Lab Manual Bio611

PLANT PHYSIOLOGY
LAB MANUAL
PLANT
PHYSIOLOGY
LABORATORY MANUAL
Authors:
Norrizah Jaafar Sidik Nor’Aishah Azani Saleh

Abu Shah
Siti Khadijah Abdul Karim Zainab Razali Abdul Manap Mahmud
2021 Universiti Teknologi MARA
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PLANT PHYSIOLOGY
LAB MANUAL
PLANT
PHYSIOLOGY
LABORATORY MANUAL
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PLANT PHYSIOLOGY
LAB MANUAL
PLANT
PHYSIOLOGY
LABORATORY MANUAL
Norrizah Jaafar Sidik Azani Saleh Nor’Aishah Abu Shah
Siti Khadijah Abdul Zainab Abdul Manap Mahmud

Karim Razali
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LAB MANUAL
CONTENTS
List of Figures
Forward
Acknowledgements
Introduction
Experiment 1 PLANT STRUCTURE AND ANATOMY
Experiment 2 STOMATAL DISTRIBUTION ON LEAVES OF

SEVERAL SELECTED PLANTS
Experiment 3 TRANSPIRATION AS A MECHANISM OF WATER

TRANSPORT IN THE CELERY XYLEM
Experiment 4 SEED GERMINATION USING HYDROPHONIC

SYSTEM
Experiment 5 GIBBERELLIC ACID AND STARCH HYDROLYSIS

IN GERMINATING BARLEY SEEDS
Experiment 6 PHOTOSYNTHESIS AND RESPIRATION
Experiment 7 STERILISATION TECHNIQUE IN PLANT TISSUE

CULTURE AND CALLUS INDUCTION
Experiment 8 EFFECTS OF PLANT GROWTH HORMONES IN

SHOOT AND ROOT INDUCTION
References
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Lab Report Guidelines
1. Reports should be typed and double-spaced. Do not include a separate title page; type the
title at the top of the first page (save trees!). Be sure to label each section (Introduction,
Materials and Methods, etc.). Figures and tables should be created using a spreadsheet or
graphics program and may be inserted into the text.
2. DO NOT PLAGIARIZE! You may NOT lift phrases or sentences from any source except
your own brain, unless you use appropriate citations. Your lab report must be your work; not
your classmate’s work. If you do plagiarize, NO CREDIT WILL BE GIVEN.
REPORTS SHOULD BE ORGANIZED IN THE FOLLOWING MANNER:
Title: Your title should convey the main concept of the lab. The title should be clear and
concise, yet descriptive.
Introduction: Provide a few statements describing the importance and/or background of the
project. Why is this work interesting? What can you tell us about the system you
investigated? State your objectives and your question clearly.
Methods and Materials: Describe what you did and how you did it. Ideally, if someone
else reads your report, they should be capable of recreating your work.
Results: Show in table and/or figure format the results obtained from your work. You must
ALSO describe these visual aids in WORDS. What does the graph say? What does the table
summarize? What kind of trends in your data do you observe? Tables and figures should be
appropriately labeled so that someone unfamiliar with your project can understand the results.
Please pay particular attention to axes labels, legends, and titles. See figure and table
examples provided below for formatting suggestions. Note: By convention, figures are
always labeled at the BOTTOM of the figure and tables are always labeled at the TOP.
Discussion: The discussion is a chance for you to briefly restate the objectives, approach, and
outcome of your project. Then you should respond to two general questions: Were your
results what you expected? Why or why not? Use your insight and knowledge of your
project to make educated interpretations of your results. Discuss whether these results were
expected. Describe why or why not these results fit your expectation.
Conclusion
Post-Lab Questions
References: If you use references, please cite them appropriately.
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EXPERIMENT 1
PLANT MORPHOLOGY AND ANATOMY
1. INTRODUCTION
The vascular plant is composed of three basic organs: roots, stems and leaves. These result
from the growth of meristems, areas of cells that retain the capability of cell division. The
structure of roots, stems and leaves reflects their basic functions in vascular plants. Roots
have a high surface area, an adaptation that facilitates the absorption of water. Stems have
evolved with cellular modifications that support the leaves in the air. Leaves have a high
surface area, an adaptation to capture the sunlight by photosynthetic tissues. All of these
structures are interconnected by vascular tissue, which transport the photoassimilates
throughout the entire plant.
There are two types of leaf morphologies that are a perfect example of structure-function
relationship. The majority of plants are so called C3 plants. These plants carry out the carbon
fixation reaction of photosynthesis in most of their cells containing chlorophyll. On the other
hand, C4 plants carry out carbon fixation into the Calvin cycle only in specialized cells that
are close to the vascular bundles and shielded as far as possible away from the surface of the
leaf to avoid contact with oxygen, since contact with oxygen diminishes overall
photosynthetic yield.
In C4 plants, the bundle-sheath cells are much larger and more regular than mesophyll cells in
C3 plants. Examples of C4 plants are sugarcane and maize. C3 plants are divided in palisade
and spongy parenchyma. Mesophyl cells are in loose contact with phloem cells in the vascular
bundle. Air spaces allow direct contact of O2 with the carbon-fixing cells. C4 plants have an
extra layer of bundle sheath cells surrounding the vasculature. Bundle sheath cells are
shielded from O2 from the outside of the leaf by tight layers of mesophyl cells.
2. OBJECTIVES
1. To examine the structures and morphology of C3 and C4 plants.
1. To identify and compare the anatomy of different plant cells and tissues.
3. MATERIALS
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Plant A and Plant B

Eosin
Prepared slaid (Cross-section of root)
Cover slip
Filter paper
Scalpel/Razor blade
Forceps
Beaker
Tiles
Microscopes
Tissue
4. PROCEDURES
4.1 Stems
1. Obtain and examine the morphology of plant A and B with leaves and root.
2. Remove soil from the plant.
3. Dip the stem and leaves specimen in water so that it is wet.
4. Wet the razor blade and fingers and with water. Water should drip from your fingers during
sectioning.
5. Slice off thin section of the stem on the tiles.
6. Put a few drops of eosin on the thin sections for 5-10 minutes.
7. Transfer carefully the stem on the slaids and put a few drops of distilled water.
8. To apply the coverslip, hold it at an angle and touch the water drop with one edge. Lower
the coverslip slowly to avoid air bubbles. Use a tissue to remove excess fluid from the slide
before you place it on the microscope or the slide will stick to the stage.
9. Observe under the microscope.
10. Draw your observation under low and high magnification.
(See if you can distinguish xylem from phloem cells. In the cross section, look for the
different tissue types. In the cross section also look for vascular bundles and cells that divide
xylem and phloem).
4.2. Leaves
1. Cut a thin slice of cross section of the the C3 and C4 leaves
2. Observe the C3 and C4 leaves under the microscope.

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3. Draw and label your observation under low and high magnification.
4.3 Roots
1. Observe the prepared slides provided (root).
(Label your drawing with each cell/tissue type and don’t forget to write on your drawing the
name of the plant, the magnification, organ, and the type of section (cross section vs.
longitudinal etc).
5. RESULTS (DRAWING WITH LABELLING)
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6. DISCUSSION
7. CONCLUSION
8. POST-LAB QUESTIONS
1. Describe the morphology characteristics of plant A and plant B.
2. How are the leaves arranged on the stem in plant A? Are they whorled (two or more at a
node)? Are they opposite (two leaves 180 degrees apart at the same node)? Or, are they
alternate (one leaf per node)?
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EXPERIMENT 2
DISTRIBUTION ON LEAVES OF SEVERAL SELECTED

PLANTS
1. INTRODUCTION
Stomatal are pores surrounded by specialized parenchymatic cells, called guard cells. Stomata
have two main functions, mainly they allow for gas exchange acting as an entryway for
carbon dioxide (CO2) and releasing the oxygen (O2). The other main function is regulating
water movement through transpiration. Stomata vary in shape and size, being able to change
to adapt to the different environmental factors, thus ensuring optimum conditions for
photosynthesis.
Stomatal density, which refers to the number of stomata per unit area of the leaf, ranges in
plants from approximately 1,400 to 40,000 stomata cm-1. The number and distribution of the
stomata plays an important role in determining the rate of gas exchange and water loss from a
leaf. For example, we might hypothesize that the more stomata, the greater the rate of
transpiration. Stomatal frequency is determined by counting the number of stomata in the
microscope field of view (after we calculate the area of view).
Most of the stomata are found in leaves, but the presence of these structures on stems,
petioles, sepals and petals is not uncommon. The numerical distribution of the stomata in a
given leaf can be calculated by stomatal index. Stomatal index is a measure of total number
of stomata per total number of epidermal cells in a given unit area. Different plants show
different stomatal index. The stomatal index number varies not only from plant to plant, but
also varies in different environmental conditions.
Stomatal density also varies between monocotyledons and dicotyledons, between plant
species, and between abaxial and adaxial surface of the leaves. Dicotyledons leaves usually
have more stomata on the abaxial (bottom) surface, whereas monocotyledonous leaves have
similar stomatal distribution on abaxial and adaxial (top) surface. On everage, fewer stomata
occur in plants adapted to dry condition.
2. OBJECTIVES
1. To compare the densities of stomata on abaxial and adaxial leaf surfaces of the same leaf
with relation to transpiration.
2. To compare the densities of stomata of different plants, with relation to transpiration.

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3. MATERIALS
Fully expanded leaves of dicot and monocots plants

Compound microscope
Microscope slides and cover-slip
Forceps
Clear nail varnish
Clear sticky tape
Digital camera
4. PROCEDURES
1. Find suitable leaves of dicot and monocts plants.
2. Identify the upper and lower saurfaces of the leaves.
3. Spread a thin layer of clear nail vanish on each surface, upper and lower side of the leaves.
4. Allow the leaves to dry for 10 to 15 minutes.
5. Place a strip of clear sticky tape over the nail varnish.
6. Press the sticky tape down to make a good connection with the nail varnish.
7. Peel off the sticky tape. The layer of nail varnish should come off with the tape.
8. Place the tape on a microscope slide and add a cover slip.
9. Label the slide with the plant name, upper and lower surface.
10.Examine under the microscope at 100x or 400x.
11.Choose a magnification that gives a number of stomata you can keep track of when
counting. Too many stomata cannaot be accurately counted.
12.Count the number of stomata in at least 3 field of views on each leaf surface.
13.Determine the area of the field of view by measuring the diameter of the lens.
14.Calculate the area of view using the formula πr2, where π =3.142 and r is the radius.
15.Record the results in a table and calculate stomatal density.
16.Capture the microscope images of the leaf surface. This greatly aids in counting stomata
and is a record of the results for future reference.
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5. RESULTS
Sample Name Magnification Surface FOV Number of Stomata density

(Ocular x (upper / stomata (Stomata/mm2)
Objective) lower)
6. DISCUSSION
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7. CONCLUSION
1. Compare the stomata density on upper and lower leaf surfaces of a leaf.
2. Contrast the stomata density between dicot and monocot plants.
3. Calculate the percentage of the stomata with open pore on upper leaf surface of dicot and
monocot plants.
9. REFERENCES
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EXPERIMENT 3
TRANSPIRATION AS A MECHANISM OF WATER
TRANSPORT IN THE CELERY XYLEM
1. INTRODUCTION
Transpiration is the process of water loss from plants through stomata. In most plants,
transpiration is a passive process largely controlled by the humidity of the atmospheric and
the moisture content of the soil. Only 1% of the transpired water passing through a plant is
used in the growth process. Transpiration also transports nutrients from the soil into the roots
and carries them to the various cells of the plant and is used to keep tissues from becoming
overheated.
The xylem carries water and minerals from the roots to the stem and leaves. The movement of
water up a plant stem is a result of several forces working together. Some movement occurs
by capillarity, the rising of water up a thin tubule cell (xylem) due to cohesive and polar
properties of water molecules and their attraction (adhesion) to xylem cell walls. Additional
force comes from root pressure, indirectly produced by active transport in the roots. Active
transport brings necessary ions into the root as water content increases and it rises up the
xylem.
Additional force is tension-cohesion (transpiration pull) that occurs as a result of water vapor
loss through stomata. As water escapes to the atmosphere it is replaced in the leaf mesophyll
by water from xylem veinlets. This continual loss and replacement of water draws water up
the xylem by cohesion. Since water molecules cohere to one another by hydrogen bonds and
adhere to xylem cell walls, water lost from the leaves by transpiration is continually replaced
by water in leaf tissue results in a “pulling” of water molecules all the way from the roots and
up through the stem. Transpiration pull can vary depending on atmospheric conditions. A
dry, hot or windy day increases transpiration by increases water loss. Little water in the soil
and high humidity reduce transpiration pull.
The rate of transpiration is measured as the amount of water lost/ square meter/ minute.
Because water evaporates through the many stomata on the leaf surface, the rate of
transpiration is directly related to the surface area. To arrive at the rate of transpiration,
therefore, you must calculate the leaf surface area of each plant: Because most stomata are
found in the lower epidermis, you will determine that surface area.
The transport of water is controlled by water potential. Water will always move from an area
of high water potential to an area with low water potential. This water potential is affected by
pressure, gravity, and solute concentration.
2. OBJECTIVES
1. To study the effect of wind, light and mist factors on transpiration in celery stalks.
2. To calculate the rate of transpiration in celery leaves.
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3. MATERIALS
Celery stalks with leaves

Eosin dye (red)
Gooseneck lamp
Plastic bag
Fan
Scalpel
Tiles
Tissue
Ruler
Graph paper
4. PROCEDURES
4.1 Effect of Transpiration
1. Cut 1 cm from the bottom end of each celery stalk as it remains submerged in water.
Cut the celery stalks at the same height. Make sure each stalk has leaves.
2. Label flask A, B, C and D which contain 200 ml eosin dye.
3. Quickly place the cut end of each celery stalk into the flasks.
4. Place flask A on a normal room temperature, flask B in front of the running fan, flask C
in front of gooseneck lamp and flask D wrap with plastic bag
5. Note the time and allow all the celery stalks to stand for less than 5 minutes in their
designated environmental conditions.
6. Stop the experiment once any of the eosin dye reach at the top end of the celery stalk
and take the time.
7. Remove the celery stalks from their flask. Rinse excess eosin dye from the stalks under
running water and place them on a tissue.
8. Use a metric ruler and scalpel to cut 1 cm segments from the bottom end of the stalks.
9. After each cut is made, examine the cut end of the stalk for the presence of eosin dye in
the xylem tissue positioned along the outer edge of the stalk.
10. Continue cutting 1 cm segments from the each stalk until the eosin dye begin to fade in
the xylem.
11. Tabulate the distance the eosin dye traveled up of each stalk in Table 4.1.
12. Determine the rate of transpiration in cellery stalk m/ min/ m2 .

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13. Sketch the cross section of celery to show the position of xylem.
Leaf area measurement
1. Lay all the celery leaves to be measured on a 1-cm grid graph paper and trace their
outlines.
2. Count the number of square centimeters. Estimate the area of the partial squares.
3. Sum up the overall leaves area from each treatment as all the leaves involve in the
transpiration process.
Example:
Below is an example of Centella leaf, which has been used to demonstrate the use of
grid paper in measuring surface. The example was hand-drawn by pressed leaf onto the
grid paper (Figure 1). The example is using 1 cm grid paper shows both basic leaf parts
(blade and petiole). The leaf blade is the primary photosynthetic surface and the petiole
is the leaf stalk connection to the stem. Do not include the area of the stem (petiole) in
your calculations.
Figure
Figure 2
In calculating surface area (refer Figure 2),

whole squares located within the leaf area drawing were identified (given number 1 to
10 on the diagram).
Squares which included half part of the leaf surface (given letter a to c) were added up
then divided by 2 since only part of the surface was included within the square. Do not
count partial squares that are less than half covered.
Whole square (1-10): 10 x 1cm2 = 10 cm2

Half square (a ,b, c): 3 x 1cm  2 = 1 ½ cm2
2
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Total surface area: 11 ½ cm2
4.2 Transpiration Rate by Potometer
Material:
a plant cutting
ring stand
clamps
clear plastic tubing approx. 60cm length
petroleum jelly
spray bottle
scale
scissors
pipette 0.1ml
Method:
1. Place tip of 0.1ml pipette to 16 in clear plastic tubing.
2. Submerge the tubing and pipette in shallow tray of water. Draw water through the
tubing until all bubbles are eliminated.
3. Carefully cut the plant stem under water. There must no air bubbles introduce into the
xylem.
4. Insert the freshly cut stem into the other open end of tubing.
5. Uplift the tubing from water and bend upward into U shape and use the clamp on ring
stand to hold both pipette and other end of tubing (refer Figure 3).
6. Use petroleum jelly to make airtight seal surrounding the stem and tubing.
7. Let the potometer equilibrate for 10 minutes before start recording the time of
experiment.
8. Read the level of water in the beginning. Continue to record the water level in the
pipette every 5 minutes for 30 minutes.
9. Determine the rate of transpiration.
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Figure 3
Plant cutting
Clamp
Pipette
Plastic tubing
Ring stand
5. RESULTS
Table 5.1 Rate of transpiration in celery stalks against diffrent physical factors
Treatments Time of dye Distance of dye Total leaves area Rate of

reach end travel (cm) (cm2) transpiration
(min.) (cm/min/cm2)
Control
Wind
Light
Mist
Table 5.2 Water lost from plant cutting in potometer
Time interval 0 5 10 15 20 25 30
(min)
Water loss
(ml)
Water loss
(/m2)
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POST-LAB QUESTIONS
1. How to estimate the true leaf surface area by using graph paper?
2. What is the key point to make the potometer work sucessfully?
3. Suggest the outcome of having thin leaf plant cutting compared to thick leaf plant.
4. Explain the role of water potential in the movement of water from soil to the plant and
into the air
5. What property of water accounts for the fact that molecules of water climb the thin
xylem vessels?
6. State the advantage and the disadvantage of close stomata to a plant when water is in
short supply.
7. Describe several adaptations that enable plants to reduce water loss from their leaves.
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EXPERIMENT 4
SEED GERMINATION USING NUTRIENT SOLUTION IN
HYDROPHONIC SYSTEM
1. INTRODUCTION
1.1 Seed Germination
Germination is the growth of an embryonic plant contained within a seed; it results in the
formation of the seedling. The seed of a higher plant is a small package produced in a fruit or
cone after the union of male and female sex cells. All fully developed seed contains an
embryo and, in most plant species some stored food reserves, wrapped in a seed coat. Some
plants produce varying numbers of seeds that lack embryos, these are called empty seeds, and
never germinate. Most seeds go through a period of quiescence where there is no active
growth, during this time the seed can be safely transported to a new location and/or survive
adverse climate conditions until it is favorable for growth. Quiescent seeds are ripe seeds that
do not germinate because they are subject to external environmental conditions that prevent
the initiation of metabolic processes and cell growth. Under favorable conditions, the seed
begins to germinate, and the embryonic tissues resume growth, developing towards a
seedling.
1.2 Nutrient Solution
Plant nutrients are dissolved in the water used in hydroponics and are mostly in inorganic and
ionic form. Primary among the dissolved cations (positivelycharged ions) are Ca2+ (calcium),
Mg2+ (magnesium), and K+ (potassium); the major nutrient anions in nutrient solutions are
NO3− (nitrate), SO42− (sulfate), and H2PO4− (dihydrogen phosphate). Commonly-used
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chemicals for the macronutrients include potassium nitrate, calcium nitrate, potassium
phosphate, and magnesium sulfate. Various micronutrients are typically added to hydroponic
solutions to supply essential elements; among them are Fe (iron), Mn (manganese), Cu
(copper), Zn (zinc), B (boron), Cl (chlorine), and Ni (nickel). Chelating agents are sometimes
used to keep Fe soluble. Plants will change the composition of the nutrient solutions upon
contact by depleting specific nutrients more rapidly than others, removing water from the
solution, and altering the pH by excretion of either acidity or alkalinity.
1.3 Hydroponics System
Hydroponics is a method of growing plants using mineral nutrient solutions, without soil.
Terrestrial plants may be grown with their roots in the mineral nutrient solution only or in an
inert medium, such as perlite, gravel, or mineral wool. Plants absorb essential mineral
nutrients as inorganic ions in water. In natural conditions, soil acts as a mineral nutrient
reservoir but the soil itself is not essential to plant growth. When the mineral nutrients in the
soil dissolve in water, plant roots are able to absorb them.
2. OBJECTIVES
1. To observe the seed germination using hydrophonic system
3. MATERIALS
Container
StyroFoam.
Pots
Threads
Seeds
Basin
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Prepared liquid fertilizer (250 ml)
4. PROCEDURES
4.1. Stage 1
1. Pour 20 liter of tap water in the container (about 1 inch from the top level).
2. Close the container surface using the StyroFoam.
3. Put the threads in the media pot and pull until the threads are outside the pot holes. Make
sure that the pot holes are fully covered to avoid mosquito.
4. Place the pots in a basin containing a tap water and then position them in StyroFoam holes.
5. Then germinate 3 seeds in each pots.
6. Leave the hydrophonic system and observe the seed germination every day.
4.2 Stage 2
1.Pour 250 ml tap water in a bottle. Add the prepared solid fertilizer into the bottle.
2. Then add all the prepared liquid fertilizer into the same bottle and mixed well (nutrient
solution)
3.Pour the nutrient solution into the hydroponic container.
4. After 7 days, add another 25 ml of the nutrient solution in the container.
5. Cover the container with the StyroFoam.
6. Harvest the plant after 3/4 weeks.
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5. RESULTS
Seed germination records
Week 1: date :__________________
Pot 1 Pot 2 Pot 3 Pot 4
(cm) (cm) (cm) (cm)
day Seed Seed Seed Seed Seed Seed Seed Seed Seed Seed Seed Seed
1 2 3 1 2 3 1 2 3 1 2 3
Week 2: date :__________________
(cm) (cm) (cm) (cm)
1 2 3 1 2 3 1 2 3 1 2 3
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Week 3: date :__________________
(cm) (cm) (cm) (cm)
1 2 3 1 2 3 1 2 3 1 2 3
Week 4: date :__________________
(cm) (cm) (cm) (cm)
1 2 3 1 2 3 1 2 3 1 2 3
6. DISCUSSION
6.1. Discuss the results of seed germination using hydrophonic system
7. CONCLUSION
7.1 Make the conclusion base on the objective
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1. Any quiescence phenomenon happen during your observation? Please explain.
2. How to prepare the nutrient solution? And how to determine the sufficent nutrient level for
seed germination?
9. REFERENCES
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EXPERIMENT 5
GIBBERELLIC ACID AND STARCH HYDROLYSIS IN GERMINATING BARLEY SEEDS
Introduction:
In this experiment you will observe barley embryo regulates the expression of the alpha-
amylase gene in the barley aleurone via the release of gibberellic acid. You will do some of
the observations made by the early investigators in plant physiology and reinforce the basics
of aseptic technique with another plant system.
Objective:
To study the role of the embryo and gibberellic acid in barley endosperm starch hydrolysis.
Materials:
• 8 sterilized petri dishes (9-cm diameter) with lids
• 50 barley seeds
• fine-mesh screen or cheesecloth large enough to cover mouth of 100-ml or 250-
ml beaker with elastic
• 100- or 250-ml beaker in which seeds will be surface sterilized
• 125-ml of sterile distilled water in 250-ml flask
• 100 ml of 95% ethanol plus burner
• scalpel with sharp blade
• forceps
• pipettors
• sterile pipette tips, both 10-200 μL (yellow) and 200-1000 μL (blue)
• household bleach
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• graduated cylinder
• starch agar culture medium (7 tubes, 13 ml media/tube)
• 0.3 mg/ml chloramphenicol solution
• 1 μM GA3 solution
• 0.15 mg/ml cyclohexamide solution
• sterile distilled water
• iodine reagent
Methods
WEEK 1
In this experiment it is essential to use sterile techniques whenever possible to prevent

contamination of your agar-containing test plates by microorganisms, especially fungi and
fungal spores (they are all over your hands and in the environment!). Begin by washing your
working area with water, wipe it with a paper towel, then wipe it again with a paper towel
soaked in 95% ethanol.
You will be provided with 7 culture tubes each containing 13 ml of autoclaved and still hot,
unsolidified starch agar solution. All such solutions contain 1% bacto-agar and 2% soluble
potato starch. These tubes are plugged with cotton to prevent contamination from air-borne
microorganisms.
Number seven of your petri dishes from 1 to 7, marking both the lid and base of each dish
and taking care to keep the lids on while marking them. Using a pipettor with a sterile tip, add
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1.0 ml of 0.3 mg/ml chloramphenicol (chloromycetin) solution to each of your seven petri
dishes.
Remove the lid of each dish only enough to the dispense the solution, then replace it.
Discard the tip.
Now add the additional components to the dishes as follows:
Petri dish No. Sterile distilled Gibberelic acid 1 Cycloheximide

water μM
0.15 mg/ml
1 1.0 ml
2 1.0 ml
3 1.0 ml 10 µl
4 1.0 ml 100 µl
5 100 µl 1 ml
6 1 ml
7 1 ml 1 ml
Swirl the covered dishes gently to mix the components. Now add a cooling (but still at 50oC
or warmer) starch agar to each dish, quickly repacing lids. To thoroughly mix all materials
present in the dishes, use the following technique:
Swirl each dish six times clockwise, six times counterclockwise, six times forward and back,
then six times left to right. Spread out the dishes on your bench top so that the agar will
rapidly cool and solidify.
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Place 50 barley seeds in a 100-ml or 250-ml beaker and cover them with 20-ml of undiluted
household bleach (about 5 percent NaOC1, sodium hypochlorite) to kill microorganisms on
their surfaces.
Swirl the flask occasionally during a period of 10 to 20 min. Cover the beaker with a screen
or cheesecloth to prevent loss of the seeds, then decant the bleach, discarding it. Rinse the
seeds with 10 parate portions of sterile water, using approximately 10 ml each time, or until
no more bleach odour remains on the seeds.
Save enough sterile water so that 10 ml may be used in the petri dish in which the seeds are
to be cut. Tap at least 6 of the seeds into the remaining unused petri dish, add 10 ml sterile
water, and replace the lid.
Dip the blade of the scalpel in 95% ethanol, burn off the alcohol in a flame, and then partly
remove the lid of the petri dish to minimally expose the seeds. Cut 6 of the seeds
transversely across their middle.
While cutting them group the half-seeds still containing embryos separately from those
without embryos.
Using alcohol-flamed forceps transfer 6 embryo-containing half-seeds into dish No. 1,

spacing them evenly around the plate about 2 cm from the edge. Force the cut ends solidly
down into the agar.
Replace the petri dish lid immediately. Similarly transfer the six non-embryo containing half-
seeds into dish No. 2.
Again alcohol-flame the scalpel and cut another group of seeds, discarding the embryo-
containing halves and adding those without embryos to dish No. 3.
Repeat until all of the dishes also contain six half-seeds without embryos. Place the dishes in
a convenient place at room temperature (20o to 23oC) for five days.
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LAB MANUAL
(After five days they may be refrigerated by the instructor until the next laboratory period.)
During this incubation period amylase secreted by the half seeds will diffuse outwardly and
digest starch in the agar.
WEEK 2
Starch digestion can be detected by looking at diffuse light through the plates. To make the
digested starch more visible, the plates can be stained with iodine.
Remove the lids and spray the dishes with a dilute iodine reagent that forms a blue color with
undigested starch (1.0 gram I2 and 2.0 grams KI in 1 liter of H2O).
Alternatively, the I2KI may be poured on the agar surfaces, then poured off after 2 or 3 min.
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PLANT PHYSIOLOGY
LAB MANUAL
Note the unstained halos around the half seeds that have secreted α-amylase. Measure and
compute (in centimeters) the average diameter of halos in each dish; list data in the report
sheet.
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PLANT PHYSIOLOGY
LAB MANUAL
EXPERIMENT 6
PHOTOSYNTHESIS AND RESPIRATION
Introduction
Photosynthesis is the process by which plants and some other organisms convert solar
energy to chemical energy in the form of sugars. While photosynthesis produces sugar with
the sun's energy, cellular respiration utilizes the chemical energy in sugar to meet the
energetic needs of plant. Not all of the sugar produce by the plant is used up in respiration
though, as some is used to create new plant biomass like roots, leaves, stems, wood, and
bark. The carbon in plant biomass is only stored temporarily, as it will return to the
atmosphere when the biomass decomposes, burns, or is eaten and metabolized.
If we place a plant in water containing dissolved gases, the plant will remove carbon dioxide
from the water as it photosynthesizes, reducing carbon dioxide concentrations in the water,
and raising the pH of the solution. If we place a plant or seed in the water and it undergoes
respiration, it will release carbon dioxide into the water, and lower the pH of the solution. So
by monitoring the changes in the pH of a solution, you can determine if the plant or seed in
the solution is photosynthesizing (pH goes up) or respiring (pH goes down).
As carbon dioxide uptake is one way to measure photosynthesis rates, and changes in
carbon dioxide concentrations cause changes in pH, we can use pH changes as an indirect
measure of photosynthesis rates. Bromothymol blue is a pH indicator solution that is blue
when its pH is basic (and carbon dioxide concentrations are low) and yellow when its pH is
acidic (and carbon dioxide concentrations are high). The solution is blue when basic,
greenish when neutral, and yellow when acidic.
Objectives
1. To observe the effect of photosynthesis and respiration process on the amount of carbon
dioxide in water.
Materials
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PLANT PHYSIOLOGY
LAB MANUAL
aquatic plant Elodea
bromothymol blue
straw
tubes
tube caps
gooseneck lamp
petri dish
jars and two jar covers
germinating mung bean (Phaseolus aureus) seeds
strainer
Methods
(I) Photosynthesis
1. Place about 75 ml of bromothymol blue into a 125 ml Erlenmeyer flask (the triangular
glass containers at your work bench).
2. Observe the color of the solution. Introduce carbon dioxide into the solution. Use a straw
and slowly blow CO2 into the solution until it just turns yellow.
3. Compare the color of the solution to the printout showing bromothymol blue at various
pHs. Find the color on the printout that best matches the color of your solution. If your color is
between those shown, use the pH value that is intermediate between them.
4. Record the color of the solution after you've introduced CO2 (Initial color) and estimated pH
(Initial pH) for each of the tubes in Table 1. When comparing or describing colors, be sure to
hold the tubes in front of a white background and be as detailed as possible when describing
the color (e.g., use terms like "greenish-blue" or "yellow-green").
5. Pour the solution into three screw cap tubes, dividing it evenly between them. Obtain two
2-inch pieces of Elodea from the open dish ("Light Elodea"). Place them in one of the tubes,
and cap it. Obtain two 2-inch pieces of Elodea from the covered dish, place them in a second
33
PLANT PHYSIOLOGY
LAB MANUAL
tube covered with aluminum foil (to prevent the entry of light), and cap it. In both tubes, make
sure the plant is completely submerged in the solution. Cap the remaining tube – it will serve
as your control.
6. Place the tubes upright on the lab bench approximately 8-10 inches from the lamp. Ensure
the light hits all three tubes equally and from the side, not from the top. Turn on the lamp.
While you allow the experiment to run, set up the respiration experiment described on the
next page.
7. Allow the plants to sit undisturbed for 50 minutes, and then carefully remove the Elodea
plants from the tubes. Determine the color of the solution against a white background, record
them in Table 1, and compare the colors to the bromothymol blue printout to determine pH.
Table 1:
Initial Final
Tube Initial color Final color
pH pH
Elodea - light
Elodea - dark
No Elodea
(II) Respiration
1. Obtain two jars and two jar covers. One of the jars should be partially filled with
germinating mung bean (Phaseolus aureus) seeds and the other should be empty.
2. Rinse out both jars thoroughly (without pouring out the seeds) to ensure that each has a
fresh supply of air then fill each jar with equal amounts of bromothymol blue to just above the
top of the seeds. Record the initial color and estimated pH of the solutions in Table 2.
3. Cover the jars and make sure that the two jars are in similar conditions. Allow the jars
undisturbed for 45 minutes.
4. Filter the seeds out of the jars by pouring the solution through the provided strainer into a
separate container. To control all your variables, do the same for the solution with no seeds
(even though there is nothing to filter out). Compare the color of the solution in each of the
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PLANT PHYSIOLOGY
LAB MANUAL
jars against a white background. Record the final color and estimated pH of the solutions in
Table 2.
Table 2:
Initial Final
Container Initial color Final color
pH pH
With seeds
No seeds
35
PLANT PHYSIOLOGY
LAB MANUAL
EXPERIMENT 7
STERILISATION TECHNIQUE IN PLANT TISSUE CULTURE
AND CALLUS INDUCTION
1. INTRODUCTION
1.1 Sterilisation technique
Plant tissue culture technique permits the growing plants in test tube or closed container in
vitro under controlled environment. This technique is devoted to solve two problems: 1) To
keep the plant cells free from microbes. 2) To grow the desired plants by providing suitable
nutrient medium and other environmental conditions. The plant materials, equipments, media,
and the laminar flow must be sterilized and free from microorganisms. Most common
methods of sterilization are dry heat, wet heat or steam, ultrafiltration, chemicals, and
ultraviolet light in laminar flow. In this laboratory session, students will be taught on surface
sterilization procedures that can be used for plant materials (tissues) such as seed, fruit, stem
and leaf.
1.2 Explant selection
Explant is a sterile excised fragment of plants from which cultures are initiated. Generally, all
types of plant cells or tissues can be used as an explant, however, it is preferable to use young
and immature tissues that is rapidly dividing (at early stage of development) as an explant
such as shoot tip, root tip, and young leaves. Another thing that is need to consider in
selecting an explant is the goal of the experiment. The choice of explant tissues is depending
on what type of response desired from the cell culture. For example, if the aim is to carry out
clonal propagation, explant of shoot or root tip is suitable to achieve it. For callus induction,
fragment of cotyledon, hypocotyl, stem, leaf, and embryo can be used as explant. For
protoplast fusion, leaf tissue from aseptically germinated seeds are preferable.
2. OBJECTIVES
1. To learn about basic sterilisation technique in plant tissue culture
2. To induce callus formation from selected explants
3. MATERIALS
70% alcohol
Gloves
Bleach containers
Sterile water
70% alcohol containers
Bleach (Clorox)
Scalpel blades
Forceps
Tiles
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PLANT PHYSIOLOGY
LAB MANUAL
Spray bottles
Seeds
MS(Murashige and Skoog) media
Hormone BAP and NAA
Laminar air flow
Pill bottles
4. PROCEDURES
1. Wash the plant tissues in running tap water for 5 minutess.
2. Sterilise the laminar flow and work area using 70% alcohol
3. Pick up the plant tissues with a forceps and immerse into 70% alcohol for 5 seconds.
4. Place the plant tissues in 70% sodium hypochlorite (bleach solution 70% v/v) and allow to
soak for 10 minutes. Stir occasionally to allow the solution gets in contact with all the tissues
surfaces.
5. Transfer the plant tissues into sterile water using a forceps. Allow the plant tissues to soak
for 5 minutes in the water.
6. Rinse the plant tissues with sterile water for 3 times.
7. Cut off the plant tissues using scalpel and forceps.
8. Loosen the caps of the pill bottle and pick up one piece of plant tissues using the forceps.
9. With the other hand, remove the cover just enough to allow space to place the plant tissues
in the pill bottle.
10. Quickly replace the cover and seal around the cap with parafilm.
11. Place the cultures in the growth chamber in growth room. Lights will be turned on for 16
hours per day. Observe callus formation at 1-week interval.
5. RESULTS
Leave it blank or Tables or Draw Graphs or Draw Figures
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PLANT PHYSIOLOGY
LAB MANUAL
6. DISCUSSION
7. CONCLUSION
1. What could be the cause of contamination in the cultures? Explain your answers by
explaining the type of contaminations, ways to identify them, and how to reduce or
prevent contaminations.
2. What is the importance of callus culture? How much is the hormone concentration
ratio are required to induce callus from an explant?
9. REFERENCES
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PLANT PHYSIOLOGY
LAB MANUAL
EXPERIMENT 8
EFFECTS OF PLANT GROWTH HORMONES IN SHOOT

AND ROOT INDUCTION
1. INTRODUCTION
1.1 Plant growth hormones
Plant growth hormones (also referred as plant growth regulator or phytohormones) such as
auxin and cytokinin are important in plant tissue culture. There are five major plant hormones
that are well-known i.e auxin, cytokinin, gibberellin, abscisic acid, ethylene and
brassinosteroid. Each of these hormones play complex interaction in regulating plant growth.
In plant tissue culture, auxin and cytokinin are the most used to initiate cell division to
produce callus from explant. Cytokinin promotes cell division and regulates growth and
development. The most widely used cytokinins are kinetin, zeatin, benzyladenin (BA or
BAP). Natural auxin, Indolacetic acid (IAA), stimulates shoot elongation. The most common
auxins are IAA, α-Naphtalene acetic acid (NAA), and 2,4-D. Gibberellins are of less
importance,however, GA3 is used in cell elongation in apical meristem.
Plant hormone with suitable concentration are added into culture medium (either solid
or liquid) to enhance cell division of explant. In this experiment we will examine the effects
of auxins and cytokinins on the cell division, growth, and differentiation of tissues from
explants when they are grown on media containing nutrients, sucrose, and different
combinations of hormones and/or growth regulators. The plant hormone can be used as sole
hormone in the medium or in combination (combination medium) to obtain optimum results.
The balance ratio between cytokinin and auxin regulates the formation of root and shoot
tissues in cell cultures. Both hormones are required to maintain cells in culture as an
undifferentiated callus. Higher ratio of auxin to cytokinin will induce root formation. In
contrast, higher ratio of cytokinin compared to auxin will induce shoot formation. Callus will
be formed in medium supplemented with equal concentration of both hormones.
2. OBJECTIVES
1. To investigate the effects of plant hormone combination concentrations in root and

shoot induction.
3. MATERIALS
Aseptically grown plantlets/seedlings

4 100 mm Petri Plates - 1 each containing: Root-forming medium,
39
PLANT PHYSIOLOGY
LAB MANUAL
Shoot-forming medium, Callus-forming medium, Empty with no medium

Bunsen Burner or Alcohol burner
Forceps
Scalpel or razor blade
Transfer hood - Laminar Flow hood
Test tube of ethanol
70% Ethanol for sterilizing work surface
Parafilm or other sealant film for the Petri plates
4. PROCEDURES
4.1 Explant culturing
This section is done and prepared by laboratory technician. All the works for sterilization are
carried out in a laminar flow. Cultured explant is neatly covered with parafilm around the pill
bottle cap. After two weeks of culture, young shoot is develops and transferred into fresh
medium containing growth hormone like BAP; starts with low concentration i.e 1 mg/L BAP,
and then gradually increased to 2 mg/L BAP, 3 mg/L BAP, 4 mg/L BAP, and 5 mg/L BAP at
every subcultures. The explant growth during this period is monitored.
4.2 Shoot and root multiplication
A strict sterilization technique is required during this experiment to avoid contamination.

Please practice a good handling technique as you have learnt in the previous experiment.
1. Every explants that have produced shoot or root will be transferred into fresh medium at
one-month interval, which termed as subculture.
2. Normally, subcultures are done within 8th to 12th week of culture depending on the number
of shoots, plant type, and the growth hormones used in the medium.
3. Pill bottles containing three different types of media have been prepared for this
experiment. The bottles containing shoot-forming medium (labeled SFM), root-forming
medium (labeled RFM), and callus-forming medium (labeled CFM). Apparatus for culturing
works such as culture flask or bottle, scalpels, and forceps, are sterilized and in aseptic
condition.
4. In the laminar flow, a scalpel and forceps are dipped into 95% ethanol and flamed using
Bunsen burner. It is then cooled for 1-2 minutes. An empty and sterile petri dish is prepared
for explant placement for the cutting process.
5. The pill bottle containing explant is gently opened, the explant is carefully removed from
the bottle and subsequently placed into the petri dish.
6. The explants are separated into two to four sections. If the plant grown with long shoot, it is
cut in smaller of 2 cm each.
7. Every section is then cultured into new bottle containing fresh medium. In total, there will
be 4 to 5 bottles of cultures according to the number of sections produced.
40
PLANT PHYSIOLOGY
LAB MANUAL
8. The bottles are capped and sealed around with parafilm. The bottles are labeled with culture
name and date.
5. RESULTS
Leave it blank or Tables or Draw Graphs or Draw Figures
6. DISCUSSION
7. CONCLUSION
1. What is the importance of root and shoot culture? What is the hormone concentration
ratio are required to induce shoot and root from an explant culture?
9. REFERENCES
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PLANT PHYSIOLOGY
LAB MANUAL
LIST OF REFERENCES
REFERENCE
http://www2.centralcatholichs.com/APbiologysite/AP%20plants/Transpiration%20lab.PDF
Bewley, J.D. 1997. Seed Germination and Dormancy. The Plant Cell, Vol. 9, 1055-1 066,
American Society of Plant Physiologists.
Sherstha,A.,&Dunn,B.2015. Hydrophonic technical report.

https://www.researchgate.net/publication/280235408
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PLANT PHYSIOLOGY

Norrizah Jaafar Sidik Nor’Aishah Azani Saleh

Siti Khadijah Abdul Karim Zainab Razali Abdul Manap Mahmud

2021 Universiti Teknologi MARA

2021 Universiti Teknologi MARA

Norrizah Jaafar Sidik Azani Saleh Nor’Aishah Abu Shah

Siti Khadijah Abdul Zainab Abdul Manap Mahmud

2021 Universiti Teknologi MARA

Experiment 1 PLANT STRUCTURE AND ANATOMY

Experiment 2 STOMATAL DISTRIBUTION ON LEAVES OF

Experiment 3 TRANSPIRATION AS A MECHANISM OF WATER

Experiment 4 SEED GERMINATION USING HYDROPHONIC

Experiment 5 GIBBERELLIC ACID AND STARCH HYDROLYSIS

Experiment 6 PHOTOSYNTHESIS AND RESPIRATION

Experiment 7 STERILISATION TECHNIQUE IN PLANT TISSUE

Experiment 8 EFFECTS OF PLANT GROWTH HORMONES IN

2021 Universiti Teknologi MARA

Lab Report Guidelines

REPORTS SHOULD BE ORGANIZED IN THE FOLLOWING MANNER:

References: If you use references, please cite them appropriately.

2021 Universiti Teknologi MARA

1. To examine the structures and morphology of C3 and C4 plants.

2021 Universiti Teknologi MARA

Plant A and Plant B

2. Remove soil from the plant.

3. Dip the stem and leaves specimen in water so that it is wet.

5. Slice off thin section of the stem on the tiles.

9. Observe under the microscope.

10. Draw your observation under low and high magnification.

1. Cut a thin slice of cross section of the the C3 and C4 leaves

2. Observe the C3 and C4 leaves under the microscope.

1. Observe the prepared slides provided (root).

5. RESULTS (DRAWING WITH LABELLING)

2021 Universiti Teknologi MARA

1. Describe the morphology characteristics of plant A and plant B.

2021 Universiti Teknologi MARA

DISTRIBUTION ON LEAVES OF SEVERAL SELECTED

2. To compare the densities of stomata of different plants, with relation to transpiration.

Fully expanded leaves of dicot and monocots plants

1. Find suitable leaves of dicot and monocts plants.

2. Identify the upper and lower saurfaces of the leaves.

4. Allow the leaves to dry for 10 to 15 minutes.

5. Place a strip of clear sticky tape over the nail varnish.

8. Place the tape on a microscope slide and add a cover slip.

10.Examine under the microscope at 100x or 400x.

15.Record the results in a table and calculate stomatal density.

2021 Universiti Teknologi MARA

Sample Name Magnification Surface FOV Number of Stomata density

2021 Universiti Teknologi MARA

2. Contrast the stomata density between dicot and monocot plants.

2021 Universiti Teknologi MARA

Celery stalks with leaves

4.1 Effect of Transpiration

2. Label flask A, B, C and D which contain 200 ml eosin dye.

12. Determine the rate of transpiration in cellery stalk m/ min/ m2 .

Leaf area measurement

In calculating surface area (refer Figure 2),

Whole square (1-10): 10 x 1cm2 = 10 cm2

2021 Universiti Teknologi MARA

Total surface area: 11 ½ cm2

4.2 Transpiration Rate by Potometer

1. Place tip of 0.1ml pipette to 16 in clear plastic tubing.