CHAPTER 1 How to Grow Arabidopsis, 1
Morphology and Anatomy 1
Seed Collection and Storage 7
Cultivation of Arabidopsis 7
General Considerations 7
Growth Environment 8
Cultivation of Plants in Soil 10
Use of Solid Media 12
Other Growth Conditions 13
Pest Management 14
Supplier's Web Sites 17
References 18
CHAPTER 2 Obtaining Mutants, 19
Obtaining Previously Identified Mutants from the Stock Centers 19
Forward Genetics:Finding Mutations That Cause Particular Phenotypes 20
EMS Mutagenesis of Seed 24
T-DNA Mutagenesis 26
Special T-DNA Vectors 26
Reverse Genetics:Finding Mutations in Particular Genes 30
Screening DNA Pools for T-DNA Insertions 31
Considerations for Phenotypic Characterization 34
Beyond Mutants:Using Natural Variation to Identify Interesting Genes 35
References 37
CHAPTER 3 Genetic Analysis of Mutants, 41
Setting up Ctosses 41
Choosing Parent Plants 41
Choosing Flowers 41
Segregation Analysis 43
Self-progeny 43
F1 Progeny 44
F2 Progeny 44
Backcrosses and Cosegregation 46
Complementation Testing 48
Strategies for Identifying Double Mutants 50
Finding Double Mutants in the F2 Generation 50
Finding Double Mutants in the F3 Generation 51
The Brute Force Approach 52
References 53
CHAPTER 4 How to Analyze a Mutant Phenotypically, 55
Growth Parameters 56
Quantitative Analysis of Root Growth 56
Hypocotyl Length 59
Flowering Time 60
Germination Rate 62
Fresh Weight Gain 62
Water Loss 62
Hormone Response 63
Gibberellin/Abscisic Acid/Paclobutrazol 63
Auxin 64
Ethylene 66
Brassinosteroids 69
Response to the Abiotic Environment 71
Root Elongation Under Salt/Hormone-induced Stress 71
Germination Rate 72
Electrolyte Leakage 73
Fresh Weight Gain 74
Water Loss 75
Others:Proline and Sugar Content 75
Heavy Metal Stress 75
Bacterial Pathogens 77
Preparation of Bacterial Cultures and Inoculation of Plants 77
Testing the Hypersensitive Response 78
Assessing Bacterial Growth 79
Oomycete Pathogens:Peronospora parasitica 81
Reviving Frozen Stocks and Inoculating Plants 81
Rating Peronospora Infections 83
Preparation of Frozen Tissue Sections 83
Maintaining Infections 84
Diaminobenzidine Stain for Hydrogen Peroxide 85
Trypan Blue Stain for Fungi,Oomycetes,and Dead Plant Cells 86
Histology 87
Preparation of Tissue Sections of Fixed Material 87
Specialized Staining Techniques 93
The PAS Reaction for Staining Cell Walls 93
Alcian Blue-PAS Reaction 95
Phloroglucinol Stain for Lignin 96
Vital Stain for Cytoplasm 97
Neutral Red Staining for Vacuoles 98
Whole-mount DAPI Staining and Measurement of DNA Content 98
Nuclear Staining for Confocal Microscopy 100
Cleared Tissue for Observation of Vascular Strands 104
Agarose Imprints of Surfaces 105
Scanning Electron Microscopy 106
Standard SEM Protocol 106
Dental Wax Impressions to be Viewed in SEM 109
SEM"Quick-and-Easy"Fixation 109
Imaging of Fresh Arabidopsis Tissues in the SEM 110
Transmission Electron Microscopy 112
Standard TEM Protocol 112
TEM Freeze Substitution 114
References 115
CHAPTER 5 How to Transform Arabidopsis, 119
Vectors and Agrobacterium Hosts 119
Agrobacterium strains 122
Transformation of Agrobacterium 122
Transformation of Agrobacterium Using Electroporation 123
Transformation of Agrobacterium Using the Freeze-Thaw Method 125
PCR Analysis of Agrobacterium 127
In Planta Transformation of Arabidopsis 128
Plant Growth 128
Floral Dip of Arabidopsis 129
Alternative Protocol 1:Vacuum Infiltration 130
Alternative Protocol 2:Spraying 131
Selecting Transformed Plants 131
Kanamycin Selection 131
Basta Selection on Soil 133
Root Transformation 134
References 140
CHAPTER 6 How to Isolate a Gene Defined by a Mutation, 143
Isolating a Gene Defined by an Insertion Mutation 143
TAIL-PCR 144
Isolating a Gene Known Only by the Phenotypes of Mutant Alleles:Positional Cloning 155
STEP 1:Determining the Approximate Map Position for yfg 155
STEP 2:Define the Map of yfg as Narrowly as Possible 161
STEP 3:Finding YFG within the Mapped Interval 162
Special Cases 162
Serious Errors 164
Purifying DNA from Arabidopsis 165
CTAB DNA Miniprep 165
Dellaporta DNA Miniprep 166
Quick DNA Prep for PCR 168
References 169
CHAPTER 7 How to Study Gene Expression, 171
RNA Expression 171
RNA Extraction for Northern Blots and RT-PCR 172
Semiquantitative Reverse Transcription followed by PCR 173
In Situ Hybridization to Tissue Sections 181
Radioactive In Situ Hybridization 182
Nonradioactive In Situ Hybridization 195
Whole-mount In Situ Hybridization 212
Protein Expression 215
Extraction of Total Protein for Western Blots 215
Organelle Preparations 216
FPLC Gel Filtration 226
Nondenaturing Gel Electrophoresis of Proteins 228
Protein Coimmunoprecipitation 233
In Situ Localization of Proteins 237
Reporter Genes 241
Whole-mount GUS Staining 243
Quantitative GUS Activity Assays 249
Subcellular Localization of GUS-and GFP-Tagged Proteins in Onion Epidermal Cells 252
Live-cell Imaging of GFP 262
Liquid LUC Activity Assays 267
LUC Imaging of Whole Plants 269
References 276
CHAPTER 8 How to Study Gene Function, 281
Reducing Gene Expression 282
Antisense RNA 282
Cosuppression/RNAi 284
Tissue-or Stage-specific Gene Knock-outs 286
Misexpression 287
Constitutive/Tissue-specific Promoter Fusions 287
Two-component Systems for Tissue-specific Misexpression 287
The Alc Gene Ethanol-inducible Switch system 291
Glucocorticoid Inducible Control of Gene Expression 293
Heat Shock Induction 295
Glucocorticoid Fusions for Transcription Factors 296
Transient Expression 300
Transient Expression in Protoplasts 300
Transgene Expression in Regenerated Roots 304
Other Gain-of-Function Strategies 308
Mosaic Analysis 309
References 313
APPENDIX 1 Where to Find Information on Arabidopsis 317
APPENDIX 2 Critical x2 Values 321
APPENDIX 3 Cautions 323
APPENDIX 4 Suppliers 341
Index, 343