Rapid-freeze fixation and substitution improves tissue preservation of microspores and tapetum in Brassica napus

Abstract 

The method of rapid freeze-fixation and substitution was used with Brassica napus floral bud material in order to improve the preservation of microspore and tapetal organelle structure. When observed using transmission electron microscopy, the appearance of the freeze-substituted material differs in a number of ways from the chemically-fixed material previously studied, in particular for the lipid-rich elaioplasts and tapetosomes in the tapetal cells. The tapetosomes have a very electron-dense, opaque appearance when visualized after rapid fixation. In addition, we were able to observe other cytoplasmic details such as pockets in the endoplasmic reticulum and cytoskeletal structures such as microfilaments. Extracellular material was also well-preserved; for example, the fibrous material in the baculae of the developing microspore exine was also visible. Finally, in the freeze-fixed sections specific structures such as elaioplasts could be labelled by antibodies, which indicates that this method preserved protein epitopes that were destroyed by chemical fixation.