Genetic manipulation of creeping bentgrass

Turfgrass plays an important role in our daily lives by adding beauty to the environment and providing the foundation for many recreational sports. In the United States, turfgrass covers over 50 million hectares, and the annual commercial value totals over 40 billion dollars. Owing to the importance of turfgrass production, research into biotic and abiotic stress resistance is increasingly important.; Genetic transformation offers unprecedented opportunities for the molecular manipulation of crops with novel traits. In this project, we performed an integrated study on turfgrass transformation using creeping bentgrass ( Agrostis palustris Huds.), an important cool-season perennial widely used for golf course putting greens. Using an Agrobacterium-mediated approach, agronomically important genes, encoding the rice thaumatin-like protein (TLPD34) and the barley late embryogenesis abundant protein (HVA1), were introduced into creeping bentgrass individually with the aid of the bar selection. Molecular studies demonstrated the genomic integration and the heterologous expression of the transgenes in both T0 and T 1 generations. Field bioassay revealed that the transgenic T0 bentgrass with overexpression of TLPD34 reduced development of dollar spot (Sclerotinia homoeocarpa) compared to the untransformed control plants. Similarly, transgenic T0 plants with the HVA1 expression showed an improved drought tolerance compared to the untransformed controls under greenhouse conditions. In addition, bentgrass transformation was performed on sole marker genes, which included a synthetic fluorescent protein gene (sgfp) and an E. coli phosphomannose isomerase gene (pmi). The co-integration and co-expression of the dual marker pmi-gfp were documented in both T0 and T1 transgenic lines. Our success using the environmentally friendly markers provides an alternative selection method without using herbicide- or antibiotic-resistant markers in the future. Finally, the study further characterized the T-DNA integration at the genomic level. Sequence analysis revealed a microsimilarity-based T-DNA integration with some accidental scenarios like T-DNA truncation, backbone integration, and multiple T-DNA arrays at a single chromosome locus.; In summary, this research has documented the success of incorporating value-added traits in creeping bentgrass through Agrobacterium-mediated transformation, and has provided the first-hand knowledge about genomic integration of the transgenes.