Plant Gene and Traits 2024, Vol.15, No.3, 141-151 http://genbreedpublisher.com/index.php/pgt 145 demonstrates the precision and effectiveness of CRISPR/Cas9 in plant genetic engineering, providing a robust method for studying gene function and improving crop traits. 4.3 Comparative analysis with non-GS2 modified rice varieties Comparative studies between GS2-modified and non-modified rice varieties have provided insights into the effectiveness of GS2 manipulation. The study by Gull et al. (2019) investigated the contribution of multiple genes, including GS2, to grain size and weight in 204 diverse rice germplasms. The results showed that GS2 was significantly associated with grain length, width, and thickness, indicating its pivotal role in determining grain size. The non-GS2 modified varieties displayed a wide range of variability in these traits, highlighting the potential for improvement through GS2 manipulation.Furthermore, the study by Ngangkham et al. (2018) examined the influence of seven known grain size-regulating genes, including GS2, in 89 rice germplasms. The findings revealed that GS2, along with other genes, showed strong associations with grain size traits. The non-modified varieties exhibited less favorable grain size characteristics compared to those with targeted gene modifications, demonstrating the advantages of GS2 manipulation in achieving desired agronomic traits. In summary, the manipulation of the GS2 gene has shown significant potential in improving rice yield through various genetic and molecular approaches. Field applications and comparative analyses further support the effectiveness of GS2 gene modification in enhancing grain size and weight, making it a valuable target for rice breeding programs. 5 Technological Advances in Studying the GS2 Gene 5.1 Recent developments in genetic engineering and CRISPR Recent advancements in genetic engineering, particularly the CRISPR/Cas9 system, have significantly enhanced our ability to manipulate genes associated with grain size in rice. CRISPR/Cas9 has been effectively used to edit multiple genes, including GS3 and GL3.1, which are closely related to grain size and weight. For instance, simultaneous editing of GS3 and GL3.1 in rice resulted in mutants with larger grains, although it also led to a reduction in grain number and overall yield, highlighting the complexity of genetic interactions in grain size regulation (Chen et al., 2020). Additionally, CRISPR/Cas9-mediated mutagenesis of the GS3 gene has been shown to increase grain length and weight by regulating proteins involved in cellular processes such as cysteine proteinase inhibitors and ubiquitin-related proteins (Usman et al. 2021). These studies underscore the potential of CRISPR/Cas9 in creating rice varieties with improved grain characteristics. 5.2 Advances in genomic sequencing techniques for GS2 The advent of high-throughput genomic sequencing techniques has revolutionized the study of genes like GS2. Techniques such as genome-wide association studies (GWAS) and single nucleotide polymorphism (SNP) arrays have been instrumental in identifying quantitative trait loci (QTLs) associated with grain size. For example, a GWAS identified the novel grain size gene OsSNB, which negatively regulates grain size, and demonstrated that knockout mutants of this gene exhibit increased grain length, width, and weight (Figure 3) (Ma et al., 2019). Similarly, chromosome segment substitution lines (CSSLs) have been used to map QTLs for grain size, leading to the identification of genes such as OsGH3.13, which influences grain length and weight (Tan et al., 2021). These genomic tools provide a comprehensive understanding of the genetic basis of grain size and facilitate the identification of candidate genes for targeted breeding. Ma et al. (2019) found that the OsSNB gene plays a critical role in rice plant development. Through the application of CRISPR/Cas9 technology, they successfully generated knockout mutant lines (KO1 and KO2) and overexpression lines (OE1 and OE2) to study the gene’s function. The relative expression levels of OsSNB were significantly altered in these transgenic lines compared to the wild type (WT). Specifically, overexpression lines exhibited elevated OsSNB expression, while knockout lines showed reduced expression. The phenotypic analysis revealed notable differences in plant morphology and growth between the transgenic lines and WT plants, highlighting the impact of OsSNB manipulation. This study underscores the importance of OsSNB in regulating key developmental processes in rice and demonstrates the potential of gene editing technologies in agricultural biotechnology.
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