Cryofixation and freeze substitution of teliospores of Gymnosporangium clavipes: an ultrastructural investigation

Teliospores of the rust fungus Gymnosporangium clavipes were cryofixed using high-pressure freezing or cold-metal slamming techniques and prepared for transmission electron microscopy by freeze substitution. Both cryofixation techniques provided excellent ultrastructural preservation of the teliospore cytoplasm. Gross cytoplasmic freeze damage was not observed in any spore frozen in a dextran solution under high pressure and only a low number had minor freeze damage. More spores exhibited cytoplasmic freeze damage using the cold-metal slamming technique. A comparison of these cryotechniques indicated that dextran induced no obvious ultrastructural artifacts during high-pressure freezing. Artifacts induced by high-pressure freezing were identified as breaks in the spore wall and disruptions of the nuclear envelope. In most teliospores, regions of the cytoplasm separated from the inner surface of the teliospore wall creating electron-transparent gaps. Teliospores consisted of two cells separated by a transverse septum. Each cell contained a single, spherical nucleus with a well-preserved nucleoplasm. A spindle pole body and oblong, fibrillar bodies were associated with the outer nuclear envelope. Ribosomes, glycogen particles, mitochondria, microbodies, lipid bodies, strands of smooth endoplasmic reticulum, complex membranous networks, double membraned cisternae and numerous vesicles were present throughout the cytoplasm. Golgi equivalents were not observed. Microtubules were observed in the cytoplasm and often seen near the outer surfaces of nuclei. Microfilaments were not detected in the cytoplasm but were observed in the nuclear matrix. The utility and fidelity of high-pressure freezing in preserving fungal cells for ultrastructural analysis are discussed.