Molecular genetic analysis of silencing in two lines of Triticum aestivum transformed with the reporter gene construct pAHC25

Unpredictable silencing of foreign genes introduced into wheat by particle bombardment transformation has been previously observed. To investigate the cause of this silencing, we selected two independent wheat lines that had been transformed via the particle gun with plasmid pAHC25 containing the genes uidA (GUS) and bar (BASTA herbicide-resistance) each controlled by the maize ubiquitin promoter-intron (Ubi1). One of these lines (WT2) had shown progressive reductions in transgenic phenotype in successive generations until GUS activity and herbicide-resistance were undetectable in all progeny of the T-3 generation tested. A second line, (WT1) exhibited the expected levels of GUS expression and herbicide-resistance up to the T-3 generation except for the loss of transgenic phenotype in a single T-1 segregant. Transgene stability, silencing and integration locus was investigated in three generations in both lines. The silencing observed in the WT2 line was found to be acting at the transcription level for both the uidA and bar genes. Analysis of transgene promoter methylation in selected T-1, T-2 and T-3 individuals revealed a positive link between methylation status and gene silencing. The silenced T-1 individual from line WT1 was also linked to promoter methylation and insertion locus rearrangement. Fluorescence in situ hybridisation (FISH) revealed highly divergent transgene insertion loci for WT1 and WT2. WT1 was detected close to the telomere whereas the WT2 insertion was located in the region of the centromere. The effect of these findings on transgene silencing and stability are discussed.