Importance of the fixative for reliable ultrastructural preservation of poikilohydric plant tissues. Observations on dry, partially, and fully hydrated tissues of Selaginella lepidophylla

Abstract

Leaves of desiccated 'resurrection plants', Selaginella lepidophylla, were hydrated either through the roots of intact plants or as isolated organs. Air-dry tissue and samples at 1, 4, 8 and 24 h (both detached and intact) of hydration were prepared for electron microscopy using aldehyde fixatives of different osmotic strengths. Both dry and hydrated tissues were also prepared using freeze substitution. Significant differences in the ultrastructural preservation of these different samples were noted. There was a direct correlation between the osmolality of both the fixative and the tissue with the quality of ultrastructural preservation. When the osmolality of the fixative was slightly (or even considerably) higher than that of the tissue, optimal preservation was achieved. Freeze substitution, however, gave the most faithful preservation of all subcellular compartments, despite the frequent presence of small ice crystals. Additionally, hydration of detached leaves for more than 4 h resulted in swelling damage of the organelles and cytoplasm, regardless of the fixation protocol. Broadly interpreted, the results of this study indicate that an optimal preservation of plant cell and organelle ultrastructure can be achieved by the use of high osmolality fixatives or, preferably, freeze substitution. These results are also important in determining the method of hydration of poikilohydric samples for physiological studies and for interpretation of functional changes as related to the structural condition of the organelles.