Preface ........................................................ xi
Acknowledgments .............................................. xiii
Scheduling and Interrelationships of Exercises ................. xv
1 History of Plant Cell Culture ................................ 1
Trevor A. Thorpe
Introduction ................................................. 1
The Early Years .............................................. 2
The Era of Techniques Development ............................ 3
The Recent Past .............................................. 5
Cell Behavior ................................................ 6
Plant Modification and Improvement ........................... 7
Pathogen-Free Plants and Germplasm Storage ................... 9
Clonal Propagation ........................................... 9
Product Formation ............................................ 9
The Present Era ............................................. 10
2 Setup of a Tissue Culture Laboratory ........................ 23
Work Areas .................................................. 24
Media Preparation/Culture Evaluation/Record-Keeping Area .... 24
Aseptic Transfer Area ....................................... 24
Environmentally Controlled Culture Area ..................... 25
3 Media Components and Preparation ............................ 31
Inorganic Salts ............................................. 32
Plant Growth Regulators ..................................... 32
Vitamins .................................................... 36
Carbohydrates ............................................... 36
Hexitols .................................................... 37
Gelling Agent ............................................... 37
Amino Acids ................................................. 38
Antibiotics ................................................. 39
Natural Complexes ........................................... 39
Media pH .................................................... 40
Medium Preparation .......................................... 40
4 Explant Preparation ......................................... 45
Explant Age ................................................. 46
Season ...................................................... 46
Explant Size ................................................ 46
Plant Quality ............................................... 46
Goal ........................................................ 46
Genotype .................................................... 48
Aseptic Technique ........................................... 48
Aseptic Germination of Seeds ................................ 49
Carrot, Sunflower, Broccoli, Cotton, or Any Seed of
Choice ...................................................... 49
Douglas Fir ................................................. 50
5 Contamination ............................................... 53
Explant ..................................................... 53
Disinfecting Agents ......................................... 54
Explant Source .............................................. 56
Internal Microbial Contamination ............................ 56
Explant Pretreatment to Control Microbial Contaminants ...... 57
Personnel ................................................... 57
Laminar Air Flow Hood ....................................... 58
Insects ..................................................... 58
Control of Insect Contamination ............................. 58
Media ....................................................... 59
Instruments ................................................. 60
Room Air Handling System .................................... 61
6 Callus Induction ............................................ 63
Callus Initiation ........................................... 63
Explant Preparation ......................................... 65
Culture ..................................................... 66
Observations ................................................ 66
Data and Questions .......................................... 66
Explant Orientation ......................................... 67
Explant ..................................................... 67
Observations ................................................ 68
Establishment of Competent Cereal Cell Cultures ............. 69
Explants .................................................... 70
Data and Questions .......................................... 70
Salt Selection In Vitro ..................................... 70
Explants .................................................... 71
Data and Questions .......................................... 72
Growth Curves ............................................... 72
Cotton ...................................................... 73
Tobacco ..................................................... 74
Cellular Variation from Callus Cultures ..................... 76
Explant ..................................................... 76
Data and Questions .......................................... 76
7 Regeneration and Morphogenesis .............................. 81
Controlled Morphogenesis: Potato Tuberization ............... 82
Explant ..................................................... 83
Data and Questions .......................................... 84
Somatic Embryogenesis ....................................... 84
Explant Preparation ......................................... 86
Procedure ................................................... 86
Data and Questions .......................................... 86
Regeneration of Rice ........................................ 87
Regeneration ................................................ 87
Data and Questions .......................................... 87
Dormancy Requirements of Explants ........................... 87
Explant Preparation ......................................... 88
Questions ................................................... 89
Primary Morphogenesis: Douglas Fir .......................... 89
Explant Preparation ......................................... 89
Data and Questions .......................................... 90
8 Woody Shrubs and Trees ...................................... 93
Brent H. McCown
Rhododendrons and Azaleas ................................... 95
Explants .................................................... 97
Advanced Studies ............................................ 97
Roses ....................................................... 97
Explant ..................................................... 98
Advanced Studies ............................................ 98
Birch Trees ................................................. 98
Explant ..................................................... 98
Advanced Studies ............................................ 99
White Cedar (Arborvitae) .................................... 99
Explant ..................................................... 99
9 Haploid Plants from Anther Culture ......................... 103
Datura Anther Culture ...................................... 105
Explant Preparation ........................................ 105
Questions .................................................. 106
African Violet ............................................. 106
Explant Preparation ........................................ 107
Observations ............................................... 107
Root Tip Chromosome Squash Technique ....................... 107
Questions .................................................. 108
Tobacco .................................................... 108
Explant Preparation ........................................ 108
Observations ............................................... 109
Questions .................................................. 109
Anther Squash Technique .................................... 109
10 Embryo Rescue .............................................. 113
Sweet Corn Embryo Culture .................................. 114
Preparation and Culture of Corn Embryos .................... 115
Questions .................................................. 116
Crabapple and Pear ......................................... 116
Explant Preparation ........................................ 116
Observations ............................................... 116
Questions .................................................. 117
11 Meristem Culture for Virus-Free Plants ..................... 119
Isolation of the Shoot Apical Meristem ..................... 121
Explant Preparation ........................................ 121
Questions .................................................. 122
Establishing Virus- and Bacteria-Free Plants ............... 122
Explant Preparation ........................................ 122
Garlic Propagation from Bulb Scales ........................ 123
Explant Preparation ........................................ 123
Data ....................................................... 123
12 In Vitro Propagation for Commercial Production of
Ornamentals ................................................ 127
Boston Fern ................................................ 129
Stage I .................................................... 130
Stage II ................................................... 131
Stage III .................................................. 132
Staghorn Fern .............................................. 133
Stage I .................................................... 134
Stage II ................................................... 134
Stage III .................................................. 135
Ficus ...................................................... 136
Stage I .................................................... 136
Stage II ................................................... 137
Stage III .................................................. 137
Kalanchoe .................................................. 137
Explant Preparation ........................................ 138
Data and Questions ......................................... 138
African Violet ............................................. 139
Explant Preparation ........................................ 139
Data and Questions ......................................... 140
Endangered Species ......................................... 140
Pitcher Plants ............................................. 141
Cactus ..................................................... 141
Explant Preparation ........................................ 142
13 Protoplast Isolation and Fusion ............................ 147
Jungeun Kim Park, Sunghun Park and James E. Craven
Plant Protoplast Isolation and Fusion ...................... 148
Supplies ................................................... 148
Protoplast Liberation ...................................... 149
Protoplast Purification .................................... 150
Vital Staining ............................................. 151
Protoplast Fusion .......................................... 151
Yield Determination ........................................ 151
Developing a Protoplast Isolation Protocol ................. 152
14 Agrobacterium-Mediated Transformation of Plants ............ 155
Jungeun Kim Park, Sunghun Park, Qingyu Wu and Stuart
Sprague
Petunia or Tobacco Leaf Disk ............................... 157
Sequence ................................................... 157
Preculture ................................................. 157
Explant Preparation ........................................ 157
Cocultivation with Agrobacterium ........................... 158
Shoot Transformation and Elimination of Agrobacterium ...... 158
Rooting and Pretesting for Transformed Shoots .............. 159
Petunia Shoot Apex ......................................... 160
Sequence ................................................... 160
Seed Germination ........................................... 160
Shoot Induction ............................................ 161
Shoot Apex Transformation .................................. 161
Rooting .................................................... 161
Tobacco Leaf Infiltration .................................. 162
Sequence ................................................... 162
Grow Agrobacterium ......................................... 162
Culture Agrobacterium ...................................... 163
Harvest Agrobacterium ...................................... 163
Infiltration Tobacco Leaves ................................ 163
Appendices .................................................... 167
Index ......................................................... 183
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