Isolation and characterization of novel Agrobacterium strains for soybean and sunflower transformation

Abstract

Although many laboratories routinely use Agrobacterium for the generation of transgenic plants, only a few bacterial strains are widely used. For this research, new Agrobacterium strains were isolated from galls and rhizospheric soil collected across the midwestern United States. Strains were isolated by plating gall and soil extracts on a semi-selective medium and screening the isolates for the presence of virG using PCR. New wild type virG positive strains as well as previously isolated, known wild type and disarmed strains were evaluated for transformation efficiency in hypocotyl and cotyledonary tissues of sunflower and soybean seedlings and in proliferative embryogenic tissue of soybean. For evaluation of transformation efficiency, a plant-expressible green fluorescent protein (gfp) gene in a binary vector was introduced into each strain, and GFP expression was quantified in plant tissues, 4–5 day post inoculation. In sunflower, seedling cotyledonary tissue was not responsive to any of the strains tested; however, hypocotyl tissues were very responsive with the highest transformation rates obtained with the widely used EHA105 strain. With sunflower hypocotyl tissues inoculated with EHA105, the vascular tissue was exceptionally responsive with >75 % of the GFP-expressing cells located in that tissues. In soybean seedling tissues, tissue specific transformation was not observed with any strain as transformed cells were evenly distributed throughout target tissue. With soybean hypocotyl, cotyledon, and embryogenic tissues, a new strain named “JTND”, isolated from soil from a soybean field gave 7–25x higher transformation rates than EHA105, while a strain called “KFOH”, from soil that was not under cultivation, gave 5–12x higher rates than EHA105.