Characterization of Plant Viruses: Methods and Protocols (Springer Protocols Handbooks)

Foreword
Preface
About the Book
Contents
About the Authors
Abbreviations
Chapter 1: Glasshouse for Maintenance of Virus and Insect Culture
	1.1 Introduction
	1.2 Construction Principles of a Glasshouse
	1.3 Preventing Contamination in the Glasshouse
	1.4 Cleaning of Equipment
		1.4.1 Glass Wares, Pestle and Mortar
		1.4.2 Pipettes
	1.5 Glasshouse for Insect Rearing (Maramorosch and Mahmood 2014)
	1.6 Notes
	References
Chapter 2: Symptoms of Virus-Infected Plants
	2.1 Introduction
		2.1.1 External Symptoms
			2.1.1.1 Local Lesion
			2.1.1.2 Systemic Symptoms
				Reduction in Growth (Stunting)
				Chlorosis of Plant Parts/Mosaic Patterns
				Ringspots
				Necrosis
				Symptoms on Stem
				Symptoms on Flower
				Symptoms on Fruit
				Symptoms in Seed/Tuber
				Malformation
		2.1.2 Internal Symptoms (Hull 2002)
			2.1.2.1 Direct Microscopic Examination (Christie and Edwardson 1987)
				Materials
				Method (Direct Microscopic Examination)
			2.1.2.2 Examination of Inclusion Bodies Through Staining with Trypan Blue
				Materials
				Method (Examination of Inclusion Bodies Through Staining with Trypan Blue)
			2.1.2.3 Examination of Inclusion Bodies Through Staining with Phloxine
				Materials
				Method (Examination of Inclusion Bodies Through Staining with Phloxine)
			2.1.2.4 Examination of Inclusion Bodies by Staining with Pyronin-Methyl Green
				Materials
				Method (Examination of Inclusion Bodies by Staining with Pyronin-Methyl Green)
			2.1.2.5 Examination by Staining with Toluidine Blue
				Materials
				Method (Examination of Inclusion Bodies by Staining with Toluidine Blue)
	References
Chapter 3: Isolation and Diagnosis of Virus Through Indicator Hosts
	3.1 Introduction
	3.2 Materials
	3.3 Method
	3.4 Notes
	References
Chapter 4: Host Range of Viruses
	4.1 Introduction
	4.2 Materials
	4.3 Method
	4.4 Notes
	References
Chapter 5: Physico-chemical Properties of Virus in Crude Sap
	5.1 Introduction
		5.1.1 Determination of the Dilution End Point (DEP)
		5.1.2 Determination of the Thermal Inactivation Point (TIP)
		5.1.3 Longevity In Vitro (LIV)
	5.2 Materials
	5.3 Methods
		5.3.1 Determination of the Dilution End Point (DEP)
		5.3.2 Determination of Thermal Inactivation Point (TEP)
		5.3.3 Determination of Longevity In Vitro (LIV)
	5.4 Notes
	References
Chapter 6: Mechanical Sap Transmission
	6.1 Introduction
		6.1.1 Methods of Mechanical Transmission
	6.2 Materials
	6.3 Methods
		6.3.1 Hand Inoculation (Kado 1972; Walkey 1991)
		6.3.2 Spray Inoculation (Mandal et al. 2008)
	6.4 Notes
	References
Chapter 7: Transmission Through Grafting and Budding
	7.1 Introduction
		7.1.1 Approach Grafting
			7.1.1.1 Materials
			7.1.1.2 Method
		7.1.2 Wedge or Top Cleft Grafting
			7.1.2.1 Materials
			7.1.2.2 Method
			7.1.2.3 Notes (Adriance and Brison 2006; Nayudu 2008)
		7.1.3 Tongue Grafting
			7.1.3.1 Materials
			7.1.3.2 Method
		7.1.4 Leaf Patch Grafting
			7.1.4.1 Materials
			7.1.4.2 Method
			7.1.4.3 Notes
		7.1.5 Bud (Shield) Grafting
			7.1.5.1 Materials
			7.1.5.2 Method
			7.1.5.3 Notes
		7.1.6 Double Budding
		7.1.7 Petiole Grafting
			7.1.7.1 Materials
			7.1.7.2 Method
			7.1.7.3 Notes
		7.1.8 Chip Bud Grafting (Wagaba et al. 2013)
			7.1.8.1 Materials
			7.1.8.2 Method
			7.1.8.3 Notes
	References
Chapter 8: Transmission Through Dodder
	8.1 Introduction
	8.2 Materials
	8.3 Method
	8.4 Notes (Hull 2002; Walkey 2012)
	References
Chapter 9: Virus Transmission Through Pollen
	9.1 Introduction
	9.2 Materials
	9.3 Method (Liu et al. 2014; Atsumi et al. 2015)
	9.4 Notes (Hull 2002; Card et al. 2007; Atsumi et al. 2015)
	References
Chapter 10: Transmission Through Seeds
	10.1 Introduction
	10.2 Materials
	10.3 Method
	10.4 Notes (Shepherd 1972; Hull 2014)
	References
Chapter 11: Transmission of Viruses by Aphids
	11.1 Introduction
		11.1.1 Rearing of Virus-Free Aphids
	11.2 Materials
	11.3 Methods
		11.3.1 Culturing of Aphids
		11.3.2 Transmission of Non-persistent Viruses by Aphids
		11.3.3 Transmission of Persistent Viruses
	11.4 Notes (Harris and Maramorosch 1980; Dijkstra and de Jager 1998; Nayudu 2008)
	References
Chapter 12: Transmission of Viruses by Leafhoppers
	12.1 Introduction
		12.1.1 Raising Insect Colonies (Maramorosch 1999)
	12.2 Materials
	12.3 Method (Transmission of Rice Tungro Spherical Virus (RTSV) by Nephotettix spp.)
		12.3.1 Raising Plants and Rearing of Insects
		12.3.2 Generating Virus-Infected Leafhopper Colonies and Virus Transmission
	12.4 Notes (Maramorosch 1999)
	References
Chapter 13: Transmission of Viruses by Whiteflies
	13.1 Introduction
	13.2 Materials
	13.3 Method
		13.3.1 Maintenance of Healthy Whitefly Culture
		13.3.2 Virus Transmission
	13.4 Notes (Muniyappa 1980; Nayudu 2008)
	References
Chapter 14: Transmission of Viruses by Thrips
	14.1 Introduction
	14.2 Materials
	14.3 Method
		14.3.1 Rearing of Thrips (Thrips palmi)
		14.3.2 Virus Transmission Studies
	14.4 Notes  (Ullman et al. 1997; Hull 2002)
	References
Chapter 15: Transmission of Viruses Through Mealybugs
	15.1 Introduction
	15.2 Materials
	15.3 Method
		15.3.1 Rearing of Mealybug (Ferrisia virgata, Planococcus sp.) and Transmission of Piper yellow mottle virus (Genus: Badnaviru...
		15.3.2 Transmission of Grapevine Leaf Roll-Associated Virus (Genus: Ampelovirus; Fam: Closteroviridae) by Planococcus ficus (T...
	15.4 Notes (Roivainen 1980; Bhat et al. 2003; Tsai et al. 2010)
	References
Chapter 16: Transmission of Viruses Through Beetles
	16.1 Introduction
	16.2 Materials
	16.3 Method
		16.3.1 Rearing Beetle Colony and Virus Culture
		16.3.2 Beetle-Virus Transmission Assays
	16.4 Notes (Smith et al. 2017)
	References
Chapter 17: Transmission of Viruses Through Mites
	17.1 Introduction
	17.2 Materials
	17.3 Method
		17.3.1 Rearing of Healthy Aceria cajani
		17.3.2 Transmission
	17.4 Notes (Slykhuis 1955; Reddy et al. 1989; Kulkarni et al. 2002)
	References
Chapter 18: Transmission of Viruses Through Fungi
	18.1 Introduction
		18.1.1 Isolation and Culturing of Fungal Vectors
		18.1.2 Transmission Studies
	18.2 Materials
	18.3 Method (Transmission of Tobacco Necrosis Virus by Olpidium sp.)
	18.4 Notes (Teakle 1972; Hull 2014)
	References
Chapter 19: Transmission of Viruses Through Nematodes
	19.1 Introduction
		19.1.1 Collection, Isolation, Handling and Culturing of Nematodes
			19.1.1.1 Sampling
			19.1.1.2 Isolation
	19.2 Materials (Isolation of Nematodes)
	19.3 Method (Isolation of Nematodes by Flegg Modified Cobb´s Decanting and Sieving Technique)
		19.3.1 Culturing Nematodes
		19.3.2 Transmission Tests
	19.4 Notes  (Barker 1985; Nayudu 2008)
	References
Chapter 20: Storage and Preservation of Plant Virus Cultures
	20.1 Introduction
		20.1.1 Desiccation of Samples
	20.2 Materials
		20.2.1 Desiccation of Samples
	20.3 Method
		20.3.1 Desiccation of Samples
		20.3.2 Preservation of Virus-Infected Rice Leaves and Recovery of the Virus Through Leafhopper Vector
			20.3.2.1 Materials
			20.3.2.2 Method
		20.3.3 Freeze Drying
			20.3.3.1 Materials
			20.3.3.2 Method
		20.3.4 Storage at -80 C
			20.3.4.1 Materials
			20.3.4.2 Method
		20.3.5 Preservation of Virus-Infected Plant Material in Liquid Nitrogen
			20.3.5.1 Materials
			20.3.5.2 Method
		20.3.6 Colour Preservation of Virus-Infected Tissues
			20.3.6.1 Materials
			20.3.6.2 Method
	20.4 Notes (Dijkstra and deJager 1998; Hull 2014)
	References
Chapter 21: Purification of Plant Viruses
	21.1 Introduction
	21.2 Stages in Purification
		21.2.1 Propagation of Virus
		21.2.2 Extraction of Virus
		21.2.3 Clarification of the Extract
		21.2.4 Concentration of the Virus
		21.2.5 Further Purification of the Virus
	21.3 Materials
		21.3.1 Purification of Potato Virus Y (PVY) (Genus: Potyvirus) (Moghal and Francki 1976; Bhat et al. 1997)
			21.3.1.1 Materials
			21.3.1.2 Method
		21.3.2 Purification of Sugarcane Mosaic Virus (SCMV) (Genus: Potyvirus) (Rao et al. 1998)
			21.3.2.1 Materials
			21.3.2.2 Method
		21.3.3 Purification of Cucumber Mosaic Virus (CMV) (Genus: Cucumovirus) (Lot et al. 1972; Bhat et al. 2004)
			21.3.3.1 Materials
			21.3.3.2 Method
		21.3.4 Purification of Piper Yellow Mottle Virus (PYMoV) (Genus: Badnavirus) (deSilva et al. 2002)
			21.3.4.1 Materials
			21.3.4.2 Method
		21.3.5 Purification of Cucumber Green Mottle Mosaic Virus (CGMMV) (Genus: Tobamovirus) (Mandal et al. 2008)
			21.3.5.1 Materials
			21.3.5.2 Method
		21.3.6 Purification of Groundnut Bud Necrosis Virus (GBNV) (Genus: Tospovirus) (Reddy et al. 1992)
			21.3.6.1 Materials
			21.3.6.2 Method
		21.3.7 Purification of Tobacco Streak Virus (Genus: Ilarvirus) (Ramiah et al. 2001)
			21.3.7.1 Materials
			21.3.7.2 Method
		21.3.8 Purification of Potato Leaf Roll Virus (PLRV) (Genus: Luteovirus) (Rowhani and Stace-Smith 1979)
			21.3.8.1 Materials
			21.3.8.2 Method
		21.3.9 Purification of Banana Bunchy Top Virus (BBTV) (Genus: Babuvirus) (Thomas and Dietzgen 1991)
			21.3.9.1 Materials
			21.3.9.2 Method
		21.3.10 Purification of Cymbidium Mosaic Virus (CymMV) (Genus: Potexvirus) (Frowd and Tremaine 1977)
			21.3.10.1 Materials
			21.3.10.2 Method
		21.3.11 Purification of Barley Yellow Dwarf Virus (BYDV) (Genus: Luteovirus) (Geske et al. 1996)
			21.3.11.1 Materials
			21.3.11.2 Method
		21.3.12 Purification of Bean Common Mosaic Virus (BCMV) (Genus: Potyvirus) (Alberio et al. 1979)
			21.3.12.1 Materials
			21.3.12.2 Method
		21.3.13 Purification of Lily Symptomless Virus (LSV) (Genus: Carlavirus) (Wang et al. 2007)
			21.3.13.1 Materials
			21.3.13.2 Method
				By Sephacryl S-1000 SF GFC
				By Superdex-2000 HR GFC
		21.3.14 Purification of Indian Tomato Leaf Curl Virus (Genus: Begomovirus) (Muniyappa et al. 1991)
			21.3.14.1 Materials
			21.3.14.2 Method
		21.3.15 Purification of Citrus Tristeza Virus (Genus: Closterovirus) (Bar-Joseph et al. 1985; Ozturk and Cirakoglu 2003)
			21.3.15.1 Materials
			21.3.15.2 Method
		21.3.16 Purification of Squash Leaf Curl Virus (Genus: Curtovirus) (Cohen et al. 1983)
			21.3.16.1 Materials
			21.3.16.2 Method
		21.3.17 Purification of Rice Tungro Bacilliform Virus (RTBV) (Genus: Tungroviurs) and Rice Turngo Spherical Virus (RTSV) (Genu...
			21.3.17.1 Materials
			21.3.17.2 Method
				Isolation and Propagation of RTSV
				Isolation and Propagation of RTBV
				Purification of RTSV and RTBV
	21.4 Notes (Francki 1972; Brakke 1960; Matthews 1991; Hull 2002)
	References
Chapter 22: Ultraviolet Absorption Spectra of Purified Virus Preparation
	22.1 Introduction
	22.2 Materials
	22.3 Method
	22.4 Notes (Noordam 1973; Wilson and Goulding 1986)
	References
Chapter 23: Electron Microscopy and Utramicrotomy
	23.1 Introduction
	23.2 Leaf Dip Method
		23.2.1 Materials
		23.2.2 Method
	23.3 Electron Microscopy of Ultrathin Sections
		23.3.1 Materials
		23.3.2 Method
	23.4 Notes (Hill 1984; Milne 1984; Roberts 1986)
	References
Chapter 24: Determination of Coat Protein Molecular Weight of Viruses
	24.1 Introduction
	24.2 Materials
	24.3 Method
	24.4 Notes (Laemmli 1970; Sambrook and Russel 2001)
	References
Chapter 25: Isolation of Nucleic Acid from Purified Virus and Determination of Its Nature
	25.1 Introduction
	25.2 Isolation of Nucleic Acid from Purified Virus Preparation
		25.2.1 Materials
			25.2.1.1 Method I
			25.2.1.2 Method II
		25.2.2 Methods
			25.2.2.1 Method I
			25.2.2.2 Method II
	25.3 Quantification of Nucleic Acid
		25.3.1 Materials
		25.3.2 Method
	25.4 Determination of the Nature of Viral Genome
		25.4.1 Materials
		25.4.2 Method
	25.5 Notes (Burrin 1986; Manchester 1995)
	References
Chapter 26: Agarose Gel Electrophoresis for Nucleic Acids
	26.1 Introduction
	26.2 Agarose Gel Electrophoresis for DNA
		26.2.1 Materials
		26.2.2 Method
	26.3 Denaturing Formaldehyde Gel for the Analysis of RNA
		26.3.1 Materials
		26.3.2 Method
	26.4 Notes (Simpson and Whittaker 1983; Sambrook and Russel 2001 )
	References
Chapter 27: In Vitro Expression of Viral Coat Protein in Prokaryotic System and Its Purification
	27.1 Introduction
	27.2 Materials
	27.3 Method
		27.3.1 Subcloning of Virus Coat Protein (CP) in Expression Vector
		27.3.2 Induction of CP Clones in Expression Cell Line
		27.3.3 SDS-PAGE Analysis
		27.3.4 Protein Purification
	27.4 Notes (Sambrook and Russel 2001; Agarwal et al. 2009)
	References
Chapter 28: Production of Polyclonal Antiserum
	28.1 Introduction
	28.2 Materials
	28.3 Method
		28.3.1 Intramuscular Injection
		28.3.2 Subcutaneous Injection
		28.3.3 Intravenous Injection
		28.3.4 Blood (Serum) Collection
		28.3.5 Processing of Antiserum
	28.4 Notes (Hampton et al. 1990; Dijkstra and deJager 1998)
	References
Chapter 29: Production of Monoclonal Antibody
	29.1 Introduction
		29.1.1 Steps Involved in Monoclonal Antibody Production
	29.2 Materials
		29.2.1 Special Reagents for Hybridoma Technology
	29.3 Immunization
		29.3.1 Materials
		29.3.2 Method
	29.4 Cell Fusion
		29.4.1 Materials
		29.4.2 Method
			29.4.2.1 Preparation of Feeder Cells
			29.4.2.2 Preparation of Myeloma Cells
			29.4.2.3 Preparation of Spleenic B Lymphocytes for Fusion
			29.4.2.4 Fusion Experiment
	29.5 Screening the Hybridoma
		29.5.1 Materials
		29.5.2 Method
			29.5.2.1 Screening Strategy
			29.5.2.2 Single Cell Cloning and Sub-cloning
	29.6 Production of MAbs from Hybridoma Clones
		29.6.1 Laboratory Methods of Hybridoma Cultivation (Batch Culture Method: Cell Culture Flask)
		29.6.2 Mouse Ascites Method
	29.7 Characterization of Monoclonal Antibodies
		29.7.1 Reactivity in ELISA
	29.8 Notes (van Regenmortel 1986; Torrance 1995)
	References
Chapter 30: Serological Tests
	30.1 Introduction
	30.2 Precipitin Tests
		30.2.1 Tube Precipitin
			30.2.1.1 Materials
			30.2.1.2 Method
		30.2.2 Micro-Precipitin Test
			30.2.2.1 Materials
			30.2.2.2 Method
		30.2.3 Chloroplast Agglutination Test
			30.2.3.1 Materials
			30.2.3.2 Method
		30.2.4 Agar Double Diffusion (Ouchterlony) Test
			30.2.4.1 Materials
			30.2.4.2 Method
	30.3 Enzyme-Linked Immunosorbent Assay (ELISA)
		30.3.1 Double-Antibody Sandwich (DAS) ELISA
			30.3.1.1 Preparation of Immunoglobulins (IgG) (Avrameas 1969)
				Materials
				Method
			30.3.1.2 Preparation of IgG-Enzyme Conjugate
				Conjugation with Alkaline Phosphatase (ALP)
					Materials
					Method
				Conjugation with Horseradish Peroxidase (HRP)
					Materials
					Method
				Conjugation with Penicillinase
					Materials
					Method
			30.3.1.3 Direct Antibody Sandwich ELISA (DAS-ELISA) Utilizing Alkaline Phosphatase
				Materials
				Method
			30.3.1.4 DAS-ELISA Using Penicillinase
				Materials
				Method
		30.3.2 F(ab´)2-ELISA
			30.3.2.1 Preparation of F(ab´)2 Fragments
				Materials
				Method [Preparation of F(ab´)2 Fragments]
			30.3.2.2 F(ab´)2-ELISA test
				Materials
				Method
		30.3.3 Plate-Trapped or Triple-Sandwich ELISA
			30.3.3.1 Materials
			30.3.3.2 Method
		30.3.4 Direct Antigen-Coated ELISA (DAC-ELISA)
			30.3.4.1 Materials
			30.3.4.2 Method
	30.4 Dot Immunobinding Assay (DIBA)
		30.4.1 Materials
		30.4.2 Method
	30.5 Tissue Blotting Immunoassay (TBIA)
		30.5.1 Materials
		30.5.2 Method
	30.6 Electro-Blot Immunoassay (EBIA)/Western Blotting
		30.6.1 Materials
		30.6.2 Method
	30.7 Immunosorbent Electron Microscopy (ISEM)
		30.7.1 Trapping
			30.7.1.1 Materials
			30.7.1.2 Method
		30.7.2 Decoration
			30.7.2.1 Materials
			30.7.2.2 Method
		30.7.3 Immunogold Labelling
			30.7.3.1 With Fresh Tissues
				Materials
				Method
			30.7.3.2 With Pre-embedded Tissues
				Materials
				Method
			30.7.3.3 With Post-embedded Tissue
				Materials
				Method
	30.8 Immunofluorescence
		30.8.1 Materials
		30.8.2 Method
	30.9 Lateral Flow Immunoassay Assay (LFIA)
		30.9.1 Preparation and Assembly of the Strip
			30.9.1.1 Test and Control Lines
			30.9.1.2 Conjugate
			30.9.1.3 Pad and Membrane Treatments
			30.9.1.4 Membrane Materials
			30.9.1.5 Material of the Sample Pad, Conjugate Release Pad and Absorbent Pad
			30.9.1.6 Labels
		30.9.2 Steps in Lateral Flow Immunoassay
			30.9.2.1 Production of Polyclonal Antibody Against Plant Virus
			30.9.2.2 Synthesis of Colloidal Gold Nanoparticles-Antibody Conjugate
				Materials
				Method
			30.9.2.3 Assembly of LFIA Strips in Polypropylene Cassettes
				Materials
				Method
		30.9.3 Standardization of LFIA Procedure
			30.9.3.1 Materials
			30.9.3.2 Method
	30.10 Notes (Clark and Adams 1977; Clark et al. 1986; Hill 1984; Banttari and Goodwin 1985; Hampton et al. 1990; Koenig and Pa...
	References
Chapter 31: Isolation of Total DNA from Plants
	31.1 Introduction
	31.2 Protocol I
		31.2.1 Materials
		31.2.2 Method
	31.3 Protocol II
		31.3.1 Materials
		31.3.2 Method
	31.4 Notes (Dellaporta et al. 1983; Murray and Thompson 1980; Zhang et al. 1998)
	References
Chapter 32: Isolation of Total RNA from Plants
	32.1 Introduction
	32.2 Acid Guanidium Thiocyanate Phenol Chloroform (AGPC) Method (Chomczynski and Sacchi 1987)
		32.2.1 Materials
		32.2.2 Method
	32.3 RNA Extraction and Column Purification
		32.3.1 Materials
		32.3.2 Method
			32.3.2.1 Extraction of Total Nucleic Acids
			32.3.2.2 Column Purification
	32.4 Notes (Chomczynski and Sacchi 1987; Sambrook and Russel 2001)
	References
Chapter 33: Isolation of Double-Stranded (ds) RNA from Virus-Infected Plants
	33.1 Introduction
	33.2 Materials
	33.3 Method
	33.4 Notes (Morris et al. 1983; Dodds et al. 1984)
	References
Chapter 34: Dot-Blot Hybridization Technique
	34.1 Introduction
	34.2 Production of Radiolabelled Probe by Random Priming Method
		34.2.1 Materials
		34.2.2 Method
		34.2.3 Notes
	34.3 Production of Non-radioactive Probe by Random Primed Labelling
		34.3.1 Materials
		34.3.2 Method
		34.3.3 Note
	34.4 Production of Radiolabelled DNA Probes by Polymerase Chain Reaction
		34.4.1 Materials
		34.4.2 Method
		34.4.3 Notes
	34.5 Production of Non-radioactive DNA Probe by PCR
		34.5.1 Materials
		34.5.2 Method
	34.6 Dot-Blot Hybridization
		34.6.1 Materials
		34.6.2 Method
			34.6.2.1 Sample Preparation
			34.6.2.2 Preparation of Membrane and Dotting Samples
			34.6.2.3 Processing and Development of Membrane (When Radioactive Probe Is Used)
				Pre-hybridization and Hybridization
				Washing
				Autoradiography
			34.6.2.4 Processing and Development of Membrane (When a Non-radioactive Probe Labelled with DIG Is Used)
				Materials
				Pre-hybridization and Hybridization
				Detection of Hybridized Bands by Colorimetric Method
				Detection of Hybridized Bands by Chemiluminescence Method
		34.6.3 Notes (When Radioactive Probe is Used)
		34.6.4 Notes (When Non-radioactive Probe is Used)
	References
Chapter 35: Polymerase Chain Reaction
	35.1 Introduction
	35.2 Steps in PCR
	35.3 Components of PCR
	35.4 Running PCR
	35.5 PCR for the Amplification of DNA Viruses
		35.5.1 Materials
		35.5.2 Method
	35.6 Reverse Transcription (RT) PCR for Detection of RNA Viruses
		35.6.1 Single-Tube RT-PCR
			35.6.1.1 Materials
			35.6.1.2 Method
		35.6.2 Two-Step RT-PCR (cDNA Synthesis Followed by PCR)
			35.6.2.1 Materials
			35.6.2.2 Method
	35.7 Immunocapture (IC) PCR and IC-RT-PCR
		35.7.1 Materials
		35.7.2 Method
	35.8 RT-PCR Using dsRNA Template
		35.8.1 Materials
		35.8.2 Method
	35.9 Multiplex-PCR
		35.9.1 Materials
		35.9.2 Method
			35.9.2.1 Isolation of Total RNA and DNA from Plants
			35.9.2.2 Single-Tube Multiplex RT-PCR (mRT-PCR)
	35.10 Nested PCR Assay
	35.11 Notes (Hadidi et al. 1995; Candresse et al. 1998; López et al. 2009; Mumford and Seal 1997; Sambrook and Russell 2001)
	References
Chapter 36: Real-Time Polymerase Chain Reaction
	36.1 Introduction
		36.1.1 Dyes Binding to the Double-Stranded DNA
		36.1.2 Melt Curve and Detection of Non-specific Amplification
		36.1.3 Fluorescent Probes Which Have Specificity for Binding to Targeted DNA
		36.1.4 Quantification
		36.1.5 Real-Time PCR Instrument
		36.1.6 Amplification Curve and its Phases
		36.1.7 Primer and Probe Design
		36.1.8 Application
	36.2 Performing Real-Time PCR/Real-Time RT-PCR Using SYBR-Green
		36.2.1 Materials
		36.2.2 Method (When RNA is Used as Template)
		36.2.3 Method (When DNA is Used as Template)
	36.3 Performing Real-Time RT-PCR Using TaqMan Assay
		36.3.1 Materials
		36.3.2 Method
	36.4 Developing Standard Curve for Quantification
		36.4.1 Materials
		36.4.2 Method
	36.5 Notes (Mackay et al. 2002; Lopez et al. 2003; Patel et al. 2016)
	References
Chapter 37: DNA Microarray for Detection of Plant Viruses
	37.1 Introduction
	37.2 Materials
	37.3 Method
		37.3.1 Design and Synthesis of Microarray Slide
		37.3.2 RNA Isolation
		37.3.3 cDNA Synthesis and Fluorescent Labelling of cDNA
		37.3.4 Hybridization and Scanning of Microarray Slide
	37.4 Notes (Boonham et al. 2003; Agindotan and Perry 2007; Bystricka et al. 2005; Hadidi et al. 2004)
	References
Chapter 38: Loop-Mediated Isothermal Amplification (LAMP)
	38.1 Introduction
	38.2 Materials
	38.3 Method
		38.3.1 Performing LAMP and RT-LAMP
		38.3.2 Visual Detection of LAMP and RT-LAMP Products
	38.4 Notes (Mori et al. 2001; Tomita et al. 2008; Zhang et al. 2014)
	References
Chapter 39: Rolling Circle Amplification (RCA)
	39.1 Introduction
	39.2 Materials
	39.3 Methods
	39.4 Notes (Dean et al. 2001; Inoue-Nagata et al. 2004; Lau and Botella 2017)
	References
Chapter 40: Recombinase Polymerase Amplification
	40.1 Introduction
	40.2 Materials
	40.3 Methods (Based on Protocol of TwistDx, Cambridge, UK)
	40.4 Notes
	References
Chapter 41: Next-Generation Sequencing for Diagnosis of Viruses
	41.1 Introduction
	41.2 Chemistry Used by Different Platforms
	41.3 General Workflow of NGS
	41.4 Application in Plant Virology
	References
Chapter 42: Cloning of PCR Product
	42.1 Introduction
	42.2 Isolation of Target DNA by Purification of PCR Product Through Low Melting Point (LMP) Agarose Gel
		42.2.1 Materials
		42.2.2 Method
	42.3 Ligation of the Purified PCR Product into Vector
		42.3.1 Materials
		42.3.2 Method
		42.3.3 Notes
	42.4 Preparation of Competent E. coli Cells
		42.4.1 Materials
		42.4.2 Method (Mendel and Higa 1970)
		42.4.3 Notes
	42.5 Transformation of E. coli
		42.5.1 Materials
		42.5.2 Method
	42.6 Selection of Transformants (Preparation of Master Plate)
		42.6.1 Materials
		42.6.2 Method
	42.7 Screening and Identification of Positive (Recombinant) Clones
		42.7.1 Rapid Disruption of Bacterial Colonies (Rapid) to Test the Size of Plasmids
			42.7.1.1 Materials
			42.7.1.2 Method
		42.7.2 Screening Putative Recombinants by Colony PCR
			42.7.2.1 Materials
			42.7.2.2 Method
	42.8 Recombinant Plasmid DNA Isolation and Restriction Analysis
		42.8.1 Plasmid Isolation by Alkaline Lysis Method (Birnboim and Doly 1979)
			42.8.1.1 Materials
			42.8.1.2 Method
		42.8.2 Plasmid Isolation by Modified Alkaline Lysis Method (Xiang et al. 1994)
			42.8.2.1 Materials
			42.8.2.2 Method
		42.8.3 Confirmation of Recombinant Clones by Restriction Analysis
			42.8.3.1 Materials
			42.8.3.2 Method
		42.8.4 Confirmation of Recombinant Clones by PCR
			42.8.4.1 Materials
			42.8.4.2 Method
	References
Chapter 43: cDNA Synthesis and Cloning
	43.1 Introduction
	43.2 First and Second Strand cDNA Synthesis
		43.2.1 Materials
		43.2.2 Method
	43.3 Ligation of DNA Fragment to the Vector DNA
		43.3.1 Materials
		43.3.2 Method
			43.3.2.1 Linearization of Vector DNA
			43.3.2.2 Dephosphorylation of Linearized Plasmid DNA
			43.3.2.3 Ligation
	43.4 Transformation
	References
Chapter 44: DNA Sequencing
	44.1 Introduction
	44.2 Maxam-Gilbert Method
	44.3 Chain Termination DNA Sequencing
	44.4 Automated DNA Sequencing
	44.5 Whole-Genome Sequencing
	44.6 Next-Generation Sequencing
	44.7 Notes
	References
Chapter 45: Sequence Analysis and Phylogenetic Studies
	45.1 Introduction
	45.2 Identification of Similar Sequences
	45.3 Sequence Retrieval from Databases
	45.4 Detecting Open Reading Frame (ORF)
	45.5 Translation of Nucleotide Sequence to Protein Sequence
	45.6 Identification of Conserved Motifs and Domains
	45.7 Determination of Similarity Between Sequences
	45.8 Multiple Sequence Alignment
	45.9 Phylogenetic Analysis
	References
Chapter 46: Development of Infectious Clone of Virus
	46.1 Introduction
		46.1.1 Construction of Infectious Clones
		46.1.2 Introduction of Infectious Clones into Plants
		46.1.3 Construction of FL-cDNA Under the Control of Bacteriophage Promoter (In Vitro Transcription)
			46.1.3.1 Materials
			46.1.3.2 Method
		46.1.4 Construction of FL-cDNA Under the Control of Cauliflower Mosaic Virus 35S Promoter (In Vivo Transcription)
			46.1.4.1 Materials
			46.1.4.2 Method
				Development of Infectious Construct of Chilli Leaf Curl Virus
				Construction of Full-Length Betasatellite Associated with Begomovirus
				Agro-Inoculation of Chilli Using Begomovirus and Betasatellite Constructs
	46.2 Notes
	References
Chapter 47: Virus Elimination by Meristem-Tip Culture
	47.1 Introduction
		47.1.1 Meristem Culture Combined with Thermotherapy
		47.1.2 Meristem Culture Combined with Chemotherapy
	47.2 Materials
	47.3 Method (Virus Elimination by Meristem-Tip Culture, Sasi and Bhat 2018)
	47.4 Method (Virus Elimination by Meristem-Tip Culture Combined with Chemotherapy, Sasi and Bhat 2018)
	47.5 Method (Virus Elimination by Meristem-Tip Culture Combined with Thermotherapy (Ramgareeb et al. 2010; Mishra et al. 2010)
	47.6 Notes (Al-Taleb et al. 2011; Fayek et al. 2009; Mink et al. 1998; Mishra et al. 2010; Sasi and Bhat 2018)
	References
Chapter 48: Virus Elimination Through Somatic Embryogenesis
	48.1 Introduction
		48.1.1 Virus Elimination Through Somatic Embryogenesis
	48.2 Materials
	48.3 Method: Virus Elimination by Somatic Embryogenesis (Sasi and Bhat 2018)
	48.4 Method: Virus Elimination by Somatic Embryogenesis Combined with Chemotherapy (Sasi and Bhat 2018)
	48.5 Method: Virus Elimination Through Somatic Embryogenesis in Sugarcane (Ramgareeb et al. 2010; Mishra et al. 2010)
	48.6 Notes (Laux and Jurgens; 1997; Raemakers et al. 1995; Ramgareeb et al. 2010; Sasi and Bhat 2018)
	References
Chapter 49: Production of Virus-Resistant Plants Through Transgenic Approaches
	49.1 Introduction
		49.1.1 Pathogen-Derived Genes for Plant Virus Resistance
		49.1.2 Post-transcriptional Gene Silencing (RNA Silencing)
		49.1.3 Viral Suppressors of RNA Silencing (VSR)
	49.2 Requirements for the Development of Virus-Resistant Transgenic Plants
		49.2.1 Development of Reliable Tissue Culture Regeneration System
		49.2.2 Transformation Methods
			49.2.2.1 Biolistic Method
			49.2.2.2 Agrobacterium-Mediated Gene Transfer
		49.2.3 Preparation of Gene Constructs in Binary Vectors
		49.2.4 Selection and Regeneration of Transgenic Plants
		49.2.5 Screening of Transgenic Plants
			49.2.5.1 GUS Assay
			49.2.5.2 Polymerase Chain Reaction (PCR) Assay
			49.2.5.3 Southern Hybridization, Northern Hybridization and Western Blotting
		49.2.6 Evaluation of Transgenic Plants
	49.3 Development of Transgenic Papaya Through Coat Protein-Mediated Approach Using Biolistic
		49.3.1 Preparation of Construct (Quemada et al. 1990; Fitch et al. 1990, 1992)
		49.3.2 Transformation, Regeneration and Confirmation of Transgene Integration
		49.3.3 Evaluation of Transgenic Papaya for Virus Resistance in the Greenhouse
	49.4 Development of Transgenic Plant Through RNAi Approach Using Agrobacterium-Mediated Transformation
		49.4.1 Preparation of Hairpin (HP) Construct
		49.4.2 Agrobacterium-Mediated Transformation of Black Pepper (Nair and Gupta 2006; Jiby and Bhat 2011)
		49.4.3 Testing of Transgenic Black Pepper for Viral Resistance in the Greenhouse
	49.5 Notes
	References
Chapter 50: Production of Virus-Resistant Plants Through CRISPR-Cas Technology
	50.1 Introduction
		50.1.1 Mechanism of CRISPR/Cas
		50.1.2 Application in Plants
		50.1.3 Virus Resístanse in Plants via CRISPR/Cas
			50.1.3.1 DNA Virus Resístanse via CRISPR/Cas9
			50.1.3.2 RNA Virus Resístanse via CRISPR/Cas9
	50.2 General Outline of CRISPR/Cas9 Genome Editing in Plants
	50.3 The CRISPR Cleavage Methodology Involves the Following Steps
	50.4 Notes
	References
Appendix: Common Conversions, Information Sources and Software of Nucleic Acids and ProteinsWeight conversion1 μg = 10-6 g1 ng...
	DNA Data
	Important DNA and Protein Information Sources and Software
	Software for Sequence Analysis
Glossary