Research Methods in Plant Physiology II
lecture + practical
1.-2. week: Fluorescence microscopy: examinatons on cytotoxicology Laboratory practice: determination of the ratio of living plant and fungal cells. Incubation of small seeds, yeast suspension and fungal spores in solutions of toxins of different concentrations. Transfer of seeds and cells into solution of vital dye and evaluation of the ratio of living organs by fluorescence microscopy. Determination of concentration- and time-dependence of effect of toxins.
3. week. Examinations on malone dialdehyde (MDA) content of various plant samples. Lecture: Characteristics and production of MDA. MDA content as a physiological indicator in plant cells. Determination of MDA content, principles of experimental work and evaluation of MDA content. Laboratory practice: Isolation of MDA: sample preparation, homogenization of plant material, centrifugation. Determination of MDA content: preparation of reagents, incubation, spectrophotometry. Determination of MDA content on the basis of absorbances and evaluation of data.
4. week: Separation of flavonoides using HPLC technique. Laboratory practice: The main aim of measurements is the isolation, qualitative and quantitative determination of flavonoides in plants under different stress conditions. Isolation and pre-separation by SepPack system, filtration by 0.45 Durapore membrane. Separation by Hypersyl ODS 5 micron column. Isocratic separation by methanol : ethylacetate : phosphate buffer (pH 2.1). Preparation of standard curves (quercetine, iso-quercetine, kaempherole). Determination of quercetine in plants, grafical evaluation.
5-6. weeks: Determination of polyamine content The aim of the measurements is the determination of qualitative and quantitative changes in free polyamine pool on the effect of different stress conditions. Laboratory practice: Extraction of free polyamines from plants exposed to various stress conditions (cold, osmotic stress, senescence, hormone treatments). Preparation of dansylated derivatives and separation by thin-layer chromarography. Indentification on the basis of Rf values using standards. Quantitative determination by fluorometry, on the basis of calibration curves.
7. week: Cytokinin activity test Laboratory practice: growing experimental plants and Amaranthus caudatus. Isolation and purification of plant extracts and incubation of test plants with extracts as well as with different concentrations of cytokinins (benzyladenine and kinetin). Extraction and determination by spectrophotmetry of betacyanine content from treated test plants. Evaluation by preparing calibration curves.
8-9. weeks: Isolation of plant RNAs The aim of experiments is to introduce into isolation and purification methods of different plant RNAs. Laboratory practice: Extraction of total RNA fraction, deproteinization, separation from polysaccharides. Separation and purification of rRNA and tRNA fractions. Evaluation of purity by spectrophotometry and gel electrophoresis. Acylation of tRNAs with labelled amino acids. Separation of isoacceptors by column chromatography. mRNA isolation using magnetic beads.
10-11. weeks: Examinations on stress-induced changes of activity of transcription. Preparation of mRNA fraction from differently treated plant samples. cDNA synthesis. Competitive PCR for quantification in differences of mRNAs.
12-13. weeks. Radioactive isotopes for measurements in plant physiology. Lecture: General rules of safety and work with radioactive isotopes. Aspects of application, general rules of planning and carrying out experiments. Laboratory practice: Liquid scintillation measurements on carbon dioxide fixation of green plants using 14CO2.
Roger M. J. R. Handbook ofPlant Ecophysiology Techniques.p. 452, Kluwer Acad. Publ. Dordrecht, etc (2001)