Processing Pollen and Spore Exines for Electron Microscopy

Abstract

A resin mixture containing Araldite M, 15 ml; Epon 812, 25 ml; dodecenyl succinic anhydride, 55 ml; and dibutyl phthlate, 2 ml, was found to be the optimal embedding resin for both fresh and acetylated pollen exines. Diamond knives greatly facilitated sectioning. Exine fine structure, and stratification patterns in fresh pollen were most clearly revealed by section staining of glutaraldehyde-fixed (2 hr), OsO₄-stained (2 hr) specimens. Acetylated exines (acetic anhydride-H₂SO₄ 9:1; 100 C, 5 min) did not require additional treatment prior to embedding, but section staining of exines so treated greatly enhanced stain differentiation of exine subunits. Successfully used section stains included Reynold's lead hydroxide, Millonig's lead citrate and aqueous KMnO₄. Additional procedures were tried but were found to have serious disadvantages, e. g. exines treated with KMnO₄ before embedding shattered badly during sectioning.