Plant Symposia and Workshops

During 30 years of plant transformation technology development, a great deal of focus has been on expanding transformable species and increasing transformation frequency through tissue culture optimization. Transgene integration quality has not been widely addressed although single-copy transgene events lacking vector backbone are desired for most biotechnology applications. We have explored molecular approaches to improve transformation frequency and transgene integration quality in Agrobacterium-mediated crop transformation. Through transformation vector modification, such as the addition of a growth retarding gene in the vector backbone, a significantly higher frequency of singlecopy, backbone-free transgenic events can be achieved. We have also demonstrated that a vector with a low copy origin of replication in Agrobacterium can significantly increase single copy, backbone-free event frequency in transformation of several crops species. Through site-directed mutation analysis of an ori pRi backbone, we identified a higher copy ori pRi vector, which abolishes the advantage of single copy transgene frequency improvement in crop transformation. In another approach, we have shown that transformation frequency in cotton can be significantly improved by using modified Agrobacterium strains with a constituitive active virG allele.