Advisor: Dr. John Finer
Monday June 23, 2014
1:30 – 2:30 pm
123 Williams Hall (Wooster) video linked to 139 Howlett Hall (Columbus)
“Isolation of Novel Agrobacterium and Transient Expression Assays in Soybean (Glycine max) and Sunflower (Helianthus annuus)”
Many laboratories routinely use Agrobacterium for the generation of transgenic plants, however only a few bacterial strains are widely available and none of these strains have been isolated and selected based on their transformation efficiency of specific plants. For this research, new Agrobacterium strains were isolated from rhizospheric, soil collected from soybean fields throughout the Midwest US and from crown galls of various plants in Ohio. These wild-type strains were isolated by plating gall and soil extracts on 1A semi-selective medium and screening the isolates for the presence of virG using PCR. The newly isolated wild-type virG positive strains as well as some previously identified wild-type strains were evaluated for transformation efficiency in sunflower and soybean. The efficiency of plant transformation was evaluated by quantifying expression of the green fluorescent protein gene, which was transformed by these strains into hypocotyl and cotyledonary tissues of sunflower and soybean seedlings, and in proliferative embryogenic tissue of soybean. 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 using strain EHA105. Sunflower hypocotyl tissues showed high tissue specific transformation with EHA105, as greater than 75% of the transformed cells were located in the vascular 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 single strain from a soybean field in North Dakota gave 5-50x higher transformation rates than EHA105, while a different strain from Ohio soil gave 5-10x higher rates.