Ebook: A Laboratory Guide for Cellular and Molecular Plant Biology

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.