The Role of virulence gene inducing factors in the improvement of in planta transformation of wheat (Triticum aestivum)

Agrobacterium tumefaciens-based plant transformation is a natural and ideal way of introducing genes into plant genomes. The system is based primarily on tissue culture techniques, including cell differentiation and plant regeneration, which might produce somatic cell mutations. In the present research, wheat plants were transformed by a non-tissue culture approach. Accordingly, mature embryos were inoculated with A. tumefaciens at an early stage of germination. A variety of different treatments, such as Agrobacterium strains (EHA۱۰۵ and LBA۴۴۰۴ harboring pCAMBIAl۱۰۵.۱R), levels of glucose, concentrations of acetosyringone (۰, ۵۰, ۱۰۰ and ۲۰۰ µM), and types of wheat cultivars (Azar۲, Alvand, and Sardari) were studied. The transformation efficiency was determined by using the number of resistant leaves to hygromycin, histochemical GUS analysis of leaf tissues, as well as PCR analysis of three transgenes located within the T-DNA region. The results showed that the maximum efficiency was obtained when ۲۰۰ µM acetosyringone was used in combination with ۱۵% glucose in the induction medium. In addition, the relative expression analysis of virulence genes by qRT-PCR revealed that VirB۲ and VirD۲ were significantly up-regulated when ۲۰۰ µM acetosyringone and ۱۵% glucose were used as inducers and carbon source, respectively. Therefore, Vir genes inducing factors as well as a three-step culture of Agrobacterium should be considered as major elements for any wheat transformation protocol. The putative transgenic T۱ seeds were soaked and germinated in hygromycin solution, and the seedlings were further analyzed with the aforementioned methods. Based on the results, a modified Agrobacterium-mediated transformation protocol is presented. The current results suggest an inexpensive, quick and efficient approach for wheat genome transformation.

کلیدواژه ها:

Acetosyringone ، glucose ، Histochemical GUS assay ، Hygromycin resistance ، Real-Time PCR