9. Place a microscope glass slide under the stereomicroscope,
harvest an emasculated mature pistil at its pedicel with fine
scissors or tweezers, and place it onto the glass slide containing
double-sided tape.
10. Take the disposable medical hypodermic syringe needles and
place the silique vertically using above needles, with the stigma
facing upward and the pedicel facing downward (Fig. 5d) (see
Note 17).
11. Use one needle with its bevel facing downward to hold the
silique on the glass slide (best position is the pedicel, or receptacle) and the other needle with the bevel facing upward to
carefully slide along the dehiscence zone visible between the
valves and the replum (as indicated in Figs. 3d and 5e) (see
Note 4).
12. Repeat the same procedure for the right valve.
13. Turn the silique 180
and proceed in the same manner at the
back side of the silique.
14. To remove valves, first hold the silique from the pedicel with
one needle with the bevel facing downward and then place the
tip of the other needle on one extreme of the valve to be
removed.
15. Next, remove the valve by holding the silique from the pedicel
and moving slowly aside the needle placed on the extreme of a
valve. Now, mature non-fertilized ovules become visible
(Fig. 5f).
16. Immediately repeat steps 14 and 15 with the other valve to
make the whole septum visible (Fig. 5g).
17. Carefully remove first the stigma and then the pedicel without
touching the ovules.
18. Perform a cut in one of the extremes of the transmitting tract
to separate both halves of the placenta (see Note 18).
19. Immediately, add 7 μL of clearing solution on top of the sample
(see Note 19). Make sure the sample is completely immersed in
clearing solution.
20. Redirect very carefully both halves of the placenta with needles
so that both rows of attached ovules become visible (Fig. 5h).
21. Place a coverslip on top and incubate the glass slide in a humid
chamber for at least 2 h to obtain good results.
22. Observe the cleared dissected pistil using a microscope
equipped with differential interference contrast (DIC) optics
and use a Â40 objective. Best results and resolution are
obtained with immersion oil using a Â60 or Â63 oil objective
(see Note 20).
384
Marı ´a Flores-Tornero et al.
harvest an emasculated mature pistil at its pedicel with fine
scissors or tweezers, and place it onto the glass slide containing
double-sided tape.
10. Take the disposable medical hypodermic syringe needles and
place the silique vertically using above needles, with the stigma
facing upward and the pedicel facing downward (Fig. 5d) (see
Note 17).
11. Use one needle with its bevel facing downward to hold the
silique on the glass slide (best position is the pedicel, or receptacle) and the other needle with the bevel facing upward to
carefully slide along the dehiscence zone visible between the
valves and the replum (as indicated in Figs. 3d and 5e) (see
Note 4).
12. Repeat the same procedure for the right valve.
13. Turn the silique 180
and proceed in the same manner at the
back side of the silique.
14. To remove valves, first hold the silique from the pedicel with
one needle with the bevel facing downward and then place the
tip of the other needle on one extreme of the valve to be
removed.
15. Next, remove the valve by holding the silique from the pedicel
and moving slowly aside the needle placed on the extreme of a
valve. Now, mature non-fertilized ovules become visible
(Fig. 5f).
16. Immediately repeat steps 14 and 15 with the other valve to
make the whole septum visible (Fig. 5g).
17. Carefully remove first the stigma and then the pedicel without
touching the ovules.
18. Perform a cut in one of the extremes of the transmitting tract
to separate both halves of the placenta (see Note 18).
19. Immediately, add 7 μL of clearing solution on top of the sample
(see Note 19). Make sure the sample is completely immersed in
clearing solution.
20. Redirect very carefully both halves of the placenta with needles
so that both rows of attached ovules become visible (Fig. 5h).
21. Place a coverslip on top and incubate the glass slide in a humid
chamber for at least 2 h to obtain good results.
22. Observe the cleared dissected pistil using a microscope
equipped with differential interference contrast (DIC) optics
and use a Â40 objective. Best results and resolution are
obtained with immersion oil using a Â60 or Â63 oil objective
(see Note 20).
384
Marı ´a Flores-Tornero et al.