Transgene inheritance and silencing in hexaploid spring wheat

Inheritance and expression of the Act1D-uidA::nptII transgene cassette inse rted 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 demonstr ated that the transgene was inherited for five generations of selfed plants . The multiple integration pattern displayed in the T-1 generation was main tained up to the T-5 generation with no evidence of transgene rearrangement . There was no cytoplasmic effect on the inheritance of the transgene as ob served by GUS histochemical assays in F-1 seeds of reciprocal crosses (T-4 crossed with untransformed 'Fielder'). Based on the histochemical GUS activ ity a clear Mendelian segregation ratio was not obtained in the F-2 seeds o f 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 lo w GUS and NPTII enzyme activities in seeds, even though Southern blot and P CR analyses indicated the presence of an intact transgene expression casset te. The transgene of these plants was methylated based on Southern blot ana lysis of genomic DNA restricted with methylation-sensitive enzymes. Norther n blot analysis revealed that the plants with the methylated transgene did not accumulate the uidA::nptII fusion gene transcript.