Nov 13, 2007

Research Results

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Optimising the biological safety of transgenic plants

“Marker-gene-free plants will soon be standard.”

New and improved gene transfer methods are being developed throughout the world. The aim is to transfer only the target gene of interest. All sequences which are no longer required once transformation is complete, especially marker genes, are to be removed from the plant’s genome. Various safety research projects are also addressing this issue. In an interview with GMO Safety Professor Reinhard Hehl outlines the current state of play.

GMO Safety: Plant transformation methods are continually being refined. The key issues include greater efficiency, stable transgene integration and expression and, not least, marker gene elimination. Can you outline the current state of play? Where has progress been made, and where do you still see weaknesses?

Reinhard Hehl: T DNA transformation is a very precise method of transforming plants. Nowadays we can transfer the transgenes of interest far more accurately than was the case just 10 years ago. Advances have also been made in the development of special procedures to eliminate marker genes. In addition, we have considerably extended the spectrum of transformable plant species. In my view weaknesses arise where methods which have been well established in model plants either fail to function in agricultural crops, or function only suboptimally.

GMO Safety: Marker gene elimination is a particular focus of your research group. Are any approaches looking promising?

Reinhard Hehl : Yes - in future it will be possible to dispense with marker genes in plants of commercial interest. One particularly promising approach is co-transformation. The principle is based on the transfer of two separate T-DNAs, one carrying the transgene and the other the marker gene. The transgene and marker gene are introduced separately into the plant genome in such a way that they are integrated at different sites in the genome. This means there is very good chance that the transgene and the marker gene can be separated during cell meiosis, through recombination processes and that plant strains which carry only the transgene can then be selected. This method is already established and is without doubt the most widely used method of marker gene elimination in easily transformable plants.

GMO Safety: Are there any other ways to produce marker-gene-free plants?

Reinhard Hehl: There are two methods worth mentioning: one, which is based on the Cre/lox system, involves excising the marker gene from the genome by activating the recombinase genes which flank the marker gene. The other method, which my team is currently working on, uses the transposon system. We exploit a well-known plant phenomenon whereby genetic elements in the plant enable specific genes to ‘jump’ to a different genome section in the plant. We introduce a transgene and marker gene together using a T-DNA vector, then generate a transposition event which causes the transgene to reintegrate at a different site on the genome, thus separating from the marker gene. Here too, the marker-gene-free plants can be separated from the marker-gene-carrying plants in the subsequent generation. We have been able to demonstrate this in sugar beet. It works, provided that the transposon system used functions efficiently in the plant species being transformed.To sum up, we can say that, thanks in part to the research group, methods of removing marker genes from transgenic plants are now available.

GMO Safety: Segregation is the final step in all the methods of marker gene elimination you have mentioned. In other words, marker-gene-free strains can be selected only in the subsequent generation. That makes the procedure very time-consuming, doesn’t it?

Reinhard Hehl: Yes - even today we spend a great deal of time identifying the transgenic marker-gene-free plants in the subsequent generation. But there are smarter research methods for obtaining marker-gene-free plants much more quickly. For example, we can dispense with the crossing stage and therefore the subsequent segregation altogether. Instead, marker-gene-free offspring are obtained from the haploid pollen of plants which have previously been successfully co-transformed.

If the target gene and marker gene are integrated on different chromosomes during co-transformation, some pollen carrying only the target gene will be produced after pollen cell division. Following artificial diploidization of the haploid chromosome set, plants carrying only the target gene can be regenerated from these pollen cells. This approach is being pursued further In a research project at the Institute of Plant Genetics and Crop Plants Research (IPK) in Gatersleben.

GMO Safety: The methods you describe, particularly co-transformation, seem to be on the verge of practical application. Are there any safety concerns?

Reinhard Hehl: The co-transformation method is ready for practical application. However, new transgene integration sites are created for each transgenic plant, and these must all undergo individual safety assessment. It must be ensured that no mutations occur at the transgene integration site and also that transgene expression takes place in the desired form.

GMO Safety: In view of the development of transgenic plants containing new and modified plant substances, such as substances of commercial or pharmaceutical interest for example, procedures are being developed to limit the ability of these plants to spread - essentially sterilisation methods. How close to practical implementation are these procedures?

Reinhard Hehl: These so-called confinement methods make sense when applied to plants which outcross easily, such as oilseed rape and certain types of cereal. The production of male-sterile plants is one biotechnological approach currently being pursued. Our research group is working on metabolic-physiological sterility, in which the pollen cells cannot metabolise carbohydrates. This inhibits pollen development, resulting in the production of male sterile plants. The results are encouraging, but the technique is not yet ready to put into practice. Chloroplast transformation, which prevents genes expressed in the chloroplast from being transferred to other plants via pollen, is a major topic of future interest.

GMO Safety: If you were to draw up a list of all the unresolved issues surrounding the optimisation of gene transfer methods, where would you focus research during the coming years?

Reinhard Hehl: The refinement of sequence-specific integration will be at the forefront, as demonstrated by the recent call for tenders from the German Federal Ministry of Education and Research (BMBF) for safety research on transgenic plants. Genes should be integrated only where we want them. In other words at specific sites in the genome which have previously been well characterised. This means excluding undesirable mutations and the ensuring optimum expression of the transgene at its integration site.

GMO Safety: Thank you for talking to us.