plant surface microbiology.pdf

11 Interactions of Microbes with Genetically Modified Plants 189
amplify a 950-bp DNA fragment. In a second PCR step, a 500-bp fragment of
rolC should be amplified from the products of the first PCR using a second
inner primer pair, again specific for rolC. Isolated DNA from transgenic aspen
leaves was used as positive control for the nested PCR, while the quality of the
fungal DNA was checked with the primer pair ITS1/ITS4 (White et al. 1990),
specific for a part of fungal rDNA clusters. The rolC gene was not detected in
any of the 24 Phialocephala colonies analyzed. The number of 24 samples was
sufficient to demonstrate that the uptake of plant DNA in Phialocephala EM
does not occur on a regular basis, as suggested for the Plasmodiophora–Brassica
interaction (Bryngelsson et al. 1988).
The second approach was with transgenic plants that contained a small
marker gene, which could confer herbicide resistance into the target organism
after HGT. The advantage of this strategy is that a large number of samples
can be simultaneously screened, but only a small number of the samples able
to grow on the selection medium have to be investigated in detail. In order to
monitor HGT in ectomycorrhizas formed between poplar and Amanita muscaria,
a 1250-bp EcoRI/XbaI fragment of pBG (Straubinger et al. 1992) containing
the Streptomyces hygroscopicus bar gene under the control of the
Cochlibolus heterostrophus GPD1 promoter was inserted into the Agrobacterium
vector pBI121 (Clontech, Palo Alto, CA, USA; Fig. 1). The function of
amp
Fig. 1. Cloning strategy for the
construction of the binary vector
pBI121/3
pBG
4.21 Kb
A
ori
XbaI
BamH1
bar
GPD1
Nos-P
RB
Nos-ter
BamH1
NPTII (Kan)
Pst1
EcoR1
EcoRV
HindIII
Cla1
Xho1
HindIII
CaMV 35S P
pBI121/3
12.27 Kb
C
Bar-Gene
Nos-ter
Nos-P
RB
XbaI
GPD1 P
NPTII (Kan)
LB
EcoRI
HindIII
CaMV 35S P
pBI121
13.00 Kb
B
GUS
XbaI
Nos-ter
LB
SstI
EcoRI