Methodologies and Results in Grapevine Research (eBook)

Preface 5
Contents 7
Contributors 10
1 Grapevine Roots and Soil Environment: Growth, Distribution and Function 17
1.1 Introduction 18
1.2 Methods and Results 19
1.2.1 Root Characteristics 19
1.2.2 Soil Environment 19
1.2.2.1 Gravimetric Method 21
1.2.2.2 Frequency Domain Reflectometry (FDR) and Time Domain Reflectometry (TDR) Sensors 21
1.2.3 Root Sampling 23
1.2.3.1 Direct Excavation 23
1.2.3.2 Trenches Trenches and Profile Root Distribution 24
1.2.3.3 Rhizotrons 25
1.2.3.4 Soil Coring 26
1.2.3.5 Ingrowth Cores 27
1.2.3.6 Minirhizotrons 28
1.2.4 Ecophysiological Measurements 28
1.2.4.1 Root Biomass 29
1.2.4.2 Root Composition 29
1.2.4.3 Hormonal Production in Roots 29
1.2.4.4 Root Water Uptake 30
1.3 General Considerations and Further Research 33
1.4 Disclaimer 33
References 33
2 Radiation Balance in Vineyards 37
2.1 Introduction: Principles of Radiation Balance 38
2.2 Materials and Methods 42
References 44
3 Vegetative Development: Total Leaf Area and SurfaceArea Indexes 46
3.1 Introduction 47
3.2 Practical Uses of LAI and SA 50
3.3 Materials and Methods 51
3.3.1 Leaf Area Index 51
3.3.1.1 Calculations 52
3.3.1.2 Example of Calculation 52
3.3.2 Surface Area 53
3.3.2.1 Measurement of SA in Bush Vines 54
3.3.2.2 Measurement of SA in a VSP Trellis 55
3.3.2.3 Measurement of SA in Curtains or Sprawl Trellis Systems 56
References 57
4 Vegetative Growth, Reproductive Development and Vineyard Balance 60
4.1 Measuring Vegetative Growth 61
4.1.1 Estimation of Annual Shoot Growth 61
4.1.2 Estimation of Annual Vine Growth 62
4.2 Measuring Reproductive Growth 63
4.2.1 Estimation of Inflorescence and Flower Number 63
4.2.2 Estimation of Fruit-Set Rate 64
4.2.3 Estimation of Fruit Load 65
4.3 Estimating Vineyard Balance 66
4.4 How to Express Results: Per Vine? Per Square Meter? Per Meter of Row? 68
References 69
5 Methodologies for the Measurement of Water Flow in Grapevines 72
5.1 Introduction 73
5.2 Invasive Methodologies 73
5.2.1 Granier Heat Dissipation Technique (hd) 73
5.2.1.1 Practical Considerations 74
5.2.2 Heat Pulse Systems 75
5.2.2.1 Practical Considerations 78
5.2.3 Other Invasive Methodologies 78
5.3 Non-invasive Methodologies 79
5.3.1 Stem Heat Balance Method (SHB) 79
5.3.1.1 Practical Considerations 81
5.3.2 Other Non-invasive Methods of Sap Flow Measures 82
References 83
6 Methods for Assessment of Hydraulic Conductance and Embolism Extent in Grapevine Organs 85
6.1 Introduction on Available Methods 86
6.2 Entire Plant 87
6.3 Leaves 88
6.4 Roots 91
6.5 Measurements on Xylem Segments 94
6.5.1 Xylem Portions 94
6.5.2 By Nuclear Magnetic Resonance 96
References 96
7 Comparison of Three Operational Tools for the Assessment of Vine Water Status: Stem Water Potential, Carbon Isotope Discrimination Measured on Grape Sugar and Water Balance 100
7.1 Introduction 101
7.2 Stem Water Potential 102
7.2.1 Impact of Soil Water Availability and Climatic Conditions on Stem Water Potential 104
7.2.2 Possible Applications of Stem Water Potential 105
7.2.2.1 Assessment of Vine Water Status in Relation to Soil Water Availability 105
7.2.2.2 Assessment of Seasonal Vine Water Status Dynamics Among Vintages 106
7.2.2.3 The Use of Stem Water Potential in Irrigation Management 106
7.3 Carbon Isotope Discrimination Measured on Grape Sugar 107
7.3.1 Possible Applications of Carbon Isotope Discrimination Measured on Grape Sugars at Ripeness 108
7.3.1.1 Assessment of Vine Water Status in Relation to the Climate of the Vintage, Soil Water Availability and Grapevine Variety 108
7.3.1.2 Spatialization of Vine Water Status at Intra-Block Scale 109
7.3.1.3 Spatialization of Vine Water Status at Estate Level 110
7.3.1.4 The Use of d13C to Assess the Necessity of Irrigation 111
7.3.2 Thresholds for d13C Values with Respect to Vine Water Deficit 111
7.4 Water Balance Modelling 112
7.4.1 The Stomatal Regulation Function 112
7.4.2 Water Fluxes and Relations of Computed Water Stress Index with d13C 114
7.4.3 Sensitivity Analysis of the Water Balance Model 115
7.4.4 Possible Applications of the Water Balance Model 117
7.5 Conclusions 117
References 118
8 Gas-Exchange and Chlorophyll Fluorescence Measurements in Grapevine Leaves in the Field 120
8.1 Introduction: Photosynthetic Primary Reactions Versus Gas Exchange 121
8.2 The Basics of Gas-Exchange Analysis: Net Photosynthesis, Stomatal Conductance, Leaf Transpiration and the Sub-stomatal Concentration of CO2 122
8.3 The Farquhar et al. Model of Photosynthesis and CO2 -Response Curves of Gas Exchange 124
8.4 Potential Errors: Influence of Leaf Temperature Recordings, Cuticular Conductance, and Heterogeneous Stomatal Closure 125
8.5 The Basics of Chlorophyll Fluorescence Analysis: The Concept of Variable Fluorescence, the Kautsky Effect and Its Interpretation 127
8.6 The Application Potential of Simultaneous Gas Exchange and Chlorophyll Fluorescence Measurements 130
References 131
9 Measuring Water Use Efficiency in Grapevines 135
9.1 Introduction 136
9.2 Defining Water Use Efficiency: From Leaf to Plants and Vineyards 137
9.2.1 Determination at the Vineyard Level 138
9.2.2 Plant Water Use Efficiency 139
9.2.3 Instantaneous Leaf Water Use Efficiency 141
9.2.4 Stable Isotope Discrimination Techniques for WUE Estimation 142
9.3 General Consideration: Future Perspectives 144
References 144
10 Use of Thermal Imaging in Viticulture: Current Application and Future Prospects 147
10.1 Introduction 148
10.2 Thermal Imaging Background 149
10.2.1 Infrared Radiation 149
10.2.2 Thermal Imaging 150
10.2.3 Important Concepts Related to Thermal Imaging 150
10.2.3.1 Emissivity and Black Body Radiation 150
10.2.3.2 The Stefan-Boltzman Law 150
10.2.4 How Does IR Radiation Become Visible to Human Eyes? 151
10.2.5 Leaf Energy Balance and Thermal Imaging 152
10.2.5.1 Leaf Energy Balance 152
10.2.5.2 Evaporative Energy Exchange 153
10.2.6 Estimating Leaf Stomatal Conductance from Thermal Imaging Measurements 153
10.2.7 Thermal Imaging and Stress Detection 155
10.3 Thermal Imaging Use in Grapevine 156
10.3.1 Stress and Stomatal Regulation in Grapevine 156
10.3.2 Stress Detection in Grapevine 156
10.3.2.1 Detection of Abiotic Stress: Monitoring of Plant Water Status and Irrigation 156
10.3.2.2 Detection of Biotic Stress 157
10.3.2.3 Breeding 158
10.4 Potential, Limitations and Future Developments of Thermal Imaging 158
References 159
11 Grapevine Fruiting Cuttings: An Experimental System to Study Grapevine Physiology Under Water Deficit Conditions 163
11.1 Description of the Technique 164
11.1.1 Plant Material 164
11.1.2 Production of Fruiting Cuttings 164
11.1.3 Technical Adaptation for PRD Studies 165
11.1.4 Water Treatments 165
11.2 Results 167
11.2.1 Main Vegetative Characteristics 167
11.2.2 Grape Development 167
11.2.3 Grapevine Responses to Irrigation Regime 168
11.3 Interest, Limitations and Progress for Our Understanding of Grapevine 173
References 174
12 Nutritional Deficiencies 176
12.1 Background 177
12.2 Mineral Element Function 178
12.3 Analytical Methods 179
12.4 Mineral Elements 180
12.4.1 Nitrogen 180
12.4.1.1 Symptoms 180
12.4.1.2 Causes and Therapy 181
12.4.2 Phosphorus 182
12.4.2.1 Symptoms 182
12.4.2.2 Causes and Therapy 182
12.4.3 Potassium 182
12.4.3.1 Symptoms 182
12.4.3.2 Causes and Therapy 185
12.4.4 Calcium 186
12.4.4.1 Symptoms 186
12.4.4.2 Causes and Therapy 187
12.4.5 Magnesium 187
12.4.5.1 Symptoms 187
12.4.5.2 Causes and Therapy 187
12.4.6 Iron 188
12.4.6.1 Symptoms 188
12.4.6.2 Causes and Therapy 189
12.4.7 Boron 192
12.4.7.1 Symptoms 192
12.4.7.2 Causes and Therapy 193
12.4.8 Zinc 196
12.4.8.1 Symptoms 196
12.4.8.2 Causes and Therapy 199
12.4.9 Manganese 199
12.4.9.1 Symptoms 199
12.4.9.2 Causes and Therapy 199
12.5 Interest, Limitation, Progress 200
References 201
13 Polyamines in Grapevine Research 203
13.1 Introduction 204
13.2 Determination of Key Enzymatic Activities Associated with Polyamine Metabolism in Grapevine 207
13.2.1 Protein Extraction 207
13.2.2 Protein Content Determination 207
13.2.3 Determination of Arginase 208
13.2.4 Determination of PA Metabolism-Involved Decarboxylases 209
13.2.5 Determination of SPDS and SPMS 211
13.2.6 Determination of DAO and PAO 212
13.3 Determination of Polyamine Titers 214
13.3.1 Polyamine Extraction 214
13.3.2 Polyamine Determination 214
References 216
14 Field Assessment and Diagnostic Methods for Detection of Grapevine Viruses 220
14.1 Viruses of Grapevine 221
14.2 Visual Symptoms and Biological Tests (indexing) 222
14.3 Serological Methods 225
14.4 Molecular Methods 227
14.4.1 Molecular Hybridization 227
14.4.2 RT-PCR 228
14.4.2.1 Nested RT-PCR 229
14.4.2.2 Polyvalent RT-PCR 229
14.4.2.3 Multiplex RT-PCR (mRT-PCR) 230
14.4.2.4 Real-Time RT-PCR 231
14.4.3 Microarray Technology 232
14.5 Conclusions 233
References 234
15 Real-Time PCR Detection Methods for Economically Important Grapevine Related Bacteria 238
15.1 Introduction 239
15.2 Real-Time PCR 240
15.3 General Guidelines for Introduction of a New Real-Time PCR Diagnostic Test 241
15.4 Development and Implementation of New Real-Time PCR Assays for Plant Pathogenic Bacteria 242
15.4.1 Grapevine Yellows Phytoplasma and X. ampelinus: Designing a New Detection System 242
15.4.1.1 Background 242
15.4.1.2 Existing Detection Methods 243
15.4.1.3 Design and Implementation of New Detection System 243
15.4.2 Aster Yellows Phytoplasma 246
15.4.2.1 Background 246
15.4.2.2 Existing Detection Methods 246
15.4.2.3 Implementation of the Detection System 246
15.4.3 Implementing an Existing Real-Time PCR Detection System: Case X. fastidiosa 247
15.4.3.1 Background 247
15.4.3.2 Existing Detection Methods 247
15.4.3.3 Implementation of the Detection System 249
15.5 Conclusions 251
References 252
16 Field Assessment and Diagnostic Methods for Detection of Grapevine Phytoplasmas 256
16.1 Phytoplasmas of Grapevine 257
16.2 Visual Symptoms 258
16.3 Molecular Methods 260
16.4 Conclusions 264
References 264
17 NICT: New Tools to Control Phytochemical Treatments and Traceability 268
17.1 Introduction 269
17.1.1 The Project Life AWARE 270
17.1.2 The Project TICSAD 271
17.1.3 Inventory of On-Board NICT Systems 271
17.2 Material and Methods 272
17.2.1 The AWARE and TICSAD Embedded Equipment 272
17.2.2 The Onboard Systems have 3 Roles 274
17.2.3 The Traceability Software 275
17.2.4 Processing of Treatment Product Data 275
17.2.5 Generation of Plot Log 275
17.3 Results 276
17.3.1 The Reduction of the Quantities of Pesticides Related to Better Practices 277
17.3.2 Model of Plant Protection Product Transfer 277
17.3.3 The Use of the NICT Embedded System 278
17.3.3.1 Role of the NICT for sprayer tuning 278
17.3.3.2 Role of NICT for Farmer Practices Improvements 279
17.3.3.3 Role of NICT for Traceability Improvement 279
17.4 Prospects 279
17.4.1 The Future of TICSAD with Scientific Approach 279
17.4.2 The POD ''Mildium'' Approach 280
17.4.3 The ''OPTIDOSE'' Approach 281
17.4.4 Volume Modulation According to the Vigour Variability of the Vine 282
17.5 Conclusions 282
References 283
18 Isolation and Use of Protoplasts from Grapevine Tissues 285
18.1 Introduction 286
18.2 Methods for the Isolation of Protoplasts from Grapevine Tissues 288
18.2.1 Protoplast Isolation from Mesocarp Tissue 288
18.2.1.1 Digestion of Mesocarp Tissue 288
18.2.1.2 Protoplast Purification 288
18.2.1.3 Protoplast Yield, Viability and Integrity 288
18.2.1.4 Visualization of the Vacuolar Apparatus with Neutral Red 290
18.2.2 Protoplast Isolation from Mesophyll Cells 290
18.2.2.1 Plant Material 290
18.2.2.2 Isolation and Culture of Mesophyll Protoplasts 291
18.2.2.3 Transport Experiments 292
18.3 Detection of Reactive Oxygen Species in Grapevine Leaf Tissue and Protoplasts 292
18.3.1 Luminol-Dependent Chemiluminescence Assay for H2O2 293
18.3.2 Lucigenin-Dependent Chemiluminescence Assay for O2.- 293
18.3.3 Assay for O2.- - Synthase 293
18.4 Assays for Antioxidant Enzyme and Antioxidant Biomolecules 294
18.4.1 Antioxidant Enzymes 294
18.4.2 Antioxidant Biomolecules 295
18.5 Final Remarks 297
References 298
19 RNA Extraction from Grapevine Woody Canes for Gene Expression Analysis by Real-Time RT-PCR 302
19.1 Introduction 303
19.2 Material and Methods 303
19.2.1 Plant Material and Sampling 303
19.2.2 RNA Extraction Protocol 303
19.2.3 RNA Analysis 304
19.2.4 Real-Time RT-PCR 304
19.3 Results and Discussion 304
References 307
20 A Method for Isolating Total RNA from Mature Buds and Other Woody Tissues of Vitis Vinifera 308
20.1 Introduction 309
20.2 Materials and Methods 310
20.2.1 Plant Material 310
20.2.2 Reagents and Solutions 310
20.2.3 Glassware and Plasticware 310
20.2.4 Procedure 310
20.2.4.1 Day 1 310
20.2.4.2 Day 2 311
20.2.4.3 Day 3 311
20.3 Results and Discussion 312
References 313
21 RNA Extraction from Young, Acidic Berries and Other Organs from Vitis Vinifera L 315
21.1 Introduction 316
21.2 Materials and Methods 317
21.2.1 Extraction Buffer 317
21.2.2 Salt Solutions 317
21.2.3 Silica Column Fixation Buffer 318
21.2.4 Sampling 318
21.2.5 Extraction and Purification Procedures 318
21.2.6 RNA Quality 319
21.2.7 Amplification of aRNA 320
21.2.8 Expected Yield 320
References 320
22 Transcriptomics Analysis Methods: Microarray Data Processing, Analysis and Visualization Using the Affymetrix Genechip Vitis Vinifera Genome Array 322
22.1 Introduction 323
22.2 A Long-Term Growth Experiment as an Example for Microarray Data Analysis 323
22.3 Long-Term Growth Experimental Design 324
22.4 Vitis GeneChip Design 324
22.5 Microarray Quality Assessment 325
22.6 Microarray Quality Assessment of the Long-Term Growth Experiment 327
22.7 Normalization of Microarray Data 329
22.8 Data Organization 331
22.9 Functional Annotation and Categorization 332
22.10 Data Visualization and Integration 333
References 336
23 Visualisation of Transcriptomics Data in Metabolic Pathways 340
23.1 Introduction 341
23.2 The Principles of MapMan Organisation 342
23.3 Specificities of Grapevine MapMan 343
23.4 Conclusions 346
References 346
24 Small RNA Extraction and Expression Analysis by Northern Blot Northern Blot 348
24.1 Introduction 349
24.2 Description of the Procedure 350
24.2.1 Extraction of Small RNAs from Grapevine Tissues 350
24.2.1.1 Materials and Reagents 350
24.2.1.2 Procedure 351
24.2.2 Small RNA Electrophoresis and Blotting 352
24.2.2.1 Materials and Reagents 353
24.2.2.2 Procedure 353
24.2.3 Small RNA Hybridization 354
24.2.3.1 Materials and Reagents 355
24.2.3.2 Procedure 355
24.3 Conclusions and Perspectives 356
References 357
25 A Rapid and Efficient Method for Isolating High-Quality Total Proteins from Mature Buds and Other Woody Tissues of Vitis Vinifera 359
25.1 Introduction 359
25.2 Materials and Methods 360
25.2.1 Plant Materials 360
25.2.2 Reagents 360
25.2.3 Glassware and Plasticware 361
25.2.4 Procedure 361
25.2.4.1 Protein Extraction 361
25.2.4.2 Gel Electrophoresis 362
25.3 Results and Discussion 362
References 363
26 Expression Analysis in Grapevine by In Situ Hybridization and Immunohistochemistry 364
26.1 Introduction 365
26.2 Historical Background 365
26.3 Methods and Materials 366
26.3.1 PCR Generation of T7 Promoter-Tailed DNA 366
26.3.2 In Vitro Transcription Labelling of RNA 368
26.3.3 Preparation of Sectioned Plant Material 368
26.3.4 Hybridization, Washes and Detection of Hybridization Sites 369
26.3.5 Immunohistochemistry 370
26.4 Results 370
26.4.1 PCR Generation of T7 Promoter-Tailed DNA 370
26.4.2 RNA Probe Synthesis 371
26.4.3 In Situ Hybridization on Various Grapevine Organs 372
26.4.4 Expression of a Class IV Chitinase Gene in the Developing Grape Berry 374
26.5 Discussion 374
References 376
27 Marker Development for Important Grapevine Traits by Genetic Diversity Studies and Investigation of Differential Gene Expression 378
27.1 Introduction 379
27.2 The Need to Develop Molecular Markers for Breeding 379
27.3 Strategies to Develop Molecular Markers 380
27.3.1 Genetic Mapping and QTL Analysis 380
27.3.2 Genetic Diversity Studies 381
27.3.2.1 SSR Markers 382
27.3.2.2 SNP Markers 382
27.3.3 Differential Gene Expression Analysis 383
27.3.3.1 Global Screening with Micro-Arrays 383
27.3.3.2 Quantitative Real Time PCR 384
27.4 Conclusions and Summary 388
References 388
28 Phenolic Maturity in Red Grapes 391
28.1 Introduction 392
28.2 Determination of Phenolic Maturity 393
28.3 Phenolic Maturity Methods Based on the Phenol Extraction 394
28.3.1 Glories Method -- Protocol for Phenol Extraction 395
28.3.1.1 Sampling 395
28.3.1.2 Procedure of Phenolic Maturity Determination 395
28.3.1.3 Calculation 396
28.3.2 Other Methods Based in Glories Protocol 396
28.3.2.1 Lamadon Method 396
28.3.2.2 Peyron Method 397
28.3.2.3 Nadal and Mateos Method 397
28.3.2.4 Iland Method 399
28.4 Helpful Hints, Results and Comments About Phenolic Maturity 400
28.4.1 Caution at Bunch and/or Berry Sampling 400
28.4.2 Interpretation of Values and Indexes 401
28.4.3 Effect of the Terroir, Variety and Water Availability on Grape and Wine Phenol Content 401
28.4.4 Stage of Ripeness Influence on Phenol Composition 407
28.4.5 Concluding Remarks 408
References 408
29 Aromas in Grape and Wine 412
29.1 Introduction 413
29.2 Analytical Methods 415
29.2.1 Analyses of Aromatic Compounds in the Past 423
29.3 Extraction Technique for Volatile Compounds in Grape and Wine 424
29.3.1 Extraction and Analysis of Volatile Compounds from Grape 424
29.3.2 Extraction and Analysis of Volatile Compounds from Wine 427
29.3.2.1 Experimental 427
29.3.3 Extraction Method for Carotenoids in Grape 436
29.3.3.1 HPLC Conditions for Carotenoids Analysis 436
29.3.3.2 Enzymes Enhancing of Primary Aroma Compounds 436
References 440
Index 444