Genetic transformation of creeping bentgrass

Turf industry has become part of the booming service sector of the economy, since their economic value has started from {dollar}4 billion in 1974 and has expected to reach {dollar}90 billion by the end of 2000. The annual seed sales of turf are {dollar}580 million–1.2 billion. Currently, 16,000 golf courses have been constructed in the US and the impact of golf courses on US economy is {dollar}20 billion annually.; Creeping bentgrass is one of the most popular cool-season grasses used on golf courses. Hot and humid summer seriously declines the vigorous of bentgrass and reduces its tolerance to stress. DNA biotechnology has provided more new and innovative genes from a diverse gene pool to enhance abiotic and biotic stress tolerance. Transformation of creeping bentgrass has been conducted using microprojectile bombardment and protoplast electroporation. Therefore, the objectives of this study are to (1) establish an alternative method— Agrobacterium-mediated transformation as a stable and efficient transformation tool for creeping bentgrass and (2) use biolistic bombardment technique to transfer a rice chitinsae gene into creeping bentgrass for disease resistance.; The 40-day-old calli induced from mature seed (cv “Crenshaw”) were used as explants for both objectives. We successfully demonstrated that the green fluorescent protein gene (gfp) has been transferred into creeping bentgrass through Agrobacterium (strain EHA 101) by GFP detection under the fluorescence-detecting stereo-microscope and confirmed by Southern blot analysis. The efficiency (GFP+ transformants/total co-cultured calli) of Agrobacterium-mediated transformation from two independent trials were 10% and 11%, respectively. We also have transferred chitinase gene (chi11) into creeping bentgrass by bombardment technique. In order to facilitate the examination of the co-bombardment efficiency, gfp has been co-transformed with chi11 . The efficiency of bombardment for different genes are 0.5% for bar+, 0.9% for gfp+, and 0.5% for both bar + and gfp+ in cotransformation, respectively. The Southern blot results showed clear bands corresponding to the chitinase coding region strongly suggesting that T-DNA has been integrated into the plant genome. However, no proteins could be detected in Western blot. We concluded that the chitinase gene could be silenced due to unknown reasons.