Optimized Agrobacterium-mediated sorghum transformation protocol and molecular data of transgenic sorghum plants

Abstract

<i>Agrobacterium</i>-mediated sorghum transformation frequency has been enhanced significantly <i>via</i> medium optimization 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 <i>Agrobacterium</i> strain LBA4404, the transformation frequency 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 <i>Agrobacterium</i> strain AGL1, the transformation frequencies were as high as 33%. Using quantitative PCR analyses of 1,182 T₀ transgenic plants representing 675 independent transgenic 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.