Genome-Wide Association Analysis of Soybean Regeneration-Related Traits and Functional Exploration of Candidate Genes

Using the cotyledonary node method, four traits related to callus induction rate were identified in 185 soybean germplasm resources. Cultivation of callus tissue is crucial for soybean (<i>Glycine max</i> (L.) Merr.) genetic transformation and functional genomics studies. Identifying genes associated with the induction rate of soybean callus tissue is therefore essential for biotechnological breeding and for understanding the molecular genetic mechanisms of soybean regeneration. The efficiency of genetic transformation impacts the breeding rate of soybeans, with its success rate dependent on the soybean regeneration system. Subsequently, whole genome association analysis (GWAS) and multidimensional functional validation were conducted. GWAS identified 66 significantly associated SNP loci corresponding to the four traits. Expression analysis in extreme phenotypes highlighted four candidate genes: <i>Glyma.12G164100</i> (<i>GmARF1</i>), <i>Glyma.12G164700</i> (<i>GmPPR</i>), <i>Glyma.02G006200</i> (<i>GmERF1</i>), and <i>Glyma.19G128800</i> (<i>GmAECC1</i>), which positively regulate callus formation. Overexpression and gene-editing assays in hairy roots confirmed that these genes significantly enhanced callus formation rate and density, with <i>GmARF1</i> exerting the most prominent effect. Hormone profiling revealed elevated levels of gibberellin (GA), auxin (IAA), cytokinin (CTK), and other phytohormones in transgenic lines, consistent with enhanced responsiveness to exogenous GA. Overall, the results suggest that these four candidate genes may promote soybean regeneration, with <i>GmARF1</i> showing the most pronounced effect. These results provide valuable genetic resources for improving soybean regeneration efficiency and accelerating genetic transformation-based breeding.