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The aim of Plant Virology Protocols is to provide a source of infor- tion to guide the reader through the wide range of methods involved in gen- ating transgenic plants that are resistant to plant viruses. To this end, we have commissioned a wide-ranging list of chapters that will cover the methods required for: plant virus isolation; RNA extraction; cloning coat p- tein genes; introduction of the coat protein gene into the plant genome; and testing transgenic plants for resistance. The book then moves on to treatments of the mechanisms of resistance, the problems encountered with field testing, and key ethical issues surrounding transgenic technology. Although Plant Virology Protocols deals with the cloning and expression of the coat protein gene, the techniques described can be equally applied to other viral genes and nucleotide sequences, many of which have also been shown to afford protection when introduced into plants. The coat protein has, however, been the most widely applied, and as such has been selected to illustrate the techniques involved. Plant Virology Protocols has been divided into six major sections, c- taining 55 chapters in total.

Plant Virology Protocols: From Virus Isolation to Transgenic Resistance

Part I. IntroductionIntroduction to Plant VirologyIntroduction to Classical CrossprotectionHistory of Coat Protein-Mediated ProtectionPart II. Virus Isolation, RNA Extraction, and Location of Coat Protein GenesGeminivirus Isolation and DNA ExtractionCaulimovirus Isolation and DNA ExtractionReovirus Isolation and RNA ExtractionProcedures for Plant Rhabdovirus Purification, Polyribosome Isolation, and Replicase ExtractionHordeivirus Isolation and RNA ExtractionFurovirus Isolation and RNA ExtractionTobravirus Isolation and RNA ExtractionTobamo virus Isolation and RNA ExtractionPotexvirus Isolation and RNA ExtractionCarlavirus Isolation and RNA ExtractionPotyvirus Isolation and RNA ExtractionTrichovirus Isolation and RNA ExtractionIlarvirus Isolation and RNA ExtractionBromovirus Isolation and RNA ExtractionCucumovirus Isolation and RNA ExtractionNepovirus Isolation and RNA ExtractionComovirus Isolation and RNA ExtractionCarmovirus Isolation and RNA ExtractionTymovirus Isolation and Genomic RNA ExtractionTombusvirus Isolation and RNA ExtractionLuteovirus Isolation and RNA ExtractionPart III. Cloning of Coat Protein GenesRNA Analysis: Size and 3' End Group DeterminationRNA Fractionation by Density Gradient CentrifugationcDNA Library Construction for Lambda ZAP®-Based VectorsPCR Cloning of Coat Protein GenesAntibody ProductionExpression Library ScreeningIn Vitro Transcription and TranslationAnalysis of Coat Expression Cassettes in ProtoplastsDNA SequencingComputer Analysis of Amino Acid Sequences: The Case of Plant Virus Capsid ProteinsPart IV. Plant Transformation and Primary AnalysisPreparation of Coat Protein-Containing Binary Vectors for Use in Agrobacterium-MediatedTransformationPotato Transformation. Transformation of TomatoTobacco TransformationGenetic Transformation of Wheat. Production of Transgenic RiceMolecular Analysis of Transgenic RicePCR Analysis of Transgenic Tobacco PlantsSouthern Analysis of Transgenic Tobacco PlantsDetection and Quantification of Transcript RNA in Transgenic Plants Using Digoxigenin-Labeled cDNA ProbesWestern Analysis of Transgenic PlantsPart V. Evaluation of ResistanceAssaying Levels of Plant Virus by ELISADetection of Plant RNA Viruses by Nonisotopic Dot-Blot HybridizationDetection and Quantification of Plant Viruses by PCRAssaying Levels of Virus with Local Lesion HostsField Testing Resistance of Transgenic PlantsAgronomic Performance of Transgenic PlantsPart VI. Mechanisms of ResistanceMechanisms of Resistance: Expression of Coat Protein. Mechanisms of RNA-Mediated Resistance to Plant VirusesDetection of Risks Associated with Coat Protein TransgenicsPotential Benefits of the Transgenic Control of Plant Viruses in the United KingdomIndex