4. Make sure that samples do not dry out by adding doubledistilled water through the corner of the coverslip.
5. Place the glass slide in a humid chamber (like in Fig. 4f) until
observation using a light microscope.
6. Purple-magenta pollen grains are viable (Fig. 4c, upper panel),
while dead pollen grains are blue-greenish (Fig. 4c, lower
panel).
3.2.4 In Vitro Pollen
Germination
1. Prepare 10 mL of liquid or solid pollen germination medium
(PGM).
2. Take a freshly opened flower and release pollen as described
above.
3. For pollen germination in liquid media, dab a fresh opened
flower on an Image Petri dish and immediately add 3 mL of
PGM (Fig. 4d, lower panel).
4. Add the lid on either the Image Petri dish containing liquid
media or standard Petri dish with solid media to prevent
evaporation.
5. Prepare a small humid chamber (e.g., a 1 mL closed pipette tip
box with wet paper on the bottom and a tip holder on top to
prevent contact between water and samples; see Fig. 4f).
6. Pollen tubes should be visible 6 h after incubation (Fig. 4g). An
inverted microscope is required to observe germination in
liquid medium and a standard microscope for studying germination on a solid surface. Â10 objectives are recommended (see
Note 13).
3.2.5 In Vivo Pollen Tube
Growth
1. Pollinate emasculated wild-type flowers as described by [21] or
as described below (see steps 1–8 in Subheading 3.3) for ovule
preparation. Select pistils 1–2 days after pollination.
2. Place pollinated pistils into a 1.5 mL centrifuge tube containing
fixing solution and incubate at least for 2 h at room temperature (see Note 14).
3. Exchange fixing solution by 70% ethanol to slowly rehydrate
the samples. Incubate for 10 min at room temperature.
4. Repeat step 3 three times by reducing ethanol to 50%, 30%,
and finally distilled water.
5. Fill a small Petri dish partially with alkaline treatment solution
and place rehydrated pistils carefully inside. Add the lid and
incubate overnight at room temperature.
6. Exchange alkaline solution carefully by distilled water and
incubate for 10 min at room temperature.
Identifying Reproductive Mutations in Arabidopsis
381
5. Place the glass slide in a humid chamber (like in Fig. 4f) until
observation using a light microscope.
6. Purple-magenta pollen grains are viable (Fig. 4c, upper panel),
while dead pollen grains are blue-greenish (Fig. 4c, lower
panel).
3.2.4 In Vitro Pollen
Germination
1. Prepare 10 mL of liquid or solid pollen germination medium
(PGM).
2. Take a freshly opened flower and release pollen as described
above.
3. For pollen germination in liquid media, dab a fresh opened
flower on an Image Petri dish and immediately add 3 mL of
PGM (Fig. 4d, lower panel).
4. Add the lid on either the Image Petri dish containing liquid
media or standard Petri dish with solid media to prevent
evaporation.
5. Prepare a small humid chamber (e.g., a 1 mL closed pipette tip
box with wet paper on the bottom and a tip holder on top to
prevent contact between water and samples; see Fig. 4f).
6. Pollen tubes should be visible 6 h after incubation (Fig. 4g). An
inverted microscope is required to observe germination in
liquid medium and a standard microscope for studying germination on a solid surface. Â10 objectives are recommended (see
Note 13).
3.2.5 In Vivo Pollen Tube
Growth
1. Pollinate emasculated wild-type flowers as described by [21] or
as described below (see steps 1–8 in Subheading 3.3) for ovule
preparation. Select pistils 1–2 days after pollination.
2. Place pollinated pistils into a 1.5 mL centrifuge tube containing
fixing solution and incubate at least for 2 h at room temperature (see Note 14).
3. Exchange fixing solution by 70% ethanol to slowly rehydrate
the samples. Incubate for 10 min at room temperature.
4. Repeat step 3 three times by reducing ethanol to 50%, 30%,
and finally distilled water.
5. Fill a small Petri dish partially with alkaline treatment solution
and place rehydrated pistils carefully inside. Add the lid and
incubate overnight at room temperature.
6. Exchange alkaline solution carefully by distilled water and
incubate for 10 min at room temperature.
Identifying Reproductive Mutations in Arabidopsis
381