Apr 21, 2006

Basic info

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Marker gene elimination

Segregation: Two genes in, one gene out

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

The first step: Separating the target gene and the marker gene

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.

However, if the two genes are integrated into the plant genome at different sites, they can be separated again in the progeny of the genetically modified plants: the progeny then receive one gene or the other but not both.

There are a number of different methods that can be used to separate the target and marker genes: one way is to make the marker gene ‘jump’ to another site in the genome using a special enzyme called a transposase; another way is to transfer the two genes using two different T-DNAs (co-transformation) so that they are integrated independently of one another in the plant genome.

The second step: Segregation

Segregation exploits the fundamental processes of inheritance. Most plants have at least a double set of chromosomes. However, the newly inserted transgenes (target and marker gene) are usually integrated only once.

The gametes carry only a single set of chromosomes. When the gametes are formed, the chromosomes are distributed at random. If the target and marker genes have been separated as described above, the gametes of the transformed plants may contain either the target gene or the marker gene, or both or neither. A large number of variations is possible among the progeny (see diagram). Statistically, 25% of the progeny will no longer contain the marker gene.

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

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.

The aim is to obtain plants that contain the target gene on both chromosomes and are also marker-gene-free.

Plant breeders need plants that are homozygous, i.e. that carry the target gene on both chromosomes of the chromosome pair in question. This ensures that the target gene will be present in all progeny. To obtain homozygous, marker-free transgenic plants, it is necessary after transgene segregation to find progeny that are marker-free on both chromosomes, yet carry the target gene twice. It is possible to identify this combination using PCR and Southern Blottechniques. It is, however, a laborious procedure because statistically, the desired combination will be found only in one in sixteen progeny.

There are ways of simplifying the search for the right progeny. One of these involves coupling the marker gene to a negative selection marker. Adding an inductor substance activates the negative marker and all the plants that carry it die. The plants that are left are those that do not carry the marker gene (in the blue box in the diagram). Statistically, of these it will now be one in four plants – instead of only one in sixteen – that carry two copies of the target gene and no marker gene.

Another way of speeding up identification of the desired progeny is androgenetic segregation. This involves intervening in the production of male gametes in the pollen. If the unripe pollen is exposed to a particular stress, the normal development path to ripe pollen can be interrupted. Replication of the chromosome set can be triggered either spontaneously or artificially.

The advantage of this method is that the plants regenerated from these cells can have one of only four possible gene combinations (see diagram). Here too, statistically it will be one in four, rather than one in sixteen, that contain the desired gene combination.

Gametes : 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.

Regenerated progeny: With androgenetic segregation, the plants that regenerate from the gametes have two identical sets of chromosomes. Like the gametes, these plants can have four different combinations of marker and target gene. It is the plants with no marker gene and two target genes (in the blue box) that are needed for varietal development.