Genetic transformation of bentgrasses for fungal disease resistance

Genetic transformation of cultivated plants is a new tool that could be used to improve plants for higher yield, better quality, and enhanced stress tolerance. To establish a stable and efficient biolistic-mediated genetic transformation system for bentgrass trait improvement, this research focused on: (1) comparing growth media for embryogenic callus induction and plantlet regeneration; (2) transferring bar, chitinase, glucanase and barnase genes into embryogenic calli of creeping and velvet bentgrasses, and regenerating transgenic plants from the transformed calli; (3) evaluating the transgenic plants by using herbicide glufosinate, polymerase chain reaction (PCR), Southern blot and northern blot hybridization assays; and (4) analyzing the transgenic bentgrass lines for resistance to dollar spot (Sclerotinia homoeocarpa) and brown patch (Rhizoctonia solani). Embryogenic calli with high regeneration rates were initiated from creeping and velvet bentgrasses on Murashige and Skoog medium supplemented with 500 mg/l casein hydrolysate, 6.63 mg/l 3,6-dichloro-o-anisic acid and 0.5–2.0 mg/l 6-benzyladenine. Microprojectile bombardment resulted in stable genetic transformation of the creeping and velvet bentgrass cultivars with transformation efficiencies up to 0.78–0.86 transgenic plants per bombardment. Co-transformation efficiency of chitinase/glucanase genes with an unlinked selectable marker bar gene was 62% in ‘Providence’ creeping bentgrass. Northern blot hybridization indicated that bar and chitinase transgenes in lines containing simple insertions were expressed at the mRNA level. Transgenic plants with simple insertion of the bar were consistently resistant to the herbicide glufosinate under greenhouse and field conditions. However, the bar and chitinase transgenes were silent in transgenic lines with complex insertion patterns. No glucanase gene was expressed at the mRNA level in any transgenic lines containing the glucanase gene. The transgenic lines expressing the chitinase gene at the mRNA level were not resistant to dollar spot and brown patch, indicating that the chitinase gene was silent or the chitinase protein was unable to defend against the pathogens under the experimental conditions used. The glufosinate-resistant transgenic lines expressing the bar gene were resistant to dollar spot and brown patch when 560 mg/l glufosinate was sprayed three hours before or one day after pathogen inoculation, indicating that glufosinate reduced fungal infection on the glufosinate-resistant transgenic grasses