Freeze-Sectioning of Plant Tissues

Techniques are described for freeze-sectioning a wide range of both fresh and fixed plant tissues. Gelatin-antifreeze media are used to support but not infiltrate the tissue during sectioning. At cryostat temperatures of -10 to -15 C, 15% gelatin (w/v) containing 0.8% dimethyl sulfoxide (DMSO), or 1.5% ethanediol (ethylene glycol), or 2% glycerol is used. Lower concentrations of gelatin and higher concentrations of antifreezes are required for sectioning at -24 C. Petri plates of media are stored at 2 C, and used by simply melting a hole in the medium. Fresh tissues can be placed directly in the hole, or prefrozen at temperature of liquid nitrogen, or equilibrated in antifreeze solution, before freeze-sectioning in the gelatin antifreeze medium. Many plant tissues have highly vacuolated cells and need equilibration in antifreeze solutions prior to freeze-sectioning. Fixed tissues are rehydrated and washed in water or buffer for 15-24 hr before equilibrating in a 10% solution of either DMSO, ethanediol or glycerol (named in order of rapidity of equilibration). Pretreatment in 10% DMSO is usually for 1-6 hr at 2 C for histochemical studies; or in 10% ethanediol or glycerol for 15-24 hr at either room temperature or 37 C for morphological studies. These methods permit serial cryostat sections free from freezing and thawing artifacts to be cut as thin as 2 μ.     

Softening Paraffin-Embedded Plant Tissues

Soaking paraffin-embedded plant specimens 2-3 days at 37°C. in a mixture of glycerol, 10 ml., Dreft, 1 g., and water 90 ml. is an effective means of softening them prior to sectioning. One side of the paraffin block must be pared away to expose the tissue before immersion in the softening solution.     

Freezing-Sectioning of Plant Tissues1

An improved and simple method is described by which serial 10µ frozen sections of plant tissues may be obtained. Thevalue of this method for use in plant histochemistry is discussed.     

Isopropyl-Alcohol-Paraffin Methods for Plant Tissues

Two methods using isopropyl alcohol for dehydration prior to paraffin infiltration of plant tissues are reported: (1) a modified Rawlins-Takahashi (1947) schedule in which the preliminary dehydration is effected by concentrating glycerol and (2) isopropyl alcohol as the sole agent for dehydration. With the latter method the fixed tissues are dehydrated successfully in 60%, 85%, and 99% isopropyl alcohol. Paraffin infiltration is accomplished by placing the tissues in isopropyl alcohol over solid paraffin in a vial and heating to 56°-58° C. The tissues settle into the melted paraffin as infiltration progresses. Several changes of pure paraffin are then made, with the last change under reduced pressure. The embedded tissues are trimmed and soaked 2-4 hours at 40° C. in either water or a glycerol, acetic acid, 70% alcohol mixture (10:15:75) to reduce static and insure uniform ribboning during microtomy.     

Ethanolamine Metabolism in Plant Tissues

Ethanolamine is readily metabolized by oat, pea, wheat, apple and carrot tissue preparations. Ethanolamine-1,2 (14)C was incorporated into the lipid fraction, and (14)C activity was distributed in the organic acid, sugar, acid volatile, carbon dioxide and insoluble residue fractions. The distribution varied with the particular tissue. Incorporation into the lipid fraction occurred in tissue homogenates in the absence of ATP by a Ca(++) activated system similar to that reported for animal preparations. The initial step in ethanolamine oxidation involves an amine oxidase. Glycolaldehyde and glyoxylic acid are metabolic intermediates, the former in the conversion of ethanolamine to carbon dioxide. No evidence was obtained for the operation of an ethanolamine transaminase or for the involvement of phosphorylated intermediates in the conversion of ethanolamine to carbon dioxide.     

Electroosmotic Phenomena in Plant Tissues

The effect of a direct electric current on electrolyte transport through plant tissues was studied by applying it to 10-mm segments of the mesocotyls of etiolated maize seedlings, similar segments of one-year linden shoots with the normal conducting system and without vascular bundles, and isolated elements of the xylem and cell wall segments. At current densities of 9–38 A/mm² (10–20 V), electrolyte solutions in plant tissues always moved toward the cathode. The results suggest that electroosmosis is one of the factors responsible for changes in solution transport through the conductive plant tissues that occur under the effect of electric current.     

Modelling Woody Plant Tissues5

An electrical impedance spectrum (20 Hz to 1 MHz) was measuredin both the bark and wood of current-year and one-year-old Scotspine (Pinus sylvestris L.) shoots. The measured impedance spectrawere analysed in reference to two lumped circuits and a distributedcircuit. It was found that neither of the two lumped circuitsfit the data from bark or wood as well as the distributed model.The lumped (double-shell) model fit fairly well for bark, butpoorly for wood. It is proposed that the good fit of the distributedmodel and the poorer fit of the lumped models are due to a widerange of cell sizes in the bark and wood. The distributed circuitused in the present study may be useful for describing differentiatedplant tissues for physiological studies. Key words: Bark, electrical impedance, equivalent circuit, Pinus sylvestris L., wood     

Freezing of Plant Tissues3

The freezing of plant cells under the microscope was studiedon three types of tissues: Tradescantia staminal hairs, fruitskin, and moss leaf. Cinemicrography was used to record stagesin the freezing processes, which are otherwise un observable.The following phenomena have been demonstrated and discussed:(1) The protective effect of persistent supercooling. (2) Ice-inoculationof cells and factors affecting it. (3) Sequence of ice formationwithin cells. (4) Modes of ice formation as affected by supercooling.(5) Freezing of cell walls and their longitudinal splitting.     

Protein Metabolism in Cultured Plant Tissues

Rates of protein synthesis in normal callus tissues (either tight or loose morphological form), in crown gall callus tissues and in cultured pith cells were measured for both the lower surface cells (those in contact with the original growth medium) and upper surface cells (those never in contact with the growth medium until labeling). Cells of both surfaces of loose and crown gall callus and the upper-surface cells of tight callus had similar rates of protein synthesis, 29–31 mg of protein synthesized × (g protein)⁻¹× h⁻¹. The lower surface cells of tight callus had a 35% lower rate of synthesis, 20 mg × g⁻¹× h⁻¹. Pulse-chase experiments suggested that rates of protein degradation for all tissues were the same, 21–23 mg protein × (g protein)⁻¹× h⁻¹. Thus, there probably was no accumulation of protein in the lower surface cells of tight callus tissue, but the other tissues had rates of accumulation equaling 10 mg × (g protein)⁻¹× h⁻¹. Autoradiography and electron-microscopic examination of cells in tight callus labeled with ³H-leucine show that: (a) the lower-surface cells were more degenerate than cells within the callus or on the upper surface; and (b) the first few cell layers nearest the medium were preferentially labeled. Pulse-chase experiments were also used to quantitate the nonprecursor pool (defined as that tritium in the soluble amino acid pool that does not equilibrate with protein during a pulse-chase experiment). The nonprecursor pool increased linearly with time at the same rate as incorporation of ³H-leucine into protein. Furthermore, the nonprecursor pool copurified with leucine and was probably either D- or L-leucine.     

Freeze Fracture of Intact Plant Tissues

A procedure for dissolving and handling replicas of cutinized leaves and other plant tissues is described. This technique yields comparatively large replicas which can include vascular tissue, epidermal cells and the cuticle. Dissolution of the tissue involves the sequential use of alcoholic KOH, sulfuric acid and chromic acid. The use of clean, uncoated grids for transfer of the tissue and replicas results in rapid, easy handling with a minimum of breakage or loss. The procedure is more efficient than previous methods and produces large replicas. This allows tissue orientation and more positive identification of cell types for better correlation of freeze fracture results with thin sections of similar material.     

Improved Paraffin Schedules for Plant Tissues

The two paraffin schedules here presented have produced less distortion in plant tissues than those commonly used. Both are modifications of the schedule described by Hemenway.¹ Schedule A is very similar to that described by Hewitt.² Schedule B usually causes less distortion than A but staining is not so bright as after A.     

Mounting Macerated Plant Tissues With Adhesives

Adhesives are important in standard paraffin methods, and they are also utilized in preparing permanent slides of diatoms (Conger, 1925a and b; Hanna and Grant, 1939) and other algae (Adams, 1940; Alcorn, 1935; Johansen, 1940). Such procedures involve the transfer of prepared materials to adhesive-coated slides or cover slips. Aldaba (1927) followed such a method for studying extremely long ramie fibers by floating them onto strips of window glass “coated with fixative”. It appears, however, that adhesives have not been employed generally in the preparation of macerated materials. The schedule to be presented was developed for handling macerated xylem elements of sorghum roots.     

Electrical Impedance Analysis in Plant Tissues8

Electrical impedance spectra (100 Hz–800 kHz) were measuredin leaves of Peperomia obtusifolia L. (a succulent) and Brassicaoleracea L. (cabbage). By measuring impedances at three or moreinter-electrode distances in a single leaf, electrode impedanceand specific tissue impedance were separated. Analysis of impedance data from B. oleracea leaves in relationto an equivalent circuit model showed that leaf developmentwas accompanied by increases in extracellular resistance, cytoplasmicresistance and vacuole interior resistance, together with decreasesin plasma membrane capacitance and tonoplast capacitance. AfterB. oleracea leaves were subjected to a –6 °C freeze-thawstress, extracellular resistance, cytoplasmic resistance andvacuole interior resistance decreased, but plasma membrane capacitanceand tonoplast capacitance did not change. These results indicatethat useful measurements of leaf parameters can be obtainedby this technique. Examination of the electrode impedance spectrum showed thatelectrode insertion produced a damaged collar, 0·4–0·5mm wide, around the electrode. This was confirmed by visualobservation of the damage in P. obtusifolia leaf. Key words: Peperomia obtusifolia L., Brassica oleracea L. (cabbage), electrical impedance, equivalent circuit, electrode polarization     

Staining Cell Constituents in Diseased Plant Tissues

Tissues of diseased plants, where embryonic cells, with dense cytoplasm, are to be studied cytologically in the same section with vacuolated cells, should best be killed with the Meves or Regaud fluids. The greatest changes in affected cells of diseased tissues are likely to occur in the vacuolar system, the contents of which should be well preserved in the killing process.

Staining with acid fuchsin and decolorizing with light green, makes it possible, in properly killed tissues, to detect the slightest alteration in tissues, and to observe the different parts of the cell, nucleus, mitochondria and plastids, even when excessive vacuolation compresses the cytoplasmic inclusions.     

Electrical Impedance Analysis in Plant Tissues11

Electrical impedance was measured at a range of AC frequenciesof 100 Hz to 1 MHz in potato tubers and carrot roots. Detailsof a method for analysing the data in relation to electricalmodels, using complex non-linear least squares (CNLS), are described.The measured data were analysed in relation to four previously-describedelectrical models for plant tissues. The results showed thatplant tissue conforms well to a double-shell model which includescomponents (resistors and capacitors) representing the vacuole,as well as cytoplasm, plasma membrane, and extracellular space.The method permitted the calculation of specific membrane capacitance,which was found to approximate l0µF cm^–2. Key words: Electrical impedance, complex non-linear least squares (CNLS), electrical modelling, membrane capacitance