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Research into horizontal gene transfer from transgenic sugar beet to bacteria

(1994 – 1997) Biologische Bundesanstalt für Land- und Forstwirtschaft (BBA) (seit 2008 Julius Kühn-Institut (JKI)), Institut für Pflanzenvirologie, Mikrobiologie und biologische Sicherheit; Braunschweig

Topic

Can DNA from transgenic plants be transferred to soil micro-organisms? In order to assess whether this kind of horizontal gene transfer is possible, it is necessary to look at more than just the uptake and integration of the transgenic DNA in micro-organisms. For horizontal gene transfer to occur, the transgenic DNA must also be present in the soil in a form available to the micro-organisms.

The project focussed on the following main questions:

  • How long can transgenic plant DNA persist in the soil?
  • Can horizontal gene transfer of transgenic plant DNA to soil bacteria be detected under laboratory or field conditions? A particular strain of bacteria was used as a model organism for these investigations.

The fate of free DNA in the soil.

Summary

  • It was possible to detect DNA from transgenic sugar beet in the soil over fairly long periods of time. It is, however, not possible to make general statements about the persistence ability of transgenic DNA in the soil because it depends on various factors.
  • It was not possible to detect gene transfer under natural conditions in the soil.
  • Under ideal laboratory conditions it was for the first time possible to demonstrate horizontal gene transfer of transgenic plant DNA to naturally competent bacteria. This even worked with ‘plant juice’ (juice from homogenised plant cells that contains DNA), although to a much lesser extent.
  • Despite this finding, the probability of horizontal gene transfer from transgenic plant DNA to bacteria in the soil appears small.

Experiment description

Soil samples are taken using a probe rod (above): 28 samples per plot (below).

Procedure of Analysis

The persistence ability of transgenic DNA was investigated in rotting fields: In two years (1993 and 1994), transgenic beet remains were ploughed into the soil after the harvest. In the spring and autumn of the following years, samples were taken and analysed according to a particular procedure.

In addition, the persistence of free DNA in the soil was investigated in the laboratory: soil samples were mixed with transgenic sugar beet DNA and observed over a period of six months using PCR.

The possibility of horizontal gene transfer was investigated both in the natural environment in the soil and in the laboratory under specific conditions conducive to gene transfer.

(1) As evidence of horizontal gene transfer in the field the researchers looked for bacteria in the soil samples that had taken up parts of the genetic construct introduced into the sugar beet. The detection methods involved PCR and hybridisation.

Since only a small proportion (0.1-1%) of soil bacteria can be cultured under laboratory conditions (culture-dependent methods), the project also investigated DNA extracted directly from the soil samples (= culture-independent), which contains fungal, plant and free DNA as well as bacterial DNA. This DNA was analysed for the presence of transgenic DNA using PCR and primers specific to the genetically engineered construct. These primers facilitate the specific and sensitive detection of the transgenic DNA.

(2) Two experimental approaches were chosen for horizontal gene transfer under optimised laboratory conditions . The bacterium Acinetobacter was used as a model organism.

In a first step, the uptake ability of bacterial cells was investigated using different types of DNA (from bacteria, plasmid DNA, transgenic plant DNA).

Horizontal gene transfer is a very rare event, which is therefore difficult to detect. For this reason, a special Acinetobacter strain was developed with ideal conditions for gene transfer: an incomplete nptII gene (kanamycin resistance) was introduced into the bacteria. The complete gene is present in the sugar beet as a marker gene. Using special mechanisms (homologous recombination), the special Acinetobacter strain can complete its nptII gene comparatively easily by taking up transgenic plant DNA.

The number of transformants and the transformation frequency were measured for both approaches.

Results

Persistence ability of transgenic DNA in the soil

  • In the 1993 rotting field, the transgenic sugar beet DNA was detected in the total DNA extracted from the soil samples (culture-independent analysis) over two years.
  • In the 1994 rotting field, however, even after only six months only small quantities of transgenic DNA were found. It is, however, not possible to say for sure whether the transgenic DNA detected is present as free DNA or ‘packed’ in rotting plant remains.
  • In the laboratory experiment (culture-dependent analysis) the transgenic DNA was still detected after one, three or six months, depending on the PCR method used.

Horizontal gene transfer

(1) Outdoor conditions (culture-dependent analysis)

  • The number of bacteria increased temporarily following the addition of the shredded beet material as a result of the addition of nutrient in both rotting fields. Around 4500 bacteria that showed resistance to kanamycin were examined more thoroughly. No instances of transgenic DNA sequences were found. The resistance trait of the bacteria is not based on the nptII gene present in the sugar beet (see Table 1)

(2) Laboratory conditions

  • No transformants that had taken up transgenic plant DNA were found under laboratory conditions either. This meant that there was no indication of horizontal gene transfer.

(3) Optimised laboratory conditions

  • Under optimised laboratory conditions, the ‘prepared’ Acinetobacter strain with an incomplete nptII gene was found to have taken up transgenic sugar beet DNA in isolated cases. Transformations with non-transgenic sugar beet DNA were not found (see Table 2).
  • It was possible to attribute the gene transfer of the transgenic DNA to a homology (similarity of DNA sequences) between the DNA of the Acinetobacter strain and the transgenic DNA.

Gene transfer from transgenic plants to Acinetobacter sp. BD413 was found only with homologous sequences and only under optimised conditions.

Table 1: Frequency of horizontal gene transfer under natural conditions in the soil (from: Nielsen et al, 1997)
(naturally occurring Acinetobacter bacteria)

DNA material used Transformants obtained
(number of micro-organisms that have taken up DNA, based on the quantity unit of the administered DNA)
Average transformation frequency
(frequency of a gene transfer based on total number of micro-organisms)
Plasmid* DNA (ring-shaped) 27000 1,9 x 10-5
1 transfer for 190.000
Plasmid DNA (linear; open ring structure) 100 2,0 x 10-8
1 transfer for 200 million
Transgenic DNA from sugar beet not detectable -11
less than 1: 100 billion
* Plasmids are ring-shaped DNA elements that are frequently exchanged between bacteria.


Table 2: Frequency of horizontal gene transfer under optimum laboratory conditions (from: Gebhard and Smalla, 1998)
(modified Acinetobacter strain with integrated incomplete nptII gene)

DNA material used Transformants obtained
(number of micro-organisms that have taken up the DNA, based on the quantity unit of the administered DNA)
Average transformation frequency
(frequency of gene transfer based on total number of micro-organisms)
Plasmid DNA 110000 9,85 x 10-5
1 transfer for 985.000 
Transgenic DNA from sugar beet 33 5,36 x 10-9
1 transfer for 5,36 billion
Non-transgenic DNA from sugar beet 0 0
Plant juice from transgenic sugar beet (homogenised plant cells) 1,5 1,5 x 10-10
1 transfer for 24 billion