Mar 26, 2009

Research Projects

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Obtaining marker-gene-free vines using the Cre/lox system

(2005 – 2008) RLP AgroScience GmbH, Neustadt / Weinstraße

Topic

Marker genes can be removed from transgenic plants using recombination systems, among others. In an earlier research project, the transient Cre/lox system was successfully used in tobacco plants. In this system, the Cre recombinase enzyme excises the marker gene flanked by two loxP recognition sequences from the plant genome. Transient means that this enzyme is active in the plant only within a specific time frame (transient expression).

The aim in this part of the project is for the Cre/lox system to be transferred to and optimised in vines. The system will be used in oilseed rape plants in the second part of the project. Another project aim is to use the recombination system with genes of commercial interest, e.g. genes that confer fungal resistance.

Summary

The aim of the project was to remove marker genes from grapevines using a temporary recombination system (Cre/loxP). The advantage of this system is that it does not require an additional crossing step to obtain marker-gene-free plants. It is therefore particularly well suited to grapevines because they are propagated vegetatively.

• The system was shown to work in principle in leaves of the grapevine.
• It was not possible to transfer the recombinase by infecting the plant with a virus clone that carried the cre gene either using the model plant tobacco or using the grapevine.
• An alternative system that uses an inducible promoter for the temporary expression the cre gene is currently being adapted for the system with the embryogenic grapevine cell culture.

Experiment description

Producing the gene constructs: Embryogenic grapevine cell cultures are obtained from the anthers of the flower stamens. These cells are transformed using Agrobacteria. The transferred gene construct contains the marker gene required for the selection process flanked by two loxP recognition sequences. Outside these there is a transferred reporter gene for a fluorescent protein. The expression of this protein is, however, prevented by the marker gene. If fluorescence is detected after the Cre-mediated recombination, this indicates that the marker gene has been successfully removed from the plant genome. Later on, a gene that could produce fungal resistance is to be inserted in place of the gene for the fluorescent protein.

Modification of the infectious viral clone. For grapevines there are currently only a few infectious viruses available as clones. The viral clone is needed for transient production of the Cre recombinase in the grapevine and to cut the marker gene out of the plant genome. For this purpose, the project is producing an infectious clone of a nepovirus that possesses the Cre recombinase gene.

Eliminating the marker gene: Young transgenic plants are infected with the virus. Molecular biological detection methods, PCR and Southern blot, are then used to check whether the marker gene has been successfully removed from the plant genome.The stable, marker-gene-free plants are then subjected to thermotherapy to eliminate the viruses and the virus-free vines are selected.

Results

Producing the gene constructs: In the first year of the project, loxP constructs suitable for the transformation of grapevines were developed. For the selection process, an antibiotic-resistance marker (nptII; canamycin resistance) was inserted between the two loxP sequences. Agrobacteria were used to transfer the loxP construct into embryogenic grapevine tissue.

In order to be able to carry out visual selection following transformation with the loxP sequences, another construct was produced. For this, a fluorescent marker gene (YFP; yellow fluorescent protein) was linked to the selection marker gene (nptII). Transformation of this modified loxP construct into embryogenic grapevine cells began in spring 2006. In addition, in 2005, tobacco cells were successfully transformed with the loxP constructs, and plants were regenerated from them. This means that a functional test of the Cre/loxP system is possible.

Modifying the infectious virus clone: The infectious virus clone was modified in the first year of the project (2005). Subsequent inoculation of the plants (2006) with the modified virus clone did not result in infection in the plants (model plants or grapevines) despite a large number of experiments with variations of the different parameters.

To improve the infectiousness, in 2005 the modified clone was also transferred to a vector system that can be used for transformation with Agrobacteria. However, when this system was used in the following year of the project, none of the plants were infected.

Eliminating the marker gene: In 2006 the Cre recombinase gene was transferred to loxP transgenic tobacco plants using Agrobacteria. With the help of PCR, recombination events were detected in the leaves. Following functional testing of the Cre/loxP system on the model plant, the marker gene was then also shown to have been eliminated in leaves of loxP transgenic grapevines.

Alternative strategy: In 2008 a gene construct was developed in which the selection marker gene and the Cre recombinase gene lie next to each other between the lox sequences. The activity of the recombinase gene is regulated by a special inducible promoter taken from maize. Following induction, both the selection marker gene and the cre gene are removed.

To monitor the success of the experiment, the gene for a red pigment (anthocyan) was integrated into the construct. This gene cannot be expressed until the selection marker gene and the cre gene have been removed. Once this has occurred, the cells turn red.

The construct has now been transferred to the model plant tobacco and to embryogenic cells of various lines of root-stock grapevines. In all cases, red areas appeared in the seedlings once the promoter had been induced. The induction conditions for the promoter are currently being tested in various tissues and at different stages of development.