Plant Gene Transfer and Expression Protocols

About this book

One stop reference for both fundamental and novel techniques.

Contents

Part I: Agrobacterium-Mediated Transformation. Tools for Expressing Foreign Genes in Plants. Introduction of Cloning Plasmids into Agrobacterium tumefaciens. Leaf Disk Transformation Using Agrobacterium tumefaciens-Expression of Heterologous Genes in Tobacco. Agrobacterium rhizogenes as a Vector for Transforming Higher Plants: Application in Lotus corniculatus Transformation. Agrobacterium-Mediated Transformation of Arabidopsis thaliana: Application in T- DNA Tagging. Agrobacterium-Mediated Transfer of Geminiviruses to Plant Tissues. Part II: Direct Gene Transfer. Stable Transformation of Barley via Direct DNA Uptake: Electroporation- and PEG- Mediated Protoplast Transformation. Gene Transfer into Plant Protoplasts by Electroporation. Transformation of Cereals by Microprojectile Bombardment of Immature Inflorescence and Scutellum Tissues. Part III: Use of Reporter Genes. The b-Glucoronidase (gus) Reporter Gene System: Gene Fusions Spectrophotometric Fluorometric and Histochemical Detection. Chloramphenicol Acetyl Transferase Assay. NPTII Assays for Measuring Gene Expression and Enzyme Activity in Transgenic Plants. Part IV: Study of Gene Organization by Southern Blotting and Inverse PCR. Gene Characterization by Southern Analysis. Isolation and Characterization of Plant Genomic DNA Sequences via (Inverse) PCR Amplification. Part V: RNA Techniques for Studying Gene Expression. Isolation of Whole Cell (Total) RNA. Poly(A)+ RNA Isolation. In Vitro Translation. Northern Analysis and Nucleic Acid Probes. Nuclear Run-On Transcription Assays. RNase A/T1 Protection Assay. Primer Extension Assay. Applications of RT-PCR. In Vitro Transcription of Class II Promoters in Higher Plants. Analysis of Plant Gene Expression by Reverse Transcription-PCR. In Situ Hybridization to Plant Tissue Sections. Xenopus Oocytes as a Heterologous Expression System. Heterologous Expression in Yeast. Part VI: Techniques for Studying Chloroplast Gene Expression. The Isolation of Intact Chloroplasts. In Vitro Protein Import by Isolated Chloroplasts. Targeting of Foreign Proteins to the Chloroplast. Part VII: Techniques for Studying Mitochondrial Gene Expression. Isolation of Mitochondria. Mitochondrial Nucleic Acid Purification and Analysis. In organello Protein Synthesis. Part VIII: Immunological Detection of Proteins. Separation of Plant Proteins by Electrophoresis. Western Blotting Analysis. ELISA Detection of Foreign Proteins. Immunocytochemical Localization of Proteins. Index.

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