Stability of transgenes in trees: expression of two reporter genes in poplar over three field seasons

High stability of transgene expression is essential for functional genomics studies using transformation approaches and for application of genetic engineering to commercial forestry. We quantified expression of two reporter genes, green fluorescent protein (GFP) and the herbicide bialaphos resistance gene (BAR), in 2256 transgenic poplar trees derived from 404 primary events, and in 106 in vitro-redifferentiated subevents, over 3 years in the greenhouse and in the field. No gene silencing (complete breakdown of expression) was observed for GFP or BAR expression in any of the primary transgenic events during the course of the study. Transgenic cassettes were physically eliminated in four subevents (2.5%) derived from three different primary events during re-organogenesis. Transgene copy number was positively correlated with transgene expression level; however, a majority of transformants (85%) carried single-copy transgenes. About one-third of the events containing two-copy inserts had repeats formed at the same chromosomal position, with direct repeats being the main type observed (87%). All events containing more than two transgene copies showed repeat formation at least at one locus, with direct repeats again dominant (77%). Loci with two direct repeats had substantially greater transgene expression level than other types of two-copy T-DNA configurations, but insert organization was not associated with stability of transgene expression. Use of the poplar rbcS promoter, which drove BAR in the transgenic constructs, had no adverse effect on transgene expression levels or stability compared with the heterologous CaMV 35S promoter, which directed GFP expression.