Ploidy variation among herbicide-resistant bermudagrass plants of cv. TifEagle transformed with the bar gene

A protocol was developed for biolistic transformation of hybrid bermudagrass cv. TifEagle using the bar gene. TifEagle is an ultradwarf used exclusively on golf greens. Herbicide resistance should serve as a useful management tool, especially if methyl-bromide is unavailable for fumigation prior to plant establishment. Hybrid bermudagrass is completely sterile, which should limit the chance of gene escape via out-crossing. Sliced nodes were used to initiate embryogenic tissue cultures on MS medium supplemented with 1 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D) and 0.01 mg/l 6-benzylaminopurine (BA). Embryogenic tissue was bombarded with the bar gene, and herbicide-resistant tissue was selected in the dark on medium supplemented with 0.75 mg/l 2,4-D, 0.01 mg/l BA and 5-15 mg/l phosphinothricin (PPT). Resistant somatic embryos were induced to germinate in the light on MS medium supplemented with 0.13 mg/l 2,4-D and 0.5 mg/l BA. Plants were transferred to the greenhouse after rooting in the presence of 10-15 mg/l PPT and testing positive in a chlorophenol red assay. A total of 89 herbicide-resistant plants were recovered from at least nine independent events from six separate bombardments, although the number of independent transformation events was not confirmed for the entire group. Flow cytometry indicated that most of the plants (82/89) were hexaploid and that the remaining seven plants were triploid. The hexaploid plants were a darker green than the triploids or TifEagle control. Other variation, present only in the hexaploids, included an increased leaf width and length. Southern blot hybridization confirmed genomic integration of the bar gene in triploid and a subset of hexaploid herbicide-resistant plants. AFLP analysis did not indicate changes in DNA profiles using [P-33] and a sample of 32 hexaploid plants recovered from a single bombardment. DNA profiles were very similar to that of the TifEagle control with a semi-automated fluorescence-based AFLP.