Importance of the Fixative for Reliable Ultrastructural Preservation of Poikilohydric Plant Tissues. Observations on Dry, Partially, and Fully Hydrated Tissues ofSelaginella lepidophylla

Leaves of desiccated ‘resurrection plants’,Selaginellalepidophylla, were hydrated either through the roots of intactplants or as isolated organs. Air-dry tissue and samples at1, 4, 8 and 24 h (both detached and intact) of hydration wereprepared for electron microscopy using aldehyde fixatives ofdifferent osmotic strengths. Both dry and hydrated tissues werealso prepared using freeze substitution. Significant differencesin the ultrastructural preservation of these different sampleswere noted. There was a direct correlation between the osmolalityof both the fixative and the tissue with the quality of ultrastructuralpreservation. When the osmolality of the fixative was slightly(or even considerably) higher than that of the tissue, optimalpreservation was achieved. Freeze substitution, however, gavethe 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 swellingdamage of the organelles and cytoplasm, regardless of the fixationprotocol. Broadly interpreted, the results of this study indicate thatan optimal preservation of plant cell and organelle ultrastructurecan be achieved by the use of high osmolality fixatives or,preferably, freeze substitution. These results are also importantin determining the method of hydration of poikilohydric samplesfor physiological studies and for interpretation of functionalchanges as related to the structural condition of the organelles.Copyright1997 Annals of Botany Company Selaginella; fixation; ultrastructure; dry; hydrated