Five years ago, the first edition of the Plant Molecular Biology Manual appeared. At that time, the editors felt that the field of plant molecular biology had matured to a point that the publication of a series of protocols in plant molecular biology was warranted. During the past five years, the field of plant molecular biology has expanded rapidly. This expansion is, among other things, reflected by the presence of several journals in the plant sciences, as well as by the increasing amount of plant sciences articles that are published in the more general journals. In 1991 approximately 3000 people attended the Third International Congress of Plant Molecular Biology in Tucson, Arizona, where more than 2000 posters were presented. It is also remarkable to see that nowadays botanical and physiological meetings pay a considerable amount of attention to plant molecular biology. Since the first edition of this manual appeared, we have published, yearly, a series of supplements to the original volume. These supplements covered new subjects and described new methods that had been developed. With time, however, the editors realized that the original manual plus supplements had become cumbersome to use, and we decided to publish a reorganized version of the manual.
Section A : In vitro methods of gene transfer to plant cells.- 1. PEG-mediated direct gene transfer and electroporation.- 2. Gene transfer to plants via particle bombardment.- Section B: Agrobacterium-mediated gene transfer to plant cells.- 1. Agrobacterium-mediated gene transfer to plant cells: cointegrate and binary vector systems.- 2. Specialized vectors for gene tagging and expression studies.- 3. Agrobacterium molecular genetics.- 4. Genetic manipulation of Agrobacterium tumefaciens strains to improve transformation of recalcitrant plant species.- 5. Transient expression assays using GUS constructs and fluorometric detection for analysis of T-DNA transfer.- 6. Agrobacterium inoculation techniques for plant tissues.- Section C: Selectable and screenable markers for plant transformation.- 1. Antibiotic-resistance markers for plant transformation.- 2. Reporter genes for plants.- 3. Opines as screenable markers for plant transformation.- Section D: Nucleic acid extraction from plant tissue.- 1. Extraction of total cellular DNA from plants, algae and fungi.- 2. Isolation and characterization of nuclear scaffolds.- 3. Isolation of plant mitochondria and mitochondrial nucleic acids.- 4. Isolation of chloroplasts and chloroplast DNA.- 5. Isolation of total, poly (A) and polysomal RNA from plant tissues.- Section E: Transcription and translation systems.- 1. Assay for gene expression using run-on transcription in isolated nuclei.- 2. Preparation of an in vitro transcription system of plant origin, with methods and templates for assessing its fidelity.- Section F: Blotting and gene detection systems.- 1. Southern, Northern and Western blot analysis.- 2. Screening of cDNA expression libraries with synthetic oligonucleotides for DNA-binding proteins.- 3. Non-radioactive nucleic acid detection systems.- Section G: In situ hybridization and immunodetection.- 1. RNA in situ hybridization in plants.- 2. In situ hybridization to plant metaphase chromosomes using digoxigenin labeled nucleic acid sequences.- Section H: Cloning and detection of DNA sequences from large DNA molecules.- 1. Methods for generating plant genomic libraries Marjory.- 2. Construction of plant yeast artificial chromosome libraries.- 3. Preparation of high molecular weight plant DNA and analysis by pulsed field gel electrophoresis.- 4. Random amplified polymorphic DNA (RAPD) markers.- Section I: Protein-nucleic acid interaction analyses.- 1. Gel mobility shift assay.- 2. Optimization of DNase I footprinting experiments.- 3. Analyses of plant chromatin and in vivo protein-DNA interactions.- 4. Expression and characterization of recombinant plant trans-acting factors.- Section J: Subcellular targeting of proteins.- 1. In vitro import of proteins into chloroplasts.- 2. In vitro targeting of proteins to mitochondria.- 3. Targeting of proteins to the vacuole.- 4. Visualizing protein import into the plant cell nucleus.- Section K: Gene tagging using transposons.- 1. Gene tagging by endogenous transposons.- 2. Heterologous transposon tagging as a tool for the isolation of plant genes.