Protoplast Technology : Methods and Protocols

Preface
Contents
Contributors
Part I: Basic Techniques and Their Applications
	Chapter 1: Physcomitrium patens Protoplasting and Protoplast Transfection
		1 Introduction
		2 Materials
			2.1 Plant and DNA Materials
			2.2 Stock Solutions
			2.3 Media
			2.4 Protoplast Handling Solutions
			2.5 Transfection Solutions
			2.6 Other Supplies
		3 Methods
			3.1 Protoplast Production
			3.2 Protoplast Transfection
			3.3 Protoplast Plating
			3.4 Selection Procedure for Integration Event by Homologous Recombination
			3.5 Selection Procedure for Transient Expression of RNAi Construct
			3.6 Selection Procedure for Transient Expression of CRISPR-Cas9 Construct
		4 Notes
		References
	Chapter 2: Protoplast Isolation and Transfection in the Single-Cell C4 Species Bienertia sinuspersici
		1 Introduction
		2 Materials
			2.1 Plant Material and Plasmid  DNA
			2.2 Buffer and Media
			2.3 Other Supplies
		3 Methods
			3.1 Protoplast Isolation
			3.2 Protoplast Transfection
		4 Notes
		References
	Chapter 3: Protoplast for Gene Functional Analysis in Arabidopsis
		1 Introduction
		2 Materials
			2.1 Plant Material, Plasmid, and Bacterial Strain
			2.2 Stock Solutions
			2.3 Media and Buffers
			2.4 Equipment and Related Supplies
		3 Methods
			3.1 Plants Preparation
			3.2 Plasmid DNA Preparation (See Note 7)
			3.3 Protoplast Isolation
			3.4 PEGCa2+-Mediated Transfection
			3.5 Protoplast Culture and Harvest
			3.6 Example 1: Functional Analysis of the Transcription Factor Activity in Mesophyll Protoplast
			3.7 Example 2: Protein Subcellular Localization Analysis-EIN2 Localization in Protoplast Cells
			3.8 Example 3: Protein-Protein Interaction-Split Luciferase Assay of TOR-FRB and FKP12 Interaction
			3.9 Example 4: Protein Kinase Activity-TOR Activated by Upstream Component ROP2 in the Protoplast System
		4 Notes
		References
	Chapter 4: Application of Protoplast Regeneration to CRISPR/Cas9 Mutagenesis in Nicotiana tabacum
		1 Introduction
		2 Materials
			2.1 Supplies and Equipment
			2.2 Plant Material and Vector Construct
			2.3 Chemicals and Stock Solutions
			2.4 Working Solution and Media
		3 Methods
			3.1 Explant Preparation
			3.2 Protoplast Isolation
			3.3 Protoplast Transfection
			3.4 Protoplast Regeneration
			3.5 Evaluate CRISPR Reagents in Protoplasts or Similar Topics
				3.5.1 Transfected Protoplast Efficiency Testing at the Initial Stage
				3.5.2 Single Protoplast Genotyping
				3.5.3 Genotyping of Regenerated Plants
		4 Notes
		References
	Chapter 5: CRISPR DNA- and RNP-Mediated Genome Editing via Nicotiana benthamiana Protoplast Transformation and Regeneration
		1 Introduction
		2 Materials
			2.1 Plant Materials
			2.2 Chemicals and Stock Solutions
			2.3 Solutions and Media
			2.4 Plant Growth Fertilizer Solutions
			2.5 Other Supplies
		3 Methods
			3.1 Choice of Target Site and Guide RNA Design
			3.2 Plasmid DNA and CRISPR-RNP Preparation
				3.2.1 Plasmid DNA Extraction
				3.2.2 RNP Complex Preparation
			3.3 Growing Plants for Explant for the Protoplast Isolation
			3.4 Protoplast Isolation (See Note 11)
			3.5 Protoplast Transfection (See Note 12)
			3.6 Protoplast Culture and Regeneration
			3.7 Analysis of Genome Editing Results
				3.7.1 Enzyme Digestion-Based Assay
				3.7.2 Sequencing-Based Assay
		4 Notes
		References
	Chapter 6: Protoplast Isolation and Transfection in Rice
		1 Introduction
		2 Materials
			2.1 Plant Material and DNA Construct
			2.2 Solutions and Media
			2.3 Other Supplies
		3 Methods
			3.1 Plant Preparation (10-12 Days)
			3.2 Protoplast Isolation (6-8 h)
			3.3 Protoplast Transfection and Culture (2 Days; See Note 14)
		4 Notes
		References
	Chapter 7: Protoplast Isolation and Transfection in Maize
		1 Introduction
		2 Materials
			2.1 Plant Materials and DNA Construct
			2.2 Stock Solutions
			2.3 Working Solutions
			2.4 Supplies
			2.5 Equipment
		3 Methods
			3.1 Plant Material Preparation
			3.2 Protoplast Isolation
			3.3 Wash the Protoplasts
			3.4 Quantification of Protoplast Yield
			3.5 Transfection
		4 Notes
		References
	Chapter 8: Isolation and Transfection of Maize Endosperm Protoplasts
		1 Introduction
		2 Materials
			2.1 Plant Material and Bacterial Strain
			2.2 DNA Constructs
			2.3 Stock Solutions
			2.4 Working Solutions and Media
			2.5 Supplies and Equipment
		3 Methods
			3.1 Preparation of Plasmid DNA
			3.2 Endosperm Dissection and Digestion
			3.3 Protoplast Isolation
			3.4 Protoplast Yield Quantification
			3.5 Protoplast Viability Determination
			3.6 Transfection
			3.7 Transfection Rate Determination
			3.8 Example 1: Analysis of Protein Subcellular Localization - ZmBT1 Localization in Endosperm Protoplast System
			3.9 Example 2: Analysis of Protein-Protein Interaction by BiFC Assay in Endosperm Protoplast System
			3.10 Example 3: Transcriptional Activity and Transient Expression Analysis of ZmMYB14 in Endosperm Protoplast System
		4 Notes
		References
	Chapter 9: Protoplast Isolation from Undifferentiated Maize Seedling Shoot Tissue
		1 Introduction
		2 Materials
			2.1 Plant Materials
			2.2 Solutions and Media
			2.3 Supplies and Equipment
		3 Methods
			3.1 Prepare Plant Material
			3.2 Dissect Maize Shoot Apex Tissue
			3.3 Isolate Protoplasts
			3.4 Determine Cell Density and Quality
		4 Notes
		References
	Chapter 10: Protoplast Isolation and Transfection in Wheat
		1 Introduction
		2 Materials
			2.1 Plasmids (See Note 1)
			2.2 Bacterial Strain and Plant Material
			2.3 Stock Solutions and Chemicals
			2.4 Working Solutions
			2.5 Supplies and Equipment
		3 Methods
			3.1 Growth of Wheat Seedlings
			3.2 Isolation of Wheat Leaf Protoplasts
			3.3 Example 1: Transfection of GFP Plasmid DNA
			3.4 Example 2: Efficiency Evaluation of Base Editors
		4 Notes
		References
	Chapter 11: Protoplast Isolation, Transformation, and Regeneration for Forage and Turf Grasses
		1 Introduction
		2 Materials
			2.1 Plant Material and DNA Construct
			2.2 Stock Solutions and Chemicals (See Note 3)
			2.3 Culture Media and Working Solutions (See Note 3)
			2.4 Supplies and Equipment
		3 Methods
			3.1 Sterilization and Callus Induction
			3.2 Establishment of Embryogenic Cell Suspensions
			3.3 Protoplast Isolation
			3.4 Protoplast Transformation
			3.5 Selection of Stable Transformed Colonies and Recovery of Transgenic Plants
		4 Notes
		References
	Chapter 12: Efficient Protoplast Isolation and DNA Transfection for Winter Oilseed Crops, Pennycress (Thlaspi arvense) and Cam...
		1 Introduction
		2 Materials
			2.1 Plant Materials
			2.2 Chemicals and Stock Solutions
			2.3 Working Solutions and Media
			2.4 Other Supplies
			2.5 Plasmid  DNA
		3 Methods
			3.1 Plant Material Preparation
				3.1.1 Growing Pennycress In Vitro
				3.1.2 Growing Camelina in  Soil
			3.2 Protoplast Isolation
				3.2.1 Leaf Explant Preparation (See Note 11)
				3.2.2 Leaf Cutting and Enzyme Digestion (See Note 14)
				3.2.3 Protoplast Separation (See Note 16)
				3.2.4 Washing
				3.2.5 Resting
				3.2.6 Cell Counting and Density Calculation
				3.2.7 Cell Viability  Test
			3.3 PEG-Calcium Transfection (See Note 31)
		4 Notes
		References
	Chapter 13: Protoplast Isolation, Transfection, and Gene Editing for Soybean (Glycine max)
		1 Introduction
		2 Materials
			2.1 Plant Materials
			2.2 Chemicals and Stock Solutions
			2.3 Solutions and Media
			2.4 Other Supplies
			2.5 Plasmid DNA
		3 Methods
			3.1 Cloning gRNA to Target Soybean Genes
				3.1.1 Design gRNA Oligonucleotide
				3.1.2 Cloning gRNA in Modular Vector
			3.2 Protoplast Isolation and Transfection
				3.2.1 Tissue Preparation and Digestion
				3.2.2 Protoplast Isolation
				3.2.3 PEG-Mediated Protoplast Transfection
			3.3 Analysis of Protoplast Transformation Efficiency
			3.4 Analysis of Gene-Editing Efficiency
				3.4.1 Cleaved-Amplified Polymorphic Site (CAPS) PCR
				3.4.2 Next Generation Amplicon Sequencing
		4 Notes
		References
	Chapter 14: Protoplast Isolation and Transformation in Oil Palm
		1 Introduction
		2 Materials
			2.1 Plant Material and DNA Constructs
			2.2 Stocks
			2.3 Solutions and Media
			2.4 Equipment and Related Supplies
		3 Methods
			3.1 Preparation of Calli for Protoplast Isolation
			3.2 Protoplast Isolation
			3.3 PEG Transfection
			3.4 DNA Microinjection
				3.4.1 Preparation of Alginate Layer
				3.4.2 Preparation of DNA Injection Solution
				3.4.3 Loading the DNA Solution into Microinjection Needle
				3.4.4 Microinjection of Protoplasts (See Note 24; Also See Fig. 3c)
				3.4.5 Alginate Layer Culture
		4 Notes
		References
Part II: Advanced Systems for High Throughput Analysis and Automation
	Chapter 15: Protoplast Preparation and Fluorescence-Activated Cell Sorting for the Evaluation of Targeted Mutagenesis in Plant...
		1 Introduction
		2 Materials
			2.1 PSB-D Cell Suspension Culture Maintenance
			2.2 PSB-D Transformation
			2.3 PSB-D Protoplasting
			2.4 Arabidopsis Growth and Sterilization
			2.5 Arabidopsis Protoplasting
			2.6 Plasmids and Bacterial Strains
			2.7 Genotyping
		3 Methods
			3.1 PSB-D Cell Suspension Culture Maintenance
			3.2 PSB-D Transformation
			3.3 PSB-D Protoplasting
			3.4 Arabidopsis Seedling Growth
			3.5 Arabidopsis Root/Cotyledon Protoplasting
			3.6 FACS
			3.7 Genotyping
			3.8 Example: CRISPR-TSKO in the Lateral Root Cap
		4 Notes
		References
	Chapter 16: Characterization of Programmable Transcription Activators in the Model Monocot Setaria viridis Via Protoplast Tran...
		1 Introduction
		2 Materials
			2.1 Stock Solutions
			2.2 Working Solutions and Media
			2.3 Supplies and Equipment
			2.4 Plant Materials
		3 Methods
			3.1 DNA Preparation
			3.2 Growing S. viridis
			3.3 Protoplast Isolation
			3.4 Low-Throughput Transfection
			3.5 96-Well Format High-Throughput Transfection
			3.6 Reporter Gene Detection
			3.7 qRT-PCR Screening
				3.7.1 RNA Isolation
				3.7.2 qRT-PCR
				3.7.3 Data Analysis
			3.8 Example 1: Guide RNA Selection for Overexpression of S. viridis Endogenous Developmental Genes
		4 Notes
		References
	Chapter 17: High-Throughput Transfection and Analysis of Soybean (Glycine max) Protoplasts
		1 Introduction
		2 Materials
			2.1 Plant Material and DNA Construct
			2.2 Stock Solutions and Media for Soybean Tissue Culture
			2.3 Stock Solutions and Media for Isolation and Transfection of Protoplasts
			2.4 Equipment and Supplies
		3 Methods
			3.1 Surface Sterilization of Soybean Seeds and Germination
			3.2 Generation of Soybean Dark-Grown Cell Suspensions
			3.3 Protoplast Isolation
			3.4 High-Throughput Protoplast Transfection Using a Tecan EVO Robotic System
			3.5 Data Collection and Analysis Using BioTek Cytation 5 Cell Imaging Multi-Mode Reader
		4 Notes
		References
Index