Transgene inactivation in Petunia hybrida is influenced by the properties of the foreign gene.

Petunia mutant RL01 was transformed with maize A1 and gerbera gdfr cDNAs, which both encode dihydroflavonol-4-reductase (DFR) activity. The same Agrobacterium vector and the same version of the CaMV 35S promoter were used in both experiments. Transformation with the cDNAs resulted in production of pelargonidin pigments in the transformants. However, the A1 and gdfr transformants showed clearly different phenotypes. The flowers of the primary A1 transformants were pale and showed variability in pigmentation during their growth, while the flowers of the gdfr transformants showed intense and highly stable coloration. The color difference in the primary transformants was reflected in the expression levels of the transgenes as well as in the levels of anthocyanin pigment. As previously reported by others, the instability in pigmentation in the A1 transformants was more often detected in clones with multiple copies of the transgene and was associated with methylation of the 35S promoter and of the transgene cDNA itself. In the gdfr transformants, the most intense pigmentation was observed in plants with multiple transgenes in their genome. Only rarely was partial methylation of the 35S promoter detected, while the gdfr cDNA always remained in an unmethylated state. We conclude that the properties of the transgene itself strongly influence the inactivation process. The dicotyledonous gdfr cDNA with a lower GC content and fewer possible methylation sites is more 'compatible' the genomic organization of petunia and this prevents it being recognized as a foreign gene and hence silenced by methylation.