Laboratory Manual AGR114 Fundamentals of Crop Physiology: (For Private Circulation Only)

LABORATORY MANUAL
AGR114
FUNDAMENTALS OF CROP PHYSIOLOGY
(For private circulation only)
Name of the Student:………………………………………………..
Registration Number/Roll No……………………………………….
Section and Group……………………………………………………..
School of Agriculture (SAGR)
Session-term: 2020-21 (Spring Term)
Page 1
General guidelines for the students
1. A Student is expected to maintain the decorum of the laboratory by maintaining proper
discipline.
2. Student is expected to be punctual in lab, should keenly perform the experiment allotted to
him without moving from one lab to another and even experimental set up should not be left
until it unavoidable.
3. Mobile phones are not allowed in the labs and should be kept in the bags in silent or switch-
off mode.
4. Keep the work area clear of all materials except those needed for your work. Extra books,
purses, bags etc. should be kept in the racks placed in the lab.
5. Clean up your work area before leaving.

Dress code:
a- Shorts and sandals should not be worn in the lab at any time. Shoes are requiredwhen
working in the laboratories.
b- Students must have lab coat, gloves and mask with them every time.
Compulsory things to be carried by the students in lab:
Lab coat, gloves, mask, calculator, butter paper, fractional weights and stationary items.
Safety Guidelines:
i- Do not use any equipment unless you are trained and approved as a user by your supervisor.
ii- Wear safety glasses when working with hazardous materials or use such materials in fuming
hood.
iii- Wear gloves when using any hazardous or toxic agent.
iv- If you have long hair or loose clothes, make sure it is tied back or confined.
Page 2
TABLE OF CONTENTS
S. No. Name of the Experiment Page

No.
1 To study about the plant cell and its structure 4-8
2 To measure the stomatal frequency and index 9-12
3 To demonstrate the endosmosis and exosmosis by using potato 13-16

osmoscope
4 To demonstrate endosmosis by using raisins and exosmosis by using 17-19

grapes
5 To demonstrate the phenomenon of imbibition 20-23
6 To demonstrate the phenomenon of plasmolysis 24-26
7 To demonstrate the effect of different wavelength of light on 27-29

photosynthesis by Ganong’s light screen.
8 To separate the chloroplast pigment by strip paper chromatography 30-33
9 To measure the rate of respiration of different respiratory substances by 34-36

pipette manometer
10 To measure the transpiration rate 37-40
11 To study about the measurement of water status in roots, stem and leaves 41-44
of the plants
12 To perform tissue test for mineral nutrients 45-49
Page 3
Experiment 1
Experiment: To study about the plant cell and its structure.

1. Materials required: Slides, cover slips, microscope, Distilled water, leaf of a plant.
2. Learning Objectives: Students will learn about the plant cell and its structure.
3. Theory: A plant cell is the structural and functional unit of a plant
Different types of plant cells
1. Parenchyma cell
2. Collenchyma cell
3. Sclerenchyma cell
4. Phloem
5. Xylem
a- Parenchyma cells are usually depicted as the typical plant cell because they are not
very specialized. These cells synthesize and store organic products in the plant. Most of
the plant's metabolism takes place in these cells.
b- Collenchyma cells have a support function in plants, particularly in young plants. These
cells help to support plants while not restraining growth due to their lack of secondary
walls and the absence of a hardening agent in their primary walls.
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i- Sclerenchyma cells also have a support function in plants but unlike Collenchyma cells,
they have a hardening agent and are much more rigid.
ii- Phloem tissue: Phloem tissue consists of: conducting cells, generally called sieve.
Elements; parenchyma cells, including both specialized companion cells or
albuminous cells and unspecialized cells; and supportive cells, such as fibers and
sclereids
iii- Xylem tissue: The most distinctive xylem cells are the long tracheary elements that
transport water. Tracheids and vessel elements are distinguished by their shape; vessel
elements are shorter, and are connected together into long tubes that are called vessels.
4. Outline of the Procedure: Take the leaf of a plant. Prepare the slide and observed under
microscope. Observe the parts of a plant cell.
Fig. Plant cell structure
Page 5
Parts of plant cell and their function:
Part Function
Nucleus Contains genetic material, which controls the activities of the cell
Cytoplasm Most chemical processes take place here, controlled by enzymes
Cell membrane Controls the movement of substances into and out of the cell
Mitochondria Most energy is released by respiration here
Ribosomes Protein synthesis happens here
Cell wall Strengthens the cell
Chloroplasts Contain chlorophyll, which absorbs light energy for photosynthesis
Permanent Filled with cell sap to help keep the cell turgid
vacuole
5. Results: Students will learn about the cell and its organelles
6. Scope of the Results: Students will acquaint with the basics about the cell and its
organelles.
7. Caution:
• Pealing of material should be very fine
• Mounting in pealed material should be in optimum concentration
• Handling of microscope should be very fine
Suggested readings for students:

Books:
1. MODERN PLANT PHYSIOLOGY by R. K. SINHA, NAROSA PUBLISHING HOUSE

2. PHYSIOLOGY OF CROP PLANTS by FRANKLIN P. GARDNER,R.B. PEARCE,R.L. MITCHELL,
SCIENTIFIC PUBLISHERS
Web links:
www.biologydiscussion.com
Page 6
Worksheet of the student
Date of performance: Registration No:
Aim: To study about the plant cell and its structure.
Observations:
Results and discussion:
Page 7
Learning Outcomes: (What I have learnt)
To be filled by faculty
Sr. Parameters Mark obtained Maximum
No marks
1 Understanding of the student 20
about the procedure/apparatus
2 Observations and analysis including 20

outcomes
3 Completion of experiment, discipline 10

and cleanliness
4 Signature of the faculty 50
Page 8
Experiment 2
Experiment: To measure the stomatal frequency and index

1. Materials Required: Microscope, stage micrometer, ocular micrometer, Plant leaf samples,
blotting paper.
2. Learning Objectives: To help the students understand the basic mechanism behind the process
of transpiration which involve movement of stomata.
3. Theory: In botany, a stoma (plural stomata) (occasionally called a stomate, plural stomates) is a
pore, found in the epidermis of leaves, stems and other organs that is used to control gas
exchange. The pore is bordered by a pair of specialized parenchyma cells known as guard cells
that are responsible for regulating the size of the opening. The term is also used collectively to
refer to an entire stomatal complex, both the pore itself and its accompanying guard cells. Air
containing carbon dioxide and oxygen enters the plant through these openings and is used in
photosynthesis in the mesophyll cells (parenchyma cells with chloroplasts) and respiration,
respectively. Oxygen produced as a by-product of photosynthesis diffuses out to the
atmosphere through these same openings. Also, water vapor is released into the atmosphere
through these pores in a process called transpiration. Stomata are present in the sporophyte
generation of all land plant groups except liverworts. Dicotyledons usually have more stomata
on the lower epidermis than the upper epidermis. Monocotyledons, on the other hand, usually
have the same number of stomata on the two epidermises. In plants with floating leaves,
stomata may be found only on the upper epidermis; submerged leaves may lack stomata
entirely.
4. Outline of Procedure:
• Start with a peel from the lower leaf surface. At 100X count the number of stomata in
the entire square grid, or, if you find the stomata too numerous.
• Count the number of stomata in 20 contiguous small squares of the grid
• Multiply by 5 to get an estimate for the entire grid.
Repeat on the upper surface peel. (In one of the oculars of your microscope you will view a
square ocular grid superimposed on the microscopic image. The grid is composed of 100
identical small squares. At 100X magnification each of these small squares has an area of
10,000 µm 2 for a total grid area of 1 mm 2).
General Calculations: The stomata included under the whole grid at 100X total magnification
represent the number of stomata in 1mm. If you count the stomata under the whole grid at
400X total magnification multiply by 16 for the final number of stomata in 1mm. If, at 400X,
you count only 20 small squares of the grid you need to multiply by 5 and by 16 for the number
of stomata per mm2.
• Calculate the number of stomata per mm2 for both the lower and the upper leafsurface.
Page 9
• Sum the upper and lower to record total stomatal density in 1 mm2 of leafarea.
• Record your lower, upper, and total stomatal density data on the class data sheet.
Post images in SAKAI. For each plant species post the best image of a cross-section and
stomatal peel taken at 100x total magnification through the eye piece of your microscope with
the grid and without zooming the camera. Be sure to name the images. These images will be
needed for your composite figure assignment.
5. Results: Stomata were observed and it was observed that number of stomata on the lower side
of the leaf is more than the upper surface in dicot leaves whereas in monocot leaves numbers of
stomata on both upper and lower surface are almost same.
6. Scope of the Results: Students will be able to understand the nature of water loss in the plants
which is brought about by the opening and closing of stomata.
7. Caution:
• Multiply factor is different for both cases.
• For more accuracy upper and lower surfaces of leaf, should be taken.

Books:
1- MODERN PLANT PHYSIOLOGY by R. K. SINHA, NAROSA PUBLISHING HOUSE
2- PHYSIOLOGY OF CROP PLANTS by FRANKLIN P. GARDNER,R.B. PEARCE,R.L. MITCHELL,
Web links
Page 10
Aim: To measure the stomatal frequency and index
Observation:
Result and discussion:
Page 11
Sr. Parameters Mark obtained Maximum marks
No

outcomes

and cleanliness
Page 12
Experiment 3
Experiment: To demonstrate the endosmosis and exosmosis by using potato osmoscope

1. Materials required: Beaker, petridishes, pin, Tubers, concentrated sugar solution, water,
capillary tubes and corks.
2. Learning Objectives: To get the students acquaint with both the process of osmosis
(endosmosis and exosmosis) using potato osmoscope.
3. Theory:
A semi-permeable membrane having very small sized pores allows only water to pass through
the pores but prevent the movement of solute across them. The tuber wall acts as a semi-
permeable membrane.
The movement of solvent molecules from the region of their higher concentration to the region
of their lower concentration through a semi permeable membrane is called osmosis.
Peel off the outer skin of three potato tubers. Cut one end flat. Make a hole or cavity in the
center of the potato, almost up to the bottom. These are osmoscope (1), (2) and (3).
Osmoscope (1)- Fill the cavity with concentrated sugar solution. Fit an airtight cork at the
mouth of the cavity. Insert a capillary tube in the hole of the cork. Mark the level of sugar
solution if any, in the capillary tube. Put this assembly in a petri dish filled with water. Allow
the experiment to remain as such.
Osmoscope (2)- Fill the cavity of potato tuber with concentrated sugar solution. Mark the level
in the cavity by piercing pin. Place this tuber in a petri dish with pure water.
Osmoscope (3)- The cavity of the other tuber s filled with water and level is marker by piercing
a pin. This tuber is placed in a petri dish containing concentrated sugar solution.
Page 13
5. Results:
1. The level begins to rise in the capillary and after sometime become stable. In this case, the
flow of solvent is along the concentration gradient, i.e. the osmotic concentration of the sugar
solution being higher and that of pure water being almost zero. The water moves through the
cells of the tube which act as semi-permeable membranes.
2. The initial level ‘A’ in this tuber rise to level ‘B’ after sometime. The increase is due the
movement of water from the outside (i.e., petri dish) through semi-permeable membranes of the
potato tuber. Since the osmosis shows the movement of water from outside (i.e., environment)
into the tuber, the process is endosmosis. There is an increase in the volume whenever
endosmosis takes place.
3. The initial level ‘A’ in this tuber to level ‘B’ after sometime. The decrease or fall in the
level is due to the movement of water from inside the tuber to the outside. The semi-permeable
membranes of the cells of the potato tuber allow this movement. The concentration of the
solvent molecule, being less in the sugar solution placed in the petri dish, the water molecules
move from the region of its higher concentration (i.e., inside the potato tuber) towards the
outside (i.e., into the petri dish) since the phenomenon of osmosis here shows the loss of water
from inside the tuber. It is called exosmosis.
6. Scope of the Results: Students will be able to understand the process of endosmosis and
exosmosis.
7. Cautions:
i. The cavity of potato must be larger to pour sufficient amount of sugar solution.
ii. The initial level of sugar solution should be marked carefully.
iii. The water level in the Petri dish should be enough to dip a major portion of potato tuber.

Books:
Web link:
Page 14
Aim: To demonstrate the endosmosis and exosmosis by using potato osmoscope
Observations:
Page 15
To be filled in by the faculty

S. No. Parameters Marks obtained Max. Marks
1 Understanding of student about 20
procedure/apparatus

learning outcomes
3 Completion of experiments, disciplines 10
and cleanliness
Signature of Faculty
Page 16
Experiment 4
Experiment: To demonstrate endosmosis by using raisins and exosmosis by using grapes.

1. Materials Required: Watch glass or beaker, Glucose, raisins or dried grapes and grapes
2. Learning Objectives: To get the students acquaint with both the process of osmosis
(endosmosis and exosmosis) using grapes and raisins.
3. Theory: A semi-permeable membrane having very small sized pores allows only water to
pass through the pores but prevent the movement of solute across them. The tuber wall acts
as a semi-permeable membrane.
The movement of solvent molecules from the region of their higher concentration to the
region of their lower concentration through a semi permeable membrane is called osmosis.
a- Put some raisins in a beaker half filled with water. Allow them to be dipped for 3-4 hrs.
b- Take concentrated sugar solution in a beaker and dip some fresh grapes in it. Leave the
grapes in sugar solution for 3-4 hrs.
5. Results:
a- The relative concentration of water (solvent molecules) is more in the beaker’s water
than in raisins. So, the water from its high concentration flows in to the raisins through
the semipermeable membrane. This experiment demonstrates the process of
endosmosis, by which the raisins swell and become turgid. Here the water is hypotonic
as compared to cell sap.
b- The sugar solution has more solute molecules or less solvent molecules as compared to
grapes, so is hypertonic. The water (solvent) molecules from its higher concentration (from
grapes) flow out to its low concentration (to the beaker).
Page 17
6. Scope of the results: Students will be able to understand the process of endosmosis
and exosmosis.
7. Caution:
i- The suitable concentration of sugar is require to get the result of exosmosis
ii- Sufficient time is requiring to get remarkable result.

Books:
Web link:
Page 18
Aim: To demonstrate endosmosis by using raisins and exosmosis by using grapes
Observations:

1 Understanding of student About 20
procedure/apparatus

learning outcomes
and cleanliness
Signature of Faculty 50
Page 19
Experiment 5
Experiment: To demonstrate the phenomenon of imbibition

1. Materials Required: Watch glass or beaker, weighing balance, three different types of seeds
(carbohydrate rich, oil rich and protein rich) ex: Pea seed or gram seeds, water beaker and NaCl
2. Learning Objectives: To get the students acquaint with the different rate of imbibition in
different kind of seeds.
3. Theory: Imbibition is the phenomenon of adsorption of water by the solid particles of a

substance without forming a solution. It is also defined as the phenomenon by which the
living or dead plant cell absorbs water by surface attraction, dynamic invasion with constant
flow rate of the displacing fluid. Substance which can imbibe or absorb a liquid without
forming a solution is known as Imbibants.
4. Outline of the Procedure:
1. Weight 5 gm of three different types of seeds separately and place in separate.
2. Pour 50 ml of distilled water in each beaker and keep at room temperature for one hour.
3. After one hour remove them and blot off the surface water.
4. Again weigh the soaked seeds separately.
5. Record the data and determined percentage of water absorption by imbibition.
5. Result: Swelling of the seed is due to a process known as imbibition in which water has
diffused from outside in to the dry seeds whose osmotic pressure is almost zero.
The process of entering of water will continue so far there is a difference in the diffusion pressure
between the liquid and Imbibants, i.e., pea seeds.
Comparison of rate of imbibition of starchy, oily and proteinaceous
Page 20
6. Scope of the results: Students will be able to understand the process of imbibition
7. Caution:
Weighing and blotting of seed before and after the dipping of seed in to experimental work
should be very precise.

Books:
Web link:
Page 21
Aim: To demonstration the phenomenon of imbibition
Observation:
Page 22

No

outcomes
3 Completion of experiment, 10
discipline and cleanliness
Page 23
Experiment 6
Experiment: To demonstrate the phenomenon of plasmolysis.

1. Materials Required: Safety blade and microscope, Tradescantia /Rhoeo discolor leaf, sugar
solution of different concentrations, cover slips, water.
2. Learning Objectives: To get the students acquaint with the phenomenon of plasmolysis.
3. Theory: Plasmolysis is the shrinkage of the protoplast of a plant cell from its cell wall due to loss
of water under the influence of hypertonic solution. If a plant cell is placed in a hypertonic solution,
the plant cell loses water and hence turgor pressure, making the plant cell flaccid. Water loss
decreases pressure to the point where the protoplasm of the cell peels away from the cell wall,
leaving gaps between the cell wall and the membrane. Plasmolysis can be reversed if the cell is
placed in a hypotonic solution.
Peel off a small segment from the lower surface of the leaf. This can be done by leaving the
leaf obliquely with a single jerk or scraping it with safety blade.
a- Mount the peel in a drop of water on a side and then place a cover slip. Observe under the
microscope.
b- Take another peel and similarly mount pieces in a drop of sugar solution of different
concentration. Observe each preparation under the microscope.
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5. Result:
a- In this condition the cell structure can be seen clearly. Note particularly the turgid cells and
colored cell sap.
b- When slightly concentrated sugar solution is used for mounting. The cell contents width
draw a little from the cell wall. Note the colorless space between cell wall and the colored cell
sap.
c- When a little more concentrated sugar solution is used for mounting. The cell contents move
appreciably away from the cell wall, leaving a considerable space between the wall and the sap.
d- When the peel is mounted in a drop of highly concentrated sugar solution, the cell contents
withdraw from the cell wall and shrink into a small, round ball-like form.
6. Scope of the Results: Students will acquaint the with basics of plasmolysis in plant cell
7. Cautions:
i. Sugar solution concentration must be correctly prepared.

ii. Peelings should be done from lower epidermis of leaf very carefully.
iii. Mount the peeling in glycerin carefully and avoid air bubbles and folding of peelings.

Books:
Web link:
Page 25
Aim: To demonstrate the phenomenon of plasmolysis.
Observations:
No
outcomes
and cleanliness
Page 26
Experiment 7
Experiment: To demonstrate the effect of different wavelength of light on photosynthesis by

Ganong’s light screen.
1. Materials Required: A large ‘Ganong’s light screen’-like box in which the leaf can be
inserted, glass top covered with blue, green and red colours, Iodine and plant twig, stand etc.
2. Learning Objectives: To get the students acquaint with the effect of different wavelength of
light on photosynthesis.
3. Theory:
Photosynthesis is the process by which the green plants convert atmospheric CO2 into organic
compounds using the energy from sunlight. Photosynthesis changes the energy from the sun
into chemical energy, splits water to release Oxygen (O2), and fixes Carbon dioxide (CO2)
into sugar. There is a linear relationship between incident light and CO2 fixation rates at low
light intensities. At higher light intensities, gradually the rate does not show further increase as
other factors become limiting. What is interesting to note is that light saturation occurs at 10
per cent of the full sunlight. Hence, except for plants in shade or in dense forests, light is rarely
a limiting factor in nature. Increase in incident light beyond a point causes the breakdown of
chlorophyll and a decrease in photosynthesis.
4. Outline of the procedure:

1. Place a potted plant in darkness for about 24 hours. It will make its leaves de-starched.
2. Fix a de-starched leaf below the glass top of the box and keep the apparatus in sunlight
3. Detach the leaf after a few hours. The chlorophyll is removed.
Page 27
4. Stain the leaf with iodine to test for the presence of starch.
5. Compare the intensity of starch in the three parts of the leaf.
5. Results:
1. Negative staining in the green region indicates that photosynthesis process has not taken
place in this region. So, green wavelength is ineffective in photosynthesis.
2. Darkest staining in the red region indicates that maximum photosynthesis has taken place in
this region. And this has finally resulted in the largest accumulation of starch in this region.
3. Second darkly-stained region is the blue region of the leaf. This indicates that photosynthesis
has taken place in this region also, but it happened at a lower rate than that of red region.
So, red wavelength is most effective, the blue wavelength comes next in order and the green is
least effective.
6. Scope of the Results: Students will get acquainted with the basics of the effect of different
wavelength of light on rate of photosynthesis using Ganong’s light screen.
7. Caution:
• Students should be very careful during the handling of instrument (Ganong’s light screen).
• Leaf sample should be insert properly inside of instrument

Books:

Web links:
Page 28
Aim: To demonstrate the effect of different wavelength of light on photosynthesis by Ganong’s

light screen.
Observations:

procedure/apparatus
learning outcomes
and cleanliness
Page 29
Experiment 8
Experiment: To separate the chloroplast pigment by strip paper chromatography.

1. Materials Required: Gas jar, sliding glass rod, hook and rubber cork, Chlorophyll extract,
Whatman No. 1 filter paper, petroleum ether, acetone, lead pencil.
2. Learning Objectives: Students will be able to understand about the separation of different
chlorophyll pigments by using strip paper chromatography method
3. Theory:
Chlorophyll is in fact only one pigment in a group of closely related pigments commonly
found in photosynthesising plants called photosynthetic pigments. This can be demonstrated
by extracting the pigments from leaves with acetone and separating them by means of paper
chromatography. With a bit of luck five pigments can be identified: chlorophyll a (blue-green),
chlorophyll b (yellow-green), xanthophylls (yellow), carotene (orange) and phaeophytin (grey,
it is a breakdown product of chlorophyll.

1. Cut fine strips of spinach leaves, place in a clean mortar and reduce it to pulp with pestle.
Add 5ml of precooled acetone. Stir well and filter through fine linen cloth. This deep green
colored acetone extract contains all the four pigments. Leave this solution at 60 degree C for 6-7
hours, uncovered in an oven for being concentrated.
2. Take a perfectly dry, long strip of Whatman No. 1 filter paper and draw a fine line with a
lead pencil, parallel to about 1.5 cm from one edge. This will indicate the bottom of your
chromatogram.
3. Draw a circle on this line just in the middle and put a drop of chlorophyll extract just in the
circle. Let it dry. (Note—avoid excess handling of the chromatography paper, since your hands
may contaminate it with amino acids. Touch it only at the edges).
4. Repeat the process of putting and drying the drop of chlorophyll extract for 2-3 times.
5. Take the rubber cork of the jar fitted with glass rod having a hook at its lower end, and
attach the upper portion of the chromatography paper with the hook.
6. About 2 cm of the jar is filled with the solvent (petroleum ether: acetone = 100:12).
Page 30
7. Insert the strip attached with the hook in the jar and sees carefully that lower edge of the
strip touches the solvent. Note carefully that chlorophyll spot should not be touched by the
solvent and paper should not touch the wall of the jar.
8. Keep the whole apparatus as such for 20 to 30 minutes and see that solvent has risen nearly
up to the top of the paper
5. Results:
Separation of different pigments on strip is based on the fact that paper chromatography
separates compounds on the basis of their different rates of migration on filter paper (cellulose).
The rate of migration depends upon the solvent which is flowing up and also on the relative
adsorption which holds the molecules more or less tightly to the paper
Page 31
6. Scope of the Results: Students will acquaint with the basics about the separation of different
pigments from the given plant sample.
7. Caution:
i- Students should be very careful during the handling of glass wares (Belzar, beaker and
measuring cylinder).
ii- Students should be very careful during the handling of chemicals

Books:
Web link:
Page 32
Aim: To separate the chloroplast pigment by strip paper chromatography
Observations:
Learning outcomes (what I have leant):

procedure/apparatus
learning outcomes
and cleanliness
Page 33
Experiment 9
Experiment: To measure the rate of respiration of different respiratory substances by pipette

manometer.
1. Materials Required: Specimen tube, small test tube, graduated pipette, cork, beaker weighing
balance and glass tube, Potassium hydroxide, respiratory substance (germinating seeds,
flower buds etc.)
2. Learning Objectives: Students will be able to understand about the measurement of

respiration rate of different respiratory substrate by pipette manometer
3. Theory:
Respiration is the process during which simple carbohydrates, like glucose, break down into
simpler substances and liberate carbon dioxide and energy. The compound used, or oxidized,
during respiration is called a respiratory substrate. Carbohydrates, fats, and proteins are
examples of respiratory substrates, and carbohydrates are the preferred respiratory substrate
among them. The rate of respiration can be measured in terms of gas exchange, that is,
consumption of the respiratory substrate oxygen, or evolution of carbon dioxide.
4. Outline of the procedure:
i. Take the respiratory material, e.g. germinating seeds or flower buds in a specimen tube or
large test tube.
ii. Fill the small test tube with KOH pellets and place into the specimen tube.
iii. Fit the graduated to specimen tube through a cork.
iv. Weight the entire apparatus.
v. Put the apparatus in a water filled glass tub in such a manner that the tip of pipette must be
above the water surface.
vi. Immerse the tip of the pipette into the water after a period of ten minutes.
vii. Record the rise in the water level of the pipette at an interval of 10 minutes for one hour.
viii.The experiment can be repeated with different respiratory substances.
5. Results:
1. Suppose the weight of respiratory substance=10gm
2. The rise of water level of the pipette (in one hour)= 2ml.
3. So the total observed oxygen =2 ml.
4. Total observed oxygen by one gram of the substance= 2ml/10gm
Page 34
5. Total absorbed oxygen by one gram of the substance per hour of time period=2ml/10gm per
hour So the respiration rate =0.2 ml. oxygen/gm/hour
6. The above calculated absorbed oxygen per gram of substance per hour is called respiration
rate of that particular substance.
6. Scope of the Results: Students will get acquainted with the measurement procedure of rate
of respiration in different respiratory substrates.
7. Caution:
• Handling of instrument should be very carefully
• Observation and calculation of respiratory substrate should be very precise
Books:
Web links:
Page 35
Aim: To measure the rate of respiration of different respiratory substances by pipette manometer
Observations:
Learning outcomes (what I have learnt):

procedure/apparatus
learning outcomes
and cleanliness
Page 36
Experiment 10
Experiment: To measure the transpiration rate.

1. Materials Required: Desiccator, hot air oven, CoCl2, potted plant, cello tape, slide,
rubber bands and filter paper strips,
2. Learning objectives: Demonstrate the rate of transpiration on different surfaces of the
leaf in different plants.
3. Theory:
Transpiration is the process of water movement through a plant and its evaporation from
aerial parts especially from leaves but also from stems and flowers. Leaf surfaces are
dotted with pores which are called stomata, and in most plants they are more numerous
on the undersides of the foliage. The stomata are bordered by guard cells and their
stomatal accessory cells (together known as stomatal complex) that open and close the
pore. Transpiration occurs through the stomatal apertures, and can be thought of as a
necessary "cost" associated with the opening of the stomata to allow the diffusion of
carbon dioxide gas from the air for photosynthesis. Transpiration also cools plants,
changes osmotic pressure of cells, and enables mass flow of mineral nutrients and water
from roots to shoots.
Mass flow of liquid water from the roots to the leaves is driven in part by capillary
action, but primarily driven by water potential differences. In taller plants and trees, the
force of gravity can only be overcome by the decrease in hydrostatic (water) pressure in
the upper parts of the plants due to the diffusion of water out of stomata into the
atmosphere. Water is absorbed at the roots by osmosis, and any dissolved mineral
nutrients travel with it through the xylem.
i- Prepare 100 ml of 5% cobalt chloride solution by dissolving 5g of cobalt chloride
in 100 ml distilled water.
ii- Cut filter paper into small strips and immerse them in cobalt chloride solution
taken in a petri dish for 3-5 minutes.
iii- With forceps, transfer the soaked strips on to the wire gauge and allow excess
CoCl2 solution to drain off.
iv- Dry the filter paper strips on hot plate/oven taking care not to burn or char the
paper.
v- The anhydrous cobalt chloride coated strips will be blue in colour. Store them in a
desiccator.
vi- Select a leaf of a potted plant growing in sunlight. If water droplets are seen blot
the leaves dry with cloth/blotting sheet.
vii- Keep one dry (strip) cobalt chloride paper on the upper surface of leaf and stick it
with cello- tape.
Page 37
viii- Similarly stick another strip of CoCl2 on the lower surface. The CoCl2 strips can
be held in position with the help of two slides and rubber bands (Fig).
ix- Place the potted plant in sunlight.
5. Results: Transpiration of the monocot and dicot leaves was measured and it was found
that in monocot leaves transpiration rate of both the sides is same whereas in dicot
leaves lower surface is more active in transpiration in comparison to upper surface.
6. Scope of the Results: To make the students familiar with the process of transpiration as
it affects many important processes in plants life.
7. Caution:
• All measurements should be taken carefully.
• All chemicals should be weighed in recommended quantity.

Books:
• MODERN PLANT PHYSIOLOGY by R. K. SINHA, NAROSA PUBLISHING HOUSE
• PHYSIOLOGY OF CROP PLANTS by FRANKLIN P. GARDNER, R. B. PEARCE, R.L.
MITCHELL, SCIENTIFIC PUBLISHERS
Web links:
Page 38
Aim: To measure the transpiration rate
Observations:
S. No. Name of plant Time taken for change in color from blue to
pink (in min)
Upper surface Lower surface
Page 39
S. Parameters Marks obtained Max. Marks

No.
1 Understanding of student About 20
procedure/apparatus
2 Observations And analysis including 20
learning outcomes
and cleanliness
Page 40
Experiment 11
Experiment: To study about the measurement of water status in roots, stem and leaves of the
plants.
1. Materials Required: Hot air oven, Petri plate, weighing scale and distilled water.
2. Learning Objectives: To learn about the methods for the measurement of water content in
different plant parts and predicting its health.
3. Theory:
Leaf water status is intimately related to several leaf physiological variables, such as leaf
turgor, growth, stomatal conductance, transpiration, photosynthesis and respiration
(Kramer & Boyer, 1995). Water content and water potential (Ψw) have been widely used
to quantify the water deficits in leaf tissues. Leaf water content is a useful indicator of
plant water balance, since it expresses the relative amount of water present on the plant
tissues. On the other hand, water potential measures the energetic status of water inside the
leaf cells (Slatyer & Taylor, 1960). Measurements of water content expressed on a tissue
fresh or dry mass basis have been mostly replaced by measurements based on the
maximum amount of water a tissue can hold. These measurements are referred to as
Relative Water Content (Barrs, 1968; Boyer, 1968). The relative water content (RWC) of
a plant tissue is expressed by RWC (%) = [(FW - DW)/(TW - DW)] * 100, where, FW,
DW and TW are the fresh, dry and turgid weight, respectively, of the tissue.
i- Relative water content (RWC) was determined by Weatherly method modified by
Slalyter and Barrs, 1965.
ii- Initially 5 cm long pieces of leaves will be weighed and fresh weight will be
recorded and placed in a petri plate having distilled water.
iii- After 6-7 hrs, pieces would be blotted dry and turgid weight will be recorded then
they would be dried in the oven at 80 - 85˚C for 24 h.
iv- After 24 hrs the dry weight would be recorded and the % relative water content
(%RWC) would be calculated using the formula.
General Calculations:
FW – DW
RWC (%) = 100
TW- DW
Where, RWC = Relative water content DW = Dry weight

FW = Fresh weight
TW = Turgid weight.
Page 41
5. Results: Relative water content of different plant parts was determined and observed that
RWC of green tissues ranges from 70-90%, of stem tissues ranges from 50-60% and in
root tissues it ranges from 50-60%.
6. Scope of the Results: The students will learn about the measurement of water balance of
plants and that water balance is the first thing affected in any type of stress which plant
faces in their day to day life.
7. Caution:
• Proper recording of fresh weight of the leaves should be done
• Careful recording of turgid weight of leaf samples should be done
• After drying of the samples their dry weight should be recorded accurately.
Books:
MODERN PLANT PHYSIOLOGY by R. K. SINHA, NAROSA PUBLISHING HOUSE
PHYSIOLOGY OF CROP PLANTS by FRANKLIN P. GARDNER,R.B. PEARCE,R.L.
MITCHELL, SCIENTIFIC PUBLISHERS
Web link:
Page 42
Aim: To study about the measurement of water status in roots, stem and leaves of the plants.
Observations:
Page 43

procedure/apparatus
learning outcomes
and cleanliness
Page 44
Experiment 12
Experiment: To perform tissue test for mineral nutrients.
1. Materials Required: Leaf, petri dish, Test tubes,Diphenylamine, sulphuric acid, sucrose,
ammonium molybdate solution, distilled water, HCl, ethyl alcohol, glacial acetic acid
2. Learning Objectives: Students will be able to perform tissue test for mineral nutrients
3. Theory:
The crop growth and productivity is conditioned by many factors of which, the nutrient status
(Content) of plant parts such as leaf, stem, etc. play a critical role. Moreover the leaf and stem
are considered as the indicator parts of plants for assessing the nutrients content of plant. Each
crop plant requires the essential element at a specific concentration at different growth stages
and it is known as ‘critical level’. When the nutrients content of plant depletes below the
critical level the plants may exhibit some symptoms. The requirement or otherwise the
availability of nutrients can be assessed by i) plant diagnosis ii) soil analysis and iii) plant
analysis by two methods a) by qualitative test and b) by quantitative estimation. Based on the
plant or soil tests, the required nutrients can be applied for crops to sustain the growth and
rectify the deficiency disorders. The rapid tissue test would pave way for rectifying the
nutritional problems for quick recovery; however the quantitative estimation of both plant and
soil for nutrients concentration will be more useful and economic for applying fertilizers either
as basal or foliar and would be the long term strategy to cope up with nutritionalproblems.
4. Outline of the Procedure

a- Nitrogen
Reagent: 1-% diphenylamine in conc. sulphuric acid.
Small bits of leaf or petiole are taken in a petri dish and a drop of 1% diphenylamine is added.
The development of blue colour indicated the presence of nitrate – nitrogen. The degree of
coloration indicates the amount of nitrogen present in that leaf.
• Dark blue: Sufficient Nitrogen
• Light blue: Slightly deficient Nitrogen
• No colour: Highly deficient Nitrogen
b- Phosphorous
Reagents:
(1) Ammonium molybdate solution,
(2) Stannous chloride powder.
Eight gm ammonium molybdate is dissolved in 100 ml of distilled water. To this, add 126 ml
of conc. Hydrochloric acid (HCL) and volume is made up to 300 ml with distilled water. This
Page 45
stock solution is kept in an amber colored bottle and at the time of use it is taken and diluted
in the ratio of 1:4 using distilled water.
A tea spoonful of freshly chapped leaf bits are taken in a test tube and 10 ml of ammonium
molybdate reagent is added and kept for few minutes. After shaking, a pinch of stannous
chloride is added. Colour development is observed.
• Dark blue: Sufficient Phosphorus

• Bluish green: Slightly deficient Phosphorus
• No colour: Highly deficient Phosphorus
c- Potassium
Reagent: (1) Sodium cobalt nitrate reagent, (2) Ethyl alcohol (95%).
Take 5 gm cobalt nitrate and mix with 30 gm of sodium nitrate in 80ml of distilled water. To
this, 5ml of glacial acetic acid is added. The volume is made up to 100 ml distilled water.
Dilute reagent prepared (5 ml) with 15 mg sodium nitrate to 100 ml using distilled water.
Finally cut leaf bits are taken in a test tube and 10 ml diluted reagent is added and shaken
vigorously for few a minutes and kept for 5 minutes. Then add 5 ml of ethyl alcohol reagent,
allowed to stand for 3 minutes. The solution is observed for the formation of turbidity.
• No turbidity: Deficiency of Potassium

• Slightly turbidity: Moderate deficient
• High turbidity: Sufficient Potassium
d- Calcium
Morgan’s Reagent: 30 ml of glacial acetic acid and 100 grams of sodium acetate are
dissolved in a little of distilled water
Procedure: 0.5 g of finally cut plant material is taken into a glass vial (both of healthy plant
and deficient plant in different vials) and 5 ml of Morgan’s reagent is added in test tube. After
allowing it to stand for 15 minutes, 2 ml of glycerin and 5 ml of 10% ammonium oxalate is
added and the solution is shaken for 2 minutes. The turbidity resembling after 15 minutes
indicate the amounts of calcium in normal plant tissue.
e- Magnesium Reagents:
a. 5% pure sucrose solution
b. 2% Hydroxylamine hydrochloride
Page 46
c. Titan Yellow
d. Sodium hydroxide
150 mg of Titan yellow is dissolved in 75 ml of 95% ethyl alcohol and 25 ml distilled water.
This solution is stored in darkness.
Procedure
To a tea spoonful of finely cut material, following reagents are added in sequence. One ml of
5% sucrose solution, 1 ml of 2 % Hydroxylamine hydrochloride and 1 ml of Titan Yellow.
Finally solution was made alkaline with 2 ml of 10% NaOH. Red colour indicates the
presence of magnesium and yellow colour indicates absence or traces of Magnesium.
f- Iron
Finely cut leaf materials (0.5g) are taken into a glass vial and 1ml of con. HCL is added in it.
After 15 minutes, 10ml of distilled water and 2-3 drops of con HNO3 are added. 10 ml of this
solution is pipetted out into a specimen tube after 2 minutes and 5ml of 20% ammonium
thiocyanate is added and stirred. Further, 2 ml of amyl alcohol is added, shake well and
allowed to stand for few minutes. The intensity of red colour in amyl alcohol layer indicates
the quantity of iron.
5. Results: As per the presence of colour for mineral nutrients, students will be able to find out
the deficiency symptoms of particular mineral element.
6. Scope of the Results: The students will learn about the method of tissue test for mineral
nutrient.
7. Caution:
• Proper recording of fresh weight of the leaves should be done
• Handling of chemical should be very careful

Books:
Web links:
www.tnau.ac.in
Page 47
Aim: To perform tissue test for mineral nutrients.
Observations:
Page 48

procedure/apparatus
learning outcomes
and cleanliness
Page 49

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LABORATORY MANUAL

(For private circulation only)

Name of the Student:………………………………………………..

Registration Number/Roll No……………………………………….

Section and Group……………………………………………………..

School of Agriculture (SAGR)

Session-term: 2020-21 (Spring Term)

5. Clean up your work area before leaving.

Compulsory things to be carried by the students in lab:

iii- Wear gloves when using any hazardous or toxic agent.

S. No. Name of the Experiment Page

2 To measure the stomatal frequency and index 9-12

3 To demonstrate the endosmosis and exosmosis by using potato 13-16

4 To demonstrate endosmosis by using raisins and exosmosis by using 17-19

6 To demonstrate the phenomenon of plasmolysis 24-26

7 To demonstrate the effect of different wavelength of light on 27-29

8 To separate the chloroplast pigment by strip paper chromatography 30-33

9 To measure the rate of respiration of different respiratory substances by 34-36

Experiment: To study about the plant cell and its structure.

Suggested readings for students:

1. MODERN PLANT PHYSIOLOGY by R. K. SINHA, NAROSA PUBLISHING HOUSE

Date of performance: Registration No:

Aim: To study about the plant cell and its structure.

Results and discussion:

2 Observations and analysis including 20

3 Completion of experiment, discipline 10

Experiment: To measure the stomatal frequency and index

Suggested readings for students:

Date of performance: Registration No:

Aim: To measure the stomatal frequency and index

Result and discussion:

2 Observations and analysis including 20

3 Completion of experiment, discipline 10

Experiment: To demonstrate the endosmosis and exosmosis by using potato osmoscope

Suggested readings for students:

Date of performance: Registration No:

Aim: To demonstrate the endosmosis and exosmosis by using potato osmoscope

Result and discussion:

To be filled in by the faculty

2 Observations and analysis including 20

Experiment: To demonstrate endosmosis by using raisins and exosmosis by using grapes.

Suggested readings for students:

Date of performance: Registration No:

Aim: To demonstrate endosmosis by using raisins and exosmosis by using grapes

Result and discussion:

Learning Outcomes: (What I have learnt)

To be filled in by the faculty

S. No. Parameters Marks obtained Max. Marks

2 Observations and analysis including 20

Experiment: To demonstrate the phenomenon of imbibition

3. Theory: Imbibition is the phenomenon of adsorption of water by the solid particles of a

4. Outline of the Procedure:

1. Weight 5 gm of three different types of seeds separately and place in separate.

4. Again weigh the soaked seeds separately.

5. Record the data and determined percentage of water absorption by imbibition.

Comparison of rate of imbibition of starchy, oily and proteinaceous

Suggested readings for students: