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  Contents  
 
  Preface XVII
  List of Contributors XXI
1 Cytometry and Cytometers: Development and Growth
Howard M. Shapiro 		1
Overview 1
1.1 Origins 1
1.2 From Absorption to Fluorescence, from Imaging to Flow 2
1.2.1 Early Microspectrophotometry and Image Cytometry 3
1.2.2 Fluorescence Microscopy and the Fluorescent Antibody Technique 3
1.2.3 Computers Meet Cytometers: The Birth of Analytical Flow Cytometry 4
1.2.4 The Development of Cell Sorting 7
1.3 The Growth of Multiparameter Flow Cytometry 8
1.4 Bench-tops and Behemoths: Convergent Evolution 11
1.5 Image Cytometry: New Beginnings? 14
References 15
2 Principles of Flow Cytometry
J. Paul Robinson and Gérald Grégori 		19
Overview 19
2.1 Introduction 19
2.2 A Brief History of Flow Cytometry 20
2.3 Components of a Flow Cytometer 21
2.3.1 Fluidics 22
2.3.2 Optics 25
2.3.3 Electronic Systems 27
2.4 Flow Cytometric Informatics 30
2.5 Spectral Compensation 33
2.6 Cell Sorting 34
2.7 Calibration Issues 37
2.8 Conclusions 37
References 39
3 Flow Cytometry with Plants: an Overview
Jaroslav Doleel, Johann Greilhuber, and Jan Suda 		41
Overview 41
3.1 Introduction 42
3.2 Fluorescence is a Fundamental Parameter 43
3.3 Pushing Plants through the Flow Cytometer 44
3.3.1 Difficulties with Plants and their Cells 44
3.3.2 Protoplasts are somewhat ``Easier'' than Intact Cells 45
3.3.3 Going for Organelles 46
3.4 Application of Flow Cytometry in Plants 47
3.4.1 Microspores and Pollen 47
3.4.2 Protoplasts 47
3.4.2.1 Physiological Processes 48
3.4.2.2 Secondary Metabolites 48
3.4.2.3 Gene Expression 48
3.4.2.4 Somatic Hybrids 49
3.4.2.5 DNA Transfection 49
3.4.3 Cell Nuclei 49
3.4.3.1 Ploidy Levels 50
3.4.3.2 Aneuploidy 51
3.4.3.3 B Chromosomes 52
3.4.3.4 Sex Chromosomes 52
3.4.3.5 Cell Cycle and Endopolyploidy 52
3.4.3.6 Reproductive Pathways 53
3.4.3.7 Nuclear Genome Size 54
3.4.3.8 DNA Base Content 55
3.4.3.9 Chromatin Composition 56
3.4.3.10 Sorting of Nuclei 56
3.4.4 Mitotic Chromosomes 57
3.4.5 Chloroplasts 57
3.4.6 Mitochondria 58
3.4.7 Plant Pathogens 58
3.4.8 Aquatic Flow Cytometry 59
3.5 A Flow Cytometer in Every Laboratory? 59
3.6 Conclusions and Future Trends 60
References 61
4 Nuclear DNA Content Measurement
Johann Greilhuber, Eva M. Temsch, and Joăo C. M. Loureiro 		67
Overview 67
4.1 Introduction 67
4.2 Nuclear DNA Content: Words, Concepts and Symbols 69
4.2.1 Replication--Division Phases 69
4.2.2 Alternation of Nuclear Phases 70
4.2.3 Generative Polyploidy Levels 70
4.2.4 Somatic Polyploidy 71
4.3 Units for Presenting DNA Amounts and their Conversion Factors 72
4.4 Sample Preparation for Flow Cytometric DNA Measurement 74
4.4.1 Selection of the Tissue 74
4.4.2 Reagents and Solutions 75
4.4.2.1 Isolation Buffers and DNA Staining 76
4.5 Standardization 80
4.5.1 Types of Standardization 80
4.5.2 Requirement of Internal Standardization -- a Practical Test 82
4.5.3 Choice of the Appropriate Standard Species 83
4.5.3.1 Biological Similarity 83
4.5.3.2 Genome Size 84
4.5.3.3 Nature of the Standard 84
4.5.3.4 Availability 84
4.5.3.5 Cytological Homogeneity 85
4.5.3.6 Accessibility 85
4.5.3.7 Reliability of C-Values 85
4.5.4 Studies on Plant Standards 86
4.5.5 Suggested Standards 88
4.6 Fluorescence Inhibitors and Coatings of Debris 89
4.6.1 What are Fluorescence Inhibitors and Coatings of Debris? 89
4.6.2 Experiments with Tannic Acid 92
4.6.3 A Flow-cytometric Test for Inhibitors 95
4.7 Quality Control and Data Presentation 95
4.8 Future Directions 98
References 99
5 Flow Cytometry and Ploidy: Applications in Plant Systematics, Ecology and Evolutionary Biology
Jan Suda, Paul Kron, Brian C. Husband, and Pavel Trávníek 		103
Overview 103
5.1 Introduction 103
5.2 Practical Considerations 104
5.2.1 Relative DNA Content, Ploidy and Flow Cytometry 104
5.2.2 General Guidelines for Ploidy-level Studies 105
5.2.3 Use of Alternative Tissues 108
5.2.3.1 Preserved or Dormant Tissue 108
5.2.3.2 Pollen 111
5.2.4 Other Considerations/Pitfalls 113
5.2.4.1 Holokinetic Chromosomes (Agmatoploidy) 113
5.2.4.2 DNA Content Variation within Individuals 113
5.3 Applications in Plant Systematics 114
5.3.1 Systematics of Heteroploid Taxa 114
5.3.1.1 Detecting Rare Cytotypes 117
5.3.1.2 Phylogenetic Inference 117
5.3.2 Systematics of Homoploid Taxa 118
5.4 Applications in Plant Ecology and Evolutionary Biology 119
5.4.1 Spatial Patterns of Ploidy Variation 119
5.4.1.1 Invasion Biology 119
5.4.2 Evolutionary Dynamics of Populations with Ploidy Variation 120
5.4.3 Ploidy Level Frequencies at Different Life Stages (Temporal Variation) 121
5.4.4 Reproductive Pathways 122
5.4.4.1 Unreduced Gametes and Polyploidy 122
5.4.4.2 Asexual Seed Production 124
5.4.4.3 Hybridization 124
5.4.5 Trophic Level Interactions and Polyploidy 125
5.5 Future Directions 126
References 128
6 Reproduction Mode Screening
Fritz Matzk 		131
Overview 131
6.1 Introduction 131
6.2 Analyses of the Mode of Reproduction 134
6.2.1 Traditional Techniques 134
6.2.2 Ploidy Analyses of Progenies Originating from Selfing or Crossing 139
6.2.2.1 Identification of BIII, BIV and MI Individuals after Selfing or Intraploidy Pollinations 139
6.2.2.2 Crossing of Parents with Different Ploidy or with Dominant Markers 140
6.2.3 Flow Cytometric Analyses of the Relative DNA Content of Microspores or Male Gametes 141
6.2.4 The Ploidy Variation of Embryo and Endosperm Depending on the Reproductive Mode 142
6.3 A Recent Innovative Method: the Flow Cytometric Seed Screen 142
6.3.1 Advantages and Limitations of the FCSS 143
6.3.2 Applications of the FCSS 146
6.3.2.1 Botanical Studies 146
6.3.2.2 Evolutionary Studies 147
6.3.2.3 Genetical Analyses of Apomixis 147
6.3.3 Methodological Implications 147
6.4 Flow Cytometry with Mature Seeds for other Purposes 149
6.5 Conclusions 150
References 151
7 Genome Size and its Uses: the Impact of Flow Cytometry
Ilia J. Leitch and Michael D. Bennett 		153
Overview 153
7.1 Introduction 153
7.2 Why is Genome Size Important? 154
7.3 What is Known about Genome Size in Plants? 155
7.3.1 Angiosperms 156
7.3.2 Gymnosperms 157
7.3.3 Pteridophytes 158
7.3.4 Bryophytes 158
7.3.5 Algae 158
7.4 The Extent of Genome Size Variation across Plant Taxa 159
7.5 Understanding the Consequences of Genome Size Variation: Ecological and Evolutionary Implications 160
7.5.1 Influence of Genome Size on Developmental Lifestyle and Life Strategy 161
7.5.2 Ecological Implications of Genome Size Variation 163
7.5.3 Implications of Genome Size Variation on Plants' Responses to Environmental Change 166
7.5.3.1 Genome Size and Plant Response to Pollution 166
7.5.3.2 Genome Size and Threat of Extinction 166
7.5.4 Consequences of Genome Size Variation for Survival in a Changing World 167
7.6 Methods of Estimating Genome Size in Plants and the Impact of Flow Cytometry 168
7.6.1 The Development of Flow Cytometry for Genome Size Estimation in Angiosperms 169
7.6.1.1 Choice of Fluorochromes 169
7.6.1.2 Internal Standardization 169
7.6.1.3 The Need for Cytological Data 170
7.6.1.4 Awareness of the Possible Interference of DNA Staining 170
7.6.2 Potential for the Application of Flow Cytometry to Other Plant Groups 171
7.6.2.1 Gymnosperms 171
7.6.2.2 Pteridophytes 172
7.6.2.3 Bryophytes 172
7.7 Recent Developments and the Future of Flow Cytometry in Genome Size Research 172
References 174
8 DNA Base Composition of Plant Genomes
Armin Meister and Martin Barow 		177
Overview 177
8.1 Introduction 177
8.2 Analysis of Base Composition by Flow Cytometry 178
8.2.1 Fluorescence of Base-Specific Dyes: Theoretical Considerations 180
8.2.2 Base Composition of Plant Species Determined by Flow Cytometry and its Relation to Genome Size and Taxonomy 185
8.2.3 Comparison of Flow Cytometric Results with Base Composition Determined by other Physico-Chemical Methods 204
8.2.4 Possible Sources of Error in Determination of Base Composition by Flow Cytometry 205
8.3 Conclusions 211
References 213
9 Detection and Viability Assessment of Plant Pathogenic Microorganisms using Flow Cytometry
Jan H. W. Bergervoet, Jan M. van der Wolf, and Jeroen Peters 		217
Overview 217
9.1 Introduction 217
9.2 Viability Assessment 218
9.2.1 Viability Tests for Spores and Bacteria 219
9.3 Immunodetection 222
9.3.1 Microsphere Immuno Assay 224
9.3.1.1 Detection of Plant Pathogenic Bacteria and Viruses 225
9.3.1.2 Paramagnetic Microsphere Immuno Assay 226
9.4 Conclusions and Future Prospects 227
References 229
10 Protoplast Analysis using Flow Cytometry and Sorting
David W. Galbraith 		231
Overview 231
10.1 Introduction 231
10.1.1 Protoplast Preparation 231
10.1.2 Adaptation of Flow Cytometric Instrumentation for Analysis of Protoplasts 233
10.1.3 Parametric Analyses Available for Protoplasts using Flow Cytometry 234
10.2 Results of Protoplast Analyses using Flow Cytometry and Sorting 237
10.2.1 Protoplast Size 237
10.2.2 Protoplast Light Scattering Properties 238
10.2.3 Protoplast Protein Content 239
10.2.4 Protoplast Viability and Physiology 239
10.2.5 Protoplast Cell Biology 243
10.2.6 Construction of Somatic Hybrids 244
10.2.7 The Cell Cycle 244
10.3 Walled Plant Cells: Special Cases for Flow Analysis and Sorting 246
10.4 Prospects 247
References 248
11 Flow Cytometry of Chloroplasts
Erhard Pfündel and Armin Meister 		251
Overview 251
11.1 Introduction 251
11.1.1 The Chloroplast 252
11.2 Chloroplast Signals in Flow Cytometry 255
11.2.1 Autofluorescence 255
11.2.2 Light Scattering 259
11.3 Progress of Research 259
11.3.1 Chloroplasts from C3 Plants 260
11.3.2 Chloroplasts from C4 Plants 261
11.4 Conclusion 263
References 264
12 DNA Flow Cytometry in Non-vascular Plants
Hermann Voglmayr 		267
Overview 267
12.1 Introduction 267
12.2 Nuclear DNA Content and Genome Size Analysis 271
12.2.1 General Methodological Considerations 272
12.2.1.1 Isolation and Fixation of Nuclei 272
12.2.1.2 Standardization 274
12.2.1.3 Fluorochromes for Estimation of Nuclear DNA Content 275
12.2.1.4 Secondary Metabolites as DNA Staining Inhibitors 276
12.2.2 DNA Content and Genome Size Studies 276
12.2.2.1 Algae 277
12.2.2.2 Bryophytes 280
12.3 Future Perspectives 283
12.4 Conclusion 284
References 285
13 Phytoplankton and their Analysis by Flow Cytometry
George B. J. Dubelaar, Raffaella Casotti, Glen A. Tarran, and Isabelle C. Biegala 		287
Overview 287
13.1 Introduction 288
13.2 Plankton and their Importance 288
13.2.1 Particles in Surface Water 288
13.2.2 Phytoplankton 289
13.2.3 Distributions in the Aquatic Environment 289
13.3 Considerations for using Flow Cytometry 291
13.3.1 Analytical Approach 291
13.3.2 Limitations and Pitfalls of using Biomedical Instruments 292
13.3.3 Instrument Modification and Specialized Cytometers 293
13.3.4 Sizing and Discrimination of Cells 295
13.3.5 More Information per Particle: From Single Properties to (Silico-) Imaging 297
13.4 Sampling: How, Where and When 301
13.4.1 Sample Preparation 301
13.4.2 Critical Scales and Sampling Frequency 302
13.4.3 Platforms for Aquatic Flow Cytometry 303
13.5 Monitoring Applications 305
13.5.1 Species Screening: Cultures 305
13.5.2 Phytoplankton Species Biodiversity 307
13.5.3 Harmful Algal Blooms 308
13.6 Ecological Applications 308
13.6.1 Population-related Processes 308
13.6.2 Cell-related Processes and Functioning 311
13.6.3 Plankton Abundance Patterns in the Sea: Indicators of Change 314
13.7 Marine Optics and Flow Cytometry 314
13.8 Future Perspectives 315
References 319
14 Cell Cycle Analysis in Plants
Martin Pfosser, Zoltan Magyar, and Laszlo Bögre 		323
Overview 323
14.1 Introduction 323
14.2 Univariate Cell Cycle Analysis in Plant Cells 325
14.3 BrdUrd Incorporation to Determine Cycling Populations 326
14.4 Cell Cycle Synchronization Methods: Analysis of Cell Cycle Transitions in Cultured Plant Cells 327
14.5 Plant Protoplasts to Study the Cell Cycle 335
14.6 Root Meristems for Cell Cycle Synchronization 335
14.7 Study of Cell Cycle Regulation by using Synchronized Cell Cultures and Flow Cytometry 336
14.8 Cell Cycle and Plant Development 338
14.9 Flow Cytometry of Dissected Tissues in Developmental Time Series 339
14.10 Cell Type-specific Characterization of Nuclear DNA Content by Flow Cytometry 339
14.11 Other Methods and Imaging Technologies to Monitor Cell Cycle Parameters and Cell Division Kinetics in Developing Organs 340
14.12 Concluding Remarks 342
References 343
15 Endopolyploidy in Plants and its Analysis by Flow Cytometry
Martin Barow and Gabriele Jovtchev 		349
Overview 349
15.1 Introduction 349
15.2 Methods to Analyze Endopolyploidy 351
15.2.1 Microscopy 351
15.2.1.1 Chromosome Counts 351
15.2.1.2 Feulgen Microdensitometry, Fluorescence Microscopy, Image Analysis 352
15.2.2 Flow Cytometry 352
15.2.2.1 Evaluation of Histograms 353
15.2.2.2 Quantification of the Degree of Endopolyploidy 354
15.3 Occurrence of Endopolyploidy 355
15.3.1 Endopolyploidy in Species 356
15.3.2 Endopolyploidy in Ecotypes and Varieties 356
15.3.3 Endopolyploidy in Different Life Strategies 357
15.3.4 Endopolyploidy in Organs 359
15.4 Factors Modifying the Degree of Endopolyploidization 362
15.4.1 Genome Size and Endopolyploidy 362
15.4.2 Environmental Factors 363
15.4.3 Symbionts and Parasites 364
15.4.4 Phytohormones 365
15.5 Dynamics of Endopolyploidization 366
15.6 Endopolyploidy and Plant Breeding 367
15.6.1 Endopolyploidy in Crop Plants 367
15.6.2 In vitro Culture and Plant Regeneration 368
15.7 Conclusions 369
References 370
16 Chromosome Analysis and Sorting
Jaroslav Doleel, Marie Kubaláková, Pavla Suchánková, Pavlína Kováová, Jan Barto, and Hana imková 		373
Overview 373
16.1 Introduction 374
16.2 How Does it Work? 375
16.3 How it All Began 377
16.4 Development of Flow Cytogenetics in Plants 379
16.4.1 Preparation of Suspensions of Intact Chromosomes 379
16.4.1.1 Biological Systems for Chromosome Isolation 379
16.4.1.2 Cell Cycle Synchronization and Metaphase Accumulation 383
16.4.1.3 Preparation of Chromosome Suspensions 383
16.4.2 Chromosome Analysis 385
16.4.2.1 Bivariate Analysis of AT and GC Content 385
16.4.2.2 Fluorescent Labeling of Repetitive DNA 386
16.4.2.3 The Use of Cytogenetic Stocks 386
16.4.2.4 Assignment of Chromosomes to Peaks on Flow Karyotypes 386
16.4.3 Chromosome Sorting 387
16.4.3.1 Estimating the Purity in Sorted Fractions 389
16.4.3.2 Improving the Sort Purity 389
16.4.3.3 Two-step Sorting 389
16.4.3.4 Purities and Sort Rates Achieved 390
16.5 Applications of Flow Cytogenetics 390
16.5.1 Flow Karyotyping 390
16.5.2 Chromosome Sorting 392
16.5.2.1 Physical Mapping and Integration of Genetic and Physical Maps 392
16.5.2.2 Cytogenetic Mapping 392
16.5.2.3 Analysis of Chromosome Structure 396
16.5.2.4 Targeted Isolation of Molecular Markers 396
16.5.2.5 Recombinant DNA Libraries 396
16.6 Conclusions and Future Prospects 398
References 400
17 Analysis of Plant Gene Expression Using Flow Cytometry and Sorting
David W. Galbraith 		405
Overview 405
17.1 Introduction 405
17.2 Methods, Technologies, and Results 406
17.2.1 Current Methods for Global Analysis of Gene Expression 406
17.2.1.1 Methods Based on Hybridization 407
17.2.1.2 Methods Based on Sequencing 408
17.2.1.3 Emerging Sequencing Technologies 409
17.2.1.4 Other -omics Disciplines and Technologies 410
17.2.2 Using Flow Cytometry to Monitor Gene Expression and Cellular States 411
17.2.2.1 Transgenic Markers Suitable for Flow Cytometry and Sorting 411
17.2.2.2 Subcellular Targeting as a Means for Transgenic Analysis 412
17.2.3 Using Flow Sorting to Measure Gene Expression and Define Cellular States 414
17.2.3.1 Protoplast and Cell Sorting Based on Endogenous Properties 414
17.2.3.2 Protoplast Sorting Based on Transgenic Markers 416
17.2.3.3 Sorting of Nuclei Based on Transgenic Markers 417
17.3 Prospects 418
17.3.1 Combining Flow and Image Cytometry 418
17.3.2 Use of Protoplasts for Confirmatory Studies 418
17.3.3 Analysing Noise in Gene Expression 419
References 421
18 FLOWER: A Plant DNA Flow Cytometry Database
Joaăo Loureiro, Jan Suda, Jaroslav Doleel, and Conceiçăo Santos 		423
Overview 423
18.1 Introduction 423
18.2 Taxonomic Representation in DNA Content Studies 425
18.3 Nuclear Isolation and Staining Buffers 427
18.4 Standardization and Standards 430
18.5 Fluorochromes 433
18.6 Quality Measures of Nuclear DNA Content Analyses 434
18.7 The Uses of DNA Flow Cytometry in Plants 435
18.8 Instrumentation 435
18.9 Where Are the Results Published? 436
18.10 Conclusion 437
References 438
Index 439

 
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