Sep 2, 2002

Research Projects

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First deliberate release in Germany – Post-release safety research into genetically modified plants (petunias)

(1990 – 1993) Max Planck Institute (MPI) for Plant Breeding Research, Department of Plant Breeding and Genetics, Cologne

Topic

In 1990 Germany’s first field trial with genetically modified plants (petunias) was conducted at the Max Planck Institute for Plant Breeding Research in Cologne. The aim of this trial was to examine the following questions:

• Can genetic information from plants persist in the soil and if so, can it become incorporated into the genetic material of soil bacteria (horizontal gene transfer)?

To answer these questions, the project aimed to develop a methodology for detecting the DNA of transgenic plants in the natural ecosystem.

A further objective was to devise an experimental approach which would provide information about the probability of a horizontal gene transfer.

Summary

With regard to the deliberate release of genetically modified plants, fears have been expressed that genes inserted into transgenic plants, which are often also bacterial in origin, could find their way back into soil bacteria.

The methods developed in the project facilitated the detection of special DNA fragments in the soil. In an examination of around 450 soil samples over a two-year period, fragments of the transgenic plants were detected in four cases, all within the first two months after ploughing in the petunias.

The gene transfer studies indicate that horizontal gene transfer can be simulated under optimized conditions. However, in view of the prerequisites outlined, horizontal gene transfer in the natural environment is regarded as highly improbable.

In the course of the project some unexpected effects came to light: Intense sunlight and high summer temperatures resulted in a “reprogramming” of the introduced maize gene in the released transgenic petunias. Many of the petunias which started off salmon pink formed new white, or red and white patterned flowers.

Experiment description

The studies used petunias as model plants.

Part 1: Development of methods for detecting transgenic DNA in the soil

In order to be able to trace the breakdown process of plant DNA in natural soil ecosystems, a marker gene (in this case an antibiotic resistance gene) was transferred to petunias. Then, at regular intervals both during and after the release, soil samples from the release site were studied for signs of the marker gene. The PCR method of detection was used.

Part 2: Horizontal gene transfer studies

Since data from the first part of the project provided no information about the precise location of the marker genes (existing freely in the soil or already absorbed by soil bacteria), the aim of this part of the project was to provoke a horizontal gene transfer so that the parameters and probability of such an event could be more closely defined.

In microcosm experiments in the laboratory and under largely natural environmental conditions, soil bacteria were brought into contact with transgenic DNA. This involved, among other things, combining ground up transgenic plant material with soil and cultivating the mixture for seven days. At the end of the cultivation period the soil bacteria were transferred to a culture medium containing antibiotics, in order to find antibiotic-resistant bacteria. Resistant bacteria were then examined with DNA probes to see if the marker genes could be detected in the bacteria, or whether they were in fact exhibiting a natural resistance.

Additional experiments were conducted to further optimise the conditions for horizontal gene transfer. For example, the integration of the transgene into the bacterial genome was to be facilitated by ensuring that the gene sequences of the transgene match the bacterial genome as closely as possible. In addition, promoters that are particularly active in bacteria were used in the transgene, and bacterial strains that are noted for their readiness to accept foreign DNA were used as potential recipients.

Results

Detection of transgenic DNA in the soil

Over a two-year period soil samples were analysed at intervals of between two weeks and one month. In four cases out of a total of 450 soil samples there were positive indications of the presence of the marker gene. Positive indications were still present two months after the transgenic plants had been ploughed in. No further positive findings were found on subsequent sampling dates.

Gene transfer studies

Results relating to the persistence of foreign DNA in the soil yielded no information as to whether the marker genes existed freely in the soil or had already been absorbed by soil bacteria. Since DNA binds to quartz and clay minerals, at least in the short term, and is thus protected against immediate breakdown, in theory there is a chance that DNA fragments could be absorbed by soil micro-organisms and incorporated into their genetic material.

However, the absorption and stable integration of foreign DNA into the bacterial chromosome depends on a large number of factors. For example the bacterial strain must be fundamentally competent, in other words it must allow the absorption of the foreign DNA. Incompetent recipient bacteria respond by immediately breaking down the DNA during absorption. Even if absorption is successful, integration and recombination, i.e. the transfer to progeny, can only ensue if the foreign and bacterial DNA possess sufficiently homologous (matching) sequences. For this reason the transgene construct was optimised and competent bacteria were selected as DNA recipients. Bearing in mind all the preconditions necessary for horizontal gene transfer, it was concluded that horizontal gene transfer is not impossible. In conclusion however, in view of the results, it was maintained that gene transfer from transgenic plants to soil bacteria under natural conditions remains highly unlikely.

Unexpected colour changes in petunia flowers

Some unexpected observations were made during the deliberate release. A large number of the transgenic petunias, which originally had salmon pink flowers, started to produce new flowers that were white, faintly coloured or red and white patterned. It transpired that intensive sunlight and high summer temperatures had disabled the gene activity of the introduced maize gene (responsible for the red colour expression). The introduced maize gene disrupts the pigment metabolism in the petunia by making the original white pigments salmon pink. The attachment of methyl groups (DNA methylation) to the DNA of the introduced maize gene in the region of the promoter effectively “switched off” the maize gene. Consequently the synthesis stage from white to salmon pink pigments was interrupted and only white or faintly coloured flowers were produced.