3. Plate the seeds on agar and keep them at 4
C for 3 days for
stratification (to enhance uniform germination).
4. Grow the plants for 2 weeks in a growth chamber with long day
conditions (16 h) (20 and 18
C, day and night, respectively)
(see Note 1).
5. Harvest the third leaf with a razor blade and cut out very small
pieces from the middle parts of the leaves (see Note 2).
6. Using fine forceps move the leaf pieces directly to the ice-cold
fixative solution.
7. Remove oxygen by placing the samples in the desiccator.
8. Leave the samples at 4
C overnight.
3.2 Embedding Leaf
Pieces in Resin and
Sectioning
1. Replace the fixative solution with distilled water.
2. Prepare 1% LMP agarose, pour it on a Petri dish and wait until
it will cool down.
3. Put leaf pieces inside the agarose and make sure they lie flat.
4. Cut small agarose cubes with the sample inside and move them
to the vials filled with distilled water (see Note 3).
5. Move the samples over different ethanol series (see Subheading
2.2, step 9) for dehydration.
6. When the samples are in 99.5% ethanol, add LRW resin drop by
drop till it will become approx. 10% of the total volume.
7. Move the samples over different dilutions of resin in ethanol
(resin 10, 25, 50, 75%) and pure resin.
8. Transfer the samples to embedding capsules and fill them up
with resin.
9. Leave the capsules in the incubator at 60
C for at least 24 h to
polymerize (see Note 4).
10. Using a razor blade, remove the capsule and trim the bottom of
the sample to obtain a cone shape with a trapezoid bottom (see
Note 5).
11. Using a microtome, cut few sections with a thickness of
0.5 μm.
12. Dip a fine brush in the water. Move the section onto a drop of
distilled water on the adhesive glass.
13. Dry the slide, apply the stain on the section, and look at it
under the microscope to make sure that the anticlinal walls are
not oblique.
14. If the sections are satisfying, cut few more (thickness 0.5 μm)
and place them on a distilled water drop in the middle of the
slide (see Note 6).
356
Mateusz Majda
C for 3 days for
stratification (to enhance uniform germination).
4. Grow the plants for 2 weeks in a growth chamber with long day
conditions (16 h) (20 and 18
C, day and night, respectively)
(see Note 1).
5. Harvest the third leaf with a razor blade and cut out very small
pieces from the middle parts of the leaves (see Note 2).
6. Using fine forceps move the leaf pieces directly to the ice-cold
fixative solution.
7. Remove oxygen by placing the samples in the desiccator.
8. Leave the samples at 4
C overnight.
3.2 Embedding Leaf
Pieces in Resin and
Sectioning
1. Replace the fixative solution with distilled water.
2. Prepare 1% LMP agarose, pour it on a Petri dish and wait until
it will cool down.
3. Put leaf pieces inside the agarose and make sure they lie flat.
4. Cut small agarose cubes with the sample inside and move them
to the vials filled with distilled water (see Note 3).
5. Move the samples over different ethanol series (see Subheading
2.2, step 9) for dehydration.
6. When the samples are in 99.5% ethanol, add LRW resin drop by
drop till it will become approx. 10% of the total volume.
7. Move the samples over different dilutions of resin in ethanol
(resin 10, 25, 50, 75%) and pure resin.
8. Transfer the samples to embedding capsules and fill them up
with resin.
9. Leave the capsules in the incubator at 60
C for at least 24 h to
polymerize (see Note 4).
10. Using a razor blade, remove the capsule and trim the bottom of
the sample to obtain a cone shape with a trapezoid bottom (see
Note 5).
11. Using a microtome, cut few sections with a thickness of
0.5 μm.
12. Dip a fine brush in the water. Move the section onto a drop of
distilled water on the adhesive glass.
13. Dry the slide, apply the stain on the section, and look at it
under the microscope to make sure that the anticlinal walls are
not oblique.
14. If the sections are satisfying, cut few more (thickness 0.5 μm)
and place them on a distilled water drop in the middle of the
slide (see Note 6).
356
Mateusz Majda