9. 70% Ethanol (v/v).
10. Small Petri dish (35 mm  15 mm).
11. Double-sided tape.
12. Stereomicroscope with a light source at the bottom.
2.2 Male
Gametophyte
Development and
Viability
1. Flowers at anthesis.
2. Two high-precision tweezers (tip width 0.1 mm  length
110 mm).
3. Standard stereomicroscope.
4. Standard glass microscope slides and coverslips.
5. DAPI working solution [3]: 0.1 M sodium phosphate pH 7.0,
1 mM EDTA, 0.1% Triton X-100, 1 μg/mL DAPI (4
0 ,6-diamidino-2-phenylindole). DAPI is a mutagen agent and must
be handled with nitrile gloves.
6. Fluorescein diacetate (FDA) stock solution: 2 mg FDA/mL
acetone.
7. BK buffer S15 MOPS [19]: 0.127 mM Ca(NO 3 ) 2 ·4H 2 O,
0.081 mM MgSO 4 ·7H 2 O, 0.1 mM KNO 3 , 15% sucrose,
10 mM MOPS pH 7.5. Aliquot it in 1 mL microcentrifuge
tubes and store at À20
C.
8. Micropipette of 20 μL capacity.
9. Fluorescence microscope with DAPI and GFP filters and highquality Â20 and Â40 microscope objectives.
2.3 In Vivo and In
Vitro Pollen Tube
Growth
1. Flowers at anthesis and pistils 12–24 h after pollination.
2. Two high-precision tweezers (tip width 0.1 mmÂlength
110 mm).
3. Standard stereomicroscope.
4. Parafilm.
5. Small Petri dish (35 mm  15 mm).
6. Distilled water.
7. Liquid Pollen Germination Medium (PGM): 18% sucrose,
0.01% H 3 BO 3 , 1 mM MgSO 4 , 1 mM CaCl 2 , 1 mM Ca
(NO 3 ) 2 . To prepare 10 mL, add 3.6 mL from 50% filtered
sterilized sucrose solution, 100 μL from a stock solution of
1% H 3 BO 3 , 1 mL from 10 mM MgSO 4 , 1 mL from 10 mM
CaCl 2 , and 1 mL from 10 mM Ca(NO 3 ) 2 . Then add 3 mL of
double-deionized sterile water, adjust the pH to 7.50 with
0.1 M NaOH, and adjust to a final volume of 10 mL. Store at
4
C.
8. Solid Pollen Germination Medium (PGM) [20]: add 50 mg of
Agar-Agar ultrapure (Merck) to 10 mL liquid PGM and melt it
in a microwave. When solution is homogeneous, pour 3 mL in
Identifying Reproductive Mutations in Arabidopsis
375
10. Small Petri dish (35 mm  15 mm).
11. Double-sided tape.
12. Stereomicroscope with a light source at the bottom.
2.2 Male
Gametophyte
Development and
Viability
1. Flowers at anthesis.
2. Two high-precision tweezers (tip width 0.1 mm  length
110 mm).
3. Standard stereomicroscope.
4. Standard glass microscope slides and coverslips.
5. DAPI working solution [3]: 0.1 M sodium phosphate pH 7.0,
1 mM EDTA, 0.1% Triton X-100, 1 μg/mL DAPI (4
0 ,6-diamidino-2-phenylindole). DAPI is a mutagen agent and must
be handled with nitrile gloves.
6. Fluorescein diacetate (FDA) stock solution: 2 mg FDA/mL
acetone.
7. BK buffer S15 MOPS [19]: 0.127 mM Ca(NO 3 ) 2 ·4H 2 O,
0.081 mM MgSO 4 ·7H 2 O, 0.1 mM KNO 3 , 15% sucrose,
10 mM MOPS pH 7.5. Aliquot it in 1 mL microcentrifuge
tubes and store at À20
C.
8. Micropipette of 20 μL capacity.
9. Fluorescence microscope with DAPI and GFP filters and highquality Â20 and Â40 microscope objectives.
2.3 In Vivo and In
Vitro Pollen Tube
Growth
1. Flowers at anthesis and pistils 12–24 h after pollination.
2. Two high-precision tweezers (tip width 0.1 mmÂlength
110 mm).
3. Standard stereomicroscope.
4. Parafilm.
5. Small Petri dish (35 mm  15 mm).
6. Distilled water.
7. Liquid Pollen Germination Medium (PGM): 18% sucrose,
0.01% H 3 BO 3 , 1 mM MgSO 4 , 1 mM CaCl 2 , 1 mM Ca
(NO 3 ) 2 . To prepare 10 mL, add 3.6 mL from 50% filtered
sterilized sucrose solution, 100 μL from a stock solution of
1% H 3 BO 3 , 1 mL from 10 mM MgSO 4 , 1 mL from 10 mM
CaCl 2 , and 1 mL from 10 mM Ca(NO 3 ) 2 . Then add 3 mL of
double-deionized sterile water, adjust the pH to 7.50 with
0.1 M NaOH, and adjust to a final volume of 10 mL. Store at
4
C.
8. Solid Pollen Germination Medium (PGM) [20]: add 50 mg of
Agar-Agar ultrapure (Merck) to 10 mL liquid PGM and melt it
in a microwave. When solution is homogeneous, pour 3 mL in
Identifying Reproductive Mutations in Arabidopsis
375