AUTHOR=Niazian Mohsen , Belzile François , Curtin Shaun J. , de Ronne Maxime , Torkamaneh Davoud 

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

JOURNAL=Frontiers in Plant Science

VOLUME=14

YEAR=2023

URL=https://www.frontiersin.org/journals/plant-science/articles/10.3389/fpls.2023.1207762

DOI=10.3389/fpls.2023.1207762

ISSN=1664-462X

ABSTRACT=<p><italic>In vitro</italic> and <italic>ex vitro Agrobacterium rhizogenes</italic>-mediated hairy root transformation (HRT) assays are key components of the plant biotechnology and functional genomics toolkit. In this report, both <italic>in vitro</italic> and <italic>ex vitro</italic> HRT were optimized in soybean using the <italic>RUBY</italic> reporter. Different parameters including <italic>A. rhizogenes</italic> strain, optical density of the bacterial cell culture (OD<sub>600</sub>), co-cultivation media, soybean genotype, explant age, and acetosyringone addition and concentration were evaluated. Overall, the <italic>in vitro</italic> assay was more efficient than the <italic>ex vitro</italic> assay in terms of the percentage of induction of hairy roots and transformed roots (expressing <italic>RUBY</italic>). Nonetheless, the <italic>ex vitro</italic> technique was deemed faster and a less complicated approach. The highest transformation of <italic>RUBY</italic> was observed on 7-d-old cotyledons of cv. Bert inoculated for 30 minutes with the R1000 resuspended in ¼ B5 medium to OD<sub>600</sub> (0.3) and 150 µM of acetosyringone. The parameters of this assay also led to the highest percentage of <italic>RUBY</italic> through two-step <italic>ex vitro</italic> 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.</p>