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

Authors: NIAZIAN, MOHSEN - Laval University; BELZILE, FRANCOIS - Laval University; Curtin, Shaun; DE RONNE, MAXIME - Laval University; TORKAMANEH, DAVOUD - Laval University

Submitted to: Frontiers in Plant Science
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 6/22/2023
Publication Date: 7/7/2023
Citation: Niazian, M., Belzile, F., Curtin, S.J., de Ronne, M., Torkamaneh, D. 2023. Optimization of in vitro and ex vitro Agrobacterium rhizogenes-mediated hairy root transformation of soybean for visual screening of transformants using RUBY. Frontiers in Plant Science. 14. Article 1207762. https://doi.org/10.3389/fpls.2023.1207762.
DOI: https://doi.org/10.3389/fpls.2023.1207762

Interpretive Summary: Identifying genetically modified plants can be labor and time intensive but the process is accelerated by using a visual reporter gene. The GUS and luciferase marker genes have been used extensively for identifying genetically modified plants; however, they are destructive or need sophisticated equipment for detection. Other reporter genes producing fluorescent red or green proteins can affect plant growth. The RUBY marker used in this report produces three enzymes required for betalain, the red color produced in beet roots. This study optimized the use of the RUBY marker with Agrobacterium rhizogenes, bacterial strains that are naturally occurring plant pathogens. The introduction of the bacteria causes a cluster of genetically modified roots to form. The RUBY marker was visible by the naked eye in the early stages of root initiation on soybean. The strain R1000 produced the highest percentage of genetically modified roots. Use of the RUBY marker with this strain increases the efficiency and reliability of identifying genetically modified soybean plants, accelerating the characterization of genes for improving soybean agronomic traits.

Technical Abstract: In vitro and ex vitro Agrobacterium rhizogenes-mediated hairy root transformation techniques are key components of the plant biotechnology and functional genomics toolkit. Here, both in vitro and ex vitro hairy root transformation techniques were optimized in soybean using a visible reporter gene, RUBY. Overall, in vitro transformation proved more efficient than ex vitro transformation in terms of the percentage of induction of hairy roots and transformed roots (expressing RUBY). Nonetheless, the ex vitro technique was deemed a faster and simpler approach. Different parameters including A. rhizogenes strain, density of the bacterial culture (OD600), inoculation duration, soybean cultivar, explant age, type of root induction media, and acetosyringone concentration were tested during in vitro transformation. The highest transformation percentage of RUBY gene was obtained by inoculation of 7-d-old cotyledons of cv. Bert with the R1000 strain at an OD600=0.3 for 30 min, followed by incubation of infected explants in ¼ B5 medium supplemented with 150 µmol/L of acetosyringone. Inoculation of 7-d-old seedlings of cv. Bert with the R1000 strain led to the highest percentage of RUBY gene delivery through two-step ex vitro hairy root transformation. Finally, using machine learning-based modeling, optimal protocols (leading to the highest percentage of transformation) were defined. For the in vitro protocol this included the following parameters: R1000 strain/OD600=0.32/29 min inoculation/cv. Bert/7-d-old cotyledons/½ B5 root induction medium/126.74 µmol/L of acetosyringone. For the ex vitro approach, this involved: the R1000 strain/cv. Bert /8.66-d-old seedlings. The presence and constant expression of RUBY were confirmed in transgenic roots by PCR and RT-qPCR. This study establishes efficient and reliable hairy root transformation protocols applicable for functional studies in soybean.