Agrobacterium-mediated transformation of polyploid cereals.: The efficiency of selection and transgene expression in wheat

Three combinations of Agrobacterium tumefaciens strains and vectors were used in the transformation of selected Polish wheat cultivars. The combinations were: two hypervirulent strains, AGLI, containing the pDM805 binary plasmid, and EHA101, containing pGAH; and the common Agro strain LBA4404, harboring the super-binary pTOK233 vector. pDM805 contained bar under the control of Ubil promoter, pGAH had nptII under nos, and pTOK233 had hpt under 35S. Additionally, pDM805 and pTOK233 carried the gus reporter gene under the Act1 promoter or 35S promoter, respectively. The highest selection rate was 12.6% and was obtained with EHA101(pGAH) on a kanamycin-containing medium. Sixty-five of the plants grown on that medium were PCR positive. The second best combination was LBA4404(pTOK233) and kanamycin selection, which gave an average transformation rate of 2.3%. Phosphinothricin selection gave 1.0% transformation efficiency, while hygromycin, depending on the strain/vector used, gave from 0.2 to 0.4%. PCR tests in T-1 revealed that 67% of the lines showed a 3:1 segregation ratio, and 11% a 15:1 ratio, while in 22%, segregation was non-Mendelian. The high number of T-0 transgenic plants containing one copy of the transgene was confirmed via Southern blot analysis. Kanamycin resistance in the T-1 generation was very low; in some lines, all the progeny were kanamycin sensitive. GUS expression, only tested in young T-1 plants, was in agreement with Mendelian segregation in three out of the twelve tested. The factors influencing the efficiency of selection and transgene expression are discussed in this paper.