Optimization of in vitro and ex vitro Agrobacterium rhizogenes -mediated hairy root transformation of soybean for visual screening of transformants using RUBY .

and -mediated hairy root transformation (HRT) assays are key components of the plant biotechnology and functional genomics toolkit. In this report, both and HRT were optimized in soybean using the reporter. Different parameters including strain, optical density of the bacterial cell culture (OD), co-cultivation media, soybean genotype, explant age, and acetosyringone addition and concentration were evaluated. Overall, the assay was more efficient than the assay in terms of the percentage of induction of hairy roots and transformed roots (expressing ). Nonetheless, the technique was deemed faster and a less complicated approach. The highest transformation of was observed on 7-d-old cotyledons of cv. Bert inoculated for 30 minutes with the R1000 resuspended in ¼ B5 medium to OD (0.3) and 150 µM of acetosyringone. The parameters of this assay also led to the highest percentage of through two-step hairy root transformation. Finally, using machine learning-based modeling, optimal protocols for both assays were further defined. This study establishes efficient and reliable hairy root transformation protocols applicable for functional studies in soybean.