Agrobacterium-mediated high-efficiency transformation of creeping bentgrass with herbicide resistance

We performed Agrobacterium-mediated genetic transformation of creeping bentgrass (Agrostis stolonifera L.) and produced herbicide-resistant transformants from commercial cultivars Crenshaw and Penncross. Seed-derived embryogenic calli were infected with A. tumefaciens EHA105 harboring pCAMBIA 3301, which includes an intron-containing gus reporter and a bar selection marker. To establish a stable system, we examined various factors that could potentially influence transformation efficiency during the pre-culture, infection, and co-cultivation steps. The addition of kinetin to the callus pre-culture media increased efficiency about three-fold. Once the optimum infection and co-cultivation conditions were identified, this protocol was used successfully to bulk-produce herbicide-resistant transgenic plants whose herbicide resistance was confirmed using the BASTA (R) resistance test. Southern blot analysis demonstrated integration and low copy numbers of the integrated transgenes, and northern blot analysis verified their expression. Thus, we have established an efficient genetic transformation system for creeping bentgrass and confirmed a high frequency of single-copy transgene integration and functional gene expression.