High-Pressure Freezing and Low-Temperature Processing of Plant Tissue Samples for Electron Microscopy - Plant Cell Morphogenesis: Methods and Protocols - page 146

seeds to germinate in the dark. Place the container at a 45°
angle so that the seedlings will grow fairly straight.
2. Leave the plates in the dark for 2 days at 25 °C.
3. Open the container, select seedlings with less than 1 mm long
roots, and transfer them onto a piece of fi lter paper wetted
with 0.1 M sucrose in distilled water.
4. Culture the seedlings further for 1 day.

1. Place a glass plate under a dissecting microscope.
2. Put a few drops of an aqueous solution of 0.1 M sucrose on
the glass plate, and make a pool of this solution ( see Note 1 ).
Prepare two brass freezer planchettes. One is for the bottom
and the other is for the top.
3. Using forceps, immerse the bottom planchette in the sucrose
pool. Be careful not to create air pockets or bubbles inside the
bottom planchette, as these would collapse during pressuriza-
tion and cause damage to the sample ( see Note 2 ; ref. [ 3 ]).
4. Select a seedling in which the length between the bending
point of the hook to the base of the cotyledon is <10 mm.
5. Pick the seedling up by gently catching the residual seed in
forceps, and immerse the seedling in the sucrose pool. Proceed
as quickly as possible from this step to the freezing step to
avoid drying of samples [ 4 ].
6. Hold the seed part with forceps in one hand, and cut a basal
part of the cotyledon 1-2 mm in length with a razor blade
using the other hand under the dissecting microscope. Split
the cotyledon section into two pieces longitudinally.
7. Put the tissue pieces in the bottom planchette. Wet the bot-
tom of the top planchette with the solution, and put it on top
of the bottom planchette. Again try to remove any air bubbles
trapped inside the bottom planchette with forceps.
8. Pick up the sample enclosed in the planchettes, and load it
into a sample holder immediately.
9. Freeze the sample using a high-pressure freezer.
10. Transfer the high-pressure frozen tissues with the planchettes
to liquid nitrogen in cryovials for storage. (Precautions for this
process are described in detail in ref. [ 25 ]; see Note 3 ).

3.2 High-Pressure
Freezing (Onion
Seedlings)

3.3 High-Pressure
Freezing (BY-2 Cells)

1. Set up a vacuum fi ltration funnel with a 30 μ m pore size nylon

fi lter.
2. Pour fresh BY-2 culture into the funnel and apply vacuum.
Vacuum out the culture medium until cells' concentrate forms
a slurry on top of the fi lter. Never dry the cells.