High Efficiency Wheat Transformation Mediated by Agrobacterium tumefaciens
Abstract 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.
Keywords Agrobacterium tumefaciens • Cereal • Genetically modification • Transformation • Triticum aestivum • Wheat
Transformation is an essential technology in both applied and basic studies in wheat. The first transgenic wheat was produced by particle bombardment method in the early 1990s (Vasil et al. 1992). Soon after efficient protocols of transformation mediated by Agrobacterium tumefaciens, which can generally transfer low copy numbers of large DNA segments with defined ends to plant chromosomes with few rearrangements, were developed in rice (Hiei et al. 1994) and maize (Ishida et al. 1996), wheat was also transformed by A. tumefaciens (Cheng et al. 1997) and quite a few reports followed. However, the progress thereafter made in wheat was slow (Przetakiewicz et al. 2004), while significant improvement in the efficiency of gene transfer and in the range of transformable genotype was made continuously in other cereals (Hiei et al. 2006; Ishida et al. 2003; Frame et al. 2006; Bartlett et al. 2008). Embryos and cultured cells of several wheat genotypes were tested, and various factors were examined in the early 2000s, however, the frequency of transformation was mostly less than 5 % of the inoculated tissue pieces. Even in the recent reports (He et al. 2010; Bińka et al. 2012), the frequency of transformation in wheat did not change much from those described in the early reports.
We examined any and all factors possibly involved in wheat transformation. Since we have an experience of development of efficient transformation protocol for rice and maize, we tried to include the factors, which were important in transformation of rice and maize and not tested in wheat. We were able to find a good combination of the parameters and to develop a highly efficient protocol for wheat transformation, which is hereby described.