success stories: field applications and yield improvements Field applications of GS2 gene manipulation have shown promising results in improving rice yield. For example, the study by Usman et al. (2021) utilized CRISPR/Cas9 to edit the GS3 gene, which is closely related to GS2, resulting in increased grain length and weight (Figure 2). The mutants exhibited a 31.39% increase in grain length and a 27.15% increase in 1 000-grain weight compared to wild-type plants. This demonstrates the potential of gene editing technologies in enhancing rice yield through targeted manipulation of genes like GS2. Additionally, the study by Zhang et al. (2020) developed functional markers for 14 genes related to grain size, including GS2. These markers were used to genotype a global collection of rice cultivars, revealing significant trait contributions fromGS2. The successful application of these markers in breeding programs has facilitated the selection of lines with superior grain yield and quality, underscoring the practical benefits of GS2 gene manipulation in field conditions. Figure 2 Schematic diagram of the procedure for CRISPR/Cas9-based generation of mutant plants and analysis of mutations and Diagramof GS3gene and positions of both target sites. ATG is the start codon (Adopted from Usman et al., 2021) Image caption: (A) Schematic diagram of the procedure for CRISPR/Cas9-based generation of mutant plants and analysis of mutations. Two sgRNAs were selected using the CRISPR-GE online web-based tool, and vector was constructed. Agrobacterium-mediated transformation was performed, and T0 plants were regenerated. Later generations were produced by self-pollination, and genotyping was performed using target-specific primers. The phenotypic data of mutant and wild-type (WT) plants were recorded and further analyzed. The proteomic analysis was also performed, and RT-qPCR was performed to assess the GS3 expression level and validate the proteomic data. (B) Diagram of GS3 gene and positions of both target sites. ATG is the start codon; TGA is the stop codon; green highlighted CGG and TGG are the PAM sequences; the white boxes at extreme left and right represent the untranslated (UTR) regions, the black boxes represents the exons; black lines in between the exon regions represent the intron regions, T1 and T2 represent target 1 and target 2, respectively (Adopted from Usman et al., 2021) Usman et al. (2021) found that the use of CRISPR/Cas9 technology effectively generates mutant plants by targeting specific genes. In their study, they selected two sgRNAs to create mutations in the GS3 gene, which influences grain size. The vector construction and Agrobacterium-mediated transformation led to the successful regeneration of T0 plants. Subsequent self-pollination produced later generations, which were genotyped to confirm the presence of mutations. Phenotypic analysis compared mutant plants to wild-type (WT) plants, and the results showed significant differences in growth and development. Proteomic analysis and RT-qPCR were used to assess GS3 expression levels, validating the impact of the mutations on plant phenotype. This approach
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