Assessment of Pollen-Mediated Intraspecific Gene Transfer between Sexual and Apomictic Cytotypes of Bahiagrass (Paspalum notatum).

Bahiagrass is the predominant forage grass in the southeastern United States. Sexual, diploid (2n=20) and tetraploid (2n=40) apomictic cytotypes of bahiagrass (Paspalum notatum Flugge) are grown.  The tetraploid cytotypes, including the commercially important cultivar ‘Argentine’ are obligate apomicts, while the sexual types are cross pollinating and self-incompatible.  The primary objective of the present study was to investigate pollen mediated gene transfer from apomictic, tetraploid bahiagrass to sexual, diploid bahiagrass using glufosinate resistance as a marker. Glufosinate resistant and fertile transgenic bahiagrass was generated by biolistic gene transfer and used as pollen donor in a field trial (USDA-Aphis permit # 05-365-01r) in Marianna, Florida with two replications. Seeds were harvested from wild type, sexual, diploid bahiagrass surrounding the transgenic glufosinate resistant bahiagrass in 1 m distance. More than 28,000 seedlings were sprayed with glufosinate (0.14%) to determine the gene transfer frequency. Primary transgenics, their apomictic seed progeny and hybrids were characterized by Southern blot analysis, immuno-chromatographic assay, herbicide resistance, flow cytometry and root-tip chromosome counting and embryo sac analysis.  Very low hybridization frequencies were observed between the apomictic transgenic and wild-type bahiagrass even if pollen receptor and donor are grown in close distance (1m) in the field. Bar gene integration and expression in primary transgenics, seed progeny and hybrids were confirmed by Southern blot and immuno-chromatographic analysis and herbicide resistance, respectively. All six of the so far analyzed hybrids have been confirmed as triploids by both flow cytometry and root-tip chromosome counting. Embryo sac analysis detected both apomictic and sexual embryo sacs, suggesting facultative apomixis. Fertility of hybrids will be evaluated. The results suggests that using apomictic bahiagrass cultivar Argentine as target for bahiagrass transformation provides a high level of transgene containment compared to open-pollinating sexual diploid turf and forage grasses.