Agrobacterium tumefaciens-mediated transformation of Pinus pinea L cotyledons: an assessment of factors influencing the efficiency of uidA gene transfer

This study is the first report of a protocol for transfer and expression of foreign chimeric genes into cotyledons excised from Pinus pinea L. embryos . Agrobacterium tumefaciens EHA105 harbouring the plasmid p35SGUSint was mo re infective than LBA4404 or C58 GV3850, as determined by the percentage of cotyledons showing uidA expression. Factors which significantly affected t he T-DNA transfer included: (1) preinduction and concentration of bacteria, (2) days of coculture and (3) the wounding procedure applied. More efficie nt transfer of the uidA gene was achieved growing the bacteria in YEP mediu m at pH 7, infecting the cotyledons according to the sonication-assisted Ag robacterium-mediated transformation procedure with a bacterial density of 1 (OD600nm) for 5 min, and coculture for 72 h. Using this protocol, 49.7% of the cotyledons showed a diffuse blue staining 7 trays after infection. How ever, all were necrotic 30 days after inoculation. Since a decrease in bact erial density to 0.01 allowed the recovery of about 4% of cotyledons formin g buds 1 month after inoculation, we conclude that the high mortality assoc iated with the infection may be related to the hypersensitive response of t he plant to bacterial infection.