plant surface microbiology.pdf

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Michael Kaldorf et al.
the GPD/bar construct in the ectomycorrhizal fungus A. muscaria was previously
verified by PEG-mediated protoplast transformation. Transgenic
poplars containing the GPD/bar construct were generated by Agrobacteriummediated
transformation. Twenty plants were isolated that originate from different
calli. PCR amplification was carried out with genomic DNA from transgenic
poplars using bar-specific primers. PCR products of the expected size
were obtained from 19 out of a total of 20 putative transgenic poplar plants
(Fig. 2). Isolated PCR-fragments of three clones were sequenced and revealed
the introduced bar gene.
For the investigation of a HGT event, 35,000 ectomycorrhizas formed
between transgenic poplars and A. muscaria were isolated and transferred to
selective agar plates.After the first round of selection, 102 putative Basta resistant
fungal colonies were obtained. However, none of these colonies was able
to grow after transfer to a fresh selection medium. Genomic DNA isolated
from fungal hyphae initially growing on the selection medium was investigated
for the presence of the bar-gene using PCR. No bar-fragment could be
obtained from any of these investigated clones. The utilization of primers for
Fig. 2. Analysis of genomic DNA isolated from putative kanamycin-resistant poplar
transformants. PCR amplification was performed on genomic DNA using primers specific
for the bar gene that amplifies an internal fragment of 550-bp length. Lanes 1 to 20
Isolated DNA from putative transformants. P Positive control with diluted DNA of
pBI121/3, K DNA isolated from a nontransformed poplar plant, M molecular size marker
(l/HindIII DNA marker)