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**List of top nine experiments on transpiration in plants:- 1. Measurement of Leaf Area 2. Demonstration of Transpirational Water Loss by Potometers 3. Determination of the Rate of Transpiration by Simple Method (Conical Flask Method) 4. Determination of the Rates of Stomatal and Cuticular Transpiration and a few others. **

**Experiment # 1. Measurement of Leaf Area****: **

The loss of water in the form of vapour from the aerial parts — particularly through leaves — is termed “transpiration”. On absorption from the soil by roots, the water is trans-located via the xylem tissue to the mesophyll cells of the leaves.

The excess water is lost through stomatal opening or through the diffusion process from leaf surface. For determination of the rate of transpiration, measurement of leaf area, i.e. transpiring surface, is highly essential. The leaf area can be measured by different methods.

**Method I: By Graph Paper Method: **

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**Requirements****:**

1. Graph paper (mm); 2. Scale, pencil, leaf, etc.

**Procedure****:**

1. Place leaf on a millimeter graph paper and draw its outline with a pencil (Fig. 3.11).

2. Then count the total area covered by the leaf from the marked outline of the leaf and express it as square centimeter.

**Observation****:**

The leaf area measurement procedure is shown in Fig. 3.11.

**Method II: By Weighing Method: **

**Requirements****:**

1. Card-board

2. Leaf, pencil, rubber, blade, balance with weight box.

**Procedure****:**

1. Place a leaf on a cardboard and draw its outline. Then cut the board along the line-mark of drawing and take its weight.

2. Now cut one square centimeter area from the board and take the weight. Observation

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The weight of the board cut to the size of leaf area is x gms. The weight of one square centimeter area of the board is y gms. Then the area of leaf is x/y sq. cm (Fig. 3.12).

**Method III: By Planimeter Method: **

**Requirements****:**

1. Planimeter — a simple instrument, having two major parts — a tracer arm having a tracing point and a carriage with a measuring wheel and also the pole arm attached to the pole, around which the instrument revolves (Fig. 3.13).

2. A platform, leaf, pencil etc.

**Procedure****:**

1. Place the leaf on a platform in a fixed position and draw the outline of the leaf by a pencil.

2. Place the pole weight close to the outline of the leaf and move the tracer point along the margin of the leaf.

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3. Record the initial reading from the scale and final reading after the tracing of leaf.

4. Compute the total leaf area by denoting the data from the main scale and also from vernier scale.

5. Record the vernier scale reading in the following ways before final computation:

First coincide the zero of the measuring wheel with zero of the vernier scale. Find out the number of divisions of the vernier scale that coincides with that of the measuring wheel.

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Suppose 10 vernier divisions = 9 divisions of measuring wheel. So, 1 vernier division = 9/10 or 0.9 division of the measuring wheel. Vernier unit = 1 – 0.9 = 0.1 sq. cm small division of the measuring wheel = 1 sq. cm and 1 div. of the counter dial = 100 sq. cm.

Thus total reading = (Counter dial reading × 100) + (Measuring wheel reading × 10) + (vernier reading × 0.10) sq. cm.

**Experiment # ****2. Demonstration of Transpirational Water Loss by Potometers:**

The water loss by the process of transpiration can be demonstrated by several types of glass apparatus, called Potometers. In most of the potometers, the rate of transpiration can be measured directly and expressed in gms per hour per sq. cm of leaf area.

But these methods are not accurate because of the fact that the amount of water absorbed by the twig (which is measured by the apparatus) is not actually transpired at the same time.

**The description and working of some potometers are given below: **

**(A) Ganong’s Proto-Meter**** (Fig. 3.14):**

This is a glass apparatus fitted with a wooden stand. It is one of the most suitable potometers used for the demonstration or determination of the rate of transpiration.

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It consists of a narrow graduated horizontal limb which holds two vertical wide-mounted tubes — one of which is fitted with a rubber cork through which passes a leafy twig while the other acts as a reservoir of water which is fitted with a stopcock in the connecting tube to control water supply. The other ends of horizontal limbs bend at right angle and at the opposite side of the vertical wide mouthed tube.

**Materials Required****:**

1. Ganong’s potometer

2. Water, beaker, leafy twig, knife, etc.

3. Graph paper, pencil, etc.

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**Procedure****:**

1. Fill the apparatus with water and insert a leafy twig (cut under water) through the cork of the vertical tube. The twig should always be cut under water to prevent air-clogging.

2. Keep some water in the reservoir funnel and close the stopcock and make all the connections air-tight by proper sealing.

3. Introduce a drop of air bubble in the horizontal limits of the apparatus.

4. Allow the twig to transpire for 1-2 hrs. under bright sunshine.

**Observation****:**

As water is lost by transpiration, the bubble will move in the horizontal graduated tube towards the transpiring twig. The rate of movement of the bubble in the horizontal tube is proportional to transpiration rate (assuming that the rates of absorption and transpiration are the same).

**Results****:**

**The rate of transpiration can be determined in the following ways: **

Initial position of the bubble on the scale — X cm

Final position of the bubble after a given time — Y cm

Therefore, the distance traversed by the bubble in time t is equal to (Y – X) cm.

Now the volume of water transpired in a given time (t) is equal to tit^{2} (Y – X) ml where ‘r’ is the radius of the bore of the horizontal tube.

So, the amount of water transpired by per unit area of the leaves of the twig per unit time is equal to

(Y – X)/ t x total leaf area * ml/min/sq. cm.

[* The leaf area can be measured by graph paper method.]

**(B) Farmer’s Potometer**** (Fig. 3.15): **

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 leafy twig can be introduced while in another a water reservoir having a stopcock is fitted. The third hole is fitted with a narrow bent tube which has a horizontal graduated tube with a centimeter scale.

**Materials Required****:**

1. Farmer’s potometer

2. Beaker

3. Water, leafy twig, pencil, graph paper etc.

**Procedure: **

1. Fill the apparatus with water and keep some water in the reservoir.

2. Introduce a freshly cut (cut under water) twig within the bottle and make all connections air-tight by proper sealing.

3. The bent end of the narrow tube is to be immersed in a beaker containing water.

4. Keep the whole set-up under bright sunshine for transpiration at a steady state.

**Observation****:**

Observe the movement of the air-bubble within the horizontal tube towards the twig. The rate of movement of air-bubble is proportional to the rate of transpiration (assuming that the rates of absorption and transpiration are equal).

**Result****:**

Same as in Ganong’s potometer.

**(C) Darwin’s Potometer**** (Fig. 3.16): **

The apparatus consists of a short glass tube from which a side tube bends upward ending in an open mouth into which a plant twig is inserted through a hole in a rubber cork.

The upper open mouth of the main tube is also closed by a cork. The lower end of the tube too is fitted with a cork through which passes a long graduated capillary tube, fitted with the help of a rubber tubing. The end of the capillary tube dip in a beaker containing.

**Materials Required****:**

1. Darwin’s potometer

2. Beaker, leafy twig, water, graph paper, pencil etc.

**Procedure****:**

1. At the beginning of the experiment, fill up the apparatus with water.

2. Insert a fresh leafy twig (cut under water) through the cork of the side tube.

3. Make all joints air-tight.

4. Introduce an air-bubble within the water column of the capillary tube.

5. Allow the whole set to transpire under bright light after fixing it with stand and clamp.

**Observation****:**

As 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, the air-bubble within the capillary tube gradually moves upward.

**Result****:**

The rate of upward movement of air bubble is recorded from the initial and final readings of the position of the air-bubble in the capillary tube. The rate of transpiration is then expressed as in case of Ganong’s Potometer (ml of water transpired per minute per unit area of the leaf).

**(D) By Garreau’s Potometer**** (Fig. 3.17): **

It consists of two small bell jars placed one above another in-between which a leaf is placed while still attached to a potted plant. At the narrow end of the two bell jars, weighed amounts of anhydrous CaCl_{2} are placed in two very small tubes. At the two ends of the bell jars, are attached two oil manometers which ensure the maintenance of constant vapour pressure within the bell jars.

**Materials and Equipment’s****:**

1. Garreau’s Potometer

2. Vaseline, Anhydrous CaCl_{2} salt

3. Potted plant, stand with clamps

4. Balance with weight box

**Procedure****:**

1. Place a leaf of a potted plant inside two bell jars and make it airtight by vase-line.

2. Clamp the whole arrangement of the apparatus in vertical position and place it in sunlight.

3. Before the onset of the experiment place measured quantities of anhydrous CaCl_{2} salt in the tubes and take the final weight after a considerable period of transpiration (at least two hours).

**Results****:**

The difference between the two weighing’s is a measure of the amount of water lost from the upper and lower leaf surfaces.

**Hence transpiration by both the surface of a leaf can be directly measured separately and simultaneously by Garreau’s potometer: **

Initial weight of CaCl_{2} in the upper tube — W_{1} gms

Initial weight of CaCl_{2} in the lower tube — W_{2} gms

Final weight of CaCl_{2} in the upper tube — W_{3} gms

Final weight of CaCl_{2} in the lower tube — W_{4} gms

Amount of transpired water by upper leaf surface — (W_{3} – W_{1}) gm.

Amount of transpired water by lower leaf surface — (W_{4} – W_{2})

Rate of upper leaf surface transpiration i.e. cuticular transpiration (in case of dorsiventral leaf) = (W_{3} – W_{1}) gm. total leaf area (sq m) and time (min)

Rate of lower leaf surface transpiration i.e. stomatal transpiration = (W_{4 }– W_{2}) gm. total leaf are (sq m) time (min)

**Experiment # ****3. Determination of the Rate of Transpiration by Simple Method (Conical Flask Method)****:**

The loss of water from the leaf surface of terrestrial plants is a normal physiological process. It is either transpirational loss, i.e., in the form of water vapour, or guttation i.e., in the form of water droplets.

Transpiration normally takes place through stomatal openings of leaves or through particular openings of stem surface or through the cuticular surface, or a combination of paths mentioned above. This transpirational water loss is a necessary evil for plant life.

Transpirational water loss can be determined by the conical flask method — a very simple method.

**Materials and Equipments****:**

1. A fresh leafy twig

2. Beaker, conical flask, knife, glass rod, thread etc.

3. Water, oil, balance with weight box

4. Graph paper, pencil, stop-watch etc.

**Procedure****:**

1. Take a 100 ml conical flask and fill it with water up to the neck.

2. Insert a freshly cut petiolate leaf (leaf cut under water within a breaker) and tie the cut end of the petiole with a glass rod by thread so that the leaf cannot be displaced from the conical flask by wind.

3. Then put some oil over the water of conical flask so that the exposed water surface will be covered.

4. Weigh the experimental set (conical flask – water-oil-leaf) in a chemical balance and record the initial weight.

5. Place the experimental set under bright sunshine for 1 hour and weight finally in a chemical balance.

6. Record the final weight and calculate the water loss by transpiration.

7. Record the total transpiring area of the leaf by Graph paper method.

8. Determine the rate of transpiration in the following way:

#### **Calculation****:**

Rate of transpiration = x gms per min per sq cm of leaf area

**Experiment # 4****. Determination of the Rates of Stomatal and Cuticular Transpiration****:**

In principle stomatal transpiration takes place through the lower surface of the leaf where maximum number of stomata are localized, whereas transpiration through upper surface of leaf is said to be of the cuticular type in dorsiventral leaves.

Thus, if one wants to determine the relative efficiency of transpiration rate by the two surfaces, then two separate experimental sets have to be prepared using the same type of leaves. In one experimental set, the upper surface of the leaf is smeared with grease so that transpiration occurs only through the lower surface (i.e. predominantly stomatal type).

In the other set the lower surface of the leaf is smeared with grease so that transpiration occurs only through the upper surface of the leaf (i.e. predominantly cuticular type). The rate of transpiration per unit time per sq cm of leaf area is calculated separately and then compared. The experimental procedure is identical with conical flask water-oil-leaf method (as in Transpiration Expt. III).

**Experiment # 5****. Determination of Stomatal Frequency, Stomatal Area, Stomatal Index, Total Number of Stomata Per Leaf and Transpiration Index****:**

The stomata constitutes the major route for the transpirational water loss to the surrounding atmosphere. Thus determination of stomatal frequency of a leaf or total stomatal area of leaf is the basic information needed to assess the rate of transpirational water loss through the stomata.

The stomatal frequency is determined by the calculation of the number of stomata per unit area (i.e. sq cm of leaf). Subsequently, the total number of stomata of a leaf and the total area covered by stomata (i.e. stomatal area) can also be calculated.

Finally, transpiration index is calculated by the relative efficiency of the rate of transpiration with that of physical evaporation. All these features vary from species to species with age of leaves and with different environmental conditions.

**Materials and Equipments Required****:**

1. A leafy twig

2. Slide, cover glass, forceps, needle, etc.

3. Microscope, stage and ocular micro-meters, graph paper, pencil etc.

4. Distilled water

**Procedure****:**

1. Prepare epidermal peelings from the lower surface of a leaf and then put on a glass slide for examination under microscope.

2. Record the number of stomata per field of vision (under high power) in random fashion by moving the slide.

3. Calculate the area of field of vision under high power by ocular and stage micrometer.

4. Then calculate the number of stomata per unit area (sq cm) of leaf, i.e., stomatal frequency.

**5. Determine the stomatal index of both leaf surfaces by the following formula: **

Stomatal Index (S.I.) = No. of stomata in a given area (S)/Total no. of cells of the area (epidermal) + S × 100

6. Determine the total area of the leaf by graph paper method and then calculate the total number of stomata of the said leaf.

7. Determine the area covered by each stomata with the help of ocular micrometer (value of each division is standardized before by the stage micrometer by the formula):

1 ocular division value = Stage div./Ocular div. × 10µ.

Then calculate the total stomatal area of the given leaf.

8. Determine the transpiration index by recording the time (in sec) required for standard change of the dry cobalt chloride paper over the evaporating surface (S) and transpiring surface (E) by using the formula:

Transpiration index = S/E × 100

In this process, take two equal pieces (2 × 2 cm) of dry cobalt chloride paper and then place one of them under the lower surface of dorsiventral leaf of a twig by cello tape. Place the other over a wire net which is kept over a petridish containing water. Record the time (in sec) for a standard colour change of the cobalt chloride papers in both the cases (the paper turns pink when it absorbs water vapour moisture).

Area of the field of vision = x sq cm (calculate it by the formula πr^{2}, where ‘r’ is the radius of the field)

Number of stomata per field = a

Total leaf area (from graph paper) = y sq cm

Stomatal frequency = a/x

Total number of stomata of the leaf = a/x × y

Stomatal index = No. of stomata in a given area(S)/Total no. of epidermal cell (E) + S = S/E + S x 100

Area of a stomata = π/4 (b x c) sq cm

Where ‘b’ and ‘c’ represent the length and the breadth of the pore.

Total stomatal area of leaf = π/4(b x c) x (a/x × y) sq cm

Transpiration index = S/E × 100

[S = Time taken to change the colour of cobalt chloride paper from a free evaporating surface; E = Time taken to change the colour of cobalt chloride paper from a free transpiring surface.]

**Experiment # 6****. Determination of the Amount ****of Water Absorbed and Transpired by a Plant:**

Absorption of water and subsequent loss of it in the form of vapour by the aerial parts of plants are two essential interlinked physiological processes. There is positive correlation between the two processes.

In normal situation the amount of water absorbed is much higher than the amount of water transpired. But under stressed conditions the amount of water transpired may be higher than the amount of water absorbed.

**The relationship between the two processes mentioned above can be determined by two experimental procedures:**

(a) Direct determination with the help of glass apparatus.

(b) By conical flask-water-oil-leaf experimental sets.

**(a) Direct Measurement Method:**

**Materials and Equipment’s:**

**1. Glass apparatus:**

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 a small plant can be introduced (Fig. 3.19).

2. A small rooted plant or a fresh twig

3. Oil, water, sealing wax, etc.

4. Balance with weight box

**Procedure**:

1. Fill the apparatus completely with water.

2. Introduce a rooted plant or a fresh twig (cut under water) in the wide-mouthed bottle through the cork).

3. Seal the cork to make air-tight.

4. Put a few drops of oil on the surface of water of the graduated side tube to check surface evaporation.

5. Record the initial weight and initial water level on the side tube.

6. Keep the whole set under sunshine for 2 hours.

7. Record the final weight and the level of water in the side tube.

8. Calculate the amount of water transpired (in gms). (Initial weight – final weight) and the amount of water absorbed (in ml) (initial reading – final reading of the side tube).

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.

**Result****:**

Initial weight of the experimental set — W_{1} gms

Final weight of the experimental set — W_{2} gms

Thus the amount of water transpired = (W_{1} – W_{2}) gm. = x gm.

Initial reading of side tube = 0 ml

Final reading of side tube = p ml

Thus the amount of water absorbed = (o – p) ml = Q ml

If the density of water is d, so Q ml of water = Q × d gm. = y gms

So the ratio of water transpired and water absorbed is x: y.

**(b) By Conical Flask-Water-Oil-Leaf Method (Fig. 3.18)****:**

**Materials and Equipment’s Required****:**

1. Conical flask, glass rod, thread etc.

2. Water, oil, leafy twig etc.

3. Balance with weight box

**Procedure****:**

1. Take a conical flask (100 ml), fill it up to the neck with water.

2. Put a very little amount of oil over the water surface and take the initial weight (W_{1}) gms.

3. Slowly incline the flask and insert a fresh petiolate leaf (cut under water) tied to a short glass rod with a thread. Care must be taken that oil does not stick to the cut surface of the leaf.

4. Take the second weight (W_{2} gms) of the set (conical flask – water-oil-leaf).

5. Place the experimental set under sunshine for 1 hour for transpiration and then take the final weight of the set with leaf – (W_{3} gms) and without leaf – (W_{4} gms).

6. Now calculate the amount of water transpired and amount of water absorbed.

**Result: **

Amount of water absorbed = (W_{1} – W_{4}) gms

Amount of water transpired = (W_{2} – W_{3}) gms

The difference between the two values gives the amount of water retained by the leaf or the excess water transpired — as the case may be.

**Experiment # 7****. Compare the Rate of Transpiration With the Rate of Evaporation****:**

The process of vaporization of water from the exposed surface of water and that from the leaf surface are called evaporation and transpiration, respectively. The former is a physical while the latter is a physiological process.

The rate of evaporation is dependent on environmental factors like temperature, humidity, wind velocity, etc. while the rate of transpiration is dependent on both environmental and plant factors, particularly the water retention capacity of the plant concerned.

**Materials and Equipments Required****:**

1. Conical flask, petridish, balance with weight box, glass rod, thread, etc.

2. A leafy twig

3. Water, oils, graph paper, pencil, etc.

**Procedure: **

1. Prepare a transpirational experimental set (conical flask-water-oil-leaf) in the usual way.

2. Take a petridish and fill it with water up to 2/3 of its volume. This is the evaporation experimental set.

3. Take the initial weight of the sets.

4. Both the sets are placed under sunshine for 1 hour.

5. Take the final weight of the sets and measure the area of transpiration surface and evaporation surface.

6. Calculate the rate of transpiration per min per sq cm of leaf area, and also the rate of evaporation per min per sq cm.

**Results****:**

**(a) Transpiration set: **

Initial weight — W_{1} gm.

Final weight — W_{2} gms

Amount of water transpired = (W_{1} – W_{2}) gm. = x gm.

Total transpiring surface (from graph paper) = y sq cm

Time — 1 hour

Rate of transpiration = x/y × 60 gm. per min per sq cm

**(b) Evaporation set: **

Initial weight — W_{3} gms

Final weight — W_{4} gms

Amount of water evaporated = (W_{3} – W_{4}) = p gm.

Total evaporating surface (from graph paper) = Q sq cm

(Apply the formula Hr^{2} to find out the total evaporating surface)

Time — 1 hour.

Rate of Evaporation = P/Q × 60 gms per min per sq cm

Ratio of Transpiration and Evaporation = X/60Y: P/60Q.

**Experiment # 8****. Determination of the Effect of Antitranspirant Chemical on Transpiration****:**

The term “antitranspirant” is used to designate any material applied to plants for the purpose of retarding transpiration. There are different groups of antitranspirant chemicals — some of them simply act as permeability barrier, some may act as metabolic inhibitors, while some may also act through permeability changes of the guard cells.

Phenyl mercuric acetate is one of the potent antitranspirant chemicals that causes the partial closure of stomatal pores and, thereby, regulates the transpiration process.

**Materials and Equipments Required****:**

1.** **Healthy leafy twig

2.** **Phenyl mercuric acetate solution (10^{2} M Stock solution)

3.** **Quick-fix.

4. Conical flasks, beakers etc.

5. Oil, water, graph paper etc.

6. Balance with weight box

**Procedure****:**

1. Prepare 4 sets of transpiration apparatus (conical flask-water-oil-leaf) using fresh petiolate leaves (leaves are cut under water).

2. Treat each set separately by spraying water (as control) or different concentrations of phenyl mercuric acetate solution (10^{-3 }M, 10^{-4} M, 10^{-5 }M) on both surfaces of leaf.

3. Place the experimental sets under sunshine for 2 hrs.

4. Record the initial and final weights before and after transpiration in each set to determine the amount of water transpired by the leaf of each set.

5. Calculate the rate of transpiration for each set separately.

**Results****:**

**Experiment # 9****. Determination of the Effect of Environmental Conditions on Transpiration Rates in Plants****:**

The rate of transpiration of a plant varies from day to day, from hour to hour and, frequently, still more rapidly. A number of environmental or internal plant factors determine the rate of water loss in the form of vapour by the plants. A few experiments can be performed to show the influence of environmental conditions on the rate of transpiration.

**(a) Effect of Atmospheric Humidity: **

**Materials and Equipment’s****:**

1. Conical flask-water-oil-leaf experimental set-up

2. Balance with weight box

3. Big bell jar

4. Blotting paper

5. Graph paper, pencil etc.

**Procedure**:

1 Prepare two conical flask-water-oil-leaf experimental sets, and take the initial weights of the sets separately.

2. Keep one set in open air and another within a bell jar whose inside is covered with water soaked blotting paper to ensure that the air inside the bell jar is humid. Apply the blotting paper to the sides of the bell jar so that the leaf can get direct sunlight from the top.

3. Record the final weight of the experimental sets after 2 hours, separately.

4. Determine the areas of transpiring leaves of both sets and calculate the.rates of transpiration separately.

**Observation****:**

Transpiration is reduced by higher humidity of atmosphere.

**Results****:**

Initial weight of Set I (kept in open air — control set): W_{1} gms

Final weight of expt. Set I: W_{3} gms

Rate of transpiration = (W_{1} – W_{3}) gms/leaf area (sq cm) and time (min)

Initial weight of expt. Set II (kept inside the bell jar): W_{2} gms

Final weight of expt. Set II: W_{4} gms

Rate of transpiration = (W_{2} – W_{4}) gms/leaf area (sq cm) and time (min)

**(b) Effect of Wind Velocity: **

**Materials and Equipment’s****:**

1. Conical flask-water-oil-leaf experimental sets

2. Balance with weight box

3. Table fan

4. Graph papers, pencil etc.

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**Procedure: **

1. Prepare two separate experimental sets for transpiration (conical flask-water-oil-leaf) and record the initial weights separately.

2. Place them separately — one in open air (control set) and another in front of the table fan (treatment set) for 1 hr and, finally, take the weights of both sets separately.

3. Calculate the rates of transpiration separately and determine the effect of wind velocity on rate of transpiration.

**Observation****:**

Transpiration is enhanced by wind flow.

**Results: **

Initial weight of control set — W_{1} gms

Final weight of control set — W_{2} gms

Total transpiring leaf area — X sq cm

Rate of transpiration = (W_{1} – W_{2}) gms/sq cm/hr

Initial weight of treatment set — W_{3} gms

Final weight of treatment set — W_{4} gms

Total transpiring leaf area —Y sq cm

Rate of transpiration = (W_{3} – W_{4}) gms/Y sq cm/hr.