Buckwheat: Methods and Protocols (Methods in Molecular Biology, 2791)

Preface
Contents
Contributors
Chapter 1: Callus Induction Followed by Regeneration and Hairy Root Induction in Common Buckwheat
	1 Introduction
	2 Materials
		2.1 Plants and Bacteria
		2.2 Equipment
		2.3 Reagents
	3 Methods
		3.1 Seed Preparation, Germination, and Sprouting
			3.1.1 Seed Preparation and Sterilization
			3.1.2 Seed Sprouting
		3.2 Callus Induction and Regeneration (See Note 1)
			3.2.1 Callus Induction (5-6 Weeks)
			3.2.2 Shoot Initiation (1-2 Months)
			3.2.3 Shoot Multiplication and Outgrowth (1-3 Months)
			3.2.4 Root Initiation (2-3 Weeks)
			3.2.5 Acclimation to Soil Conditions (1-2 Weeks)
		3.3 Hairy Root Induction
			3.3.1 Transformation
			3.3.2 Cocultivation
			3.3.3 Selection
		3.4 Callus Induction in Hairy Roots
	4 Notes
	References
Chapter 2: Immunostaining for Epigenetic Modifications in Fagopyrum Calli
	1 Introduction
	2 Materials
		2.1 Tissue Preparation
		2.2 Tissue Fixation
		2.3 Wax Embedding
		2.4 Tissue Sectioning
		2.5 Dewaxing
		2.6 Immunostaining
	3 Methods
		3.1 Tissue Preparation
		3.2 Material Fixation
		3.3 HYRAX M40 Rotary Microtome Slicing
		3.4 De-embedding and Immunostaining
	4 Notes
	References
Chapter 3: Quantitative Analysis of Epigenetic Modifications in Fagopyrum Nuclei with Confocal Microscope, ImageJ, and R Studio
	1 Introduction
	2 Materials
	3 Methods
		3.1 Image Acquisition
		3.2 Image Analysis
		3.3 Statistical Analysis and Visualization with  R
	4 Notes
	References
Chapter 4: Phenolic Determination in Proembryogenic Cell Complexes of Buckwheat Morphogenic Cell Culture with Osmium Tetroxide...
	1 Introduction
	2 Materials
		2.1 Plant Material
		2.2 Materials
	3 Methods
		3.1 Fixation Material (See Note 2)
		3.2 Postfixation
		3.3 Dehydration
		3.4 Embedding and Polymerization
		3.5 Staining of Section with Toluidine Blue O Dye
			3.5.1 Identification of Phenol in PECC by Toluidine Blue O Dye with Prefixation in Karnovsky´s Fixative (See Note 6)
			3.5.2 Identification of Phenol in PECC Prefixed with FeCl3 and Fixed in Karnovsky´s Fixative (See Note 7)
	4 Notes
	References
Chapter 5: Protoplast Isolation, Culture, and Regeneration in Common and Tartary Buckwheat
	1 Introduction
	2 Materials
		2.1 Solutions and Equipment
		2.2 Media and Solutions
	3 Methods
		3.1 Hypocotyl-Derived Protoplasts of Common Buckwheat
			3.1.1 Seed Sterilization and Germination
			3.1.2 Protoplast Isolation and Culture
			3.1.3 Protoplast-Derived Callus Multiplication and Plant Regeneration
		3.2 MC-Derived Protoplasts of Common Buckwheat
			3.2.1 Protoplast Isolation and Culture
			3.2.2 Protoplast-Derived Callus Multiplication and Plant Regeneration
		3.3 Hypocotyl-Derived Protoplasts of Tartary Buckwheat
			3.3.1 Seed Sterilization and Germination
			3.3.2 Protoplast Isolation and Culture
			3.3.3 Protoplast-Derived Callus Multiplication and Plant Regeneration
		3.4 Protoplasts Derived from MC of Tartary Buckwheat
			3.4.1 Isolation and Culture of Protoplasts
			3.4.2 Callus Multiplication and Plant Regeneration of Tartary Buckwheat
	References
Chapter 6: Immunohistochemical Detection of the Wall Components on the Example of Shoot Apical Meristem of Fagopyrum esculentu...
	1 Introduction
	2 Materials
		2.1 Culture
		2.2 Fixation
		2.3 Dehydration
		2.4 Infiltration and Embedding
		2.5 Tissue Sectioning
		2.6 Immunostaining
	3 Methods
		3.1 Culture
		3.2 Fixation
		3.3 Dehydration
		3.4 Embedding
		3.5 Sectioning
		3.6 Immunostaining
		3.7 Observations
	4 Notes
	References
Chapter 7: Immunodetection of Cell Wall Components in Studies on Cell Wall Rebuilding in Fagopyrum esculentum and Fagopyrum ta...
	1 Introduction
	2 Materials
		2.1 Protoplast Staining
		2.2 Protoplast Selection
		2.3 Protoplast Culture
		2.4 Fixation
		2.5 Immunostaining
		2.6 Microscopical Observations
	3 Methods
		3.1 Protoplast Staining
		3.2 Protoplast Selection
		3.3 Protoplast Culture
		3.4 Fixation
		3.5 Immunostaining
		3.6 Microscopical Observations
	4 Notes
	References
Chapter 8: Visualization of Fagopyrum esculentum and Fagopyrum tataricum Chromosomes and Micronuclei
	1 Introduction
	2 Materials
		2.1 Plant Material, Disposables, and Equipment
		2.2 Solutions
	3 Methods
		3.1 Seed Germination
		3.2 Pretreatment and Slide Preparation
	4 Notes
	References
Chapter 9: Embryo Rescue Technique in Buckwheat Fagopyrum esculentum Moench
	1 Introduction
	2 Materials
		2.1 Nutrient Media Composition
		2.2 Sterilizing Solution
		2.3 Instruments and Equipment
	3 Methods
		3.1 Preparing of Nutrient Media
		3.2 Culture of 4-6 Days´ Embryos (I Group)
		3.3 Culture of 7-10 Days´ Embryos (II Group)
		3.4 Culture of 11-15 Days´ Embryos (III Group)
		3.5 Seedling Acclimatization
	4 Notes
	References
Chapter 10: Comprehensive Embryological Analyses of Common Buckwheat (Fagopyrym esculentum Moench)
	1 Introduction
	2 Materials
		2.1 Solutions and Stains
		2.2 Equipment
	3 Methods
		3.1 Paraffin Method for Observation of Microsporogenesis and Megasporogenesis Run, Pollen and Female Gametophyte Development, ...
			3.1.1 Fixation and Sections Preparation
			3.1.2 Double Staining of Paraffin Sections with Ehrlich´s Hematoxylin and Alcian  Blue
			3.1.3 Double Staining of Paraffin Sections with Heidenhain´s Iron Hematoxylin and Alcian  Blue
		3.2 Alexander´s and Acetocarmine Tests for Pollen Viability Analysis
		3.3 Staining for Callose in Pollen Tubes of Germinating Pollen Grains (According to)
	4 Notes
	References
Chapter 11: Phenol-Based Protein Extraction Method for Plant Proteomic Studies
	1 Introduction
	2 Materials
		2.1 Equipment
		2.2 Solutions and Reagents
	3 Methods
		3.1 Protein Extraction
		3.2 Protein Precipitation
		3.3 Protein Purification
		3.4 Protein Solubilization
		3.5 Sample Aliquoting
	4 Notes
	References
Chapter 12: Two-Dimensional Gel Electrophoresis in Studies of Flower and Leaf Proteome of Common Buckwheat
	1 Introduction
	2 Materials
		2.1 Equipment
		2.2 Solutions and Reagents
		2.3 Software
	3 Methods
		3.1 IPG-Based IEF
		3.2 SDS-Page
		3.3 Gel Staining
		3.4 Gel Digitalization
	4 Notes
	References
Chapter 13: Measurement of the Kinetics of Chlorophyll a Fluorescence by an LED-Light Source Fluorimeter, Handy PEA
	1 Introduction
	2 Materials
	3 Methods
	4 Notes
	References
Chapter 14: Measurements of Leaf Gas-Exchange Parameters Using Portable CIRAS-3 Infrared Gas Analyzer, with a Parkinson Leaf C...
	1 Introduction
	2 Materials
	3 Methods
	4 Notes
	5 Interpretation of the Results
	References
Chapter 15: Measurement of the Light Intensity and Spectrum Influence on Plant Growth and Secondary Metabolites of Common Buck...
	1 Introduction
	2 Materials
	3 Methods
		3.1 Measurements
		3.2 Data Transfer
	4 Notes
	References
Index