Using biolistics and hybridization to combine multiple glycosidase inhibitor transgenes in wheat

Several approaches are available to achieve multi-transgene-stacking in plants for the introduction of complex or multiple traits. We used co-transformation with multiple plasmids using particle bombardment and hybridization of plants carrying separate transgenes in wheat. In the co-transformation approach, four constructs containing the defence genes Pvpgip2, Acpmei, Taxi-III or Xip-III and the bar gene were co-bombarded into immature embryos of durum or common wheat. The four transgenes integrated into the wheat genome at co-transformation frequencies of 58 and 27 % in durum and common wheat, respectively. Segregation analysis of the T-1 and T-2 generations showed that 45-90 % of the progeny inherited all four transgenes together in both durum and common wheat. In the hybridization approach, pyramiding of Pvpgip2, Acpmei and Taxi-III was achieved by crossing durum wheat plants containing the Pvpgip2 and Acpmei transgenes with transgenic plants carrying Taxi-III. Segregation analysis showed that only 4.5 % of the progeny inherited all four transgenes together, including the bar gene. Co-expression analyses of Pvpgip2, Acpmei and Taxi-III or Xip-III showed loss of mRNA or protein activity in the progenies of both transgenic and hybrid wheat lines. Particle bombardment yielded progenies with either tightly linked transgenes or with different transgene combinations that could be useful in research applications. However, its efficiency was constrained by the occurrence of transgene silencing.