Molecular Plant Breeding 2025, Vol.16, No.1, 63-72 http://genbreedpublisher.com/index.php/mpb 67 4 Case Study: Gene Editing for Enhancing Drought Tolerance 4.1 Overview of CRISPR/Cas9 technology in plant breeding CRISPR/Cas9 technology has revolutionized plant breeding by enabling precise and efficient genome editing. This system utilizes a guide RNA (gRNA) to direct the Cas9 nuclease to specific DNA sequences, where it introduces double-strand breaks. These breaks are then repaired by the plant’s natural repair mechanisms, leading to targeted mutations. The simplicity, adaptability, and wide applicability of CRISPR/Cas9 make it a powerful tool for developing crop varieties with enhanced traits, including drought tolerance (Joshi et al., 2020; Kar et al., 2022; Kumar et al., 2023). 4.2 Application of CRISPR/Cas9 in modifying drought-responsive genes in soybeans One notable example of CRISPR/Cas9 application in soybeans is the editing of the GmHdz4 gene, a homeodomain-leucine zipper transcription factor. This gene plays a crucial role in drought stress response. By using CRISPR/Cas9 to create gmhdz4 mutants, researchers observed enhanced drought tolerance in soybean plants. The edited plants exhibited better growth, improved root system architecture, and higher activity of antioxidant enzymes, which helped in maintaining turgor pressure and reducing oxidative stress under drought conditions (Zhong et al., 2022). Another study targeted the GmFAD2 genes, which are involved in fatty acid desaturation. By editing these genes, researchers were able to produce soybean plants with a high oleic acid content, which is associated with improved drought tolerance. The dual gRNA CRISPR/Cas9 system used in this study demonstrated high efficiency in creating targeted mutations, resulting in significant changes in the fatty acid profile of the soybean seeds (Do et al., 2019). 4.3 Potential and challenges of CRISPR/Cas9 for developing drought-resilient varieties While CRISPR/Cas9 offers tremendous potential for developing drought-resilient soybean varieties, there are challenges that need to be addressed. One major concern is the off-target effects, where unintended genomic regions may be edited, leading to undesirable traits. Efforts to mitigate these effects include the use of shorter gRNAs and dual Cas9 nickases, which increase the specificity of the editing process (Erdoğan et al., 2023). Regulatory considerations also play a significant role in the adoption of CRISPR/Cas9-edited crops. The regulatory landscape varies across countries, with some viewing CRISPR-edited plants as genetically modified organisms (GMOs) and others not. This inconsistency can affect the commercialization and acceptance of CRISPR-edited crops. Therefore, it is crucial to develop clear and consistent regulatory frameworks to facilitate the use of CRISPR/Cas9 technology in agriculture (Joshi et al., 2020; Erdoğan et al., 2023). In conclusion, CRISPR/Cas9 technology holds great promise for enhancing drought tolerance in soybeans by enabling precise modifications of drought-responsive genes. However, addressing off-target effects and navigating regulatory challenges are essential for the successful implementation of this technology in developing drought-resilient crop varieties. 5 Integrative Approaches in Studying Drought Tolerance 5.1 Systems biology approaches and integrative analysis The integration of multiple omics approaches, such as transcriptomics, proteomics, metabolomics, and phenomics, provides a comprehensive understanding of the complex mechanisms underlying drought tolerance in soybean. For instance, transcriptome profiling has identified numerous differentially expressed genes (DEGs) associated with drought tolerance, including those involved in water and auxin transport, cell wall/membrane integrity, antioxidant activity, and secondary metabolism (Aleem et al., 2020; Shahriari et al., 2022). Proteomic and metabolomic analyses further elucidate the biochemical pathways and metabolic networks that are activated in response to drought stress, such as photosynthesis and cytokinin dehydrogenase activity. Phenomics, which involves high-throughput phenotyping, allows for the assessment of drought tolerance traits at the whole-plant level, providing valuable data for breeding programs (Dubey et al., 2019; Fatema et al., 2023). Network analysis and predictive modeling are essential tools for understanding the regulatory networks that govern drought responses in soybean. Gene co-expression analysis and protein-protein interaction (PPI) networks have identified key hub genes and transcription factors that play central roles in drought tolerance (Shahriari e al.,
RkJQdWJsaXNoZXIy MjQ4ODYzNA==