CHLOROPLAST BIOTECHNOLOGY : methods and protocols.

Dedication
Preface
Contents
Contributors
Part I: Background
	Chapter 1: Plastid Genomes of Flowering Plants: Essential Principles
		1 Introduction
		2 Characteristics of the Angiosperm Plastome
		3 Gene Content
		4 The Large Inverted Repeat
			4.1 Genes Are Duplicated in the Inverted Repeat
			4.2 The IR and Recombination
			4.3 Speculation Regarding the Origin of the IR
			4.4 IR Persistence and Loss
			4.5 The IR and Plastome Stability
		5 Synteny Across Plastomes
			5.1 Genomic Rearrangements
		6 Plastome Gene Loss
			6.1 Defining Gene Losses
			6.2 Old and New Gene Loss
			6.3 Gene Retention
		7 Plastid Genes in the Nucleus and Other Promiscuous Sequences
			7.1 Transcriptional Activation in the Nucleus
			7.2 Promiscuous ptDNA and Experimental Artifacts
			7.3 ``Foreign´´ DNA in Wild-Type Plastomes
		8 Recombination and the Plastome
			8.1 DNA Replication, Repair, and Recombination
				8.1.1 RecA Protein
			8.2 DNA Replication, Physical Form and Copy Number
			8.3 Maintaining Plastome Integrity
				8.3.1 Plastid Targeted Whirlies
				8.3.2 Double Strand Break Repair in Plastids
		9 Plastome Evolution: Phylogenetic and Biotechnological Considerations
			9.1 Targeting Plastome Insertion
			9.2 Phylogenetic Utility of ``Noncoding´´ Sequences
				9.2.1 Small Inversions in Regulatory Elements
				9.2.2 Nucleotide Substitution in Noncoding Regions
		10 Plastome Inheritance
			10.1 Biparental Inheritance
			10.2 Coevolved Complexes and Plastome-Genome Incompatibility
		11 Conclusion
		References
	Chapter 2: Plastid Gene Transcription: An Update on Promoters and RNA Polymerases
		1 Introduction
		2 NEP: Nuclear-Encoded Polymerase
		3 PEP: Plastid-Encoded Polymerase
			3.1 Sigma Factors
				3.1.1 Arabidopsis thaliana
				3.1.2 A Look to Sigma Factors Beyond Arabidopsis
			3.2 PTK: Pastid Transcription Kinase
		4 Plastid Promoters
			4.1 NEP Promoters
			4.2 PEP Promoters
				4.2.1 A Closer View of the rrn Promoters
				4.2.2 BLRP, the Blue Light Responsive Promoter of  psbD
		5 Summary and Perspectives
		References
	Chapter 3: Engineering Chloroplasts for High-Level Constitutive or Inducible Transgene Expression
		1 Introduction
		2 Choice of Expression Elements
			2.1 Promoter Strength
			2.2 Choice of the 5′  UTR
			2.3 Choice of the 3′  UTR
		3 Coding Region
			3.1 Codon Usage
			3.2 Downstream Boxes
		4 Choice of the Integration Site in the Genome
		5 Protein Stability and Folding
		6 Protein Localization
		7 Multigene Expression from Operons
		8 Other Considerations
		9 Summary and Guidelines for Maximizing Expression Levels
		10 Methods for Inducible Transgene Expression from the plastid Genome of Seed Plants
		References
	Chapter 4: Marker-Free Transplastomic Plants by Excision of Plastid Marker Genes Using Directly Repeated DNA Sequences
		1 Introduction
		2 The Arguments in Favor of Removing Marker Genes from Plastids
		3 Marker Gene Excision from Plastids
		4 Native DNA Recombination Pathways in Plastids
		5 Excision of Marker Genes by Recombination Between Direct DNA Repeats
		6 Factors Influencing Marker Excision by Direct Repeats
			6.1 Cis-Acting DNA Components Present in the Excision Cassette
				6.1.1 Length of DNA Direct Repeats
				6.1.2 Number of DNA Direct Repeats
				6.1.3 Influence of Genes Within the Direct Repeats on Excision Frequency
				6.1.4 Marker Excision Linked to Restoration of Gene Function
			6.2 Trans-Acting Recombination Proteins Acting on Direct Repeats
		7 Conclusions
		References
	Chapter 5: Fluorescent Labeling and Confocal Microcopy of Plastids and Stromules
		1 Introduction
			1.1 Fluorescent Labels
			1.2 Selection of the Fluorescent Protein for Labeling
			1.3 Introduction of Fluorescent Protein-Encoding Genes into Plants
		2 Materials
			2.1 Plants, Plasmids, and Bacterial Strains
			2.2 Disposables
			2.3 Buffers, Media, and Solutions
		3 Methods
			3.1 Bombardment of Aseptic Leaves and Onion Explants
			3.2 Agroinfiltration of Leaves
			3.3 Monitoring of Expression
		4 Notes
		References
Part II: Nicotiana Tabacum: The Model Species of Chloroplast Biotechnology
	Chapter 6: Transformation of the Plastid Genome in Tobacco: The Model System for Chloroplast Genome Engineering
		1 Introduction
			1.1 Marker Genes
			1.2 Vectors Targeting Insertions at Alternative Sites
			1.3 Need for Cultivar-Specific Protocols
		2 Materials
			2.1 Plant Propagation
			2.2 Preparation of DNA-Coated Gold Particles
			2.3 DNA Delivery by the Biolistic Process
			2.4 Identification of Transplastomic Events
			2.5 Transfer of Plants to Greenhouse
		3 Methods
			3.1 Plant Propagation
			3.2 Preparation of DNA-Coated Gold Particles
			3.3 DNA Delivery by the Biolistic Process
			3.4 Identification of Transplastomic Events
			3.5 Transferring Plants to Greenhouse and Testing Maternal Inheritance
		4 Notes
		References
	Chapter 7: Nicotiana tabacum: An Update on PEG-Mediated Plastid Transformation
		1 Introduction
		2 Materials
			2.1 Culture of Plant Material
			2.2 Protoplast Isolation
			2.3 PEG-Mediated Plastid Transformation
			2.4 Protoplast Culture and Regeneration
		3 Methods
			3.1 Germination of Nicotiana tabacum
			3.2 Protoplast Isolation from Nicotiana tabacum Leaves
			3.3 PEG-Mediated Plastid Transformation
			3.4 Protoplast Culture and Regeneration
		4 Notes
		References
	Chapter 8: Transformation of the Plastid Genome in Tobacco Suspension Cell Cultures
		1 Introduction
		2 Materials
			2.1 Tissue Culture Media
			2.2 Particle Bombardment
		3 Methods
			3.1 Initiation and Maintenance of Tobacco Suspension Cell Cultures
			3.2 Preparation of Suspension Cells for Bombardment
			3.3 Preparation of Microparticles for Particle Bombardment
			3.4 Coating Microcarriers with  DNA
			3.5 Loading DNA-Coated Microparticles onto the Macrocarrier
			3.6 Selection of Plastid-Transformed Colonies and Plants
		4 Notes
		References
	Chapter 9: Plastid Marker Gene Excision in the Tobacco Shoot Apex by Agrobacterium-Delivered Cre Recombinase
		1 Introduction
		2 Materials
			2.1 Plant Materials
			2.2 Agrobacterium Culture and Induction of T-DNA Transfer
			2.3 Bud Injection
			2.4 PCR Analyses
			2.5 DNA Gel Blot Analyses
		3 Methods
			3.1 Cultivation of Plants in the Greenhouse
			3.2 Agrobacterium Culture and Induction of T-DNA Transfer
			3.3 Agrobacterium Bud Injection to Induce Marker-Free Green Sectors
			3.4 PCR Analyses to Confirm Transgene Integration
			3.5 DNA Gel Blot Analyses to Confirm Marker Excision
		4 Notes
		References
	Chapter 10: Rubisco Engineering by Plastid Transformation and Protocols for Assessing Expression
		1 Introduction
		2 Materials
			2.1 Rubisco Gene Amplification and Plasmid Cloning
			2.2 Plant Tissue Culture
			2.3 Leaf Protein Extraction and Polyacrylamide Gel Electrophoresis (PAGE)
			2.4 Plant Growth in  Soil
			2.5 [14C]-CABP Synthesis
			2.6 Gel Filtration Chromatography
			2.7 Coomassie and Western Blot Densitometry for Detecting Rubisco Expression
		3 Methods
			3.1 Cloning Rubisco Genes into pLEV Plastome Transforming Plasmids
			3.2 Culture of cmtrL and Transformed Tobacco
			3.3 Identification of Transformed Plants by Nondenaturing PAGE
			3.4 Preparation of Leaf Samples for Rubisco Analysis
			3.5 14C-CABP Synthesis
			3.6 Quantifying Leaf Rubisco Content by 14C-CABP Binding
				3.6.1 Forming the 14C-CABP-Rubisco Complexes
				3.6.2 Quantify Rubisco Content by Gel Filtration Separation of [14C]-CABP Bound Enzyme
			3.7 Densitometry Analysis of Rubisco Expression
				3.7.1 Relative Rubisco Content from Protein Staining with Coomassie  Blue
				3.7.2 Western Blot Analysis of Rubisco Expression
		4 Notes
		References
Part III: Crop Specific Plastid Transformation Protocols
	Chapter 11: Plastid Transformation in Tomato: A Vegetable Crop and Model Species
		1 Introduction
		2 Materials
			2.1 Plant Material and Growth Conditions
			2.2 Chemicals and Stock Solutions for Plant Culture Media
			2.3 Plant Culture Media
			2.4 Materials for Particle Preparation
		3 Methods
			3.1 Vectors for Tomato Plastid Transformation
			3.2 Gold Particle Preparation for Biolistic Plastid Transformation
			3.3 Biolistic Bombardment
			3.4 Selection of Transplastomic Tomato Plants
			3.5 Analysis of Transplastomic Tomato Plants
		4 Notes
		References
	Chapter 12: Stable Plastid Transformation of Petunia for Studies in Basic Research
		1 Introduction
		2 Materials
			2.1 Plant Material
			2.2 Culture Media Components (See Table 1)
			2.3 Preparation of Gold Microcarriers
			2.4 Other Chemicals
		3 Methods (See Notes 10 and 11)
			3.1 Preparation of Culture Media
			3.2 Germination of Seedlings In Vitro (See Note 11)
			3.3 Propagation of Plants In Vitro
			3.4 Preparation of Leaf Explants for Particle Bombardment
			3.5 Coating Gold Micro Carriers with Vector  DNA
			3.6 Operation of Particle Delivery System
			3.7 Antibiotic-Based Selection of Resistant Shoots
			3.8 Inheritance of Transgenic Plastids
		4 Notes
		References
	Chapter 13: Plastid Transformation in Potato: An Important Source of Nutrition and Industrial Materials
		1 Introduction
		2 Materials
			2.1 Plant Material and Consumables
			2.2 Equipment
			2.3 Media
			2.4 PCR Analysis
		3 Methods
			3.1 Precultivation of Potato Leaves
			3.2 Microcarrier Stock Preparation
			3.3 Sterilization and Preparation of Consumables
			3.4 DNA Precipitation onto the Microcarriers
			3.5 Leaf Bombardment
			3.6 Regeneration of Transplastomic Calli and Shoots
			3.7 PCR Analysis of Transplastomic Plants
		4 Notes
		References
	Chapter 14: Plastid Transformation in Poplar: A Model for Perennial Trees
		1 Introduction
		2 Materials
			2.1 Plant Material, Tissue Culture, and Plant Generation
			2.2 Construction of Poplar-Specific Plastid Transformation Vector
			2.3 DNA and Gold Particle Preparation
			2.4 Biolistic Delivery System
			2.5 Southern Blot Analysis of Transplastomic Plants
		3 Methods
			3.1 Preparation of Leaf Explants for Particle Bombardment
			3.2 Construction of Plastid Transformation Vector
			3.3 Preparation of Plasmid DNA for Plastid Transformation
			3.4 Particle Preparation and DNA Coating (See Note 3)
			3.5 Bombardment Procedure
			3.6 Selection and Regeneration of Transplastomic Plants
			3.7 Transfer to Soil and Cultivation
		4 Notes
		References
	Chapter 15: Biolistic Plastid Transformation in Lettuce (Lactuca sativa) for Oral Delivery of Biopharmaceuticals
		1 Introduction
		2 Materials
			2.1 Construction of Lettuce Specific Transformation Vector
			2.2 Growth of Wild-Type Lettuce Plants
			2.3 Optimization of Regeneration and Selection Media
			2.4 Biolistic DNA Delivery
			2.5 Regeneration and Confirmation of Transplastomic Lines
			2.6 Transfer to Soil, Cultivation and Seed Production
		3 Protocols
			3.1 Construction of Lettuce Specific Transformation Vector
			3.2 Growth of Wild Type Lettuce Plants
			3.3 Optimization of Culture Conditions for Regeneration from Leaf Explants
			3.4 Preparation of Consumables for Biolistic DNA Delivery
				3.4.1 Preparation of Gold Suspension
				3.4.2 Preparation of Lettuce Leaves for Bombardment
				3.4.3 Precipitate Plasmid DNA onto Gold Particles
				3.4.4 Loading Gold Suspension onto Macrocarrier  Disk
			3.5 Bombardment Procedure
			3.6 Regeneration and Screening for Transplastomic Lines
			3.7 Transfer to Soil and Seed Production
		4 Notes
		References
	Chapter 16: Plastid Transformation in Sugar Beet: An Important Industrial Crop
		1 Introduction
		2 Materials
			2.1 Seed Sterilization and Germination
			2.2 Sample Preparation
			2.3 Gold Particle Coating
			2.4 Biolistic  Gun
			2.5 Sugar Beet Regeneration
		3 Methods
			3.1 Sowing Seeds
			3.2 Petioles from Leaves
			3.3 Gold Preparation and Coating
			3.4 Petiole Bombardment
			3.5 Transplastomic Sugar Beet Plants
		4 Notes
		References
Part IV: Plastid Transformation in Algae
	Chapter 17: A Simple Technology for Generating Marker-Free Chloroplast Transformants of the Green Alga Chlamydomonas reinhardt...
		1 Introduction
		2 Materials
			2.1 Chlamydomonas Strains and Transformation plasmids
			2.2 Growth of Chlamydomonas
			2.3 Glass Bead Transformation
			2.4 Genomic Extraction and PCR Analysis
		3 Methods
			3.1 Preparation of Chlamydomonas Gametolysin
			3.2 Preparing Cells for Transformation
			3.3 Glass Bead Transformation
			3.4 Isolating Homoplasmic Transformants (See Note 10)
		4 Notes
		References
	Chapter 18: Regulated Chloroplast Gene Expression in Chlamydomonas
		1 Introduction
		2 Materials
			2.1 Growth Media
			2.2 Materials
		3 Methods
			3.1 Strains and Growth Conditions
			3.2 Construction of Strains for Repressing Any Chloroplast Gene
			3.3 Inducible/Repressible Expression of Chloroplast Genes
			3.4 Inducible Expression of Foreign Genes in the Chloroplast
		4 Notes
		References
Part V: Plastid Transformation in Bryophyte
	Chapter 19: Plastid Transformation in Physcomitrium (Physcomitrella) patens: An Update
		1 Introduction
		2 Materials
			2.1 Plant Material and Plasmids
			2.2 Stock Solutions for Culture Media
			2.3 Culture Media
			2.4 Solutions and Media for Transformation
		3 Methods
			3.1 Preparation of Moss Protoplasts
			3.2 Introduction of DNA into the Moss Protoplasts
			3.3 Screening Moss Plastid Transformants
			3.4 Analysis of Transformants
				3.4.1 One-Step PCR Analysis
				3.4.2 Southern Blot Analysis of Total Cellular  DNA
		4 Notes
		References
	Chapter 20: Plastid Transformation of Sporelings from the Liverwort Marchantia polymorpha L.
		1 Introduction
		2 Materials
			2.1 Media and Buffer
			2.2 Preparation of Surface-Sterilized Sporangia
			2.3 Transformation Vector
			2.4 Particle Bombardment
			2.5 Selection and Identification of Plastid Transformants
		3 Methods
			3.1 Preparation of Sporelings for Bombardment
			3.2 Particle Bombardment
			3.3 Identification of Homoplasmic Transformants by  PCR
		4 Notes
		References
	Chapter 21: Rapid and Modular DNA Assembly for Transformation of Marchantia Chloroplasts
		1 Introduction
			1.1 Plastid Transformation Vectors
			1.2 Chloroplast Transformation
		2 Materials
			2.1 DNA Assembly
				2.1.1 Agarose Gel Electrophoresis and Gel Extraction
				2.1.2 PCR
			2.2 Growing Sporelings for Transformation
				2.2.1 Growth of Spores
			2.3 Transformation and Sequencing
			2.4 Equipment
		3 Methods
			3.1 DNA Assembly
				3.1.1 Level 0 Reaction Protocol
				3.1.2 Level 1 Reaction Protocol
				3.1.3 Odd Level from Level 3 Onwards Reaction Protocol
				3.1.4 Even Level Reaction Protocol
			3.2 Spore Production
			3.3 Growing Sporelings for Transformation
			3.4 Transformation
				3.4.1 Plasmid DNA Preparation
				3.4.2 Nanoparticle Preparation
				3.4.3 Bombardment
				3.4.4 Selection of Successful Transformants
			3.5 Genotyping
		4 Notes
		References
Index