The following points highlight the seventeen experiments on process of transpiration in plant cell. Some of the experiments are: 1. Determination of Stomatal Frequency (Or the Number of Stomata per Unit Area) of a Leaf 2. Measurement of Stomatal Pore 3. Determination of Changes of Stomatal Opening in Light, Dark and Under Desiccation 4. Effect of pH on Stomatal Opening and Closing and Others.
Contents:
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Determination of Stomatal Frequency (Or the Number of Stomata per Unit Area) of a Leaf
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Measurement of Stomatal PoreDetermination of Changes of Stomatal Opening in Light, Dark and Under Desiccation
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Effect of pH on Stomatal Opening and Closing
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Determination of the Area of Leaves by Different Methods
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Determination of the Percentage of Total Stomatal Aperture in Relation to the Area of the Whole Leaf
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Demonstration of Law of Diffusion Through Small PoresDemonstration of the Phenomenon of Transpiration
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Determination of Transpiration Index
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Determination of the Rate of Transpiration
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Quantitative Determination of the Differential Rates of Transpiration from the Two Surfaces of a Leaf (Cuticular and Stomatal)
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Simultaneous Determination of the Amount of Water Absorbed, Retained and Transpired by a Plant
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Demonstration of Suction Force Due to Transpiration or Transpiration Pulls
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Determination of Stomata-Bearing Surface of a Leaf without Using a Microscope
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Determination of the Effects of Environmental Factors on the Rate of Transpiration
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Quantitative Determination of Transpiration Under Conditions of Different Experimental Errors Generally Encountered
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Demonstration of Guttation
Contents
Experiment # 1
Determination of Stomatal Frequency (Or the Number of Stomata per Unit Area) of a Leaf:
Experiment:
In order to determine the dimension of stomata and the area of field of vision, the ocular is first standardized with the help of a stage micrometer. One stage micrometer division is generally 10µ (it is indicated on the stage itself).
The length of the scale of the stage micrometer is equal to 1 mm and 1 mm is equal to 1000µ. For standardization of the ocular micrometer, it is first placed inside the eye-piece and the stage micrometer on the stage of a microscope at a particular magnification. Now it is observed how many divisions of the ocular coincide exactly with those of stage micrometer.
Three such readings are taken and the average is determined. The value of one ocular division is calculated as follows:
If X stage divisions are equal to Y ocular divisions, then one ocular division becomes equal to 10X/Yµ (1 stage division = 10µ). Standardization in both low and high power of microscope is separately done.
The diameter of the field of vision of the microscope is determined with the help of the ocular or the stage. The area of the field of vision is calculated from the formula πr2, where r is the radius of the field of vision, i.e., half of the diameter (convert µ into centimeter and calculate area of the field of vision in square centimeter).
Epidermal peelings from the lower surface of Basella leaf are mounted on a slide and the number of stomata within the field of vision is counted. Three such readings are taken by moving the slide. Such readings are taken from the apical, middle and the basal portions of the leaf and the average number is calculated.
Results:
The number of stomata within the area of field of vision is determined and from it the number of stomata per square centimeter is calculated. This gives the stomatal frequency of the leaf.
Discussion:
Stomatal frequency is defined as the number of stomata per unit area of the leaf surface. Different species of plant have different stomatal frequencies and this varies with environment and age of the leaves.
In leaves stomata may occur in both upper and lower surfaces. In woody plants with dorsiventral leaves they are located mainly on the lower epidermis and in herbaceous plants with isobilateral leaves they occur on both the surfaces, though more abundant on the lower side.
In an individual leaf stomata are more numerous near the apex and minimum near the base, the middle region having an average distribution. Stomatal frequency generally ranges between few thousands to a hundred thousands. In determining stomatal frequency of an isobilateral leaf the stomata of both the surfaces should be taken-into consideration.
Experiment # 2
Measurement of Stomatal Pore:
There are several methods for determination of the state of stomatal opening. A few important methods are given below.
(a) Lloyd’s method:
By this method stomatal pore of leaves can be measured by using ocular (standardized) and the microscope. The epidermal peelings of leaf of a suitable species are taken and immediately fixed by immersing in hot alcohol.
The alcohol fixes the stomata preventing any further movement of the guard cells. The peelings are examined under the microscope and the opening of the guard cells is then measured by a standardized ocular.
The area of the pore is determined. Since the area of the pore is somewhat ellipsoidal in nature, the area could be very approximately taken as equivalent to (πr/4) (ax b), where a and b represent the length and the breadth of the pore, and π = 3.142.
(b) Impression method:
The leaf is taken from a healthy potted plant and its lower surface is smeared with Durofix or Collodian (1gm of pyroxylin in 6 ml of alcohol and 20 ml of ether). It is kept for some time. After about half an hour the Durofix or Collodian is dried up into a thin papery film.
The film is now stripped off and the impressions of the stomata in. such films can be observed under a microscope. The exact area of the stomatal opening is calculated by the above method.
(c) Darwin’s porometer method:
Darwin’s porometer is a useful apparatus for following the changes in the stomatal aperture, i.e., the degree of opening of stomata. This is an indirect method for knowing the degree of opening of stomatal aperture.
The Principle of the method is that the water vapour, which comes out of the stomatal pores, produces a pressure on the level of water column in a vertical tube pushing the level down at a rate at which water vapour comes out (Figure 10).
The figure shows a T-tube with two side arms A and B at top and a graduated vertical tube C. The arm A is connected to a rubber tube on which a pinch cock is fitted to cut off the flow of air when desired.
The arm B is connected to a cup by rubber tubing. The mouth of the cup is attached to the lower surface of a leaf by some adhesive or Vaseline. Care should be taken so that adhesive or Vaseline does not adhere to the leaf surface thereby closing the stomata. The fittings must be perfectly air-tight. The lower open end of the stem tube G is immersed in a beaker containing water.
At the start, the pinch cock is opened and the air is sucked out by mouth so that water level rises in the stem tube C.
The level of water is adjusted at the desired height and the pinch cock is closed. If the stomata on the leaf surface within the cup are open, then air will enter through the leaf surface via leaf tissue and will come out of stomatal pore along with transpired water vapour.
This air will move via the cup chamber, rubber tube and finally it will exert pressure on the level of water in the stem tube pushing it down at a rate at which air is coming out of the stomatal pore, depending upon their degree of opening. The experiment can be done under different environmental conditions to observe their effects on the degree of opening of stomata.
Experiment # 3
Determination of Changes of Stomatal Opening in Light, Dark and Under Desiccation:
Experiment:
Darwin’s porometer is used in this experiment (Refer Expt. 2. c). The potted plant is subjected to different environmental conditions after setting up of the whole apparatus, such as different intensities of light, V darkness and desiccation.
In each case the rate of fall of water level in stem tube C is noted after, 15 minutes of equilibrium. For the purpose of desiccation the potted plant is subjected to desiccation by keeping the plant in water-stressed condition.
Results:
The rate of fall of water level in the stem tube C under each environmental condition is recorded and graphically represented.
Discussion:
The rate of fall of water level in the stem tube C is an indication of the state of opening of the stomatal pore. The stomata remain open in light and the opening gradually widens with increase in light intensity up to a certain limit.
In dark since stomata remain closed, the rate of fall of water level is almost nil and in case of desiccation the rate of fall is similar to the dark effect.
Experiment # 4
Effect of pH on Stomatal Opening and Closing:
Experiment:
Different grades of pH solutions using phosphate buffers are prepared by mixing 0.1 M disodium mono-hydrogen phosphate (Mol. wt. 14216) and 0-1 M monosodium di-hydrogen phosphate (Mol. wt. 156) solutions in the following proportions:
If Na2HPO4 contains 2 mols of water of crystallization, then the molecular weight will be 178 16. Sodium phosphate buffer may be substituted by potassium phosphate buffer (Mol. wt. of K2HPO4 = 174-20 and KH2P04 = 136-20. If water of crystallization is present, it should be taken into account.)
Six watch glasses are taken and 2 ml of each buffer solution is poured in each watch glass separately. Another watch glass containing 2 ml of distilled water serves as control. Epidermal peelings from suitable leaf are taken and put in each watch glass.
After 10 to 15 minutes the peelings arc taken out and observed under the microscope. The area of stomatal aperture is measured as in Expt. 2a. At least three such readings are taken for each set and average area of the pore is determined.
Results:
Results arc plotted graphically taking pH as abscissa and area of the pore as ordinate. The pH at which the area is maximum is noted from the graph.
Discussion:
The extent of stomatal opening is greatly influenced by pH. Stomata remain closed in acidic pH whereas in alkaline pH stomata open. At higher pH (pH 7 to 7 20) and in presence of enzyme and inorganic phosphate, starch is converted to glucose-1-phosphate.
At lower pH (pH 5) the synthesis of starch takes place from glucose-1-phosphate. Since starch is osmotically inactive and glucose osmotically active the above reaction shows the possible explanation of the effect of pH on stomatal opening and closing.
N.B. The effect of temperature on stomatal opening and closing may also be studied. Under suitable environmental conditions, with the rise of temperature up to 30°C opening of stomata increases.
The effect of potassium ions on the opening and closing of stomata may be studied by taking different concentrations of KNO3 and putting the epidermal peelings in these solutions under bright sunlight. The effect of some metabolic inhibitors like DNP, cyanide, etc. may also be examined to show that the mechanism of opening and closing of stomata is controlled by the metabolic activity of the guard cells.
Experiment # 5
Determination of the Area of Leaves by Different Methods:
(a) By polar planimeter method:
The Polar Planimeter was invented by Professor Amesler in 1856. This is a simple instrument for determination of areas of unspecified surfaces. It is a very useful apparatus as areas could be read out from readings in a small vernier attached to the instrument (Figure 11).
It consists of two principal parts, a tracer arm having tracing point and a carriage with a measuring wheel, and the pole arm attached to the pole, around which the instrument revolves.
The main operation of the apparatus in determining the area of a leaf is to place the leaf on a paper or on a board (outline of the leaf may also be drawn on it), to keep the point of the instrument near one margin of the leaf and to pass it around the margin of the outline.
The result is those computed 6.0 ml the reading of the graduated measuring wheel and the counter dial which regbters the number of its revolutions. The measuring wheel is with a vernier; vernier unit is 10 sq. mm.
Direction for use:
1. First coincide the zero of the measuring wheel with the zero of the vernier scale. Find out the number of divisions of the vernier scale that coincides with that of the measuring wheel.
2. Before taking actual measurement of area from the outline drawn on a paper, zero of the lever vernier should be fixed at 35 mark of the tracer arm.
(b) By weighing method:
A leaf is placed on a card board or on a century board and its outline is drawn. The leaf area on the board is accurately cut and weighed. Now one square centimeter area is cut out from the board and is weighed. If weight of the area drawn on the board is X. gm and that of one square centimeter is Y gm, then the area of the leaf is X/Y sq. cm.
(c) By graph paper method:
This is the most convenient method which is generally used in laboratory experiments. The leaf is placed in a millimeter graph paper and its outline is drawn. The total number of large square blocks (one large square block is equal to one square centimeter) is counted.
The number of small squares (one small square is equal to one square millimeter) in the remaining area of the outline of the leaf is then counted and converted into square centimeter. All the values are added in order to get the total area of the leaf.
Experiment # 6
Determination of the Percentage of Total Stomatal Aperture in Relation to the Area of the Whole Leaf:
Experiment:
Stomatal frequency (X) of the experimental ‘leaf is determined as in Expt. 1 and the area of the leaf (Y) is found out as in Expt. 5c. Now the average area (Z) of the stomatal pore is determined as in Expt. 2.a.
Results:
The product of X (stomatal frequency) and Y (area of the leaf) gives the total number of stomata. This product when multiplied by Z (area of the aperture) gives the total area of the stomatal aperture of the leaf. The percentage of the area of the stomatal aperture in relation to the entire leaf area is calculated.
Discussion:
Stomatal frequency varies with different species of plants, age of leaf of the same species and the conditions under which it is grown. When there are a few stomata per unit area of the leaf, they are usually large, if there are many they are generally small.
Calculations of stomatal areas as percentages of leaf areas, however, show no constant relation. The range is not very great and is usually between 0.4 to 0.2 percent of the leaf area calculated on the basis of one surface in case of dorsiventral or both the surfaces in case of isobilateral leaves.
Experiment # 7
Demonstration of Law of Diffusion Through Small Pores:
Experiment:
The law of diffusion through small pores can be studied by taking equal amounts of alcohol in two wide-mouthed bottles. The mouths of the bottles are covered with two polythene sheets having holes of different diameters.
Care should be taken that the holes are more or less widely spaced. The rate of evaporation is measured by weighing the bottles from time to time. The total diameter and perimeter in centimeter and area in square centimeter of all the pores are separately determined in the two sheets.
Results:
The area of the pores and the perimeter of the pores in each case are determined by applying the formulae πd2/4 and π × d respectively, where d is the diameter of the pore. The mean change in weight per unit time per unit area and per unit linear dimension of the pore (perimeter) gives the rate of evaporation through pores of different diameters. The results are entered in tabular form.
Discussion:
In quiet air the rate of diffusion is more nearly proportional with the linear dimension, i.e., the perimeter of the pores than to their areas. For this reason the rate of diffusion per unit surface of pore is higher in the smaller pores.
If the pores are uniformly scattered over a surface, the actual open area is highly reduced but there is slight reduction in diffusion rate. Thus the diffusion through a multi-perforate septum is higher as compared with that of a single opening with an area equal to the aggregate area of the pores.
Closely spaced pores bring interference in diffusion due to overlapping of humidity layers. It has been estimated that if pores are over 8 or 10 times diameter apart, interference is minimum and each pore allows for its maximum diffusion.
Interestingly, stomata are usually further apart than 8 times of their diameter. This is why the rate of diffusion through stomatal apertures is high even though the total area of stomatal opening is small.
Although, the open area of the stomatal pore only represents 1 % of the total leaf area, the diffusion of water vapour through the pores often exceeds 50% of that evaporating from free ‘ water surface.
N.B. In order to establish the degree of correlation between:
(i) The rate of evaporation and the perimeter of pores and
(ii) The rate of evaporation and the area of pores, results are tabulated and subjected to statistical analysis for degree of correlation.
Experiment # 8
Demonstration of the Phenomenon of Transpiration:
(a) Bell jar method (qualitative):
Experiment:
A small healthy potted plant is taken and its soil is covered with a polythene sheet to check evaporation from the soil surface. The stem and leaves remain uncovered. The plant is now placed under a bell jar and the rim of the bell jar is sealed with Vaseline. The set-up is kept in a lighted place. Another set-up is similarly maintained where all the leaves from the plant are removed previously.
Observation:
In the first set-up droplets of water are seen on the inner walls of the bell jar. In the second set-up there is no condensation of water vapour on the inner wall of bell jar.
Inference:
When leaves transpire water the water vapour comes out and after saturating the inner atmosphere of the bell jar it condenses in the form of droplets on the inner walls. In the case where all the leaves are removed from the plant before covering with bell jar, no condensation of water vapour takes place because the transpiring organs, that are leaves, are absent.
N.B. Instead of removing the leaves, Vaseline may be applied on both the surfaces of the leaves of the plant to prevent transpiration.
Effects of the presence of some poisonous gases (CO, CO2, HCL vapour, etc.) on transpiration may be studied by introducing the gas into the bell jar before covering the plant. The effect of light and darkness may also be studied by this method.
(b) Cobalt chloride paper method (qualitative):
Experiment:
A few pieces of filter paper are soaked in 5 % aqueous cobalt chloride (COCl2) solution for a few minutes. The pieces are then taken out, excess solution is removed by hanging and pieces are dried in hot air oven at 60°C.
These are then cut into squares (2×2 cm) and kept in a desiccator containing fused CaCl3. The original colour of the wet papers is pale pink, when dried to a standard uniform shade, the colour becomes intense blue.
Now a piece of cobalt chloride paper thus prepared is placed at the lower surface of a leaf of a potted plant and the paper is covered with glass slides from both surfaces of the leaf the slides are made air-tight with Vaseline.
Observation:
After some time the blue colour of the cobalt chloride paper turns pink.
Inference:
As water vapour comes out of the stomatal pores (if they are open) it moistens the cobalt chloride paper changing its colour to pink. The treated blue dry paper is thus a moisture detector, turning pink when left in the air or when placed in contact or near an evaporating surface.
(c) By conical flask-water-oil-leaf method (quantitative):
Experiment:
A 250 ml conical flask is filled up to its neck with water and a-suitable petiolate leaf (cut under water in order to maintain continuity of water column) is inserted within the conical flask in such a way that its petiole remain under water.
A sinker may be used to keep the leaf in position. Now oil is poured on the surface of water to check evaporation from water surface. The whole set-up is weighed and allowed to stand in open air or in a well ventilated room having sufficient light. After 2 hours the set-up is reweighed.
Results:
The difference between the first and the second weights gives the amount of water transpired by the leaf in 2 hours. The results may be expressed in per unit time.
Discussion:
The loss in weight is due to loss of water in the form of water vapours through the leaf. Since the free evaporating surface is checked by oil film, the sole loss of water is only due to transpiration through the leaf.
(d) Hanging leaf method:
Experiment:
Two healthy leaves of a suitable plant are selected. One of the leaves is smeared with Vaseline on both the surfaces and to the petiole to check transpiration. The second leaf is left as such. The leaves are then weighed and kept hanging in bright sunlight for an hour. These are then taken out and reweighed.
Results:
After one hour it is seen that the first leaf remains turgid and fresh while the second leaf wilts. In the first leaf the difference between the initial and the final weights are negligible, but in the second leaf considerable loss in weight takes place.
Discussion:
Results indicate that the first leaf in which transpiration is checked, the loss of water is insignificant and the leaf remains turgid and fresh. While in the second leaf loss of water due to transpiration takes place in, appreciable quantity and the leaf wilts.
Experiment # 9
Determination of Transpiration Index:
Experiment:
Transpiration index is defined as the ratio of the time (in seconds) required for a standard change of colour of dry cobalt chloride paper over a free evaporating surface (S) and the transpiring surface (E) and can be expressed as follows:
Transpiration index = S × 100/E
(The index is multiplied by 100 to obtain the loss of water from the leaf surface as a percentage of evaporation from free water surface).
Two equal pieces (2×2 cm) of cobalt chloride papers are taken. One is attached to the lower surface of a dorsiventral leaf as in Expt. 8.b and another is placed on a wire net which is kept over a petridish containing water. Time taken in seconds for a standard colour change of the cobalt chloride papers in both the cases is noted.
Results:
Transpiration index is calculated from the above mentioned formula.
Discussion:
Transpiration index gives an indication of the relative efficiency of the rate of transpiration with that of physical evaporation. It varies from species to species, with age of leaves and with different environmental conditions.
N.B. Transpiration index of upper and lower surfaces of a leaf, in light and dark, in young and old leaves and in different ecological types of leaves may be compared. The participation of conducting tissues in transpiration may also be studied from this experiment by separately removing xylem and phloem tissues.
Experiment # 10
Determination of the Rate of Transpiration:
(a) Cobalt chloride paper method (qualitative):
Two pieces of cobalt chloride papers are affixed on both the surfaces of a dorsiventral leaf as in Expt. 8.c. The length of time required for the papers to turn pink indicates the rate of transpiration from the two surfaces of the leaf.
(b) Conical flask-water-oil-leaf method (quantitative):
Experiment:
Two dorsiventral leaves of equal and comparable sizes are taken. The upper surface of one leaf and the lower surface of the other are smeared perfectly with Vaseline. The experiment is then set-up as in Expt. 8.c.
Results:
The amount of water transpired per unit time divided by the area of the leaf (determined by graphical method as in Expt. 5.c) gives the rate of transpiration per unit time per unit area of the leaf.
Discussion:
The rate of transpiration is higher in lower surface than in upper surface because stomata are more abundant in lower surface than upper surface which is cuticularised. The rate of transpiration from the lower surface indicates stomatal transpiration and that from the upper surface indicates cuticular transpiration.
N.B. The rate of transpiration may be correlated with:
(i) Stomatal frequency,
(ii) A single stoma (amount of water transpired per unit time divided by the total number of stomata of the leaf),
(iii) Area of stomatal aperture (amount of water transpired per unit time by the total stomatal aperture),
(iv) Number of xylem vessels present in the petiole (a cross section of the petiole may be cut for estimation of total number of xylem; the total area of xylem vessels as determined from the formula πr2) and correlated with the rate of transpiration), and
(v) Simple physical evaporation (rate of evaporation of water per unit time per unit area of a petridish is to be calculated taking area of petridish to be πr2) and this may be compared with the rate of transpiration per unit area of the stomatal aperture.
(c) Potometer method (quantitative):
The rate of transpiration (expressed in gm per hour per square centimeter of the leaf surface) can be measured with the help of an apparatus known as potometer. Determination of the rate of transpiration by different potometers, excepting Garreau’s, is an indirect one where both absorption and transpiration have been taken into consideration.
Potometers are designed on the principle that the rate of transpiration is nearly proportional to the rate of absorption of water by the plant, although it is not the general rule. There are many types of potometers; all working on the same principle excepting Garreau’s which gives a direct measurement of transpiration. The potometers described below are generally used in laboratory experiments.
(i) Darwin’s potometer:
Description:
This apparatus consists of a short glass tube from which a side tube bends upward ending in an open mouth (manometer tube) into which a plant twig is inserted through a cork.
The upper open mouth of the main tube is closed by a cork. The lower end of the tube is also fitted with a cork through which passes a long graduated (in ml) capillary tube. The end of the capillary tube dips in a beaker containing water (Figure 12).
Experiment:
At the beginning of the experiment, water is filled up in all the tubes maintaining continuity. A twig (cut under water) having some leaves are inserted through the cork of the side tube. All joints should be air-tight.
A small bubble is introduced through the lower end of the capillary tube. As the transpiration occurs from leaves of the twig, water is absorbed by the twig from the side tube and this produces a suction force which sucks up water from the capillary tube. As a result, air bubble within the capillary tube gradually moves upward.
Results:
The rate of upward movement of air bubble is recorded by noting the height by which it ascends in a given time. The rate of transpiration is expressed as the ml of water transpired per minute per unit area of the leaves.
Discussion:
See Expt. 10c (iii).
(ii) Ganogo’s potometer:
Description:
This apparatus is most suitable for determination of the rate of transpiration. It consists of a narrow graduated horizontal limb which holds two vertical wide-mouthed tubes-one of which is fitted with a cork through which passes a leafy twig (cut under water) while the other acts as a reservoir which is fitted with a stopcock in the connecting tube to control water supply.
The other end of the horizontal limb bends at right angle and at the opposite side of the vertical wide-mouthed tube (Figure 13).
Experiment:
At the outset, water is filled in all parts of the tube by opening the stopcock of the reservoir. A leafy twig is inserted through the cork of the vertical tube. Keeping the stopcock closed, all the connections are made air-tight.
The twig is then allowed to transpire for a short period as a result of which water moves upward in the lower bent tube which dips in a beaker of water. An air bubble is now introduced through the lower end of the bent tube and its initial position is noted.
The apparatus is fixed in this position with a stand and clamp. As water moves due to transpiration, the bubble also moves in the horizontal graduated limb. When the bubble comes to one end of the limb, it can be pushed back to its original position by opening the stopcock of the reservoir and the, experiment can be repeated several times.
Results:
The rate of movement of the bubble in the horizontal tube is followed by measuring the distance it moves along the graduated scale within a certain period of time. This is considered as proportional to transpiration rate.
The rate of transpiration can be obtained by the following calculations:
Initial position of the bubble on the scale— X cm.
Final position of the bubble after a given time (t) — Y cm.
Therefore, the distance traversed by the bubble in time t is equal to (Y—X) cm. Now, volume of water transpired in time t is equal to πr2 (Y—X) ml, where r is the internal radius of the bore. Thus the volume of water transpired by per unit area of the leaves of the twig per unit time is equal to {πr2(Y—X)/t} × area of leaves] ml.
(iii) Farmer’s potometer:
Description:
It has the same principle as that of Ganong’s potometer and is only a modification of the latter. The apparatus consists of a wide-mouthed bottle fitted with a rubber stopper having three holes.
The bottle is filled with water up to the neck. In one hole a leafy twig (cut under water) is introduced in such a way that its lower cut end remains well under water. In the second hole, a water reservoir having a stopcock in its connecting tube is fitted so as to control the supply of water into the bottle.
In the third hole, a narrow bent tube is fitted so that its lower end is well below the water surface of the bottle. The bent part of this tube is horizontal in position and either graduated or fitted with a centimeter scale. The outer end of the horizontal tube is again bent downward and immersed in water (Figure 14).
Experiment:
At the beginning, the bottle is filled with water by opening the stopcock of the reservoir up to the mouth and also in the horizontal tube. The stopcock is then closed. An air bubble is now introduced into the outer bent end of the horizontal tube in the usual manner.
As transpiration occurs from the leaves of twigs, water is absorbed from the lower cut end of the twig and a suction force is produced which sucks water from the horizontal tube. As a result air bubble moves inward. The rate of movement of air bubble is noted, which is considered proportional to the transpiration rate.
Results:
As in Expt. 10c (ii).
Discussion:
With the help of potometers (Darwin’s, Ganong’s & Farmer’s) the rate of transpiration can be easily determined. This is an indirect method because the idea of this experiment is based on the assumption that the amount of water transpired by a twig is almost equal to the amount of water absorbed.
In fact this does not generally happen in plants and the amount of water absorbed by a plant is not fully transpired but some amount is retained by it for its normal metabolic functions Hence, the potometer methods described above for determination of rate of transpiration are hot fully accurate.
(iv) Garreau’s potometer:
Description and Experiment:
This potometer is conveniently used for quantitative measurement of differential rates of transpiration from both upper and lower surfaces of a leaf. It consists of two wide-mouthed cups which are placed face to face keeping a widely expanded dorsiventral leaf; of a twig from a potted plant in between them.
Before keeping the leaf in between; the cups, some anhydrous CaCl2 contained in two small vials are weighed and placed in both the cups. The ends of the cups are closed with corks through which two mercury manometers are connected in order to keep the vapour pressure within the cups constant.
The broad rims of the cups in contact with leaf surface are made Potted air-tight by applying Vaseline carefully. A change in vapour pressure within the cups shown by the manometers is indicative of the fact that either the connections are not air-tight or that all the vapour given out by the leaf surface is not being absorbed by the CaCl2 within the vials. The whole arrangement is clamped vertically on a stand (Figure 15).
Results:
After a few hours, CaCl2 vials are taken out and weighed again. The difference between the two weighing’s is a measure of the amount of water loss from the two leaf surfaces. As the areas are the same, this is also a quantitative measure of the differential rates of transpiration.
Discussion:
By Garreau’s potometer method, not only the rate of transpiration but a comparative assessment of the rate of transpiration of upper and lower surfaces of a leaf, i.e., cuticular and stomatal transpiration respectively, can be simultaneously determined. This is a very convenient and accurate method for direct and quantitative measurement of the rate of transpiration in contrast to other potometer methods.
N.B. It is easily possible to set up potometer experiments under different environmental conditions such as at different temperatures or light intensities or relative humidities or wind velocities and to observe their effects on transpiration rate.
Experiment # 11
Quantitative Determination of the Differential Rates of Transpiration from the Two Surfaces of a Leaf (Cuticular and Stomatal):
Experiment:
This experiment is performed with the help of a conical flask-water-oil-leaf method as in Expt. 10b. Three dorsiventral leaves (betel or Hibiscus leaf) of almost uniform size and age are selected.
Three conical flasks filled with water up to the neck are taken:
(i) With untreated leaf,
(ii) With leaf whose upper surface is thinly smeared with Vaseline, and
(iii) With leaf whose lower surface is similarly smeared with Vaseline.
Oil is poured in each flask to make a thin film on the water surface to check evaporation. The initial weights are noted separately in each case.
Results:
The rates of transpiration of all the three leaves are determined and differential rates of transpiration from either surface are obtained. In each case area of the evaporating surface is determined and results are expressed as gm per unit area per unit time.
Discussion:
As the dorsiventral leaves have a very few number of stomata on the upper surface which is cuticularized, the rate of transpiration from this surface is termed cuticular transpiration. That the cuticular transpiration is insignificant is clear when it is compared with the rate of transpiration of the leaf whose lower surface has been smeared.
The rate of transpiration from the lower surface, having abundant stomata, gives the Stomatal transpiration.
It is generally found that the rate of stomatal transpiration is nearly equal to that of control leaf this indicates that in case of dorsiventral leaf the major loss of water takes place through stomata, i.e. , through the under surface of the leaf.
N.B. A qualitative approach regarding this experiment may be made by cobalt chloride paper method (Expt. 10a).
If a quantitative estimation of amount of water lost per stoma in a leaf is desired, the total number of stomata in both upper and lower surfaces is determined as also the total amount of water lost. The amount of water lost from the transpiring surface divided by the total number of stomata gives an approximate measure of the amount of water given out per stoma.
Experiment # 12
Simultaneous Determination of the Amount of Water Absorbed, Retained and Transpired by a Plant:
(a) By direct measurement method:
Experiment:
This is a simple apparatus consisting of a wide-mouthed bottle with a graduated side tube (in ml) attached to its base through a cork. The mouth of the bottle is fitted with a cork through which is introduced a small leafy plant. The bottle is filled up with water and all connections are made air-tight (Figure 16).
The evaporation from the side tube is prevented by pouring a thin film of oil on the exposed water surface. The level of water in the side tube is recorded. The whole arrangement is weighed and kept in bright sunlight. After 2 hours or so the level of water in the side tube is again recorded and the whole arrangement is reweighed.
Results:
The difference between the initial and final levels of water if the side tube is a measure of the amount of water absorbed in ml by the plant and the difference between the first and the second weight evidently shows the amount of water transpired in gm from the leaf surface.
The volume of water absorbed may be converted to gm by multiplying the density of water at that temperature from a standard temperature-density table (see Appendix II). Now the difference between the amount of water absorbed and the amount of water transpired indicates the amount of water retained.
Discussion:
See Expt. 12(b).
(b) By conical flask-water-oil-leaf method:
Experiment:
The above relation can also be experimentally determined by conical flask-water-oil-leaf method (Expt. 10b). The weight of the conical flask plus water plus oil is taken (W1).
Care should be taken to avoid excess addition of oil. There should be a thin film of oil evaporating surface. Now the petiole of a leaf from a suitable plant is carefully introduced through the open water surface by tilting the conical flask so that no oil adheres to the petiolar end (see N.B.).
The weight of the conical flask plus water-plus oil plus leaf (W2) is noted. The whole arrangement is exposed to a well-ventilated lighted place for 2-hours. The set-up is then reweighed (W3). The leaf is taken out carefully so that no water or oil adheres to it. The weight of the conical flask plus water plus oil is again noted (W4).
Results:
Now, (W1—W4) is equal to the amount of water absorbed by the leaf (i). (W2—W3) is equal to the amount of water transpired by the leaf (ii). The difference between (i) and (ii) gives the amount of water retained by the leaf.
Discussion:
The above experiment clearly demonstrates that the amount of water absorbed by a twig or a leaf is not fully transpired under normal environmental conditions. Some amount of water is retained by it for its metabolic functions. This is a useful method for simultaneous determination of water absorption, retention and transpiration when the controlling factor acts chiefly through the leaves.
N.B. Instead of pouring oil in the conical flask before introducing the leaf into it, a vial containing oil may be weighed along with the flask and water. The leaf is then introduced and some amount of oil is poured from the vial on the surface of water. The vial should be always weighed in every step of weighing. This method will avoid the possibility of adherence of oil to the cut end of the petiole,
Experiment # 13
Demonstration of Suction Force Due to Transpiration or Transpiration Pulls:
Experiment:
A narrow-bored glass tube is taken and a suitable leafy twig (cut under water) is inserted into it with the help of rubber tubing and the connection is made air-tight with sealing wax. The tube is completely filled with water and holding it with the thumb, the lower end is carefully dipped below the surface of mercury contained in a small beaker.
The whole arrangement is vertically clamped with the help of a stand and is kept in a well-ventilated lighted place for transpiration. The rise in mercury column is noted at an interval of 15 minutes and final height is recorded after 1 hour.
Results:
The suction force due to transpiration or the weight of the transpiration pull is calculated by the formula
πr2 X h X p X g dynes per cm, where πr2 is the area of the bore of the glass tube, h is the increase in the height of mercury column in the glass tube at a given time, p is the specific density of mercury (13.6) and g is the acceleration due to gravity (981 dynes per sec.2).
Discussion:
The rise of mercury within the glass tube indicates the suction or the transpiration pull exerted on the water in the xylem vessels which is transmitted to the water column in the glass tube and ultimately to the mercury level in the beaker causing the mercury to rise in the tube.
N.B. This experiment can be further modified by taking into consideration:
(i) Stomatal and cuticular transpiration by smearing either surface with Vaseline,
(ii) The variation of the leaf area either by removing some leaves or smearing Vaseline on both the surfaces of some leaves,
(iii) Herbaceous and woody twigs having comparable leaf area, and
(iv) Different environmental factors.
Experiment # 14
Determination of Stomata-Bearing Surface of a Leaf without Using a Microscope:
Experiment:
Four dorsiventral leaves of almost similar size and age are selected and Vaseline is applied as follows:
(i) Vaseline is smeared on both the surfaces of the first leaf,
(ii) Vaseline is smeared on the upper surface of the second leaf,
(iii) Vaseline is smeared on the lower surface of the third leaf, and
(iv) The fourth leaf is kept as such.
All the leaves are then weighed separately and kept hanging from threads which are tied to stands in an open space for a few hours. Now, each leaf is weighed again.
Results:
In case of the first leaf no wilting is observed and there is no loss in weight also. The second leaf wilts and loses weight. The third leaf, however, shows least wilting and minimum loss in weight. On the other hand the fourth leaf wilts considerably losing maximum weight.
Discussion:
Leaves lose water depending on the rate of transpiration. It is clear from the results that smearing of the upper surface of the leaves affects the rate of transpiration least.
While smearing of the leaves on the lower surface considerably reduces the rate of transpiration, the upper surface in case of dorsiventral leaf is responsible for cuticular transpiration which is insignificant compared to stomatal transpiration through the lower surface. Hence, stomata are present on the lower surface in case of dorsiventral leaf.
Experiment # 15
Determination of the Effects of Environmental Factors on the Rate of Transpiration:
(a) Effect of Humidity:
Experiment:
Conical flask oil water-leaf arrangement is to be used for this experiment. Two such arrangements having identical leaves are made. One such arrangement is weighed and kept in laboratory condition having relatively low relative humidity.
The second weighed flask with the leaf is kept under a bell jar which is previously made humid by sprinkling water on the inner wall of the jar and by placing petridish containing steaming water which ensures almost saturated vapour pressure condition.
The bell jar is placed on a glass plate and the contact is made air-tight with Vaseline. After 2 hours both the flasks are weighed and areas of leaves are determined.
Result:
The rate of transpiration in each ease is calculated and expressed as gm per sq. cm. per hour.
Discussion:
The internal atmosphere of the leaf is considered to be saturated or nearly so. The external atmosphere, on the other hand, is usually in an unsaturated condition. The vapour pressure gradient exists, therefore, between internal and external atmospheres and water vapour diffuses through the stoma from the region of high vapour pressure to the region of low vapour pressure.
The lower the vapour pressure gradient the less is the rate of transpiration at a constant temperature. If the vapour pressures of internal and external atmospheres are the same, no transpiration will-take place. From the above experiment it is clear that the rate of transpiration under humid condition (high vapour pressure) is much less than the control.
(b) Effect of wind velocity:
Experiment:
Conical flask oil water-leaf arrangement is to be used for this experiment. Two such arrangements having identical leaves are made. The weighed control flask with the leaf is kept under laboratory condition in still air.
The second weighed flask with the leaf is placed under a fan at a considerable distance from the first set. The leaf may be tied to the flask with thread to avoid its displacement. The experiment is continued for 2 hours. The flasks with leaves are reweighed. The areas of the leaves are determined.
Results:
Results are calculated and expressed as gm per sq. cm. per hour.
Discussion:
Air movements tend to hasten transpiration because it lowers the humidity of the external atmosphere of the leaf and thus make greater difference between the vapour pressure inside and outside of the leaf by mechanically removing the accumulated moisture.
Whether the abrupt change in vapour pressure, which is one of the controlling factors of transpiration, is due to wind velocity is not known. The results of this experiment show that the rate of transpiration is much higher under air current than that under still air.
N.B. The wind velocity may be controlled by regulating the speed of the fan with the help of its regulator and the rate of transpiration under each air current may be compared.
(c) Effect of light:
Experiment:
Conical flask oil water-leaf arrangement can be conveniently used for this experiment. Three such arrangements having identical leaves are made. One weighed conical flask with leaf is kept under direct sunlight.
The second weighed conical flask is kept under shade and the third weighed conical flask is kept in a dark room. The experiment is continued for 2 hours. The flasks with the leaves are re- weighed and areas of the leaves are determined.
Results:
Results are calculated and expressed as gm per sq. cm. per hour.
Discussion:
Light and especially direct sunlight as contrast to shade or darkness has a marked effect in bringing about an increase in transpiration.
This in most cases is chiefly due to the fact that much of the radiation is absorbed by the leaves. The temperature of the leaves in direct sunlight is, therefore, always higher than the surrounding temperature of the atmosphere.
The second factor that might be of some importance is the opening of the stomata in the light. The results show that the rate of transpiration is maximum under direct sunlight and minimum under darkness.
N.B. Effects of different intensities of light may also be studied. Temperature is an important factor which controls the rate of transpiration. Effect of temperature may be studied by the conical flask arrangement keeping the flask in different temperatures.
Experiment # 16
Quantitative Determination of Transpiration Under Conditions of Different Experimental Errors Generally Encountered:
Experiment:
During different transpiration experiments the following experimental errors are generally come across:
(i) The twig or petiole of the leaf not cut under water.
(ii) Adherence of Vaseline to the surface of leaf through which transpiration is to occur.
(iii) Taking of aged or yellowing leaves as experimental materials for normal transpiration experiments.
(iv) Taking of leaves of different ages for similar experiments.
(v) Keeping up of experiments under closed environment,
(vi) Adherence of oil at the cut end of the petiole.
The same conical flask-oil-water-leaf-arrangement may be conveniently used in performing these experiments. Six such arrangements are made and each arrangement involves one of the above errors. Leaves from a same species of plant should be used for better comparison of results.
A separate set-up is also made in which a healthy medium-aged leaf is taken avoiding all the above errors. All the flasks with leaves are separately weighed and kept in places which will suit the experiment most. The experiments are continued for about 2 hours. The flasks with the leaves are reweighed. The area of the leaves is measured.
Results:
Results are determined and expressed as gms per sq. cm. per hour.
Discussion:
The petiole of the twig is cut under water in order to maintain the continuity of water column within the xylem vessels. If this continuity is broken air bubble gets entry and the transpiration pull steps.
Vaseline is generally applied to the leave surface to check transpiration by avoiding contact of the stomatal pore with the atmosphere, thus, if vaseline adheres to a surface from which transpiration is to be determined, the transpiration is greatly affected.
The rate of transpiration in case of aged or yellowed leaf is less because of its slow metabolic activity. Similarly different aged leaves affect transpiration in comparative studies owing to difference in metabolic activities as well as stomatal frequency.
The rate of transpiration in a closed environment is affected by less light intensity and higher humidity. Adherence of oil at the cut end of the petiole hampers the entry of water through the conducting tissues. All these experiments show that precuations must be taken in performing experiments on transpiration to minimise the errors in results.
Experiment # 17
Demonstration of Guttation:
Experiment:
Phenomenon of guttation can be seen in the early morning in plants such as Bryophyllum, Tropeolum (garden nasturtium), grasses, etc. especially after one humid night.
Now a small potted plant of tomato which has been previously well watered is taken and kept under a bell jar and sealed from all sides. The bell jar may be partially evacuated with the help of an aspirator fitted to the outlet of the bell jar. It is then placed in a warm room for some time.
Observation:
Drops of guttation fluid oozing out at the margin of the leaves at its vein ending, are observed (Figures 17),
Inference:
Guttation is the exudation of liquid containing water from the margins of leaves of a real shoot. This occurs when the roots of such plants absorb water actively but transpiration is checked or lowered due to high relative humidity in atmosphere developing thereby a high root pressure. This produces a positive upward force in the xylem sap and causes exudation of liquid or guttation.