Sep 25, 2005

Research Projects

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Using a negative selection marker to produce marker-gene-free transgenic plants

(2001 – 2004) University of Rostock, Department of Agroecology, Institute of Soil Science

Topic

The marker gene (an antibiotic-resistance gene) used for the transformation of plant cells was coupled with a negative selection marker. It was thought that this would make it possible to eliminate all plants carrying a marker gene from the progeny of the transgenic plants without the need to conduct costly analyses. Plants with the marker gene die out and only marker-free transgenic plants survive.

The project focussed primarily on the following two objectives:

• Optimisation of the independent integration of both marker genes (the closely coupled antibiotic-resistance gene and negative marker) and the target gene on different chromosomes.
• Refinement of the procedure for selecting plants which carry a marker gene. It should be applicable to different plant species.

Summary

The transformation method using one agrobacterial strain and two plasmids resulted in high co-transformation rates, whilst the method using two agrobacterial strains, each with one plasmid, resulted in lower rates. It was possible to balance out this difference by modifying the procedure.

It was demonstrated that the selection system is suitable for plants. However, the inductor substance is not yet pure enough for wider use. Work on optimising the inductor substance is underway with the aim of greatly reducing the cost of analyses required to identify plants with the target gene.

Experiment description

Co-transformation. Initially, new vectors were constructed to transfer the target gene and marker genes independently of one another and integrate them separately into the plant genome of a plant. Both marker genes were joined in a single cassette and arranged on a different vector from the target gene that conveys a new trait.

If the marker genes and the target gene really are integrated at different sites in the genome of the plant, they will be distributed in a range of combinations among the offspring. This occurs as a result of the new combination of the maternal and paternal chromosomes during fertilisation of the egg cell. Only plants which contain the target gene, but not the marker gene, should survive.

Selection markers. A gene for the enzyme deacetylase from E.coli (argE) was used as the negative selection marker. If it is present in a plant, the plant reacts to an otherwise harmless substance (inductor) and dies off. The inductor substance was a herbicide derivative (N-acetyl-phosphinothricin), which is converted into an active toxic herbicide (phosphinothricin) by the enzyme deacetylase.

• The procedure was developed for transgenic oilseed rape. Since a suitable inductor substance had not yet been found for oilseed rape, the aim was to obtain it by biotechnological methods, a technique which has proved successful with tobacco plants.
• The selection was tested and optimised initially in the greenhouse and then later in field trials. The plants were also studied using molecular biological techniques (PCR, Southern hybridisation).

Results

Co-transformation. The first step was to separately insert the target gene and the two coupled marker genes. First a plant transformation vector was designed, which contained the two very closely coupled marker genes. The target gene was located on another vector, which was then used to transform the oilseed rape either in the same agrobacterial strain or in a second strain.

The co-transformation rates vary depending on whether the two vectors are used in one bacterial strain or in two different bacterial strains.

As expected, the transformation method using one agrobacterial strain and two plasmids yielded high co-transformation rates (71% in oilseed rape, 68% in tobacco), whilst the method using two agrobacterial strains each with a single plasmid resulted in lower rates of 48% in oilseed rape and only 35% in tobacco. However, by modifying the procedure it is possible to compensate for this disadvantage (co-transformation rate 47%), so that both the co-transformation rates and the probability of separate integration are high enough to obtain marker-free transgenic offspring.

Selection: To identify these plants, the deacetlyase gene from E.coli (argE) was used as a negative marker. The enzyme deacetlyase, the product of this gene, generates the herbicide phosphinotricin from N-acetyl-phosphinothricin, a substance that is non-toxic to plants. It was demonstrated that this substance is suitable for selecting argE-free plants. Consequently, the cost of analyses required to identify plants with the target gene can be dramatically reduced. The inductor substance is not yet pure enough for wider use, but there are some promising purification approaches available.