Cotton Genomics and Genetics 2025, Vol.16, No.1, 12-20 http://cropscipublisher.com/index.php/cgg 16 difference between this and traditional breeding? In fact, the biggest difference is speed. The traditional method of selecting from generation to generation is so slow that it makes people worry; and this fixed-point editing technology is efficient and accurate in direction. In a few years, a batch of new cotton varieties that are truly "drought-resistant" can be screened out. 5.2 Knockout of negative regulators like GhABI1 enhances water-use efficiency In addition to enhancing positive regulatory genes, CRISPR/Cas9 can also be used to knock out negative regulatory genes such as GhABI1. These genes hinder cotton's ability to cope with drought. After knocking them out, cotton can more easily close stomata and reduce water loss, which can improve water use efficiency (Shinwari et al., 2020). This method allows cotton to grow normally and maintain a certain yield even in an environment with little water. This shows that CRISPR/Cas9 can regulate multiple links and is a good tool for dealing with complex stress responses. 5.3 Phenotypic and transcriptomic validation of edited lines It is not enough to just assume that the edited cotton has been "trained". Whether it is "trained" or not, we still have to see the actual performance. The researchers went to the fields to see that under drought conditions, these cottons did live longer, grew stronger, and the yield did not drop significantly. At least from the phenotype, the difference is quite obvious. But it is not enough to just look at the appearance. Whether the genes behind it move with it, there must be data to speak. The results of transcriptome analysis showed that these edited cottons did activate more gene pathways related to stress resistance (Rai et al., 2023). In other words, not only "live well", but also the mechanism level is right (Figure 2). It is worth mentioning that these genetic changes did not cause confusion in other important traits, and the main traits such as yield and plant type were not significantly affected (Kumar et al., 2023). This is very critical, otherwise the changes will "hurt the vitality". In the final analysis, comparing the field measurements and molecular verification, CRISPR/Cas9 is indeed reliable in improving the drought resistance of cotton. We have already obtained many good results in the experiments, and it is worth looking forward to whether it can be popularized and applied in the future. 6 Challenges in Implementing Gene Editing for Abiotic Stress Tolerance 6.1 Off-target effects and genome instability concerns Things don’t always go as planned. Gene editing is said to be “precise”, but it can also “go off-target” sometimes - this is what we often refer to as off-target effects. The target is one, but the editing tool changes something else. It’s not intentional, but the consequences can be troublesome, such as genome disruption and even unexpected trait problems. The cotton genome is large and complex, making this risk of “accidental damage” even more difficult to fully control (Erdoğan et al., 2023). Of course, researchers are not sitting back and watching. The current common approach is to optimize the design of the guide RNA and switch to smarter CRISPR tools to reduce bias - but to be honest, it is still technically difficult to achieve zero off-target effects. In this case, the accuracy of the editing is not only important for the scientific research itself, but also has a direct impact on whether it can pass regulatory approval (Khan et al., 2023). In other words, if you can’t ensure that the right place is changed and other areas are not disturbed, many subsequent links may be stuck. 6.2 Regulatory hurdles and public perception of gene-edited crops Currently, gene-edited crops have to pass the supervision of many countries, and each country has different definitions and approval rules for such crops. Some CRISPR technologies do not require the addition of foreign DNA, so they are not classified as genetically modified, which has eased the regulatory pressure to a certain extent. However, many people are still worried about its safety, environmental impact and ethical issues, and public acceptance is not high (Rahman et al., 2022). If gene-edited cotton is to be promoted, the public must be informed of the real situation, and there must also be a clear safety testing process and clear regulatory rules. 6.3 Limitations in cotton transformation and tissue regeneration efficiency Cotton is a relatively difficult crop to transform, and its tissue culture and regeneration processes are inefficient, which has become a major problem in the application of gene editing. Methods such as Agrobacterium-mediated transformation have a low success rate in cotton, which makes it difficult to breed stable edited varieties in large
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