A method for polyethyleneglycol (PEG) mediated direct DNA transfer int
o protoplasts was successfully established for transient and stable tr
ansformation of Triticum aestivum L. cell cultures. Transgenic cell li
nes, which had been transformed with the neomycin phosphotransferase I
I gene (nptII) fused to different promoters, were selected and integra
tion and expression of the marker gene was shown by Southern analysis
and enzyme activity test. For investigation of expression stability, f
ive nptII positive cell lines maintained under selection were protopla
sted and clonal callus lines were cultivated from the genetically iden
tical single cells without selection pressure. Marker gene activity of
271 clonal callus lines was determined and compared with the correspo
nding 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 marke
r 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 differe
nces in marker gene expression could be observed regardless of copy nu
mber and position of the integrated nptII gene. The significance of su
ch observations for a stable expression of foreign genes in plant cell
s is discussed.