Experiments on Respiration in Plants | Botany

List of top five experiments on respiration in plants:- 1. Demonstration Experiments on Aerobic Respiration, Anaerobic Respiration and Alcoholic Fermentation 2. Quantitative Estimation of Co₂ Released During Aerobic Respiration 3. Determine the Effect of Inhibitors and Un-Couplers on Respiration and a few others.

Experiment # 1. Demonstration Experiments on Aerobic Respiration, Anaerobic Respiration and Alcoholic Fermentation:

Respiration is a catabolic process in which energy is released involving oxidation of organic compounds with molecular oxygen which serves as an ultimate electron acceptor.

The overall reaction mechanism involves the oxidation of carbohydrates and subsequent production of CO₂, H₂O and energy:

C₆H₁₂O₆ + 6H₂O + 6O₂ → 6CO₂ + 12H₂O

Measurement of respiratory gas exchange provides the most convenient means of determining respiration rates.

There are two principal categories of respiration: aerobic respiration in which complete oxidation of respiratory substrate takes place in presence of O₂ through EMP-linked TCA cycle resulting in end- products of CO₂ and water; anaerobic respiration in which respiration occurs in absence of O₂ when the respiratory substrate is partially oxidized to form some carbonic compounds like ethyl alcohol, acetic acid, lactic acid etc., along with CO₂ and H₂O. However, anaerobic breakdown of sugar in microorganisms is termed fermentation.

(A) Aerobic Respiration [Fig. 3.25 (a), (b)]:

Materials and Equipments Required:

1. Round-bottomed flask fitted with a graduated glass tube passing through a cork, or Respiroscope or bent flask

2. Germinating seeds or flower petals

(In all experiments on respiration, non-green plant tissue should be taken, otherwise simulta­neous occurrence of photosynthesis will give erroneous results.)

3. Cotton

4. KOH pellets, mercury or oil

5. Stand with clamp, beaker, graph paper, pen­cil etc.

Procedure:

1. Put a measured quantity of germinating seeds or petals in the respiroscope or round-bottomed flask or bent flask (Fig. 3.25 a, b, c).

2. Place a cotton plug followed by some KOH pellets resting on another cotton plug in the stem of the flask.

3. Make all connections air-tight with vase-line and invert it on a trough of mer­cury oil, immersing the free end of the stem of the flask in mercury leaving a slight gap in between the free end and the bot­tom of the trough. Fix it with stand and clamp.

4. After 1-2 hrs. record the size of mercury column in the stem of the flask or glass tube to denote the volume of O₂ consumed by the respiring living materials.

Observation:

The rise of mercury or oil in the vertical tube of the apparatus proves the production of partial vacuum inside due to consumption of O₂ in respiration.

Result:

The amount of O₂ consumed by respiring material/hr can be calculated by the formula:

Rate of Respiration = Final mercury column reading (ml) – Initial mercury column reading (ml)/Amount of respiring materials (mgs)

Inference:

Due to respiration of seeds, CO₂ has been released along with consumption of O₂. The CO₂ is then absorbed by KOH pellets and a partial vacuum is produced due to O₂ utilization. As such, mercury or oil is drawn upward into the tube. The rise corresponds to the volume of O₂ consumed.

(B) Anaerobic Respiration:

Materials and Equipments Required:

1. Graduated test tube, Petridish

2. Germinating seeds

3. Mercury

4. Stand and clamps, Forceps, etc.

Procedure:

1. Fill the graduated test tube with mercury. Insert a measured quantity of germinated seeds inside the mercury column of the tube placed by forceps over a petridish containing mercury (Fig. 3.26).

2. Hold up the experimental set-up vertically with the help of a clamp stand for 1 or 2 hrs.

Observations:

After a period of 1 or 2 hrs., mercury level falls within the tube with the production of CO₂ by anaerobic process. This CO₂ accumulates at the top of mercury column which can be verified by KOH pellet introduction into the tube.

Result:

The amount of CO₂ formed per gram of seeds per hour is the rate of anaerobic respiration (ml per gram per hour).

Inference:

Here the respiration of germinating seeds takes place in the complete absence of O₂ and the gas thus produced is CO₂ as evidenced by KOH sensitivity test.

(C) Fermentation:

Materials and Equipments Required:

1. Kuhne’s vessel

2. Sucrose solution (10%), Yeast suspension

3. Cotton plug

Procedure:

1. Fill the Kuhne’s vessel with 10% sucrose solution and then mix with it a small quantity of yeast suspension (Fig. 3.27).

2. Plug the mouth of the apparatus by cotton for 12-18 hrs.

Observations:

After the desirable period is over, CO, will be collected within the graduated arm of the vessel by replacing sucrose yeast soln. and there will be smell of alcohol on opening the cotton plug.

Inference:

This process is known as fermentation when sugar is bro­ken down into alcohol and CO, by yeast.

Experiment # 2. Quantitative Estimation of Co₂ Released During Aerobic Respiration:

The rate of aerobic respiration can be measured by determining either O₂ consumed or CO₂ released during the process. The latter can easily be done by absorbing the evolved CO₂ in known volume of Barium hydroxide [Ba (OH) ₂] soln. As a result, Barium carbonate [Ba (CO) ₃] is formed which precipitates out and the volume of residual Ba (OH) ₂ present can be estimated by titration against HCl.

From these data, one can easily determine the amount of turnover of Ba (OH) ₂ to BaCO₃ during aerobic respiration:

Ba (OH) ₂ + CO₂ → BaCO₃ + H₂O

Materials and Equipments Required:

1. Glass Apparatus — 7 conical flasks connected in a series by glass tubes

2. Potato tubers

3. Chemicals: 20% KOH solution, N/10 Ba (OH) ₂ soln; N/10 HC1; Methyl green indicator

4. Burette, Pipette, Balance weight box, Cork Borer, etc.

5. Aspirator or suction pump

Description of Equipments:

The specially designed glass apparatus contains 7 conical flasks in a series connected by glass tubes for continuous air circulation when fitted with aspirator or suction pump (Fig. 3.28).

Procedure:

1. Fill the flasks with different solns. in the following manner:

(a) 1st and 2nd flasks are filled with 50 ml of 20% KOH soln. to absorb the CO₂ from the air entering the apparatus.

(b) 3rd flask is filled with 50 ml of distilled water.

(c) 4th flask is filled with a measured quantity of plant tissue suspended in 50 ml distilled water.

(d) 5th and 6th flasks are filled with 50 ml of freshly prepared N/10 Ba (OH) ₂ soln.

(e) 7th flask remains empty and is connected with aspirator or suction pump.

2. All the flasks are made air-tight to ensure the circulation of CO₂-free air through the circulating channels during respiration, except that produced by living tissues.

3. Maintain the circulation of air through this set at regular rate of 10 bubbles per minute for 30 or 60 minutes. After that Ba (OH) ₂ of 5th and 6th flasks are taken out and mixed together and finally 10 ml of this liquid is titrated against N/10 HCl, using methyl green as indicator. A blank titration of 10 ml of N/10 Ba (OH) ₂ is also made against N/10 HC1.

Results:

Calculation:

1,000 ml of (N/10) HCl = 1,000 ml of (N/10) Ba (OH) ₂

= 1,000 ml of (N/10) CO₂ soln.

= 22 gms of CO₂

1ml of (N/10) HCl = 22/1000 × 10 ⁼ 0.022 gm. of CO₂

The amount of CO₂ evolved = Difference in volume of N/10 HC1 (From titration) × 22 mgm of CO₂

= x × 22 mgm of CO₂

= x × 22 mg y gm. fresh tissue per hr.

Precaution:

1. Apparatus must be air-tight.

2. Colour changes during titration persists for 10 sec. This should be considered for end-point.

3. Ba (OH) ₂ soln., when used, should be freshly prepared and the experiment should be com­pleted quickly so that it cannot absorb atmospheric CO₂.

Experiment # 3. Determine the Effect of Inhibitors and Un-Couplers on Respiration:

During the terminal phase of respiration the electron transport system is coupled with a number of elec­tron carriers localized in the mitochondrial membrane. This process is associated with oxidative phosphorylation. There are several chemicals which can inhibit the electron transport or uncouple the phosphory­lation process i.e. inhibit ATP synthesis.

Materials and Equipments Required:

1. Plant materials — germinating seeds

2. Inhibitor chemicals — Sodium salt of azide (10^-2, 10^-3, 10^-4 M soln.)

3. Un-coupler — 2, 4 dinitrophenol (10^-2, 10^-3, 10^-4 M soln.)

4. Respiroscope

Procedure:

1. Soak the germinating seeds (5 gms) with an inhibitor or an un-coupler separately and then place them in the respiroscope for the determination of O₂ consumption during respiration.

2. Allow respiration to occur for 1 hr.

3. Calculate the rate of respiration from the data thus obtained.

Results:

Experiment # 4. Determination of Respiratory Quotient (R.Q. = CO₂/O₂) Ganong’s Respirometer:

The term ‘Respiratory Quotient’ or R.Q. is the ratio of the volume of CO₂ evolved to the volume of O₂ used in respiration by u given mass of tissue in a given time.

Thus R.Q. = Volume of CO₂ evolved/Volume of O₂ consumed

The value of R.Q. of a tissue depends upon the nature of substance which acts as the respiratory substrate and is oxidized in respiration.

For instance, when carbohydrate is the respiratory substrate, R.Q. is unity:

Materials and Equipments Required:

1. Ganong’s respirometer:

The apparatus consists of three parts:

(i) The bulb for the respiring material which ends in a smaller bulb at the bottom,

(ii) a graduated manometer fitted with the bulb, and

(iii) A leveling tube connected with the manometer tube by rubber tubing. The whole apparatus is clamped on a stand (Fig. 3.29).

2. Germinating seeds of different kinds

3. KOH solution, Mercury

Procedure:

1. Put germinating seeds in the bulb of the Ganong’s respirometer.

2. Fill the manometer with mercury.

3. Make the whole set-up air-tight and hold it in vertical position by stand and clamp.

4. Take the initial reading of the mercury in the graduated tube.

5. Allow respiration to continue for 1 or 2 hours and record the level of mercury.

6. Put some KOH solution into the open end of the tube and allow the solution to move down and absorb CO₂.

7. Record the final level of mercury from the graduated tube.

8. Perform separate experiments for different kinds of seeds.

Observations:

Initially O₂ is consumed and CO₂ is released by respiration. If the volume of O₂ consumed is more than CO₂ released, then the mercury column rises due to partial vacuum created inside. But after the addition of KOH soln., when the total volume of CO₂ is absorbed as recorded from the 2nd and final readings, the total volume of O₂ thus consumed can be calculated.

Results:

Initial reading = Xml

Second reading = Yml

Final reading = Zml

Volume of CO₂ released = (Z – Y) ml

Volume of O₂ consumed = (Z – X) ml

Thus R.Q. = Z – Y/Z – X 

Record the R.Q. of different kinds of seeds.

Inference:

The relation between R.Q. and the nature of seed used can thus be established.

Experiment # Study of Mitochondrial Respiration and Determination of Oxidative Phosphorylation:

When mitochondria are carefully isolated and suspended in an isotonic medium in the presence of substrate, only a slow rate of respiration is observed.

On adding ADP, the respiration rate increases until all the ADP is phosphorylated, when the rate of respiration returns to the original slow rate. The quantity of ADP that is added is known and the amount of O₂ consumed is measured (X); so it is possible to arrive at a P/O ratio for the particular substrate used.

P/O ratio = µ mol ADP added/µ atoms O₂ utilized (X).

Materials:

1. Isolated rat liver mitochondrial

2. Isolation medium (0.3 mol/lit sucrose 2.5 m mol/lit tris – HC1, pH 7.4, 0.5 m mol EDTA)

3. Incubation medium (Sucrose — 150 m mol/lit, KC1 — 20 m mol/lit, MgCl₂ — 20 m mol/lit,

K₂PO₃ — 1 m mol/lit, pH 7.4)

4. Sodium malate (200 m mol/lit, pH 7.4)

5. Sodium succinate (200 ml mol/lit, pH 7.4)

6. ADP (20 m mol/lit, pH 7.4)

Procedure:

Isolation of rat liver mitochondria:

A rat liver is isolated from a fresh killed rat and the tissue washed free of blood in ice-cold sucrose. Then lightly blot the same and place in a washed beaker to weigh. Liver is now cut into small fragments and homogenized in sucrose (20 g/100 ml), at 2,000 rpm in glass homogenizer.

Finally, the suspension was centrifuged as per the scheme stated below:

The mitochondrial pellet is washed twice with the isolation medium by re-suspension and centrifugation at 8,000 rpm for 10 min. Finally, the pellet is re-suspended in 5 ml of sucrose and stored on ice until required.

Immediately before use dilute the suspension so that 0.1 ml added to the KCl tris solution gives an initial extinction in the range 0.4 – 0.7. When a suitable dilution is formed add 0.1 ml to the mixtures below and follow the extinction at 520 nm with time.

The oxygen electrode is set up as described earlier and a small volume of the mitochondrial suspen­sion (50-100 µ1) followed succinate and ADP is added.

Finally, calculate the P/O ratio as per the formulae stated earlier.