Autoradiography of Water-Soluble Materials in Plant Tissues

Samples of tissue with water-soluble substances are lyophilized and doubly-infiltrated with parlodion and paraffin. Sections are mounted on an adhesive-coated cover slip with chloroform. The reverse side of the cover slip is coated successively with a thin layer of Harleco resin and a thick layer of Kodak “Opaque”. A corner of the cover slip is broken off as a marker. The cover slip is assembled with a covering glass slide on a nuclear track plate (Kodak NTB-2) with protective coating, the section being in contact with the emulsion, and held in place during radioactive exposure. Before development, the cover slip and plate are exposed briefly to light and then disassembled. Following development of the plate and removal of the layer of resin and opaque from the cover slip, staining of the section is optional. Lining up of the section with its autoradiograph can be accurate within 1-5μ.     

Autoradiography of Tracks from Beta-Particle Emitters in Tissues

Mounted paraffin sections, 2-4μ thick, ˙were stained, dehydrated, allowed to air dry, and given a thin coating of 1 % Plexi-glas solution in chloroform. The chloroform was allowed to evaporate completely in a dry atmosphere. An emulsion whose dried thickness was 100-150μ, was prepared from Ilford G5 type in gel form and glued to the section by means of a 15% solution of shellac in absolute alcohol. The surface of the emulsion was then cleaned with absolute ethyl alcohol, to remove the impermeable shellac layer. The exposure for radiation reaction was made at about 2°C and required, in the conditions of our experiment, about 24 hrs. The emulsions were processed by the “temperature-development method.” With the described procedure, autoradiographs have been obtained of various organs of albino rats, labeled with P³², S³⁵ and other radioisotopes, and very precise localizations of the origin of electron tracks was attempted. This technic has allowed the fixing and staining of the tissues by means of all the reagents commonly employed in histology, without any damage to the emulsion and the obtaining of good adhesion and minimum separation between specimen and emulsion, thus permitting reliable extrapolations of electron tracks. Due to the fact that the emulsion is fully sensitized when placed in contact with the preparation the limits of the exposure times were well defined. The uniform development at all depths of the emulsion achieved by the temperature-development method facilitated the work with fast electron tracks.     

Preservation and Long-Term Storage of Feulgen-Stained Mouse Tissues for Subsequent Autoradiography

Tissue of the jejunal crypts of mouse intestine was fixed for 24-48 hr in acetic-ethanol, 1:3, and stained en bloc by the Feulgen reaction. The stained preparations were then stored 4 mo at -25 C in either 45% acetic acid alone or in dimethyl sulfoxide (DMSO) or glycerol to which 45% acetic acid had been added to make 15% of the total volume. Such storage preserved not only the stain but allowed autoradiographs to be made. No loss of silver grains or a decrease of labeling index was observed. The procedures are equally successful when used with double-labeling experiments. Solid, transplantable, experimental carcinomas can be preserved in a manner identical to that suggested for the epithelial cells of the crypts.     

Spot Diameter Method of Quantitative Autoradiography Of Ruthenium106 Particles in Lung Tissues

Ruthenium¹⁰⁶ dioxide particles were administered to mice by intravenous or intratracheal injections in doses ranging from 1.1 to 50 μc. The animals were sacrificed after 100 to 420 days exposure to beta radiation. The lung tissue was examined by autoradiographic methods using Eastman Kodak NTB 10 μ film and a standardized exposure and development time. A quantitative straight line function exists between the sum of the spot diameters or a single spot diameter and the total activity of each autoradiogram.     

Mordanting Plant Tissues

Selected current ideas on the mordanting of plant tissues are presented in the hope that they will be of practical assistance, especially to the beginner. The nature of the mordanting process and the results which may be expected following mordanting are briefly described. Specific technics are presented for the mordanting of natural dyes, synthetic dyes generally, the basic synthetic dyes and the acid synthetic dyes.     

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.     

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.     

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.     

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.     

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.     

Regulators of Cell Division in Plant Tissues

Procedures were devised for purification of the cytokinins in the milk of mature coconuts (Cocos nucifera fruits). One cytokinin isolated in crystalline form was unequivocally identified as 9-β-D-ribofuranosylzeatin. This compound appeared to account for a large proportion of the cytokinin activity in n-butanol extracts of coconut milk. The activity which was not extracted by n-butanol was largely due to unidentified compounds which could be partially purified by adsorption onto and elution from charcoal.     

Regulators of Cell Division in Plant Tissues

The cytokinins, 6-benzylaminopurine and kinetin, markedly enhanced the yield of both free and membrane-bound 80S ribosomes per unit weight of radish (Raphanus sativus) cotyledon tissue. The response was observed only after the induction of growth by cytokinin; during the lag period preceding cytokinin-induced growth, ribosome yields from both control and cytokinin-treated cotyledons were below detectable levels. Mannitol depressed both growth and ribosome yield to the same degree. The enhanced ribosome yield appeared to be an indirect effect of cytokinin and was probably a consequence of cytokinin-induced growth. The effect of 6-benzylaminopurine on ribosome yield was not reflected in enhanced levels of cytoplasmic ribosomal RNA, while recently synthesized ribosomes were found to be more readily recovered from cytokinin-treated tissue than from control tissue. It was concluded that cytokinin-enhanced ribosome yield resulted from enhanced ribosome recovery or extractability and that ribosome yield is an unreliable indication of ribosome level in plant tissue.     

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     

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.     

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.