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Journal of Electron Microscopy Advance Access originally published online on August 25, 2005
Journal of Electron Microscopy 2005 54(4):393-402; doi:10.1093/jmicro/dfi054
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© The Author 2005. Published by Oxford University Press on behalf of Japanese Society of Microscopy. All rights reserved. For permissions, please email: journals.permissions@oxfordjournals.org

Effects of different fixation and freeze substitution methods on the ultrastructural preservation of ZYMV-infected Cucurbita pepo (L.) leaves

Bernd Zechmann*, Maria Müller and Günther Zellnig

University of Graz, Institute of Plant Sciences, Schubertstrasse 51, 8010 Graz, Austria

* To whom correspondence should be addressed. E-mail: bernd.zechmann{at}uni-graz.at

Different fixation protocols [chemical fixation, plunge and high pressure freezing (HPF)] were used to study the effects of Zucchini yellow mosaic virus (ZYMV) disease on the ultrastructure of adult leaves of Styrian oil pumpkin plants (Cucurbita pepo L. subsp. pepo var. styriaca GREB.) with the transmission electron microscope. Additionally, different media were tested for freeze substitution (FS) to evaluate differences in the ultrastructural preservation of cryofixed plant leaf cells. FS was either performed in (i) 2% osmium tetroxide in anhydrous acetone containing 0.2% uranyl acetate, (ii) 0.01% safranin in anhydrous acetone, (iii) 0.5% glutaraldehyde in anhydrous acetone or (iv) anhydrous acetone. No ultrastructural differences were found in well-preserved cells of plunge and high pressure frozen samples. Cryofixed cells showed a finer granulated cytosol and smoother membranes, than what was found in chemically fixed samples. HPF led in comparison to plunge frozen plant material to an excellent preservation of vascular bundle cells. The use of FS-media such as anhydrous acetone, 0.01% safranin and 0.5% glutaraldehyde led to low membrane contrast and did not preserve the inner fine structures of mitochondria. Additionally, the use of 0.5% glutaraldehyde caused the cytosol to be fuzzy and partly loosened. ZYMV-induced ultrastructural alterations like cylindrical inclusions and dilated ER-cisternae did not differ between chemically fixed and cryofixed cells and were found within the cytosol of infected leaf cells and within sieve tube elements. The results demonstrate specific structural differences depending on the FS-medium used, which has to be considered for investigations of selected cell structures.

Keywords     freeze substitution, Cucurbita pepo, high pressure freezing, transmission electron microscopy, Zucchini yellow mosaic virus

Received      4 May 2005, accepted 15 July 2005


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