New marker genes

Negative markers: fatal to those that have them

The aim: to achieve genetically modified plants which no longer contain the marker genes necessary for transformation . The idea: negative selection markers.

Once the transformed cells have been identified with the help of the marker genes , these are no longer required in the genetically modified plant. However, until now it has been difficult to remove marker genes. One possible way of achieving this aim is to use negative selection markers. The idea is that these would, on a particular "chemical signal“, separate out plants that carry the marker gene.

Diagram: Gametes of plants with independently integrated target gene and marker gene cassette

The gametes contain only a single chromosome set, which is otherwise usually double. Various combinations of target gene (Z) and marker gene cassette are possible (+ = antibiotic resistance gene,- = negative selection marker). Some gametes have neither the target gene nor the marker gene cassette on their chromosomes.

Some progeny carry only the target gene. In all others the marker gene cassette is present.

The first step: Cotransformation. The target gene and a marker cassette are inserted into plant cells independently of each other so that they are integrated at different sites in the plant genome, ideally as far away from each other as possible (cotransformation). The marker cassette contains the marker gene which is required in the first instance, and the negative selection marker.

The second step: Progeny. Once the target gene and marker cassette have been integrated separately into the plant genome they are distributed in a variety of different ways in the progeny (segregation ). There will be plants that contain only the target gene, but also plants with both target gene and marker cassette.

Third step: Activating the negative selection marker. Now it is the turn of the negative selection marker. A signal (= inductor substance) activates the negative marker so that all plants carrying the marker die. Those that are left are plants that carry only the target gene and those which, during segregation, have lost all foreign genes.Application. A negative selection system of this type was refined during a project within the SiFo programme that ended in 2003 and tested for its practicability. It was demonstrated that this selection system is suitable for plants. The procedure – in particular the purity of the inductor substance – does, however, need further refinement before it will be ready for a broad application.

In the system under investigation, the enzyme N-acetylphosphinothricin deacetylase (= DEA) serves as the negative selection marker. This converts a substance which is non-toxic to the plant into one which is toxic. In the case of DEA, an inactive form of a herbicide (N-acetylphosphinothricin) is converted into an active herbicide (phosphinothricin), which attacks the plant’s central metabolism and kills it (see diagram below).

If the transgenic progeny are treated with the inactive herbicide, those that carry the negative DEA selection marker will die. Plants that are not sensitive to the inactive herbicide remain unaffected. They are marker-gene-free.

Inducible cell death system. The plant which carries the DEA gene dies when an inductor substance is applied. This substance is a derivative of the herbicide phosphinothricin (= glufosinate, Liberty) and is non-toxic to plants and biodegradable. The plants take in the inductor through flowers and leaves and transport the substance primarily towards the shoot tips. Here the substance remains unchanged until it is converted into the active herbicide by the DEA enzyme. Because the DEA gene is linked to the marker gene which is to be eliminated, all plants containing both marker genes are killed.

Negative markers: fatal to those that have them

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