8. Visualization of seed set may provide important hints about the
nature of reproductive defects. If well-developed seeds are only
found in the stigmatic region of the silique, pollen tubes may
not be capable to reach the ovules located at the base due to
tube growth problems, transmitting tract and/or pollen tube
guidance defects.
9. The use of a stereomicroscope is strongly recommended when
dabbing freshly dehiscent anthers on the surface to release
pollen. It is important to observe the quantity and quality of
pollen grains released from anthers in comparison to those of a
wild-type plant. Differences could indicate problems in anther
and/or pollen development, or in pollen dehiscence.
10. Sometimes sperm cells and vegetative nuclei are barely visible
because the exine of the pollen coat interferes with stain penetration at different stages of development. In that case, samples
could be incubated in a humid chamber at 4
C for longer time
periods [2] or pollen coat of mature pollen could be removed
to increase signals [24].
11. We suggest to use a modified Alexander stain [25]. The classical staining protocol [5] uses toxic components including
phenol.
12. Alternatively, a Bunsen burner can be used and heating of the
sample could be controlled by shortly moving the glass slide in
and out of the flame [25].
13. When observing germination on solid media, be extremely
careful not to touch the PGM with the Â10 objective. To
observe pollen tubes at higher magnification, carefully place a
coverslip on top of the solid media containing germinated
pollen grains to observe samples.
14. Instead of using a small Petri dish, 12 mm diameter 48-well cell
culture plates can be used.
15. To avoid random dispersion of pollen tubes, we recommend to
increase the glycerol concentration till 1 M in DABS [7].
16. See also Note 3. First and last flowers are often sterile.
17. The direction of sliding along the pistils and siliques position
with hypodermic syringe needles for dissection may be modified according to individual preferences. Another detailed dissection protocol supported by audiovisual content has been
described recently [24]. Alternative diagrams describing the
preparation of pistils and how to remove ovary walls can be
found [26, 27].
18. It is recommended to separate the placenta in two halves.
Afterward, this improves the individual visualization and analysis of ovules. Otherwise, ovules tend to overlap with each
Identifying Reproductive Mutations in Arabidopsis
387
nature of reproductive defects. If well-developed seeds are only
found in the stigmatic region of the silique, pollen tubes may
not be capable to reach the ovules located at the base due to
tube growth problems, transmitting tract and/or pollen tube
guidance defects.
9. The use of a stereomicroscope is strongly recommended when
dabbing freshly dehiscent anthers on the surface to release
pollen. It is important to observe the quantity and quality of
pollen grains released from anthers in comparison to those of a
wild-type plant. Differences could indicate problems in anther
and/or pollen development, or in pollen dehiscence.
10. Sometimes sperm cells and vegetative nuclei are barely visible
because the exine of the pollen coat interferes with stain penetration at different stages of development. In that case, samples
could be incubated in a humid chamber at 4
C for longer time
periods [2] or pollen coat of mature pollen could be removed
to increase signals [24].
11. We suggest to use a modified Alexander stain [25]. The classical staining protocol [5] uses toxic components including
phenol.
12. Alternatively, a Bunsen burner can be used and heating of the
sample could be controlled by shortly moving the glass slide in
and out of the flame [25].
13. When observing germination on solid media, be extremely
careful not to touch the PGM with the Â10 objective. To
observe pollen tubes at higher magnification, carefully place a
coverslip on top of the solid media containing germinated
pollen grains to observe samples.
14. Instead of using a small Petri dish, 12 mm diameter 48-well cell
culture plates can be used.
15. To avoid random dispersion of pollen tubes, we recommend to
increase the glycerol concentration till 1 M in DABS [7].
16. See also Note 3. First and last flowers are often sterile.
17. The direction of sliding along the pistils and siliques position
with hypodermic syringe needles for dissection may be modified according to individual preferences. Another detailed dissection protocol supported by audiovisual content has been
described recently [24]. Alternative diagrams describing the
preparation of pistils and how to remove ovary walls can be
found [26, 27].
18. It is recommended to separate the placenta in two halves.
Afterward, this improves the individual visualization and analysis of ovules. Otherwise, ovules tend to overlap with each
Identifying Reproductive Mutations in Arabidopsis
387