Stability and Expression of Chimeric Genes in Populus

Abstract

We have employed Populus as a model system to investigate the stability and expression of transgenes at the morphological, physiological, and molecular levels. Four clones from European aspen (Populus tremuld) and hybrid aspen (P. tremula x P. tremuloides) clones were genetically transformed, using an Agrobacterium binary vector system, with different gene constructs, in particular roIC. Two types of promoters, namely 35S from cauliflower mosaic virus, and rbcS from potato, were employed for control of roIC gene expression. An improved transformation method employing leaf discs was developed, and putative transformants were regenerated on a kanamycin-containing medium. More than 1000 transgenic aspens have been raised in the greenhouse during the last two years. The morphology and physiology of the transgenic plants seems to be influenced by the type of promoter employed. Transgenic aspens carrying the 35S-ro/C gene construct exhibited much smaller leaves, as compared to the untransformed controls. On the other hand, rbcS-ro/C carrying transgenic aspens showed only slightly smaller leaves, as compared to the controls. However, transgenic plants carrying the chimeric gene 35S-ro/C or rbcS-ro/C exhibited yellowish-green leaves, as compared to dark green leaf color of the untransformed controls. A number of leaf abnormalities, chimeras, revertants to normal state have been observed in the transgenic aspens. In order to silence the roIC gene expression, two constructs carrying the transposable element Ac from maize, along with roIC, were employed for genetic transformation in aspen. Transgenic aspens carrying the transgenes 35S-Ac-rolC and rbcS-Ac-rolC were morphologically similar to the untransformed aspens, in terms of leaf size, shape and color, indicating that at least the phenotypic expression of the roIC gene has been repressed by Ac. The Ac excision events were monitored by variegated spots on the leaves. There were significantly more Ac excisions in the transgenic plants carrying 35S-Ac-rolC than in rbcS-Ac-rolC transgenes. These observations suggest that the type of promoter and/or gene construct seems to influence the phenotype and physiology in the transgenic plants. Transformation was confirmed by PCR amplifications of the roIC and Ac coding regions, and Southern blot analysis was carried out to determine the copy number of the transgenes and their relationship to the morphogenetic response, as well as genetic stability in the transgenic aspens.KeywordsTransgenic PlantGene ConstructTransgenic PotatoUntransformed ControlHybrid AspenThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.