Development of an efficient marker-free soybean transformation method using the novel bacterium Ochrobactrum haywardense H1.

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.

An Improved Variant of Soybean Type 1 Diacylglycerol Acyltransferase Increases the Oil Content and Decreases the Soluble Carbohydrate Content of Soybeans.

Kinetically improved diacylglycerol acyltransferase (DGAT) variants were created to favorably alter carbon partitioning in soybean (Glycine max) seeds. Initially, variants of a type 1 DGAT from a high-oil, high-oleic acid plant seed, Corylus americana, were screened for high oil content in Saccharomyces cerevisiae Nearly all DGAT variants examined from high-oil strains had increased affinity for oleoyl-CoA, with S0.5 values decreased as much as 4.7-fold compared with the wild-type value of 0.94 µm Improved soybean DGAT variants were then designed to include amino acid substitutions observed in promising C. americana DGAT variants. The expression of soybean and C. americana DGAT variants in soybean somatic embryos resulted in oil contents as high as 10% and 12%, respectively, compared with only 5% and 7.6% oil achieved by overexpressing the corresponding wild-type DGATs. The affinity for oleoyl-CoA correlated strongly with oil content. The soybean DGAT variant that gave the greatest oil increase contained 14 amino acid substitutions out of a total of 504 (97% sequence identity with native). Seed-preferred expression of this soybean DGAT1 variant increased oil content of soybean seeds by an average of 3% (16% relative increase) in highly replicated, single-location field trials. The DGAT transgenes significantly reduced the soluble carbohydrate content of mature seeds and increased the seed protein content of some events. This study demonstrated that engineering of the native DGAT enzyme is an effective strategy to improve the oil content and value of soybeans.