Preface The object of this book is to bring into focus the practical application of the theoretical knowledge of crop physiology. It has been chiefly designed in accordance with the prescribed syllabus of practical part of crop physiology. It is very often seen that students familiar with the theoretical aspects of crop physiology fail to interpret the same in practice. The precise knowledge of the crop physiological processes is of paramount importance for the undergraduate students of agriculture. This manual of Crop Physiology brought out by the Department of Plant Physiology, S.K.N. College of Agriculture, Jobner, is an appreciable step which fulfils a long felt need. This manual includes all the practical exercises of Crop Physiology (PPHYS-4221) course, offered to B.Sc. (Hons.)Agriculture students.

Object: Preparation of solutions.  Material required: Volumetric flask, measuring cylinder, beakers, pipettes, balance, distilled water and chemicals.  Theory: The use of solution is common in experimental procedures of plant physiology. It is very important for a student to be familiar with their preparation. The substance dissolved in a solution is called solute and the medium in which it is dissolved is known as solvent. Solutions of two liquids (e.g. alcohol in water) and solid in liquid (e.g. NaCl in water) are most common. Solutions are thus a homogenous mixture of a solute in a solvent. 

Object: To demonstrate the phenomenon of osmosis using potato osmoscope.  Material required: A potato tuber, sugar solution, beaker, water, petridish, knife, measuring cylinder, and stand. Theory: Osmosis is a phenomenon in which water molecules diffuse through the semi permeable membrane from region of its higher concentration to the region of lower concentration.

Object: To demonstrate the phenomenon of endosmosis and exosmosis by using grapes and raisins. Material required: Grapes, raisins, beaker or petridish, water and sugar solution.

Object: To demonstrate the process of diffusion. Material required: Beakers, copper sulphate, distilled water and sugar solution.

Object: Methods of measurement of water status and water saturation deficit in plant parts.  Material required: Leaf samples, leaf borer, water, petridishes, forceps, oven and blotting paper.  Theory: Plant water status strongly influences plant growth and biomass production through its effects on leaf and root expansion and on photosynthesis. Therefore, measurement of plant water status is an important component of understanding plant processes and maximizing yield under different ecosystems.  Method: Plant water status can be measured and computed by any one of the following methods

Object: Estimation of water potential of plant tissue by Chardakov’s method. Material required: Test tubes, sucrose or polyethylene glycol (PEG) 6000, methylene blue and dropper. Principle: Concentration of the solution in which a plant tissue is immersed changes. If the solution is hypertonic (with lesser yw) the tissue loses water and the solution becomes dilute. If the solution is hypotonic (with higher yW) the tissue absorbs water and the Solution becomes concentrated. If the solution is isotonic (with yw equal to the tissue sap) the tissue neither loses nor absorbs water and the solution concentration does not change. Thus the yw of this solution gives the yw of the tissue.

Objective: Estimation of photosynthetic pigment from plant leaves. Material required: Plant leaves, test tube, test tube stand, spectrophotometer, balance, centrifuge machine, mortar and pestle, acetone and distilled water. Theory: Photosynthetic pigments, namely, the chlorophylls are present in green leaves and shoots of plants. There are at least five types of chlorophylls in plants. Chl’a’ and ‘b’ occur in higher plants, ferns and mosses. Chl ‘c’,‘d’ and ‘e’ are found only in algae and in certain bacteria. Chlorophylls are extracted from plant leaves. The absorption peaks of chl ‘a’ at 663 nm and ‘b’ at 645 nm are characteristic measures from which amount of chlorophylls can be calculated.

Object: Measurement of absorption spectrum of chloroplastic pigments. Material required: Spectrophotometer, plant leaves, ethanol, electric balance, beaker, test tubes and water.

Object: To demonstrate chlorophyll fluorescence. Material required: Spectrophotometer, fresh green spinach, mortar, pestle, acid washed sand, acetone, funnel, filter paper and test tubes. Principle: Chlorophyll molecule absorbs light in the blue (450nm) and red (660nm) region and get into excited state and emits an electron. The electron drops back to the ground state within 10-9 seconds and some energy (which is not used in biochemical reaction) are given out as the observed fluorescent far red light. Procedure and observation: Take some soft green spinach leaves. Grind these with a pestle -mortar in acetone adding a pinch of acid washed sand. Make a fine paste. Filter to get a clear solution in a test tube. Take the test tube in bright sun light.

Object: Measurement of leaf area by graph paper method. Material required: leaves, graph paper and pencil or pen.

Object: Estimation of stomatal index and stomatal frequency with replica method. Material required: Leaf sample, blue/red correction fluid, transparent finger nail polish, brush glass slide and microscope. Principle: The number of stomata per unit area of the leaf is called as Stomatal Frequency. The stomatal index is defined as the percentage number of Stomata as compared to all the epidermal cells in a unit area of leaf i.e.

Object: Demonstration of leaf anatomy or structure of C3 and C4 Plants (demonstration by already prepared slides). Material required: Already prepared slides of corn and wheat leaves and microscope.

Object: To demonstrate that water is lost from a living plant during transpiration.  Material Required: A potted healthy plant, a bell jar, a rubber sheet or oil cloth, a glass sheet and grease. Theory: Transpiration: Loss of water in the form of water vapour from the internal tissues of living plants through aerial parts of the plant is known as transpiration.

Object: To measure rate of transpiration of potted plant by gravimetric (Lysimeter) method.  Material required: Potted plants, Polythene sheet, Platform and electric balance etc. Method: Select same size and same type well watered potted intact plants. Seal the pots and the root system with a sheet of polythene so that the water loss to the atmosphere occurs only from the shoot system not from the soil (To check evaporation losses). Immediately record the weight of the potted plant and keep them in different environment such as (1) warm environment (2) cool environment (3) Calm environment.

Object: To measure the rate of transpiration by using Ganong’s potometer Material required: Ganong’s potometer, beaker, wax or vaseline, stop watch and twig or a branch of a plant. Theory: The amount of water absorbed is almost equal to the amount of water transpired. The difference between the two amounts is negligible.  Method: Potometer consists of wide mouthed tube fitted with a hard cork in which a twig or branch of a plant is inserted. Other end of tube is dipped in beaker. One half of the tube is graduated. The apparatus is filled with water using the reservoir. An air bubble is inserted in the tube as shown in figure.

Object: To find out the relation between transpiration and absorption.  Material Required: Wide mouthed glass bottle, with a narrow graduated side tube, split rubber cork, water and a plant with intact root system. Theory: The loss in weight of plant indicates the amount of water transpired. And fall in water level of the side tube indicates the amount of water absorbed. This shows that amount of water transpired is normally equal to the amount of water absorbed by the plant roots.

Object: To demonstrate unequal transpiration by the two leaf surfaces from four leaf method.  Material required: four leaves of china rose or any dorsiventral leaves, two stands, thread and grease or vaseline.  Theory: Transpiration is a process in which loss of water occurs in the form of water vapour from the aerial parts of the living plant.  Method: Take four leaves of equal size from a plant (e.g. China rose). Now apply or smear the vaseline on the four leaves as follows:

Object: To demonstrate that light and carbon dioxide are essential for photosynthesis (Moll’s half leaf experiment).  Material Required: A potted plant, a wide mouthed bottle, caustic potash, splits of rubber Cork and vaseline etc. 

Object: To demonstrate that O2 is evolved during photosynthesis.    Material required: Hydrilla plant, beaker, test tube, funnel, pond water and pyrogallol.    Theory: Oxygen is a byproduct of photosynthesis, which is evolved in presence of light and chlorophyll during photosynthesis.

Object: To demonstrate anaerobic respiration.  Material required: Soaked pea or gram seeds, mercury, test tube stand, forceps and KOH pellets.  Theory: Generally, plants at certain stage have the capacity to respire in absence of oxygen i.e. anaerobic respiration. In this process, oxidation of carbohydrate takes places, ethyl alcohol and CO2 being end products and only a little amount of energy is liberated-

Object: To demonstrate that oxygen is used up during respiration.  Material required: A conical flask, some germinating seeds, glass tube having two bends at right angles, mercury, beaker, small tube and caustic potash (KOH). Theory: Plants at some stage have a capacity to respire more rapidly in presence of oxygen i.e. aerobic respiration. In this process, oxidation of carbohydrate occurs resulting in conversion of carbohydrate into carbon dioxide (CO2) and water with release of large amount of energy.

Object: To determine respiratory quotient of different types of respiratory substrates.

Object: To demonstrate the optimum conditions for seed germination.  Material required: Beaker, glass slide, bean seeds, thread and water. Theory: Germination is a process by which the embryo in the seed becomes activated and begins to grow into a new seedling under favorable conditions. Food is stored in the seeds in dry conditions but the developing embryo cannot utilize this dry food. Food can be utilized in liquid form only and seeds can utilize only dissolved oxygen. Excess water stop germination because once all the dissolves oxygen is utilized by the seed, further germination is not possible as the life supporting oxygen is lacking. Suitable temperature is necessary because low temperature retards the embryo activity and high temperature destroys the delicate embryo tissue. Seed usually germinate between 0oC to 50oC temperature and the optimum temperature is lies between 25 to 30oC. During germination rapid cell division takes place. Energy is required for cell division. This energy is obtained from oxidation. The oxygen is required for oxidation is supplied by the air.

Object: Breaking of seed dormancy to facilitate germination. Material required: Seeds, water, sharp needle, sharp razor, sandpaper, acids, Potassium nitrate and gibberellic acid.  Definition: Inability of a viable seed to respond the favorable environmental conditions for germination is known as dormancy.

Object: Determination of the viability of seeds by tetrazolium test.  Material required: Healthy seeds, tetrazolium solutions, beakers, water and petridishes.  Theory: Viability of seeds shows their potential towards germination.

Object: To demonstrate the measurement of growth in plants with the help of Arc auxanometer.  Material required: Potted plant, Arc auxanometer, rope and weight. Theory: Growth is a permanent change in plants with respect to its size, form, weight and volume.

Object: Growth analysis: Calculation of growth parameters.  Material required: Primary data collected from individual plants or derived from whole canopies.  Theory: The growth analysis study provides information on the growth of plants or of a plant community in a precise way with the available’ raw data. The study also provides precise information on the nature of plant and environment interaction in a particular habitat.

Object: Yield estimation of kharif crops. Material required: Polythene bags, balance, gunny bags or baskets, pans and harvesting equipment like sickle, knife etc. Theory: Yield is the ultimate objective of cultivating agricultural plants. Different plant parts constitute the economic yield in different crops, like grain in cereals, pulses and oil seeds, vegetative parts or fruits in record yield at the correct maturation stage. Do not allow excessive wilting or drying of the material. Estimate yield in a sufficiently large sample. Materials required only a representative crop to estimate yields. (Do not select plants of any particular type. Do not damage any plant part vegetable crops; bark or out growth in fiber crops (jute, cotton); and the entire plant in fodder crops. The yield per unit area (per M2 or ha) is the product of yield per plant multiplied by the number of plants per unit area. It is, therefore, essential to know the yield components per plant as well as per unit area.

Object: Effect of ethylene on regulation of stomata. Material required: Plant leaves, ethaphon, ACC, double distilled water, measuring cylinder, beaker and electric balance. Theory: Ethylene is a plant hormone that regulates many aspects of growth and development. Despite the well-known association between ethylene and stress signalling, its effects on stomatal movements are largely unexplored. Here, genetic and physiological data are provided that position ethylene into the Arabidopsis guard cell signalling network, and demonstrate a functional link between ethylene and hydrogen peroxide (H2O2). In wild-type leaves, ethylene induces stomatal closure that is dependent on H2O2 production in guard cells, generated by the nicotinamide adenine dinucleotide phosphate hydrogen (NADPH) oxidase AtrbohF. 

Colour Plates Exercise 9: To Demonstrate Chlorophyll Fluorescence