Plant cytogenetics

549 Pages·2017·39.944 MB·English

by  Ram J. Singh

Plant
Cytogenetics
Third Edition

Plant
Cytogenetics
Third Edition
Ram J. Singh
USDA-Agricultural Research Service
Soybean/Maize Germplasm, Pathology, and Genetics Research Unit
Department of Crop Sciences
University of Illinois, Urbana-Champaign
Urbana, Illinois, USA
Boca Raton London New York
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Library of Congress Cataloging‑in‑Publication Data
Names: Singh, Ram J., author.
Title: Plant cytogenetics / author: Ram J. Singh.
Description: Third edition. | Boca Raton : Taylor & Francis, 2017. | Includes
bibliographical references and index.
Identifiers: LCCN 2016017670 | ISBN 9781439884188 (alk. paper)
Subjects: LCSH: Plant cytogenetics.
Classification: LCC QK981.35 .S56 2017 | DDC 572.8/2--dc23
LC record available at https://lccn.loc.gov/2016017670
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To my wife, boys, and grandson (Kingston)

Contents
Preface..............................................................................................................................................xv
Acknowledgments ..........................................................................................................................xvii
Author .............................................................................................................................................xix
Chapter 1 Introduction ..................................................................................................................1
Chapter 2 Handling of Plant Chromosome ...................................................................................7
2.1 Introduction .......................................................................................................7
2.2 Squash Technique: Mitotic and Meiotic Chromosomes ....................................7
2.2.1 Collection of Roots ...............................................................................7
2.2.2 Pretreatment of Roots ...........................................................................7
2.2.2.1 Ice-Cold Water ......................................................................8
2.2.2.2 8-Hydroxyquinoline ..............................................................8
2.2.2.3 Colchicine .............................................................................9
2.2.2.4 α-Bromonaphthalene ............................................................9
2.2.2.5 Para-Dichlorobenzene ..........................................................9
2.2.3 Fixation .................................................................................................9
2.2.3.1 Carnoy’s Solution 1 ...............................................................9
2.2.3.2 Carnoy’s Solution 2 ...............................................................9
2.2.3.3 Propionic Acid Alcohol Solution ..........................................9
2.2.4 Staining of Chromosomes ..................................................................10
2.2.4.1 Aceto-Carmine Staining .....................................................10
2.2.4.2 Feulgen Staining .................................................................10
2.2.4.3 Alcoholic-Hydrochloric Acid-Carmine ..............................12
2.2.4.4 Lacto-Propionic-Orcein ......................................................13
2.2.4.5 Carbol Fuchsin Staining .....................................................13
2.2.4.6 Giemsa Staining..................................................................15
2.3 Smear Technique for Plant Chromosomes ......................................................21
2.3.1 Chromosome Preparation from Roots................................................21
2.3.2 Chromosome Preparation from Cell Suspension and Callus .............22
2.3.2.1 Suspension Culture of Celery (Apium graveolens) .............22
2.3.2.2 Callus Culture of Brassica carinata ...................................22
2.3.3 Chromosome Preparation from Flowers ............................................23
2.3.4 Chromosome Preparation from Shoots ..............................................23
2.4 Pollen Staining ................................................................................................24
2.4.1 Pollen Fertility ....................................................................................24
2.4.2 Chromosome Count in Pollen ............................................................25
2.4.3 Differential Staining of Pollen ...........................................................26
2.4.3.1 Ingredient ............................................................................26
2.4.3.2 Staining of Pollen with Thin Walls ....................................26
2.4.3.3 Staining of Pollen with Thick and Spiny Walls, or Both .....26
2.5 Pollen-Stigma Incompatibility.........................................................................27
2.5.1 Staining Pistils by Aniline Blue .........................................................27
2.5.2 Cleared-Pistil Technique ....................................................................27
2.5.3 Modified Cleared-Pistil Technique ....................................................29
vii
viii Contents
2.6 Fluorescence In Situ Hybridization (FISH) .....................................................29
2.6.1 Chromosome Preparation ...................................................................30
2.6.1.1 Collection, Pretreatment, and Fixation of Roots ................30
2.6.1.2 Preparation of Buffer ..........................................................30
2.6.1.3 Enzyme Solution .................................................................30
2.6.1.4 Preparation of Slides ...........................................................30
2.6.2 Fluorescence In Situ Hybridization Procedure (FISH) ......................31
2.6.2.1 Prehybridization Method ....................................................31
2.6.2.2 Hybridization ......................................................................31
2.6.2.3 Observation and Photography .............................................33
2.6.3 Genomic In Situ Hybridization (GISH)..............................................33
2.6.4 Multicolor Genomic In Situ Hybridization (McGISH) .....................34
2.6.5 Primed In Situ (PRINS) DNA Labeling ............................................34
2.6.6 Fluorescence In Situ Hybridization on Extended DNA Fibers:
Fiber-Fish............................................................................................36
2.6.6.1 Protocol-I: Fiber-FISH on Extended Nuclear
DNA Fibers ......................................................................36
2.6.6.2 Protocol-II: Fiber-FISH Using BAC and Circular
Molecules as Targets ...........................................................39
2.6.6.3 Protocol-III: Staining Fibers (Yo-Yo Staining) ..................40
2.6.6.4 Source of Chemicals ...........................................................40
2.7 Total DNA Extraction: Plant Genomic DNA ..................................................40
2.7.1 Protocol-I ............................................................................................40
2.7.1.1 Solutions .............................................................................40
2.7.1.2 Procedure ............................................................................40
2.7.2 Protocol-II ..........................................................................................42
2.7.2.1 Solutions .............................................................................42
2.7.2.2 Procedure ............................................................................42
2.7.3 Protocol-III .........................................................................................42
2.7.3.1 Buffer/Tissue ......................................................................42
2.7.3.2 Procedure ............................................................................43
2.7.3.3 Determination of DNA Yields and Susceptibility
to Restriction Enzymes .......................................................43
2.7.4 Protocol-IV .........................................................................................44
2.7.4.1 Background .........................................................................44
2.7.4.2 Buffer/Tissue ......................................................................45
2.7.4.3 Procedure ............................................................................45
2.8 Determination of Nuclear DNA Content of Plants by Flow Cytometry .........46
2.8.1 Introduction ........................................................................................46
2.8.2 Protocol-I ............................................................................................47
2.8.2.1 Stock Solutions ...................................................................47
2.8.2.2 Final Solutions ....................................................................47
2.8.2.3 Procedure ............................................................................47
2.8.3 Protocol-II ..........................................................................................49
2.8.3.1 Flow Cytometry after DAPI Staining .................................49
2.8.3.2 Flow Cytometry after Propidium Iodide (PI) Staining ......50
2.8.4 Protocol-III .........................................................................................51
2.8.4.1 Procedure ............................................................................51
2.8.4.2 Fluorescent Staining of Nuclear DNA ................................51
2.8.4.3 Cell Cycle in Higher Plants ................................................51
2.8.4.4 Nuclear DNA Content and the Cell Cycle ..........................52
Contents ix
2.8.4.5 Determination of Nuclear Genome Size .............................52
2.8.4.6 Resolution of DNA Content Histograms ............................53
2.8.4.7 Instrument Calibration ........................................................53
2.8.4.8 Preparation of Fixed CRBC Nuclei for Instrument
Alignment ...........................................................................53
2.8.4.9 CRBC Buffers .....................................................................53
2.8.4.10 Determination of Ploidy in Plants by Flow Cytometry ......54
2.8.4.11 Identification of Interspecific Hybrids ................................54
2.8.4.12 Identification of Aneuploid Lines .......................................54
2.8.5 Protocol-IV .........................................................................................54
2.8.5.1 Chemicals ...........................................................................54
2.8.5.2 Extraction Buffers ...............................................................55
2.8.5.3 Stain Solution A (10 mL) ....................................................55
2.8.5.4 Stain Solution B (10 mL) ....................................................55
2.8.5.5 Isolation and Staining of Nuclei .........................................55
2.8.6 Validity of Genome Size Measurements by Flow Cytometry ...........56
2.9 Karyotyping and Sorting of Plant Chromosomes by Flow Cytometry ...........57
2.9.1 Protocol-I ............................................................................................57
2.9.1.1 Accumulation of Mitotic Metaphase ..................................57
2.9.1.2 Analysis of the Degree of the Metaphase Synchrony .........58
2.9.2 Protocol-II ..........................................................................................59
2.9.2.1 Suspensions of Plant Chromosomes ...................................59
2.9.2.2 Alignment of Flow Cytometer for Chromosome
Analysis and Sorting ...........................................................60
2.9.3 Protocol-III .........................................................................................61
2.9.3.1 Univariate Flow Karyotyping and Sorting of Plant
Chromosomes .....................................................................61
2.9.3.2 Bivariate Flow Karyotyping and Chromosome Sorting .....63
2.9.4 Protocol-IV .........................................................................................64
2.9.4.1 Physical Mapping of DNA Sequences Using PCR .............64
2.9.4.2 Two-Step Sorting ................................................................65
2.9.4.3 Reagents and Solutions .......................................................65
2.10 Production of Wide Hybrids through In Vitro Technique ...............................66
Chapter 3 Methods in Plant Cytogenetics ...................................................................................67
3.1 Introduction .....................................................................................................67
3.2 Mendelian Genetics .........................................................................................67
3.2.1 Monohybrid Inheritance .....................................................................67
3.2.2 Dihybrid Inheritance ..........................................................................69
3.2.2.1 Gene Interaction and Expression ........................................71
3.2.2.2 Genetic Mapping of Chromosomes ....................................76
3.3 Rise and Decline of Plant Cytogenetics ..........................................................83
Chapter 4 Cell Division ...............................................................................................................85
4.1 Introduction .....................................................................................................85
4.2 Mitosis .............................................................................................................85
4.2.1 Process of Mitosis ..............................................................................85
4.2.1.1 Interphase ...........................................................................85
4.2.1.2 Prophase ..............................................................................85