Alternatives to antibiotic-resistance genes

New marker genes: there is another way

Marker genes – so far these have usually been antibiotic-resistance genes. But there are alternatives.

Marker genes are needed following the transformation of a large number of plant cells to help find those that have integrated the desired new genes. Possible alternatives to the resistance genes are various physiological markers – genes that influence the natural metabolism of the plants.

With the help of these physiological markers, a plant can break down substances that would normally be toxic. Unlike the antibiotic resistance genes , however, their use as a marker gene is confined to certain plants. It is necessary to understand fully the metabolism of the plant in question and to be sure that the marker gene will not intervene in it and produce new or modified metabolites. For many plants, this required knowledge is not yet available.

Some projects within the SiFo programme that ended in 2003 refined some of these new physiological markers and tested them for their practicability.

2-DOG system

• The system is based on the fact that the sugar 2-deoxyglucose (2-DOG) is usually toxic to plants, since it inhibits respiration and cell growth. It is known from 2-DOG-resistant yeast cells that their resistance stems from an over‑expression of the gene for a particular enzyme (2-deoxyglucose-6-phosphate phosphatase).

• This gene was introduced into potato plants: the 2-DOG system was effective under field conditions. No differences were found between the transgenic plants and the control plants, so a practical application of the gene as a marker for the selection of transgenic plants seems possible in principle.

• Whether the 2-DOG system can actually be applied without safety concerns is to be investigated further.

Palatinose system

• Plant cells cannot do anything with saccharose isomers – chemical relatives of our household sugar which include palatinose. Palatinose is not taken up or used by plant cells.

•  By contrast, transformed plant cells which contain a certain enzyme (= palatinase), which splits palatinose into fructose and glucose, can live on palatinose as their only source of carbohydrate. Normal plants die if they receive only palatinose.

• The research project succeeded in inserting the palatinase gene into tobacco plants. However, although it was expressed, no functioning protein was produced. Even with different variations of the gene/gene construct it was not possible to trigger palatinase enzyme activity in the transgenic tobacco plants.

These physiological marker gene systems are, however, not the only systems being worked on.

Mannose system: If an enzyme gene, which does not occur naturally in many plants (mannose-6-phosphate isomerase) is introduced, the plant can grow on a nutritive medium containing mannose. Mannose is, like palatinose, a special type of sugar which cannot be metabolised by many plants. However, there are several species of plant, like tobacco, which can use mannose as a source of carbohydrate. The mannose marker cannot be used with these species.

Dye markers: There are also visual markers, which trigger colour reactions (formation of anthocyans or carotinoids). However, the intervention in the plant’s metabolism that this involves is not always compatible with the objectives of the genetic modification. A green fluorescence gene (gfp) can also be used as a marker gene. Since the dye is only visible under the microscope, this method is very labour-intensive because the cells all have to be viewed individually. This gene cannot be used as a marker for crop plants with a low transformation efficiency rate.

More from GMO Safety

• Research project (2001-2004): Development of new marker genes for the selection of transgenic plants; Sungene, Gatersleben