This laboratory guide comes at a time when several other method books have already been published in this field. Is this one different from the others? Yes and no. There was no attempt made to be comprehensive. Rather, data were brought to bear on areas where enough competence has been gathered in our laboratories and to complement recent method books (many of which cover extensively various aspects of molecular biology) in those matters which appeared to us somewhat neglected. There was a constant preoccupation and effort to provide miniaturized proce­ dures that are both simple and time-saving. Interest was devoted to standardized procedures and culture conditions, avoiding dogmas such as those giving excessive importance to sophisticated culture media with endless adjustments for local or personal considerations. The key to success is the quality of the plant material serving as a source of cells. Consequently, isolation. extraction or culture techniques can be simplified and standardized. This is symptomatic for our times as it marks the end of a period when methodological matters were frequently above the biological problems. The times of "methods above all" is basically over, despite the fact that many of us still believe that, say, tissue culture is a "science" per se. By presenting a few original techniques we believe that one seriously reduces the empiricism still prevailing in this area of research.

Inhalt

Foreword.- 1 Cellular Techniques - General Introduction.- 1.1 Isolation and Culture of Protoplasts.- 1.2 Mutagenic Treatments on Isolated Cells.- 1.3 Somatic Hybridization.- 1.3.1 Protoplast Fusion - The PEG System.- 1.3.2 Electrofusion of Protoplasts.- 1.4 Dilution Series as a Tool to Improve Medium Composition.- 1.5 Plating Efficiency Evaluation in a Peroxidase Assay.- 1.6 Production of Haploid Plants.- 1.6.1 Pollen Culture for Haploid Production in Tobacco.- 1.6.2 Haploid Induction Via Anther Culture as a Tool to Study Developmental Processes.- 1.7 Isolation of Viable Microspores and Immature Pollen Grains from Cereal Inflorescences.- 1.8 Isolation of Viable Sperm Cells from Corn (Zea mays) Pollen Grains.- 1.9 Embryo Rescue in Nicotiana plumbaginifolia.- 1.10 Use of Iodide Ions for Chemical Reduction of the Oxidative Agent H2O2 and Hypochlorites after Application as Decontaminating Agents for Plant Tissues.- 2 Transformation Techniques - General Introduction.- 2.1 Agrobacterium Transformation of Various Arabidopsis Expiants.- 2.2 Direct Gene Transfer.- 2.2.1 Direct Gene Transfer into Protoplasts - The Chemical Approach.- 2.2.2 Direct Gene Transfer - Electroporation for Transient Expression in Protoplasts.- 3 Extraction Techniques - General Introduction.- 3.1 Isolation of DNA and RNA from Arabidopsis thaliana.- 3.1.1 Large-Scale Extraction of Arabidopsis Genomic DNA.- 3.1.2 Mini-Scale DNA Extraction Procedure.- 3.1.3 Extraction of Nuclei and Nuclear DNA.- 3.1.4 RNA Extraction Procedure.- 3.1.5 Isolation of Poly (A+)-RNA.- 3.2 Total DNA Extraction - Alternative Protocols.- 3.3 Characterization of Mitochondrial-DNA from Minute Quantities of Plant Material.- 3.4 Generation of Large Amounts of cDNA by Polymerase Chain Reaction from Small Amounts of Total RNA.- 3.5 Isolation of Nuclei from Plant Tissues.- 3.6 Extraction of Amino Acids from Plant Samples and Their Analysis Using Ion-Exchange Chromatography.- 3.7 Electroelution of Proteins from Plant Tissues.- 3.8 Extraction, Purification and Analysis of Endogenous Indoleacetic Acid and Abscisic Acid.- 4 Aspects of Structural and Functional Analysis of Genomes and Genes - General Introduction.- 4.1 Southern Blot Analysis of Transgenic Nicotiana sp.- 4.2 Northern Blot Analysis of ADH (Alcohol Dehydrogenase) Mutants in Arabidopsis.- 4.3 Western Blot Detection of Proteins Synthesized Transiently in Transfected Plant Protoplasts.- 4.4 Cloning Nuclear Single Copy Sequences for RFLP Analysis.- 4.5 Run-on Transcription in Isolated Plant Nuclei.- 4.6 Preparation of Nuclear Extracts, Gel-Retardation Assay and DNAase I Footprinting.- 4.6.1 Preparation of Nuclear Extracts from Plant Nuclei.- 4.6.2 Gel Retardation Assay Using Large DNA Probes.- 4.6.3 DNAse I Analysis of Retarded Complexes.- 4.6.4 Gel Retardation Using Oligonucleotide Probes.- 4.7 Pulsed-Field Gel Electrophoresis of Plant DNA.- 4.8 Assessing Methylation of Inserted DNA by Restriction with Isoschizomeric Enzymes and Inducing Demethylation with 5-Azacytidine.- 5 Cytological Techniques - General Introduction.- 5.1 Karyotyping with Protoplast Procedures.- 5.2 In situ Hybridization.- 5.2.1 Gene targeting in Plant Metaphase Chromosomes by In situ Hybridization with Tritiated Probe DNA.- 5.2.2 Protocols for In situ Hybridization - The Biotinylation Technique.- 5.3 How Cytometry of Nuclei for Ploidy and Cell Cycle Analysis.- 5.4 Control of Cell Cycle Progression.- 5.5 Induction and Isolation of Micronuclei and Microprotoplasts.- 6 Appendices.- 6.1 Culture Media and Basic Stock Solution.- 6.4 Extraction and Purification of IAA and ABA.- 7 Subject Index.