Development of an efficient marker-free soybean transformation method using the novel bacterium Ochrobactrum...

We have discovered a novel bacterium, Ochrobactrum haywardense H1 (Oh H1), which is capable of efficient plant transformation. Ochrobactrum is a new host for Agrobacterium-derived vir and T-DNA-mediated transformation. Oh H1 is a unique, non-phytopathogenic species, categorized as a BSL-1 organism. We engineered Oh H1 with repurposed Agrobacterium virulence machinery and demonstrated Oh H1 can transform numerous dicot species and at least one monocot, sorghum. We generated a cysteine auxotrophic Oh H1-8 strain containing a binary vector system. Oh H1-8 produced transgenic soybean plants with an efficiency 1.6 times that of Agrobacterium strain AGL1 and 2.9 times that of LBA4404Thy-. Oh H1-8 successfully transformed several elite Corteva soybean varieties with T0 transformation frequency up to 35%. In addition to higher transformation efficiencies, Oh H1-8 generated high-quality, transgenic events with single-copy, plasmid backbone-free insertion at frequencies higher than AGL1. The SpcN selectable marker gene is excised using a heat shock-inducible excision system resulting in marker-free transgenic events. Approximately, 24.5% of the regenerated plants contained only a single copy of the transgene and contained no vector backbone. There were no statistically significant differences in yield comparing T3 null-segregant lines to wild-type controls. We have demonstrated that Oh H1-8, combined with spectinomycin selection, is an efficient, rapid, marker-free and yield-neutral transformation system for elite soybean.