plant surface microbiology.pdf

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Thomas F.C. Chin-A-Woeng et al.
phase contrast microscopy and, when the protoplastation nears completion,
the protoplasts are collected on three layers of Miracloth, transferred to a new
tube and washed with a sterile cold sorbitol solution (1 M sorbitol, 50 mM
CaCl 2 , 10 mM Tris-HCl, pH 7.4). The protoplasts are sedimented by centrifugation
at 850xg (2100 rpm) at 4 °C and the number of protoplasts is determined
with a haemocytometer.
5.1.1.3 Transformation of Protoplasts
Protoplast are transformed by addition of up to 15 mg of DNA to 200 ml of protoplast
suspension and incubated on ice for 15 min, or stored at 4 °C.A volume
of 1.0 ml PEG solution (60 % (w/v) polyethylene glycol 6000, 50 mM CaCl 2,
10 mM Tris-HCl, pH 7.4) is slowly added while shaking gently. The mixture is
incubated on ice for 30 min after which the protoplasts are washed with a
magnesium sulphate/potato dextrose broth solution at 4 °C. The protoplasts
are sedimented by centrifugation at 2500 rpm at 4 °C for 10 min and resuspended
in the remaining fluid after discarding the supernatant. The protoplasts
are incubated 30 min at room temperature and portions (50–1200 ml)
are plated onto selective media containing 0.8 M sucrose, 10 mM Tris-HCl pH
7.4, 100 mg/ml hygromycin, and 1.5 % (w/v) agar. Plates are incubated for 2 or
3 days at the appropriate growth temperature.
5.2 Marking Rhizosphere Bacteria with Autofluorescent Proteins
The green fluorescent protein (GFP) of the jellyfish Aequorea victoria has
been rapidly and successfully adopted as an important marker for investigating
processes in the rhizosphere. GFP is a 27-kDa polypeptide which converts
the blue chemiluminescence of the Ca 2+ -sensitive photoprotein
aequorin into green light. The active chromophore is a tripeptide, the formation
of which is oxygen-dependent and occurs gradually after translation
by undergoing an autocatalytic reaction. GFP emits bright green light
(l max =510 nm) when excited with ultraviolet (UV) or blue light
(l max=395 nm) in vivo and in vitro.
GFP allows the non-destructive localisation and monitoring of individual
cells on the root surface and does not require, unlike other biomarkers, exogenously
added substrates, energy sources, or cofactors other than molecular
oxygen. GFP fluorescence is stable, species-independent, requires no processing
by the cells and fixing or staining is not necessary so artefacts cannot be
introduced. However, if required, GFP allows fixation since it is unaffected by
paraformaldehyde treatment. It is also stable under many other denaturing
conditions such as the presence of denaturants or proteolytic enzymes, high
temperatures (65 °C), and pH levels (6–12). Expression can be easily detected
using epifluorescence or confocal laser scanning microscopy. Other optical