Agrobacterium tumefaciens-mediated transformation of maize embryos using a standard binary vector system

We have achieved routine transformation of maize (Zea mays) using an Agrobacterium tumefaciens standard binary (non-super binary) vector system. Immature zygotic embryos of the hybrid line Hi 11 were infected with A. tumefaciens strain EHA101 harboring a standard binary vector and cocultivated in the presence of 400 mg L-1 L-cysteine. Inclusion Of L-cysteine in cocultivation medium lead to an improvement in transient P-glucuronidase expression observed in targeted cells and a significant increase in stable transformation efficiency, but was associated with a decrease in embryo response after cocultivation. The average stable transformation efficiency (no. of bialaphos-resistant events recovered per 100 embryos infected) of the present protocol was 5.5%. Southern-blot and progeny analyses confirmed the integration, expression, and inheritance of the bar and gus transgenes in R-0, R-1, and R-2 generations of transgenic events. To our knowledge, this represents the first report in which fertile, stable transgenic maize has been routinely produced using an A. tumefaciens standard binary vector system.