Chromosome and Genomic Engineering in Plants: Methods and Protocols (Methods in Molecular Biology, 1469)

✍ Scribed by Minoru Murata (editor)

• Publisher: Humana
• Year: 2016
• Tongue: English
• Leaves: 222
• Category: Library

✦ Synopsis

This volume assembles protocols for chromosome engineering and genome editing in two recently developed approaches for manipulating chromosomal and genomic DNA in plants. The first approach is a “plant chromosome vector” system, which allows the introduction of desired genes or DNA into target sites on the chromosome vector, particularly by sequence-specific recombination. The second approach is “genome-editing,” which makes it possible to introduce mutations into any of the genes of DNA that we wish to change. In addition, this book also covers other related techniques used to accelerate progress in plant chromosome and genome engineering. Written in the highly successful Methods in Molecular Biology series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls.

Cutting-edge and thorough, Chromosome and Genomic Engineering in Plants: Methods and Protocols provides a comprehensive source of protocols and other necessary information to anyone interested in this field of study.

✦ Table of Contents

Preface
Contents
Contributors
Chapter 1: Production of Engineered Minichromosome Vectors via the Introduction of Telomere Sequences
1 Introduction
2 Materials
2.1 Materials for In-Gel Ligation into Agrobacterium Competent Vector
2.2 Equipment for In-Gel Ligation into Agrobacterium Competent Vector
2.3 Recipes for In-Gel Ligation into Agrobacterium Competent Vector
2.4 Materials for Preparation of Telomere for Particle Bombardment
2.5 Equipment for Preparation of Telomere for Particle Bombardment
3 Methods
3.1 Insertion of Telomere Through In-Gel Ligation
3.1.1 Screening Potential Colonies for Telomere Size
3.2 Production of Telomere Fragments via Polymerase Chain Reaction
3.3 Transgene Delivery into Plants with Telomere Arrays
4 Notes
References
Chapter 2: Method for Biolistic Site-Specific Integration in Plants Catalyzed by Bxb1 Integrase
1 Introduction
2 Materials
2.1 Rice and Tobacco Target Lines
2.2 DNA Constructs
2.3 Stock Solutions
2.4 Medium Composition
2.4.1 Rice Media Composition
2.4.2 Tobacco Medium Composition
2.5 Materials for Bombardment
2.5.1 Biolistic Gun
2.5.2 Materials for Gold Particles Preparation (See Note 1)
3 Methods
3.1 Explants Preparation
3.1.1 Rice Callus Initiation and Proliferation
3.1.2 Tobacco Seed Sterilization and Plant Maintenance
3.2 Bxb1-Mediated Biolistic Transformation
3.2.1 DNA Preparation
3.2.2 Gold Particle Preparation
Wash Gold and Make Aliquots
Coating the Gold with DNA
3.2.3 Performing Bombardment (See Note 9)
3.3 Resting and Selection
3.4 Regeneration and Rooting
3.5 GUS Staining and GFP Observation
3.5.1 GUS Staining
3.5.2 GFP Expression
3.6 PCR Analysis
3.7 Southern Blot Analysis
4 Notes
References
Chapter 3: Protocol for In Vitro Stacked Molecules Compatible with In Vivo Recombinase-Mediated Gene Stacking
1 Introduction
2 Materials
2.1 Strains
2.2 Antibiotics and Mediums
2.3 Solutions
2.4 Enzymes, Recombinant DNA
3 Methods
3.1 Preparation of Recombinase E. coli Cell Extracts
3.2 Recombinant DNA
3.3 Example of In Vitro Gene Stacking of Five Genes
3.3.1 First Round Stacking In Vitro
3.3.2 Following Three Rounds of In Vitro Gene Stacking
3.3.3 Gene Stack Integrated into Agrobacterium Gene Transfer Vector
4 Notes
References
Chapter 4: Generation and Analysis of Transposon Ac/Ds-Induced Chromosomal Rearrangements in Rice Plants
1 Introduction
2 Materials
2.1 Tissue Culture for Regeneration of Plants
2.2 Genomic DNA Extraction
2.3 Primers for PCR Reactions
2.4 Southern Blot Analysis
3 Methods
3.1 Tissue Culture for Transgenic Plants
3.2 Preparation to Grow Rice Plants in Paddy Field
3.3 Genomic DNA Extraction
3.4 PCR Reactions
3.5 Southern Blot Analysis
4 Notes
References
Chapter 5: One-Step Generation of Chromosomal Rearrangements in Rice
1 Introduction
2 Materials
2.1 Plant Materials
2.2 Agrobacterium Strain and the T-DNA Vector
2.2.1 Agrobacterium Strain
2.2.2 Binary Vector
2.2.3 Stock Solutions
2.2.4 Media for Callus Formation
2.2.5 Agrobacterium Culture
2.2.6 Regeneration Media
2.3 PCR Reagents and a Restriction Enzyme
2.4 Cytological Reagents
3 Methods
3.1 Agrobacterium-
3.1.1 Callus Induction
3.1.2 Agrobacterium Infection
3.1.3 Regeneration of Transformants
3.2 Analysis of T1 Transgenic Plants
3.2.1 PCR Analysis of Marker Genes
3.2.2 T-DNA Mapping
3.3 Analysis of T2 Transgenic Plants
3.3.1 PCR Analysis
3.3.2 Cytological Analysis
4 Notes
References
Chapter 6: Genome Elimination by Tailswap CenH3: In Vivo Haploid Production in Arabidopsis thaliana
1 Introduction
2 Materials
3 Methods
3.1 Growing of A. thaliana Plants in Soil Medium [20]
3.2 Plant Growth Conditions
3.3 Growing, Identification, and Maintenance of Haploid-­Inducing GFP-­tailswap Plants
3.4 Crossing GFP-tailswap as Female Parent to Produce Paternal Haploids
3.5 Preselection of Haploid Seeds/Seedlings
3.5.1 Surface Sterilization of Seeds
3.5.2 Root-Based Prescreening for Haploid Seedlings
3.6 Identification of Haploid Plants
3.6.1 DNA Isolation for Genotyping of Haploids
3.6.2 PCR Genotyping of GFP-­tailswap Plants
3.7 Converting Sterile Haploids to Fertile Double Haploids (DH)
3.8 Colchicine Treatment of Haploid Seedlings to Convert Them to Doubled Haploids
3.9 Crossing GFP-tailswap as a Pollen Parent to Produce Maternal Haploids
4 Notes
References
Chapter 7: Gametocidal System for Dissecting Wheat Chromosomes
1 Introduction
2 Materials
3 Methods
3.1 Plant Cultivation
3.2 Production of AABBDD + 2C by Crossing
3.3 Production of a Population Including Chromosome Breakage in Hemizygous State
4 Notes
References
Chapter 8: CRISPR/Cas-Mediated Site-Specific Mutagenesis in Arabidopsis thaliana Using Cas9 Nucleases and Paired Nickases
1 Introduction
2 Materials
2.1 Plant Material
2.2 Vectors
2.3 Reagents
3 Methods
3.1 Cloning of sgRNAs
3.2 Transfer of sgRNA to Binary Vector by Conventional Cloning
3.3 Transfer of sgRNA by Gateway Cloning
3.4 Cloning of Constructs with Paired Nickases
3.5 Mutant Screening in A. thaliana
4 Notes
References
Chapter 9: Targeted Mutagenesis in Rice Using TALENs and the CRISPR/Cas9 System
1 Introduction
2 Materials
2.1 Vector Construction
2.2 Agrobacterium-
2.2.1 Plant and Agrobacterium Materials
2.2.2 Media for Agrobacterium-­Mediated Transformation
2.3 Selection and Regeneration of TALENs or Cas9- and gRNA-Transformed Calli
2.3.1 Media for Selection of Transgenic Calli
2.4 Detection of Mutations
2.4.1 DNA Extraction
2.4.2 Polymerase Chain Reaction
2.4.3 Cleaved Amplified Polymeric Sequences (CAPS) Analysis
2.4.4 Analysis of Mutation Patterns in Calli by Sequencing
2.4.5 Guide-It Resolvase Assay
2.4.6 Heteroduplex Mobility Shift Assay (HMA)
3 Methods
3.1 Selecting Target Sequence
3.1.1 TALENs
3.1.2 CRISPR/Cas9
3.2 Vector Construction
3.2.1 TALENs
3.2.2 CRISPR/Cas9
3.3 Agrobacterium-­Mediated Transformation Using Primary Callus of Rice
3.4 Detection of Mutation in Rice Calli (See Note 3)
3.4.1 DNA Isolation
3.4.2 Amplification of the Targeted Sequence
3.4.3 CAPS Assay
3.4.4 Estimation of the Precise Mutation Frequency
3.4.5 Analysis of Mutation Patterns in Calli by Sequencing
3.5 Guide-It Resolvase Assay
3.6 Heteroduplex Mobility Shift Assay (HMA)
4 Notes
References
Chapter 10: Seamless Genome Editing in Rice via Gene Targeting and Precise Marker Elimination
1 Introduction
2 Materials
2.1 Agrobacterium-
2.1.1 Plant and Agrobacterium Materials
2.1.2 GT Vector
2.1.3 piggyBac Transposase Expression Vector
2.1.4 Media for Agrobacterium-­Mediated Transformation
2.1.5 Stock Solutions
2.2 Identification of GT Calli
2.2.1 DNA Extraction
2.2.2 Polymerase Chain Reaction
2.2.3 Purification of PCR Products for Sequencing Analysis
2.3 Selection of Marker-­Free Plants
2.3.1 Southern Blot Analysis
2.3.2 Polymerase Chain Reaction
3 Methods
3.1 Agrobacterium-­Mediated Transformation of GT Vector into Rice Calli
3.2 Identification of GT Calli Using PCR Analysis
3.3 Marker Excision via piggyBac Transposition
3.4 Selection of Marker-­Free Plants
4 Notes
References
Chapter 11: Development of Genome Engineering Tools from Plant-­Specific PPR Proteins Using Animal Cultured Cells
1 Introduction
2 Materials
2.1 Instruments
2.2 Cell Culture
2.3 Transient Transfection
2.4 Luciferase Assay
3 Methods
3.1 Vector Construction
3.2 Cell Culture
3.2.1 Starting Cell Culture from the Frozen Cell Stock
3.2.2 Subculturing for Cell Maintenance
3.2.3 Freezing Cell Stocks for Future Use
3.3 Transient Transfection
3.4 Luciferase Assay
3.5 Data Analysis
4 Notes
References
Chapter 12: Chromosomal Allocation of DNA Sequences in Wheat Using Flow-Sorted Chromosomes
1 Introduction
2 Materials
2.1 Plant Material
2.2 Laboratory Reagents and Solutions
2.2.1 Laboratory Reagents and Solutions for Chromosome Sample Preparation and Flow Cytometry
2.2.2 Laboratory Reagents and Solutions for FISH
2.2.3 Reagents and Solutions for FISHIS
2.2.4 Laboratory Reagents and Solutions for PCR
2.2.5 Laboratory Reagents and Solutions for Single Chromosome Amplification
2.3 Consumables, Laboratory Equipment, and Instruments
3 Methods
3.1 Sample Preparation
3.2 Chromosome Labeling Using FISH in Suspension (FISHIS)
3.3 Chromosome Flow Sorting
3.4 FISH Analysis with Sorted Chromosomes
3.5 PCR Analysis with Sorted Chromosomes
3.6 Single Chromosome Amplification and PCR
3.7 Chromosomal Allocation of DNA Sequences
4 Notes
References
Chapter 13: Image Analysis of DNA Fiber and Nucleus in Plants
1 Introduction
2 Materials
2.1 Software and Equipment
2.2 Reagents
3 Methods
3.1 Preparation of Nuclei
3.2 CHIAS: Straight
3.3 CHIAS-
4 Notes
References
Chapter 14: Detection of Transgenes on DNA Fibers
1 Introduction
2 Materials
2.1 Stock Buffers
2.2 Reagent and Equipment for Preparation of DNA Fibers
2.3 Reagent for Probe Labeling
2.4 Reagent and Equipment for Hybridization
2.5 Reagent for Detection
3 Methods
3.1 Preparation of DNA Fibers from A. thaliana Leaves (See Note 4)
3.1.1 Preparation of Protoplasts
3.1.2 Preparation of DNA Fibers
3.2 Probe Labeling
3.3 Hybridization
3.4 Detection
4 Notes
References
Chapter 15: Three-Dimensional, Live-Cell Imaging of Chromatin Dynamics in Plant Nuclei Using Chromatin Tagging Systems
1 Introduction
2 Materials
2.1 The lacO/LacI-EGFP System in A. thaliana
2.2 Seeding for Live-­Cell Imaging in Roots
2.3 Seedling for Live-Cell Imaging in Cotyledons
2.4 Live-Cell Imaging of Chromatin Dynamics
3 Methods
3.1 Seeding for Imaging of Chromatin Dynamics in the Nuclei of Roots
3.2 Time-Lapse Imaging of Chromatin Dynamics in the Nuclei of Roots
3.3 Seeding for Imaging of Chromatin Dynamics in the Nuclei of Guard Cells of a Cotyledon
3.4 Preparation of Samples for Time-Lapse Imaging of Chromatin Dynamics in the Nuclei of Guard Cells of a Cotyledon
3.5 Time-Lapse Imaging of Chromatin Dynamics in the Nuclei of Guard Cells of a Cotyledon
4 Notes
References
Chapter 16: Chromatin Immunoprecipitation for Detecting Epigenetic Marks on Plant Nucleosomes
1 Introduction
2 Materials
2.1 Fixation
2.2 Isolation of Nucleosome
2.3 Chromatin Immunoprecipitation
3 Methods
3.1 Fixation
3.2 Isolation of Nucleosome
3.3 Immuno-precipitation of Nucleosomes
4 Notes
References
Chapter 17: Mapping of T-DNA and Ac/Ds by TAIL-PCR to Analyze Chromosomal Rearrangements
1 Introduction
2 Materials
2.1 DNA Extraction from Plants
2.2 Thermal Asymmetric Interlaced PCR (TAIL-PCR)
3 Methods
3.1 DNA Extraction from Plants
3.2 Thermal Asymmetric Interlaced PCR (TAIL-PCR)
4 Notes
References
Index