To provide estimates of horizontal gene transfer from transgenic
crops to indigenous soil bacteria, transformation frequencies were obtained
for naturally transformable Acinetobacter baylyi
BD413 using a chromosomally integrated plant transgene. The transgene
comprised sequences for two phenotypic markers: kanamycin resistance
(npt II) and green fluorescent protein
(gfp), expressed from their own bacterial
promoters. Recipient bacteria carried a copy of these two genes, with
deletions in their 3'-termini abolishing the marker activity, these genes
were integrated into a 16S rRNA gene in the bacterial chromosomal genome or
carried on a broad host range plasmid. Successful recombination between the
plant transgene and the bacterial genome resulted in restoration of the
markers, allowing detection through antibiotic selection and fluorescence.
Transformation parameters of increasing complexity, without any enrichment
steps, were used to approach the field conditions, while still obtaining
measurable transformation frequencies. In pure culture filter experiments,
transformation was detected using ground, chopped and whole leaves, as well
as whole sterile seedlings, and ground roots. In sterile soil microcosms,
transformation was detected using pure plant DNA (3.6 × 10-8
transformants per recipient) and ground leaves (2.5 × 10-11).
Transformation was also detected for the first time in non-sterile soil
using pure plant DNA (5.5 × 10-11). Since the same constructs
were used throughout, these data allow predictions of even more complex
environmental systems where measurable frequencies are not easily
obtainable.