7. Exchange distilled water carefully by decolorized aniline blue
solution (DABS), wrap Petri dish with aluminum foil, and
incubate for at least 2 h at room temperature.
8. Transfer each pistil onto a standard glass microscope slide, add
extra DABS, and carefully place a cover slip on top from the end
of pistil to avoid bubble formation.
9. Observe pistils under a fluorescence microscope with DAPI
filter (see above) and determine whether pollen tubes reached
ovules (Fig. 4h) (see Note 15).
3.3 Ovule and
Embryo Sac
Development
If seed set is affected, ovule and/or embryo sac development as well
as fertilization could be defective. We recommend to first study the
presence of all female gametophyte (embryo sac) cells with nuclei at
correct positions [8]. Embryo sacs within the ovules are less accessible; thus, high-quality dissection and DIC microscopy of wellcleared ovules is critical for the success of these investigations.
1. Use closed flower buds that do not yet show white petals
peeking out of the sepals but where the petals reach the height
of the long stamens (Stage 12) [22] for emasculation (see Note
16).
2. Place a microscope glass slide under a stereomicroscope and
add a piece of double-sided tape.
3. Place the pot with the single plant horizontally and select an
inflorescence under the stereomicroscope.
4. Immobilize the inflorescence by gently sticking it on the
double-sided tape and work on the 2–3 buds of the inflorescence which are around flower stage 12 [22] (white arrow in
Fig. 5a). Remove open flowers and the remaining too small
flower buds from this inflorescence by using disposable medical
hypodermic syringe needles.
5. Carefully open selected buds with needles by moving upper
sepals and petals downward to get access to the pistil and
stamens without damaging the pistil (Fig. 5b).
6. Carefully emasculate the flower by removing all six stamens
without damaging the pistil (Fig. 5c).
7. After 2 days, take the plant back to the stereomicroscope.
Emasculated mature pistils are recognized by their long papilla
cells on the stigma (Fig. 5d). These pistils should contain
mature but unfertilized ovules. Do not use pistils if papilla
appear collapsed or dry.
8. Before ovule isolation, 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, like in Fig. 4f).
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Marı ´a Flores-Tornero et al.
solution (DABS), wrap Petri dish with aluminum foil, and
incubate for at least 2 h at room temperature.
8. Transfer each pistil onto a standard glass microscope slide, add
extra DABS, and carefully place a cover slip on top from the end
of pistil to avoid bubble formation.
9. Observe pistils under a fluorescence microscope with DAPI
filter (see above) and determine whether pollen tubes reached
ovules (Fig. 4h) (see Note 15).
3.3 Ovule and
Embryo Sac
Development
If seed set is affected, ovule and/or embryo sac development as well
as fertilization could be defective. We recommend to first study the
presence of all female gametophyte (embryo sac) cells with nuclei at
correct positions [8]. Embryo sacs within the ovules are less accessible; thus, high-quality dissection and DIC microscopy of wellcleared ovules is critical for the success of these investigations.
1. Use closed flower buds that do not yet show white petals
peeking out of the sepals but where the petals reach the height
of the long stamens (Stage 12) [22] for emasculation (see Note
16).
2. Place a microscope glass slide under a stereomicroscope and
add a piece of double-sided tape.
3. Place the pot with the single plant horizontally and select an
inflorescence under the stereomicroscope.
4. Immobilize the inflorescence by gently sticking it on the
double-sided tape and work on the 2–3 buds of the inflorescence which are around flower stage 12 [22] (white arrow in
Fig. 5a). Remove open flowers and the remaining too small
flower buds from this inflorescence by using disposable medical
hypodermic syringe needles.
5. Carefully open selected buds with needles by moving upper
sepals and petals downward to get access to the pistil and
stamens without damaging the pistil (Fig. 5b).
6. Carefully emasculate the flower by removing all six stamens
without damaging the pistil (Fig. 5c).
7. After 2 days, take the plant back to the stereomicroscope.
Emasculated mature pistils are recognized by their long papilla
cells on the stigma (Fig. 5d). These pistils should contain
mature but unfertilized ovules. Do not use pistils if papilla
appear collapsed or dry.
8. Before ovule isolation, 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, like in Fig. 4f).
382
Marı ´a Flores-Tornero et al.