plant surface microbiology.pdf

11 Interactions of Microbes with Genetically Modified Plants 187
rying genes from GMPs. The exchange of genetic information between different
bacterial species by transformation, transduction or conjugation seems to
be common in nature (Krishnapillai 1996; Wöstemeyer et al. 1997 and references
therein). The detailed analysis of DNA and amino acid sequence data
has indicated that horizontal transmission of genes, even between bacteria
and eukaryotes or between eukaryotes from different systematic kingdoms,
probably occurred in rare cases during evolution (Dröge et al. 1998 and references
therein), but there is no experimental access to further investigation or
verification of such horizontal gene transfer (HGT) events.
The focus of the experimental work on HGT has been the question whether
antibiotic resistance genes, used as selectable markers in GMPs, can be transferred
to bacteria, enhancing the frequency of antibiotic-resistant bacteria of
medical importance (Nielsen et al. 1998). Beside the relevance of this question
for the risk assessment of GMPs, the transfer of antibiotic-resistance genes is
easy to detect compared to a possible horizontal transfer of genes which cannot
be used as a selectable marker for the isolation of transformed bacteria.
Therefore, experimental data about the possible HGT of other genes are
scarce.
The transformation of different bacterial species has been demonstrated
under optimized laboratory conditions using isolated plasmid DNA, total
DNA from GMPs and even homogenized plant material from GMPs as the
source for antibiotic-resistance genes (Schlüter et al. 1995; Gebhard and
Smalla 1998). The efficiency of the integration of the nptII gene, causing resistance
to kanamycin, into the genome of Acinetobacter sp. strongly depended
on the presence of homologous sequences in the bacterial DNA (Nielsen et al.
1997). This observation was confirmed by de Vries et al. (2001) using Acinetobacter
sp. and Pseudomonas stutzeri as well as by Bertolla et al. (2000) using
the plant pathogenic bacterium Ralstonia solanacearum as recipient for
antibiotic-resistance genes.
While transformation of bacteria is common under optimized laboratory
conditions, all experiments under natural conditions indicated that the frequency
of HGT is drastically reduced compared to optimized conditions.
Although DNA from transgenic plants can persist in soil for up to 2 years
(Gebhard and Smalla 1999), the availability of DNA from GMPs could be a
limiting factor for HGT. Even under otherwise optimized conditions (e.g., use
of purified DNA from transgenic sugar beet as source for the nptII gen, construction
of an Acinetobacter strain carrying a deleted nptII gene to allow
homologous recombination in the recipient bacteria), the frequency of HGT
was low in sterilized soil microcosms and below the detection limit in nonsterilized
soil (Nielsen et al. 2000). In a field release experiment with nptIItransgenic
sugar beet, a total of 4000 kanamycin-resistant colonies of soil bacteria
isolated from the field release site was checked for the presence of the
nptII gene from the transgenic plants by dot blot hybridization and PCR.
None of the isolates carried the nptII gene, indicating a natural kanamycin