Biology Reference
In-Depth Information
3.3 Imaging the
Meristem
1. To image the meristem with water-dipping lenses, pour dis-
tilled water into the box containing the cut apices or NPA-
grown seedlings so that the meristems are fully covered with
water (Fig.
1
). Since the solid growth medium absorb the
water and can swell, we recommend to keep the water in the
dish for at least 10 min before image acquisition.
2. The stack of images is obtained with a resolution of 0.5-1
m
along the
Z-
axis (an Arabidopsis meristematic cell is about
5
μ
m long in width and height).
3. If the meristem is moving during image acquisition, because
the medium does not adhere to the apex enough, it is useful
to add a few drops of more concentrated agarose (e.g., 2 %)
around the apex to immobilize it. To check if the initial posi-
tion of the object has changed during the stack acquisition,
restart an acquisition and check whether the position has
changed. Although the signal would still be visible, make sure
not to cover the meristem with agarose.
4. Confocal settings have to be adapted to the object and the
software for data processing. For time-lapse imaging, it is bet-
ter to reduce both time of scanning and laser irradiation as
much as possible.
5. After imaging, water is removed and the boxes containing the
meristems are returned to a growth chamber (
see
Note 7
).
Depending on the time interval between successive time
points, restaining with FM4-64 is not always necessary.
μ
4
Notes
1. Short day conditions are not absolutely required in this protocol,
and live imaging of the meristem can be achieved from plants
grown in long day and continuous white light conditions too.
2. MS medium or any other kind of standard medium can be used
here. The most important thing is to have a relatively concentrated
agar medium to maintain the stem in a vertical orientation.
3. Dissection can be done in air or in water. It is preferable to dis-
sect in water, as the meristem may dry out fast. If the dissection
only last for a few minutes, dissection in air can provide good
results and can be easier to start with, as dissection of the meri-
stem in water under the binocular can prove challenging because
of optical aberrations.
4. In tomato, the transition from vegetative to reproductive phase is
autonomous and the meristem stays vegetative until 6-12 leaves
are formed [
26
]. The time of transition can be modulated by
exogenous stimuli (light, temperature) and is different in differ-
ent backgrounds. The transition from vegetative to infl orescence
meristems can occur in vitro, when too old seedlings are used.
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