Plant Embryogenesis: Methods and Protocols (Methods in Molecular Biology, 2122)

Preface
Contents
Contributors
Part I: Mutagenesis and Genetic Screens
Chapter 1: Genetic Screens to Target Embryo and Endosperm Pathways in Arabidopsis and Maize
	1 Introduction
	2 Designing Genetic Screens for Seed Phenotypes
		2.1 Use of Morphological Criteria
		2.2 Use of Specific Genetic Backgrounds
		2.3 Imaging Embryos
		2.4 Screens Involving Crosses
	3 Example of Screening Protocol: Use of a Fluorescent Marker for Identifying Patterning Defects in Embryos Produced by Mutagen...
	4 Example of Screening Protocol: Crossing-Based Schemes for Identifying Parent-of-Origin Effects in Maize Seed Development
	References
Chapter 2: EMS Mutagenesis of Arabidopsis Seeds
	1 Introduction
	2 Materials
		2.1 Laboratory and Safety Equipment
		2.2 Glass and Plasticware
		2.3 Arabidopsis Seeds
		2.4 Reagents and Solutions
	3 Methods
		3.1 EMS Mutagenesis of Arabidopsis Seeds
		3.2 Arabidopsis M1 Generation: Sectors, Mutation Rate, and Seed Pooling
	4 Notes
	References
Chapter 3: EMS Mutagenesis of Maize Pollen
	1 Introduction
	2 Materials
		2.1 Laboratory and Safety Equipment
		2.2 Supplies and Plasticware
		2.3 Chemicals
		2.4 Plant Material
		2.5 Field Supplies
	3 Methods
		3.1 EMS Mutagenesis of Maize Pollen
		3.2 Maize M1 Generation: Nonconcordant Kernels, Production of M2
	4 Notes
	References
Part II: Microscopic Imaging and Phenotypic Analysis of Arabidopsis Embryos
Chapter 4: Live-Cell Imaging of Zygotic Intracellular Structures and Early Embryo Pattern Formation in Arabidopsis thaliana
	1 Introduction
	2 Materials
	3 Methods
		3.1 Preparation of PDMS Micropillar Array Device
		3.2 Silique Dissection to Extract Ovules
		3.3 Live-Cell Imaging
	4 Notes
	References
Chapter 5: Analyzing Subcellular Reorganization During Early Arabidopsis Embryogenesis Using Fluorescent Markers
	1 Introduction
	2 Materials
		2.1 Tools
		2.2 Mounting Media
	3 Methods
		3.1 Transferring Seeds from the Silique to the Microscope Slide
		3.2 Embryo Extraction
		3.3 Imaging the Subcellular Structure Using an Upright Confocal Microscope
	4 Notes
	References
Chapter 6: From Stained Plant Tissues to Quantitative Cell Segmentation Analysis with MorphoGraphX
	1 Introduction
		1.1 Information in 3D
		1.2 Comparison Between Different Staining Techniques
	2 Materials
		2.1 Chemicals and Solutions
		2.2 Equipment
	3 Methods
		3.1 Preparation for Microscopy
			3.1.1 Schiff Staining
			3.1.2 Renaissance Staining
		3.2 Confocal Microscopy
		3.3 Image Processing and Analysis Using MorphoGraphX
			3.3.1 Getting Started with MorphoGraphX
			3.3.2 Analysis of Embryo Data
		3.4 Additional Analysis Tools Using MorphoGraphX
			3.4.1 Changing the Color (and the Cell Label) of a Cell in a Mesh
			3.4.2 Changing the Color of the Main Stack
		3.5 Image Analysis of Root Tissue Using MorphoGraphX
			3.5.1 Segmentation of Root Images
			3.5.2 Root Cell Analysis Using 3D Cell Atlas
	4 Notes
	References
Part III: Expression Analysis and Transcriptomics
Chapter 7: Small RNA In Situ Hybridizations on Sections of Arabidopsis Embryos
	1 Introduction
	2 Materials
		2.1 Reagents and Solutions
		2.2 Equipment
	3 Methods
		3.1 Probe Design
		3.2 Tissue Fixation and Dehydration (Days 1-3)
		3.3 Clearing, Sectioning and Embedding (Days 3-5)
		3.4 Proteinase K Digestion, EDC Fixation and Probe Hybridization (Days 5-6)
			3.4.1 Dewaxing
			3.4.2 Hydration
			3.4.3 Proteinase K Digestion
			3.4.4 EDC Fixation
			3.4.5 Dehydration
			3.4.6 Probe Hybridization
		3.5 Washing and Antibody Reaction (Day 6)
			3.5.1 Coverslip Removal and High Stringency Washes
			3.5.2 Blocking and Antibody Reaction
		3.6 Colorimetric Reaction and Mounting (Days 6-7)
			3.6.1 Colorimetric Reaction
			3.6.2 Mounting
	4 Notes
	References
Chapter 8: Manual Isolation of Living Early Embryos from Tobacco Seeds
	1 Introduction
	2 Materials
		2.1 Plant Materials
		2.2 Reagents
		2.3 Equipment
		2.4 Solutions for Cell Isolation
	3 Methods
		3.1 Hand Emasculation and Pollination
		3.2 Seed Collection and Embryo Isolation (Before 32-Cell Embryo Stage)
		3.3 Embryo Isolation (After 32-Cell Embryo Stage)
		3.4 Estimation of Cell Viability
		3.5 mRNA Extraction and cDNA Amplification
	4 Notes
	References
Chapter 9: Profiling Transcriptomes of Manually Dissected Arabidopsis Embryos
	1 Introduction
	2 Materials
		2.1 Early Embryo Isolation
		2.2 RNA Extraction, cDNA Construction, and mRNA-Seq Library Generation
		2.3 mRNA-Seq Data Quality Control Workflow with Tissue Enrichment Test
	3 Methods
		3.1 Early Embryo Isolation (See Note 2)
			3.1.1 Hand Dissection (Modified from)
			3.1.2 Bulk Rupture Method (Modified from)
		3.2 RNA Extraction, cDNA Construction, and mRNA-Seq Library Generation
			3.2.1 RNA Extraction (See Note 7)
			3.2.2 cDNA Construction and NGS Library Preparation (Modified from Smart-seq2 Protocol)
		3.3 mRNA-Seq Data Quality Control Workflow with Tissue Enrichment Test
			3.3.1 Alignment and Quantification (All Resources Are Listed in Table 8)
			3.3.2 Tissue Enrichment Test
	4 Notes
	References
Chapter 10: Laser-Assisted Microdissection of Plant Embryos for Transcriptional Profiling
	1 Introduction
	2 Materials
		2.1 Removal of RNAses from Working Material and Equipment
		2.2 Tissue Collection and Fixation
		2.3 Tissue Embedding, Blocking, and Slide Preparation
		2.4 Laser-Assisted Microdissection (LAM)
	3 Methods
		3.1 Removal of RNAses from Working Material and Equipment
		3.2 Tissue Collection and Fixation
		3.3 Tissue Embedding, Blocking, and Slide Preparation
		3.4 Laser-Assisted Microdissection (LAM) on the Laser Dissection Microscope (LCM)
		3.5 RNA Extraction, cDNA Synthesis, and Library Preparation for Illumina RNA Sequencing
	4 Notes
	References
Chapter 11: Cell Type-Specific Transcriptomics in the Plant Embryo Using an Adapted INTACT Protocol
	1 Introduction
	2 Materials
		2.1 Seed Isolation System
		2.2 Purification of biotin-Tagged Nuclei
		2.3 RNA Isolation and Transcriptome Profiling
	3 Methods
		3.1 Isolation of Seeds with Embryos of a Predetermined Developmental Stage
		3.2 Purification of biotin-Tagged Nuclei
		3.3 RNA Isolation and Transcriptome Profiling
	4 Notes
	References
Chapter 12: Transcriptomic Profiling of the Arabidopsis Embryonic Epidermis Using FANS in Combination with RNAseq
	1 Introduction
	2 Material
		2.1 Nuclear Fixation and Extraction
			2.1.1 General Material
			2.1.2 Embryos Until Heart Stage and Inaccessible Tissues
			2.1.3 Embryos from Torpedo Stage and Accessible Tissues
		2.2 FANS
		2.3 RNA Extraction
		2.4 Reverse Transcription (See Note 4)
		2.5 cDNA Amplification (First PCR, Called PCR Preamplification)
		2.6 Tagmentation and PCR Amplification (Second PCR)
	3 Methods
		3.1 Tissue Collection and Fixation
			3.1.1 Early Embryos and Inaccessible Tissues
			3.1.2 Later-Stage Embryos and Accessible Tissues
		3.2 Nuclear Extraction
			3.2.1 Early Embryos and Inaccessible Tissues
			3.2.2 Later-Stage Embryos and Accessible Tissues
		3.3 FANS
		3.4 RNA Extraction
		3.5 Reverse Transcription
		3.6 cDNA Amplification (First PCR, Called PCR Preamplification)
		3.7 Tagmentation and PCR Amplification (Second PCR)
	4 Notes
	References
Part IV: Protein Interaction Studies
Chapter 13: Visualizing Protein Associations in Living Arabidopsis Embryo
	1 Introduction
	2 Materials
	3 Methods
		3.1 Establishing Plant Material
		3.2 Embryo Dissection
		3.3 Checking Fluorescence Intensity
		3.4 Confocal Imaging
		3.5 Instrument Calibration
		3.6 Instrument Response Function (IRF)
		3.7 FLIM Imaging
			3.7.1 Data Fitting
			3.7.2 Cell Type Specific Fluorescence Lifetime Quantification
			3.7.3 Fluorescence Intensity Image
			3.7.4 Representation of Lifetime Reduction
	4 Notes
	References
Part V: Embryogenesis in Other Model and Non-model Species
Chapter 14: Imaging of Embryo Sac and Early Seed Development in Maize after Feulgen Staining
	1 Introduction
	2 Materials
		2.1 Fixing and Storage of Whole Maize Ears
		2.2 Fixing and Storage of Dissected Maize Ovaries
		2.3 Rehydration
		2.4 Feulgen Staining
		2.5 Dehydration
		2.6 Clearing
	3 Methods
		3.1 Fixing and Storage of Whole Maize Ears
		3.2 Fixing and Storage of Dissected Maize Ovaries
		3.3 Rehydration
		3.4 Feulgen Staining
		3.5 Dehydration
		3.6 Clearing
		3.7 Microscopy
	4 Notes
	References
Chapter 15: Using Giant Scarlet Runner Bean (Phaseolus coccineus) Embryos to Dissect the Early Events in Plant Embryogenesis
	1 Introduction
	2 Materials
		2.1 Planting Scarlet Runner Bean Seeds
		2.2 Hand-Pollinating Scarlet Runner Bean Flowers
		2.3 Collecting Globular Stage Scarlet Runner Bean Seeds
		2.4 Dissecting Whole-Mount Globular Stage Scarlet Runner Bean Embryos
		2.5 Fixing Globular Stage Scarlet Runner Bean Seeds for LCM or in situ Hybridization
		2.6 Dehydration for LCM
		2.7 Dehydration for in situ Hybridization
		2.8 Infiltration and Embedding for LCM or in situ Hybridization
		2.9 Sectioning for LCM or in situ Hybridization
		2.10 LCM
	3 Methods
		3.1 Planting Scarlet Runner Bean Seeds
		3.2 Hand-Pollination of Scarlet Runner Bean Flowers
		3.3 Collecting Scarlet Runner Bean Seeds Containing Embryos at the Globular Stage
		3.4 Dissecting Scarlet Runner Bean Globular Stage Whole-Mount Embryos
		3.5 Fixing Seeds with Globular-Stage Embryos for LCM or in situ Hybridization
		3.6 Seed Dehydration for LCM
		3.7 Seed Dehydration for in situ Hybridization
		3.8 Infiltration and Embedding of Seeds for LCM or in situ Hybridization
		3.9 Sectioning Paraffin-Embedded Seeds for LCM or in situ Hybridization
		3.10 LCM of Scarlet Runner Bean Globular Stage Embryos
	4 Notes
	References
Chapter 16: Microscopical Detection of Cell Death Processes During Scots Pine Zygotic Embryogenesis
	1 Introduction
	2 Materials
		2.1 Timing of Cone Collection, Seed Preparation, and Fixation
		2.2 Dehydration and Paraffin Infiltration
		2.3 Sectioning
		2.4 Toluidine Blue Staining
		2.5 Dewaxing and Rehydration of Slide
		2.6 Acridine Orange (AO) Staining
		2.7 TUNEL Assay
	3 Methods
		3.1 Timing of Cone Collection, Seed Preparation and Fixation
			3.1.1 Timing of Cone Collection
			3.1.2 Protocol for Making Fixative
			3.1.3 Seed Preparation and Fixation
		3.2 Dehydration and Paraffin Infiltration
		3.3 Sectioning
		3.4 Toluidine Blue Staining
		3.5 Dewaxing and Rehydration
		3.6 Acridine Orange Staining of Nucleic Acids
		3.7 Detection of DNA Fragmentation with TUNEL Assay
	4 Notes
	References
Part VI: In Vitro Systems to Study Embryogenesis
Chapter 17: Regulation of Somatic Embryo Development in Norway Spruce
	1 Introduction
	2 Materials
		2.1 Standard Procedures for Stimulating Development and Maturation of Somatic Embryos
		2.2 Establishment of Transgenic Embryogenic Cell Lines
		2.3 Phenotyping
		2.4 Confocal Microscopy
		2.5 Histological Analysis of Somatic Embryos
		2.6 Progression of Somatic Embryo Development
		2.7 Time-Lapse Tracking of Somatic Embryo Development
		2.8 GUS Reporter Gene Expression
	3 Methods
		3.1 Standard Procedures for Stimulating Development and Maturation of Somatic Embryos
		3.2 Establishment of Transgenic Embryogenic Cell Lines
		3.3 Phenotyping Early and Late Somatic Embryos
		3.4 Confocal Microscopy of Early and Late Somatic Embryos
		3.5 Histological Analysis of Somatic Embryos
			3.5.1 Fixation
			3.5.2 Dehydration
			3.5.3 Embedding
		3.6 Progression of Somatic Embryo Development
		3.7 Time-Lapse Tracking of Somatic Embryo Development
		3.8 GUS Reporter Gene Expression
	4 Notes
	References
Chapter 18: In Vitro Production of Zygotes by Electrofusion of Rice Gametes
	1 Introduction
	2 Materials
		2.1 Isolation and Transfer of Gametes
		2.2 Fusion of Gametes
		2.3 Culture of Zygotes into Embryo-Like Structure and Plantlets
	3 Methods
		3.1 Isolation of Gametes
		3.2 Fusion of Gametes
		3.3 Culture of Zygotes into Embryo-Like Structure and Plantlets
	4 Notes
	References
Chapter 19: Isolated Microspore Culture in Brassica napus
	1 Introduction
	2 Materials
		2.1 Plant Material
		2.2 Equipment
		2.3 Disinfection, Isolation, Culture, Ploidy Analysis, and Acclimation
		2.4 Culture Media and Chemicals
	3 Methods
		3.1 Donor Plant Growth Conditions
		3.2 In Vitro Culture of Isolated Microspores
		3.3 MDE Germination and Acclimation
		3.4 Analysis of Ploidy Level
	4 Notes
	References
Chapter 20: Anther Culture in Eggplant (Solanum melongena L.)
	1 Introduction
	2 Materials
		2.1 Plant Material
		2.2 Equipment
		2.3 Materials
		2.4 Solutions for Anther Culture
		2.5 Culture Media
	3 Methods
		3.1 Donor Plant Growth Conditions
		3.2 In Vitro Culture of Anthers
		3.3 Analysis of Ploidy Level
	4 Notes
	References
Correction to: Small RNA In Situ Hybridizations on Sections of Arabidopsis Embryos
Index