THE IMPORTANCE OF DNA METHYLATION FOR STABILITY OF FOREIGN DNA IN BARLEY

We developed a system using 'passive' reporter genes driven by aleurone tissue-specific promoters to test the effect of methylation of foreign DNA on its stability after introduction into barley plants. The foreign DNA was introduced into barley cells and was able to persist through at least two plant generations. Transformation of barley cells was defined by showing initiation of transcription at the proper site on the barley promoter for the chimeric gene in aleurone tissue from both a primary transformant and its progeny, and by tissue-specific expression (aleurone > leaf) in the progeny. This persistence through many multiples of cell division is formally equivalent to transformation, regardless of whether the DNA was chromosomally integrated or carried as an episome, but did not necessarily represent stable integration into the genome since the foreign DNA was frequently rearranged or lost. The foreign DNA was most stable when plasmid DNA used in transformation lacked dA methylation but had complete methylation of dC residues in the CG dinucleotide at Hpa II sites; dA methylation alone was associated with marked foreign DNA instability. Our results are consistent with the presence of a previously undefined system in barley that identifies DNA lacking the proper methylation pattern and causes its loss from actively dividing cells. These results may be important when applied to different strategies using selectable markers for cereal transformation.