One‐Step ex Vitro Hairy Root Transformation of Soybean Using the RUBY Visible Reporter Gene

ABSTRACT Rhizobium rhizogenes (formerly Agrobacterium rhizogenes )‐mediated hairy root transformation is one of the most efficient genetic engineering methods in soybean. 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 four soybean genotypes (20SS01, Bert, Jack, 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 in‐root genetic modifications in soybeans.