Plant Virology Protocols: From Virus Isolation to Transgenic Resistance / Edition 1

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A comprehensive collection of state-of-the-art techniques for generating transgenic plants that are resistant to plant viruses via the cloning and expression of the coat protein gene. Its unfailingly reproducible methods, perfected by hands-on masters, cover the entire process from virus isolation, RNA extraction, and cloning coat protein genes, to the introduction of the coat protein gene into the plant genome and the testing of transgenic plants for resistance. Methods for testing for transformation by PCR and Southern blotting, the detection of RNA transcripts by Northern blotting, and the production of protein by Western analysis are provided, as are methods for challenging the transgenic plants produced and for detecting and measuring the levels of virus.

"...state-of-the-art protocols for cloning coat protein genes, an up-to-date review of plant resistance & current opinions about its mechanisms, & methods for generating & identifying cDNA clones representing the coat protein gene."

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Editorial Reviews

A theoretical and hands-on guide to generating transgenic plants that are resistant to viruses via the cloning and expression of the coat protein gene. One hundred contributors cover plant virus purification protocols, protocols for cloning coat protein genes, methods for plant transformation and primary analysis, plant resistance and its mechanisms, techniques for analyzing viral genes and nucleotide sequences, methods for generating and identifying cDNA clones representing the coat protein gene, and RNA extraction. Annotation c. by Book News, Inc., Portland, Or.
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Product Details

  • ISBN-13: 9780896033856
  • Publisher: Springer-Verlag New York, LLC
  • Publication date: 2/24/1998
  • Series: Methods in Molecular Biology Series, #81
  • Edition description: 1998
  • Edition number: 1
  • Pages: 588
  • Product dimensions: 9.21 (w) x 6.14 (h) x 1.25 (d)

Table of Contents

Part I. Introduction

Introduction to Plant Virology

Introduction to Classical Crossprotection

History of Coat Protein-Mediated Protection

Part II. Virus Isolation, RNA Extraction, and Location of Coat Protein Genes

Geminivirus Isolation and DNA Extraction

Caulimovirus Isolation and DNA Extraction

Reovirus Isolation and RNA Extraction

Procedures for Plant Rhabdovirus Purification, Polyribosome Isolation, and Replicase Extraction

Hordeivirus Isolation and RNA Extraction

Furovirus Isolation and RNA Extraction

Tobravirus Isolation and RNA Extraction

Tobamo virus Isolation and RNA Extraction

Potexvirus Isolation and RNA Extraction

Carlavirus Isolation and RNA Extraction

Potyvirus Isolation and RNA Extraction

Trichovirus Isolation and RNA Extraction

Ilarvirus Isolation and RNA Extraction

Bromovirus Isolation and RNA Extraction

Cucumovirus Isolation and RNA Extraction

Nepovirus Isolation and RNA Extraction

Comovirus Isolation and RNA Extraction

Carmovirus Isolation and RNA Extraction

Tymovirus Isolation and Genomic RNA Extraction

Tombusvirus Isolation and RNA Extraction

Luteovirus Isolation and RNA Extraction

Part III. Cloning of Coat Protein Genes

RNA Analysis: Size and 3' End Group Determination

RNA Fractionation by Density Gradient Centrifugation

cDNA Library Construction for Lambda ZAPĀ®-Based Vectors

PCR Cloning of Coat Protein Genes

Antibody Production

Expression Library Screening

In Vitro Transcription and Translation

Analysis of Coat Expression Cassettes in Protoplasts

DNA Sequencing

Computer Analysis of Amino Acid Sequences: The Case of Plant Virus Capsid Proteins

Part IV. Plant Transformation and Primary Analysis

Preparation of Coat Protein-Containing Binary Vectors for Use in Agrobacterium-MediatedTransformation

Potato Transformation. Transformation of Tomato

Tobacco Transformation

Genetic Transformation of Wheat. Production of Transgenic Rice

Molecular Analysis of Transgenic Rice

PCR Analysis of Transgenic Tobacco Plants

Southern Analysis of Transgenic Tobacco Plants

Detection and Quantification of Transcript RNA in Transgenic Plants Using Digoxigenin-Labeled cDNA Probes

Western Analysis of Transgenic Plants

Part V. Evaluation of Resistance

Assaying Levels of Plant Virus by ELISA

Detection of Plant RNA Viruses by Nonisotopic Dot-Blot Hybridization

Detection and Quantification of Plant Viruses by PCR

Assaying Levels of Virus with Local Lesion Hosts

Field Testing Resistance of Transgenic Plants

Agronomic Performance of Transgenic Plants

Part VI. Mechanisms of Resistance

Mechanisms of Resistance: Expression of Coat Protein. Mechanisms of RNA-Mediated Resistance to Plant Viruses

Detection of Risks Associated with Coat Protein Transgenics

Potential Benefits of the Transgenic Control of Plant Viruses in the United Kingdom


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