Handling Germinated Pollen on Millipore Membrane During the Feulgen and Autoradiographic Procedures

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Abstract
To prevent loss of pollen during the Feulgen''s procedure, the pollen was grown on an autoclaved membrane filter (Millipore AA WP 025 00) in contact with a sterilized medium containing agar 0. 5-1%, sucrose according to the genus (Malus 0. 3-0. 5 [image]; Persica and Tulipa 0. 4 M), and H3BO3, 0. 01%. To fix the germinated pollen of most species, the membrane was placed for 2 hr. to overnight at 2-4[degree]C on filter paper wet with the following mixture: OSO4, 1 gm; CrO3, 1. 66 gm; and distilled water, 233 ml. To fix Persica pollen, 10% of glacial acetic acid had to be added to the fixative. Washing with distilled water and bleaching with a mixture of 3% H2O2 and sat. aq. ammonium oxalate, 1: 1, were performed also on filter paper. Similarly, the preparation was processed for Feulgen staining by use of pieces of filter paper wet with the required fluids. Hydrolysis preceding the Schiff''s reagent was performed at room temperature with 5 [image] HC1 for 18 min. The differentiation after the Schiff''s action was with 2% K2S2O5 buf-fered to pH 2. 3 with 9 ml of phosphate buffer (KH2PO4, 1. 4 gm; conc. HC1, 0. 35 ml and distilled water to make 100 ml). The stained pollen was floated off the membrane with a drop of glacial acetic acid to a gelantinized or an albumenized slide, and squashed. When the cover-slip is removed the preparation may be either dehydrated and mounted or coated with autoradiographic film.