small sterile Petri dishes (35 mm  15 mm) under a sterile
bench and let the medium solidify for exactly 1 h. Cover with a
lid and seal plated with parafilm to prevent evaporation. Store
upside down at 4
C (see Note 1).
9. Fixing solution: ! 99.8% ethanol, p.A., absolute, acetic acid,
(3:1) (v/v).
10. 70% Ethanol (v/v).
11. 50% Ethanol (v/v).
12. 30% Ethanol (v/v).
13. Alkaline treatment solution: 8 M NaOH.
14. Filter paper.
15. Active carbon powder.
16. Glycerol.
17. Decolorized aniline blue solution (DABS): prepare 0.1% (w/v)
aniline blue in 108 mM K 3 PO 4 at pH 11 and incubate at 4
C
overnight. In the following day, make a funnel with filter paper
and add a teaspoonful active carbon powder. Then, take the
aniline blue solution from 4
C and filter it. Finally, add glycerol to the filtrate to a final concentration of 2% (v/v) and store
it in the fridge at 4
C.
18. Aluminum foil.
19. Image Petri dish 1.5.
20. Standard coverslips and glass microscope slides.
21. Humid chamber (e.g., a 1 mL closed tip box with wet paper at
the bottom and a tip holder on top; see Fig. 4f).
22. Fluorescence microscope with DAPI filter and Â20 and Â40
microscope objectives.
2.4 Female
Gametophyte and
Embryo Development
1. A pot with a single plant of interest with first open flowers
(anthesis).
2. Two high-precision tweezers (tip width 0.1 mmÂlength
110 mm).
3. Disposable medical hypodermic syringe needles (0.4 mm diameter and 13 mm length).
4. Double-sided tape.
5. Standard stereomicroscope.
6. Distilled water.
7. Standard glass microscope slides and coverslips.
8. Clearing solution (see Note 2): 0.02 M chloral hydrate, 20%
glycerol. Add 20 mL double-distilled water to a beaker. Weigh
80 g chloral hydrate and transfer to the beaker. Add 10 mL
376
Marı ´a Flores-Tornero et al.
bench and let the medium solidify for exactly 1 h. Cover with a
lid and seal plated with parafilm to prevent evaporation. Store
upside down at 4
C (see Note 1).
9. Fixing solution: ! 99.8% ethanol, p.A., absolute, acetic acid,
(3:1) (v/v).
10. 70% Ethanol (v/v).
11. 50% Ethanol (v/v).
12. 30% Ethanol (v/v).
13. Alkaline treatment solution: 8 M NaOH.
14. Filter paper.
15. Active carbon powder.
16. Glycerol.
17. Decolorized aniline blue solution (DABS): prepare 0.1% (w/v)
aniline blue in 108 mM K 3 PO 4 at pH 11 and incubate at 4
C
overnight. In the following day, make a funnel with filter paper
and add a teaspoonful active carbon powder. Then, take the
aniline blue solution from 4
C and filter it. Finally, add glycerol to the filtrate to a final concentration of 2% (v/v) and store
it in the fridge at 4
C.
18. Aluminum foil.
19. Image Petri dish 1.5.
20. Standard coverslips and glass microscope slides.
21. Humid chamber (e.g., a 1 mL closed tip box with wet paper at
the bottom and a tip holder on top; see Fig. 4f).
22. Fluorescence microscope with DAPI filter and Â20 and Â40
microscope objectives.
2.4 Female
Gametophyte and
Embryo Development
1. A pot with a single plant of interest with first open flowers
(anthesis).
2. Two high-precision tweezers (tip width 0.1 mmÂlength
110 mm).
3. Disposable medical hypodermic syringe needles (0.4 mm diameter and 13 mm length).
4. Double-sided tape.
5. Standard stereomicroscope.
6. Distilled water.
7. Standard glass microscope slides and coverslips.
8. Clearing solution (see Note 2): 0.02 M chloral hydrate, 20%
glycerol. Add 20 mL double-distilled water to a beaker. Weigh
80 g chloral hydrate and transfer to the beaker. Add 10 mL
376
Marı ´a Flores-Tornero et al.