The following points highlight the top sixteen experiments on respiration in plants. Some of the experiments are: 1. Demonstration of Aerobic Respiration in Plants 2. Demonstration of Anaerobic Respiration 3. Demonstration of Alcoholic Fermentation 4. Determination of Rate of Respiration 5. Comparison of Rate of Respiration in different Plant Parts and Others.
Contents
Experiment # 1
Demonstration of Aerobic Respiration in Plants:
Experiment:
Aerobic respiration in plants can be experimentally proved with the help of a simple apparatus like:
(i) Respiroscope which consists essentially of a stout vertical tube which is bent into a bulb at the one end (Figure 19a), or
(ii) With the help of a long-necked round-bottomed flask fitted with a centrally-bored cork at the mouth through which passes a glass tube (Figure 19b).
The respiroscope or the inverted flask is fixed vertically to a stand and a few germinating gram seeds or flower petals are placed in the bulb of the respective or in the inverted flask plugged with cotton at the base the vertical tube of the respiroscope or inverted flask is dipped just below the surface of water or mercury in a beaker.
A few caustic potash (KOH) pellets are introduced in the bent portion of the respiroscope or in the long neck of the round bottom flask and kept in position with loosely held cotton wool Care should be taken that respiratory materials and KOH pellets do not come in contact.
Precautions should be taken that the free end of the tube does not touch the bottom of the water or mercury trough. Fittings must be air-tight to avoid any leakage.
Observation:
The apparatus is allowed to stand for a few hours when it is seen that water or mercury has risen in the vertical tube of the apparatus proving the production of partial vacuum.
Inference:
Due to respiration of germinating seeds or flower petals CO2 has been released which is at once absorbed by KOH pellets. Thus the partial vacuum produced by the absorption of O2 by the respiring material could not be filled up by the released CO2. Hence, water or mercury is drawn upward into the tube.
Experiment # 2
Demonstration of Anaerobic Respiration:
Experiment:
A few germinating gram seeds are taken in a test tube which is completely filled with mercury and is then inverted just below the surface of mercury in a trough. It is then vertically held with a clamp and stand (Figure 20).
Observation:
Observation from time to time reveals that a gas is formed within the test tube by the displacement of mercury in the test tube. A few KOH pellets arc introduced through the open end of the test tube when mercury again rises filling the test tube.
Inference:
Here the respiration of germinating seeds takes place in complete absence of O2 supply and the gas produced is CO2 as evidenced by its absorption by KOH. This proves that anaerobic respiration has taken place.
Experiment # 3
Demonstration of Alcoholic Fermentation:
Experiment:
A fermentation tube or Kuhne’s vessel (Figure 21) is filled with 10% sucrose solution and mixed with a small quantity of Baker’s yeast or a few millilitres of suspension of yeast cells. The open end of the apparatus is plugged with cotton wool.
Observation:
Occurrence of fermentation or anaerobic respiration and collection of CO2 gas in the back arm of the Kuhne’s tube are observed. When the cotton wool is taken off smell of alcohol may be perceived.
Inference:
In alcoholic fermentation the sugar and yeast soln. broken down to alcohol and carbon dioxide liberating certain amount of energy. The enzyme, ‘zymase complex’ present in the yeast brings about this reaction through a number of steps. Alcoholic fermentation takes place in absence of atmospheric oxygen. Hence it is an anaerobic process.
Experiment # 4
Determination of Rate of Respiration:
Experiment:
The rate of respiration can be measured with the help of Ganong’s respirometer (Figure 22).
Description and Experiment:
The apparatus consists of three Levelling tube parts:
(i) The bulb for the respiring material which ends in a 10% KOH win smaller bulb at the bottom. The bigger bulb is provided with a stopper having a lateral hole, through which atmospheric connection can be made by turning the stopper,
(ii) A graduated manometer fitted with the bulb, and
(iii) A levelling tube connected with the manometer tube by rubber tubing. The whole apparatus is clamped on a stand.
Two ml of respiring material (measured by displacement of water) like germinating seeds or flower petals are placed into the bigger bulb of the respirometer. A 10% solution of KOH is taken in the manometer tube. At the beginning of the experiment the air around the material is brought to the atmospheric pressure by turning the stopper of the bulb until its hole coincides with that of the neck of the bulb.
The levelling of the reservoir tube on the right is so adjusted that the KOH solution in the tube is at the 100 ml mark at the bottom of the manometer. Two ml of respiring material is now surrounded by 100 ml of air. The experiment is started by turning the glass stopper at the top and thus cutting off connection with the atmospheric air.
Results:
As the respiration takes place in a closed space, the solution in the manometer tube rises up gradually. The reading should be taken up to 80 ml mark, i.e., up to 20 ml volume (since atmospheric oxygen is 20%) at an interval of 10 minutes, each time bringing the liquid in both the tubes at the same level, i.e., the liquid in the closed tube is brought under atmospheric pressure.
Results are expressed as millilitre of CO2 evolved per minute by the given respiring material.
Discussion:
The released CO2, on coming in contact with KOH solution, is absorbed by it, oxygen is consumed and as a result KOH solution rises up in the manometer tube. The rate of rise of KOH solution is taken as a measure of rate of aerobic respiration in terms of volume of O2 consumed per unit time per 2 ml of respiring material.
One-fifth volume of atmospheric air is O2. Hence out of 100 ml of enclosed air within respirometer there is 20 ml of O2. Hence reading should be taken up to 20 ml rise in volume of KOH solution. After that anaerobic respiration will start.
Experiment # 5
Comparison of Rate of Respiration in Different Plant Parts:
Experiment:
Flower buds, roots and leaves of a suitable herbaceous plant are collected 2 ml of each is measured by displacement of water and placed in the bulbs of three different Ganong’s respirometers. Rate of respiration is measured in each case following Expt. 4.
Results:
The volume of CO2 evolved at an interval of 10 minutes is recorded in each case and rates of respiration are graphically plotted for each sample of plant material and compared.
Discussion:
The rate of respiration is always higher in younger actively growing meristematic tissues than that of older and mature parts. There is a direct relationship between the amount of protoplasm and the rate of respiration the greater the protoplaim, the higher is the respiration rate.
The hydration of protoplasm and quantity of respiratory enzymes are always greater in young cells compared to mature and vacuolated cells. Hence the respiratory rate is always higher in young cells which are rich in protoplasm.
Vigorous respiration of root takes place if the space surrounding the roots and root hairs has ample supply of oxygen. In this experiment the maximum rate of respiration is expected in case of flower buds than in roots and leaves.
N.B. This experiment may also be performed with different types of seeds (starchy, proteinaceous and fatty).
Experiment # 6
Quantitative Estimation Of CO2 Evolved During Respiration:
(a) By Barcroft-Warburg’s constant volume micro-respirometer:
i. Principle and Description:
A convenient method of measuring the respiration of tissues in minute quantities has been developed by Warburg (1926).
The tissue whose respiration is to be measured is placed in a closed container with an attached manometer which records changes in gas pressure as a result of oxygen consumption or carbon dioxide production.
The apparatus is shown in Figure 23. Each respirometer consists of two main parts, a glass flask f and a manometer m, separable at a ground glass joint j. The tissue is placed in the flask f. When only oxygen consumption is to be measured, Ba (OH)2 or NaOH solution is added to the well w at the centre of the flask.
But when both oxygen consumed and carbon dioxide produced are to be measured, an HCL solution is placed in the side arm a with stoppered neck n in addition to the alkali in w.
The manometer fluid is contained in a rubber bulb b and can be added to or withdrawn from the manometer by adjusting the screws. This enables one to return the right side of the manometer to the starting point during making a reading and also to read the pressure on the left arm of the apparatus at constant volume.
A mirror behind the manometer reduces parallax in reading. The manometer is provided with a two-way tap t at the closed end. The reaction chamber is kept in a bath of constant temperature. The entire apparatus is shaken to facilitate gas exchange and temperature equilibrium.
ii. Standardization:
It is necessary to know the volume of the apparatus including the manometer to the manometer fluid in order to calculate gas volumes from changes in pressure. The manometer is detached and filled with clean mercury from the 15 cm mark to a marked point about 2 cm above the flask connection.
Now mercury is poured into a beaker and the dry reaction chamber f is filled with clean mercury until it just rises to the marked point on the manometer when the manometer and flask are connected.
This mercury is collected in a beaker and the temperature and weight of the metal are determined. The weight of the mercury in milligrams divided by its density at the observed temperature gives the volume of the apparatus in cubic millimeters.
Brodie solution (23gm NaCL, 5gm sodium cholate, 500 ml water, 5 drops conc. thymol in alcohol, as a preservative, and a few crystals of neutral red to colour) is used in the manometer to increase the sensitivity of readings, and to avoid sticking and other difficulties. This solution has a density of 1.0336 and gives a manometric pressure of one atmosphere (760 mm Hg) at 10,000 mm.
If in addition to these two values, the volume of the apparatus and the normal barometric height of the manometer fluid, we know the temperature, the volume of the material whose respiration is being measured, the volume of fluids (water, NaOH, etc.) added to the reaction chamber, and the solubility of the gas being measured in these contained liquids, the change in volume of the contained gases in cubic millimeters under standard conditions can be calculated with the equation
where X is the volume of gas absorbed (- X) or evolved (+ X) in cubic millimeters (cu. mm) under standard temperature and pressure; h is the manometer reading in millimeters (reading of left arm minus right); Vg is the free volume of gas in flask and manometer to manometer fluid (total volume of apparatus less volume of sample, liquids, etc., added to reaction chamber); T is the absolute temperature of the water around the reaction flask; if is the volume of all fluids in which the measured gas might dissolve (ordinarily not including volume of solid samples); a is the Bunsen coefficient of the solubility of the gas being measured in the contained fluids at the temperature T (see below); it is to be noted that Vf x a gives the volume of dissolved gas and that this is added to the free gas (Vg) to give the total volume; Po is the normal pressure in terms of the manometer fluid (for Brodie solution 10,000 mm).
In its simplest terms the equation states that the change in gas volume during the experiment is equal to the fractional change in pressure h/Po times the total volume of the gas Vg, with corrections for temperature and the solubility of the gas in the fluids present.
iii. Thermo-barometer:
The above equation assumes that the barometric pressure, the temperature, and the vapour pressure of the contained liquids remain constant during the experiment and therefore cancel out.
In practice, the great sensitivity of the manometer makes it necessary to set up an apparatus with the liquids, but without a sample, and to correct the experimental readings by the changes in the manometer of this blank apparatus, which are due to temperature or to barometric variations during the course of the experiment.
Experiment:
This method is most suitable for measuring respiration of minute quantity of respiring material. The reservoir b of the manometer is filled with Brodie solution. 1 ml of the respiring material is placed in the outer part of the flask f 0.2 to 0.4 ml of CO2 free 2N KOH solution is taken in the central cup w and 0.3 to 0.6 ml 2.5 N HCL in the side arm a.
The manometer connections are greased lightly but uniformly. The flask is secured in place with springs or rubber bands keeping the stopcock t open. The temperature of the water bath is kept constant at 30°C.
If only O2 consumption is to be measured, the HGL is omitted from the side arm and one or more samples are set up as desired in different flasks. If both O2 and CO2 are to be measured to obtain RQ, the HCL is included in the side arm and all experiments are set up in duplicate. In either case a flask is set up with a sample but with KOH and other fluid to serve as thermo-barometer.
The stopcock t is left open. The assembled manometers and flasks are set in the water bath and shaken for 15 minutes to attain temperature equilibrium. Now with screw is the right arm of the manometers is adjusted to 250 point for which they are calibrated (150 to 250 mm), the stopcocks are closed and the time is recorded as the beginning of the experiment.
When CO2 production is to be measured, the duplicate flask for each sample is quickly removed, a finger is held tightly over the open end of the manometer to prevent the manometer fluid being blown out or sucked into the flask, the flask is tipped to mix the KOH and HCL solutions thoroughly from the cup w and arm a.
These flasks are returned to the bath the manometer is carefully released, shaken for 5 to 8 minutes and manometer reading is recorded. This is corrected by changes in the thermo-barometer, as CO2 present or produced before experimental time.
The remaining flasks are shaken for 100 to 130 oscillations per minute for one hour or more. Intermediate readings for O2 consumption may be made as desired by stopping the shaker and adjusting the right arms of the manometers to the original point and recording the manometer readings including that of the thermo-barometer.
At the end of the experiment, the total O2 consumption is recorded and absorbed CO2 is liberated by the method used for the control samples taking care to protect the manometer fluid against changes in pressure and to bring the gases at water bath temperature with vigorous shaking before reading the CO2 pressure.
The change in pressure upon the mixing of KOH and HCL gives the ‘h’ reading for CO2. The right side of the manometer arm is always returned to its original setting before taking a reading since all of the calculations are based upon a constant volume of gas within the apparatus. The thermo-barometer pressure is always recorded along with each reading.
Calculations:
The proper terms are substituted in the equation
The gas absorbed or evolved is calculated in cu. mm, or in ml per gram of dry tissue per hour. The value Vg varies with the flask and with the volume of added samples or other fluids.
The volume of bacterial cultures and of KOH and HCL solution is obtained by pipetting; the volume of seeds, tissues, etc., by displacement. Vf is usually the volume of sample and other fluids for bacterial cultures but does not include the volume of seed tissue.
In experiments in which a constant volume of sample is run at constant temperature, the value of the quantity within the brackets remains constant and can be assigned a value K (flask constant) so that the equation becomes X = AK, in which K is calculated for each flask at each temperature.
The result may be expressed in mg by multiplying the volume of CO2 in ml by the density of CO2 at i particular temperature and pressure.
The effects of temperature or other factors upon the respiration and RQ of seeds and plant tissues may be measured by this apparatus.
(b) By Pettenkoffer’s gas stream method of CO2 estimation Principle and description of apparatus:
The principle of this method is that CO2 liberated by respiratory tissue is removed from its chamber along with CO2 free gas stream and absorbed in baryta (Barium hydroxide solution) taken in Pettenkoffer’s tube to form barium carbonate.
This is then titrated by a standard acid (HCL) to know its CO2 content. CO2 free air is drawn through a system as shown in Figure 24.
Description:
At the extreme left there is a soda lime tower containing soda lime for absorbing CO2 of the air entering through its opening at the mouth. The end of the tower is fitted with a tube through which CO2 free air passes into a U-tube containing KOH solution (30%).
The U-tube in turn is connected with another tower containing lime water or B(OH)2 solution which is connected to the respiration chamber by means of a tube. The respiration chamber is again connected to one end of the horizontally placed Pettenkoffer’s tube containing 50 ml of standard N/10 B(OH)2 solution (8-567gm/litre).
The other end of this tube is connected to an aspirator or suction pump. Thus on applying suction at the extreme right, air current enters the system of towers and tubes through the inlet at the top of the soda lime tower.
A mercury trough may be introduced in between Pettenkoffer’s tubes and the aspirator to regulate air flow. All connections should be made air-tight.
Experiment:
At the beginning of experiment, weighed amount of plant tissue is taken in the respiration chamber. All the towers and Pettenkoffer’s tubes are connected as described and made air-tight. The inlets of KOH tube and baryta tower must be dipped into the solution but the exit tubes should remain well above the surface of the solution.
Now the aspirator or suction pump is started. Air is sucked out through the end of the Pettenkoffer’s tube at the aspirator end causing the air to bubble into the solution of baryta contained in the Pettenkoffer’s tube through the respiration chamber and other towers successively.
Thus air first comes through the soda lime tower and then through KOH tube and baryta tower, thus becoming completely free of CO2. This CO2 free air containing O2 comes in contact with the plant tissue in the respiration chamber and aerobic respiration takes place as a result of which CO2 is evolved.
This CO2 produced by plant tissue during aerobic respiration then passes through the standard baryta solution of Pettenkoffer’s tube and is completely absorbed by it forming BaCO3.
After allowing respiration to occur for a particular time Pettenkoffer’s tube is taken out and the excess baryta is titrated against Standard N/10 HCl using phenolphthalein as an indicator to estimate the quantity of CO2 from the following relations.
Results:
The baryta and the BaCO3 solution of the Pettenkoffer’s tube is quantitatively transferred in a flask, a drop or two of phenolphthalein solution is added and titrated against N/10 HCL until the pink colour is just discharged.
This gives the volume of residual Ba (HO)2 (not utilised by the CO2 formed). This titration value is subtracted from the titration value of fresh 50 ml sample of Ba (OH)2 solution and weight of CO2 liberated in respiration is calculated from the following equation:
mg CO2 = V × N × 22
Where Vis the difference between blank and experimental titration values in millilitres, N is the normality of acid used in titration and 22 is the equivalent weight of CO2 in BaCO3. Since molecular weight of CO2 is 44 and it is absorbed by Ba (OH)2 as H2CO3 the equivalent weight is to be calculated by dividing the molecular weight by 2.
Discussion:
This method sometimes called the “gas-stream method”, has the advantage of accuracy and convenience and of permitting a study of the cell material for an indefinite period under constant conditions.
Here the CO2 yield is obtained in milligrams; the result may be divided by thousand to give grams or divided by 1-977 (density of CO2) to change to ml of CO2 at 0°C and 760 mm. Hg. It is to be noted that CO2 is calculated directly rather than as the carbonic acid which is actually measured.
Experiment # 7
Determination of Respiratory Quotients (RQ) by Ganong’s Respirometer:
Experiment:
From this experiment, volume of CO2 evolved and O2 consumed during aerobic respiration can be directly obtained and RQ,, i.e., (volume of CO2/volume of O2) can be calculated.
The experiment can be conveniently performed by Ganong’s respirometer described in Expt. 4. The manometer tube and reservoir are filled with brine solution (saturated NaCl solution) and by adjusting the reservoir, the surface of the brine solution is brought to 100 ml mark of manometer.
The weighed equal quantities (2 ml) of tissues whose RQ, is to be determined are taken in the bulbs of two similar types of Ganong’s respirometer. In the bulb of one respirometer 1 ml of 40% KOH solution contained in a small vial is kept along with the tissue.
Total volume of tissue plus KOH solution plus vial should be noted (let it be k ml). The other respirometer contains only tissue in the bulb and its volume is noted (let it be t ml). Now the air within the bulb is brought to atmospheric pressure by turning the stopper.
The level of brine solution is brought to 100 ml mark at the bottom of manometer. This is done in both the respirometers and the connection with the outside air is cut off by turning the stoppers. Now the bulb and the manometer tube up to the level of brine solution contain a definite volume of air having 20% 02 and 0.03% of CO2 on an average.
The setup is placed in dark or covered with black paper for a certain period of time (say two hours) and any change in the reading of brine level is noted. The volume of air in case of the respirometer containing plant tissue only is 100—t and this multiplied by 20/100 gives the volume of O2 in this air.
Similarly the volume of air in case of the respirometer containing KOH vial and the plant tissue is 100 —k; and this multiplied by 20/100 gives the O2 in this closed atmosphere.
During the time for which the setup is kept, respiration occurs in the tissues of both the respirometers by absorbing O2 and giving out CO2. In the respirometer containing KOH via), CO2 given out is absorbed by KOH solution but this does not happen in the respirometer without KOH3. Therefore, the brine level rises in the respirometer with KOH and either rises or falls or remains stationary in the respirometer without KOH.
In the respirometer containing KOH, CO2 is absorbed reducing the pressure in the manometer tube and brine level rises because O2 has been used up by the tissue. The more O2 is used up, the more raises the brine level.
Now by adjusting the reservoir the brine level is brought to the same level in both the arms, thus bringing the volume of air in the manometer tube in atmospheric pressure. At this stage the brine level in manometer tube is noted and the difference between the final arid initial readings gives the volume of O2 used by the tissue (let it be x ml).
Now in a respirometer without KOH, O2 is also used up giving out CO2. But in this case CO2 is not absorbed since KOH is not present. If CO2given out is less than O2 used (when fat is a substrate) the brine level will rise in the manometer tube.
If CO2 given out is more than O2 used (when acid is a substrate) the brine level will fall down in the manometer tube and when CO2 given out is equal to O2 used up (when carbohydrate is a substrate), the level of brine remains stationary. Before taking readings the brine level is adjusted at the same level in both the arms.
The difference between the final and initial readings gives the net gas exchange which has taken place (let it be y ml). If the rise of brine level upward is considered as negative change and fall of brine level downward is considered as positive change, the volume of CO2 evolved will be (x – y) ml when brine level rises up and (x + y) ml when brine level falls down. It may also remain stationary when RQ, is unity.
Unity when the brine level in the respirometer without KOH remains stationary indicating that the respiring substrate is carbohydrate.
In this way the RQ for any given tissue may be calculated. It is to be particularly noted that the volume of the closed air within the bulb and the manometer should be equal in both the cases. The respirometers should be of the same size and volume and equal volumes of tissue should be taken in each case.
Since KOH vial is kept in one bulb only, a similar vial containing equal quantity of brine solution may be kept in the other bulb so that same volume of air is present in both the bulbs. Temperature greatly affects the volume of gases and hence both respirometers should be placed at the same temperature.
Experiment # 8
Demonstration of Liberation of Heat Energy During Respiration:
Experiment:
Three thermos-flasks are taken. One contains water-soaked seeds, second dry seeds and the third boiled seeds (dead) and all the lots are of equal weight. The mouths of the thermos-flasks are corked through which passes a thermometer in each flask so that the bulb of the thermometer remains within the seeds. The flasks are left for 24 hours.
Observation:
Rise of temperature is observed in all the cases.
Inference:
The rise in temperature of the flasks containing soaked seeds indicates that heat is produced during respiration of seeds. Temperature remains nearly unchanged in dry seeds and completely unchanged in case of boiled seeds. This is because the respiration of dry seeds is almost negligible and in case of boiled seeds nil.
N.B. The mathematical evaluation of heat loss during respiration of one gram mole of glucose is as follows. Every mole of ATP formed from ADP and phosphate requires about 12 K. Cal. of energy. But complete combustion or chemical oxidation of one mole of glucose yields 684 K. Cal. of energy as heat. Therefore, 684—456 = 228 K. Cal. is lost as heat energy. Thus in aerobic respiration of each mole of glucose about
= 67 % energy of each glucose mole is carried into 38 mole of ATP. This is called as the “efficiency of energy conservation” in aerobic respiration.
Experiment # 9
Demonstration of Loss of Weight in Respiration:
Experiment:
About 50 seeds are surface sterilised with, 1% HgCL2, washed thoroughly and 5 such seeds are separately placed in ten petridishes containing soaked filter paper. The pertidishes are kept in dark and seeds are allowed to germinate. The fresh and dry weights of each lot of seedlings are taken every alternate day.
Results:
The percentage loss in dry weight is calculated in each case and the results are graphically plotted taking loss in weight as ordinate and days as abscissa.
Discussion:
Respiration is a catabolic process which takes place by breaking down of stored carbohydrate and liberating CO2, H2O and energy. Since the seedlings are grown in dark the anabolic process, i.e., photosynthesis, cannot take place and as a result of which loss in weight occurs as the plants grow.
Experiment # 10
Effect of Wounding on Respiration:
Experiment:
Experiment can be conveniently performed either with Warburg’s method or Pettenkoffer’s method. Two lots of potato tubers are taken; one lot is slightly larger than the other.
The larger potatoes are peeled off and surface area is made comparable with the other lot. Approximately the equal weights of these two lots are washed and dried and the two samples are placed in two respiration chambers of the Pettenkoffer’s apparatus or in the flasks of Warburg’s apparatus. The rate of respiration is determined in both the cases at a constant temperature.
Results:
Respiration rates in millilitres of CO2 per gram dry tissue per hour are plotted. The dry weights of the samples are determined at the end of the experiment.
Discussion:
Injury to a given plant tissue often causes the respiratory activity to increase. Generally, in case of injured plant tissues the rate of respiration increases for the time being and this increase gradually rises to a maximum point and then the rate decreases.
Wounding generally initiates meristematic activity in the area of the wound, resulting in the development of “wound callus”. It has been shown that a considerable increase in sugar content (about 70%) around the injured cells takes place.
Perhaps the increase in respiration due to wounding is caused by increased availability of respiratory substrate and meristematic cells.
Experiment # 11
Effect of Pre-treatment with Carbon Dioxide on the Rate of Respiration:
Experiment:
Equal weights of three lots of potato tubers are taken. Two lots are taken in two petridishes placed on, two ground glass plates and each is covered with a 1litre bell jar having an outlet at the top (Figure 25).
The lower rims of the bell jars arc made air-tight with grease. The bell jars are partially evacuated with the help of a suction pump. Now CO2 is passed from a Woulfe’s bottle (CaCO2+HCL) into one bell jar for 10 minutes and to the other for 20 minutes. The outlets are then closed and tubers are kept in these atmospheres for about three hours.
The control lot is also placed under a bell jar for the same period of time under normal atmosphere. A centrally placed thermometer records the temperature. After the stipulated time the tubers are taken out and rates of respiration are determined with the help of Ganong’s respirometer.
Results:
The rate of respiration in each case is graphically plotted and compared.
Discussion:
Increasing concentration of CO2 has a definite repressing effect on respiration. Since the direct measurement of respiration rate in terms of CO2 evolution and simultaneous increase in CO2 concentration is difficult with ordinary apparatus; here the effect of pre-treatment of CO2 is studied. CO2 input raises the internal concentration of CO2 considerably and limits respiration by its toxic effect.
Experiment # 12
Effect of Moisture Content on Respiration of Grains:
Experiment:
About 300 grams of dehusked rice or wheat seeds are taken in a beaker and dried in an oven at 40°C for 24 hours. These seeds are divided into 5 lots. The initial moisture content of one lot is determined. Water is added to the other 4 lots and each lot is soaked for 5, 10, 15 and 30 minutes separately.
The moisture content of each lot is then determined by taking a portion of seed from each lot. Equal weights of seeds from each lot including un-soaked one are taken in Ganong’s respirometer and rate of respiration is measured.
Results:
The rate of respiration is expressed in terms of millilitre of CO2 evolved per gram of dry seeds per hour and graphically plotted in each case.
Discussion:
Within certain limits, moisture content of tissue affects its respiratory rate. In dormant seeds water content is generally less than 10% and their respiratory rate is very slow.
Seed, when come in contact with moisture imbibe water and swell and their respiratory rate gradually increases. The moisture content of the tissue increases the amount of soluble respiratory substrate and also the activity of the protoplasm by the enzymes.
Experiment # 13
Effect of Food Supply on Respiration:
Experiment:
One lot of leaf sample is collected from rice plants which were previously kept in dark for 24 hours and another lot is collected from rice plants previously kept in light for 24 hours. The rate of respiration of equal weights of each sample is measured in a Pettenkoffer’s tube or in Ganong’s respirometer. The dry weight of each sample is determined.
Results:
The results are expressed as millilitre of CO2 evolved per gram of dry tissue per hour.
Discussion:
The rate of respiration of a given tissue is governed by the concentration of the soluble respirable substrates. Since organic materials are oxidised during respiration, the amount and kind of materials present in the cells appreciably affect both the rate and course of respiration.
The rate of respiration increases due to increased carbohydrate production as a result of photosynthesis and decreases due to lower carbohydrate content in the dark. Thus the concentration of respirable substrate may limit the rates of CO2 production or O2 uptake.
N.B. Increased respiration is also observed when various sugars (Especially sucrose, glucose, fructose or maltose) are supplied to floating leaves or other tissues in solutions.
Experiment # 14
Determination of Rates of Respiration and Nature of Substrates by McDougal Respiroscopes:
Experiment:
This is a simple apparatus fixed in a wooden frame (Figure 26) which consists of a pair of vertical tubes. The upper end of the tube is wider than the lower end which is graduated. The lower end of the graduated tube dips into a beaker containing brine solution.
The upper bulb-like wider ends of the respiroscopes are closed by means of corks through which pass two small tubes having bent ends. The upper ends, i.e., the outside ends of the bent tubes are fitted with stopcocks.
From the lower bent ends of the two tubes, two small vials one containing KOH pellets hang within the bulbs. Equal amounts of germinated starchy or fatty seeds (seed coat removed) are placed within the wider bulbs of the respiroscope on a soaked cotton plug. A thermometer may be centrally placed to record temperature.
The stopcock is then turned to make connection with atmospheric air. The brine solution rises through the graduated end of the respiroscope and becomes stationary at the brine level of the beakers. The stopcock is closed and the level of brine solution in both the tubes is recorded. The rise of brine columns in the tubes is recorded at an interval of 15 minutes for 2 hours.
Results:
The rate of rise of water column indicates the rate of respiration.
Discussion:
The partial vacuum created due to absorption of O2during respiration cannot be filled up by CO2 released and this vacuum is then filled up by the brine solution. The volume of CO2evolved during respiration by starchy seeds is equal to the volume of O2consumed (RQ = 1). But in case of fatty seeds the volume of CO2 liberated is less than the volume of O2 used up (RQ, < 1).
Hence the rate of respiration as measured in terms of O2consumption is less in case of fatty seeds than starchy seeds. The depression (when RQ,> 1) or elevation (when RQ, < 1) column of brine solution in the control McDougal respiroscope (without KOH) indicates the nature of Substrate. If the brine level is stationary (RQ, = 1) then the substrate is carbohydrate.
Experiment # 15
Respiration of Roots of Intact Plants:
Experiment:
The adventitious roots of rice or wheat plants are carefully washed with water and placed in a bottle containing water which is made slightly alkaline with dilute NaOH solution. This is coloured pink with addition of a few drops of phenolphthalein.
A second bottle b prepared in the same way, stoppered tightly and left without a plant. Both the bottles are allowed to stand in diffused light and the solutions are examined from time to time and change in colour is carefully noted in each case.
Observation:
The pink colour of the solution in the bottle containing the plant gradually fades and ultimately becomes colourless after a considerable time. The solution of the other bottle remains as such.
Inference:
During respiration of roots CO2 is released which is converted to H2CO3when comes in contact with water. This acid neutralises the dilute NaOH solution and pink colour of the solution fades.
When a portion of this neutralised solution is gently boiled for few minutes the pink colour reappears because CO2comes off on boiling leaving the solution alkaline again.
Experiment # 16
Demonstration of Continuity of Intercellular Spaces:
Experiment:
A conical flask is taken and half-filled with water. The mouth of the flask is fitted with a cork having two holes, through one of which is inserted a long petioled leaf of arum (Colocasia) so that the cut end of the petiole remains well under water.
Through another hole is inserted a bent glass tube which is connected to a suction pump. The end of this bent tube remains well above the water surface. All connections are made air-tight. The air within the flask is drawn out by the suction pump.
Observation:
Bubbles are seen to come out from the cut end of the petiole into the water of the flask.
Inference:
The experiment shows that as the air is sucked, the atmospheric air enters through the stomata of the leaf and through the intercellular spaces ultimately comes out through the cut end of the petioles. This shows that stomata and intercellular spaces form continuity and are involved in gaseous exchange.
N.B. A similar experiment can also be set up by introducing a woody leafless twig instead of arum. All the cut ends of the woody twig are sealed with paraffin wax excepting the lower end which dips in water. The atmospheric air in this, case passes through the lenticels to the intercellular spaces and ultimately comes out in water through the lower cut end of the twig and submerged lenticels.