PLANT HORMONES and GROWTH-REGULATING SUBSTANCES
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METHODS OF STUDYING PLANT HORMONES and GROWTH-REGULATING SUBSTANCES Agriculture Handbook No. 336 Agricultural Research Service UNITED STATES DEPARTMENT OF AGRICULTURE METHODS OF STUDYING PLANT HORMONES AND GROWTH-REGULATING SUBSTANCES By John W. Mitchell, Research Plant Physiologist, Crops Research Division and George A. Livingston, Senior Staff Associate, Institutional Relations, National Science Foundation Agriculture Handbook No. 336 Agricultural Research Service UNITED STATES DEPARTMENT OF AGRICULTURE ACKNOWLEDGMENTS The authors greatly appreciate efforts by all who contributed to this book. The assistance given by the late Paul C. Marth, who was coauthor of the ñrst edition, is gratefully acknowledged. While it is impracticable to provide a complete list of dealers offering chemicals, apparatus, and materials mentioned in this compilation, a few sources are given to aid the reader in obtaining items that may be difficult to purchase. Trade names and the names of commercial companies are used solely to provide specific information. Mention of a trade product or manu- facturer does not constitute a guarantee or warranty of the product by the U.S. Department of Agriculture or an endorsement by the Department over other products not mentioned. This handbook supersedes Agriculture Handbook No. 126, 'Test Methods With Plant-Regulating Chemicals" Extra care should be exercised in experimenting with new chemicals until it has been determined that they are not harmful to man or animals. The uses of chemicals mentioned in this book are for experi- mental purposes. These chemicals are not recommended, on the basis of these experimental methods, for application to plants to be used as food. V^ashington, D.C. Issued February 1968 For sale by the Superintendent of Documents, U.S. Government Printing Office Washington, D.C. 20402 — Price 55 cents CONTENTS Page Page Biological methods—Continued Introduction , 1 Growth modification 66 Biological methods 1 Sterile nutrient method 66 Abscission 1 Water culture method 67 Apple petiole method 1 Field method 68 Attached petiole method 2 Growth retardation 68 Coleus petiole method 4 Vegetative sprout method 68 Expiant method 5 Wheat seedling method 69 Lanolin paste method 7 Root growth 72 Absorption and translocation 8 Agar slant method 72 Agar block polar transport method 8 Cone container method 73 Bean root method 10 Impregnated filter paper method 74 Bean stem curvature method 11 Nutrient mist method 75 Angle and elongation of branches 13 Root induction 76 Lanolin method 13 Rooting cofactor method 76 Cell division or multiplication 13 Root primordia method 78 Endosperm method 13 Stem-cutting method 79 Microculture method 15 Seed formation 81 Root culture method 16 Lanolin paste method 81 Single isolated cell method 18 Detection of exogenous growth regulators in plants— 82 Soybean callus tissue method 19 Graft method 82 Stem tissue culture method 20 Detection of exogenous plant growth regulators in Submerged cultivation method 21 animals 82 Tobacco pith method (micro) 22 Bean stem method 82 Cell elongation 23 Oat coleoptile section method 23 Detection of regulators in water 84 Bean hypocotyl method 25 Root growth method 84 Bean second internode method 26 Penetration of soil by regulators 84 Oat first internode method 29 Seed method- 84 Pea stem section method 30 Residue of regulators in soil 86 Radish cotyledon method 31 Successive crop method 86 Split pea stem method 32 Some physical methods 87 Tomato hypocotyl method 34 Introduction 87 Wheat coleoptile straight growth method 36 Radioautographs of entire plants 87 Young wheat coleoptile method 40 Radioautograph method 87 Cell enlargement 41 Translocation of radioactively tagged compounds Citrus petal method 41 (See also ''Absorption and translocation/' p. 8.)-- 89 Leaf disk enlargement method 42 Ground tissue method 89 Chlorophyll retention 43 Volatility of plant regulators 90 Barley leaf method 43 Disposable container method 90 Cocklebur leaf disk method 44 Germinating seed method 91 Dock leaf disk method 45 Activated charcoal method 92 Wheat leaf senescence method 46 References to other physical methods 93 Effect of one compound on the absorption and trans- Chromatography 94 location of another 47 Supplementary techniques 94 Bean stem curvature method 47 Treating plants 94 Enzyme production 48 Absorption cell for introducing compounds into Barley endosperm method 48 limited portions of roots 94 Exudation of regulators from roots 49 Absorption through a leaf flap 95 Bean method 49 Agar block preparation and use 96 Root exudation method 52 Application of aqueous mixtures quantitatively to Flowering 54 stems 97 Biennial or perennial plant method 54 Application of a measured amount of regulating Form or growth modification 55 chemical to a known area of leaf surface 98 Bean leaf method 55 Application of regulators as a part of a coating on Lanolin paste method 56 seeds 98 Underground growth method 57 Application of growth substances with capillary Fruit growth 58 tubes 99 Ovary culture method 58 Easy method of impregnating dust carriers with Fruit set 60 regulating chemicals 100 Aerosol method 60 Convenient method of dispersing relatively in- Grape method 62 soluble organic compounds in water 100 Tomato method 62 Segment cutters 101 Fruit size 64 Device that delivers measured amounts of regu- Grape method 64 lating chemical 102 Fruit thinning 64 Direct measurement of volatility 102 Apple method 64 Fractionated lanolin as a carrier for growth-regu- Peach and prune method 65 lating compounds 104 m Page Page Supplementary techniques—Continued Supplementary techniques—Continued Treating plants—Continued Observing and measuring plants—Continued Injecting a regulating substance or hormone in- Formative effects of regulating chemicals 119 to a single, living cell 104 Interferometer method of detecting minute growth Injecting substances into trees 105 movements 120 Introduction of chemicals directly into xylem ele- Freehand sections for studying cell responses to ments in stems of herbaceous plants 105 regulators 120 Lanolin as a carrier for regulating chemicals 106 Measurement of root response 121 Movement of regulating substances through plant Radioautographs of sectioned plant material 121 membranes or tissues 106 Separation of leaf areas treated with a regulating Plastic culture vessel (^'plasticon") for small plants. 107 chemical from the remaining untreated portions Simulated spray-droplets 109 of the leaf 123 Technique for studying absorption and translocation Slide chamber for the study of living cells 123 of regulating substances by aquatic plants 109 Terminal growth measurements 124 Use of dwarf corn to detect and identify gibberellins Observation chamber for continuous applicati(m of and gibberellin-like substances 111 hormones or growth regulators to living cells 124 (îrowing plants 112 Motion detector for recording plant growth 124 Highly sensitive test plants 112 Supplementary information 128 References to methods of m.iintaining constant Measures, equivalents, and abbreviations 128 relative humidity 113 Temperature conversions 128 Microisolation device 113 Short-cut method of converting from Centigrade to Suggestions concerning the production of greenhouse Fahrenheit degrees 128 plants for experiments with regulating chemicals _ 114 Conventional method of converting from Centigrade Storage of seeds used as a plant source for the study to Fahrenheit degrees 128 of hormones and regulators...^ 1 116 Conventional method of C(mverting from Fahrenheit Observing and measuring plants 117 to Centigrade degrees 128 Aphid honey dew technique for study of sieve tube Met hods of making nut rient solut ions 129 content _' 117 Nutrient solution made with tapwater 129 Aphid stylet technique for studying regulating Nutrient solution made with distilled water 129 substances in sieve tubes 118 Potassium phosphate buffer mixtures 129 Extraction of hormones and growth-regulating Descriptive index 131 substances from plants 118 iv METHODS OF STUDYING PLANT HORMONES AND GROWTH-REGULATING SUBSTANCES INTRODUCTION The purposes of this compilation are, first, to The index is arranged so methods can be provide scientists with ready access to methods found by approaching the handbook from dif- useful in the study of plant hormones and ferent viewpoints. For example, from a chemi- growth-regulating substances, and, second, as cal or biochemical viewpoint, methods useful for our knowledge in this field advances, to stimu- detecting gibberellic acid, gibberellin-like com- late scientists to devise new methods that will be pounds, kinetin, or auxin can be readily found. necessary to understand more fully how hor- From the viewpoint of the response induced by mones and regulators control the behavior of a hormone or regulator, methods useful in de- plants. tecting substances that cause cell elongation, Information in this handbook is presented in abscission, growth retardation, cell division, and three sections: biological methods that are es- other responses can be located. Methods can be sential in the study of hormones, some very found readily that involve the use of specific useful physical methods, and many supplemen- plant parts or that help accomplish a specific tary techniques that facilitate research in this task, such as injecting a regulator into a living field. A few very useful methods, such as the cell. well-known oat coleoptile method, have not been Progress in the accumulation of new scien- included, since these are widely used and de- tific knowledge