Plant Physiology experimental protocols

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CONTENTS CHAPTER 1 - CARBOHYDRATES 1.1. Estimation of reducing sugars 1.2. DNS Method 1.3. Estimation of total sugars 1.4. Phenol-sulphuric acid method 1.5. Estimation of non-reducing sugars 1.6. Estimation of starch 1.7. Estimation of cellulose 1.8. Estimation of hemicellulose 1.9. Estimation of crude fiber 1.10. Estimation of pectin substances - Colorimetric method CHAPTER 2- NITROGEN 2.1. Estimation of nitrogen 2.2. Estimation of non-protein nitrogen 2.3. Estimation of amino acids 2.4. Estimation of Protein - Biuret method 2.5. Estimation of protein - Lowry's Method 2.6. Estimation of soluble protein by Dye-binding method (Bradford's method) 2.7. Polyacrylamide-sodium dodecyl sulphate gel electrophoresis (SDS-PAGE) of proteins 2.8. Isolation of plant DNA 2.9. Extraction of RNA from plant 2.10. Estimation of DNA 2.11. Estimation of RNA 2.12. Isolation of Plasmids 2.13. Estimation of proline 2.14. Estimation of methionine 2.15. Estimation of lysine 2.16. Estimation of tryptophan 2.17. Pathogenesis-Related Proteins (PR-Proteins) 2.18. Estimation of Chloroplast Protein 2.19. Estimation of Heat Shock Proteins 2.20. Nitrogen Fractions

CHAPTER 3 - PHOSPHORUS 3.1. Phosphorus Fractions 3.2. Phytin Phosphorus 3.3. Estimation of Phytic Acid

CHAPTER 4 – FATS

4.1. Extraction of total lipids 4.2. Estimation of oil or crude fat 4.3. Isolation and estimation of free fatty acids 4.4. Estimation of free fatty acids or acid value of oil 4.5. Estimation of free fatty acids by colorimetry 4.6. Estimation of peroxide value of an oil or fat 4.7. Estimation of Iodine value of oil 4.8. Estimation of Saponification value / Saponification number of an oil or fat 4.9. Estimation of Oil by NMR Method

CHAPTER 5 - ENZYMES 5.1. Extraction of enzyme 5.2. Amylases 5.3. Invertase (-fructofuranosidase) 5.4. Deoxyribonuclease (DNase) 5.5. Urease 5.6. Phosphoenolpyruvate carboxylase (PEP carboxylase) 5.7. Glutamine Synthetase (GS) 5.8. Glutamate Dehydrogenase (GDH) 5.9. Glutamate Synthase (GOGAT) 5.10. Nitrate Reductase (NR) 5.11. Catalase 5.12. Polygalactouronase (PG) 5.13. Pectin Methyl Esterase (PME) 5.14. L-Phenylalanine Ammonia Lyase (PAL) 5.15. Polyphenol Oxidase (PPO) or DOPA oxidase 5.16. Indoleacetic Acid Oxidase (IAA oxidase) 5.17. Ascorbic Acid Oxidase 5.18. Pyruvate decarboxylase 5.19. Lipoxygenase (lipoxidase) 5.20. Nitrite reductase (NiR) 5.21. Peroxidase (POD) 5.22. Acid phosphatase 5.23. Succinate dehydrogenase 5.24. Estimation of nitrogenase activity 5.25. Estimation of Rubisco by Elisa 5.26. Estimation of Total Dehydrogenase Activity 5.27. Estimation of PEP – Carboxylase 5.28. Estimation of Ribonuclease Activity 5.29. Estimation of Super Oxide Dismutase, Catalase And Peroxidase 5.30. Estimation of Glycolate Oxidase Activity 5.31. Estimation of Lipid Peroxidation

5.32. Starch phosphorylase 5.33. Sucrose synthase (sucrose-6 phosphate ) 5.34. Isozymes in Plant Samples

CHAPTER 6 - MINERAL NUTRITION 6.1. Estimation of Phosphorus 6.2. Estimation of Potassium 6.3. Estimation of Calcium and Magnesium 6.4. Estimation of Iron and Manganese 6.5. Plant Tissue Tests 6.6. Hydroponics CHAPTER 7 - PLANT GROWTH REGULATORS 7.1. Estimation of auxins (indole 3 acetic acid or IAA) 7.2. Bioassay of IAA 7.3. Estimation of Gibberellins by Calorimetry 7.4. Bioassay of Gibberllins - Amylase release test from seeds 7.5. Extraction and estimation of cytokinins by Chromotography 7.6. Bioassay of Cytokinin - Radish cotyledon test 7.7. Estimation of Cytokinin by ELISA 7.8. Abscisic acid 7.9. Bioassay of ABA - Inhibition of α-amylase synthesis in barley endosperm 7.10. Estimation of Abscissic Acid by ELISA 7.11. Estimation of Ethylene by Gas Chromotography 7.12. Estimation of Ethylene by Colorimetry 7.13. Bioassay of ethylene by Epinastic response 7.14. Triple pea test 7.15. Pea stem swelling test 7.16. Anthocyanin inhibition test 7.17. Chlorophyll retention test 7.18. Chlorophyll formation test 7.19. Specific protocols for ABA and Cytokinin extractions

CHAPTER 8 - SECONDARY METABOLITES

8.1. Estimation of curcumin 8.2. Estimation of anthocyanin 8.3. Estimation of oxalic acid 8.4. Estimation of gossypol

8.5. Identification and Determination of Polyamines (By TLC) 8.6. Analysis of Polyamines By HPLC 8.7. Estimation of HCN 8.8. Estimation of total carotenoids 8.9. Estimation of lycopene 8.10. Estimation of Chloroplast Pigment Composition 8.11. Estimation of Chlorophyll Content without Homogenisation 8.12. Estimation of anthocyanin 8.13. Estimation of leuco-anthocyanin 8.14. Estimation of lignins 8.15. Estimation of capsaicin 8.16. Estimation of Quinones 8.17. Estimation of Tannins 8.18. Estimation of ascorbic acid (vitamin C) 8.19. Estimation of ascorbic acid by colorimetry 8.20. Estimation of total Phenols 8.21. Estimation of ortho-dihydric phenols CHAPTER 9 - PHYSIOLOGY 9.1. Measurement of Hill reaction 9.2. Estimation of mitochondria 9.3. Isolation of chloroplasts 9.4. Sullivan‟s heat tolerance test 9.5. Measurement of loss of membrane permeability 9.6. Chlorophyll Stability Index (For Drought Tolerance in Plants) 9.7. Plant Tissue Culture 9.8. Equipment and other Requirements for Tissue Culture Laboratory 9.9. Plant Tissue Culture Technique: Steps 9.10. Cation Exchange Capacity (CEC) of Roots 9.11. Physiological Disorders and Corrective Measures 9.12. Leaf Area Determination 9.13. Plant Growth Analysis 9.14. Determination of Light Extinction Coefficient 9.15. Measurement of Light Interception 9.16. Measurement of Rate of Germination 9.17. Measurement of Respiratory Rates of Foliage 9.18. Measurement of Aerobic Respiration 9.19. Estimation of Relative Water Content (RWC) 9.20. Determination of Leaf Epicuticular Wax 9.21. Effect of Osmotic Potential up on Imbibition 9.22. Determination of Chemical Oxygen Demand (COD) in a Water Sample 9.23. Testing for Heat Tolerance in Plants 9.24. Desiccation Tolerance Test for Drought Resistance 9.25. Seed Germination Test for Drought Tolerance 9.26. Chlorophyll Stability Index for Drought Tolerance in Plants

9.27. Measurement of Leaf Water Potential (Thermocouple Psychrometer) 9.28. Measurement of Plant/Canopy Temperature with Infrared Thermometers and Use in Irrigation Scheduling 9.29. Measuring Stomatal Conductance, Transpiration and Leaf Temperature (Li-1600 Steady State Porometer) 9.30. Construction of Pressure-Volume Curve and Estimation of Water Potential  and  with Pressure Vessel 9.31. Water Potential of Polyethylene Glycol and Stress Imposition in Water Culture 9.32. Procedure to Evaluate Photosynthesis on intact single leaf 9.33. Photosynthesis Estimates with Labelled CO2 9.34. Measuring Photosynthesis and Transpiration in whole plant by Constant Flow System 9.35. Measurement of Light Intensity 9.36. Estimation of Stomatal Index and Stomatal Frqeuency 9.37. Test for Pollen Viability 9.38. Soil Analysis

CHAPTER 10 - GENERAL PROCEDURES 10.1. Chromatographic Separation of Plant Extracts 10.2. Radio Tracer Techniques for 14CO2 Studies 10.3. Chromatography 10.4. Atomic Absorption Spectrophotometry 10.5. Low Voltage Paper Electrophoresis 10.6. The Tracer Technique 10.7. Poly Acrylamide Gel Electrophoresis 10.8. Infra Red Gas Analysis for CO2 Studies (IRGA) 10.9. Enzyme - Linked Immunosorbant Assay (ELISA) (General) 10.10. The Double Antibody Sandwich Technique 10.11. Preparation for Light Microscopy : Wax Embedding Techniques 10.12. Scanning Electron Microscopy Techniques

1. CARBOHYDRATES 1.1. Extraction and Estimation of reducing sugars Alcohol is highly effective in penetrating tissues and stopping the enzymatic activity. Boiling alcohol is more effective than cold alcohol. Hence, it is advisable that tissues used for analysis be extracted in boiling 80% alcohol. Reagent 1. 80 % Alcohol Procedure 1. Grind a known weight of material in boiling 80% ethyl alcohol (5-10 ml/g material) thoroughly in a mortar with pestle or in a blender for 5-10 min. And cool under cold water. 2. Pass the extract through two layers of cheese cloth. 3. Re-extract the ground material as in step 1. 4. Pool both the extracts together and filter through Whatman No.41 filter paper. Collect the filtrate, note the volume and store in sealed vials at 0-4C. The preserved alcohol extract and residue can be used for analysis of various compounds. 1.1.1. Estimation of reducing sugars Sugars with the prescence of potentially free aldehyde or keto group are able to reduce metal ions under alkaline conditions. Such sugars are called as reducing sugars. Some of these are glucose, galactose, lactose and maltose. Reducing sugars may be estimated following either by Nelson-Somogyi method (Somogyi, 1952) or the Dinitrosalicylic acid (DNS) method (Miller, 1972). Nelson Somogyi's method Principle The reducing sugars when heated with alkaline copper tartrate reduce the copper from the cupric to cuprous state and thus cuprous oxide is formed. When cuprous oxide is treated with arsenomolybdic acid, the reduction of molybdic acid to molybdenum blue takes place. The blue colour developed is compared with a set of standards in a colorimeter at 620 nm. Reagents 1. Alkaline copper tartrate: a). Dissolve 2.5 g of anhydrous sodium carbonate, 2 g of sodium bicarbonate, 2.5 g of potassium sodium tartrate and 20 g of anhydrous sodium sulphate in 80 ml water and make up to 100 ml. b) Dissolve 15 g of copper sulphate in a small volume of distilled water. Add one drop of sulphuric acid and make up to 100 ml. Mix 4 ml of (b) and 96 ml of solution (a) before use. 2. Arsenomolybdate reagent: Dissolve 2.5 g ammonium molybdate in 45 ml water. And 2.5 ml sulphuric acid and mix well. Then add 0.3 g disodium hydrogen

arsenate dissolved in 25 ml water. Mix well and incubate at 37C for 24 to 48hours. 3. Standard Glucose solution (stock): 100 mg of glucose in 100 ml distilled water. 4. Working standard: Dilute 10 ml of stock solution to 100 ml with distilled water (100 g/ml). Procedure 1. Weight 100 mg of the sample and extract the sugars with hot 80% alcohol twice (5ml each time). 2. Collect the supernatant and evaporate on water bath. 3. Add 10 ml of water and dissolve the sugars. 4. Pipette out aliquots of 0.1 or 0.2 ml of alcohol-free extract to separate test tubes. 5. Pipette out 0.2, 0.4, 0.6, 0.8 and 1 ml of the working standard solution into a series of test tubes. 6. Make up the volume in both sample and standard tubes to 2 ml with distilled water. 7. Pipette out 2 ml distilled water into a separate tube to serve as a blank. 8. Add 1 ml of alkaline copper tartrate reagent to each tube. 9. Place the tubes in boiling water for 10 minutes. 10. Cool the tubes and add 1 ml of arsenomolybdic acid reagent to all the tubes. 11. Make up the volume in each tube to 10 ml with water. 12. Read the absorbance of blue colour at 620 nm after 10 min. 13. From the graph drawn, calculate the amount of reducing sugars present in the sample. Calculation Absorbance corresponds to 0.1ml of test = x mg of glucose 10 ml contains

=

 x 10 mg of glucose = % of reducing sugars 0.1

Reference Sadasivam, S. and A. Manickam, (1992). In: Biochemical Methods for Agricultural Sciences, Wiley Eastern Limited. New Delhi. pp.5-6. 1.2. Estimation of reducing sugar by Dinitrosalicylic acid method Several reagents have been employed which assay sugars by their reducing properties. One such compounds is 3, 5 dinitrosalicylic and (DNS) which in alkaline solution is reduced to 3-amino-5-nitrosalicyclic acid. Reagents 1. Dinitrosalicyclic acid (DNS) reagent: Dissolve simultaneously 1 g of dinitrosalicylic acid, 200 mg of crystalline phenol and 50mg of sodium sulphite in 100 ml of 1% NaOH solution by stirring. Store the reagent in a stoppered bottle at 4C. During storage the reagent deteriorates, due to atmospheric oxidation which takes place by the prescence of sodium sulphite. If required to be stored, prepare the reagent without adding sodium sulphite and add it just before use.

2. 40% Rochelle salt solution (Sodium-potassium tartrate solution). 3. Standard sugar solution: (See under Nelson-Somogyi's method). Procedure 1. Follow the steps 1 to 3 as in Nelson-Somogyi's method to extract the reducing sugars from the sample. 2. Pipette out 0.5 to 3 ml of alcohol-free extract into test tubes and make up the volume to 3 ml with water in all the tubes. 3. Add 3ml of DNS reagent and mix. 4. Heat for 5 minutes in a boiling water bath. 5. After the colour has developed, add 1 ml of 40% Rochelle salt solution (when the contents are still warm) and mix. 6. Cool the tubes under running tap water and measure the absorbance at 510 nm using reagent blank adjusted to zero absorbance. 7. Calculate the amount of reducing sugar in the sample using a standard graph prepared from working standard glucose solution (0 to 500 g) in the same manner. Reference Miller, G.L. 1972. Anal. Chem. 3: 426.

1.3. Estimation of total sugars The amount of total soluble sugars can be estimated using either anthrone or phenol-sulphuric acid method colorimetrically. Anthrone method Principle Carbohydrates are dehydrated by conc. H2S04 to form furfural. Furfural condenses with anthrone (10-keto- 9, 10-dihydroanthracene) to form a blue green coloured complex which is measured colorimetrically at 630 nm. Reagents 1) 2.5N HCL. 2) Anthrone reagent: Dissolve 200mg anthrone in 100ml of ice cold 95% H 2SO4 prepare fresh before use. 3) Standard glucose (stock): Dissolve 100mg in 100ml water 4) Working standard: 10ml of stock diluted to 100ml with distilled water. Store refrigerated after adding a few drops of toluene. Method 1) Weigh 100mg of the sample into a boiling tube. 2) Hydrolyze by keeping it in a boiling water bath for 3 hours with 5ml of 2.5N HCL and cool to room temperature 3) Neutralize it with solid sodium carbonate until the effervescence ceases. 4) Make up the volume to 100ml and centrifuge. 5) Collect the supernatant and take 0.5 and 1ml aliquots for analysis. 6) Prepare the standards by taking 0, 0.2, 0.4, 0.6, 0.8 and 1ml of the working standard. „0‟ serves as blank.

7) Make up the volume to 1ml in all the tubes including the sample tubes by adding distilled water. 8) Then add 4ml of anthrone reagent. 9) Heat for 8 min in a boiling water bath. 10) Cool rapidly and read the green to dark green colour at 630 nm. 11) Draw a standard graph by ploting concentration of the standard on the X-axis versus absorbance on the Y-axis. 12) From the graph calculate the amount of carbohydrates present in the sample tube. Calculation Sugar value from Total vol. of extract (ml) Amount of carbohydrate graph (mg) present in sample (% mg) = ------------------------ X --------------------------- X 100 Aliquot sample used Wt. of sample (mg) (0.5 or 1ml) Reference Hedge, J.E. and B.T. Hofreiter. 1962. In Carbohydrates Chemistry, 17 (eds. Whistler, R.L. and BeMIller, J.N.) Academic Press, New York.

1.4. Phenol-sulphuric acid method for total carbhohydrate Simple sugars, oligosaccharides, polysaccharides and their derivatives give green colour when treated with phenol and conc. H2SO4. The reaction is sensitive and the colour is stable. Principle It hot acidic medium glucose is dehydrated to hydroxymethyl furfural. This forms green coloured product with phenol and has absorption maximum at 490 nm. Reagents 1. 5% Phenol: Dissolve 50 g of redistilled (reagent grade) phenol in water and dilute to one litre. 2. 96% Sulphuric acid (reagent grade). 3. Standard glucose (stock): 100 mg in 100 ml of water. 4. Working standard: 10 ml of stock diluted to 100 ml with distilled water. Procedure 1. Follow the steps 1 to 4 as given in anthrone method for sample preparation. 2. Pipette out 0.2, 0.4, 0.6, 0.8 and 1 ml of working standard into a series of test tubes. 3. Pipette out 0.1 and 0.2 ml of the sample solution in two separate test tubes. Make up the volume in each tube to 1 ml with water. 4. Set a blank with 1 ml of water. 5. Add 1 ml of phenol solution to each tube. 6. Add 5ml of 96% sulphuric acid to each tube and shake well. 7. After 10 min. shake the contents in the tubes and place in a water bath at 25-30C for 20 min. 8. Read the colour at 490 nm. 9. Calculate the amount of total carbohydrate present in the sample solution using the standard graph. Calcualtion Absorbance correspondsto 0.1ml of the test =  mg of glucose 10 ml contains

=

 x 10 mg of glucose = % of total cabhohydrate present 0.1

Reference Dubois, M., K.A. Gilles, J.K. Hamilton, P.A. Robers and F. Smith. 1956. Anal. Chem. 26: 350.

1.5. Estimation of non-reducing sugars Principle Non-reducing sugars present in the plant extracts are first hydrolyzed with either sulphuric acid or formic acid to reducing sugars. Then, the total reducing sugars are estimated either by Nelson-Somogyi's or DNS method. Reagents 1. 1N H2SO4 2. 1N NaOH 3. Methyl red indicator Procedure 1. Follow the steps 1 to 3 as given in Nelson-Somogyi's method for sample preparation. 2. Pipette out 1 ml of extract and add 1 ml of 1N H2SO4 3. Hydrolyze the mixture by heating at 49C for 30 min. (the acid hydrolysis is effective in splitting the sucrose-type linkages). 4. Cool the tubes and add 1 or 2 drops of methyl red indicator. 5. Neutralize the contents by adding 1N NaOH drop wise from a pipette. Maintain appropriate reagent blanks. 6. Estimate the total reducing sugars by either Nelson-Somogyi‟s or DNS method as described earlier. 7. The content of non-reducing sugars can also be calculated by subtracting the reducing sugars from total carbohydrate content. Reference Malhotra, S.S. and S.K. Sarkar. (1979). Physiol. Plant. 47, 223-228.

1.6. Estimation of starch Principle The sample is treated with 80% alcohol to remove sugars and then starch is extracted with perchloric acid. In hot acidic medium starch is hydrolyzed to glucose and dehydrated to hydroxymethly furfural. This compound forms a green coloured product with anthrone. Reagents 1) 2) 3) 4)

Anthrone: Dissolve 200mg anthrone in 100ml of ice-cold 95% sulphuric acid. 80% Ethanol. 52% Perchloric acid. Standard glucose: Stock – 100ml water. Working standard -10ml of stock diluted to 100ml with water (100µ g/ml).

Method 1) Homogenize 0.1 to 0.5g of the sample in hot 80% ethanol to remove sugars. Centrifuge and retain the residue. Wash the residue repeatedly with hot 80% ethanol till the washings to not give colour with anthrone reagent. Dry the residue well over a water bath. 2) To the residue add 5.0ml of water and 6.5 ml of 52 % perchloric acid. 3) Extract at 0oC for 20min. Centrifuge and save the supernatant. 4) Repeat the extraction using fresh perchloric acid. Centrifuge and pool the supernatants and make up to 100ml. 5) Pipette out 0.1 or 0.2 ml of the supernatant and kale up to the volume to 1ml with water. 6) Prepare the standards by taking 0.2, 0.4, 0.6, 0.8 and 1ml of the working standard and make up the volume to 1ml in each tube with water. 7) Add 4ml of anthrone reagent to each tube. 8) Heat for 8 min in a boiling water bath. 9) Cool rapidly and read the intensity of green to dark green colour at 630nm. Calculation Find out the glucose content in the sample using the standard graph. Multiply the value by a factor 0.9 to arrive at the starch content. Reference Hodge, J.E and B.T. Hofreiter. 1962. In: Methods in Carbohydrate Chemistry (eds. Whistler, R.L and BeMiller, J.N.), Academic Press, New York.

1.7. Estimation of cellulose Cellulose undergoes acetolysis with acetic/nitric reagent to form acetylated cellodextrins which get dissolved and hydrolyzed to form glucose units on treatment with 67% H2SO4. On dehydration with H2SO4, glucose forms 5-hydroxymethyl furfural which on reaction with anthrone gives a green coloured product. The colour intensity can be measured at 630 nm. Reagents 1. Acetic/nitric reagent: Mix 150 ml of 80% acetic acid with 15 ml of conc. HNO 3. 2. Anthrone reagent: 200 mg anthrone / 100 ml of conc. H 2SO4 (Prepare fresh and chill for two hours before use). 3. 67% H2SO4 4. Standard cellulose solution: Add 100 mg of cellulose in 10 ml of 67% H2SO4 and leave for 1h. Dilute 1 ml of the solution to 100 ml (100 g/ml). Procedure 1. To about 0.5-1.0 g of sample, add 3 ml of acetic: nitric reagent and mix using a vortex mixer. 2. Place in a water bath at 100C for 3 min. 3. Cool and centrifuge for 15-20 min. Discard the supernatant. 4. Wash the residue with water, add 10 ml of 67% H2SO4 and leave it for 1 h. 5. Dilute 1ml of this solution to 100 ml. To 1ml of the diluted solution, add 10 ml of anthrone reagent and mix well. 6. Heat the tubes in a boiling water bath for 10 min, cool and measure the absorbance at 630 nm. 7. Prepare the blank with anthrone reagent and water. 8. Prepare standard curve by taking 0.4 to 2ml of standard cellulose solution (corresponding to 40-200 g of cellulose), equalize the volume and procced from step no. 4 for standard and develop the colour as above. 9. Calculate the amount of cellulose in the sample from the standard graph. Reference Updegroff, D.M. 1969. Anal, Biochem. 32: 420.

1.8. Estimation of hemicellulose Hemicelluloses are non-cellulosic, non-pectic cells wall polysaccharides. They are regarded as being composed of xylans, mannans, glucomannans, galactans and arabinogalactans. Principle The sample is refluxed with neutral detergent solution to remove the water-soluble and materials other than the fibrous component. The left out material is weighed after filtration and expressed as neutral detergent fiber (NDF). Reagents 1. Acetone 2. Sodium sulphite 3. Decahydronaphthalene 4. Neutral detergent solution: Dissolve 18.61 g of disodium ethylenediamine tetraacetate and 6.81 g of sodium borate decahydrate in about 200 ml of water by heating. To this, add about 100-200 ml of a solution containing 30 g of sodium lauryl sulphate and 10 ml of 2-ethoxy ethanol. Then add about 100 ml of a solution containing 4.5 g of disodium hydrogen phosphate. Adjust the pH to 7.0 and make up to one litre. Procedure 1. Take 1g of the powdered sample in a refluxing flask add 10 ml of cold neutral detergent solution. 2. Add 2ml of decahydronaphthalene and 0.5 g of sodium sulphite. 3. Heat to boiling and reflux for 60 min. 4. Filter the solution through sintered glass crucible (G-2) by suction and wash with hot water. 5. Wash twice with acetone, transfer the residue to a crucible and dry at 100C for 8h. 6. Cool the crucible in a desiccator and weigh. Calculation Hemicellulose = Neutral detergent fiber (NDF) - Acid detergent fiber (ADF) Reference Goering, H.D. and P.J Vansoest. 1975. Forage fiber Analysis, US Dept. of Agriculture, Agricultural Research Service, Washington.

1.9. Estimation of crude fiber The crude fiber content is commonly used as a measure of the nutritive value of livestock feeds and also in the analysis of various foods and food products to detect quality, quantity and adulteration. The crude fiber consists largely of cellulose, lignin (97%) and some mineral matter. It represents only 60-80% of the cellulose and 4-6% of the lignin. Principle During the acid and subsequent alkali treatment, oxidative hydrolytic degradation of the native cellulose and considerable degradation of lignin occur. The residue obtained after final filtration is weighed, incinerated, cooled and weighed again. The loss in weight gives the crude fiber content. Reagents 1. Sulphuric acid solution: 1.25g concentrated sulphuric acid diluted to 100ml (concenteration must be checked by titration). 2. Sodium hydroxide solution : 1.25g sodium hydroxide in 100 ml distilled water (concentration must be checked by titration with standard acid) Procedure 1. Extract 2g of ground sample with ether or petroleum ether to remove fat (initial boiling temperature 35-38C and final temperature, 52C). If fat content is less than 1% extraction may be omitted. 2. Boil 2g of dried sample with 200 ml of H2SO4 for 30 min with bumping chips. 3. Filter through muslin cloth and wash with boiling water until washings are free of acid. 4. Boil the residue with 200 ml of NaOH for 30 min. 5. Filter through muslin cloth again and wash with 25 ml of boiling H2SO4, three 50 ml portions of water and 25 ml of alcohol. 6. Remove the residue and transfer to pre-weighed ashing dish (W1g). 7. Dry the residue for 2h at 130 + 2C. Cool the dish in a desiccator and weigh (W2 g). 8. Ignite for 30 min at 600 + 15C. 9. Cool in a desiccator and reweigh (W3g). Calculation Loss in weight x (W2 - W1) - (W3 - W1) on ignition

% Crude fiber content =

x 100 Weight of sample (g)

Reference Maynard, A.J. 1970. Methods in Food Analysis, Academic Press, New York, p. 176.

1.10. Estimation of pectin substances – Colorimetric method Principle Galacturonic acid is reacted with carbazole in the presence of H2SO4 and the colour developed is measured at 520 nm. Reagents 1. 60% Ethyl alcohol (Mix 500 ml 95% alcohol and 300 ml water) 2. 95% Ethyl alcohol 3. Purified ethyl alcohol (Refluxes 1 litre of 95% ethyl alcohol with 4g zinc dust and 2ml conc. H2SO4 for 15 h and distills in all glass distillation apparatus). Redistill with 4 g zinc dust and 4 g KOH. 4. 1N and 0.05N Sodium hydroxide. 5. H2SO4 (Analytical grade) 6. 0.1% Carbazole reagent: Weigh 100 mg recrystallized carbazole, dissolve and dilute to 100 ml with purified alcohol. Standard Weigh 120.5 mg galacturonic acid monohydrate (from a sample vacuum dried for 5h at 30C) and transfer to a 1 litre volumetric flask. Add 10 ml 0.05 N NaOH and dilute to volume with water. After mixing, allow it to stand overnight. Dilute 10, 20, 40, 50, 60 and 80 ml of this standard solution to 100 ml with water. Take 2ml of these solutions for colour developing and proceed as in the case of the sample. Draw a standard curve-the absorbance versus concentration. Procedure 1. Weigh 100 mg of pectin and dissolve in 100 ml of 0.05 N NaOH. 2. Allow it to stand for 30 min, to deesterify the pectin. 3. Take 2ml of this solution and make up to 100 ml with water. 4. Pipette out 2ml of deesterified pectin solution and add 1ml of carbazole reagent. A white precipitate will be formed. 5. Add 12ml conc. H2SO4 with constant stirring. 6. Close the tubes with rubber stopper and allow standing for 10 minutes to develop the colour. 7. To set a blank add 1ml of purified ethyl alcohol in the place of carbozole reagent. 8. Read the colour intensity at 525 nm against blank, exactly 15 min. after the addition of acid. Calculation Read the concentration of the anhydrogalacturonic aid corresponding to the reading of the sample, and calculate as follows: % Anhydrogalacturonic acid g of anhydrogalacturonic acid in the aliquot x dilution x 100 = ml taken for estimation x wt. of pectin sample x 1,000,000

Reference Ranganna, S. 1979. Manual of Analysis of Fruit and Vegetative Products, Tata McGrawHill Publ. Co. Ltd., New Delhi, p.634.

2. NITROGEN 2.1. Estimation of nitrogen In most proteins, nitrogen constitutes nearly 16% of the total composition and hence, the total nitrogen content of the sample is multiplied by 6.25 to calculate the crude protein content. Principle The sample is digested with conc.H2SO4 in the presence of a catalyst to convert the nitrogen in protein or any other organic material to ammonium sulphate. By steam distillation of this salt in the presence of a strong alkali, ammonia is liberated and collected in boric acid solution as ammonium borate which is estimated against a standard acid by titration. (Note: 1ml of 0.1N acid = 1.40 mg N). Reagents 1. Conc. H2SO4 2. Mercuric sulphate 3. Sodium hydroxide-sodium thiosulphate solution: Dissolve 600g of NaOH and 50g of Na2SO3. 5H2O in water and make up to 1 litre. 0.02N standard HCL or H2SO4 4. 5. 4% Boric acid solution : Dissolve 4g of H3BO3 in warm water and dilute to 100ml 6. Mixed indicator solution: Mix 2 parts of 0.2% methyl red in ethanol with 1 part of 0.2% methlyene blue in ethanol (or mix 1 part of 0.2% methyl red in ethanol with 5 pats of 0.2% bromocresol green in ethanol). Method 1. Weigh 100 mg sample into a 30ml digestion flask. 2. Add 0.1g K2SO4 and, 2 ml of Conc. H2SO4 and mix well. 3. Add boiling chips/glass beads and digest the sample over digestion rack. 4. Cool and add minimum quantity of water along the sides of the flask to dissolve solids and transfer to the distillation apparatus. 5. Place a 100ml conical flask containing 5 ml of boric acid solution with a few drops of mixed indicator in such a way that the tip of the condenser dipping inside the solution. 6. Add 10ml sodium hydroxide-sodium thiosulphate solution to digest in the apparatus through the funnel and rinse with water. 7. Distill and collect the ammonia absorbed (collect at least 15-20 ml of distillate). 8. Rinse the tip of the condenser with water and titrate the distilled sample against the standard H2SO4 (0.02N) until the appearance of original violet colour as the end point. 9. Run a blank digested similarly with an equal volume of water after washing the distillation apparatus by back suction with excess of water. Calculation The nitrogen content of the sample can be calculated based on any one of the following formulate as the case may be:

(ml H2SO4 in sample) – (ml H2SO4 in blank) x normality of acid x 14.01 x 100 %N = Weight of sample (mg) Reference Humphries, E.C. 1956. Modern methods of plant analysis. p.468-502 2.2. Estimation of non-protein nitrogen Principle The proteins from the sample are precipitated in the presence of 10% TCA. Then the protein-free aliquot is distilled, titrated against the standard acid and the nitrogen content is calculated as described earlier. This gives the percentage of non-protein nitrogen. Reagents 1. 10% TCA (chilled) 2. Other reagents for estimation of total nitrogen Procedure 1. Extract 100mg of powdered material with 10ml of ice-cold 10% TCA to precipitate proteins 2. Centrifuge, wash the precipitate with TCA, recentrifuge, pool all the supernatants and make up to a known volume (25 or 50ml). This contains non-protein nitrogen. 3. Distill an aliquot as described earlier 4. Titrate against the standard acid and calculate the percentage of non-protein nitrogen. Estimation of Protein nitrogen When the total nitrogen content estimated by the Micro-Kjeldhal method is multiplied with the conversion factor, a value of crude protein content is obtained which also includes non-protein nitrogen. However, to get true protein content, deduct the nonprotein nitrogen from the total nitrogen and then multiply with the factor. Estimation of amino-nitrogen 1) Estimate the total free amino acid content 2) Then multiply the percentage equivalent of leucine with 14/131 to get the percentage of amino-nitrogen. Reference FAO Nurtitional Studies No. 24 1970. Amino Acid Content of Foods and Biological Data on Protiens, FAO, Rome.

2.3. Estimation of amino acids The best solvent for extraction of free amino acids from the biological samples is 70 80% ethanol in water. The amino acids may be determined by colorimetric method using ninhydrin or by chromatographic procedures or in an amino acid analyzer. Extraction of free amino acids Reagents 1. 70 - 80% Ethanol in water or 0.01 M phosphate buffer (pH 7.0) 2. 0.01 N HCl Procedure To a known weight of powdered sample, add warm (60C) 70-80% ethanol or 0.01 M phosphate buffer (pH 7.0), extract and filter or centrifuge.Repeat extraction 3-6 times, pool the supernatants and evaporate in a rotary vacuum evaporator to dryness. Dissolve the residue in 110 ml of 0.01N HCl or in a suitable sample dilution buffer. Colorimetric estimation of total free amino acids Principle Ninhydrin (triketohydrindene hydrate), a powerful oxidizing agent reacts with  amino acids between pH 4 and 8 and decarboxylates to given an intensely bluish-purple coloured compound which is measured colorimetrically at 570 nm. The amino acids proline and hydroxyproline give a yellow colour. Reagents 1. 80% Ethanol 2. 0.2 M Citrate buffer, pH, 5.0 3. Ninhydrin reagent: Dissolve 0.8g of stannous chloride (SnCl2.2H2O) in 500 ml of 0.2M citrate buffer (pH. 5.0). Add this solution to 20g ninhydrin in 500 ml of methyl cellosolve (2-methoxyethanol). Prepare fresh and store in a brown bottle (care: carcinogenic). 4. Diluent solvent : Mix equal volumes of water and n-propanol. 5. Stock standard leucine solution. 50 mg of leucine / 50 ml water. 6. Working standard leucine solution: Dilute 10 ml of stock leucine solution to 100 ml with water. Procedure Sample extraction 1. Grind a known weight of sample (500 mg) in a pestle and mortar with a small quantity of acid-washed sand. 2. Add 5 - 10 ml of 80% ethanol (if the tissue is tough, use boiling 80% ethanol). Filter or centrifuge. 3. Repeat the extraction twice and pool all the supernatants. 4. Reduce the volume if required by evaporation and use the extract for estimation of total free amino acids.

Procedure 1. To 0.1 ml of extract, add 1 ml of ninhydrin reagent and mix. 2. Make up the volume to 2ml with water. 3. Heat in a boiling water bath for 20 min. 4. Add 5 ml of the diluent while still on the water bath and mix. 5. After 15 min of boiling, cool the tubes under running tap and read the absorbance of the purple colour against a reagent blank (Prepare by taking 0.1 ml of 80% ethanol instead of extract) at 570 nm (Green filter). 6. Calculate the amount of total free amino acids using standard curve prepared from leucine by pipetting out 0.1 - 1.0 ml (10 - 100 g range) of working standard solution. Express the results as percentage equivalent of leucine. Reference Moore, S. and W.H. Stein. 1948. In: Methods Enzymol. (Eds. Colowick, S.P. and Kaplan, N.D.), Academic press, New York, 3, 468.

2.4. Estimation of Protein - Biuret method This is one of the first colorimetric protein assay methods developed. It is most often used in applications requiring a fast but not highly accurate determination. This method is commonly used to estimate the protein in the range from 0.5-5 mg. Principle It is based on the fact that the CO-NH- groups (peptide bonds) of protein form a purple complex with copper ions in an alkaline solution. The intensity of the purple complex is measured at 520 nm colorimetrically. Reagents 1. Biuret reagent: Dissolve 3 g of CuSO4. 5H2O and 9g of sodium potassium tartrate in 500 ml of 0.2N NaOH solution. To this solution, add 5g of potassium iodide and make up to 1 litre with 0.2 N NaOH. 2. Standard protein solution: 5mg of Bovine Serum Albumin/ml water. Prepare fresh. Procedure 1. Make up 1 ml of sample to 4 ml with water. 2. Add 6 ml of biuret reagent and mix well. 3. Keep at 37C for 10 min. 4. Cool and read the absorbance at 520 nm (green filter) against a reagent blank (prepare similarly with 4ml of water). 5. Draw the standard graph by pipetting out 0.1, 0.2, 0.4, 0.6, 0.8 and 1.0 ml of standard protein solution into a series of test tubes and making up the volume in each to 4ml with water. Carry out steps 2 to 4. 6. Calculate the protein content in the sample using a standard graph. 7. In order to overcome interfering substances, a modified biuret method may be followed. For this, make up the volume of sample to 1 ml with water. Add 1ml of 10% TCA, mix well and centrifuge at 3000 rpm for 10 min. To the precipitate, add 2ml of ethyl ether mix and recentrifuge. Dissolve the final dry precipitate in 4ml of water, mix and carry out steps from 2 to 6. Reference Layne, E. 1957. Methods Enzymol. (Eds. Colowick, S.P. and Kaplan, N.O.) Academic Press, New York - 3 pp. 447-466.

2.5. Estimation of Protein - Lowry's Method Principle Protein reacts with the Folin-Ciocalteau reagent (FCR) to give a blue coloured complex. The colour so formed is due to the reaction of the alkaline copper with the protein and the reduction of phosphomolybdic phospotungstic components in the FCR by the amino acids tyrosine and tryptophan present in the protein. The intensity of the blue colour is measured colorimetrically at 660 nm. Reagents 1. Solution A 2% sodium carbonate (anhyd.) in 0.1N NaOH. 2. Solution B : 0.5% Copper sulphate (CuSO4.5H2O) in 1% sodium potassium tartrate (prepare fresh) 3. Solution C (alkaline copper solution): Mix 50 ml of solution A with 1ml of solution B just prior to use. 4. Folin-Ciocalteau reagent (FCR): Dilute the reagent with an equal volume of water on the day of use. Stock standard protein solution: 50 mg of Bovine Serum Albumin / 50ml of water. Working standard solution: Dilute 10 ml of the stock solution to 50 ml with water to obtain 200 g protein/ml. Procedure Extraction of protein from sample 1. Grind 0.5 g of the sample with a suitable solvent system (water or buffer) in a pestle and mortar. 2. Centrifuge and use the supernatant for protein estimation. Estimation of Protein 1. Pipette out 0.2, 0.4, 0.6, 0.8 and 1.0 ml of the working standard solution into series of test tubes. 2. Pipette out 0.1 ml and 0.2 ml of the sample extract into two other test tubes. 3. Make up the volume to 1ml with water in all the tubes. A tube with 1 ml of water serves as the blank. 4. Add 5ml of solution C, mix well and incubate at room temperature for 10 min. 5. Add 0.5 ml of FCR, mix well immediately and incubate at room temperature in dark for 30 min. 6. Read the absorbance at 660 nm against the blank. 7. Draw a standard graph and calculate the amount of protein in the sample and express the results as mg/g or mg/100g sample or percentage. 8. If the sample has high phenolic or pigment content, extract should be prepared with a reducing agent preferably cysteine and NaCl. Precipitate the protein with TCA, separate and dissolve in 2N NaOH and proceed for estimation of protein. Reference Lowry, O.H., N.J. Rosebrough, A.L. Farr, and R.J. Randall. 1951. J. Biol. Chem., 193: 265.

2.6. Estimation of soluble protein by Dye-binding method (Bradford's method) Reagents 1. Standard protein solution: Dissolve 25 mg of Bovine Serum Albumin in 0.15 M NaCl and make up the volume to 25 ml (1mg / ml). 2. 0.01% Protein reagent: Dissolve 100 mg of Coomassi Brilliant Blue G-250 in 50 ml of 95% alcohol and add 100 ml of 85% (w/v) phosphoric acid and dilute to 1 litre with water. Prepare fresh before use. Method 1. Pipette out 0.01, 0.02, and 0.04, 0.1 ml of standard protein solution into a series of test tubes. 2. Make up volume in each tube to 0.1 ml with an appropriate buffer, 0.1 ml of buffer alone serves as the blank. 3. Add 5ml of protein reagent and mix thoroughly by inversion or vortexing. 4. Measure the absorbance at 595 nm after 2 min. and before 1 h against a reagent blank. 5. Plot a standard graph and calculate the amount of protein in the un known sample treated in the same manner. Reference Bradford, M.M. 1976. Anal, Biochem.72: 248-254.

2.7. Polyacrylamide-sodium dodecyl sulphate gel electrophoresis (SDS-PAGE) of proteins Many proteins are oligomeric proteins containing two or more subunits. By a modification of PAGE called SDS-PAGE an oligomeric protein may be dissociated into its subunits and the molecular weight of the subunits determined. SDS-PAGE of proteins is carried out in the presence of sodium dodecyl sulphate (CH3(CH2)10 CH2OSO3-Na+), an animoic detergent readily binds and dissociates oligomeric proteins in the presence of a reducing agent, 2-mercaptothanol into their subunits. Under these conditions, most proteins bind about 1.4g of SDS/g of protein which completely masks natural charge of the protein giving a constant charge to mass ratio. Marker proteins of different molecular weight are run and a calibration curve is drawn from which the molecular weight of unknown protein is determined. Analysis and comparison of proteins in a large number of samples is easily made on polyacrylamide gel slabs containing SDS under denaturing conditions. Principle SDS is an anionic detergent which binds strongly and denatures proteins. The number of SDS molecules bound to a polypeptide chain is approximately half the number of amino acid residues in that chain. The protein – SDS complex carries net negative charges, hence moves towards the anode and the separation is based on the size of the protein. Reagents 1. Acrylamide solution: Dissolve 22.2g of acrylamide and 0.6g of bisacrylamide in water and dilute to 100ml or dissolve 22.8g of cyanogum in water and dilute to 100ml. filter and store at 0-4oC in brown bottles. 2. Gel buffer (0.2M phosphate buffer, pH7.0): Dissolve of Na2HPO4.H2O and 38.6g of Na2HPO4.7H2O and 2g of SDS in water and dilute to 1 litre. 3. Sample buffer: 0.01M phosphate buffer of pH 7.0 containing 1% SDS (w/v) and 1% mercaptoethanol (w/v) 4. Reservoir buffer : Dilute gel buffer 1:1 5. Ammonium persulphate solution: 15mg/ml in water, prepare fresh. 6. 0.055 (w/v) Bromophenol blue solution in sample buffer 7. Staining solution: Dissolve 1.25g of Coomassie brilliant Blue in a mixture of 454 ml of 505 methanol and 46ml of glacial acetic acid. 8. Destaining solution: Mix 75ml of glacial acetic acid, 50ml of methanol and 575 ml of water. 9. Standard marker proteins. Method 1. Dissolve about 3mg of protein in 1ml of sample buffer and incubate at 37 oC for 2hr or heat for 3min at 100oC (in case of sample extract, adjust the protein concentration to 1mg/ml of sample buffer). 2. To 100 µ l of 0.055 bromophenol blue solution, add 1 drop of glycerol and 1 drop of mercaptoethanol and mix.

3. For preparation of gels (10%), mix 13.5 ml of acrylamide solution, 15ml of gel buffer, 1.5 ml of ammonium persulphate solution, and 0.045ml of TEMED or DMAPN. 4. Quickly pipette out gel mixture into 10cm long gel tubes and allow it to polymerize under a layer of water. Remove the water completely after polymerization. 5. Apply the prepared protein sample to the gels and layer 1:1 diluted gel buffer over the samples. Similarly, load a few gels with standard marker proteins in the sample buffer. 6. Carry out electrophoresis with 1:1 diluted gel buffer with the positive electrode in the lower chamber at 8 mA/tube until the tracking dye is almost at the end of the gel. 7. Rim and remove the gels using a fine hypodermic syringe and wash with distilled water. 8. Measure the length of each gel and the distance migrated by the tracking dye. 9. Immerse the gels in tubes containing staining solution overnight (16-18h). 10. Destain the gels in destaining solution until the background is clear. 11. Measure the length of the gel after destianing and the position of each protein band. 12. Calculate the mobility relative to the tracking dye using the relationship: Distance moved by protein (mm) Mobility = --------------------------------------------------Distance moved by tracking dye (mm) 13. Plot mobility of marker proteins against mol. wts. On asemilogarithmic scale or against log10 molecular weight and calculate the molecular weight of the unknown form calibration curve.    

In 10% polyacrylamide gels, the low molecular weight (≈10000 daltons) polypeptides may diffuse; for resolution of these polypeptides use gels of higher (15%) acrylamide concentration. Any band of 0.1 µ g protein is visulised by Coomassie Brilliant Blue staining in SDS-PAGE; for visualizing protein of lower concentration below 0.1 µ g use silver staining method (highly sensitive) The gels prepared should be free from air bubbles. The molecular weights obtained are those of the denatured proteins subunits rather than the native proteins.

Reference Laemmli, U.K. (1970). Nature, 227, 680.

2.8. Isolation of plant DNA Reagents 1. Seedling or any other fresh tissue 2. Liquid nitrogen 3. Extraction buffer: 100mM Tris-HCl (pH 7.8) 6.06 g 10 mM Na2 EDTA 9.30 g 500 mM NaCl 14.61g Water 500 ml 4. 5M Potassium acetate: Dissolve 24.55g in 50 ml water, sterilize and use. 5. 3 M Sodium acetate: Dissolve 2.46g anhydrrous salt in 10 ml water, sterilize and use. 6. Suspension buffer: 50mM Tris-HCl (pH 8.0) 0.61 g 10 mM Na2 EDTA 0.37 g Water 100 ml Procedure 1. Weigh 5g of plant tissue, quickly freeze in liquid nitrogen and grind to a fine powder in a pestle and mortar. 2. Add 75 ml of extraction buffer in small aliquots and grind thorougholy. 3. Transfer the homogenate to a 250 ml flask. Add 5ml of 20% SDS and mix thoroughly using a magnetic stirrer for 15-20 min. Then incubate the contents at 65C for 10 min. 4. Add 50 ml potassium acetate solution, mix and incubate at 0C for 30min in order to precipitate proteins and polysaccharides. 5. Remove the precipitate by centrifuging at 2500 rpm for 15 min. To the supernatant add six tenth volumes of isopropanol and stand - 20C for at least 30 min to precipitate DNA. 6. Pellet DNA at 20,000 g fro 15 min, discard the supernatant and drain off any liquid by inverting the tubes on filter paper for 2-3 min. 7. Redissolve DNA pellet in suspension buffer (3ml). Add 1.8 ml isopropanol and 180l 3M sodium acetate solution and let stand at - 20C for 1h. 8. Repellet DNA by centrifugation, wash with ice-cold 80% ethanol and gently dry in vacuo or streaming nitrogen gas. 9. Redissolve the DNA pellet in a suitable volume of (0.5-5ml) TE buffer. 10. Estimate the DNA content and check purity by UV spectrometry.

2.9. Extraction of RNA from plant Reagents 1. 0.01 M Tris-HCl buffer, pH 7.4 containing 50 mM NaCl and 1% paminosalicylic acid. 2. Water-saturated butanol containing 0.1% 8-hydroxyquinoline and 14% m-cresol 3. 1.3 M NaCl. 4. Phenol-cresol. 5. Absolute and 80% ethanol 6. 0.15 M Sodium acetate containing 0.25% SDS. Procedure 1. Homogenize a known weight of fresh plant tissue (100 mg) in 4ml of tris-HCl buffer. 2. Add equal volume of water-saturated phenol and centrifuge. 3. Remove the phenol layer and add 1ml of 1.3 M NaCl, 4ml of phenol cresol reagents and recentrifuge. 4. Remove the Tris layer and rextract with 5ml of phenol-cresol. 5. Make up the final Tris layer to 0.02 M with respect of sodium acetate and precipitate RNA with 3 volumes of ethanol at 0C Centrifuge. 6. Wash the precipitate with 80% ethanol, cetnrifuge and dissolve in 2ml of 0.15 M sodium acetate containing 0.25% SDS. 7. Precipitate finally the pure RNA by adding 6ml of ethanol at - 20C and standing for 1h. Reference: Cherry, J.H. 1972. Nucleic acid determination in storage tissues of higher plants. Chloroplast proteins. Plant physiol., 37: 670-675. Bradford, M.M. 1976. A rapid and sensitive method for the quantification of microgram quantities of protein dry binding. Anal Biochem., 72: 248-254.

2.10. Estimation of DNA Principle Under extreme acid conditions, DNA is initially depurinated quantitatively followed by the dehydration of sugar to w-hydroxylevulinyladehyde. This aldehyde condenses, in acidic medium, with diphenylamine to produce deep-blue coloured condensation products with absorption maximum at 595 nm. Reagents 1. DNA Standard (0.5 mg/ml). 2. Saline citrate (0.15 M NaCl, 0.015 M Na3 citrate) solution. 3. Diphenylamine reagent: Mix 5g fresh or recrystallized diphenylamine, 500 ml glacial acetic acid and 13.75 ml conc. H2SO4. STable for six months at 2C. Warm to room temperature and wirl to mix before use.

Procedure 1. Prepare separate marked tubes containing 1ml, 2ml and 3ml aliquots of the isolated DNA dissolved in standard saline citrate and similar aliquots of a 0.5 mg DNA / ml standard. 2. Take all sample tubes and a separate blank, up to 3ml with H2O. 3. Add 6ml of diphenylamine reagent to each tube, and after mixing, heat the tubes in a boiling water bath for 10min. Cool the tubes. 4. Read the absorbance of blue solution at 600nm against the blank. 5. Construct a standard graph A600 (ordinate) versus quantity of DNA (abscissa) and then calculate the saline citrate solution. Reference Burton, K. 1956. Biochem, J. 62: 315

2.11. Estimation of RNA Principle The method depends on conversion of pentose, ribose in the presence of hot acid to furfural, which then reacts with orcinol to yield green colour. Reagents 1. Standard RNA 2. Orcinol acid reagent: Add 2ml of a 10% solution (w/v) of FeCL3, 6H2O to 400 ml of conc. HCl. 3. 6% Alcohol orcinol: Dissolve 6g orcinol in 100 ml 95% ethanol. Refrigerate in a brown bottle until use. Stable for one month. Procedure 1. Prepare a standard RNA (50 g RNA/ml) solution in 10mM Tris-acetate. 1mM EDTA buffer (pH 7.2). 2. Prepare a series of tubes containing 0.5 ml, 1ml, 1.5 ml, and 3 ml of isolated RNA, 0.5 ml, 1ml, 1.5 ml and 3ml of 50 g standard RNA /ml. 3. Make up each tube to 3ml with water. In addition set a blank containing 3ml of water. 4. Add 6ml of orcinol acid reagent to each tube. 5. Add 0.4ml of 6.0% alcoholic oricinol to each tube. Shake the tubes to mix the contents, and then heat all the tubes in a boiling water bath for 20 min. 6. Cool the tubes and read the absorbance at 660nm against the blank. 7. Draw a standard curve using A660 and the concentration of standard RNA. Calculate the amount in the isolated RNA solution. Reference Ashwell, G. (1957). In: Method Enzymol: 3 (eds. Colowick, S.P. and Kalpan, N.O.). Academic Press, New York, p.87. Determination of phosphorus content in nucleic acid The phosphorus in nucleic acids is in a bound, organic form and as such cannot be estimated directly. It is converted to inorganic phosphate by digestion with acid and then estimated. Principle The nucleic acid is oxidized with perchloric acid to give inorganic phosphate which is then estimated by the usual methods. Reagents 1. 60% (v/v) Perchloric acid 2. Reagents for estimation of phosphorus Method 1. Pipette out 2ml of nucleic acid solution (0.1mg/ml) into a digestion flask 2. Add 0.5ml of perchloric acid and digest in the heating rack or oven a sand bath for 1 hour until all the inorganic matter disappears (care ! explosion risk) 3. Cool to room temperature and add 1ml of water 4. Estimate the phosphorous and express the results as mg P/mg nucleic acid. Reference Plummer,D.T. (1990). In: An Introduction to Practical Biochemistry (3rd edn.), Tata McGraw-Hill Publishing Company Limited, New Delhi, pp. 221-222.

2.12. Isolation of Plasmids Plasmids are extrachromosomal, self-replicating double stranded, circular DNA molecules found in most prokaryotes. These molecules carry genetic information for a variety of special functions such as resistance to antibiotics, nitrogen fixation, ability to utilize novel substances etc. the plasmids can be transferred from one cell to another and therefore function as vectors or carries in genetic engineering techniques. A number of plasmids used in genetic engineering have a relaxed mode of replication. This means that the plasmid replicates independently of chromosome control, accumulates upto one-third of the cellular DNA content when cell protein synthesis is inhibited by a drug. Thus milligram quantities of plasmid DNA may be isolated from a single litre of cells. For cloning work the plasmid DNA is required in substantially pure form and involves elaborate procedure. On the other hand, a large number of samples can be examined by a rapid method for quantitative information. Principle The bacterial cells are grown to stationary phase, harvested and gently lysed by weakening the cell walls with lysozyme treatment followed by use of the detergent SDS. As a result the cells release their DNA in high molecular weight form which is removed by high-speed centrifugation leaving the plasmid DNA in the cleared lysate. This fraction is deproteinized and nucleic acids are then precipitated by ethanol. Purification of the plasmid is performed by equilibrium density centrifugation incesuim chloride. Materials and reagents 1. Bacterial stain carrying the plasmid (eg. E. coli. JA 221 carrying pBR 328) 2. LB Broth: Yeast extract 5g NaCl 10g Tryptone 10g Water 1L 3. TE + sucrose (pH 8.0) 0.05M Tris 0.61g 25% (w/v) sucrose 25g Water 100ml 4. Lysozyme solution : 5mg/ml in 0.25M Tris-HCL (pH 8.0) 5. Phenol-chloroform mixture: 1:1 (v/v) 6. Saline sodium citrate (SSC) solution: 0.15M NaCl 0.88g 0.015M Sodium citrate 0.44g Water 100ml Dilute it ten times to get 0.1 SSC 7. Ethidum bromide : 5mg/ml in 0.1 SSC solution 0.25M EDTA solution TES buffer (pH 8.0) 30mM Tris 0.36g 5mM Na2EDTA 0.19g 50mM NaCl 0.28g Water 100ml

8. High speed refrigerated centrifuge 9. Ultracentrifuge with suitable rotors 10. Polycarbonate ultracentrifuge tubes 11. UV Lamp (longwave length) 12. Pasteur pipettes Method A. Harvesting cells 1. Grow the bacterial strain in 250ml LB broth + antibiotic (ampicillin) at 37oC with shaking (vigorous aeration) to stationary phase. 2. Harvest the cells by centrifugation at 5000rpm in a refrigerated centrifuge for 10min at 4oC 3. Wash the cells by resuspending in the TES buffer and centrifuging at 6000 rpm for 10 min. and repeat the washing steps. 4. Resuspend the cells in a small volume of TE + sucrose buffer (the cells can be stored frozen at this stage, if necessary). Make up the volume of the suspension to 3.75 ml by adding TE+sucrose buffer. B. Lysing cell and DNA isolation 1. Transfer the cell suspension to a precooled 100ml flask 2. Add 0.75 ml of lysozyme solution followed by 1.25 ml of 0.25M EDTA (pH 8.0) solution and shake the contents on ice for 10min 3. Add 0.75ml of 20% SDS (final concentration 2%) and ensure uniform mixing 4. Incubate without shaking at 37oC in a water bath until the suspension clears (cell lysis; 10-60 min). cool on ice 5. Centrifuge the lysate in thick walled polycarbonate tubes on an ultracentrifuge at 40000rpm for 1 hour at 20oC (use 0.25 Tris pH 8.0 for balancing the centritubes, if necessary). This will clear the lysate and the supernatant will contain most of the plasmids with RNA and proteins as contaminants. High molecular weight chromosomal DNA is removed in the pellet. 6. Carefully decant the supernatant into a measuring cylinder, note its volume and transfer into a 100ml flask. Add 0.1 volume supernatant 2.0 M Tris base (pH unadjusted). 7. Add an equal volume of phenol:chloroform and shake thoroughly at room temperature for 4min. 8. Centrifuge the emulsion in a bench centrifuge at 5000rpm for 10min to separate the aqueous and organic phases. 9. Transfer the upper aqueous phase and note its volume. Add 0.25 times the volume of 4.5M potassium acetate to give a 0.9 M solution to ensure quantitative precipitation of DNA. 10. Add two volumes of chilled ethanol and place in freezer for 60min to allow complete precipitation of DNA. 11. Centrifuge the contents at 10000rpm for 010min at 0 oC to pellet the DNA. Decant the supernatant and drain off any liquid by inverting the tubes on paper towels. Dry gently in a vaccum desiccator or using a stream of nitrogen gas.

12. Dissolve the precipitate in 0.4ml 0.1 SSA and withdraw 20 µ l for testing by electrophoresis; then make up the remaining solution to 3.6 ml with 0.1 SSC. C. Purification by cesium chloride centrifugation 1. Dissolve 3.9g CsCl in the preparation completely. Then add 0.4ml of ethidium bromide 2. load the sample into ultracentrifuge tubes to within a few mm of the top and balance the tubes in pairs 3. centrifuge at 140000g for 40h at 20oC in a swing out rotor 4. After centrifugation view the tubes under long-wave UV light. The DNA ethidium bromide complex fluoresces and two defined bands could be seen near the middle of the tube. The more intense lower band consists of supercoiled, circular plasmids and the top band consists of linear plasmids and fragment of nuclear DNA. 5. The plasmid band can be recovered by a number of ways. First deaw-off the upper part of the gradient and the top DNA band using a Pasteur pipette. Then suck the plasmid band into a sterile syringe fitted with a wide-bore needle carefully. Alternatively, a long needle fitted to a syringe is carefully lowered to the plasmid band and carefully drawn into syringe 6. Remove the ethidium bromide from the plasmid fraction by extracting thrice with two volumes each of isopropyl alcohol. Cesium chloride and ethdium bromide are removed by dialysis for 16h against several changes of 0.1x SSC or any other suitable buffer for further analysis. 7. Following dialysis, transfer the plasmid solution to sterile tubes. Measure the absorbance at 260 and 280 nm. The A260 should be nearly two fold of A280 for a good preparation. Calculate the concentration of plasmid DNA using the relationship: 8. A260 of 1.0 = 50 µ l/ml of DNA The preparation can be stored frozen for several weeks. If a more concentrated preparation is required concentrated by precipitation with ethanol (step 4-16). Reference Clewell, D.B. and D.R. Helinski, (1971). Biochemistry, 9, 4428.

2.13. Estimation of proline Proline is a heterocyclic amino acid found abundantly in basic proteins. Free proline in plants is said to play a role under induced, cold, drought, salt and pathological stess conditions. Free proline from plant tissues may be selectively extracted in aqueous sulphosalicylic acid and its amount is estimated by ninhydrin method. Principle Proteins are precipitated as a protein-sulphosalicylic acid complex during extraction of tissue with sulphosalicylic acid. The extracted proline is made to react with ninhydrin under acidic conditions to form a red colour which is measured colorimetrically at 520nm. Reagents 1. Acid ninhydrin reagent: Dissolve 1.25 g of ninhydrin in a mixture of warm 30 ml of galcial acetic acid and 20 ml of 6 M phosphoric acid (pH 1.0) with agitation until it is dissolved. Store at 4°C and use within 24h. 2. 3% Aqueous sulphosalicylic acid. 3. Glacial acetic acid 4. Toluene 5. Standard proline solution. Procedure 1. Homogenize 0.5 g of tissue in a pestle and mortar with 10 ml of 3% aqueous sulphosalicylic acid and filter through Whatman No. 2 filter paper. 2. Repeat the extraction and pool the filtrates. 3. To 2 ml of filtrate, add 2 ml each of glacial acetic acid and ninhydrin and mix. 4. Keep in boiling water bath for 1 h and then terminate reaction by placing on ice bath. 5. Add 4 ml of toluene, mix vigorously for 20-30 sec. 6. Aspirate the chromophore (toluene) layer and warm to room temperature. 7. Measure the absorbance of red colour at 520 nm against a reagnet blank. 8. Calculate the amount of proline in the sample using a standard curve prepared from pure proline (range 0.1-36 µ mole) and express on fresh weight basis of sample. Calculation

μ moles of proline/g tissue 

g proline / ml x ml toluene 115.5

x

5 g sample

Where, 115.5 is the molecular weight of proline. Reference Bates, L.S., R.P. Waldeen, and I.D. Teare. 1973. Plant Soil, 39, 205.

2.14. Estimation of methionine Methionine is one of the sulphur-containing and essential amino acids. It is a limiting amino acid in grain legumes. Principle The protein in the sample is first hydrolyzed under mold acidic condition. Under alkaline condition, the liberated methionine gives a yellow colour with nitroprusside solution and turns red on acidification. The colour formed by other amino acids is inhibited by adding glycine to the reaction mixture. Reagents 1. 2. 3. 4. 5. 6.

10N NaOH (40%) 10% NaOH 2N HCl 10% Sodium nitroprusside: Prepare fresh every few days. 3% Glycine Standard methionine: Dissolve 0.1 g of DL of L-methionine in 4 ml of 20% HCl and dilute with water to 100 ml (1mg/ml). Procedure

1. To 0.5g of defatted sample in conical flask, add 6 ml of 2N HCl and autoclave at 15 lb pressure for 1 h. 2. Add a pinch of activated charcoal to the autoclaved sample (hydrolyzate) and heat to boil. Filter when hot and wash the charcoal with hot water. 3. Adjust the pH of filtrate to 6.5 with 10 N NaOH and make up the volume to 50 ml with water after cooling to room temperature. 4. Transfer 25 ml of the made up solution into a 100 ml conical flask. 5. Add 3 ml of 10% NaOH and 0.15 ml of sodium nitroprusside and mix well. Add 1 ml of Glycine solution after 10 min. 6. After another 10 min add 2 ml of othrophosphoric acid and mix vigorously. 7. Read the absorbance of red colour after 10 min at 520 nm against a blank prepared similarly without nitroprusside. 8. Draw the standard curve by pipetting out 0, 1, 2, 3, 4 and 5 ml of standard methionin esolution and making up to 25 ml with water. 9. Develop colour following steps 5 to 8 the 'o' level serves as a blank. 10. Calculate the methionine content from the graph. Calculation Methionine content in sample (mg/g) = Methionine content x 4 from graph Usually methionine is expressed as percentage of protein or g/16g N. Methionine content of the sample (g/16g N) =

Methionine content from graph x6.4 % N ins ample

Reference Horn, M.J., D.B. Jones, and A.E. Blum. 1946. J. Biol. Chem. 166: 313.

2.15. Estimation of lysine Lysine is another important essential amino acid. It is a limiting amino acid in cereal grains. Principle The protein in the grain sample is hydrolyzed with a proteolytic enzyme, papain. Then the -amino groups of the derived amino acids are made to form a complex with copper. The є amino group of lysine which does not couple with copper is made to form є-dinitropyridyl derivative of lysine with 2-chloro-3, 5-dinitropyridine. The excess pyridine is removed with ethyl acetate and the colour of є –dinitropyridyl derivative is read at 390 nm. Reagents 1. Papain solution: Dissolve 400 mg of technical grade papain (Sigma Co., USA) in 100 ml of 0.1 M sodium acetate buffer, pH 7.0. Filter if necessary. 2. 0.05 M sodium Carbonate buffer, pH 9.0 3. 0.005 M sodium borate buffer, pH 9.0 4. Copper phsophate reagent : 1. Solution A: 2.89g of CuCl2. 2H2O in 100 ml water. 2. Solution B: 13.6g of Na3PO4. 12 H2O in 200 ml water. Mix solution A (100 ml) with solution B (200 ml), centrifuge at 3000g for 5 min and discard the supernatant. Wash the precipitate 4 times with 0.005 M sodium borate buffer, pH 9.0 followed by centrifugation. Resuspend the pellet in 80 ml of borate buffer. Prepare the reagent fresh for every seven days. 5. 3% 2-Chloro-3, 5-dinitropyridine solution in methanol or ethanol. Prepare fresh just prior to use. 6. 1.2 N HCl. 7. Amino acid mixture : Grind in a mortar 30 mg each of alanine, histidine, isoleucine, threonine and tyrosine; 20 mg each of cysteine and methionine; 40 mg each of glycine, phenylalanine and valine; 50 mg each of arginine and serine; 60 mg of asparatic acid; 80mg each of leucine and proline and 300 mg of glutamic acid. Dissolve 100 mg of this mixture in 10 ml of sodium carbonate buffer (0.05M, pH 9.0). 8. Ethyl acetate. 9. Standard lysine solution: 62.5 mg of lysine monohydrochloride / 50 ml of carbonate buffer (1 mg/ml). Procedure 1. To 100 mg of finely ground and defatted sample, add 5 ml of papain solution and incubate at 65°C overnight (shake the sample after 1 h of incubation and before 1h

from removing from incubator). Cool to room temperature, centrifuge at 3000g for 5 min and collect the supernatant. 2. To 1 ml of supernatant in a centrifuge tube, add 0.5 ml of carbonate buffer and 0.5 ml of copper phosphate suspension. 3. Mix in a vortex mixer for 5 min and centrifuge. 4. To 1 ml of supernatant add 0.1 ml of 2-chloro-3, 5-dinitrophyridine solution, mix well and shake for 2 h at room temperature and 5 ml of 1.2 N HCl and mix. 5. Extract three times with 5 ml of ehtyl acetate using a separatory flask and discard the ethyl acetate (top) layer. 6. Read the absorbance of aqueous layer at 390nm, against a reagent blank (prepare with 5 ml of papain alone and repeat steps from 1 to 7). 7. Prepare a standard curve by pipetting out 0.2, 0.4, 0.6, 0.8 and 1.0 ml of standard lysine solution and make up to 1 ml with carbonate buffer. Add 4 ml of papain to each tube and mix. Pipette out 1 ml from each tube and add 0.5 ml of amino acid mixture and 0.5 ml of copper phosphate suspension. Follow the steps 3 to 7 (The standard curve represents for 40, 80, 120, 160 and 200 µg of lysine, respectively). Calculation

Lysine content of sample (g/16gN) = or Lysine in protein (%) =

Lysine value fromgraph in g x 0.16 % N in sample

% Lysine in sample x100 % Protein in sample

Reference Tsai, O.Y., L.W. Hansel, and O.E. Nelson. 1972. Cereal Chemistry, 49(5): 572-579.

2.16. Estimation of tryptophan Tryptophan is a heterocyclic and one of the essential amino acids for human beings and albino rats. Cereals such as maize and sorghum are deficient in tryptophan in addition to lysine. The method described here under may be adopted for estimation of tryptophan in foods, feeds and cereal grains etc. Principle Under strongly acidic conditions, the indole ring of tryptophan gives an orangered colour which is measured at 545 nm. Reagents 1. Reagent A: Dissolve 135 mg of FeCl3. 6H2O in 0.25 ml of water and dilute to 500 ml with glacial acetic acid containing 2% acetic anhydride. 2. Reagent B: 30N H2SO4. 3. Reagent C: Mix equal volumes of reagents A & B about one hour before use. 4. Papain solution: Dissolve 0.4 g of technical grade papain in 100 ml of 0.1 N sodium acetate buffer, pH 7.0 Prepare fresh on the day of use. 5. Standard tryptophan solution: 5 mg tryptophan / 100 ml water (50 µg/ml). Procedure 1. To 0.1g of air-dried, powdered and defatted grain sample, add 5 ml of papain solution shake well and close the tube. 2. Incubate at 65°C overnight. Cool to room temperature, centrifuge and collect the supernatant. 3. To 1 ml of supernatant, add 4 ml of reagent C and mix in a vortex mixer and incubate at 65°C for 15 min. 4. Cool to room temperature and read the absorbances of orange-red colour at 545 nm against a blank (prepare with 5 ml of papain and repeat steps from 2 to 4). 5. Calculate the tryptophan content in the sample using the standard curve prepared from tryptophan (from 0-50µ range, in each case make up the volume to 1 ml with water and develop colour following steps from 3 to 4) and report on a per cent basis or as g/16g N using the following formulae.

% Tryptophan in protein =

Tryptophanin sample x100 % Protein in s ample

Tryptophan in sample (g / 16g N) =

Tryptophanvalue from graph ( g) x 0.096 x100 % N in s ample

Readings Mertz, E.T., R. Jambunathan, and P.S. Misra. 1975. In: Protein Quality, Agricultural Research Station Bull No. 7, Purdue Univ., USA, p. 9.

2.17. Pathogenesis-Related Proteins (PR-Proteins) Plant pathogens such as viruses, bacterial, fungi and nematodes elicit the induced synthesis of host proteins which help in restricting the multiplication and spread of pathogens in the healthy tissue. These proteins are called pathogenesis-related proteins (PR-Proteins). Analysis of PR-Proteins Reagents 1. 0.1M Sodium phosphate buffer, pH 7.0 2. 25mM Tris-HCL buffer, pH 7.8 containing 0.5M sucrose, 10mM MgCl2, 10mM CaCl2, 0.5mM PMSF and 5mM 2-mercaptoethanol 3. Citrate-phosphate buffer: 84mM citrate and 32mM disodium hydrogen phosphate, pH 2.8 containing 0.1% 2-mercapitaethanol and 0.1% L-ascorbic acid. 4. Sephadex G-50 column. 5. 50mM Tris-HCL containing 1mM EDTA, pH 8.0. 6. Reagents for estimation of protein by Lowry‟s or Bradford‟s method. 7. Growth chamber Procedure Intercellular fluid extraction 1. Cut the freshly collected leaves with scissors into pieces of 4-5 cm2 2. Infiltrate the pieces in vacuo with gentle agitation for three periods of 30 seconds each, with a large excess of the following cold (4 oC) mixture: 25mM Tris-HCL buffer, pH 7.8 containing 0.5M sucrose, 10mM MgCl2, 10mM CaCl2, 0.5mM PMSF and 5mM 2-mercaptoethanol, (alternatively, only distilled water can be used instead of buffer). 3. Gently blot dry, roll up and place the pieces in a 20ml plastic syringe. 4. Place the syringe in a centrifuge tube and centrifuge at 1000 x g for 10 min at 4 oC 5. Collect the intercellular fluid at the bottom of the tube in a Eppendorf tube and centrifuge for clarification (normal yield = 0.5ml/g tissue = 1mg protein/ml). Protein extraction from infected leaves 1. Extract sample of 5g leaves in 5ml of citrate phosphate buffer 2. Centrifuge the homogenate for 5min at 10000xg and apply the supernatant to a column (25x1cm) of Sephadex G-50 equilibrated in 50mM Tris –HCL, 1Mm EDTA, pH 8.0 3. Collect the fractions eluate showing 280nm absorption and concentrate (alternatively, the extract is dialyzed against the column buffer). Determination of protein 1. Determination of protein content of the concentrated sample either by Lowry‟s or Bradford‟s ,method 2. Other analysis of PR-Proteins such as separation of basic proteins using acid gel electrophoresis, 2-dimensional electrophoresis, enzyme function and the nature of intercellular PR-Proteins may be analysed as per the methods described elsewhere. Reference Parent, J.G. and A. Asselin. 1984. Can. J. Bot. 62: 564-569.

2.18. Estimation of Chloroplast Protein (Nagata and Ishii, 1979) Principle The leaf tissue was homogenised with suerage PO4 buffer and the chloroplasts were taken in PO4 buffer. The proteins were precipitated by TCA and quantitatively estimated according to Lowry et al, 1952. Reagents 1. Phosphate buffer (pH 6.2) Solution A

: 0.05M NaH2PO4 (7.8 g of NaH2 PO4 in 1000ml distilled water)

Solution B

: 0.05M Na2 HPO4 12 H2O (17.925 gram of Na2 HPO4.12H2O in 1000ml distilled water

Phosphate buffer (pH 6.2) is prepared by mixing 81.5ml of a solution and 18.5 ml of B solution and diluted with distilled water to 200ml. 2. Sucrose 0.4M

: 136.920 g in water and volume made up to 100ml distilled

water. Procedure Isolation of Chloroplast (dark) Chloroplasts are extracted from 5 gram of leaf material homogenized with 0.4M sucrose in 0.05M phosphate buffer (pH 6.2), centrifuged at 3000 rpm for 5 minutes and the residue is discarded. The supernatant is centrifuged again at 5000 rpm for 90 Sec. The residue containing the chloroplast is suspended in 2ml of phosphate buffer. Chloroplast Protein estimation In (one ml of) chloroplast suspension, 10ml of acetone / ethanol / methanol is added and centrifuged at least four times at 3000 rpm for 3 minutes to remove the chlorophyll. Then 10 ml of 10 per cent TCA is added to the residue and centrifuged twice at 5000 rpm for 5 minutes. Then 10 ml of acetone is added to the residue and centrifuged again at 2000 rpm for 3 minutes. The residue is suspended in dimethyl ether and centrifuged twice at 3000 rpm for 5 minutes and the resultant residue is air dried to get chloroplast dry protein power. After weighing the Chloroplast protein dry powder, a known measure of protein dry powder is dissolved in 5 ml distilled water which is known as chloroplast protein extract. The protein content of chloroplast protein extract is

determined by Lowry‟s method using alkaline copper tartarate and folin phenol reagent and expressed as mg protein per gram of chloroplast dry protein powder. 2.19. Estimation of Heat Shock Proteins Principle Proteins are synthesized in response to higher temperature are called heat shock proteins. An increase in shift of temperature from 8 to 10C above the normal growth temperature decreases the synthesis of normal cellular proteins but increased the ion molecular weight HSPs. Reagents 20 mm Tris – Hcl (pH 7.5) 50 mm HEPS – KOH pH 8.33 35 S – Methionine, 4mm MgCl2 Procedure Seedling growth: Pre-soaked seeds of Vigna sinensis L. cv. Walp were germinated in the dark at 30C for 2 days. When the primary leaves had fully developed. 2-day-old seedlings were given appropriate treatment in environmental growth chambers maintained at 10, 20, 30 and 40C. Leaves were collected after 48 hours of treatment under different conditions. In vivo labeling and protein extraction : Leaf segments (200 mg) were placed in glass tubes containing 20 mM Tris – HCl, pH 7.5, and incubated at 25C under white light in the presence of 1.85 MBq of (35s) – methionine. At the end of 60 min. incubation period, the leaf segments were washed with 1 mM methionine (non-radioactive) and the proteins were extracted. In vitro labeling and protein extraction : For protein labeling, chloroplasts equivalent to 500 g Chl were resuspended in 1.0 ml of medium containing 300 mM sorbitol, 50 mM HEPES – KOH, pH 8.3, and 4 mm MgCl2 and incubated at 25C under white light (450 mol m-2 s-1) in the presence of 1.85 MBq of (35s) – methionine. At the end of the 60 – min. incubation period, the chloroplasts were diluted in resuspension buffer, pelleted and washed. Extraction of protein was done. SDS-PAGE and Fluorography: SDS-PAGE was run using an 8-16% polyacrylamide gradient gel and fluorography was done for identification and quantification (Refer Appendix).

2.20. Nitrogen fractions (Pregl, 1945) Principle Ammoniacal nitrogen Hot water boiling of the samples with diluted sulphuric acid at 100C convert ammonical Nitrogen into inorganic form which was distilled using alkali. The ammonia evolved is absorbed in weak boric acid and back titrated against standard acid. Known weight of dried powered plant samples were taken and extracted with 10 ml of 10 per cent sulphuric acid. The extract was heated in a water bath at 100C for 15 minutes and filtered through Whatman No.41 filter paper. This was made upto 50 ml with distilled water. 10 ml of the aliquot was taken for estimation by microjeldahl method described for total nitrogen content estimation. Expressed the content of ammoniacal nitrogen as percentage on dry weight basis. Principle Non-protein nitrogen and protein nitrogen 1. The non-protein part of the total protein in taken in trichloro acid by homogenisation and the ammonia is distilled and back titrated against standard acid. 2. The microkjeldahl method for estimation of non-protein nitrogen and protein nitrogen was employed (Pregl, 1945). 3. Dried powered plant material of 0.1 gm was ground with 10 ml of 5 per cent trichloro acetic acid (TCA) in a glass nortar and filtered. The filtrate was made upto a known volume with trichloro aceticacid. 4. A quantity of 10 ml of aliquot was taken and used for the estimation of nitrogen as done for total nitrogen. The direct value represents non-protein nitrogen and the amount was expressed as percentage on dry weight basis. 5. Protein nitrogen was calculated as the difference between total nitrogen and nonprotein nitrogen and expressed in percentage. Amide, Nitrite and Nitrate Nitrogen 1. Hundred mg of powdered plant sample was taken and ground well in a pestle and mortar with 10 ml of 5 per cent trichloro acetic acid. Filtered through Whatman filter paper No.41 and made upto 50 ml with trichloro acetic acid. 2. Adjusted 10 ml of the extract to pH 10 with 40 per cent sodium hydroxide and one ml of borax buffer was added (Dissolved 3.84 gm of sodium tetra borate in 1 litre of distilled water. Transferred this solution to microkjeldahl unit. 10 ml of 40 per cent sodium hydroxide was added to the flask. 3. Amide nitrogen was removed by steam distillation at 100C, collected in 20 ml of 2 per cent boric acid. Check for the end of distillation by red litmus. 4. 5ml of 20 per cent ferrous sulphate solution was added and continued the distillation to remove nitrate nitrogen. 5. 5 ml of saturated silver sulphate was added thereby nitrite nitrogen was removed. At every stage, collected the respective fractions of nitrogen in 20 ml of 2 per cent boric acid and titrated against N/50 sulphuric acid. Expressed the nitrogen fractions as percentage on dry weight basis.

Refernce Pregl. 1945. Quantitative organic micro analysis. J.A. Churchill Ltd. London. 3. PHOSPHORUS 3.1. Phosphorus Fractions (Hogue et al., 1970) Reagents 1. Perchloric acid 0.2 N 2. Nitric acid 3. Alcohol, Ether, Chloroform 4. Perchloric acid 5% 5. KOH 0.5 N 6. Plant tissue (500nmg fresh weight or 100 mg dry weight) Homogenised in 2 ml of 0.2 N Perchloric acid and centrifuged at 10,000 xg, washed the pellet three times with 2 ml of 0.2 N Perchloric acid. Perchloric acid

Supernatant Fraction I : Acid soluble Phosphorus (organic & inorganic)

Supernatant

Fraction 2

Supernatant Fraction 3 RNA phosphorus

Residue Washed tow times with 3ml of alcohol

Residue Extracted with 6 ml Ethyl alcohol : ether : Chloroform (2:2:1) at 50C for one hour. Then washed it with 4 ml. of cold Ether. Residue Extracted with 5 ml of 0.5 M KOH at 37C for 17 hours

Residue Extracted with 5 ml of 5 % Perchloric acid for one hour at 70C Fraction Phosphoprotein Autoclaved with 6 N Hcl for 1½ hours at 15 lb pressure Supernatant

Residue (Rejected)

The various phosphorus fractions were determined according to Sumner, (1944).

Fraction I was made upto 20 ml. Volume and 10ml was used for measurement of acid soluble inorganic P (Pi) and 10ml was digested by hydrolyzing it with concentrated nitric acid and poerchloric acid to determine total acid soluble P (Pt). The difference (Total P (Pt) – Pi = Sol Po) represent the soluble organic phosphate. All the other fractions were acid digested directly and all the colour development for phosphorus in Systronix colorimeter at 630 nm.

Reference Sumner, J.B. 1944. A method for calorimetric determination of phosphorus. Science, 100: 413.

3.2. Phytin Phosphorus (Dickmen and Bray, 1940) Principle Acid hydrolysis of the sample release phytin P and flocculate the Ferrie phytate and then to Na phytate. Under acidic medium, the phosphomolybotic complex with stannous chloride and the red chromophore obtained was read colorimetrically. Hydrochloric Ammonium molyblate solution Reagents 15 g ammonium molybdate in 300 ml warm water. Filter, cool and add 350 ml of 10 N HCl. Cool and make up the volume to 1000 ml. Ferric chloride reagent 10 per cent with 1 ml of 7.5 N H 2SO4. Stannous chloride (0.012 per cent). Reagents HCl N/2, N/6, NaOH 40%, Phenolphthalein H2SO4, Perchloric acid 65% and Ammonium molybdate 6.6%. Procedure One gram of powdered grain material was taken to which 100 ml N/2 HCl was added. It was taken for 2 hours to extract phytic acid and filtered. The filtrate was neutralized, with sodium hydroxide using phenolphthalein as indicator and made upto 100 ml. From that 20 ml of aliquot was taken and 4 ml of ferric chloride reagent was added and boiled for 15 minutes to flocculate Ferric phytate. It was cooled, centrifuged and the supernatant liquid was discarded. The residue was washed with 5ml of N/6 HCl. To the ferric phytate precipitate, 2 ml of distilled water was added and stirred for 2 minutes. Then added 2 ml of 2 per cent sodium hydroxide and boiled for 15 minutes to bring ferric phytate 10 sodium phytate forms. It was filtered, collected in the Kjeldahl flask. The precipitate was washed in hot water twice. One ml of concentrated H 2SO4 and one ml of 65 per cent perchloric acid were added and slightly heated at first and then heated for one hour. About 20 ml of water was added and neutralized with 40 per cent sodium hydroxide, and the volume was made upto 100 ml. A quantity of 5ml of this aliquot was pipetted to which one ml of concentrated H2SO4 was added and made upto 10 ml. To this 10 ml of Hydrochloric Ammonium Molybdate reagent and 5 ml of stannous chloride solution were added and left for 20 minute for colour development. The colour was read at 660 mm. The value was referred to the standard curve prepared from different concentrations from potassium dihydrogen phosphate.

3.3. Estimation of phytic acid Phytic acid (inositol hexaphosphoric acid) is the stored form of phosphorus in seeds and also considered as an antinutritional factor. Phytin is the stored form of phorphorus in plant material. Principle The phytate is extracted with trichloroacetic acid and precipitated as ferric salt. The iron is determined colorimetrically and the phytate phosphorus content calculated from this value assuming a constant 4Fe:6P molecular ratio in the precipitate. Reagents 1. 3% Trichloroacetic acid (TCA) 2. 3% Sodium sulphate in 3% TCA 3. 1.5N NaOH 4. 2 N HNO3 5. FeCl3 solution : Dissolve 583mg FeCl3 in 100ml of 3% TCA 6. 1.5M Potassium thiocyanate (KCSN) : Dissolve 29.15g in 200ml water. 7. Standard Fe (NO3)3 solution Procedure 1. Extract sample in 50ml 3% TCA for 30min with mechanical shaking or with occasional swirling for 45min. 2. Centrifuge and transfer a 10ml aliquot of the supernatant to a 40ml conical centrifuge tube 3. Add 4ml of FeCl3 solution to the aliquot. 4. Heat the contents in a boiling water bath for 45min. 5. Centrifuge (10-15 min) and decant the clear supernatant. 6. Wash the precipitate twice with 20 to 25 ml 3% TCA, heat in boiling water for 5 to 10 min and centrifuge. 7. Repeat washing with water. 8. Disperse the precipitate in a few ml of water and add 3ml of 1.5N NaOH with mixing 9. Bring volume to approximately 30ml with water and heat in boiling water for 30 min. 10. Filter hot through a Whatman No.2 11. Wash the precipitate with 60-70ml hot water and discard the filtrate. 12. Dissolve the precipitate with 40ml hot 3.2N HNO3 into a 100ml volumetric flask. 13. Cool flask and contents to room temperature and dilute to known volume with water 14. Transfer a 5ml aliquot to a volumetric flask and dilute to approximately 70ml. 15. Add 20ml of 1.5M KSCN, dilute to volume, and read colour immediately (within 1min) at 480nm. 16. Run a reagent blank with each set of samples.

Standard Dissolve 433 mg Fe (NO3)3 in 100ml distilled water in a volumetric flask. Dilute 2.5ml of this stock standard and make up to 250ml. Pipette out 2.5, 5, 15 and 20ml of the working standard. Calculation Find out the µ g iron present in the test from the standard curve, and calculate the phytate P as per the equation. µ g Fe x 15 Phytate P (mg/100g sample) = Weight of sample (g) Reference Wheeler, E.L. and R.E. Ferrel. 1971. Cereal Chem., 48: 312.

4. FATS 4.1. Extraction of total lipids Principle The tissue is extracted in chloroform: methanol mixture (10:20, v/v) filtered. The chloroform is evaporated to dryness, weighed and the per cent total lipids are calculated. Reagents 1. Chloroform : methanol, (10:20, v/v) 2. Chloroform Procedure 1. Homogenize about 5 g material in a blendor for two minutes in a mixture of chloroform: methanol. 2. Add 10 ml of chloroform and homogenize for a minute. 3. Add 10ml water and homogenize further for a minute. 4. Filter and wash the precipitate with 10 ml of chloroform, refilter and transfer to a separating funnel. 5. Remove the upper methanol-water layer by aspiration and a small volume of the chloroform layer is also removed to ensure complete removal of the upper layer. 6. Record again the volume of chloroform layer ('y' ml), transfer quantitatively into a preweighed conical falsk ('a' g). 7. Evaporate in a 40-50 C water bath. 8. Cool and dry the residue over phosphoric anhydride in vacuum desiccators. 9. Weigh the flask second time ('b' g.) 10. Add t ml of chloroform 3 times to dissolve the lipids. Evaporate and dry the flask as in steps 9 and 10. 11. Weigh the flask a third time ('c‟ g). Weight of lipids (g): (b-a) - (c-1) =„d‟ g Total vol. of chloroform layer ('x' ml) Total lipids (g) = Weight of lipids (d)

X Vol. of chloroform layer evaporated ('y' ml) Total lipids (g)

% Total lipids =

X 100 Weight of sample (g)

'a' is the weight of empty flask. Reference Bligh, E.G. and W.J. Dyer. 1959. Can. J. Biochem. and Physiol., 37: 911.

4.2. Estimation of oil or crude fat Principle Ether is continuously volatilized, condensed and then allowed to pass through the sample to extract ether soluble materials. When the process is completed, the ether is distilled, collected in another container, remaining crude fat is dried, weighed and per cent oil is calculated. Reagent Petroleum ether or ethyl ether or hexane Procedure 1. Weigh 2-3 g of dried sample in a thimble and place it in the Soxhlet apparatus. 2. Add the required volume of solvent (petroleum ether, Boiling point of 40-60C or ethyl ether or hexane) and connect to condenser and extract for 16 hours. 3. Remove the thimble and evaporate the excess of ether from the solvent flask on a hot water bath and dry the flask at 105C for 30 min. 4. Cool the flask in desiccators and weigh ('b' g). (b - 1) x 100 Crude fat or oil content in sample (%) dry wt.basis = Weight of sample (g).

Reference Sadasivam, S. and A. Manickam. 1992. In: Biochemical Methods for Agricultural Sciences, Wiley Eastern Limited, New Delhi, pp. 26-27.

4.3. Isolation and estimation of free fatty acids Principle The sample is initially extracted in 4N HCl and followed by chloroform which is then separated and dried. Reagents 1. 4N HCl 2. Chloroform Procedure 1. Take 10mg of sample, add 1ml of 4N HCl in a small glass tube. 2. Heat for 5 hours in boiling water with intermittent shaking. 3. Extract the content three times with 1ml of chloroform. 4. Remove the bottom chloroform layer using a separating funnel. 5. Dry the flask in vacuum desiccators over P2O5 until a constant weight is obtained. Reference Keleti, G. and W.H. Lederer. 1974. In: Handbook of Micro methods for the Biological Sciences, Van Nostrand Reinhold Company, New York, pp.74.

4.4. Estimation of free fatty acids or acid value of oil The unpleasant effects of hydrolytic rancidity are noticeable in fats containing the volatile butyric and caproic acids. Hydrolytic rancidity is usually measured by the acid number. The acid value is the number of mg of KOH required to neutralize the free fatty acids present in 1g of fat. The amount of free fatty acids gives an indication of the age and quality of the fat. Principle The free fatty acid in oil is estimated by titrating it against KOH in the presence of phenolphthalein indicator. The free fatty acid content is expressed as oleic equivalents. Reagents 1. 1% Phenolphthalein in 95% ethanol 2. 0.1N Potassium hydroxide 3. Neutral solvent: Mix 25 ml ether, 25 ml 95% alcohol and 1ml of 1% phenolphthalein solution and neutralize with N/10 alkali. Procedure 1. Dissolve 1-10 g of oil or melted fat in 50 ml of the neutral solvent in a 250 ml conical flask. 2. Add a few drops of phenolphthalein. 3. Titrate the contents against 0.1N potassium hydroxide. 4. Shake constantly until a pink colour which persists for fifteen seconds. Titre value x Normality of KOH x 56.1 Acid value (mg KOH/g)

= Weight of the sample (g)

The free fatty acid is calculated as oleic acid using the equation: 1ml N/10 KOH = 0.028g oleic acid. Reference Cox, H.E. and D. Pearson. 1962. The Chemical Analysis of Foods Chemical Publishing Co, Inc., New York, p.420.

4.5. Estimation of free fatty acids by colorimetry Reagents 1. 1.0 N Triethanolamine 2. Copper reagent : Mix 18ml of 1N triethanolamine + 2ml of 1N acetic acid + 20 ml of 6.45% Cu (NO3)2.3H2O. 3. 0.1% Sodium diethyldithiocarbamate in distilled 2-butanol. 4. Standard palmitic acid: 4g / ml (linear upto 20g) 5. Chloroform Procedure 1. Take 1ml of sample in chloroform from 'Isolation of free fatty acids' add 500 l of copper reagent and shake for 2min. 2. Centrifuge at 2000 rpm for 10 min. at 4C. 3. To the lower chloroform layer, add 150 l of 0.1% sodium diethyldithiocarbamate solution and mix. 4. Read the absorbance at 440 nm. 5. Calculate the amount of free fatty acids in the sample using a standard curve prepared from palmitic acid. Reference Duncombe, W.G. 1963. Biochem. J., 88:7.

4.6. Estimation of peroxide value of an oil or fat When lipids contain unsaturated fatty acids, oxidation takes place at the unsaturated linkage. Oxidative rancidity of corn and cottonseed oils is believed to be due to the photochemical action of light upon a component of the oils giving rise to peroxides. Principle Peroxide value is a measure of the peroxides contained in the oil. The peroxides present are determined by titration against thiosulphate in the presence of Potassium Iodide. Starch is used as indicator. Reagents 1. Acetic acid - chloroform mixture: Mix glacial acetic acid: chloroform in the ratio 3:1, v/v. 2. Saturated Potassium Iodide solution: Dissolve excess of Potassium Iodide in freshly boiled water. Excess solid should remain. Store in the dark. 3. Standard 0.01N Sodium thiosulphate: Prepare appromixately 0.1N sodium thiosulphate solution by dissolving 13 g of Na2S2O3. 5H2O in 500 ml of water. Standardize Potassium dichromate solution and dilute to obtain 0.01 N solution. 4. 1% Starch indicator. Procedure 1. Take 5 g of oil into a 500 ml conical flask, add 30 ml acetic acid - chloroform mixture and dissolve the oil completely. 2. Add 0.5 ml of saturated Potassium Iodide solution, mix well and allow stand for one minute. 3. Add 430 ml of water, 3-4 drops of starch indicator and mix well. 4. Titrate against standard 0.01 N sodium thiosulphate with vigrorous shaking to liberate all iodine from chloroform layer until the blue colour just disappears. 5. Treat the blank similarly in the absence of oil. Reference Cox, H.E. and D. Pearson. 1962. The chemical Analysis of Foods, Chemical Publishing Co., New York, p. 421.

4.7. Estimation of Iodine value of oil The iodine value is a measure of the degree of unsaturation of an oil or fat. It is constant for particular oil for fat. It is a useful parameter in studying oxidative rancidity of oils. Principle Iodine monochlororide is allowed to react with the fat in the dark. Iodine gets incorporated into the fatty acid chain wherever the double bonds exist. The amount of iodine consumed is then determined by titrating the iodine released (after adding KI) with standard thiosulphate and comparing with blank in which that fat is omitted. Hence the measure of iodine absorbed by an oil or fat gives the degree of unsaturation. The iodine value or number is defined as the number of grams of iodine absorbed by 100 g of the oil/fat. Reagents 1. Hanus Iodine solution: dissolve 6.8g of iodine in 500 ml of glacial acetic acid and heat to dissolve. Cool and add 1.5 ml of bromine. 2. 15% Potassium iodide solution: Prepare in water. 3. Standard - 0.1 N sodium thiosulphate solution: Dissolve 6.2 g of Na2S2O3.5H2O in 250 ml of water. Standardise against standard Potassium dichromate solution and dilute to get exactly 0.1N Na2S2O3 solution. 4. 1% starch indicator. 5. Chloroform. Procedure 1. Take 0.2-0.3 g of oil or fat into 500 ml conical flask. 2. Add 20 ml of chloroform and dissolve the oil completely. 3. Add 25 ml of Hanus iodine solution, mix well, stopper the flask and keep in dark for 30 min. 4. Add 20 ml of KI solution and mix well. 5. Titrate against standard 0.1N Na2S2O3 solution using starch as in indicator. 6. Run blank similarly in the absence of oil. A x N x 0.1269 x 100 Iodine number

=

gI2/100 g oil or fat Weight of oil (g)

Where,

A N

= =

ml of Na2S2O3 (Blank - Test) Normality of Na2S2O3 Solution

Reference William Horowitz. 1975. Official Methods of Analysis of AOAC (Association of Official Analytical Chemists), Washington (12th edn.), p. 488.

4.8. Estimation of Saponification value / Saponification number of an oil or fat This value gives an indication of the nature of fatty acids in the fat since the longer the carbon chain the less acid is liberated per gram of fat hydrolysed. Thus, this value is useful for a comparative study of the fatty acid chain length in oils. Principle A known quantity of oil is refluxed with an excess amount of alcoholic Potassium hydroxide. After saponification, remaining Potassium hydroxide is estimated by titrating it against a standard acid. Reagents 1. 0.5 N Alcoholic Potassium hydroxide solution: hydroxide in one litre of 90% alcohol. 2. Standard 0.5 N HCl. 3. 1% Phenolphthalein solution in alcohol.

Dissolve 28 g of Potassium

Procedure 1. Accurately weigh 1 -2g of oil in to a 250 ml conical flask, add 25 ml of alcoholic Potassium hydroxide and dissolve the oil completely. 2. Connect air condensor to the flask and boil for about 30 min on a boiling water bath. 3. Cool to room temperature; add 2-3 drops of phenolphthalein indicator and mix. 4. Titrate against standard 0.5 N HCl until the pink colour disappears. 5. Treat the blank similarly without of oil. (Blank - Titre) x 28.06 Saponification value of an oil (mg KOH)

= Weight of oil (g)

1ml of 0.5 N HCl = 28.06 mg Potassium hydroxide Reference William Horowitz. 1975. Official methods of Analysis of AOAC (Association of Official Analytical Chemists), Washington, 12th edition, p.490.

4.9. Estimation of Oil by NMR Method It is rapid and nondestructive method of oil and fat detection. No sample preparation is required. Principle „H‟ content in the seed is measured. In an atomic nucleus, protons are charged particles and (+) are associated with magnetic field. Protons behave like small magnet and are moving (spinning) both clockwise and anti-clockwise. In a sample, there are „H‟ molecules. A sample containing „H‟ is kept in magnetic field. When put into strong electro-magnet (NMR), a strong electromagnetic field is created in poles (Fig.9.5). Sample is kept between the poles. The sample H is forced to move in the direction of magnetic force. Most of the nuclei follow the magnetic field, while few move in opposite directions (few in number) which are at high energy level. More portons are with low energy. The difference in an energy level is proportional to the number of protons. There are two methods of measuring proton number 1. Pulsed NMR 2. Continuous NMR. The oil is in liquid phase. The „H‟ is relatively mobile in liquid phase than a solid phase that reduces the contribution of solid „H‟ and water is avoided by taking further long distance signals. Decay for solid and water is quick as compared to the oil. The sample should be brought to 4% moisture, before taking readings on NMR.

5. Enzymes 5.1. Extraction of enzyme Different procedures are followed to extract enzyme from tissues: grinding in (a) cold distilled water, (b) chilled buffer and (c) chilled acetone. The crude enzyme extract prepared by these methods contains a mixture of several enzymes. Preparation of acetone powder from tissues offers many advantages. Acetone power can be stored in sealed containers at -20oC for a long time without the loss of enzymatic activity. Extraction of enzymes in water/buffer Method 1. Weigh a known amount of tissues and cut into 1-2cm pieces. 2. Grind the tissue thoroughly either in a pre-chilled pestle and mortar or using a blender with cold distilled water of suitable buffer Use 5ml for each g of tissue; if blender is used grind at low speed for 2-3 min; stir the contents and grind at high speed for 2-3 min). 3. Squeeze the extracts through 3 layers of cheese cloth to remove the pulp. 4. Centrifuge at a suitable speed and for optimal time (depends upon the nature and source of enzyme) at 4oC. 5. Use the clear extract as enzyme source for an assay by suitable method and determine the protein content of the extract to calculate the specific activity per mg of protein Preparation of acetone powder Method 1. Wash the residue again with diethyl ether 2. Dry the powder on the funnel with suction, spread the powder on What man No.1 filter paper and air dry for about I hour. 3. Store the powder in sealed containers at 0 – 4oC until use. 4. For extraction on enzyme from acetone powder, use a suitable buffer with an optimal pH

5.2. Amylases Starch is the principal storage polysaccharide in plant cells. It is made up of about 10-20% of amylase and about 80-90% if amylopectin. Amylose is a linear homopolymer of D-glucose units linked by α – 1, 4-glucosidic bonds without any branches. While amylopectin is a branched polymer of D-glucose units linked by both α -1,4 and at brancheing with α-1,6 –glycosidic bonds Principle The reducing sugars produced by the action of α-and / or β–amylase react with dinitrosalicyolic acid and reduce it to a brown colouring product nitroaminosalicylic acid. Reagents 1. 0.1M sodiuma acetate buffer, pH 4.7 2. 1 Starch solution; 1g starch in 100ml acetate buffer. Slightly warm. 3. Ditrosalicylic acid solution (potassium sodium tartrate) 4. Maltose solution: Dissolve 50mg maltose in 50ml distilled water. Method Extraction of amylases: Extract 1g of sample material with 5-10 volumes of ice-cold 10mM calcium chrode solution overnight at 4oC or for 3h at room temperature. Centrifuge the extract at 54000g at 4oC for 20min. the supernatant is used as enzyme source Extraction of β-amylases (free and bound): The free β-amylase is extracted form acetone defatted sample material in 66mM phosphate buffer (pH 7.0) containing 0.5M NaCl. The extract is centrifuged at 20000rpm for 15min. the supernatant is used as a source of free β-amylase. The pellet is then extracted with phosphate buffer containing 0.5% 2mercaptoethanol. The clear extract is used as source of bound β-amylase. All operations are carried out at 4oC. Enzyme Assay 1. Pipette out 1ml of starch solution and 1ml of properly diluted enzyme in a test tube. Incubate it at 27oC for 15 min 2. Stop the reaction by the addition of 2ml of dinitrosalicylic acid reagent 3. Heat the solution in a boiling water bath for 5min 4. Add 1ml potassium sodium tartrate solution 5. Then cool it in running tap water 6. Make up the volume to 10ml by addition of 6ml water 7. Read the absorbance at 560nm 8. Terminate the reaction at zero time in the control tubes 9. Prepare a standard graph with 0-100µ g maltose. Reference

Hawk, P.B., B.L. Oser and W.H. Summerson. 1954. Practical Physical Chemistry. Mc.Graw Hall Book Company, Inc. New York. Pp.1439.

5.3. Invertase (-fructofuranosidase) The enzyme, invertase cleaves, sucrose to glucose and fructose. Principle Invertase catalyzes the hydrolysis of cane sugar (sucrose): Sucrose + H2O fructose + Glucose The amount of reducing sugar is measured colorimetrically either by the Nelson's or dinitrosalicyclic acid method. Reagents 1. 2.5% sucrose solution 2. 1M sodium acetate buffer, pH 5.0 3. 20% Glycerol 4. Toluene 5. Other reagents for estimation of reducing sugars Procedure Enzyme extraction 1. Homogenize 5.0 g of plant tissue in a chilled mortor placed in an ice bath with precooled 20% glycerol. 2. Filter through several layers of cheese cloth and make up the volume with 20% glycerol to 100 ml. 3. Add about 1-2ml of toluene above the mark to preserve the extract. 4. Store at 0-4C. Enzyme assay 1. Pipette out 5 ml of the enzyme solution into 100 ml conical flask. Add 10ml of buffer and 5ml of sucrose solution. 2. Incubate at 37C for 24h 3. Pipette out aliquots of 1ml of the reaction mixture and stop the reaction by adding 1ml of either Nelson's or dinitrosalicyclic acid (DNS) reagent.] 4. Estimate the reducing sugars present in the reaction mixture either by Nelson's or DNS method. 5. Estimate the protein content in the enzyme extract by Lowry's method. 6. Express the enzyme activity as mg glucose released / h/mg protein. Reference Sridhar, R. and S.H. Ou. (1972). Phillippine Phytopath., 8, 52-56.

5.4. Deoxyribonuclease (DNase) Principle Deoxyribonuclease (DNase) acts upon DNA and deoxyribonuclotides and releases deoxyribose. The increase in acid-soluble nucleotides can be measured spectrophotometrically. Reagents 1. 0.1M Phosphate buffer, pH 6.5 2. 0.05% Calf thymus DNA 0.05M phosphate buffer, pH 6.5 3. 0.2% mg Cl2 4. 25% HCIO4 Method Enzyme extraction Follow the steps from 1-3 as described under method for ribonuclease extraction Enzyme assay 1. Pipette aliquots of 2 ml of the enzyme extract into test tubes 2. Add 0.5ml of 0.05% calf thymus DNA solution, 0.5ml of 0.25 MgCl2 and mix. 3. Incubate at 37oC for 4 hour in a water bath 4. Add 0.5ml of chilled 25% HCIO4 to terminate the reaction and precipitate the unhydrolysed nucleic acid 5. Maintain a zero time blank through the above steps from 2 to 5 6. Cool under running tap water and centrifuge at 2000g for 15min 7. Measure the absorbance of the supernatant (dilute, if necessary) at 260nm in a spectrophotometer 8. Estimate the protein content of the enzyme extract by Lowry‟s method 9. Consider an increase of 1.0 optical density at A260/h as one unit of DNase and express the results as DNase units/h/mg protein. Reference Naito, K.A., L.H. Suzuki and H.T. Suji (1979). Physiol. Plant, 46, 50-53.

5.5. Urease Urease is present in large amounts in biologically active soil, since many microorganisms hydrolyze urea enzymatically. Principle Urease catalyzes the reaction: H2N-CO-NH2 +H2O

CO2 + 2NH3

The enzyme activity is easily determined by measuring the amount of NH 3 formed and estimated colorimetrically at 630nm or 580nm. Reagents 1. 10% Urea. 2. Citrate buffer (pH 6.7) : Dissolve 368g of citric acid in 800 ml of water Dissolve 295 g of KOH in 300 ml of water. Combine the two solutions, cool, adjust to pH 6.7 with 1N KOH and dilute to 2000ml with water. 3. 12.5% Sodium phenate: a) Dissolve 62.5g of phenol in the smallest volume of ethanol, add 2 ml of methanol and 18.5 ml of acetone. Dilute to 100 ml with ethanol and store refrigerated. b) Dissolve 27 g of NaOH in water and make up to 100ml. Just before use mix 20 ml of solutions a) and b) and make up to 100 ml with water. 4. Sodium hypochlorite (NaOCI) : Dilute the commercial solution with water so that it contains 0.9% active chlorine. 5. Standard ammonium sulphate solution (1.21 mg NH3/ml) : Dissolve 4.717 g of (NH4)2SO4 in water and make up to 1000ml. Dilute 10 ml of this solution to 1000 ml with water. 1ml of this solution contains 10 µ g N. Method Enzyme extraction 1. Finely grind 5 g of plant material with 2 ml of toluene, 10 g of sand and a little 20% glycerol in a mortar. 2. Transfer to a 100ml volumetric flask after washing with glycerol solution. 3. After the addition of 1 ml of toluene, shake for 1h. dilute to the mark with 20 % glycerol solution mix thoroughly and filter through a fluted filter paper. Store the filtrate as enzyme source. Enzyme assay 1. Pipette out 1 ml of filtrate, 9ml of water, 4ml phenate solution and 3 ml of sodium hypochlorite solution into 50 ml volumetric flask. 2. Mix well and allow to stand for 20 min until the maximum color is obtained. 3. Dilute to 50 ml with water and mix well.

4. Read the optical density at 630 or 580nm within 60 min against the reagent blank prepared in the same manner. 5. Construct the standard curve by pipetting 0, 1, 2, 4, 6, 8 and 10 ml of diluted standard ammonium sulphate solution (10µ gN/ml) into a series of 50 ml volumetric flasks, make up the volume in each flask to 10 ml with water, add 4 ml of phenate solution and 3 ml of NaOCL solution and proceed through steps 2 to 4. Measure the absorbance against the blank i.e., zero flask and plot optical density vs concentration. 6. Measure the protein content of the enzyme source by Lowry‟s method. From the standard graph calculate the enzyme activity in terms of µg ammonia N (as NH3) liberated and express the specific activity as µ g N liberated /min/mg protein. Reading Hofman, E. (1965). In: Methods of Enzymatic Analysis (ed. Bergmeyer, H.), Academic Press, New York, pp. 915-916.

5.6. Phosphoenolpyruvate carboxylase (PEP carboxylase) Phoshoenolpyruvate carboxylase (PEP carboxylase) (EC 4.1.1.31), the enzyme fixes CO2 into oxaloacetate. The enzyme also provides oxalo acetate required in the pathway of ammonia assimilation. Principle The enzyme is determined in the presence of NADH and malate dchydrogenase by following either the rate of incorporation of [ 14C] HCO3- into malate (acid-stable radioactivity) or the rate of NADH oxidation spectrophotometrically at 340nm (christeller et al., 1977; Lane et al., 1969). Reagents 1. Extraction medium : 100mM Tris-HCl, pH 7.2, 1mM EDTA, 10mM MgCl2, 3% (w/v) PVP and 25% (v/v) glycerol, pH 7.2 2. 100mM Tris-HCI in 25% (v/v) glycerol, pH 7.2 3. [14C]NaHCO3 (55mCimmol-1) 4. 20mM Phosphoenolpyrvuate 5. Reaction mixture : Containing 100mM Tris-HCI (pH 8.0), 20mM MgCl2, 10mM dithiothiothreitol, 2mM NADH, 20mM [14C] NaHCO 3 (4µ Ci/ml) and malate dehydrogenase (40 units/ml) (Note: for malatc dehydorgenase preparation accordingly see under aspartate aminotransferase) 6. Scintillation fluid : Containing 30g p-terphenyl, 6 litres of xylene and 3 litres of Trition X-100 7. 2M HCI. Method Enzyme extraction 1. Grind 0.5-0.1g of leaf tissue in a prechilled mortar with sand, a small amount of (0.1g) of insoluble PVP and 5-7.5ml of extraction medium. 2. Centrifuge at 26200g for 10min and desalt the supernatant through a Sephadex G25-300 column (1x 20cm) equilibrated with 100mM Tris-HCI in 25% glycerol (v/v), pH 7.2 Use the eluate as source of enzyme. Enzyme assay [14C] HCO3- fixation assay 1. In scintillation wial incubate 0.25ml of the reaction mixture, 50µ l of phosphonenopyruvate and water to a final volume of 0.5ml at 25ºC. 2. Start the reaction by adding 10-100µ l of enzyme and incubate for 5min. 3. Stop the reaction by adding 100µ l of 2M HCI. 4. Prepare the blank (background vial) similarly byt without phosphoenolpyruvate in place of which add 50µ l of water. 5. Dry the vials at 95ºC, cool and dissolve the residue in 1ml of water. 6. Add 10ml of scintillation fluid, cap the vials and shake vigorously to suspend the aqueous phase.

7. Determine theacid-stable 14C activity (as [4C] malatc) with a lliquid cintillation counter. 8. Determine the total counts per minute per ml of the reaction mixture by adding 5µ l aliquots of the reaction mixture to 1ml of 10% ethanolamine in scintillation vials. Add 10ml scintillation fluid and count. 9. Convert counts per minute into disintegrations per minute before calculation the results. Total moles of HCO3/ml of reaction mixture Conversion factor (CF) = dpm /ml of reaction mixture HCO3- fixed (mole/min) = [ (dpm of sample ) – (dpm of background) ] X

CF

= „x‟ mole / min

5min 1000 Reaction rate

=

1 X

x

mg protein in the enzyme assay sample

(µ mole / min /mg protein)

1. [14C] is incorporated into oxaloacetate by PEP carboxylase. Oxaloacetate is unstable and highly inhibitor to PEP carboxylase activity. Both these problems can be overcome by adding excess of malate dehydrogenase and NADH to convert rapidly all of the oxaloacetate formed into malate. 2. Carry out a range of assay times and enzyme concentrations to check that a linear response is always obtained. 3. Mix the sample well at the end of the assay to ensure that all of the reaction mixture in the vial has been acidified. Spectrophotmetric assay The reaction mixture and conditions are modified from those described for the [14C] HCO3- fixation assay in that unlabelled NaHCO3 and less NADH (0.15µ mole/ml of assay solution ) are used. Procedure 1. In a test tube incubate 0.25ml of the reaction mixture (prepare as per above conditions), 10-100µ l of the enzyme and appropriate volume of water to a final volume of 0.45ml at at 25ºC. 2. Initiate the reaction by adding 50µ l of phosphoenolpyruvate at different time intervals 1,2,3,4 and 5min.

3. Terminate the reaction by adding 100µ l of 2M HCI 4. Carry out the blank similarly without phosphoenolpyruvate in place of which add 50µ l of water. 5. Measure the rate of NADH oxidation at 340nm (1cm light path, 25ºC ) against the blank. 6. Plot the graph (OD vs time ) and calculate the decrease in absorbance Reference Stamatakis, K., N.A. Gavalas and Y. Manetas (1988). Aust. J. Plant Physiol., 15,621631.

5.7. Glutamine Synthetase (GS) Ammonia produced during N2- fixation in bacteroids is assimilated in the cytoplasm of host cells and then transported either as ureides or amides to various plant parts. It is now well established that most of the tropical legumes e.g., cowpea, pea and soybean are ureide producing legumes while lupin, cluster bean and alfalfa translocate their fixed N predominantly as amides. In nodules, ammonia produced during N2fixation is primarily assimilated via glutamine synthase (GS) and glutamate synthase (GOGAT).). Glutamine synthetase (EC, 6.3.1.2.) has higher affinity for ammonia and it catalyzes the following reaction. L – Glutamate + NH3 + ATP

Mn2+ L - glutamine + ADP + Pi

Principle The enzyme is assayed by measuring the formation of y-glutamyl hydroxamate which reacts with ferric chloride and the brown colour in the acid medium measured at 540nm. Reagents 1. Grinding medium: 0.1M Potassium phosphate buffer, pH 7.8 containing 0.4M sucrose, 10mM DTT, KCI, 1mM Mgcl2 and 10mM EDTA. 2. 1M Tris-maleate buffer, pH 7.5 3. 100 mM hydroxylamine (556mg/10ml) 4. 0.1M L-Glutamine (360mg/12ml) 5. 10mM ATP 6. 20mM EDTA 7. 50mM MgCl2 8. FeCl3 mixture: (10g tricuhloroacetic acid, and 8g ferric chloride in 250ml of 0.5N HCI). 9. Standard Y-glutamylhydroxamate : Prepare in 50mM Tris-maleae buffer (500µ g/ml) Method Enzyme extraction (at 4ºC, Sawhney et at., 1985) 1. Macerate 500 mg of the nodules or 200mg of leaf in chilled pestle and mortar in 5.0ml of grinding medium. 2. Squeeze through 4 layers of cheesecloth and centrifuge at 10000g for 20min. 3. Desalt the supernatant by passing through a column of Shepherded G-25 (12 x 2.5cm) and use for determining the activities of glutamine synthetase, aspartate aminotransferase,and glutamate dehydrogenase

Enzyme assay (Boland et al., 1978) 1. Reaction mixture : 0.75ml containing 50mM Tris-maleate buffer, pH7.5, 67mM hydroxylamine, 80mM l-glutamine, 8mM ATP, 4mM EDTA, 50µl of crude enzyme and 33mM Mg2+ as MgCI2. 2. Prepare the blank without glutamine using buffer. 3. Incubate for 10min at 25˚C. 4. Stop the reaction by adding 0.2ml of a FeCl3 mixture. 5. Centrifuge and measure the absorbance of brown color at 540nm. 6. Prepare the standard curve with commercial y-glutamylhydroxmate (100-500μg range) and develop the color similarly with ferric chloride reagent. Calculate the enzyme activity as nmole y-glutamylhydroxmate formed/min and specific activity as n mole/min/mg protein. Reference 1. Sawhney, V., Amarjit and R. Singh (1985). Plant soil. 86, 241-248.

5.8. Glutamate Dehydrogenase (GDH) Nitrogen fixation in root nodules is to a large extent controlled by ammonia assimilation. The enzymes glutamate dehydrogenase (GDH) is implicated in efficacy of NH3 assimilation. GDH (EC. 1.4.1.3.) which catalyzes the reductive amination of α-KG and considered to be the most important enzyme in NH3 assimilation in root nodules. The reaction catalyzed by the enzyme is as follows: L – Glutamate + H2O + NAD+ (P) ------- 2 – oxoglutarate + NH4+ + NAD (P) H + H+ Principle GDH activity is measured by following the oxidation of the reduced coenzyme, NADH at 340nm. Reagents 1. 2. 3. 4. 5.

Extraction medium: 0.05M Tris-HCL (pH7.5) containing 0.4M sucrose and 0.01M β-mercaptoethanol 0.1M Tris-HCI (pH 7.5) 0.33M 2-Oxoglutarate (pH 6.0) in water 3M NH4CI: Dissolve 160.5g in water and make up to one litre 10-3M NADH in water.

Method Enzyme extraction 1. Grind a known weight (0.5g) of tissue with 3ml of extraction medium in the presence of polyclar AT (0.5g/fresh weight) 2. Squeeze through 4 layers of cheese cloth and centrifuge at 100 x g for 2min. 3. Recentrifuge at 20,000 x g for 30min to produce a pellet containing mitochondria (in roots and bacteroids in case of nodules). Separation of nodules my be accomplished by a second centrifugation of 3000 x g for 10min to produce a bacteroid rich fraction and a third centrifugation of 20,000 x g for 30min to produce a mitochondria rich fraction. 4. Suspend each pellet in 2-5ml extraction medium with a glass tissue grinder and then freeze-thaw to solubilize enzymes. 5. Use both the pellet and supernatant fraction for assay of GDH. Enzyme assay 1. Prepare the reaction mixture (3ml) consisting of 1.6ml of 0.1M Tris-HCI buffer (p 7.5), 0.1ml of 0.33M 2-oxoglutarate (pH 6.0), 0.1ml of 3M NH4CI, 0.2ml of 103M NADH and 1.0ml of enzyme extract.

2. Add 0.1ml of water in the blank instead of 2-ozoglutarate and incubate at37˚C for 15-30 min. 3. Measure the change in absorbance at 340nm. Calculate the amount of NADH oxidized from the molar extinction coefficient which is 6.22 x 103 for NADH at 340nm (indicates that 1μmole of NADH/ml will have an absorbance of 6.22) and express the specific activity as n mole NADH oxidized /min /mg protein using the formula: A340 x Vol. Of assay solution x 1000 6.22 x Incubation from (min) time x mg protein in the enzyme extract used The enzyme specific activity may be also expressed as n moles NADH oxidized/min/g fr. Wt. of sample. Reference Duke. S.H. and G.E. Ham (1976). Plant and Cell Physiol. 17,1037-1044.

5.9. Glutamate Synthase (GOGAT) Glutamate synthase (GOGAT) (EC.2.6.1.53) is another important NH 3 assimilating system. GOGAT, which transfers an amino group from glutamine to αketoglutarate converting both to glutamate requires glutamine synthetase (GS) for substrate production. . The enzyme catalyzes the following reaction: GS Glutamic acid + NH3 ATP

glutamine + ADP + Pi GOGAT

Glutamine + 2-oxoglutarate + NADH

2 glutamic acid + NAD +

Principle The GOGAT activity is measured following the oxidation of NADH at 340nm. Reagents 1. 2.

0.3M L-Glutamine (pH 7.0) Other reagents as for „Glutamate dehydrogenase‟.

Method Enzyme extraction See under „Glutamate dehyrogenase‟. Enzyme assay 1. The reaction mixture (3ml) : 0.7ml of 0.1M Tris-HCI buffer (pH 7.5), 1ml of glutamine (pH 7.0), 0.1ml of 0.33M 2-oxoglutarte, 0.2ml of 10-3M NADH and 1ml of enzyme. 2. Add 0.1ml of water in the blank instead of 2-oxoglutarate. 3. Incubate at 37˚C for 15-30min. 4. Record the change in absorbance at 340nm. For calculation see under „Glutamate dehydrogenase‟. Reference Duke, S.H. and G.E. Ham (976). Plant and Cell Physiol., 17, 1037-1044.

5.10. Nitrate Reductase (NR) The enzyme nitrate reductase (NR) occupies a control points in the path way of nitrate assimilation. In plants, the nitrate reducing system consists of nitrate reductase and nitrite reductase catalyzes stepwise reduction of nitrate to nitrite and then to NH3.

NO3- + AH2

NO2- + A + H2O

The NADH-dependent NR (EC.1.6.6.1) is most prevalent in plants. In vivo assay method Recent studies indication that nitrate reductase could be measured in green leaf disks vacuum infiltrated with a nitrate containing phosphate buffer. Leaf disks incubated in the dark rapidly accumulate nitrite. Nitrate is reduced to nitrite in the dark using endogenous NADH as the reductant. The reaction is carried out in the dark to prevent the further reduction of nitrite to NH3. The nitrite is estimated spectrophotometrically at 540nm, Surfactant such as propanol is used to increase the permeadility of be tissue to nitrate and nitrite. Reagents 1. 2. 3. 4. 5. 6. 7.

0.1M Potassium phosphate buffer, pH 7.5 0.02M KNO3 in water 5% Prophanol Chloramphenicol 1% Sulfanilamide in 3M HCI (w/v) 0.02% N-1-naphthyl-ethylenediamine HCI: Dissolve 20mg in 100ml water Standard KNO2 solution.

Enzyme assay 1. Prepare the leaf punches (1cm diameter) or leaf slices or use the whole leaves if desired as long as the tissue is completely submerged in incubation medium. 2. Suspend approximately 200mg of leaf punches in 5ml of a medium consisting of 0.1M phosphate buffer, 0.02M KNO3, 5% propanol and two drops of chloramphenicol (0.5mg/ml). 3. Keep in the dark at 25°C for desired incubation periods. 4. Determine the nitrite released into the medium at zero time and at various time intervals thereafter by treating 0.4ml aliquots with 0.3ml each of 1% sulfanilamide and 0.02% N-1-naphthyl-ethylenediamine HCI. 5. After 20min, dilute the solutions with 4ml of water and measure the absorbance at 540nm. 6. Draw the standard curve with standard KNO2 solution in a series of test tubes making up the volume in each to 2ml with water and proceed from steps 5 to 6. Express the enzyme activity as μmoles of nitrite formed/g fr. Wt. of sample/h. Reference Jaworski, E.G. (1971) Biochim, Biophys. Res. Commun., 43, 1274-1279.

5.11. Catalase Catalase (EC.1.11.1.6.) is an enzyme present in nearly all animal and plant cells, which catalzes the breakdown of H2O2 to water and molecular oxygen (catalytic) or peroxidatively oxidation of H donors (methanol, formic acid, phenol, etc.,) in the presence of H2O2. H2O2 2H2O + O2 (catalytic) ROOH + AH2 H2O + ROH + A (peroxidative) It is not clear whether the role of catalase in the organism as to decompose H 2O2 or to catalyze a peroxidation reaction. Increases in catalase activity have been reported in a few host-parasite interactions. Principle The enzyme activity is assayed by estimating the residual H 2O2 in the reaction mixture, which is then determined by oxidation with KMnO4 titrimetrically. Reagents 1. 0.1M Potassium phosphate buffer (pH .0) 2. 0.005M H2O2: Prepare fresh 3. 0.7N H2SO4 4. 0.01N KMnO4 Enzyme extraction 1. Grind the sample (0.1g) with 0.1M phosphate buffer, pH 7.0 in a prechilled mortar and pestle. 2. Centrifuge at 15,000g for 30min at 4˚C 3. Use the supernatant as enzyme source. Enzyme assay 1. Pipette out 3ml of phosphate buffer, 2ml of H2O2 and 1ml of enzyme extract into at 20˚C for 1min 2. Incubate at 20˚C for 1min 3. After 1min stop the reaction by adding 10ml of 0.7N H2SO4. 4. Titrate the reaction mixture against 0.01N KMNO4 to find out the residual H2O2 until a faint purple color persists for at least 15sec. 5. Prepare the blank by adding the enzyme extract to an acidified solution of reaction mixture at zero time Express the enzyme activity as units/min and specific activity as units/min/mg protein or per g weight of sample. One unit of catalase of defined as that amount of enzyme, which breaks down 1μmole of H2O2 under the assay conditions. Calculate the concentration of H2O2 using the extinction coefficient 0.036/μmole/ml. The activity of the enzyme may be also expressed as nmoles of H2O2 used/min/g weight of sample. Reference Barber, J.M. (1980). Z.Pflazen., 97.135.

5.12. Polygalactouronase (PG) The pectic substances of fruit tissues change during the ripening period. The increased polygalacturonates (PG) activity during ripening of a number of fruits suggests that suggests that depolymerization commonly contributes to increased solubility. The enzyme (EC 3.2.1.15.) catalyzes the hydrolytic cleavage of α-1,4 galacturonan linkage in polygalacturoni acid polysaccharide of pectin. Principle The enzyme is assayed as the amount of reducing sugars formed using polygalacturonic acid as the substrate. Reagents 1. 0.017M Tris-HCI containing 5mM 2-mercaptoethanol, pH 10 2. Extraction buffer: 1.7M NaCI, 50ml sodium citrate and 15mM EDTA, pH 5.5 3. 2M NH4CI 4. 1% Polygalacturonic acid, pH 4.2 5. 5% TCA 6. Standard α-D-galacturonic or D-glucose 7. Reagents for estimation of reducing sugars by DNS or Somogyi‟s method – see under „Carbohydrates‟ Method Enzyme extraction (0-4˚C) 1. Homogenize 10g of material in 13ml of Tris-HCI buffer. 2. Centrifuge at 15000g for 1.5min 3. Terminate the reaction by adding 0.3ml of 5% TCA. 4. Centrifuge at 2000g for 30min and collect the supernatant. 5. Estimate the reducing sugars formed by the DNS or Somogyi‟s (1952), method as described under „Carbohydrates. 6. Draw the standard curve using α-D-galacturonate or E-glucose as a standard. Express the enzyme activity as moles of reducing sugars formed or katals and specific as moles/sec/mg protein. Reference Zainon, M.L and C.J. Brady (1982) Aust J.Plant Physiol., 9, 155-169.

5.13. Pectin Methyl Esterase (PME) Pectin methyl esterase (PME) (EC 3.1.1.11) catalyzes the demethoxylation of carboxyl groups from the galacturonosyl residues of pectin. Pectin degradation plays an important role in plant disease, fruit ripening, nutrition and food product stability. Pectin

COOCH3 + H2O

pectin – COO- + H+ + CH3OH

Principle The enzyme activity is determined at 25˚C in a continuous spectrophotometer assay at pH 7.5according to Hagerman and Austin (1986). Reagents 1. 0.5% (w/v) Citrus pectin: Prepare in water by heating the mixture to 40˚C while continuous stirring. Store at 4˚C, it can be used for 1 month. 2. 0.01% (w/v) Bromothymol blue: Prepare in 0.003M potassium phosphate buffer, pH 7.5 3. 0.003M Potassium phosphate buffer, pH 7.5 4. 8.8% (w/v) HCI 5. 0.02N NaOH Enzyme extraction 1. Homogenize 4 to 5g in 15ml of cold (4˚C) 8.8% NaCI using pestle and mortar. 2. Centrifuge at 200g for 10min 3. Collect the supernatant and adjust to pH 7.5 with NaOH and use for assay. Enzyme assay 1. Mix 2ml pectin with 0.15ml of bromothymol blue and 0.83ml of water an incubate at 25˚C with a circulating water blank. 2. Determine the initial absorbance at 620nm (A620) against water blank. 3. Start the reaction by adding 100μl of enzyme solution and measure the rate of decrease at 620nm at 20,40,60, and 80 sec intervals. 4. Calculate the activity of enzyme from the linear part of the curve by subtracting the initial absorbance value obtained at step 2, Express the enzyme activity as A620/min and specific activity A620/min/mg protein. Reference Hagerman, A.E. and P.J. Austin (1986). Agric. Food Chem., 34(3) 440-444.

5.14. L-Phenylalanine Ammonia Lyase (PAL) Phenylalanine ammonia lyase (PAL) (EC 4.3.1.5) catalyzes deamination of phenylalanine to trans-cinnamic acid. In disease resistance mechanism, the enzyme plays an important role in the conversion of phenylalanine to coumaric acids. Principle The enzyme may be assayed by measuring the appearance of trans-cinnamic acid from phenylalanine. Reagents 1. 2. 3. 4. 5. 6. 7.

0.2M Sodium borate buffer, pH 8.7 0.01M L-Phenylalanine (pH*.7) : Prepare in sodium borate buffer 0.05M Tris-HCI buffer, pH 8.8 IN HCI Peroxide-free ether 0.05N NaOH : Prepare fresh 1N NaOH Standard cinnamic acid : Prepare in borate buffer

Method 1. Grind 3.0g of material in 2.6ml of sodium borate buffer containing 2mercaptoethanol (0.8ml/liter). 2. Filter through cheesecloth and add acetic acid (1M) to bring the filtrate to pH 5.5. 3. Add protamine sulfate solution (0.002g + 0.008ml of and centrifuge at 7000g for 10min. Use the supernatant for assay. Enzyme assay 1. Incubate 1ml of 0.05M Tris –HCI buffer, pH 8.8, 0.5ml of 0.01M Lphenylalanine and 0.4ml of water at 30˚C for 5min. 2. Initiate the reaction by adding 0.1ml of enzyme and incubate for 60min at 30˚C. 3. Run a blank without phenylalanine. 4. Stop the reaction by adding 0.5ml of 1N HCL 5. Extract the mixture twice with 3.5ml of ether. 6. Remove the ether phase, pool and dry under a stream of air. 7. Dissolve the residue in 3ml of 0.05N NaOH. 8. Read the absorbance at 268nm. 9. Draw the standard curve using cinnamic acid similarly. Express the specific activity of the enzyme as μ moles of cinnamic acid produced/min/mg protein. Reference Subba Rao, P.V. and G.H.N. Tower (1970). In ; Methods Enzymol., (eds. S.P. Colowick and N.O. Kaplan), Vol. XVIIA p. 581.

5.15. Polyphenol Oxidase (PPO) or DOPA oxidase Polyphenol oxidase (PPO) (EC 1.14.18.1) is one of the enzymes involved in the oxidation of phenolic compounds to brown pigments. In plants the enzyme is involved in resistance to infection, in synthesis of plant constituents and as an oxygen scavenger in photosynthetic tissue. The enzyme is also known as phenol oxidase, tyrosinase, DOPA oxidase. Catechol oxidase, e.t.c., The enzyme catalyzes the oxidation of monophenols and o-diphenols. Principle The enzyme activity is measured as rate of increase in absorbance colorimetrically at 410nm with the oxidation of catechol as the substrate. Reagents 1. 2. 3.

Chilled acetone 0.2M Potassium phosphate buffer, pH 6.8 0.05M Catechol : Prepare in 0.2M phosphate buffer, pH 6.8

Method Enzyme extraction 1. Prepare acetone powder from the plant material as described earlier. 2. Suspend 5g of acetone powder prepared from plant tissue in 200ml of 0.2M potassium phosphate buffer, pH 6.8 and stir for 30min at 2˚C 3. Centrifuge at 11000g for 20min at 2˚C and dialyze the supernatant against 0.2M phosphate buffer for 2 days with 2 changes of buffer. Use the dialyzate for assay. Enzyme assay 1. Incubate 1ml of 0.05M catechol and 4.5ml of 0.2M phosphate bufferm pH 6.8 at 30˚C. 2. Initiate the reaction by adding varying amounts of enzyme extract in a final volume of 5ml 3. Measure the rate of increase in absorbance at 410nm against the blank (prepared in the absence of enzyme ) at ever 30sec up to 3min. 4. Plat the changes in absorbance between 30 to 180sec of incubation and calculate the enzyme activity from linear part of the curve. Express the enzymatic activity as units/min at 410nm considering one enzyme unit as the change in absorbance of 0.001/min and the specific activity as units/min/mg protein. Reference Augustin, M.A. H.M. Ghazil and H. Hashim (1985) J.Agric. Food Chem., 36, 12591265.

5.16. Indoleacetic Acid Oxidase (IAA oxidase) The accumulation or destruction of auxin is frequently associated with IAA oxidase, an enzyme which destroys the auxin. Inhibition of IAA oxidase leads to the accmulation of IAA and vice versa. AA oxidases belong to a class of enzymes known as peroxidases which catalyse the oxidation of a wide variety of substrate including IAA in vivo. Principle The in vivo enzyme activity is determined by estimating the amount of residual indoleacetic acid present in the reaction mixture at 540nm. Reagents 1. 2. 3. 4.

10-5M 2, 4-Dichlorophenol Standard indole acetic acid Stock citric acid-phosphate buffer (pH 5.6) Salkowski reagent : Mix 50ml of 35% perchloric acid and 1ml of 0.5M FeCI3 reagent.

In vivo assay 1. Take fifty 5mm sections of the material (root or leaf or cotyledon) and quickly place in incubation flasks containing 10ml of 0.05mM 2, 4-dicholrophenol, 40μg.ml IAA and 150mM citric acid-phosphate buffer (pH5.6). 2. Place the flasks in a water bath shaker (30˚C_+ 1 at 130rpm ) for 1h 3. Remove the flasks from water bath and estimate the residual IAA in the aliquots of solution in the flasks. 4. Place aliquots of solution in test tubes with Salkowski reagent and allow to develop for 1h.and measure the absorbance at 540nm. 5. Draw the standard curve similarly using IAA from step 4 onwards and calculate the residual IAA. 6. Remove the leaf discs from the flasks, dry in an oven at 81ºC_+ 1 and weigh to the nearest 0.1mg. Express the enzyme activity as μg IAA destroyed/h/mg dry ewight sample. Reference Bohansack, C.W. and L.S. Albert (1977). Plant Physiol., 59, 1047-1050.

5.17. Ascorbic Acid Oxidase Ascorbic acid oxidase (EC. 1.10.3.3.) is a copper-containing enzyme which has been extracted from several plant tissue. The enzyme catayzes the direct oxidation of ascorbic acid by molecular oxygen. Principle The enzymic activity is assayed by measuring the decrease in absorbance at 265nm spectrophotometically due to the disappearance of ascorbic acid. Reagents 1. Grinding medium: 0.15M Macllvaine buffer, pH 5.0 (0.2 M sodium hydrogen phosphate –0.1M citric acid) containing 10 -3 M cysteine and BSA (1mg/ml) 2. Dilute the homogenate appropriately with the grinding medium and centrifuge at 17000 g for 20min at 0˚C. Use the supernatant for assay. Method Enzyme extraction 1. Incubate an appropriate volume of enzyme extract with grinding medium in a total volume of 2.5 ml at 25˚ 2. Start the reaction by adding 50μl of 10mM ascorbic acid substrate. 3. Measure the decrease in absorbance at 265nm at different time intervals over one minute. 4. Plot a graph (OD vs time in sec) and calculate the decrease in OD from initial part of the curve. The enzyme activity can be calculated from the molar extinction coefficient of ascorbic acid, which is 13.79 x 103 at 265nm (indicates the 1μ mole of ascorbic acid/ml will have an absorbance of 13.79) and express the specific activity as μ mole ascorbic acid disappeared/min/mg protein using the formula: A265 x Vol. of assay solution x 1000 13.79 x Incubation time (min) x mg protein in the enzyme extract used The enzyme may also be assayed by determining the residual ascorbic acid left behind in the reaction mixture Reference Drumm, H., K. Bruning and H. Mohr (1972). Planta ., 106, 259-267.

5.18. Pyruvate decarboxylase Principle It is assayed by a method in which the decarboxylase is coupled with alcohol dehydrogenase in the presence of thiamine pyrophosphate (TPP) and NADH. The rate of disappearance of NADH is followed spectrophotometrically at 340 nm. Reagents 1. 2. 3. 4. 5. 6. 7.

0.25 M Histidine – HCl buffer, pH 6.5 at 25°C. 14 mM TPP (Store at – 20°C) Yeast alcohol dehydrogenase : 5 mg/ml (store at –20°C) 10 mM MgCl2 0.67M pyruvate (sodium salt): Store at – 20°C 6.7 mM NADH: Prepare fresh daily. 20 mM potassium phosphate buffer, pH 7.5 containing 0.7 M mannitol and 1 mM EDTA.

Procedure Enzyme extraction (carry out at 0-4°C) 1. Homogenize 1.0 g to tissue with 3 ml of chilled (4°C) phosphate buffer and 0.03g of potassium isoascorbate and 0.2g of polyclar AT. 2. Squeeze the homogenate through 4 layers of cotton gauze and centrifuge at 20,000 g for 30 min at 4°C. 3. Use the supernatant as enzyme source. Enzyme assay 1. Place 0.008 of histidine – HCl buffer, 0.02 ml of pyruvate, 0.005 ml of enzyme solution and water to make the final volume of 0.4 ml in a reference quartz cell (0.7 ml, 1 cm light path). 2. In a test quartz cell (0.7 ml 1 cm light path) place 0.08 ml of histidine – HCl buffer, 0.01 ml of TPP, 0.003 ml of alcohol dehydrogenase, 0.014 ml of MgCl 2, 0.01 ml of NADH, 0.005 ml of enzyme and water to make the final volume of 0.38 ml. 3. When the decrease in absorbance at 340 nm ceases, initiate the reaction by adding 0.02 ml of pyruvate and measure the decrease in absorbance at 340 nm in a recording spectrophotometer. 4. Estimate the protein content of enzyme extract by Lowry's method. 5. Considering one unit of enzyme activity as the amount of enzyme required to produce 1 n mole of acetaldehyde / min under the above assay condition based on the fact that 1n mole yields 1n more of NAD+ by oxidation of NADH in coupled assay. Calculated the sepcific activity and express the results as number of enzyme units/mg protein. Reference Ullrich, J., J.M. Wittorf and C.L. Gubler (1966). Biochem. Biophs. Acta, 113, 595.

5.19. Lipoxygenase (lipoxidase) Lipoxygenase (EC 1.13.11.12) is also lipoxidase is a dioxygenase that catalyzes, , the hydroperoxidation by molecular oxygen of linoleic acid and other polyunsaturated lipids. Principle The formation of the conjugated diene from linoleate is measured at 234 nm. Reagents 1. 2. 3. 4. 5. 6. 7. 8.

Acetone Diethyl ether 0.1 M Tris-HCI buffer containing 0.1% Triton X – 100 at pH 7.3 Absolute ethanol Tween 20 0.05M Na2HPO4 1N NaOH Substrate : Stock A : 1% linoleic acid (w/v) in absolute ethanol

Stock B : To 7.1ml of stock A, add 0.25 ml of Tween 20. Evaporate the ethanol under vaccum in a rotary evaporator. Dissolve the residue in 100ml of Na 2HPO4 and adjust the pH to 9.0 using 1N NaOH. This stock solution contains linoleic acid and Tween at levels of 2.5 c 10-3 M and 0.25% respectively. Procedure 1. Cool the material in a dry ice bath (-30°C) and grind to a fine powder. 2. Defat with acetone and diethyl ether and suspend 1.0g of defatted material in 3 volumes of Tris-HCI buffer with mechanical stirring for 16h. 3. Centrifuge at 48000g for 30 min and discard the pellet. 4. Subject the supernatant to two additional centrifugation steps (30 min, 48000g). Discard the pellet. 5. Resubject the supernatant to three repeated ultracentrifugation at 200000g for 3h and discard the pellet. Use the supernatant as enzyme source. Enzyme assay 1. Dilute stock solution B ten fold with 0.2M citrate-phosphate buffer of the desired pH (the pH of the final substrate solution is that of the diluting buffer). 2. Take 2.4ml of substrate solution and 0.1ml of water in the control cuvette. 3. Zero the UV recording spectrophotometer with both reference and sample cuvette filled with the control solution. 4. Empty the sampel cuvette and fill with 2.4ml of substrate solution. At zero time add 0.1ml of enzyme extract, mix rapidly and start the recorder so that the time

interval between addition of the enzyme and start of the recorder is not more than 10sec. 5. Compute the rate of increase of optical density (OD234/min) from the initial linear potion of the graph. 6. Express the enzyme activity as OD234/ min/mg protein or per g weight of material. Reference Grossman, S. and R. Zakut (1979). In : Methods of Biochemical Analysis, (ed. D. Glick), Vol. 25, John Wiley and Sons, New York, pp. 303-329.

5.20. Nitrite reductase (NiR) The enzyme which catalyzes the reduction of nitrite to ammonium is called nitrite reductase (NiR). The enzyme, located within the chloroplast accepts electrons directly from light-induced ferredoxin. NO-2 + 6e- + 8H+

NH4+ + 2 H2O

Principle The enzyme is assayed by following the rate of removal of nitrite in the presence of an artificial electron donors sodium dithionite and the dye methyl viologen. Reagents 1. 0.2M Potassium phosphate buffer, pH 7.0 2. 5mM Sodium nitrite : Prepare in water 3. 1.5mM Methyl violgoen or benzyl viologen 4. Dithionite reagent : Dissolve 40mg of sodium dithionite and 40mg of sodium hydrogen carbonate in 10ml of water just prior to use. Procedure Enzyme extraction 1. Grind 7g of plant material (leaf) in 2 volumes of 20mM potassium phosphate buffer, pH 7.5 containing 5mM cysteine HCl and 0.05mM EDTA for 90 sec. 2. Filter, add charcoal (0.5g/8ml), stirr and centrifuge soon at 5000g. Use the supernatant for assay. Enzyme assay 1. Incubate 0.2ml of phosphate buffer, 0.1ml of sodium nitrite, 0.1ml of methyl viologen or benzyl viologen, desirable volume of enzyme and water to 0.8ml at 30°C. 2. Start the reaction by adding 0.2ml of the dithionite reagent and incubate for 10min. 3. Stop the reaction by vigorously shaking the mixture until the diothionite is completely oxidized and the dye color has disappeared. 4. Run the blank similarly without the enzyme and the boiled enzyme. 5. Determine the amount of nitrite in a suitably diluted aliquot of the reaction mixture by the procedure given under nitrate reductase assay. Express the specific activity of NiR as µ moles of nitrite removed /min/mg protein.

Reference Miflin, B.J. (1967). Nature, London, 214 : 1133.

5.21. Peroxidase (POD) Peroxidase catalyzes the oxidation of many organic compounds by hydrogen peroxide : amines (O – phenylendiamine, p-phenylenediamine, benzidine), phenols (pyrogallol, guaicol, O-cresol), hydroquinones, etc. Peroxidase (EC 1.11.1.7) catalyzes the reaction AH2 + H2O2 2H2O + A Principle The enzyme activity is assayed using O-dianisidine as hydrogen donor and H2O2 as electron acceptor. The rate of formation of yellow range coloured dianisidine dehydrogenation product is a measure of the POD activity and can be assayed spectrophotometrically at 430nm. O-Dianisidine + H2O2

oxidized O-dianisidine + 2H2O

Reagents 1. 0.1M Potassium phosphate buffer, pH 6.0 2. 0.01M O-Dianisidine in methanol : Prepare fresh 3. 0.02M H2O2 (20mM) : Dilute 0.227ml of 30% H2O2 to 100ml with water. Prepare fresh. 4. 2N H2SO4 Enzyme extraction 1. Homogenize the material in ice-cold 0.1M phosphate buffer, pH 6.0 (1:10, w/v). 2. Strain through two folds of muslin cloth and centrifuge the homogenate at 16,000g for 20 min at 4°C. Use the supernatant as enzyme source. Enzyme assay (modified Summer and Gjessing, 1943) 1. Pipette out 1ml of O-dianisidine, 0.5ml of H2O2, 1 ml of phosphate buffer and 2.4ml of distilled water into a test tube. 2. For blank exclude, H2O2 but add additional volume of water. 3. Incubate at 30°C and start the reaction by adding 0.2ml of enzyme. 4. After 5min, stop the reaction by adding 1ml of 2N H2SO4. 5. Read the absorbance at 430nm. Express the specific activity of enzyme as units/min/mg protein or per g weight of sample considering one unit of enzyme as an increase in OD by 1.0 under standard conditions. Reference Summer, J.B. and E.C. Gjessing (1943). Arch. Biochem., 2 : 291.

5.22. Acid phosphatase Phosphatases are the enzymes that catalyze the hydrolysis of phosphate monoesteres with consequent release of inorganic phosphate. Acid phosphatases (EC 3.1.3.2) catalyze the hydrolysis of a variety of phosphate esters and exhibit pH optima below 6.0. Principle The enzyme activity is measured using the substrate p-nitrophenol phosphate and measuring and amount of p-nitrophenol formed by hydrolytic activity of the enzyme spectrophotometrically at 405nm. Reagents 1. 10mM p-Nitrophenyl phosphate (PNPP) substrate 2. Standard p-nitrophenol (100nmoles/ml) 3. 100mM Tris-maleate buffer, pH 5.2 4. 200mM Sodium carbonate Enzyme extraction 1. Grind 2.0g of material in 5ml of Tris-maleate buffer, pH 5.2 for 30 min at 5°C. 2. Centrifuge at 10,000 rpm for 15 min and use the supernatant for assay. Enzyme assay 1. Pipette out 0.5ml of PNPP and 0.4ml of Tris-maleate buffer into a test tube and incubate at 37°C for 5 min. 2. Add 0.1ml of approximately diluted enzyme and incubate for further 5min. 3. Stop the reaction by adding 2ml of sodium carbonate. 4. For blank add the sodium carbonate before adding the enzyme. 5. Measure the absorbance at 405nm. 6. For the standard curve-pipette out standard p-nitrophenol into a series of the tubes (10-100 n moles range), make up the volume in each to 1ml with water. Add 2ml of sodium carbonate and read at 405nm against the blank prepared without pnitrophenol. Express the enzyme activity in n katals where 1 katal is the amount of enzyme that hydrolyzes 1mole of PNPP/sec under the assay conditions. The enzyme may be also assayed by measuring the amount of inorganic phosphate released from sodium dihydrogen orothophosphate as substrate. Reference Parfsh, R.W. (1974). J. Histochem. Cytochem., 9 : 542.

5.23. Succinate dehydrogenase Succinate dehydrogenase is one of the key enzymes of TCA cycle catalyzing the conversion of succinate into fumarate. Dehydrogenases catalyze transfer of hydrogen atoms from a substrate to hydrogen acceptor, changes in dehydrogenase activity are reported in a few host-parasite interactions. Principle The enzyme activity is measured in terms of amount of dye, triphenyl tetrazolium chloride (TTC) reduced which is measured colorimetrically at 460nm. Reagents 1. 0.1M Phosphate buffer, pH 7.0 2. Acetone (reagent grade) 3. 0.2M Sodium succinate in water 4. 0.1% Triphenyl tetrazolium chloride (TTC) Enzyme extraction 1. Grind the tissue in a mortar with sand in 2 volumes of 0.1M phosphate buffer, pH 7.2 2. Centrifuge for 20min at 1500g. Use the supernatant for assay. Enzyme assay 1. Pipette out 2ml of sodium succinate, 1ml of phosphate buffer, 1ml of TTC and 2ml of enzyme extract 2. Incubate in a water bath at 30°C. 3. At various time intervals, remove one tube and add 7ml of acetone to stop the reaction. 4. Centrifuge at 2000g for 30min to remove the precipitate. 5. Measure the absorbance of supernatant at 460nm 6. For blank, add acetone reagent before adding the enzyme at zero time. 7. Plot a standard curve with TTC reduced by suspending a few crystals of sodium hydrosulphite into the dye solution and measure the absorbance at 460nm Express the specific activity of the enzyme as µg of dye reduced/min/mg protein or per g weight of sample. Reference Kun, E and L.G. Abood (1949). Science, 109, 144-146.

5.24. Estimation of nitrogenase activity The key enzyme systems which catalyzes the reduction of atmospheric dinitrogen to ammonia is called notrogenase. Principle Acetylene is reduced to ethylene by nitrogenase. The ethylene produced is measured in a gas liquid chromatography (GLC) and the activity is expressed as n mole ethylene produced for unit time per g dry nodules. Materials and reagents 1. Gas chromatography with Flame Ionization Detector (FID) 2. Air-tight syringes 3. Conical flasks (100ml) with small mouth to fit serum caps 4. Acetylene gas 5. Standard ethylene gas 6. GLC operating conditions : Carrier gas-nitrogen/helium/argon with a flow rate 30 to 45ml/min Gas for detector-hydrogen and air Column – Porapak N, R, T and Q or silica gel

Oven/column temperature Intector temperature Detector temperature Retention time for ethylene (min) Retention time for acetylene (min)

Porapak 60oC 65 oC 85 oC 1.3 1.8

Silica gel 150 oC 160 oC 175 oC 1.5 3.0

Method 1. Remove the plants from the soil without disturbing the root nodules 2. Excise the roots with nodules 3. Place the root systems into a 100ml conical flask 4. Seal the flask with rubber septum (serum cap) 5. Remove 10ml of air from the flask with an air tight syringe 6. Inject 10ml of acetylene into the flask. 7. Incubate for 30-60 min at room temperature 8. Remove 0.5 to 1ml gas mixture from the flask with an air-tight syringe 9. Inject the gas mixture into a pre-conditioned GLC 10. Look for acetylene and ethylene peaks and measure the ethylene peak/height 11. At the end of the experiment, detach the nodules and take the dry weight 12. For standard, inject 10 µ l (Z) of pure ethylene into a 100ml sealed conical flask to satisfy identical assay conditions. Remove 0.5 to 1ml (same volume as of the sample) and inject into the GLC and measure ethylene peak height. Calculation 1. Standard amount of ethylene (E) in µ mol

0.0446 x Z µ l = Peak height in mm x Attenuation 2. Amount of ethylene produced in µ mol in the sample = E x Peak height of sample ethylene x attenuation in mm 3. Activity of nitrogenase = n mol or µ mol ethylene per unit sample per unit time (unit sample may be g dry weight nodules or mg dry weight or mg protein). Reference Turner, G.L. and A.H. Gibson, (1980). In : Methods for Evaluating Biological Nitrogen Fixation (ed. Bergerson, F.J.) John Wiley and Sons, New York, p. 111.

5.25. Estimation of Rubisco by Elisa Antisera were raised in Newzealand rabbit using intra dermal injection of Large subunit of Rubisco antigen (1mg/ml of PBS) mixed with 1 ml of Freund‟s complete adjuvant). 15 days after the first injection, a booster injection was given followed by second and third boosters at the same time interval with the same level of the antigen and the adjuvant. Ten days after the last booster, the rabbit was bled and blood was collected. It was kept at room, temperature for 2 hours and then over night at 4C. The serum was decanted and centrifuged at 3,500 rpm for 10 minutes to remove residual red cells. Crude serum was stored at 20 and was used for further work. Dot blot technique for qualitative analysis of serum for antibodies. Nitro cellulose membranes by virtue of their peculiar structure, can capture proteins by non-covalent interactions. The use of such membranes is an important component of immuno detection systems. The dot immuno binding assay is on such sensitive system which enables characterization and identification of an immobilized antigen by the use of antibody probes. This can subsequently be visualized by enzyme conjugated secondary antibodies. In dot immuno blot, antigen are immobilized on nitro cellulose membranes. Reagents 1. 2. 3. 4.

Nitro cellulose membrane Primary antibodies (1:50, 1:100, 1:500) Milk casein in PBS (2%) (Blocking solution) Labelled anti – immunoglobin (secondary antibody conjugated with alkaline phosphatase) 5. Substrate 3M Tris pH 8.8 Nacl IM Mgc12 (5-brmo 4-Chloro – 3indolyl PO4) NBT (Nitro Blue Terazolium)

0.67 ml 0.17 g 10.00 ul 2 mg * 6 mg *

* (Prepared in Di-methyl formamide 200 l volume made up to 206 ml phosphate buffer saline (pH 7.4) Na2 HPO4 K2H2PO4 Volume made up to

1.44 g 0.24 g 1000 ml

Around 5-6 l of antigen was initially applied on to the nitro cellulose membrane and dried immediately limiting the binding of antigen in a small area. Other binding sites

were saturated with blocking agent by keeping the membrane strips in 1% casein prepared (pH 7.4). These were shaken gently for about 1 hour. Membranes were then immersed in antibody solution diluted in PBS (pH 7.4). Repeated washing was done with PBS after two hours. Then the membranes were put in the labeled anti immuno-globulin (see antibody) diluted to 1:2000 for 1-2 hours. Washing was repeated and membranes were put in substrate solution. Using this, qualitative analysis of the serum was done to know the antigen antibody specifity. The formation of an insoluble, deep blue colour due to the reaction of the conjugated enzyme substrate colour indicated the positive reaction. Often intensity of colour formed is used as a basis of the degree of the antigen – antibody reaction. Reference Wareing, P.F., M.M. Khalifa and K.J. Trehern. 1968. Rate limiting processes in photosynthesis at saturating light intensisties. Nature, 220: 453-457.

5.26. Estimation of Total Dehydrogenase Activity Principle Dehydrogenases catalysis transfer of H+ atoms from a substrate to a hydrogen acceptor. The rate of reaction is measured according to the degree of reduction of the dye, usually methylene blue or Triphenyl Tetrazolium chloride. Reagents Tetrazolium chloride (0.02%) Acetone or methyl cellosolve Phosphate buffer (pH 7.00) Procedure 1. 2.

3. 4. 5. 6.

Take known weight of plant tissue, cut into small pieces and submerge in 0.2 per cent aqueous solution of tetrazolium chloride. Incubate in dark until well stained the leaf bits (usually for 2 or 3 hours). Immediately wash the leaf bits and submerge in solvent for extraction of the water – insoluble, red coloured 1, 3, 5 triphenyl formazan (formazan or reduced TTC). Homogenize leaf bits in acetone or methyl cellulose for extraction of the formazan. Measure optical density of all formazan solution at 480 nm. Plot a standard curve with TTC reduced by suspending a few crystals of sodium hydrosulphide into the dye solution. Express the enzyme activity interms of microgram of dye reduced per unit time per gram of fresh weight or protein content.

Reference: Kottack, D.A. and A.G. Law. 1968. Relationship of seedling vigour to respiration and tetrazolium chloride reduction by germinating wheat seeds. Agron. J., 60: 286288.

5.27. Estimation of PEP – Carboxylase (Singh et al., 1974) In tropical grasses (sugarcane, Sorghum, corn. etc.) it has been observed that the first product of CO2 fixation is C4 dicarboxylic acid – oxaloacetate which is converted to either malate or aspartate. Hence, the tropical grasses are known as C 4 plants. These plants have a highly active PEP – carboxylase which is about 50 – 100 times more active than that present in C3 plants. The enzyme is believed to be loosely attached to the mesophyll chloroplasts and may be located in the chloroplast envolop membrance. The reaction catalysed by PEP – carboxylase can be written as follows : Extraction of enzyme About 5 g leaves are ground well in about 20 ml medium containing 50 mM HEPES, pH 7.8, 10 nM MgCl2 and 10 mM MnCl2 using mortar and pestle. The homogenate is centrifuged at 20,000 x g for 10 min and the supernatant assayed for PEP – carboxylase activity. Assay of PEP-carboxylase Method-I Carboxylase is assayed radiometrically using NaH14 CO3. Oxaloacetate produced in this reaction is allowed to form a condensation produce with acidified DNP, which is counted for radioactivity. Assay The reaction mixture containing the following constituents is incubated at 30 for 5 min. 0.10 ml 0.05 ml 0.05 ml

HEPES buffer, 0.25 mM, pH 7.8 PEP, 50 mN MgCl2

The reaction is initiated by the addition of NaH14 CO3 (sp. Act. 1 uCi/lumole) and 50 ul aliquots are with drawn at intervals of 1 min for 8 min. Each aliquot is added to 20 ul of saturated DNP in 2 N HCL, mixed well, left in cold, dried on Whatman No.3 filter paper discs and counted. Method-II PEP – carboxylase can also be assayed spectrophoto metrically by following the oxidation of NADH at 340 nm in the presence of exaloacetate and malate dehydrogenase. Assay The reaction mixture containing 0.5 ml 0.1 ml 0.1 ml

Tris – HCL, 100 mM, pH 8.0 PEP, 20 mN NADH, 1.5 mN

0.1 ml 0.1 ml

MDH, 5 units and enzyme extract

is incubated at 25C for 5 min. The reaction is initiated by the addition on 0.1 ml 50 mM NaHCO3 and the change in absorbance at 340 nm is recorded. Assay of RuBP carboxylase About 5 g of leaves are ground with a mortar and pestle in about 20 ml of 50 mM HEPES buffer, pH 7.8 containing 10 mM MgCl2, 10 mM MnCl2 and 10 mM DTT. The homogenate is centrifuged at 20,000 x g for 10 min and the supernatant is taken for the assay of RuPP – carboxylase. Assay RuBP – carboxylase is assayed radiometrically using NaH14 CO3. The reaction mixture contains 0.10 ml HEPES buffer, 0.25 mM, pH 7.8 0.05 ml RuBP, 1 mM 0.05 ml MgCl2, 100 mM 0.05 ml DTT, 50 mM and 0.20 ml enzyme extract The reaction mixture is illuminated at 25C for 2 min and the reaction is initiated by the addition of NaH14 CO3 (sp. act. 1 uCi/umole) and is stopeed after 10 min by adding 0.1 ml of 10% acetic acid. Aliquots are transferred on to Whatman No.3 dilter paper discs, dried and counted for radioactivity. Reference: Wareing, D.H., J.L. Ozbun and M.H.Munger. 1972. Physiolo. Wareing, P.F., M.M.Khalifa and K.J. Treharne. 1968. Rate Limiting process in photosynthesis at saturating light intensities Nature. 220: 453-457.

5.28. Estimation of Ribonuclease Activity (Phillips and Fletcher, 1969) Principle The activities of specific degradative enzymes increase during senescenece leading to the breakdown of cell organelles and macromolecules and to the subsequent mobilization of soluble products from the senescing tissue. RNA levels in the senescing tissue is regulated by the activities of ribonuclease at the initial stage it self. Reagents 1. 2. 3. 4.

0.05 M phosphate buffer (pH 6.8) Ribonuleic acid (0.15 per cent) 25 per cent perchloric acid 0.75 per cent Uranyl acetate

Reaction termination mixture 25 per cent perchloric acid containing 0.75 per cent Uranyl acetate Procedure 1.

2.

3.

4. 5. 6.

Homogenize freeze leaf tissue with 0.05 M phosphate buffer (pH 6.80). The homogenate is made up to 10 ml with cold phosphate buffer of pH 6.8 and use the extract as enzyme aliquot. One ml of enzyme aliquot of the supernatant is incubated with 2 ml of 0.15 per cent (W/V) ribonucleic acid in 0.1 M phosphate buffer at pH 6.8 and kept at 30C for the one hour. The reaction is terminated and the unhydrolysed RNA precipitates at 0 C with 0.5 ml reaction terminating mixture (25% perchloric acid containing 0.75% Uranyl acetate) for one hour. After centrifugation, the supernatant is diluted (0.5 to 13 ml) with 0.5 M phosphate buffer and the Absorbance at 257 nm measured in the spectrophotometer. One unit of enzyme activity represents the generation of an absorbance change of 0.01 by 1 ml of initial homogenate. The ribonuclease activity is expressed as unit or ribonuclease activity per gram of fresh plant tissue per hour.

5.29. Estimation of Super Oxide Dismutase, Catalase And Peroxidase (Giannopolitis and Ries, 1977) Principle The activity of SOD was assayed by measuring its ability to inhabit the photochemical reduction of nitro blue - tetrazolium (NBT) Reagents 1. 2. 3. 4. 5. 6.

HEPES - KOM Buffer (pH 7.8) 50 mm EDTA 0.1 mm Na2CO3 (pH 10.2) 50 mm L.Methionine 12 mm NBT 75 m (Nitro blue tetrazolium chloride) Ribo Hann 1 m

Procedure Homogenized tissue in 10 ml HEPS - KOH buffer containing 0.1 mm EDTA. Centrifuge at 15000g for 15min. All operation was done at 4C. Use the supernatant for SOD assay. Reaction mixture (3ml consisting of 50 mM hEPES - HOH, 0.1 mM EDTA, 50 mM Na2CO3, 12 mM L.methionine, NBT 75 m, diluted enzyme extract (0 to 300 w) and 1 m riboflavin. One unit SOD activity was defined as the amount of enzyme required to result a 50% inhibition of the rate of NBT reduction at 560 nm. The results are expressed as units / g fresh weight and as unit / mg protein. Assay for catalase Reaction mixture 25 mM potassium phosphate buffer (pH 6.8), 10 mm H 2O2 and diluted enzyme extract in a total volume of 1ml. The decomposition of H 2O2 was followed by a decline in CD at 240 nm. The results expressed as units/g fresh weight.

5.30. Estimation of Glycolate Oxidase Activity Reagents 0.1m PO4 buffer (pH 7.5); 0.1 m Sodium glycolate; 0.1 m Phenyl hydrazine chloride; 0.1 m Cystein; In HCI; Potassium ferricyanide 0.25%. Procedure 1. The ice cooled sample leaves were washed with distilled water and blotted out with filter paper. 2.

The leaves were then cut into small pieces and 250 mg of samples were ground in a pre chilled glass mortor using 0.1 M phosphate buffer (pH 7.5).

3.

The homogenate was later centrifuged at 12000 rpm at 0C for 20 minutes. The enzyme extract of 0.1 ml was added to the assay mixture containing 0.1 M sodium glycolate, 0.1 M phenyl hydrazine chloride, 0.1 M of cystein, 0.1 M of phosphate buffer in a final volume of 3 ml.

4.

A blank, without the enzyme extract was used. Incubation was done for 30 minutes maintaining at 35C.

5.

The reaction was stopped by adding 3 ml of IN HCI of the reaction mixture. Out of which 2ml was taken out and mixed with 1 ml of 0.25% potassium ferricyanide solution, allowed for 15 minutes and final volume was made upto 5 ml when the optical density at 520 nm was read in spectrophotometer.

Standard The stock solution of one mole ml-1 sodium glycolate was used to prepae a series of solutions containing 0.05, 0.1, 0.15, 0.2, 0.6 moles. An aliquot of 0.1 ml was takenfrom each of these to which 0.2 ml of phenyl hydrazine chloride was added and then shaken vigorously. Finally 1 ml each of IN HCI and potassium ferricyanide were added and volume was made upto 5 ml. After 15 minutes optical density at 520 nm was read using spectronic - 20. A standard curve was then plotted with optical density and the corresponding concentration of sodium glycolate. The amount of glycolate oxidase was estimated by reading against the standard curve and expressed as the glycollate oxidase activity.

5.31. Estimation of Lipid Peroxidation (Heath and Pacber, 1968) Principle The level of lipid peroxidation in the leaf tissue was measured in terms of malondialdehyde (MDA) content determined by the thiobarbituric acid (TBA) reaction. Reagents 0.1% Trichloro acetic acid 0.5% thio barbituric acid Free-radical-induced lipid peroxidation is considered to be an important mechanism of membrane deterioration during abiotic stress period. Superoxide dismutase (SOD) and catalase destroy the superoxide radical (O 2) and hydrogen peroxide respectively and control the level of lipid peroxidation. Procedure A 250 mg root sample was homogenized in 5 ml 0.1% TCA. The homogenate was centrifuged at 10000 g for 5 min. To 1ml aliquot of the supernatant 4 ml 20% TCA containing 0.5% TBA were added. The mixture was heated at 95C for 30 min. And quickly cool it in an ice bath. Centrifuge at 10000 rpm for 10 min. The absorbance of the supernatant at 532 nm was read and the value for the non-specific absorption at 600 nm was subtracted. The concentration of MDA was calculated using its extinction coefficient of 155 mm-1 cm-1.

5.32. Starch phosphorylase This enzyme sequentially removes glucose units in presence of inorganic phosphate (Pi) attacking α-1, 4 glycosidic bonds at the non-reducing end resulting phosphate: (Glucose) n + Pi

(glucose) n-1 + glucose –1- phosphate

Principle The enzyme activity is measured by the amount of inorganic phosphate released from glucose-1-phosphate added as substrate and amylopectin as primer. Reagents 1. 2. 3. 4. 5. 6. 7.

0.1M Tris-maleate buffer, pH 6.5 1mm NaF Amylopectin Glucose-1-phosphate solution 5% Perchloric acid PVP Reagents for estimation of inorganic phosphorus

Procedure Enzyme extraction 1. Grind a known weight of sample in chilled mortar with 0.1M Tris-maleate buffers, pH 6.5 and 1% PVP. 2. Centrifuge at 20,000g for 20min. Use the supernatant for assay. Enzyme assay 1. Prepare the reaction mixture (3ml) consisting of Tris-maleate buffer: 1mM NaF, 50 μmole, pH, 6.3: 25mg amylopectin: 0.2ml of enzyme preparation and 25μmole of glucose-1-phosphate. 2. Incubate at 29˚C for 2h. 3. Terminate the reaction by adding 1ml of 5% perchloric acid. 4. Run the control by adding glucose-1-phosphate only after incubation and addition of 5% perchloric acid. 5. Estimate the amount of release of inorganic phosphorus. 6. Estimable the protein content in the extract by the method of Lowry et at. (1951) Express the enzyme activity as μmole phosphorus released/h/mg protein. Reference Dhaliwal. A.S. and H.L. Sharma (1986). Aust. J. Plant Physiol., 13,249-255.

5.33. Sucrose synthase (sucrose-6-phosphate) Sucrose synthase is involved in sucrose synthesis. Principle The enzyme is assayed by measuring the amount of sucrose synthesized by measuring the hexoses colorimetrically at 520nm. Reagents 1. Extraction medium : 50mM HEPES buffer, pH 7.5, 7.5mM MgCI 2, 2mM EGTA, 5mM DTT, 2% PEG 1000, 2% PVP 2. 200mM HEPES buffer, pH 7.5 3. 50mM MgCI2 4. 100mM UDP-Glucose 5. 100mM Fructose 6. 1N NaOH 7. 1% Resorceinol in ethanol 8. 30% HCI Method Sample extraction 1. Grind 1g of material in 8ml of ice-cold extraction medium for 30min. 2. Filter through nylon mesh (60μm). 3. Centrifuge at 17000rpm for 10min. Use the supernatant for enzyme assay. Enzyme assay 1. Incubate 0.9ml of extract, 0.5ml of 200mM HEPES (pH 7.5) Buffer, 0.2ml of 50mM MgCL2 0.2ml of 100mM UDP-glucose and 0.2mlof 100mM fructose 30min at 30˚C in a test tube. 2. Terminate the reaction with 2ml of IN NaOH, heat for 10min at 100˚C to destroy unreacted fructose and cool. 3. To 0.4ml of aliquot sample, add 0.5ml of 1% resorcinol, 1.5ml of 30% HCI and incubate for 8min at 80˚C. 4. Centrifuge at 1500g for 5min and measure the absorbance at 520nmagainst the blank made in the absence of enzyme. 5. For standard curve use sucrose by taking different volumes of standard sucrose in a series of test tubes and make up the volume to 0.4ml with water. Add 0.5ml of 1% resorcinol, 1.5ml of 30% HCl, incubate for 8min at 80˚C. Again incubate on ice and measure the absorbance at 520nm. 6. Estimate the protein content in the extract by the method of Lowry et al. (1951) Express the enzyme activity as μmole sucrose synthesized/h/g wt. of sample of per mg protein. Reference Wardlaw, I.F. and J. Willenbrink (1994). Aust. J. Plant Physiol, 21,255-271.

5.34. Isozymes in Plant Samples The term Isozyme was first introduced by Market and Moller in 1959 to refer to multiple molecular forms of an enzyme with similar or identical substrate specificity occurring within the same organisms. Multiple forms of enzymes may rise due to various reasons like, genetic control, chemical or physical environments. One genetic mechanism by which isozymes might arise is gene duplication with subsequent mutations at daughter and parental loci. Selection of Tissue Enzymes can be extracted from a wide variety of plant tissues. Availability of material is often a foremost consideration and for this reason extracts are usually obtained from vegetative tissues, such as the lamina or petioles of leaves, succulent stems, terminal portions of roots etc. Sometimes, entire germinating seedlings are used, where seed supply is not a limiting factor. Several parts of seeds have been used, including entire embryos, endosperms, imbibed cotyledons, hypocotyls and radicals. In most cases, germinating seedlings or young metabolically active leaves are used. Extraction buffers for different isozymes S. No 1. 2. 3 4

5

6 7 8 9

10

11

Isozyme Esterase (EST)

Extraction buffer 0.2M Tris HCl buffer (pH 6.0) containing 0.006M Mercaptoethanol Peroxidase (PRX) 0.1M phosphate buffer pH 7.0 Polyphenol Oxidase (PPO) 0.01M Potassium phosphate buffer pH 7.0 containing 1% non-ionic detergent (Tween 8.0) Superoxide dismutase Phosphate buffer pH 7.0, 50ml, containing 37mg (SOD) KGl, 10mg MgCl2, 18mg Sodium EDTA, 25mg Polivinyl pyrrolidone and 150µl of mercaptoethanol Aspartate aminotransferase Tris HCl buffer pH 8.5, 50ml containing 37mg (AAT) KCl, 10mg MgCl2, 18mg tetrasodium EDTA, 25mg Polivinyl Iso citrate dehydrogenase As that of Aspartate amino transferase (ICD) Shikimic acid As that of Aspartate amino transferase dehydrogenase (SKD) Acid phosphatase (AP) 50mM Citrate buffer (pH 5.3) Glutamine synthetase (GS) 50mM imidazole acetate buffer pH 7.8 containing 0.5mM EDTA, 1mM dithioerithriol, 2mM MnCl2 and 20% glycerol. Phosphoenol pyruvate 100mM Tris HCl (pH 7.9), 10mM MgCl2 10mM carboxylase (PEP) Sodium bicarbonate, 2% polyvinyl pyrolidone, 1mM EDTA and 15mM mercaptoethanol Glutamate dehydrogenase 100mM Tris HCl (pH 7.9), 10mM MgCl2 10mM (GDH) Sodium bicarbonate, 2% polyvinyl pyrrolidone, 1mM EDTA and 15mM mercaptoethanol

12

13

Indoleacetic Acid Oxidase Prepare acetone powder from the frozen tissue by (IAA oxidase) blender homogenising 25g tissue in two successive 100ml adiquots of cold acetone. The homogenate was air dried until free of acetone odour, the resulting dry powder was weighed and freezer stored in cold containers. One gram of acetone powder was ground in two successive 20ml aliquots of 25mM phosphate buffer (pH 6.2) in a mortar chilled in an ice bath. Collect the extract by Buchner filtration through Whatman No.1 paper after each grinding. Combine the filtrates and dilute to 50ml with phosphate buffer. Malate dehydrogenase 50mM Tris HCl (pH 8.0), 50mM MgCl2, 5m (MDH) mercaptoethanol and 1mM EDTA.

Superior results are obtained from freezing enzyme extracts rather than tissue samples and from freezing in an ultra cold (-70°C) rather than conventional (-20°C) freezer. Electrophoresis is the process of moving charged molecules in solution by applying an electrical field across the mixture. Because molecules in an electrical field move with a speed dependent on their charge, shape and size, electrophoresis has been extensively developed for molecular separations. Electrophoresis of macromolecules is normally carried out by applying a thin layer of a sample to a solution stabilized by a porous matrix. Under the influence of an applied voltage, different species of molecule in the sample move through the matrix at different velocities. At the end of the separation, the different species are detected as bands at different positions in the matrix. The matrix can be composed of a number of different materials, including paper, cellulose acetate or gels made of polyacrylamide, agarose or starch. Preparation of stock solution 1. Stock acrylamide solution Acrylamide 30% w/v Bisacrylamide 0.8% w/v Distilled water 2. Separating gel buffer pH 8.8 1.875 M Tris-HCl Distilled water 3. Stacking gel buffer pH 6.8 0.6M tris HCl Distilled water 4. Polymerising agents Ammonium per sulphate 5% w/v 0.05 g per ml TEMED (N,N,N.N-Tetramethylene diamine)

- 30g - 0.8 g - 100 ml - 22.7 g - 100 ml - 7.26 g - 100 ml - Prepared freshly - Fresh from the refrigerator

5.

6.

Electrode buffer 0.05M Tris – HCl 0.192M Glycine Water Sample gel buffer Stacking gel buffer (pH 6.8) Sucrose Bromophenol blue (0.5% w/v)

- 12.0g - 28.8g it can be used for 2-3 times. - 21 ml - 5.0 g (Stored in small aliquots and - 5.0 g (diluted to 1 x concentration - 1.0 ml (before use)

Protocol for electrophorosis (Vertical) 1. Thoroughly clean the glass plates (18x9 cm) and spacers. Firmly fix with bull dog clips and clamp upright. Two percent agar melted in boiling water bath should be applied along the edges of the spacers to hold them in place and seal the chambers between the glass plates. (Fig.13.1 and 13.3). 2. Gently mix the separating gel mixture (Table 2) and cast the gel carefully by pouring the same in the chamber between the glass plates and layer the top of the gel with distilled water to prevent the entry of oxygen and to facilitate polymerization. Leave it for 30-60 minutes for polymerization. 3. Remove water from the top of the gel and cast the stacking gel mixture. A comb of 0.1mm thickness and with 100µ l well capacity should be placed in the stacking gel and allow to polymerize for 30-60 minutes. 4. Remove the comb without disturbing the shape of the well after the stacking gel is polymerized. Carefully assemble the gel in the vertical slab gel unit, after removing the clips and agar. Fill it with electrode buffer solution. The electrode buffer and the plates should be kept cooled by using a cooling apparatus so that heat generated during the run is dissipated. 5. Load the wells with samples. After loading the wells, the apparatus is connected to current source and run the gel at a constant current of 25mA until the tracking dye reaches the bottom of the gel. 6. After completing the run, transfer the gel carefully and to a staining tray/box and incubate in the staining solution. 7. Place the gel over the trans-illuminator and draw the electrophoresis by keeping a transparency on the gel. Take the photograph of the gel.

Solutions for Tris-Glycine Polyacrylamide gel electrophoresis 6% gel Volume of Separating gel Distilled water 30% Acrylamide mix 1.5 M Tris (pH 8.8) 10% APS TEMED

8% gel Volume of Separating gel Distilled water 30% Acrylamide mix 1.5M Tris (pH 8.8) APS TEMED

10% gel Volume of Separating gel Distilled water 30% Acrylamide mix 1.5M Tris (pH 8.8) APS TEMED

12% gel Volume of Separating gel Distilled water 30% Acrylamide mix 1.5M Tris (pH 8.8) 10% APS TEMED

5ml

10ml

15 ml

20 ml

25 ml

30 ml

40 ml

50 ml

2.7

5.3

8.0

10.6

13.3

15.9

21.4

26.5

1.0

2.0

3.0

4.0

5.0

6.0

8.0

10.0

1.3

2.5

3.8

5.0

6.3

7.5

10.0

12.5

0.05 0.004

0.1 0.008

0.15 0.012

0.2 0.016

0.25 0.02

0.30 0.024

0.40 0.032

0.50 0.04

5ml

10ml

15 ml

20 ml

25 ml

30 ml

40 ml

50 ml

2.3

4.6

7.0

9.3

11.6

13.9

18.6

23.2

1.3

2.7

4.0

5.3

6.7

8.0

0.7

13.4

1.3

2.5

3.8

5.0

6.3

7.5

10.0

12.5

0.05 0.003

0.1 0.006

0.15 0.009

0.0 0.012

0.25 0.015

0.3 0.018

0.4 0.02

0.5 0.024

5ml

10ml

15 ml

20 ml

25 ml

30 ml

40 ml

50 ml

2.0

4.0

5.9

7.9

9.9

11.9

15.8

20.0

1.7

3.3

5.6

6.7

8.3

10.0

13.3

16.6

1.3

2.5

3.8

5.0

6.3

7.5

10.0

12.5

0.005 0.002

0.1 0.004

0.15 0.005

0.2 0.006

0.25 0.01

0.3 0.012

0.4 0.016

0.5 0.02

5ml

10ml

15 ml

20 ml

25 ml

30 ml

40 ml

50 ml

1.7

3.3

5.0

6.6

8.3

9.9

13.2

16.4

2.0

4.0

6.0

8.0

10.0

12.0

14.0

20.0

1.3

2.5

3.8

5.0

6.3

7.5

10.0

12.5

0.05 0.002

0.1 0.004

0.15 0.006

0.2 0.008

0.25 0.01

0.3 0.012

0.4 0.016

0.5 0.02

10ml

15 ml

20 ml

25 ml

30 ml

40 ml

50 ml

2.3

3.5

4.8

5.7

6.0

9.2

11.4

15% gel Volume 5ml of Separating gel Distilled water 1.2 30%

Acrylamide mix 2.5 1.5M Tris (pH 8.8) 10% 1.3 APS TEMED 0.05 0.002 Volume of 1ml Separating gel Distilled water 0.66 30% Acrylamide mix 0.17 1.5M Tris (pH 8.8) APS 0.13 TEMED 0.01 0.001

5.0

7.5

10.0

12.5

15.0

20.0

25.0

2.5

3.8

5.0

6.3

7.5

10.0

12.5

0.1 0.004

0.15 0.006

0.2 0.008

0.25 0.01

0.3 0.012

0.4 0.016

0.5 0.02

2ml

3 ml

4 ml

5 ml

6 ml

8 ml

10 ml

1.4

2.1

2.7

3.4

4.1

5.5

6.8

0.33

0.5

0.67

0.83

1.0

1.3

1.7

0.25

0.38

0.60

0.63

0.75

1.0

1.25

0.02 0.002

0.03 0.003

0.04 0.004

0.05 0.005

0.06 0.006

0.08 0.008

0.1 0.01

The two important factors are temperature and light. Most procedures indicate incubation at room temperature (25-30°C) and a few indicate 37°C, or even higher. Light is also an important factor. Many stains are extremely sensitive being inactivated by exposure to light (Tetrazolium salts are photo reduced diazonium salt fast blue BB is inactivated when exposed to light. Staining Systems and Procedures 1. AZO coupling system : (To detect phosphatases) In this system diazonium salts are used to produce as AZO dye, when the salt combined wit aryl group. The aryl group is liberated because of the reaction of phosphatase on arylphosphate. Eg. Arylphosphate + H2O  Arylalcohol phosphatase Aryl alcohol (Substrate) _ Diazonium salt*  AZO Dye 2. Tetrazolium system: (To detect oxido reductases) In this system oxido reductase involve the transfer of electrons from the substrate to an electron acceptor (NADPH+). The electron acceptor after the acceptance of electrons acts as a reducing agent to convert tetrazolium salt into formazan. Phenazine methosulphate (PMS) is used as an electron carrier. Oxido reductase Susbstrate  Product + NAD(p) NAD(p)H PMS (reduced) PMS (oxidized) Tetrazolium Formazan (soluble) (coloured precipitate) 3. Starch Iodine system In this system starch iodine chlorophere is formed. This can use for the detection of activity of amylases, phosphorylases and catalases which is be mostly used method for

negative staining. Since the reaction catalysed by phosphorylases can be return in either direction, a positive staining is also possible. 4. Redox dyes : This group includes dyes other than tetrazolium salts. Although they represent a diverse group of chemicals they all have a common characteristic, a visible change in the physical, chemical properties, such as a shift in the wavelength of absorption and/or a charge in solubility concomitant with changes in their state of oxidation.

Staining procedure for different isozymes S. N Isozyme Extraction buffer 1 Esterase (EST) Incubate the gel in a solution given below at 37°C for 20-30 min., preferably in dark. Sodium dihydrogen phosphate 2.8g Disodium hydrogen phosphate 1.1g Fast blue RR salt 0.2 g Alpha-naphthyl acetate 0.03 g Water 200 ml Stop the enzyme reaction by adding a mixture of methanol : water : acetic acid : ethyl alcohol in the ration 10:10:2:1 2 Peroxidase (PRX) Incubate the gel in the following solution for 30 min in dark Benzidine 2.08 g Acetic acid 18.0 ml Water 80.0 ml Then add 1ml Hydroen peroxide drop by drop till the bright blue colour bands appear in gel. When the bands are stained sufficiently, arrest the reaction by immersing the gel into a large volume of 0.67% sodium hydroxide or 7% acetic acid solution for 10 min. 3. Polyphenol Oxidase (PPO) Incubate the gel for 30 min in 0.1% p-phenylene diamine in 0.1M potassium phosphate buffer (pH 7.0) followed by 10mM catechol in the same buffer. 4. Superoxide dismutase Incubate the gel for 30 min in the dark at 30-35°C (SOD) in a solution prepared by mixing the following Potassium phosphate buffer (250 mM) 20 ml EDTA (10 mM) 1 ml TEMED 200µl Riboflavin (300 mM) 1 ml Nitroblue tetrazolium (10 mM) 2.5 ml After incubation the gel is kept under light with shaking until the bands appeared.

5.

6

7.

8.

Aspartate aminotransferase Incubate the gel in the following solution for 30 (AAT) minutes in dark 0.1M Tris HCl pH 8.5 50 ml Alpha ketoglutarate 18 mg L. Aspartic acid 65 mg Pyridonal – 5 PO4 5 mg Polyvinyl pyrolidone 4 OT 250 mg Na2HPO4 710 mg Fast blue BB salt 200 mg Just before pouring, add 5mg of Pyridonal 5 PO4 Iso citrate dehydrogenase Incubate the gel in the following solution for 30 (ICD) min. in dark 0.1 m Tris HCl pH 8.0 50 ml Trisodium isocitrate 50.0 ml NADP 10.0 mg MTT 10.0 mg PMS 3.0 mg Acid phosphatase (AP) Incubate the gel at 37°C for 2-3 hr in the following solution 1-napthyl phosphate 0.05g Fast blue RR 0.05g Sodium Chloride 1.00g 10% MgCl2 0.50ml 0.1M Acetate buffer (pH 5.0) 50.00 ml Glutamine synthetase (GS) Mix the following solutions Tris Sigma 7.9 (Sigma T.1328) 0.1M 36.0 ml Imidazole HCl 1M 10.0 ml Hydroxylamine HCl, 0.8M 10.0 ml Magnesium 3M 0.4 ml Monosodium glutamate, 0.8M 2.0 ml Adjust to pH 7.7 with NaOH. Incubate the gel at 32°C for 10 min. Add 14.5 ml of 0.2M ATP Solution to the above mixture and continue the incubation for 50 min. Terminate the reaction by adding 7ml of a mixture consisting of ferric chloride, hexahydrate (55 g/l_ tricvhloracetic acid (20 g/l) and concentrated HCl (21 ml/l). The isozymes appear as purple bands against yellow background and are not permanent unless stored at 4°C.

9.

10.

11.

12.

Phosphoenol pyruvate Incubate the gel in the following solution for 30 carboxylase (PEP) min. at 40°C 100mM Tris-HCl (pH 8.0) 10mM MgCl2 200mM CaCl2 10mM Sodium bicarbonate 5mM Phosphoenol pyruvate During the incubation, white bands of precipitate develop indicating the position of enzymes on the gel. The phosphate released by the enzyme action is precipitated as white calcium phosphate in the gel and is stable for many hours. Glutamate dehydrogenase Incubate the gel in the following solution for 30 (GDH) min. 0.2 mM Methylthiazol tetrazolium (MTT) or 0.2 mM Nitroblue tetrazolium (MB) 0.1 mM Phenazine methosulphate 0.2 mM NAD 1.2 mM Sodium cyanide 200 mM Tris HCl buffer pH 7.5 Indolacetic Acid Oxidase Incubate the gel in staining mixture at 30°C for (IAA oxidase) sufficient time preferably over night 2,3,6-trichlorophenol 0.08 mg Fast blue BB 2.00 mg 60mM Phosphate buffer (pH 6.0) 50.0 ml Malate dehydrogenase First preincubate the gel for 15-20 min. in 200 (MDH) mM Tris HCl (pH 7.5) buffer. Then transfer the gel to a solution containing the following mixture 16mM L-Malate 0.2mM NADP 0.25mM Methylthiazol tetrazolium (MTT) 0.8mM Phenazine methosulphate 4 mM MgCl2 1.2 mM Sodium cyanide 200 mM Tris HCl (pH 7.5 buffer)

Trouble shooting and remedies S.N 1

2 3 4

5 6 7

8

Trouble Cause Failure or slow Presence of oxygen polymerization of Absence of catalyst the gel

Remedy Degas the solutions sufficiently Check if all solutions are mixed Use fresh solutions Degrease the plates with ethanol

Stock solutions aged Glass plates Poor sample wells Stacking gel and comb Fit and or remove the comb carefully Long duration of the Air bubbles interference Flush air bubbles run Bands are Insufficient Run for longer time inadequately electrophoresis resolved separation gel Bands are wavy Excess per sulphate Use optimum concentration of persulphate Stain migration is Gel is partly insulated Remove air bubbles before not even by air bubbles electrophoresis The band-lane Sample density Load equal volume of samples broadens at the in each well, equal strength bottom of sample buffer, leave no empty separation gel wells in the middle. Sample diffuses Low density of sample Increase the concentration of while loading the sucrose or glycerol in the wells sample buffer

6. Mineral nutrition 6.1. Estimation of Phosphorus Triple acid extract A quantity of 500 mg of dried and powdered plant sample was taken in a pyrex flant bottomed flask and digested with a mixture of concentrated nitric acid, sulphuric acid and perchloric acid (9:2:1 v/v). Initial digestion was done in a cold state and then digested over sand both until an ashy white digest was obtained. The digest was filtered and made upto a knwon volume. The triple acid aliquot was used for estimation of minerals. Phosphorus Estimation Reagents 1. Ammonium molybdate metavanadate reagent – 5g of ammonium molybdate dissolved in 100ml water and 1.25ml of 0.25per cent ammonium metavanadate (1.25 gm of ammonium metavenadate dissolved in 200 ml of hot water and 250 ml of concentrated nitric acid and added 50 ml of distilled water) Procedure 1. 2. 3.

Five ml of the triple acid aliquot was taken in a test tube and to this 1.25 ml of 5 per cent ammonium molybdate reagent. And 1.25 ml of 0.25 per cent ammonium metavenadate, were added for colour development. The colour developed was read in colorimeter at 470 nm. The amount of phosphorus was calculated by referring to a standard curve prepared using potassium dihydrogen phosphate and expressed as percentage on dry weight basis.

Reference Piper, B.S. 1966. Soil and Plant Analysis. Hans Publishers, Bombay.

6.2. Estimation of Potassium Principle Potassium is estimated by Flame photometry. It is known that potassium gives lilac colour due to emission on the flame. The intensity of emission is proportional to the quantity of K in the solution. Reagents Ammonium hydroxide (1:4 ammonia: water) Procedure Take 5 ml of triple acid extract and to this, add 5 ml of ammonium hydroxide (1:4) for neutralization. Feed the contents directly to the flame photo meter after adjusting the flame photometer to zero with blank and standardizing with 100 ppm of potassium solution with 100 galvanometer readings. Note the galvanometer readings. Read the corresponding ppm from the standard curve drawn for the meter. From the ppm, calculate the percentage of total potassium of the given sample. Preparation of Standard Curve Accurately weigh 1.907 g of potassium chloride and transfer it to 1000 ml volumetric flask, and make up the volume with distilled water. This solution contains 100 ppm potassium. From the above stock solution, a series of dilutions can be prepared as follows. Transfer 50 ml of the 1000 ppm K standard solution to a 100 ml flask and dilute to 100 ml with distilled water. This solution contains 5000 ppm potassium Transfer 10 ml, 20 ml, 30 ml, 40 ml and 50 ml aliquot of the 500 ppm K solution to 250 ml standard flasks. Dilute to 250 ml with distilled water and mix well. These solutions contain 20, 40, 60, 80 and 100 ppm and drawn the standard curve. Weight of the sample taken = Volume made upto = Content of K in plant material with refernce to the standard graph ppm Therefore the percentage of total K on moisture free basis 

PPM

10

6

xV x

„W‟g „V‟ ml.

100 100 x 100  M W

Where M = Moisture content of the plant sample. Reference Sumner, J.B. 1944. A method for colorimetric determination of phosphorus, Science 100: 413.

6.3. Estimation of Calcium and Magnesium Principle Calcium and magnesium get commplexed by EDTA in the orde Ca first and Mg afterwards. In this experiment Ca is estimated first by using an indicator murexide at pH 12 in the presence of sodium hydroxide. The calcium + Magnesium is estimated by using an indicator Eriochrome black T at pH 10 in the presence of ammonium chloride and ammonium hydroxide buffer solution Materials required 1. 0.02 N EDTA 2. 10% sodium hydroxide 3. Ammonium Chloride ammonium hydroxide buffer solution 4. Murexide indicator 5. Eriochrome black – T indicator Procedure Calcium alone Pipette out 25 ml of triple acid extrafct into a porcelain basin. Add 10% Sodium hydroxide drop by drop to neutralise the acidity (red litmus turns blue) and add another 5 ml excess to maintain the pH at 12. Add a pinch of murexide indicator and titrate against 0.02 N EDTA till red colour changes from pinkish red to purple or violet. Calcium & Magnesium Pipette out 25 ml of triple acid extract into a porcelain basin. Add ammonium chloride - ammonium hydroxide buffer solution drop by drop to neutralise the acidity and add 5 ml excess to maintain the pH at 10. Add 2-3 drops of Eriochrome black Tindicator and tirate against 0.02 N EDTA till the colour changes from purplish red to sky blue. Observations and Calculations Weight of plant sample taken = 'Wg' Volume of triple acid extract prepated = 'V' ml Volume of triple acid extract pipette out for titration = 25 ml Volume of 0.02 N EDTA used for Ca + Mg = 'A' ml Volume of 0.02 N EDTA used for Ca alone = 'B' ml Volume of 0.02 N EDTA used for Mg alone= 'A-B' ml

1 ml of 0.02 N EDTA = 0.0004 g of calcium Percentage of calcium in the given sample on moisture free basis  o.ooo 4 x B x

250 100 x  M) 25 3

1 ml of 0.02 N EDTA = 00024 g of Mg. Percentage of Magnesium in the given sample on moisture free basis 250 100 100  o.ooo 4 ( A  B x x x ) 25 3 100  m Where M = Moisture percentage in the given sample.

Reference Jackson, M.L. 1973. Soil chemical analysis. Prentice Hall of Indian Pvt. Ltd. New Delhi.

6.4. Estimation of Iron and Manganese (Jackson, 1973) Iron Ten ml of the plant extract (triple acid extract) was taken in a 250 ml beaker and added 0.5 ml of concentrated HCl. Two ml of hydroxylamine hydrochloride and one ml or orthophenanthroline, pH ajusted between 1.3 - 2.7 were added and made up to 25 ml. The colour developed was measured in Systronic colorimeter using 490 m. This was compared against the standard. Manganese Ten ml of the plant extract was evaporated to dryness in a beaker. Five ml of HNO3 and 2 ml of H2O2 were added to destroy the organic matter and again evaporated to dryness. 20 ml of phophoric acid solution (H3PO4 9 per cent - V/V) and 0.3 g of potassium periodate (KIO4) were added. This solution was boiled for 3 minuted for full colour development, cooled and made up to 100 ml. The colour intensity was measured in colorimeter (540 m) and compared with standard. Reference Jackson, M.L. 1973. Soil chemical analysis. Prentice Hall of Indian Pvt. Ltd. New Delhi.

SULPHATE CONTENT Procedure: (0.2g) of dried ground leaf sample is taken in a 50ml kjeldahl flask and is digested with amixture of 0.1g selenium oxide, 10ml of concentrated HNO 3 and one ml of concentrated HCL.To the residue 0.2ml of dilute HCL is added and again evaporated to dryness.One ml of conc.HCL and 25ml distilled water is added and then boil and filter in 100ml volumetric flask.The filtrate is made to 50ml by 5ml 3%glycerol and distilled water. 10ml of aliquot is taken in a test tube and 0.5ml of 2% barium chloride solution is added. Sulphate is precipitated as barium sulphate and the absorbance is read at 420nm against blank. The actual sulphatr content is calibrated from the bstandard curve prepare by potassium sulphate solution and is expressed as mg/g dry wt. Reference: McDonald and Chen. 1965. Anal Biochem., 10: 175-177.

Plant Tissue Tests Nitrate nitrogen (NO3-N) Reagents

1% Dipenylamine Dissolve 1 g of diphenylamine in 100 ml of H2SO4. Prepare afresh every time

Procedure

Small bits of tissues are taken in porcelain dish and a drop of diphenylamine is added. Blue colour formation indicates the NO3-N presence. Degree of colouration indicates the amount present. Dark blue – Sufficient nitrogen Light blue – Deficient No colour – Highly omogenat

Phosphorus Reagent

Ammonium molybdate solutionDissolve 8 g of ammonium molybdate in 100 ml of distilled water and slightly warm. To this add 120 ml of conc. HCL and the volume made up to 250 ml. It is the stock solution. It should be diluted whever it is be used.

Dilution : 1 ml diluted to 4 ml Procedure

A teaspoon full of finely chopped leaf bits are taken in a test tube and 10 ml of diluted reagent is added and shaken for about 30 seconds. After shaking a pinch of stannous chloride is dded and shaken. Blue colour develops Dark Bluish green No colour -

Sufficient Slightly omogenat Deficient

Comparison of colours are made from the standards prepared from KH2PO4.

Potassium Reagent

Sodium cobaltinitrite solution 5 g of sodium cobaltinitrite and 30 g of sodium nitrite are dissolved in 80 ml of distilled water to which 5 ml of glacial acetic acid are added and the volume made up to 100 ml. The solution is to be stored in amber coloured bottle. Dilution : Take 5 ml of the above solution and 15 mkg of NaNO2 is added and made up to 100 ml with distilled water

Procedure

Take 10 ml of the diluted reagent and add ½ teaspoon of leaf tissue (finely chopped) and shake it for one minute. After one minute add 5 ml of ethyl alcohol. Intensity of turbidity indicates the presence or absence. Clear Intense -

K absent K sufficient

Iron Iron is first converted from ferrous to ferric form and estimated Reagent

1. Concentrated hydrochloric acid 2. Concentrated nitric acid 3. Ammonium thiocyanate (20%) 4. Amyl alcohol

Procedure

0.5 g of the material to be tested is taken and 1 ml of conc. Hydrochloric acid is added and allowed to stand for 15 minutes. After this add 10 ml of distilled water followed by 2-3 drops of concentrated nitric acid. After 2 minutes take 10 ml of this solution and add 5 ml of 20% ammonium thiocyanate. Brick red colour Faint pink colour

- Fe present - No iron

To quantity, use FeSO+ standards. Comparison should be done within 30 minutes after colour formation, drops of amyl alcohol is added.

Reference: Jackson, M.L. 1962. Soil chemical analysis. Constable and Co Ltd. London., pp. 498.

7. Plant growth regulators 7.1. Estimation of auxins (indole 3 acetic acid or IAA) IAA is an important hormone involved in plant growth and development. It stimulates cell enlargement, cell division, root growth and inhibits lateral buds. It is synthesized from tryptophan as a precursor of aminoacids. Colorimetric method Gorden and Paleg (1957) standardized a method of measure IAA. This method permits the detection of IAA in crude extract. The methanol extract from the culture filtrates of microorganisims can be directly estimated for IAA as there are no interfering substances. However, the plant extracts should be purified. Reagents 1. IAA extract from culture filtrate of microorganisims or chromatograms of plant extract 2. Salper reagent: Mix 1ml of 0.5M FeCl3 in 50ml of 35% HCIO : Prepare fresh. Procedure 1. Remove the spot containing IAA, from the chromatogram corresponding to the authentic IAA and elute in 1 or 2 mol of methanol or ethanol (elution time, 30min). 2. To 1ml of extract containing 1AA, add 2ml of Salper reagent. Add the reagent dropwise but rapidely with continuous agitation. 3. Incubate the samples in the dark (35 min for diethyl ether dissolved IAA; 60min for methanol sample). The pink colour is stable. 4. Measure its absorbance at 535 nm against a solvent-reagent blank. If the intensity is deep, dilute the reaction mixture with the solvent. 5. From a standard curve drawn from known concentrations of IAA, find out the quantity of IAA in the extract. Reference Gorden, S.A. and L.G. Paleg (1957). Physiol. Plant, 10, p.37-48.

Bioassay of IAA There are numerous biological assays to measure auxin activity. Some of them require special rooms to raise the seedlings while a few tests are relatively easy to perform. Avena straight growth test, the pea curvature test and rice root inhibition assay are easy to conduct in the laboratory and are described. Straight Growth Test for Auxin Assay Procedure The eliolated pea seedlings grown in dark and 3 hours exposed to light daily for 8 days after germination were used in this experiment. The material required in this test is similar as in slit pea test. The uniform sections of stem having 2 cm length were cut after discarding apical 5cm epicolyte for greater sensitivity. The uniform sections were then kept in petridishes containing various concentrations of I.A.A. as used in slit peat test i.e. 5,10,25,50 and 100 ppm respectively after labeling properly. All these manipulations were carried out in red light only and further the petridishes were kept in dark for 24 hours. Before keeping sections in solutions, they were washed thoroughly with distilled water with a view to make it free from native auxin. Minimum segments were maintained in each petridish as far as possible. After 24 hours, the length of pieces was measured. Period can be increased depending on response. Observational Data Particulars S. N. Con. Of IAA in ppm 1 Control 2 5 ppm 3 10 ppm 4 25 ppm 5 50 ppm 6. 100 ppm

1

2

3

4

5

6

Mean

2.0 1.9 2.5 2.0 2.0 2.0

2.0 2.5 2.0 2.0 2.0 2.0

1.8 2.0 2.0 2.0 2.0 2.0

1.9 2.0

2.0 2.0

2.0 2.0

1.95 2.13 2.16 2.0 2.0 2.0

Result

The straight growth in etiolated pea segments is increased in case of 5 x 10 ppm con. Of IAA solution and in all rest of the treatments, it remained unaffected as compared to the control : Concluding remarks

Microscopic examinations of treated material can be taken up for cell elongation studies. Dimension can be recorded. Number of cells/unit is to be recorded to know auxin affect on cell division. Preparation of dilutions The dilutions of stock solution can be made following the equation given below:

Where

M x V = M‟ x V‟ M = Con. Of stock solution V = Volume of stock solu. To be diluted M‟ = Con. Of Required solution V‟ = Volume of required solution

Example

Con. Of stock solution = 100 ppm (M) Con. Of Required solution = 50 ppm (M‟) Vol. of Required solution = 100 ml (V‟) M‟ x V‟ Find V = -------M 50 x 100 V = ---------- = 50ml. 100

Estimation of Gibberellins by calorimetry

In 1938 crystalline gibberellin A was isolated by the Japanese workers form the culture filtrate of Gibberalla fujikuroi, a fungus that was responsible for disease of rice. Extraction of free gibberellins-like substances from plants Gibberellins also occur in plants in bound and free form. Free Gibberellins Reagents 1. Absolute and 80% methanol 2. Ethyl acetate 3. 1.6M HCL, and 3.2M HCL 4. 0.48M NaHCO3 5. PVP Method 1. Homogenize 250g of shoot portions in belndor at 4 oC with suitable aliquot of 80% chilled methanol (10ml/g fresh tissue) and leave overnight at 4oC. 2. Filter through Whatman No.41 filter paper and re extract the residue twice with ethanol in cold 3. Combine the methanol extracts and evaporate the methanol at 40 oC using a rotary evaporator. 4. Freeze the water phase of the extract, thaw it to the room temperature and filter. 5. Stir the aqueous phase of the extract with PVP, acidity it with 3.2M HCL to pH 2.5 and extract thrice with equal volumes of acetate using separatory funnel and save the aqueous phase for bound gibberellins. 6. Combine the ethyl acetate extracts and partition it with 0.48M NaHCO 3 7. Adjust the ph of the bicarbonate phase to 2.5 with 1.6 M HCl. 8. Combine the ethyl acetate fractions and evaporate the dryness in a rotary evaporator at 40oC. 9. Dissolve the residue in minimum quantity of methanol (containing free acidic fractions of gibberellin-like substances). Estimation of gibberellins Spectrophotometric method Principle The amount of GA produced can be estimated based on the conversion to gibberellic acid followed by the measurement of its absorption at 254nm. Reagents 1. Gibberellin extract 2. 5% and 30%HCL 3. Standard GA solution : 5 -50 µ g/ml 4. Zinc acetate solution: Dissolve 21.9g of Zinc acetate in 80ml distilled water, add 1ml of glacial acetic acid and make up the volume to 100ml with distilled water

5. Potassium ferrocyanide solution: Dissolve 10.6g of A.R. Grade potassium ferrocyanide in 100ml of water. Procedure 1. Pipette 15ml of the culture filtrate or the extract containing GA to the test tube and add 2ml of zinc acetate to it. 2. After 2min, add 2ml of potassium ferrocyanide 3. Centrifuge at low speed of 15min. 4. To 5ml of the supernatant, add 5ml of 30% HCL in a test tune and incubate the mixture at 20oC for 75min 5. Treat the blank sample with 5% HCL. 6. Measure the absorbance of the sample and the blank at 254nm. Reference Holbrook. A.A., W.J.W. Edge and F. Bailey (1961). Spectrophotometric method for determination of gibberellic acid In: Gibberellin, A.C.S. Washington, D.C. pp.159-167.

Bioassay for Gibberllins – Amylase release test from seeds

The barley endosperm assay has been modified by Ogawa (1966) using rice seeds and Jones and Warner (1967) using barley seeds to avoid interference by the endogenous reducing sugars present in the extracts and the residues of some of the organic solvents used. In this test the enzyme activity is determined and is insensitive to solvent residues. The method of Ogawa (1966) is described here. Materials 1) Rice seeds, any variety 2) Gibberellin extract 3) GA3 standards 4) Pipettes, 1 ml, 5 ml 5) Seitz filter or autoclave 6) Glass mortar and pestle 7) Whatman No.1 filter paper 8) Incubatore at 30 C 9) Water bath at 40 C 10) Colorimeter Reagents 1) Sodium hypochlorite solution (1% active chlorine) 2) 0.2% (w/v) Calcium chloride 3) Sodium acetate buffer, pH 4.8 containing 0.25% (w/v) starch (potato starch) 4) 0.5 M Acetic acid 5) 0.03 N Iodine solution Procedure Cut the dehulled rice seeds transversely with a razor. Sterilize the apical halves without embryo with sodium hypochlorite solution for 10-20 min and rinse in several changes of sterile distilled water. Weight them in groups of five. Sterilize the test solution (gibberellin extract / GA standard) by filtering through Seitz filter or by steaming for 15 min twice at 24 hr interval in an autoclave. Pipette 1 ml of the test solution into test tubes and transfer the weighed embryoless seed halves under aseptic conditions to the test solution. Incubate the seeds for 4 days at 30° C. Discard the test solution and rinse the endosperm in small volumes of distilled water and grind them with 5 ml of 0.2 per cent calcium chloride in a small glass mortar. Filter the homogenate through Whatman No.41 filter paper (Alternatively, it can be centrifuged at 2,000 g for 10-15 min to discard the suspended materials). Pipette 1 ml of the filtrate to test tubes containing 4 ml of sodium acetate buffer, pH 4.8 containing 0.25 per cent starch and incubate the reaction mixture at 40 C in a water bath for 30 min. Pipette 0.5 ml of the reaction mixture into another test tube containing 5 ml of 0.5 M acetic acid to stop the reaction. Add 5 ml of 0.03N iodine to the mixture and measure the absorbance at 700 nm in a colorimeter. Maintain a zero hour control. Calculate the -amylase activity using the formula :

C–E ------- x 100 (%); where, C is the optical density of the initial starch C Solution at the end of the reaction. Express the enzyme activity per unit wt of the seeds. Use a dosage response curve prepared with GA3 to calculate the GA content in the test solution.

Extraction and estimation of cytokinins by omogenate hy Extraction Extraction of cytokinins from the plant tissues poses some problems due to the presence of interfering substances and due to losses occurring during purification. Reagents 1. Seedlings (15-20 days old seedlings) 2. 1N HCL 3. 1N NaOH 4. Mellvaine‟s buffer, pH 5.6 5. Ethanol 6. Ether 7. n-Butanol Procedure 1. Homogenize 25g of fresh tissues in 25ml of buffer, in a blendor 2. Add 150ml of ethanol and after 6-8h, centrifuge at 2000g for 20min 3. Decant the supernatant and evaporate it to 10ml under reduced pressure on a rotary evaporate at 40oC. 4. Adjust the pH of the solution to 2.9 with 1N HCL 5. Shake the extract in a separatory funnel thrice with 10ml of ethyl ether to remove chlorophylls, auxins and gibberellins 6. Discard the ether layer. Adjust the pH of the aqueous solution to 7.8 with 1N NaOH 7. Extract with 5ml portions of n-butanol six times in a separatory funnel. 8. Pool the butanol layers and evaporate to dryness under reduced pressure at 40 oC. 9. Dissolve the residue in 2ml of buffer and use it for chromatography. Reference Sridhar, R. S.K. Mohanty and A. Anjaneyule (1978). Physiol. Plant 43, pp.363366.

Estimation of cytokinins – Radish cotyledon test The amount of omogenat present in the extracts is measured by using bioassays. However, gas chromatography may be employed only with highly purified extracts. Cytokinins may be assayed using leaf senescence, chlorophyll production, cotyledon enlargement or callus growth bioassays. Callus bioassay is the most specific and dependable, but takes more time and needs a well organized laboratory. The radish cotyledon test is described below. Radish cotyledon test Cytokinins reduce the growth of radish cotyledons which is primarily due to cell expansion rather than cell division (Letham, 1971). This test is sensitive to kinetin (10 µ g to 25mg/L) and 2ip and is insensitive to IAA. Materials 1. Cytokinin extract 2. Kinetin standards (10 µ g to 25mg/L) 3. Radish seedlings (2-3 days old) grown in dark Procedure 1. Select uniform sized cotyledons 2. Float the preweighed cotyledons in groups of 5 in 7ml of either water (control) or test solutions in 10cm petridishes or place them on filter paper moistened with test solutions 3. Incubate the leaves under fluorescent light for 3 days 4. After incubation, remove the leaves, blot them dry and determine their fresh weight or measure the leaf area. 5. Correspondingly, run a standard with known concentrations of kinetin and draw a dose response curve. Reference Letham, D.S. (1971). Physiol. Plant 25, pp. 391-396.

Estimation of cytokinins by ELISA Collection of xylem sap Xylem omogena was collected 50 DAS and 70 Das i.e. just after the time of panicle initiation (50 DAS) and before flowering (70 DAS). Sap collection was done in the morning time from 7.30 a.m. to 10.00 a.m. because of more root pressure in morning time. The plants were cut 5 cm above the soil surface. The detopped plants with roots and soil intact were used to collect xylem exdate. The xylem omogena exuded was collected with the help of capillary tubes from the cut portion with natural root omogena. About 0.14 ml to 0.6 ml of omogena was collected from the plants in 20 minutes and sap collected was transferred to sterile vials after determining the volume. For all the samples betylated omogena toluene (BHT) an I (10 mg/l) and EDTA a chelating agent (10 ml/l were added to the omogena before they were stored at -20ºC. Determination of CK content in xylem sap Cytokinin content in xylem omogena was determined using a competitive omogenate by ELISA technique using polyclonal antibodies. Enzymes immuno assay, sometimes called the enzyme linked immunosorbant assay (ELISA), combines the specificity of antibodies with the sensitivity of simple spectrophotometric enzyme which also possess a high turn over number. The unknown concentration of antigen can be determined from a reference dose response curve constructed with unknown concentration of standard antigen. Antibodies were raised against t-zeatin riboside ELISA may be used for assaying antigens by either a competitive method or a double antibody method and for a specific antibody by an indirect method. The competitive method (Director ELISA) A mixture of a known amount of enzyme – omogen antigen and an unknown amount of unlabelled antigen is allowed to react with a specific omogena attached to a solid phase. After the complex has been washed with buffer, the enzyme substrate is added and the enzyme activity measured in the conventional way. The difference between this value and that of a sample lacking unlabelled antigen is a measure of the concentration of unlabelled antigen. Materials required 1. Tracer (CK – Alkaline phosphatase conjugate) 2. Fractional – G 3. ELISA plates 4. Buffers

Reagents Coating buffer (Carbonate buffer / bicarbonate buffer, 0.5 m pH 9.5) Na2CO3 NaHCO3 NaN3 H20

1.59 g 2.93 g 0.20 g 100 ml

can be stored at 4ºC for minimum 14 days 2. Washing buffer (PBS / Tween pH 7.4) NaCl 8.0 g KCl 0.20 g KH2PO4 0.2 g Na2HPO4.H2O 1.15 g Tween - 20 0.50 ml H2O 1000 ml can be stored at 4ºC for minimum 14 days 3. Dilution buffer (blocking buffer) Casein 1.0 g Washing buffer 100 ml (should be prepared just before use) Tracer One of the essential components of competitive binding assay is the tracer. Preparation of CK - Alkaline Phosphatase (CK - Alpase ; Tracer) conjugate 0.5 mg t-zR was dissolved in a solution of 100 l Dimethyl formamide, 15 l of double distilled water and 15 l 0.1 M NaIO4. The solution was stirred for 30 minutes at room temperature. Unreacted NaIO4 was then destroyed by adding 5 l 0.1 Methylene glycol and stirring was continued for another 30 minutes at room temperature. This solution was added in portions of 10 l to an ice-cold preparation of 0.25 mg Alpase disolved in 50 l NaHCO3 buffer (50m, pH 9.6). The solution was incubated for 16 hours 4ºC in the dark with continuous stirring, and then the pH was adjusted to 9.3 with 0.1 M NaHCO3. NaBH4 (0.1 mg) was added and the solution is stirred for another hour at 4ºC in the dark. After this the pH was brought to 6.5 with 0.1 N HCl and the enzyme conjugate was dialyed for 3 days against PBS buffer (50 m pH 7.8) containing 0.01% (W/V) NaN3. the dialyzed conjugate was stored in 50% (V/V) glycerol at - 18ºC. ELISA procedure Coating the plate with antibody 1. Antibody was diluted using coating buffer and 200 l of it was added to each well and the plate was incubated at 4ºC overnight.

2.

The contents in the well were discarded and the plates were washed 3 times with washing buffer keeping 3 minutes each time at room temperature.

Blocking step This step is important to reduce the reaction of BSA specific antibodies which cause interference by using blocking buffers. 3. The wells were filled to full capacity with blocking solution (PBS + tween + 1% casein) and the plate was incubated for 30 minutes at 37 deg C. 4. Plate was washed thrice with washing solution Addition of the subsequent reagents 5. 200 l of dilution buffer was added to the wells and to this 50 l of pre-diluted tracer was added 6. Plate was incubated for 1 hour at 37ºC 7. It was washed 3 times as described earlier. Detection 1. Para nitro phenol phosphate (PNPP) was prepared in coating buffer (lmg/ml), 200 l of which was added to each well. 2. Plate was incubated for 1 hr at 37 C 3. Reaction was stopped by added 25 ml of 5 N JOH 4. PNPP was added to two empty wells and was taken as control 5. Absorbance was read at 405 nm using ELISA reader Construction of standard curve Tracer solution (1: 1200) : Initial cheque board titration showed that the optimum dilution of enzyme labelled antigen (Alp-tZR Ag) i.e., the tracer was 1:1200 at an antibody (t-ZR-Ab0 dilution of 1:1000. A concentration range of 0-10p, mol/0.1 ml may be tried using pure standard (tZR-Ab) dilution of 1:1000 A concentration range of 0.10 p.mol/0.1 ml may be tried using pure standard t-ZR in a competitive binding direct ELISA. tZR standards 0, 0.02, 0.05, 0.2, 0.5, 2.5, 10 p moles - Preparation of tZR stock (5 mM solution) a. b. c.

17.57 mg tZR in 10 ml dimethyl formamide Diluted this to 1:50 with DHI to have a 0.1 mM stock 0-10 P.moles and 100 P.mole t-ZR solution were prepared from this using PBS, pH 7.5.

Buffers a. b. c. d.

Coating buffer Diltion buffer Washing buffer Blocking solution / PBS + Tween + 1% casein

Procedure Instead of 200 w of dilution buffer and 50 of 1:1200 tracer and 100 l of tZR standards were added in duplicates. For finding out 100 per cent binding 100 l PBS pH 7.4 and 100 l tracer were added to the wells and for 0 per cent binding 100 l 100 p.mole tZR standard and 100 l tracer were added to the wells. Rest of the procedure was as described earlier for determination of working titre. Calculations 1. Optical density was recorded 2. Averages of optical density of duplicate standards were taken 3. Per cent binding of each standard was calculated using the formula Standard OD NSB OD % Binding

=

x 100

Bo OD-NSB OD = 100 per cent binding = Non-specific binding per cent binding Per cent binding was converted to logit B/Bo Logit B/Bo = Ln (B/Bo %) (100 - B/Bo) Standard curve wad drawn using t-ZR (P mole / 0.1 ml) against logit B/Bo values. The crude xylem sap was used as such without processing for determining the CK content in xylem sap. The cytokinin content was expressed in picomole / 0.05 ml. Bo NSB

Procedure 100 l of 1:1200 tracer and 50 l of xylem sap were added to the wells replacing standards (rest of the procedure was followed as described earlier). The cytokinin concentration in plant sap was determined by extrapolation of the sample per cent binding from the best fit standard curve.

7.8. Abscisic acid Asbscisic acid (ABA), a sesquiterpenoid is considered as the only naturally occurring growth inhibitor. It was referred to forming and abscisin II. ABA is present in almost all plants. Extraction from plant tissues Reagent 1. 70% Methanol 2. 1N NaHCO3 3. 1N HCL Method 1. Homogenize the fresh leaves in methanol using a blendor 2. Filtrate the homogenate through Buchner funnel using Whatman No. 41 filter paper and wash the residue with excess methanol 3. Pool the filtrate and washings and reduce its volume to 15 per cent of the original volume at 40oC 4. Adjust the pH of the concentrated extract to 8.% with 1N NaHCO3 5. Partition the alkaline extract with petroleum ether thrice and discard the petroleum ether fraction 6. Readjust the pH of the aqueous fraction to 2.7 with 1N HCL 7. Partition the acidic extract twice with ethyl acetate 8. Pool the ethyl acetate fractions and evaporate to dryness at 40 oC in a rotary evaporator. 9. Dissolve the residue in minimum volume of methanol and use for chromatographic separation of ABA and for subsequent bioassays Reference Mohanty, S.K., A. Anjeneyulu and R. Sridhar (1979). Physio. Plant, 45, pp.132-136.

7.9. Estimation of ABA - Inhibition of α-amylase synthesis in barley endosperm ABA inhibits the synthesis of α-amylase in the aleurone layers which is triggered by gibberellins. A bioassay has been developed to determine ABA activity. Mohanty et al. (1979) used this test to detect ABA like activity in extracts of diseased plants. Materials and reagents 1. ABA extract 2. ABA standard solutions 3. Barley seeds 4. 1% Sodium hypochlorite solution 5. Streptomycin sulphate solution (1mg/ml) 6. GA solution, 10-7 M. Method 1. Sterilize the seeds by soaking in hypochlorite solution for 20min and rinse in several changes of sterile distilled water. 2. Cut the seeds transversely with a razor 3mm from the distal end and discard the piece with embryo. 3. Pipette out 1ml of GA solution, 0.5ml of streptomycin sulphate, 0.2ml of ABA extract and 0.8ml of distilled water to make up the voloume to 2.5ml into 5ml glass lidded vials. 4. Weigh the embryoless barley seed halves in group of 4 and transfer them to the vials. 5. Maintain controls with distilled water and standards with known concentrations of ABA. 6. Incubate the vials for 32hours at 28 ±2oC in the dark 7. Estimate the reducing sugars released into the ambient solution by the action of αamylase on the endosperm starch, employing either Nelson‟s or DNS method. 8. Express the results as amount of reducing sugars released per mg dry weight of the endosperm. This test may also be performed with rice or wheat seed. Instead of assaying the reducing sugars released, the enzyme activity may be measured. Reference Mohanty, S.K., A. Anjeneyulu and R. Sridhar (1979). Physio. Plant, 45, pp.132136.

7.10. Estimation of Abscissic Acid by ELISA Preparation of plant sample: The seedlings were washed with Tris buffer (150 mm Tris - HCl. pH 7.0 and homogenised in the same. The homogenate taken into method of appropriate volume and stirred with magnetic stirbass over night at 4C in dark. Extract were centrifuged for 10 min at the super natrients concentrated. The Aba contents of the sample was measured by ELISA using monocbenal antibody for Aba (Walker & Simmom 1987). Root ABA estimation The method used for tissue analysis was a modification of the methanolic extraction method of Weiler (1990) as described by Wolf et al., 1990. Tisse sample were freeze-dried in liquid nitrogen and extracted over night at 4 C in 80% aqueous methanol. The methanolic extracts of tissues were passed over a Sap-pak C18 cartridge to remove pigments and other lipophilic impurities. The methanol was removed by vacuum distillation and the aqueous reside was partitioned three times against ethyl acetate at pH 3.0. The combine ethyl acetate fractions were reduced to dryness under reduced pressue, the reside taken up in saline Tris buffer (TBS) (50 mol. m Tris, 150 mol m NaCI, 1.0 mol m Mgcl, pH 7.8). Recovery determinations were done by adding an internal standard (10,000 dpm of high specific activity 3 (G ABA) to the methanolic extracts. Recovery of ABA after purification was between 95-97%. ABA immunoassay Polyclonal antibodies were raised against the C carbonyl site of cis-trans ABA after conjugating it to a carrier protein. Discrimination between ABA free acid and its conjugates was much less with antibodies raised to ABA coupled to an immunogenic carrier through the carboxyl (C-4) site as compared with the carboxyl group, an observation made earlier by Weiler (1980) and Quarrie and Galfre (1985). Specificity studies showed that the cross-reaction of the C antibodies to the naturally occurring conjugate ABA-glucose ester was less than 0.5% on a molar basis and less than 0.4% with phaseic acid or the synthetic conjugate ABA methyl ester. A direct ELISA was adopted for estimation of ABA on samples with minor modifications. Nonspecific binding was nullified by using 1% casein in PBS - Tween in the blocking step. Tracer (ABA - Alkaline phosphatase) conjugation was done by the method of Weiler (1982) per cent binding of each standard point or sample was calculated by the following : Standard or sample O.D - NSB O.B % binding

= Bo o.D - NSB O.D.

Bo NSB

= =

100 l buffer + 100 l tracer = 100% binding nonspecific binding = 100 pmole / 0.1 ml ABA + tracer = 0% binding.

Estimation of Ethylene by Gas Chromotography Principle The ethylene evolved is measured in a gas chromatograph based on adsorption principle on activated silica gel or porapak. Materials 1. Conical flasks or cylinders with facility to seal with rubber gaskets 2. Air tight syringes 3. Ethylene gas (standard) 4. GLC-see „Nitrogenase‟ enzyme for opening conditions Method 1. Place the leaves/fruits in conical flasks or cylinders 2. Seal the mouth with rubber septum or gasket 3. Incubate for 1 hour at 20oC 4. Withdraw gas samples with hypodermic syringe and inject into GLC 5. For standard, inject pure ethylene into empty conical flasks or cylinders of same volume and satisfy identical assay conditions. Remove the same volume of internal atmosphere as that of the sample from the flask. Inject into GLC and measure ethylene peak height. Calculation Refer „Nitrogenase‟ The quantity of ethylene produced is expressed as µ l ethylene per hour per kg material. Reference Teitel, D.C., Ahavoni, Y. and Barkai-Golan, R. (1989). J. Hortic. Sci., 64, 367. 7.12. Estimation of Ethylene by Colorimetry Ethylene trapped in aqueous mercuric chloride may be released and oxidized to formaldehyde to measure its concentration colorimetrically (LaRue and Kurz, 1973). The probable first products of oxidation of ethylene are ethylene glycol and some glyoxal. These are oxidized to formaldehyde, formic acid and CO 2. The amount of formaldehyde formed is determined with Nash reagent which is based on the Hantzsch reaction between acetylacetone, ammonia and formaldehyde which forms 3, 5 – diacetyl1, 4-dihydrolutidine. The reaction is specific for formaldehyde. Materials 1. Gas (ethylene) sample 2. Conical flask, 10 ml sealed with rubber serum bottle cap 3. Pipettes, 1 ml, 2 ml 4. Syringe 5. Rotary shaker 6. Clinical centrifuge 7. Colorimeter

Reagents 1. Sodium metaperiodate, 0.05 M : Dissolve 16.5 g of NaIO4 in 1 liter of water. Store in a dark bottle. For keeps reagent 4 weeks. 2. Potassium permanganate 0.005 M : Dissolve 0.79 g of KMnO 4 in one liter of water and store in dark and well stoppered bottle. A slight precipitate usually forms after 1 or 2 days. If more precipitate occurs, use fresh reagent. 3. Sulphuric acid, 4 N 4. Sodium arsenite 4M : Dissolve 52 g of NaAsO2 in 100 ml of water. 5. Nash reagent : Dissolve 150 g ammonium acetate in water and add 3 ml of acetic acid and 2 ml of distilled acetyl acetone (2, 5-pentanedione) and dilute the mixture to 1 liter with water. Procedure Place 1.5 ml of oxidant solution in a 10 ml conical flask and seal it with a rubber serum bottle cap. Transfer 1 to 5 ml of gas sample containing up to 1 mole of C2 H4 from a chamber in which the plants are incubated or from reaction vials used to release (use section on manometric estimation of ethylene) trapped ethylene using a syringe. If necessary, remove some of the atmosphere in the conical flask with a syringe prior to injection of the gas sample. Agritate the conical flask vigorously on a rotary shaker at 300 r.p.m. for 90 min at room temperature (22 C). Add 0.25 ml of 4 M NaAsO 2 to the flasks and mix well to destroy the excess of oxidant. Add 1 ml of Nash reagent to the contents of the flash and after 60 min determine the absorbance of the solution at 412 nm in a colorimeter. Calculate ethylene concentration from a standard curve.

7.13. Bioassay of Ehtylene - Epinastic response Ethylene induces epinasty of leaves of many plant species and this physiological character of the gas was used by Crocker et al., (1932) to devise the bioassay. Materials 1. Tomato seedlings, 12-15 days old (older plants respond slowly) 2. Bell jar or air – tight glass chamber with vent sealed with rubber serum caps 3. Diseased plants or gas sample Procedure Place the tomato plants and diseased plant materials on a table. Cover them with a bell jar. Observe the epinastic response within 2 to 3 days. Alternatively, keep the tomato plants in an air – tight glass chamber provided with a vent sealed with rubber serum cap and inject the gas samples collected from the above experiments and observe the plants after 2 to 3 days for epinastic response.

7.14. Bioassay of Ehtylene - Triple pea test Based on the physiological effect of ethylene to cause subapical thickening of the stem, reduction in the rate of elongation and horizontal nutation of the stem in etiolated pea seedlings the triple pea test has been developed (Pratt and Biale, 1941). This test response to ethylene concentrations between 0.025 and 0.1 ppm of ethylene. Materials 1. Etiolated pea seedlings 2. Petri dishes 3. Bell jar or air – tight glass chamber with vent sealed with rubber serum caps Procedure Place the etiolated pea seedlings (4 cm height) in Petri dishes. Cover the dishes containing pea seedlings and diseased plants kept on a table with a bell jar. After 2-4 days depending upon ethylene concentration, observe the plants and photograph them. Alternatively, place the etiolated pea seedlings in an air-tight glass chamber provided with a vent sealed with a rubber serum cap and inject the gas samples collected from the above experiments and observe the plants after 2-4 days. Treat the etiolated pea seedlings with known concentration of ethylene under similar conditions and photograph them. In the presence of ethylene, epicotyls show increased growth in thickness and reduced rate of longitudinal and horizontal growth. Compare the response with the test plants.

7.15. Bioassay of Ehtylene - Pea stem swelling test Ethylene markedly increases the stem swelling of pea seedlings and this can be used to measure ethylene concentration (Cherry, 1973). Materials 1. Pea seeds, local cultivar 2. Vermiculite / peat moss 3. Ethrlenmeyer flasks 4. Bell jar with vent 5. India ink Procedure Plant pea seeds in vermiculite / peat moss and allow them to germinate in the dark at room temperature. After 4 days, the stems should have a height of about 2 or 3 cm. Remove the roots by cutting just below the cotyledons. Mark the epicotyl with an India ink pen at a position 1 cm below the apex. Place 10 of the derooted cuttings in a petri dish and place in an air-tight bell jar provided with a vent sealed with a rubber serum cap containing diseased cotton plants. Incubate the set-up under a bank of light. Swelling is measured by cutting the epicotyls off at the India ink mark and measuring individual lengths and fresh weight of the group of apical pieces. Incubate the samples for 24 hr. The initial weight of the 1 cm apex is found by cutting ten epicotyls at the India ink mark and weighing. The presence of the cotyledons is essential for good swelling response to ethylene. The results are expressed as a ration of weight to length. Ratios for a control cutting (no ethylene) should be about 3.0 ; 1 ppm of ethylene should increase this ratio to about 4.0.

7.16. Bioassay of Gibberellin - Anthocyanin inhibition test GA inhibits anthocyanin synthesis in young tomato seedlings and Khan (1980) developed a test. The method is sensitive for GA3 concentrations from 10 -5 to 10 mg per litre. The extraction and estimation of anthocyanin are based on the method described by Rabino et al., (1977). Materials 1. Gibberellin extract 2. GA standards 3. Tomato seeds 4. Petri dishes, 10 cm 5. Beakers, 100 ml 6. Whatman No.41 filter paper 7. Filter paper sheets 8. White fluorescent light source 9. Incubator at 20 C 10. Refrigerator 11. Colorimeter Reagents 1. 0.1% Mercuric chloride solution 2. 1% HCl in methanol (w/v) Procedure Surface sterilize tomato seeds with 0.1 per cent mercuric chloride solution and wash them several times with distilled water. Germinate the seeds in the dark at 20 C in Petri dishes lined with moist filter paper. Transfer 20 uniformly germinated seeds into 10-cm Petri dishes lined with filter paper moistened with 3 ml of test solution. Incubate the Petri dishes containing growing plants inside sealed plastic boxes lined with moist filter paper in the dark for 7 days at 30 C. Expose the plants to continuous illumination (white fluorescent light giving 2000 lux at seedling level) at 20 C for 40 hr. Select 15 seedlings of uniform growth, weight them intact and plunge them (whole seedlings with root and shoot portions) in 10 ml of 1 per cent HCl in methanol kept in a 100 ml beaker. Extract for two days at 3-5 C with continuous shaking (occasional shaking also results in complete extraction of anthocyanins), Filter the extracts through Whatman No.41 filter paper and measure the absorbance of the filtrate at 530 and 657 nm in a colorimeter. Express the anthocyanin content as A530 – 0.33 A657 (formula used to correct for the contribution of fcarotinoid and its degradation products in acid solution to the absorbance of the extracts at 530 nm). Calculate the GA content of the test solution from a dosage response curve prepared with known GA3.

7.17. Bioassay of Cytokinin - Chlorophyll retention test This test is based on the fact that cytokinins delay sence of leaves (Osborne and McCalla, 1961). Since the introduction of chlorophyll retention test, screening an extract for cytokinin activity is relatively simple. This test is more reliable than root inhibition. The method is sensitive between 0.1 and 10 mg knietin per liter. Materials 1. Cytokinin extract 2. Kinetin standards (0.1 to 10 mg/l) 3. Rice seedlings 4. Petri dishes, 4 cm diameter 5. Pipettes, 0.1 ml, 1 ml 6. Test tubes 7. Whatman No. 41 filter paper 8. Razor or cork borer 9. Enamel tray 10. Hot water bath 11. Aluminium foil 12. Colorimeter Reagent 80% Ethanol Procedure Collect the first or second leaves from the plants, float them in Petri dishes containing distilled water and incubate in dim light for 24 hr. Select the leaves with uniform light green colour and cut out 3 cm a pical sections of the leaves or prepare 1 cm leaf discs with a cork borer. Float them in groups of five with their adaxial side up (the lower side of the blade contacting the solution or the filter paper) on 5 ml of test solution kept in 4 cm Petri dish which was previously moistened with 0.5 ml of either the test solution or water (control). Arrange the dishes in trays lined with moist filter paper. Cover them with aluminium foil and incubate for 48 hr at room temperature. Pool the leaf bits from each dish separately and transfer them to test tubes containing 5 to 6 ml of 80 per cent ethanol. Extract the chlorophyll from the tissues by boiling in a hot water bath. Cool the tubes in a pan of cold water, filter and dilute the content to 10 ml with 80 per cent ethanol. Measure the absorbance of this solution at 665 nm (chlorophyll a). Plot the absorbance of the standards against logarithm of the concentration of kinetin and calculate the amount of cytokinins present in the extract in terms of kinetin from the standard curve.

7.18. Bioassay of Cytokinin - Chlorophyll formation test Cytokinins stimulate chlorophyll production (Fletcher and McCullagh, 1971). phenomenon is used to bioassay the cytokinins present in the extract.

This

Materials 1. Cytokinin extract 2. Kinetin standards 3. Cucumus sativus seedlings, 6 day old, use the cotyledons 4. Petri dishes, 4 cm diameter 5. Pipettes, 1 ml, 5 ml 6. Test tubes 7. Hot water bath 8. Foam rubber disks 9. Whatman No.41 filter paper 10. Colorimeter Reagents Acetone or ethanol, 80% Procedure Place the foam rubber disks in 4-cm Petri dishes and saturate withknown volume of cytokinin extract or standard solutions. Maintain water control. Place the etiolated cucumber cotyledons over the foam rubber disks. Arrange the disks in a tray lined with moist filter paper and incubate for 17 hr in dim white light. After incubation, extract the chlorophyll from the cotyledons with acetone or 80 per cent ethanol as described in the chlorophyll retention test and measure the absorbance of the chlorophyll extract at 665 nm in a colorimeter. From a dosage response curve with kinetin calculate the amount of cytokinin present in the extract.

7.19. Specific protocols for ABA and Cytokinin extractions 1. Root ABA quantification Root ABA was quantified by Indirect ELISA, by a solvent extraction as described by Wolf et al., (1988).+The procedure is briefly described below Root ABA extraction All extractions were carried out in dim, indirect light. Extracts were stored at 4C in dark and analysed within 2 days. The clear extract was then passed through a Sep-Pak C-18 Cartridge (Millipore Corporation, Milford, MASS, USA) to remove pigments and lipids from the extract. Before using the Sep-Pak Cartridge, the column was wetted with 5 ml methanol and 5 ml of H2O. The last trace of water in the column was removed by passing air. The methanolic extract was then passed through the column, followed by 4 ml 80% methanol. To regenerate the Sep-Pak cartridge, the column was washed with 2 ml each of hexane, chloroform, methanol and water successively. The pooled washings were evaporated under vacuum. The residue was used for further purification. Ethyl acetate Fractionation The residue was partitioned three times against an equal volume of ethyl acetate pH 3.0 (Ryu and Li, 1992). This on fractionation gives an organic phase which contains free ABA and the aqueous phase contains conjugated ABA. The organic phase was evaporated and the residue redissolved in 2 ml of TBS buffer pH 7.5. Aliquots from this was used in an immunoassay for quantification of free ABA using the ABA-C1 – antibody. 2. Extraction of cytokinins in plants The procedure of Hansen et al., (1984) was used. The tissue was ground using pestle and mortar under cold conditions. While grinding a pinch of Betylated hydroxy toluene (BHT) was added to prevent oxidation of cytokinins. The ground tissue was transferred to 5 volumes of chilled distilled ethanol and using a rotary evaporator at room temperature. The residue was dissolved in distilled water and the extract was used for assay.

8. Secondary metabolites 8.1. Estimation of curcumin The turmeric rhizome contains a variety of pigments. It is used as a natural dye in food industries and in cosmetic and pharmaceutical products as an antimicrobial principle, curcumin. Principle Curcumin is extracted spectrometrically at 425nm.

by refluxing

with

alcohol

and

is

estimated

Reagents/Apparatus 1. Absolute alcohol 2. Stoppered flask and air condenser Procedure 1. Dissolve 0.2-0.5g of turmeric powder in 250ml of absolute alcohol. 2. Reflux the contents in the flask fitted with an air condenser over a heating mantle for 3-5hours. 3. Cool and decant into a volumetric flask and make up the volume 4. Dilute a suitable aliquot (1-2ml) to 10ml with absolute alcohol. Measure the intensity of yellow colour at 425nm. Curcumin content (g/100g) = 0.0025 x A425xVolume made up x dilution factor x 100 0.42 x weight of the sample (g) x 100 (0.42 absorbance at 425nm = 0.0025 g curcumin) Reference Sadasivam, S. and A. Manickam (1992). In : Biochemical Methods for Agricultural Sciennces, Willey Eastern Ltd, New Delhi, pp. 185-186. 8.2. Estimation of anthocyanin

8.3. Estimation of oxalic acid Oxalic acid is an important toxic plant acid present in Oxalis shoots, Spinach and Begonia leaves. Principle The acid treated sample is defatted with ether and extracted with NaOH and water. The water layer is treated with calcium chloride buffer (pH 4.5) and centrifuged. The pellet is treated with acetic acid saturated with calcium oxalate, centrifuged and the residue is dissolved in H2SO4. Finally, the extract is titrated against standard 0.02N potassium permanganate solution and the amount of oxalic acid is calculated. Reagents 1. 2. 3. 4.

4N H2SO4 1N NaOH Diethyl ether Calcium chloride-acetate buffer: Dissolve 25g of anhydrous calcium chloride in 500ml of 50% acetic acid. Dissolve 330g of sodium acetate trihydrate in water and make up the volume of 550ml. combine the two solutions and adjust the pH to 4.5 if necessary. 5. 5% acetic acid saturated with calcium oxalate 6. 4N H2SO4 7. Standard 0.002N Potassium permanganate solution

Procedure Sample extraction and Estimation 1. Weigh 500mg of dry sample and mix with 1g of asbestos and 1.5ml of 4N H2SO4. 2. Fill the extraction thimble (prepared from Whatman filter paper) to a depth of 2cm with clear ground glass (20 mesh). Place two circular pieces of cheese cloth. 3. Transfer the sample mixed with asbestos and H2SO4 to the thimble. 4. Place the thimble with contents in soxhlet extraction apparatus and extract with 500ml of pure diethyl ether for 48h. 5. Add 5ml of 1N NaOH and 7ml of water. 6. Evaporate the ether layer in a rotary evaporator. 7. Transfer the water phase to centrifuge tube, add 4ml of calcium chloride- acetate buffer and allow standing overnight. 8. Centrifuge at 3000g for 10min 9. Discard the supernatant and wash the pellet with 5ml of 55 acetic acid saturated with calcium oxalate and centrifuge. 10. Dissolve the residue in 5ml of 4N H2SO4 and heat at 80-90oC on a water bath. 11. Filter while hot and titrate against standard 0.02N potassium permanganate solution.

12. Calculate the amount of oxalic acid (mg/100g sample) present in the sample using the relationship. 13. 1ml of 0.02N Potassium permanganate = 1.2653 mg of oxalic acid Reference Mahadevan, A. (1979). The concept of impeding, In: A. Mahadevan (ed.) Physiology of Host Parasite Interaction, Today and Tomorrow‟s, New Delhi, pp.167-171.

8.4. Estimation of gossypol Gossypol is a yellowish phenolic pigment in cotton seed kernels, flowers, and bark of roots. Its content varies widely depending upon species, variety, climatic and agronomic factors. It confers resistance to the cotton plant against a number of pests and diseases. Principle Gossypol reacts with phloroglucinol under highly acidic condition to produce a reddish brown product which is measured at 550nm. Reagent 1. 90 % Ethanol 2. Phloroglucinol reagent : Dissolve 5g phloroglucinol in 100ml of 80% ethanol Procedure 1. Weigh 5g of fresh tissue, place in boiling 95% ethyl alcohol (15-20ml) for 5 min. 2. Collect the extract by filtration. 3. Repeat the extraction with residue and combine the extracts. 4. Dilute the extract with 40% ethanol and adjust the extract with 1N Hcl to pH 3.0 5. Mix the contents with 1.5 volumes to diethyl ether at 10 oC using a separating funnel. 6. Save the ether phase and wash with two changes of distilled water. 7. Evaporate the ether extract in vivo for dryness and redisslove the residue in a known volume of 90% ethanol. 8. Pipette out different aliquots (1,2ml) of the gossypol extract in ethanol in test tubes. Add 0.5ml of phloroglucinol reagent followed by 1ml of conc. Hcl to each tube. 9. Incubate for 30min with occasional shaking at room temperature. 10. Make the volume of solution to 10ml with 90% ethanol. 11. Read the absorbance of colour at 550nm against a reagent blank. 12. Prepare a standard graph with gossypol acetate. Reference Bell, A.A. (1967). Phytopathol. 57, 759.

8.5. Identification and Determination of Polyamines (By TLC) Principle The amino groups were dansylated under alkaline condition and separated by TLC and identified based on RF values Reagents Perchloric acid 5%; Ethonal; Dansyl chloride; Saturated sodium corbonate; Proline; Benzene; Chloroform; Triethylamine; Cyclohexane; Ethyl acetate. Standard Putrescine, Spermidine, Spermine (20 nmole each dissolved in 0.01 N Hcl.) Procedure Extraction: Plant tissue were extracted in 5% cold HC104 (100 mg plant tissue / ml of Hc104). After extraction for 1 hr in an ice bath, the contents were centrifuged at 48,000 g for 20 min. The supernant fraction contains free ployamines and stored at - 20 in plastic vial. Dansylation and TLC analysis 1. Mix two hundred l of HC104 extract were mixed with 400 l of dansyl chloride (5 mg/ml in acetone, prepared fresh), and 200 l pf saturated sodium carbonate. 2. After brief vortexing, incubate the mixture in darkness at room temperature overnight. Excess dansyl reagents were removed by reaction with 100 l (100 mg/ml) of added proline, and incubate for 30 min. 3. Extract dansylpolyamines in 0.5 ml benzene, and vortex for 30 seconds. 4. The organic phase was collect and store in glass vials at -20. Dansylated extracts were stable for upto 1 month. Standards were process in the same way, and 20 nmol were dansylated for each alone or in combination. 5. TLC performs on high resolution. LK6D silica gel plates (Whatman). Up to 50 l dansylated extract load on the pre adsorbent zone, and develop the chromatogram for about 1 h with either of two solvent systems: chloroformtriethylamine (25:2 v/v) or cyclohexane, ethylacatate (5.4 v/v). Identification of the unknowns was done by comparison of the Rfs in these two solvent systems, fluorescence spectra, and at least partially by mass spectrometric analysis. After TLC, the dansylpolyamine bands were scraped, eluted in 2 ml ethylacetate, and quantified with spectrophotofluorimeter, with excitation at 250 nm and emission at 495 nm. Analysis of the dansyl derivatives had to be performed immediately because of the rapid fading of fluorescence after TLC.

Reference

8.6.Analysis of Polyamines by HPLC Principle The polyamines were benzoylated and analysed by HPLC Reagents 2N NaOH, Benzoyl chloride, Saturated NaCl, Diethyl ether and Methanol. Procedure 1. The plant extracts was obtained as detailed in extraction procedure for polyamines with HC104. 2. Mix one ml 2 N NaOH with 250 to 500 l of HC104 extract. 3. After addition of 10 l benzoyl chloride, vortexing for 10s, incubate for 20min at room temperature. 4. Add 2 ml saturated NaCl and Benzoylpolyamines were extracted in 2 ml diethyl ether (anhydrous grade; Baker). 5. Centrifuge at 1500 g x 5 min, 1 ml of the ether phase was collected, evaporate to dryness under a stream of warm air, and redissolved in 100 l methanol (Baker, HPLC grade). 6. Treat the standards in a similar way, with up to 50 nmol of each polyamine in the reaction mixture. 7. The benzoylated sample was store at -20ºC. Except for Agm and Spd, the benzoylpolyamines were stable under these conditions for several months. HPLC analysis was done with the solvent system of methanol water, run at 60 to 65% methanol, at a flow rate of 1 ml/min.

8.7. Estimation of HCN Principle Hydrocyanic acid which is evolved from the sample forms a red coloured compound with sodium picrate and the intensity is measured at 625 nm. Reagents 1. Chloroform 2. Whatman No.1 Filter Paper: Cut filter paper into strips 10-12 cm long and 0.5 cm wide, and saturate them with alkaline picrate solution. 3. Alkaline picrate solution : Dissolve 25 g sodium carbonte and 5g picric acid in one litre of water 4. Standard Hydrogen Cyanide Solution: Dissolve 0.241 g of KCN / litre of water. This gives a solution containing 100 g hydrogen cyanide / ml. Procedure 1. Homogenize 1 g of the sample in 25 ml water with 3-4 drops of chloroform. 2. Place the homogenate in 500 ml conical flask and place the saturated filter paper in the hanging position with the help of a cork stopper inside the conical flask. 3. Incubate the mixture at room temperature (20C) of 20-24h. The sodium picrate present in the filter paper is reduced to reddish compound in proportion to the amount of hydrocyanic acid evolve. 4. Elute the colour by placing the paper in clean test tube containing 10 ml distilled water and compare it with standards at 625 nm. 5. Preparation of the standard curve: (KCN is poisonous and use automatic pipettes or burettes for transfer of cyanide solution). 6. Place 5 ml of the alkaline picrate solution and 5 ml of the Potassium Cyanide solution in a test tube. 7. Heat for 5 min in boiling water. Deliver the following volumes from the above KCN alkaline picrate solution to six test tubes: 0.1, 0.2, 4, 0.6, 0.8 and 1 ml. Bring the value of each test tube to 10 ml by adding distilled water. 8. Press rubber stopper to the tubes and keep them in cool place. Measure the absorbance at 625 nm. 9. Prepare a blank with 10ml of distilled water.

8.8. Estimation of total carotenoids Carotenoids are tetraterpenoid (C40) compounds widely distributed in plants. They function as accessory pigments in photosynthesis and as colouring matters in flowers and fruits. Some of the commonly occurring carotenoids are simple unsaturated hydrocarbons based on lycopene and their oxygenated derivatives known as xanthophylls. -Carotene is the most common pigment Reagents Acetone (80%) Procedure Sample extraction 1. Cut the fresh plant material and grind a known amount (100mg) in a mortar with 20ml of either distilled acetone. 2. Acetone and apinch of clean, fine sand. 3. The extract is centrifuged at 2500rpm for 10 minutes and the clear supernatant is made up to 10ml with 80% acetone. 4. The absorbance of the extract is read at 480nm and 510nm. Make suitable dilution of the acetone extract of the plant tissues and measure the absorbance at 480 and 510nm in a colorimeter. If A450 is 10 times, the carotenoids present in the extract may be estimated from the A450 values using the following formula: C = (7.6  OD at 480)-(1.49  OD at 510) 

V

1000 X W C V W

= Total amount of carotenoids (mg/g) = Final volume of supernantant in ml = Weight of the leaf sample taken in gram

Reference Jensen, A. (1978). Chlorophylls and Carotenoids. In : Hellebust, A and J.S. Crargie (eds.) Handbook of Phytological Methods, Cambridge Univ. Press, London, pp. 59-70.

8.9. Estimation of lycopene Lycopene is responsible for the red colour of tomato and the fleshy part of water melon. It is a carotene having the formula C40H56. Principle The carotenoids in the sample are extracted in acetone and then taken up in the petroleum ether. Lycopene has absorption maxima at 473nm and 503 nm. One mole of lycopene when dissolved in one litre light petroleum (40-60°C) and measured in a spectrophotometer at 503nm in 1cm light path gives an absorbance of 17.2 x 10 4. Therefore, a concentration of 3.1206µg lycopene/ml gives unit absorbance. Reagents 1. Acetone (AR grade) 2. Petroleum ether b.p. 40-60°C (AR) 3. 5% sodium sulphate Procedure 1. Take 3-4 tomato fruits (sample) and pulp it. 2. Weigh 5-10g of this pulp and extract repeatedly with acetone until the residue is colourless. 3. Pool the acetone extracts and transfer to a separating funnel containing about 20ml petroleum ether and mix gently. 4. Add about 20ml of 5% sodium sulphate solution and shake the separating funnel gently. Add 20ml more of petroleum ether to the separating funnel for clear separation of two layers. Most of the colour will be noticed in the upper petroleum ether layer. 5. Separate the two phases and re-extract the lower aqueous phase with additional 20ml petroleum ether until the aqueous phase is colourless. 6. Pool the petroleum ether extracts and wash once with a little distilled water. 7. Pour the washed petroleum ether extract into a brown container about 10g anhydrous sodium sulphate. Keep it aside for 30min or longer. 8. Decant the petroleum ether extract into a 100ml volumetric flask through a funnel containing cotton wool. Wash sodium sulphate slurry with petroleum ether until it is colourless and transfer the washings to the volumetric flask. 9. Make up the volume and measure the absorbance in a spectrophotometer at 503nm using petroleum ether as blank. Absorbance (1 unit) = 3.1206µg lycopene/ml mg lycopene in 100g sample =

31.206 x Absorbance Wt. of sample (g)

Reference Ranganna, S. 1976. In : Manual of Analysis of Fruits and Vegetable Products, McGraw Hill, New Delhi, p. 77.

8.10. Estimation of Chloroplast Pigment Composition (Yoshida et al., 1971) Principle Pigment composition of chloroplast has major impact to capture the solar energy and funneling to the reaction centers of chloroplasts where the photoenergy is converted into chemical energy. These pigment composition altered due to ageing as senescence effect. Reagent 80 % Acetone Procedure Known measure of leaf tissue is homogenized with 80 per cent acetone and centrifuged at 3000 rpm for ten minutes. Repeat the extraction till all the chlorophyll was extracted in the solvent. The combine supernatant was collected and made up to known volume with 80 per cent acetone. The optical density of the extract is measured at 645, 663 and 652 for chlorophyll „a‟, chlorophyll „b‟ and total chlorophyll respectively using 80 per cent acetone as blank. Total chlorophyll and its fractions are computed by using the formula and expressed accordingly as adopted by Arnon (1949). A. V Chlorophyll „a‟= 12.7 (O.D. value at 663)– 2.69 (O.D. value at 645) ---------------1000 x W and expressed as mg of chlorophyll „a‟ per gram of fresh weight B. V Chlorophyll „b‟= 22.9 (O.D. value at 645) – 4.68 (O.D. value at 663) ---------------1000 x W and expressed as mg of chlorophyll „a‟ per gram of fresh weight

C. Total Chlorophyll =

O.D. value at 652 x 1000 V ------------------------------- x -----------34.5 1000

and expressed as mg total chlorophyll per gram of fresh weight V

= Final volume of acetone extract

W

= Fresh weight in gram

8.11. Estimation of Chlorophyll Content without Homogenisation Principle Chlorophyll content of the chloroplast is also estimated without homogenization by using dimethly sulfoxide as per the method suggested by Hilscox and Israelstam (1979). Procedure 1. Known measure of leaf tissue place in a vial containing 7ml of dimethyl sulfoxide (DMSO) and extract chlorophyll into the solvent (DMSO) without grinding by incubating overnight at 65C. 2. The extracted liquid is transfer to a graduated tube and made upto a total volume of 10 ml with DMSO. 3. Measure at 645, 652 and 663 nm for their absorbance against DMSO as blank in a colorimeter. 4. Chlorophyll „a‟, „b‟ and total chlorophyll content calculate as milligram of chlorophyll per gram of fresh weight by using the formula given for a, b and total chlorophyll described earlier.

8.12. Estimation of anthocyanin Anthocyanins are the most important and widespread groups of colouring matters in plants. There are six common anthocyanidins (anthocyanin aglycones formed when anthocyanins are hydrolyzed with acid) which differ in the nature of the sugar. The method described by Swain and Hillis (1959) to measure anthocyanin is sensitive and avoids the interference by other pigments such as chlorophylls. Principle The alcohol extract of the sample is treated with HCI in aqueous methanol followed by anthocyanin reagent. The colour intensity is measured colorimetrically at 525nm. Reagents 1. Alcohol 2. 0.5N HCI in 80-85% methanol (HCI in aqueous methanol) 3. Anthocyanin reagent: Mix 1 ml of 30% H2O2 with 9ml of methanolic HCI (5:1, 3N). Procedure 1. Grind a known weight of fresh plant material in alcohol and Filter or centrifuge and collect the extract. 2. Pipette 1ml of the alcohol extract into the test tube and add 3ml of HCI in aqueous methanol and add 1ml of anthocyanin reagents to the samples. 3. Prepare the blank in the same manner by adding 1ml of methanol – HCI instead of anthocyanin reagent. 4. After 15 min of incubation in the dark, measure the absorbance at 525nm against the blank. 5. Calculate the amount of anthocyanins present in the sample from a standard curve prepared with cyanin hydrochloride. 10µg of cyanin hydrochloride / ml in methanol – HCI – absorbance of 0.405 in a 1.0cm cell at A525. Alternatively, the anthocyanin content may be expressed as A525 values. Reference Swain, T. and W.E. Hillis (1959). J. Sci. Food. Agric. 10, 63-68.

8.13. Estimation of leuco-anthocyanin Leuco-anthocyanidins are the colourless polymeric colouring matters present in wood and leaf of woody plants, and in the flowers. Principle The sample is treated with methanol or ethanol to extract the leuco-an-thocyanins. Then the alcohol extract is treated with leuco-anthocyanin reagent and measured at 550nm. Reagents 1. Methanol or ethanol (distilled) 2. Leuco-anthocyanin reagent: Dilute 25ml of 36% Hcl to 500ml with n-butanol. Procedure 1. Grind a known weight of tissue in methanol or ethanol filter or centrifuge and collect the supernatant and pipette 1ml of the extract into a test tube. 2. Reduce the volume to 0.5ml on a hot water bath so that the sample does not contain more than 0.5ml of methanol or ethanol. 3. Add 0.5ml of water and 10ml of leuco-anthocyanin reagent. Mix thoroughly and heat the tubes in a water bath at 97 ± 1°C for 3 min. 4. Cover the tubes with glass stoppers and continue heating for a total of 40 min and cool under a running tap. 5. Maintain the blank similarly with the extract but without heating and measure the absorbance at 550nm. 6. If chlorophyll is present, measure the absorbance at 650nm and express the results as A650 values. Reference Harborne, J.B. (1973). Phytochemical Methods. Chapman and Hall, London.

8.14. Estimation of lignins Lignins are phenolic polymers present in the cell walls of plants which are responsible together with cellulose, for the stiffness and rigidity of plant stems. Spectrophotometric method The sample is extracted in NaOH solution and the aliquot samples are adjusted to pH 7.0 and 12.3. The amount of lignin is calculated by a difference between A245 (pH 7.0) and A350 (pH 12.3). Reagents 1. Diethyl ether 2. 0.1 M Sodium phosphate buffer, pH 7.0 3. 0.1 and 0.5N NaOH 4. 2N HCI Procedure 1. Grind known weight of dry material with ether and centrifuge at 2000g for 5min and decant the supernatant. 2. Wash the sediment with water, recentrifuge and discard the supernatant. Repeat washings twice and add 2ml of NaOH to the residue and extract at 70-80°C for 12-16 hours. 3. Cool, add 0.45ml of 2N HCI and adjust the pH to 7 or 8 with NaOH. 4. Make up the volume to 3ml with water. Centrifuge at 2000g for 5min. Collect the supernatant. 5. To 0.8ml of extract, add 0.8ml of 01M sodium phosphate buffer, pH 7.0. 6. To another aliquot of 0.8ml extract, add 0.8ml of 0.1N NaOH (pH 12.3). 7. Measure the absorbance at 245 and 350nm. 8. Derive the lignin concentration from the difference between A245 and A350 ( E350) on pH 7.0 and 12.3 samples with buffer and NaOH, respectively. Express the amount of lignin as  E350 / sample. Use a conversion factor, 32 to calculate the lignin content (32 x mg pehnol = mg lignin, calculated from bagasse sample). Reference Stafford, H.A. (1960). Plant Physiol., 35, pp. 108-114.

8.15. Estimation of capsaicin Principle Capsaicin is a protoalkaloid present in chilli fruits. The phenolic group is capsaicin reduces phosphomolybdic acid to lower acids of molybdenum imparting blue in colour and is read at 650nm. Reagents 1. 0.4% sodium hydroxide. 2. 3% Phosphomolybdic acid. 3. Dry acetone: Add about 25g anhydrous sodium sulphate to 500ml acetone of analytical grade at least one day before use. 4. Stock standard capsaicin solution: Dissolve exactly 50mg capsaicin in 50ml of 0.4% sodium hydroxide solution (1000µg/ml). 5. Working standard: (200 µg/ml). Procedure 1. Weigh 0.5g dry chilli powder into a glass-stoppered test tube or volumetric flask. 2. Pipette out 10ml dry acetone into the flask and shake it for 3h in a mechanical shaker. Let the contents settle down or centrifuge (10,000 rpm for 10min). 3. Pipette out 1ml of the clear supernatant into a test tube and evaporate to dryness in a hot water bath and dissolve the residue in 5ml of 0.4% sodium hydroxide solution. 4. Add 3ml of 3% phosphomolybdic acid and shake the contents and let it stand for 1h. 5. Filter the solution and centrifuge at about 5,000 rpm for 10-15 min. 6. Transfer the clear blue coloured solution directly into the cuvette and read the absorbance at 650nm. 7. Run a reagent blank along with the test samples and prepare a standard graph using 0200µg capsaicin. Reference Quagliotti, L. 1971. Hortic. Res., 11, p. 93.

8.16. Estimation of Quinones

Principle Quinones are coloured and contain the same basic chromophore, that of benzoquinone. Quinones may be estimated by the quinone reduction method which involves the measurement of decrease in extinction value at 400nm following the addition of a reducing substance (ascorbic acid). Reagents 1. 0.1M sodium phosphate buffer, pH 6.6. 2. Standard catechol or caffcic acid (5 x 10-3 M in water) 3. 0.5N Trichloroacetic acid in 60% ethanol. 4. 0.5N Trichloroacetic acid in 60% ethanol containing 0.05M ascorbic acid. Procedure 1. Grind a known weight of tissue in a mortar pestile with chilled phosphate buffer (5ml for each g of tissue). 2. Collect the supernatant by centrifugation at 2000g for 30 min at 4°C. Use this as enzyme extract. 3. Pipette out 3ml of buffer, 3ml of standard catechol or caffeic acid and 1.5ml of enzyme extract into a test tube. 4. Shake gently and incubate in the water bath and at different intervals withdraw 2ml of samples in duplicate. 5. Add 4ml of TCA reagent (without ascorbic acid) to one sample and to the other corresponding sample add 4ml of TCA containing ascorbic acid and filter off the precipitate. 6. Record the absorbance at 400nm. 7. Calculate the amount of quinone produced by the enzyme extract from ortho-dihydric phenols. Reference Mahadevan, A. (1996). Phytopath, Z. 57, 96-97.

8.17. Estimation of Tannins

Vanillin hydrochloride method Principle Hydrolyzable tannins contain a polyhydric alcohol usually glucose esterified with gallic acid or with hexahydroxydiphenic acid. Condensed tannins are mostly flavonols. The vanillin reagent will react with any phenol that has an unsaturated resorcinol or pholoroglucinol nucleus and forms a coloured product which is measured at 500nm. Reagents 1. Vanillin hydrochloride reagent: Mix equal volumes of 8% hydrochloric acid in methanol and 4% vanillin in methanol. 2. Catechin-stock standard solution: l mg catechin/ml methanol. 3. Working standard: 10ml to 100ml (100µg/ml). Procedure Extraction 1. Extract 1g of ground seed in 50ml methanol. After 20-28h, centrifuge and collect the supernatant and pipette out 1ml of the supernatant into a test tube. 2. Quickly add 5ml of vanillin hydrochloride reagent and mix and read in a spectrophotometer at 500nm after 20 min. 3. Prepare a blank with vanillin hydrochloride reagent alone. 4. Prepare a standard graph with 20-100µg catechin using the diluted stock solution and express the results as catechin equivalents. Reference Robert, E.B. (1971). Agro. J. 63, p. 511.

8.18. Estimation of ascorbic acid (vitamin C) Principle Ascorbic acid reduces the 2, 6-dichlorophenol-indophenol dye to a colour less leuco-base. The ascorbic acid gets oxidized to to dehydroascorbic acid. Though the dye is a blue coloured compound, the end product is the appearance of pink colour. Reagents 1. 4% Oxlic acid 2. Dye solution: Weigh 42mg sodium bicarbonte into a small volume of distilled water. Dissolve 52mg 2, 6-dichlorophenol in it and make up to 200ml with distilled water. 3. Stock standard solution: Dissolve 100mg ascorbic acid in 100ml of 4% oxalic acid solution in a standard flask (1mg/ml). 4. Working Standard: Dilute 10ml of the stock solution 10 100 ml with 4% oxalic acid. The concentration of working standard is 100 μg/ml. Procedure 1. Take 5ml of the working standard solution into a 100ml conical flask. 2. Add 10ml of 4% oxalic acid and titrate against the dye (V1 ml). End point is the appearance of pink colour which persists for a few minutes. The amount of the dye consumed is equivalent to the amount of ascorbic acid. 3. Extract the sample (0.5-5g depending on the sample) in 4% oxalic acid and make up to a known volume (100ml) and centrifuge. 4. Pipette out 5ml of this supernatant, add 10ml of 4% oxalic acid and titrate against the dye (V2 ml.). Amount of ascorbic acid (mg/100g sample) 0.5mg =

V2 x

V1

100ml x

15 ml

x 100 Wt. of the sample

Reference Harris, L.J. and S.N. Ray (1935). Lacet 1, 462

8.19. Estimation of ascorbic acid by colorimetry Principle Ascorbic acid is also determined calorimetrically. The dehydroascorbic acid alone reacts quantitatively and not the other reducing substances present in the sample extract. Thus, this method gives an accurate analysis of ascorbic acid content than the dye method. Ascorbic acid is first dehydrogenated by bromination. The dehydroascorbic acid is then reacted with 2, 4 dinitirophenyl hydrazine to form osazone and dissolved in suplphuric acid to give an organge-red color solution measured at 540mm. Reagents 1. 4% Oxalic acid 2. 0.5N Sulphuric acid 3. 2% 2, 4 Dinitrophenyl hydrazine (DNPH) reagent: Dissolve by heating 2g DNPH in 100ml 0.5N H2SO4. Filter and use. 4. 10% Thiourea solution 5. 80% Sulphuric acid 6. Bromine water: Dissolve 1-2 drops of liquor bromine in 100ml cool water. 7. Ascorbic acid stock solution: See previous procedure. Procedure Extraction 1. Grind 0.5-5g of sample material cither mechanically or using a pestle and mortar in 25-50ml 4% oxalic acid solution and centrifuge or filter. 2. Transfer an aliquot (10ml) to a conical flask and add bromine water dropwise with constant mixing. 3. When the extract turns orange yellow due to excess bromine, expel it by blowing in air. 4. Make up to a known volume (25 or 50ml) with 4% oxalic acid solution. 5. Similarly, convert 10ml stock ascorbic acid solution into dehydro form by bromination and pipette out 10-100μg standard dehydroascorbic solution into a series of tubes. 6. Similarly, pipette out 0.1ml –2ml sample extract and make up the volume in each tube to 3ml with distilled water. 7. Add 1ml of DNPH reagent followed by 1-2 drops of thiourea to each tube. 8. Set a blank as above but with water in place of ascorbic acid solution. 9. Mix the contents of the tubes and incubate at 37˚C for 3 hours. 10. Dissolve the orange-red osazone crystals formed by adding 7ml of 80% sulphuric acid and measure absorbance at 540nm. 11. Plot a graph of ascorbic acid concentration versus absorbance and calculate the ascorbic acid content in the sample. Reference Roe, J.H. and C.A. Kuether (1942), Science, 95,77.

8.20. Estimation of total Phenols Principle Total phenols estimation can be carried out with Folin-Ciocalteu reagent. Phenols react with an oxidizing agent phosphomolydate in Folin-Ciocaltu reagent under alkaline conditions and result in the formation of a blue coloured complex, the molybdenum blue which is measured at 650nm colorimetrically. Reagents 1. 2. 3. 4.

80% Ethanol Folin-Ciocalteu reagent (FCR) 20% Na2 CO3 Standard (100mg catechol in100ml of water). Dilute 10 times for a working standard.

Procedure 1. Weigh exactly 0.5 to 1g of the sample and grind it with a pestle and mortar in 10-time volume of 80% ethanol. 2. Centrifuge the homegenate at 10,000 rpm for 20min. Save the super natant. Re-extract the residue with five times the volume of 80% ethanol, centrifuge and pool the supernatant and evaporate the supernatant to dryness. 3. Dissolve the residue in a known volume of distilled water (5ml) 4. Pipette out different aliquots (0.2 to 2 ml) into test tubes and make up the volume in each tube to 3ml with water. 5. Add 0.5ml of Folin-Ciocalteu reagent. 6. After 3min, add 2ml of 20% Na2CO3 solution to each tube and mix thoroughly. Place the tubes in a boiling water for exactly one min, cool and measure the absorbance at 650nm against a reagent blank. 7. Prepare a standard curve using different concentrations of catechol. From the standard curve find out the concentration of phenols in the test sample and express as mg phenols/100g material. If any white precipitate is observed on boiling, the colour may be developed at room temperature for 60 minutes. Express the results in terms of catechol or any other phenol equivalents used as standard. Reference Bray, H.G. and W.V. Thorpe (1954). Meth. Biochem. Anal. 1: 27-52

8.21. Estimation of ortho-dihydric phenols Principle Ortho-dihydric phenols are important in disease resistance reactions, they are easily oxidized by phenol oxidases and the resulting quinines are highly reactive and toxic to pathogens and their enzymes. Arnow‟s reagent specifically reacts with orthodihydric phenols and produces a pink coloured complex, which is measured at 515nm. Reagents 1. 2. 3. 4.

80% Ethanol 0.5N HCI 1N NaOH Arnow‟s reagent: Dissolve 10g of sodium nitrite (NaNO2) and 10g of sodium molybdate (NaMoO2) in 100ml of water. Store in a brown bottle (stable for a year). 5. Standard catechol: 100mg Catechol/100ml of water 6. Working standard: Dilute the standard catechol solution, 1:10 with water (100 μg/ml). Procedure 1. Follow the steps from 1 to 4 under the estimation of total phenolics (see 14.1). 2. Pipette out different aliquots (0.2 to 1ml) into test tubes. 3. Make up the volume in each tube to 1ml with water 4. Add 1ml of 0.05N HCI, 1ml of Arnow‟s reagent, 10ml of water and 2ml of 1N NaOH. Mix thoroughly (pink colour appears). 5. Maintain the reagent blank similarly without the extract. 6. Measure the absorbance at 515nm and calculate the amount of ortho-dihydric phenols present in the sample using the standard catechol solution and express as mg /100 g materials. If the colour intensity is high, dilute the solution appropriately (25ml) with water and measure the absorbance. Reference Mahadevan, A. and R.Sridhar (1986). In : Methods in Physiological Plant Pathology (3 rd edn) Sivakami Publications, pp. 183-184.

9 . Physiology 9.1. Measurement of Hill reaction In Hill reaction water is split into oxygen and hydrogen through photosynthetic phosphorylation. Principle The Hill reaction is measured by incubating a suspension of isolated chloroplasts with dichlorophenol-indopehnol (DEPIP) dye which loses its blue colour on reduction. 2DCPIP + 2H2O → 2DCPIPH2 + O2 Reagents 1. 2,6 Dichlorophenol-indophenol dye solution containing 0.1mM of the dye and 0.01M KCL in 0.04M potassium phosphate buffer, pH 6.5 Procedure  Pipette out 0.2ml of chloroplast suspension (prepared as described under isolation of chloroplasts) into test tube and add 4ml of water followed by 2ml of 2,6 Dichlorophenol-indophenol dye solution  Measure the absorbance at 620nm  Illuminate the tubes with 500W tungsten lamp for 5 min and again measure the absorbance at 620nm  Determine the chlorophyll content of the suspensions as described earlier  Express the Hill activity as changes in absorbance as ΔA620/mg chlorophyll/5ml. Reference Garg, I.D. and C.L. Mandahar (1976). Phytopath. Z. 85, pp.298-307.

9.2. Estimation of mitochondria Principle The fresh tissue is gently homogenized to disrupt the cells and release the contents and the mitochondria are pelleted by differential centrifugation. Further purification is carried out by sucrose gradient centrifugation. Materials and reagents  Isolation medium (pH 7.8) containing 30mM 3-(N-Morpholino) ethane sulfonic acid (MOPS), 0.3M mannitol, 4mM cysteine, 1mM EDTA and 0.1% (w/v) defatted BSA adjusted to pH 7.8. For green leaf tissue, 0.6% (w/v) insoluble polyvinylpyrrolidone (acid-washed) is included and the BSA concentration increased to 0.2% (w/v).  Re-suspension medium: The above medium but without 4mM cysteine.  Nonlinear sucrose gradient: Sucrose solution of 1.8M (52.1%, w/v), 1.5M (43.1%), 1.2M (35.6%) and 0.6M (19.1%) separately prepared in 10mM MOPS or phosphate buffer (pH 7.2), 0.1% (w/v) BSA.  Tissue Homogenizer. Procedure 1. Take 100-200g of etiolated fresh tissue and add two volumes of chilled isolation medium (4oC) and blend for 2-3sec at low speed. 2. Squeeze the homogenate through six layers of cheese. 3. Centrifuge at 700-1000g for 10min and decant the supernatant. 4. Centrifuge the supernatant fraction at 10000g for 20min or alternatively at 39000g for 5min and discard the resultant supernatant. 5. Gently disperse the pellet in 40-50 ml of resuspension medium. 6. Centrifuge the suspension at 250g for 10min. 7. Centrifuge the supernatant at 10000g for 15min. Suspend the mitochondria in the pellet in 1-2ml of resuspension medium. 8. Prepare step gradients in a suitatble centrifuge tube by carefully pipetting 6ml of 1.8M, 6ml 1.2M and 3ml 0.6M sucrose solutions successively, load 1ml of crude preparation (40-50 mg protein) on to the gradient. 9. Centrifuge the gradient at 40000g for 45min in an ultracentrifuge. 10. The mitochondria band at the 1.5M – 1.2M interface. 11. Collect the band by side-puncturing the tube using a hypodermic needle slightly below the band. 12. Dilute the gradient fraction containing mitochondria to isotonic conditions (0.3M) by slow, careful addition of buffer. 13. Pellet the mitochondria by centrifuging at 10000g for 15min and finally suspend in a small volume of relevant medium. The isolation medium should contain cation chelating agents (EDTA) and phenolics scavengers such as BSA and PVP in a suitable osmoticum such as mannitol (0.3M) to maintain membrane structure. Mitochondria from green leaf tissue can be isolated in a similar way described above. The leaves are deribbed. The medium is identical to that used for etiolated tissues except

for the addition 0.6% (w/v) acid washed insoluble PVP and increasing the defatted BSA concentration to 0.2% (w/v). After filtration through cheese cloth, chloroplasts are sedimented at 3000g for 5min, and the mitochondria are collected from the supernatant by centrifuging at 12000g for 20min. The pellets are resuspended in approximately 50ml of medium except for addition of 0.2% defatted BSA as described previously. Following a low speed centrifugation at 1500g for 10min, mitochondria are sedimented form the supernatant by centrifugation at 11000g for 15min. Purification by centrifugation is also carried out in sucrose/percoll gradients either linear or non-linear. Reference Douce, R, E.L. Christensen, and W.D. Bonner Jr. (1972). Biochem. Biophys. Acta, 275, 148.

9.3. Isolation of chloroplasts Isolated chloroplasts are required to study the electron transport system of the photosynthetic apparatus. Reagents Isolation medium: Weigh 2.42g of Tris (20mM); 72.8 g sorbitol (0.4M); 1.168 g NaCl (20mM); 0.610g MgCl2, 6H2O (3mM) and dissolve in one litre of distilled water. Adjust to pH 7.8. Procedure 1. Cut 5 to 10g of leaf tissues into small bits. Add 20ml of the prechilled isolation medium 2. Homogenize with an omni mixer (three five-seconds with five seconds intervals). 3. Filter the brie through eight-layered cheese cloth 4. Centrifuge at 3000g for 2 min 5. Discard the supernatant and suspend the pellets in the isolation medium 6. Centrifuge at 3000Xg for 2min 7. Discard the supernatant and resuspend the pellet in a small volume of the grinding medium and store on ice. 8. Since any further advanced study on isolated chloroplasts is expressed on the basis of chlorophyll, estimate the chlorophyll by diluting 0.1-0.2 ml of chloroplast suspension to a total volume of 4ml with 80% acetone. Calculate the chlorophyll content as follows: (12.7 x A663) – (2.69 x A645) = chl. A (µ g/ml) (22.9 x A645) – (4.68 x A663) = chl. b (µ g/ml) (20.2 x A645) + (8.02 x A663) = total chl. (µ g/ml) Calculate the chlorophyll concentration of the stock chloroplast suspension. An alternative grinding medium, most useful for many purposes is : 330 mM sorbitol 10mM Na4P2O7 (sodium pyrophosphate) 5mM MgCl2 2mM Na isoascorbate Adjust to pH 6.5 with HCL. Centrifuge at 6000rpm Reference Walker, D.A. (1980). In: Methods Enzymol., 69 (eds. Colowick, S.P. and Kaplan, N.O.), Academic Press, p. 64.

9.4. Sullivan’s heat tolerance test In Sullivan‟s test, the total inorganic ions (mainly K‟) leaked out, is measured in terms of electrical conductivity (EC) of the bathing medium before and after the treatment, using a conductivity bridge. Procedure Leaf discs (about 20) of the test sample are taken in a test tube or a 50 ml beaker with about 10 ml of deionised water. The test tubes are covered and subjected to two high temperature regimes (45C and 55C) for 30 minutes, usually in a water bath. In practice, an initial EC, just before transfer to the high temperature treatment is done as there is a small degree of leakage by the discs caused by the punching treatment. The EC determined is recorded (Eca). After 30 minutes, the EC is again measured (E cb). After this the tubes are autoclaved or boiled at 100C for 10 minutes and the EC again recorded (Ecc) Ecb - Eca Percent leakage = x 100 Ecc Reference Sullivan, C.Y. 1971. Techniques for measuring plant drought stress. In. (Eds.K.L. Larson and J.D. Eastin): Drought injury and resistance in crops. Crop Sci. Soc. A.M. Madison. Wis.

9.5. Measurement of loss of membrane permeability One of the primary effects and kind of stress is on the cell membrane system. Moisture stress alters the permeability of plasma membrane. As a consequence of altered permeability cell losses compartmentation and leak its contents. There are two major reasons for the leakage. a. Leakage due to physical changes in membrane configuration, b. Leakage due to chemical / biochemical changes at the membrane site. Principle Amino acids, sugars, sugar alcohols organic acids, growth regulators, phenolics, nucleic acids and inorganic ions are leaked out from the cells to dehydrating media. This heterocyclic compounds like nucleic acids, cyclic AMP etc., have absorption maxima at 273 nM (UV range). The extent of leakage is measured by recording optical density at 273 nM. Procedure Fully expanded leaves from three genotypes / species are excised with their petioles intact in water and allowed to region turgidity by incubating in distilled water for 45 minutes. After this initial turgid weight of leaves recorded by using petioles. These leaves are then allowed to live wilt under shade. After leaves have lost 40 per cent and 60 per cent of their fresh weight, leaf punches of 1 cm diameter are taken. These punches are washed for 1 to 2 minutes to leach out their solutes from the cut ends and blotted on clean filter paper. Ten leaf punches of each genotype / species are incubated in a beaker containing 20 ml distilled water for 3 hours. Then leakage of solutes in this bathing medium is estimated by recording its absorbance at 273 nm. This is refered to as the initial leakage of solutes. Following this, the beakers are incubated in hot water bath (100C) for minutes. After suitable dilution the absorbance of the bathing medium is again read at 273 nm to indicate the final absorbance due to the total solutes contained in the tissue. The percentage leakage of solutes which is a direct reflection of the extent of loss of membrance integrity is calculated as Initial absorbance of bathing medium Dilution factor =

x 100 Final absorbance of bathing medium

Reference Leopold, A.C., M.E. Musgrave and K.M. Williams. 1981. Solute leakage resulting from leaf dessication. Plant Physiol., 68: 1222 – 1225.

9.6. Chlorophyll Stability Index (For Drought Tolerance in Plants) (Murthy and Majumdar, 1962) Principle Green plant pigments are thermosensitive and degradation occurs when it is subjected to higher temperature. This method is based on pigment changes induced by heating. The chlorophyll destruction commences rapidly at critical temperature of 55 to 56C. Thus, chlorophyll stability is a function of temperature. Materials / Reagents / Materials 1. Larged sized glass tubes of 2.5 cm in diameter 2. Acetone (80%) and distilled water 3. Mortar and pestle or blender 4. Balance 5. Waterbath with thermostatic control Procedure Two clean glass tubes are taken and five grams of representative leaf sample is placed in them with 50ml distilled water. One tube is then subjected to heat in waterbath at 56C + 1C for exactly 30 minutes. Other tube is kept as control. The leaves are then ground in a mortar for five minutes with 100ml of 80 per cent acetone. The slurry is then filtered with Whatman No.1 filter paper. This chlorophyll extract is further examined immediately for light absorption with colorimeter. A parallel leaf sample of 5g in another tube is then estimated for chlorophyll content without heating simultaneously and light adsorption is measured with colorimeter at 660mm. The difference in two readings (reading without heating and m reading after heating 50C = R) is defined as chlorophyll stability index (CSI). This CSI is found to be correlated with drought tolerance. Thus, CSI is inversely related with drought tolerance efficiency.

Reference: Koleyoreas, A.S. 1958. A new methode of determining drought resistance. Plant Physiol., 33:232-233. Marty, K.S and S.K. Majumder. 1962. Modification of the technique for determination of Chlorophyll Stability Index is relation to studies of drought resistance in rice. Curr. Sci., 31: 470-471.

9.7. Plant Tissue Culture (Aneja, 1993)

Plant tissue culture technology can be divided into five classes, based on the type of materials used; (i)

Callus culture the culture of callus (cell masses) on agar media produced from the explant of seedling or other plant source.

(ii)

Cell cultue the culture of cells in liquid media, usually aerated by agitation.

(iii)

Organ culture the aseptic culture of embryos, anthers (spores), ovaries, roots, shoots or other organs on nutrient media.

(iv)

Meristem culture the aspetic culture of shoot meristems or other explant tissue on nutrient media to get complete plants.

(v)

Protoplast culture the aseptic isolation and culture of plant protoplasts from cultured cells or plant tissue.

9.8. Equipment and other Requirements for Tissue Culture Laboratory The cultivation of plant tissue in vitro requires simple unexpensive equipment like pressure cooker and a few jam jars. However, to investigate ultrastructural changes occurring in the course of growth and differentiation of a particular system, sophisticated apparatuses like electron microscope and dark room facilities will be reqquired. For biochemical studies, high-speed centriguge, spectrophotometer, freezedrier etc. will be required. The basic organisation and facilities of most tissue culture laboratories are as follows : (i) (ii) (iii) (iv) (v) (vi) (vii)

Large sinks (some lead-lined to tolerate strong acids and alkalies) and draining areas. Cabinet and shelf space for storage of chemicals and dust free storage space for clean glassware. Transfer areas / chamber / or laminar air - flow set for aseptic manipulation , if possible fitted with ultraviolet lighting. Culture rooms or incubators with controlled light, temperature and humidity to incubate cultures. Essential services such as electricity, water, gas and compressed air and vacuum in the working areas. Washing machines to wash glassware in bulk. Hot-air cabinets, to dry washed glassware.

Instruments 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16.

Balance, one to weight small quantities (electronic analytical to 5 decimal places) and the other to weigh comparatively large quantities (to 2 decimal places) Pressure cooker and / or autoclave for steam sterilization of media and apparatuses Over for dry-heat sterilization of glassware Hot plate-cum-magnetic strirrer, to dissolve chemicals Refreigerator, to store chemicals, stock solutions, and plant materials Heater or heat-regulated hot plate for heating purposes Exhaust pump, to facilitate filter sterilisation pH meter, to adjust pH of solutions and media Shaker, to grow suspension cultures Steamer to dissolve agar and melt media Low-speed centrifuge, to sediment cells and cleaning of protoplasts Atomiser, to spray spirit in the inoculation chamber Deep freeze, to store sock solutions, certain enzymes and coconut milk etc. Water distillation apparatus or demineralisation apparatus, to obtain distilled or deionized water. Filter membranes and their holders, to filter sterilize solutions Instrument's stand, to keep sterilized instruments

17. 18. 19. Tools 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16.

Metal trays and carts for transport of culture flasks, racks of tubes etc. Disseccting (ow-power) microscope, for dissecting out microscopic explants and overall observation of tissue development Bright-field (compound) microscope, to observe cells and tissues.

Spirit lamps or Bunsen burners, to flame instruments Large forceps with blunt ends, to inoculate and subsculturing Forceps with fine tips to peel leaves Fine needles for dissections Scalpels, to shred the tissues Cork-borer, to remove tissue cylinders of standardised size Hemocytometer, to determine cell counts Cavity slides, for handling drop cultures. Microscope slides and cover-glass Plastic or steel buckets, to soak labware for washing Large plastic carboys (e.g. 10 or 25 litre) for storing high quality water Hypodermic syringes, to filter sterilise solution Screw-cap bottles, to sterilizer plant material Culture tube rack slanted to prepare medium slants Culture tube rack, upright to store cultures. Stainless steel or Teflon sieve of various pore size (40-100 mesh screens, i.e. with 0.38 openings to 0.0737 openings), to separate cell clumps.

Glassware for Media 1. 2. 3. 4. 5. 6. 7.

Erlenmeyer flasks (50, 100, 125, 500 ml. 1l, 5l capacity) Volumetric flasks (500 ml 1, 2 & 3l capacity) Measuring cylinders (10, 25, 100, 500 ml and 1l capacity) Graduated pipettes (1, 2, 5, 10 ml capacity) Pasteur pipettes and tests for them Culture vials Petri dishes (50 x 17,000 x 17, 150 x 20 mm), culture tubes (25 x 150 mm), screw-cap bottles of various sizes. Buchner funnel.

9.9. Plant Tissue Culture Technique: Steps (i)

(ii)

(iii) (iv) (v)

(vi) (vii) (viii) (ix)

(x)

Collection of explant materials (pieces of seedlings, swelling buds, stem or storage organs, leaf materials, and for cereals, immature embryos, mesocotyl or basal stem sections of young plants) in a screw-cap bottle. Sterilisation of the materials by submerging in dilute solution of the surface sterilants (e.g. calcium or sodium hypochlorite, hydrogen peroxide, bromine water, silvernitrate, bleaching powder, alcohol), containing a small amount of a suitable surfactant (Teepol or Tween - 80) which is to be shaken two to three times after closing the vial Removal of the sterilants by thoroughly rinsing the material with several changes of sterile distilled water Transferring the material to a sterile petri dish Preparation of suitable explants from the surface sterilized materials using sterilised (by dipping in 95% alcohol and flamming and cooled instruments (Scalpels, needles, cork-borer, forceps, etc.) Transferring the inoculum into a suitable medium Incubation at 26-28C in the dark (or low light) for 3-4 weeks for the development of the callus Transfer the callus into a liquid medium Incubation of the flasks on a shaker at 150 rpm in continuous light at 26C for 4-6 weeks by decanting and replacing the volume with fresh medium at two weeks intervals Regeneration of plants from cell suspension cultures.

Sterilization of Plant Materials Surfaces of plant parts carry a wide range of microbial contaminants, fungi and bacteria being the common. To avoid the mocrobial growth which is detrimental to culture growth, explants must be surface sterilized in disinfectant solutions before planting these in the nutrient medium. The most common surface sterilants are listed below with the concentration and exposure times that preserve the explant but at the same time kill any microbial contaminant. Commonly used surface sterilants for plant tissue cultures Disinfectant Calcium hypochlorite Sodium hypochlorite Hydrogen peroxide Ethyl alcohol Bromine water Silver water Mercuric chloride Antibiotics

Concentration (%) Used 9-10 0.5 – 1.5 * 3-12 70-95 1-2 1.0 0.1 – 1.0 4-50 mg

Exposure time (min) 5-30 5-30 5-15 0.1 – 5.0 2-10 5-30 2-10 30-60

Effectveness Very good Very good Good Good Very good Good Satisfactory Fairly good

*

Commerical bleach contains about 5% sodium hypochlorite and thus may be used at a concentration of 10-20% (v/v) of the commercial preparation that is equivalent to 0.5-1.05% sodium hypochlorite.

Requirements Explant materials Commercial bleach / calcium hypoclorite Ethanol (70%) Surfactant (Triton-X / Tween 20 or 80) Mild detergent Screw-cap bottles Sterile forceps Scalpels Sterile distilled water Procedure 1. Make small pieces of explant materials (seedlings, swelling buds, stem or storage organs, leaf materials) using scalpel 2. Wash explants in a mild detergent (Herbaceous materials may not be treated) 3. Rinse explants under running tap water for 10-30 min. 4. Rinse explants in 70% ethanol for 30 seconds and material left exposed in the sterile hood for evaporation of alcohol. 5. Aseptically transfer explants into a vial containing a mixture of wetting agant (surfactant) to reduce surface tension and for better surface contact and 20% commerical bleach (5% sodium hypoclorite), a disinfactant. 6. Keep the explants submerged in the above solution for 5-30 minutes, shaking the vial 2-3 times, for sterilization. 7. Decant the liquid 8. Pour an adequate amount of sterile distilled water into the vial and replace the cap. 9. Shake the vial, a few times and discard the water 10. Repeat steps 9 and 10 to rinse explants 4-5 times. Observations and Results : Explant materials are surface sterilized and ready for inoculation on a nutrient medium. Preparaion of Tissue Culture Media Requirements Constituents of the MS medium Erlenmeye flasks (100, 250, 500 ml capacity) Measuring cylinders (100, 1000 ml capacity) Pipettes (1, 5, 10 ml) Distilled or demineralised water pH meter 1.0 N NaOH Autoclave

Procedure 1. Prepare macronutrients solution in 100 ml distilled water 2. Prepare micronutrients solution in 100 ml distilled water 3. Add macronutrient and micronutrient solutions while stirring into 700 ml distilled water taken in 1 litre Erlenmeyer flask. 4. Add the other heat stable constituents (e.g. sucrose, vitamins and hormones) and agar powder (if desired at a concentration of 0.8-1.0% (vitamins and auxins can be added after autoclaving for better results) 5. Make the final volume of the medium by the addition of more distilled water 6. Adjust pH of the medium to 5.7, while stirring, using 0.1 NaOH or 0.1 N HCl. 7. If solid medium is desired, i.e. agar is used, heat the solution while stirring until agar is dissolved. 8. Pour the medium into the desired culture vessels (15 ml in a 25 x 150 mm culture tube and 50 ml in a 150 ml flask) 9. Plug the culture vessels with non-absorbent cotton wool wrapped in cheese cloth, or with any other suitable closure 10. Transfer the culture vessels to appropriate baskets covered with aluminium foil to brevent wetting of plugs during autoclaving. 11. Transfer the baskets to autoclave 12. Sterilize the medium by autoclaving at 121C (1.05 kg/cm) (15 psi) for the time period depending upon the volume of the medium in the vessel (e.g for 25, 50, 100, 250, 500, 1000, 2000, 4000 ml capacity time required is 20, 25, 28, 31, 35, 40, 48 and 63 minutes, respectively) 13. The medium is allowed to cool at room temperature Plant Regeneration from Callus or Plant Tisse The Murashige - Skoog (MS) medium is the most suitable for plant regenaration from tissue and callus. The hormones are the important compounds in the plant regenerating medium. The capacity for plant regeneration of tissues varies widely in different species. In some species the morphogenesis is readily induced (e.g., carrot, coffee, celery) and develop into complete plants while in others it fails to occur. Requirements Callus (raised from cells or pith tissue of tobacco) MS liquid medium Microhamber (A sterile microscope slide enclosing single cell within the mineral oil, surrounded by two cover glasses, placed on oil drop on eachside and finally covered with a third cover glass bridging the two cover glasses) 1. Rotary shaker that moves in a circle or spiral way 2. Forceps 3. Sterile Petri Plates 4. Glass tubes or jars 5. Pots and Green house or grwoth chamber Procedure 1. Transfer callus into flasks each containing 20 ml MS liquid medium

2. 3. 4. 5. 6.

7. 8. 9. 10.

Shake the culture flasks on a notary shaker at 150 rpm to dissociate callus into single cells Transfer cell from the flask and place it in a drop of MS medium in a microchamber Place the micro-chamber in a sterile Petri dish Incubate the Petri plate at 26 C in continuous light for the development of tissue / callus Remove the cover glass of the micro-chamber and aseptically transfer the small callus (tissue) into MS medium with 4.5 m 2, 4-D and 1-2-grams of casein hydrolysate Incubate at 25-27C light (ca. 1000 lux) to develop the tissue into a large callus and shoot for 4-5 weeks. When shoot appears, transplant these on half-strength MS medium or hormone free MS medium Transfer the rooted plants to pots containing soil and water with a nutrient solution Keep the pots in a green house or growth chamber where high humidity is maintained.

Observations and Results Observe the micro-chamber and culture tubes for the developing calli and plantlets and pots for the nature plants. Appearance of callus and shoot takes place after 4 weeks of incubation on MS medium supplemented with hormones. Root appears later from the callus with shoots in half strength MS medium. The plants in pots will grow to maturity, flower and set seeds similar to the plants growing in nature. Somatic embryogenesis or organogenesis In this process, embryos are formed either directly on the surface of the explants or indirectly, with the help of callus or cell masses. In certain cases like citrus, adventitious embryos (direct budding of embryos) are possible. A. Protocols to Meristem culture 1. Collect rapidly growing apex of a shoot. 2. Remove all the leaves except the smallest leaves, wash thoroughly under running water with a drop of tween 20. 3. Disinfect the working area of the laminar flow cabinet with 70% alcohol or rectified spirit. 4. Dip forceps, scalpels with removable blades in ethanol and sterilize by flaming after each operation. 5. Dip the explants after washing in 0.1% mercuric chloride solution for 5-7 min for surface sterilization, and wash with sterile distilled water thoroughly for 3-4 times. 6. Place the shoot tips on sterile filter paper to remove moisture.

7. Hold the stem firmly on a sterile tile/petri plate with forcep and remove the young leaves with a needle. 8. Remove the underlying leaf primordial. 9. Remove up to the 3rd and 4th leaf primordial and keep intact the 1st and 2nd leaf primordial carefully without damaging the fragile dome shaped apical meristem. 10. With a surfical scalpel, remove the apical dome (0.1-0.3 mm) using stereomicroscope. 11. Transfer the dome to the culture tubes containing medium. 12. The culture tubes maintain at 25±2°C for 12h light (3000 lux) / 12h dark cycle. 13. Within 2-3 weeks of inoculation, apical meristem grows and forms shoots, each single shoot undergoes proliferation. There shoots are separated and cultured in rooting media. Root initiation occurs within 18-21 days of inoculation. Once the plantlets have two to three leaves and one strong root, they are be transferred to liquid basal media with the help of paper bridge. Then after about 8 weeks, the plantlets are soaked in 0.2% Bavistin, a fungicide for 10 min, and they are potted in vermiculite mixture till they grow. Seed, ovule and embryo culture 1. Collect mature or immature fruits. 2. Dip the fruit in alcohol and flame them in laminar flow cabinet. 3. Peel the fruit and remove the seeds. Wash the seeds in alcohol for ½ a min. Remove and wash with sterile water. If it is seed culture, then surface sterilize the seeds with 0.1% mercuric chloride or sodium hydroxide solution for 5-7 min. and wash thoroughly in sterile distilled water for 2-3 times. (if it is very small seeds like orchid seeds then use filter paper and funnel for washing the seeds). 4. In case of embryo culture, after surface sterilization, remove the seed coat with the help of a sharp forceps and take out the embryo carefully and inoculate. 5. In case of nut fruits like coconut, oilpalm etc. first break the nut, take out the embryo with the help of a scoop or forceps and do surface sterilization. 6. The germination time varies from type to type, but generally shoot appears before root. Anther and Microspore Culture Anther culture is used to produce haploid plants so it depends on the stage of development of the microspore, donor plants, genotype and physiology. Though anther culture technique is relatively simple, microspore culture has certain advantages. The problem of mixed ploidy calli can totally be eliminated by microspore culture. The inhibitory effect of the anther walls in some species can also be eliminated by culturing microspore. Protocol 1. Collect buds of appropriate size, young shoots are exposed to sunlight. 2. Treat the buds with a few drops of tween 20 for 5 min. and then wash under running water.

3. In the laminar flow cabinet, the buds are surface sterilized with mercuric chloride (0.1%) for 10 min. Wash 3 times with sterile distilled water. 4. Keep the buds on sterile filter paper. 5. Open the buds one boy one with the help of forceps and needle, collect the anthers in a sterile petriplates. 6. Inoculate the anthers on media gelled s slants. 7. Inoculate in dark at 27°C. Callus Culture Callus tissue is an unorganise proliferation of parenchymatus cells from the segments of plant organs. Callus can be initiated from any parts of plant like root, hypocotyls, leaf, stem, embryo, seed, anther etc. Callus cultures are usually grown o a semisolid basal media containing sucrose and 2,4-D as one of the growth regulator. Protocol 1. Collect soft immature leaves or stem from young seedlings or segments of carrot root tissues. 2. Wash them with Teepol under running water. 3. Take out the material from water and bring them to laminar flow cabinet. 4. Surface sterilizes them with 0.1% HgCl2 for 5 min. Wash with sterile water 3 to 4 times. 5. Keep the explants gradually (2-3 numbers) on a sterile filter paper which will absorb the water. 6. With the help of sterile scalpel, cut the leaves to 4-5 mm size. 7. Transfer the pieces into the culture media. 8. Incubate at 25±2°C in dark. (Composition of callus media: MS basal supplemented with BAP (0.5mg – 1mg/l) and 2,4 D (2 mg-30 mg/l) depending upon the crops.

Embryogenesis of calus After 45 days of formation of subculture, the callus grown in regeneration media. (Regeneration media generally supplemented with BAP (0.25 mg – 0.75 mg/l), GA3 (0.5 mg – 2mg/l), and Adenine (0.5 mg – 2 mg/l) but not 2,4-D). Protoplast Culture For protoplast culture from mesophyll protoplasts in vitro grown seedlings or plants are used, leaves are to be sterilized as described in meristem culture. (Ahuja et al., 1990). 1. Remove fully expanded young leaves from the top portion of the stem. 2. Peel the lower epidermis or slice the leaves into thin pieces. 3. Transfer the leaf pieces to CPW solution (10ml of 13M) for about half an hour or more.

4. Carefully pipette the CPW solution from the petriplates and add enzyme solution. There are many enzyme mixtures being used. The suitable ones for the plant species and the tissue can be found from the literature. For example the commonly used mixture for the leaves are 1.5% meicelase, 0.05% Macerozyme R 10in CPW 13M medium at a pH of 5.8. 5. Incubate for 16 hours in dark at 25°C 6. Sieve the suspension through 75µ sieve into fresh petriplate. 7. Transfer the suspension to sterile centrifuge tubes and spin at 80g for 5 min. 8. Pippette of the supernatant and add CPW 21S solution in the tube and spin at 100g for 10 min. (The green band at the surface of the solution is the proptoplast) 9. The protoplast are transferred carefully to another tube containing CPW 13M solution. 10. Count the protoplasts through a haemocytometer and spin the tube for 5 min. at 80g. 11. Remove CPW 13M and add CPM. 12. Adjust the density of protoplast suspension to 1x104 protoplast/ml and plate in 90mm petriplates pouring 10ml of the protoplast suspension/plate. The density of culture varies with plant species and seals the petriplates with para film and incubates in dark at 25±2°C. 13. The isolated protoplasts can be cultured in many ways viz., liquid layer, liquid over agar, hanging drop culture, microdrop array etc. For example in liquid over agar method, the following method is employed. a. Add measured amount (10ml) of agar solidified PCM in petriplates and allow solidifying as thin layer. b. 10 ml of the protoplast suspension is poured over the agar layer. The cultures are initially maintained in dark for 10 to 15 days at 25°C. Cell wall formation takes place within 48 hours and cells divide after 3 to 7 days. The protoplasts along with agar are lifted as blocks and cultured in medium with lower osmoticum. These protoplasts attain callus stage. CPW Solution (Frearson et al. 1973) Composition Culture. S. No.

Chemical

Quantity (mg/l)

1

KH2PO4

27.2

2

KNO3

101.0

3

CaCl22H2O

1480.

4

MgSO47H2O

246.0

5

KI

0.16

6

CuSO45H2O

0.025

7

Mannitol

13% (w/v)

8

Sucrose

21% (w/v)

Make the volume to 1 litre and adjust pH to 5.8.

CPW 13M and CPW 21S solutions contain the above with 13% (w/v) mannitol and 21% (w/v) sucrose respectively. Protoplast culture medium (PCM) MS salts + 3% sucrose + 9% mannitol + 1.0 mg/l NAA + 0.2 mg/l 1,2,4-D + 0. mg/l BAP. Adjust pH to 5.8. Autoclave and store in dark.

9.10. Cation Exchange Capacity (CEC) of Roots (Crooke, 1964) Principle The ion exchangeable sites in the root were replaced with H irn by treatment with dilute HCl. Addition of excess K ions through KCl, replaces the exchangeable H+ ions into solution from root and lower the pH of the solution. Back titration with standard alkali to raise the pH to original pH indicates the cations exchange for H ions by K ions. Reagents Nacl 0.01N KCl 1 M KOH.0.01 N Procedure The soil block containing the root system was loosened and removed. The roots were freed from the bulk of the soil by gentle blow of water. Final separation was achieved by placing roots on the sieve under running water. Roots were dried at 80C overnight and milled to pass through one mm sieve. After through mixing, sample of 0.2 g was withdrawn for the estimation of CEC of roots. The milled roots were placed in a 400 ml beaker and moistened with a few drops of distilled water to prevent floating during the next stage. A quantity of 200 ml of 0.01 N hydrominutes. The root material was allowed to settle and the bulk of acid was decanted quickly through Whatman No.1 filter paper. The root was washed into a funel using distilled water until the washings were free of chlorides. The filter paper was pierced and the root material was washed into a 250 ml beaker using a total of 200 ml of 1 M potassium chloried (adjusted to pH 7.0). The pH of root - HCl suspension was determined using glass electrode and enough 0.01 N potassium hydroxie was added to the suspension to restore the pH 7.0 and it was maintained there during the arbitrary five minutes titration time. From the amount of 0.01 N alkali consumed, root CEC was calculated and expressed as me/100 g of dry roots. Reference: Crooke, W.M. 1964. The Measurement of Cation Exchange Capacity of plant roots. Plant and Soil, 21; 43-49.

9.11. Physiological Disorders and Corrective Measures

Crop

Malady

Corrective Measurement

Rice Rice

Severe chlorosis of leaves

Rice

Irregular flowering and chaffiness Multiple deficiency of nutrients

Maize

Tip drying, and marginal scorching and browning

Maize

Chlorosis

Maize

Maize

'White bud' yellowing in the bud leaves only Tip drying and marginal scorching Pinkish colouration of lower leaves Marginal scorching and yellowing

Cholam

Irregular drying of tips and margins Chlorosis of younger leaves

Maize

1 per cent phosphate extract with and 0.5 per cent ferrous sulphate 1 per cent super hosphate extra magensium sulphate and 0.5 per cent 1 per cent super phosphate extra cent zinc sulphate and 0.1 per cent plant protection sprays A spray mixture containing 0.5 per cent sulphate aned 0.5 per cent urea 0.5 per cent urea with teepol 0.5 per cent zinc sulphte spray urea 1 per cent super phosphate extra zinc sulphate 0.5 per cent ferrous sulphate + sulphate and 1 per cent urea 6 kg of zinc sulphate per acre Spraying of 0.5 per cent ferous with 0.5 per cent urea of 0.5 per cent ammonium sulphate if available. Ferrous ammonium sulphate if avilable situation ferrous ammonium sulphate be useful

Pulses Cowpea

Water soaked necrotic spots on leaf surface. Root growth very much restricted in 10-12 day old seedlings

Sprays containing copper sulphate zinc sulphate 0.1 per cent and

Oilseeds Groundnut

Chlorosis of terminal leaves

Groundnut

Poor pod formation

Coconut

Lower portions of leaves dried

Coconut

Button shedding

Hollow nut

0.5 per cent ferrous sulphate with urea Gypsum application 80 kg/ac to nutrient spray on 25 & 30 DAS Application of potassium sulphate after regulating the irrigation Application of potassium sulphate with ¼ kg of (zinc) sulphate Application of potassium sulphate with ¼ kg of Borax

Fibre crop Cotton

Ill developed bolls and shedding

Cotton

Pesticide induced toxicity. Irregular leaves and flower drop Reddening of leaves and boll shedding

Cotton

Urea 1 per cent spray with plano concentration Literal water sprays followed by spray Magnesium sulphate 5 per cent 0.1 per cent boron sprays

Vegetables Brinjal

Yellowing of leaves in both old and young

Onion Fruits

Bulbs affected

Guava

Mis-shaped fruits, cracs and corky out growth 1. Marginal scorching and necrosis 2. Chlorosis of younger leaves

Grape / vine

Foliar sprays of 5.0 per cent 1 per cent urea with 0.5 ml / litre 0.2 per cent calcium nitrate spray Sprays of 0.3 per cen boron, sulphate and 0.5 per cent copper 0. 1 Per cernt borax sprays 0.3 per cent ferrous sulphate with urea mixture sprays.

Ornamentals Jasmine

Chlorosis

Rose

Flower bud rot

0.3 per cent ferrous sulphate with u the addition ml/litre of water Bavistin sprays for the control followed by 1 per cent super photo sprays.

9.12. Leaf Area Determination Several methods have been developed for the estimation of leaf area. Among them the most simple, inexpensive and accurate are (1) Graphic method; (2) Planimeter; (3) Electronic method; and (4) Dry weight method. Relatively simple, time saving and non-destructive method for estimating the leaf are aby statistically defined mathematical relationship linear dimensions of the leaf was first proposed by Montgomery (1911) using the following relationship. Leaf area (A) = K x L x W Where, A = Leaf area per leaf B = Leaf area constant L and W are maximum length and width respectively. The value of leaf area constant (K) is the ratio between actual leaf area and apparent leaf area and is generally less than one Apparent leaf / area can be calculated by measuring length and width at the maximum regions of a leaf. Actual leaf area can be measured by using electronic leaf area meter. With regard to regression, the regression equation V = a plus b was developed for calculating the regression constants a and bx. 'Y' represents leaf area, 'x' represents the linear measurements of length and breadth. Crops Equation Rice

Maize

‘r’ Value

A = 0.73 (LxW) dry season A = 0.75 (LxW) wet season

0.690 0.537

A = 0.796 (LxW)  0.01

0.88

References Palanisamy and Gomez (1974) - do – Balakrishnan et al. (1987 a)

A = 0.747 (LxW)

0.994

Stickler et al. (1961)

A = 0.629 (LxW)

0.998

Balakrishnan et al. (1987 b)

0.90

Rajappa et al. (1972)

0.90

Richards (1983)

0.99

Bathla and Sharma (1978)

0.99

Kemp (1960)

0.945

Padalia and Patel (1980)

0.998

Rawsen et al. (1980)

0.99

Sepashkhan (1977)

0.978

Ashley et al. (1963)

0.99

Patil et al. (1989)

0.89

Manina et al. (1986)

0.970

Yeboah et al. (1983)

0.979

Hughes et al. (1979)

0.983

Wiersma and Bailey (1975)

sorghum Cumbu

Ragi

LxWx8.773

LAI =

-----------------  0.1254

Unit land area Leaf length (L) breadth (B) of 5th leaf

Wheat

A = 0.70 (LxW)

Sugarcane

A = 0.6274 (LxW)

Greasses

A = 0.905 (LxW)

Groundnut

A = 0.70 (LxW)

Sunflower

A = 0.73 

Safflower

Y = 0.88 + 0.664 (LxW)

Cotton

A = 0.775  0.978

Castor

A = LxW x 0.516

0.021

Greengram A = 1.84  0.027 (LxW) length and width of theterminal leaflet

Cowpea

Pigeonpea

Soybean

A = 2.325 (LxW) length and width of the terminal leaflet A = 1.705 (LxW)  0.0316 L and W of terminal leaflet

A = 6.532 + 2.045 (LxW) L and W of terminal leaflet

Banana

A = 0.756  0.024 (L x W)

Papaya

Y = 106 x – 1050

Grapes

A = L x W x 0.81

Y = -0.40 + 0.211

0.9764

Balakrishnan et al. (1986)

0.862

Karikari (1973)

0.90

Gill and Brar, 1980

0.90

Garg and Mandahar (1972)

0.897

Asif (1977)

0.976

Hoffman (1971)

0.952

Shrestha and Balakrishnan (1980)

Tomato

Bhendi

Y = 115 x 1050 X = Length of the midrib

Onion

Y = 2.794 + 1.686 (X) X = leaf length

Acid lime

A = 0.608 (LxW)

REFERENCES Ashley, D.A., B.D.Doss and O.L.Bennett (1963). Agron. J. 55:584-6585. Asif, T.M. (1977). Trop. Agric. 54 : 192. Bathia, A.V.L. and H.L. Sharma (1978). Indian Sugar Crops J. 16-17. Balakrishnan, K., K.M.Sundaram, N.Natarajaratham and H.Vijayaraghavan. 1978a. Madras agric. J. 74 : 240-241. Balakrishnan, K., L.Veerannah and M.Kulasekaran (1986). Madras agric. J. 73 : 717719. Garg, I.D. and C.L.Mandahar. (1972). Indian J. agric. Sci. 42 : 950-959. Hoffman, G.J. (1971). Agron. J. 63 : 948-949. Huges, G., J.D.H. Keatinge and S.P. Scott. 91979). Trop. Agric. 56 : 371-374. Karikari, S.K. (1973). Trop. Agric. 60 : 346. Kemp, C.D. (1960). Ann. Bot. 24 : 491-499. Manian, K., K.Balakrishnan and N.Natarajaratnam (1986). J. Agron. and Crop Sci. 159 : 764-767. Padalia, M.R. and Patel, C.C. (1980). Indian J.Agric. Sci. 50 : 880-882. Palaniswamy, K.M. and K.A. Gomez (1974). Agron. J. 66 : 430-433. Patil, S.J., P.R.Reddy, P.V.K. Gounder and S.A.patil. 1989. J.Oilseed Res., 6 : 384-385. Puttasamy, S., S.T.Gowda and K.Krishnamurthy (1976). Curr. Res. 5 : 43-58. Rajappa, M.G., M.K.Jagannath, B.G.Rajasekar and K.N.Mellana (1972). Mysore J.agric. Sci. 6 : 102-106.

Rawson, H.M., G.A.Constable and G.N.home. (1980). Aust. J. Plant Physiol. 7 : 313315. Richardds, R.A. (1983). Aust. J.Agric. Res. 34 : 23-31. Sepaskhah, A.R. (1977). Agron. J. 69 : 783-785. Stickler, F.C., S.Wearden and A.W.Pauli. (1961). Agron. J. 53 : 187-188. Shrestha, T.N. and K.Balakrishnan. (1985). South Indian Hort. 33 : 393-394. Wiersma, J.V., and T.B.Bailey. (1975). Agron. J.67 : 26-30. Yeboah, S.O., J.L.Lindsay and F.A.Gumbs. (1983). Trop. Agric. 60 : 149-150.

9.13. Plant Growth Analysis Absolute and Relative Growth Rate Absolute growth rate (AGR) is a plain and simple measure of rate of increase in weight. RGR provides more informative comparison of the plant's relative performances. Fisher (1921) suggested instantaneous RGR equation as : 10geW2 – 10geW1 RGR T2 – t 1 Where, loge = natural logarithms ln. W1 and W2 are dry weight at time (day) t 1 and t2 respectively. Scope and Significance Thus, RGR is sensitive linkage to all environmental variables and hence changes in RGR are the expression of ontogenetic drifts. Inter specific and intra-specific differences exists in RGR (Blackman, 1919; Crime and Hunt, 1975). RGR provides integrtion of the various plant parts. It is useful for comparison of species and treatment differences on a uniform basic. Leaf Area Ratio (LAR) Briggs et al. (1920) suggested a measure of leaf area ratio (LAR). It is defined as the ratio of total leaf area to whole plant dry weight. In broad sense, LAR represents the ratio of photosynthesizing to respiring material within plant.

LAR

=

(LA1/W1) + LA2/W2) ---------------------------2 (LA2-LA1) x 2.303 (log 10w2 - log10 W1)

LAR =

---------------------------------------------------2.303 (log10 LA2 - log10 LA1) (W2-W1)

Where, LA1 and LA2 are leaf area and W1 and W2 are whole plant dry weight at initial and final time. Net Assimilation Rate Gregory (1926) used the term 'net assimilation' rate (NAR) for ULR. Williams (1946) provided a formula for estimating mean NAR or ULR over a period of time. W2 - W1 NAR = (g/dm2/day)

-----------t 2 - t1

log2 LA2 - log2 LA1 X

------------------------LA2 - LA1

Where, LA1 and LA2 W2 and W1

= =

leaf area (cm2) and total dry weight of a plant (g) at time interval t 1 and t2 (days) respectively.

Specific leaf are and Leaf weight ratio (Pearce et al., 1969) Growth efficiency is associated with leaf area and its weight that mostly reflects leaf thickness. Thickness of leaf is now an important character in relation to the boundary layer and aerodynamic resistances. The mechanism governing leaf expansion considers the analysis of two components of LAR i.e., specific leaf area (SLA) and leaf weight ratio (LWR). LAR is further subdivided into two quantities LA ---W

=

LA ---LW

=

LW ---W

LAR = SLA x LWR Where, (i) LA/LW is the specific leaf area i.e. the mean area of leaf displayed per unit of leaf weight (in a sense measure of leaf density or relative thickness) and (ii) LW/W is the leaf weight ratio, a dimensionless index of the plant on weight basis (i.e. leafliness of the plant area / weight basis). LW Specific leaf weight SLW = ------LA Where, LW = Leaf weight (g) and LA - leaf area (dm2) expressed in same units. SLW decreases as LAI increases which reduces respiration per unit of leaf area. Relative Leaf Growth Rate RLGR involves consideration of physiological activity like photosynthesis, respiration, mineral uptake and metabolic balance, as far as supply of new material is concerned. LA2 - LA1 1 RLGR -----------------x ---t 2 - t1 LA Where, LA1 and LA2 are leaf area at time interval t1 and t2, respectively.

Leaf Area Index (Walson, 1947) Watson introduced more croporiented concept of leafiness in relation to land area. This he named as leaf area index (LAI) and defined as leaf area (LA) per unit of land area (P). LA LAI

=

------P

Crop Growth Rate (CGR) (Watson, 1958) CGR is a simple and important index of agricultural productivity on rate of dry matter production. It was first given a name, crop growth rate (CGR) CGR = NAR x LAI Leaf Area Duration (LAD) (Power et al., 1967) Leaf area duration (LAD) is a measure of the ability of plant to produce and maintain leaf area and its whole opportunity for assimilation during growing season. (LAI1 + LAI2) LAD =



-------------------2

x

(t2 - t1)

Unit Production Rate (Dudney, 1973) Dudney devised unit production rate (UPR) with following equation as : 1 W2 - W1 UPR = -------- x --------------WP t2 - t1 Where, WP = was of perennating structure, W1 and W2 are total dry weights of plant at time interval t1 and t2 respectively. This equation provides a measure of the efficiency with which perenating mass of this provided further dry weight. Growth Efficiency (GE) W GE

=

-----S Where, W = S =

x 100 increase in plant weight (W1 - W2) Weight of substance used for growth (S1 - S2)

Translocation percentage (TP ) Straw weight at flowering - Straw weight at harvest -------------------------------------------------------------------------Panicle weight at flowering - Panicle weight at harvest Biomass duration (BMD) (Hunt, 1978) TP =

W1 + (Wi +1) BMD = ----------------- x t2 - t1 2 Where, W1 and Wi + 1 are total biomass production at t 1 and t2 respectively (between subsequent stages) Biomass density (BD) (Jolliffe et al., 1982) Number of plants x Dry weight per plant BD = --------------------------------------------------Land area Potter and Jones (1977) described the following partitioning factors Leaf area partitioning (LAP) dA /dt LAP

=

--------

dW /dt Leaf weight partitioning (LWP)

LWP =

dL/dt -------dW/dt

Stem weight partitioning (SWP)

SWP =

dS/dt --------dW/DT

Root weight partitioning (RWP)

RWP =

dR/dt -------dW/dt

dm2.day-1 =

-----------g.day-1

Where, W A L S R

= = = = =

total weight at any given time leaf area at any given time leaf weight at any given time stem weight at any given time Root weight at any given time

LAP was much better correlated with growth than NAR. Generally LAP coefficients are extremely sensitive to temperature. It reveals that leaf area expansion is stroungly influenced by temperature. Dry Matter Efficiency (DME) Krishnamurthy et. al., 1973 Dry matter efficiency is a measure of dry matter utilisation for economic yield over a period of time of crop growth can be calculated by using the formula suggested by Krishnamurthy. Seed yield 100 DME = -------------x --------------------------Total DMP Duration of genotypes Specific absorption rate Rate of intake (mineral nutrient content of plant) Williams (1948)

Or

Specific absorption rate (SAR) Welbank 1964)

1 M2 – M1 ---- x ------------- = SAR RW t 2 – t1 where M1 and M2 is mineral content of plant under consideration as time t 1 and t2 respectively and RW is dry wt. of root system. Rate of intake =

Specific utilization rate Hunt (1978) described specific utilization rate (SUR) for mineral nutrients as given below – 1 W2 – W1 SUR = ----- X ----------M t2 – t1 Growth Efficiency (GE) GE = W/S x 100 Where, W = increase in plant weight (W2 – W1) S = weight of substance used for growth (S 1 – S2) Extinction Coefficient -loge I/I0

K=

----------LAI Where I0 & I are the light intensity at top & bottom of a population with leaf area index (LAI). Light Transmission ratio (TR) LTR = I / I0 Where, I = light interrupted at ground level of population. I0 = light incident on top of population Light intensity is expressed in klux or Wm-2. Translocation percentage (TP) Straw wt. at flowering – straw wt. at harvest TP = -------------------------------------------------------Panicle wt. at flowering – panicle wt. at harvest Reference: Girija, T. 1998 Physiological investigation on the Recalcitrancy Behaviour of Mango (Mangifera indica L.) AND RATTAN (Calamus spp) seeds by Department of Crop Physiology.

9.14. Determination of Light Extinction Coefficient (Verhagen et al., 1963) Plant with prostrate arrangements have higher extinction coefficient (K) within the canopy than those with erect leaves. Verhagen et al. (1963) related light extinction coefficient to leaf area index, where K value was low, the intensity of light available at depths in the foliage is high. In general, low light extinction coefficient (K = 0.3) is efficient in allowing more light penetration at critical leaf area index in pigeonpea. The light extinction coefficient (K) was worked out by using the formula adopted by Monsi and Saeki (1953). I

K

Where, I = Io

=

e k

= =

=

IOe-KL

= -

I ----L

loge

I ( -------- ) IO

-

I ----L

loge

I ( -------- ) IO

Light flux density (photosynthetically active radiation) to a horizontal surface below L units of the leaf area index. Light flux density (photosynthetically active radiation) ave the canopy The base to the natural logarithms extinction coefficient

Reference: Monsi, M. and T. Sacki, 1953. Concerning the light factor in plant communities and its significance for dry matter production. Jpn. J. Bot 14:22-52. Chlorophyll. Yoshida, SDA. Forno, J.H.Cock and K.A.Gome Z. 1972. Laboratory. Manual for physiological studies of rice. Int. Rice. Res Instt. Los. Banos, Phillippines. p,70.

9.15. Measurement of Light Interception (Nelliat et al., 1974) Percentage interception of light is calculated by comparison with a solarimeter placed in the open integrated every day. Generally, it can be measured between mid-day and 2.00 p.m. Light intensity in the open - Average of light intensity at the middle of the canopy and ground level Light interception -------------------------------------------------------- x 100 Light intensity in the open Measurement of Light Transmission Ratio (Ltr) (Trenbath, 1979) It will differ on cloudy and clear days. This will help to now the ground coverage of the crop. Place on photometer above the canopy and the other at the ground surface. Read the light intensity on both instruments at the sametime. I1 LTR (%)

=

------I0

x 100

Where, I0 = Light intensity above the canopy I1 = Light intensity at the ground surface Make 5 to 10 measurements in the one canopy and average values obatained. Measurement of LTR will give an idea about the canopy growth of crops. Generally LTR increases as the leaf area index decreases. Reference Trenbath, 1979. Proc. Int. Workshop on Intercropping held at ICRISAT. Pp.141-154.

9.16. Measurement of Rate of Germination Maguire (1962) Rate of germination is carried out in sterlized blotting paper medium. The germination of normal seedlings is counted daily from the third day and the mean percentage of germination recorded in each day and the rate of germination is calculated. X1 X2 – X1 Xn – Xn – 1 Rate of germination = ------ + ---------- = ---------------Y1 Y2 Yn Where, Xn = Percentage germination of nth count Yn = Number of days from sowing to nth count Measurement of Vigour Index Abdul – Balki and Anderson, 1973 On the seventh day, besides germination percentage, mean dry weight of seedling was taken and vigour index was calculated using the formula suggested by Abdul-Baki and Anderson. Vigour Index

=

Germination percentage x mean dry weight of a normal seedling (mg) from standard germination test.

9.17. Measurement of Respiratory Rates of Foliage (Dixon, 1958) Materials Warburg Constant Volume Respirometer Reagents KOH 20% Sorensen – PO4 Buffer Procedure Twenty five numbers of uniform sized leaf bits formed the sampling unit for measuring the oxygen uptake. The leaf bits were suspended in 2 ml. of Sorensen‟s phosphate buffer, adjusted to pH 7.0. fluted filter papers (Whatman No.40) soaked in 0.3 to pH 7.0. fluted filter papers (Whatman No.40) soaked in 0.3 ml. of 20 per cent potassium hydroxide solution were placed in the central well to absorb the carbon dioxide released during the experiment. Then the flask were fitted to the monometers and were allowed for equilibrium inside the water, bath, the temperature of which was maintained at 30C. After the temperature equilibration of the flask for 15 minutes, the experiment was run for two hours in darkness taking readings at intervals of 30 minutes. The plant materials were washed well and dried at 80C for 48 hours. The dry weights were recorded. The oxygen absorbed was expressed as l. of oxygen per hour per mg. dry weight of the tissue. Sorensen’s Phosphate buffer for respiration Solution A

: 0.2 M solution of Monobasic sodium phosphate (27.8 g. in 1000 ml of water)

Solution B

: 0.2 M solution of Dibasic sodium phosphate (53.65 g. of Na2HPO4. 7H2O in 1000 ml of x ml. of solution A + Y ml. of solution B diluted to a volume of 200 ml. A

B

39.0

61.0

PH = 7.0

9.18. Measurement of Aerobic Respiration (Umbreit et al., 1964) Measurement rate of oxygen uptake by plant tissues and the influence of various chemicals on this rate. Procedure In the experiment with the Warburg apparatus, the rate of oxygen utilization, or oxygen, will be measured. Respiration rates are often expressed in terms of the QO 2. This value is equal to the l.O2 used/gm. Dry weight tissue per hour (ul-micro-liter). In any manometric method for measuring gas volume changes at a given temperature, the measurement may be made of either the change in pressure, with he volume of the gas maintained as a constant. This technique used here is the constant volume method the pressure. Thus with each pressure change the original volume is restored in the manometer and flask by admitting or removing fluid from the reservoir at the bottom of the tubes. The new pressure may then be read in mm. Of height of fluid on the outside arm of the tubes. The temperature of the water bath is kept constant by a sensitive thermostat and any variation is compensated for by the use of a flask with no tissue in it which acts as a control for changes either in temperature or barometric pressure. Thus if the TB manometer increases in pressure, the increase decreases, this value should be substracted from the experimental flasks. Each (meaning change) value should be corrected for the TB reading. In order to determine the Qo 2 of the tissue used in the experiment the dry weight of the tissue rust be known. To determine this, the tissue is placed (after running respiration determination) in a small, previously weighed stainless steel cup, numbered to distinguish it on the side. The original weight of the cup is the “tare” weight. The cup and tissue may be dried over night at 95 - 100C cooled and weighed, the difference being the dry weight of the tissue. The flask constants (K) have been calculated for each set of flasks and manometers and should be remarked on the data sheet. Multiplying the pressure in mm of manometer fluid, by the vessel constant (K) yields the equivalent of volume change calculated to 0C and one atmosphere of pressure. A standard procedure recommended by Umbreit et al., 1964 1. Clean, dry, Warbug flasks equipped with a center well, add materials (excent cells) to the main compartment of flask. 2. Add materials (if any) to the side arm. 3. Add 0.2 cc. Alkali (usually 5, 10 or 20 HOH) 4. Grease attachment joint on manometer and grease and insert plug for sidearm, Grease top of alkali cup of alkali cup of desirable 5. Add cells 6. Add filter paper strip to alkali in center cup (see absorption of carbon dioxide) 7. Attach flask to manometer 8. Place in constant temperature bath

9. 10. 11. 12. 13. 14.

Adjust and tighter flask after about 5 minutes shaking in bath. (This is done since sometimes the grease becomes softer and the flask tends to crop slightly) Allow to equilibrate, with shaking, for (10-15) minutes Adjust manometer fluid to zero point (closed side of manometer) Close stopcock Measure readings (Set times for ten minutes at beginning of each reading series, them re-set immediately as it rings and read each flask in same order as the first time)

Reference Umbreti, W.W., R.H. Burris and J.F. Stauffer. 1964. Mnometric techniques. Bull. Nat. Agriculture Science. Tokyo: 1-120.

9.19. Estimation of Relative Water Content (RWC) (Weatherly, 1950) A reduction of RWC from 95 to 90 per cent causes 60 per cent reduction in photosynthesis. Growth is also reduced in cotton sorghum and peanut, when RWC fell to 90 per cent (Slatyer, 1955) The procedure given by Barrs and Weatherley (1962) is explained below. Procedure Normally, a leaf that is physiologically functional (Third from top) is selected for RWC estimation. The fresh weight of leaf is immediately recorded after it is excised. The leaf samples can be brought to the lab after careful sealing in double walled thermocole chamber or plastic bag in order to prevent from water loss and fresh weight (WF) is recorded. The petiole may be detached keeping only leaf lamina. Secondly, keep these leaf samples floating on water under diffused light to get turgid weight (Wt) for 4- 6 hrs depending upon the degree of imbition. Finally the same leaf is kept in an overn at 75C for assessing dry weight (Wd). Further, the values are plugged in the following formula : Fresh wt. (W f) – Dry wt. (w d) RWC(%) = x 100 Turgit wt. (W t) – Dry wt. (w d) The relative turgidity technique consists in comparing the initial and turgid water content on a percentage basis. The RWC was primarily estimated with whole leaf method but most workers were unable to obtain values as high as 98%. The procedure given by Barrs and Weatherley (1962) is explained below. (RWC > 90%  No stress, 80-90%  Mod. Stress. 1.0 suggests relative drought resistant and an index of < 1.0 relative drought susceptibility. The observed differences in stress was correlated with drought index. Further, the drought tolerance efficiency (DTE) can be worked out by multiplying with 100 and ranking genotypes as resistant, tolerant, moderately tolerant and susceptible.

Yield under stress Drought Tolerance Efficiency (%) = -------------------------- x 100 Yield under no-stress Procedure Twelve cotton genotypes were tested for their drought tolerance efficiency (DTE) under rigorous severe stress conditions during 1986 and under moderate stress conditions during 1987. Soil depth was less than 20 cm during former while it was less than 60cm during later year of testing. The water holding capacity ranged from 30-40% in silty loam soil condition. (Stress) as against 50% in clayey vertisol (Normal). The crop received stress during boll development period and onward rainfall receded in Sept. under both the seasons. Normal conditions allowed gradual release of sol water during the late stages of floral development lower water holding capacity of silty loam soil allowed plants to experience adequate stress during continued reproductive stage as was evident from drastic reduction in biomass production and yield.

9.26. Chlorophyll Stability Index for Drought Tolerance in Plants Green plant pigments are thermosensitive and its degradation occurs when it is subjected to higher temperature. This method is based on pigment changes induced by heating. The chlorophyll destruction commences rapidly at critical temperature of 55 to 56°C. Thus, chlorophyll stability is a function of temperature. This base has been formerly used in pine needles immersed in water and heated gradually in a temperature regulated water bath at 58°C. This property of chlorophyll stability was found to correlate well with drought resistance. Materials 1. Large size glass tubes of 2.5 cm in diameter 2. Acetone (80%) and distilled water. 3. Mortar and pestle or blender. 4. Balance 5. Water bath with thermostatic control 6. Colorimeter with red filter (No.66) 7. Whatman filter paper. Procedure Two clean glass tubes are taken and five grams of representative leaf sample is placed in them with 50ml of distilled water. One tube is then subjected to heat in water bath at 56°C ± 1°C for exactly 30 minuets. Other is kept as control. The leaves are then ground in a mortar for five minutes with 100ml of 80% acetone. The slurry is then filtered with Whatman No.1 filter paper. This chlorophyll extract is further examined immediately for light absorption with photoelectric colorimeter using red filter (No.66). a parallel leaf sample of 5g in another tube is then estimated for chlorophyll content without heating simultaneously and light adsorption is measured with colorimeter as explained. The difference in two readings (Reading without heating and reading after heating 56°C = R is defined as chlorophyll stability index (CSI). This CSI is found to be correlated with drought tolerance. High CSI corresponded with low drought tolerance. Thus, CSI is inversely related with drought tolerance efficiency. Effects of antitranspirants on chlorophyll stability index (Readings on Klett) Antitranspirant Cetyl alachol (Soil Application) Phenyl Mercuric ocetate (Foliar Application)

Control 129 -

C1 16 (30 Kg/ha)

122

64

-

(1 ppm)

Concentrations C2 R 113 50 (60 Kg/ha

R 79

58

46

76 (5 ppm)

Results Lower values in treated plants indicate induction of drought tolerance in plants treated with antitranspirants. The values are difference between two readings taken on Klett Summerson colorimeter having an expanded scale. This index has no units of

measurements. The readings on Klett Summerson can be converted to optical density by multiplying it with a factor 0.02 and then R between two readings can be found out. Field Screening Of Crop Plants For Photoefficiency Based on drought intensity studies we have developed a technique to screen cultivars for the photoefficiency and it can also be computed by exposing plants to low light intensity by suitable cloth tents. Photoefficiency index (PEI) can be worked out as : Yield under stress ------------------------- x 100 Yield under control A light stress can be of 20% less of 60% less than normal (100%) light intensity. Increased plant height under low light intensity is a marked feature and researcher may impose light stress in a range of 20 to 40%. Normally a light cut by 45 to 50% is observed during complete cloud cover. PEI (%) =

Procedure The normal light intensity (100%) was interrupted by shading plants with a temporary cloth tents of 4m x 2m x 1.6m size. Tents were prepared with fine mesh cloth (muslin) and rough mesh cloth (majarpath). The light intensity inside & outside the tents was measured with Lux meter and a cut in light intensity was computed. By fine mesh muslin cloth there was 22% cut in light intensity while by rough cloth it was 60%. The plants were covered with tents at 35 days after planting till 75 days, with shaded light along with 100% control.

9.27. Measurement of Leaf Water Potential (Thermocouple Psychrometer) This instrument is used to measure the water potential of leaf tissue, soil or any related material. Thermocouple psychrometer mainly consists of : 1. Microvoltmenter 2. Control unit to supply current to the thermocouple for cooling 3. Recorder for measuring the slope. Advantages of Thermocouple Psychrometer 1. Accurate at fairly low water potential (-25 to –40 bars) 2. Measures the total potential no matter what is in the system such as high xylem potential. 3. Good accuracy 4. Can measure O.P. of soil solution 5. Can measure osmotic potential directly after freezing and correcting the bound water.  Measured after freezing  = --------------------------------1–ß where, ß= 0.2  Total =  + ( + m)  = Osmotic potential negative value (Bars)

p = Pressure potential (Bars)

Precautions and Problems 1. Need to establish a plateau. If too dry, not enough water will condense on the junction and no plateau is established. (Not good for –40 to –60 bars). 2. Probably does not work well above -3 to -4 bars. Growth and respiration become relatively more troublesome, if equilibrium time is increased. Operation of thermocouple Psychrometer 1. Set voltmeter scale to 1 millivolt. 2. To test temperature of psychrometer cup, depress silver toggle switch downward. Needle should deflect to the left about 0.8 to .9 millivolt. (~20°C) 3. Set voltmeter scale to 30 microvolt. 4. Turn on the recorder. 5. Adjust recorder pen to a zero starting point with toggle switch engaged in upward position. 6. As soon as recorder pen is zeroed, start chart paper movement and depress black cooling button for 10sec. Toggle switch should not be engaged during this operation.

7. Release cooling button (Black) and immediately engage toggles switch to upward position. Calculations

Suppose we have to measure total leaf water potential of corn leaf at 20°C. Follow the instructions for operating the instrument step by step. Cut leaf with cork borer of chamber size and keep it in thermocouple. Adjust the recorder pen to zero. Start the chart paper. You will get a curve as shown in Fig.4.3 Extrapolate the parallel line on „Y‟ coordinate and measure units on pen recorder. It comes to 22.5. Refer the standard curve made for known concentration for KCl as shown in Fig.4.4 and get the interest value of water potential () against the measured units of 22.5. It is seen as –13.2 bars. Thus, the total water potential () of corn leaf is – 13.2 bars at 20°C. In order to know the osmotic potential in bars : first, the slope is measured in units using known concentrations of KCl or NaCl. A standard curve is plotted taking known concentration of KCl at 20°C in molar or bars against the slope units obtained on the recorder. This calibration gives the values of unknown further. Measure the water potential of same leaf by freezing it in defreeze in refrigerator for few minutes. A sap is extracted and a filter paper disc of smaller size (0.5 cm dia). Made out of whatman filter for thermocouple chamber and allow it to equilibrate for some time. Graph units are recorded as explained earlier. It comes to 16.5 which directly corresponds with –8.6 bars osmotic potential on graph paper. Knowing these two potentials, we can calculate turgor potential (p) using following equation by substituting the value for each : T = p +  -13.2 = p + (-8.6) p = 13.2-8.6 = 4.6 bars. Thus, corn leaf has total water potential as –13.2 bars, osmotic potential as –8.6 bars and turgor potential as 4.6 bars at 20°C.

Reference: Meidner, H. 1984. Class experiments in plant physiology. George allen and unwin, London, p.169. Girija, T. 1984. Ratooning in Forage Sorghum (Sorghum bicolor (L.) Moench. A Physiological Investigation.

9.28. Measurement of Plant/Canopy Temperature with Infrared Thermometers and Use in Irrigation Scheduling

Principle of Operation The energy flux of an object is a function of its temperature. The IRT senses longwave radiation and converts this value to a temperature scale. Irrigation Scheduling Using The Infra Red Thermometer One of the important possible uses for the IRT will be in scheduling irrigations for maximum water efficiency. When plants begin to undergo water stress, their leaf temperature will start to rise. As the leaf temperature rises, the amount of energy radiated from the leaf increases. Using the IRT, we can measure these changes in energy and thus leaf temperature. There are two main methods of using the IRT for irrigation scheduling. The first is to use the variance in temperature over a crop field. The IRT is used to take several readings over the field. The second method of using the IRT is by calculating the difference in canopy temperature from the true air temperature. If the value is negative, then the canopy temperature is lower than the air temperature. This indicates a high transpiration rate and therefore, a sufficient plant water status. If the value is positive, this indicates a higher canopy temperature than air temperature. Both of these methods need to be used on a day basis. Measurements can not be readily compared from one day to the next. This is due to incoming radiation differences caused by clouds, precipitation etc. A daily observation of evapotranspiration measurements can aid in timing and applying correct amounts of water.

9.29. Measuring Stomatal Conductance, Transpiration and Leaf Temperature (Li-1600 Steady State Porometer) This porometer has set the standards for measurements of stomatal resistance and is used for wide range of application in plant sciences. By utilizing the steady state technique, the Li-1600 (USA) eliminated calibration difficulties. The measurement is rapid thus minimizing potential modification of stomata since observations are recorded in 10 seconds. It facilitates the following measurements. 1. Stomatal conductance (cm.s-1) 2. Leaf temperature (°C) 3. Rate of transpiration (µ.g.cm-2.s-1) 4. Instant photosynthetic Active Radiation (PAR) (µE. cm-1s-1) The measurements can be made without shaping the leaf and a ventilation radiation shields maintain the cuvette temperature within 3°C of ambient. The cuvette fan circulates and mixes vapor and incoming dry air in a systematic pattern, creating only a shallow, constant boundary layer. Leaf temperature is measured using a thermocouple in contact with the leaf. Data can b recorded and transferred to a cassette tape. Operation 1. The aperture area and atmospheric pressure are initially entered in to the porometer. 2. After the cuvette is acclimated to ambient condition, the null adjust valve is used, to bring the flow rate within the dynamic range of the internal automatic flow controller. The amount of adjustment is dependent on the resistance of the sample. This process can be eliminated on subsequent measurements under similar conditions. 3. The sample is clamped into place on the senor head. Immediately, the humidity setting switch (Hum.SET) is pressed to store the cuvette RH I into the memory as the null point humidity (typically ambient). The humidity other than ambient can also be as the null point. This process need not be repeated if the same null point is to be used for subsequent measurements. 4. The null adjust indicator meter is observed. If the indicator has stabilized within the range of the meter, the cuvette has reached equilibrium within 1% of the user, set null balance humidity, reading can then be taken. The hold switch can then be used to transfer the data to cassette tape or a computer, and or hold the parameters in memory of the display. If the indicator is not within the range of the meter and several seconds have elapsed, the null adjust value should be turned in the direction indicated on the meter. This is repeated until the indicator stabilizes within the meter range. A reading can then be taken. Theory of operation Water loss from a leaf determined by maintaining a constant vapour density in a cuvette that is in contact with the transpiring leaf. This is achieved by pumping dry air in to the cuvette at an appropriate, measured rate to obtain a balance (at predetermined humidity) between the flow of water transpired by the leaf and the flow of moist air out of the cuvette. Stomatal resistance is determined directly from the measured parameters.

Operation and maintenance of Humidity sensor 1. In handling, do not touch or scratch the surface of the sensor. 2. Do not store below 10% RH or above 90% RH. 3. Do not store in a confined environment with desiccant that may vaporize. 4. While installing a new sensor, install with a surface having a thin film on it towards the cuvette wall. This is the surface with the two lads soldered to it. 5. Clean only with dust free air or rinse with the distilled water. If rinsing with water, allow the sensor to re-equilibriate for one day at approximate humidity at which you will be using it before making measurements. 6. Do not expose the sensor to solvents, cigar smoke, sulphurous pollutants. It affects polymer coating on the sensor and cause failure.

9.30. Construction of Pressure-Volume Curve and Estimation of Water Potential  and  with Pressure Vessel Procedure The pressure chamber techniques have long been used to measure the various potentials of leaf tissue since its discovery by Scolander et al. (1965). The water potential can directly be measured by applying a balancing pressure till xylem exudates oozes out instantly provided cells adjoining xylem are at equilibrium and xylem sap has negligible  selected turgid leaf is covered in a plastic bag for preventing vessel with its stalk projecting outside. It is good if excision to a leaf or a stalk is given while it is immersed in water in order to maintain its xylem continuity and preventing xylem cavitation. Gradually, compression of the air around the leaf cell is attempted by pressure knob with an eye on the cut stump. Cells are squeezed and a small drop of liquid is seen (use lense) on the excised portion of the leaf or stalk. A pressure (p) that is compressed is numerically equal to newly imposed xylem sap tension when leaf is at atmospheric pressure. Collect the expressed water (V1 & V2) and weigh it or get the value by subtracting initial wt. (W1) or from leaf after squeeze (W2). Record pressure on the gauge required to oozout the liquid. Continue this operation till you end up with no exudates. This time you may need very high pressure (>400 psi) that is quite dangerous to handle. Discontinue this operation and keep this leaf in oven to get the dry wt. By now, we have 6-8 exudation pressure points (p) to plot a pressure-volume curve with corresponding expressed water, V3, V4, V5 and V6 etc. For the cell protoplast, pv = constant, so far protoplast, of I/P against V is linear. If the linear position is extrapolated to zero potential when I/V = O, this gives the OP of vacuolar sap at full turgor. Divide psi by 14.7 to get values in bars and reciprocal values (I/P) are obtained. Calculate the total water content (VT) by subtraction from graph (Fig.4.14).

9.31. Water Potential of Polyethylene Glycol and Stress Imposition in Water Culture A water stress is imposed with polyethelene glycol (PEG) of various molecular weight under water culture experiment. The stress so created though does not directly correspond to the stress under field conditions is often used by researchers to investigate fundamental mechanism of drought tolerance in various crops. This osmoticum (PEG) is now widely used to induce water stress. Te creation of stress conditions with PEG does not parallel with field conditions since it is complex phenomenon a plant can experience. However, basic information about stress can be gained by artificially creating conditions close to the field conditions. An osmoticum such as PEG is not harmful to the plant growth. Association of PEG-600 and PEG-1000 of various concentrations with water potential (w). *Gram PEGMos/kg Calculated w bars 600/100 ml. 6.15 187 -5 7.52 225 -6 8.94 255 -7 10.11 299 -8 11.38 331 -9 12.65 368 -10 14.52 482 -13 17.25 600 -16 Gram PEG-1000/10 ml. 8.23 174 -5 10.20 218 -6 11.46 247 -7 13.10 288 -8 14.50 332 -9 15.56 366 -10 17.94 494 -13 20.70 612 -16 * Solution brought to volume with nutrient solution

Actual w -5.22 -6.22 -7.02 -8.18 -9.03 -10.01 -13.03 -16.16 -4.87 -6.04 6.81 -7.89 -9.06 -9.96 -13.35 -16.50

How to create a stress? The water potential (bars) of PEG-600 and PEG-1000 in percentage is furnished in Table 4.7 along with its corresponding osmolality (m.Os/Kg). A graph can be plotted taking percentage PEG (wt/vol) on X‟ axis and water potential in bars (w) on „Y‟ coordinate for both PEG-600 and PEG-1000. A water stress of desired bars can then be imposed through water culture technique. Example Suppose, one is interested to impose a w of –8 bars with PEG-600. As per table 4.7, the corresponding PEG-600% comes to 10.11 (Wt/Vol.). The density of PEG.600 is

1.114 g/ml. Thus, 10.11 g of PEG-600 equals 9.0754 ml/100ml volume. If the volume of the nutrient solution in a pot is 11 liters, then one has to add 998.29 ml of PEG-600 (11000 ml x 9.0754/10 = 998.29) in a nutrient solution to induce water stress equivalent to –8 bars. The water stress of greater magnitude >-5 bars can also be imposed from the values that can be obtained from a graph. Caution The calculated quantity of PEG-600 may be added slowly in a span of 3-4 days to induce water stress step by step. Imposition of stress immediately in a day may be fatal to the seedlings. Gradual induction of stress is always advised.

9.32. Procedure to Evaluate Photosynthesis on intact single leaf Such method of measuring rate of photosynthesis on intact attached leaf is devised by Clegg et al. (1978). A plexiglass chamber has been developed to conveniently incorporate the leaf of intact plant. An additional advantage of this method is that one can measure the photosynthetic rate while leaf is attached to the plant and reliable estimates can be obtained without modification of stomatal conductance. The samples of air from this plexiglass can be made in few seconds with the syringes within some definite time interval and therefore, there can not be any major modification in the boundary layer resistance of the leaf. The method is quick and reliable. The technique to evaluate PSR on a single leaf basis made use of a portable, 1.8 dm3 plexiglass assimilation chamber tightly attached to the central portion of the youngest fully expanded leaf (Marked leaf). Two 10.0 cm3 air samples were drawn for every determination, one after the other in a time interval of 45 seconds. Common glass syringes were used for this purpose. After all the plants in one growth room were sampled, the paired air samples were fed to an infrared gas analyzer (Backman 865) to quantitate their CO2 concentration in parts per million (PPM). Materials required 1. IR gas analyzer. 2. flow meter 3. mV Recorder, 4. Drying columns, 5. Plexiglass chamber 6. Nitrogen gas cylinder, 7. Alkathine tubings, 8. Syringes with cap. Procedure 1. Grow the plants in growth chamber at specific temperature you are interested. 2. allow the plants to equilibrate with the temperature gradually. It takes roughly 45 minutes. 3. Get the plexiglass chamber of known area (1.8 dm3, Fig.7.2). 4. Insert the leaf inside the chamber and support seal for getting the air sample, 7 ml each. 5. Two syringes should already be in the rubber seal for getting the air sample, 7ml each. 6. Start the internal fans by switching on the button with a thumb. 7. Note the time and get the initial sample of 7 ml air. 8. Allow it to run for 30 to 60 seconds (calibrate your sample) with fans on, and get the air sample with another syringe which is already inside the chamber. 9. Mark the leaf that was inside the chamber (Photosynthesizing surface). 10. Detach the leaf and obtain the leaf area on area meter. 11. Take the syringes to the IRGA which is already on for half an hour and allow the 7ml air sample in the samples-in. Know the CO2 content in ppm. With N2 gas as reference. (0.6 LPM flow rate). 12. Incorporate another air sample similarly into the IRGA and get the CO 2 concentration in ppm. 13. The step 11 and 12 should only be commenced after calibrating the IRGA with known CO2 concentrations that can be obtained from the companies. 14. The CO2 can be obtained / unit time and the values can be plugged in the following formula given by Eastin and Sullivan, (1969) :

PSR = (F x CO2 x CF) / LA F = air flow rate, in dm-3, h-1 = (1.8 dm3 x 3600 sec h-1) / 45 sec. CO2 = CO2 uptake by the enclosed leaf in mg of CO2 dm3 i.e (CO2 at initial stage – CO2 at 45 seconds) x (44000 mg CO2 per mole / 22.4 dm-3 per mole) (10-6). CF = Correction factor for temperature and atmospheric pressure (unitless) = (270°k / air temp, °k) x (97.3 Kpa / 101.3 KPa) = 262.22/temp, in °K LA = Leaf area in dm2, of the leaf portion enclosed in the assimilating chamber

Where,

Calculations 7 ml of 380ppm CO2 gave 22 units, on IRGA for 1 unit on IRGA = 17.273 ppm, CO 2. Data Collected Air Crop Sample (ml.) Corn 7

Leaf Area Initial cm2 Reading

Final Reading

128.06

36.5

32.0

Soybean

35.15

36.5

35.5

7

Pn (corn) = =

Pn (Soybean) = =

CO2 Difference

Time

4.5 (0.3599/s) 120 sec. 1.00 180 sec. (0.09546/sec.)

44000mg CO2 / mol x 10-6 x 3.943(1) --------------------------------------------- x 0.3569 CO2 x 3600 22.41/mole -------------------------------------------------------------------------1.2806dm-2 -2 -1 7.836mg CO2.dm hr = 0.22 mg CO2m-2.s-1. 44000mg CO2 / mol x 10-6 -------------------------------- x 3.943(1) x 0.09596 x 3600 22.41/mole -------------------------------------------------------------------------0.3515dm2 7.614mg CO2.dm2h-1 = 0.21 mg CO2m-2.s-1.

Result The photosynthetic rate in corn and soybean did not differ at seeding stage.

9.33. Photosynthesis Estimates with Labelled CO2 Material Plexiglass chambers, (Fig.7.2); Radioactive NaHCO 3 (100µ ci); 0.2 N HCl, syringes with sealing bids, leaf area meter, Nitrogen gas cylinder, IRGA, Scientilation counter. Procedure 1. Equilibrate the plans with the temperature you are interested to initiate the operation in a growth chamber. 2. Insert a part of the attached leaf into the photosynthetic chamber having a vial to support radioactive NaHCO2 (Fig.7.2). 3. Close the chamber and allow fans to run for few seconds. 4. Get two air samples with syringes at an interval of one minute to obtain CO2/unit time. 5. Insert a syringe with 0.2 N HCl though a rubber seal and add one ml acid in a vial containing NaHCO2. Note the time. 14CO2 will start liberating. Continue to rune for three minutes. 6. Mark the leaf area which was photosynthesing inside the chamber and cut the leaf to get its area and dry matter. 7. Keep the leaf in an oven at 75°C for dry matter and radioactivity data. 8. Plug in the values in the equation and obtain Net photosynthesis (P n) in mg CO2/m2/s. Data Collected 1. Seven ml of 380 ppm CO2 gas reads sample 21.8 units on IRGA. Thus, 1 unit amounts to 17.43 ppm. 2. (a). Initiatl readings of IRGA for 7ml gas sample was = 53.9 units (b). Second sample = 49.8 unit. (c). Difference of reading = 4.1 units. (d). CO2 thus is = 4.1 x 17.43 = 71.46 ppm/60 seconds. 3. Leaf area is = 164 cm2 = 1/64 dm2 4. Total dry weight of leaf = 0.814g. 5. Volume of the assimilating chamber = 1.51 litres 6. Radioactivity (dpm) = 1539/50 mg tissue

Pn =

44000mgCO2/molx10-6 1 ---------------------------- x Chamber volume x ppm CO2/s x 3600

sec. x -----22.41/mol 2

dm

= =

1964.285 x 10-6 x 1.51 x 1.191 ppm/Sec x 3600 ---------------------------------------------------------1.64 7.75 mg CO2 / dm-2 / hr = 0.215 mg CO2 / m2 / s.

leaf

area

Pn calculation on radioactivity Total activity = 1539 dpm/50mg 814 mg = 25054.92 dpm/leaf = 1.1285 x 10-2 µ ci 1 µ ci = 2.22 x 106 dpm 25054.92 dpm = 1.1286 x 10-2 µ ci 58 mci / m. mol Na2HCO3 0.1 mci = 0.0017241 m. mol. CO2 1 m mol = 46 mg CO2 0.0017241 m. mol. CO2 = 0.0793 mg 14CO2 0.0793mg 14CO2 is in 100µ ci = 1.1286 x 10-2 µ ci = 8.949 x 10-6 mg 14CO2 14 Since uptake of C is 80% as efficient as 12C uptake thus is, 8.949 x 10-6 --------------- = 1.1187 x 10-5 12CO2 0.8 1.1187 x 10-5 + 8.949 x 10-6 = 2.0136 x 10-1 mg CO2 2.0136 x 10-5 mg CO2 = 180 sec. (3 minutes) 1 sec. = 1.1187 x 10-7 mg CO2 / s / 164 cm2 164 cm2 = 1.187 x 10-7 10,000 cm2 = 6.82 x 10-6 mg CO2/m2/s A. Net photosynthesis measured with  CO2 = 0.215 mgCO2/m2/s B. Pn measured with 14CO2 = 6.82 x 10-6 mg CO2/m2/s Result The Pn measured with 14CO2 radio active technique is much lower than the one measured with direct CO2 data. The techniques of labeled C14 is more accurate.

9.34. Measuring Photosynthesis and Transpiration in whole plant by Constant Flow System Materials required 1. Plexiglass chamber of (10x10x2 dm) size = dm3 Area with built-in fan. (can be modified as per requirement). 2. Infra Red Gas Analyser. (Beckman-865). 3. Air pumps. 4. Cooling Tower 5. Flow meters of varying capacity. 1 LPM to 200 LPM. (LPM = Liters per minute) 6. Pen chart recorder. 7. Growth chamber to monitor photosynthetic active radiation and temperature. 8. Dew point hygrometer 9. Tygon tubings. 10. Leaf area meter. Procedure The Constant flow system is designed to monitor the net photosynthetic rate (PSR) and transpiration of the entire aerial part (shoot) of sorghum plants and consisted of : a) 200 dm3 (10x10x2 dm) plexiglass assimilation chamber, able to enclose the whole shoot of even a fully developed sorghum hybrid plant; b) two pressure vacuum pumps connected in parallel to force outdoor air into the plexiglass box; c) a cooler in between the pumps and the assimilation box to offset the heating effect of the air pumps; d) a fan inside the box, located slightly above the incoming fresh air (inlet), that provided internal circulation and mixing of the air : e) several gas flow meters conveniently allocated to regulate the flow rate of the air passing through the assimilation chamber, and f) an exit (outlet) for the „used‟ air, which was located 20c below the internal fan. Air is sampled at 2 LPM rate through dew point hygrometer to measure a transpiration. The assimilation chamber is attached to an infrared gas analyzer, Beckman 865 (IRGA) by two air sampling lines, one pulling fresh air from the tinlet and the other line pulling „used‟ air from the outlet. Both lines had a flow rate of 1.0 dm3 min-1 and were activated by a small pressure-vacuum pump. The IRGA was previously calibrated with two different standard gas whose CO2 concentrations are known. So the number of ppm of CO2 per unit of IRGA is was determined. Once the system reached its equilibrium, the IRGA meter of the chart recorder indicated the net CO2 uptake by the enclosed plant at any interval of time. To monitor the net photosynthesis of entire sorghum shoot at different air temperatures but at constant light intensity, the assimilation chamber was assembled inside a growth room keeping the instruments outside the growth room. As in the method based on a single leaf, the calculations to estimate net carbon dioxide exchange rate (PSR) of the whole plant in mg of CO 2 dm-2 h-1, were also based on the expression given by Eastin and Sullivan (1969).

Thus, Equation and Computation of results : PSR = F x CO2 x 1.9643 x 10-3 CF/LA Where, F = flow rate of the air passing through the assimilation, chamber, dm-3 h-1 (in most cases, F = 10800 dm-3 h-1) CO2 = net CO2 uptake by the enclosed plant, in ppm 1.9643 x 10-3 = constant to convert ppm of CO2 into mg of CO2, regarding CO2 as an ideal gas. CF = 262.22 / air temperature, in K (K = °C + 273) LA = functional leaf area of the enclosed plant, dm2, as measured with a portable leaf area meter (LI-3000). In these calculations the leaf blade is considered as the unique photosynthetic tissue, neglecting the possible contribution of other green tissues, such as leafsheaths and exposed stalks. This technique required 20 to 30 minutes to monitor one plant. The dew point hygrometer (E G C –400) measures with precision the dew point temperature of gas samples flowing through its sensor. Two readings were obtained for every plant one from the fresh air entering the assimilation chamber (inlet) and the other from the „used‟ air exiting the system (outlet). Therefore, difference in water vapor density between the outlet and the inlet represents the amount of water lost by transpiration from the enclosed plant at any interval of time. As such no difference is humidity of incoming and outgoing air was detected without a plant in a assimilating chamber. Then, to calculate mean rate of transpiration (TR) per unit of leaf area in µg. H 2O lost/cm of areas, the following expression was computed : TR = (f x  WVD) / LA Where, F = flow rate of the air passing through the assimilation chamber (m3/s). WVD= (WVD outlet – WVD inlet) x (106,) in µ g/m3 WVD = Water vapor density of the air in (g/m3) as given in Table 108 of Smithsonian meterological Table. LA = Functional leaf area / plant in cm2 as measured with portable leaf area meter (LI-3000). Obviously, the estimates of TR with this method are including both sides of the leaves (upper & lower epidermis). So the values obtained will not necessarily match with those obtained with the steady, state promoter which measures TR on the lower side of leaf. 2

9.35. Measurement of Light Intensity The measurement of light intensity in visible wave length region is normally done by photographic type light meters. These meters work on the principle that light on the photographic plate or film coated with photo sensitive material e.g. selenium cell produces an electric current which is proportional to the intensity of light. There is relationship between the number of molecules photochemically changed in the photosynthesis and the number of photons absorbed, regardless of energy of photon, in as long as the photon I within the wave length band 400-700 nm. The spectroradiometer, a most versatile and useful instrument has now been developed for measurement of light quality which gives a direct readout and scanning of light intensity in 380 to 1550nm wave length region of solar radiation. The wedgeinterference filter system in the spectroradiometer gives continues wave length scan and fibre-optic extension head is specially suitable for light quality measurements in plant canopies. The instruments, when used in inverted position can measure the light quantity as intensity of reflected radiation. PAR  A full sunlight, midsummer, cloudless sky is 2000 µEm-2S-1 PI  B. Full sunlight, midsummer, cloudless sky or 1000 Wm-2 Light intensity with various sensors and sources Sources a) Room light b) Growth chamber c) Walk-in growth chamber d) Research green house (sorghum canopy) e) Outside (winter day)

38.0

7.9

Reduction(%) Quantum (PAR) 98.1

225

53.0

88.75

94.70

1050

460

47.5

54.00

135

71

93.25

92.9

290

150

85.5

85.0

Quantum PAR Pyranometer µ Em-2 sec-1 (PI) Wm-2

Pyranometer (PI) 99.21

Conditions : a) Sensor 1m away from light source (scale = 100), b) Sensor near sorghum canopy, approximately 1m away from light source (scale used 1000), c) Sensor above soyabean canopy, roughly. 1.5m away from light source. d) very diffused light, glass fibre roof is covered with snow. Sensor near sorghum canopy. Plant height 30cm cloudy day. E) cloudy day of winter, time 4 p.m. measured outside the green house.

Conversion Factors 1a. Photosynthetic Photon Flux Density (PPFD) (LI* - 190SB, Li – 191SB, LI-192SB Quantum Sensors). Photosynthetic Photon Flux Fluence Rate (PPFFR) (Li-193SB, LI194SB spherical Quantum Sensors. Units (instantaneous) 1µ E s-1 m-2

= 1.0µ mol s-1 m-2 = 6.02 . 1017 quanta s-1 m-2 = 6.02 . 1017- photons s-1 m-2 Full sunlight = 2000 µ E s-1 m-2 for the 400-700mm wave band 1b. Photosynthetic Photon Exposure (LI * - 190SB, LI-191SB, LI-192SB, LI-193SB, LI194SB) Units (Integrated) = 3600 µ E m-2 = 3600 µ mol m-2 1 µE s-1 m-2 = 2.17 x 1023 quanta m-2 = 2.17 x 1021 photon m-2 One day‟s integration = 60µ E m-2 Solar Irradiance (LI-200SB Pyranometer, LI-201SB Pyrheliometer) Photosynthetic Irradiance (LI-190SEB, LI-192SEB Photosynthetic Irradiance sensors) Near Infrared Irradiance (LI-220SB Near Infrared sensor) Units (Instantaneous) = 1.433 x 10-3 cal cm-2 min-1 = 1.433 x 10-3 langley min-1 = 0.100 m W cm-2 1W m-2 = 1000 erg cm-2 s-1 = 5.285 x 10-3 BTU ft-2 min= 0.317 BTU ft-2 h-1 = 1000 W m-2 when using Pyranometer and Pyrheliometer sensors Full sunlight = 500 W m-2 when using Photosynthetic Irradiance sensor = 60 W m-2 when using Near Infrared sensor Temperature : Temperature is closely related to the radiant energy. The advection of a different air mass into the region may cause a appreciable change in temperature. For example, advection of land and sea breezes of forced wind too much affect the climate of the whole region.

Measurement of Temperature Air temperature can be measured by making use of various sensors like mercury or alcohol in glass-thermometers, thermo-couples, thermistors etc. The copper-constantan thermocouples are widely used in micrometerological measurement of temperature. It is referenced against ice bath to generate e.m.f. (electron motive force) that can be related to the temperature. Thermocouples may also be used to measure temperature difference with greater accuracy by wiring differentially. The instrument used for measurement of surface temperature in this method is called as radiation thermometer. The working of radiation thermometer, is based on Stefen‟s law, the intensity of outgoing radiation from the black body (earth surface behaves approximately as black body surface) is proportional to fourth power of its temperature (in degree absolute) i.e. I = E..T4 T = (I/E)1/4

9.36. Estimation of Stomatal Index and Stomatal Frqeuency This method has been developed by Wolf and his associates (1979). It is simple to use and can have useful information not only on stomatal index but also its frequency / unit leaf area. Procedure The applicator brush attached to bottle lid is dipped into the fluid. The brush is stroked across the area to be replicated with a single motion. A little practice is required to gauge the proper amount of fluid needed on brush. After a few seconds when the fluid was firm, the replica was ready to be removed. A small piece of double adhesive celluloides tape was secured to a glass slide. The exposed sticky surface is then placed over the replica and with slight pressure the film is removed from the leaf surface. Then the replica is mounted for viewing as a imprint on reverse of the image of leaf surface. Using a microscope with calibrated grid, the number of stomata and epidermal cells can be counted. Take a circular piece of paper of the size of eyepiece with a rectangular cut in the center. Insert this paper in an eyepiece. 2. Your microscopic field now appears rectangular and now you are ready to scan small area of leaf easily‟ using the low power objective 3. Count the number of epidermal cells (E) and the stomata (S) in a specified field. 4. Plug in the values in following equation and get the values of stomatal index (SI). SI (%) = S/S + E X 100 This is the proportion of stomata to the epidermal cells. This has relevance in studies on plant water relations. The observation on stomatal frequency can be obtained following few further steps. 1. Mount the stage micrometer on the stage of the microscope. The scale is of one mm that is divided into 100 equal divisions and each division is of 10µ. 2. Consider the stage of divisions with a rectangle / square that is in the eyepiece (Ocular) and count the divisions for the length and breadth of a rectangle / square. Know the area of the rectangle / square in µ 2 using low power objective; 3. Count the number of stomata within the area of rectangle / square. Half cut stomata may also be counted. 4. Now you know the scanned area and the stomatal number, compute the stomatal frequency in mm2 per unit leaf area. A transparent finger nail polish (Colodion) can be used to get the impressions. Dissolved compounds such as colodion or celeries acetate when spread on the leaf will dry and replica can be peeled of by means of transparent tape that can be stored for examination.

9.37. Test for Pollen Viability Iodine or acetocarmine stains indicate the amount of starch development in pollen grains, but this may not always indicate the viability of pollen. Watkins and Curtis (1968) developed a staining technique to determine viability of wheat pollen which was highly correlated with actual germination tests. This method is likely applicable to pollen of other crop species. They used an aqueous solution of sugar gelation, and MTT, i.e. 3 (4,5 dimethyl thiazolyl 1-2) 2, 5-diphenyl tetrazolium bromide. The medium is prepared a follows : Add 50ml. of distilled water to 2.75g of gelatin (such as Knox unflavored). Heat in a water bath, stirring until gelatin is completely dissolved. Dissolve 28.5 g cane sugar in the gelatin solution. Add 0.0075g MTT for each 10ml. of sugar-gelatin solution. Mixing must be accomplished while solution is hot but do not boil. Place hot standing medium on 1.5mm. – deep depression slides at the rate of one drop per slide. Let medium cool 1 or more minutes until a semi-hard gel forms. Use cover slips to prevent drying. Staining solution which is not used can be refrigerated and reheated for later use. To test viability, dust fresh pollen on medium, which has been held at room temperature, replace cover slip. After one hour, viable pollen grains have a dark purple color while non-viable grains are colorless. A 100 x magnification (low power) is sufficient. Tetrazolium chloride(2, 3, 5-triphenyl tetrazolium chloride) has also been used to test for pollen viability. Munoz (1963) used a solution of 0.5% tetrazolium chloride in 5% sucrose (in water) to test fresh sorghum pollen for viability. Pollen was left I the staining solution for at least 30 minutes. A red colour in the pollen grains indicated viability whereas lack of color indicated non-viability. Reference:

Kushavamurthy, M.N., 1995. Male gametophytic selection in identification and development of moisture stress tolerant lines in sunflower (Helianthus annus L.) M.Sc.(Ag) Thesis submitted to University of Agricultural Sciences, Bangalore, India.

9.38. Soil Analysis There are number of physico-chemical properties of soils which may be used to assess their potential to assimilate certain levels of specific pollutants without affecting their intended use for plant growth and nutrition. This presentation starts with soil sampling in the field followed by only the most essential laboratory tests. Soil Sampling In the event of a suspected soil pollution, it is necessary to collect surface soil samples (top 20-30 cm) from the affected and control areas using the standard procedures of sampling (De, 1962; Black, 1965a). Apparatus The apparatus required for soil sampling includes spades, soil augers-a small one for examination of the soil and a large one for taking deeper samples, if required (Jackson, 1958); polythene bags of appropriate site to hold about 500 to 3500g, or more soil (depending upon the specific purpose and number of parameters); a cloth bag to hold the filled up (polythene bag; and a marker for labeling. To guard against accidental confusion of samples, it is desirable to place an identification tag inside the bag in addition to using an external marking or tag. Soil Sample Processing Sample processing includes transport of the soil samples from the sampling site to the soil testing laboratory and subsequent handling of the soil material for various parameter-specific analytical procedures. After reaching the laboratory, the soil sample is divided into three parts; one-about 2.5 kg, only in case of the samples for which the soil permeability and hydraulic conductivity are to be determined, is placed in an enamel plate of the required size and kept in a hot air oven for drying at 100-110°C, this part is designated as Stock A; second part-abut 300g- is air dried or oven dried at 30 to 50°C, this part is designated as Stock B; a third part-about 150 g, -is kept in a suitable polybag in a refrigerator for microbiological parameters. Material required for this phase of sample processing thus include a hot air oven with heating range of 30 to about 120°C enamel plates, polythene bags and the usual tagging material. Processing of the dried sample involves gent grinding followed by sieving. For these two steps, the material required are agate mortar and pestle, iron mortar and pestle (for certain physical properties where interference by metallic contamination is of little significance) and a 10 mesh sieve used for sieving the soil sample to the upper size limit of 2.0mm. The sieved soil can be safely stored in a polythene bag of an appropriate size for the usual period required for the desired analysis. The soil from the Stock A is used for the determination of bulk and particle densities, texture, water retention properties, permeability and hydraulic conductivity. The air dried sample is used for the determination of pH, electrical conductivity. The air dried sample is used for the determination of pH, electrical conductivity, individual dissolved cations and anions, exchangeable cations, nutrient elements (nitrogen, phosphorus and potassium), metals and pesticides. The microbiological properties of the soil (to be preserved in a refrigerator) have not been

covered here; detailed procedures have been published by the American Society of Agronomy (Black, 1965b). Bulk and Particle Densities, Porosity The material required for determination of these parameters include a 25ml graduated glass cylinder, a Mettler balance, glazed-smooth paper sheets, a 50ml volumetric flask, a 20ml bulb pipette, a 10ml bulb pipette, a graduated 10ml ordinary pipette, a conical flask with distilled water. For routine analysis of soils for these parameters in pollution studies, the conventional methods (Black, 1965a), such as the core method, the excavation method, and the clod method used for determination of soil bulk density are not suitable as these methods do not exclude rat/insect/organism holes and also gravel and other heavy material exceeding 2mm size, if present in the sample. For determination of soil bulk density, b, expressed as the bulk weight in grams per cubic centimeter of the soil, the soil from the Stock A, passed through a 2mm sieve and kept in a polythene bag within a tagged cloth bag, is filled in a dry pre-weighed (W1) 25ml graduated cylinder, bit by bit with intermittent tapping gently, upto the 20ml mark and weighed again (W2) on the Meter balance. The soil bulk density, b, is then determined as follow :

b=

Weight of 20ml bulk soil W2 – W1 ------------------------------- = ------------- g/cm3 Bulk volume of the soil 20

The cylinder containing 20ml soil is then carefully emptied on a glazed paper sheet and the soil is then transferred completely and carefully to a 50ml volumetric flask. Now, 20ml distilled water is added to the soil in this flask. After about 5 to 10 minutes, additional 10ml distilled water is again added to the soil in the flask. After some time, all the air voids in the soil will be filled up with water. Then, with the graduated 10ml pipette, add distilled water to fill up the flask upto the 50ml mark and measure this quantity of additional water (say x ml); this quantity of additional water is equal to the volume that was occupied by the air voids in the bulk 20ml soil. The soil particle density, p, is determined as follows : Weight of 20ml bulk soil W2-W1 ------------------------------ = ------------- g/cm3 (20-x) (20-x) In this expression, the weight of the air occupying the voids in the bulk soil, which being extremely small, is neglected. p=

Using the particle and bulk densities, soil porosity is determined as follows : (p-b)100 Soil porosity (percent) = ----------P As an example, the relevant values determined on a soil sample, using the above procedure, have been given below :

W1 = 45.0115g; W2 = 73.1487g; b = (W2 – W1)/20 or (73.1487 – 45.0115)20 i.e. 28.1372/20 = 1.406 g/cm3. This sample required 7.6ml water to fill up the air voids. Thus, the volume of the soil particles alone works out to be 20 –7.6 or 12.4 ml, and the particle density, p is equal to 28.1372/12.4, or 2.269 g/cm3. The porosity works out to be (2.269-1.406) x 100/2.269, or 38.034, say 38%. Soil bulk density is used for conversion of the contents of nutrient elements (% ppm etc). or that of any soil amendment into per hectare basis assuming a plough layer of 17cm. This has been explained later. Soil Texture Apparatus The material required for determination of soil texture includes a Mettler balance, glazed paper, a 400ml ordinary glass beaker (for each sample), a Soil Hydrometer with graduations on the stem from –5 and –4, -3, -2, -1, 0, 1, to 60, a heavy glass or polythene beaker to keep the soil hydrometer over a soft paper fold in the beaker with the hydrometer bulb resting on the paper, two glass rods-one 15cms long and another 60 cms long, a 1000 ml graduated glass cylinder, distilled water, a watch glass to cover the beaker, a wash bottle with a fine delivery, a 3-cycle semilogarithmic graph paper and a copy of Triangular Texture Diagram based on the international particle size fractions with effective diameters of 0.002, 0.02 and 2.0 mm for upper limits of the clay, silt and sand fractions, respectively. Regent Sodium hexametaphosphate Procedure Take 40grams soil from the Stock A, ground and sieved through a 2.0 mm sieve in the 400 ml glass beaker. Add 50ml of saturated of sodium hexametaphosphate (42 g/l) and about 100ml distilled water. Mix using the 15cm glass rod, rinse the glass rod with distilled water into the same beaker, cover the beaker with a watch glass and keep overnight at the room temperature. Next day in the morning, mix again using the glass rod and transfer quantitatively to a 1000ml graduated glass cylinder, rinsing the beaker and the glass rod into this cylinder. Immediately, add distilled water to this cylinder upto almost the 1000ml mark. Stir the soil suspension with the 60cm glass rod, rinse it in the cylinder and make up the volume to the 1000 ml mark. Immediately, but gently, insert the soil hydrometer into the cylinder, with the bulb down and holding the stem carefully, note the time. Take the first hydrometer reading after 30 seconds. Without removing the hydrometer, read it at the end of 1 and 3 minutes. After this, remove the hydrometer carefully, rinse the surface using minimum quantity of water (with the fine delivery wash bottle), wipe it dry with a soft towel and keep it with the bulb on a soft paper in the heavy beaker (Fig.16.1). Without remixing the suspension between subsequent measurements, lower the hydrometer carefully into the suspension about 10s. before each measurement and take hydrometer readings at 10, 30, 90, 270, 400, 600 and if possible, 720 minutes. In these 9 hydrometer

readings (excluding the 720min. reading), the first reading will be highest and the last lowest. Against each hydrometer reading, the value of  is recorded from Table 16.1.

Values of  for determination of particle size from observed hydrometer readings, R R R R    -5 50.4 -4 50.1 11 46.4 26 42.2 -3 49.9 12 46.2 27 41.9 -2 49.6 13 45.9 28 41.6 -1 49.4 14 45.6 29 41.3 0 49.2 15 45.3 30 41.0 1 48.9 16 45.0 31 40.7 2 48.7 17 44.8 32 40.4 3 48.4 18 44.5 33 40.1 4 48.2 19 44.2 34 39.8 5 47.9 20 43.9 35 39.5 6 47.7 21 43.7 36 39.2 7 47.4 22 43.4 37 38.9 8 47.2 23 43.1 38 38.6 9 47.0 24 42.8 39 38.3 10 46.7 25 42.5 40 38.0 The particle size in microns is given by  divided by the square root of the time in minutes. This has to be converted into mm unit by dividing by 1000 or by multiplying by 10-3; the later is convenient for plotting the particle size on a semilog 3-cycle graph paper. A control hydrometer reading taken with only the 50ml saturated solution of sodium hexametaphosphate and distilled water made upto 1000ml mark is also taken each time using another 1000ml cylinder; this reading is designated as R L. The concentration of suspension for each reading is then calculated from the equation c=R-RL, and the summation percentage, P = 100(c/40). The particle size in mm (taken on the X-axis of the semilog graph paper) and the corresponding percentage are then plotted on the semilog paper. From this plot, using 2 x 10 -3, 2 x 10-2, 5 x 10-2 and 2.0mm as the particle size limits for the clay, silt, fine sand and sand fractions, respectively, the percent contents of clay, silt fine and coarse sand are determined in the soil sample, as the cumulative percentage is taken on the Y-axis. With these values, and using the Triangular Texture Diagram (this diagram is usually available with most of the State and Central Soil Testing Labs), the texture of the soil is determined. References Statyer, R.O.1955. Studies of the water relations of crop plants grown under natural rainfall in Northern Australia. AustP2. J. Agri. Res. 6:365. Thll, D.C.; R.D. Schirman; A.P. Appleby. 1979. Osmotic stability of polyethelene gycol20,000 used as seed germination media. Agron. J. 71:105-108. Clegg, M.D., C.Y. Sullivan and J.D.Eastin. 1978. A sensitive technique for the rapid measurement of CO2 concentrations. Plant Physiol. 62: 924-926. Austin, R.B. and Longden. P.C. 1967. A rapid method for measurement of rates of photosynthesis using 14CO2 Ann. Bot. 31:245-353.

10. General procedures 10.1. Chromatographic Separation of Plant Extracts Chromatography is probably the most powerful tool to separate substances from a mixture according to their partition coefficients between two immiscible phases. The liquid-liquid chromatography (paper chromatography) and solid liquid chromatography (thin-layer chromatography) are widely used. Ion exchange chromatography Iron-exchange materials are practically insoluble in water and in organic solvents. They are all high molecular-weight branched polymers, containing ionic groups, either cations (cation exchange resins) or anions (anion exchange resins) and they exchange reversibly with other ions in the surrounding medium. Cation-exchangers contain weakly acidic groups, carboxyls or strongly acidic groups, sulphonic acids Weak cation-exchangers are ionized only above pH 7 while strong cation-exchangers are ionized at all pH levels. Anion-exchangers are of two types : weakly basic groups such as amino groups or strong types containing quarternary ammonium groups. The former is ionic at low pH while the latter at all pH levels. A cation-exchanger is used for the adsorption of cations and ananion-exchanger, for anions. Materials 1. Glass column (8 cm height and 2 cm diameter) 2. Sintered glass disc or glass wool 3. Beaker 4. Glass rod 5. Fraction collector (if available) Reagents 1. Selected resin 2. Ethanol 3. Suitable developer (an acid or a base) 4. Suitable buffer Method Use glass columns of 8 cm height and 2 cm diameter; convenient for the separation of small aliquots. Plug the lower end with glass wool or sintered glass disc. Fill up the column with water (Fig 3). Mix the selected resin with water and allow standing for at least one hr to swell. Add 0.5 ml of ethanol to eliminate air bubbles. Stir the slurry with a glass rod often-on

and pack the column with great care; pack the resin uniformly. In a column, about 60 per cent of the packed volume is due to the ion exchange material and the other 40 per cent due to “voids” filled by fluid. Do not allow the column to go dry; have it filled with distilled water or weak acid or alkali. The extract to be separated is allowed to flow slowly through the column at a suitable pH value. Wash the column with distilled water, once at twice. Develop the column with a developer, an acid or a base, depending upon the mixture being chromatographed. If accurate control of the pH is required use cation buffers on anionexhangers and anion buffers on cation-exchangers. The developer may be of one uniform concentration or of gradually increasing concentration. If a fraction collector is available, individual fractions of 0.2 ml or 0.5 ml may be collected and analysed. The flow rate may be speeded up by increasing the temperature or applying vacuum. Reference: Wardlaw, I.F. and H.K. Porter. 1967. The redistribution of stem sugars in wheat during grain development. Aust. J. Boil. Sci., 20: 309-318.

10.2. Radio Tracer Techniques for 14CO2 Studies This apparatus for incorporating 14CO2 to each leaf. The leaf or ear was enclosed in perspexcontainer, the mouth of which was sealed by a rubber plug which had been cut into two. The leaf was placed between the two pieces of the rubber plug. The rubber plug was sealed with a small amount of water, acidified with hydrochloric acid for preventing the leakage of the gas. Beeswax with cotton wool labeled with 14 C solution (obtained from BARC, Bombay) was taken in a vial by means of a Hemilton syringe. The cock of the thistle funnel was opened, a certain amount of 50 per cent lactic acid was poured in, then closed. 14 CO2 was expelled from the Sodium carbonate solution and was dispersed uniformly by means of an aspirator. The leaf or ear was illuminated under natural sunlight. The 14C assimilation was initiated at about noon for all the stages. At the end of the assimilation (30 minutes), excess saturated solution of Barium hydroxide was added and precipitated the unused 14 CO2 as Barium hydroxide was added and precipitated the unused 14 CO2 as Barium carbonate. The samples were taken out and separated into individual organs, at different sampling time. From the date of ear emergence, the stages were fixed for 14 CO 2 assimilation. After feeding, in one set, plants allowed to translocate for varying durations (0, 4, 8, 24 hours and at harvest) under natural conditions. Plants intended for „O‟ hour reading were immediately killed in an over at 80C. The proportion of radiocarbon in the plant organs was determined by a direct assay technique similar to o‟Brien and Warlaw (1961). The plant tissues after killing at 80C for 3 days were weighed and plunged into 80 per cent ethanol. The plant tissue to be examined was pulverized with pestle and mortar and the pulverized material was extracted with 80 per cent ethanol, centrifuged and the volume of the supernatant (alcohol soluble portions) was measured. The residue was treated with 2N Hydrochloric acid and autoclaved for 60 minutes at 15lbs per square inch pressure (120C). The hydrolysate was neutralized with 2N Sodium hydroxide and volume was measured. The ethanol soluble and insoluble (HC1extract) samples were transferred to 16mm planchets, dried under infra-red lamp and their radioactivity was determined in Geiger Counting system under thin mica (1-2 mg per cm2 thickness) end window GM tube (Type No:I.1031 of Electronic Corporation of India Limited, Hyderabad). The activity was expressed in cpm on dry weight basis, unit area basis and unit volume basis. Refernce Brien, T.P. and I.F. Wardlaw. 1961. The direct assay of 14 C in dried plant materials. Aust. J. Biol. Sci., 14: 361-367.

10.3. Chromatography Three different chromatographic techniques are far more commonly employed in the college laboratories. They are : a) Adsorption chromatography b) Partition chromatography c) Ion exchange chromatography

a) Adsorption chromatography Adsorption is the adhesion of a gas, liquid or solid dissolved in a liquid to the surface of a solid. In adsorption chromatography this property is used to bringing about separation and then, identification of a substance. The most widely employed adsorbents include Magnesia, Kieselghyr, Sucrose, Charcoal and alumina and the most widely employed solvents are acetone, petroleum ether, water, butanol, acetic acid, chloroform, benzene, phenol and ammonia. In fact, many hundreds of organic solvents are in use nowadays

b) Partition chromatography This technique is evolved on the basic of solubility of a substance in different solvents. Currently, two types of partition chromatography are in extensive use : paper chromatography and Thin layer chromatography. There are two types of paper chromatography : Unidimensional and 2dimensional. In the first type a small aliquot of the mixture from which the separation of the compounds is sought to be effected is applied to the chromatographic paper at a spot called the Origin : It is the loading spot. A micropipette with a capillary will do excellently to do this, though a glass rod serves quite well. The spot should not be spread out : it must be small and yet contain sufficient quantity of the mixture. Two or three application at intervals long enough to permit drying of the previously applied mixture are recommended. The paper so loaded is developed inside a glass jar usually referred to as chromatographic chamber. For the success of the experiment it is essential that you do not leave any impressions from your hand on the paper : and tht the chromatographic chamber is suffused with the vapours of the solvent. After pouring sufficient quantity of the solvenet close the mouth of the chromatographic chamber (Jar) by ground glass lids smeared with Vaseline. Another important precaution to be taken while conducting the experiment is to see that the paper (the one on which the development takes place) does not touch the walls of the jar. It is held in the jar in such a way that though in physical contact with chromatographic solvent the loading spot itself is slightly above the solvent surface. The essential idea is that as the solvent ascends or descends through the length

of paper, depending upon the experimental arrangement, it carries with it the components of the mixture as it passes through the loading spot. Since the movement of the solvent is along the length of the paper only it is called unidimensional chromatography. When far better separation is envisaged, two dimensional chromatography is employed. In this technique, after the development of the chromatogram with one solvent is completed in one direction, the paper is dried, turned at right angles and the process repeated with another solvent. When two dimensional chromatography is planned the paper is marked with two faint pencil lines, one a little above the bottom edge of the paper and the second a little inside the left margin of the paper. The areas where the loading spots are meant to be prepared are indicated by faint circles made in pencil a little above each line. In such a chromatogram the individual components of the mixture are spread over the entire surface instead of appearing in a line as in the case of unidimensional chromatography. Thin layer chromatography A glass plate coated with an adsorbent is used instead of paper in this technique. A glass plate of the required size is taken and a slurry of Silica gel, plaster of paris and a small quantity of glue is evenly poured over it. It is than dried on a hot air oven. The rest of the procedure for development a chromatogram is the same as in paper chromatography. The TLC has two advantages over paper chromatography. First, the quality of the adsorbing surface can be prepared to suit the fastidious demands of the experimenter. In paper chromatography no such control is possible, at any rte in the laboratory. Second, in TLC the elute can be scraped out in its entirely. Whether it is paper chromatography or TLC, indeed in any chromatographic setup, it may be noted that there is a stationary phase and a mobile phase. Ready-made chromatographic kits, consisting of all the items necessary to run a chromatogram are available. It is better that we use them for these experiments. They are time saving and labour saving. The procedure for identifying the compounds so separated differs depending upon the properties of the compound. The method employed may be any one or a combination of those given below. 1.

If the compound is colourless, it is inspected under U.V. light. Substances such as gibberellins and some phenoic acids fluoresce under U.V. light.

2.

Often, a reagent is sprayed on to the chromatogram. The compounds develop colour on interaction with the reagent. For example, aminoacids turn deep violet when ninhydrin is sprayed on them.

3.

The substances are eluted serially and physical properties such as pH, optical rotation, refracted index, electrical conductivity etc. are determined : all of which would well from a basis for identification of the compund.

4.

If the material is radioactive, use a Gieger – Muller counter

5.

If the substance is biologically active, employ bioassay.

6.

Martin and his associates found that a compound in a chromatogram can be identified by arriving at its Rf value, Rf standing for Resolution front. The Rf value is arrived at as follows : divide the distance moved by the spot by the spot by the distance move by the advancing front of the solvent. Let us say, three different compounds, A, B and C traveled distances of 3.4, 6.9 and 12.6 cms. Respectively along with adsorbent surface and that the solvent itself had traveled 15 cms. From the origin. Then, the Rf values for A, B and C would be respectively. 5.4

6.9 = 0.36,

15

12.6 = 0.46 and

15

= 0.84 15

Note that the Rf value is always less than 1. The Rf value is the same for given compound, if temperature, solvents employed and the time of development are maintained uniformly. Paper chromatography for sugars In this experiment, chromatograms are prepared for a set of known sugars as also for two or three unknown mixtures of sugars. The component sugars of the unknown mixtures are identified by an educated guess, made on the basis of a comparison of Rf values of known and unknown sugars. The unknown sugar mixtures are obtained from nectarines of some of the flowers from the college garden. Plants like Asclepias, Poinsettia, Thevetia, Turners etc. will serve very well. Collect the fluid and make it upto 1% (w/v) concentration. Prepared 1% (w/v) Sugar solutions of glucose, fructose, arabinose, ribose and galactose. Prepare the two solvents required : solvent I, prepared from t-butanol, glacial acetic acid and water in the ratio of 3:1:1 and Solvent 11, prepared from phenol and water in the ration of 4:1. About 300 ml. of each solvent will be required. Get glass jars, preferably 10” by 18” to serve as chromatographic chambers. Have suitable ground glass lids with rims along which grease may be applied. So that the

jars are made airtight on being covered with these lids. Two jars, one for each solvent will be needed. Pour each solvent into its respective jar, label it suitably and cover it with the greased lid. Leave them for several hours so that the atmosphere inside the jars is suffused with the vapours of the solvent in it. Whatman No.1 chromatographic paper cut to suitable size will serve as the stationary phase of the chromatogram. Prepare two sheets, one to serve in each solvent. Lay the paper on a clean surface with the long axis parellel to the edge of the table. Draw a pencil line 2” above the bottom edge. Then make a series of pencil dots at one inch intervals. Each dot will serve to indicate the identify of the sugar mixture with which the loading spot is made a little above that position. Label suitably. In between the known sugars, the loading points for the unknown sugar mixtures are prepared. In the preparation of the loading spots and in handling the chromatography paper observe the precautions metioned in the general account. After all the spots on each sheet are dry, staple it in such a way so as to form a cylinder with the row of spots near the base. The paper cylinder must be so prepared that it can stand upright in the jar, containing the solvent. Now, put each paper cylinder into the chromatographic chamber assigned to it. You need about 10-12 hours for the development to be completed. After development, remove the paper cylinders, unfasten the staples and mark the solvent front in each. Hang the papers to dry. When completely dry, spray p-anisidine reagent* and place them in an oven at 80C for 3 minutes. Circle the resulting spots with a pencil. Note the colours. * - anisidine Reagent : Add 2 ml. of phosphoric acid to 50 ml. of 95% methyl alcohol. Dissolve 0.5 gms. Of P-anisidine in the mixture and then add excess of concentrated hydrochioric acid until the solution is purple. (Should be freshly prepared). Paper chromatography for aminoacids Two points need to be remembered in particular as one plants to obtain a chromatogram of aminoacids. First, it has to be two dimensional and second, the colourless aminoacids can be brought into relief only by spraying a reagent on to the chromatographic paper. Usually, Ninhydrin is employed for the purpose. In this experiment also the unknown aminoacids of an extract are identified by comparing the Rf values with those of the known. Two solvents are employed. In each solvent, two chromatograms, one for the known mixture of aminoacids and another for

the unknown aminoacids of the extract prepared are to be developed. Therefore, four chromatographic chambers are required, two for each solvent. Preparation of a known aminoacid mixture : Available standard aminoacids such as alanine, arginine, glutamic acid, lysine, tyrosine, valine etc. are dissolved in 10 ml. of 10% isoprophyalcohol. 0.5% solution is alright for purposes of this experiment. Prepared aminoacid solutions properly labeled may be stored in a cool chamber. Preparation of an unknown aminoacid mixture : Take 5 gms of fresh been seedlings and grind in a mortar, adding 25 ml. of 80% ethanol. Filter it through Whatman No.1 filter it through Whatman No.1 filter paper. The residue on the filter paper is then repeatedly washed with 80% ethanol. This is then evaporated on a waterbath to dryness. The dried extract is then mixed with 10% isoprophyl alcohol and centrifuged at 3000 g. for 5 minutes. The supernatant is a mixture of aminoacids and can be used for our experiment. Preparation of Chromatographic chambers and solvents : Four glass jars (10” x 18”) with lids of ground glass plate will serve as excellent chromatographic chambers. Number them 1,2, 3 and 4. Into the Jars 1 and 2 pour solvent 1 and into the Jars 3 and 4, pour solvent II. Chromatograms for known aminoacids are developed in Jars 1 and 3, first in Jar 1 in one direction and then in jar 3 in the other direction. Chromatograms for the unknown aminoacids are developed in jars 2 and 4, first in Jar 2 in one direction and then in Jar 4 in the other direction. After you are satisfied that the papers are fully dry, turn them through 90C such that th 2nd pencil line (the one drawn inside the left hand margin) on each sheet is parallel to the edge of the table. Prepare the loading spot, taking care to see that the source is the same the loading spot, taking care to see that the source is the same for each paper. For purposes of easy stapling, you may trim " off the first stapled edge. Repeat the procedure with Solvent II. Take care to see that the paper carrying the loading spot from the known aminoacid mixture goes into Jar 3 and that the paper carrying loading spot from the bean extract goes into jar 4. it will be 10-12 hrs before the development of this second chromtogram is completed. Now, take out the papers. Unstaple and mark the resolution front on each paper. Dry them. When throughly dry, the paper is sprayed uniformly and lightly with ninhydrin reagent. Heat the sprayed paper at 90C for 5 min. Then outline the spots with a pencil. Note the colours. Most of the aminoacids give purple spots. However, phenylalanine, tyrosine and aspartic acid give blue colour; tryptophan, olive brown; aspargine, cystine and cysteine, brown; and proline yellow. Calculate the Rf values for the aminoacids of the known and unknown mixtues. Compare and identify.

Solvent I is water-saturated Phenol. Solvent II is made up of n-Butanol, glacial acetic acid and water mixed in 3:1;1. About 350 ml. of each solvent is required. Pour our Solvent I into the Jars 1 and 2 such that there is " of it in each Jar and pour out solvent II into Jars 3 and 4 similarly. Label the Jars appropriately in terms of the Solvent present in each. Leave them with the solvent sufficiently long so that the atmosphere in each jar is suffused with the vapours of the solvent. A large sheet of chromatographic paper is taken and cut into two sheets of a suitable size. Two faint pencil lines, one about a cm. above the bottom edge and another about a cm. inside the left hand margin are drawn on each paper. The spots where the loading spots are to be prepared are indicated by a small circle above each line. While handling the paper, do be caredul so as not to leave any impressions from your hand especially in that part of the paper through which the solvent is going to rise. On one paper, prepare the loading spot from the mixture of known aminoacids and on the other, do the same using the extract prepared from the Bean seedlings. The loading spot is prepared, keeping in mind all the precautions mentioned in this regard. Label each sheet appropriately to indicate the source of the loading spot. Each sheet is then made into a cylinger by stapling the margins together. Then the paper cylinder is made to stand upright in the chromatographic chambers or Jars 1 and 2. The paper carrying loading spot from the known aminoacid mixture is put into Jar 1 and the paper carrying loading spot from the Bean extract into jar 2. Development takes about 10 to 12 hours. Remove the papers, mark the solvent front and dry them after the development is complete. Reference: Moore, W. and D. Johnson. 1967. Procedures for chemical analysis of wood and wood product. Nadison, W.I. US forest product laboratory, US Department of Agriculture.

10.4. Atomic Absorption Spectrophotometry Atomic absorption is a quantitative technique of the determination of elements in analytical samples. The analytical samples must be in solution or suspension. The sample in solution or suspension is hostd to a high temperature by burning it in a flame. The flame breaks up the chemical bonds between the molecules and enables the individual atoms to float freely in the sample area. In this condition the atoms (unexvited) absorb ultraviolet or visible radiation. The wave length bonds which each element can absorb are narrow. Hence at a particular wave length the absorption is to be measured. The amount of light absorbed gives a direct indidation of the amount of metal that is present (concentration). Nitrous oxide - acetylene flame gives a better atomization Detection limit 5 l/ml. only. Summary of Procedure in routine operation 1.

Switch on the instrument, set the lamp at the desired current, and allow to stabilise for 10-15 minutes.

2.

Set the indicator unit in the 'TRANSMISSION' mode with the select swithch in 'Normal'

3.

Set the monochromator to the wave length required with the relevant slit opening and using a gain setting to give approximately 80% Treading, adjust the 'SET' until a maximum reading is obtained on the indicator Unit. Adjust the gain so that meter reads 100% T.Check that the meter reads Zero when zero adjustment may be made with the backing control.

4.

Select the desired meter of operation on the indicator unit (i.e) 'ABSORBANCE' or 'TRANSMISSION'

5.

Select the 'Auto 100' mode and trim the 'Set 100' to read 0.000 Absorbance or 100.0% transmission depending on which mode is being used.

6.

Light the flame

7.

Nebulize a sample into the flame and adjust the burner position and gas mixture until the desired sensitivity is obtained.

Types of flames A.

Air/Acetylene : For Tin, molybdenus and chromium estimations, Copper, Iron, Potassium, Magnesium. Absorbance between 0.1to 0.2.

B.

Nitrous oxide - Acetylene For Magnesium, calcium, strontium, barium, tin, chromium and Molybdenus.

C.

Air - Hydrozen : For Arsenium, selenium, tellurium and tin.

D.

Nitrogen - Hydrozen - Entrained Air : Arsenic, Selenium, Cadmium, Mercury, Tin, Fellurium, Lead, Calcium and zinc.

10.5. Low Voltage Paper Electrophoresis The design depends upon the magnitude of the heat produced by the applied current. It the potential gradient along the paper strip is less than 20 V/cm. (low voltage paper electrophoresis) an adequate control of evaporation can usually achieved simply by means of a tight fitting lid. If it is higher than 20 C/crv - 100 V/ca (H.V.Z) direct cooling of the paper strip is necessary. Horizontal or flat bed electrophoresis tank Strips of paper are clampld between two shoulder places (upto 30 cm apart) and supported in the middle by a thin nylon thread or on a series of pointed rods. It has the advantages of being adaptable for gel electrophoresis. The Buffer In general the buffer has two functions 1. 2.

To wet the strip with electrolyte so that current will flow To maintain the correct pH throughout the experiment so that the conditions of migration remain as constant as possible. Some exampls of buffers used in paper electrophosis (to make 1:0:1 in water)

1. For serum proteins

2.

Barbiturte pH 8.6

1.84 g. diethyl barbituric acid 10.3 g. medium barbiturate

Borate pH 8.6

8.8 g. sodium borte 4.65 g. boric acid

Borate pH 8.6

7.65 g. sodium borate 0.62 g. boric acid

Phosphate

0.6 g sodium dihydrogen PO4 (H2O)

pH 7.4

2.2 g disodium hydrogen PO4 (Analyses)

For amino acids Pthalate pH 5.9

3.

For carbohydrates

5.10 g. pot hydrogen pthalate 0.86 g sodium hydroxide

Borate pH 10.0

7.44 g. boric acid 4.0 g sodium hydroxide

Experimental procedure 1.

Equalise the buffer levels in opposite compartments

2.

Filter paper cut into 3 to 5 cm width

3.

Mark the origin line - depends on the expected spread of separtion (1.3) Blood serum 1/3 distance between the shoulder piece from the cathode end.

4.

Dip the filter paper strip in buffer, blott, position in the electrophorosis tank

5.

Apply a potential difference between the electrodes (either at constant current or constant voltage of upto 1 hour. The voltage across the paper should be 5-10 V for every contimeter strips of length.

7.

When separation over remove the paper and dry it

8.

Stain with appropriate dye a. Protein : Bromophenol blue, naphaleine black, Axocarmine Amide schwartx, Lissamine green b. Lipides and Lipoprotein - Sudan black, oil Red , oil blue c. Glycoproteins - Diphengglamine d. Carbohydrates - Schiff - periodic acid reagent e. Amino acid - Ninhydrin, various specific dyes

After staining the excess dye is washed from the strip with suitable solvent. The amount of dye taken by the various components can be estimated by elution or by photoelectric scanning. Elution gives the more accurat results. Scanning method gives a series of peaks which can be resolved into component areas. In calculating the percentage compositions allowance must be made since (1) th dye uptake is lessthan proportional to the concentration of a component and 2. at a given concentrations, different components will take up different amounts of dye (eg) the globulins components. For the protein standing, Radiotracer such as 32 P, 35S and 131I have been successfully for detection purposes. Application 1. Analysis of protein mixtures, protein can be more easily separated. 2. 3.

Low vol. Elec. is of limited value in the study of aminoacids since only separation into basic, acidic and neutral groups are possible. Nucleic acid derivatives and carbohydrates can be studid by LVE. Better separation by HVE. LVE is a valuable technique for separating protein and other high molecular weight substance.

10.6. The Tracer Technique On certain occations, it is necessary to follow the pathway of the compounds without disturbing the material. In such cases, photograph of the treated part of the organism is taken as the radiations from the radio isotopes have the same effect on the photographic plate as that of light. Taking advantage of this properly the technique of autoradiography is adopted for tracing the pathway of various elements in the organism. There are different methods like apposition method, coating method, shipping method etc. and one of these is used depending upon the purpose, clarity required, type of film used, period of exposure etc. In this process, a qualitative study of the pathway of the compounds can be made. In addition, to the radio isotopes, certain of the stable isotopes are also used for studying the various processess. The stable isotopes which have been widely used in agriculture are, Nitrogen 15, denterium etc. When the stable isotopes are used, then the same has to be counted with the help of mass spectrometer. However, in view of its high cost, only a few of the institutions have this facility and as such the amount of work done with stable isotopesare comparatively less than that with radio isotopes. Autoradiography The fagging of photographic film by various radiations is exploted in autoradiography to study the distribution of radio activity in the material under study. For this, the plant is fed with the isotope, washed free of any contaminating external radioactivity after the specific period of administration and killed instantaneopusly by pressing in between two hot copper plates. The dried plant is pressed against an x-ray film in dark. For about 10-15 days depending upon the nature and energy of the radiations and the amoutn of radioactivity concentrated in the material. The regions of the plant that come to have the isotope emit raditions that fog the film much similar to viable light. On development, the irradiated areas appear on the film as darkened areas corresponding to the distribution of the tracter. This 'self written image" of the plant is called the autoradiograph or radioautograph and the corresponding technique is autoradiography or radioautography.

10.7. Poly Acrylamide Gel Electrophoresis A principal virture of this method is that protein components are directly visualised. Relative migration of proteins on non-denaturing gels is a function of molecular mass and charge. Procedure for making slabs Assemble the glass sandwich according to the manufacturer's instructions. If these are unavilable, take a notched glass plate and border the straight sides with 3 plastic spacer strips that have been coated on both sides with a continuous bead of petroleum jelly. Complete the sandwich with an unnotched plate and hold together with clamps or binder clips spaced along the 3 unnotched edges. Hold upright, fill to the notch with water and examine for leaks (particularly at the bottom corners). Decant the water and dry inside the sandwich with flter paper strips. Measure the distance between notch and bottom spacer; using a marker, draw a horizontal line on the notched plate, about 1/8 this destance from the notch. Fill the sandwich to this level with resolvinggel. Carefully cover the surface with a water layer to give an even, horizontal boundary. Afte polymerisation (10 minutes), decant the water. Rinse the surface of the resolving gel with a few millilitres of stacking gel. Insert the comb (lightly coated with petroleum jelly) until the teeth are 1-2 mm short of being fully submerged in gel, taking care not to trap air bubbles below the teeth. After the gel has set (10 minutes), it may be used directly or stored in a refrigerator for upto 3 days in a sealed plastic bag. Preparation of samples Sample preparation affects the results markedly. For denaturing gels, protein is commonly precipitated with 6 - 10% w/v cold trichloroacetic acid. After centrifugation, the precipitated protein is washed in 1:1 ethanol/ether and dissolved approx. 15 l 0.1 M Na2 CO3, 10% v/v mercaptoethanol using agitation or sonication if needed; then 15 l 24% w/v sucrose, 4% w/v SDS, 0.1 w/v bromopheol blue is added. The effect of heating the samples at this stage for 2minutes at temperature from 40 - 100C should be investigated, as resolution may be enhanced or diminished. Each land (or "well") on the gel will be loaded with 10-100 l of fluid consisting of a control, a molecular weight marker or a solubilised sample containing 10-50 g of protein. the lower values are used for "mini-gels", or when only a few polypeptides are expected to be present. It follows that some method is needed for determining approximate protein content; on the case of thylakoids, this can be estimated assuming 1.0 g chlorophyll represents 5 g protein.

Loading the samples Remove the sandwich from the apparatus. Insert a straight edge between the glass plates at the bottom and gently prises the plates apart. Rinse the gel with water and transfer to a plastic box. Wear gloves when doing this. Cover the slab with stain solution and gently agitate for about 3 hours. Pour off the stain (this can be filtered and reused), rinse the slab and box in water, then cover the slab with enough destain solution to halffill the box. Agitate as before for 1 hour, decant and discard the solution and agitate with destain solution for a further 2 hours. Decant and rinse with destain solution. Mount on a gel-drying apparatus, with the slab up, cover with cellulose acetate film and apply vaccum. Preparation of solutions Rubber gloves should be worn while preparing and handling solutions. Ensure that ventilation of the work area is adequate. Acrylamide monomer is very toxic. Replace 10% mercaptoethanol with 0.1 M dithiothreitol, if available, to minimise risk of allergic sensitization to thiols. Acrylamide/Bis stock Dissolve 300 g acrylamide completely in less than 1.0 1 water, add 4.0 g Bis (N, N‟ - methylenebisacrylamide), adjust to 1.0 1, decolourise with 0.5 g activated charcoal, filter, store in refrigerator. 1.5M Tris - Cl, pH 8.8 Dissolve 18.15 g Tris in about 60 ml water, adjust to pH 8.8 with HCl, make up to 100 ml. 0.5 M Tris - Cl, pH 6.7 Dissolve 3 g Tris in about 30 ml water, adjust to pH 6.7 with HCl, make up to 50 ml. Other solutions needed 10% w/v SDS; 0.2 M Na2 Na2EDTA; 10% w/v ammonium persulphate (freshly made); 50% w/v sucrose. Resolving gel Acrylamide/Bis 1.5 M Tris/Cl, pH 8.8

15 ml 3 ml

10% SDS 50% sucrose 0.2 M EDTA water

0.3 ml 1.0 ml 0.3 ml 10 ml

Evacuate and agitate in small Buchner flask, to remove air, immediately before use, gently mix in TEMED ** 10% ammonium persulphate 0.3 l

15 l

Stacking gel acrylamide / Bis o.5 M Tris - Cl, pH 6.7 10% SDS 0.2 M EDTA water

15 ml 1.25 ml 0.1 ml 0.1 ml 4.95 ml

Deaerate as for resolving gel; immediately before use add; TEMED ** 4 l 10% ammonium persulphate 0.1 ml Running buffer Tris glycine 10% SDS 0.2 M EDTA

3g 14.4 g 10 ml 10 ml

Make up to 1.01 with water. The pH should be 8.3 Destain solution Dissolve 0.25% w/v coomassie Brilliant Blue R250 in methanol: glacial acetic acid : water (50:7:43 by volume), then filter. Reference Laemmli, U.K. 1970. Nature, 77: 680. Manual on techniques in Molecular Biology (Proteins). 1986. Workshop held at the Department of Biochemistry, Tamil Nadu Agricultural University, Coimbatore. pp. 16.

10.8. Infra Red Gas Analysis for CO2 Studies (IRGA) (Coombs et al., 1987) Infra - red gas analysis is by far the most popular method of determining changes in CO2 concentration, resulting from the photosynthetic and respiratory CO2 exchange of plants. Infrared gas analysis has been used for the measurement of a wide range of heteratomic gas molecules, including CO2, H2O NH3, CO, SO2, N2O, NO and gaseour determination of CO2 concentration, but also H2O vapour in transpiration studies and CO, SO2, NO, etc. in studies of atmospheric pollution. The infra-red gas analyser consists of three basic parts, the I. R. source, the sample chamber, and the detector. Gas analysis systems IRGs can be incorporated into three basic types of gas analysis system for the measurement of photosynthetic and respiratory CO 2 exchange by plants: closed, semiclosed, and open systems. In a closed system air is drawn from a chamber enclosing the leaf or plant into the analysis tubes of the IRGS, which is calibrated in absoluted mode. The air is then recycled from the IRGA back to the chamber. Thus, no air will leave the system and no air willenter it from outside. If the leaf enclosed in the chamber is phtosynthesising then the CO2 concentration in the system will decline, and continue to decline until the CO 2 compensation point of photosynthesis ( ) is reached. Rate of photosynthetic CO 2 assimilation (F CO2) can be calculated from the equation (1). CO2

=

Ca.V ------t.A

.

. . . . . (1)

where Ca = change in CO2 concentration over time interval of length t . (mg m-3) V t A

= = =

volume of system (M3) length of interval over which CO2 concentration (s) leaf area (m2)

Thus to determine CO2 in a closed system the only requirements are: that the IRGA is calibrated accurately on absolute mode and that V and A are accurately determined. The major disadvantage of this type of system is that since the CO 2 concentration of the system will be changing, CO2 cannot reach steady state.

10.9. Enzyme - Linked Immunosorbant Assay (ELISA) (General) (Wim Gaastra, 1984)

The ELISA technique is used for a semiquantitiative determination of the concentration of certain antigens / antibodies. It is already used in medicine to detect the antigen or antibodies in serum samples. At present, it has application in the immunodiagnosis of several infectious diseases. In developed countries, ELISA kit has become a market commodity for early diagnosis of plant diseases. It is also being standardized to measure the plant growth regulators at the level of prts per billion. The general tecnique is described here and for each specific purpose it has to be standardized. Principle The ELISA tecnique was first introduced in early 1970s by Enginvall and perlmann. The principle underlying the double antibody is washed. The antigen is now added and binds to the absorbed antibodies. Then, an enzyme - linked antibody molecule called the conjugate is added which also binds to the antigen. A chromogenic substrate for the enzyme is added and the coloured product generated is measured, the intensity of the colour is proportional to the bound enzyme and thus to the amount of the bound antigen. Hence, the intensity of the colour produced by a series of standard antigens allows the calculation of the amount of antigen in an unknown sample. Materials 



 







Flat-bottomed polystyrene Microtitere Plates with 96 Wells Micropipettes (Gilson / Finnpipettes) 0-250 l

Multi-channel Pipette 0-250 l for pipetting of all reagents (if available) ELISA reader (Multiscanphotometer), if available 0.01M Carbonate Buffer (pH 9.6) : Prepare 0.1 M Na2 CO3 solution and adjust to pH 9.6 with NaOH. The chemicals should be of the highest quality and water double distilled. Wash Solution : Mix 90 ml Tween 80 with 910 ml water BST : 0.2% (w/v) Bovine serum albumin, 0.01% Tween 80 and 0.9% (w/v) sodium chloride in distilled water



 



Substrate solutions : It depends upon the enzyme that is coupled to the conjugte. Two widely used enzymes in ELISA technique are horseradish peroxidase (HRP) and alkaline phosphatase. For HRP, there are tow substrate solutions and are prepared as below : Solution 1 Dissolve 80 mg 5 amino-salicylic acid (purple - red brown colour) in 100 ml 0.05M potassium phosphate buffer (pH 6.0) containing 0.01M EDTA. Add 20 ml H2O2 (30%) and mix. Solution 2 mix 24. 3ml 0.1M citric acid 25.7ml 0.2M Na2HPO4 50ml H2O 40 mg ortho-phenylenediamine (Yellow colour) 40l H2O2 (30%) Stop Solution 0.3M NaOH (in the case of solution 1) or 1M H2SO4 (in case of solution 2) is used.

(When alkaline phosphatase is the enzyme coupled, the substrate is then pnitrophenyl phosphatte and is released as yellow colored - p-nitrophenol).  



A diluted solution of IgG against the antigent to be measured. The dilution is usually 1500-2500 fold depending of the titre of 1gG. dilutions are made in 0.1 M carbonate buffer. Antigen solutions to be tested and standard antigen solutions Enzyme (HRP) labelled diluted 500-2000 times in BST.

Procedure 10.10. The Double Antibody Sandwich Technique Pipette 150 l of the diluted IgG solution to each of the wells of a microtitre plate manually or using multichannel pipette. Cover the plate and incubate overnight at room temperature. Wash the plates with wash solution. The wells can be emptied by tapping the plate over a sink and then beating th plate upside down against a filter paper. The plates can be stores several moths, covered and cooled. An appropriate volume of wash solution is pipetted into the wells and left for a couple of minutes. The wells are then emptied as described above. Washing is repeated a few times to ensure good results. After washing and 100 l BST to each well. Add 100 l of an antigen solution to be tested to the first well of each row. Mix carefully and thoroghly. Avoid air bubbles or splashing of small drops. Take 100 l from the first wells and transfer them to the second wells in each row. Repeat the mixing procedure (step 5). Take 100 l from the

second wells and add to the third wells and so on. By this way a two-fold dilution series from wells 1-12 is created. Finally, remove the 100 l excess from the last wells. Incubate the plate for 2 h at 37 C, to allow the antigen bind to the coated antiserum, and then wash thoroghly. Add 100 l of the diluted conjugate solution to each well and incubate for 2 h at 37C, then wash the plate thoroughly. Add 100 l of substrate solution to each well and incubate for 1-2 h at 37C in the dark. Stop the reaction by adding 100 l of stope solution. Read the titre of the antigen solutions. This can be done by either using an ELISA Reader or visually by observing the last well that still gives some colour with the naked eyes. Preparation of Conjugate A conjugate is the covalent comple of IgG and an enzyme. The coupling of HRP is described below : 1.

Dissolve 5 mg of HRP in 1 ml 0.3 M Na2CO3 (pH 8.1). This solution should be prepared fresh.

2.

Add 0.1 ml of 1% flurorodinitroenzene in pure ethanol. If the HRP used is not pure, a precipitate may be formed that must be removed by centrifugation (10 min, 18,000 rpm).

3.

Mix thoroughly and incubate for 1 h at room temperature

4.

Add 1 ml of 0.16 M ethylene glycol, mix, and incubate for another hour at room temperature. the total volume is now 2.1 ml.

5.

Dialyze the mixture against 0.01 M sodium carbonate buffer (pH 9.5) for 25 h. The buffer should be changed at least three times.

6.

Add 1gG dissolved in 0.01M sodium carbonate buffer (pH 9.5) to the peroxidase aldehyde solution in the following ratio : one volume of IgG solution to one volume activated peroxidasealdehyde or 5mg purified IgG protein to 3 ml peroxidase solution.

7.

Mix well and incubate 2-3h but not longer at room temperature. If any precipitate is formed, clarify by centrifugation (10 min. 10,000 rpm).

8.

Dialyze extensively against 0.01M phosphate buffer (pH 7.2) containing 0.9% NaCl at 4C. Store the conjugate in a refrigerator or freezer in small aliquots and use once only.

Reference: Wim Gaastra. 1984. In: Methods in Molecular Biology Vol I proteins (Ed J.M. Walker). Humana Press, New Jersy. Pp.349.

10.11. Preparation for Light Microscopy: Wax Embedding Techniques Materials Samples were collected from field. Stem samples of sorghum were collected from top third internode and leaf samples from the last fully expanded leaf at 60 days after planting. Fixation : Materials were fixed for 48 hours in FAA (4%) formalin, propionic acid, 78% ethyl alcohol, proportion 5:5:90 by volume). Dehydration and embedding in wax The material was then dehydrated in graded water: alcohol solutions (to 70% alcohol) followed by an absolute alcohol and tertiary butanol series (Johnson 1940) and finally brought into pure tertiary butanol. The material was then kept in mixture of tertiary butanol and paraffin wax (BP 60°C) for a period of 24 to 72h, for infiltrations and was finally embedded in paraffin wax. Sectioning and slide preparation Sections of the wax-embedded material were cut with a Cambridge Rotary Microtome at a thickness of 8 to 10 microns. Some stem sections were cut. The ribbons of serial sections were floated on warm water (50°C) before placement on a slide smeared with Haupt‟s adhesive (1g Knox gelatine, 2g phenol and 15ml glycerol in 100ml water). With a few drops of 4% formalin, the slides were kept on a hot plate at 50°C. Sections were dewaxed by passing the slides through pure xylene for 30 minutes, followed by immersion in a 1:1 mixture of xylene and absolute alcohol for 5 minutes. The slides were then passed through a series of alcohol solution (100%, 90%, 70% 50% and 30%) and finally (if aqueous stains were to be used) distilled water before staining. For alcoholic stains, rehydration was not done. Stains Toluidine Blue : Staining was done with 0.05% aqueous solution as described by Feder‟s and O‟Brien (1968). Safranin and Aniline Blue : Sections were first stained with safranin (1% in 70% alcohol) and destained in 90% alcohol and counter stained with aniline blue (1% in absolute alcohol) Heamatoxylin Sections were first immersed in a 3% aqueous solution of iron alum for 30 to 60 minutes, washed, placed in 1% aqueous solution of hematoxylin for 30 to 60 minutes and then washed and differentiated in iron alumn or in a saturated solution of picric acid in 70% alcohol. Some sections were counterstained with safranin.

10.12. Scanning Electron Microscopy Techniques Scanning techniques are designed primarily to identify surface details of nonbiological material (conductive or nonconductive) at microlevel. Surface of leaf, another stomata, floret primordial pollen and any part of a plant can be scanned to desired restoration and can be photographed. Anatomical details of leaf with proper care can be had with scanning techniques. Precaution is however, necessary to avoid any artifacts arising due mishanding, of plant parts. [Fig.11.5[3]]. Techniques reveal beautifully the internal as well as external cellular details of any plant organ. Following ancillary instruments for heading ahead the scanning techniques. 1. Scanning Electron microscope [Fig. 11.2[2]. Critical point drying apparatus 3.Diode sputter for metal coating 4.Vaccum pump. 5.Drak room attachment for photography 6.Refrigirater for storing sample specimen. Scanning Electron Microscope (SEM) It scans a file beam of electrons across the specimen surface and displays an images of that surface on a cathode Ray Tube (CRT) using any of the radiation that are generated by incident electrons. Many types of material specimens can be examined on SEM with no preparation but to obtain optimum results, it is often necessary or desirable to coat the surface with a thin conductive layer. Commercial SEM was developed in 1964 with resolution of 10 nm. Major breakthrough in SEM technique was in1979 with fine resolution of 3nm and the magnification could be obtained from 20x to 100,000x.

APPENDIX CONTENTS OF ACIDS AND BASES Acid or Base Formula Molecular weight

Acetic acid Ammonium hydroxide Formic acid Hydrochloric acid Nitric acid Perchloric acid Phosphoric acid Sulphuric acid

CH3COOH NH4OH HCOOH HCL HNO3 HCIO4 H3PO4 H2PO4

60.1 35.0 46.0 36.5 63.0 100.5 80.0 98.1

Commercial concentrated reagent Specific gravity 1.05 0.89 1.20 1.18 1.42 1.67 1.70 1.84

% By weight Molarity (M) 99.5 17.4 28 14.8 90 23.4 36 11.6 71 16.0 70 11.6 85 18.1 96 18.0

PREPARATION OF BUFFERS Acetate buffer (0.2M) Stock solutions a. 0.2M Acetic acid solution = Dilute 11.55 ml of acetic acid to 1000ml with water b. 0.2M Sodium acetate solution (C2H3O2 Na.3H2O) = 22.22g in water and make up to 1000ml Mix x ml of A with y ml of B Xml 9.25 8.8 8.2 7.35 6.3 5.1 4.1 3.0 2.1 1.4 0.9 0.6 Yml 0.75 1.2 1.8 2.65 3.7 4.9 5.9 7.0 7.9 8.6 9.1 9.4 pH 3.6 3.8 4.0 4.2 4.4 4.6 4.8 5.0 5.2 5.4 5.6 5.8

Aconitate buffer Stock solutions A. 0.5M Solution of aconitic acid = 87.05g in water and make up to 1000ml B. 0.2M NaOH 20ml of A is mixed with x ml of B and diluted to 200ml with water Xml 15.0 21 28 36 44 52 50 68 76 83 90 97 103 108 113 119 126 pH 2.5 2.7 2.9 3.1 3.3 3.5 3.7 3.9 4.1 4.3 4.5 4.7 4.9 5.1 5.3 5.5 5.7 Gomori, G. (unpublished) Barbitone buffer (0.07M, pH 8.6) Dissolve 2.58g of diethyl barbituric acid and 14.42g of sodium diethylbarbiturate in water and make up to 1.0 litre with water. Boric acid-borax buffer (0.2M borate) Stock solutions A. 0.05M Solution of borax (Na2B4O7. 10H20) = 19.05g in water and make up to 1000ml (=0.2M tetrasodium borate) B. 0.2M Solution of boric acid = 12.37g in water and make up to 1000ml

Mix x ml of A (borax) with Y ml of B. X ml 1.0 1.5 2.0 3.0 Y ml 9.0 8.5 8.0 7.30 pH 7.4 7.6 7.8 8.0 Holmes, W. (1943). Ant. Record. 86, 163.

3.5 6.5 8.2

4.5 5.5 8.4

6.0 4.0 8.7

8.0 2.0 9.0

Borax-NaOH buffer (0.05M borate) Stock solutions A. 0.05M Solution of borax = 19.05g in water and make up to 1000ml (=0.2M tetrasodium borate) B. 0.2M NaOH Mix x ml of A and Y ml of B and dilute to 200ml with water x ml 50 50 50 50 50 50 y ml 0 11 23 34 43 46 pH 9.3 9.4 9.6 9.8 10 10.1 Clark. W.M. and H.A. Lubs, (1917). J. Bacteriol, 2,1. Borate buffer (0.3M, pH 8.6) Dissolve 18.55g of boric acid and 3.65g of sodium bydroxide in water and make up to 1.0 litre with water. Citrate buffer (0.1M) Stock solutions A. 0.1M solution of citric acid = 21.01 g in water and make up to 1 litre B. 0.1M Solution of sodium citrate (C6H5O7Na3. 2H2O) = 29.4g in water and make up to 1litre Mix x ml of A with y ml of B X ml 16.4 14.6 12.7 10.8 9.9 7.0 5.1 3.2 6.1 Yml 3.6 5.4 7.3 9.2 11.1 13.0 14.9 16.8 18.4 pH 3.0 3.4 3.8 4.2 4.6 5.0 5.4 5.8 6.2 Citrate phosphate buffer (phosphate-citrate buffer) or Mcllvaine’s buffer Stock solutions A. 0.1M solution of citric acid = 21.01g in water and make up to 1 litre B. 0.2M solution of dibasic sodium phosphate (35.61g of Na2HPO4.2H2O) or 53.65g of Na2HPO4.7H2O or 71.7g of Na2HPO4.12H2O in water and make up to 1 litre) X ml of A is mixed with y ml of B and diluted to 100 ml with water Xml 44.6 42.2 39.8 37.7 35.9 33.9 32.3 30.7 29.4 27.8 26.7 Yml 5.4 7.8 10.2 12.3 14.1 16.1 17.7 19.3 20.6 22.2 23.3 pH 2.6 2.8 3.0 3.2 3.4 3.6 3.8 4.0 4.2 4.4 4.6 Xml 24.3 23.3 22.2 21.0 19.7 17.9 16.9 15.4 13.6 9.1 Yml 25.7 26.7 27.8 29.0 30.3 32.1 33.1 24.6 36.4 40.9 pH 5.0 5.2 5.4 5.6 5.8 6.0 6.2 6.4 6.6 6.8 Mcllvaine, T.C. (1921). J. Biol. Chem. 49.183.

25.2 24.8 4.8 6.5 43.6 7.0

Citrate phosphate buffer (0.2M pH 4.5) Mix 27.3 ml of 0.2M citric acid with 45.4 ml of 0.2M disodium hydrogen phosphate. Carbonate/bicarbonate buffer Stock solutions A. 0.2M solution of anhydrous sodium carbonate = 21.2g in water and make up to 1000ml B. 0.2M solution of sodium bicarbonate = 16.8g in water and make up to 1000ml X ml of A is mixed with y ml of B and diluted to 200ml with water Xml 4.0 7.5 9.5 13 16 19.5 22 25 27.5 30 33 35.5 38.5 40.5 Yml 46 42.5 40.5 37 34 30.5 28 25 22.5 20 17 14.5 11.5 9.5 pH 9.2 9.3 9.4 9.5 9.6 9.7 9.8 9.9 10.0 10.1 10.2 10.3 10.4 10.5 Delory, G.E. and J. King (1945). Biochem. J. 39, 245. Glycine-HCL buffer Stock solutions A. 0.2M Solution of glycine = 15.01g in water and make up to 1000ml B. 0.2M HCL 50ml of A is mixed with x ml of B and diluted to total of 200ml with water Xml 5.0 6.4 8.2 11.2 16.8 24.2 32.4 pH 3.6 3.47 3.2 3.0 2.8 2.6 2.4 Sorenson, S.P.L., (1909). Biochem. Z., 21:131; 22: 352.

44.0 2.2

Glycine-NaOH buffer Stock solutions A. 0.2M solution of glycine = 15.01g in water and make up to 1000ml B. 0.2M NaOH 50ml of A is mixed with xml of B and diluted to 200ml with water X ml 4.0 6.0 8.8 12.0 16.8 22.4 27.2 32 38.6 pH 8.6 8.8 9.0 9.2 9.4 9.6 9.8 10 10.4 Sorenson, S.P.L., (1909). Biochem. Z., 21:131; 22: 352. Hydrochloric acid-potassium chloride buffer Stock solutions A. 0.2M solution of KCL = 14.91 in water and make up to 1000ml B. 0.2M HCL 50ml of A is mixed with xml of B and diluted to 200ml with water Xml 97.0 78.0 64.5 51.0 41.5 33.3 26.3 20.6 16.6 13.2 10.6 8.4 pH 1.0 1.1 1.2 1.3 1.4 1.5 1.6 1.7 1.8 1.9 2.0 2.1 Clark, W.M. and H.A. Lubs (1917). J. Bacteriol. 2:1

45.5 10.6

6.7 2.2

Phthalate-hydrochloric acid buffer Stock solutions A. 0.2M solution of potassium acid phthalate = 40.84g in water and make up to 1000ml B. 0.2M HCL

50ml A is mixed with xml of B and diluted to 200ml with water Xml 46.7 39.6 33.0 26.4 20.2 14.7 pH 2.2 2.4 2.6 2.8 3.0 3.2 Clark, W.M. and H.A. Lubs (1917). J. Bacteriol. 2:1.

9.9 3.4

6.0 3.6

Phthalate (0.1M, pH 6.0) buffer Add 43ml of 0.1 NaOH to 50ml of 0.2M potassium hydrogen phthalate and make up to 100ml with water. Phthalate- NaOH buffer Stock solutions A. 0.2M Solution of potassium acid phthalate = 40.84g in water and make up to 1000ml B. 0.2M NaOH 50ml of A is mixed with xml of B and diluted to 200ml with water Xml 3.7 7.5 12.2 17.7 23.9 30.0 35.5 39.8 43.0 45.5 pH 4.2 4.4 4.6 4.8 5.0 5.2 5.4 5.6 5.8 6.0 Clark, W.M. and H.A. Lubs (1917). J. Bacteriol. 2:1 Sodium phosphate buffer (0.1M) Stock solutions A. 0.2M Solution of Na2HPO4 .7H2O = 35.61g in water and make up to 1000ml B. 0.2M Solution of Na2HPO4 .7H2O = 31.21g in water and make up to 1000ml Mix x ml of A with y ml of B and dilute to 200ml with water Xml 8.0 12.3 18.5 26.4 37.5 49 61 72 81 87 91.5 94.7 Yml 92 87.7 81.5 73.5 62.5 51 39 28 19 13 8.5 5.3 pH 5.8 6.0 6.2 6.4 6.6 6.8 7.0 7.2 7.4 7.6 7.8 8.0 Sorenson, S.P.L., (1909). Biochem. Z., 21:131; 22: 352.] Potassium phosphate buffer (0.1M) Stock solutions A. 0.2M Solution of KOH = 11.22g in water and make up to 1000ml B. 0.2M solution of KH2PO4 = 27.22g in water and make up to 1000ml Mix xml of A with y ml of B and dilute to 100ml with water Xml 3.5 5.8 9.1 13.0 18.0 24.0 30 35 40 43 45 Yml 50 50 50 50 50 50 50 50 50 50 50 pH 5.8 6.0 6.2 6.4 6.6 6.8 7.0 7.2 7.4 7.6 7.8 Sorensons’s phosphate buffer (pH 7.0) Stock solutions A. KH2PO4 = 9.078g in water and make up to 1000ml B. Na2HPO4 = 11.86g in water and make up to 1000ml Mix solutions A and B in ratio 4:6

47 50 8.0

Potassium phosphate-NaOH buffer (0.05M) Stock solutions A. 0.2M KH2PO4 = 27.22g in water and make up to 1000ml B. 0.2M NaOH Mix xml of A and y ml of B and dilute to 20ml with water Xml 5 5 5 5 5 5 5 5 5 Yml 0.36 0.56 0.81 1.16 1.64 2.24 2.91 3.47 3.91 pH 5.8 6.0 6.2 6.4 6.6 6.8 7.0 7.2 7.4

5 4.24 7.6

5 4.45 7.8

5 4.61 8.0

Maleate buffer Stock solutions A. 0.2M Solution of acid sodium maleate = 8.0g of NaOH plus 23.2g of maleic acid or 19.6g of maleic anhydride in water and make up to 1000ml B. 0.2M NaOH Mix 50ml of A and x ml of B and dilute to 200ml with water Xml 7.2 10.5 15.3 20.8 26.9 33.0 38.0 41.6 44.4 pH 5.2 5.4 5.6 5.8 6.0 6.2 6.4 6.6 6.8 Temple, W. (1929). J. Am. Chem. Soc., 5 1:1754. Succinate buffer A. 0.2M Solution of Succinic acid = 23.6g in water and make up to 1000ml B. 0.2M NaOH Mix 25ml of A and x ml of B and dilute to 100ml with water Xml 7.5 10.0 13.3 16.7 20.0 23.5 26.5 30.3 34.2 37.5 pH 3.8 4.0 4.2 4.4 4.6 4.8 5.0 5.2 5.4 5.6 Gomori, G. (unpublished).

40.7 5.8

Tris (hydroxymethyl) aminomethane-HCL buffer (0.1M) Stock solutions A. 0.2M Solution of Tris (hydroxymethyl) aminomethane – 24.23g in water and dilute to 1000ml B. 0.2M NaOH Mix 50ml of A and x ml of B and dilute to 100ml with water Xml 43 41 39 34 29 24 18 13 9.5 pH 7.2 7.4 7.6 7.8 8.0 8.2 8.4 8.6 8.8

Tris (hydroxymethyl) aminomethane-maleate (Tris-maleate) buffer Stock solutions A. 0.2M Solution of Tris acid maleate = 24.2g of Tris (hydroxymethyl) aminomethane plus 23.2g maleic acid or 19.6g of maleic anhyddie in water and make up to 1000ml B. 0.2M NaOH Mix 50ml of A and x ml of B and dilute to 200ml with water Xml 7.0 10.8 15.5 20.5 26.0 31.5 37.0 42.5 45 pH 5.2 5.4 5.6 5.8 6.0 6.2 6.4 6.6 6.8 Xml 48.0 51.0 54.0 58.0 63.5 69.0 75.0 81.0 86.5 pH 7.0 7.2 7.4 7.6 7.8 8.0 8.2 8.4 8.6 Gomori, G. (1948). Proc. Soc. Exptl. Biol. Med., 68, 354. Preparation of standard solutions Standard HCL solution (0.1M or 0.1N) Reagents 1. Approx. 0.1M/0.1N HCL solution: Dilute 9.0ml (sp. gr. 1.18, 35%) or 8.5ml (sp. gr. 1.19, 37 %) of pure concentrate HCL to 1.0 litre with distilled water in a volumetric falsk. Invert several times and transfer to a clean, dry bottle. 2. Anhydrous Na2CO3 solution (0.1N) : Dry the pure anhydrous Na2CO3 on a watch glass in the oven at 110oC for an hour. Accurately weigh 0.53g of dry sodium carbonate and transfer quantitatively to a beaker and dissolve in about 20ml of water. Transfer to 100ml volumetric flask with washings, up to the mark and mix. 3. Methyl orange indicator solution : Dissolve 0.5g of methyl orange in 1.0L of water. Method Fill a clan, dry burette with HCL solution (approx. 0.1M/0.1N) and titrate against a known volume (10ml) of sodium carbonate solution (0.1N) using 2 drops of methyl orange indicator until an orange or faint pink colour end point is obtained. Note the titre value and repeat titration 2-3 times. Calculation Using N1V1 = N2V2 relationship, the exact normality of HCL can be calculated. Where, N1 – Normality of HCL to be calculated N2 - Normality of Na2CO3 = 0.1N V1 - Volume of HCL (titre value) V2 - Volume of Na2CO3 (10ml) Note Methyl red may also be used in place of methyl orange as an indicator provided the CO2 in solution is expelled by boiling before the end point is reached. Standard H2SO4 solution (0.05M/0.1N) Reagents 1. Approx. 0.05M/0.1N H2SO4 solution : Dilute 3.0ml of pure concentrated H2SO4 solution (sp. gr. 1.82, 96%) to 1.0L with water in a volumetric flask and mix thoroughly.

2. Anhydrous Na2CO3 solution (0.1N) : Prepare as described under standard HCL. 3. Methyl orange indicator : Prepare as described under standard HCL. Method Fill a clean, dry burette with H2SO4 solution (0.05M/0.1N) and titrate against a known volume (10ml) of anhydrous Na2CO3 solution using 2 drops of methyl orange as an indicator until an orange or faint pink colour end point is obtained. Repeat titration 2-3 times Calculation Calculate the exact normality of H2SO4 using the relationship, N1V1 = N2V2 as described before. Standard NaOH solution (0.1M/0.1N) Reagents 1. Approximately 0.1N NaOH solution : Prepare a saturated solution of sodium hydroxide as follows. Add in small portions bout 100g of AR Grade NaOH to 100ml of water in a flask, stopper and allow to stand for a few days or until the Na2CO3 settles to the bottom. The clear solution which is carbonate-free contains about 50g of NaOH/100ml (approx. 50% solution or 12.5M). From this solution, pipette out 5.3ml of clear supernatant liquid and transfer to a 1.0 litre rubber stoppered bottle and dilute upto the mark. 2. Standard HCL solution (0.1N) : Prepare and standardize as described before. 3. Phenolphthalein solution: Dissolve 0.5g of phenolphthalein in 100ml of 9.5% alcohol. Method Using a pipette, transfer 20ml of standard HCL solution into 150ml beaker. Add 25ml of water and one drop of phenolphthalein indicator. Titrate with the sodium hydroxide (apprx. 0.1N) until a faint pink colour is obtained. Repeat titration 2-3 times. Calculate the exact normality of NaOH using the relationship N1V1 = N2V2. Standard sodium thiosulphate solution (0.1N) Reagents 1. Approx. 0.1N Na2S2O3.5H20 solution : Dissolve 24.82g of sodium thiosulphate in freshly boiled and cooled water and dilute to 1.0L 2. 0.1N potassium dichromate : Dissolve 4.903g of potassium dichromate in water and dilute to 1.0L 3. Starch indicator : To 1.0g of starch add 10ml of water and pour the suspension into 90ml of boiling water. Boil for 1-2 min and cool. 4. 15 % JI solution : Dissolve 15g of KI in water and dilute to 100ml 5. HCL solution Method Place in a glass-stopped flask 20ml of 0.1N potassium dichromate and 10ml of 15% KI solution. Add 5ml of HCL. Dilute with 100ml of freshly boiled and cooled water and titrate against the 0.1N Na2S2O3.5H20 until the yellow color has almost disappeared. Add a few drops of starch indicator and continue to add Na2S2O3 until the blue colour just disappear. Calculate the exact normality of Na2S2O3 using the relationship N1V1 = N2V2. Standard potassium permanganate solution (0.1N)

Reagents 1. Potassium permanganate solution (0.1N approx.) : Dissolve about 3.2g of pure crystals of potassium permanganate in 200ml of hot water and allow to stand for 60min at about 100oC. The mouth of the container is well closed with a glass-stopper to disallow any addition of dust particles into the permanganate solution. The solution is then filtered after 2 days standing through either a sintered filter or a filter of glass wool but never filter through a filter paper because the latter reduces the strength of permanganate considerably. The solution is kept in dark because of its sensitivity to light. 2. Oxalic acid (H2C2O4 .2H20) or sodium oxalate (COONa2)2 (0.1N) : Dissolve 6.3g of pure crystals of oxalic acid in 200ml of water and transfer to a litre flask. Make up with water or slowly heat 6.7g of pure sodium oxalate salt in a platinum crucible until the flame of carbon monoxide formed disappears. Dissolve the contents of crucible with water and make up to 1.0L. 3. 4N H2SO4 Method 4. Dilute 10ml of standard oxalic acid or oxalate solution to 20ml with water and treat with 15ml of 4N H2SO4. Heat the solution to 70-80oC and titrate against permanganate solution until the solution becomes slightly pink. The color is permanent. Calculate the normality of permanganate solution using the relationship N1V1 = N2V2. Standard iodine solution (0.1N) Reagents 1. Iodine solution (0.1N) : Dissolve 12.7g of iodine (A.R.) and 40g of iodate-free potassium iodide in 50ml of water. Stir the mixture until whole of iodine dissolves completely. Transfer it to a litre flask and make up to the mark with water. Keep in a dark cool place. 2. Standard arsenious oxide solution (0.1N) : Dry about 8.0g of arsenious oxide (AS2O3) for 2 hour at 105-110oC in a platinum crucible and cool in a desiccator. Weigh accurately 4.945g of dried sample and dissolve in 10ml of 4N NaOH solution. The solution may be heated if required. Transfer to a litre and treat with 4 drops of phenolphthalein. The alkalinity of medium is neutralized by carefully adding just enough dilute HCL (disappearance of pink colour). Then mix the solution with another 50ml of Dil. HCL and make up to volume to the lite mark. 3. Starch solution: Dissolve 1.0g of starch and 5g of HgCl2 in 1.0ml of water and add the suspension to 500ml of boiling water. Boil until the clear solution is obtained and store in a glass-stoppered bottle. Method Take 25ml of standard arsenious oxide solution in 250ml of flask and mix with 50ml of water, 1.0g of sodium bicarbonate and 1.0ml of starch solution. Titrate against prepared I2 solution until blue colour appears. Calculate the normality of I 2 using the relationship N1V1 = N2V2.

Note Best results are obtained if the starch solution is added just before the end point is reached. Standard hydrogen peroxide solution (0.1N) Reagents 1. 0.1N H2O2 : Take about 7.0g of chemically pure H2O2 solution (ca.7.0 P.C.)in a 500ml measuring flask and dilute to the mark with water. Shake, keep in a dark, cool place. 2. Standard Na2S2O3 solution (0.1N) : Prepare and standardize as described before. 3. Standard potassium permanganate solution : Prepare and standardize as described before. Method 1. Standardization with thiosulphate solution Take 25ml of H2O2 in a 250ml flask, treat with 10ml of 4N H2SO4, 1.0g of KI and 4 drops of 3% ammonium molybdate to catalyze the reaction. Titrate against standard thiosulphate using starch as an indicator until the blue colour just disappears. 2. Standardisation with potassium permanganate Take 25ml of H2O2 in a 250ml flask titrate with 0.1N permanganate solution in presence of 10ml of dilute H2SO4 (1:5) until a light permanent pink colour is obtained. Calculate the normality of H2O2 using the relationship N1V1 = N2V2. 3. Standard sodium nitrate solution (0.1N) Method 50ml of 0.1N permanganate solution and 5ml of dilute H2SO4 (1:5) are mixed together and treated with 25ml of approx. 0.1N NaNO2 solution. The mouth of flask is closed with a glass stopper and allowed to stand for 25min. Then the excess of permangangate is titrated back by procedure described under preparation of standard potassium permanganate solution or iodometrically after adding 2.0g of KI and then titrating with standard thiosulfate solution. Calculation Note Volume of 0.1N KMnO4 consumed by nitrite = (50-volume of 0.1N standard solution used for back titration) = actual volume of permanganate = V1 Calculate normality of NaNO2 using the relationship N1V1 = N2V2. Standardisation of the Fehling’s solution Reagents 1. Fehling‟s solution A (copper sulphate solution) : Dissolve 34.636g of CuSO4.5H2O (pure recrystallized) in water, dilute to 500ml and filter. 2. Fehling‟s solution B (alkaline tartrate solution) : Dissolve 173g of Rochelle salt (dodium tartrate) and 50g of NaOH in water, dilute to 500ml. Allow to stand for 2 days and filter. Mix equal volumes (5ml each) both immediately before use.

3. standard glucose solution (0.5%) : Dissolve accurately 0.5g of pure anhydroys glucose powder in water and dilute to 100ml with water. 4. 1.0% Methylene blue : Dissolve 1.0g in 100ml 95% alcohol. Method Into a conical flask, pipette out 5.0ml of each of Fehling‟s solutions A and B. Dilute with about 10ml of water and add glass beads to it. Keep the flask over a wire gauze or hot plate and bring to boil. Add from the burette the standard glucose solution slowly, 1ml at a time till the colour just turns from blue to red. Then add 3.-4 drops of 1.0% methylene blue indicator which gives a blue colour to the solution and continue to add the glucose solution dropwise till a brick red colour end point of solution is obtained. Again in another conical flask, take 5.0ml of each of the Fehling‟s solutions A and B, dilute with about 10ml of water and add 3 glass beads into it. Boil the contents as above and add from the burette the standard glucose solutions 2.0ml less than the quantity required in the first titration. Then add 3-4 drops pf methylene blue indicator and continue the titration till the brick red coloured end point is obtained. Repeat the whole process of titration 3-4 times and calculate the mean of the last 3 readings (excluding the first reading). From the mean titration reading, calculate the amount of anhydrous glucose corresponding to 10ml of the mixed Fehling‟s solutions A and B. The titration should be completed within a total boiling time of about 3 min. Note down the first burette readings. Calculation Let „a‟ ml be the mean titration reading. 10ml of Fehling‟s A & B together = „a‟ ml pf std. glucose solution. Now, 100ml of std. glucose solution = 0.5g of glucose „a‟ ml of std. glucose solution = 0.5 x a g of glucose ― 100 100ml of Fehling‟s solutions = 0.5 x a g of glucose ― 100 A and B contain. PREPARATION OF INDICATORS 1. 0.04% Bromophenol blue : Grind 0.1g of dry bromophenol blue powder in a mortar with 14.9 ml of 0.01M NaOH. Dilute to 250 ml with water. 2. 0.04% Bromothymol blue : Grind 0.1g of bromothymol blue dry powder in a mortar with 16ml of 0.01M NaOH. Dilute to 250ml with water. 3. 0.5% Methyl Red : Dissolve 0.5g of methyl red in 100ml of 95% ethyl alcohol. 4. Methylene blue-methyl red mixed indicator : This solution consists of 1 part of 0.2% methyl red in alcohol and 1 part of 0.1% methylene blue in alcohol. The pH

is at 5.4. The colour is green in alkaline (pH 5.6), colourless at pH 5.4 (end point) and reddish pink in acid solution (pH 5.2). 5. 0.02% Methylene blue : Dissolve 0.2g of „certified‟ methylene blue in 1 litre of water. 6. 0.04% Phenol red : Grind 0.1g of dry phenol red powder in a mortar with 28.2ml of 0.01M NaOH. Dilute to 250ml with water. 7. 0.5% Phenolphthalein solution: Dissolve 0.5g phenolphthalein in 100ml of 95% alcohol. 8. 1% starch : Mix 1g of soluble starch with a little water, stir and add 20 100ml of boiling water. 9. 0.1% Bromocresol green : Dissolve 20mg in 20ml water 10. 0.5% Congo red : Dissolve 5g of congo red in 900ml of water and dilute to a litre with 95% alcohol. 11. Methyl orange : Dissolve 0.5g of methyl orange in 1.0L of water 12. Litmus : Dissolve 1.0g of azolitmin in 1.0L of water 13. Potassium chromate : Dissolve 5.0g of potassium chromate in 100ml of water. Hoagland solution (for plant culture) Solution1: Compound Weight (g/l) KHEPO4 0.136 KNO2 1.02 Ca(NO6)2 0.492 MgSO4.7H20 0.49

LIGHT FILTERS FOR COLORIMETERS Colour Wavelength range (nm) Klett-Summerson Colorimeter 42 Blue 400-465 54 Green 500-570 66 Red 640-700 Systronix colorimeter 621 Violet 380-510 (420) 622 Blue 400-530 (440) 623 Blue-green 460-540 (490) 624 Green 490-560 (520) 625 Yellow-green 510-590 (540) 626 Yellow 540-610 607 Orange 570-700 (600) 608 Red 630 and above (720) ( ) Represents wavelength peak Filter No.