Sep 2, 2002

Research Projects

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Release of DNA from transgenic sugar beet and horizontal gene transfer in the soil

(1995 – 1999) University of Oldenburg; Department of Biology; Genetics Working Group

Topic

During release trials with transgenic sugar beet, the following questions were investigated:

• What is the extent of DNA release from transgenic plants?
• How long does the DNA survive in the soil? How significant is the binding of DNA to soil particles? How is the DNA broken down?

A further question arose when transgenic plant DNA was found in soil on which transgenic plants had never been grown.

• By what method can DNA be introduced?

Background: If free transgenic DNA from sugar beet is present in the soil, it could in principle be taken up by micro-organisms in the soil. Whether horizontal gene transfer does actually occur was studied in a different research project.

Summary

• Genetic information transferred to sugar beet by means of genetic engineering was detected in the soil. The DNA transfer takes place via leaf material and beet remains, but chiefly as a result of pollen dispersal.
• Both free transgenic DNA and transgenic DNA bound to soil particles were present in the soil. The bound DNA breaks down more slowly. DNA can even survive the winter in the soil.
• The spread of transgenic DNA to areas where transgenic plants have never been grown was traced to pollen dispersal.

Experiment description

The studies were initially conducted with virus-resistant sugar beet at two release sites. Soil samples were taken both before and during cultivation of the transgenic plants. One site had a viral infection (_Rizomania_), but not the other.

DNA Release: In each soil sample the total amount of DNA present was isolated as well as just the free DNA, i.e. the DNA outside the plant cells. PCR analysis was used to investigate the isolates for traces of transgenic DNA.

Persistence of DNA in the environment in the soil: The breakdown of a marked DNA was tracked in different soil types (brown earth, loam, podzol) and the DNase activity was measured. Observations were conducted over a ten-day period to see if the DNase activity and the bacteria count changed. (A DNase is an enzyme which breaks down DNA and is generally released by soil bacteria as they grow.)

Further soil samples were studied in order to retrace the route by which transgenic DNA is introduced: They were taken both during cultivation of the transgenic sugar beet and after harvesting, and in addition from adjacent areas with and without pollen development. A nursery where sugar beet was coming into flower served as a control with pollen development.

(Sugar beet is a biennial plant. In the first year after sowing the beet itself is formed, in the second year it flowers and forms pollen. Beet plants are usually harvested before they flower. It is quite common for individual plants – bolters – to flower prematurely.)

Results

DNA release from transgenic plants: The transgenic DNA increased in the soil whilst the beets were growing and decreased again at the end of the vegetation period. Presumably the plant material disperses in the environment first, and the transgenic DNA is not released into the soil until later. The lifting of the sugar beet leaves significantly less transgenic DNA in the soil than pollen dispersal.

Persistence in the environment

• Free DNA, which was not bound to soil particles, was rapidly broken down by naturally occurring DNases. The soil type had no significant effect on this process.
• At both sites – with and without the virus – both the bacterial count and the DNase activity increased over the ten-day observation period; the latter increased over the entire vegetation period.
• DNA bound to soil particles can persist for longer than free DNA. Despite the presence of the enzyme which breaks down DNA in the soil sample, DNA can remain intact for a longer period of time, once it has been released from the cells of the plant material.

Dispersal methods for transgenic DNA. Transgenic DNA was found unexpectedly at some sites even before transgenic sugar beet was grown there. It is assumed that the transgenic DNA was introduced from adjacent areas, where pollen had formed in the previous year. Apart from the beet fields with adjacent pollen formation, no transgenic DNA was discovered at the trial sites. This was also the case after harvest and at the end of the winter period.

However, transgenic DNA was detected in several soil samples from sites where pollen formation had occurred nearby. It is clear that the prime cause of the spread of transgenic DNA is pollen dispersal.