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Histology ceil wall staining

Alcian Blue staining, 95-96 PAS reaction materials, 94 principles, 93

Schiff's reagent preparation,

93-94 slide incubations, 95 DAB staining for hydrogen peroxide, 85 DNA content staining

DAP1 staining of whole mounts, 99

propidium iodide staining of nucleus apex dissection, 102-103 clearing, 102 confocal microscopy, 103 fixation, 101 materials, 100-101 mounting, 103 staining, 101-102 rationale, 98-99 electron microscopy. See Scanning electron microscopy; Transmission electron microscopy lignin staining with phloroglucinol, 96-97

plastic embedding of sections advantages, 87 dissection, 87-88 fixation and dehydration, 88 methacrylate infiltration, 90-91 mounting for section* lg, 91-92 resin selection, 89 sectioning, 92-93 staining, 88-89 Trypan blue staining, 86 vacuole staining with Neutral Red, 98

vital staining fluorescein diacetate staining of live cells, 97 propidium iodide staining of dead cells, 97

Hydrogen peroxide, assay with diaminobenzidine staining, 85

Hygromycin, limitations in transformation screening, 133

Hypocotyl green fluorescent protein imaging, 266

length analysis of mutants, 59-60

Immunohistochemistry alkaline-phosphatase-coupled secondary antibodies, 240-241 horseradish-peroxidase-coupied avidin-biotin complex antibody incubations, 239 color development, 240 deparaffinization, 238 endogenous avidin and biotin blocking, 239 endogenous peroxidase blocking, 238 fixation, 238 kits, 237-238 materials, 238 principles, 237 protease treatment, 239 rehydration, 238 overview, 237

Insecticides, guidelines for use, 16-17 In situ hybridization, RNA

digoxygenin-labeled probe hybridization, alternat- /e protocol detection, 210-211 embedding, 203-205 fixation, 203-204 hybridization, 209 pretreatment of sections, 208209

probe synthesis, 205-208 sectioning, 203-205 washing, 210-211 digoxygenin-labeled probe hybridization detection, 200-201 double-labeling, 201-202 embedding, 195-196 fixation, 195-196 hybridization, 199-200 pretreatment of sections, 198199

probe synthesis and hydrolysis,

196-198 sectioning, 195-196 washing, 200-201 overview, 181 radioactive hybridization autoradiography, 192-195 embedding, 184 fixation, 182-184 hybridization, 190-191 pretreatment of sections, 188— 189

probe synthesis and hydrolysis, 186-188 sectioning, 185 washing, 191-192 troubleshooting, 202-203 whole-mount in situ hybridization fixation and dehydration, 213 hybridization, 214 materials, 212 overview, 212 pretreatment, 213-214 washing, 214

Kanamycin, Arabidopsis transformant selection, 131-132

Leaf development, 3-5 green fluorescent protein imaging, 266

LhG4:pOp, expression system, 290291

Lignin, staining with phloroglucinol, 96-97

Lineage analysis overview, 310-311 recombinases, 309-310

Luciferase advantages as reporter, 242 applications, 241-242, 266 liquid assays extracts, 267-268 intact tissue, 268-269 whole-plant assay

CCD camera imaging data acquisition, 273-274 image analysis, 274—275 instrumentation, 273 luciferin concentrations, 274 detection overview, 269 microtiter plate assay materials, 270 Packard TopCount assay,

271-272 seedling growth, 271 vectors, 269-270

Methacrylate, embedding of sections advantages, 87 dissection, 87-88 fixation and dehydration, 88 methacrylate infiltration, 90-91 mounting for sectioning, 91-92 resin selection, 89 sectioning, 92-93 staining, 88-89

Misexpression, genes

Ale, ethanol-inducible switch system advantages, 291 background expression, 292 duration of induced expression, 292

ethanoi application methods,

292-293 principles, 291 applications, 287 constitutive promoters, 287 glucocorticoid receptor fusion system advantages, 296 construct design, 296 dexamethasone treatment, 297298

glucocorticoid types for induction, 297 induction testing, 297 principles, 296 protein synthesis inhibition studies, 299 transgenic line selection, 297 GVG glucocorticoid-mediated transcriptional induction system construct design, 294 dexamethasone treatment, 294295

overview, 293-294 transgenic line selection, 294 heat shock induction systems, 295296

tissue-specific promoters, 287 two-component expression systems GAL4:C1, 289 GAL4:VP16, 289 LhG4:pOp, 290-291 overview, 287-289 Mitochondria isolation centrifugation, 220 enrichment assays, 221 materials, 219 Morphology, Arabidopsis, 1, 3-5 Mosaic analysis approaches, 311-313 recombinases, 309-310 Mutants abiotic stress mutants, overview, 71

analysis. See Complementation testing; Crosses; Histology; Phenotypic analysis; Segregation analysis double mutant identificat an brute force approach, 52-53 considerations, 50 F2 mutant identification, 50-51 F3 mutant identification, 51-52 gene identification. See DNA

purification, Arabidopsis; Positional cloning; TAIL-PCR

mutagens. See Ethylmethane sulfonate mutagenesis; T-DNA mutagenesis natural variant analysis, 35-37 stock center availability, 19-20

Native gel electrophoresis applications, 228-229 casting of gradient gel, 230-232 electrophoresis conditions, 233 materials, 230-231 pH selection, 229 protein extraction from seedlings, 229-230 Neutral Red, vacuole staining, 98 Nucleus extraction for electrophoretic mobility shift assay, 223-225 isolation, 222-223

Oomycete pathogen response hydrogen peroxide assay with diaminobenzidine staining, 85 pathogen types, 81 Peronospora parasitica assays frozen fungal stock reviving, 81-82

frozen tissue stock preparation, 83-84

OomyCete pathogen response (icontinued) infection maintenance, 84-85 inoculation, 82 rating of infection, 83 Trypan blue sta.ning, 86-87

Paclobutrazol, response analysis of mutants, 63-64 PAS reaction, cell wall staining materials, 94 principles, 93

Schiff's reagent preparation, 93-94 slide incubations, 95 Pathogen response. See Bacterial pathogen response; Oomycete pathogen response

PCR. See Polymerase chain reaction Pest management, Arabidopsis cultivation, 14-17 Phenotypic analysis agarose imprinting of surfaces, 105

arsenic stress assay materials, 76

oxidation state dependence, 75 root-bending assay, 76-77 bacterial pathogen response bacterial culture, 77 bacterial growth monitoring, 79-80

hypersensitive response, 78-79 inoculation, 77-78 materials, 77-78 pathogen types, 77 brassinosteroid response brassinazole inhibitor utilization, 69 materials, 70 plate assay, 69-70 spray assay, 70 ethylene response gas treatment of seedlings, 68 materials, 67-68 sources of hormone, 66

flowering time cultivation, 62 definition, 61 environmental influences, 60-61 leaf production, 61 materials, 62 freezing stress response with electrolyte leakage assay conductivity measurement, 74 freezing, 74 materials, 73 fresh weight gain, 62, 74 genetic linkage of phenotypes, 55, 143

germination time abiotic stress assays, 72-73 abscisic acid response, 63 cultivation, 64 gibberellin response, 63 overview, 62

paclobutrazol response, 63-64 histologic analysis. See Histology hypocotyl length, 59-60 oomycete pathogen response hydrogen peroxide assay with diaminobenzidine staining, 85 pathogen types, 81 Peronospora parasitica assays frozen fungal stock reviving, 81-82

frozen tissue stock preparation, 83-84 infection maintenance, 8485

inoculation, 82 rating of infection, 83 Trypan blue staining, 86-87 prediction of phenotypes, 55 proline and sugar content assays for osmotic stress, 75 root growth auxin response, 64-66 considerations, 57 cultivation, 57 documentation, 56

Image software analysis, 56,

58-59 materials, 56

salt/hormone-induced stress response assay, 71-72 vascular strand imaging, 104-105 water loss, 62, 75 Phloroglucinol, lignin staining, 96-97 Pollen, development, 5-6 Polymerase chain reaction (PCR) Agrobacterium tumefaciens transformant selection, 127 Arabidopsis mutant screening amplification reactions, 33 gel electrophoresis of products,

33-34 overview, 30-31 primers, 32 DNA preparation. See DNA

purification, Arabidopsis insertion site identification. See

TAIL-PCR RNA analysis. See Reverse transcriptase-polymerase chain reaction Positional cloning gene finding within mapped interval, 162 map position approximation cleaved amplified polymorphic sequence markers generation, 156-157 mapping, 159-160 polymerase chain reaction and analysis, 157-158 principles, 155-156 simple-sequence-length polymorphisms mapping, 159-160 marker utilization, 158 polymerase chain reaction and analysis, 158-159 positioning within 50-kb interval, 161

rationale, 155 special cases dominant genes, 162

ecotypes versus mutations, 163 homozygotes, incorrect scoring, 164

nonsegregation as single locus in mapf ng cross, 164 recessive lethal mutations, 163 sterility induction, 163 Posttranscriptional gene silencing. See

RNA interference Primordia, green fluorescent protein imaging on shoot apex, 265

Proline, assays for osmotic stress, 75 Propidium iodide dead cell staining, 97 DNA content staining of nucleus apex dissection, 102-103 clearing, 102 confocal microscopy, 103 fixation, 101 materials, 100-101 mounting, 103 staining, 101-102 Protoplast, transient expression materials, 300-301 protoplast extraction, 302 reporter assay, 304 transformation, 303

Quantitative trait loci (QTL), natural variant analysis, 36-37

Recombinant inbred line (RIL), natural variant analysis, 36-37

Reporter genes. See ^-Glucuronidase;

Green fluorescent protein; Luciferase Reverse transcriptase-polymerase chain reaction (RT-PCR) noncompetitive RT-PCR amplification, 175 annealing reaction, 174 gel electrophoresis of products,

176 kits, 174 materials, 174

Reverse transcriptase-polymerase chain reaction (RT-PCR) [continued) quantification standards, 176 reverse transcription, 175 principles, 173

semiquantitative RT-PCR using nun es amplification reactions, 179180

calculations, 180-181 DNA concentration determination, 179 overview, 176-177 reverse transcription, 177-179 RIL. See Recombinant inbred line RNAi. See RNA interference RNA in situ hybridization. See In situ hybridization, RNA RNA interference (RNAi)

construct preparation, 285-286 double-stranded RNA production,

284-285 gene suppression advantages, 286 posttranscriptional gene silencing discovery, 284 RNA purification, Arabidopsis extraction and pelleting, 172-173 kits, 172 materials, 172 Root development, 3

green fluorescent protein imaging cultivation, 264 microscopy, 264-266 growth analysis of mutants auxin response, 64-66 considerations, 57 cultivation, 57 documentation, 56 Image software analysis, 56,

58-59 materials, 56

salt/hormone-induced stress response assay, 71-72 transformation callus induction and Agrobac terium inoculation, 137 efficiency, 134 materials, 134-136 seed sterilization and culture, 136

selection and regeneration,

138-139 soil transfer of transformants, 139

transient expression

Agrobacterium transformation,

306-307 cultivation, 306 materials, 304-305 regeneration of roots, 307 RT-PCR. See Reverse transcriptase-polymerase chain reaction

Scanning electron microscopy (SEM) coating, 108

critical point drying, 107-108 dental wax impression preparation, 109 fixation, quick-and-easy, 109 fresh tissue imaging image quality, 110 materials, 111 sample preparation, 111 materials, 106 mounting, 108 tissue preparation, 106-107 Seed collection and storage, 7 germination time analysis of mutants abiotic stress assays, 72-73 abscisic acid response, 63 cultivation, 64 gibberellin response, 63 overview, 62

paclobutrazol response, 63-64 germination, 8, 11, 13-14 mutagens. See Ethylmethane sulfonate mutagenesis sterilization, 13

Segregation analysis backcrosses and cosegregation,

46-48 Ft progeny, 44 F2 progeny, 44-46 overview, 43 self-progeny, 43-44 SEM. See Scanning electron microscopy Simple-sequence-length polymorphisms (SSLPs), positional cloning mapping, 159-160 marker utilization, 158 polymerase chain reaction and analysis, 158-159 SSLPs. See Simple-sequence-length polymorphisms Stocks, ordering, 19, 318-319 Sugar content, assays for osmotic stress, 75

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