Stability of Herbicide Resistance and GUS Expression in Transgenic Hybrid Poplars (Populus sp.) During Four Years of Field Trials and Vegetative Propagation

We assessed the stability of transgene expression in 40 transgenic lines (i.e., transformation events) of hybrid poplars during four years of field trials. The population consists of 40 lines of hybrid cottonwood (Populus trichocarpa x P. deltoides) grown at three field sites. All lines were transformed with a binary construct that included two genes conferring resistance to glyphosate (GOX and CP4), a gene encoding resistance to the antibiotic kanamycin (NPTll), and a visible marker gene (GUS). Agrobacterium tumefaciens was used for transformation; callogenesis and organogenesis occurred under kanamycin selection. To test the stability of transgene expression, we began repeated applications of herbicide on all lines shortly after outplanting, and challenged ramets from 40 previously untreated lines during their fourth season of vegetative growth. Here we describe how maintenance of herbicide resistance and GUS expression is indicative of transgene stability, and how we will use growth, leaf morphology, and microsatellites to investigate somaclonal variation. Preliminary data reveal that all lines that were highly resistant in year 1 continued to be highly resistant in year 4. Since the early reports of transgene silencing by Matzke and Matzke (1991), Jorgensen (1992), and others (Finnegan and McElroy, 1994; Kooter and Mol, 1993), there has been increasing concern about the stability of transgene expression. This is especially important for trees, which undergo numerous dormancy cycles and are often exposed to extreme environmental changes during their long lives. Unstable expression has taken on greater significance given recent reports that environmental changes can trigger transgene silencing (Brandle et al., 1995; Ellis et al., 1996). Sexual reproduction can also affect the stability of transgene expression (Metzet al., 1997). This issue is of interest to the Tree Genetic Engineering Research Cooperative (TGERC) because one of its main projects, engineering reproductive sterility, relies on stable transgene expression. The Animal and Plant Health Inspection Service (APHIS) is one of the federal agencies involved in regulating the commercialization of transgenic plants. APHIS’ role with regard to agricultural biotechnology is to manage and oversee regulations to ensure the safe and rapid development of the products of biotechnology. Implicit in this statement is that the risk of transgene spread into the environment must be minimized. The easiest way to accomplish this objective is to deploy transgenic trees that are reliably sterile. Some of our recent work has been motivated by a report from Wang et al. (1996). The authors described a population of transgenic poplars that was engineered for insect resistance, using a Cry1A gene. They detected high levels of somaclonal variation in leaf morphology (Fig.1), and evidence of genetic mutation using a variety of molecular tools (i.e., random amplified polymorphic DNA, RAPD; restriction fragment length polymorphism, RFLP; amplified fragment length polymorphism, AFLP; and simple sequence repeat, SSR). Although we have observed no obvious changes in plant morphology in our transgenic populations, their findings prompted us to more carefully search our oldest field trial for similar effects. In this paper, we describe two field studies in which we investigated the stability of transgene expression and somaclonal variation. The first is a screening trial that was conducted during the 1996 and 1997 growing seasons. The primary objectives of this trial were to select lines that had commercially useful levels of resistance to glyphosate (the active ingredient in the herbicide Roundup®) and to look at the effects of glyphosate treatment on growth. This study was continued for a second year to determine the stability of glyphosate tolerance. Before terminating the screening trial, we took hardwood (i.e.,dormant) cuttings from all untreated individuals in each transgenic line at one of the test sites. These vegetative propagules were used to establish a second study in which we continued to assess the stability of transgene expression, and to look for somaclonal variants.