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

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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.

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Acknowledgments

Salaries and research support were provided by the United Soybean Board, Hiram College and by State and Federal funds appropriated to Kent State University and The Ohio State University/Ohio Agricultural Research and Development Center. The authors thank Alex Butcher, Hannah Petcovic, Chris Shalaty, and Lauren Shunkwiler, undergraduates at Hiram College who participated in the initial isolation of Agrobacterium strains from soil and gall samples. Mention of trademark or proprietary products does not constitute a guarantee or warranty of the product and also does not imply approval to the exclusion of other products that may also be suitable.

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Authors and Affiliations

  1. Department of Horticulture and Crop Science, The Ohio State University, 1680 Madison Avenue, Wooster, OH, 44691, USA

    Kyle A. Benzle & John J. Finer

  2. Department of Biological Sciences, Kent State University, Canton, OH, 44720, USA

    Kim R. Finer

  3. Department of Plant Pathology, The Ohio State University, Wooster, OH, 44691, USA

    DeeMarie Marty & Christopher G. Taylor

  4. Department of Horticulture and Crop Science, The Ohio State University, Columbus, OH, 43210, USA

    Leah K. McHale

  5. Department of Biology, Hiram College, Hiram, OH, 44234, USA

    Brad W. Goodner

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  1. Kyle A. Benzle
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  2. Kim R. Finer
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  4. Leah K. McHale
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Correspondence to John J. Finer.

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Benzle, K.A., Finer, K.R., Marty, D. et al. Isolation and characterization of novel Agrobacterium strains for soybean and sunflower transformation. Plant Cell Tiss Organ Cult 121, 71–81 (2015). https://doi.org/10.1007/s11240-014-0679-x

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  • DOI: https://doi.org/10.1007/s11240-014-0679-x

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