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A novel Agrobacterium rhizogenes-mediated transformation method using a primary-node explant from Dairyland cultivar 93061 was developed for soybean using the disarmed Agrobacterium strain SHA17. Transformed plants regenerated from explants inoculated with SHA17 were fertile and phenotypically normal. In a comparative experiment, regeneration frequencies were not significantly different between explants inoculated with A. rhizogenes strain SHA17 and Agrobacterium tumefaciens strain AGL1; however, a 3.5-fold increase in transformation efficiency [(number of Southern or TaqMan-positive independent events/total number of explants inoculated) × 100] was found for explants cocultured with SHA17 compared to AGL1 (6.6 and 1.64%, respectively). Southern analysis of 48 T0 plants suggested that 37.5, 23, and 39.6% of the T0 plants contained 1, 2, and 3 or more T-DNA fragments integrated into the genome, respectively. Additionally, T1 progeny analysis of 8 independent events resulted in typical Mendelian inheritance of T-DNA genes. Of seven T0 plants that had two or more T-DNA fragments, six contained multiple loci segregating in T1 progenies. Further analysis of four lines confirmed the presence of PAT, GUS, and/or DsRED2 proteins in transgenic plants that were encoded on the T-DNA into the T2 generation.