The following points highlight the six experiments on respiration in plants. The experiments are: I. To Prove that the Intercellular Spaces in Plants form a Continuous System II. Demonstration of Aerobic Respiration in Plant Tissues III. Quantitative Estimation of CO2 Released in Aerobic Respiration IV. Demonstration of Anaerobic Respiration in Plants and Others.
Experiment # I. To Prove that the Intercellular Spaces in Plants form a Continuous System:
Take two wide-mouthed bottles and fill about three-fourths of them with water. Fit in rubber corks, each with two holes and fit them air-tight on the mouths of the bottles. In Fig. 726, insert the petiole of a Nasturtium or any other suitable leaf with a long petiole through one of the holes and allow it to dip into water of the bottle. In Fig. 727, insert a freshly cut twig, the cut ends of which are closed. In both the bottles, horizontally bent glass tubes as shown in the figures are inserted whose short vertical ends lie just below the corks above the surface of water.
Experiment # II. Demonstration of Aerobic Respiration in Plant Tissues:
Aerobic respiration in plants can be experimentally proved very simply by the use of a respiroscope, (Fig. 728) which consists essentially of a stout vertical tube which is bent into a bulb at the one end. The experiment can be done as well with a long- necked round-bottomed flask or simply a very wide test tube (Fig. 729).
The respiroscope (or the inverted tube) is fixed vertically to a stand and a few germinating seeds or flower buds (from which all green parts have been removed) are placed in the bulb of the respiroscope, the vertical tube of which is dipped just below the surface of water in a beaker (instead of water, mercury may also be used as in Fig. 729).
The air enclosed in the apparatus is thus cut off from external air. Introduce a piece or two of caustic potash pellets in the bent portion of the respiroscope, preventing its fall with loosely held cotton wool as shown in the figures.
Allow the apparatus to stand for a few hours (2-3 hrs.); it will be seen that water has risen in the vertical tube of the apparatus proving the production of partial vacuum (due to absorption of evolved CO2 in respiration by KOH) which draws water (or mercury, Fig. 729) upwards into the tube. Instead of water, it is better to use brine for CO2 is highly soluble in pure water.
In Fig. 730, there is a pair of respiroscopes fixed on a vertical stand. In the tube A, the swollen portion contains germinating seeds but no caustic potash whereas in B, a small tube of pellets of KOH is hung from the top inside the apparatus. Both the tubes are dipped below the surface of water in two vessels. It will be seen after sometimes, that water rises in the respiroscope tube B due to absorption of CO2 evolved by the seeds by KOH whereas the level of water both outside and inside the tube in A remains stationary.
In the tube A, the volume of oxygen utilised is approximately equal to the volume of co2 liberated; hence there is no vacuum inside the tube. If seeds or other materials, storing fats and oils as reserve food in the endosperm, e.g., castor seeds are used in the tube A instead of carbohydrate-storing seeds, rise of level of water could be seen in the tube A without any KOH, indicating that when fats are utilised as respiratory substrate, more oxygen is absorbed than CO2 evolved in respiration. This will certainly give an R. Q. less than unity.
Experiment # III. Quantitative Estimation of CO2 Released in Aerobic Respiration:
Set up the apparatus as diagrammed in Fig. 728, and test to ensure freedom from all leaks. The apparatus is designed to pass a current of CO2-free air through the respiring chamber, D. As the suction is applied by a filter pump in the right-hand side, atmospheric air is sucked in by the left-hand opening of the tube. The ‘U’ tube A, containing soda lime removes the major portion of CO2 from the incoming air before it passes through the apparatus.
The bottle B, containing N Ba(OH)2 solution absorbs any CO2 left over from soda lime tube, A. The bottle C, containing a N/10 solution of baryta serves as a check and as long as the solution in it remains clear, it shows that the stream of incoming air passing through D is CO2-free. The respiration chamber, D, contains an weighed amount of fresh plant material (e.g., potatoes, etc.). The bubbling of air is continued for 1-2 hrs. (time should be noted accurately), the respiring material absorbs oxygen from air and releases CO2 which is absorbed in a known volume
Ba(OH)2 (or of any other known concentration, e.g., N/50, N/100) coloured red with phenolphthalein in the bottle E. The indicator serves as a test for excess alkali. If the solution in E becomes colourless during a run, stopping the gas stream, a further 10—20 ml of N/10 baryta may be added in the bottle E. The bottle F (also containing N/10 baryta) serves as a final safety check and as long as the solution in this bottle remains clear, it shows that all the CO2 evolved in respiration by the material in the chamber D, is being completely absorbed by the solution in test bottle E.
The CO2 released by the respiring material partially precipitates baryta solution as BaCO3 in the test bottle E. The residual Ba(OH)2 is titrated against N/10 HC1 and mgs of CO2 evolved are easily calculated for a particular weight of the material in a given time, preferably 1 hour. This titration value should be subtracted from the titration of a fresh 50 ml sample of N/10 baryta with acid. The weight of CO2 released in respiration could be calculated from the following equation;
mgs. CO2= V x N x 22.0 where V is the difference between the blank and experimental titration; N, the normality of the acid used for titration and 22.0 the equivalent weight of CO2.
Experiment # IV. Demonstration of Anaerobic Respiration in Plants:
(a) Take a few soaked gram seeds from which the seed coats have been removed carefully without damaging the embryos (the seed coats are removed to facilitate diffusion of gases). Fill a test tube completely with mercury and invert it just below the surface of mercury contained in a petridish. Pass the seeds under the open end of the tube in the mercury by means of forceps when they will naturally float up to the top of the test tube (Fig. 732A).
Observe that in a day or two, the mercury level falls in the test tube in B and the test tube is half or more full of a gas. Introduce a little caustic potash pellet through the open end of the tube. Caustic potash absorbs the gas and the mercury level in the test tube again rises. This proves that the gas is CO2. That the gas produced by respiration of germinating seeds in complete absence of oxygen supply is CO2 can also be proved by inserting a lighted match stick in the test tube which is at once extinguished.
(b) Fill three fermentation tubes (Kuhne’s vessel, Fig. 730) with mixtures of 10% solutions of glucose, sucrose and maltose respectively mixed with small quantities of bakers’ yeast (or a suspension of living yeast cells). Plug the open end of the apparatus with cotton wool and observe the Occurrence of fermentation (anaerobic respiration) and the collection of CO2 gas in the back arm of the tube. One with a sensitive nose may even detect faint smell of alcohol produced when the cotton wool is taken off.
Experiment # V. Ganong’s Respirometer:
1. The apparatus (Fig. 734) consists of three parts: (i) the bulb for the respiring material, with a second smaller bulb at the button of the first and provided with a stopper with a lateral hole which can be opened or closed by turning the stopper, (ii) a graduated manometer tube and (iii) a levelling or reservoir tube connected with the manometer tube by a stout rubber tubing.
2 ml of respiring material are put into the bigger bulb of the respirometer and a 10% solution of KOH is placed in the manometer tube. At the outset the air around the material is brought at the atmospheric pressure by turning the stopper of the bulb until the hole coincides with the neck of the tube. The levelling tube on the right is so adjusted that the potash solution in the tube is at the 100 ml mark at the bottom. The 2 ml of respiring material is thus surrounded by 100 ml of air.
Now the experiment is started by turning the glass stopper at the top and thus cutting off all connections with the external air. Respiration now takes place in a closed space and the absorption of CO2 liberated by KOH is shown by the rise of solution in the graduated tube. When the solution rises up to 80 mark, the solution is so adjusted that it is in the same level in both the graduated and the reservoir tubes. It remains stationary at this level, i.e., one-fifth of the enclosed air (% O2 in air is about 21%) is utilised during aerobic respiration. This fraction represents O2 absorbed in respiration.
Hence the only inference is that; in aerobic respiration CO2 output is accompanied by an equal volume of oxygen absorption. The rate of ascent of KOH solution in the tube can be taken as a measure of the rate of O2 absorption by the respiring material. Respirometers may also be conveniently used to determine the R.Q, of a material over a period of time. In order to do this a saturated solution of NaCl is at first placed in the manometer tube. (Pure water cannot be used as CO2 evolved is absorbed by water which is, however, only slightly soluble in a strong solution of brine.)
If carbohydrate-storing seeds are used as respiring material, the level of sodium chloride solution in the graduated tube will remain more or less unaltered showing that the volumes of CO2 evolved and oxygen absorbed are the same and R.Q. = 1. If solid KOH pellets are now added to the salt solution in the tube, the accumulated CO2 is absorbed and can be measured from the reading in the tube. This certainly also represents the volume of O2 utilised.
In another experiment, instead of potash or brine, the reservoir-leveling tube can be filed with mercury (as shown in Fig. 734, depicted here). Observe that after a time the mercury in the two tubes maintains a constant level showing, R.Q. = 1. Another experiment may be performed in which alkaline pyrogallol solution (which absorbs oxygen readily) is taken in the manometer tube exactly up to 100 marks.
As this promptly absorbs all the oxygen in the tube, the volume of oxygen in air in the tube can be found out by the rise of pyrogallate solution which at its maximum may attain 80 mark in the manometer tube, showing that approximately one-fifth of the atmospheric air is oxygen and this is available for respiration of the experimental plant material.
If instead of carbohydrate-storing seeds, fatty seeds, e.g., castor, mustard, linseed, etc., are used as respiring material, and saturated sodium chloride solution is taken in the tube, the salt solution rises in the graduated tube without any addition of KOH, indicating that R.Q. is less than unity, i.e., greater volume of oxygen is absorbed compared to CO2 liberated. Suppose that this excess oxygen is V1 ml. KOH pellets are now added to the salt solution in the graduated tube; there is a further rise of the solution in the graduated tube, say about V2 ml. V2ml is really the volume of CO2 evolved and V1+V2 then represent the volume of O2 absorbed by the fatty seeds in respiration. R.Q. is evidently, V2/(V1+V2).
Experiment # VI. Demonstration of Liberation of Heat Energy during Respiration:
Surface sterilized germinating seeds placed in thermos flasks show rise in temperature for a few days in the flask A (Fig. 735). In the flask B, in which seeds have been previously killed by boiling water the temperature did not rise for about 2 days. The rise of temperature was, however, noticed in flask B after 2-3 days as micro-organisms began to grow and multiply in the dead-seed medium in the flask, leading to a rise in temperature due to bacterial respiration.
In the flask C, in which the seeds were killed by immersing them in 1 % mercuric chloride, the temperature did not show any increase, remaining constant throughout the experiment. The HgCl2 kept the killed seeds sterile. The results of the three experiments have been graphically represented in the lower part of the figure 735.
