Title: Phenotypic Expression and Stability in a Large-Scale Field Study of Genetically Engineered Poplars Containing Sexual Containment Transgenes

Genetic engineering (GE) has the potential to help meet demand for forest products and ecological services. However, high research and development costs, market restrictions, and regulatory obstacles to performing field tests have severely limited the extent and duration of field research. There is a notable paucity of field studies of flowering GE trees due to the time frame required and regulatory constraints. Here we summarize our findings from field testing over 3,300 GE poplar trees and 948 transformation events in a single, 3.6 hectare field trial for seven growing seasons; this trial appears to be the largest field-based scientific study of GE forest trees in the world. The goal was to assess a diversity of approaches for obtaining bisexual sterility by modifying RNA expression or protein function of floral regulatory genes, including LEAFY, AGAMOUS, APETALA1, SHORT VEGETATIVE PHASE, and FLOWERING LOCUS T. Two female and one male clone were transformed with up to 23 different genetic constructs designed to obtain sterile flowers or delay onset of flowering. To prevent gene flow by pollen and facilitate regulatory approval, the test genotypes chosen were incompatible with native poplars in the area. We monitored tree survival, growth, floral onset, floral abundance, pollen production, seed formation and seed viability. Tree survival was above 95%, and variation in site conditions generally had a larger impact on vegetative performance and onset of flowering than did genetic constructs. Floral traits, when modified, were stable over three to five flowering seasons, and we successfully identified RNAi or overexpression constructs that either postponed floral onset or led to sterile flowers. There was an absence of detectable somaclonal variation; no trees were identified that showed vegetative or floral modifications that did not appear to be related to the transgene added. Surveys for seedling and sucker establishment both within and around the plantation identified small numbers of vegetative shoots (root sprouts) but no seedlings, indicative of a lack of establishment of trees via seeds in the area. Overall, this long term study showed that GE containment traits can be obtained which are effective, stable, and not associated with vegetative abnormalities or somaclonal variation.