Methods and Techniques in Plant Physiology
Methods and Techniques in Plant Physiology
By
A. Bhattacharya M.Sc. (Botany, Allahabad University), Ph.D. Plant Physiology Indian Agricultural Research Institute New Delhi
Vijay Laxmi M.Sc., Ph.D. Plant Physiology Indian Agricultural Research Institute New Delhi
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Preface
It is the valid experimentation that generates universal hypothesis in science. Theoretical information undoubtedly originates from practical and therefore a scientist through in modern practical orientation can be a scientist of high academic accomplishment. Heavy emphasis on practical teaching is trusted upon with the understanding that mere theory won’t help building personal career of an individual. We believe that teachers must exercise what they are supposed to demonstrate to their taught in a laboratory. The science is advancing fast and the knowledge based on swinging hypothesis becomes outdated. Today, we are in an electronic age of computer science and this leads us to unveil mysteries of various complex phenomenons. The older concepts are subject to change to the ever changing demands of the day. Therefore, a teacher or a student has to be toned up with the time. Our attempts are to make both Post Graduate student as well as teachers familiar with such captive world. We do not claim current furnishings of techniques in any way exhaustive, but we do hope that it may certainly be to value to our readers. Techniques related to various physiological phenomenon are subject of tremendous interest and importance to plant physiologist, agronomist, horticulturist, ecologist, and biochemists. This book is intended to provide recognised methods related various plant processes in a comprehensive form. Text begins with what a researcher is primarily supposed to know before he actually goes for estimations. Further, you may see techniques on crop physiology such as hydroponics and plant nutrition, test for various stresses, water potential and water flow in plants, canopy gas measurements (Photosynthesis, Respiration and Transpiration), basic equations for growth studies and methods for estimations of plant products, microclimate. Efforts were also made to incorporate the topic like Climate Change and theory of phytotron as well as rhizotron in this book. In
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Preface
this book research worker must be familiar with latest procedure to elucidate the problems. The validity of the results based on fundamentals principles of physics. This book is meant to be used in conjunction with a standard text of plant physiology though elementary principles relating to the techniques are briefed. The subjects on hormones, tissue culture and seed technology are useful for students. Hope this book shall serve the need of students, teachers and researchers. The suggestions and advice are most welcome for the improvements in structural and for additions of current techniques. We shall feel successful in our efforts publishing this book if we know how this text has become meaningful to our readers in planning their research strategies. We would appreciate suggestions from teachers and students who use the book. Hope this book shall serve the need of students, teachers and researchers. The suggestions and advice are most welcome for the improvements in structural and for additions of current techniques. We shall feel successful in our efforts publishing this book if we know how this text has become meaningful to our readers in planning their research strategies. We would appreciate suggestions from teachers and students who use the book on how we could improve upon it.
A. Bhattacharya Vijay Laxmi
Contents Preface .............................................................................................................. v
1. Importance of Plant / Crop Physiology in Agriculture .................. 1 2. Plant Physiology Laboratory ............................................................. 5 2.1 Microscopy ....................................................................................... 5 2.2 Analytical balance and weighing accuracy ....................................... 6 2.3 Chemistry and solution dilutions ....................................................... 7 2.4 Protein assay and spectrophotometer use ........................................ 9 2.5 Standard curves and graphing formats ........................................... 11 2.6 Preparation of solution .................................................................... 13 2.6.1 Normal solutions .................................................................. 14 2.6.2 Molar solutions .................................................................... 14 2.6.3 Parts per million (ppm) solution .......................................... 15
3. Osmotic Pressure Potential by Plasmolytic Method .................. 17 3.1 Physiological importance of water ................................................. 17 3.2 Methods of measuring osmotic potential ........................................ 19 3.3 Measurement of the water potential of plant tissue by weight method ........................................................................... 20 3.4 Tissue water relations ..................................................................... 22 3.5 Leaf water, osmotic and turgor potentials and bulk density elastic modules of tissues .............................................................. 22 3.6 Effect of osmotic concentration and temperature on imbibition of water by seed ............................................................ 25 3.7 Chardakov method for water potential measurement ..................... 26 3.8 Relative water content (RWC) ........................................................ 29
4. Separation of Plant Pigments by Chemical Method .................... 33 4.1 Estimation of chlorophyll and other chloroplast pigments ............. 34 4.2 Separation of chloroplast pigments by solvent partitioning technique ........................................................................................ 36
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4.3 Paper chromatography separates plant pigments .......................... 37 4.3.1 Paper chromatography method ........................................... 38 4.3.2 Identifying pigments isolated by paper chromatography .... 40
5. Determination of Stomatal Frequency and Stomatal Index ........ 43 5.1 Measurement of stomatal frequency and stomatal index. .............. 45
6. Transpiration ..................................................................................... 47 6.1 Influence of anti transpirant ............................................................ 48
7. Oxygen Evolution During Photochemical Reaction ................... 51 8. Measurement of Photosynthetic Rates by Gas Exchange Techniques...................................................................... 55 8.1 Measurement of photosynthetic rate by Infra-red gas analyzer (IRGA) ............................................................................. 56 8.1.1 Measurement of photosynthetic rate using a closed IRGA system ........................................................................ 59 8.2 Measurement of respiration rate by gas exchange approach ......... 59 8.3 Measurement of respiration rate ..................................................... 60 8.4 Measurement of respiration rate by determining the increase in CO2 concentration in a chamber ................................................. 60
9. Growth Regulators ........................................................................... 63 9.1 Auxins ............................................................................................. 64 9.2 Gibberellins ..................................................................................... 66 9.3 Cytokinins ....................................................................................... 69 9.4 Ethylene ........................................................................................... 70
10. Seed Physiology ................................................................................ 71 10.1 Morphological and physiological changes during seed development: ......................................................................... 73 10.2 Biochemical changes during seed development ............................ 74 10.3 Morphological and physiological changes during physiological maturity ........................................................................................ 75 10.4 Seed viability and vigor ................................................................. 76 10.5 Seed dormancy ............................................................................. 78 10.5.1 Factors of seed dormancy ................................................. 78 10.5.2 Breaking of seed dormancy ............................................... 79 10.6 Methods of testing seed viability and vigor .................................. 80 10.7 Mobilization and utilization of seed reserves ................................. 82 10.8 Changes during seed germination: morphological, physiological & biochemical ........................................................ 83
Contents
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11. Tissue Culture Techniques ............................................................ 87 11.1 Demonstration of callus induction in tobacco leaves .................... 91 11.2 Growing plants in tissue culture ................................................... 92
12. Growth Analysis ................................................................................ 97 12.1 Measurement of growth ............................................................. 100
13. Hydroponics and Plant Nutrition ................................................. 107 13.1 Sand Culture ................................................................................ 116
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Test for Various Stresses in Crop Plants .................................... 121 14.1 Testing for heat tolerance in plants ............................................. 121 14.2 Desiccation tolerance test for drought resistance ...................... 125 14.3 Seed germination test for drought tolerance ............................... 126 14.4 Field screening of plant for drought tolerance efficiency .......... 127 14.5 Temperature tolerance in crop plants - Solar energy traps ........ 127 14.6 Chlorophyll stability for drought tolerance in plants ................... 128
15. Analysis for Carbohydrates .......................................................... 131 15.1 Determination of reducing sugarsby nelson-somogyi method ... 132 15.2 Estimation of reducing sugar by dinitrosalicylic acid method .... 136 15.3 Determination of total carbohydrate by anthrone method .......... 136 15.4 Phenol sulphuric acid method for total carbohydrate ................. 138 15.5 Estimation of starch by anthrone reagent ................................... 139 15.6 Determination of amylose: .......................................................... 140 15.7 Estimation of cellulose ................................................................ 141 15.8 Estimation of hemicellulose: ........................................................ 143 15.9 Determination of fructose and inulin: ......................................... 144 15.9.1 Inulin ................................................................................ 145 15.10 Estimation of pectic substances ............................................... 145 15.11 Extraction and purification of pectin ......................................... 147 15.12 Estimation of crude fibre .......................................................... 148 15.13 Estimation of pyruvic acid ........................................................ 149 16.
Nitrogen and Protein Analysis ..................................................... 151 16.1 Nitrogen estimation by kjeldahl method ...................................... 154 16.2. Determination of ammonia nitrogen .......................................... 155 16.2.1 Distillation procedure ....................................................... 155 16.2.2 Colorimetric method ........................................................ 156 16.3 Determination of organic nitrogen .............................................. 158 16.4 Protein estimation by Lowry’s method ....................................... 158
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16.5 Extractable nitrates ...................................................................... 160 16.6 Quantitative estimation of total amino acid through ninhydrin ... 161
17. Analytical Methods for Fats and Fatty Acids ............................. 165 17.1 Determination of lipid content by ether extraction ..................... 168 17.2 Acid-Ether extraction of lipids .................................................... 169 17.3 Esterification of fatty acids ......................................................... 169 17.4 Lipid extraction ........................................................................... 170 17.5 Gas-Liquid chromatography for lipids ........................................ 170
18. Micronutrient Analysis .................................................................. 173 18.1 Determination of boron ............................................................... 174 18.2 Determination of extractable chloride ......................................... 176 18.3 Determination of total sulfur ....................................................... 177 18.4 Determination of iron .................................................................. 177 18.5 Determination of molybdenum ................................................... 180 18.6 Determination of zinc .................................................................. 181 18.7 Determination of manganese ....................................................... 184 19.
Determination of Phytochemicals ................................................ 185 19.1 Determination of alkaloids .......................................................... 186 19.2 Determination of total phenols .................................................... 186 19.3 Determination of flavonoids ........................................................ 186 19.4 Determination of saponins .......................................................... 187 19.5 Determination of riboflavin ......................................................... 187 19.6 Determination of niacin ............................................................... 187 19.7 Determination of ascorbic acid ................................................... 188 19.8 Alternate method for Ascorbic acid ............................................ 189
20. Climate Change, Phytotron and Rhizotron ................................ 191 20.1 Phytotron .................................................................................... 192 20.2 Rhizotron ..................................................................................... 196
References ...................................................................................... 201
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