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A Method for Preparing Difficult Plant Tissues for Light and Electron Microscopy

Published online by Cambridge University Press:  20 July 2015

Peta L. Clode
Peta L. Clode*
Affiliation:
Centre for Microscopy, Characterisation & Analysis, The University of Western Australia, Crawley, WA 6009, Australia
*
*Corresponding author. [email protected]
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Abstract

Although the advent of microwave technologies has both improved and accelerated tissue processing for microscopy, there still remain many limitations in conventional chemical fixation, dehydration, embedding, and sectioning, particularly with regard to plant materials. The Proteaceae, a family of plants widely distributed in the Southern Hemisphere and well adapted to harsh climates and nutrient-poor soils, is a perfect example; the complexity of Proteaceae leaves means that almost no ultrastructural data are available as these are notoriously difficult to both infiltrate and section. Here, a step-by-step protocol is described that allows for the successful preparation of Banksia prionotes (Australian Proteaceae) leaves for both light and transmission electron microscopy. The method, which applies a novel combination of vibratome sectioning, microwave processing and vacuum steps, and the utilization of an ultra low viscosity resin, results in highly reproducible, well-preserved, sectionable material from which very high-quality light and electron micrographs can be obtained. With this, cellular ultrastructure from the level of a leaf through to organelle substructure can be studied. This approach will be widely applicable, both within and outside of the plant sciences, and can be readily adapted to meet specific sample requirements and imaging needs.

Keywords

ultrastructurehistologysectionsleavessample preparation
Type
Biological Applications and Techniques
Information
Microscopy and Microanalysis , Volume 21 , Issue 4 , August 2015 , pp. 902 - 909
Copyright
© Microscopy Society of America 2015 

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References

Bowes, B.G. & Mauseth, J.D. (2008). Plant Structure: A Colour Guide, 2nd ed. Collingwood: CSIRO Publishing.CrossRefGoogle Scholar
Cavalier, A., Spehner, D. & Humbel, B.M. (2009). Handbook of Cryo-Preparation Methods for Electron Microscopy. Boca Raton, FL: CRC Press.Google Scholar
Giberson, R.T. & Demaree, R.S. (Eds.) 2001). Microwave Techniques and Protocols. Totowa, NJ: Humana Press.Google Scholar
Hassiotou, F., Evans, J.R., Ludwig, M. & Veneklaas, E.J. (2009). Stomatal crypts may facilitate diffusion of CO2 to adaxial mesophyll cells in thick sclerophylls. Plant Cell Environ 32, 1596–1611.Google Scholar
Hayat, M.A. (2000). Principles and Techniques of Electron Microscopy: Biological Applications, 4th ed. New York, NY: Cambridge University Press.Google Scholar
Hibbs, A.R. (2004). Confocal Microscopy for Biologists. New York, NY: Kluwer Academic/Plenum Publishers.CrossRefGoogle Scholar
Knott, G., Rosset, S. & Cantoni, M. (2011). Focussed ion beam milling and scanning electron microscopy of brain tissue. J Vis Exp 53, e2588.Google Scholar
Kuo, J. (2007). Processing plant tissues for ultrastructural study. Methods Mol Biol 369, 35–45.Google Scholar
Lambers, H., Brundrett, M.C., Raven, J.A. & Hopper, S.D. (2010). Plant mineral nutrition in ancient landscapes: High plant species diversity on infertile soils is linked to functional diversity for nutritional strategies. Plant Soil 334, 11–31.Google Scholar
Lambers, H., Cawthray, G.R., Giavalisco, P., Kuo, J., Laliberté, E., Pearse, S.J., Scheible, W.-R., Stitt, M., Teste, F. & Turner, B.L. (2012). Proteaceae from severely phosphorus-impoverished soils extensively replace phospholipids with galactolipids and sulfolipids during leaf development to achieve a high photosynthetic phosphorus-use-efficiency. New Phytol 196, 1098–1108.Google Scholar
Lambers, H., Colmer, T.D., Hassiotou, F., Mitchell, P.M., Poot, P., Shane, M.W. & Veneklaas, E.J. (2014). Carbon and water relations. In Plant Life on the Sandplains in Southwest Australia: A Global Biodiversity Hotspot, Lambers, H. (Ed.), pp. 129–146). Perth: UWA Publishing.Google Scholar
Leduc, E.H. & Bernard, W. (1967). Recent modifications of the glycol methacrylate embedding procedure. J Ultrastruct Res 19, 196–199.Google Scholar
Lersten, N.R. & Horner, H.T. (2005 a). Development of the calcium oxalate crystal macropattern in pomegranate (Punica granatum, Punicaceae). Am J Bot 92, 1935–1941.CrossRefGoogle ScholarPubMed
Lersten, N.R. & Horner, H.T. (2005 b). Macropattern of styloid and druse crystals in Quillaja (Quillajaceae) bark and leaves. Int J Plant Sci 166, 705–711.CrossRefGoogle Scholar
Maksimovic, S., Layne, J.E. & Buschbeck, E.K. (2011). Spectral sensitivity of the principal eyes of sunburst diving beetle, Thermonectus marmoratus (Coleoptera: Dytiscidae), larvae. J Exp Biol 214, 3524–3531.Google Scholar
Mensi, I., Vernerey, M.S., Gargani, D., Nicole, M. & Rott, P. (2014). Breaking dogmas: The plant vascular pathogen Xanthomonas albilineans is able to invade non-vascular tissues despite its reduced genome. Open Biol 4, 130116.Google Scholar
Terng, H.J., Gessner, R., Fuchs, H., Stahl, U. & Lang, C. (1998). Human transferrin receptor is active and plasma membrane-targeted in yeast. FEMS Microbiol Lett 160, 61–67.Google Scholar
Weston, P.H. (2007). Proteaceae. In The Families and Genera of Vascular Plants, vol. IX Kubitzki, K. (Ed.), pp. 364–404. Heidelberg: Springer.Google Scholar
Zechmann, B. & Zellnig, G. (2009). Microwave-assisted rapid plant sample preparation for transmission electron microscopy. J Microsc 233, 258–268.Google Scholar