Marker gene elimination

Cotransformation and segregation:
Two genes in, one gene out

When target and marker genes are introduced separately into a plant genome, breeding methods can be used to produce marker-gene-free progeny.

The first step: Cotransformation

In the classic methods used to genetically modify plants, the target gene, which confers the desired trait, and the marker gene are coupled together. They are usually passed on to the progeny as a unit.

To change this, the target and marker gene must be introduced into the genome in such a way that they are integrated at different sites and are situated on different chromosomes . These are then called independent, uncoupled genes.

For cotransformation using Agrobacteria various arrangements of marker gene (red) and the gene of interest (target gene, green) are possible on the vector :

two different strains of Agrobacteria, each with a transformation vector; one vector contains the marker gene, the other the target gene (top diagram);
one bacterial strain with two vectors, each with one gene (centre);
one bacterial strain with one vector, on which the two genes are located at separate sites (bottom).

Cotransformation is successful if both genes are integrated independently of each other

Results: Several research projects pursued the aim of testing the different cotransformation approaches in practice. A further aim was to find rules for their application in different plant species. The results indicate that all three strategies lead to cotransformation. However, the degree of success varied between the different plant species included in the experiments - Arabidopsis (thale cress), barley, oilseed rape and tobacco.

The second step: Segregation

Once the target and marker gene have been integrated independently into the plant genome, they can be separated again in the progeny of these genetically modified plants: the progeny receive one gene or the other but not both.

The segregation process exploits the fundamental processes of inheritance. Most plants have at least a double set of chromosomes . However, the newly inserted transgenes (target or marker gene) are located on only one chromosome set. The other contains no transgene or only one of them.

In the gametes , the chromosomes are redistributed among the progeny, so these may contain transgenes in both chromosome sets or in only one. A large number of variations is possible (see diagram). Statistically, 25% of the progeny will no longer contain the marker gene.

In this way, a marker gene that initially facilitates the identification of successfully transformed plants during transformation , can be removed again once its work is done.

Diagram: Elimination of marker genes using segregation

Parents: Gametes (sex cells) contain only one chromosome set. It may carry only the marker gene (M, red), the target gene (Z, green), both genes or neither gene.

Progeny: During sexual reproduction, the gametes of the maternal and paternal lines combine and their chromosomes come together to form a double chromosome set.

Various combinations of marker and target genes are possible in the progeny generation. The plants that do not carry a marker gene (blue) can be used for further breeding.

Mathematically, every fourth plant of this marker-gene-free group of progeny will carry neither marker gene nor target gene.

Cotransformation and segregation: Two genes in, one gene out

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Cotransformation and segregation:
Two genes in, one gene out