Working with Ferns (eBook)

Ashwani Kumar Professor, Department of Botany, University of Rajasthan, Jaipur. The author’s repertoire of published works spreads across 150 research articles in various national and international journals. He has an experience of over three decades in his field of research, namely, tissue culture and biochemistry, being awarded with prestigeous V. Puri Medal in 2008 for his services to advancement of Botany. Helena Fernández Associate Professor, Department of Biology of Organisms and Systems, Oviedo University, Spain. Her research focuses on micropropagation and reproduction in ferns during the two last decades. In 2002 she obtained the award “Ramón y Cajal” by The Ministry of Science and Technology, being engaged to the Oviedo University as tenurer, full time, in the Area of Plant Physiology since then. M. Angeles Revilla Plant Physioloy Assistant Professor in the Biology Faculty at the Oviedo University (Spain) since 1987. Twenty years experience in plant tissue culture. She has also worked in cryopreservation and genetic stability for the last ten years, mainly in the development of protocols for in vitro shoot apices in agronomic species.

Preface 6
Preface 8
About the Book 10
Contents 12
Contributors 16
Chapter 1: Introduction 22
References 29
Part I:Contribution of Ferns to Understanding of Plant Development 30
Chapter 2: Cellular, Molecular, and Genetic Changes During the Development of Ceratopteris richardii Gametophytes 31
2.1 Introduction 31
2.2 Regulation of Differentiation in Multicellular Gametophytes 32
2.3 Genes and Genomic Studies of C. richardii Spores 35
2.4 Spores as a Tool for the Study of Cellular Gravity Response 37
2.5 Resources Available and on the Horizon for Use in C. richardii Research 41
References 42
Chapter 3: Laboratory-Induced Apogamy and Apospory in Ceratopteris richardii 45
3.1 Introduction 45
3.2 Alternation of Generations 45
3.3 Apogamy and Apospory in Ferns 48
3.4 Comparison of Apogamy and Apospory in Ferns with Apomixis in Angiosperms 49
3.5 Induction of Apogamy and Apospory in C. richardii 52
References 54
Chapter 4: Sexual Reproduction in Ferns 57
4.1 Introduction 57
4.2 Looking for New Experimental Systems 58
4.3 Sexual Reproduction in Pteridophyta 59
4.3.1 Antheridiogens 59
4.3.2 Plant Growth Regulators and Sex Determination in Blechnum spicant L 60
4.4 New Tools to Study the Molecular Basis of Sex Determination 62
4.5 Sexuality in the Laboratory and in Nature 65
References 66
Chapter 5: Gibberellic Acid and Ethylene Control Male Sex Determination and Development of Anemia phyllitidis Gametophytes 69
5.1 Main Aspects of A. phyllitidis Gametophyte Development 69
5.2 Spore Germination, Growth, and Differentiation of A. phyllitidis Gametophytes 71
5.3 The Antheridiogens – Epigenetic Aspects of Development of A. phyllitidis Gametophytes 73
5.4 Precocious, GA3-induced, Antheridia Formation and the “Three-zonal Model” of Structure and Function in Development of A. phyllitidisGametophytes 74
5.5 ACCF and DDG Disturb GA3-Induced Antheridiogenesis and Development of A. phyllitidis Gametophytes 75
5.6 How Does Ethylene Participate in Development and Male Sex Determination in A. phyllitidis gametophytes? 76
5.7 Regulation of Antheridia Formation 77
5.8 Regulation of Cell Growth and Cell Division Cycleduring GA3-induced Antheridiogenesis 77
5.9 A. phyllitidis Gametophytes Produce ACC 80
5.10 Main Aspects of Antheridiogenesis 80
References 83
Chapter 6: The Sporophytes of Seed-Free Vascular Plants – Major Vegetative Developmental Features and Molecular Genetic Pathways 86
6.1 Introduction 86
6.2 Six Sporophyte Body Plans 88
6.3 Embryogeny 91
6.4 Apical Meristem Structure 93
6.5 Branching 94
6.6 Radial Patterning of Sporophyte Axes 96
6.7 Leaf Development 98
6.8 Developmental Genes 101
6.8.1 KNOX and ARP Genes 103
6.8.2 HD-ZIP Genes 105
6.8.3 MIKC-type MADS-box Genes 106
6.8.4 AP2 and ANT Genes 107
6.8.5 FLORICAULA/LEAFY 107
6.8.6 Other Genes 107
6.8.7 MicroRNA Regulation of Genes 108
6.9 Conclusion 109
References 110
Part II:Propagation, Conservation and Control of Genetic Variability in Ferns 114
Chapter 7: From Spore to Sporophyte: How to Proceed In Vitro 115
7.1 Introduction 115
7.2 Spore Culture 116
7.3 Gametophyte Culture: Nutritional and Environmental Conditions 119
7.4 Gametophyte Multiplication 120
7.4.1 By Natural Means 120
7.4.2 Homogenised Cultures 120
7.5 Sporophyte Formation 121
7.5.1 Sexual Reproduction 121
7.5.2 Apogamy 122
7.6 Sporophyte Multiplication 122
References 125
Chapter 8: In Vitro Regeneration Systems of Platycerium 129
8.1 Introduction 129
8.2 In Vitro Regeneration Systems of Sporophytic Tissue 131
8.2.1 Direct Shoot Organogenesis 132
8.2.1.1 Shoot and Rhizome Culture 132
8.2.1.2 Leaf Culture 134
8.2.1.3 Culture of Bud Scales 135
8.2.1.4 Homogenization of Sporophytes 137
8.2.2 Indirect Shoot Organogenesis 137
8.2.3 Apospory and Apogamy 138
8.3 In Vitro Cultures of Platycerium in Developmental and Physiological Studies 139
8.4 Conclusion 140
References 141
Chapter 9: Stipule Propagation in Five Marattioid Species Native to Taiwan (Marattiaceae Pteridophyte)144
9.1 Introduction 144
9.2 Materials and Methods 145
9.3 Results 146
9.4 Discussion 149
References 151
Chapter 10: Tree Ferns Biotechnology: From Spores to Sporophytes 152
10.1 Introduction 152
10.2 Media Most Often Used 154
10.3 Plant Growth Hormones 155
10.4 Origin of Spore 155
10.5 Spore Sterilization 155
10.6 Spore Germination 156
10.7 Gametophyte Growth and Development 157
10.8 Gametophyte Multiplication 158
10.9 Sexual Determination of Gametophyte 159
10.10 Sporophyte Production 160
10.11 Conclusion 161
References 162
Chapter 11: In Vitro Propagation of Rare and Endangered Serpentine Fern Species 165
11.1 Introduction 165
11.2 Materials and Methods 166
11.2.1 Initiation of Culture 166
11.2.2 Growth of Gametophytes 167
11.2.3 Gene Bank of Prothalli 167
11.2.4 Culture of Sporophytes 168
11.2.5 Regeneration of Shoot Buds 168
11.2.6 Acclimation of Plants Ex Vitro 168
11.2.7 Measurements of Stomata Cells and Spores Size 168
11.3 Results and Discussion 169
11.3.1 Sporophyte Formation 173
11.3.2 Sporophytes Multiplication 173
11.3.3 Regeneration from Sporophytes 175
11.3.4 Plant Preparation Before Transfer to Greenhouse 176
11.3.5 Comparison of Plants from Natural Stands and Originated In Vitro 177
References 179
Chapter 12: Conservation of Fern Spores 181
12.1 Introduction 181
12.2 Background: Fern Spore Longevity and Storage 181
12.3 Water Content of Fern Spores and Storage Stability 182
12.4 Fern Spores: Orthodox or Recalcitrant Storage Behavior? 184
12.5 Conclusions 185
References 185
Chapter 13: Exploration of Cryo-methods to Preserve Tree and Herbaceous Fern Gametophytes 189
13.1 Introduction 189
13.1.1 Fern Gametophytes as Germplasm for Genebanks 189
13.1.2 Current Status of Gametophyte Cryopreservation 190
13.1.3 Brief Summary of Habitat for Fern Species in This Study 191
13.2 Materials and Methods 193
13.2.1 Plant Material 193
13.2.2 Cryoprotection Procedures 194
13.2.3 Thawing of Gametophytes 194
13.2.4 Survival Assessments 195
13.3 Results 195
13.3.1 Gametophyte Survival Following Various Cryo-methods 195
13.3.2 Preculture Effects on Detectable Damage 197
13.3.3 Recovery of Gametophyte in Culture and Sporophyte Production 199
13.4 Discussion 201
13.4.1 Cryoprotection and Gametophyte Survival 203
13.4.2 Autofluorescence as a Simple Methods Assessment of Gametophyte Viability 204
13.4.3 Cellular Damage to Gametophytes and Totipotency 204
13.4.4 Desiccation and Cold Tolerance and Cryopreservability 205
13.5 Conclusions 205
References 206
Chapter 14: Pteridophyte Spores Viability 209
14.1 Concept of Spore Viability – Biological Importance 209
14.2 Viability Variation Among Species 211
14.3 Factors Affecting Viability 211
14.3.1 Genotype 212
14.3.2 Age 212
14.3.3 Temperature 214
14.4 Physiology of Fern Spores Viability 215
14.5 Viability Detection Techniques 216
14.6 Viability and Conservation 218
References 218
Chapter 15: Microsatellites: A Powerful Genetic Marker for Fern Research 222
15.1 Genetic Molecular Markers 222
15.2 Microsatellites 224
15.2.1 What and Where Are They? 224
15.2.2 How to Use Them? 226
15.3 Microsatellites in Ferns 228
15.3.1 Previous Microsatellite Works on Ferns 228
15.3.2 A Particular Study Case: Dryopteris aemula 229
15.4 Advantages and Disadvantages of Microsatellites for Fern Studies 230
15.4.1 Why to Use microsatellites? 230
15.4.2 Problems and Limitations 231
15.5 Future Prospects 232
15.5.1 Potential Applications 232
15.5.2 Next-Generation DNA Sequencing 232
15.6 Conclusion 232
References 233
Chapter 16: Diversity in Natural Fern Populations: Dominant Markers as Genetic Tools 236
16.1 Introduction 236
16.2 Why Choose a Dominant Marker? 237
16.3 Measuring Molecular Diversity in Complex Genomes 238
16.4 Critical Steps in AFLP Protocols 240
16.5 Measure of Genetic Diversity Using Dominant Markers in Natural Fern Populations 241
16.6 Perspectives: Dominant Markers as an Effective Way to Develop Specific Markers 245
16.7 Conclusion 246
References 247
Part III:Environmental Biotechnology:Ecotoxicology and Bioremediation in Ferns 250
Chapter 17: Mitochondrial Activity of Fern Spores for the Evaluation of Acute Toxicity in Higher Plant Development 251
17.1 Introduction 251
17.2 Characteristics of Toxicity Tests 252
17.3 Current Challenges of Ecotoxicology 252
17.4 Ferns and Ecotoxicology 253
17.5 Candidate Ferns for Toxicity Testing 256
17.6 Application of Fern Spores to Toxicity Testing 258
17.7 Conclusion 260
References 261
Chapter 18: Chronic Phytotoxicity in Gametophytes: DNA as Biomarker of Growth and Chlorophyll Autofluorescence as Biomarker of Cell Function 262
18.1 Introduction 262
18.2 Current State of Phytotoxicity Testing 263
18.3 Limitations of Traditional Acute Toxicity Tests 264
18.4 Limitations of Traditional Terrestrial Plant Toxicity Tests 265
18.5 Usefulness of Ferns in Phytotoxicity Testing 265
18.6 DNA as a Measurement of Cellular Proliferation 266
18.7 Chlorophyll a Autofluorescence as Surrogate of Plant Physiological State 267
18.8 Applications of the Bioassay of Chronic Toxicity Based on Fern Spores 268
18.9 Protocols 270
18.9.1 Toxicity Tests 270
18.9.1.1 Plant Material 270
18.9.1.2 Chronic Toxicity Bioassay 271
DNA Quantification 271
Chlorophyll Quantification 271
18.10 Conclusions 271
References 272
Chapter 19: Arsenic Hyperaccumulator Fern Pteris vittata: Utilities for Arsenic Phytoremediation and Plant Biotechnology 274
19.1 Arsenic – A Toxic Metalloid Widespread in the Environment 274
19.2 Discovery of Arsenic Hyperaccumulation Trait in Ferns 275
19.3 Biological Roles for Arsenic Hyperaccumulation 276
19.4 Arsenate Uptake 277
19.5 Arsenate Reduction 277
19.6 Arsenite Uptake 278
19.7 Arsenic Transport within the Plant 278
19.8 Oxidative Stress Tolerance 278
19.9 Utilities of Brake Fern for Phytoremediation 279
19.10 Brake Fern as a Source of Genes for Biotechnology 279
References 279
Chapter 20: Aerobiology of Pteridophyta Spores: Preliminary Results and Applications 283
20.1 Introduction 283
20.2 Methodology in Aerobiology Studies 284
20.3 Aerobiological Notes of Pteridophyta 284
20.4 Perspectives and Applications 290
References 291
Part IV:Therapeutical/Medicinal Applications 294
Chapter 21: Studies on Folk Medicinal Fern: An Example of “Gu-Sui-Bu” 295
21.1 Introduction 295
21.2 Comparative Analysis of Different Phenolic Compounds and Their Activities in Six Different “Gu-Sui-Bu” 297
21.2.1 Antioxidant Activity 297
21.2.2 Antioxidant Activity by ABTS Assay 297
21.2.3 Antioxidant Activity by Dot-Blot and DPPH Staining 297
21.2.4 Determination of “Reducing Power” 299
21.2.5 Scavenging Activity Against DPPH Radical 300
21.2.6 Total Polyphenols Content (Flavonoids, Flavonols, Condensed Tannin, and Proanthocyanidin) Determination in “Gu-Sui-Bu” 301
21.2.6.1 Flavonoids Content 303
21.2.6.2 Flavonols Content 303
21.2.6.3 Condensed Tannins/Proanthocyanidin Content 303
21.3 In Vitro Studies on D. fortunei 304
21.3.1 Effects of MS Basal Medium, Sucrose Concentration, and Sugars on Germination of Spores and Development of Gametophytes 304
21.3.2 Effects of pH on Spore Germination, Development of Gametophytes, and Reproductive Organs 305
21.3.3 Effects of Light Spectra on Spore Germination, Development of Gametophytes, and Reproductive Organs 306
21.4 Conclusion 311
References 312
Chapter 22: Ecdysteroids in Ferns: Distribution, Diversity, Biosynthesis, and Functions 315
22.1 Introduction 315
22.2 Distribution and Diversity 315
22.3 Biosynthesis 320
22.4 Regulation of Ecdysteroid Biosynthesisand Accumulation 323
22.5 Function(s) 324
References 325
Chapter 23: Ferns: From Traditional Uses to Pharmaceutical Development, Chemical Identification of Active Principles 330
23.1 Introduction 330
23.2 Traditional Medicine Uses of Ferns 330
23.3 Fern Bioactive Components 339
23.3.1 Terpenoids 340
23.3.2 Triterpenoids 340
23.3.3 Diterpenoids 340
23.3.4 Sesquiterpenoids 345
23.3.5 Phenolic Compounds 347
23.3.6 Flavonoids 348
23.3.7 Alkaloids 348
23.4 Pharmaceutical Development 349
23.4.1 Huperzine A 349
23.4.2 Pharmaceutical Preparations from Polypodium leucotomos 350
References 350
Chapter 24: Functional Activities of Ferns for Human Health 356
24.1 Introduction 356
24.2 Natural Antioxidant 357
24.3 Foods 360
24.4 Natural Antimicrobial Agents 361
24.5 Cosmetic Ingredient 363
24.6 Air Purifier 363
24.7 The Future of Ferns and Fern Allies 364
References 365
Chapter 25: Toxicological and Medicinal Aspects of the Most Frequent Fern Species, Pteridium aquilinum (L.) Kuhn 369
25.1 Introduction 369
25.1.1 Chemical Aspects of the Plant 370
25.2 Toxicological Aspects 370
25.2.1 Compounds of Anti-thiamine Character 370
25.2.2 Cyanogen Glycosides 371
25.2.3 Compounds of Illudane Skeleton 371
25.2.3.1 Ptaquiloside 371
25.2.3.2 Pterosins 373
25.2.3.3 Other Chemical Components 373
25.3 Effects on Animals 374
25.3.1 Non-ruminant Animals 374
25.3.1.1 Swine 374
25.3.1.2 Rabbit 374
25.3.1.3 Guinea Pig 375
25.3.1.4 Rat and Mice 375
25.3.2 Ruminant Animals 375
25.3.2.1 Cattle 375
The Bovine Enzootic Haemature 375
Haemorrhagic Syndromes (Acute Haemorrhagic Disease) 376
Upper Alimentary Carcinoma 376
25.3.2.2 Sheep 377
25.4 Effects in Humans 377
25.5 Working Mechanism of Ptaquiloside 377
25.6 Presence of Ptaquiloside in Soil and Water 378
25.7 Medicinal Uses of P. aquilinum 379
25.8 Conclusions 379
References 380
Index 384