3. Fill a small Petri dish with 70% ethanol and place it under a
stereomicroscope equipped with a light source from the
bottom.
4. Pull fixed siliques out of the tube (see Note 6) and immediately
place them into the Petri dish filled with 70% ethanol (Fig. 3h).
Siliques will sink and stay at the bottom. It is important to
avoid drying.
5. Place siliques in a way that ovules/seeds of both ovary chambers become visible (Fig. 3i). Tweezers can be used to place
siliques properly (see Note 7).
6. Switch on the light source at the bottom of the stereomicroscope and adjust it to obtain a proper contrast to visualize seed
(Fig. 3i) (see Note 8).
3.2 Male
Gametophyte
Development
If seed set is defective and mature ovules did not develop further
after pollination—only tiny structures are visible attached to the
placenta of the silique—we next recommend to study male gametophyte (pollen grain) development and function. First, pollen
grain morphology and content should be studied, then its viability,
and afterward in vitro pollen germination capability as well as
in vivo pollen tube growth.
3.2.1 Pollen Morphology
and Content
1. Prepare a microscope glass slide under a stereomicroscope.
2. Take a freshly opened flower (like the example shown in
Fig. 1b) by catching its pedicel with tweezers.
3. While holding the flower under the stereomicroscope, dab it
onto the glass slide so you can check how pollen grains are
released to the surface (see Note 9).
4. Immediately add 7 μL of DAPI working solution on pollen
grains. DAPI is a mutagen agent and must be handled with
nitrile gloves.
5. Carefully place a coverslip on the drop of DAPI solution and
incubate the glass slide in a humid chamber for 5 min in
the dark.
6. Observe pollen using a fluorescence microscope with UV
settings.
7. Investigate pollen grains morphology with bright field (Fig. 4a,
upper panel) as well as the presence of the sperm cells and
vegetative nucleus with DAPI filter (Fig. 4a, lower panel) (see
Note 10).
3.2.2 FDA Staining for
Pollen Viability
1. Thaw an aliquot of BK buffer S15 MOPS.
2. Add 1 μL of FDA stock solution in a 1 mL aliquot of BK. This
FDA-BK mixture can be used up to 2 h after preparation [19].
Identifying Reproductive Mutations in Arabidopsis
379
stereomicroscope equipped with a light source from the
bottom.
4. Pull fixed siliques out of the tube (see Note 6) and immediately
place them into the Petri dish filled with 70% ethanol (Fig. 3h).
Siliques will sink and stay at the bottom. It is important to
avoid drying.
5. Place siliques in a way that ovules/seeds of both ovary chambers become visible (Fig. 3i). Tweezers can be used to place
siliques properly (see Note 7).
6. Switch on the light source at the bottom of the stereomicroscope and adjust it to obtain a proper contrast to visualize seed
(Fig. 3i) (see Note 8).
3.2 Male
Gametophyte
Development
If seed set is defective and mature ovules did not develop further
after pollination—only tiny structures are visible attached to the
placenta of the silique—we next recommend to study male gametophyte (pollen grain) development and function. First, pollen
grain morphology and content should be studied, then its viability,
and afterward in vitro pollen germination capability as well as
in vivo pollen tube growth.
3.2.1 Pollen Morphology
and Content
1. Prepare a microscope glass slide under a stereomicroscope.
2. Take a freshly opened flower (like the example shown in
Fig. 1b) by catching its pedicel with tweezers.
3. While holding the flower under the stereomicroscope, dab it
onto the glass slide so you can check how pollen grains are
released to the surface (see Note 9).
4. Immediately add 7 μL of DAPI working solution on pollen
grains. DAPI is a mutagen agent and must be handled with
nitrile gloves.
5. Carefully place a coverslip on the drop of DAPI solution and
incubate the glass slide in a humid chamber for 5 min in
the dark.
6. Observe pollen using a fluorescence microscope with UV
settings.
7. Investigate pollen grains morphology with bright field (Fig. 4a,
upper panel) as well as the presence of the sperm cells and
vegetative nucleus with DAPI filter (Fig. 4a, lower panel) (see
Note 10).
3.2.2 FDA Staining for
Pollen Viability
1. Thaw an aliquot of BK buffer S15 MOPS.
2. Add 1 μL of FDA stock solution in a 1 mL aliquot of BK. This
FDA-BK mixture can be used up to 2 h after preparation [19].
Identifying Reproductive Mutations in Arabidopsis
379