High-Pressure Freezing and Low-Temperature Processing of Plant Tissue Samples for Electron Microscopy - Plant Cell Morphogenesis: Methods and Protocols - page 150

were germinated in the presence of 0.05 M sucrose for 2 days.
Germinated seedlings were then grown in the presence of
0.1 M sucrose for 1 day and were then processed for high-
pressure freezing. Tobacco seedlings were grown in the pres-
ence of 3.5 % (w/v) sucrose before root tips were removed for
HPF [ 18 ]. In general, non-penetrating cryoprotectants are
preferred because they are considered to disrupt cell structure
less than penetrating cryoprotectants [ 4 ]. 1-Hexadecene [ 29 ]
can be used for plant tissue [ 30 ]; we have obtained good
results with 1-hexadecene for onion cotyledon. However, after
treating 1-hexadecene-protected specimens with OsO 4 , the FS
medium turns black, making it diffi cult to see specimen pieces.
Be careful not to discard specimens when removing the OsO 4
solution and when washing with acetone.
2. Plant cells are surrounded by a dense meshwork of cell wall
polysaccharides. Aerial organs of plants are covered with hydro-
phobic waxes. These protective layers on plant tissues interfere
with the penetration of chemical cross-linkers. The large vacu-
oles that most plant cells contain also pose problems for chemi-
cal fi xation. Chemical fi xatives sometimes break vacuoles, and
the leakage of acidic vacuolar contents could disrupt cellular
structures and can also dilute the fi xative. These shortcomings
of conventional chemical fi xation can be overcome by the use of
HPF. However, plant tissues are often too large to fi t into HPF
planchettes. When dissecting plant tissues, stress and damage to
the tissues prior to freezing should be minimized. Therefore, it
is crucial to dissect rapidly and carefully. It is important to prac-
tice dissecting material and transferring samples to planchettes
before attempting to freeze critical samples.
3. Poor preservation of a high-pressure frozen sample is primarily
due to damage to the fi ne structure of subcellular components
caused by the formation of ice crystals in the cytoplasm or
nucleus during freezing. Examples of poor freezing are shown
in refs. [ 25 , 26 , 31 ]. Not all plant samples are suitable for HPF
processing. Waxy and hard tissues, like tree leaves, are diffi cult
to dissect and to submerge in cryoprotectant. For such sam-
ples, it is better to resort to conventional fi xation [ 32 ]. It is
also diffi cult to freeze plant cells in which the volume is occu-
pied almost entirely by vacuoles, because of the high water
content in the vacuoles.
4. Dextran is a glucose polymer that binds water molecules with
its hydroxyl group. Dextran serves as a cryoprotectant because
it suppresses ice crystal growth outside BY-2 cells. Another
role of dextran is to hold BY-2 cells together throughout the
FS and resin-embedding steps.