Date of this Version
Thesis (M.S.)—University of Nebraska—Lincoln, 1962. Department of Botany (Plant Pathology).
This study was undertaken to devise an easy, accurate method to detect and separate various components in extracts of healthy bean, tobacco, and barley plants by using techniques common to virus purification and to determine the effect of some of the more common denaturing and adsorbing agents on these healthy plant components.Conditions for density-gradient centrifugation were selected to give the best separation of normal components, and these conditions were then used as the basis of an analytical method to evaluate the denaturation procedures.
Protoplasmic components of healthy bean, tobacco, and barley plants were isolated and studied by use of techniques common in plant virus purification. Three major types of components were separated by density-gradient centrifugation in columns prepared with 0.01 M neutral phosphate buffer.Material that did not sediment in high speed centrifugation or through gradient columns consisted mainly of phenols and low molecular weight proteins. Ribosomal components sedimented through density-gradient columns at rates similar to some small spherical plant viruses and to previously reported sedimentation rates for ribosomes from E. coli and pea seedlings.These particles appeared spherical in the electron microscope, and strongly absorbed ultraviolet light.The integrity of the particles depended upon the concentration of magnesium in the system, with larger particles predominating in higher concentrations of magnesium.Three to five distinct ribosomal components differing in sedimentation rate, density, and ultraviolet absorption spectra were observed.Material that sedimented more rapidly through rate density gradient columns could be separated into three components by equilibrium zonal centrifugation in sucrose gradient columns.These components were larger, less dense, and absorbed less ultraviolet light than the ribosomes.
Denaturation treatments tested included heating, various organic solvents, calcium phosphate, acidification, and freezing.Scanning patterns of treated extracts were compared to those of untreated resuspended high speed pellet from the same preparation of leaves.Effectiveness varied among the plant species tested, but in general, heating, long-term freezing, and calcium phosphate adsorption removed more of the normal plant components than did other treatments. Treatments of the crude sap followed by high speed centrifugation were more effective than the same treatments on solutions of the resuspended high speed pellet. Components were also effectively removed from extracts by aggregation with high magnesium concentrations and dispersion with EDTA.
Advisor: M.K. Brakke