Wheat is one of the most important cereals for humans but quite recalcitrant in transformation. We have thoroughly examined every aspect of the wheat transformation protocols mediated by Agrobacterium tumefaciens and we were able to identify and optimize the key factors. Immature embryos isolated from healthy plants grown in a greenhouse were pre-treated with centrifuging and co-cultivated with A. tumefaciens. The frequency of transformation (independent transgenics/explant) was between 50 % and 60 % were routinely observed and higher than 90 % were recorded in the best cases. Not surprisingly, the key factors did not differ much from those in other cereal plants such as rice and maize. Both bar and hpt genes were good as selection markers. Fielder, a spring wheat cultivar, constantly showed high efficiency of transformation by our protocol. We have been able to obtain transgenic plants from the embryos harvested from the greenhouses throughout the year. Most of the transformed plants were normal in morphology and fully fertile. More than 40 % of the transformants had a single copy of the transgenes, which were inherited in a Mendelian fashion in most of the lines analyzed. Transgenic wheat has been generated at high frequency by several research groups by our protocol by now. Therefore, wheat has finally joined the list of cereals that can be efficiently transformed.
