STTR Phase I: Enhancing plant genetic transformation by Agrobacterium

The broader impact/commercial potential of this Small Business Technology Transfer (STTR) project is the improvement of plant genetic transformation (the introduction of new genes via biotechnology approaches). The past two decades of research and commercialization of genetically modified plants have resulted in crop species with improved agronomic traits, such as herbicide tolerance and pest resistance, which have benefitted both farmers and consumers. Whereas the vast majority of corn and soybeans currently grown in the US is genetically modified, transformation of cotton and many other crops, including sunflower, remains difficult. In addition, it has been challenging for scientists to introduce desirable genes into plants without the concomitant introduction of extraneous DNA, resulting in both technical and regulatory complications. These technical problems have limited production of important products, including rubber, in transformation-recalcitrant plants. Technology developed in this proposal will enable widespread transformation, and thereby commercialization, of a greater number of minor and industrial crop species by reducing product development costs and regulatory barriers. These transformation techniques will be applied toward the expression of industrial products including the sustainable domestic production of natural rubber in non-traditional crops, as well as enabling the creation of more efficient food crops. This STTR Phase I project proposes to develop a novel Agrobacterium-based plant transformation platform to improve the efficiency and quality of transformation events in recalcitrant crops. This technology combines the use of plant histone genes or proteins, which increases transformation efficiency, with "launching" Agrobacterium T-DNA from the bacterial chromosome, which decreases integrated transgene copy number and eliminates integration of extraneous vector sequences that present regulatory problems. Specially designed Agrobacterium strains will be tested that can transfer a histone H2A cDNA or protein simultaneously with T-DNA containing selection and reporter genes. Transformation will be conducted on Arabidopsis (because of the ease of obtaining large numbers of transformants to analyze) and on tomato (a transformation-recalcitrant but agronomically important crop species). The goal is to determine whether these novel Agrobacterium strains are able to double the transformation frequency. In addition, the plan is to analyze the genomes of hundreds of transgenic events, using qPCR and DNA blotting strategies, to determine whether transformation by these bacterial strains results in a high percentage of transgenic plants containing a single copy of the introduced transgenes without extraneous DNA sequences flanking the T-DNA (vector backbone or chromosomal DNA sequences). The best strain will be advanced for additional testing in Phase II.