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ARS Home » Midwest Area » St. Paul, Minnesota » Plant Science Research » Research » Publications at this Location » Publication #411636
Research Project: Gene Discovery and Trait Improvement in Soybean, Alfalfa, and Other Legumes by Gene Editing

Location: Plant Science Research

Title: One-step ex vitro hairy root transformation of soybean using the RUBY visible reporter gene

Author
item NIAZIAN, MOHSEN - University Of Laval
item BELZILE, FRANÇOIS - University Of Laval
item Curtin, Shaun
item TORKAMANEH, DAVOUD - University Of Laval

Submitted to: Genome
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 1/12/2026
Publication Date: 2/15/2026
Citation: Niazian, M., Belzile, F., Curtin, S.J., Torkamaneh, D. 2026. One-step ex vitro hairy root transformation of soybean using the RUBY visible reporter gene. Genome. 8(1). Article e70081. https://doi.org/10.1002/leg3.70081.
DOI: https://doi.org/10.1002/leg3.70081

Interpretive Summary: An essential tool in crop improvement is to generate genetically modified plants, but the process for introducing genes is labor- and time-intensive. For soybean, producing a genetically modified plant can take 12 months or more. To facilitate the evaluation of genes important in root function, genetically modified roots can be produced on young shoots but a simple method for identifying the modified roots is needed. We report an optimized tissue-culture free, one-step protocol for producing genetically modified roots coupled with a visual reporter called RUBY that produces a red color in modified roots. Using the optimized assay, a transformation efficiency of 78% was obtained on 7-d-old soybean seedlings. The improved efficiency and reliability of this new assay will facilitate the characterization of genes for improving soybean agronomic traits.

Technical Abstract: Rhizobium rhizogenes (formerly Agrobacterium rhizogenes)-mediated hairy root transformation is one of the most efficient genetic engineering methods in soybean. The ex vitro hairy root induction, coupled with a visually detectable reporter gene, represents a convenient technique for achieving successful hairy root transformation. In this study, we explored the feasibility of using the RUBY reporter gene in a one-step ex vitro (hypocotyl cutting) transformation protocol in soybean. The inoculated seedlings were cultivated in growth pouches, facilitating easy monitoring of transformation efficiency and visualization of the resulting red transgenic roots. The R. rhizogenes strains R1000, ARqual, and K599 demonstrated a 100% hairy root induction in three soybean genotypes (20SS01, Bert, and Williams). The highest transformation efficiency (78.33%) was obtained by inoculation of 7-d-old seedlings of cv. Bert with the R1000 strain. This streamlined method offers a rapid and cost-effective means for real-time, high-throughput phenotyping of transgenic roots. The utilization of the RUBY reporter gene in conjunction with ex vitro hairy root transformation represents a promising avenue to accelerate genetic modifications in soybeans.