High efficiency transformation of tall fescue with Agrobacterium tumefaciens

An efficient genetic transformation system for tall fescue (Festuca arundinacea Schreb.), using Agrobacterium tumefaciens-mediated T-DNA delivery, is described. Seed-derived embryogenic calli were infected with Agrobacterium tumefaciens strain EHA105 harboring plasmids pTOK47 and pCAMBIA1301. Infected calli were selected at 250 mg L−1 hyg B and the regenerated plantlets at 50 mg L−1. Using the protocol developed, 34% of the calli infected were hyg B resistant, and the overall plant transformation frequency (number of independently transformed plants over number of calli infected) was 8%. Stable integration of transgene into plant genome and GUS reporter gene expression were confirmed. Low transgene copies (1 or 2 as estimated) were observed in all the transgenic plants analyzed, and the transgene was transmitted to the progenies. Further experiments suggest an elevated 2,4-D concentration (5 mg L−1) used during callus culture and co-cultivation was partially attributable to the observed high transformation efficiency, while the inclusion of plasmid pTOK47 in Agrobacterium was not. The whole process from callus induction to transplanting green plantlets to the soil takes about six months, significantly shorter than the suspension culture approach often used in tall fescue transformation.