Biolistic DNA Delivery: Methods and Protocols

Foreword
Preface
Contents
Contributors
Part I: Tribute to Professor Diter von Wettstein
	Chapter 1: Diter von Wettstein, Professor of Genetics and Master of Translating Science into Applications
		1 Family History
		2 A Brief Biography
		3 A Glimpse into Diter´s Personality and Research
			3.1 Fundamental Research
			3.2 Interphase Research
				3.2.1 Breeding for Root and Crown Rot Resistance
				3.2.2 Production of Nutritionally Enhanced ``Celiac-Safe´´ Wheat Genotypes
				3.2.3 Production of Wheat Genotypes with Improved Bioavailability of Dietary Fibers
			3.3 Applied Research
				3.3.1 Breeding for Imidazolinone Tolerance
				3.3.2 Breeding for Proanthocyanidin-Free Malting Barley
				3.3.3 Breeding for Increased Bioavailability of β-Glucans
		4 Closing Remark
		References
	Chapter 2: Diter von Wettstein and The Meiotic Program of Pairing and Recombination
		References
Part II: Background and Overview
	Chapter 3: A Short History and Perspectives on Plant Genetic Transformation
		1 Introduction
		2 Agrobacterium-Mediated Plant Genetic Transformation
		3 Biolistics or Particle Bombardment-Mediated Genetic Transformation of the Nuclear Genome
		4 Biolistics-Mediated Chloroplast Transformation
		5 Bioactive Beads-Mediated Gene Transfer
		6 Pollen and Pollen Tube Pathway-Mediated Plant Transformation
		7 Silicon Carbide Whisker- and Nanoparticle-Mediated Plant Transformation
		8 Electrophoretic Transfection and Electroporation-Mediated Plant Transformation
		9 Microinjection-Mediated Plant Transformation
		10 Progress in Recalcitrant Plant Transformation
		11 Gene Integration Through Homologous Recombination
		12 CRISPR/Cas9-Mediated Genome Editing and Beyond
		13 Conclusions
		References
	Chapter 4: Choice of the Promoter for Tissue and Developmental Stage-Specific Gene Expression
		1 Introduction
		2 Materials
			2.1 Fruit-Specific Promoters
			2.2 Flower-Specific Promoters
			2.3 Anther/Pollen-Specific Promoters
			2.4 Seed-Specific Promoters
			2.5 Root-Specific Promoters
			2.6 Developmentally Regulated Promoters
		3 Notes
		References
	Chapter 5: Choice of Explant for Plant Genetic Transformation
		1 Introduction
		2 Plant Genetic Transformation
			2.1 General Strategies for Transgene Delivery
			2.2 Biolistics or Particle Bombardment Method
		3 Different Types of Explants Used in Plant Genetic Transformation
		4 Factors Influencing the Choice of Explants
			4.1 Explant Age
			4.2 Explant Size
			4.3 Explant Excise Position
			4.4 Explant Orientation
			4.5 Explant Genotype
		5 Explant Pretreatment
		6 Explant Preparation
		7 Application of Particle Bombardment Method in Plant Genetic Transformation
		8 Conclusion
		References
	Chapter 6: Biolistic Approach for Transient Gene Expression Studies in Plants
		1 Introduction
		2 Advantages and Limitations of Transient Expression Mediated by Biolistics
			2.1 Range of Species and Tissue Targets
			2.2 Target Organelles
			2.3 Plant Sample Preparation
			2.4 Vector Preparation
			2.5 Invasiveness
			2.6 Early Events Following Microparticle Bombardment
			2.7 Time-Frame of Biolistic Experiments
		3 Examples of Biolistic-Mediated Transient Expression Studies
			3.1 Transcriptional Regulation and Promoter Activity
			3.2 Protein Subcellular Localization
			3.3 Cell-to-Cell Protein Movement
			3.4 Virus Inoculation
			3.5 RNA Silencing Induction
			3.6 Targeted Genome Editing
		4 Discussion and Conclusion
		References
	Chapter 7: Nanobiolistics: An Emerging Genetic Transformation Approach
		1 Introduction
		2 Discussion
			2.1 Synthesis and Characterization of Nanoparticles for Nanobiolistics
				2.1.1 Synthesis of Mesoporous Silica Nanoparticles
				2.1.2 Synthesis of Gold Nanoparticles
				2.1.3 Nanoparticle Characterization
			2.2 Testing Nanobiolistics in Animals
			2.3 Emerging Studies of Nanobiolistics in Plants
		3 Conclusions
		References
Part III: Protocols
	Chapter 8: Biolistic Transformation of Japonica Rice Varieties
		1 Introduction
		2 Materials
			2.1 Plasmid Constructs
			2.2 Plant Material (See Note 2)
			2.3 Stock Solutions (See Note 3)
			2.4 Culture Media (See Note 5)
			2.5 Equipment and Related Supplies
		3 Methods
			3.1 Microprojectile Preparation (See Note 7)
				3.1.1 Plasmid Confirmation
				3.1.2 Gold Microprojectile Preparation
				3.1.3 DNA/Gold Coating and Loading to Macrocarriers
			3.2 Embryo Explant Preparation
				3.2.1 Seed Sterilization
				3.2.2 Explant Preparation
				3.2.3 Osmotic Treatment, Bombardment and Resting
			3.3 Selection of Transformed Calli
			3.4 Regeneration and Rooting
			3.5 Growing Plants in Growth Chambers
			3.6 Transgene Inheritance Analysis
		4 Notes
		References
	Chapter 9: Biolistic DNA Delivery in Maize Immature Embryos
		1 Introduction
		2 Materials
			2.1 DNA Constructs (See Note 1)
			2.2 Plant Material
			2.3 Stock Solutions
			2.4 Culture Media
			2.5 Equipment and Related Supplies
		3 Methods
			3.1 Microprojectile Preparation (See Note 8)
				3.1.1 Preparing of Gold Microprojectiles
				3.1.2 Coating DNA on the Gold Particles
				3.1.3 Loading the DNA/Gold onto the Macrocarrier
				3.1.4 Preparing the Biolistic Gun Device for Bombardment
			3.2 Maize Embryo Explant Preparation
				3.2.1 Embryo Dissection
				3.2.2 Pre-bombardment Osmotic Treatment
			3.3 Bombardment
				3.3.1 Launch the Biolistic Device
				3.3.2 Microprojectile Bombardment of DNA (See Note 25)
				3.3.3 Post-bombardment Biolistic Gun Care
				3.3.4 Post-bombardment Osmotic Treatment and Transient Assays
			3.4 Selection and Regeneration
				3.4.1 Selection of Stable Transgenic Events
				3.4.2 Callus Maturation
		4 Notes
		References
	Chapter 10: Biolistic DNA Delivery and Its Applications in Sorghum bicolor
		1 Introduction
		2 Sorghum Tissue Culture
			2.1 Explants
			2.2 Media
			2.3 Environment
		3 Biolistic Delivery Parameters
			3.1 Nanoparticles
			3.2 Selection
		4 Applications
			4.1 Expression of Transgenes
			4.2 RNAi
			4.3 Genome Editing and CRISPR/Cas9
		5 Future of Particle Bombardment in Sorghum
		References
	Chapter 11: Biolistics-Mediated Gene Delivery in Sugarcane
		1 Introduction
		2 Materials
			2.1 Target Tissue
			2.2 Stock Solutions and Media
			2.3 DNA for Bombardment into Sugarcane Cells
			2.4 Basic Equipment and Reagents for Bombardment
		3 Methods
			3.1 Preparation of Target Tissue
			3.2 Preparation of Gold Particles
			3.3 Microcarrier Preparation for Bombardment
			3.4 Microprojectile Bombardment
			3.5 Post Bombardment
			3.6 Molecular Analysis of Regenerants
		4 Notes
		References
	Chapter 12: Genetic Transformation of Common Wheat (Triticum aestivum L.) Using Biolistics
		1 Introduction
		2 Materials
			2.1 Media
				2.1.1 Stock Solutions
				2.1.2 Plant Culture Media Stock Solutions
				2.1.3 Final Culture Media
			2.2 Isolation of Target Explants
				2.2.1 Growth of Donor Plants
				2.2.2 Sterilization of Immature Caryopses
			2.3 Components for Particle Bombardment
		3 Methods
			3.1 Isolation of Target Explants
				3.1.1 Collection and Sterilization of Immature Caryopses
				3.1.2 Isolation of Immature Scutella
			3.2 Preparation of Gold Microcarriers for Particle Bombardment
				3.2.1 Preparation of Gold Microcarriers Stock
				3.2.2 Coating of Gold Microcarriers with DNA
			3.3 Transformation Via Particle Bombardment
				3.3.1 Preparation of Particle Gun and Components
				3.3.2 Assembling the Gun
				3.3.3 Firing the Gun
				3.3.4 Disassembling the Gun
				3.3.5 Conclusion of Bombardment
			3.4 Regeneration and Selection of Transgenic Plantlets Following Bombardment
				3.4.1 Induction of Embryogenic Callus
				3.4.2 Introduction of Selection
				3.4.3 Increasing Selection Pressure
				3.4.4 Regeneration of Plantlets
				3.4.5 Rooting of Plantlets
				3.4.6 Potting Putatively Transformed Plantlets to Soil
				3.4.7 Analysis of Plants and Further Growth
		4 Notes
		References
	Chapter 13: Biolistic DNA Delivery in Turfgrass Embryonic Callus Initiated from Mature Seeds
		1 Introduction
		2 Materials
			2.1 Plant Material
			2.2 Plasmids
			2.3 Culture Media and Solutions
				2.3.1 Chemical Stock
				2.3.2 Initiation of Embryogenic Callus from Mature Seeds
				2.3.3 Plant Selection and Regeneration Media
		3 Methods
			3.1 Initiation and Proliferation of Embryonic Callus from Mature Seeds
			3.2 Callus Preparation Prior to Bombardment
			3.3 Biolistic Transformation of Creeping Bentgrass Embryogenic Calli with pSBUbibar-35SGUS
			3.4 Selection and Regeneration of Transformed Colonies
			3.5 Regenerated Plants Maintenance
			3.6 Molecular Analysis of Transgene Integration and Expression in the Transformed Plants
				3.6.1 PCR Analysis
				3.6.2 Southern Blot Analyses
				3.6.3 Northern Blot Analyses
		4 Notes
		References
	Chapter 14: Use of Microspore-Derived Calli as Explants for Biolistic Transformation of Common Wheat
		1 Introduction
		2 Materials
			2.1 Plants
			2.2 Stock Solutions
			2.3 Specific Laboratory Equipment and Supplies
		3 Methods
			3.1 Growth Conditions and Selection of Spikes
			3.2 Pretreatment
			3.3 Isolation of Microspores
			3.4 Preparation of Plant Material for Microprojectile Bombardment
			3.5 Preparation of Gold Particles Stock Solution for Biolistic Transformation of Microspore-Derived Calli
			3.6 Preparation of Gold Particles for Bombardment
			3.7 Coating the Macrocarrier
			3.8 Microprojectile Bombardment
			3.9 Regeneration
			3.10 Colchicine Treatment
			3.11 Determination of Ploidy by Chromosome Analysis
		4 Development of Wheat Genotypes with Reduced Immunogenicity
			4.1 Construction of dTALE Repressor (Donor) and CRISPR/Cas9 Nuclease Constructs
			4.2 Identification of Candidate Transformants with Adduct Lesions in the Wheat Dre2 Gene Homoeologs
		5 Notes
		References
	Chapter 15: Plant Transformation Techniques: Agrobacterium- and Microparticle-Mediated Gene Transfer in Cereal Plants
		1 Introduction
			1.1 State-of-the-Art Transformation Techniques in Cereals
		2 Agrobacterium-Mediated Transformation
			2.1 Materials
			2.2 Plant Material and Growth Conditions
			2.3 Preparation of A. tumefaciens Electrocompetent Cells
			2.4 Surface Sterilization of Immature Barley Seed
			2.5 Isolation of Immature Embryos and Inoculation with A. tumefaciens
			2.6 Generation of Plants from Calli
			2.7 Determination of GFP in Transformed Tissue
		3 Biolistic Transformation
			3.1 Materials
			3.2 Collection and Sterilization of Wheat Caryopses
			3.3 Isolation of Immature Wheat Embryos
			3.4 Coating of Gold Particles with DNA (for Six Shots)
			3.5 Delivery of DNA-Coated Gold Particles
			3.6 Histochemical Assays for GUS Detection
		4 Notes
		References
	Chapter 16: Proteolistics: A Protein Delivery Method
		1 Introduction
		2 Materials
			2.1 Plant Materials
			2.2 Stock Solutions and Media
			2.3 Proteins (See Note 4)
			2.4 Equipment and Related Supplies
			2.5 Other Supplies
		3 Methods
			3.1 Gold Particle Preparation
			3.2 Protein-Gold Particle Suspension Preparation
			3.3 Drying the Suspension on the Macrocarrier
				3.3.1 Air-Drying
				3.3.2 Freeze-Drying
				3.3.3 Storage of Macrocarrier-Set Until Bombardment and Post-Drying Protein Check
			3.4 Plant Target Tissue Preparation
				3.4.1 Preparation of Onion Epidermis Tissue
				3.4.2 Preparation of Maize Immature Embryos
			3.5 Bombardment Using PDS-1000/He Biolistic Device (See Note 18)
			3.6 Monitoring Intracellular Delivery
		4 Notes
		References
	Chapter 17: Biolistic Delivery of Programmable Nuclease (CRISPR/Cas9) in Bread Wheat
		1 Introduction
		2 Materials
			2.1 Materials for Designing and Validating CRISPR/Cas9 RNPs
				2.1.1 sgRNA Cloning
				2.1.2 In Vitro Transcription of sgRNA
				2.1.3 Cas9 RNP Synthesis and Validation
			2.2 Material for Biolistic Delivery of Cas9 RNPs and Mutant Screening
				2.2.1 Plant Material
				2.2.2 Sterilization Material
				2.2.3 Particle Bombardment
				2.2.4 Regeneration of Embryos
				2.2.5 Mutant Screening
		3 Methods
			3.1 Designing and Validation of CRISPR/Cas9 RNPs
				3.1.1 Designing and Synthesis of Single-Guide RNA (sgRNA) for Cas9
				3.1.2 Designing ssDNA oligos (Only for Homology Directed Repair)
				3.1.3 Cloning of sgRNA into Scaffold Vector
				3.1.4 In Vitro Transcription of sgRNA
				3.1.5 Production of Cas9 Protein
				3.1.6 Validation of Cas9 RNPs Activity
					In Vitro Assay
					In Vivo Assay (Using Protoplast)
					Assessment of Cas9 RNP Activity
				3.1.7 Production of Cas9 RNPs
			3.2 Biolistic Delivery of CRISPR/Cas9 RNPs and Mutant Screening
				3.2.1 Wheat Growth Conditions
				3.2.2 Harvesting Spikes and Sterilization
				3.2.3 Isolation of Immature Embryo
				3.2.4 Preparation of Gold Particle Suspension
				3.2.5 Coating of Cas9 RNPs on Gold Nanoparticles
				3.2.6 Biolistic Delivery of Cas9 RNPs
				3.2.7 Verification of Cas9 RNP Delivery
				3.2.8 Tissue Culture Regeneration of Bombarded Embryos
				3.2.9 Mutant Screening
		4 Notes
		References
Index