glycerol, mix well by magnetic steering, and adjust to 50 mL
with double-distilled water. Store at 4
C for up to 1 month.
9. 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).
10. Microscope equipped with differential interference contrast
(DIC) optics.
3 Methods
3.1 Silique
Dissection and
Observation
When observing short siliques, problems in seed set, or segregation
distortions in the progeny of mutant plants, we suggest to first
investigate the siliques and seeds. For that purpose, it is possible
to fix and clear siliques to analyze seed set without damaging the
siliques, and to perform a fast dissection of developing siliques to
investigate the shape and size of the seeds in more detail.
3.1.1 Dissection of
Siliques for Microscopy
1. Prepare a stereomicroscope (Fig. 3a), two high-precision tweezers, and two disposable medical hypodermic syringe needles
(Fig. 3b).
2. Harvest siliques of defined developmental stages with fine-tip
scissors or high-precision tweezers at the pedicel and collect the
siliques in a 1.5 mL microcentrifuge tube (see Note 3).
3. Place a glass slide under a stereomicroscope, pick one silique at
its pedicel, and place it on the slide.
4. Place the silique vertically using above needles, with the stigma
facing upward and the pedicel facing downward (Fig. 3c).
5. Use one needle with its bevel facing downward to hold the
silique softly on the glass slide (best position is the pedicel, or
receptacle).
6. Use the other needle with its bevel facing upward to slice the
silique open. Carefully slide the needle along the dehiscence
zone visible between the valves and the replum (dotted lines in
Fig. 3d) (see Note 4).
7. Turn the silique 180
and proceed in the same manner at the
back side of the silique.
8. Remove dissected valves with a needle (Fig. 3e).
9. After removing both valves, pictures should be taken as fast as
possible as seeds get dehydrated very quickly.
3.1.2 Fixation and
Clearing of Siliques for
Microscopy
1. Harvest siliques as described above (see also Fig. 3f) and collect
them upside down in 1.5 mL microcentrifuge tube filled with
1–1.2 mL of fixing/clearing solution. Place tube overnight at
room temperature (Fig. 3g, left tube) (see Note 5).
Identifying Reproductive Mutations in Arabidopsis
377
with double-distilled water. Store at 4
C for up to 1 month.
9. 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).
10. Microscope equipped with differential interference contrast
(DIC) optics.
3 Methods
3.1 Silique
Dissection and
Observation
When observing short siliques, problems in seed set, or segregation
distortions in the progeny of mutant plants, we suggest to first
investigate the siliques and seeds. For that purpose, it is possible
to fix and clear siliques to analyze seed set without damaging the
siliques, and to perform a fast dissection of developing siliques to
investigate the shape and size of the seeds in more detail.
3.1.1 Dissection of
Siliques for Microscopy
1. Prepare a stereomicroscope (Fig. 3a), two high-precision tweezers, and two disposable medical hypodermic syringe needles
(Fig. 3b).
2. Harvest siliques of defined developmental stages with fine-tip
scissors or high-precision tweezers at the pedicel and collect the
siliques in a 1.5 mL microcentrifuge tube (see Note 3).
3. Place a glass slide under a stereomicroscope, pick one silique at
its pedicel, and place it on the slide.
4. Place the silique vertically using above needles, with the stigma
facing upward and the pedicel facing downward (Fig. 3c).
5. Use one needle with its bevel facing downward to hold the
silique softly on the glass slide (best position is the pedicel, or
receptacle).
6. Use the other needle with its bevel facing upward to slice the
silique open. Carefully slide the needle along the dehiscence
zone visible between the valves and the replum (dotted lines in
Fig. 3d) (see Note 4).
7. Turn the silique 180
and proceed in the same manner at the
back side of the silique.
8. Remove dissected valves with a needle (Fig. 3e).
9. After removing both valves, pictures should be taken as fast as
possible as seeds get dehydrated very quickly.
3.1.2 Fixation and
Clearing of Siliques for
Microscopy
1. Harvest siliques as described above (see also Fig. 3f) and collect
them upside down in 1.5 mL microcentrifuge tube filled with
1–1.2 mL of fixing/clearing solution. Place tube overnight at
room temperature (Fig. 3g, left tube) (see Note 5).
Identifying Reproductive Mutations in Arabidopsis
377