Aug 5, 2005

Research Projects

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Using a recombination system to produce marker-gene-free plants

(2001 – 2004) Bavarian State Research Center for Agriculture; Institute for Plant Cultivation and Breeding; Freising

Topic

This project investigated a novel method for producing marker-gene-free transgenic crop plants.

A sequence-specific recombination system (γδ resolvase/res system) was used to remove the marker genes. Once the system had been tested, it was to be applied to potatoes with altered starch composition.

Summary

The test system was tested on potato protoplasts.

The functionality of the γδ resolvase/res system was demonstrated. It works both when the DNA segments to be removed are located on plasmids, and when they are integrated in the genome.

It was also possible to optimise the system for the specific conditions found in potato plants.

Experiment description

Before starting the experiments it was not known whether the γδ resolvase/res recombination system derived from bacteria was also suitable for plant applications. To find this out, the system was first tested on plant protoplasts. This method allowed the system to be optimised more quickly by bypassing the time-consuming production of transgenic plants.

• The γδ resolvase gene is transferred to potato protoplasts. At the same time, DNA sequences carrying a promoter and a reporter gene are introduced. The activity of the reporter gene is suppressed by the insertion of additional DNA segments, which are flanked by special recognition sites – “res sites”. The γδ resolvase recognises these “res sites” and removes the DNA segments in between. The promoter switches the reporter gene on only once these segments have been cut out: a visible blue dye forms.
• The second step involves the production of transgenic potatoes using Agrobacterium-mediated transformation. These contain marker genes flanked by two res sites and the gene for the altered starch composition (anti-sense GBBS). They facilitate control of both the successful insertion of the genes into the plants and of the removal of the marker genes.

The potato tubers undergo a starch test to determine whether the marker-gene-free plants have the desired altered starch composition. Potatoes, into which the GBSS target gene has been transferred by conventional means, are grown as a control.

Various varieties of potato were transformed. The first stages were tested using various molecular biological procedures. The marker-free plants were subsequently tested for stability and starch composition in the greenhouse.

Results

• The γδ resolvase gene was produced (gene synthesis) and subjected to PCR analysis.
• This involved the insertion via suitable recognition sequences (primers) of mutations which could result in an optimised expression and recombinase activity in the potato plants.
• The γδ resolvase gene sequences were inserted into expression constructs, which allow production of the corresponding γδ resolvase protein in potato plants. In addition, DNA substrates were produced which contain res sites and activate a reporter gene once recombination has occurred. The test system was tested on potato protoplasts.The functionality of the system was demonstrated, both when the DNA segments to be removed were located on plasmids and when they were integrated in the genome.
• γδ resolvase with the original amino acid sequence can recognise even drastically shortened res sites. On this basis, minimised T-DNA constructs were produced which carry a marker gene cassette flanked by res sites and which, it is hoped, will result in the expression of altered starch composition especially in potato tubers. In transformation experiments the new T-DNA sequences were transferred into potato plants. It was shown, that it is possible to reduce the original res-sequence of 114 base pairs whilst retaining the functionality. Furthermore, optimisation of the DNA sequence of the resolvase for the specific conditions associated with potato plants resulted in improved effectiveness.