Biology Reference
In-Depth Information
Sections were stained with periodic acid-Schiff's reagent (PAS) for insoluble
carbohydrates and with PAS/Toluidine Blue for general histological observations
[64]. Sections were also stained for cutine and exine with 0.01% auramine in 0.05
M phosphate buffer [65] and for cellulose with 0.007% calcofluor in water [66].
Intine and exine were observed with a 3:1 mixture of 0.01% auramine in water
and 0.007% calcofluor in water.
To observe nuclei during pollen development, anthers collected from flow-
ers at the same developmental stages ranging from 9 mm long to anthesis were
also fixed in 3:1 (V1/V2) ethanol-acetic acid, embedded as described above,
sectioned at 5 µm and stained with a solution of 0.25 mg/ml of 4',6-diamidi-
no-2-phenylindole (DAPI) and 0.1 mg/ml p-phenylenediamine (added to re-
duce fading) in 0.05 M Tris (pH 7.2) for 1 hr at room temperature in a light-
free environment [67]. Preparations were observed under an epifluorescent
Leica DM LB2 microscope with 340-380 and LP 425 filters for auramine,
calcofluor, and DAPI.
For the study of pollen morphology and pollen size, dehisced anthers were
sieved through a 0.26 mm mesh sieve and the pollen was placed in glacial ace-
tic acid for acetolysis. Pollen grains were transferred to a mixture of 9:1 acetic
anhydride:concentrated sulphuric acid at 65°C for 10 minutes, then washed with
glacial acetic acid and washed again three times with water following a modifica-
tion of the method by Erdtman (1960) [68].
Scanning Electron Microscopy
Pollen for scanning electron microscopy (SEM) was fresh dried with silica gel and
directly attached to SEM stubs using adhesive carbon tabs and observed with a
JSM-840 scanning electron microscope (JEOL) operated at 10 kV.
Immunocytochemistry
Immunocytochemistry was performed on Technovit 8100 (Kulzer & Co, Weh-
rheim, Germany) embedded semithin sections and revealed by fluorochromes, as
described previously [69,70]. Anthers from three flowers per developmental stage
with petals 6, 9, 12, 16, 22, 24 and 30 mm long and at anthesis were fixed in 4%
paraformaldehyde in phosphate buffered saline (PBS) at pH 7.3 overnight at 4°C,
dehydrated in an acetone series, embedded in Technovit 8100 (Kulzer), polymer-
ized at 4°C and sectioned at 2
µ
m. Sections were placed in a drop of water on a slide
covered with 3-Aminopropyltrietoxy-silane 2% and dried at room temperature.
Different antibodies were used to localize specific cell components: an an-
ti-RNA mouse monoclonal antibody, D44 [71,72], for total RNA detection;
