Plant Gene Transfer and Expression Protocols

1. Front Matter

   Pages i-xiii

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2. Agrobacterium-Mediated Transformation

   1. Tools for Expressing Foreign Genes in Plants

      • François Guerineau

      Pages 1-32

   2. Introduction of Cloning Plasmids into Agrobacterium tumefaciens

      • Charles H. Shaw

      Pages 33-37

   3. Leaf Disk Transformation Using Agrobacterium tumefaciens-Expression of Heterologous Genes in Tobacco

      • Patrick Gallois, Paulo Marinho

      Pages 39-48

   4. Agobacterium rhizogenes as a Vector for Transforming Higher Plants

      • Jens Stougaard

      Pages 49-61

   5. Agrobacterium-Mediated Transformation of Arabidopsis thaliana

      • Jennifer F. Topping, Wenbin Wei, Michael C. Clarke, Paul Muskett, Keith Lindsey

      Pages 63-76

   6. Agrobacterium -Mediated Transfer of Geminiviruses to Plant Tissues

      • Margaret I. Boulton

      Pages 77-93

3. Direct Gene Transfer

   1. Stable Transformation of Barley via Direct DNA Uptake

      • Paul A. Lazzeri

      Pages 95-106

   2. Gene Transfer into Plant Protoplasts by Electroporation

      • Heddwyn Jones

      Pages 107-112

   3. Transformation of Cereals by Microprojectile Bombardment of Immature Inflorescence and Scutellum Tissues

      • Pilar Barcelo, Paul A. Lazzeri

      Pages 113-123

4. Use of Reporter Genes

   1. The β-Glucuronidase (gus) Reporter Gene System

      • Gillian A. Hull, Martine Devic

      Pages 125-141

   2. Chloramphenicol Acetyl Transferase Assay

      • Michael R. Davey, Nigel W. Blackhall, J. Brian Power

      Pages 143-148

   3. NPTII Assays for Measuring Gene Expression and Enzyme Activity in Transgenic Plants

      • Ian S. Curtis, J. Brian Power, Michael R. Davey

      Pages 149-159

5. Study of Gene Organization by Southern Blotting and Inverse PCR

   1. Gene Characterization by Southern Analysis

      • Paolo A. Sabelli, Peter R. Shewry

      Pages 161-180

   2. Isolation and Characterization of Plant Genomic DNA Sequences via (Inverse) PCR Amplification

      • Remko Offringa, Frederique van der Lee

      Pages 181-195

6. RNA Techniques for Studying Gene Expression

   1. Isolation of Whole Cell (Total) RNA

      • Rod J. Scott

      Pages 197-202

   2. Poly(A)+RNA Isolation

      • Rod J. Scott

      Pages 203-206

   3. In Vitro Translation

      • Rod J. Scott

      Pages 207-211

   4. Northern Analysis and Nucleic Acid Probes

      • Paolo A. Sabelli, Peter R. Shewry

      Pages 213-228

   5. Nuclear “Run-On” Transcription Assays

      • John Gatehouse, Andrew J. Thompson

      Pages 229-238

The development of recombinant DNA technology and methods for transferring recombinant genes into plants has brought about significant advances in plant science. First, it has allowed investigation, using reporter genes, into the transcriptional regulation of plant genes—a key to the under­ standing of the biochemical basis of growth and development in plants. Second, gene transfer technology has facilitated the molecular cloning, by tagging genomic sequences, of important genes (e. g. , homeotic genes) whose gene products control the normal pattern of growth and differentia­ tion of plants. Third, overproducing foreign or endogenous proteins in plants can often lead to a better understanding of biochemical and physiological processes. Fourth, gene transfer technology has allowed the improvement of plant agricultural productivity. For example, plants have been engineered with improved viral resistance or the ability to withstand herbicide attack, therefore allowing a more effective use of herbicides to kill weeds. Fifth, there have been recent successes that demonstrate the potential use of plants as biotechnological chemical factories. For example, it is possible to use plants in the production of human antibodies and antigens of medical importance. It has been demonstrated recently that plants can be engineered to produce modified oils and even plastics! This paves the way to redirect agriculture from the production of surplus foods to the production of bio­ technological products of industrial importance.

The chapters were written by outstanding scientists and [the book] can be recommended to any laboratory working with plants, providing tips and alternatives in case of problems. -Journal of Plant Physiology

• University of Hertfordshire, Hatfield, UK

  Heddwyn Jones

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