Optimization of Agrobacterium-mediated Transformation Conditions for Egyptian Bread Wheat cv. G164

Abstract

Agrobacterium-mediated transforma- tion was firstly used to transform dicot species. Nevertheless, this highly efficient and successful method was not used for cereal transformation as mono- cots were widely considered to be outside the natural host range of Agrobacterium. Fortunately, a few individuals continued to defy the conventional wisdom, and were finally rewarded with success, first with rice and then all of the major cereals. One of the major barriers to the use of Agrobacterium to transform cereals was the absence of wound response and the associated activation of virulence genes. These problems were overcome with the use of actively dividing, embryogenic cells, such as immature embryos and calli induced from scutella, which are co- cultivated with Agrobacterium in the presence of acetosyringone, which is a potent inducer of virulence genes (Vasil, 2005).
Wheat is one of the most important field crops worldwide, with the largest harvested area and production levels. As a monocotyledonous plant, wheat has lagged behind dicotyledonous plants in ease and efficiency of transformation
using Agrobacterium-based technique. Immature embryos have long been known to be a good regenerable explant source for wheat and there are effective protocols using biolistics for transformation and regeneration of this tissue. However, de- spite considerable interest there are few publications describing successful Agro- bacterium-mediated transformation of wheat (Cheng et al., 1997; Weir et al., 2001; Wu et al., 2003). However, research to make the Agrobacterium- based transformation method amendable to cereal crops has continued as the system is perceived to possess several advantages over other forms of transformation including: the ability to transfer large segments of DNA with minimal rearrangement; the precise insertion of transgenes resulting in fewer copies of inserted genes; and it is a simple technology with lower cost (Amoah et al., 2001). Moreover, it allows for the stable integration of a defined segment of DNA into the plant genome and generally results in an improved stability of expression over generations than the direct DNA delivery methods (Smith and Hood, 1995). In addition, Agrobacterium transformation may facilitate removal of plant selectable marker genes by segregation. These are important considerations, particularly when creating genetically manipulated lines in crop species for field testing; when the presence of unnecessary DNA and transgene arrangement/copy number are scrutinized as part of the regulatory processes (Wu et al., 2003).
The majority of wheat transforma- tion investigations that have been reported (Vasil et al. 1992, 1993; Weeks et al. 1993; Nehra et al. 1994; Becker et al. 1994; Zhou et al. 1995; Zhang et al. 2000, Bahieldin et al., 2000) utilized microparti- cle bombardment technology. Cheng et al. (1997) first reported the success of Agrobacterium-mediated transformation in wheat with transformation efficiency 1-4%, but these results were limited to small-scale experiments and selection for the neomycin phosphotransferase II gene (nptII). Since then, some advances have been achieved (Jones et al., 2005). Agro- bacterium-mediated wheat transforma- tion, however, has not yet become an established and robust method of genetic transformation because the ability to rou- tinely transform wheat using Agrobac- terium tumefaciens is currently restricted to a few well-resourced laboratories worldwide (Jones et al., 2005).
A critical step in the development of Agrobacterium tumefaciens-mediated transformation is the establishment of optimal conditions for T-DNA delivery into tissues from which whole plants can be regenerated (Amoah et al., 2001). Success in wheat transformation using Agrobacterium requires the identification of a model tissue culture system with a high capacity for producing regenerable cells, the optimization of parameters for gene transfer into those cells and tailoring selection and regeneration procedures to recover transgenic plants (Jones et al., 2005). In the present investigation, we optimized the conditions for genetic transformation of wheat cv. Giza 164 by examining three variables influencing T- DNA delivery and the regeneration of fertile plants in one of the commercially important Egyptian wheat variety.