BOTANY Lab Manual BSc.-III Medical Semester V 212
Experiment 1 Aim: Determine Water Potential of Vacuolar Sap by Plasmolytic Method. Requirements: Leaves of Tradescantia solutions of different concentrations, slides, microscope, coverslip, watch glass, blade brush etc. Theory: Plasmolysis is defined as shrinkage of protoplast of cells when they are placed in hypertonic solution. Osmotic potential is defined as the hydrostatic pressure which must be applied on solution to prevent further entry of water into it. OP = CRT [OP: Osmotic Potential, C: Concentration, R: Gas Constant, T: Temperature] 1. Peeled a small segment of epidermis from lower surface of leaf and placed in water. 2. Prepared different concentrations of sugar solution i.e. 0.1, 0.2, 0.3, 0.4 & 0.5 M. 3. Put 5 ml of different concentrations of sugar solution in respective watchglass and used water as control or blank. 4. Kept small pieces of epidermal peels in different sugar solutions for about 15 min. 5. Mounted different epidermal peels in their respective solution on clean glass slide and put coverslip. 6. Observed all peels under microscope and noted down observations. 1. Fully turgid leaves should be used for this experiment. 2. Sugar solution should be prepared carefully. 3. The epidermal peel should be single layered. 4. Observed different stages of Plasmolysis carefully. 213
Diagrams: Observations: Limiting Plasmolysis Incipient Plasmolysiss Evident Plasmolysis 214
Aim: Determine water potential of any tuber. Experiment 2 Requirements: Peeled Potato, Sugar solution of different concentrations, methylene blue, ten test tubes, pipette, dropper, cork borer, blotting sheet, blade, petridish etc. Theory: Water potential is the chemical potential of water which is equivalent to diffusion pressure deficiet (DPD) with negative sign. It is the difference between free enrgy of water molecules in pure water and that of water in a system or solution. It is equal to Ψ w = Ψ s + Ψ m + Ψ p [Where Ψ w is water potential, Ψ s is solute potential, Ψ m Matric potential and Ψ p Pressure potential] 1. Ten test tubes were taken and labeled as 0.1, 0.2, 0.3, 0.4 and 0.5 M in duplicate set. 2. Prepared different concentrations of sugar solutions viz. 0.1, 0.2, 0.3, 0.4 and 0.5 M in distilled water and put 10 ml of each concentration in two test tubes each. 3. Cut cylinders of potato tubers with the help of cork borer and trimmed to 1cm in length. 4. Dried potato tubers into folds of blotting sheet and immediately transferred two pieces in each test tube of one set. 5. Put a drop of methylene blue to same set and shook well. 6. Kept the set as such for about 30 min and a small drop of sugar solution from this set was withdrawn with the help of dropper and released in second tube of same concentration. 7. Observed the movement of drop carefully. 1. Potato pieces should be of equal length. 2. Sugar solutions should be prepared carefully. 3. The movement of drop should be observed against white background. 4. The tubes with potato tubers should be mixed thoroughly before taking observations. Observations 215
Concentration (M) Movement of Drop Nature of Solution 0.1 0.2 0.3 0.4 0.5 Hypotonic Solution Hypertonic Solution (Drop moves down) (Drop moves upward) 216
Experiment 3 Aim: To study the extraction and separation of chlorophyll pigments by solvent method. Requirements: Pestle and mortar, Cassia leaf powder, separating funnel, conical flask, measuring cylinder, stand, filter paper, weighing balance, potassium hydroxide, acetone, petroleum ether, diethyl ether, distilled water, funnel, pipette, blotting sheet, glass rod. Theory: Chlorophylls are green coloured pigments present in the thylakoids of chloroplasts along with carotenoids. These can be extracted from leaves using organic solvents. 1. 8g of Cassia leaf powder was mixed with 100 ml of 80% acetone and stirred for 10 min. 2. Solution mixture was filtered twice using blotting sheet twice. 3. Transferred 40 ml of the filtrate in separating funnel and added 50 ml of petroleum ether along wall. 4. Rotated separating funnel gently for 5 min and added 60 ml of distilled water along sides of separating funnel. 5. Shook the mixture gently for 5-10 min and left undisturbed with the help of a stand till two layers separated. 6. Discarded lower layer and poured 50 ml of distilled water along sides, stirred gently and removed lower water layer carefully. 7. Added 40 ml of 92% methanol and mixed well. 8. Allowed separating funnel to rest for 2-3 min till two layers separated. 9. Removed lower layer in other separating funnel and performed further experiment for both layers separately. Upper layer: 10. Added 15 ml of 30% Methanolic KOH and 20 ml of distilled water along sides and shook well. 11. Two layers separated; upper light yellow coloured containing carotenes and lower bluish green containing chl a. 217
Lower layer: 12. Added 50 ml of diethyl ether to lower methanolic layer and shook well. 13. Added 20 ml of distilled water along sides and mixed well. 14. Allowed two layers to separate and added discarded lower methanolic layer. 15. Added 15 ml of 30% methanolic KOH and 20 ml of distilled water along sides carefully and shook separating funnel well. 16. Allowed two layers to separate; upper yellowish brown coloured containing xanthophylls and lower olive green coloured containing chl b. 1. The chlorophyll extract should be concentrated enough. 2. Distilled water should be added along sides to avoid emulsification. 3. Various reagents should be prepared carefully. 4. The separating funnel should be handled carefully. 5. Thorough mixing of the contents should be done before discarding any layer. Observations: Xanthophyll Chl b Carotene (Upper layer) Chl a (Lower layer) 218
Experiment 4 Aim: separation of pigments by paper chromatography. Requirements: Cassia leaves, 80% acetone, petroleum ether, 95% acetone, chromatography jar, whatman filter paper, pestle and mortar, aluminum foil, measuring cylinder, capillary tube, pencil, scale etc. Theory: Chromatography is a technique through which the components of a mixture are separated on the basis of colour differentiation. Most common methods include paper chromatography, which is two dimensional and various components are identified on the basis of Rf (Retention Factor) values: Rf = Distance travelled by organic substance Distance travelled by solvent front 1. Prepared a paste of cassia fresh leaves with 80% acetone. 2. Cut Whatman filter paper strip approximately 5 cm wide. 3. Draw a line 2 cm above one end of strip with the help of pencil as reference line. 4. Loaded concentrated solution of photosynthetic pigments in the centre of line with the help of capillary tube 8-10 times to increase the density of pigments. 5. Put solution of petroleum ether and 95% acetone in the ratio of 100: 12 in chromatography chamber. 6. Suspended the filter paper strip loaded with photosynthetic pigments in the jar with the help of aluminum foil. 7. The jar was left undisturbed for about 2 hours till the solvent front travelled enough to cover 2/3 rd of filter paper. 8. Took out the whatman filter paper strip and marked solvent front with the help of pencil. 9. Determined the distance travelled by various pigments from reference line and calculated Rf values. 1. The pigment extract should be concentrated enough. 2. The chromatogram should be developed till full separation of pigments. 219
3. The solvent mixture should not touch the spot of photosynthetic pigments. 4. The chromatographic chamber should be left undisturbed. 5. The filter paper should not touch the walls of chromatographic chamber. Observations: Calculations: Chromatogram 220
Experiment 5 Aim: Separation of amino acids in a mixture by paper chromatography. Requirements: Whatman filter paper, n-butanol, glacial acetic acid, chromatographic chamber, distilled water, alanine, lysine, 0.2% ninhydrin solution, capillary tubes, hot air oven, hot plate, weighing balance, aluminum foil, atomizer etc. Theory: Paper chromatography is a form of two dimensional chromatography where a mixture is separated into its components on a paper using a particular mixture of solvents as mobile phase. In this experiment amino acids are separated and visualized using ninhydrin which forms a blue coloured complex with them. 1. Cut Whatman filter paper strip of required size as the size of chromatographic chamber. 2. Prepared a mixture of n-butanol, glacial acetic acid and distilled water in the ratio of 3:1:1. 3. Poured the solvent mixture in chromatographic chamber and covered it with aluminum foil. 4. Draw a reference line of filter paper strip approx. 2 cm above the edge. 5. Spotted filter paper with pure amino acids and their mixture carefully about 8-10 times so as to load sufficient amount for chromatographic development. 6. Dried spots over hotplate and suspended the filter paper in TLC chamber carefully and left the set up undisturbed for about 2 hours. 7. Took out the strip and marked the level of solvent front. 8. Allowed filter paper to dry completely and sprayed with ninhydrin solution using an atomizer. 9. Heated the filter paper strip in oven at 90º C for about 5 min. 10. Marked the positions of amino acids and determined distance travelled by them from reference line. 11. Determined Rf values for amino acids according to formula. 1. Sufficient amino acids should be loaded on filter paper by repeated application. 2. Do not touch filter paper with hands to avoid contamination with amino acids in our sweat. 221
3. Don not dip spot of amino acids in mixture solvent. 4. Cover the chromatographic chamber with glass lid or aluminum foil to make the chamber saturated. 5. Determine Rf values carefully. Diagrams: Calculations: Rf = Distance travelled by amino acid Distance travelled by solvent front 222
Aim: To demonstrated Phototropism. Experiment 6 Requirements: Potted plant, phototropic chamber. Theory: Phototropism is defined as the plant curvature movement in response to light. Shoot is positively phototropic whereas root is negatively phototropic. 1. A wooden phototropic chamber painted black from inner side having a hole on one side with removable top was taken. 2. Placed a small potted plant inside this chamber. 3. Placed the chamber in sunlight or an artificial source of light. 4. Left the set up as such for few days. 5. Observed the orientation of different plant organs after few days. 1. The chamber should be painted black from inside. 2. The plant should be placed upright near the source of light. 3. There should be only one opening for entering light in the chamber. Diagrams: Observations: The plant shoot moved towards the source of light because shoot is positively phototropic. Experiment 7 Aim: Demonstrate Ascent of Sap using a dye. 223
Requirements: Fresh leafy shoot of balsam or petunia, blade, eosin dye, distilled water, scissor, stand, beaker, cotton, weighing balance, cotton etc. 1. A leafy shoot of petunia or balsam was cut under water obliquely. 2. Dipped the cut end in 2% eosin solution contained in a beaker. 3. Adjusted the shoot erect using a stand, cotton and left the set up undisturbed for an hour. 4. Observed the colour of veins on leaves, stem and flower after sometime. 5. Cut T.S of stem and V.S of leaf for observing the path of ascent of sap. 1. The leafy shoot should be cut under water obliquely. 2. Eosin solution should be prepared carefully. 3. The transverse section should be thin enough. Diagrams & Observations: Experiment 8 Aim: To demonstrate imbibitions by plaster of paris method. Requirements: Plaster of paris, water, petridish, funnel, filter paper, gram seeds, tripod stand, glass rod etc. 224
Theory: Imbibition is the process of adsorption of liquid on the surface of solid without forming solution. It generates huge imbibitions pressure designated by ψ m (Matric Potential). Its value may reach 1000 atm. 1. Prepared the slurry of plaster of paris using water and glass rod. 2. Poured the POP paste in glass funnel lined with filter paper to fill half. 3. Added number of dry gram seeds over it and more POP paste to cover them completely. 4. Allowed POP to harden for 10-15 min. 5. The cone of POP was taken out along with filter paper, remover paper and placed cone in a petri dish filled with water with broad base downwards. 6. Observed POP cone after an hour. 1. The slurry should be made fresh and Seeds should be dry and viable. 2. The POP paste should be allowed to set properly before taking it out of funnel. Observations: 225
Experiment 9 Aim: To demonstrate the evolution of oxygen during photosynthesis. Requirements: Hydrilla plant, test tube, funnel, beaker, water, sodium bicarbonate, glass rod etc. Theory: Photosynthesis is the process of synthesis of carbohydrates from carbon dioxide and water in the presence of sunlight, chlorophyll and enzymes. This results in evolution of oxygen as byproduct which can be observed in the form of bubbles. 1. A fresh Hydrilla plant was taken and put in funnel. 2. Placed the funnel inverted in beaker containing distilled water. 3. Added a pinch of sodium bicarbonate in the beaker and mixed well with glass rod. 4. Inverted a test tube filled with water over the stem of funnel carefully. 5. Kept the set up in bright sunlight and observed carefully the evolution of bubbles after sometime. 1. Stem of the funnel should be completely immersed under water. 2. Place the test tube carefully over stem of funnel avoiding any spillage of water. 3. Only fresh Hydrilla plant should be used for this experiment. 4. The set up should be kept in sunlight for some time. 226
Diagrams: Observations: Hydrilla Plant Photosynthesizing 227
Experiment 10 Aim: To study the effect of different concentrations of organic solvents on permeability of plasma membrane. Requirement: Beet root, distilled water, acetone, test tubes, cork borer, knife, petri dish, blotting paper, beaker, measuring cylinder, scale, pipettes, distilled water etc. Theory: Plasma membrane is selectively permeable which allows the movement of some substances and not all. In response to different concentrations of organic solvents the membrane shows differential permeability. In this experiment cell membrane of beet root shows outward movement of anthocyanins, red coloured pigments from vacuolar sap due to injury caused by organic solvent in dose dependent manner. 1. The beet root cylinders of 1 cm length were cut using a cork borer and blade. 2. The cylinders were washed repeatedly using distilled water till the pigment stopped diffusing out. 3. Different concentrations of acetone i.e. 25, 50, 75 and 100% were prepared using distilled water. 4. The test tubes were filled with 10 ml of each concentration and water as control. 5. Put two pieces of beet root cylinders in each test tube and left the set up as such 30 min. 6. The observations were taken visually to determine maximum leaching of anthocyanins by comparing the intensity of colour. 1. The beet root pieces should be of equal length. 2. The concentration of organic solvents should be accurate. 3. Beet root pieces should be washed frequently till the leaching of pigment stops. 4. The test tubes should be shaken to allow maximum diffusion. Observations: 228
(% Conc. of acetone) 0 25 50 75 100 Maximum leaching at 75% acetone 229