23. After the first minute vacuum, it is recommended to gentle
agitate the vacuum desiccator to resuspend the Agrobacterium
in the infiltration buffer and make sure that it is in contact with
the seedlings.
24. The range of pressure usually applied is 300–500 mbar. If less
pressure (more vacuum) is used, the efficiency is higher but the
seedlings could be damaged and cell death will be observed in
some areas of the cotyledons.
25. Agrobacterium suspension after vacuum infiltration is removed
from the plates with a pipette. It is also recommended to use a
micropipette to remove the bacterial solution that remains on
the cotyledons (one by one) and try to stand up each seedling
to avoid the contact of cotyledons with the MS agar medium of
the plate to prevent contamination.
26. Agrobacterium motility seems to be reduced with exposure to
light.
27. When a marker is highly expressed, it is also possible to image
after 2 days.
28. Try to choose the healthiest and non-stressed seedlings to get
reliable results. Seedlings of growth mutants can be more
affected to the vacuum exposure than wild-type seedlings. On
the other hand, immunity mutant seedlings could have higher
expression efficiency than wild-type seedlings. This has to be
taken into consideration.
29. It is very useful to screen the cotyledons in an epifluorescence
microscope mode before the confocal microscope mode in
order to localize the areas where the marker is expressed.
30. It is also important to detect in the epifluorescence microscope
mode the dying cell areas (chloroplasts are still viable) because
cells in these areas may produce fluorescent compounds. This
unspecific fluorescent signal can be confused with the green
fluorescent signal of the markers. Dead cell areas with no signal
of chloroplast fluorescence appear as dark areas.
31. Some cells might overexpress the fluorescent marker, so it is
important to have into consideration that an overexpressed
marker can appear in unexpected organelles of the cell. For
example, if it is a secretory protein and it is overexpressed, it
could appear in the ER or in the Golgi bodies besides the
apoplastic space. In addition, Agrobacterium is often still alive
for a few days after infiltration and it may appear as fluorescent
dots clumped together which are observed in the apoplastic
space or on the top of the cells.
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Ce ´ sar Bernat-Silvestre et al.
agitate the vacuum desiccator to resuspend the Agrobacterium
in the infiltration buffer and make sure that it is in contact with
the seedlings.
24. The range of pressure usually applied is 300–500 mbar. If less
pressure (more vacuum) is used, the efficiency is higher but the
seedlings could be damaged and cell death will be observed in
some areas of the cotyledons.
25. Agrobacterium suspension after vacuum infiltration is removed
from the plates with a pipette. It is also recommended to use a
micropipette to remove the bacterial solution that remains on
the cotyledons (one by one) and try to stand up each seedling
to avoid the contact of cotyledons with the MS agar medium of
the plate to prevent contamination.
26. Agrobacterium motility seems to be reduced with exposure to
light.
27. When a marker is highly expressed, it is also possible to image
after 2 days.
28. Try to choose the healthiest and non-stressed seedlings to get
reliable results. Seedlings of growth mutants can be more
affected to the vacuum exposure than wild-type seedlings. On
the other hand, immunity mutant seedlings could have higher
expression efficiency than wild-type seedlings. This has to be
taken into consideration.
29. It is very useful to screen the cotyledons in an epifluorescence
microscope mode before the confocal microscope mode in
order to localize the areas where the marker is expressed.
30. It is also important to detect in the epifluorescence microscope
mode the dying cell areas (chloroplasts are still viable) because
cells in these areas may produce fluorescent compounds. This
unspecific fluorescent signal can be confused with the green
fluorescent signal of the markers. Dead cell areas with no signal
of chloroplast fluorescence appear as dark areas.
31. Some cells might overexpress the fluorescent marker, so it is
important to have into consideration that an overexpressed
marker can appear in unexpected organelles of the cell. For
example, if it is a secretory protein and it is overexpressed, it
could appear in the ER or in the Golgi bodies besides the
apoplastic space. In addition, Agrobacterium is often still alive
for a few days after infiltration and it may appear as fluorescent
dots clumped together which are observed in the apoplastic
space or on the top of the cells.
154
Ce ´ sar Bernat-Silvestre et al.