Transgenic Christmas trees regenerated from Agrobacterium tumefaciens-mediated transformation of zygotic embryos using the green fluorescence protein as a reporter

Mature zygotic embryos of recalcitrant Christmas tree species Fraser fir [Abies fraseri (Pursh) Poir], and Nordmann fir (Abies nordmanniana L.k.), and Virginia pine (Pinus virginiana Mill.) were used as explants for Agrobacterium tumefaciens strain GV3850-mediated transformation using the gfp (green fluorescent protein) gene as a reporter. Factors including media used for inoculation and co-cultivation, concentrations of acetosyringone, and antibiotics in tissue culture media have been evaluated. A high transformation frequency was obtained on TE medium containing 50 mu M acetosyringone and using 500 mg/l timentin to eliminate bacteria. Transient gene expression was observed in all three Christmas tree species, but transgenic plants were only produced from Virginia pine. Stable integration and expression of transgenes in the plant genome of Virginia pine was confirmed by polymerase chain reaction (PCR), Southern and northern blot analyses. These results demonstrated that a stable transformation system has been established in Virginia pine and this system would provide an opportunity to transfer economically important genes into Christmas tree species.