A high throughput Agrobacterium tumefaciens-mediated transformation method for functional genomics of perennial ryegrass (Lolium perenne L.)

A robust and high throughput Agrobacterium genetic transformation procedure has been developed for perennial ryegrass (Lolium perenne L.). Embryogenic callus lines were selected and maintained as plants in vitro. Embryogenic calli derived from meristematic regions of the vegetative tillers were co-cultivated with Agrobacterium tumefaciens strain EHA101 carrying the plasmid pCAMBIA 1305.1 in the presence of acetosyringone for 3–4 days. The calli were grown under 94.8 and 151.6 μM hygromycin selection, respectively for two cycles of 2-weeks each, followed by transfer to regeneration medium with 47.4 μM hygromycin. Regenerated plants were rooted and successfully transferred to soil. The transgenic nature of the regenerated plants was confirmed by DNA gel blot analysis and gene expression demonstrated by GUS histochemical assay and/or reverse transcription PCR. After development of the transformation procedure, we used Agrobacterium strain EHA101 carrying a modified binary plasmid pMH bearing genes of interest. In the past 2 years, we have produced more than 1,000 plants with constructs encoding different genes of interest from perennial ryegrass.