STABILITY OF TRANSGENES AND PRESENCE OF N-6 METHYLADENINE DNA IN TRANSFORMED WHEAT CELLS

A number of stably transformed wheat cell lines were obtained using tw o different direct gene transfer techniques. When integration of the f oreign genes was investigated in DNA samples taken at 10 months post-s election, complicated profiles of transgene bands were observed in Sou thern blot analysis. Among these, a number of common bands were identi fied showing similar hybridization patterns between independently tran sformed cell lines. This type of hybridization pattern has been a comm on observation and is usually interpreted as concatameric rearrangemen t of integrated DNA. However, when integration was reinvestigated with DNA samples taken at 30 months postselection, the hybridization patte rn changed and most of the common bands had disappeared. Further analy sis using a set of methylation-sensitive enzymes revealed that the DNA represented by the common bands was N-6-adenine methylated ((m6)A DNA ), and there was even (m6)A DNA in some 30 month samples. Although the source of (m6)A DNA in the wheat cultures was not clearly established , the data indicate that transformation of an endophyte (e.g. a mycopl asmalike organism) may have occurred at the same time as the transform ation of wheat cells. The integration pattern of undermethylated trans genes in the transformed cells became simpler and clearer after treati ng the DNA preparations with Dpnl, which only cuts (m6)A DNA. The impl ications of these data for other methods of inoculating cereals with D NA are discussed.