Triticeae Genomics and Genetics, 2025, Vol.16, No.2, 72-78 http://cropscipublisher.com/index.php/tgg 76 5.3 Field evaluation of mutants by Nagoya University, Japan The team at Nagoya University in Japan conducted field trials. They introduced TaGW2 mutations into tetraploid and hexaploid wheat, such as splice acceptor site mutations in TaGW2-A1. These mutations significantly increased wheat grain weight, grain width and grain length under various environments. Moreover, they did not affect the number of spikelets or grains. This shows that editing TaGW2 can not only increase yield, but also has no negative effects (Simmonds et al., 2016). Editing TaGW2 does have a significant effect. However, the performance will vary between different varieties and different homologous genes. From global research, editing multiple genes together will have a better effect (Simmonds et al., 2016; Wang et al., 2018; Zhang et al., 2018). 6 Integration of CRISPR/Cas9 with Breeding Pipelines for Grain Weight Improvement 6.1 Combination with genomic selection (GS) for selection accuracy CRISPR/Cas9 can accurately edit key genes such as TaGW2. But if it is used with genomic selection (GS), the breeding effect will be better. GS uses genome-wide markers to predict which plants have potential. In this way, breeders can select plants that not only have the target edited gene but also have a good genetic background. This combination can find materials with larger grains and higher yields more quickly. This method has been proven to be effective in other crops (Awan et al., 2022; Ahmar et al., 2023). 6.2 Integration with high-throughput phenotyping (HTP) platforms High-throughput phenotyping (HTP) can quickly measure the grain size, weight and other agronomic traits of many wheat plants using machines and sensors. If HTP is combined with CRISPR/Cas9 editing, the best performing mutants can be quickly screened. This will allow for faster evaluation of the effects of these edits in different environments and genetic backgrounds. This approach will make the breeding process simpler and more efficient, and ensure that only the most promising lines advance to the next stage of promotion (Awan et al., 2022; Ahmar et al., 2023). 6.3 Introgression of edited traits into commercial cultivars Once we have found CRISPR-edited lines with heavy grains and good traits, we can use conventional hybridization methods to introduce these good genes into existing commercial varieties. In this process, marker-assisted selection can also improve efficiency. Rapid breeding and accelerated generation advancement technology can also make this process faster. This approach has been successful in other cereals. This shows that this strategy is also promising for wheat, and can help us breed high-quality non-GMO wheat varieties suitable for large-scale planting (Liang et al., 2017; Awan et al., 2022). 7 Concluding Remarks Site-directed editing of the TaGW2 gene homolog in wheat using CRISPR/Cas9 technology has been shown to significantly affect grain size and thousand-grain weight (TGW). The study found that the more sites that were mutated, the more obvious the improvement in TGW. TGW increased in single, double, and triple mutations, with the triple mutant increasing TGW by 16% to 21%. However, this phenotypic effect also varies depending on the wheat variety and the location of the edit in the genome. This suggests that the expression level of TaGW2 may be different in different varieties. Overall, these results prove that TaGW2 is a gene that suppresses grain size, and "knocking it out" can help increase yield. CRISPR/Cas9 technology can quickly and stably change key yield genes like TaGW2. It can also reduce the possibility of off-target effects, allowing the modified traits to continue in future generations. This technology can also edit multiple genes at once, which is very helpful for improving complex traits. These advances also show that gene editing technology can be gradually applied to conventional wheat breeding, helping us breed varieties with strong adaptability and high yield. In order to maximize the advantages of CRISPR/Cas9, future breeding work will also need to combine it with genomic selection, high-throughput phenotyping, and efficient gene introduction methods. This combined strategy
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