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

Abstract

<i>In vitro</i> and <i>ex vitro Agrobacterium rhizogenes</i>-mediated hairy root transformation (HRT) assays are key components of the plant biotechnology and functional genomics toolkit. In this report, both <i>in vitro</i> and <i>ex vitro</i> HRT were optimized in soybean using the <i>RUBY</i> reporter. Different parameters including <i>A. rhizogenes</i> strain, optical density of the bacterial cell culture (OD₆₀₀), co-cultivation media, soybean genotype, explant age, and acetosyringone addition and concentration were evaluated. Overall, the <i>in vitro</i> assay was more efficient than the <i>ex vitro</i> assay in terms of the percentage of induction of hairy roots and transformed roots (expressing <i>RUBY</i>). Nonetheless, the <i>ex vitro</i> technique was deemed faster and a less complicated approach. The highest transformation of <i>RUBY</i> was observed on 7-d-old cotyledons of cv. Bert inoculated for 30 minutes with the R1000 resuspended in ¼ B5 medium to OD₆₀₀ (0.3) and 150 µM of acetosyringone. The parameters of this assay also led to the highest percentage of <i>RUBY</i> through two-step <i>ex vitro</i> 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.