Background and Objective

In contrast to agriculturally used plants, well-documented knowledge about GM trees is only available in a few databases. However, trees differ in many characteristics such as complex habitat, long lifespan, low degree of domestication compared to agricultural plants. The aim of the work in this project is divided into a theoretical and a practical part. As part of a literature study, information generated in various European countries and relevant to biosecurity protocols for GM trees will be viewed and evaluated. This information could provide the scientific basis for future EU regulations on environmental risk assessment of GM trees to ensure safe development and use. In the practical part of this project, the possibilities of a targeted integration of foreign genes into the genome of the trees as well as an elimination of marker genes with the help of recombination systems are to be examined. In the context of research into biological safety, these investigations are extremely important in order to be able to carry out a risk assessment when using such systems.

Approach

This project is based on preliminary with using recombination systems, which has been successfully tested in annual plants. Also for aspen it has been shown, that the recombination systems Cre/lox as well FLP/FRT work well. For risk assessment studies it has to be analysed whether pleiotropic effects can be observed in transgenic trees carrying recombination systems.

In order to generate transgene-free pollen in poplar plants, two constructs must be produced that carry the two recombination systems, antibiotic resistance and the "gene of interest".
The first construct carries the hyg antibiotic resistance gene. This gene construct also serves as a plant selection marker. This is followed by Cre recombinase under the control of a heat shock inducible promoter (HSP). A loxP recognition sequence is located between the HSP promoter and the coding region of the FLP recombinase. The second loxP sequence is behind the terminator sequence of the selection marker gene. The left loxP sequence is followed by the coding region of FLP recombinase and the right loxP sequence by a pollen-specific promoter from Cuphea lanceolata (Töpfer, unpublished) followed by a gene of interest. The functionality of the Cuphea promoter in poplar was demonstrated in a previous BMBF-funded project. The uidA gene (GUS) should only be used as a "gene of interest" for the work to be carried out here. Optionally, a second selection marker gene (nptII) follows under the control of a constitutive promoter, which can be used to detect the second recombination that has taken place.
The second construct is structured like the first, but contains the exactly inverse arrangement of the recombinase genes and the recognition sequences. The purpose of this is to examine which system might work more efficiently.

Early flowering 35S::FT transgenic male aspen plants are available for transformation.

Results

This project is based on preliminary with using recombination systems, which has been successfully tested in annual plants. Also for aspen it has been shown, that the recombination systems Cre/lox as well FLP/FRT work well. For risk assessment studies it has to be analysed whether pleiotropic effects can be observed in transgenic trees carrying recombination systems.