Antibiotic-resistance genes as markers

Once necessary, now undesirable

The use of antibiotic-resistance genes in genetically modified plants is strictly controlled. But what are they used for and why are they controversial? Some questions and answers.

Infectious diseases. More and more pathogens have become resistant to antibiotics. The fact that antibiotics today are increasingly losing their effect has nothing to do with genetically modified plants and their marker genes. (Photograph: Streptococci)

What are marker genes actually used for?

There are various procedures available for inserting novel genes into the DNA of plants. Parts of a soil bacterium (Agrobacterium tumefaciens) can be used as a ” ferry”, but it is also possible to insert genes directly into a cell or a “cell without a cell wall” (protoplast), using a gene gun for example. The problem is that only a few of the treated plant cells actually become transformed, so that the foreign gene is integrated in their genome.

Even with a plant which is as easy to transform as Arabidopsis (thale cress), the model plant of genetic engineers, only around 5 in every 1000 treated cells are actually genetically modified, and fewer still in most plant species.

Markers are needed to find or “select” the transformed cells among the multitude of untransformed ones.

Marker genes are often also required in an earlier phase, when the transformation tools are constructed and subsequently amplified. These are generally bacteria or plasmids (DNA rings), which serve as “packaging” (vectors) for the foreign gene which is to be transferred.

Why are most marker genes antibiotic-resistance genes?

The aim of marker genes is to mark transformed plant cells - and the more easily, quickly and reliably the better. For a long time genetic engineers believed they had found the perfect solution: antibiotics - those medicines that doctors prescribe to fight a large number of infectious diseases.

Antibiotics are natural antibodies produced by certain moulds to combat bacteria, their competitors. Some species of bacteria have “learnt” to deactivate the toxins aimed at them: they have a gene which makes them resistant to the antibiotic.

The mode of action of the marker is based on this system of “toxin” and “antidote”. An antibiotic-resistance gene, generally of bacterial origin, is coupled with the target gene, which is to confer the desired new trait on the plant. Each transformed cell then contains the resistance gene as well as the target gene: unlike normal plant cells, the relevant antibiotic cannot harm them. If the plant cells are placed on a nutritive medium soaked with the antibiotic, all the cells die except those which carry the marker gene. Only the transformed cells survive.

Why have antibiotic-resistance genes aroused so much criticism?

The very success of antibiotics in medicine has now become a problem. Many bacteria, including pathogens of infectious diseases, are already resistant and can no longer be controlled with the particular antibiotic.

Antibiotics have been used too frequently in human and animal medicine - but of far greater significance is the fact that for a long time they were added to animal feed as a performance enhancer. This practice is now largely outlawed, but the pervasive antibiotics have given a survival advantage to those bacteria that have a corresponding resistance gene. And they have made the most of it. Moreover, resistance genes in bacteria are often located on mobile DNA units, which can be exchanged between different species.

Against this background there are fears that bacteria could absorb marker genes from transgenic plants. This could eventually result in pathogens, against which antibiotics currently being prescribed are ineffective.

The prerequisite for such a scenario is horizontal gene transfer - the absorption of plant genes by bacteria. Such an event is basically possible under natural conditions, especially where decomposed or rotting plant material encounters large quantities of bacteria: in the intestinal tract of humans and animals or in maize silage, for instance.

Are the fears justified?

Bacteria are capable of absorbing plants genes, but under natural conditions such an event is extremely rare. From various studies we now know that a range of conditions must be fulfilled to enable bacteria to not only incorporate foreign genes into their own genome, but also to express the genetic information and convert it to a protein. Only then does an antibiotic-resistance gene become effective.

Many factors must come together before horizontal gene transfer actually occurs. Normally the probability of a bacterium absorbing a plant gene is around 1:1 billion to 1: 100,000 billion.

Nevertheless, such a gene transfer cannot be completely ruled out, especially if certain conditions apply which increase the likelihood. In such a case, pathogens could absorb antibiotic-resistance markers from rotting transgenic plants for example.

Instances of resistance are in fact widespread in bacteria anyway. Many bacteria found in arable soil are resistant to the antibiotic kanamycin. Ampicillin-resistant bacteria were found in three-quarters of all samples isolated from pigs and cattle.

When assessing the potential risks it should also be considered that the marker gene systems are based on various different antibiotic agents. Some of them are used in animal and human medicine, but many of them have long since ceased to be prescribed.

Undesirable antibiotic-resistance markers

Whatever the actual risk may be - antibiotic resistance markers have been the focus of controversy for some time now. Albeit for different reasons, authorities and experts now recommend that in future genetically modified plants should be produced without them.

  • As early as 1997 the ZKBS (Central Committee for Biological Safety), a committee of experts which advises the German authorisation authorities, spoke in favour of restricting the transfer of genes to those which code for the new, desirable trait.
  • The EU Deliberate Release Directive, which has been in effect since 2002, requires “the phasing out of the use of antibiotic-resistance markers in GMOs which may have a harmful impact on human health or the environment” from autumn 2002.

The drawbacks of traditional markers are becoming apparent even in practical research.

  • Two different marker gene systems are required if plants which have already been genetically modified are to be transformed again. But there are only a few available for each crop species.
  • If several marker genes left over from various developmental phases accumulate in a plant, the stability of the genetically engineered trait can be impaired.
  • The more genes and marker genes are transferred, the greater the probability of unforeseen effects occurring in the plant (pleiotropic effects).

In the next generation of transgenic plants, antibiotic-resistance markers will be the exception rather than the rule. But there is still a long way to go before sufficient new procedures and strategies for practical plant breeding become available.