Transgene inheritance and silencing in hexaploid spring wheat

Inheritance and expression of the Act1D-uidA::nptII transgene cassette inserted into the genome of a spring wheat cultivar, 'Fielder', was studied in T-4 and T-5 transgenic wheat lines. Southern blot and PCR analyses demonstrated that the transgene was inherited for five generations of selfed plants. The multiple integration pattern displayed in the T-1 generation was maintained up to the T-5 generation with no evidence of transgene rearrangement. There was no cytoplasmic effect on the inheritance of the transgene as observed by GUS histochemical assays in F-1 seeds of reciprocal crosses (T-4 crossed with untransformed 'Fielder'). Based on the histochemical GUS activity a clear Mendelian segregation ratio was not obtained in the F-2 seeds of the crosses, although there was a tendency towards a two-locus insertion ratio. For one cross (Al//FD/Al), some of the transgenic plants produced low GUS and NPTII enzyme activities in seeds, even though Southern blot and PCR analyses indicated the presence of an intact transgene expression cassette. The transgene of these plants was methylated based on Southern blot analysis of genomic DNA restricted with methylation-sensitive enzymes. Northern blot analysis revealed that the plants with the methylated transgene did not accumulate the uidA::nptII fusion gene transcript.