BIOTECHNOLOGY/GENETIC TRANSFORMATION/FUNCTIONAL GENOMICS Optimized Agrobacterium-mediated sorghum transformation

Abstract Agrobacterium-mediated sorghum transformation frequency has been enhanced significantly via medium opti-mization using immature embryos from sorghum variety TX430 as the target tissue. The new transformation protocol includes the addition of elevated copper sulfate and 6-benzylaminopurine in the resting and selection media. Using Agrobacterium strain LBA4404, the transformation frequen-cy reached over 10 % using either of two different selection marker genes, moPAT or PMI, and any of three different vectors in large-scale transformation experiments. With Agrobacterium strain AGL1, the transformation frequencies were as high as 33%. Using quantitative PCR analyses of 1, 182 T0 transgenic plants representing 675 independent trans-genic events, data was collected for T-DNA copy number, intact or truncated T-DNA integration, and vector backbone integration into the sorghum genome. A comparison of the transformation frequencies and molecular data characterizing T-DNA integration patterns in the transgenic plants derived from LBA4404 versus AGL1 transformation revealed that twice as many transgenic high-quality events were generated when AGL1 was used compared to LBA4404. This is the first report providing molecular data for T-DNA integration patterns in a large number of independent transgenic plants in sorghum