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

(2008 - 2011) University of Hamburg, Department of Biology, Developmental Biology and Biotechnology Division

Topic

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 project is to develop a new confinement system for maize that will enable the production of transgenic maize plants which are capable of reproduction, but produce transgene-free pollen. This will be 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 at any time during the life cycle of the plant, scientist can ensure that the transgene remains in the plant genome during breeding. After applying a simple heat treatment, the breeder can release the seed obtained from these plants to the farmer. The seed will then produce plants which carry the genetic modification in all tissues except the pollen.

Experiment description

Maize plants after transformation. Left: herbicide-resistant plants where transformation has been successful. Right: non-resistant plants.

Each recombinase recognises a specific DNA sequence. To enable a specific sequence to be removed from the genome using a recombinase, the sequence must be flanked by two recognition sequences. 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 element (promoter ) for the recombinase gene. In this project maize strains will be produced which employ two recombinases with different promoters. The gene for the first recombinase is controlled by a heat-inducible 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 gene. 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, maize plants will be transformed with a gene construct containing a reporter gene to detect the second recombination, in addition to the two recombination systems. The gene construct will also contain a herbicide-resistance marker gene to detect transformation .

The gene construct will be integrated into a vector which will be introduced into the plant genome via the medium of Agrobacterium tumefaciens .

Producing transgenic maize strains

The maize strains will be transformed and grown to maturity in the greenhouse. They will be sprayed with the complemantary herbicide of the introduced herbicide-resistance gene to identify the transformed plants. Their ability to grow under these conditions indicates that transformation has been successful.

1. Recombination system – Activating the heat-inducible promoter

A heat shock (16 hrs, 37 °C) 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, the pollen will be treated with a substance which 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.

Non-transgenic maize plants will be pollinated with pollen from the transgenic plants and their offspring will be sprayed with the herbicide. These serves as an additional measurement of the efficiency of recombination. Plants that are able to grow under these conditions still carry the herbicide-resistance gene, i.e. recombination has not taken place in these plants.