SUBJECT

Research Methods in Plant Physiology I

practical

master

Faculty of Science

Biology MSc

4

Semester 2

Spring semester

Weeks 1 and 2. Plant culturing – exercises in plant mineral nutrition

Lecture: Methods in plant culture, nutrient solutions, installing hydroponic cultures. Climate-controlled plant growth chambers, setting the parameters.

Practical: Germination of the test plants (determination of germination %), planting and growth (measurement of pH, growth parameters-fresh and dry weight). (Optional topics: Effect of essential and toxic elements, deficiency solutions, and inhibitory concentrations. Role of iron nutrition, and the effect of chelating agents. Effect of nitrogen forms on plants.)

Week 3. Exercises in plant gas exchange

Porometry: measurement principle, structure of the Delta-T porometer, measured parameters (resistance, conductance, gas flow velocity), measuring methods. Relationship between stomatal conductance and transpiration. Measuring transpiration in test plants. Infrared gas analysis: measurement principle, set up of the device and the measuring cells, measuring methods. Measuring the CO2 fixation of the test plants at different light intensity.

Week 4. Fluorescence microscopy

Lecture: Definition of fluorescence and chemiluminescence. Luminescence phenomena in nature. Utilization of luminescence in biochemistry. Characteristics of most important fluorophores. Application of fluorophores for marking the nucleus, membranes, and organelles, nucleic acids, in vivo staining, detection of metals (Ca) and heavy metals, measurement of redox potential and pH. Operation principles of the fluorescent microscope.

Practical: Studying the water movement in plants by berberine sulphate. Control or inhibitor treated, excised or intact wheat seedlings of different age grown in hydroponics are placed into berberine sulphate solution for different times. Studying the microscopic picture of xylem the rate of water movement in the differently treated seedlings is determined and interpreted.

Week 5. O2 electrode measurements of respiration and photosynthesis

Lecture: Overview of physiological processes of oxygen production and consumption. Methods and equipments for measuring oxygen production and consumption in biological systems. Theory of O2 electrode measurements. Set-up and operation of different types of O2 electrodes. Clark-type O2 electrode. Sample preparation. Conditions of measurement. Possible errors. Evaluation of results.

Practical: Demonstration of different types of O2 electrodes. Main parts of a Clark-type O2 electrode (Hansatech) and its supplements. Computer program for evaluation of measurements. Calibration process of O2 electrode. Sample preparation for in vivo measurement for respiration rate and photosynthetic oxygen evolution. In vitro measurement of oxygen evolution in chloroplast suspension. Measurement of the rate of respiration and photosynthetic oxygen evolution in differently treated samples. Evaluation of results.

Week 6. Fluorescence induction and fluorescence imaging techniques

Lecture: The relation of fluorescence and photosynthesis. The phenomenon of fluorescence induction. Connection of fast and slow phase of fluorescence induction with photosynthetic processes. Measurement of fluorescence induction with PAM fluorometer. The physiological meaning of the most important fluorescence induction parameters. Fluorescence quenching mechanisms. The application of fluorescence imaging in detection of plant stress.

Practical I: Changes of quenching parameters in plants exposed to various abiotic/biotic stresses.

Practical II: The measurement of 440, 520, 690 and 740nm fluorescence emitted by leaves or other plant organs and diagnostical evaluation of fluorescence imaging data.

Weeks 7 to 10. Identification of wheat varieties studying storage protein genes by PCR

Lecture: Therory and practice of PCR used for marker based breeding. General principles of primer planning, primers suitable for tracing point mutation (SNP), deletion, and insertion.

Practical: DNA extraction from grains and leaves of different wheat varieties. Measurement of DNA concentration and checking the purity of the extract. DNA quality control is carried out by inner standard: making PCR reaction mixture, running the reaction, and separation of products by agarose gel electrophoresis. Determination of the composition of wheat strorage proteins by PCR markers: making the PCR reaction mixture for inner standard, running the reaction, and separation of products by agarose gel electrophoresis. Markers used are known from literature. They are suitable for identification of varieties, and for finding SNPs, deletions, and insertions. Evaluation of results.

Week 11. Separation of protein complexes by density gradient centrifugation

Lecture: Principles of centrifugation. Centrifugation techniques: differential, density gradient centrifugation (rate-zonal, isopicnic centrifugation). Properties and usage of gradient materials. Types and application of centrifuges, rotors. Methods for preparation of gradients. Sample application. Extraction of fractions from the gradient.

Practical: Separation of thylakoid complexes by sucrose density gradient centrifugation. Solubilization of isolated thylakoids. Preparation of gradient, application of samples, centrifugation by ultracentrifuge. Extraction of the separated fractions. Characterization of the complexes by their chlorophyll a/b ratio.

Week 12. Separation of plant growth regulators (PGR-s) by HPLC

Lecture: The methodology of HPLC, application to the separation of plant metabolites.

Practical: Comparison of cytokinin content of different plant organs of different age. Extraction in methanol:water:formic acid buffer, pre-separation on Baker solid phase column, filtration on Durapore (0.45) membrane, and separation on Hypersil Elite C18 column by gradient elution (methanol:water). PGR content is measured by UV absorption (275 nm), and calculated from a calibration curve (standards: trans-zeatin, trans-zeatin riboside, isopentenyl adenosine).

Weeks 13 and 14. RNA isolation from plants and studying translation in vitro

Lecture: Methods of RNA isolation, peculiarities in the isolation procedures of plant RNA. Conditions necessary for operation of cell-free protein synthesizing system.

Practical: Isolation of total RNA from differently treated plants: etiolated and illuminated for different periods. Purification of the extract, removal of polysaccharide contamination. Separation of RNA fraction of low molecular weight. Checking the purity of the extracts.

Setting-up the in vitro translation system. Isolation of polysome fraction. Preparation of reaction mixture of different tRNA fractions and radioactive amino acids. Detection of labeled proteins by liquid scintillation method.

• Handbook of Plant Ecophysiology Techniques (MJR Roger, ed.) ISBN 0-7923-7053-8, 2001, Kluwer Acad. Publ., Dordrecht, The Netherlands.

• Photosynthesis Energy Transduction: A Practical Approach (MF Hipkins & NR Baker, eds.), 1986, ISBN 0-947946-51-9, IRL Press Lim., Oxford, England

• Centrifugation: A Practical Approach (D Rickwood, ed.), 1984, IRL Press Lim., Oxford, England