BBYCL-138

PLANT PHYSIOLOGY AND

Indira Gandhi METABOLISM: LABORATORY
National Open University
School of Sciences

1 Demonstration of Bolting 5

2 Demonstration of the Effect of Auxins on Rooting 8

3 Demonstration of Root Respiration 11

4 Demonstration of Suction Due to Transpiration 13

Experiment 1
To Determine the Osmotic Potential of Plant Cell Sap by
Plasmolytic Method 15
Experiment 2
To Study the Effect of Two Environmental Factors
(Light and Wind Velocity) on Transpiration by an Excised 20
Experiment 3
To Determine the Stomatal Index and Stomatal Frequency in a
Mesophyte and Xerophyte 24
Experiment 4
To Demonstrate Hill Reaction 27
Experiment 5
To Demonstrate the Activity of Catalase and Study the Effect
of Ph and Enzyme Concentration 31
Experiment 6
To Study the Effect of Light Intensity and Bicarbonate
Concentration on O2 Evolution in Photosynthesis 35

Experiment 7
To Compare the Rate of Respiration in any Two Parts of a Plant 40
Experiment 8
To Separate Amino Acids by Paper Chromatograph 43
Course Design Committee
Prof. G.C. Srivastava (Retd.) School of Sciences, IGNOU
Former Head,
Prof. M.S. Nathawat, Director, (Ex.)
Department of Physiology,
IARI, Pusa, Prof. Amrita Nigam
New Delhi-110012
Prof. Jaswant Sokhi (Retd.)
Prof. Vijay Paul Prof. Bano Saidullah (Retd.)
Principal Scientist,
Prof. Neera Kapoor
Division of Plant Physiology
IARI, Pusa,
New Delhi-110012

Block Preparation Team

Prof. Amrita Nigam Editor
SOS, IGNOU, New Delhi-110068
Prof. G.C. Srivastava (Retd.)
Dr. Eklavya Chauhan Former Head,
Sr. Consultant, Department of Physiology,
SOS, IGNOU, New Delhi-110068 IARI, Pusa,
New Delhi-110012

Course Coordinator: Prof. Amrita Nigam

Production
Mr. Hemant Kumar
SO(P), MPDD, IGNOU

Acknowledgement
• Dr. Kumkum Chaturvedi, Dr. Bhupinder Dhir for giving useful inputs.

• Sh. Manoj Kumar, Assistant for word processing and CRC preparation.

March, 2021
Indira Gandhi National Open University, 2020
ISBN :
All rights reserved. No part of this work may be reproduced in any form, by mimeograph or
any other means, without permission in writing from Indira Gandhi National Open University.
Further information on Indira Gandhi National Open University courses may be obtained from
the University’s office at MaidanGarhi, New Delhi-110 068 or IGNOU website
[Link].
Printed and published on behalf of Indira Gandhi National Open University, New Delhi by the
Registrar, MPDD, IGNOU.
Printed at
 PLANT PHYSIOLOGY AND METABOLISM: LABORATORY
This practical course is designed to give you hands on experience on various aspects of
Plant Physiology which you have studied in theory course. These experiments will be related
to the concept of water relations, photosynthesis, respiration and enzymes. The course is of
2 credits. The course consists of 4 demonstration experiments and 8 practical experiments.
Demonstrations are for water relations and plant hormones while experiments pertain to
photosynthesis, water relations, respiration and enzymes.

You would be able to successfully conduct the practical sessions within the prescribed time
frame if you are well prepared with theoretical background, a detailed list of materials used,
procedure to be followed and the observations that you need to make. You should study the
lab manual carefully before joining the lab work.

After performing these practical exercises, your theoretical concepts will be clearer and you
will have a better understanding of the processes such as transpiration, plasmolysis,
photosynthesis, respiration, working of enzymes and plant hormones. You will also be able to
tell about the importance of all the physiological events in relation to growth of plants. The
questions asked at the end of each demonstration will be useful to you as they will provide
you an insight of the processes operative in plants. The course is aimed to develop your
practical skills as you will note the observations after performing the practical exercises.

You need to be both physically and mentally alert while performing the laboratory exercises.
By the end of the course it is expected that you will get trained and develop the ability to work
independently. You will also gain confidence after noting down the observations by yourself.
It is necessary that you observe demonstration experiments carefully and note down the
observations during the practical exercises, analyze them and reach a conclusion. It is
advised that you discuss your results with the counsellor as this will help in clearing your
doubts and help you to reach a logical conclusion.

You should realise that conducting practical session is an expensive exercise. It requires
participation of a counsellor and technical staff. Therefore, the whole practical session should
be taken seriously and utilised maximally to develop scientific temperament. Laboratory
exercises are interesting and enjoyable. We hope that you will enjoy the practical session of
Plant physiology. It is once more suggested that you go through the theoretical background
required for conducting the practical exercise.

Objectives
After completing the exercises, you should be able to :

describe the importance of water and hormones in the growth of plants through
demonstration experiments;

discuss the concept of plasmolysis, osmotic potential, and transpiration;

provide information about the variations in distribution of stomata in plants;

appreciate the process of photosynthesis and the environmental factors affecting it;

be aware of the process of respiration;

recognise the working of enzymes and factors affecting their activity; and

discuss the basic principle involved in the technique of paper chromatography. 3

Instruments and other requirements
Before you start the laboratory course, you should carry some of these items along with you:

• sharpened pencils

• observation notebook

• an eraser

• a lab coat

• sanitizer

Instruments such as Compound microscope, Lux meter, Potometer, and Centrifuge will be
provided to you from the laboratory. Glassware, chemicals and other miscellaneous items
will also be provided to you from the laboratory.

Laboratory Etiquettes
One should be keen, hardworking, sincere, and with an analytical frame of mind to attain
maximum knowledge from the laboratory courses. The present laboratory course is designed
in such a way so that your knowledge about the subject will be enhanced and you will be
able to develop analytical and observational skills. One needs to follow the following points
before coming to the laboratory :

• read the laboratory manual exercises along with theory given in the course book.

• plan your experiment in such a manner that you complete the assigned work in the
suggested time frame .

• use the facilities judiciously.

• strictly follow the instructions given by the counsellor.

• get the observations checked regularly by the counsellor after completing the
experiment.

• handle the laboratory instruments and materials carefully.

• always discuss the doubts with the counsellor.

By following these points, the success and satisfaction will be yours. We wish you best of
luck.

4
Demonstration 1 Demonstration of Bolting

1
DEMONSTRATION OF
BOLTING

Structure
1.1 Introduction 1.3 Procedure

1.2 Requirements

1.1 INTRODUCTION
Gibberellins are a group of naturally occurring hormones having many
physiological effects in plants. The effects are generally growth promotive.

One of the most remarkable effects of gibberellins is in converting a

genetically dwarf plant into a plant of normal height. Addition of gibberellins to
a cabbage plant converts the ‘head’ or dwarf stem into a stem which is 6-8 feet
tall. Rosette plants of sugarbeet is an extensive case of dwarfing. Lang(1956)
showed that such a stem can undergo rapid growth or ‘bolting’ if it is treated
with gibberellins. Bolting is the elongation of the floral axis stalk in some dwarf
biennial plants to produce flowers. The plant for one season grows
vegetatively and in other season produces floral axis and fruits subsequently.
The application of gibberellins to the plant at vegetative phase causes the
plant to produce floral axis prematurely. This property has been exploited by
agriculturalists in getting the crop within short span of time.

Many worker shave demonstrated the stimulation of cell division in the sweet
peas. It is believed that the dwarfism in the mutant variation of a plant is due to
blocking of the gene responsible for controlling the capacity for normal
gibberellin production.

1.2 REQUIREMENTS
Plant Material: Two groups of potted plants of Launaea (Rosette habit) of
same age (Four in each group).

Chemicals: GA3 solutions {0.1 mg/L (0.1 ppm); 1 mg/L (1 ppm); 5mg/L (5ppm)
and 10 mg/L(10ppm)}.

Miscellaneous: Sprayer/ cotton swab. 5

BBYCL 138 Plant Physiology and Metabolism: Laboratory
1.3 PROCEDURE
Expose the shoot apex of the rosette plants and

1. Select 4 weeks old potted plants of Launaea or Lactuca sativa (lettuce)

of roughly equal size.

2. A total of 18 plants are divided into six groups of 3 plants each to

demonstrate the effect of different concentrations of GA3.

3. The six groups are categorized as i) Control (no GA3) but only distilled
water; ii) 0.01mg/L GA3); iii) 0.1 mg/L GA3; iv) 1 mg/L GA3; v) 5 mg /L
GA3 and vi) 10 mg/ L GA3.

4. Prepare 100ml solution of each [Link] shift the leaves to

expose the shoot apex and apply the specific GA3 concentration with the
help of a cotton swab or spray the GA solution to runoff level. Repeat the
application of hormone every third day for two weeks.

You will observe that the control plants retained their dwarf habit where as the
plants sprayed with GA3 showed elongation of internodes and bolting.

Fig. 1.1: Demonstration of bolting.

Now answer the following questions

1. What does the above experimental set-up demonstrate?

2. What causes the floral axis elongation?

3. What is the commercial advantage of extending the internode length?

4. Which chemicals promote dwarfing? Is dwarfing of any commercial

6 importance?
Demonstration 1 Demonstration of Bolting
Hints

1. Demonstration of the effect of GA3 on rosette plants to induce bolting.

2. Bolting occurs due to increase in cell number as well as cell elongation.

3. For example, grape plants can be treated with gibberellins to extend the
internode length. In this way the flowers are spaced further enabling the
fruits to grow freely/with more room.

4. Dwarfing is promoted by antigibberellins e.g., AMO-1618, Cycocel

(CCC), Phosphon D and Ancymidol. These chemicals make the plant
short thereby preventing lodging. The antigibberellins block the
gibberellin biosynthesis.

7
BBYCL 138 Plant Physiology and Metabolism: Laboratory

2
DEMONSTRATION OF THE
EFFECT OF AUXINS ON
ROOTING

Structure
2.1 Introduction 2.3 Procedure
2.2 Requirements

2.1 INTRODUCTION
Auxins are one of the most important groups of plant hormones because of
their many-sided roles in plants. These substances were also the first growth
factors to be identified as plant hormones. F.W. Went succeeded in isolating
these growth substances and named them as auxins. Auxins are synthesized
from the amino acid tryptophan.

In nature root formation by a plant has been shown to be possible only if there
are developing buds or leaves on them. Dormant buds fail to induce rooting.
Ringing the cuttings immediately below developing buds from a normal plant
also prevents rooting. Evidently the rooting in all such cases depends on the
presence of a hormone. Thimann and his collaborators have shown that root
priming substance and auxins are identical. The auxins have been found to
increase the rate of formation and final number of root initials. This property of
auxins has been taken advantage of in propagation of plants by stem cuttings
in plants.

Thimann and Went (1930) found that indole acetic acid (IAA) and other
growth substances are essential for initiating adventitious root formation in
cuttings. They are applied at concentrations ranging from 100-1000 ppm (parts
per million).

2.2 REQUIREMENTS
Glassware : 4 conical flasks (250 ml).

Plant Material : Stem cuttings of Morus alba.

Chemicals : i) IAA solutions (10-3M, 10-4M, 10-5M). ii) Distilled water.

8 Miscellaneous : Cork stoppers.

Demonstration2 Demonstration of the Effect of Auxins on Rooting

2.3 PROCEDURE
Firstly four conical flasks were taken and one of them was filled with distilled
water (control).In other three conical flasks, IAA solutions of different
concentrations (10-3M, 10-4M, 10-5M) were filled. All the flasks were sealed with
cork stopper. Freshly cut pieces of the branches of Morus alba were inserted
in the holes of cork stoppers so that lower ends of cuttings were touching the
solution. These conical flasks were left for one week.

You will observe that in control flask having only distilled water there was no
root initiation, but the flask having 10-4 M IAA solution had maximum root
initiation. On the other hand, the cutting in flask having 10-5 M IAA solution
showed poor root initiation. You will also observe that the stem cutting of flask
having 10-3 M IAA solution showed no root initiation.

In the control flask without IAA, no root initiation was observed because
endogenous auxins were present in extremely low concentrations thus, having
no significant effect. In flask having 10-5 M 1AA solution, little root initiation
occurred as the amount of auxin was less than the optimum concentration. In
flask having 10-4 M IAA solution maximum root initiation was observed
because it was having optimum concentration of IAA. In flask with 10-3 M 1AA
solution the stem cutting got deformed due to supraoptimal concentration of
IAA causing damage.

Fig. 2.1: Demonstration of effect of auxin on rooting in stem cuttings 9

BBYCL 138 Plant Physiology and Metabolism: Laboratory
Now answer the following questions

1. What does this experimental set-up demonstrate?

2. What will happen if the concentrationof IAA in the flask is increased ten-
fold?

3. Name six synthetic auxins.

Answers

1. Demonstration of root induction by IAA in stem cuttings.

2. Supraoptimal concentrations of auxins are lethal to a plant.

3. NAA, IBA , 2,4-D, 2,4,5-T, MCPA, PCPA .

10
Demonstration 3 Demonstration of Root Respiration

3
DEMONSTRATION OF ROOT
RESPIRATION

Structure
3.1 Introduction 3.3 Procedure
3.2 Requirements

3.1 INTRODUCTION
Cellular respiration is vital for organisms and consists of a series of pathways.
The stored/reserve materials act as respiratory substrates and get oxidized to
release ATP. Since all cells respire, roots are no exception. Usually the aerial
parts of a plant are used to demonstrate rate of respiration or respiratory
quotient (RQ). Roots also respire and thus, aerated soils are essential for
normal plant growth. Waterlogging for long time chokes the roots and results
in death of the plant.

3.2 REQUIREMENTS
Plant Material : Two small rooted plants of Tagetes with adventitious roots
Chemicals : Dilute NaOH solution, phenolphthalein
Miscellaneous : cork stopper with a hole.

3.3 PROCEDURE
In this demonstration experiment, a small, rooted plant (e.g., Tagetes, or
wheat) with intact adventitious roots was taken and placed in a flask which had
slightly alkaline water (with dilute NaOH solution) and coloured red with
phenolphthalein. A second flask was taken which served as control and was
without any plant but had only red coloured alkaline [Link] was fixed
tightly. Both flasks were allowed to stand in diffuse light and the solutions in
them were examined after some time.

You will observe that the control flask does not show change in the colour
while the one with the roots becomes colorless as the colour of the solution
fades. The respiring roots release CO2, which reacts with water to form
carbonic acid (H2CO3)
CO2 + H2O H2CO3 11
BBYCL 138 Plant Physiology and Metabolism: Laboratory
Carbonic acid neutralizes NaOH present in the flask and the alkalinity of the
solution starts decreasing, thus fading the red colour (phenolphthalein is
colourless in the neutral medium).

H2CO3 + 2NaOH Na2CO3 + H2O

Fig. 3.1: Demonstration of root respiration

Questions
1. Explain the physiological mechanism involved in this set up, giving an
equation.
2. What would happen if the solution in flask contained a buffer solution of
pH 7.8?
3. What will happen if a drop of HCL is added to the experimental flask?
4. What results do you expect, if the roots in the experimental flask are pre-
boiled?
5. In which way is root respiration helpful in the nutrient uptake from the
soil? Explain.
Hints
1. See procedure
2. Solution will not turn neutral or acidic. Pink colour does not fade away.
3. Pink color will fade.
4. No change in colour as the roots are killed.
5. For active uptake of nutrients energy in the form of ATPis required,which
12 is provided by the respiring roots.
Demonstration 3 Demonstration of Root Respiration

4
DEMONSTRATION OF
SUCTION DUE TO
TRANSPIRATION

Structure
4.1 Introduction 4.3 Procedure
4.2 Requirements

4.1 INTRODUCTION
You know that the plant transpires actively in nature and water is lifted
upwards as a continuous column. You can see that water column does not
collapse because of strong cohesive force among the water molecules as well
as great adhesive force between water molecules and the hydrophilic walls of
the tracheary elements. The continuous water column exists between the
roots and the transpiring parts of the plant which are leaves. Thus, due to
transpiration, a suction force or transpiration pull develops in the leaves of the
plant, which is transmitted below to the roots via stem resulting in water uptake
from the soil. The water lost by the plant during transpiration is compensated
by the water absorbed by it from the capillary tube of the potometer. This
results in rising of the mercury column.

4.2 REQUIREMENTS
Plant Material: Two small rooted plants of Tagetes (Marigold)

Apparatus: A simple or H-shaped potometer

Chemicals: Mercury

Miscellaneous: Cork stopper with a hole

4.3 PROCEDURE
This set upcomprises a simple potometer which includes a hollow glass tube
with one end submerged in a trough containing mercury, and the other end
fitted with the shoot of an actively transpiring plant (such as guava, marigold,
sunflower or Geranium) under airtight conditions. Put the setup under the fan 13
BBYCL 138 Plant Physiology and Metabolism: Laboratory
to increase transpiration .You will observe a rise in the level of mercury column
after some time (30 minutes or so), which indicates suction due to
transpiration.

Fig4.1: Simple potometer to show suction due to transpiration.

Now try to answer these Questions

1. List the factors affecting the process being demonstrated.

2. What will happen if the leaves are smeared with grease or wax?

3. Why is mercury used in the set up?

4. What will happen if the set up is shifted to a humid environment?

5. Which plant hormone regulates stomatal closure during water deficit

conditions?
Answers

1. Transpiration is affected by many factors like: sunlight, temperature,

atmospheric humidity,air movement, CO2,and water availability.

2. Stomates will not transpire and so mercury column will not rise.

3. It does not react with water.

4. Rate of transpiration will be low due to less vapour pressure deficit.

5. ABA (Abscisic acid).

14
Experiment 1 To Determine the Osmotic Potential of Plant Cell Sap by Plasmolytic Method

EXPERIMENT 1
TO DETERMINE THE
OSMOTIC POTENTIAL
OF PLANT CELL SAP BY
PLASMOLYTIC METHOD

Structure
1.1 Introduction 1.5 Observations

1.2 Materials Required 1.6 Results

1.3 Principle 1.7 Precautions

1.4 Procedure

1.1 INTRODUCTION
One of the important conditions for maintenance of the physiological active
state of a plant is the optimum water balance. Water is an important
constituent of the plant cell. It is the solvent for entry and transport of
substances and for metabolic reactions. In Unit 1 you have learnt about the
physical principles that govern the net water fluxes from one cell to the next
cell and the bulk movement of water in soil-plant atmospheric system. Water
potential(Ψw)is the driving force which causes water to move in plant system.
Osmotic potential (Ψs/Ψ࣊) is an important component of water potential and is
related to it by the equation.

Ψw = Ψs + Ψp

1.2 MATERIALS REQUIRED

• Leaves of Rhoeo discolor

• Sucrose solution (0.25 M), distilled water

• Petri dishes, glass slides, cover slips, beakers andpipettes(1ml,10ml)

• Microscope, forceps, needle, razor blade, stopwatch, graph paper 15

BBYCL 138 Plant Physiology and Metabolism: Laboratory
1.3 PRINCIPLE
You know that biological membranes are differentially permeable to different
solvents. Osmosis is the movement of the solvent molecules from region of
higher concentration to lower concentration through semipermeable
membrane.

Osmotic potential is the amount by which water potential is reduced as a

result of the presence of the solute .It is also called solute potential. The
osmotic potential of cell-sap is considered to be equal to that of solution at
which 50% of the cells show plasmolysis and osmotic potential can be
calculated by the formula given below

Osmotic potential = - CRT

Where C = molarconcentration at incipient plasmolysis

R = Universal gas constant(0.082 atm/mol)

T = absolute temperaturein 0K (t + 273)

1.4 PROCEDURE
1. Prepare a series of sucrose solutions ranging from 0.15 molar to 0.35
molar by using 1 molar sucrose as a stock solution. The solutions can be
prepared with the help of dilution table given below.

2. Take leaves of Rhoeo discolor and peel out the epidermis. This plant is
used because they have anthocyanin pigments in their vacuolar sap.
Anthocyanin pigments are water –soluble and respond to change in pH
values of external medium.

Fig1.1: Plotting of graph between concentration of sucrose solution and degree

16 of plasmolysis.
Experiment 1 To Determine the Osmotic Potential of Plant Cell Sap by Plasmolytic Method
3. In each petri plate, place three peels after a time gap of five minutes.
Wait for ten minutes.

4. Take out the peel and mount it on the slides in their respective solutions
and observe under microscope (6x, 40x).

5. Count total number of pigmented cells visible and number of cells that
had undergone partial or complete plasmolysis. Plot a graph between
the percentage plamolysed cells (Y-axis) against the sucrose
concentrations (X-axis). From this graph we can find out the
concentration at which 50% of the cells are plasmolysed – the stage of
incipient plasmolysis.

6. Calculate osmotic potential of the cells by the formula given above.

Dilution Table
Stock solution = 1.0M (Molar) sucrose (342g sucrose in some distilled water.
Raise the final volume to 1000 ml).

Concentration Amount of 1 M Amount of Total Volume

of Sucrose Sucrose Solvent
Solution (M) (distilled water)
Control − 10.0 ml 10 ml
0.15 M 1.5 ml 8.5 ml 10 ml
0.20 M 2.0 ml 8.0 ml 10 ml
0.22 M 2.2 ml 7.8 ml 10 ml
0.24 M 2.4 ml 7.6 ml 10 ml
0.26 M 2.6 ml 7.4 ml 10 ml
0.30 M 3.0 ml 7.0 ml 10 ml
0.35 M 3.5 ml 6.5 ml 10 ml

1.5 OBSERVATIONS
Observation Table
Sucrose No. of No. of cells Degree of
concentration pigmented cells plasmolysed plasmolysis
(M) examined (mean)
(mean)
Control a) a)
b) b)
c) c)
0.15 M a) a)
b) b)
c) c)
0.20 M a) a)
b) b)
c) c) 17
BBYCL 138 Plant Physiology and Metabolism: Laboratory
0.22 M a) a)
b) b)
c) c)
0.24 M a) a)
b) b)
c) c)
0.26 M a) a)
b) b)
c) c)
0.30 M a) a)
b) b)
c) c)
0.35 M a) a)
b) b)
c) c)

Calculations
I. For given plant material

C = isotonic concentration = 0.32 M (from the graph)

R = gas constant = 0.082

T= absolute temperature = 306.5 °K (say at 33°C)

Ψs/Ψ࣊ = - CRT

= - 0.32 × 0.082 × 306.5

Ψs/Ψ࣊ = - 8.0425 bars

1.6 RESULTS
No plasmolysis is observed in the control (distilled water). With an increase in
sucrose concentration, the number of plasmolysed cells increases because
the external medium becomes hypertonic with respect to cell sap. As the
plasmolysiscontinues, the cytoplasm along with cell membrane starts
shrinking. A space is created between the cell wall and the cell membrane,
which gets filled up with the plasmolysing solution (external sucrose solution
and water from the cell sap). The sucrose concentration where 50% of the
cells are plasmolysed, i.e. isotonic concentration, is determined from the
graph. The concentration of an unknown solution can also be determined from
the graph and used for calculating osmotic potential of that solution.

1.7 PRECAUTIONS
1. The epidermal peels should be one-celled thick and free from green
18 tissue, and air bubbles.
Experiment 1 To Determine the Osmotic Potential of Plant Cell Sap by Plasmolytic Method
2. All cells of the peel should invariably contain anthocyanin pigment.

3. Before use, keep the peels fully immersed in water to avoid the entry of
air bubbles.

4. The peels should be examined after a fixed duration of time, the time to
be adjusted before starting the experiment (trial run for fixing time period
may be done).

5. Mounting of the peels should be done in their respective concentrations,

and not in water to prevent deplasmolysis.

6. The petri dishes containing different sucrose solutions should be

covered to prevent evaporation.

7. Even if the cells are slightly plasmolysed, they should be categorized as

having undergone plasmolysis.

19
BBYCL 138 Plant Physiology and Metabolism: Laboratory

EXPERIMENT 2
TO STUDY THE EFFECT OF
TWO ENVIRONMENTAL
FACTORS (LIGHT AND WIND
VELOCITY) ON
TRANSPIRATION BY AN
EXCISED

Structure
2.1 Introduction 2.5 Observations

2.2 Materials Required 2.6 Results

2.3 Principle 2.7 Precautions

2.4 Procedure

2.1 INTRODUCTION
Plant absorbs water from its surroundings (especially roots) but only a small
fraction of the absorbed water is actually utilized by them. The bulk of the
water absorbed is not retained and is evaporated into the air from the leaves
and other aerial parts of the plant. The loss of water in the form of vapour from
the aerial parts of the plant is known as transpiration.

Loss of water can take place from any part of the plant which is exposed to air.
Leaves are, however, the principal organs of transpiration. The transpiration
which occurs from the leaves is known as foliartranspiration. Most of the
foliar transpiration takes place through the stomatal openings and is therefore,
known as stomatal transpiration. A small fraction of the foliar transpiration
also takes place from the general surface of the leaf through the cuticle and is
known as cuticular transpiration. Loss of water vapours also takes place
through the lenticels of woody stems and fruits, and is called lenticular
20 transpiration.
Experiment 2 To Study the Effect of Two Environmental Factors (Light and Wind Velocity)
on Transpiration by An Excised
2.2 MATERIALS REQUIRED
• Plant material – Twigs of Tecomaplant

• Apparatus - Potometer

• Glassware – i) A graduated pipette (1 mL)

ii) 1 Beaker (1000 mL)

• Miscellaneous – Rubber tube, Stopwatch, Iron stand (with two holders),

Sharp blades, Lux meter, Anemometer (optional) and modelling clay

2.3 PRINCIPLE
The rate of transpiration is influenced directly or indirectly by a number of
factors, of which light intensity and wind velocity are among the chief ones.
Light affects the rate of transpiration directly by opening the stomata. In the
absence of light the stomata are closed and the stomatal transpiration is
completely checked. High light intensity causes more stomata to open hence
high rate of transpiration. It also affects the transpiration by heating the surface
of leaf, hence more vaporisation of water present in cells of leaf.

Wind velocity also has remarkable effect on rate of transpiration. If wind is

not blowing the water vapours are assumed to accumulate above the
transpiring leaves. It lowers the steepness of the vapour- pressure
gradient,which decreases the rate of transpiration. When wind is blowing, the
water vapours quickly move away from the leaf surface resulting in increase in
the steepness of vapour pressure gradient, hence rapid rate of transpiration. A
gentle breeze is more effective in increasing the rate of transpiration than wind
of greater velocity. It is believed that the wind has a cooling effect on the
evaporating surface of leaf. This lowers the vapour pressure gradient, which
reduces the rate of transpiration. High wind velocity also reduces the rate of
transpiration by increasing the rate of loss of water from the mesophyll cells
which eventually results in water stress- the flaccidity and closure of stomata.

Thus, wind velocity, like light, also affects the rate of transpiration as a
combination of positive and negative influences.

2.4 PROCEDURE
1. First take a simple potometer with one end of the rubber tube attached to
a graduated pipette.

2. Then fill it with water till the water level rises to the top most mark on the
scale of the pipette.

3. Now cut a twig of Tecoma plant obliquely under water with a sharp blade
and insert it at the free end of the rubber tube under water to avoid entry
of air bubbles in the twig.

4. Then place the apparatus in sunlight for about 10 minutes (Lag period)
and record the fall in water level in the pipette. 21
BBYCL 138 Plant Physiology and Metabolism: Laboratory
5. You have to repeat this procedure two more times.

6. Then place the apparatus in the shade but diffused sun light should be
present there.

7. Keep the apparatus for a lag period of 10 minutes and again record the
rate of fall in the water level. This process should be done three times.

8. Now repeat the same process in the room and take three readings.

9. Record the light intensity by a luxmeter or photometer. Now put the

apparatus under very low wind velocity produced by a table fan.

10. Give a lag period of 10 minutes and record the fall in water level per
minute.

11. Repeat the procedure for medium wind velocity and high wind velocity
by giving the lag period in each case.

12. Compare the readings and plot the graphs.

13. You can measure the wind velocity with the help of an anemometer.

Fig 2.1: Measurement of the rate of transpiration with the help of a potometer.

2.5 OBSERVATIONS
Observation Table

a) Effect of Light Intensity on Rate of Transpiration

Sl. Light Initial Level Final Level Volume of Rate of

No. Intensity of Pipette of Pipette H2O Transpiration
(mL) (mL) Absorbed
(mL/min)

1. Lux 1)

2)

3)
22
Experiment 2 To Study the Effect of Two Environmental Factors (Light and Wind Velocity)
on Transpiration by An Excised
2. Lux 1)

2)

3)

3. Lux 1)

2)

3)

b) Effect of wind Velocity on Rate of Transpiration

Sl. Wind Initial Level Final Level Volume Rate of

No. Velocity of Pipette of Pipette of H2O Transpiration
(mL) (mL) Absorbed
(mL/min)

1. Low 1)

2)

3)

2. Medium 1)

2)

3)

3. High 1)

2)

3)

2.6 RESULTS
The rate of transpiration is maximum when the potometer is kept under
highlight intensity but decreases as it is shifted to a region of low light intensity.

The rate of transpiration is low at low wind velocity but increases at the
medium wind velocity. The rate of transpiration again comes down at high
wind velocity.

2.7 PRECAUTIONS
1. A healthy twig must be used for the experiment.

2. An oblique cut must be made in the stem while it is under water.

3. The apparatus must be air tight.

4. Constant log period must be given before changing the environmental

conditions. 23
BBYCL 138 Plant Physiology and Metabolism: Laboratory

EXPERIMENT 3
TO DETERMINE THE
STOMATAL INDEX AND
STOMATAL FREQUENCY IN A
MESOPHYTE AND XEROPHYTE

Structure
3.1 Introduction 3.5 Observations
3.2 Materials Required 3.6 Results
3.3 Principle 3.7 Precautions
3.4 Procedure

3.1 INTRODUCTION
The number, distribution and behaviour of stomata vary in plant leaves
belonging to different habitats. These parameters directly affect the rate of
transpiration. Stomata play an important role in the exchange of carbon
dioxide and oxygen gases and loss of water in a [Link] are present in
the epidermal layer of the leaf. The stomatal pore is surrounded by a pair of
guard cells which control its opening and closing. When the turgor pressure in
guard cells increases, stomata opens and when it decreases the guard cells
collapse and close the stomata. In this exercise you will determine stomatal
index and stomatal frequencyin a mesophyte and xerophyte leaf.

Objectives
After performing this exercise, you should be able to:

compare stomatal index and stomatal frequency in mesophytes and

xerophytes and appreciate its ecological importance.

3.2 MATERIALS REQUIRED

• Leaves of a mesophyte : Withania somnifera or any other mesophyte

• Leaves of xerophytic plant: Bryophyllum or anyother xerophytes

24 • Distilled water
Experiment 3 To Determine the Stomatal Index and Stomatal Frequency
in a Mesophyte and Xerophyt
• Stage micrometer

• Ocular micrometer

• Petri dishes, slides, and cover slips

• Compound microscope, razor blade, blotting paper, brush and needle

3.3 PRINCIPLE
The epidermal surface of a leaf has large number of stomata. You can see
stomata onlyunder a microscope. The number of stomata varies from species
to species and it ranges from 1000 to 60,0000 per square centimeter. You
can find stomata on both the sides of leaf, but in many species, they are
present on either side. Stomata playan important role in regulating gaseous
exchange. Generally, they are open in the light and closed in the dark. You will
calculate stomatal frequency by the help of micrometry.

S
Stomatal Index (%) SI = × 100
E+S

S = Number of stomata in field of vision

E = Number of epidermal cells in the same area

Stomatal Frequency SF = Number of stomata per unit area (per cm2)

= Number of stomata in a given microscopic field

Area of microscopic field

Area of microscopic field = πr2 [r = radius of the microscopic field]

Salisbury concluded that stomatal frequency differs with environmental

conditions, especially on availability of water.

Micrometry technique is used to determine dimensions of any microscopic

material. Micrometers are of two types – stage and ocular micrometer.

i) Stage Micrometer : It is in the form of a glass slide with markings etched

which can measure upto an accuracy of 100th part of a mm. i.e., 100
divisions = 1 mm. Stage micrometer is placed on the stage of the
microscope and is used to calibrate the ocular micrometer and to
calculate area of microscopic field.

ii) Ocular micrometer : It is in the form of a circular glass disc with 100
equidistant divisions but of unknown dimensions. It is placed between the
ocular and the objective lenses. Since the ocular micrometer is not
calibrated, it is done using the stage micrometer for calculating the least
count.

3.4 PROCEDURE
1. Take out epidermal peels from the lower surface of the leaves of
Withania and Bryophyllum.

2. Stain them with dilute safranin, mount in glycerine and observe under
high power (6 X × 30 X) of the compound microscope. 25
BBYCL 138 Plant Physiology and Metabolism: Laboratory
3. Calculate the least count, i.e., dimension of each small division on the
ocular micrometer by coinciding it to the divisions of the stage
micrometer.

4. Count the number of stomata and number of epidermal cells in the

microscopic field. Record three concordant readings.

5. Calculate the ‘stomatal index’ and ‘stomatal frequency’ using the

appropriate formulae.

3.5 OBSERVATIONS
Record your observations in the following table:

Observation table

Least count = µm

Plant Number of Number Stomatal (SI) Area of Stomatal

Material stomata (S) of Index the field Frequency
Epidermal of Vision S/A
S
Cells (E) × 100 (A)
S+E
Number Mean
Withania A a
somnifera
Bryophyllum B b
C c

3.6 RESULTS
Withania leaves show higher stomatal index and stomatal frequency as
compared to Bryophyllum leaves. The latter being a succulent,hasa smaller
number of stomata to reduce the rate of transpiration.

3.7 PRECAUTIONS
1. Peels should be taken from the same leaf by applying unequal pressure.

2. Avoid counting of stomata near edges of the field of vision (border

effect).

3. Peels should be one-celled thick and free from green mesophyll tissue.

4. Magnification of the microscope should remain the same throughout the

experiment.

5. Peels should be immediately kept in water to prevent the entrance of air

bubbles.

26
Experiment 4 To Demonstrate Hill Reaction

EXPERIMENT 4
TO DEMONSTRATE
HILL REACTION
REACTION

Structure
4.1 Introduction 4.5 Observations
4.2 Materials Required 4.6 Results
4.3 Principle 4.7 Precautions
4.4 Procedure

4.1 INTRODUCTION
The process of photosynthesis is one of the most remarkable activities of a
living green plant. Carbohydrates produced through photosynthesis
contributeto the basic raw materials which directly or indirectly give rise to all
organic compounds of virtually all plants and animals. The total global CO2
available to plants for photosynthesis is about 11.2×1014tons.

Photosynthesis consists of two phases, - light phase and dark phase. Light
phase is photochemical phase called “Hill reaction”. It occurs inside
thylakoids especially in grana region.

Dark phase is biochemical phase and occurs in the stroma. It is called

Blackman’s reaction. During light reaction water is photolyzed (split in the
presence of light) and releases oxygen and electrons. These electrons travel
through a series of carriers: cytochromes, plastoquinone and ferredoxin, to
finally reduce NADP+ TO NADPH.

Robin Hilland his colleagues (1937) first demonstrated the evolution of O2 by

illuminated suspension of isolated chloroplasts of Stellaria media when
provided with artificial electron acceptors like ferricyanide in the absence of
CO2. Ferricyanide is reduced to ferrocyanide by photolysis of water.

The photoreduction of the dye by isolated chloroplasts is commonly referred to

as Hill Reaction.
Light
2 A + 2H2O   → 2AH2 + O2
Chloroplas ts

A is the hydrogen acceptor and also called as Hill’s oxidant. 27

BBYCL 138 Plant Physiology and Metabolism: Laboratory
4.2 MATERIALS REQUIRED
Glassware : Test tube, Funnels, Beakers (25 mL), Measuring
cylinder 25 mL), Pipette (5 mL), Pipette (1 mL),
Centrifugation tubes

Plant Material : Fresh spinach leaves

Chemicals : 0.5 M sucrose solution

0.1 M Na2HPO4

0.01M DCPIP (2, 6-dichlorophenol indophenol)

Distilled water

Miscellaneous : Blender

Ice-cubes and ice bucket

Muslin cloth

Test tube stand

Black paper

Spectrophotometer

Centrifuge and table lamp

4.3 PRINCIPLE
Hill reaction demonstrates that the artificial electron acceptors can substitute
naturally occurring acceptors. In this exercise you will observe photoreduction
of a dye 2,6-Dichlorophenol indophenol (DCPIP) which is blue in an oxidized
state (quinone form) but becomes a colourless compound when reduced
(phenol form)
Light
DCPIP (blue) +H2   → DCPIP-H2 (Colourless) + ½ O2.
Chloroplas ts

After performing this experiment you should be able to:

• Isolate chloroplasts from leaves, and

• show photoreduction of dye (Hill oxidant) by illuminated suspension of

chloroplasts.

4.4 PROCEDURE
i) Preparation of dye: Dissolve 10 mg of dye in 100 mL of distilled water
to make 0.01 M DCPIP.

ii) Preparation of buffer: Mix 68.5 mL of 0.2 M NaH2 PO4 [Sodium

dihydrogen orthophosphate] and 31.5 mL of 0.2 M Na2 HPO4 [disodium
hydrogen phosphate] to make 100mL of 0.2 M buffer (Phosphate), pH 6.5

28 iii) Isolation of Chloroplasts

Experiment 4 To Demonstrate Hill Reaction
A) Homogenization

1. Pre-chill the glassware and reagents.

2. Take 50 g of spinach leaves (refrigerated in dark), remove petioles and

midrib.

3. Add 100 mL of cold 0.5 m sucrose solution, and blend contents at top
speed for about 15 sin a glass blender and stop for few seconds.

4. Then, again blend at low speed for 10 seconds.

B) Filtration

Filter the homogenate through muslin cloth in a beaker with the help of pre-
chilled funnel.

C) Centrifugation

1. Now pour the filtrate into two pre-chilled centrifuge tubes in equal
amounts and centrifuge at low speed (500 × g) for 3 min.

2. Take out the supernatant solution in another centrifuge tube and again
centrifuge at high speed (2000 × g) for about 7 minutes.

3. Take out the pellet in a test tube and discard the supernatant solution.

4. Resuspend the pellet in 5 mL of cold 0.5 M sucrose solution. Keep it in

dark in an ice bucket.

Demonstration of Hill reaction

Take 3 test tubes and pour into them the following materials:

Test tube I : 1 mL of chloroplast suspension + 2 mL of phosphate buffer + 2

mL distilled water + 1 mL DCPIP

Test tube II : 1 mL of chloroplast + 1 mL of phosphate buffer + 2 mL distilled

water + 1 mL DCPIP

Test tube III : 1 mL phosphate buffer + 3 mL distilled water + 0.5 mL DCPIP

(control).

1. Keep test tube I under light conditions.

2. Keep test tube II under light deficient conditions by wrapping black paper
or aluminum foil around it.

3. Keep test tube III as control test tube.

4. Leave test tubes undisturbed for some time.

5. Take readings for optical density and % transmission in a

spectrophotometer 29
BBYCL 138 Plant Physiology and Metabolism: Laboratory

Fig.4.1: Pictoral depiction of the protocol for Hill reaction.

4.5 OBSERVATIONS
Test Tube Spectrophotometer Reading
I 0 minOD/T 5 min OD/T 10 min OD/ T
II - - -
III - - -

Plot a graph between Time (X-axis) and Optical Density –OD(Y axis).

4.6 RESULTS
The test tube I will show maximum reduction of dye while test tubes II and III
will show very low or no reduction of dye.

4.7 PRECAUTIONS
1. Homogenization should be carried out carefully so that chlorophyll does
not get mechanically crushed and denatured.

2. All equipments and reagents should be pre-chilled.

3. Only fresh leaves should be used.

4. All reagents should be freshly prepared.

5. Healthy spinach leaves must be frozen and their petioles and mid veins
removed.

30
Experiment 5 Activity of Catalase and Study the Effect of pH and Enzyme Concentration

EXPERIMENT 5
TO DEMONSTRATE THE
ACTIVITY OF CATALASE AND
STUDY THE EFFECT OF
pH AND ENZYME
CONCENTRATION

Structure
5.1 Introduction 5.5 Observations
5.2 Materials Required 5.6 Results
5.3 Principle 5.7 Precautions
5.4 Procedure

5.1 INTRODUCTION
All the living cells carry out huge variety of biochemical reactions, yet they are
able to rapidly construct very large and complicated molecules, or regulate the
flow of materials through complex metabolic pathways with absolute precision
and accuracy. Enzymes make this possible without any error. Enzymes are
biological catalysts which make possible the conversion of substrate
molecules to products, but they themselves are not permanently changed by
the reaction .Cells contain thousands of enzymes, each one catalyzing a
particular reaction or specific for a particular reaction.

The most remarkable characteristics of enzymes are their catalytic power and
specificity. Catalytic activity takes place at a particular site on the enzyme
which is called active site. Enzymes accelerate reactions by factors of as
much as a million or more without affecting its equilibrium position.

In the present experiment, you will study the effect of two parameters, viz., pH
and enzyme concentration on the activity of the enzyme catalase.

5.2 MATERIALS REQUIRED

• Potato tuber (Solanum tuberosum)

• 3% H2O2 , standard buffer solution (of pH 1.0,4.0,7.0,9.0 and 12.0),

Distilled water 31
BBYCL 138 Plant Physiology and Metabolism: Laboratory
• Y-shaped apparatus, burettes (2), beaker, measuring cylinder.

• Clamp stands (2)

5.3 PRINCIPLE
Photosynthesis results in splitting of water (photolysis) into protons and
hydroxyl ions during light reaction.

H2O H+ + OH-

H+ is utilized in forming NADPH which is used in Calvin Cycle. The OH-

undergoes reaction to form hydrogen peroxide (OH- + OH- + 2e- H2O2)
which is highly toxic and may cause degradation of membranes and generates
free radicals. Catalase enzyme scavenges or removes H2O2 by breakingit into
water and oxygen.

2H2O2 Catalase
 → 2H2O+O2.

Catalase is an important enzyme for photosynthetic cells (It can be obtained

from green leaves and also from potato tubers).

5.4 PROCEDURE AND OBSERVATIONS

1. Activity of catalase enzyme

• For testing the activity of catalase enzyme, you must take 5mL of
catalase enzyme extract and add 2mL of 3% H2O2 in a test tube.

• In another lot boiled potatoes extract should be taken.

• Observe the reaction as follows:

Observation Table
S. Nature of Observations Inference
No. Enzyme
Initial Final

1. Active No froth Froth Froth formation because of evolution

of O2 gas.

2. Denatured No froth No froth No froth because O2 gas is not

(Boiled evolved as the enzyme is killed.
potato
extract)

3. Control No froth No froth No froth because reaction mixture

(No potato lacks the enzyme.
extract)

2. Effect of pH on enzyme activity

i) Set up the experiment as shown in the figure.

32 ii) Fill the burettes with water.

Experiment 5 Activity of Catalase and Study the Effect of pH and Enzyme Concentration
iii) Weigh approximately 5g of potato tuber and add 1-2 mL of water in
pestle and mortar and crush it. Keep the potato extract on one side
of the Y–shaped apparatus.

iv) Add 5mL of 3% H2O2 and 5mL of buffer of pH 1.0 on the other
side. Connect the apparatus to the burette.

v) Note down the initial level of water in the burette towards left side.

vi) Mix the contents of both the sides of apparatus and keep it for
about 5 minutes. Record final level of water in the burette. Repeat
the same procedure for other pH ranges, i.e. 4.0, 7.0, 9.0 and
12.0.

vii) Plot a graph with pH (X-axis) against the amount of water

displaced in the burette (Y-axis).

Fig 5.1: Estimation of catalase activity by Y –shaped apparatus.

ObservationTable

a) Effect of pH
S. pH of Substrate Enzyme Burette reading Volume of
No. buffer (5 mL) extract (5g) water
solution displaced
Initial Final
(mL)
(5 mL) (mL) (mL)
1. 1.0 3 % H2 O2 Potato tuber
2. 4.0 3 % H2 O2 ,,
3. 7.0 3 % H2 O2 ,,
4. 9.0 3 % H2 O2 ,,
5. 12.0 3 % H2 O2 ,,
33
BBYCL 138 Plant Physiology and Metabolism: Laboratory
b) Effect of enzyme concentration

Prepare a crude enzyme extract as in the above experiment. Make similar

enzyme extracts. Dilute the stock solution to 4%, 3%, and 1% by pipetting out
accurate amounts of enzyme and buffer of pH 7.0.

S. Substrate Enzyme Buffer Burette reading Volume

No. (5 mL) Extract pH 7.0 of water
Initial Final (mL)
displaced
(mL)
(mL)
1. 3 % H2 O2 1 mL 4 mL
2. 3 % H2 O2 2 mL 3 mL
3. 3 % H2 O2 3 mL 2 mL
4. 3 % H2 O2 4 mL 1 mL
5. 3 % H2 O2 5 mL 0 mL

5.5 RESULTS
The amount of water displaced in the burette indicates the amount of O2
evolved which in turn depends upon the catalase activity. Discuss your results
at every pH and find out how much water was displaced. Plot a graph between
pH (X-axis) and the amount of water displaced (Yaxis) to show the pH at
which maximum activity is recorded.

Likewise, plot a graph between enzyme concentration (X-axis) and the amount
of water displaced (Y-axis) to record the pattern of catalase activity as
influenced by its concentration. Rate of reaction increases with an increase in
the enzyme concentration. However, after a certain level the reaction rate
becomes constant due to lack of substrate for the active sites.

5.6 PRECAUTIONS
1. Buffer solution and enzyme extract should be freshly prepared

2. Reaction time should be constant for all readings.

3. Initial level of water must be recorded before mixing of contents in the Y-

shaped apparatus.

34
Experiment 6 To Study the Effect of Light intensity and Bicarbonate concentration
on O2 Evolution in Photosynthesis

EXPERIMENT 6
TO STUDY THE EFFECT OF
LIGHT INTENSITY
AND BICARBONATE
CONCENTRATION ON
O2 EVOLUTION IN
PHOTOSYNTHESIS

Structure
6.1 Introduction 6.5 Observations
6.2 Materials Required 6.6 Results
6.3 Principle 6.7 Precautions
6.4 Procedure

6.1 INTRODUCTION
Photosynthesis is an anabolic, endergonic, oxido-reductive process and can
be defined as the formation of carbohydrates from CO2 and H2O by illuminated
green (chlorophyll containing) cells with O2 and H2O being the by-products.

6CO 2 + 12H 2 O Light

→ C 2 H 12 O 6 + 6O 2 + 6H 2 O
Chlorophyl l
Carbohydra te

Various external factors affecting the process of photosynthesis are: light,

CO2, O2, temperature, and amount of available water. Since photosynthesis is
controlled by many factors which interact, it is also governed by the Law of
Limiting Factors (Blackman).

According to the concept of three cardinal points introduced by Sachs (1880),

there is minimum, optimum and maximum for each factor in relation to
photosynthesis.
For example, any species has a minimum temperature below which no
photosynthesis takes place, an optimum temperature at which the highest rate
takes place, and a maximum temperature above which photosynthesis ceases
altogether. 35
BBYCL 138 Plant Physiology and Metabolism: Laboratory
6.2 MATERIALS REQUIRED
Plant material : Fresh twigs of Hydrilla verticillata; Family-
Hydrocharitaceae
Chemicals : Filtered pond water, sodium bicarbonate

Glass apparatus : Measuring cylinder (100 mL), glass rod, beaker, pipette,
a large petri dish.
Miscellaneous : Meter rod, blade, thread, stop watch, taly counter, lux
meter, table lamps, electric balance, graph paper.

6.3 PRINCIPLE
Effect of light: Light affects photosynthesis through its intensity, quality and
duration. A direct relationship between the rate of photosynthesis and light
intensity is shown at the lower light intensities. As intensity of light is
increased, beyond a certain level, there is a decline in the photosynthetic rate
because of some other limiting factor or the destructive effects of high light
intensity (photo-oxidation). Also, the point of saturation may be achieved, after
which the rate of photosynthesis will remain constant.
Effect of CO2 concentration: The amount of CO2 in air, although small, is
relatively constant. It provides a steady and adequate supply of carbon dioxide
to the plant world. With an increase in the concentration of CO2 there is an
increase in the rate of photosynthesis at the lower levels of CO2 concentration.
A decline in rate is noticed at higher concentrations, as it may inhibit
respiration and also cause cell damage.
Bubble-counting method is used for measurement of photosynthesis in which
the rate of evolution of bubbles by a submerged aquatic plant is considered to
be proportional to the rate of apparent photosynthesis.

6.4 PROCEDURE
1. Cut a healthy or actively growing twig of Hydrilla, about 15 cm length
(with apex intact), under water. An oblique cut must be given to the
stem.

2. Place it in a measuring cylinder containing 100 mL of filtered pond water.

3. With the help of glass rod, tie the twig loosely so that when placed in
water it stays submerged under the water. The cut end of the twig should
face upwards whereas the intact shoot apex downwards.
4. When the twig starts producing bubbles uniformly, count the number of
bubbles given out from the cut end at an interval of one minute, using a
taly counter. Repeat counting at least thrice.
a) Effect of Light Intensity

Repeat the bubble counting under different intensities of light achieved by

varying the distance between the light source and the plant material.

Allow the plant to remain in each intensity of light for 5 minutes before starting
36 the count. Record the light intensity using lux meter.
Experiment 6 To Study the Effect of Light intensity and Bicarbonate concentration
on O2 Evolution in Photosynthesis

Fig.6.1: Experimental setup for studying effect of light and CO2 on

photosynthesis.

6.5 OBSERVATIONS
Observation Table
S. Environmental Light intensity Oxygen bubbles Mean
No. Factor (Lux) per minute
1. Diffuse Sunlight i)
ii)
iii)
2. Diffuse Sunlight i)
+ 1 Lamp ii)
iii)
3. Bright Sunlight i)
+ 2 Lamps ii)
iii)

Graph : Plot your recorded observations (Light Intensity -X-axis) vs Number of

bubbles evolved per minute -Y-axis) on a graph paper.

b) Effect of CO2 concentration

NaHCO 3 ⇌ Na + + HCO 3−
H 2 O → H + + OH −
Na + + OH − → NaOH
H 2 CO 3 H 2 O + CO 2
H + + HCO −
3 → →
Carbonic acid Both act as reactants in photosynthesis
37
BBYCL 138 Plant Physiology and Metabolism: Laboratory
The set up for this experiment is the same as explained in 6.4a. Effect of
different concentrations of NaHCO3 (source of CO2) on the rate of
photosynthesis is studied at a constant light intensity.
Dissolve 0.01 g NaHCO3 in 100 mL of filtered pond water (Sol.A). Wait for 5
minutes and record the number of oxygen bubbles evolved from the cut end of
Hydrilla twig per minute (3 concordant readings). Take out 5 mL of solution A,
and add 0.09 g of NaHCO3. Dissolve and then put the solution back into A.
This is now the solution B. Wait for 5 minutes and record the number of
oxygen bubbles evolved from the cut end of Hydrilla twig per minute
3 concordant readings). Similarly to prepare solution C, take out 5 mL of
solution B, add 0.9 g of NaHCO3 dissolve and then put the solution back into
B. Record the observations as done for solutions A and B. Sodium
bicarbonate powder is not added directly into the flask as it will not disperse
uniformly.

Sol. A : 0.01% - 0.01 g dissolved per 100 ml of pond water

Sol. B : 0.1% - 0.09 g + Sol. A (0.1 g/100 ml)

Sol. C : 1% - 0.9 g + Sol. B (1 g/100 ml)

Fixed Environmental Factor = Diffuse Sunlight + 1 Lamp (at a distance of

30cm)

S. Concentration of O2 bubbles evolved per Mean

No. NaHCO3 minute
1. 0.01 % i)
ii)
iii)
2. 0.1 % i)
ii)
iii)
3. 1% i)
ii)
iii)

Graph: Plot your recorded observations (NaHCO3 concentration (X-axis) vs

number of bubbles evolved per minute (Y-axis) on a graph paper.

6.6 RESULTS
With an increase in light intensity, the rate of photosynthesis goes on
increasing till some other factor (CO2 or temperature) becomes limiting. Very
high light intensity results in decline in photosynthetic rate.

With an increase in CO2 concentration, the rate of photosynthesis increases till

some other factor (light/temperature) becomes limiting. Also, higher CO2
concentrations are toxic to a plant.
38
Experiment 6 To Study the Effect of Light intensity and Bicarbonate concentration
on O2 Evolution in Photosynthesis
6.7 PRECAUTIONS
1. Hydrilla twig should be healthy, dark green, injury-free and with intact
shoot apex.

2. Oblique cut should be given to the twig, under water.

3. Twig should not touch the side walls of the cylinder and can be
supported by using glass rod.

4. Same twig should be used throughout the experiment.

5. The twig should be allowed to remain in each light intensity for 3-5
minutes before starting the bubble count.

6. The twig should be allowed to remain in each NaHCO3 concentration for

3-5 minutes before starting the bubble count.

7. A sufficient quantity of NaHCO3 (approx 0.1 g/100 ml) should be added

to the filtered pond water so that CO2 does not become a limiting factor.
It is desirable not to add different concentrations of NaHCO3 directly into
the solution in the cylinder but should be first dissolved in 5 ml of solution
withdrawn from the cylinder, using a pipette and then added back.

39
BBYCL 138 Plant Physiology and Metabolism: Laboratory

EXPERIMENT 7
TO COMPARE THE RATE OF
RESPIRATION IN ANY TWO
PARTS OF A PLANT
PLANT

Structure
7.1 Introduction 7.5 Observations
7.2 Materials Required 7.6 Results
7.3 Principle 7.7 Precautions
7.4 Procedure

7.1 INTRODUCTION
We all know that during photosynthesis, light energy is converted into
chemical energy which is stored in carbohydrate molecules in form of glucose
and starch. Living organisms use this energy for other activities by oxidising
these molecules into simple ones i.e., carbon dioxide and water. This reaction
is known as respiration. You can define respiration as a process by which
living cells break down complex high energy molecules into simple low energy
molecules, CO2 and H2O, releasing the energy trapped within the chemical
bonds. This energy is made available for various activities through an
intermediate compound known as adenosine triphosphate (ATP).

7.2 MATERIALS REQUIRED

• Whole plant of Ageratum conizoides
• Brine solution (saturated solution of NaCl); KOH pellets
• Buchner’s flasks (two sets), measuring cylinder (1000ml), beakers (250)
ignition tubes
• Pestle and mortar, electric balance, clamp standand black paper.

7.3 PRINCIPLE
You know that cellular respiration undergoes a series of independent
pathways through which carbohydrates and some other molecules are
oxidized for the purpose of retrieving the energy stored in photosynthetic
40 products.
Experiment 7 To Compare the Rate of Respiration in Any Two Parts of a Plant
The reaction which takes place in respiration may be summed up as follows:
C6H12O6 +6O2 6CO2 + 6H2O+ Energy
The rate of respiration depends upon amount of protoplasm and its state of
activity. Younger cells have highest protoplasmic activity because of active
growing regions while spores and dry seeds or drying leaves have low
respiration rates. Environmental conditions also affect the rate of respiration

7.4 PROCEDURE
1. First weigh equal amounts (7-10 g) of two organs of the same plant :
leaves and roots.

2. Make two set-ups consisting of Buchner’s flask with a rubber cork, an

ignition tube which contains a thick paste of KOH pellets and a beaker
containing brine solution as shown in Fig 7.1.

3. Place roots and leaves in a separate Buchner’s flask. Now take the flask
with green leaves and make the apparatus air tight, while the side tube is
immersed in the measuring cylinder containing brine solution. Cover this
flask with black paper to avoid photosynthesis.

4. In both set-ups suspend the side tube in a 250 ml beaker containing

brine solution. Note the initial level of brine solution and then record the
rise in level in the setup at 5.10,15 min intervals respectively. Repeat the
procedure with roots of the plant.

Fig 7.1: Setup to estimate respiration in plant parts.

7.5 OBSERVATIONS
Observation Table
Plant Rise in the level of brine solution Rate of respiration
material
d\t (mL)
Initial 5min 10 min 15 min

Roots
Leaves
41
BBYCL 138 Plant Physiology and Metabolism: Laboratory
7.6 RESULTS
The CO2 released during the respiration of roots and leaves is absorbed by
KOH in the ignition tube and a vacuum is created in the flask causing brine
level to rise. The rise in the level of brine in the side tube after 5, 10 and 15
minutes can be calculated by the formula as given. Compare the values
obtained from leaves and roots and see which organ respires more.

7.7 PRECAUTIONS
• You have to put same amount of plant material in both the sets.

• You should keep the ignition tube open throughout the experiment.

• Keep the graduated side tube untouched by the wall of the beaker.

• The apparatus should be vertical and air tight.

• Do not wash plant material before starting the experiment

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Experiment 8 To Separate Amino Acids by Paper Chromatograph

EXPERIMENT 8
TO SEPARATE AMINO ACIDS
BY PAPER CHROMATOGRAPH

Structure
8.1 Introduction 8.5 Observations

8.2 Materials Required 8.6 Results

8.3 Principle 8.7 Precautions

8.4 Procedure

8.1 INTRODUCTION
Living organisms contain thousands of different molecules in a single cell. If
we want to study a single molecule it is necessary to separate it from rest of
them. Chromatography is one of the most effective and widely used
techniques for separation and identification of biomolecules. Different kinds of
chromatography techniques have been evolved and improved in recent years.

In this exercise you will learn how to separate amino acids from a given
mixture by paper chromatography.

Objectives
After performing this exercise you should be able to:

• separate amino acids in a given mixture

• learn chromatography technique

• use the paper chromatography technique for separation of leaf pigments.

8.2 MATERIALS REQUIRED

• Amino acids, ninhydrin (0.2 % in acetone)

• Organic solvent (butanol:aceticacid:distilled water [Link])

• Chromatography jar with a lid, glass plate, capillary tube or microsyringe

• Whatman paper (No.1), atomizer, hair dryer, oven, pencil.

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BBYCL 138 Plant Physiology and Metabolism: Laboratory
8.3 PRINCIPLE
The solute separation is based on liquid – liquid partitioning in this technique.
The principle of paper chromatography technique is based on the difference in
solute partition between two immiscible phases – a stationary aqueous phase
which is strongly bound to the cellulose fibers of the paper, and mobile
organic phase passing through the paper through capillary action. The more
soluble a solute in the mobile phase, the farther the molecules will travel along
the paper. The migration rate of a substance may be expressed according to
its Rf (Resolution front/Relative front) value.

Rf is more or less constant for a compound provided the solvent system,

solute concentration, temperature and pH are carefully controlled.

Rf = Distance travelled by solute

Distance travelled by solvent front

8.4 PROCEDURE
1. Cut a sheet of Whatman no.1 paper to a convenient size. Draw a line 2.5
cm with the help of a pencil from the edge of the paper.

2. Now load the sample using fine capillary tubes, keeping the diameter of
each spot as small as possible. This process should be repeated 5 to 6
times. After each application you should dry the first spot with the help of
a hair –dryer.

3. In the mean time you should pre-saturate the jar in which you are going
to run the chromatographic paper with the vapours of organic solvent by
closing the lid of the jar so that the jar is saturated with the vapours of
the solvent.

4. Now lower the chromatography paper into the solvent to place the strip
in the centre of the jar. Take a thick square size paper, a few inches
bigger in size than the mouth of the jar. Fold it and place the Whatman 1
filter paper strip between the folds perpendicular to the line of fold. Pin
them together with a paper clip. Open the folded side. The strip can be
hanged in the jar keeping the thicker paper above the mouth. You may
use some other device to hang the paper. Make sure that its lower edge
is dipped in the solvent layer but the spot remains well above it. The
paper should not touch the walls of the jar.

5. Leave the jar undisturbed. Note the solvent front from time to time and
allow the solvent to run about 2/3rd of the length of the paper.

6. Remove the paper from the jar and immediately mark the position of the
solvent front with the help of a pencil.

7. Dry the chromatogram with hair-dryer. Now spray the paper with the
ninhydrin solution using an atomizer.

8. Dry the paper for 5 min at room temperature followed by at 100°C in an

44 oven for 2-3 min.
Experiment 8 To Separate Amino Acids by Paper Chromatograph

Fig.8.1: a) fully developed chromatogram showing various amino acids; b)

Experimental setup for descending paper chromatography.

8.5 OBSERVATIONS
Observation table

Solvent Front = cm

Distance travelled by Distance travelled by Rf value

amino acid solvent(s)

A A/S

B B/S

C C/S

8.6 RESULTS
Trace the chromatogram and show the amino acidswhich appears in blue,
purple or yellow spots (use pencil). 45
BBYCL 138 Plant Physiology and Metabolism: Laboratory
Make an outline of the spots visible on the chromatogram. Measure the
distance from baseline to the centre of each spot. Calculate the Rf for each
amino acid.

8.7 PRECAUTIONS
1. Hold the chromatogram from an edge only so as to avoid finger marks.

2. Spots should be kept small for maximum resolution.

3. The jar must be saturated with solvent. Therefore, do not leave it without
lid except while you hang chromatogram.

4. Do not disturb the apparatus once you have set it.

5. Always prepare the ninhydrin solution fresh.

6. Avoid contact with ninhydrin (preferably use a ventilated chamber).

7. You must mark the solvent front with pencil while the paper is wet.

8. Chromatography paper strip must always be cut in the machine

direction.

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