Download PREFACE Botany is a fundamental course for the specialty of

Anatomy experiment in botany
Han Lihong
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PREFACE
Botany is a fundamental course for the specialty of biology. The
objectives of the course are to have students know morphological
characteristics and functions of plant cells, tissues and organs, and master
basic knowledge and skills of morphological anatomy associated with
vegetative and reproductive organs after students complete the course.
Students are required to have a preliminary understanding of various
plant groups and their relationship. These will lay a foundation for
students to learn Plant Taxonomy, Plant Physiology, Plant Ecology, Plant
Resources, Genetics, Cytology, Molecular Biology, etc. in the future.
Through the study of Botany, it is expected that students will grasp the
current tendency of botanical research at home and abroad, have the
capacity of referring to references themselves, and strengthen their
enthusiasm about plant sciences. It is hoped that study of Botany will lay
a foundation for students to have the potential to become an
internationally professional biologist with a wide view.
What value is plant anatomy and how does it relate to other fields of
study? Plant anatomy, or the development and comparative study of plant
cells, tissues, and organs, is a botanical discipline with a long tradition.
Many individuals have emphasized the fact that anatomy is both a
descriptive and an experimental science. In other words, anatomists
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employ critical and extensive observations, resulting in the compilation,
codification, and analysis of descriptive data, but they also use the
analytical methods of the experimental sciences. Each methodology has
different advantages and limitations, and each requires distinct skills on
the part of the investigator.
You do not need to remember the names of these plants, however, I
do try to use a few plants to illustrate many different structures, and you
will become familiar with their names.
This should give you some
depth of knowledge as well as some breadth. Observation is often the first
step in developing a research problem. Anatomical observations can
lead to ecological, physiological and even molecular hypotheses. We will
expose you to several different types of microscopy which you can use in
your class project. You will also learn photomicroscopy. We will also
teach you how to use the freezing microtome to make uniform 20 - 50m
sections. Once you have some slides we will show you ho to digitize
and modify them with computer programs.
The course will be taught bilingually. It is expected to nurture
students to think about Botany-related issues in an English-Chinese
atmosphere and communicate with others in English.
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ONLINE
Experimental general rule
Experiment report format
The purpose of the experimental
Proper use of the Compound Microscope
EXPERIMENT ONE
The epidermis
EXPERIMENT TWO
Xylem & phloem
EXPERIMENT THREE
EXPERIMENT Four
The seed of the plant
Mitosis of the plant cell
EXPERIMENT Five Mature tissue of the plant cell
EXPERIMENT Six The morphology of the root
EXPERIMENT SEVEN The morphology of the stem
EXPERIMENT EIGHT The morphology of the leaf
EXPERIMENT NINE The morphology of the flower
Table 1 List of references
Table 2 List of methods to make reagent
Table 3 Sampling for Plant Analysis
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EXPERIMENTAL GENERAL RULE
The scientific laboratory is a place of adventure and discovery. Some of
the most important events in scientific history have happened in
laboratories. The antibiotic powers of penicillin were discovered in a
laboratory. The plastics used today for clothing and other products were
first made in a laboratory. The list is endless.
One of the first things any scientist learns is that working in the
laboratory can be an exciting experience. However, the laboratory can
also be quite dangerous if proper safety rules are not followed at all times.
In order to prepare yourself for a safe year in the laboratory, read over the
following safety rules. Then read them a second time. Make sure you
understand each rule. If you do not, ask your teacher to explain any rules
you are unsure of. You may even want to suggest further rules in the
section labeled "Other Rules". When you are satisfied that you
understand all the rules on this list, sign and date the contract in the place
provided. Signing this contract tells your teacher that you are aware of the
rules of the laboratory.
A. Dress Code
1. Many materials in the laboratory can cause eye injury. To protect
yourself from possible injury, always wear safety goggles or
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glasses whenever you are working with chemicals, burners, or any
substance that might get into your eyes.
2. Laboratory aprons or coats should also be worn whenever working
with chemicals or heated substances.
3. Tie back long hair in order to keep it away from any chemicals,
burners, and candles, or other laboratory equipment.
4. Any article of clothing or jewelry that can hang down and touch
chemcials and flames should be removed or tied back before
working in the laboratory. Sleeves should be rolled up.
5. Sandals will not protect the feet.
B. General Safety Rules
1. Read all directions for an experiment several times. Follow the
directions exactly as they are written. If you are in doubt about any
part of the experiment, ask your teacher for assistance.
2. Never perform activites that are not authorized by your teacher.
Always obtain permission before "experimenting" on your own.
3. Never handle any equimpent unless you have specific permission.
4. Take extreme care not to spill any material in the laboratory. If
spills occur, ask your teacher immediately about the proper
clean-up procedure. Never simply pour chemicals or other
substances into the sink or trash container.
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5. Never eat or drink in the laboratory. Wash your hands before and
after each experiment.
6. There should be no loud talking or horseplay in the laboratory.
7. When performing a lab, make sure the work area has been cleared
of purses, books , jackets, etc.
8. Know the location and use of all safety equipment (goggles, aprons,
eyewash, fire blanket, fire extinguishers, etc.)
9. Read your asignment before coming to class and be aware of all
safety precautions. Follow directions.
10.Never work alone in the lab.
C. Heating and Fire Safety
1. Again, never use any heat source such as a candle or burner
without wearing safety goggles.
2. Never heat any chemical that you are not instructed to heat. A
chemical that is harmless when cool can be dangerous when
heated.
3. Always maintain a clean work area and keep all materials away
from flames. Never leave a flame unattended.
4. Never reach across a flame.
5. Make sure you know how to light a Bunsen burner. (Your teacher
will demonstrate the proper procedure for lighting a burner.) If the
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flame leaps out of a burner towards you, turn the gas off
immediately. Do not touch the burner. It may be hot. And never
leave a lighted burner unattended!
6. Always point a test tube that is being heated away from you and
others. Chemicals can splash or boil out of a heated test tube.
7. Never heat a liquid in a closed container. The expanding gases
produced may blow the container apart, injuring you or others.
8. Never pick up any container that has been heated without first
holding the back of your hand near it. If you can feel the heat on
the back of your hand, the container may be too hot to handle.
Always use a clamp or tongs when handling hot containers. Hot
glassware looks the same as cool glassware.
D. Using Chemicals Safely
1. Never mix chemicals for the "fun of it." You might produce a
dangerous, possibly explosive substance. No unauthorized
experiments should be performed.
2. Never touch, taste, or smell any chemical that you do not know for
a fact is harmless. Many chemicals are poisonous. If you are
instructed to note the fumes in an experiment, always gently wave
your hand over the opening of a container and direct the fumes
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toward your nose. Do not inhale the fumes directly from the
container.
3. use only those chemicals needed in the activity. Keep all lids
closed when a chemical is not being used. Notify your teacher
when chemicals are spilled.
4. Dispose of all chemicals as instructed by your teacher.
5. Be extra careful when working with acids or bases. Pour such
chemicals over the sink, not over your work bench.
6. When diluting an acid, always pour the acid into water. Never pour
water into the acid.
7. Rinse any acids off your skin or clothing with water. Immediately
notify your teacher of any acid spill.
8. Never pipet by mouth.
9. Be sure you use the correct chemical. Read the label twice.
10.Do not return any excess back to the reagent bottle.
11.Do not contaminate the chemical supply.
12.Keep combustible materials away from open flames (alcohol,
carbon disulfide, and acetone are combustible).
13.Do NOT use the same spatula to remove chemicals from two
different containers. Each container should have a different spatula.
14.When you remove the stopper from a bottle, do NOT lay it down
on the desk, but place the stopper between your two fingers and
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hold the bottle so the label is in the palm of your hand so drips
won't ruin the label, etc. Both the bottle and the stopper will be
held in one hand. Be sure and rinse any drips that might have
gotten on the outside of the bottle.
15.Be careful not to interchange stoppers from two differnt containers
16.Replace all stoppers and caps on the bottle as soon as you finish
using it.
17.Mercury spills must be cleaned up immediately. Use the new
mercury sponge clean up kits put out by various companies.
E. Using Glassware Safely
1. Glass tubing should never be forced into a rubber stopper. A
turning motion and lubricant will be helpful when inserting glass
tubing into rubber stoppers or rubber tubing. Your teacher will
demonstrate the proper way to insert glass tubing.
2. When heating glassware, use a wire or ceramic screen to protect
glassware from the flame of a Bunsen burner.
3. If you are instructed to cut glass tubing, always fire polish the ends
immediately to remove sharp edges.
4. Never use broken or chipped glassware. If glassware breaks, notify
your teacher and dispose of the glassware in the proper trash
container.
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5. Never eat or drink from laboratory glassware. Always thoroughly
clean glassware before putting it away.
F. Using Sharp Instruments
1. Handle scalpels or razor blades with extreme care. Never cut any
material towards you: always cut away from you.
2. Notify your teacher immediately if you are cut in the laboratory.
3. Properly mount, dissecting specimens to the dissecting pan before
making a cut.
G. Electrical Equipment Rules
1. Batteries should never be intentionally shorted. Severe burns can
be caused by the heat generated in a bare copper wire placed
directly across the battery terminals. If a mercury type dry cell is
shorted, an explosion can result.
2. Never deliberately shock yourself or another person. Susceptibility
to shock and possible resulting injury is unpredictable because of
the many physical and physiological variables.
3. Turn off all power when setting up circuits or repairing electrical
equipment.
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4. Never use such metal articles as metal rulers, metal pencils or pens,
nor wear rings, metal watchbands, bracelets, etc. when doing
electrical work.
5. When disconnecting a piece of electrical equipment, pull the plug
and not the wire.
6. Use caution in handling electrical equipment which has been in use
and has been disconnected. The equipment may still be hot enough
to produce a serious burn.
7. Never connect, disconnect, or operate a piece of electrical
equipment with wet hands or while standing on a wet floor.
H. End-of-Experiment Rules
1. When an experiment is completed, always clean up your work area
and return all equipment to its proper place.
2. Wash your hands after every experiment.
3. Make sure all candles and burners are turned off before leaving the
laboratory. Check that the gas line leading to the burner is off as
well.
I. Other Safety Rules
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1. Do not use hair spray or hair mousse during or even before coming
to laboratory class. These are highly flammable and might cause
automatic ignition when in close proximity to a heat source.
2. Synthetic fingernails are also highly flammable and should not be
worn in the lab.
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EXPERIMENT REPORT FORMAT
Department of biologic resource and environmental
sciences (Plant experiment)
Name
Class
Number
Date
Experiment title
Experiment
purpose
Materials
Instrument
Reagent
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Methods:
Plot：
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Results and Analysis：
Remark and grade：
Faculty adviser:
year
16
month
day
THE PURPOSE OF THE EXPERIMENT
Scientists use an experiment to search for cause and effect relationships
in nature. In other words, they design an experiment so that changes to
one item cause something else to vary in a predictable way.
These changing quantities are called variables. A variable is any factor,
trait, or condition that can exist in differing amounts or types. An
experiment usually has three kinds of variables: independent, dependent,
and controlled.
The independent variable is the one that is changed by the scientist.
To ensure a fair test, a good experiment has only one independent
variable. As the scientist changes the independent variable, he or she
observes what happens.
The scientist focuses his or her observations on the dependent
variable to see how it responds to the change made to the independent
variable. The new value of the dependent variable is caused by and
depends on the value of the independent variable.
For example, if you open a faucet (the independent variable), the
quantity of water flowing (dependent variable) changes in response--you
observe that the water flow increases. The number of dependent variables
in an experiment varies, but there is often more than one.
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Experiments also have controlled variables. Controlled variables are
quantities that a scientist wants to remain constant, and he must observe
them as carefully as the dependent variables. For example, if we want to
measure how much water flow increases when we open a faucet, it is
important to make sure that the water pressure (the controlled variable) is
held constant. That's because both the water pressure and the opening of a
faucet have an impact on how much water flows. If we change both of
them at the same time, we can't be sure how much of the change in water
flow is because of the faucet opening and how much because of the water
pressure. In other words, it would not be a fair test. Most experiments
have more than one controlled variable. Some people refer to controlled
variables as "constant variables."
In a good experiment, the scientist must be able to measure the
values for each variable. Weight or mass is an example of a variable that
is very easy to measure. However, imagine trying to do an experiment
where one of the variables is love. There is no such thing as a
"love-meter." You might have a belief that someone is in love, but you
cannot really be sure, and you would probably have friends that don't
agree with you. So, love is not measurable in a scientific sense; therefore,
it would be a poor variable to use in an experiment.
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Proper use of the Compound Microscope
Introduction
Although the light microscope is the most commonly used biological
instrument, it is often used improperly. This may not matter so much with
very thin commercial slides but proper alignment of the illumination
system is essential for viewing thick sections and whole mounts. It is also
crucial for photomicroscopy. You will be using microscopes throughout
this class and for years to come. If you learn the simple lessons we will
teach you today you will do much better in your work and see the
exciting world of microscopy in a new light! (Sorry about the pun). The
procedure we follow was developed by the German scientist, August
Kohler (1866-1948), and it bears his name. Recently his ideas were used
to make an advanced Electron Microscope by Zeiss. Thus, this procedure
which was introduced in 1893 has been of lasting and value.
The Compound Microscope
Because the lens systems in a microscope are composed of many lenses it
is called compound. The typical illumination of specimens in which light
passes through the specimen and travels to your eye is called Bright Field
microscopy. Light has the following path.
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We will be using Leitz microscopes in this class, however, the
instructions for correct alignment of the condenser will be applicable to
other microscopes with adjustable condensers.
Light is provided by a built in bulb which is reflected through the field
iris diaphragm, the condenser, the condenser iris diaphragm, the specimen,
the objective, the tube and the ocular.
There are various control knobs on the microscope which affect the light
path. In addition, there are knobs for coarse and fine focus, as well as
knobs to move the stage.
Focusing the Objectives
Locate the coarse and fine focusing knobs on each side of your scope.
Each knob does coarse and fine focusing. There is no separate knob for
fine focusing. You will see how this works later. The rotation of this knob
focuses the objective onto the specimen.
Mechanical Stage The knobs which control the mechanical stage are on
the right side of the microscope as it faces you.
Condenser
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The condenser aligns and focuses light on the specimen. It has a long
vertical knob on its left side as you face the scope. Rotation of the small
knob at its tip, raises and lowers the condenser to achieve focus.
Swinging Lens
Directly above this at a right angle to the condenser adjusting knob you
will find a rod which controls a lens which can be swung in or out of
place. This swinging lens is left out for low-power illumination, and
swung into the light path for objectives of 10X or greater magnification.
Failure to use this lens properly is the most common mistake that most
people make. If you fail to use this properly you will not be able to see
much, especially when we use thick sections or whole mounts.
Centering Screws
In addition, there are two small knobs on the front of the condenser, set at
45o which are used to center it.
Aperture Iris Diaphragm
Finally, there is a lever which controls the aperture iris. This improves
contrast (difference between light & dark) especially at intermediate and
high magnifications.
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Field Diaphragm
The light source is housed in the base of the microscope. It passes
through the field iris diaphragm. The size of the field diaphragm is
controlled by rotating a knurled ring which is concentric with it. The field
diaphragm controls the area of illumination.
Objectives
The magnification of an image is primarily controlled by the objectives
which are housed in a rotating nose piece. To change objectives you
rotate the nosepiece, starting with the 4X objective. Do not start viewing
by swinging in the 20 - 100 X objectives. These may be damaged if they
hit the specimen.
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The magnification is indicated by a number on each objective.
Furthermore there is a progression in size such that the longest objectives
have greatest magnification. The distance between the objectives and the
cover slip (working distance) decreases dramatically as the magnification
of the objective increases.
The 100 X objective is an oil immersion lens. Note the black line near the
tip of the objective. This is used to identify an oil immersion lens.
Place a small drop of oil on the objective lens.
A small drop of oil must also be placed on the cover slip.
The lens should be carefully lowered into the oil prior to focusing.
Observe this with your naked eyes focusing on the objective and the
specimen. Do NOT look through the oculars.
Oil improves the optics because it unites the glass cover slip and the
objective. It replaces air with oil. The oil has the same refractive index as
glass. Thus less light scattering & refraction occurs.
Be sure that the specimen was in focus at 40X before switching to 100X.
Avoid focusing down on the specimen with an oil immersion lens.
Change the focus so that the objective is traveling away from the slide. If
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the image does not come into focus, reverse the direction until it does.
When in doubt, STOP!!! & ask for HELP!!!
The lens might be dirty or there may be some other problem.
The oil also protects the objective lens from scratching.
Notice that we have 4, 10, 20, 40 & 100 X objectives. Always start with
the 4 X objective to prevent damage to the other objectives which may
collide with the specimen.
Once one of the lenses is focused an a specimen, the others should also be
in focus when they are swung into place. This property is referred to by
the term parafocal. However, in actual practice some adjustment is
required when you switch from one objective to another. This usually
presents little difficulty. However, you must be especially careful when
switching from 10X to 40X and from 40X to 100X.
We will often be using fresh sections and whole mounts in the class.
These can be thick and irregular. Consequently, greater care must be
taken when changing objectives. When in doubt, play it safe and ask for
help until you get acquainted with the material you are studying.
Oculars
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The oculars should be adjusted to suit both of your eyes. Note that there
is a scale on the tube holding both objectives. We will label microscopes
so that each student can work with the same instrument throughout the
course.
Grasp the adjustable knurled ring below each ocular with your thumb and
forefinger and gently rotate it so that each is set at 64 which is its
midpoint.
Before you make any adjustments, place a slide on the stage and focus on
part of the specimen.
The best resolution occurs when all elements of the microscope are in
perfect alignment and the iris diaphragms are properly adjusted to the
best aperture. On simple microscopes you may not be able to alter the
alignment of the different parts, but on these Leitz microscopes it is
possible to align and focus the condenser to achieve "Kohler
Illumination".
Because we will be using a lot of thick hand-sections in this class, it is
vital that you learn how to achieve Kohler illumination. Otherwise, you
will not be able to analyze your specimens.
1] Place a commercially prepared slide on the stage.
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2] Make sure the swinging lens is in the light path (facing up) and focus
on the specimen using the 10X objective.
3] Use only one eye [right eye with right ocular or left eye with left
ocular] and focus the specimen with the coarse/fine focusing knob.
4] Use the knurled ring below the other ocular to focus it while looking
through it with your other eye. You may not need to change the focus.
However, experiment by rotating the knurled focusing ring to see its
effect. My German friends have told me that the correct way to focus the
second ocular is to make it more negative so it is out of focus, then rotate
it in a positive direction until it is focused.
5] Having the oculars focused will improve image quality and will
decrease eye strain. Once this is done it need not be changed during a
given session. However, it is a good habit to do this at the beginning of
each lab. It is best done at 10X because there is less chance for errors at
this magnification compared to 4 X.
6] Make sure the aperture iris is completely open [rotated all the way
counter-clockwise].
7] Reduce the field of illumination by rotating the knurled ring on the
field diaphragm completely clockwise. Be gentle with the field
diaphragm. It should close without any effort.
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8] You should see a small circle of light. If you are lucky, it will be in the
center of the field. However, it will most likely be off-center and out of
focus. Let us know if you can't find it!!
9] Use the vertical condenser adjusting knob to make the circle as small
as possible by gently rotating it. This moves the condenser up and down.
Do this carefully so that the circle of light is not pushed laterally. As you
focus the field diaphragm you will notice that its halo turns from blue to
red and red to blue. The best focus occurs when you adjust the condenser
so that the halo is just between red and blue. This is a little hard to do so
don't be too worried if you have some red or blue in the halo.
10] Expand the field diaphragm by rotating its knurled ring
counter-clockwise, until the light touches one edge of the field. If the
light is perfectly centered it should touch the entire circumference of the
field. This is unlikely.
11] Center the circle of light by using the two small adjustable knobs on
the front of the condenser. When you are satisfied, expand the field so
that the light fills it completely. However, do not fully open the field
diaphragm. Open it just enough to extend beyond the field of view.
12] Repeat this with the 20 or 40 X objective. For critical work this
should be done for each objective. This is especially important for taking
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photographs and for examining minute, translucent specimens like fungi
and algae. For our labs, it will be good to do this for the 10X objective at
the start of each session. You need not do this for 4X and 40X. However,
if you are having some problems resolving details, check to be sure that
you have the condenser aligned and focused.
It may be difficult to do this with the 100 X objective. However, if you
achieve proper alignment with the 40 X objective, the 100 X will be
similar.
13] When working at 20 - 100 X it is important to adjust the condenser
aperture iris. This is especially important for translucent structures.
Closing this iris increases contrast. Thus something fuzzy becomes
smooth and something faint becomes dark. It is usually possible to close
the iris and judge its effects subjectively. However, there is a "tried &
true" procedure which you should know.
14] Remove one of the oculars and look directly down the tube at the
light field. Close the iris so that it occludes 1/4 - 1/3 of the area. This
should give the best contrast. Examine a specimen before and after
adjusting the aperture iris. This should be done for each objective for
critical viewing. In practice, you can experiment with this while viewing
a specimen and adjust it without removing the ocular. Closing the
aperture iris also increases depth of focus up to a point. Thus, more areas
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of a three dimensional specimen will be in focus If it is closed to much, a
flat indistinct image results.
The example shows part of a diatom frustule. There is little detail when
the iris is wide open (top). When it is fully closed (middle) the contrast is
increased but there are aberrations which make the small holes appear
larger than they are in actuality. The outline of the small holes is also
indistinct. When the iris is closed 25 - 30 % there is improved contrast
and less aberration.
15] Experiment with the aperture iris while viewing a prepared slide.
Once you have achieved what you think gives the best image quality,
remove one of the oculars and see how much of the field is occluded.
As part of the first lab, we will be using different stains to study their
effects an fresh specimens. Experiment with the aperture iris as you study
these. Fresh sections are usually too thick for detailed examination at high
magnification, but the aperture iris can be used to great effect with this
type of material.
While these procedures may seem tedious, they will become routine as
you progress in the course.
Hand Sections
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The ability to make free hand sections will allow you to quickly analyze
plant organs without resorting to laborious procedures. A tremendous
amount of information can be derived from hand sections. These do not
need to be extremely thin to be of use. In addition, hand sections of a
structure do not need to be complete or uniformly thin to be useful. Your
initial attempts at hand sectioning will probably be frustrating, however,
you will quickly become proficient. Hand sections also provide 3-D
information which is not available with most commercial slides.
Instructions (Right-Handed)
1] Place a Band-Aid on the thumb of your left hand. Have the cotton
portion on the bottom of your thumb. The thumb is a backstop for this
operation.
2] Place another on the end of your index finger. The index finger will
control the height go the specimen, and thus its thickness.
3] Grasp the plant structure between your thumb and forefinger so that
the top of the specimen extends above the level of your forefinger.
4] Take a single-edge razor blade in your right hand. Be sure that it is
wet.
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5. Rest the blade on your forefinger and use a slicing motion to cut off the
top of the specimen.
6. Try to avoid cutting your thumb with the blade!!!
7] Raise the specimen slightly by manipulating it with your fingers and
repeat the slicing motion.
8] Thin sections can often be obtained by pressing the blade down on
your forefinger and then slicing through the specimen several times.
9] After several sections have accumulated on the blade, wash them off in
a Petri dish of water.
10] Keep on slicing until you have some thin sections. These will appear
translucent when seen against the dark background of your lab bench. In
most cases, the sections will have thin and thick regions. As long as part
of the section is thin, you may be able to use it, and thick sections are
frequently OK.
11] Sections can be removed with forceps and placed in a drop of water
or stain on a microscope slide.
12] It is a good idea to view unstained sections prior to staining. Proper
use of the aperture iris is important for this.
Staining
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Adding Coverslips
It is essential that the sections be completely immersed in water so that
air is excluded.
Air bubbles or spaces will interfere greatly with your observations. To
avoid these when adding a coverslip do the following.
a] Place your sections in 2-3 drops of water or stain in the center of the
slide.
b] Use a fine forceps to pick up a large cover slip (20 x 40 or 20 x 50)
c] Place one end of the coverslip on the slide (near boundary with frosting)
without touching the solution containing the specimens.
d] Steady this end with the fingers of your left hand.
e] Slowly lower the forceps until it touches the slide. By this time the
coverslip should have touched the solution on the slide.
f] Slowly remove the forceps so that the coverslip is gently lowered into
its final resting position.
g] Remove excess solution by touching the side of a Kimwipe or paper
towel to the narrow edges of the coverslip. Be careful not to drag out your
sections with the excess solution.
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h] if you have been using a stain, add water to one end of the coverslip
while withdrawing the stain at the opposite end with a Kimwipe or towel.
In most cases you do not need to get all of the stain out.
i] Wipe excess fluid from the bottom of the slide or it will stick on the
stage and make your life more miserable than it already is.
Stains
Phloroglucinol-HCl - This stains lignified and suberized cell walls
red-orange. The stain is colorless until it reacts with lignin or suberin. The
reaction may take several minutes and works best without a coverslip. If
you are going to use several stains or look at several specimens, stain
with Phloroglucinol first, and set these aside until you have finished other
things. Add a cover slip and observe. This stain contains 20% HCl.
Consequently, clean up any spills, especially on the microscope stage,
and avoid getting this on yourself.
Toluidine Blue - This is the stain that we will use most frequently, so be
sure you learn how to use it in the first few labs. It is a metachromatic
(many colors) stain, and stains lignified walls blue-green. Unlignified
walls with lots of pectin stain cherry red. However, if you over-strain (too
long) with Toluidine blue, everything will be blue. Add several sections
to a drop of water on a slide. Add a drop of Toluidine blue to this.
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a] Quickly add a coverslip.
b] Remove the excess stain by blotting with a Kimwipe. Wipe excess
fluid from the bottom of the slide.
c] View right away.
Caution: Toluidine Blue is hard to get out of clothing, so use it carefully
and clean up any spills with lots of water. In addition, it is poisonous, so
avoid getting it on your skin as much as possible. We will have surgical
gloves available if you want to protect your hands. Be sure to wash your
hands well if they become stained.
IKI - This will stain starch blue-black to orange depending on the type of
starch present. It will also stain nuclei a golden color. Cell walls also stain
light yellow with IKI. It is frequently not necessary to remove IKI before
viewing the sections.
Sudan - This stains suberized cell walls and oil in cells. The stain is
dissolved in alcohol. when the specimen is rinsed with water, waxy
materials which have taken up the stain remain red, other areas are
colorless. Place several drops of stain on the slide and add sections to it.
The alcohol evaporates rapidly, so it is best to add a coverslip right away.
It takes a few minutes for the stain to work, so you will need to add stain
periodically to the edge of the coverslip to prevent the formation of air
34
bubbles. Wash the stain out with water as described above. Look for
red-orange areas.
Slide Preservation - Slides can be saved for short periods by sealing the
edges of the coverslip with freezing medium. This is used to stabilize
tissues for cryosectioning and works well for saving slides. However, it
only lasts for a day or two. Apply one generous coat and allow to dry.
Then add a second coat. Staining intensity will degrade over time but
slides can be saved for a long time in this state.
Polarizing Filters - These cause light to vibrate in one plane. Light
traveling along a straight line vibrates in all possible planes. Imagine
many radii emanating from a common center. These would represent the
many vibrational planes of the light beam. A polarizer cuts out all but one
of these.
if two polarizers are oriented at 90o to one another, no light will pass
through the second one in the series. Verify this by holding one polarizer
while looking at a bright object. Take a second polarizer in your other
hand and superimpose it on the first. Turn either one until the light is
completely blocked.
If a crystalline or paracrystaile object is placed between crossed
polarizers, it will depolarize the light which passes through it. This
35
property is known as birefringence. Consequently, the birefringent
material will be visible while all else will remain dark. Cell walls, crystals
and some starch grains are birefringent, and become apparent using
polarized light. This works with unstained and stained sections.
Lab Materials
You will be given several types of plant material to section and stain in
this lab.
a] Make sure you learn how to properly set up your microscope.
b] You will have many opportunities to work on your sectioning and
staining in subsequent labs, so don't panic if you have trouble today. On
the other hand, make the most of this first lab because you will have lots
to do in the remaining labs.
36
EXPERIMENT ONE
The epidermis
Teaching periods: 3
Teaching purpose and requirement:
To know the types and structure of the epidermis.
1. Experiment theory
The Epidermis is in direct contact with the external environment. It
contains many important adaptations which allow plants to survive &
reproduce on land. We will observe the most general adaptations as well
as some exotic ones. The functions of many types of epidermal cells are
well known but there are some specialized cells with unknown functions.
The epidermis is important in both vegetative and reproductive organs. It
is treated here in a broad sense as the superficial layer (or rarely layers)
on all differentiated parts of the plant in the primary state of growth.
During secondary growth the epidermis is often replaced by Periderm.
Many features of the epidermis can be seen in whole mounts at low
magnification with the compound microscope.
2. Experiment steps
Many features of the epidermis can be seen in whole mounts at low
magnification with the compound microscope.
Mount a piece of a Coleus leaf on a microscope slide.
Be sure to have the 4X objective in place.
Use tape to secure the leaf at each end. Do NOT cover the leaf with tape!
37
Place the slide on the microscope stage.
Move it so that the leaf is under the objective.
Be sure that the condenser iris is wide open.
Flip UP the swinging lens and turn the illuminator up. You may need to
use maximal illumination.
Observe!
Note the differences between the upper and lower surfaces as well as
the veins vs the lamina. You may be able to use the 10X objective but be
careful NOT to bring it into contact with the leaf. THICK paradermal
(parallel to the surface) sections or peels may be needed to observe
epidermis from stems because of their thickness.
Epidermal Peels: Epidermal features can also be captured by making
peels. In many plants, particularly succulent ones, the epidermis may be
easily stripped from fresh leaves. Such epidermal peels are useful for the
study of the shape of epidermal cells and their arrangement as well as the
distribution and structure of stomata. To make epidermal peels you
should do the following.
Make a clean cut at one end of the structure.
Use fine forceps to clamp down on a thick spot along the cut which
includes the epidermis as the top layer.
38
Pull the forceps forward. At first you will have a thick wad of tissues.
However, as you pull the subepidermal layers will remain on the
specimen and you will get an area that contains only epidermis.
Place this face up on a microscope slide and crop it so that some of the
thin strip is retained.
Add water and observe!
Epidermal Windows: There is a method of looking at the epidermis of
leaves that I call the window method.
Place a Band-Aid on your index finger.
Roll the leaf over the Band-Aid.
Use a razorblade to scrape away the overlying tissues without cutting all
the way through.
A window of translucent tissue should be left. Cut this out.
Turn it over, place on a slide, add water, a cover slip and observe.
Epidermal Replicas: Finally, it is possible to make replicas of the
Epidermis with Nail Polish. This works well with a smooth epidermis like
Agave or Rhoeo but may not work well with highly pubescent one.
Coat the surface with Nail Polish.
Let it dry.
Place clear plastic wrapping tape over the nail polish and press it against
the surface.
Gently remove the tape. The nail polish should come with the tape.
39
Cut out part of the tape with nail polish.
Place it on a microscope slide such that the sticky side of the tape faces
UP.
View at 4X and 10X.
Experiment by adding clear oil to the exposed surface & adding a cover
slip. This should improve the visibility of fine details.
The Cuticle is a complex waxy layer secreted by Epidermal cells. It forms
a barrier to water loss and the entrance of pathogens. Because it is waxy,
it will stain with Sudan dissolved in ethanol.
Cut out a 1 x 2 cm piece from the upper side of an Agave leaf.
Cut cross sections & place them in water.
Select the best ones and transfer them to a microscope slide containing
Sudan.
Allow a few minutes for the stain to set. You may need to add more
Sudan as the ethanol will evaporate.
Add a cover slip and observe!
Leaves of Dicots:
Leaves of Hibiscus. Use the whole mount technique to view the upper
and lower surfaces. Which of the pictures on page 1 is from Hibiscus?
Make epidermal peels of Bryophyllum or Kalanchoe leaves.
Leaves of Monocots:
Examine prepared slides of Lilium.
40
Make epidermal peels of Rhoeo leaves.
Make epidermal peels from the upper surface of Agave leaves.
Use the polarizers to see if there are any ergastic substances in the
epidermal cells. Then examine the lower epidermis.
What do the polarizers indicate regarding the ORGANIZATION of the
ergastic substance?
In what part of the cell is it probably located?
What might be the function of the ergastic material? Agave grows in dry
sites which receive intense solar irradiation. Does this suggest a potential
function?
Uniseriate (Single Layer) Epidermis:
This is the most common type of epidermis.
Examine commercial slides of leaf cross sections.
The following features should be noted:
shape and size of epidermal cells
variation in size and structure of cells in different parts of the leaf (upper
and lower sides, over and between veins)
structure of walls and the presence of cuticle
cell contents.
Stomata should receive special attention. Sections may reveal Guard
Cells cut in more than one plane.
41
Note whether guard cells are in the same plane as rest of epidermis, or if
they are raised or sunken.
Subsidiary Cells may or may not be present. The arrangement of
subsidiary cells and guard cells can be used to identify plants.
We will study Pyrus (pear) leaves as an example of a dicot.
Sugarcane (Saccharum) or ko will be used as an example of a monocot.
A special feature to note is the prominent bulliform cells. These are
involved in leaf expansion and in the folding of leaves subjected to
drought.
Ancient Hawaiians brought many varieties of ko with them. The
stems were sucked or eaten raw. Sugarcane eventually became a major
plantation crop in Hawaii but this epoch is coming to an end.
Trichomes are treated here in a broad sense to designate unicellular
and multicellular appendages that develop from epidermal cells.
Examine whole mounts with a dissecting microscope, and the
compound scope, and prepare proper sections for observation with higher
magnifications on the microscope. Trichomes can be unicellular or
multicellular; glandular or nonglandular.
Unicellular, multicellular and glandular hairs can be seen on petioles
of Pelargonium (geranium), Passiflora foetida (Passionflower) plus stems
& petioles of Pentas & Widelia stems.
42
Examine cross sections of Pentas or Widelia stems containing glandular
trichomes.
Make cross sections of pubescent leaves from ohi'a lehua (Metrosideros
polymorpha). Use your polarizers.
Are these unicellular or multicellular? Are they dead or alive?
Stinging Hairs - Observe commercial slides of Urtica, (Stinging Nettle)
leaves. Find the stinging hairs. These work like hypodermic syringes and
inject a powerful chemical upon contact.
Observe fresh sections of Urtica stem or leaves (if available).
Closely related species in Hawaii have hairs that closely resemble their
mainland cousins but lack the noxious secretion. This is probably due to
the absence of herbivores in these islands. What is the advantage gained
by NOT producing the secretion?
Scales or Peltate Hairs are large complex structures which can resemble
umbrellas or shields.
Scrape scales from the surface of Spanish Moss (Tillandsia) with a razor
blade and mount in water to observe special absorbent trichomes.
Add a drop of Toluidine Blue to the side of the coverslip and watch the
wild staining reaction!!!!! Hawaiians called this plant ‘umi’umi-o-Dole or
Dole’s Whiskers.
Similar Trichomes can be observed on Olive Leaves. These account for
the Silver appearance of these leaves when they are dry.
43
What function does this suggest for the scales?
Hint, Hint, the leaves become green when the scales are wet.
What process is associated with green pigments in plants?
How is this related to the water relations of the leaf? Is water required for
photosynthesis?
What else is required and must enter the leaf directly from the outside
environment?
The leaves become silver white when they dry. How will this affect the
amount of light reaching the chloroplasts?
Chloroplasts can be damaged by light if they are missing key components
of photosynthesis, like water, and can't photosynthesize.
You may have noticed that kukui leaves are lighter than other leaves in
the forest. This is due to the presence of multicellular nonglandular
trichomes.
Observe the lower surface of kukui leaves with a dissecting scope, or at
4X with your microscope. Locate the trichomes!
Scrape some onto a drop of water on a microscope slide add a cover slip
and observe!!
Cotton Fibers:
Observe DEMO slide of Gossypium (Cotton) fruit that shows young
fibers which are epidermal hairs of the seeds.
Observe whole fruits and seeds if available.
44
Cotton is the most important vegetable fiber and has staged a commercial
resurgence recently.
Hawaiian cotton has been useful in the genetic improvement of
commercial cotton cultivars.
The presence of a multiple epidermis is rare and is restricted to the leaves
of certain families like the Moraceae (Breadfruit & Figs) , and to orchid
roots. The multiple layers can be traced back to the Protoderm which is
the primary meristem for the Epidermis. Thus, two or more cell layers are
derived from the protoderm. A good example of multiple epidermis is
found in Ficus (Fig) leaves.
Make free-hand cross sections of Ficus leaves. Note the many layers of
achlorophyllous cells on the upper side of the leaf. These constitute a
multiple epidermis. You would need to do a developmental study to be
certain about this.
The epidermis of Ficus is also known for crystals called cystoliths which
are found in certain epidermal cells called lithocysts.
Find these in the Ficus leaf sections and compare with commercial slides.
The Velamen of orchid roots is another example of a multiple epidermis.
The cells are dead at maturity and can store free water. This is important
for epiphytes because their roots may be directly exposed to the
atmosphere. There is an Endodermal-like layer between the Velamen and
the living parenchyma of the root. This suggests that the root in a manner
45
similar to that which occurs at the Endodermis which separates the cortex
from the vascular tissues may take up water.
Make hand sections, Stain with Toluidine Blue and observe with and
without polarizers.
46
EXPERIMENT TWO
Xylem & phloem
Teaching periods: 3
Teaching purpose and requirement:
To understand the structure and composition of xylem and phloem.
1. Experiment theory
The purpose of this lab is to continue our study of individual cell types, in
this case Tracheary and Sieve Elements. In later labs we will study more
closely the ways in which these cell types relate to other cells in the same
tissues.
Cells in the primary xylem develop while the organ is still
elongating. Thus, they must be sturdy enough to form an uninterrupted
conduit for water, and extensible enough to avoid being ruptured. Indeed
the initial strands of tracheary elements are ripped to pieces during
elongation.
Primary xylem contains tracheary elements, which show a
centrifugal sequence of secondary wall patterns. These are annular
(hoop-like), helical (spiral), scalariform (ladder-like) and reticulate
(net-like). The patterns progress from the center -> outside (Centrifugal),
and from the simple to the complex. Furthermore, the relative area of
secondary wall also increases progressively. As the area of secondary
47
wall increases it becomes impossible to classify tracheary elements as one
of the preceding types.
The relatively small areas of thin primary wall that remain are called
pits. These can be broadly elliptical (scalariform), or circular. The
circular pits can be simple or bordered. Bordered pits predominate on
most tracheary elements. The pattern and types of pits are important for
translocation and are also useful for wood identification.
Treachery Elements in the Primary Xylem.
2. Experiment steps
A variety of young stems (stems without secondary growth) will be
used to show many of the variations in xylem development. Helical
thickenings are the most common and may show various degrees of pitch
and coiling.
The extendibility of the helical thickened tracheary elements is
readily demonstrated by making a circular incision in a petiole or stem of
Ricinus (Castor Bean) and then breaking the material in two. The helical
thickenings become exposed and can be extended by pulling.
This is also well demonstrated by Rose Flowered Jatropha.
Suitable material for the study of primary xylem are Coleus, Castor
Bean (Ricinus), Widelia, and geranium stems, or petioles of celery
(Apium) or kukui leaves.
48
The Xylem and Sclerenchyma should stain Blue-Green while the
Phloem stains Pink with Toluidine Blue. The Phloem may be unstained or
may become pink.
Xylem and Sclerenchyma stain red-orange with Phloroglucinol
while the Phloem is always unstained.
Make stem cross sections of Widelia to locate the primary vascular
bundles by staining with Toluidine Blue and Phloroglucinol.
Compare with commercial slides of sunflower (Helianthus).
Make longitudinal sections through celery petiole or Coleus stems,
stain with Toluidine Blue and Phloroglucinol to observe the primary
xylem in profile.
The major vascular bundles are at the corners of Coleus stems. Make
your longitudinal sections here.
Tracheary Elements in Secondary Xylem
The basic difference between tracheids and vessel members is the
presence of a perforation plate on the end walls of vessel members and its
absence on tracheids. The perforation plate has openings that are larger
than the pits that are present in tracheids. A linear series of vessel
members is called a vessel.
The secondary xylem (wood) is highly complex and will be studied
in greater detail in later labs. For now it is sufficient to be introduced to
the basic difference between Tracheids and Vessel Members.
49
Make or observe free-hand cross sections of Podocarpus which is a
Gymnosperm and Coffee (Coffea) which is an Angiosperm.
Examine unstained with polarizers. Locate the xylem that should be
highly birefringent.
Stain with phloroglucinol. The xylem is a large continuous zone and
it should stain red-orange. The phloem of Coffea has fiber bands that are
birefringent, but are discontinuous. How can you locate the phloem in
Araucaria?
Compare the xylem in Podocarpus and Coffea. Can you see any
differences in the size of cell diameters within each? In other words,
which is more homogenous in cross section?
Observe prepared slides of Pinus wood and find the bordered pit
pairs of the tracheids in cross & radial sections. The torus is more darkly
stained and fairly easy to spot.
View macerated Pinewood and note the relative uniformity of the
cells which are all Tracheids. Perforation plates are NOT present on the
end-walls of the Tracheids. However, large pits may be clustered where
tracheids overlap.
For Vessels, look at slides of Tilia wood. Unlike tracheids, the vessel
members have large openings in their end walls; in this case, Simple
Perforation Plates (one opening per end wall). Find these openings.
50
Use longitudinal sections to see the numerous bordered pits on the
sidewalls of the vessels. These have simple perforation plates.
Phloem
The detailed structure of sieve elements in the phloem cannot be
observed easily without the use of special staining techniques.
Consequently, some of the material used in this exercise will be fresh.
Sections of living material are usually more difficult to interpret than
commercial slides. Therefore certain prepared slides will be used for
orientation, and to demonstrate the arrangement of cells in the phloem, as
well as the associations of phloem & xylem.
Primary Phloem of squash (Cucurbita)
Study prepared slides of cross and longitudinal sections of Cucurbita
stems.
Locate the xylem and phloem. Does the phloem occur on one side of
the xylem (collateral bundle) or on both sides (bicollateral)????
Prepare a hand section and stain with Toluidine Blue. Compare this
with the commercial slide.
High-power study shows the three components of the phloem tissue:
Sieve Elements (here Sieve Tube Members), Companion Cells (small
cells accompanying the sieve elements), and Phloem Parenchyma cells
(intermediate in size between sieve elements and companion cells). The
end walls of the sieve elements seen from the surface in cross sections,
51
bear highly differentiated Sieve Areas. These end walls are called Sieve
Plates. The protoplasts of adjacent sieve tube members form a continuum
through the sieve plates.
These connections are the Connecting Strands. Each is encased in
Callose, a carbohydrate wall substance chemically distinct from the
cellulose that lines the sieve pores.
Staining shows that sieve elements appear end to end in longitudinal
series and thus, form Sieve Tubes.
The lateral walls of sieve tubes bear relatively undifferentiated Sieve
Areas. The pores in the sieve areas are much larger than typical pits and
resemble those in the sieve plate.
The callose will stain blue. However, Aniline Blue will also stain
other materials in the section so you need to locate the xylem which is
auto-fluorescent, then the phloem.
Look for concentrations of the stain in the phloem region, and locate
the presence of sieve plates in the highly stained areas.
Callose accumulates at the Sieve Plates due to the pressure that
exists in the Phloem.
Observe these sections with a fluorescence microscope that clearly
shows the sieve plates because of aniline blue fluorescence. These will
appear white or light blue against a dark background. Plastids will
52
fluoresce red. Xylem fluorescence will also be blue but you can easily
identify it due to the characteristic secondary wall thickenings.
The sieve plates will be the most fluorescent areas because callose
accumulates there normally and becomes more concentrated after
wounding.
The sieve plates vary in their orientation. Some are perpendicular to
the long axis of the stem while others may have 45O angles of inclination.
The latter can be seen in face view in longitudinal sections.
This allows you to see the sieve pores.
53
EXPERIMENT THREE
The seed of the plant
Teaching periods: 3
Teaching purpose and requirement:
To understand the course of how seed form seedling. To know the
types of the seed.
1. Experiment theory
Students watch seeds sprout roots and measure the progress in this simple
experiment that can be easily accomplished in a week.
Measuring the rate of root growth is an experiment that introduces
students to collecting data and determining plant growth rates. Folding
the seeds in a moist paper towel and sealing it in a plastic bag can
increase the growth rate so the activity can be accomplished in a week.
The activity can also be extended to include other skills, such as
technology and report writing.
2. Materials and instrument
Materials Needed to Grow Seeds
54
3. Experiment steps
Students will each need a plastic bag, paper towel or coffee filter, and two
or three plant seeds. Multiple seeds are used in case one does not
germinate, or grow.
First, fold the paper towel into quarters or the coffee filter in half.
Next, dampen the paper so it is moist but not dripping wet. Then, gently
open the paper along its last fold and place the seeds inside, putting them
about one inch apart so the sprouts do not become tangled.Place the moist
paper with the seeds into the plastic bag and seal about halfway. Mark the
bag with the student's name, date, and sealing time using a permanent
marker or masking tape. Each seed should also be given a number which
is marked clearly on the bag. Then, put the bag in a warm place in the
classroom.
Students should prepare a chart or table on which to record their data.
This should include their name, type of seed, time of observation, seed
number and length of roots. If seeds grow more than one root, students
can either continue to measure the first one or further divide the data
section for their observation so it contains space for the additional roots.
If students begin the activity on a Monday they can usually end their data
collection on Friday. Since the environment create can cause sprouting to
55
occur faster than if placed in soil, it is possible to make more than one
observation each day. Once the data recording is complete students can
use it to make comparisons between each other, different growing
environments, and even look at variations between the seeds in the same
bag.
Some extensions can be added while performing the experiment.
Students can use a digital camera to create a visual record in addition to
their physical measurement. They can also prepare multiple bags of seeds
and place them in different environments, such as different temperatures
or light levels, and compare the collected data to see if the environments
affected the growth rates.
Once the collecting and recording of data from the growing roots is
complete it can be used for other educational lessons. Students can type
their data into a spreadsheet and create a graph or chart. They can use the
data to make a poster of the experiment to display around the classroom
or a public area of the school. The seedlings can also be transplanted to
small soil pots to continue their growth.
Growing seeds in a moist, soil-free environment makes it easier for
students to monitor the root growth. The plastic can increase the growth
rate, making it possible to complete the data collection in the span of a
week. The seedlings can then be transplanted to pots for further
observations and the data used for a variety of comparison activities.
56
4. plotting
5. Questions
(1)What environment cause the seed become seedling?
(2)How long will it take to germinate?
Dandelion seeds
The inside of a Ginkgo seed, showing a well-developed embryo, nutritive
tissue.
57
EXPERIMENT
Four
Mitosis of the plant cell
Teaching periods: 3
Teaching purpose and requirement:
To have a general knowledge of cells, the cell structure, functions
and types of cell division.
1. Experiment theory
Onion root tips are more popular for the understanding and viewing
various stages of mitosis since the chromosome are large and when these
chromosomes are stained, they are very dark and are easily viewed
through a light microscope. The apical meristem, area of the plant where
cell division takes place at a rapid rate of onion roots are used for the
study of mitosis.
2. Materials and instrument
The root tip that is responsible for the root’s downward growth is one of
the regions where the plant cells are actively elongating and dividing.
Hence, the root tip is and excellent source for the study of process of
cytokinesis (cell division) and mitosis (nuclear division). The
chromosomes of onion are large and only a few chromosomes are
present.
58
3. Experiment steps
Mitosis is nuclear division plus cytokinesis, and produces two identical
daughter cells during prophase, prometaphase, metaphase, anaphase, and
telophase. Interphase is often included in discussions of mitosis, but
interphase is technically not part of mitosis, but rather encompasses
stages G1, S, and G2 of the cell cycle.
Interphase: The nuclei is intact and apparent but the chromosomes
are not apparent. The nucleus contains the chromatin that consists of the
stretched out chromosomes and hence the individual chromosomes are
not visible. Interphase cell typically have one or more number of nucleoli.
Prophase: The nucleus starts to break down during the prophase and the
chromosomes start coiling up in the center of the cell. The chromosomes
becoming condensed are observed. The nuclear envelope disperses and
the nucleoli disappear.
Metaphase: It is the middle stage at which all the double chromatid
chromosomes line up on the metaphase equator of the cell along the
spindle fibers that are pulled to either side of the cell.
Anaphase: In the anaphase, the centromeres divide and a group of
single-chromatid chromosome, the spindle fibers in the chromosomes
becomes short pulling each chromosome pair apart to the opposite ends
of the cell.
Telophase: This is the final stage of replication. The formation of
59
cell plate is initiated in the process of Cytokinesis. Groups of
single-chromatid chromosomes reach the poles of the cell and the nuclear
envelope and nucleoli are formed again. Two new daughter cells are
formed that are separated by the cell wall that is created at the center of
the cell.
Since the chromosomes present in onion are fairly large and they
look when stained, it is widely used for the mitotic cell division study.
Since the cell division takes place at a rapid rate at the root tips, the apical
meristem, onion root tips are popularly used for viewing the various
stages of mitosis.
Plants use a similar process with a few differences. For example,
although a plant cell creates a mitotic spindle and has a centrosome, it
lacks centrioles. The other major difference in plants is the way in which
cytokinesis occurs. In animal cells, the plasma membrane pinches in
along the midline of the cell, creating a cleavage furrow that will separate
the cytoplasm in two. Plant cells have rigid cell walls that prevent this.
Instead, they use two different approaches for cytokinesis. The plasma
membrane and cell wall grow inward together, eventually separating the
parent cell into two. Alternatively, the cell wall that will separate the two
daughter cells starts growing in the middle of the cell between the two
nuclei and continues toward the periphery. This is known as the cell plate.
60
It continues growing until its edges reach the cell's outer surface,
separating the parent cell into two daughter cells.
4. plotting
5. Questions
(1) How many cells are made in mitosis?
(2) Are there chloroplasts in the onion cells?
61
EXPERIMENT Five
Mature tissue of the plant cell
Teaching periods: 3
Teaching purpose and requirement:
To have a general knowledge of tissues.
To understand the types and
function of different tissue.
1. Experiment theory
This lab is designed to give you information on the primary nonvascular
tissues. These are relatively simple compared to xylem and phloem.
However, we will see that there is a considerable amount of variation
within these tissues. In addition, you will observe the major components
of the protoplast that are visible with the light microscope.
Study cell shape, contents, and wall structure, the relation of cells to
one another for intact tissues, the presence of intercellular connections via
pits, and the presence or absence of intercellular spaces. The cell walls
and air spaces constitute the Apoplast. The Plasmalemma and all within it
constitute the Symplast. These are Extremely Important concepts, which
must be appreciated to understand Plant Physiology!
62
Within the Symplast, look for the cytoplasm, nuclei, chloroplasts,
other plastids, crystals, and vacuoles colored with anthocyanins. Use
polarizing filters to locate starch grains and crystals. Also use polarizers
and stains to study cell wall organization and composition.
A mature vascular plant (any plant other than mosses and liverworts),
contains several differentiated cell types. These are grouped together in
tissues. Some tissues contain only one type of cell. Some consist of
several.
Protective tissue covers the surface of leaves and the living cells of
roots and stems. Its cells are flattened with their top and bottom surfaces
parallel. The upper and lower epidermis of the leaf are examples of
protective tissue.
The cells of parenchyma are large, thin-walled, and usually have a
large central vacuole. They are often partially separated from each other.
They are usually stuffed with plastids. In areas not exposed to light,
colorless plastids predominate and food storage is the main function. The
cells of the white potato are parenchyma cells. Where light is present, e.g.,
in leaves, chloroplasts predominate and photosynthesis is the main
function.
The walls of these cells are very thick and built up in a uniform layer
around the entire margin of the cell. Often, the protoplasts die after the
cell wall is fully formed. Sclerenchyma cells are usually found associated
63
with other cells types and give them mechanical support. Sclerenchyma is
found in stems and also in leaf veins. Sclerenchyma also makes up the
hard outer covering of seeds and nuts.
Collenchyma cells have thick walls that are especially thick at their
corners. These cells provide mechanical support for the plant. They are
most often found in areas that are growing rapidly and need to be
strengthened. The petiole ("stalk") of leaves is usually reinforced with
collenchyma
Xylem conducts water and dissolved minerals from the roots to all
the other parts of the plant. Xylem vessels arise from individual
cylindrical cells oriented end to end. At maturity the end walls of these
cells dissolve away and the cytoplasmic contents die. The result is the
xylem vessel, a continuous nonliving duct. The vessels carry water and
some dissolved solutes, such as inorganic ions, up the plant. Sieve
elements have no nucleus and only a sparse collection of other organelles.
They depend on the adjacent companion cells for many functions.
Companion cells move sugars and amino acids into and out of the sieve
elements. In "source" tissue, such as a leaf, the companion cells use
transmembrane proteins to take up — by active transport — sugars and
amino acids from the cells manufacturing them. Water follows by
osmosis. These materials then move into adjacent sieve elements by
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diffusion through plasmodesmata. The pressure created by osmosis drives
the flow of materials through the sieve tubes.
2. Materials and instrument
Tissue System
Component Tissues
and Its Functions
Systems
Dermal Tissue System
Epidermis
• protection
Periderm (in older stems
• prevention of water
and roots)
loss
Ground Tissue System
Parenchyma tissue
• photosynthesis
Collenchyma tissue
• food storage
Sclerenchyma tissue
• regeneration
• support
• protection
Vascular Tissue System Xylem tissue
• transport of water and
Location of Tissue
Phloem tissue
minerals
• transport of food
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3. Experiment steps
Observe free-hand cross sections, and mount in water. The Cortex occurs
between the epidermis and the vascular tissues. It contains some
Collenchyma near the epidermis and Parenchyma near the vascular
tissues.
Parenchyma consists of relatively large, thin-walled cells. The cells
are arranged loosely, that is, there are intercellular spaces among them.
The protoplasts of these cells contain chloroplasts. Some of these cells
may have amyloplasts and crystals. Pereskia is a member of the cactus
family. It has spines but it also has normal leaves. Its flowers are
extremely beautiful like those of most cactaceae. Stain cross sections with
Toluidine Blue. What colors are visible in parenchyma cell walls? What
does this indicate in terms of cell wall composition?
Mount an entire leaf in a drop of water. Study cells in the region
halfway between middle and margin. These leaves are only two cells
thick, except at the midrib, and there is little tissue differentiation. This is
good material to demonstrate cytoplasmic streaming. The general term
Chlorenchyma is used to describe photosynthetic Parenchyma regardless
of its location.
Use the condenser iris to observe the cytoplam.
Observe demo with phase contrast optics to study the cytoplasm.
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Observe Macerated Pith of Begonia (prepared by boiling in dilute
KOH): Note numerous faces of individual cells. What term is used to
describe cells which have this shape?
Locate Stellate Parenchyma cells in petioles and midribs of Canna
(ali’iope) leaves. Cut hand sections and examine with a dissecting scope
before observing with a compound microscope. Do these have 3D
branching?
Examine the Parenchyma in Papyrus (Cyperus papyrus) stems by
making transverse sections. Find the Aerenchyma with a dissecting scope
and examine with a compound microscope. What is the shape of the
individual cells which comprise the Aerenchyma? Are they branched in
2D or 3D?
Stain these with IKI and look for starch containing Amyloplasts.
Examine the demonstration slide of persimmon or palm (niu)
endosperm. This material will also show fine lines traversing the thick
walls from cell lumen to cell lumen. These lines are pits, which connect
the symplast of adjacent cells.
Examine fresh sections of unroasted coffee "beans" or palm fruits to
observe the thick walls and their pits.
Observe chromoplasts and pigment bodies in free-hand sections of
bell pepper fruits, various flower petals, and the root of carrot.
Chloroplasts are Chromoplasts, as well!
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Observe these in cells of the lower epidermis from leaves of Rhoeo,
or Zebrina. These are best seen in epidermal peels. This will be
demonstrated for you.
Amyloplasts are filled with starch, which sometimes occupies the
entire organelle. They are also regarded as Leucoplasts because they lack
color.
Observe thin free-hand sections of Papyrus and stain with IKI. We
will have a demo of potato amyloplasts.
Observe an unstained specimen and use the polarizers.
View a stained slide and then use the polarizers.
Make slides of Rhoeo or Zebrina epidermis (see above for
Leucoplasts). These demonstrate vacuoles, which contain anthocyanin.
The pigmentation in many flower petals, like Erithrina (wiliwili), is
also contained in vacuoles. This is best observed by looking at fresh cross
sections of the petals. How can you tell if the color is due to chromoplasts
or vacuolar pigments?
Observe star-like (Druses) in Begonia or Pereskia stems. Druse
crystals are very common. The other commonly observed crystals are
spear-like Raphides. Both are birefringent (bright) in polarized light.
They probably deter herbivory and are more abundant in plants that grow
in dry environments.
Observe Raphides in sections of taro (kalo) leaves, petioles of
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Heliconia, or stems of Impatiens, Dieffenbachia and Pereskia.
Locate Collenchyma in hand sections of Widelia stem, Celery or
Water Lily (Nymphia) petioles. Determine cell shape by observing cross
sections and a demo of a longitudinal section.
Mount fresh sections in water. After examining them, stain with
Toluidine Blue and then examine again. What does the pink color of the
cell walls indicate?
Observe free-hand cross sections of celery petioles:. The cell wall
thickenings are in the corners where adjacent cells meet. Lamination in
the wall may be discernible. It results from a centripetal deposition of
wall layers differing chemically and physically from one another. What
type of Collenchyma is this??????
Observe cross sections of Widelia stem. Be sure to use the
polarizers.
Observe (prepared slides) of Sambucus stems. The thickenings are
chiefly on the tangential walls. Tangential in these case means walls
oreiented parallel to the surface of the structure. What type of
Collenchyma is this?
Study cross sections from the stem of Hoya (wax plant). Sclereids
occur between the cortex and the vascular region, and in the pith. They
resemble parenchyma cells in shape but have thick walls. A comparison
of the sclereids with the adjacent parenchyma cells illustrates the two
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extremes in the variation of plant cell walls
Stain transverse sections with Toluidine Blue. Use older stems for
lignified sclereids. The parenchyma cells have thin primary, nonlignified
walls. The sclereids have a thick lignified secondary wall deposited inside
the thin primary wall. The secondary wall obscures the primary wall and
shows concentric lamination because it is deposited in successive layers.
It also shows prominent canal-like pits. To observe details of the pits,
focus up and down while examining them. The primary walls of adjacent
sclereids, and the middle lamella are tightly joined and obscured by lignin
deposition. Lignin makes the cell walls very strong and waterproof.
Examine partly macerated leaves of Monstera, Olive (Olea),
Osmanthus, or bulb scales of garlic (Allium sativum). Tease these apart in
a drop of water on slide, using dissecting needles.
Mount small pieces of a garlic bulb scales in phloroglucinol-HCl.
These contain elongated sclereids that occur in groups. They resemble
fibers except for their large pits.
Examine cross sections of Podocarpus leaves and locate the large
sclereids with polarizing filters!
Mash a small piece of pear (Pyrus) flesh and mount in
phloroglucinol-HCl. These sclereids are stone cells or brachysclereids.
Note the ramiform pits.
Examine the partly macerated seed coats of peas and beans.
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Brachysclerids, Macrosclereids and Osteosclereids are present.
Observe the sclereids in the olive leaf occur in the mesophyll and are
long and fiber-like Trichosclereids. Tricho means hair!!!
Observe Macrosclereids in Osmanthus leaves. These are columnar
and ramified at each end.
Look for Astrosclereids in fresh sections of Nymphaea (water lily)
leaves.
4. plotting
5. Questions
Describr the types of the mature plant tissue.
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EXPERIMENT Six The morphology of the root
Teaching periods: 3
Teaching purpose and requirement:
To understand the structure and function of the root.
1. Experiment theory
Plants, unlike the majority of animals, remain fixed in one place
absorbing food from their environment. Roots play an important role in
this way of life. They serve as organs of attachment, anchoring the plant
to the ground, and also as organs of absorption and transport for water
and dissolved salts.
Roots are the first organs to emerge from the seed. They penetrate
the soil and are responsible for the uptake of water and minerals from the
rhizosphere. Roots contain a tissue called Endodermis. The endodermis is
one of the most important vegetative adaptations of terrestrial plants
because it asserts biological control over water and mineral uptake in the
root.
Roots form symbiotic associations with soil microbes. Virtually all
plants have root symbioses. The most famous symbioses are those that
involve
nitrogen-fixing
microorganisms.
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However,
mycorrhizal
associations between plants and fungi are more universal and
consequently more important in the biosphere.
Roots are the Rodney Dangerfields of the plant world because they
are subterranean for the most part. Out of sight … However, the root
system may be as great as or greater than the shoot system in terms of
biomass and complexity. This is especially true for the roots of desert
plants. I am somewhat prejudiced because I studied strange Cycad roots
for my Ph. D.
The roots of epiphytes are exposed directly to the atmosphere and
have special adaptations to accommodate the consequent environmental
insults that accrue from this. Prop roots help certain plants like Pandanus
to remain erect. Because the prop roots of Pandanus originate from the
stem (not from pre-existing roots) they are called Adventitious. Many
vines produce adventitious roots which help them adhere to their
substrate. Ie' Ie (Freycinetia arborea) is an excellent example of this. Its
stem produces strong adventitious roots which encircle tree trunks, and
allow the vine to grow to the top of the canopy where it can intercept
maximal levels of light for photosynthesis. These roots are extremely
fibrous and strong. Cell walls in the roots are heavily lignified. This
makes them resistant to decay and mechanical damage.
Ancient Hawaiians learned how to use these roots for fish traps and in
house construction. It is a mystery how they got the roots to be so straight.
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I have some ideas about this and I think this would be a good
undergraduate research project.
2. Materials and instrument
Microscope, dissector, nipper, burette, scissors, knives, pledget, beaker, et
al.
3. Experiment steps
The tip of the root, called the root cap, has the task of forcing its way into
solid earth. For this purpose the root cap is made up of layers of flattened
and hardened cells.
Behind the root cap is a thick white down consisting of thousands of
tiny hairs. This part of the root has the function of absorbing water, with
mineral salts dissolved in it, from the soil.
The water is absorbed by a process called osmosis. The liquid inside
the root hairs is a rather strong solution of sugars and mineral salts; that
outside them, i.e., the water in the soil, is a weak solution of mineral salts.
The outer skin of the root hairs is a membrane, of a type known as a
semipermeable membrane, with the special property of enabling weaker
solutions pass through into stronger ones. Therefore, so long as the
solution inside the root hairs is stronger than that outside, they will take in
water.
The liquid absorbed by the root passes from cell to cell to its center.
Here it is carried up to the above-ground parts of the plant through
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slender tubes of xylem and phloem.
As well as taking in water and salts, roots sometimes give out
substances in solution. For instance, if the root meets a hard substance
such as marble, which is insoluble in water, it gives out carbon dioxide.
When mixed with water, this carbon dioxide acts on the calcium
carbonate in the marble and turns it into soluble sodium bicarbonate,
which the root can then absorb.
The root contains the same sort of tissues as the stem but the
strengthening tissues of the root are centrally placed, reflecting the
pulling strain suffered by the root as opposed to the bending strain
imposed on the stem.
Cross sections of the root hair region of a typical root are shown in
the illustrations. The inner layer of the wide cortex is called the
endodermis. The walls of this layer become thickened with a corky
substance. Within the endodermis is the stele whose outer layer is of
parenchyma cells and is called the pericycle. The protoxylem is on the
outside of the xylem tissue (exarch condition) which is star-shaped as a
rule. Monocotyledons (e.g., grasses) usually have more "arms" than
dicotyledons (e.g., buttercups). A central pith occurs in some species.
Secondary growth (with very few exceptions) occurs only in
dicotyledons. A strip of parenchyma inside the phloem becomes active,
forming a cambium which grows and makes contact with the pericycle. A
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continuous wavy ring of cambium is thus formed. The secondary tissues
are produced from this. Opposite the xylem groups a ray of parenchyma
is produced instead of vascular tissue. If secondary growth is excessive
(e.g. in trees) the whole pericycle becomes active and produces a layer of
cork outside the stele. The cortex then dies away leaving this corky
covering.
Root of an Herbaceous Dicot
The classic example of such a root is found in the genus Ranunculus
(buttercup).
Observe prepared slides of roots of three ages are available.
Study slides 0 & 1.
Identify proto & metaxylem.
Determine whether the developmental sequence for the xylem is
centrifugal (endarch) or centripetal (exarch)?
Locate the phloem sectors.
The following details should be observed as you scan the tissues
from the outside (epidermis) to the center (xylem).
The epidermis may be present or absent, because it is often poorly
preserved, especially in older stages.
In some cases you will be able to see a Casparian strip in the
outermost layer of the cortex, just beneath the epidermis. This is an
Exodermis (or hypodermis). What is its function?
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A wide cortex, the cells of which usually contain starch.
Endodermis with Casparian strips in younger roots with thick
secondary walls and passage cells in older roots.
Single layer of thin-walled pericycle cells.
The primary xylem. The term primary means all of the xylem, which
develops directly from the procambium.
The strands of primary phloem alternate with the primary xylem
ridges.
Root of a Monocot
Smilax (cat's brier) is a vine and these may be aerial roots. Thus
some details of their anatomy, especially regarding the epidermis and
hypodermis may be due to their aerial environment.
The following details should be noted.
Uniseriate epidermis with relatively thick walls.
Exodermis beneath the epidermis. Cortex Most of the cortex consists
of starch-containing parenchyma cells, with numerous intercellular
spaces.
The inner layer of the cortex is the endodermis, the cells of which
are in the tertiary state of development and exhibit thick secondary walls
which cover the primary radial and inner tangential walls.
A thick-walled pericycle, two to six cells in depth.
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Vascular cylinder with 20 or more separate strands of primary xylem
alternating with an equal number of phloem strands. The xylem strands
are embedded in thick-walled parenchyma similar to that constituting the
pericycle. In each xylem strand the smallest, oldest tracheary elements
occur next to the pericycle. These elements constitute the protoxylem.
The intermediate and large tracheary cells constitute the metaxylem.
Similarly, the smallest sieve elements next to the pericycle, compose the
protophloem, the larger sieve elements located farther inward make up
the metaphloem. This means that they have exarch or endarch maturation.
Choose one arch.
The center of the root contains parenchyma which have developed
thick secondary walls. This area is often called the pith. However, these
cells arise from the procambium and are part of the xylem.
In the case of stem pith, the parenchyma cells originate from the
Ground Meristem. Big deal you might say! In corn (monocot) a large
vessel member occupies the center. This clearly demonstrates the origin
of these cells from the procambium.
Origin of Branch Roots
Examine cross sections of Salix (willow) roots.
The branch roots originate in the pericycle. In these slides the branch
roots are so large that it may not be evident they originated from this
layer, although their origin is clearly endogenous (from within).
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Study various preparations, which illustrate different stages of lateral
root initiation.
4. plotting
5. Questions
79
80
EXPERIMENT SEVEN
The morphology of the stem
Teaching periods: 3
Teaching purpose and requirement:
To understand the structure and function of the stem.
1. Experiment theory
The stem is a part of the plant that holds up other structures such as the
leaves and flowers. This is important as the leaves need to be held up to
the sun to get its light for photosynthesis and the flowers need to be held
up to be available for pollination. Stems also carry water and minerals up
from the roots to the leaves to help with photosynthesis and take food
back down to be stored and distributed to the plant as it has need. The
tubes in the stem that take the water and minerals up into the plant are the
xylem and the tubes that carry the food back down are called the phloem.
Stems vary in structure and may be classified into types such as
woody vs. herbaceous. This classification is artificial, because there is no
sharp line of demarcation between these two, and sometimes the
differences are of a purely quantitative kind. The chief differences
between stems are based on the spatial relationship between the vascular
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and nonvascular tissues, and by the relative amounts of secondary
growth.
Stems are more complex in structure than roots mainly because of
the complexity of the primary vascular system. This is due to the fact that
several vascular bundles may diverge into the leaves. These bundles
which are outside of the central ring of vascular bundles are called leaf
traces. The size and complexity of leaves is correlated with the
complexity of the vascular system in the stem. This lab will consider
stems which trace their origin directly to the apical meristem. This is
known as primary growth. Some plants stop growing at this point but
others many continue to grow due to the activity of lateral meristems.
This type of growth is called secondary.
During secondary growth the resemblance between roots and stems
increases as the secondary xylem and phloem of roots and stems show
many similarities.
2. Materials and instrument
Microscope, dissector, nipper, burette, scissors, knives, pledget, beaker, et
al.
3. Experiment steps
The internal structure of the stem reflects the function. There are
conducting and supporting tissues. These are basically the same in all
parts of the plant and the description given here will cover roots and
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leaves as well. It is in the arrangement of the tissues that roots and stems
mainly differ.
The internal structure is studied by cutting very thin sections and
examining them under a microscope. A suitable, easily obtained
dicotyledon stem for study is that of the sunflower. A section of an
internode cut a short distance from the tip shows the mature primary
structure. The epidermal cells are regular and have a waxy cuticle on the
outside. The cortex cells are simple and of the type known as parenchyma.
In the outer cortical region the cells have thicker walls for extra support
and are known as collenchyma cells. The inner layer of the cortex is the
starch sheath whose cells have starch deposits within them. Inside this
sheath is the stele, made up of vascular tissue and pith. The conducting
(vascular) tissue is arranged in vascular bundles. Each bundle has on the
outside a number of sclerenchyma fibers. These are elongated cells whose
walls have been impregnated with various chemicals known collectively
as lignin. The fibers have no cytoplasm and are therefore not living cells.
They are tough and elastic, enabling the plant to stand bending by the
wind. The phloem is the tissue through which manufactured food is
transported. It consists of elongated sieve-tubes with large vacuoles. The
ends of the tubes are perforated (sieve-plates), allowing passage of the
food solutions. Each sieve tube has a companion cell alongside it which
regulates its functioning. There is usually some ordinary parenchyma
83
tissue too.
The dicotyledon bundles are said to be open (i.e., they contain
cambium). Cambium is the name given to the actively dividing cells or
meristems in the center of the bundle. These play an important part in
secondary growth. The inner part of the bundle consists of the
water-conducting tissue called xylem. These are elongated vessels and
shorter tracheids, although the latter are not common. Xylem tissues are
dead and have lignified walls. They are not evenly thickened however.
The thickness is spiral or ringed in the first-formed xylem (protoxylem).
The later vessels have numerous unthickened pits through which the
water passes to neighboring vessels. The pith and the medullary rays are
normally of simple parenchyma cells.
Monocotyledon stems differ considerably from the foregoing. The
bundles are not arranged in any regular pattern and are of the closed type,
i.e., there is no cambium. Strengthening tissue may occur, especially near
the outside. The bundles are frequently surrounded by sclerenchyma
fibers.
Apical Meristems of Vegetative Shoots
Gross characteristics of shoot tips. We can study fresh preparations
of shoot tips from Elodea, Artocarpus (breadfruit), Ficus, Apium (celery)
or Coleus species. Dissect the shoot tip of Elodea under a stereo scope.
84
Take a vigorous stem and remove a terminal stem of approximately
4-cm.Remove the leaves from the basal 2-3 cm.Blot this dry and embed it
in a piece of modeling clay. Attach the clay to a plastic Petri plate base so
that the stem is erect, and place this on the stage of a dissecting scope.
Start removing the most apical leaves carefully with forceps. The leaves
should get smaller and smaller as you approach the shoot tip. Increase
your magnification as you proceed.
You may want to switch to a dissecting needle to remove the
smallest leaves by applying gentle pressure.You should eventually notice
a smooth, shiny, rounded apical meristem with tiny leaf primordia.
This reveals the uppermost region of tissue initiation called the
apical meristem and the subtending zone of leaf initiation.
These are collectively known as the shoot tip. The uppermost leaves
are called primordia because they do not possess leaf-like morphology.
More well developed leaves can be seen in the basipetal direction
(basipetal = away from & towards the base).
Depending on the species, the primordia are arranged spirally, in
pairs or in whorls. They appear close to one another because the
internodes are not yet extended in the youngest portion of the shoot.
Examine a commercial slide of Elodea and identify the structures cited
above.
The Apical Meristem and the Origin of Tissues
85
Commercial longitudinal sections of shoot tips illustrate the
beginning of tissue differentiation beneath the apical meristem. The
following tissues and structures may be distinguished:
1. apical meristem; 2. leaf primordia; 3. protoderm; 4. procambium; 5.
ground (rib) meristem; 6. axillary buds
Observe the size increases of successively older cells. Note
differences between cells of the procambium and ground meristem in
terms of shape, size, and contents. Also find the rib-meristem which
produces the pith.
The term Rib Meristem is applied to Ground Meristem which
produces the Pith. It is characterized by transverse divisions which
produce very orderly cell files. The Ground Meristem that produces the
Cortex is not always easy to pinpoint. In my mind both are Ground
Meristem but you should understand the term Rib Meristem.
Species with opposite leaves, such as Coleus are particularly
convenient for the study of apical differentiation. It is not possible to see
all of these features in one shoot tip. Examine the illustrations below to
help you identify the major regions and structures in the shoot tips we
have displayed in the lab.
Apical Meristems with Apical Cells
Apical meristem with a single Apical Cell: Equisetum (horsetail)
Seed plants have multicellular apical meristems. However, non-seed
86
plants like ferns can have a large Apical Cell which acts as the source of
all cells.
Apical Meristems with Tunica-Corpus Organization. The tunica, is
one or more superficial layers that show only anticlinal (perpendicular to
the surface) divisions.
The corpus is a group of cells covered by the tunica. The corpus is
characterized by divisions in many planes. This adds volume to the stem
as its derivatives enlarge. The actual number of tunica layers is not
always clear. It will be sufficient to discern meristems with One or
Several tunica layers.
The corpus can also be hard to delimit. However, when there is a
prominent rib meristem, the corpus is circumscribed. Elodea has a
single-layered tunica. Ricinus has two layers in tunica. (No Illustration
shown)Forsythia, has at least three layers in tunica.
The terms cytohistochemical zonation mean that different groups of
cells (cyto) respond in a distinct way to various stains (histochemical) .
Gymnosperms like Norfolk Island Pine (Araucaria) best exemplify
this type of apical meristem. The zonation seen in some plants is due to
differences in the staining density of the cells and in perceived patterns of
cell division. There are a large number of terms which are used to
describe different zones in these meristems. The basic feature of these
meristems is the presence of central mother cells. These are centrally
87
located at the summit of the SAM, and include parts of the tunica and
corpus. The cells are larger and more vacuolate than the surrounding
meristematic cells. Consequently, they stain lightly compared to their
neighbors. Finally, they seem to divide infrequently. The overall
organization has a tunica and corpus.
Observe demo of Ginkgo and locate the central mother cells.
Dicot Stems
Dicot stems usually have one ring of vascular
tissue in stems. The
vascular cylinder is usually composed of individual vascular bundles.
Study Helianthus (sunflower) stems in two stages of development. The
epidermis is typical and stomata may be present. The cortex is composed
of parenchyma with abundant intercellular spaces. Discrete vascular
bundles occur in the young stem.
Note the immature fiber bundles towards the outside of the phloem.
These fibers arise from the same part of the procambium as the primary
phloem. The fibers eventually develop lignified secondary walls.
Note the degree of fiber development in the older stem. The
Interfascicular parenchyma is present between the vascular bundles. The
bundles are collateral and contain primary phloem and primary xylem.
Mature and immature tracheary elements may be present. The
immature tracheary elements occur between the differentiated cells of the
phloem and xylem.
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Compare the degree of vascular development in young and old stems.
In young stems the vascular meristem is called procambium. Later,
undifferentiated procambium may divide periclinally (parallel to the
surface). This is called the vascular cambium. The vascular cambium is a
lateral meristem. It produces secondary xylem and phloem. The
accumulation of secondary xylem increases the girth of the stem.
Secondary vascular development is limited or absent in Helianthus.
However, the vascular cambium may spread to the interfascicular areas.
The fascicular and interfascicular cambia can unite and form a continuous
ring which produces cylinders of secondary xylem and phloem. The
primary xylem is displaced by the secondary xylem and is pushed into the
pith. The pith is composed of parenchyma cells. Study hand sections from
the upper and lower parts of Widelia stems. Stain with Phloroglucinol and
Toluidine Blue.
Intercalary Meristems
Many monocots have Intercalary Meristems. Intercalate means to
insert between. In this case a meristematic layer occurs BETWEEN the
Node and the Apical Meristem.
The Apical Meristem produces a small number of derivative cells
towards the base. Those closest to the apical meristem enlarge and
differentiate. Those more distal continue to divide and produce
derivatives towards the apical meristem. This is the Intercalary Meristem.
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These occur just above the nodes and function for a limited period
and are thus said to be determinate. They are similar to the basal
meristem which produces the strap like leaves of monocots. These are
responsible for the re-growth of grass after it is mowed!
Examine prepared slides of Equisetum. Locate the youngest nodes
follow the cell files acropetally (towards the tip). You should see that the
least differentiated cells near the node and the most differentiated cells
towards the apex. Study the Illustrations on the next page to help you
visualize how the intercalary meristem works.
Follow the cell files indicated by the stars. Start at the node (bottom
of the image) and follow cell files towards the top. You should see that
cells enlarge as you scan from the bottom to the top. Note the different
shapes which occur in the different cell files. Some undergo extensive
elongation parallel to the vertical axis. Others enlarge in the transverse
plane and some cells enlarge equally in both directions.
Nodal Anatomy
The study of nodes reveals the association between the vascular
system of the stem and the vascular bundles of the leaves. The part of a
vascular bundle in the stem that diverges from the vascular cylinder and
enters the leaf is called leaf trace. In the location where the leaf trace is
bent towards the leaf, a relatively wide parenchymatous area appears in
the vascular cylinder of the stem. This area is the leaf gap. Different
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plants have different numbers of leaf traces per leaf and different number
of leaf gaps per node.
Make serial sections of Nerium (Oleander), Coleus or Hibiscus. Cut
thick serial sections through a node.
These transverse sections must include some of the internode above
and below the node as well as the node itself. Arrange the sections in
series on a microscope slide or Perti dish. Observe with a dissecting
microscope. Stain these with Phloroglucinol. This will emphasize the
location of the xylem and make the vascular bundles easier to see.
One-year-old stems (slide B) have completed primary growth. Tissues
barely evident in the youngest sections have now matured. The vascular
cambium should be prominent between the primary xylem and phloem,
and the interfascicular cambium is just forming.
Two- three year old stems (slide C) exhibit considerable secondary
growth. The Vascular Cambium and the Cork Cambium (phellogen)
produce secondary growth. These produce internal pressures due to the
production and enlargement of the new cells they produce. During
enlargement the ring of perivascular (extraxylary) fibers often becomes
broken. The gaps in the ring are filled with parenchyma cells, some of
which develop into thick-walled sclereids. In this case, pressure has
induced meristematic activity in cells.
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The epidermis is still intact except in a few places where lenticels
are forming from the phellogen. Since the interfascicular cambium is
composed only of ray initials, the vascular bundles remain distinct, and
are separated by wide rays.
The xylem shows two growth layers. Many pith cells are crushed, as
a consequence both of movement of the vine and of inward pressure of
developing xylem tissue. The fact that many vines have isolated vascular
bundles or wide areas of parenchyma in the xylem suggests that this
might have functional significance. Considering the manner in which
vines grow, can you imagine what function this tissue organization may
have?
Older stems (slide D) clearly show periderm formation. In
Aristolochia the periderm develops first in isolated vertical strips. Thus,
as seen in cross sections, parts of the stem's circumference have an intact
epidermis while other parts have a well developed periderm with several
layers of cork cells (phellem), cork cambium (phellogen) and a relatively
wide phelloderm.
The Phellogen (Cork Cambium) produces the Phellem (Cork) to the
outside and the Phellogen towards the Inside. The Phellogen can be
located by following the two files of vacuolate cells towards the centrally
located meristematic cells. Remember that meristematic cells are densely
cytoplasmic and lack prominent vacuoles. The phelloderm is usually not
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prominent and may consist of a single layer of parenchyma.Note the
lenticels in the periderm. These are areas of hyperactive cell division
compared to the rest of the Phellogen.
The xylem shows several growth layers. As the individual wedges of
vascular tissues increase in size, new rays develop within them.The pith
and some of the inner parts of the interfascicular areas are almost
completely crushed.
The development of secondary growth in stems is readily seen by
making a series of cross sections from a Coleus stem.The large primary
bundles are conveniently located in the corners of its square stem. By
taking sections from more mature parts of the stem the pattern of
development is readily discerned!Examine the series of photos below,
then observe fresh sections.
Woody Gymnosperms – Araucaria, Podocarpus or Pinus stems.
The initial events in vascular development for gymnosperms is
similar to that of Tilia. However, Secondary Xylem in gymnosperms is
more homogenous than that in most Angiosperms. The only Tracheary
Elements present are Tracheids. However, in environments which have
distinct seasons (Cold/Warm or Wet/Dry) large thin-walled tracheids are
produced during optimal growth periods while narrower, thick-walled
cells are produced under stressful conditions.
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This produces "Growth Rings". Furthermore, there are more pits in
the thin-walled tracheids compared to the thick-walled tracheids.
Consequently, there is some degree of structure/function variation in the
xylem. What does this last sentence mean?
Observe the following from commercial slides and fresh sections
stained with Phloroglucinol. Work from the outside towards the inside of
the stem! Locate the Periderm, which will replace the Epidermis. The
Cortex has large resin canals. These are tube-like cavities originating as
schizogenous intercellular spaces. They are lined with epithelial cells that
excrete resinous material into the cavity. Many cortical parenchyma cells
contain tannins.
The outermost phloem occurs just inside the resin canals. The sieve
cells in the outer part of the phloem are crushed. The functional phloem
occurs next to the vascular cambium. The secondary xylem stains red due
to the presence of lignin. It is fairly homogenous in appearance, but
differences in diameter and wall thickness produce alternating growth
rings.The pith occupies the center of the stem.
4. plotting
5. Questions
What is the types and structure of the plant stem？
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EXPERIMENT EIGHT
The morphology of the leaf
Teaching periods: 3
Teaching purpose and requirement:
Compare and contrast the structures of monocot and dicot internal leaves.
1. Experiment theory
In botany, a leaf is an above-ground plant organ specialized for the
process of photosynthesis. For this purpose, a leaf is typically flat
(laminar) and thin. As an evolutionary trait, the flatness of leaves works
to expose the chloroplasts to more light and to increase the absorption of
carbon dioxide at the expense of water loss. In the Devonian period, when
carbon dioxide concentration was at several times its present value, plants
did not have leaves or flat stems. Many bryophytes have flat,
photosynthetic organs, but these are not true leaves.
2. Materials and instrument
Microscope, dissector, nipper, burette, scissors, knives, pledget, beaker, et
al.
Prepared slides of Zea mays or corn leaves(monocot),and Lingustrum or
sunflower (dicot),compound microscopes,and leaf samples.
3. Experiment steps
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A structurally complete leaf of an angiosperm consists of a petiole (leaf
stalk), a lamina (leaf blade), and stipules (small processes located to
either side of the base of the petiole). The petiole attaches to the stem at a
point called the "leaf axil." Not every species produces leaves with all of
the aforementioned structural components. In certain species, paired
stipules are not obvious or are absent altogether. A petiole may be absent,
or the blade may not be laminar (flattened). The tremendous variety
shown in leaf structure (anatomy) from species to species is presented in
detail below under Leaf morphology. Periodically (i.e. seasonally, during
the autumn), deciduous trees shed their leaves. These leaves then
decompose into the soil.
A leaf is considered a plant organ and typically consists of the
following tissues: An epidermis that covers the upper and lower surfaces ,
An interior chlorenchyma called the mesophyll, An arrangement of veins
(the vascular tissue). The epidermis is the outer layer of cells covering the
leaf. It forms the boundary separating the plant's inner cells from the
external world. The epidermis serves several functions: protection against
water loss by way of transpiration, regulation of gas exchange, secretion
of metabolic compounds, and (in some species) absorption of water. Most
leaves show dorsoventral anatomy: the upper (adaxial) and lower (abaxial)
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surfaces have somewhat different construction and may serve different
functions.
The epidermis is usually transparent (epidermal cells lack
chloroplasts) and coated on the outer side with a waxy cuticle that
prevents water loss. The cuticle is in some cases thinner on the lower
epidermis than on the upper epidermis, and is thicker on leaves from dry
climates as compared with those from wet climates.
Most of the interior of the leaf between the upper and lower layers of
epidermis is a parenchyma (ground tissue) or chlorenchyma tissue called
the mesophyll (Greek for "middle leaf"). This assimilation tissue is the
primary location of photosynthesis in the plant. The products of
photosynthesis are called "assimilates".
The veins are the vascular tissue of the leaf and are located in the
spongy layer of the mesophyll. They are typical examples of pattern
formation through ramification. The pattern of the veins is called
venation.
Part I “Internal Structures of a Monocot Leaf”
(1)View the prepared Zea leaf slide under scanning power(40 X).
(2)Find the largest vein on the leaf and center the vein.Set
magnification to low power(100 X)
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(3)Draw and label the leaf on 100 X.
(4)Include in your drawing: the xylem, phloem, sclerenchyma of the
vein, the mesophyll and chloroplasts, the epidermis and cuticle, and the
guard cell and stoma.
(5)While viewing the slide or making your drawing, answer
questions.
Part II “Internal Structures of a Dicot Leaf”
(1)Examine the Lingustrum leaf slide under scanning(40 X).
(2)Locate and draw and label the structures from the below
diagram.It might be necessary to switch to 100 X.Include the following:
upper & lower epidermis, cuticle, palisades layer, mesophyll layer,
stomata, sclerenchyma, xylem and phloem.
(3)This illustration might be helpful. But you need to look at the real
thing.
(4)Answer questions.
Part III “The Stomata”
(1)Obtain a geranium leaf and using forceps, peal a small amount of
epidermis from the lower surface of the leaf.
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(2)It should be clear and thin, if it is green then it is too thick.
(3)Place this piece of epidermis on a slide and make a wet mount out
of it.
(4)Examine under the microscope and draw it on at least 400x.
4. plotting
5. Questions
(1)How many cell layers make up the upper epidermis?
(2)What is the function of the cuticle?
(3)What type of plant tissue is the mesophyll composed of?
(4)What type of tissues make up the vein of the leaf?
(5)How can you tell the difference between the three tissues of the vein?
(6)What are the functions of the tissues that make up a vein?
(7)Where are most of the guard cells and stoma locate on the leaf?
(8)What is the function of the stomata?
(9)Why are air spaces near most of the stomata?
(10)How thick(how many cell layers)is the palisade layer?
(11)What structures are present inside the palisades cells?
(120Why are these found in this layer?
(130If you were small enough to travel down the xylem of the leaf where
do you think you would eventually end up?
(14)What color is the leaf phloem?This might help you answer question.
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(150What is the function of the phloem of the leaf?
(160What color is the xylem of the leaf?
(17)What is the function of the xylem of the leaf?
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EXPERIMENT NINE
The morphology of the flower
Teaching periods: 3
Teaching purpose and requirement:
To grasp the composition of the flower; to know the development of the
embryo-sac and pollen.
1. Experiment theory
Flowers are the plant's reproductive structures. Angiosperms are types of
plants that bear fruits and flowers. Flowers are usually both male and
female, and are brightly colored to attract insects to help them carry
pollen used for sexual reproduction. Not all flowers are colorful, though.
These flowers usually use the wind for pollination.
Flowers are the reproductive structures produced by plants which
belong to the group known as Angiosperms, or 'Flowering Plants'. This
group includes an enormous variety of different plants ranging from
buttercups and orchids to oak trees and grasses. There are about 250,000
known species.
A flower is basically made up of four concentric rings of structures.
There is an outer ring of modified leaves called sepals. These provide
protection to the flower before it opens and are usually green. This outer
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ring is known as the calyx. Inside the sepals is another ring of modified
leaves called petals which are often brightly coloured. This layer is
known as the corolla. Within the corolla are one or more stamens
containing pollen, which are the male reproductive structures. In the very
centre of the flower are the female reproductive organs.
2. Materials and instrument
Microscope, dissector, nipper, burette, scissors, knives, pledget, beaker, et
al.
3. Experiment steps
The receptacle is the part of the branch on which a flower forms. Color
the receptacle (B) brown. Sepals are leaf like structures that surround and
protect the flower before it blooms. Color the sepals (C) green. Petals are
the colorful part of the flower that attracts insects and even other small
animals, such as mice, birds, and bats. Color the petals (D) a bright color
of your choice. All flowering plants have flowers, but some are not
brightly colored. The petals of these flowers are reduced or absent and the
plant relies on the wind or water for pollination.
The flower has both male and female reproductive parts. The female
reproductive structures are called carpels. In most flowers, the carpels are
fused together to form a pistil. Color the pistil (P) pink. The pistil has
three parts, which can be seen, in the box labeled "pistil". The stigma at
the top is often sticky and is where the pollen attaches. Color the stigma
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(J) purple. The style is the long tube that attaches the stigma to the ovary.
Sperm from the pollen will travel down this tube to the ovules. The
ovules, or eggs, are stored in the ovary until they are fertilized. Plants can
only fertilize eggs of the same species. Special chemicals prevent sperm
from fertilizing the eggs of flowers that are not the same kind. Color the
style (K) red, and the ovary (L) pink. Color the ovules (O) black.
The male reproductive structures are called the stamens. Color the
stamens (H) blue. Each stamen consists of an anther (A), which produces
pollen, and a filament (F), which supports the anther. In the box labeled
"stamen" color the anther dark blue, and the filament light blue. Pollen
produced by the anther is carried by insects or other animals to the pistil
of another flower where it may fertilize the eggs.
The other flowers in the picture follow the same plan, although they
come in many different colors and styles. Color each of the flowers
according to the colors above (blue for stamen, pink for pistil, bright
colors for the petals. etc.). Note that in some of the flowers, not all the
structures are visible.
Sexual reproduction in plants occurs when the pollen from an anther
is transferred to the stigma. Plants can fertilize themselves: called
self-fertilization. Self-fertilization occurs when the pollen from an anther
fertilizes the eggs on the same flower. Cross-fertilization occurs when the
pollen is transferred to the stigma of an entirely different plant.
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When the ovules are fertilized, they will develop into seeds. The
petals of the flower fall off leaving only the ovary behind, which will
develop into a fruit. There are many different kinds of fruits, including
apples and oranges and peaches. A fruit is any structure that encloses and
protects a seed, so fruits are also "helicopters" and acorns, and bean pods.
When you eat a fruit, you are actually eating the ovary of the flower.
Flowers do not always have the two outer layers of calyx and corolla.
These two layers are most noticeable in plants which are pollinated by
insects. The corolla, or petals are often brightly coloured with markings
attractive to insects. The flowers may also be scented. For instance,
Honeysuckle has showy, attractive flowers which attract insects by day.
However, in the dark, their colourful show is not much use, and their
heady scent then helps to attract night-flying moths.
In insect-pollinated plants, there are also usually nectaries which
secrete sugary nectar, located within the flower. These provide an
incentive to insects to visit the flowers. In the search for nectar, the
insects will often get pollen grains caught on their bodies. This may then
brush off onto the stigma of the next flower visited and in this way the
flowers are pollinated.
Many flowers have evolved very specific associations with a
particular insect species or group of species. In these cases flower
structure may be very specialized (e.g. orchids). Foxgloves (Digitalis
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purpurea) and Primroses (Primula vulgaris) show a range of adaptations
to insect pollination.
Wind pollination is also common in flowering plants. In this case,
because there is no necessity to attract insects, the calyx and corolla of the
flowers may be absent.
Fertilization
Once a pollen grain reaches the stigma of another flower of the same
species, it will produce a pollen tube. This grows down through the style
until it reaches an ovule. Fertilization then takes place, resulting in a seed.
When pollen from one flower fertilizes the ovule of another flower,
it is called cross pollination. If an ovule is fertilized by pollen from the
same flower, it is called self fertilization. In evolutionary terms, this is
generally not particularly favourable, as it leads to inbreeding. Most
species therefore tend to be cross pollinated. In this case they need
something to transfer the pollen from one flower to another. This might
be insects, birds, wind or water. This
need to use an outside agent to
transfer the pollen has led to the extraordinary variety of shapes, colours,
scents and arrangements of flowers seen today.
4. plotting
5. Questions
(1)If the petals of a flower are reduced or absent, how is the plant
pollinated?
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(2)Where are the ovules stored?
(3)In many flowers, the pistils and stamens reach maturity at different
times. Considering (4)what you know about pollination, why would this
be an advantage to the plant?
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Table 1 List of references
Botanica North America: The Illustrated Guide to Our Native Plants,
Their Botany, Their History and the Way They Have Shaped our World.
Marjorie Harris. 2003. HarperCollins, Toronto.
Native Trees of Canada
R.C. Hosie. 1990. Fitzhenry and Whiteside, Markham, On
Newcomb’s Wildflower Guide
Lawrence Newcomb and Gordon Morrison. 1997. Little Brown and Co.,
Toronto
Plants of Carolinian Canada
Larry Lamb and Gail Rhynard (illustrated by Judie Shore). 1994.
Federation of Ontario Naturalists, Don Mills, On.
Peterson Field Guide to Eastern Trees
George A. Petrides and Janet Wehr.1998. Houghton Mifflin Co., NY Rare
and Endangered Species of Grey and Bruce Counties
The Bruce-Grey Plant Committee (Owen Sound Field Naturalists). 2001.
Stan Brown Printers Ltd., Owen Sound, On
Shrubs of Ontario
James H. Soper and Margaret L. Heimburger. 1994. Royal Ontario
Museum, Toronto, On.
A Field Guide to California and Pacific Northwest Forests
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John C. Kricher. 1993/98. Houghton Mifflin Co, New York.
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Table 2
List of methods to make reagent
Barfoed's reagent
A mixture of copper (II) acetate and acetic acid, used to show the
presence of strongly reducing sugars in solution. After boiling,
monosaccharides cause the formation of a red precipitate of copper(I)
oxide. Disaccha-rides are not such powerful reducing agents and will not
show a positive reaction.’
Schiff's reagent
A colourless solution that is produced by the reduction of basic fuchsin (a
magenta dye) with sulphurous acid. It is used in histochemical tests to
detect aldehyde and ketone groups in certain compounds, which oxidize
the reagent and restore its magenta colour. See also Feulgen's test.
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Table 3
Sampling for Plant Analysis
Plant analysis assesses nutrient uptake while soil testing predicts nutrient
availability. The two tests are complementary as crop management tools.
Plant analysis will detect unseen hidden hunger and confirm visual
deficiency symptoms. Toxic levels may also be detected. If it is done
early, plant analysis will allow a corrective fertilizer application in the
same season.
A basic knowledge of plant structure is necessary before collecting
samples. A leaf is made up of a leaf "blade" and a "petiole". The petiole is
the stalk attached to the blade. A compound leaf may have several
"leaflets" attached to it. In some cases, only terminal "leaflets" may be
sampled, as in the case of walnuts and pistachios. A common error in
tomatoes is when only leaflets are sampled instead of the whole
compound leaf. This shows the importance of understanding proper
sampling technique.
The most recent mature leaf (MRML) is the first fully expanded leaf
below the growing point. It is neither dull from age nor shiny green from
immaturity. For some crops, the most recent mature leaf is a compound
leaf. The most recent mature leaf on soybean and strawberry, for example,
is a trifoliate compound leaf: three leaflets comprising one leaf.
For cotton, grape, potato and strawberry, petioles provide an
additional indication of nitrogen status. When sampling these crops,
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collect most recent mature leaves and their petioles. Detach leaves from
petioles in the field to stop the translocation of nutrients. Put petioles in a
separate bag. "Midribs" are the middle ribs to large leaves such as corn,
lettuce, and cabbage, and would equate to a petiole sampling.
Deciding When to Sample
To monitor plant nutrient status most effectively, sample during the
recommended growth stages for your specific crop Take samples weekly
or biweekly during critical periods, depending on management intensity
and crop value. However, to identify a specific plant growth problem,
take samples whenever you suspect the problem.
The best time to collect samples is between mid-morning and
mid-afternoon. Nitrate nitrogen varies with time of day and prevailing
conditions but generally not enough to alter interpretation. Sampling
during damp conditions is okay but requires extra care to prevent tissue
from decomposing during shipping. Keep samples free of soil and other
contaminants that can alter results.
The appropriate part of the plant to sample varies with crop, stage of
growth, and purpose of sampling. When sampling seedlings less than 4
inches tall, take whole plants from 1 inch above the soil line. For larger
plants, the most recent mature leaf is the best indicator sample.
Taking A Representative Sample
Proper sampling is the key to reliable plant analysis results. A
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sample can represent the status of one plant or 20 acres of plants. In
general, a common-sense approach works well.
When problem solving, take samples from both "good" and "bad"
areas. Comparison between the two groups of samples helps pinpoint the
limiting element. Comparative sampling also helps factor out the
influence of drought stress, disease, or injury. Take matching soil samples
from the root zones of both "good" and "bad" plants for the most
complete evaluation.
When monitoring the status of healthy plants, take samples from a
uniform area. If the entire field is uniform, one sample can represent a
number of acres. If there are variations in soil type, topography, or crop
history, take multiple samples so that each unique area is represented by
its own sample.
Choosing Sample Size
The actual laboratory analysis requires less than one gram of tissue.
However, a good sample contains enough leaves to represent the area
sampled. Therefore, the larger the area is, the larger the sample size needs
to be.
Sample size also varies with crop. For crops with large leaves, like
tobacco, a sample of three or four leaves is adequate. For crops with
small leaves, like azalea, a sample of 25 to 30 leaves is more appropriate.
For most crops, 8 to 15 leaves is adequate. For crops requiring petiole
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analysis, collect at least 15 to 20 leaves.
Submitting the Sample
Send the completed information sheet and proper fee with each
sample. Use permanent ink or pencil on sample forms and bags Avoid
numbering samples simply as 1,2,3 … as it may lead to confusion later.
Give each sample a unique identifier that will help you remember the
plants or area it corresponds to-such as HOUSE1, 15B, GOOD, or BAD.
You can use up to six letters and/or numbers. Put the identifier on both
the information sheet and the sample envelope. Pay attention to detail
when filling out the information sheet. Note any conditions-drought,
disease, injury, pesticide or foliar nutrient applications-that might be
relevant. Indicate the analysis desired and provide very specific
information on stage of growth and plant part if an interpretation is
required. The laboratory does not automatically provide an interpretation,
as some clients prefer to make their own.
Diagnostic interpretations require more details than predictive.
When sending matching soil, solution, or waste samples, indicate the
matching sample ID in the designated areas on the information sheet. Be
sure the grower name and address are exactly the same on all matching
information sheets. Ship all matching samples in the same container. Ship
the tissue sample in a paper envelope or cardboard box so it can begin
drying during transport. Samples put in plastic bags will rot, and
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decomposition may alter test results.
If samples are very wet, air-dry to a workable condition before
packaging. Otherwise, decomposition or molding will occur. Include a
completed plant analysis information sheet or cover letter with
instructions within the same package. Processing will be delayed if sent
separately. Also, include payment if you do not have an established
account. Samples should be shipped by a carrier such as UPS or FEDEX,
or by first class mail.
Interpreting the Report
Samples are analyzed the next day of their arrival. The prompt
turnaround makes it possible for growers to take any corrective action
needed to salvage the current crop. The report can be emailed or post
mailed to the grower.
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