Somatic Embryogenesis: Methods and Protocols

Preface
Contents
Contributors
Chapter 1: Overview of Somatic Embryogenesis
	1 Introduction
	2 Overview of Somatic Embryogenesis
	3 Types of Somatic Embryogenesis
		3.1 Direct Somatic Embryogenesis
		3.2 Indirect Somatic Embryogenesis
	4 Secondary Somatic Embryogenesis
	5 Concluding Remarks
	References
Part I: Somatic Embryogenesis: Advances in Recalcitrant Species
	Chapter 2: Internal and External Regulatory Elements Controlling Somatic Embryogenesis in Catharanthus: A Model Medicinal Plant
		1 Introduction
		2 Steps of Embryogenesis
			2.1 Preparation of Explants
			2.2 Induction of Non-embryogenic and Embryogenic Callus from Explants
			2.3 Embryo Differentiation and Proliferation
			2.4 Maturation and Germination of Somatic Embryos
			2.5 Plantlet Development and Transfer to Outdoor Conditions
		3 Key Elements/Factors Regulating Somatic Embryogenesis
			3.1 Genotype, Variety, Type of Explants
			3.2 Herbaceous vs. Woody Genera and Embryogenesis
			3.3 Light Quality and Intensity
			3.4 Aeration and Dissolved Oxygen (DO) Level in Medium
			3.5 The Role of Energy or Carbon Source
			3.6 PGRs as Signaling Elements
			3.7 Cytokinin Signaling and Embryogenesis
			3.8 Some Genes Related to Somatic Embryogenesis
			3.9 Proteins and Somatic Embryogenesis
		4 Conclusions
		References
	Chapter 3: Advances in Somatic Embryogenesis in Vanilla (Vanilla planifolia Jacks.)
		1 Introduction
		2 Overview of Vanilla planifolia
		3 In Vitro Morphogenesis
		4 Organogenesis
			4.1 Direct Organogenesis
			4.2 Indirect Organogenesis
		5 Protein and Molecular Studies on Recalcitrance to Somatic Embryogenesis
		6 Conclusions and Perspectives of Somatic Embryogenesis in Vanilla
		References
	Chapter 4: Somatic Embryogenesis in Pines
		1 Introduction
		2 Materials
			2.1 Plant Material
			2.2 Media
				2.2.1 Initiation, Proliferation
				2.2.2 Maturation
				2.2.3 Germination
				2.2.4 Cryopreservation
		3 Methods
			3.1 Media Preparation
			3.2 Initiation and Proliferation
			3.3 Maturation
			3.4 Germination and Acclimatization
			3.5 Cryopreservation
		4 Notes
		References
Part II: Somatic Embryogenesis in Agroindustrial Species: Protocols
	Chapter 5: Use of Thin Cell Layer Technique for Induction of Somatic Embryogenesis of Agave fourcroydes
		1 Introduction
		2 Materials
			2.1 Plant Material
			2.2 Instrumentation
			2.3 Culture Dishes or Glassware/Plasticware
			2.4 Other Materials
			2.5 Reagents
				2.5.1 Stock Solutions
				2.5.2 Culture Media
				2.5.3 Sterilization of Media
		3 Methods
			3.1 Plant Material
			3.2 Maintenance of Plant Material and Source of Explant
			3.3 TCL Technique
			3.4 Induction of Somatic Embryogenesis
		4 Notes
		References
	Chapter 6: Theobroma cacao: Somatic Embryogenesis
		1 Introduction
		2 Material
			2.1 Instrumentation, Glassware, and Other Materials
			2.2 Reagents, Solutions, and Culture Media
			2.3 Culture Media
			2.4 Greenhouses
			2.5 Plant Material
		3 Method
			3.1 Culture Media
			3.2 Culture Conditions
			3.3 Direct Somatic Embryogenesis
				3.3.1 Bud Disinfection
				3.3.2 Somatic Embryogenesis Induction
			3.4 Indirect Somatic Embryogenesis
				3.4.1 Onto Solid Culture Medium
					Embryogenic Calli Production
					Embryo Expression
				3.4.2 Into Liquid Culture Medium
					Embryogenic Calli Production
					Embryo Expression
			3.5 Embryo-to-Plant Conversion
				3.5.1 Embryo Development
				3.5.2 Plantlet Acclimatization
			3.6 Process Efficiency
		4 Notes
		References
	Chapter 7: Somatic Embryogenesis in Sugarcane (Saccharum spp.)
		1 Introduction
		2 Materials
			2.1 Explant Source Preparation  Step
			2.2 Culture Medium Preparation and Components
			2.3 Laboratory Structure
		3 Methods
			3.1 Explant Source Preparation
			3.2 Induction Culture Medium Preparation
			3.3 Multiplication Culture Medium Preparation
			3.4 Pretreatment of Maturation Culture Medium Preparation
			3.5 Maturation Culture Medium Preparation
			3.6 Semisolid Conversion Culture Medium Preparation
			3.7 Liquid Conversion Culture Medium Preparation
			3.8 Embling Elongation Culture Medium Preparation
			3.9 Callus Embryogenic Induction, Multiplication, and Pretreatment of Maturation
			3.10 Callus Multiplication and Pretreatment of Maturation
			3.11 Maturation and Somatic Embryo Differentiation
			3.12 Somatic Embryo Conversion in Semisolid Culture Medium or Liquid Culture Medium in a RITA Temporary Immersion System
			3.13 Embling Elongation and Acclimatization
		4 Notes
		References
	Chapter 8: Somatic Embryogenesis in Banana (Musa spp.)
		1 Introduction
		2 Materials
			2.1 Culture Medium
			2.2 In Vitro Establishment and Incubation Materials
			2.3 Acclimatization
		3 Methods
			3.1 Obtaining Explants to Induce Somatic Embryogenesis
				3.1.1 Shoot-Tip Establishment
				3.1.2 Shoot-Tip Multiplication
			3.2 Induction of Somatic Embryogenesis from Callus Formation in Semisolid Culture Medium or in Liquid Culture Medium
			3.3 Multiplication of Embryogenic Cell Suspensions
			3.4 Formation of Somatic Embryos in Semisolid or Liquid Culture Medium
			3.5 Maturation of Somatic Embryos in Semisolid or Liquid Culture Medium
				3.5.1 Maturation of Somatic Embryos in Semisolid Culture Medium
				3.5.2 Maturation of Somatic Embryos in Liquid Culture Medium
			3.6 Germination of Somatic Embryos in Semisolid Culture Medium or in Temporal Immersion System, Type Rita
				3.6.1 Germination of Somatic Embryos in Semisolid Culture Media
				3.6.2 Germination of Somatic Embryos in Temporal Immersion System, Type Rita
			3.7 Conversion into Plants of Somatic Embryos
		4 Notes
		References
	Chapter 9: Citrus Protoplast Isolation and Plant Regeneration Through Somatic Embryogenesis
		1 Introduction
		2 Materials
			2.1 Physical Material and Equipment
			2.2 Medium Stock Solutions
			2.3 Plant Growth Regulator Stocks
			2.4 Enzymes Stock Solutions
			2.5 CPW Solution
			2.6 Polyethylene Glycol (PEG) Solutions
			2.7 Callus-Induction and Cell Suspension Maintenance Medium
			2.8 Protoplast Isolation and Culture Medium
			2.9 Protoplast Culture and Plant Regeneration Medium
		3 Methods
			3.1 Initiation and Maintenance of Embryogenic Cultures
			3.2 Explant Preparation, Protoplast Isolation and Purification
				3.2.1 Preparation and Enzymatic Incubation of Embryogenic Callus
				3.2.2 Preparation and Enzymatic Incubation of Leaves
				3.2.3 Protoplast Isolation and Purification
			3.3 Polyethylene Glycol (PEG)-Induced Protoplast Fusion or Transformation
			3.4 Protoplast Culture and Plant Regeneration
		4 Notes
		References
	Chapter 10: Somatic Embryogenesis in Citrus (Citrus spp.), var. Valencia
		1 Introduction
		2 Materials
			2.1 Plant Material and Equipment
			2.2 Culture Media
		3 Methods
			3.1 Callus Induction from Nucellus and Callus Multiplication
			3.2 Callus Sieving
			3.3 Somatic Embryo Germination and Regeneration
		4 Notes
		References
	Chapter 11: Somatic Embryogenesis from Immature Olive Zygotic Embryos
		1 Introduction
		2 Materials
			2.1 Collection and Disinfection of Immature Olive Fruits
			2.2 Culture Media and Culture Conditions
		3 Methods
			3.1 Fruit Disinfection and Embryos Preparation
			3.2 Callus Culture
			3.3 Somatic Embryos Multiplication and Maturation
		4 Notes
		References
	Chapter 12: Somatic Embryogenesis in Papaya (Carica papaya L.)
		1 Introduction
		2 Materials
			2.1 Plant Material
			2.2 Disinfection of Immature Fruits
			2.3 Extraction of Immature Zygotic Embryos
			2.4 Formation of Somatic Embryos from Zygotic Embryos
			2.5 Secondary Multiplication of Somatic Embryos
			2.6 Maturation of Somatic Embryos
			2.7 Morpho-Anatomical Characterization of Somatic Embryos
				2.7.1 Scanning Electron Microscopy (SEM)
				2.7.2 Histological Analysis
			2.8 Germination of Somatic Embryos
			2.9 Plant Growth In Vitro
			2.10 Sex Determination by PCR of Papaya In Vitro Shoots
			2.11 Ex Vitro Acclimatization
		3 Methods
			3.1 Disinfection of Immature Fruits and Extraction of Zygotic Embryos
			3.2 Formation of Somatic Embryos from Zygotic Embryos
			3.3 Secondary Multiplication of Somatic Embryos
			3.4 Maturation of Somatic Embryos
			3.5 Morpho-Anatomical Characterization of Somatic Embryos
				3.5.1 Scanning Electron Microscopy (SEM)
				3.5.2 Histological Analysis
			3.6 Germination of Somatic Embryos
			3.7 Plant Growth In Vitro
			3.8 Sex Determination by PCR of Papaya In Vitro Shoots
			3.9 Ex Vitro Acclimatization
		4 Notes
		References
Part III: Somatic Embryogenesis in Ornamental Species: Protocols
	Chapter 13: Somatic Embryogenesis of Anthurium andraeanum Linden., -A Tropical  Florists' Plant
		1 Introduction
		2 Materials
			2.1 Mother Plant Maintenance and Handling of Microplants
			2.2 Constituents of Culture Medium
			2.3 Medium Composition for the Different Stages of Somatic Embryogenesis
			2.4 Surface Sterilization and Culture Establishment
			2.5 Histology and SEM Analysis
			2.6 Glassware and Stationaries
			2.7 Instruments and Infrastructure
		3 Methods
			3.1 Mother Plants Maintenance
			3.2 Media Preparation
			3.3 In Vitro Culture Establishment
			3.4 Embryogenic Callus Induction
			3.5 Multiplication of Embryogenic Callus
			3.6 Induction of Somatic Embryogenesis
			3.7 Maturation and Germination of Somatic Embryos
			3.8 Confirmation of Somatic Embryogenesis Through Histological and Scanning Electron Microscopic (SEM) Analysis
			3.9 Hardening of Somatic Plants
		4 Notes
		References
Part IV: Strategies for Obtaining Somatic Embryogenesis
	Chapter 14: Use of Thin Cell Layer (TCL) to Obtain Somatic Embryogenesis
		1 Introduction
		2 Description
		3 Contributions
		4 Orchids
		5 Herbaceous Plants
		6 Woody and Recalcitrant Species
		7 Perspectives on the Use of TCLs
		8 Conclusion
		References
	Chapter 15: Inorganic Compounds that Aid in Obtaining Somatic Embryos
		1 Introduction
		2 Media Culture
			2.1 Murashige and Skoog Medium
			2.2 Gamborg B5 Medium
			2.3 LP and Litvay´s Media
			2.4 DKW Medium
			2.5 N6 Medium
			2.6 Nitsch Medium
			2.7 Yasuda Medium
			2.8 SH Medium
			2.9 DCR Medium
			2.10 WPM Medium
		3 Use of Main Inorganic Elements in SE
			3.1 Magnesium
			3.2 Iron
			3.3 Manganese
			3.4 Zinc
			3.5 Calcium
			3.6 Boron
			3.7 Copper
			3.8 Silver
			3.9 Silicon
		References
	Chapter 16: In Vitro Stress-Mediated Somatic Embryogenesis in Plants
		1 Introduction
		2 Stress in Plants and Morphogenesis
			2.1 Osmotic Stress in Somatic Embryogenesis
		3 Oxidative Stress in Somatic Embryogenesis
			3.1 Plant Growth Regulators Stress Mediating Somatic Embryogenesis
			3.2 Heavy Metal Stress in Somatic Embryogenesis
		References
Part V: Biotechnological Strategies for the Study and Use of Somatic Embryogenesis
	Chapter 17: Histological Procedures Carried out During Coconut Somatic Embryogenesis
		1 Introduction
		2 Materials
			2.1 Phosphate Buffer
			2.2 Paraformaldehyde Fixative
			2.3 JB-4 Embedding  Kit
			2.4 Schiff´s Reagent
			2.5 Periodic Acid (1%)
			2.6 Naphthol Blue Black (1%)
		3 Methods
			3.1 Fixation
			3.2 Embedding
			3.3 Staining with PAS-SCHIFF-Naphthol Blue Black
		4 Notes
		References
	Chapter 18: Somatic Embryogenesis of Brachiaria brizantha (Syn. Urochloa brizantha) Analyzed by In Situ Hybridization
		1 Introduction
		2 Materials
			2.1 Common Chemicals and Materials
			2.2 Probe Synthesis
			2.3 Fixation
			2.4 Paraffin Embedding
			2.5 BMM Embedding
			2.6 Pre-treatment
			2.7 In Situ Hybridization
			2.8 Post-hybridization
		3 Methods
			3.1 Probe Synthesis
			3.2 Tissue Processing
			3.3 Paraplast Embedding and Sectioning
			3.4 BMM Infiltration, Embedding, and Sectioning
			3.5 In Situ Hybridization (See Note 7)
			3.6 Post-hybridization
			3.7 Whole-Mount In Situ Hybridization
		4 Notes
		References
Part VI: General Conclusions and Perspectives
	Chapter 19: Perspectives of Somatic Embryogenesis: Concluding Remarks
		1 Introduction
		2 Concluding Remarks
		3 Perspectives
		References
Index