other on the glass slide after placing a coverslip on top and thus
are difficult to be observed individually.
19. Clearing solution should be stored at 4
C and used only for a
few days; otherwise clearing of ovules does not give satisfactory
results. Try to avoid an overflow of clearing solution beyond
the limits of the coverslip; otherwise it might get into contact
with the immersion oil, forming precipitates.
20. When observing sample using an oil immersion objective, it is
very important to avoid contact between immersion oil and
clearing solution that could be overflowing from the edges of
the coverslip. If that happens, a white solution is formed that
strongly compromises the quality of the image. This can be
easily removed from the microscope objective with isopropanol, but it is difficult to remove from the coverslip without
damaging the sample.
21. If samples are not clear enough to identify all structures
described in Fig. 5i, preparations could be incubated in a
humid chamber for a longer time (up to 1 day).
22. To study different embryo and endosperm developmental
stages, it is recommended to start with almost mature but
still green siliques harvested from the lower part of the inflorescence. For younger developmental stages, successively harvest younger siliques further up of the inflorescence and place
them in 1.5 mL microcentrifuge tubes labeled accordingly.
23. When reproductive processes are studied, wild-type and
mutant plants should be grown at identical conditions and
ideally in the same tray, as differences in watering and light
conditions will affect fertilization and seed development.
Silique development can be followed from flower buds at
stage 12 [22] till mature siliques turns yellowish, which occurs
approximately 15 days later.
24. Valves from almost mature siliques are easily separated shortly
after the beginning of dissection. It is important to avoid
removal of seeds that may remain attached to separated valves.
25. If siliques are almost mature, seeds tend to spread around the
glass slide. To make sure that seed do not get lost, fast or
abrupt movements during dissection should be avoided.
26. When almost mature seeds are cleared, it is difficult to fully
immerse them in clearing solution after placing a coverslip on
top. Integuments are quite hard and could break the coverslip.
In that case, it is recommended to add more clearing solution
under the coverslip by using a micropipette. Avoid overflow.
388
Marı ´a Flores-Tornero et al.
are difficult to be observed individually.
19. Clearing solution should be stored at 4
C and used only for a
few days; otherwise clearing of ovules does not give satisfactory
results. Try to avoid an overflow of clearing solution beyond
the limits of the coverslip; otherwise it might get into contact
with the immersion oil, forming precipitates.
20. When observing sample using an oil immersion objective, it is
very important to avoid contact between immersion oil and
clearing solution that could be overflowing from the edges of
the coverslip. If that happens, a white solution is formed that
strongly compromises the quality of the image. This can be
easily removed from the microscope objective with isopropanol, but it is difficult to remove from the coverslip without
damaging the sample.
21. If samples are not clear enough to identify all structures
described in Fig. 5i, preparations could be incubated in a
humid chamber for a longer time (up to 1 day).
22. To study different embryo and endosperm developmental
stages, it is recommended to start with almost mature but
still green siliques harvested from the lower part of the inflorescence. For younger developmental stages, successively harvest younger siliques further up of the inflorescence and place
them in 1.5 mL microcentrifuge tubes labeled accordingly.
23. When reproductive processes are studied, wild-type and
mutant plants should be grown at identical conditions and
ideally in the same tray, as differences in watering and light
conditions will affect fertilization and seed development.
Silique development can be followed from flower buds at
stage 12 [22] till mature siliques turns yellowish, which occurs
approximately 15 days later.
24. Valves from almost mature siliques are easily separated shortly
after the beginning of dissection. It is important to avoid
removal of seeds that may remain attached to separated valves.
25. If siliques are almost mature, seeds tend to spread around the
glass slide. To make sure that seed do not get lost, fast or
abrupt movements during dissection should be avoided.
26. When almost mature seeds are cleared, it is difficult to fully
immerse them in clearing solution after placing a coverslip on
top. Integuments are quite hard and could break the coverslip.
In that case, it is recommended to add more clearing solution
under the coverslip by using a micropipette. Avoid overflow.
388
Marı ´a Flores-Tornero et al.