Variability of transgene expression in clonal cell lines of wheat

A method for polyethyleneglycol (PEG) mediated direct DNA transfer into protoplasts was successfully established for transient and stable transformation of Triticum aestivum L. cell cultures. Transgenic cell lines, which had been transformed with the neomycin phosphotransferase II gene (nptII) fused to different promoters, were selected and integration and expression of the marker gene was shown by Southern analysis and enzyme activity test. For investigation of expression stability, five nptII positive cell lines maintained under selection were protoplasted and clonal callus lines were cultivated from the genetically identical single cells without selection pressure. Marker gene activity of 271 clonal callus lines was determined and compared with the corresponding parental line. A reduction or loss of marker gene expression in up to 50% of the clonal cell lines was observed. Detailed analysis of randomly selected clones showed that the observed variability in marker gene expression occurred due to a reduction in the nptII transcript level and was associated with hypermethylation of the integrated DNA. The silencing effect was reversible by a 4 week culture phase on media supplemented with the demethylation agent 5-azacytidin. These differences in marker gene expression could be observed regardless of copy number and position of the integrated nptII gene. The significance of such observations for a stable expression of foreign genes in plant cells is discussed.