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United States Department of Agriculture

Agricultural Research Service

Title: Importance of the Fixative for Reliable Ultrastructural Preservation of Poikilohydric Plant Tissues. Observations on Dry, Partially, and Fully Hydrated Tissues of Selaginella Lepidophylla

item Oliver, Melvin

Submitted to: Annals Of Botany
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: June 11, 1997
Publication Date: N/A

Interpretive Summary: The authors present new work on the analysis of desiccated plant tissues. Previous work in the field has led to confusion as to the actual state of cells when dry. The main question being, when does drying cause cellular damage? Is it during drying, in the dried state, or when water is added back during rehydration? Most of the confusion has come from the inappropriate use of fixatives for electron microscopy. Most fixatives are aqueous and dilute and their use immediately rehydrates the dried tissues to be examined causing artifactual damage as the chemicals act. This study has explored these methods and demonstrated the artifactual nature of the data obtained. This study then describes procedures which are free of artifact and are reliable methods for future studies. The paper also establishes that damage of dried tissues occurs only during rehydration and only if, as would occur in nature, the rehydration medium is dilute.

Technical Abstract: Leaves of desiccated "resurrection plants", Salaginella lepidophylla, were hydrated either through the roots of intact plants or as isolated organs. Air-dry tissue and samples at 1, 4, 8 and 24h (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 tissues 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 4h 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 structure 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 to the structural condition or the organelles.

Last Modified: 4/22/2015
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