Gene Stability in Transgenic Aspen‐ Populus ‐ III. T‐DNA Repeats Influence Transgene Expression Differentially among Different Transgenic Lines

Transgene silencing mechanisms have been confirmed in many crop plants, however, few reports are currently available for long‐lived tree species. Three transgenic aspen lines carrying the rolC gene of Agrobacterium rhizogenes, under control of the 35S promoter of the cauliflower mosaic virus, but showing very different types of reversion from rolC ‐phenotype to wild type are investigated in detail. A complete suppression of rolC phenotype was observed after one year of in vitro culturing in one transgenic line, whereas the second transgenic line showed gradual decrease in rolC expression, and the plants were comparable to control plants after three to four years in the greenhouse. In the third transgenic line, however, transgene inactivation was observed only in some shoots of the greenhouse‐grown plants which reverted to the rolC phenotype in subsequent years. Southern blotting experiments using a rolC probe revealed the integration of one or two tandem copies of the foreign genes into the host genome. All three transgenic lines contain in/complete T‐DNA repeats, as indicated by reverse primer (rp) PCR. In the reverted tissues of two transgenic lines loss of rolC protein was observed despite the presence of the rolC transcript. The third transgenic line, showing a completely suppressed rolC phenotype, was found positive for the rolC transcript and protein. On the basis of the molecular data presented for the three unstable lines, including Southern, Northern and Western blotting experiments, as well as a survey of transgenic methylation patterns, new insights were obtained with respect to structure and inactivation of transgenes following their transfer in a host tree species.