Hostname: page-component-78c5997874-94fs2 Total loading time: 0 Render date: 2024-11-10T22:53:57.337Z Has data issue: false hasContentIssue false
  • English
  • Français

A comparison of anhydrous fixation methods for the observation of pea embryonic axes (Pisum sativum L. cv. Alaska)

Published online by Cambridge University Press:  22 February 2007

Jodie L. Ramsay  and
Karen L. Koster
Jodie L. Ramsay
Affiliation:
Department of Biology, The University of South Dakota, 414 E. Clark Street Vermillion SD 57069 USA
Karen L. Koster*
Affiliation:
Department of Biology, The University of South Dakota, 414 E. Clark Street Vermillion SD 57069 USA
*
*Correspondence Fax: +1–605–677–6557 Email: kkoster@usd.edu
Get access Rights & Permissions [Opens in a new window]

Abstract

To develop an effective protocol for observation of dry pea embryonic axes (Pisum sativum L. cv. Alaska), several fixation methods were compared for ease of infiltration and sectioning and for quality of sections. These methods included osmium vapour fixation, freeze-substitution followed by an aqueous wash, and freeze-substitution using anhydrous methanol. Axes fixed with osmium vapour were brittle and difficult to section. Cells fixed using vapour from an aqueous OsO4 solution had cell walls separated from plasma membranes, while cells fixed using osmium crystals exhibited folded walls tightly appressed to plasma membranes. Axes fixed using freeze-substitution followed by an aqueous wash sectioned easily, but appeared plasmolysed, with cell walls completely separated from plasma membranes. Even a brief (10 min) rinse in 90% acetone led to separation of the walls from the membranes. Tissues infiltrated with anhydrous methanol and Spurr’s resin were difficult to section, but showed extensive folding of the cell walls, with plasma membranes appressed to the walls. Additional modifications to the infiltration protocol led to improved sectioning. The highest-quality sections were obtained using freeze-substitution with anhydrous methanol as a solvent, followed by infiltration using methanol and Spurr’s resin, with the catalyst (dimethylaminoethanol) omitted from the infiltration protocol until the final step. Through comparison of several protocols recommended in the literature for the preparation of dry tissues, it is apparent that the presence of water, even in the vapour phase, at any step can cause swelling of the cell walls and the artefactual separation of the walls from the fixed protoplast.

Keywords

anhydrous fixationcell wallselectron microscopymembranesPisum sativumultrastructure
Type
Research Article
Information
Seed Science Research , Volume 12 , Issue 2 , June 2002 , pp. 83 - 90
Copyright
Copyright © Cambridge University Press 2002

Access options

Get access to the full version of this content by using one of the access options below. (Log in options will check for institutional or personal access. Content may require purchase if you do not have access.)

References

Berjak, P., Farrant, J.M. and Pammenter, N.W. (1989) The basis of recalcitrant seed behaviour. pp 89–108. Taylorson, R.B. (Eds) Recent advances in the development and germination of seeds. New York, Plenum Press.Google Scholar
Buttrose, M.S. (1973) Rapid water uptake and structural changes in imbibing seed tissues. Protoplasma 77, 111–122.CrossRefGoogle Scholar
Chabot, J.F. and Leopold, A.C. (1982) Ultrastructural changes of membranes with hydration in soybean seeds. American Journal of Botany 69, 623–633.CrossRefGoogle Scholar
Farrant, J.M., Cooper, K., Kruger, L.A. and Sherwin, H.W. (1999) The effect of drying rate on the survival of three desiccation-tolerant angiosperm species. Annals of Botany 84, 371–379.CrossRefGoogle Scholar
Horowitz, J. (1981) A new preparative technique for studying dry seeds of Pisum sativum with the aid of transmission electron microscopy. Micron 12, 139–146.Google Scholar
International Seed Testing Association. (1996) International rules for seed testing. Seed Science and Technology 24 Supplement. pp 335.Google Scholar
Lancelle, S.A., Callaham, D.A. and Hepler, P.K. (1986) A method for rapid freeze fixation of plant cells. Protoplasma 131, 153–165.Google Scholar
Oparka, K.J. (1994) Plasmolysis: new insights into an old process. New Phytologist 126, 571–591.CrossRefGoogle Scholar
Öpik, H. (1980) The ultrastructure of coleoptile cells in dry rice (Oryza sativa L.) grains after anhydrous fixation with osmium tetroxide vapour. New Phytologist 85, 521–529.Google Scholar
Öpik, H. (1985) The fine structure of some dry seed tissues observed after completely anhydrous chemical fixation. Annals of Botany 56, 453–466.Google Scholar
Perner, E. (1965) Elektronenmikroskopische Untersuchungen an Zellen von Embryonen im Zustand völliger Samenruhe. Planta 65, 334–357.CrossRefGoogle Scholar
Platt, K.A., Oliver, M.J. and Thomson, W.W. (1997) Importance of the fixative for reliable ultrastructural preservation of poikilohydric plant tissues. Observations on dry, partially, and fully hydrated tissues of Selaginella lepidophylla. Annals of Botany 80, 599–610.CrossRefGoogle Scholar
Quartacci, M.F., Forli, M., Rascio, N., Dalla Vecchia, F., Bochicchio, A. and Navari-Izzo, F. (1997) Desiccation-tolerant Sporobolus stapfianus: lipid composition and cellular ultrastructure during dehydration and rehydration. Journal of Experimental Botany 48, 1269–1279.CrossRefGoogle Scholar
Ristic, Z. and Ashworth, E.N. (1993) New infiltration method permits use of freeze-substitution for preparation of wood tissues for transmission electron microscopy. Journal of Microscopy 171, 137–142.Google Scholar
Ristic, Z. and Ashworth, E.N. (1994) Response of xylem ray parenchyma cells of red osier dogwood (Cornus sericea L.) to freezing stress. Microscopic evidence of protoplasm contraction. Plant Physiology 104, 737–746.CrossRefGoogle ScholarPubMed
Singh, J., Blackwell, B.A., Miller, R.W. and Bewley, J.D. (1984) Membrane organization of the desiccation-tolerant moss Tortula ruralis in dehydrated states. Plant Physiology 75, 1075–1079.CrossRefGoogle ScholarPubMed
Smith, M.T. (1991) Studies on the anhydrous fixation of dry seeds of lettuce (Lactuca sativa L.). New Phytologist 119, 575–584.CrossRefGoogle Scholar
Spurr, A.R. (1969) A low-viscosity epoxy resin embedding medium for electron microscopy. Journal of Ultrastructure Research 26, 31–43.CrossRefGoogle ScholarPubMed
Thomson, W.W. (1979) Ultrastructure of dry seed tissue after a non-aqueous primary fixation. New Phytologist 82, 207–212.CrossRefGoogle Scholar
Thomson, W.W. and Platt, K.A. (1997) Conservation of cell order in desiccated mesophyll of Selaginella lepidophylla ([Hook and Grev.] Spring). Annals of Botany 79, 439–447.CrossRefGoogle Scholar
Tiwari, S.C., Polito, V.S. and Webster, B.D. (1990) In dry pear (Pyrus communis L.) pollen, membranes assume a tightly packed multilamellate aspect that disappears rapidly upon hydration. Protoplasma 153, 157–168.CrossRefGoogle Scholar
Venable, J.H. and Coggeshall, R. (1965) A simplified lead citrate stain for use in electron microscopy. Journal of Cell Biology 25, 407–408.CrossRefGoogle ScholarPubMed
Vigil, E.L., Steere, R.L., Wergin, W.P. and Christiansen, M.N. (1984) Tissue preparation and fine structure of the radicle apex from cotton seeds. American Journal of Botany 71, 645–659.CrossRefGoogle Scholar
Vigil, E.L., Steere, R.L., Wergin, W.P. and Christiansen, M.N. (1985) Structure of plasma membrane in radicles from cotton seeds. Protoplasma 129, 168–177.CrossRefGoogle Scholar
Webb, M.A. and Arnott, H.J. (1982) Cell wall conformation in dry seeds in relation to the preservation of structural integrity during desiccation. American Journal of Botany 60, 1657–1668.Google Scholar
Webster, B.D. and Leopold, A.C. (1977) The ultrastructure of dry and imbibed cotyledons of soybean. American Journal of Botany 64, 1286–1293.CrossRefGoogle Scholar
Wesley-Smith, J. (2001) Freeze-substitution of dehydrated plant tissues: artefacts of aqueous fixation revisited. Protoplasma 218, 154–167.CrossRefGoogle ScholarPubMed
Wesley-Smith, J., Pammenter, N.W., Berjak, P. and Walters, C. (2001) The effects of two drying rates on the desiccation tolerance of embryonic axes of recalcitrant jackfruit (Artocarpus heterophyllus Lamk.) seeds. Annals of Botany 88, 653–664.CrossRefGoogle Scholar
Yatsu, L.Y. (1983) Electron microscopy of dry peanut (Arachis hypogaea L.) seeds crushed for oil removal: Fixation and embedding of anhydrously prepared tissues. Protoplasma 117, 1–6.CrossRefGoogle Scholar