Doubled Haploid Technology: Volume 1: General Topics, Alliaceae, Cereals (Methods in Molecular Biology, 2287)

Preface
Contents
Contributors
Part I: General Topics and Transversal Methods in DH Technology
	Chapter 1: Overview of In Vitro and In Vivo Doubled Haploid Technologies
		1 Introduction
		2 In Vitro Methods
			2.1 Methods Based on In Vitro Androgenesis
				2.1.1 Anther Culture
				2.1.2 Isolated Microspore Culture
					Methods Based on In Vitro Gynogenesis
		3 In Vivo Approaches
			3.1 Haploidization by Wide-Crosses
			3.2 Haploidization by Intra-specific Crosses
		4 Concluding Remarks
		References
	Chapter 2: Applications of Doubled Haploids in Plant Breeding and Applied Research
		1 Introduction
		2 Recombination and Fixation of Genetic Variance
		3 Use of Haploid and DH Technologies to Develop Wide Crosses and Use of Hybridization
		4 DHs for Mapping and Marker-Assisted Selection
		5 DHs for Genomic Selection and Genomic Prediction
		6 DHs and Genetic Transformation
		7 Use of Haploid Cells and Tissues for Increasing Genetic Variation by Mutation
		8 Genome Editing Using DH Technologies
		9 Epigenetics and  DHs
		10 Reverse Breeding
		11 Future Perspectives
		References
	Chapter 3: Species with Haploid or Doubled Haploid Protocols
		1 Introduction
		References
	Chapter 4: Analysis of Ploidy in Haploids and Doubled Haploids
		1 Introduction
		2 Overview of Techniques for Ploidy Level Determination
		3 Ploidy Level Determination by Flow Cytometry
			3.1 Methodology
			3.2 Advantages and Limitations
			3.3 Representation of Flow Cytometry  Data
			3.4 Stains and Standards
			3.5 Flow Cytometry as a Diagnostic Tool Beyond Ploidy Estimation
		4 Chromosome Counting
			4.1 Chromosome Staining Prior to Squashing
				4.1.1 Aceto-orcein/Acetocarmine Stains
				4.1.2 The Feulgen Method
			4.2 Chromosome Staining Following Squashing
				4.2.1 Fluorescence Methods
				4.2.2 Nonfluorescent Stains
				4.2.3 DNA Image Analysis
		5 Techniques for Ploidy Level Determination not Requiring Dividing Cells
			5.1 Chloroplast Counting in Stomatal Guard Cells
			5.2 Stomatal Size and Length
			5.3 Counting of Nucleoli
			5.4 Pollen Formation and Viability
			5.5 Analysis of Cell  Size
			5.6 Analysis of Morphological Markers
		6 Concluding Remarks
		References
	Chapter 5: Methods for Chromosome Doubling
		1 Introduction
		2 Importance of Chromosome Doubling in Haploid Systems
		3 Spontaneous Genome Doubling
		4 Induced Genome Doubling
		5 Antimicrotubular Agents
		6 In Vivo Induced Chromosome Doubling
		7 In Vitro Induced Chromosome Doubling
		8 Methods to Verify Chromosome Doubling
		9 Conclusion
		References
Part II: Doubled Haploids in Alliaceae
	Chapter 6: Doubled Haploid Onion (Allium cepa L.) Production Via In Vitro Gynogenesis
		1 Introduction
		2 Materials
			2.1 Plant Material
			2.2 Equipment
			2.3 Laboratory Materials
			2.4 Greenhouse/Growth Chamber Materials
			2.5 Solutions
			2.6 Culture Media
		3 Methods
			3.1 Donor Plant Growth Conditions
			3.2 Establishment of Gynogenesis Induction Cultures and Obtaining Gynogenic Plants
			3.3 Determination of Ploidy of Androgenic Regenerants with Flow Cytometry Analysis
			3.4 Chromosome Doubling
				3.4.1 Induced Chromosome Doubling Through Treatment of Whole Basal Explants with Antimitotic Agents
				3.4.2 Spontaneous Chromosome Doubling in Somatic Regenerants from Cultured Flower Buds of Haploid Plants
				3.4.3 Recovery of Diploid Plants from Tetraploid Gynogenic Plants by Second Cycle Gynogenesis
			3.5 Acclimatization to Ex Vitro Conditions
			3.6 Production of Seeds and Checking for Plant Uniformity in DH Lines
		4 Notes
		References
	Chapter 7: In Vitro Gynogenesis in Leek (Allium ampeloprasum L.)
		1 Introduction
		2 Materials
			2.1 Plant Material
			2.2 Equipment
			2.3 Laboratory Materials
			2.4 Greenhouse/Growth Chamber Materials
			2.5 Solutions
			2.6 Culture Media
		3 Methods
			3.1 Donor Plant Growth Conditions
			3.2 Establishment of Gynogenesis Induction Cultures and Obtaining Gynogenic Plants
			3.3 Determination of Ploidy of Androgenic Regenerants with Flow Cytometry Analysis
			3.4 Acclimatization to Ex Vitro Conditions
		4 Notes
		References
Part III: Doubled Haploids in Cereals
	Chapter 8: Barley Isolated Microspore Culture
		1 Introduction
		2 Materials
			2.1 Lab Tools
			2.2 Equipment and Facilities
			2.3 Plant Tissue Media
		3 Methods
			3.1 Donor Plants and Growth Conditions
			3.2 Harvesting of Spikes
			3.3 Pretreatment of Anthers
			3.4 Microspore Isolation
			3.5 Microspore Culture
			3.6 Transfer of Embryos
			3.7 Plant Transfer to Magenta Boxes
			3.8 Plant Transfer to Soil
		4 Notes
		References
	Chapter 9: Site-Directed Mutagenesis in Barley Using RNA-Guided Cas Endonucleases During Microspore-Derived Generation of Doub...
		1 Introduction
		2 Materials
			2.1 Plant Material
			2.2 Equipment
			2.3 Laboratory Materials
			2.4 Glasshouse and Growth Chamber Materials
			2.5 Stock Solutions and Chemicals
			2.6 Culture Media
		3 Methods
			3.1 Vector Construction and Bacterial Strains
			3.2 Growth of Donor Plants
			3.3 Isolation of Microspores
				3.3.1 Spike Pretreatment
				3.3.2 Isolation, Purification and Precultivation of Microspores
			3.4 Agrobacterium-Mediated Gene Transfer to Embryogenic Pollen
				3.4.1 Preparation of A. tumefaciens Stocks
				3.4.2 Co-Cultivation of Embryogenic Pollen Cultures and A. tumefaciens
			3.5 Regeneration of Transgenics
			3.6 Analysis of Putative Transgenic Plants
				3.6.1 Ploidy Determination and Colchicine-Induced Whole-Genome Duplication
				3.6.2 Molecular Analyses: DNA Isolation, PCR and Sequencing
		4 Notes
		References
	Chapter 10: Generation of Doubled Haploid Barley by Interspecific Pollination with Hordeum bulbosum
		1 Introduction
		2 Materials
			2.1 Plant Material
			2.2 Equipment
			2.3 Laboratory Materials
			2.4 Glasshouse and Growth Chamber Materials
			2.5 Stock Solutions
				2.5.1 Mineral Salts
				2.5.2 Organic Supplements
				2.5.3 Selective Agents
				2.5.4 Gelling Agent
				2.5.5 Other Solutions
			2.6 Culture Media
				2.6.1 Embryo Rescue
				2.6.2 Plant Regeneration
		3 Methods
			3.1 Plant Growth Conditions
				3.1.1 Barley
				3.1.2 Hordeum bulbosum
			3.2 Generation of Haploid Barley
				3.2.1 Emasculation
				3.2.2 Pollination of Barley with H. bulbosum Pollen
				3.2.3 Auxin Treatment
				3.2.4 Embryo Rescue
				3.2.5 Colchicine-Induced Whole-Genome Duplication
		4 Notes
		References
	Chapter 11: Bread Wheat Doubled Haploid Production by Anther Culture
		1 Introduction
		2 Materials
			2.1 Plant Material
			2.2 Equipment
			2.3 Mother Plant Growth/Disinfection/Sterilization, Culture, Acclimation and Ploidy Analysis
			2.4 Culture Media
			2.5 Chemicals and Solutions for Anther Culture, Ploidy Analysis and Watering of the Plants
		3 Methods
			3.1 Growth Conditions of the Mother Plants
			3.2 Harvest of Spikes and Sterilization
			3.3 Ovary Pre-conditioned Medium
			3.4 Anther Pre-Treatment and Culture
			3.5 Vernalization and Transfer to  Soil
			3.6 Ploidy Analysis, Chromosome Doubling and Seed Production
		4 Notes
		References
	Chapter 12: Unpollinated Ovaries Used to Produce Doubled Haploid Lines in Durum Wheat
		1 Introduction
		2 Materials
			2.1 Equipment and Laboratory Materials
			2.2 Glassware
			2.3 Solutions
			2.4 Culture Media
		3 Methods
			3.1 Donor Plants and Growth Conditions
			3.2 Spike Selection and Pretreatment
			3.3 Ovary Culture and Plant Regeneration
			3.4 Chromosome Studies
			3.5 Chromosome Doubling
			3.6 Transfer of Plants to  Soil
		4 Notes
		References
	Chapter 13: In Vitro Anther Culture for Doubled Haploid Plant Production in Spelt Wheat
		1 Introduction
		2 Materials
			2.1 Equipment
			2.2 Culture Containers
			2.3 Solutions and Culture Media
		3 Methods
			3.1 Growing of Plant Materials
			3.2 Pre-treatment of Donor Tillers
			3.3 Preparation of Anther Cultures
			3.4 Plant Regeneration of Anther Culture-Derived  ELS
			3.5 Acclimatization of Anther Culture-Derived Green Plantlets
			3.6 Flow Cytometry
			3.7 Growing of Anther Culture-Derived Plantlets in DH Nursery
			3.8 Integration of DH Lines into Breeding Program
		4 Notes
		References
	Chapter 14: Production of Wheat Doubled Haploids Through Intergeneric Hybridization with Maize
		1 Introduction
		2 Materials
			2.1 Greenhouse
			2.2 Laboratory
		3 Methods
			3.1 Donor Plants and Growth Conditions
			3.2 Hybridization and Post-pollination Treatment
			3.3 Embryo Culture
			3.4 Sampling and Chromosome Doubling
			3.5 Seed Collection, Multiplication and Breeding
		4 Notes
		References
	Chapter 15: Isolation of Staged and Viable Maize Microspores for DH Production
		1 Introduction
		2 Materials
			2.1 Plant Material
			2.2 Equipment and Materials
			2.3 Reagents and Stocks
			2.4 Sucrose-Based Media for Microspore Isolation and Culture (See Note 4)
			2.5 Maltose-Based Media for Microspore Isolation and Culture (See Note 4)
			2.6 Media for Plantlet Regeneration from Embryos
			2.7 Reagents for Cytological Assays
		3 Methods
			3.1 Tassel Sampling and Pretreatment
			3.2 Microspore Isolation and Purification
			3.3 Microspore Culture
			3.4 Plantlet Regeneration and Transfer to  Soil
		4 Notes
		References
	Chapter 16: Triticale Isolated Microspore Culture for Doubled Haploid Production
		1 Introduction
		2 Materials
			2.1 Surface Sterilized Materials
			2.2 Growth Chamber Materials
			2.3 Stock Solutions
			2.4 Culture Media
			2.5 Disinfecting Agents
		3 Methods
			3.1 Handling of Donor Plants
			3.2 Collection of Donor Spikes
			3.3 Cold Pretreatment of Donor Spikes
			3.4 Selection of Spikes for Ovary Donors
			3.5 Disinfection of Spikes
			3.6 Microspore Isolation Procedure
			3.7 Isolated Microspore Culture Method
			3.8 Regeneration of Doubled Haploid Plantlets
			3.9 Rooting and Soil Transfer of Plantlets
		4 Notes
		References
	Chapter 17: Oat (Avena sativa L.) Anther Culture
		1 Introduction
		2 Materials
			2.1 Equipment
			2.2 General Materials
			2.3 Solutions and Media
		3 Methods
			3.1 Donor Plant Growth
			3.2 Collecting Tillers and Cold Pretreatment
			3.3 Determination of Microspore Developmental Stage
			3.4 Tiller Disinfection
			3.5 Anther Isolation
			3.6 Regeneration of Embryogenic Structures and Rooting of Haploid Plants
			3.7 Acclimatization of Haploid Plants
			3.8 Determination of Ploidy Level
			3.9 Chromosome Doubling and Growth of DH Plants
		4 Notes
		References
	Chapter 18: Oat Doubled Haploid Production Through Wide Hybridization with Maize
		1 Introduction
		2 Materials
			2.1 Equipment
			2.2 General Materials
			2.3 Solutions and Media
		3 Methods
			3.1 Donor Plant Growth
			3.2 Panicles Emasculation and Pollination
			3.3 Disinfection of Enlarged Ovaries and Isolation of Haploid Embryos
			3.4 Haploid Embryos Isolation
			3.5 Regeneration of Haploid Embryos and Acclimation of Haploid Plants
			3.6 Ploidy-Level Determination
			3.7 Chromosome Doubling
			3.8 Growth of DH Plants
		4 Notes
		References
	Chapter 19: Anther Culture and Chromosome Doubling in Mediterranean Japonica Rice
		1 Introduction
		2 Materials
			2.1 Plant Material
			2.2 General Labware
			2.3 Equipment
			2.4 Solutions, Culture Media and Rice Substrate
		3 Methods
			3.1 Donor Plant Growth Conditions
			3.2 Tiller Collection and Cold Pretreatment
			3.3 In Vitro Anther Culture
			3.4 Ploidy-Level Determination
			3.5 Chromosome Doubling of Haploid Plantlets
			3.6 Acclimation and Seed Recovery
		4 Notes
		References
	Chapter 20: High-Throughput Doubled Haploid Production for Indica Rice Breeding
		1 Introduction
		2 Materials
			2.1 Plant Materials
			2.2 Equipment
			2.3 Infrastructures
			2.4 Glassware
			2.5 General Labware
			2.6 Chemicals and Reagents
			2.7 Culture Media and Solutions
		3 Methods
			3.1 Development of Crosses
				3.1.1 Hybridization
				3.1.2 Raising F1 Plants
				3.1.3 Test of Hybridity
			3.2 Anther Culture for DH Production
				3.2.1 Preparatory Stage: Stage  I
				3.2.2 Initiation of Anther Culture: Stage  II
				3.2.3 Callus Induction and Proliferation: Stage  III
				3.2.4 Green Plantlet Regeneration: Stage  IV
				3.2.5 Production of DHs: Stage  V
				3.2.6 Acclimatization and Plant Establishment: Stage  VI
				3.2.7 Test for Homozygosity and Recovery of DHs: Stage  VII
		4 Notes
		References
Index