Vacuum infiltration of Petunia hybrida pollen with Agrobacterium tumefaciens to achieve plant transformation

Genetic transformation of Petunia hybrida with a reporter gene and selectable marker gene (35S-bar) was achieved in similar frequencies by pollinating flowers with pollen vacuum-infiltrated with Agrobacterium tumefaciens or applying a drop of Agrobacterium suspension to the stigma immediately prior to pollination. Nine percent of the T-1, and 5% of the T-2 progeny germinated in nutrient medium with 3 mgl/l Basta (R). Polymerase chain reaction assays indicated that of the Basta (R)-resistant plants, 66% of the T-1 plants, and 61% of the T-2 plants harboured the GUS gene. Histochemical assays showed that 10% of the putatively transformed T-1 plants and 5% of their progeny expressed GUS in leaf tissue, pistils and young anthers. Southern hybridization confirmed genomic integration of the bar gene in one to three places in selected T-1 and T-2 progeny.