Main description:

Modem Methods of Plant Analysis When the handbook Modern Methods of Plant Analysis was fIrst introduced in 1954 the considerations were: 1. the dependence of scientifIc progress in biology on the improvement of existing and the introduction of new methods; 2. the difficulty in fInding many new analytical methods in specialized journals which are normally not accessible to experimental plant biologists; 3. the fact that in the methods sections of papers the description of methods is frequently so compact, or even sometimes so incomplete that it is difficult to reproduce experiments. These considerations still stand today. The series was hIghly successful, seven volumes appearing between 1956 and 1964. Since there is still today a demand for the old series, the publisher has decided to resume publication of Modern Methods of Plant Analysis. It is hoped that the New Series will be just as acceptable to those working in plant sciences and related fIelds as the early volumes undoubtedly were. It is difficult to single out the major reasons for success of any publication, but we believe that the methods published in the fIrst series were up-to-date at the time and presented in a way that made description, as applied to plant material, complete in itself with little need to consult other publications. Contributing authors have attempted to follow these guidelines in this New Series of volumes.

Contents:

Plant Hormone Immunoassays Based on Monoclonal and Polyclonal Antibodies.- 1 Introduction.- 2 Synthesis of Immunogens.- 2.1 Synthesis of ABA Conjugates.- 2.1.1 Procedure 1: Synthesis of ABA-(C1)-BSA Conjugates (Modified from Weiler 1980).- 2.1.2 Procedure 2: Synthesis of ABA-C4?-BSA Conjugates (According to Weiler 1980).- 2.2 Synthesis of Indole-3-Acetic Acid. Conjugates.- 2.2.1 Procedure 3: Synthesis of IAA-BSA Conjugates Via the Mixed Anhydride.- 2.3 Synthesis of Gibberellin Conjugates.- 3 Radio- and Enzyme-Labeling of Plant Hormones.- 3.1 Radiolabeled Immunotracers.- 3.2 Enzyme-Labeled Plant Hormones.- 3.2.1 Procedure 4: Synthesis of Alkaline Phosphatase-Labeled IAA (Modified from Weiler et al. 1981).- 3.2.2 Procedure 5: Coupling of ABA-C4? -Hydrazones to Alkaline Phosphatase.- 4 Immunological Procedures.- 4.1 Immunization of Rabbits.- 4.2 Immunization of Mice.- 5 Antiserum Processing.- 6 Production of Monoclonal Antibodies.- 6.1 Generation of Hybridomas.- 6.2 Monoclonal Antibody Production and Characterization.- 7 Radioimmunoassay Procedures.- 7.1 Procedure 6: Radioimmunoassay of Plant Hormones.- 8 Enzyme Immunoassays Based on Monoclonal Antibodies.- 8.1 Procedure 7: Enzyme Immunoassay of Plant Hormones Based on Mouse Monoclonal Antibodies.- 9 Plant Immunoanalysis.- 9.1 General Remarks.- 9.2 Validation of Immunoassays.- 9.3 Processing of Plant Material.- 9.3.1 Procedure 8.- 9.3.2 Procedure 9.- References.- Radioimmunoassay and Gas Chromatography/Mass Spectrometry for Cytokinin Determination.- 1 Introduction.- 2 Theoretical Considerations for Analytical Methods.- 3 Radioimmunoassay.- 3.1 General Principles.- 3.2 Cytokinin Antisera.- 3.3 Cytokinin Determination in the Primary Extract.- 3.4 Cytokinin Determination After Preliminary Purification of the Primary Extract.- 3.5 Methods.- 3.5.1 General Isolation Protocol.- 3.5.2 Synthesis of Cytokinin Protein Conjugates.- 3.5.3 Immunization.- 3.5.4 Synthesis of 3H-Cytokinin Riboside-Dialcohols.- 3.5.5 RIA Protocol.- 4 Gas Chromatography/Mass Spectrometry.- 4.1 General Principles.- 4.2 Identification of Cytokinins.- 4.3 Quantitation of Cytokinins.- 4.4 Methods.- 4.4.1 General Isolation Procedure and GC/MS.- 4.4.2 Synthesis of Deuterium-Labeled Cytokinins.- 4.4.3 Practical Approach.- 5 Concluding Remarks.- References.- Immunodetection of Phytochrome: Immunocytochemistry, Immunoblotting, and Immunoquantitation.- 1 Introduction.- 1.1 Antibody Specificity.- 1.2 Immunodetection Methods.- 2 Immunocytochemistry.- 2.1 Rationale.- 2.2 Method.- 2.3 Controls.- 2.4 Evaluation of Method.- 3 Immunoblotting.- 3.1 Rationale.- 3.2 Method.- 3.3 Controls.- 3.4 Evaluation of Method.- 4 Immunoquantitation.- 4.1 Rationale.- 4.2 Method.- 4.3 Controls.- 4.4 Evaluation of Method.- 5 Concluding Remarks.- References.- Radioimmunoassay for a Soybean Phytoalexin.- 1 Isolation of Glyceollin I.- 2 Synthesis of Glyceollin I-BSA Conjugate.- 2.1 Preparation of p-Aminohippuric Acid Substituted BSA.- 2.2 Coupling of Glyceollin I to p-Aminohippuric Acid Substituted BSA.- 3 Generation of Antisera.- 4 Preparation of the [125I]-Glyceollin I Tracer.- 5 Radioimmunoassay for Glyceollin I.- 6 Specificity of the Radioimmunoassay.- 7 Application of the Radioimmunoassay for Glyceollin I to the Quantitation of Phytoalexins in Infected Soybean Tissue.- References.- The Measurement of Low-Molecular-Weight, Non-Immunogenic Compounds by Immunoassay.- 1 Introduction.- 1.1 The Analytical Role of the Immunoassay.- 1.2 Types of Immunoassay.- 1.3 Automation.- 2 Development of an Immunoassay.- 2.1 Synthesis of Immunogen.- 2.1.1 Methods of Conjugation.- 2.1.1.1 Carboxylic Acid-Directed Reagents.- 2.1.1.2 Alcohol-Directed Reagents.- 2.1.1.3 Phenolic-Directed Reagents.- 2.1.1.4 N-in-Aromatic-Ring-Directed Reagents.- 2.1.1.5 Amine-Direct Reagents.- 2.1.1.6 Ketone-Directed Reagents.- 2.1.1.7 Sugar-Directed Reagents.- 2.1.2 Bridge Conjugates.- 2.1.2.1 The 6-Amino-N-Hexanoic Acid Method.- 2.1.2.2 Alcohol-Directed Reagents.- 2.1.2.3 The 4-Aminohippuric Acid Method.- 2.1.3 Choice of Protein.- 2.2 Production of Antiserum.- 2.2.1 Immunisation Procedure.- 2.3 Synthesis of Labelled Tracers.- 2.3.1 Radiolabelled Tracers.- 2.3.1.1 Aromatic Ring Substitution.- 2.3.1.2 Incorporation at the Point of Conjugation.- 2.3.1.3 Reduction with Sodium [3H]-Borohydride.- 2.3.1.4 [125I]-Incorporation.- 2.3.2 Enzyme-Linked Tracers.- 2.4 ELISA Procedure.- 2.4.1 Microtitration Plates.- 2.4.2 Buffers.- 2.4.3 Initial Titre Measurement.- 2.4.4 Coating Concentration.- 2.4.5 Standard Curve.- 2.4.6 Serum Dilution.- 2.4.7 Second Antibody Dilution.- 2.4.8 Second Antibody Development.- 2.4.8.1 Alkaline Phosphatase.- 2.4.8.2 Peroxidase.- 2.4.8.3 ?-Galactosidase.- 2.4.9 Assay Validation.- 2.4.10 Future Developments.- 2.4.10.1 Fluorescent Enzyme Assays.- 2.4.10.2 The Biotin-Streptavidin System.- 2.4.10.3 The Enzymatic Radioimmunoassay.- 2.4.10.4 Chemiluminescent-Labelled Antibodies.- 2.4.11 Speed of Assay.- 3 Immmunoassays.- 3.1 Alkaloids.- 3.1.1 Nicotiana Alkaloids.- 3.1.1.1 Nicotine.- 3.1.1.2 Cotinine.- 3.1.2 Indole Alkaloids of Catharanthus roseus.- 3.1.2.1 Vincristine and Vinblastine.- 3.1.2.2 Vindoline.- 3.1.2.3 Ajmalicine and Serpentine.- 3.1.3 Tropane Alkaloids.- 3.1.4 Quinoline Alkaloids.- 3.1.4.1 Quinine.- 3.1.4.2 Quinidine.- 3.1.5 Papaver Alkaloids.- 3.1.5.1 Morphine and Codeine.- 3.1.5.2 The Benzylisoquinoline Pathway.- 3.2 Terpenoids.- 3.2.1 Litnonin.- 3.2.2 Bruceantin.- 3.2.3 Quassin.- 3.2.4 Iridoid Glucosides.- 3.3 Flavonoids.- 3.3.1 Naringin.- 3.4 Anthrones.- 3.4.1 Sennosides.- 3.5 Steroids.- 3.5.1 Digitalis Cardenolides.- 3.5.1.1 Digoxin.- 3.5.1.2 Digitoxin.- 3.5.2 Solanum Steroidal Glycoalkaloids.- 3.5.2.1 Solanidine-Derived Glycoalkaloids.- 3.5.2.2 Solasodine-Derived Glycoalkaloids.- 3.6 Miscellaneous.- 3.6.1 Physiologically Active Metabolites.- 3.6.1.1 Colchicine.- 3.6.1.2 Chloramphenicol.- 3.6.1.3 Paraquat.- 3.6.2 Contamination of Food and Feedstuffs.- 3.6.2.1 Aflatoxin B1.- 3.6.2.2 Sterigmatocystin.- 3.6.2.3 Ochratoxin A.- Appendix: Sources of Materials and Equipment for ELISA.- References.- Radioimmunoassay and Western Blot Analysis of Acyl Carrier Protein Isoforms in Plants.- 1 Introduction.- 2 Production of ACP Antibody.- 2.1 Purification of Spinach ACP Isoforms.- 2.2 Immunization Procedure.- 3 Radioimmunoassay.- 3.1 Preparation of Radiolabeled ACP.- 3.2 Radioimmunoassay Procedure.- 3.3 Applications.- 3.3.1 ACP Levels During Soybean Seed Development.- 3 3 2 Immuno-Cross-Reactivity Among ACPs.- 4 Immunological Analysis of ACP Isoforms.- 4.1 Immunorelationship of Spinach ACP Isoforms.- 4.2 Western Blot Analysis of ACP Isoforms in Plant Tissue.- 4.2.1 Preparation of Plant Tissue Extracts.- 4.2.2 Western Blot Analysis.- 5 Conclusion.- References.- Immunofluorescent Labelling of Enzymes.- 1 Introduction.- 2 General Methodology.- 2.1 Staining Sequences.- 2.2 Antibody Preparation.- 2.3 Specimen Preparation.- 2.4 Fluorescence Microscopy.- 2.5 Controls.- 3 Examples for Localization of Enzymes in Plant Tissue by Immunofluorescent Labeling.- 3.1 In Situ Immunofluorescent Labeling of Ribulosebisphosphate Carboxylase in Leaves of C3 and C4 Plants.- 3.2 In Situ Immunofluorescent Labeling of a ?-Glucosidase for Coniferin and of UDP-Glucose: Coniferyl Alcohol Glucosyltransferase.- 3.2.1 Localization of ?-Glucosidase.- 3.2.2 Localization of Glucosyltransferase.- References.- Quantitative Immunochemistry of Plant Phosphoenolpyruvate Carboxylases.- 1 Purification of PEPC - Preparation of Immune Sera.- 1.1 Extraction of PEPC.- 1.2 Purification of PEPC.- 1.3 Preparation of Antisera.- 1.4 Purification of Antisera.- 1.4.1 Fractionation by Ion-Exchange Chromatography.- 1.4.2 Affinity Chromatography Utilizing Protein A Immobilized on a Support.- 1.4.3 Affinity Chromatography Utilizing Enzyme Immobilized on an Activated Support.- 2 Quantitative Immunoprecipitation in Gels.- 3 Immunotitration Coupled to Enzyme Activity.- 3.1 Measurement of PEPC Residual Activity in the Supernatant..- 3.2 Measurement of PEPC Activity in the Immunoprecipitates.- 3.3 Applications.- 3.3.1 PEPC During Establishment of CAM and C4-Type Photosynthesis.- 3 3 2 Immunotitration of PEPCs in Relation to Plant Photosynthethic Types.- 3.3.3 Miscellaneous.- 4 Quantitative Immunoprecipitation in Extracts.- 4.1 HPLC Techniques.- 4.2 SDS-PAGE Techniques.- 5 Immunoaffinity Chromatography.- 6 Concluding Remarks.- References.- Immunochemical Methods for Higher Plant Nitrate Reductase.- 1 Introduction.- 2 Nitrate Reductase Purification.- 2.1 Seedling Growth Conditions.- 2.2 Extraction.- 2.3 Affinity Chromatography.- 2.4 Assays.- 3 Production of Nitrate Reductase Antiserum.- 3.1 Preparation of Nitrate Reductase.- 3.2 Immunization.- 3.3 Serum Collection and Isolation.- 4 Characterization of Nitrate Reductase Antiserum.- 4.1 Antiserum Titration.- 4.2 NR Antiserum Monospecificity.- 4 2 1 Immunodiffusion.- 4.2.2 Crossed Immunoelectrophoresis.- 5 Applications.- 5.1 Quantification of Nitrate Reductase Antigen.- 5.1.1 Protection of Nitrate Reductase Inactivation.- 5.1.2 Rocket Immunoelectrophoresis.- 5.1.3 Other Methods.- 5.2 Western Blot.- 5.2.1 Native Polyacrylamide Gel Electrophoresis.- 5.2.2 Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE).- 5.2.3 Electrophoretic Transfer of Proteins from Polyacrylamide Gels to Nitrocellulose Sheets.- 5 2 4 Immunodetection of Nitrate Reductase.- 5.3 Immunopurifcation of Nitrate Reductase.- 5 3 1 Immuneline Electrophoresis.- 5.3.2 Antibody Column.- 6 Summary.- References.- Immunological-Cytochenlical Localization of Cell Products in Plant Tissue Culture.- 1 Introduction.- 2 The Laticifer.- 2.1 Laticifer Morphology and Differentiation in the Asclepiadaceae.- 2.2 Methods of Preparing Anti-Latex Antibodies.- 2.3 Section Preparation.- 2.4 Laticifer Identification in Zygotic Material.- 3 The Tissue Culture System for the Asclepiadaceae.- 3.1 Culture Techniques.- 3.2 Embryoid Differentiation and Laticifer Identification.- 4 Future Uses of Immunological-Cytochemical Techniques in Tissue Culture.- 4.1 Embryogenesis and Plant Hormones.- 4.2 Embryogenesis and Embryogenic Protein.- 4.3 Cell Sorting.- References.- Measurement of Oat Globulin by Radioimmunoassay.- 1 Introduction.- 1.1 Theory of the RIA.- 1.2 Oat Globulin.- 2 Requirements for the Radioimmunoassay.- 2.1 Preparation of Pure Antigen.- 2.2 Preparation of Radiolabeled Antigen.- 2.3 Antibody Production.- 2.4 Serum and IgG Preparation.- 2.5 Preparation of Oat Seed Extracts.- 3 Designing the RIA.- 3.1 Selecting the Amount of Radiolabeled Antigen.- 3.2 Titration of the Antibody.- 3.3 Precipitation of the Antigen/Antibody Complexes.- 3.4 Conducting the RIA.- 3.5 Preparing the Standard Curve.- 3.6 Data Treatment.- 4 Summary.- References.- Immunocytocheniistry of Chloroplast Antigens.- 1 Introduction.- 2 Light Microscopy.- 2.1 Procedures for Indirect Immunofluorescence.- 3 Electron Microscopy.- 3.1 The Peroxidase Anti-Peroxidase (PAP) Methods (Post-Embedding).- 3.2 Peroxidase Anti-Peroxidase Procedure.- 3.3 Colloidal Gold.- 3.4 Post-Embedding Labeling with Colloidal Gold on Lowicryl-Embedded Tissue.- 4 Conclusions.- References.