Production of marker-gene-free plants using a recombination system (Cre/lox)

Topic

The aim was to develop a novel method for producing marker‑gene -free transgenic crop plants.

The idea was for the introduced marker gene to be removed from the transgenic plant using a bacterial recombination system (cre/lox system).

The system was first established on transgenic tobacco plants. The plan was to transfer it to the (vegetatively propagated) potato later on. The aim is to achieve a marker-free potato with reduced amylose content.

Information on recombination system:

• Recombination: New arrangements of genes

Summary

A new method of marker gene elimination was established using transient expression of recombinase (cre):

• The recombinase, which “cuts out” the marker gene, is active in transgenic plant cells. Transfer of the recombinase works both with a plant virus (PVX, TMV) and with Agrobacterium tumefaciens .

• There were two possible strategies for developing the recombined plant cells into whole plants: regeneration and self-pollination.

• Transfer of the transgenic trait to the next generation: progeny from self-pollination were marker-free. A PVX-cre vector was used on potatoes. The first experiments showed promising results.

Experiment description

In order to eliminate the marker gene, a cre/lox recombination system was used.

A marker gene (bar) that imparts herbicide resistance was first transformed between the recognition sequences (= lox sites) in tobacco. The marker gene prevents the expression of the reporter gene (gfp), which was also introduced.

The introduction of the recombinase (cre) results in elimination of the marker gene and fluorescence of the gfp. The gene for the recombinase required for cutting was introduced temporarily (transiently) into the plants in a variety of ways:

• Via two different viral clones. The viruses used are not seed-borne. This means that the progeny of these plants are virus-free and no longer possess the recombinase gene either.
  Further Information on the process:

• Viral full-length clones: Viruses as Trojan horses

• Via agrobacteria (agroinfiltration). This path is particularly interesting for asexually reproduced plants, since the viruses can only be removed via seeds.

The elimination of the marker genes is tested using PCR and Southern analysis .

The recombination events triggered by the recombinase cre can be transmitted to the next generation via regeneration or self-pollination strategies.

• Regeneration: Infected leaves and cells transformed using agrobacteria were used for regeneration. Marker-free regenerated plants were self-pollinated for segregation analysis of the progeny.

• Self-pollination: In addition, infected plants were self-pollinated. Since the viruses used are not seed-borne, the progeny of these plants – and those of the regenerates from infected leaves – are virus-free.

Segregation analysis of the progeny was carried out using germination tests. The seedlings were tested for herbicide resistance and gfp fluorescence.

Results

An A.tumefaciens vector and viral vectors (PVX and TMV) were produced which stably carry the recombinase gene (cre) needed to cut out the marker. The recombinase gene was capable of being transiently expressed in the plant – i.e. the gene was only temporarily active and was not integrated into the plant genome.

Of 45 possible transformants, three independent strains were used for the marker gene elimination following Southern analysis. All three vectors successfully eliminated the marker gene from the whole plant.

Regeneration: With both virus vectors (PVX and TMV) between 47 and 61 per cent of the regenerated plants were marker-free. The elimination of the bar gene was transmitted to the following generation. Most of the tested seedlings were gfp-positive.

With the A. tumefaciens vector, 34 per cent of the regenerated plants were marker-free. None of the tested seeds exhibited herbicide resistance. 109 of 114 tested seeds were gfp-positive. This means that marker gene elimination can be achieved with the A. tumefaciens vector using regeneration.

Self-pollination: Progeny of six of the eight PVX-cre plants produced by self-pollination exhibited successful recombination. By contrast, with the TMV-cre vector only a few marker-free plants were found, of which none showed any gfp activity. This means that this method is very effective for the PVX-cre vector, but cannot be used for the TMV-cre vector.

Practical application: The success with the tobacco plant model makes a practical application appear feasible.

As a first step in the production of low-amylose, marker-free potato plants, a vector with a gbss gene and an nptII gene surrounded by lox sites was produced. Using agrobacterial transformation, over a hundred transgenic potato strains were obtained and tested for their amylose content. Three of the strains with reduced amylose content were selected for further tests.

It was possible to demonstrate that the recombinase (with PVX-cre virus) works in infected potato leaves. Regenerated plants from infected leaves are currently undergoing molecular analysis.