Biology Reference
In-Depth Information
3. In tomato ( see Note 4 ), 11-12-day-old seedlings are cut. The
apex is put vertically in a petri dish fi lled with a solid agarose
medium (e.g., 1 % agarose) to prevent drying of apex during
the dissection. Most of the preexisting leaf primordia are
removed, but it is reasonable to leave the youngest primordia.
If organs are covering the meristem, their most apical part can
be cut, although removing too much could damage the meri-
stem or affect its development through the indirect effects of
wounding. The dissected apices of a subapical region ca. 5 mm
long are transferred to the tomato apex culture medium. It is
recommended to put only one apex per dish, to minimize time
that each the apex will be under the CLSM during imaging.
It is usually better to dissect the apex a few hours before imag-
ing, to let the apex recover from the wound stress.
To reconstruct meristem geometry from CLSM stacks and/or seg-
ment the cell topology for further analysis, cell outlines must be
visualized. The signal-to-noise ratio (from either plasma mem-
branes or cell walls) must be high enough to ensure the best results
from the segmentation tools. One can use a transgenic line, in
which fl uorescent protein is fused with plasma membranes (e.g.,
the Arabidopsis p35S::GFP-LTI6b line), or fl uorescent dyes for
either plasma membranes (FM4-64) or cell walls (PI).
3.2 Staining the
Meristem Before
Imaging
1. Staining plasma membranes: before imaging, the meristem is
immersed for at least 5 min in distilled water ( see Note 5 ).
This will notably fully hydrate the gel and prevent the stem
from moving too much in the Z direction while imaging. This
is notably crucial when stacks of images are obtained. The
immersion in water may also hydrate the meristem surface and
facilitate the uptake of dyes, like the membrane marker FM4-
64. If membranes need to be stained, the water is removed,
infl orescences are treated with about 2
g/ml stock
in water of FM4-64 (Invitrogen) for 1-5 min ( see Note 6 ) and
the infl orescences are immersed in water again.
2. Staining cell walls: a 0.1 % PI solution is used. Generally, the
meristem has to be stained each time immediately before the
imaging. To stain the meristem, pour the PI solution to the
petri dish or other culture dish, so that the whole apex is
immersed in the solution. If the meristem is stained for the
fi rst time, incubate for ca. 5 min. For the following times
2-3 min is enough. After removing the PI solution (the solu-
tion can be reused again a few times), wash the apex in the
water at least twice. Note that although PI is light sensitive, it
can be handled under regular room light conditions.
μ
l of a 330
μ
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