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SummaryA simple modi®cation to standard binary vector design has been utilized to enrich an Agrobacteriumtransformed population for plants containing only T-DNA sequences. A lethal gene was incorporated into the non-T-DNA portion of a binary vector, along with a screenable marker. The resulting class of vectors is designated as NTL T-DNA vectors (non-T-DNA lethal gene-containing T-DNA vectors). The lethal gene used here is a CaMV 35S-barnase gene with an intron in the coding sequence (barnase-INT); the screenable marker is a pMAS-luciferase gene with an intron in the coding sequence (LUC-int). To evaluate the utility of this vector design, tobacco plants were transformed with either the NTL T-DNA vector or a control vector from which most of the barnase-INT gene was deleted. Populations of 50 transgenic plants were scored for LUC expression. The results indicated a dramatic reduction in the presence of non-T-DNA sequences in the transgenic population using the NTL T-DNA vector. Only one transgenic plant was found to be LUC+ using the NTL vector, compared with 42 of 50 plants using the control vector. Importantly, the ef®ciency with which transformed tobacco plants was obtained was reduced by no more than 30%. The reduction in LUC+ transgenics was partially reversed when a barstar-expressing tobacco line was transformed, indicating that barnase expression was responsible for the reduced frequency of incorporating non-T-DNA sequences. Similar transformation results were obtained with tomato and grape. The incorporation of a barnase-INT gene outside the left border appears to provide a generally applicable tool for enriching an Agrobacterium-transformed population for plants containing only T-DNA sequences.

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We have developed an efficient method for transformation and regeneration of plants from carnation, Dianthus caryophyllus L. Whole leaves from in vitro shoot cultures were mixed with Agrobacterium, cocultivated for 5 days and then plated on 2 #g/1 chlorsulfuron (CS). Regenerated shoots and shoot clusters were divided into smaller sections and plated on 3 #g/1 CS for selection to produce fully transformed shoots. Geneticin (G418) and kanamycin used were not as effective selective agents as CS. All regenerated shoots were vitrified. These were normalized, rooted and transferred to the greenhouse. 100~'o ®enerated plants were transformed based on rooting assay, GUS assay, PCR and Southern analysis.

Summary 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.