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During the past ten years, great advances have been made in the area of plant molecular biology. Such formerly esoteric techniques as gene transfer and plant regeneration are now routinely performed, making the dissection of regulatory elements of genes a common practice in many laboratories. Along with this new technology has come an almost bewildering array of rapidly changing techniques, often making it difficult for the novice to select and perform the technique most appropriate for answering a given biological question. In 1986, some of us felt that many of these techniques had become routine enough to warrant the publication of a laboratory manual. The manual is designed both for advanced college level laboratory courses and as a 'bench guide' for use in the scientific laboratory. Recognizing the rapidly changing nature of plant molecular biology technology, the editors have designed a laboratory manual that is both easy to use in the laboratory and which will be updated as the techniques change and new technologies are devised. Additional chapters that can replace or be added to this first edition will be published periodically. The editors recognize that many of the techniques described in this manual depend upon specialized plant genetic material, microbial strains, or recombinant plasmids. Those people desiring such material should contact the relevant authors directly. A list of the various contributors to this manual, including their addresses, is included.
List of contents
Section A: Introduction of DNA into Plant Cells.- 1. Direct DNA transfer to protoplasts with and without electroporation.- 2. Use of cointegrating Ti plasmid vectors.- 3. Binary vectors.- 4. Agrobacterium molecular genetics.- 5. Leaf disc transformation.- 6. Extraction of DNA from plant tissues.- 7. Procedures for contructing ds-cDNA clone banks.- 8. Procedures for constructing genomic clone banks.- 9. Selectable and screenable markers.- 10. Transformation of plant pathogenic fungi.- Section B: Expression of Genes in Plants.- 1. Use of reporter genes to study gene expression in plant cells.- 2. Assays for studying chromatin structure.- 3. Assays for studying DNA methylation.- 4. Analysis of gene expression in transgenic plants.- 5. Subcellular targeting of proteins in vivo and in vitro.- 6. Isolation of total and polysomal RNA from plant tissues.- 7. Translation in Xenopus oocytes of mRNAs transcribed in vitro.- 8. Use of the firefly luciferase gene as a reporter of gene expression in plants.- 9. In situ RNA hybridization in plant tissues.- 10. Light-regulated promoter sequences in the multigene Cab and rbcS gene families.- 11. In vitro DNA footprinting.- 12. In vivo detection of protein-DNA interactions.- Section C: Fate of Introduced Genes.- 1. Stability of introduced genes and stability in expression.- 2. Restriction fragment length polymorphism.
