Wine research: long drawn-out processes

The tour of the ultra-modern laboratory ends in the cellar, although anyone expecting a quick taster will be disappointed… but immediately consoled with a look through the microscope. Lights off, spotlight on: Under UV light the delicate leaf of a grapevine glows fluorescent green. “It worked,” comments Goetz Reustle, head of the Grapevine Biotechnology Department. He is referring to the method used by the institute to remove antibiotic‑resistance genes from genetically modified plants. These genes are often used as markers and linked to the gene that is to be inserted into the plant. They show whether the genetic modification has taken place or not. Only around five out of every thousand cells will actually integrate the new genes and survive the treatment with antibiotics. Most of the marker genes used come from antibiotics that no longer play a significant role in human or animal medicine. Nevertheless, marker genes as a technical aid are political dynamite: there is a fear that the antibiotic resistance could be transferred by gene transfer to pathogenic bacteria if the genetically modified plants are cultivated. Even though such a gene transfer from the plant to a bacterium is highly unlikely and has not yet been shown to have occurred under natural circumstances, as a precaution, biological safety research is looking at methods that can be used to remove the marker gene from the plant genome.

The Cre/lox recombination system achieves this using the ‘Cre recombinase’ enzyme . It recognises the marker gene by means of two flanking recognition sequences, the ‘lox sites’, which are introduced into the plants during transformation . The enzyme is inserted into the young transgenic plants by infecting them with a virus that contains the Cre recombinase gene. This means that Cre recombinase is temporarily produced in the plant.

In order to establish whether the method has been successful and whether the marker gene has been removed, the researchers use a special trick: They insert a reporter gene outside the lox sites that codes for green fluorescent protein (gfp). However, the protein is formed only once the marker gene has been removed, thereby bringing the reporter gene into direct contact with a promoter – a DNA sequence that acts as a ‘gene switch’, enabling the reporter gene to be expressed. “The recombination, i.e. the reorganisation of the DNA, activates the gfp gene, which glows green, indicating that the marker gene has been removed,” explains Reustle.

From cell mass to transgenic shoot

His aim is to transfer the method, which has already been used successfully on tobacco, to grapevines as part of the biosafety research programme. Because they are long-lived, grapevines have a particularly long-term effect on the environment. But before success comes a long drawn-out process that demands a lot of patience from the scientists. Reustle explains why: On the laboratory bench in front of him are Petri dishes and test-tubes with grapevines of various sizes and at different stages of development. The raw material for the transformation is an embryogenic callus , a milky-white cell complex produced from the anthers of vine flowers. After around six months of cultivation and tending, if enough callus material is present, Agrobacteria are used as a vehicle to transfer the desired genes. “And then the tedious selection work begins,” says Reustle, pointing to a Petri dish. When plant hormones are added to the cell mass, it produces tiny shoots which will survive being transferred to a culture medium containing antibiotics only if they have integrated the lox gene construct complete with marker gene into their genome. Anything that manages to grow here is tended with loving care in climate chambers and in the greenhouse. However, it is not known how many of the plants will survive: stress often causes the plants to lose their regeneration ability. “We are pushing masses of material through. Sometimes it produces a lot, sometimes nothing at all. If we are really lucky, after one year we will have ten transformed grapevines,” explains Reustle.

Tobacco – a model plant

According to Reustle, the system is not very efficient and every laboratory has to refine the method itself, but it is still a tedious process. “If we are unlucky, it will be five to ten years before we are successful in the scientific sense,” the researcher says regretfully, looking at the tobacco plants standing between all the grapevines on the laboratory bench. Tobacco is often used as a model plant in biotechnology to test out new procedures. It has the advantage of being easy to transform and to regenerate into transgenic plants, it has a short generation time and is a practical size. Scientists call this “proof of concept”. “You can do lots of great things with tobacco,” says Reustle. Applied research on grapevines is incomparably more difficult

Mature developments

In addition, the researchers are restricting themselves to four established wine-growing varieties – and what works for one variety, by no means works for another. Reustle explains that the researchers feel under an obligation to the seed stock producers who helped establish the grapevine biotechnology research area years ago. In the long term, the Cre/lox system is to be linked to genes of commercial interest, e.g. a virus- or fungus-resistance gene. From the perspective of the researcher, the latter could well prove an opportunity to cut down on pesticides, which are used in large quantities in wine-growing. Even organic wine-growers are looking for reliable systems to combat the dreaded fungi. They make use of copper preparations, which contaminate the soil with heavy metals. Nevertheless, Reustle does not believe that genetically modified wine will be on the market in the near future. Wine is, after all, a “very emotional product”, a luxury product. “We may carry on researching for a few more years without anyone taking an interest in our work. It may be that what we are doing today will be of interest only in twenty years’ time and then we will be happy that we have done this preliminary work – whether it is developing a method or a product,” says the scientist with conviction. One has to think long-term, especially with long-lived crops.