Preventing the spread of genetically modified poplars through transgene-free pollen

(2008 - 2011) Johann Heinrich von Thünen-Institut, Institute for Forest Genetics, Großhansdorf

Topic

The use of genetically modified poplars in commercial plantations, as a renewable raw material for example, has been under discussion for a number of years. A key consideration in the safety assessment of GM crops is the potential spread of the newly inserted genes via pollen or seed. For this reason, scientists throughout the world are developing strategies aimed at the targeted prevention of gene transfer via pollen or seed (biological confinement).

The aim of this sub-project is to generate transgenic poplars which are capable of reproduction, but which produce transgene-free pollen. This is achieved using recombinases, special enzymes which can be used to cut targeted DNA segments out of the plant genome.

The transgene will be excised from the genome during pollen formation using the approach described here. Since this excision can be induced by heat treatment at any time during the life cycle of the plant, scientists can ensure that the transgene remains in the plant genome during breeding. The seed, which is destined for commercial cultivation, undergoes heat treatment before being released. It then produces poplars which carry the genetic modification in all tissues except the pollen.

Experiment description

Leaf of a genetically modified poplar that carries a reporter gene. These plants are the starting material for this experiment. The reporter gene carries the information for an enzyme that can convert a colourless substance that is applied to the leaf into a blue dye.

Each recombinase recognises a specific DNA sequence. To enable a specific sequence to be removed from the genome using recombinases, the sequence must be flanked by two recognition sequences. The recombinase and the recognition sequence together are referred to as a recombination system.

The site or time of recombination can be determined by selecting the control elements (promoters ) for the recombinase gene. In this project poplar strains will be produced which employ two recombinases with different promoters. The gene for the first recombinase is controlled by a heat-activated promoter. In other words, only after heat treatment, which can take place at any time, is the gene expressed and the first recombinase produced. It cuts from the genome a DNA sequence that blocks the promoter for the second recombinase. Only then can the second recombinase form. The second promoter is pollen-specific, i.e. the second recombinase is produced only in developing pollen, where it excises the transgene from the plant genome.

To test the method described above, aspens will be transformed with a gene construct containing a reporter gene to detect the second recombination, in addition to the two recombination systems. The reporter gene will be used here instead of a transgene to give a visible indication of the system's success. The gene construct also contains two marker genes : a herbicide‑resistance gene to detect the transformation and an antibiotic‑resistance gene to detect the second recombination.

Producing transgenic poplar strains

Leaf sections will be transformed and cultivated on a special culture medium in a climate chamber. Each leaf section forms a callus (an undifferentiated cluster of plant cells) which is capable of developing into a poplar plant under favourable conditions.

1. Recombination system – Activating the heat-inducible promoter

Heat shock activates the first recombinase gene in the plants, which cuts out the DNA section between its recognition sequences. This then brings the second recombinase gene under the influence of the pollen-specific promoter. This is verified using molecular-biological analysis (PCR and Southern Blot ).

2. Recombination system – Activating the pollen-specific promoter

Plants which have successfully undergone the first recombination are grown on in the greenhouse to the point of flowering. The second recombinase is then activated during pollen formation. Once again, molecular-biological methods (PCR, Southern Blot) are used to verify that the reporter gene has been successfully excised. In addition, a substance is added to the pollen that is converted to a blue dye in the presence of the reporter gene. The greater the quantity of undyed pollen, the more effective the second recombination has been.