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

The method of rapid freeze-fixation and substitution was used with Brassica napus floral bud material in order to improve the preservation of microspo re and tapetal organelle structure. When observed using transmission electr on 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 c ells. The tapetosomes have a very electron-dense, opaque appearance when vi sualized after rapid fixation, In addition, we were able to observe other c ytoplasmic details such as pockets in the endoplasmic reticulum and cytoske letal structures such as microfilaments. Extracellular material was also we ll-preserved; for example, the fibrous material in the baculae of the devel oping microspore exine was also visible. Finally, in the freeze-fixed secti ons specific structures such as elaioplasts could be labelled by antibodies , which indicates that this method preserved protein epitopes that were des troyed by chemical fixation.