THE EFFECT OF T-DNA COPY NUMBER, POSITION AND METHYLATION ON REPORTER GENE-EXPRESSION IN TOBACCO TRANSFORMANTS

Inter-transformant variability in the expression of introduced genes was studied in the R1 and R2 generations of 10 tobacco transformants, produced by Agrobacterium-mediated transformation. In replicated and physiologically equivalent material, transformants showed considerable variability in the expression of the reporter gene uidA as shown by transcript levels and beta-glucuronidase (GUS) activity. However, homozygous R2 material could be investigated for seven of the transformants and among these, and in one line in which two inserts could segregate independently, this inter-transformant variability was reduced to simple bimodal expression. The two levels of expression for GUS activity in leaves were high or low (approximately 2.5 or 0.3 nmol cm-2 min-1 respectively), with no continuous variation. Transformants in the high group had single T-DNA insertions, while those in the low group had multiple T-DNA insertions, at the same or different loci. Within each group, although T-DNA was apparently integrated at different sites in the plant genome, there was no evidence of position effects. GUS activity levels of the transformants were very similar in the field and in environmentally controlled conditions under high or low light. Plants with multiple insertions and low expression also tended to have increased methylation of the integrated T-DNA.