1

Laboratory 1
Microscopes
Introduction
This laboratory will concentrate on the compound light microscope, the simplest, most versatile tool
for examination of very small objects. If these exercises seem elementary because of your past
experience, be very sure that you can understand and can perform the procedures covered. If some
seem difficult or confusing, be sure to ask for assistance.
Now, and in future laboratories, share your knowledge and your questions with one another. Learning
is often most effective when it comes informally from a peer, and there is probably no better way to
solidify your knowledge than teaching what you know to someone else. On the other hand, be sure to
do instrument manipulations yourself. Our labs introduce and reinforce skills that, like driving a car,
you cant really learn second hand.

A. Use of the Compound Microscope

To become familiar with the basic workings of your compound microscope, examine the colored
strings, graph paper squares, and typed letters that should be on the first prepared slide in your set.
(1) Lower the stage with the coarse focus knob.
(2) Be sure the 4X objective lens is in place, rotating the turret if necessary to bring that lens into
position.
(3) Clip the prepared slide onto the stage.
(4) Raise the stage as far as possible.
(5) Use the left/right and backward/forward stage adjustment knobs to center the examination material
under the objective.
(6) While looking through the microscope, lower the stage with the coarse focus knob until material on
the slide comes generally into focus.
(7) Focus on the letters that are part of the slide, and compare their orientation in the field of view with
their actual orientation on the slide.
(8) Use the stage adjustment knobs to move the slide right and left, and forward and backward, and
note how the image movement compares with the slide movement. It is possible, of course, to produce
lens systems which show images that match the material in orientation and direction of movement, but
getting used to the inversion is so easy that few will pay the increased expense.
(9) Rotate the turret to bring successively higher-powered objectives into position, and note the changes
in magnification, clearness of image, and number of letters that you can see. At higher powers, practice
adjusting the fine focus to increase visible detail. Look for inconsistencies in focus in different parts of the
field of view, especially when using the higher-powered objectives. A research microscope would have
higher quality lenses, and come closer to consistent focus across the entire field. People who use
microscopes a lot constantly adjust the fine focus as they work. Changes in appearance of an object as
one focuses up and down give clues to three-dimensional shape. Later exercises in this lab will offer
increased opportunity to demonstrate that for yourself.
(10) Use the graph paper to note changes in size of field with changes in magnification. What is the
maximum number of entire 1-mm squares that you can see at each power? Complete . Compound
Microscope Magnification.. Note that the total magnification is determined by multiplying the power of the
objective by the power of the eyepiece (10X).

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Table 1-1. Compound Microscope Magnification.

Objective
Power

Total Power

Approximate Number of
Squares

4X
10X
20X
40X
(11) Examine the intersections of the colored strings using high power (40X objective) and with the
diaphragm opened wide (and the lamp brightness adjusted to a comfortable level). This minimizes the
depth of field (the depth of the region that is in focus). Focusing up and down, determine the order in
which the strings occur from top to bottom. Fill in . Depth Perception with a Compound Microscope..
Table 1-2. Depth Perception with a Compound Microscope.

Color of Top String

Color of Middle String
Color of Bottom String
B. Calibration of the Eyepiece Micrometer
The eyepiece micrometer is the scale visible in the left eyepiece of your microscope. It is divided
into 100 equal graduations. How big a distance does each graduation represent?

Actually, the size represented by each graduation depends on the magnification you're using.
The stage micrometers are mounted on microscope slides, and can be obtained from the instructor.
They are precisely etched scales, also of 100 equal graduations, but their size is known and constant.
The total size of the stage micrometer is 1 millimeter (mm), so each graduation is .01 mm. That's
equal to 10 micrometers (m). See the summary of metric distances at the end of this chapter.
The objective of this exercise is to calibrate the eyepiece micrometer at each of our four possible
magnifications by viewing it and the stage micrometer at the same time and matching up the scales.
(1) Obtain a stage micrometer from the instructor, handle it very carefully, and clip it onto your
microscope stage.
(2) View it at 40 power (4X objective). If necessary, rotate the eyepiece so that both micrometers are
parallel. Use the stage adjustment knobs to line both micrometers up so that their left edges coincide.
Which micrometer is which? The stage micrometer, of course, is the one that moves when you move the
stage. The eyepiece micrometer is the one that turns when you rotate the left eyepiece. See for what
you should be able to see.
Once the micrometers are in position, determining their sizes is relatively straightforward. We know
the stage micrometer is exactly 1 mm in total length. Notice that at this magnification, the eyepiece
micrometer looks far bigger than the stage micrometer. Exactly how much bigger?
Note your result in . Estimating the Size of the Eyepiece Micrometer at Different Magnifications.,
being careful to use the measurement units specified, and being as precise as you can.

Figure 1-1. Eyepiece Micrometer (Above) and Stage

Micrometer (Below) at 40X.
Not everyone will get exactly the same answer, because the actual magnification varies slightly from
one microscope to another.
(3) Repeat the procedure at 100X, 200X, and 400X. Doubling of magnification should result in doubling
the apparent size of each item in the field of view. The eyepiece micrometer, however, looks exactly the
same. Enter your estimations of the apparent size of the eyepiece micrometer in . Estimating the Size of
the Eyepiece Micrometer at Different Magnifications. and be sure they make sense. Does the size
represented by the eyepiece micrometer increase or decrease at higher magnifications?

Be sure you understand why. The reason for going through this calibration procedure is so that the
eyepiece micrometer can be used as a measuring device to estimate the size of cells and cell parts
during the remainder of the semester.
The calibration of the eyepiece micrometer for a 100X objective is covered in the next section.

Table 1-3. Estimating the Size of the Eyepiece Micrometer at Different Magnifications.
Stage Micrometer
Objective Eyepiece
Power
Power

Total
Power

Total
Length

Size of Each of the

100 Divisions

Eyepiece Micrometer
Entire
Length

Size of Each of the

100 Divisions

(multiply)

mm

mm

4X

10X

1.00

0.01

10.0

10X

10X

1.00

0.01

10.0

20X

10X

1.00

0.01

10.0

40X

10X

1.00

0.01

10.0

100X

10X

1.00

0.01

10.0

mm

mm

C. Oil Immersion Lens

As objective lenses become more and more powerful, a point is reached where it is impossible to
achieve good focus if there is any gap at all between the lens and the slide. The problem results from
refraction, which is bending of light that occurs when it passes from one medium to another in which
its velocity is different, such as from glass to air. The solution to the refraction problem is to replace
the air gap with very clear oil, which has optical characteristics similar to glass. This procedure is
referred to as oil immersion. Typical oil immersion lenses magnify 100 times, increasing the total
magnification of the microscope to 1000X. This is particularly useful when viewing bacteria or other
microbes, and for small structures of larger cells such as flagella.
If the 100X lens is not currently on the microscope, carefully exchange it with the 4X lens, being
careful to put the 4X lens in the plastic lens container. The condenser unit should be adjusted to just
below the specimen slide in order to concentrate light in the very small area that is being viewed. The
iris diaphragm should be nearly closed if the specimen is unstained and opened more if the specimen
is stained. Material should always be observed at a lower magnification before switching to the oil
immersion lens.

1. Eyepiece Micrometer Calibration (Again!)

Although the image will not be as good, please do not use oil for this operation. The extra cleaning
that it would entail increases the chances of breaking the stage micrometer. Follow the same
procedure outlined in Part B and obtain the relevant measurements. Enter your data in . Estimating
the Size of the Eyepiece Micrometer at Different Magnifications..
Carefully place the stage micrometer in its case and return it to the instructor.

2. Using Oil Immersion to Examine Bacteria

(1) Position a prepared slide of bacteria on the microscope stage and locate bacteria at low power. The
slide has three separate areas for each of three bacterial shapes (round, rod, and spiral). Select any one
of the areas.
(2) Increase the magnification to 400X (known as high dry because greater magnification requires oil
immersion).
(3) When bacteria are centered in the field, without altering the focus, rotate the nosepiece to a point
halfway between the 40X and 100X lenses.
(4) Add a drop of immersion oil to the slide, right where the immersion lens will be when it is rotated into
place.
(5) Continue rotating the nosepiece until the immersion lens is in viewing position. If all has gone well,
the lens will be in contact with the oil. If not, either reapply oil or very carefully lower the lens with the
fine focus knob until contact is made with the oil.
(6) Your microscope is parfocal, which means there should be little or no focus change when rotating
from one lens to another. Focus as needed, slowly, with the fine focus knob. The depth of focus is so
small at this magnification that the changes into and out of focus are very rapid.
(7) Measure an individual bacterial cell and record your results in . Bacterial Cell Measurements..
Table 1-4. Bacterial Cell Measurements.

Bacterial Shape
mm
Length
Width

3. Cleanup
It is necessary to clean oil from the lens, from the slide, and sometimes from the stage after viewing.
Oil that remains on the lens housing can work its way onto the cement that anchors the lens. This
shortens the useful life of the lens. Wipe away all oil from the microscope with lens paper. Be sure to
check the 40X objective too, for sometimes it picks up oil when the lens is rotated. Clean oil from the
slide with soap and water. Swap back the oil immersion and 4X lenses. Put the oil immersion lens
back into the plastic container.

D. Review of metric units:

1 cm (centimeter) = .01 meter (2.5 cm = about one inch)
1 cm = 10 mm (millimeters)
1 mm = 1,000 m (micrometers) (This page is about 15 m thick)
1 m = 1,000 nm (nanometers)

Exercises in metric measurements:

If a cell is 40 m wide, how wide is it in millimeters?
In nanometers?
If a cell membrane is 70 nm thick, how thick is it in micrometers?

KEY WORDS
eyepiece micrometer
stage micrometer
millimeter
micrometer
calibrate
oil immersion lens
parfocal
centimeter
nanometer

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Laboratory 2
Laboratory 3
The Plant Cell
Introduction
The development of microscopes, and the development of our understanding of the cellular nature of
living things have gone hand in hand. While there are a few extraordinary cells that can be seen
without magnification, for practical purposes studying cells requires using compound microscopes. At
least.
Actually, the physical structure of cells has a fineness of detail greater than even the finest light
microscopes can show. Much of what we know about cells was only revealed when the electron
microscope was developed. Electron microscopes have easily doubled what we know about cell
structure.
Do electron microscopes show all there is to see? Not at all. Studying the diffraction of x-rays that
have passed through cells hints at structural detail far beyond anything that can be shown in a
photographic image. The more detailed models and charts on display in the lab are based on
information from the whole spectrum of cellular investigation techniques.
As anyone would guess, cells of living things show enormous variation in size, form and function. Yet
they also exhibit many basic similarities. Most of the parts, or organelles, of the plants studied in
today's laboratory can also be found in animals or fungi. Most can not be found in bacteria. Bacterial
cells are so simple that biologists routinely classify them as prokaryotic and the cells found in all the
other kingdoms as eukaryotic.
As you work with microscopic views of cells in this laboratory, be aware of special problems in
interpreting what you see:
(1) Dimensionality: A compound light microscope or transmission electron microscope shows a
flat, two-dimensional view, but the organelles themselves are all three-dimensional. The models on
display should help in visualizing the three-dimensional structure of organelles.
(2) Size: Keep aware of the magnification involved in each organelle you examine. The electron
micrographs are typically magnified much more than what you see through your light microscope.
(3) Contrast: With the notable exception of chloroplasts, most cell organelles are frustratingly
neutral in color on an unstained slide. Keep experimenting with different light intensities as you look
for significant detail. Special staining procedures are needed to obtain clear views of many cell
organelles. Phase contrast microscope systems exaggerate small differences in light absorption to
produce false colors. This enables seeing details in living cells otherwise only shown with stains.
(4) Life: Unless you kill them with rough handling, slides of onion epidermis and the Elodea leaf will
show living cells. Most staining procedures for the light microscope and all preparation of material
for the transmission electron microscope, however, result in the death of the cells. That makes it
especially difficult to relate the function of cell organelles to their form, and to determine what moves
and what doesn't. The problem is particularly great in electron micrographs, which show us still
views of cell ultrastructure, some of which may be in constant movement.

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During the course of the laboratory, complete . Organelle Views: *** = Easily Seen. * = Seen with
Difficulty. 0 = Not Seen..
Table 2-5. Organelle Views: *** = Easily Seen. * = Seen with Difficulty. 0 = Not Seen.
VIEW

Slides

ORGANELLE

middle lamella
cell wall
plasma membrane
nucleus
nucleolus
chromosome
vacuole
chloroplast
granum
mitochondrion
crista
endoplasmic reticulum
Golgi body
ribosome

Onion
Epidermis

Elodea

Models
Cauliflower

Electron Micrographs
#1

#2

#3

Exercises
A. Cell Study With a Light Microscope.
1. Cells of Onion Epidermis.
Make a wet-mount of a small piece of the inner epidermis of an onion leaf. To do this, clean a slide

and place a drop of water in the center. Using a pair of forceps, peel off a thin piece of the inner
epidermal surface of an onion scale. Be sure it is very thin or you will not be able to resolve the
internal parts of the cell. You would like it to be one cell thick! Place it in the drop of water and add a
clean coverslip. Blot off excess water with a paper towel or tissue fragment, for any water that
adheres to the slide will interfere with slide movement across the microscope stage. Examine under
the microscope at low power and then later at higher powers. You may need to adjust the amount of
light coming through the specimen.
Make notes in . Organelle Views: *** = Easily Seen. * = Seen with Difficulty. 0 = Not Seen.
concerning the parts you can see. (These cells from the outer cell layer of the onion bulb segment
are likely to contain clear crystals, which are not included in the table.) Notice how many of the listed
parts are not visible! In , try to make an accurate labeled sketch of the cell showing exactly the detail
that your microscope reveals in the best cells from your slides.

Figure 2-2. Onion Epidermis Cell (Sketch).

After thorough study of the cell, make another wet mount of onion epidermis by mounting it in a drop
of 0.1% solution of neutral red instead of plain water. This stains the protoplasm but leaves the cell
wall clear. Study cells for individual parts again.
Which parts could you see better with the neutral red solution?

How large are these cells?

2. Photosynthetic Cells of the Leaf of Elodea

Select a young leaf from near the tip of an active growing shoot. Make a wet mount and observe
under low power. For more practice in locating microscopic structures, move the stage and look for
cells that project from the margin of the leaf (spur cells). Then scan the leaf interior, searching for
cells that show internal movement.
a. Cytoplasmic Streaming (Cyclosis)
Observe the motion of the chloroplasts.
What is their pattern of movement?

Is the direction of movement the same for all cells?

What causes the chloroplasts to move?

Do they move from one cell to another?

What characteristics of the Elodea slide make it possible for us to see cytoplasmic streaming?

Why is cytoplasmic streaming never observed with an electron microscope?

b. Cellular Detail
Select cells that are especially easy to see. Make notes in about the parts you can detect.
Again, notice how many organelles are not detectable. If you cant find the nucleus, it may be
revealed by where chloroplasts pile up as they are swept along by cyclosis. Label .

Figure 2-3. Microscopic View of Elodea Leaf. a. Cells near Edge. b. Detail of a Cell.

c. Organelle Location, Size, Shape and Position

Open the microscope diaphragm wide to reduce depth of focus to a minimum and adjust the light
level with the lamp intensity control. By focusing up and down, see how the appearance of the
chloroplasts differs throughout the cell. Since chloroplasts are in the cytoplasm but outside the
vacuole, you can figure out the size and location of the vacuole indirectly by where the
chloroplasts occur. Conversely, since the chloroplasts are often squeezed between the vacuole
and the cell wall, you can find both side and top views, and figure out their shape.
Finally, focus up and down, keeping track of how the cell changes, and try to determine the
number of cell layers in the leaf. One? Two? Three? More than three?

Figure 2-4. Guard Cells Containing

Chloroplasts (Much Enlarged).

d. Viewing the Cell Membrane

Normally, the cell membrane cannot be detected with a light microscope. We can reveal its
presence, though, with a physiological trick. Before discarding your Elodea slide, remove it from
the microscope and put a few drops of saline solution (NaCl) at one edge of the cover slip. Use a
paper towel to take up water on the opposite side of the cover slip, so that the salt water is drawn
under the cover slip and across the cell. Be sure to draw a lot of water across. Blot off any
excess salt water and examine the cells. Be careful not to get salt water on the microscope.
If all goes well, the cell should lose so much water that it will shrink away from its wall. The cell
membrane should then be detectable as the line between the clear salt water and the cell
contents, which appear green from the chloroplasts. This shrinking of the cell contents away from
the wall is called plasmolysis, and will be studied again in Laboratory 20. It is invariably fatal to
the cell.

3. Measurement of Cells and Organelles

Make a wet-mount of a small piece of the lower epidermis of a Bryophyllum leaf. Examine the cells of
this tissue with the compound microscope and use the eyepiece micrometer to estimate of the length
and width of the largest cell you can clearly see. Enter the result in . Bryophyllum Cell
Measurements..
Table 2-6. Bryophyllum Cell Measurements.

mm
Length
Width

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Most epidermis cells lack chloroplasts and are transparent. Still, your material is likely to include
some green cells with chloroplasts. If the chloroplast-containing cell is one of a matched pair, the
cells are guard cells, and the chloroplasts will be very small (see ). If the green cell is one from
deeper in the leaf, and adhered to the epidermis when you peeled it away, the cell is probably a
mesophyll cell, and both the chloroplasts and the cell itself will be larger. Use the eyepiece
micrometer to estimate the diameter of a chloroplast. Enter your results in . Measurement of
Chloroplast..

Table 2-7. Measurement of Chloroplast.

Type of cell (guard or mesophyll)

Diameter of chloroplast in m

4. Hyperactive Cells from Cauliflower

Transfer small pieces of stained cauliflower tissue to a microscope slide. Cover with a coverslip and
squash with pressure on the coverslip. Observe with the microscope at high power. In this
preparation, mitochondria appear as very small black spots.

B. Demonstrations of Cell Organelles

1. Prepared slide(s)
a. Mitochondria
Observe the prepared slide showing mitochondria in a cell.
b. Plasmodesmata
Plasmodesmata are membrane-bound tubes of cytoplasm that connect cells across walls and
the middle lamella. Observe the plasmodesmata in the prepared slide of persimmon seed.
Judging from their location and appearance, speculate on the function of plasmodesmata:

2. Models
Observe the models of the cell. Use our familiar . Organelle Views: *** = Easily Seen. * = Seen with
Difficulty. 0 = Not Seen. as a checklist to verify your recognition of organelles.
a. Organelle Detail
Study the special organelle models: chloroplast (grana, thylakoids, stroma (matrix),
mitochondrion (cristae) and Golgi complex (dictyosome, cisternae, vesicles). Note the names
and appearance of subunits of the cell organelles that are detailed in special models.

Chloroplast:

Mitochondrion:

Golgi Body:

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b. Organelle Function
What is the function of each cell organelle? Complete . Functions of Eukaryotic Cell Organelles..
Table 2-8. Functions of Eukaryotic Cell Organelles.

Organelle
middle lamella
cell wall
plasma membrane
nucleus
nucleolus
chromosome
vacuole
chloroplast
mitochondrion
endoplasmic reticulum
Golgi Body
ribosome

Function(s)

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C. Electron Micrographs
Study the electron micrographs that are available on the lab tables. Collaborate with one another in
locating each organelle listed in . Organelle Views: *** = Easily Seen. * = Seen with Difficulty. 0 =
Not Seen., above, and complete . Cell Organelles, Transmission Electron Micrographs..
Use those micrographs and any other materials available in lab to identify the numbered organelles or
organelle parts in , , and and enter the name of each part in . Cell Organelles, Transmission
Electron Micrographs..

Figure 2-5. Resin Secreting Gland of Populus deltoides (Eastern Cottonwood)Transmission

Electron Micrograph 15,500X.

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Table 2-9. Cell Organelles, Transmission Electron Micrographs.

1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17

oil body
cristae
Golgi body vesicle (dictyosome vesicle)
microtubules

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Figure 2-6. Same Source as Figure 2-4. 95,000X.

Figure 2-7. From Leaf Mesophyll Cell of Populus Deltoides (Eastern Cottonwood)Transmission
Figure 2-8. Same Source as Figure 2-4. 95,000X.
Electron Micrograph 43,000X.

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KEY WORDS
organelle
prokaryotic
eukaryotic
middle lamella (pl. middle lamellae)
cell wall
plasma membrane
nucleus (pl. nuclei)
nucleolus (pl. nucleoli)
chromosome
vacuole
chloroplast
granum (pl. grana)
thylakoid
stroma or matrix
mitochondrion (pl. mitochondria)
crista (pl. cristae)
matrix
endoplasmic reticulum
Golgi body
ribosome
wet mount
cyclosis
plasmolysis
plasmodesma (pl. plasmodesmata)
thylakoid
dictyosome
cisterna (pl. cisternae)
vesicle

Laboratory 4
Cell Division, Mitosis
Introduction
A mature pear tree contains an estimated 15,000,000,000,000 cells. However, this tree began its life
as a single cell. This tremendous amount of growth is made possible by the process of cell division
in combination with the expansion of cells between successive divisions.
Cell division begins with precise duplication of chromosomes, then mitosis, or the segregation of
chromosomes into two daughter cells that are genetically identical to the mother cell. In order to
complete its division, a cell also equally partitions its cytoplasmic components so each of the two
daughter cells can function independently. This step is called cytokinesis. Following cytokinesis,
daughter cells enter interphase, during which they grow and prepare for another division. In plants,
cell division is mostly confined to specific regions, called meristems. For example, plant stems grow
in length by cell division at the tips, or shoot apical meristems.
Mitosis provides the mechanism for a cell to equally partition its genetic material (genes) into daughter
cells. Genes are segments of DNA that carry the information necessary for a cell to function. A
chromosome is basically a molecule of DNA containing many genes in a linear arrangement. Before
mitosis, each DNA molecule is copied. The two identical molecules that result, called chromatids,
are attached together at only a single place, the centromere. After DNA has been copied, a
chromosome is composed of two chromatids and one centromere. During mitosis, the chromatids
separate so each ends up in a different daughter cell. After the chromatids separate at mitosis, each
chromatid is considered to be a chromosome. A chromosome, therefore, may contain one or two
DNA molecules, but it always has only one centromere.

Exercises
A. Sequence of Events in Mitosis
You will receive a set of yellow cards illustrating stages (phases) of mitosis. The cell in this example
has four chromosomes. Arrange the events in the most logical sequence. The first card should show
one cell with four chromosomes; the last step should show two cells, each with four chromosomes.
Check your sequence with to make sure it is correct. Note and learn the order, names, and events of
the phases of mitosis: prophase, metaphase, anaphase, telophase and interphase. It is important
to realize that the four chromosomes move independently of each other during mitosis. This is a
major feature that distinguishes mitosis from meiosis. In meiosis similar chromosomes pair with one
another.

B. Chromosomal Movement at Mitosis

Use plastic straws to represent chromosomes.

1. Behavior of a Single Chromosome in Mitosis

Begin with a yellow straw, which represents a single chromosome. During interphase, the period
between cell divisions, the DNA in each chromosome is copied. Add a second yellow straw of equal
length to indicate this replication. The chromosome is now composed of two genetically identical
chromatids (straws) held together at the centromere (Velcro). The duplication has occurred during
interphase and now it and every other chromosome in the cell are ready for mitosis.
On a large piece of paper, draw a rectangle to represent a cell. Place two yellow chromosomes (each
with two chromatids) in the center of the cell. Outline the steps that must be taken to produce two
identical daughter cells. If necessary, refer to the sequence of events you assembled in exercise A.
These two chromosomes carry identical or different versions (alleles) of a gene which controls a
particular characteristic (trait) and are, therefore, homologous. In laboratory 5, you will

Figure 3-9. Chromosome Changes during Mitosis.

3
learn why cells contain two copies of each chromosome. After mitosis, does a chromosome contain
one or two DNA molecules?

2. Behavior of Two Different Chromosomes during Mitosis

A single chromosome does not carry all the genes needed by a eukaryotic organism. Every species
has a characteristic number of chromosomes to carry its complete complement of genes. In corn, for
example, the complete set of genes is partitioned into 10 different chromosomes, so we say its basic
chromosome number is 10. The basic chromosome number of humans is 23. We have two sets of
chromosomes so each human cell contains 46 chromosomes. Again using straws to represent
chromosomes, carry out the stages of mitosis for an organism with a basic chromosome number of
only two. The two chromosomes in the basic set will probably differ in length and centromere position
(in this exercise, the model chromosomes differ in color); more importantly, they carry different genes.
Remember that the mother and the two daughter cells must each have four chromosomes (two each
of the two chromosomes in the basic set). Chromosome counts are based on the number of
centromeres, since a chromosome may consist of one or two chromatids. All chromosomes in a cell
move simultaneously during each stage of mitosis.

3. Mitosis Models
Observe the mitosis models and slide set on demonstration. What is the basic chromosome number
of the organism represented by the model?

C. Onion Root Tip Squash for Chromosomes

Use the method recommended by the instructor to prepare cells for chromosome viewing. Cell
divisions are abundant only in youngest part: the terminal 1 mm of each root.
METHOD 1
(1) Remove an entire young onion root with a razor blade. This prevents other students from
going through the procedure with an apparent tip that will have no cell divisions.
(2)

Cut off 1 mm of the growing tip and place it on a slide.

(3) Cover root tip with 2 drops of 1 N HCl (the HCl dissolves the middle lamella so the cells can
separate).
(4) Warm gently (do not boil) over the flame of an alcohol lamp, keeping slide warm for 60 seconds
(this enhances the action of the HCl and also causes the chromosomes to swell).
(5)

Blot off excess HCl with a paper towel.

(6)

Add coverslip. Fold paper towel over slide and crush gently.

(7)

Remove coverslip and add two drops of toluidine blue.

(8)

Warm gently (do not boil) over the flame of an alcohol lamp for one minute.

(9)

Blot off excess stain with paper toweling.

(10) Add one drop of water and apply a coverslip.

(11) Fold a paper towel over the slide and coverslip and crush gently with thumb.
(12) Apply cover slip and observe under relatively low power of microscope. Look for cells that are
well separated, not elongated, and have conspicuous nuclei. In these regions, look for dividing
cells, which have darkly stained chromosomes. Look at dividing cells under high power to
determine which stages of division are present.

4
METHOD 2
(1) Remove an entire young onion root with a razor blade. This prevents other students from
going through the procedure with an apparent tip that will have no cell divisions.
(2)

Cut off 2 mm of the growing tip and place it in the hollow of a depression slide.

(3)

Cover with acetocarmine stain.

(4)

Wait 60 seconds.

(5)

Heat for 60 seconds without boiling or drying. Add additional drop of stain, if needed.

(6)

Wait 60 seconds.

(7)

Transfer root tip to flat slide.

(8)

Cover with drop of fresh stain.

(9)

Macerate with end of glass rod, about 40 taps.

(10) Apply cover slip and observe under relatively low power. Look for cells which are wellseparated, not elongated, and have conspicuous nuclei. In these regions, look for dividing cells,
which have darkly stained chromosomes. Look at dividing cells under high power to determine
which stages of division are present.
Compare your slide with the prepared slide of mitosis. If the slide you stained has abundant cell
divisions, use it to observe 100 cells. Otherwise, use the prepared slide. Count the number of cells in
each stage of mitosis and in interphase.
A cell cycle (the interval from one cell division to the next) in an onion root tip is typically takes
approximately 16 hours! This is the amount of time required to carry out one round of mitosis,
cytokinesis, and all the growth involved in the next interphase. You can use the following formula to
calculate the amount of time a cell spends in each stage:

# of cells in stage
Duration of stage (min)
total # of cells

) ( )(

Use your data and calculations to complete . Estimated Duration of Phases of Mitosis..
Table 3-10. Estimated Duration of Phases of Mitosis.

Your Count
Number
Prophase
Metaphase
Anaphase
Telophase
Interphase
TOTAL

Duration

Class Count
Number

Duration

16 hr
cycle

60min
hr

5
Do your results seem logical, considering the events that must occur in each stage of mitosis?

D. Asexual Reproduction
In the next lab, well study the processes involved in sexual reproduction, including the special kind of
cell division known as meiosis and the union of gametes in fertilization. A notable consequence of
sexual reproduction is offspring that combine characteristics from two parents. Many plants have the
ability to reproduce with neither meiosis or fertilization: asexual reproduction.
Observe the demonstration of asexual reproduction. Potato tubers, tulip bulbs, quackgrass rhizomes,
and strawberry runners are examples of plant parts, resulting from mitosis and cell growth only, which
will regenerate into new plants. All asexually produced plants are genetically identical to the mother
plant and are called clones. What might be some advantages of asexual reproduction over sexual
production involving pollination and the production of seeds?

Although asexual reproduction is common in nature, it is also widely used in commercial propagation
of plants. For example, all 'Red Delicious apple trees originated from one tree. They are asexually
propagated by grafting their branches or buds onto apple seedlings. Many plants, such as rose,
grape, willow, lilac, African violet, and black raspberry, are cloned by inducing root formation on pieces
of stems or leaves (cuttings). Observe the demonstrations of asexual propagation by cuttings. What
environmental conditions would encourage successful propagation by cuttings?

Why do you suppose some plants are propagated by cuttings more easily than others?

A method of asexual propagation that is becoming increasingly popular is tissue culture. Segments of
actively dividing plant tissue are aseptically cultured into new plants. Because each cell contains all
the genetic information to regenerate a complete plant, even a single cell can often be cultured if the
correct development environment is provided. Genetic engineering techniques often involve inserting
DNA into a single cell. When that cell divides by mitosis, the new DNA will be distributed to all cells in
the regenerated plant along with the preexisting genes. Observe the demonstrations of tissue culture.
What might be some advantages of tissue culture over other methods of asexual reproduction?

What might be some disadvantages?

E. DNA Replication
Mitosis separates chromatids into daughter cells. Therefore, each chromosome in a daughter cell
contains only one DNA molecule (one chromatid). If this cell is to divide again, that DNA molecule
must be copied to produce two chromatids. Study the DNA molecule on demonstration. Note that a
single DNA molecule consists of a double, twisted strand of nitrogen bases. There are only four kinds
of bases in DNA, adenine (A), thymine (T), guanine (G) and cytosine (C). Does its structure
suggest a simple way in which the molecule may be copied?

During interphase, each DNA molecule (chromosome) "unzips" at the hydrogen bonds which connect
adenine to thymine and guanine to cytosine. Then, new are inserted. Since adenine always pairs
with thymine and guanine always pairs with cytosine, the new strand can be synthesized using the old
one as a pattern, or template. After DNA replication, each DNA molecule is composed of one old
strand and one new one.
Given the following template, list the sequence of bases that would be inserted to build a new strand:

ATT G CG TACCG AATCAG G T

New strand
How would the shape of a DNA molecule differ if A-G and T-C bonds existed?

F. Questions
1. Mitosis appears to biologically produce the mathematically impossible. That is, a cell with 20
chromosomes can split into two cells, each with 20 chromosomes. Explain how this happens.

8
Table 3-11. Chromosome Base
Number Examples

Organism

Chromosome #
(haploid set)

bacterium

some sunflowers

onion

6 or 9

sequoia

11

yeast

17

bread wheat

21

human

23

marsh grass

30

raven

39

mallard duck

40

goldfish

47

some ferns

600

2. Do you think that complexity of an organism is related to its chromosome number?

Given the information in , would you reconsider your answer?

How might you explain the discrepancy between chromosome number and organism complexity?

9
3. In animals, cytokinesis occurs by the formation of a constriction in the cell membrane of the mother
cell. In plants, a cell plate is formed between the two daughter nuclei. It grows outward to form two
cell walls and a middle lamella. These partition the cytoplasm and nuclei into two daughter cells.
Why do you suppose cytokinesis is so different between the two groups of organisms?

4. What cellular events do you suppose must occur before a daughter cell can undergo mitosis?

10
5. In which regions of a plant would you expect to find a large proportion of cells undergoing mitosis?
How would you verify your hypothesis?

11

KEY WORDS
cell division
mitosis
daughter cell
mother cell
cytokinesis
interphase
meristem
gene
DNA (deoxyribonucleic acid)
chromosome
chromatid
centromere
phase
prophase
metaphase
anaphase
telophase
allele
trait
homologous
basic chromosome number
cell cycle
asexual reproduction
clone
DNA replication
adenine
thymine
guanine
cytosine
template
haploid

12

(blank page)

Laboratory 5
Meiosis and Gene Segregation
Introduction
When multicellular organisms grow, the cell divisions involved in the growth are always mitosis.
When single celled organisms reproduce, the division is often mitosis. As stressed in the previous
lab, this type of cell division produces genetically identical daughter cells. Cell division by meiosis,
on the other hand, occurs only in connection with sexual reproduction. In flowering plants, meiosis
occurs only in the female and male reproductive organs: within the ovaries and anthers, respectively.
The essential accomplishment of meiosis is to produce haploid cells (cells with a single set of
chromosomes) from diploid cells (cells with two sets of chromosomes). In higher animals, the
products of meiosis are gametes, which will be directly involved in fertilization. In plants and fungi,
however, meiosis produces spores that will typically develop into many celled haploid plants by
mitosis before gametes are produced and fertilization occurs. In any organism, sexual reproduction
produces genetically unique offspring that express characteristics of both parents. The sexual
recombining of genes that results from meiosis followed by fertilization produces genetic variability
upon which natural selection can act. Organism which lack meiosis
Meiosis must achieve the following:
(1) reduce the chromosome number in the gametes to half the normal number.
Why?

(2) create gametes which are genetically different from the mother cell.
Why?

During fertilization, two gametes combine to form a zygote. Each gamete carries a basic
chromosome set, so the zygote contains two of each chromosome. A new plant will develop through
mitosis of the zygote. The plants cells will be diploid, carrying two of every chromosome. The bodies
of animals and higher plants are diploid. Cells that are produced by meiosis, on the other hand, have
one of every chromosome. These are said to be haploid cells. In plants, haploid cells are often able
to undergo mitosis to produce identical (haploid) daughter cells.

Exercises
A. Sequence of Events during Meiosis
Use the green cards to study the process of meiosis. As in the previous lab, the cell in this example
has four chromosomes. Arrange the events in the most logical sequence. The first card should show
one cell with four chromosomes; the last step should be four cells, each with two chromosomes.
Check your sequence with to make sure it is correct. Note the names of the stages of meiosis.
How do the steps of meiosis differ from those of mitosis?

How are the steps of mitosis and meiosis similar?

B. Chromosomal Movement during Meiosis

Use plastic straws again to represent chromosomes.
Any body cell of a plant whose basic chromosome number is one would actually have two
chromosomes, one which came originally from its mother's gamete and one from its father's. This is a
diploid cell because it has two of every type of chromosome.

1. Prophase I
Put two yellow "chromosomes", each consisting of two chromatids attached at the centromere, into a
square outline of a cell to simulate the first appearance of the chromosomes. Align the chromosomes
next to each other along their length to simulate the pairing that occurs. This pairing is where meiosis
differs dramatically from mitosis. The chromosomes that seek one another out are referred to as
homologous.

2. Metaphase I
Bring the paired chromosomes to an imaginary central metaphase plate, with one on each side of the
center. Cells undergoing mitosis often also have two copies of every chromosome, but the
chromosomes do not pair up.

3. Anaphase I
Separate the pair of chromosomes so each chromosome moves to a different pole. Note that, unlike
mitosis, chromatids have not separated. This is the stage that begins the reduction in chromosome
number per cell. Spindle fibers are involved in moving the chromosomes.

4. Telophase I
Draw a line between the chromosomes to produce two cells. Note that each cell now has only one
chromosome and each chromosome consists of two chromatids. A second division, similar to mitosis,
will occur next.

5. Prophase II
From this point on, follow only one of the two cells. Remember that the same events are occurring at
the same time in the other cell.

6. Metaphase II
Bring the chromosome to an imaginary metaphase II plate.

7. Anaphase II
Separate the chromatids and begin to move them toward opposite ends of the cell.

8. Telophase II
Draw a line between the chromosomes (chromatids) to represent cytokinesis. Adjust the
chromosomes to complete the division of the cell weve been ignoring and note that meiosis has
produced four cells, each with one of the beginning chromatids.

9. Meiosis with 4 Chromosomes

Try to simulate the steps of meiosis for a diploid plant that has a haploid chromosome number of two.
How many chromosomes are in the mother cell?

How many chromosomes are in each of the gametes?

If this had been a corn cell with a base chromosome number of 10, the mother cell would have had 20
chromosomes, and each gamete would end up with 10.

10. Mitosis Review

For review, carry out the steps of mitosis with a diploid plant that has a basic chromosome number of
two.

Figure 4-10. Chromosome Appearance during Meiosis.

11. Questions:
a. Why is meiosis sometimes referred to as "reduction division?"

b. Why is it important for homologous chromosomes to be paired at metaphase I? Why aren't they
paired at metaphase II?

c. What would happen if meiosis didn't exist but gametes still combined to form a zygote?

d. Peas have a basic chromosome number of 7.

How many chromosomes are in a diploid somatic (body) cell?
How many chromosomes are in each gamete?
How many chromosomes are in each zygote?

C. Determination of Gamete and Offspring Types

Recall that genes are segments of DNA on chromosomes. Now that you know how chromosomes
move at meiosis, you can determine the genetic composition of gametes. With this information, you
can predict the genetic makeup of offspring that result from the random union of gametes at
fertilization.
You have probably noticed the letters "P" and "Y" printed on your straw chromosomes. These letters
symbolize genes on the chromosomes. The upper and lower case letters indicate alternative forms of
the gene, alleles. Alleles code for slightly different types of the same gene product. For example, the
gene for flower color may have alleles for white color and red color.

6
A plant that carries the same allele on both of its homologous chromosomes is homozygous. If it
carries one allele on one chromosome and a different allele on the homologous chromosome, it is
heterozygous. A plant may be homozygous for one gene and heterozygous for another on the same
chromosome. When a plant is heterozygous, one allele often masks the expression of another. The
allele that is expressed is called the dominant allele (and is indicated by an upper case letter), while
the allele that is not expressed (indicated by a lower case letter) is called the recessive allele. The
physical appearance of a plant is called its phenotype, while its genetic makeup is its genotype.
(1) Choose a "Y" straw chromosome (with two identical chromatids) and a homologous "y"
chromosome. Complete meiosis with this pair. What is the genetic composition (genotype) of each of
the four gametes (products of meiosis)?

(2) If five cells from step 1 undergo meiosis, how many "Y" and how many "y" gametes would be
produced?

Choose white foam blocks labeled "Y" and "y" to represent this pool of gametes. Once you have
collected the correct number of each foam block, turn the blocks over and put them in a pile. These
will represent the gametes produced by the female parent.
(3) Repeat step (2) with yellow foam blocks to represent the gametes from the male parent.
(4) Randomly choose one male and one female gamete to unite at fertilization. Use . Genetic
Combinations Resulting from Random Gamete Pairing, Yy x Yy Cross. to note the genetic
combinations you have created. Continue until all gametes have been united.

Table 4-12. Genetic Combinations Resulting from Random Gamete Pairing, Yy x Yy Cross.

Yy x Yy Cross

Genotype

Trial 1

Number
Proportion

Trial 2

Number
Proportion

Total

Number
Proportion

YY

Yy

yy

7
Repeat step 4 and compare your results with your first trial. If they are similar, what proportion of
each genetic type did you obtain?

If they are not similar, then repeat until you obtain consistent results. You are determining the
probability of obtaining various genetic combinations following the random union of gametes. As your
sample size increases, so does the accuracy of your results.
You have just completed a cross between two parents, each with genotype Yy, and you have
determined the genetic makeup of their offspring.
What is the genotypic ratio (YY : Yy : yy) expected of offspring from a Yy x Yy cross?

If Y is responsible for purple color and y results in yellow color, what is the phenotypic
(purple : yellow) ratio?

(5) Repeat steps 1 to 4 with the following cross: Yy x yy. Record your results in . Genetic
Combinations Resulting from Random Gamete Pairing, Yy x yy Cross..
Table 4-13. Genetic Combinations Resulting from Random Gamete Pairing, Yy x yy Cross.

Yy x yy Cross

Genotype

Trial 1

Number
Proportion

Trial 2

Number
Proportion

Total

Number
Proportion

YY

Yy

yy

8
What genotypic and phenotypic ratios did you obtain among the offspring of this cross? How does
this differ from the results of your Yy x Yy cross?

Would the results differ if one parent produced twice as many gametes as the other?

(6) A simple way to predict offspring genetic combinations is to use a Punnett square. With this
method, you list expected proportions of male gametes along one axis and female gametes along the
other. This allows you to systematically combine gametes to determine offspring proportions. For
example, a Punnett square for a Yy (female) x yy (male) cross would look like this:

Gametes

0.50 Y

0.50 y

1.00 y
To complete the square, multiply gamete proportions and add alleles. This will determine the
expected proportion of each offspring type. Remember, every gamete has one copy of every gene
and each product of fertilization has two.

Gametes

0.50 Y

0.50 y

1.00 y

0.50 Yy

0.50 yy

Complete the following Punnett square for a Yy x Yy cross:

Gametes

0.50 Y

0.50 Y

0.25 YY

Yy

Yy

D. Segregation of Genes
Every chromosome in a plant contains thousands of genes. Therefore, if you are studying genetic
ratios of two genes, you need to know whether they are on the same or different chromosomes. Two
genes on the same chromosome are said to be linked, and the occurrence of genes on the same
chromosome is referred to as linkage.

1. Genes Which Occur on Different Chromosomes

Use the straw chromosomes to determine the gametes produced from a plant that is heterozygous for
genes Y and P. Because these genes are on different chromosomes, they are unlinked.
You should determine that a YyPp plant will produce the following gamete proportions:
25% YP, 25% Yp, 25% yP, and 25% yp. Ask your instructor for help if you did not obtain these results.
Set up a Punnett square to determine the offspring ratios expected from a YyPp x YyPp cross:

Gametes

What offspring genotypic ratio did you obtain?

If Y purple, y yellow, P plump, and p shrunken, then what is the offspring phenotypic ratio?

2. Linked Genes
Use the straw chromosomes to determine the gametes produced by a heterozygote with linked
genes. Notice that one chromosome may carry both dominant or both recessive alleles, or it may
have one of each. If one chromosome carries both dominants and the homologue carries both
recessives, the genotype is indicated YT / yt. If each chromosome carries one dominant and one
recessive allele, it is Yt / yT.
What gametes are produced by a YT / yt plant?

By a Yt / yT plant?

10
Set up a Punnett square to determine the offspring ratios expected from a YT / yt x Yt / yT cross:

Gametes

E. Genetics Problems
(1) In tomatoes, red fruit is dominant to yellow. What offspring genotypes and phenotypes would you
expect from?
(a) homozygous red x yellow
(b) homozygous red x heterozygous
(c) heterozygous x heterozygous
(2) If you crossed a red fruit plant with a yellow fruit one and obtained 50 red fruit and 45 yellow fruit
offspring, what was the genotype of the red fruit parent?

11
(3) Another gene in tomato is responsible for stem color. A plant with purple stems is crossed with
another of the same phenotype. In the offspring generation, 74 purple and 23 green plants are
observed.
(a) Which trait is dominant?

(b) What were the genotypes of the purple parents?

(4) What offspring phenotypic ratios would you expect from:

(a) a cross between two tomato plants, both heterozygous for fruit color and stem color (the
genes are not linked)?

(b) a cross between a homozygous tomato with yellow fruit and green stems, and a heterozygous
plant for both genes?

12
(5) Supersweet sweet corn is homozygous for the recessive alleles of genes S and H. These genes
are not linked. Regular sweet corn is homozygous recessive for s, but contains either one or two
dominant H alleles. All other genotypes are not sweet. What phenotypic offspring proportions would
you expect from the following crosses?
(a) SShh x ssHH

(b) ssHh x ssHh

(c) SsHh x SsHh

KEY WORDS
meiosis
haploid
diploid
gamete
fertilization
spore
zygote
homologous chromosomes
homozygous
heterozygous
dominant
recessive
phenotype
genotype
Punnett square
linked genes (linkage)

13

Answers to Genetics Problems

(1) In tomatoes, red fruit is dominant to yellow. What offspring genotypes and phenotypes would you
expect from:
(a) homozygous red x yellow

YY x yy all Yy; red

(b) homozygous red x heterozygous

(c) heterozygous x heterozygous

YY x Yy 1/2 YY, 1/2 Yy; all red

Yy x Yy 1/4 YY, 1/2 Yy, 1/4 yy; 3/4 red, 1/4 yellow

(2) If you crossed a red fruit plant with a yellow fruit one and obtained 50 red fruit and 45 yellow fruit
offspring, what was the genotype of the red fruit parent?
Y x yy 1/2 Y , 1/2 yy; Y must be Yy
(3) Another gene in tomato is responsible for stem color. A plant with purple stems is crossed with
another of the same phenotype. In the offspring generation, 74 purple and 23 green plants are
observed.
(a) Which trait is dominant?

purple x purple 3/4 purple, 1/4 green;

purple must be dominant

(b) What were the genotypes of the purple parents?

Both were Gg

(4) What offspring phenotypic ratios would you expect from:

(a) a cross between two tomato plants, both heterozygous for fruit color and stem color (the
genes are not linked)?
YyGg x YyGg

9/16 red fruit, purple stems

3/16 red fruit, green stems
3/16 yellow fruit, purple stems
1/16 yellow fruit, green stems

(b) a cross between a homozygous tomato with yellow fruit and green stems, and a heterozygous
plant for both genes?
yygg x YyGg
1/4 red fruit, purple stems
1/4 red fruit, green stems
1/4 yellow fruit, purple stems
1/4 yellow fruit, green stems
(5) Supersweet sweet corn is homozygous for the recessive alleles of genes S and H. These genes
are not linked. Regular sweet corn is homozygous recessive for s, but contains either one or two
dominant H alleles. All other genotypes are not sweet. What phenotypic offspring proportions would
you expect from the following crosses?
note:

sshh = supersweet
ssHh or ssHH = sweet
all others - not sweet

(a) SShh x ssHH

all SsHh, not sweet

(b) ssHh x ssHh

1/4 ssHH, 1/2 ssHh, 1/4 sshh

3/4 sweet, 1/4 supersweet

(c) SsHh x SsHh

12/16 S , 3/16 ssH , 1/16 sshh

14
12/16 not sweet, 3/16 sweet, 1/16 supersweet

Laboratory 6

(blank page)

Laboratory 7
Bacterial Study Techniques
Introduction
These simple unicellular and microscopic organisms are the most abundant and widely distributed of
all life forms. Most taxonomists place these organisms in one or even two separate kingdoms.
Bacteria are prokaryotic; that is, they lack a well-defined nucleus and other membrane-bound
cellular organelles, and their chromosome structure is fundamentally different from that of other,
eukaryotic, organisms. The majority of bacteria are saprophytes and live by decay. Of course, many
others obtain their nutrition from living plants or animals as parasites or pathogens. Many, notably
the cyanobacteria, are autotrophic.
The best way to observe bacterial activity is to culture them in the laboratory; that is, grow them
under optimal conditions, often until they produce visible colonies. Most of this activity can be seen
in a relatively short time since bacteria multiply very rapidly by asexual binary fission. Reproduction
by fission simply means that cell division produces two individual organisms from one. The reactions
that these organisms carry out on various food sources (media, singular: medium) also provide one of
the primary means of identifying them since they are so much smaller and simpler than most other
organisms.

Sterile Technique
Special procedures are necessary for working with cultured microorganisms for two main reasons:
(1)

to prevent the escape of pathogenic organisms from cultures to the surroundings.

(2)

to prevent contaminating an established culture with microbes from the surroundings.

In many circumstances, including most of our use of bacterial, fungal, and algal cultures in these
botany labs, the second is really the most critical. For your sake, and for the sake of your fellow
students, be sure to use sterile technique when specified by the lab instructions! (next page)
summarizes techniques for sterile transfer of microorganisms from one place to another.

Exercises
A. Preparation of a Hanging Drop Mount
The motility of bacteria can be readily demonstrated in a hanging drop mount. Prepare the mount at
the demonstration table using a depression slide. Using a wire loop and sterile technique, take a
loopful of bacteria from the flask and place it in the center of a square coverslip. Invert the coverslip
with the bacterial drop and place it quickly over the depression. A diagram of this setup is shown in
Figure 6 -11.

Figure 6-11. Depression slide with hanging drop mount.

Examine the hanging drop mount under the microscope. Locate the edge of the drop on the slide
with the low power objective lens. Now switch to high power (400X), increase the depth of focus by
closing the diaphragm, and if necessary, adjust the amount of light by increasing lamp brightness.
Keep adjusting the light until the bacteria become visible. Try to distinguish motility from Brownian
movement. The latter is the bombardment of bacterial cells with electrons of the solution and appears
as random shaking. Flagellated bacteria will be actively moving. What are the shapes of the motile
bacteria?

Clean your slide by dipping it in the disinfectant, rinsing it with water, and drying it. Please return the
clean slide and coverslip to the demonstration table.
Table 6-14. Sterile Technique for Transfer of Cultured Micororganisms.
(1) Identify the material you want to transfer, which will differ enormously depending on what
organism is to be transferred: a drop of liquid, clear or cloudy; semi-solid paste from a colony on agar,
or a green scum from an algal culture. In some cases, youll be encouraged to agitate a liquid to get
the organisms into suspension, in other cases, exactly the opposite!
(2) Assemble transfer materials:
(a) transfer implement, such as a bacterial loop or needle
(b) culture source, such as a test tube, Erlenmeyer Flask, or Petri plate.
(c) source of open flame, such as a Bunsen Burner
(d) clean surface to receive the transfer, such as a microscope slide or a cover slip for a hanging
drop mount (see below).
(3) Perform the transfer:
(a) Flame transfer implement (loop, needle, etc.) until it glows.
(b) Allow transfer implement to cool, taking care not to set it on any surface, else it can become
contaminated.
(c) Remove stopper or cover from culture vessel (flask, test tube, Petri Plate, etc.) 3rd and 4th
digits from same hand that holds the transfer implement. If your manual dexterity is marginal,
work cooperatively with another student so you have more than two hands available.
(d) Flame the mouth of the culture vessel if it is a flask or test tube. If it is a Petri Plate, keep the
cover substantially above the plate to reduce the chance of airborne contamination.

3
(e) Pick up a loopful, scraping, or snagging of the microorganism. Note: If picking up from a
solid agar surface, scrape gently! A tiny amount of material will provide millions of bacteria, so
you dont need much! As many as a hundred students may be sharing the same source, so you
shouldnt take much.
(f) If you need an additional material from a liquid source, always re-flame and re-cool the loop
before returning to the source culture.
(4) Put away materials.
(a) When the transfer is completed, re-flame the mouth of the culture vessel if it is a flask or test
tube.
(b) Replace the (uncontaminated) cover. If a cover becomes contaminated it should be replaced
with a sterile one (see instructor).
(c) Re-flame the transfer implement a last time before returning it to its place.
(d) Follow or seek directions for ultimate disposal of transferred material.
(e) Inform the instructor about probable culture contamination.

COCCI

BACILLI

SPIRILLA

Figure 6-12. Bacterial Shapes.

B. Shapes of Bacteria
Examine a prepared slide showing the three general shapes of bacteria. A spherical bacterium is a
coccus (plural: cocci), a rod-shaped bacterium is a bacillus (plural: bacilli), and a curved, twisted,
rod-shaped bacterium is a spirillum (plural: spirilla). Make drawings of your observations in Figure
6 -12.

C. The Gram Stain

Bacteria tend to have one of two basic types of cell walls. Differences in the chemistry of these
accounts for differences in response to a standard procedure called a Gram stain. This provides a
fundamental means of identification since the stain will also show the arrangement, size and shape of
the bacterial cells.
Those bacteria that tend to retain a crystal violet-iodine chemical dye complex appear purple and are
termed Gram positive (G+). Bacteria that lose the initial dye complex and become counterstained by
another dye are termed Gram negative (G-). These bacteria stain red.
Prepare a slide from a mixed culture of bacteria and check them for their Gram stain properties. Your
instructor will demonstrate proper sterile technique for handling bacteria. Take several loopfuls of
bacteria from the flask or culture tube and make a smear on the center of a clean slide. The smear
should be about the size of a nickel and not too thick. Allow the slide to air dry and then fix the smear
by gently passing it over a flame a few times. Let the slide cool. Place your slide on the staining tray
and carefully follow the staining procedure.
(1) Stain the smear with crystal violet solution for 1 minute: then wash with water for a few seconds.
Drain off excess water on paper toweling and blot gently.
(2) Apply Gram's iodine solution for 1 minute; wash with water, drain and blot.
(3) Decolorize with alcohol (95%) until free color has been washed off (approximately 30 seconds).
Tilt the slide when applying the alcohol so that it runs over the smear. Wash the slide with water,
drain and blot.
(4) Counterstain the smear for 30 seconds with safranin; wash and blot dry.
Examine the slide under 200 power and try to locate the stained area. Without a coverslip, add a
drop of immersion oil directly to the slide and view the preparation with the 100X lens. The instructor
will help you find the bacteria.
Two genera in the mixed culture are Gram positive cocci arranged in chains or grape-like clusters.
Another bacterium is a large Gram positive bacillus which occurs singly, paired or in chains. The last
bacterium is a small Gram negative bacillus occurring singly or in pairs.

Figure 6-13. Appearance of Bacteria Prepared by Negative Staining.

Make drawings in Table 6 -15 of your observations. Be sure to note color differences and variations
in cell arrangements and shapes (chained, clustered, in two's bacilli, cocci, etc.).
Table 6-15. Gram Stain.

Escherichia

Staphylococcus

Streptococcus

Bacillus

Gram Stain
Shape and
Arrangement
of Cells
D. Negative Stain
Unstained bacteria can be made visible when they are prepared as a film in nigrosin. The nigrosin
provides a dark background within which the cells remain clear, essentially a negative stain. A
special benefit of negative staining is lack of cell distortion compared with other staining procedures.
Place a drop of water about 1/2" from one end of a slide. Add a small amount of fluid from the mixed
culture used in Gram staining to the water drop. Add a drop of nigrosin. Mix well and spread out with
the flat of the loop over the entire slide to give thick and thin areas of smear. Air Dry.
Examine the preparation under 400X. You will have to view the light areas toward the edges. The
cell areas will appear bright in an otherwise dark field. Make drawings of the cells in Figure 6 -13.

E. Influence of a Physical Factor on Bacterial Growth

Ultraviolet radiation (UV) has shorter wavelength and higher energy than visible light. It can has
adverse effects on biological tissues by increasing the reactivity of normally stable chemicals.

6
Infrared radiation (IR) has longer wavelength and lower energy than visible light and is not
considered to be damaging to organisms.

Short-wave Ultra-violet

Long-wave Infrared

Figure 6-14. Appearance of Plates Exposed to Ultraviolet and Infrared Radiation Respectively.

Two plates of the same bacterial species have been partially masked with a cardboard cutout, then
exposed to 5 minutes of UV and IR radiation respectively. Clear zones corresponding to the
unmasked part indicate radiation-induced failure of bacterial growth. Observe the plates and record
your observations in Figure 6 -14.

F. Influence of Chemical Factors on Bacterial Growth

The effects of some common chemical substances are on demonstration.
Sterile paper discs have been soaked with chemicals chosen for their demonstrated or alleged ability
to kill bacteria. The discs are then placed on agar plates that have been surface inoculated with gram
positive bacterium (Staphylococcus) or a gram negative bacterium (Pseudomonas). Observe the
Table 6-16. Influence of Chemicals on Bacterial Growth.

Chemical Agent

Zone of Inhibition (mm)

G+
G-

phenol*
Lysol
Scope
Bactine
H2O2 (hydrogen
peroxide)
70% alcohol
* Traditional standard in tests of chemical effects.
plates and measure the diameters (mm) of growth inhibition (clear zones) around the paper discs

(include the disc in your measurement). Measure the culture plate itself, not the lid. Is there an
overall difference in resistance between gram negative and gram positive bacteria?

Record your results in Table 6 -16

G. Culturing Microbes from the Environment

We have already observed the growth of microbes in liquid and on solid culture media. The
solidifying agent added to liquid media is agar (a substance obtained from red algae). Most media
contain plant or animal extracts often supplemented with sugars. Briefly opening sterile culture plates
in various places, then incubating them for a day or two in a warm place will readily show the
presence of bacteria and fungi (molds) in those environments from the visible colonies which develop.
Bacterial colonies are usually small and round with a slimy consistency. Fungal colonies, on the other
hand, tend to have a cottony, filamentous growth pattern.
Record your observations on colony type and frequency in a systematic way in Table 6 -17
(none few many very numerous).
Table 6-17 Colony Count, by Type and Exposure Site.

Greenhouse
Bacteria
Fungi

Lab

Outdoors

10

KEY WORDS
prokaryotic
saprophyte
parasite
pathogen
culture
colony
binary fission
sterile technique
hanging drop mount
depression slide
wire loop
Petri plate
bacillus (pl. bacilli)
coccus (pl. cocci)
spirillum (pl. spirilla)
Gram stain
negative stain
ultraviolet radiation (UV)
infrared radiation (IR)
culture medium (pl. culture media)
agar

11

(blank page)

Laboratory 8
Bacteria
Cyanobacteria
Viruses
I. Bacteria
Introduction
We will now examine some of the cellular, chemical and cultural features of bacteria in more detail.

Exercises
A. Bacterial Anatomy
1. Bacterial Endospores
An endospore is a heat, water, and chemical resistant survival structure produced by a few genera of
bacteria. Endospores tend to be formed when conditions become unfavorable. Typically, only a
single round or oval endospore develops per cell. The endospore matures and eventually becomes a
free spore as the cell that produces it disintegrates. This spore may remain dormant for many years
but can readily germinate into a vegetative cell with the return of suitable environmental conditions.
Bacillus and Clostridium are common bacterial genera that form endospores. Botulism, lockjaw, and
anthrax are examples of human and animal diseases caused by these organisms but not all
endospore-forming bacteria are pathogenic.
Use the following procedure to differentially stain the endospores and free spores of Bacillus
megaterium.
(1) Prepare a thin smear from the provided culture of B. megaterium onto a clean slide. Air dry and
heat fix.
(2) Flood the smear with malachite green/Tergitol 7 and allow to stand for 30 minutes or more.
Tergitol 7 is a detergent-like compound that facilitates the entry of malachite green into the spore.
(3) Rinse the preparation with water. Use the plastic wash bottle provided and gently wash the
stain starting at the top part of the slide.
(4)

Counterstain the preparation with safranin for 1 minute. Wash gently with water and blot.

(5)

Examine the preparation under the microscope with the oil immersion lens.

Both the endospores and free spores that are no longer enclosed by the original cell wall should be
green. The vegetative part of the cell should be red. Not every cell will form an endospore. Make
measurements of the vegetative cells, endospores, and free spores and record your results on Figure
7 -15.

Figure 7-15. Bacteria with Endospores. (Label and give approximate cell dimensions.)

Polar (Monotrichous)

Peritrichous

Figure 7-16. Bacterial Flagella Patterns. (Sketch from microscope view.)

Figure 7-17. Bacterial Capsules. (Sketch and notes.)

2. Flagella
Observe the demonstration slides showing the two most common arrangements of flagella in
bacterial cells. Draw the arrangement of flagella on the cells in Figure 7 -16.

3. Capsules
The cell wall of many species of bacteria is surrounded by a mucilaginous substance referred to as a
capsule or slime layer. This capsule is usually composed of polysaccharides. These slime layers
may protect the bacteria against desiccation because they contain a lot of water and also help
bacteria attach to surfaces. Certain pneumonia-causing bacteria produce large capsules which helps
them to resist phagocytosis by a host immune response. Examine a demonstration slide of bacteria
producing capsules. Make drawings of your observation in Figure 7 -17.

B. Growth and Cultural Characteristics of Bacteria

The growth appearance of bacteria on various media can be used in their identification. Color,
abundance of growth and even odor along with optimum temperature and oxygen requirements are
important criteria in the separation of bacteria.

1. Chromogenesis (Production of Color)

Examine various cultures of bacteria displaying chromogenesis (the production of pigment). Watersoluble pigments tend to diffuse into the medium where you may see color changes. Fat-soluble
pigments tend to be retained by the cells.

2. Isolation and Cultural Characteristics:

a. Streak Plate
A streak plate is a method of obtaining a pure culture from a mixed culture of bacteria or to
determine cultural characteristics (colony size, density, etc.) of a particular microbe. The bacteria
are streaked with a loop from an initial inoculating point until they become isolated into single
cells, which produce colonies through fission. It is then easy to transfer the isolated bacteria to
suitable media. Examine the streak plate showing two species of bacteria. Note differences in
colony size and color.
b. Pour Plate
To make a pour plate the original source of the bacteria is diluted in order to obtain a
concentration level that is suitable for isolation. The dilution is placed in melted agar so surface
and subsurface growth occurs when the agar solidifies. Examine the demonstration plate.

C. Biochemical Activities of Bacteria

Bacteria utilize chemical substances in a variety of ways. This metabolism often produces byproducts
that can be used in the identification of these microbes.

1. Litmus Milk

5
Powdered skim milk with a litmus pH indicator and water provides an excellent medium for the
growth of bacteria. If a particular bacterium can break down the primary sugar (lactose) in milk, a
drop in pH occurs because of the release of acids and gas. The litmus indicator turns pink below a
pH of 5.
The major protein component in milk is casein, which exists as large molecules in colloidal
suspension. Casein gives milk its white-turbid appearance. Another bacterium may produce
protein-degrading enzymes which cause casein to be broken down with the subsequent release of
ammonia. This increases the pH of the litmus indicator above 8 and turns the solution blue-violet.
Observe the demonstrations and record your observations in Figure 7 -18. Note the lavender color in
the control tube, intermediate between the two contrasting test responses.

Figure 7-18. Litmus Milk Response to Sugarand Protein-Utilizing Bacteria. (Sketch and
notes.)

2. Fermentation of Carbohydrates
The ability of bacteria to ferment different sugars and the type(s) of byproducts formed during the
fermentations can be used in their identification. A given carbohydrate may be utilized to form a
single end product or a number of different end products (acids, alcohols, other organic molecules or
gases) depending on the particular bacterium.
A bacterium inoculated to a suitable medium containing one sugar and a pH indicator (phenol red)
may produce an acid(s) and sometimes gas if the microbe has the necessary enzymes. The acid(s)
turns the pH indicator from red to yellow.
Each test tube contains an inverted gas collecting tube (Durham tube). If gas is produced during the
fermentation reaction by the bacterium, some of the fluid inside the Durham tube will be displaced.
This traps a gas bubble.
Observe the demonstration where a particular bacterium has been inoculated to four different sugars.
Look for acid (A) or acid-gas (AG) changes. Each bacterium is likely to have its own characteristic set
of food using abilities. These correspond to the enzymes that each species produces. Record your

Figure 7-19. Bacterial Fermentation. (Sketch and label.)

observations in Figure 7 -19.

3. Fat and Protein Utilization

The degradation of fats to glycerol and fatty acids and proteins to amino acids by bacterial
enzymes can be readily demonstrated on solid media. Clear zones surrounding bacterial growth
indicate enzymatic degradation of the substrate. Observe the demonstrations and record your
observations in Figure 7 -20.

7
a.

b.

Figure 7-20. Lipid and Protein Digestion by Bacteria. a. Lipid (Fat). b. Protein. (Sketch and
notes on color changes.)

4. Antibiotics
Bacteria produce the majority of antibiotics. The advantage to the bacteria that produce them is
decreasing the number of competitors for the food supply. These substances do not appear to be
essential for the growth of the organism. Observe the effects of various antibiotics on a Gram positive
bacterium and on a Gram negative bacterium. It is not necessary to remember all of the antibiotics
but note the contrast in sensitivity or resistance of the two bacteria to the different antibiotics. Record
general results in Figure 7 -21.

Gram +

Figure 7-21. Antibiotic Sensitivity. (Notes.)

Gram

8
A culture of Streptomyces is on demonstration. Members of this genus produce most of the
antibiotics used in this demonstration.

D. Pathogenic Bacteria
1. The TB Organism
The genus Mycobacterium includes the causal organisms for tuberculosis and leprosy. Observe the
2 x 2 slide showing the red-stained, rod-like cells found in sputum from an infected tuberculosis
patient.
After a time when the disease was considered under control, tuberculosis has been on the increase in
the United States. Highly contagious strains have been isolated from AIDS patients.

Figure 7-22. Growth of Streptococcus

Colonies on Blood Agar. (Sketch.)

2. Streptococcus on Blood Agar

Blood serum added to a medium provides nutrients that favor the growth of certain pathogenic
bacteria. Some species of Streptococcus degrade (hemolyze) red blood cells. Hold the Petri Dish
towards a light source and observe (from the bottom) the clearing around the bacterial colonies.
Hemolysis usually indicates that the organism is pathogenic. Record your observations in Figure 7
-22.

3. Agrobacterium tumefaciens on Tomato Plants

Galls on plants are not uncommon. They are caused by many types of stimuli including bacteria,
insects, viruses and fungi. Agrobacterium tumefaciens is the only bacterium that incites gall-like
tumors (cancer cells) in plants. The disease is worldwide in distribution and also has a wide host
range.
Compare a tomato plant inoculated with A. tumefaciens by wounding with an uninoculated control.
Note the tumor growth. Once the tumor has been initiated by the bacterium, the causal organism no
longer has to be present. The bacterium has a plasmid gene governing tumor induction. Plasmids
are small circular DNA packets separate from the main chromosome. The gene becomes
incorporated into the host plant chromosome and then is transcribed.
It is possible to remove the tumor-inducing region from the plasmid and then incorporate genetic
material from a plant into that site. The bacterial plasmid could then function as a vector for
transferring beneficial genes between different plants. Theoretically, genetic traits that influence plant
resistance to diseases or insects can be transferred.

10

Figure 7-23. Legume Root Nodule with Bacteria-Containing Cells.

In practice, this is complicated by lack of knowledge of the chromosome locations of these beneficial
genes, as well as by the same gene functioning differently in plants that are not closely related.
Sketch an infected plant:

E. Nitrogen Fixation
Nitrogen-fixing bacteria are able to reduce nitrogen gas from the soil atmosphere into ammonium
ions and subsequently incorporate the nitrogen into amino acids. A few select groups of prokaryotes
are the only living things that have the enzyme system to accomplish this critical conversion.
Members of the bacterial genus Rhizobium that can fix nitrogen occur in nodules formed by
leguminous plant roots. As a result legumes have a competitive advantage on nitrogen-deficient soils.
Observe the demonstration slide of the bacteria in these nodules. Label Figure 7 -23, identifying
normal root tissue, the nodule and the enlarged cells filled with nitrogen-fixing bacteria. Whole plant
roots with nodules are also on demonstration.

II. Cyanobacteria or Blue-Green Algae

Originally, this large, widespread group was classified with other algae but now they are generally
included with the bacteria because of their prokaryotic cells, gram-negative cell walls, and antiquity in
the fossil record. There also has been a taxonomic revolution within the group. The total number of
species has been reduced from 6,000 to less than 600 by recognizing that many were
environmentally induced growth forms. Probably the most widely dispersed algae, the blue-green

11
algae occur worldwide in salt, fresh, brackish waters and hot springs. They also are found in or on
soil, vegetation and rocks. Blooms of blue-green algae can produce problems in human water
supplies and stock ponds.
The blue-green color is derived from pigments known as phycocyanins. Under some conditions
bluegreens may actually be red from closely related pigments known as phycoerythrins. Both
groups of pigments are water-soluble and function as accessory pigments in photosynthesis.
Accessory pigments absorb light at different wavelengths than chlorophyll and transfer the energy to
chlorophyll a for use in photosynthesis. The food storage product of blue green algae,
cyanophycean starch, is chemically similar to the glycogen produced by animals. A gelantinized
sheath usually surrounds cyanobacterial cells. Reproduction is asexual by fission or fragmentation
(breaking up of a colony or filament).

Exercises
A. Nostoc
This alga grows in colonial forms made up of many filaments surrounded by a gelatinous mass. It
aggregates into marble-shaped colonies, which often wash up on the shore of fresh water lakes and
streams. Look at the demonstration slide showing akinetes, which are what endospores have
traditionally been called by phycologists (scientists who study algae). In the Nostoc slide in your slide
set, distinguish between vegetative cells (ordinary non-reproductive photosynthetic cells) and
heterocysts (thick-walled but with a clear center), which fix nitrogen. Observe the living or preserved
material by making a wet mount and label Figure 7 -24.

12

Figure 7-25. Oscillatoria.

Figure 7-24. Nostoc. a. Colony (Macroscopic). b. Small Portion of a Colony (400X). c. Single
Filament Showing Detail of Cell Differences.

Note: Anabaena, which may be on display, is a genus whose species are very similar to Nostoc.
Anabaena, however, does not form gelatinous masses.

B. Oscillatoria
This filamentous blue-green displays a curious type of waving motion. Make a wet mount of living
material to see the blue-green color and this movement. A prepared slides should show separation
discs, where fragmentation of filaments occurs (The small segments are hormogonia and serve as
agents of asexual reproduction). Label Figure 7 -25.

13

Figure 7-26. Gleocapsa.

C. Gleocapsa
This small-celled alga can occur singly or in colonies but each cell or cells may be surrounded by a
gelatinous mass. Look at the prepared slide and label Figure 7 -26. Observe the living or preserved
material by making a wet mount.

D. Other Blue-greens
Observe specimens on demonstration.

14

III. Viruses
Viruses represent a unique group of infectious agents whose chemical make-up consists of one or
more molecules of either DNA or RNA encased in a coat of protein. The more complex viruses may
have additional substances such as lipids, carbohydrates and other proteins. These viruses differ
from living cells because of: their simple organization; having only DNA or only RNA, never both, no
food relationships and the inability to reproduce independently of living cells. Within host cells, the
virus nucleic acids serve as a source of genetic information and induce host cells to synthesize viral
components. The newly synthesized virus components are then assembled and viruses are released
from the host cells.
Viruses attack animals, plants, fungi, protozoans, algae and bacteria. Bacterial and animal viruses
tend to be highly specific, often only able to infect a single species. Many of the higher plant viruses
infect a single host but others are able to infect a number of related species. Fungal, algal and
protozoan viruses exist, but are not very well known.
There are over 2,000 known viruses and probably extremely many more that have not been
discovered. Viruses cause a variety of diseases in animals and plants including tumors. We will
briefly review some of the shapes of viruses along with the diseases and symptoms they induced in
various hosts.

Exercise
A. Virus shapes and sizes
Examine demonstration material showing the shapes and sizes of some representative viruses.
Figure 7 -27 shows the relative size of some viruses compared to a bacterial cell measuring
1 micrometer (1m). Viral measurements are usually given in nanometers (nm). A nanometer is
equivalent to 1/1,000 of a micrometer or 1/1,000,000 of a millimeter.
Observe viral symptoms induced on various hosts from 2 X 2 slides and photographs. Fill in Table 7
-18 with some representative examples. What are some ways to prevent human viral disease?

Disease resistant varieties are found in many species of higher plants.

Table 7-18. Some Representative Viruses.

Virus

Host

General Symptoms

15

Figure 7-27. Comparative Sizes and Shapes of Representative Viruses. Bacterial Cell Included for
Scale.

Figure 7-28. Virus-Induced Plaques.

16

B. Bacteriophages
The viruses that attack bacteria are called bacteriophages. The bacterial cells are lysed (broken
open) when the virus replication has been completed. The released viruses can then re-infect other
living cells.
Bacteriophages can be easily isolated and cultivated on young, actively growing cultures of bacteria.
Examine an agar plate covered with the slimy growth of Escherichia coli (a common intestinal
bacterium). Note the appearance of clear circles scattered over the plate. These clear areas are
called plaques and represent areas of lysed bacteria. Make drawings and notes from your
observations in Figure 7 -28.

17

KEY WORDS
endospore
flagellum (pl. flagella)
capsule
chromogenesis
streak plate
pour plate
litmus
lactose
casein
colloidal suspension
ferment (verb)
fermentation (noun)
glycerol
amino acid
fatty acid
antibiotic
hemolysis
plasmid
nitrogen fixing
phycocyanin
phycoerythrin
accessory pigment
chlorophyll a
cyanophycean starch
glycogen
akinete
vegetative cell
heterocyst
separation disc
hormogonium (pl. hormogonia)
bacteriophage
lyse (verb)

lysis (noun)

Laboratory 9
Zygote Fungi
Sac Fungi
Haploid Genetics
Introduction
The more than 100,000 species of true fungi comprise a large and diverse group whose taxonomy is
based almost entirely on morphological characteristics. They obtain their nutrition by being parasites
and saprophytes since they lack chlorophyll.
These organisms play a major role in the decomposition of organic matter in soil, increasing fertility in
the process. Fungi are used in industry to carry out fermentation reactions to produce organic acids,
alcohols and vitamins. They are the source of some antibiotics. Certain cheeses have their aroma
and flavor enhanced by fungal activity. On the other hand, fungi cause the destruction of food,
fabrics, leather goods and other commercial and industrial products. Fungi cause diseases in plants,
animals and man. And finally, there are the mushrooms. These common and sometimes colorful
forms may be edible to highly poisonous but the majority are really neither.
We will briefly review the general morphological features of the fungi before we proceed with a study
of the different taxonomic groupings. The thallus that results from the non-reproductive or
vegetative growth of fungi, usually consists of microscopic threads known as hyphae. These hyphae
are literally tubes of protoplasm. In the more primitive fungi the hyphae are continuous, without
crosswalls (coenocytic or aseptate). Hyphae separated at irregular intervals with cross-walls
(septa) are considered more advanced. The mass of vegetative hyphae is collectively called
mycelium.
Fungi typically reproduce both asexually and sexually. Asexual reproduction is important in the
propagation of many individuals. The most common method of asexual reproduction is by asexual
spores formed from the mycelium, typically on or in a specialized sporangium. Sexual reproduction
involves fusion of compatible haploid nuclei, karyogamy. This is usually followed directly by meiosis
that again reduces the chromosome number to the haploid condition in the newly formed spores or
mycelium. Meiotic divisions in fungi, like all organisms, are the major source of genetic variation. The
sexually produced spores are usually associated with a fruiting body, which contains or bears
spores.

I. Zygote Fungi
Biologically, these fungi range from saprobes (live by decay) to weak plant parasites, to specialized
animal parasites, and finally to obligate parasites of other zygote fungi. They are common soil and air
inhabitants that grow rapidly, so they can be troublesome as contaminants in laboratories and on
food. Some are used industrially to produce chemicals. The distinctive morphological feature of the
zygote fungi is the production of a sexual resting spore called a zygospore. Asexual reproduction is
usually by spores borne in large numbers on sporangia. The mycelium is coenocytic (non-septate)
except at the point where reproductive structures are formed.

A. The Genus Rhizopus

This is a representative terrestrial genus commonly called black bread mold.
(1) Examine a demonstration plate of Rhizopus stolonifera under the dissecting microscope. The
mycelium is growing extensively over the plate. The asexually produced dark sporangia are very

Figure 8-29. Rhizopus. Vegetative Hyphae and Asexual Spores.

conspicuous. These sporangia contain numerous sporangiospores. The stalk-like structure bearing
each sporangium is the sporangiophore.
(2) Prepare a wet mount from a culture plate. The sporangia tend to break and you may see the
sporangial wall folded over. Note the numerous dark spores. Look for the swollen tip of the
sporangiophore (inside the sporangial wall). This is the columella. The mycelium develops stolons
(runners) which connect the bases of the different sporangiophores. Rhizoids (root-like hyphal
branches) develop from the base of the sporangiophores. Label the structures in Figure 8 -29.

3
(3) Examine a prepared slide of Rhizopus showing sexual reproduction. Rhizopus is an example of a
fungus that is heterothallic. This means that two different strains of mycelia (designated as + and strains) are needed in order for sexual reproduction to occur. Organisms that have no such restriction
are referred to as homothallic. Strands of the + and - strains send out a lateral projection
(progametangium) toward one other. After contact is established, the progametangia become walled
off and are then termed gametangia. The walls between the two then dissolve and fertilization
occurs. A thick-walled zygospore is formed. The zygospore is a resting stage that typically persists
after the hyphae have died. Label the structures in Figure 8 -30.

Figure 8-30. Sequence of Events in Rhizopus Sexual Reproduction.

Figure 8-31. Phycomyces Sexual

Reproduction.

Figure 8-32. Pilobolus Sporangium.

B. The Genus Phycomyces

(1) Phycomyces is heterothallic. Examine the demonstration plate showing + and - strains inoculated
on opposite sides. The line of fusion between the two gives rise to zygospores. Take particular note
of the hooked gametangia (sometimes called suspensors) and the projections that surround the
zygospore. See Figure 8 -31.
(2) A flask culture of Phycomyces showing asexual reproduction is on demonstration. Note the long
sporangiophores and the dark sporangia at the tips.

C. The Genus Pilobolus

Pilobolus is commonly found on dung of grazing animals. It has evolved a remarkable mechanism for
spore dispersal by discharging its jet-black sporangia explosively toward a light source. The

Figure 8-33. Entomophthora muscae.

mycelium forms a basal swelling called a trophocyst. A sporangiophore grows from the trophocyst,
elongates and forms a subsporangial swelling and a dark sporangium. The subsporangial wall
ruptures at maturity and the sporangium is shot off (propelled by the liquid in the sporangiophore).
The sticky sporangium adheres to whatever it hits.
Cultures may be started by placing fresh horse manure in an open glass container. Sporangiophores
of the fungus usually develop in 4 to 7 days. Observe the dish with dissecting microscope. Label
Figure 8 -32.

D. The Genus Entomophthora

A few of the more advanced zygote fungi are parasitic on insects. Entomophthora muscae is
commonly called the fly fungus. Diseased flies are sometimes attached to the glass of a windowpane
and the flies become surrounded by a white halo of discharged sporangia. The insect will show a
distended abdomen with white bands of sporangiophores protruding from its exoskeleton after the
mycelium of the fungus has permeated the insect body. Each sporangiophore has only a single
sporangiospore instead of the numerous spores typical of most zygote fungi. The spore is forcibly
discharged and can infect another host. Sexual spores (zygospores) are also produced.
Examine a prepared slide of a housefly infected with Entomophthora muscae. Note the
sporangiophores protruding from the abdomen along with their single spores. Sexual structures are
not likely to be present on the slide. Label Figure 8 -33.

II. Sac Fungi

Sac fungi are a large group of plants whose taxonomy, like most fungi, is based on sexual
reproduction. These fungi produce asci that are usually in or on a fruiting body termed an ascocarp.
Primitive sac fungi may lack ascocarps. The ascus is a sac-like structure with a specific number of
haploid ascospores. Most species also reproduce asexually. The majority of these fungi are
saprophytes, getting their food by causing decay. Dutch elm disease, apple scab and chestnut blight
are examples of destructive plant diseases caused by sac fungi. A few forms are human pathogens.
The brewing and baking industries use yeasts to produce fermentation byproducts. Antibiotics result
from the biochemical activities of certain genera. Sac fungi also play important roles in food spoilage
and in certain food preparations.

A. Physically Simple Sac FungiThe Yeasts

These are simple single cells that lack mycelium and ascocarps. Most yeasts reproduce asexually by
an asymmetrical fission called budding. A few yeasts multiply by normal, symmetrical fission.
Sexual reproduction involves the formation of an ascus containing ascospores.

Figure 8-34. Saccharomyces cerevisiae

1. Asexual reproduction
a. Saccharomyces cerevisiae is the yeast used in the majority of commercial fermentation reactions.
Make a wet mount from a sugar solution to which a moist yeast cake has been added. Examine the
mount under high power. The yeast cells are reproducing by forming lateral projections called buds.
Look for the large vacuoles in mature cells.

Figure 8-35. Schizosaccharomyces octosporus, Asexual Reproduction.

b. Schizosaccharomyces octosporus is a fission yeast. The cells divide in a symmetrical fission

rather than by budding. Make a wet mount from the material provided.

2. Sexual reproduction

Figure 8-36. Schizosaccharomyces octosporus,

Sexual Reproduction.

9
a. Schizosaccharomyces forms an ascus containing eight ascospores. Prepare a wet mount from
the provided culture and label Figure 8 -36.

10

Figure 8-37. Ascocarp of Powdery Mildew.

B. Powdery Mildews
These fungi cause a group of plant diseases commonly known as powdery mildews. The name
comes from the white, powdery growth produced by the asexual stage of the organism. Powdery
mildews are all obligate parasites. In late summer the sexual stage is initiated and enclosed
ascocarps (cleistothecia) are formed. Generic separation is based upon the number of asci in the
cleistothecium and on morphological differences in appendages that extend out from the
cleistothecium.
Examine various leaves infected with the organism. Look for the dark, round cleistothecia that occur
atop the surface of the host. Scrape these ascocarps onto a slide. Add a drop of water to the mount
and a coverslip. Examine the preparation under the microscope. Press down on the coverslip and
break the ascocarps, which will display asci with ascospores. Some of the cleistothecia may not be
mature. The asexual (conidial) stage of the fungus is also present but it tends to break up when you
make a wet mount. Conidia are spore-like, but not associated with a well-developed sporangium.
They sometimes consist of more than one cell. Label Figure 8 -37. On the left is a broken ascocarp
with asci protruding. On the right are variations in appendages shown by different species.

C. The Genus Venturia

Apple scab, the most important disease of apple, is caused by Venturia inaequalis. The fungus
causes a size reduction and premature drop in leaves and fruits along with poor fruit bud development
for the next year. Note the demonstrations showing symptoms on leaves and fruits.
Ascospores are the primary source of apple infection in the spring on buds and emerging leaves.
Ascocarps develop in the fall, overwintering on fallen leaves. As the fungus grows on the surface of
the host, it begins to produce conidia, which continue to develop on apple leaves and fruits over the
entire growing season. The disease is favored by cool, moist weather. Periodic applications of
fungicides and the use of disease resistant varieties are the primary approaches to controlling apple
scab.

11
Examine a prepared slide of the conidial stage and look for the dark conidiophores bearing the flameshaped conidia on the plant surface. Label Figure 8 -38.

Figure 8-38. Conidia of Apple Scab.

12
Figure 8-39. Ascocarp of Apple Scab

Figure 8-40. Peziza Ascocarps.

Look for the sexual stage of Venturia on another prepared slide. Note the embedded ascocarps
(perithecia) containing asci with unequal sized ascospores. Note Figure 8 -39.

D. The Genus Peziza (Cup Fungus)

These fungi produce cup or disc-shaped fruiting bodies called apothecia. The asci are borne on the
surface or in large, open cavities. The spores are released in "clouds. The apothecia may be
brightly colored and produced on the ground, on wood or leaves and on animal dung.

13

1. Examine the fruiting body of Peziza or of other cup fungi on demonstration. The exposed surface
of the apothecium bears the asci and ascospores. Label the habit sketch of Peziza shown in Figure
8 -40.
2. Observe the fruiting layer bearing the asci and ascospores in greater detail. This layer is called
the hymenium. Examine a prepared slide of Peziza. Note the cylindrical asci (many of which contain
ascospores) and the presence of slender hairs between the asci. These sterile structures are
paraphyses. A portion of a hymenium is illustrated in Figure 8 -41.

Figure 8-41. Detail of the Hymenium of a Peziza Ascocarp, showing asci and ascospores.

14

Figure 8-43. Black Knot Fungus on Plum or Cherry.

Figure 8-42. Morel Ascocarp.

E. Morels
The morels (sponge mushrooms) are one of the most edible and delicious of all fungi. The apothecia
have a thick stalk and a pitted cap (or pileus) which give the morel a sponge-like appearance. The
asci and ascospores line the pits of the cap. This is a readily recognized genus which fruits in the
spring. Observe demonstration material. Label the general features of the ascocarp in Figure 8 -42.

F. Other Common Plant Diseases Caused by Sac Fungi.

You should be able to identify these diseases by common name, as well as apple scab (above).

1. Brown Rot of Stone Fruits

This is a common disease of peaches, plums and cherries. Major losses occur due to blossom
destruction and fruit rot. Note the demonstration material of the disease on fruits. The fruit blemishes
result from the conidial stage of the fungus. In addition to conidia, these infected fruits bear cupshaped ascocarps. Both ascospores and conidia infect blossoms in the spring during wet weather.
The fungus can also persist in the cankers.
Control of the disease is with fungicide applications to the trees or ground. It is important to control
the fungus in residual fruits left from harvesting. Chemical dips of harvested fruits and careful
handling of fruits during harvesting to avoid bruising are other ways to slow the spread of brown rot.

2. Black Knot of Plum and Cherry

The fungus causes conspicuous black-knotty swelling on twigs and branches. Infected trees become
worthless after a few years as stunting and limb death occur. Conidia and ascospores produce
hyphae that invade both healthy and injured wood. The disease can be controlled with pruning to
remove infected parts or by spraying with protectant fungicides. Observe the demonstration material.
Note Figure 8 -43.

15

Figure 8-44. Sclerotia Formed by Ergot on Rye Heads.

3. Ergot of Rye
The disease is most prevalent on rye but also occurs on other grasses. When the grass heads out,
the fungus invades the floral heads. The result is a sticky, sugary mass of conidia, which attracts
insects. Splashing rain and the insects disseminate the conidia. By the time the seed mature, the
fungus has developed dark, resistant structures called sclerotia or ergots. These sclerotia eventually
develop perithecia with asci and ascospores, typically in the next spring. The ascospores are capable
of infecting new rye plants.
The economic losses from the disease are usually minimal but the sclerotia are poisonous to humans
or animals that eat the contaminated grain or even bread made from it. The toxic substance is a
strong alkaloid, specifically a lysergic acid derivative (LSD). Although the sclerotia can be removed
with modern cleaning machinery, livestock poisoning still remains a problem.
Examine a mount of an infected rye plant. Note the elongated sclerotia developing on the grain head.
Label Figure 8 -44.

4. Dutch Elm Disease

This may be the ultimate example of a plant disease imported from another continent with
catastrophic results. Introduced into the United States from Europe, it is the most destructive shade
tree disease in this country. All elm species are affected but the fungus is most severe on American
elm. The disease may kill branches and entire trees in anywhere from a few weeks to a few years
from the time of infection. The fungus is a vascular parasite that plugs the water conducting system
(xylem) of the tree, resulting quickly in defoliation.
Although the fungus is responsible for the disease itself, bark beetles carry the causal organism from
dying or dead trees and logs to healthy trees. The beetles establish brood galleries in diseased or
dead trees, in which they lay their eggs. When larvae develop into mature insects, dissemination of
the fungus continues. The fungus can also be spread from infected trees to healthy ones via root
grafts.

16
The asexual (conidial) stage plays the major role in the disease cycle. Sexual reproduction is rare in
nature.
Systemic fungicides offer some control of Dutch elm disease, but are not very dependable. Disease

Figure 8-45. Tar Spot on an Elm Leaf.

resistant varieties have been developed but they have neither the desirable shade nor size traits of
American elm. Observe demonstration material of the disease.
5. Tar Spot of Elm
The fungus is a common parasite on elm leaves. This is an example of an infection where the
pathogen is not highly destructive to the host. Perithecia embedded in the leaves protrude their black
beaks to the surface, producing the spots that account for the name. Observe an infected leaf. Note
Figure 8 -45.

17

III. Haploid Genetics

In the Kingdom Fungi, diploid cells can divide only by meiosis. In the zygote fungi, this meiosis
occurs in the diploid zygotes inside the zygospore. In the sac fungi, it occurs in the developing ascus.
In the club fungi (treated in the next lab), it occurs in the developing basidium, with each haploid
nucleus migrating into one of the four basidiospores.
Does this mean that members of the fungus kingdom are effectively haploid in their biology? Not
quite. Dikaryotic hyphae have two haploid nuclei per cell, and have two sets of chromosomes
providing information for every cell. This is the functional equivalent of diploidy, but the nuclei
themselves are haploid.

A. Gene Segregation Demonstrated by Ascospore Pattern in Hybrid Asci.

The fungus, Sordaria fimicola, is useful for demonstrating segregation. The products of meiosis are
arranged in an orderly fashion that reflects the precise order in meiotic events.
The organism grows readily on artificial media and completes its life cycle pattern in a week or so.
Perithecia are formed during sexual reproduction. The perithecia contain asci in ascospores are
encased. These ascospores are the result of meiotic and mitotic divisions. Ascospore color mutants
have been obtained by irradiating cultures of the fungus. We will work with a mutant that produces a
tan-colored ascospore and the normal (wild-type) strain which has a dark-green ascospore.
If the parent culture and the mutant are inoculated on opposite sides of the same Petri dish, there will
be a hybridization effect where the two cultures meet on the plate. Hybrid perithecia and perithecia
from both the original cultures will be present. Most of the asci from hybrid perithecia contain four
dark-green and four tan ascospores. Six arrangements of the two kinds of ascospores formed in the
hybrid are possible (see Figure 8 -46 and Figure 8 -47). Asci which have four dark and four tan
ascospores in sequence are those in which the gene for spore color segregated at the first meiotic
division. In those with two dark or two tan ascospores at the tip of the ascus, segregation of the gene
for spore color occurred at the second meiotic division. First division segregation results from the
absence of a crossover between the gene and the centromere, while the second division segregation
results from a single crossover between gene and centromere. Since the likelihood of crossing over
depends on the distance between the gene and the centromere, the frequency of the two kinds of
segregation can be used in mapping the position of the gene for spore color.
Inoculated plates of Sordaria are available under dissecting microscopes on the lab bench. You will
be able to see color differences on each side of the plate. We will designate the tan isolate with G'
and the green isolate with G. Examine a plate for the sexual stage. Mature perithecia will appear
black. Look for the line of overlap between the two strains.
(1) Select 4-5 mature perithecia from the line of fusion using a teasing needle and transfer them to a
slide. You will have to dig into the medium to remove unbroken perithecia. Place a small drop of
water on the preparation and add a coverslip. Examine the mount under the medium power of your
microscope. Figure 8 -46 shows the relationship between a perithecium, the asci, and the
ascospores. Label those three components.

18
(2) Gently press down on the coverslip with your needle until the perithecia rupture and discharge the
mass of asci. Manipulation of the coverslip will help to get asci to fan out for easier examination.
(3) The following arrangements of ascospores may be observed:
Asci containing only G spores (all green)no hybridization.
Asci containing only G' spores (all tan)no hybridization.
Asci containing only G and G' sporeshybridization.
Note: You may have to make 3 or 4 slide preparations to succeed in finding hybrid asci.
(4) Tabulate ascospore arrangements in all hybrid asci. Count from the center out. The spores can
only be counted if they are in a linear sequence (still contained in the ascus). Figure 8 -47 illustrates
the effects of segregation and crossover on the appearance of a hybrid ascus.
(5) Count at least ten asci showing hybridization. Spore sequences should be assigned to:
Category 1
Pattern A or B
Category 2
Pattern C, D, E, or F [see Figure 8 -47]

Figure 8-46. Perithecium of Sordaria Sectioned to Show Hybrid Asci inside.

(6) Fill in Table 8 -19. The instructor may tabulate class results for Table 8 -20.
Table 8-19. Ascus Pattern Counts.

Spore Sequences

10

Total

A and B
C, D, E and F
Table 8-20. Class Results.

Number
% of Total

A and B

C, D, E & F

Total

19

Figure 8-47. Segregation Patterns and Ascospore Arrangements.

20

KEY WORDS
thallus (pl. thalli)
vegetative
hypha (pl. hyphae)
coenocytic (aseptate)
septum (pl. septa)
mycelium (pl. mycelia)
sporangium (pl. sporangia)
karyogamy
fruiting body
zygospore
stolon
rhizoid
heterothallic
homothallic
ascus (pl. asci)
ascocarp
ascospore
budding
cleistothecium (pl. cleistothecia)
conidium (pl. conidia)
perithecium (pl. perithecia)
apothecium (pl. apothecia)
hymenium (pl. hymenia)
wild type

21

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Laboratory 10

Figure 9-48. Uredium of Wheat Rust, on Wheat, with Uredospores.

Club Fungi
Imperfect Fungi
I. Club Fungi
Club fungi are considered the most highly evolved group of fungi. Rusts and smuts are primitive club
fungi that can be very destructive as plant pathogens. Higher club fungi include mushrooms,
puffballs, bracket fungi and other lesser-known forms. Diseases of forest and shade trees and the
destruction of lumber and railroad ties result from their degradation processes. The majority of these
higher fungi are saprophytic.
Club fungi differ from all other fungi in that they produce their spores, called basidiospores, on the
outside of a specialized, microscopic spore-producing body, the basidium. Karyogamy and meiosis
occur in the basidium. The higher club fungi produce their basidia in highly organized fruiting bodies
of various shapes and sizes. These fruiting bodies are basidiocarps. A mushroom, therefore, is a
basidiocarp that contains basidia and basidiospores. The mycelium of all club fungi is septate.
Asexual reproduction in these fungi is not as common as other fungal groups although conidia and
other spores are produced by some. A few forms reproduce by budding.

Exercises
A. Lower club fungi
Rusts and smuts make up the majority of these fungi. They differ from the more advanced club fungi
because they lack basidiocarps and their basidia do not have the typical club shape.
Rusts are obligate plate parasites that can cause extensive damage on many cultivated hosts.

1. Wheat Rust
One of the best known rusts is Puccinia graminis f. tritici. It is the causal organism for the disease
black stem rust of wheat. The life cycle of this fungus illustrates the high degree of specialization that
a particular parasite may evolve. Wheat rust produces five kinds of spores and grows on two different
hosts. A rust that infects two different hosts is termed heteroecious. A one-host rust is referred to as
autoecious.
a. Red Rust Stage

2
Examine wheat leaves and stems bearing rusty-brown pustules. These pustules contain masses
of spores called uredospores inside structures called uredia. Uredospores are one-celled but
contain two nuclei (binucleate). Uredia only develop after the mycelium has perpetuated
throughout the host. This stage keeps reinfecting wheat. Examine a prepared slide. Label
Figure 9 -48.

Figure 9-49. Telium of Wheat Rust, on Wheat, with Teleospores.

Figure 9-50. Stages in Development of Wheat Rust Basidium: a. Binucleate Cells in Teleospore.
Figure
9-51. Wheat
Rust, Onand
Barberry:
a.
b. Karyogamy.
c. Basidium
Basidiospores.
Spermagonia with Spermatia and Receptive
Hyphae. b. Aecia with Aeciospores.

b. Black Rust Stage

In late summer, uredia on wheat darken from rusty red to black, and are now referred to as telia.
On close examination, the color turns out to derive from spores of a very different appearance,
teliospores. These spores are two-celled and each cell is binucleate although the nuclei fuse
later on. Examine a prepared slide showing telia and teliospores. Telia overwinter on wheat
stubble. Figure 9 -49.
b. Sexual Reproduction
In spring, each cell of a teliospore may germinate to produce a primitive basidium. The nucleus
undergoes meiosis to form four nuclei, each of which migrates into one of four basidiospores.
Since the rust is heterothallic, two + and two - basidiospores result. These spores cannot infect
wheat, but they do have the ability to invade the alternate host, barberry. Examine a
demonstration slide showing basidia and basidiospores developing from teliospores. Label the
appropriate structures in Figure 9 -50.
d. Barberry Infection
After the basidiospores germinate and develop a monokaryotic mycelium in the barberry leaf,
small flask-shaped structures called spermogonia appear on the upper leaf surface.
Spermagonia produce spermatia which cross-fertilize (+ to -) (- to +) to female receptive
structures called receptive hyphae. Subsequent development of the fungus takes the form of
dikaryotic hyphae (two nuclei per cell), followed by eruption of cup-like structures on the undersurface of the barberry leaf. These are aecia that produce aeciospores. Infection of wheat
occurs from these spores, and would not have been possible without the infection of barberry.
Examine demonstration material of infected barberry. Observe a prepared slide showing a crosssection of a barberry leaf and label the appropriate structures (Figure 9 -51).

2. Other Rusts
There are two other examples of heteroecious rusts on display. You are not responsible for specific
spore stages but should be able to recognize white pine blister rust and cedar apple rust on the
different hosts from 2 x 2 slides. The host where the rust produces teliospores is considered
primary.

White Pine Blister Rust

Cedar Apple Rust

Primary
Host
Alternate
Host
Asparagus rust is an example of an autoecious rust. You can readily see the telial (black stage) under
the dissecting microscope.

3. Smuts
Smuts are normally parasitic on grasses but they are not obligate parasites (they can live by decay
as well).
Examine specimens of corn smut. The mycelium of the fungus develops extensively in the host
(particularly in the ears and tassels). Large, black and dusty spore balls eventually appear. These
contain masses of spores (teliospores). These spores germinate to produce a primitive basidium with
basidiospores. Prepare a wet mount of teliospores. Note the texture of the wall.
Make a drawing of your observations:

Figure 9-52. Fruiting Body (Basidiocarp).

B. Higher Club Fungi

The great variation in color, shape, size and texture of the fruiting bodies (basidiocarps) make these
fungi conspicuous to everyone. A mushroom is a basidiocarp that tends to be fleshy, umbrellashaped and decays easily. There are about 5,000 mushroom species and maybe 100 are poisonous.
The number of edible forms outnumbers the number of poisonous forms by a ratio of about 4:1.

1. Fruiting Body
We will now examine the fruiting body (basidiocarp) of a mushroom. Bear in mind that this is the
quick-developing, reproductive part of the fungus. The enduring fungus consists of a mycelium
extending thorough its substrate. Note the expanded cap (pileus) bearing gills on its lower surface.
A stalk or stipe supports the cap. There may be a ring or annulus on the stipe. The young
mushroom develops from a ball of interwoven subsurface mycelia. This ball increases until the
"button" stage appears. The "button" expands into the mushroom with favorable environmental
conditions. Label Figure 9 -52.

6
a. Gill Fungi.
There is a prepared slide showing the cross-section of the gill area of Coprinus (Figure 9 -53a). The
gills contain the typical club-shaped basidia. Identify the hymenial layer (spore-bearing layer. The
dark-stained, haploid basidiospores are attached to the less conspicuous basidia by prong-like
projections called sterigmata. Apply the necessary terms to Figure 9 -53b, which shows a portion of
a gill enlarged.

Figure 9-53. Gill Fungus (Coprinus) Fruiting Body Detail. a.

Cap Cross Section. b. Enlargement of Gill.

Figure 9-54. Pore Fungus Fruiting Body. a. Underside of Cap. b. Detail of Pore.

b. Pore Fungi
There are also basidiocarps that are penetrated by pores instead of being segmented into gills.
Basidia and basidiospores line these pores. Observe the demonstration slides and specimens. The
gross morphology of a pore fungus is shown in Figure 9 -54a. Label Figure 9 -54b which illustrates
a single pore.
c. Spore Prints
Some mushrooms can be readily identified from macroscopic features of the basidiocarp but most of
the taxonomic keys used to separate them rely on spore color along with microscopic and
macroscopic structures. Masses of spores can easily be obtained by making a spore print. Cut off
the stalk and place the cap with the exposed gills or pores down on a piece of black or white paper.
Spores will usually be deposited in a few hours if the specimen is mature or fertile. Examine
specimens on demonstration that have been chosen to show spore color variations.

2. Mushroom Identification
We will now examine some of the many variations that occur in basidiocarps. You should know the
following fungi by common or generic names.
a. Amanita
This is a genus with some highly poisonous members. Although there are some edible species,
these mushrooms are to be avoided. Note the swollen base (volva) and annulus. The
basidiocarp has white gills and spores. Very common.

Figure 9-55. Amanita.

Figure 9-57. Boletus.

Figure 9-59. Puffball.

Figure 9-56. Agaricus.

Figure 9-58. Coprinus.

b. Agaricus
This genus has an annulus and gills that are pinkish when young but become chocolate-brown
with age. These are the typical grocery store mushrooms (both fresh and canned). There are
many edible species but even this safe genus may cause vomiting and diarrhea. Very common.
c. Boletus
This mushroom has pores instead of gills. A few boletes are very good edibles while others are
average in terms of edibility. Those that stain blue and have reddish pores are poisonous. Very
common.

d. Inky caps and shaggy manes (Coprinus)

These mushrooms have black spores. The basidiocarp breaks down very quickly and forms an
"inky" mass (hence the name). Very common. Many are edible when immature.

e. Puffballs
These are common and highly edible fungi although hard-rind puffballs are slightly poisonous.
They are not edible when the spores are being "puffed-out." There are no gills or pores present,
which helps to distinguish them from mushroom-like fungi.

10

Figure 9-62. Coral Fungus.

Figure 9-61. Stinkhorn.

f. Earth stars
These basidiocarps are modified puffballs. The name comes from the star-like appearance. The
outer layer of the fruiting body folds out. Common in open fields and woods. Non-edible.

g. Stinkhorns
These fungi are characterized by foul-smelling basidiocarps which tend to attract swarms of
blowflies to the slimy spore mass. The insects disseminate the spores. Stinkhorns are common
on ground or rotten wood. The fruiting body emerges from a round to oval egg. These fungi
also have a volva.

Figure 9-60. Earth Star.

11

Figure 9-63. Birds Nest Fungus.

h. Coral fungi
The basidiocarps resemble clusters of coral. Some are edible. Common in woods.

i. Bird's nest
The name comes from the basidiocarps which resemble a nest filled with eggs. Common in
lawns, fields, gardens and on decaying wood.

Figure 9-64. Fomes.

12

j. Fomes
This is a common form of bracket fungus that grows on trees as a perennial (year after year) and
as a parasite. Note the "woody" texture, pores and "rings."

13

Figure 9-66. Alternaria with Conidia.

Figure 9-65. Polyporus.

k. Polyporus
This is another very common bracket fungus. It grows mostly as an annual saprophyte on wood.
The basidiocarp may be fleshy or corky. Note the pores. A few Polyporus species are edible.

II. Imperfect Fungi

The imperfect fungi are fungi whose sexual stages either dont exist or have not been found.
Reproduction takes place by means of conidia of various types. The classification of the imperfects is
based on the color, shape and size of the conidia and conidiophores that are sometimes borne in or
on specialized structures. A few of the imperfect fungi are yeast-like. Some of the imperfect fungi
cause serious diseases of plants, animals and man. The majority are saprophytes. These fungi
appear to be related to sac fungi because the conidial stages are similar.

Exercises
A. The Genus Alternaria
This is a common saprophytic and plant parasitic fungus. The conidia are so distinctive and abundant
during the summer that Alternaria "counts" are given as a "pollen-like" indicator for people suffering
from allergies.

14
The organism causes a plant disease called early blight. Observe infected tomato leaves (a
common host). Examine a prepared slide showing the numerous dark-multicelled conidia on the plant
surface. Note Figure 9 -66.

B. The Genus Geotrichum

This organism is a common contaminant on pickles, silage, sauerkraut and dairy products. Certain
species are human pathogens. Geotrichum produces a well-developed mycelium that fragments into
specialized conidia called arthrospores. Examine a demonstration slide and label Figure 9 -67.

C. The Genus Aspergillus

Figure 9-67. Geotrichum with Arthrospores.

15
Species of this fungus cause spoilage of many fruits and vegetables. The genus is important
industrially in the production of various
organic acids and antibiotics. Some
members cause lung infections of higher
vertebrates. They are the source of toxic
and carcinogenic substances called
aflatoxins on grains and peanuts in
storage.

Figure 9-68. Asexual Reproduction in Aspergillus.

These fungi have septate mycelium. Asexual (conidial) reproduction involves the development of
aerial growth from a specialized foot cell. The apex of the conidiophore (spore bearing branch) swells
to form a vesicle. Surrounding the vesicle are sterigmata. Spiny conidia are connected in chains to
these sterigmata and fan out in all directions on the vesicle that forms their base. The conidia will
readily germinate to produce mycelia of the fungus. Some species have a sexual (perfect) stage and
are classified as sac fungi.
Examine a culture of Aspergillus under the dissecting microscope. You can readily see the masses of
conidia borne on the conidiophores. View a prepared slide and label the appropriate structures in
Figure 9 -68.

16

D. The Genus Penicillium

These fungi also cause spoilage of many fruits and vegetables. Some strains are used in cheese
ripening. One of the best known antibiotics, penicillin, results from one of their chemical byproducts.
The septate vegetative mycelia penetrate their substrate and produce aerial hyphae, on which
specialized conidiophores produce conidia. The conidia develop from specialized branches
(sterigmata) of the conidiophore. We will only examine the conidial stage of Penicillium, although
loosely woven ascocarps are formed by some species.
Examine a culture of Penicillium chrysogenum under the dissecting microscope. This is the isolate
from which the majority of the antibiotic is obtained. These molds are typically blue and green in
color. Look for the asexual reproductive structures on a prepared slide and label Figure 9 -69.

E. Fungi Pathogenic on Humans and Other Animals

Birds and mammals are susceptible to fungal infections. The majority of human pathogenic fungi are
found in the imperfects or are sometimes conidial or yeast-like stages of certain sac fungi. These
fungi cause dermal infections, systemic infections or infections which range in severity between the
two (intermediate). We will examine infections from the first two types of diseases.

1. Dermatophytic Diseases

Figure 9-69. Asexual Reproduction in Penicillium.

17
Dermatophytes are fungi that attack the skin or its appendages. These fungi are widely distributed in
soil. The spores and mycelia are abundant in skin, hair, and feathers shed from their infected hosts.
Athlete's foot and ringworm are common dermatophytic infections that also tend to be contagious.
Treatment of these diseases
usually involves the use of
an oral antibiotic such as
griseofulvin or ointments and
solutions of sulfur-containing
compounds.

Figure 9-70. Microsporum: a. Mycelium. b. Macroconidium.

Examine 2x2 slides showing some common skin infections. Observe a demonstration slide of
Microsporum (a common dermatophytic fungus) showing the large macroconidia and occasional
microconidia.

2. Systemic Infections
Systemic mycoses are systemic infections caused by fungi that exist primarily as soil saprophytes.
Some infections occur because of occupational exposure to the fungus-containing feces of birds
(including domestic fowl) and bats. Air-borne spores enter the host through inhalation or occasionally
by puncture wounds. Inhalation may result in ramification of the fungus into the lungs while infection
of puncture wounds result in pit-like ulcers. In either case, the parasite can be disseminated by the
blood and lymph to other internal organs or into the nervous system. Infections range from mild to
chronic to fatal but are rarely transmitted directly from one host to another. Systemic fungal infections
are relatively rare except among individuals whose immune system has been compromised by the
effects of AIDS or other decline in normal immunity. Treatment includes bed rest along with
supplemental nutrients to stimulate natural immunity, surgery and antibiotics.
Sporotrichosis is a chronic infection that sometimes becomes epidemic. The fungus is found in soil
and on living plants. Most infections occur from accidental inoculations of the fungus into the skin or
subcutaneous tissues. The organism may disseminate to the bones and internal organs but this is
rare. Observe 2X2 slides of the disease.
Blastomycosis, a disease of man and animals (particularly dogs), is present in humans as
cutaneous lesions, an acute or chronic pneumonia, or a systemic infection. The disseminated
disease can affect almost any tissue or organ. The fungus persists in soil enriched with decaying
organic material and infection occurs through inhalation. Observe the demonstration slides.

18

KEY WORDS
basidiospore
basidium (pl. basidia)
basidiocarp
rust
heteroecious
autoecious
uredospore
uredium (pl. uredia)
telium (pl. telia)
teliospore
spermogonium (pl. spermogonia)
spermatium (pl. spermatia)
receptive hyphae
dikaryotic hyphae
aecium (pl. aecia)
aeciospore
smut
pileus
gill
stipe
annulus
sterigmatum (pl. sterigmata)
pore
spore print
volva
early blight
arthrospores
dermatophytes
systemic mycoses
sporotrichosis
blastomycosis

19

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Laboratory 11

Plantae

Fungi
Animalia

PROTISTA
Bacteria

Archaebacteria
Figure 10-71. Kingdom Relationships.

Protists:
Slime Molds; Egg Fungi; Euglenoids; Dinoflagellates; Yellow-Green
Algae; Diatoms
Introduction
This lab deals with a diversity of plants which can be referred to as protists. The Protozoa studied in
most zoology classes are also considered to be protists. Protists are similar to one another in being
physically simple, but they differ enormously in cell structure, lifestyle, nutrition, and ecology. Look for
these differences as you proceed through the laboratory.
The relationships among these creatures are far from obvious, but evolutionists believe that protists
are likely to have evolved from bacterial ancestors, and that higher plants, higher fungi, and higher
animals all are likely to have evolved from ancestors who were protists. Figure 10 -71 shows one
scheme.

A. Slime Molds
Plasmodial slime molds are heterotrophic organisms that possess both plant-like and animal-like
characteristics. The plasmodium that constitutes the body of the organism is a multinucleate mass
of protoplasm with no cellular divisions or walls. It literally moves over its substrate, ingesting and
digesting bacteria, protozoa, fungi and small particles of decaying organic material as it goes. When
food or water becomes scarce, the slime mold changes character. Movement ceases, and
distinctively shaped fruiting bodies containing spores are produced. About 500 species of slime
molds are recognized.

Figure 10-72. Plasmodium of a Slime Mold.

Cellular slime molds, apparently not closely related to plasmodial ones, live their vegetative life as
single amoeba-like cells. In reproduction, the individual cells coalesce into a pseudoplasmodium,
which proceeds to produce a fruiting body and spores. About 70 species of cellular slime molds are
recognized.

1. Plasmodium
Remove the cover from a plate and observe the protoplasmic strands under the dissecting
microscope. Note the contrast between the flowing streams of cytoplasm and the gel-like nature of
that on the periphery. Watch the movement for long enough to observe the sudden reversal of
direction which takes place. Measure or estimate the amount of time between direction changes:

2. Fruiting bodies

Figure 10-73. Fruiting

Body of a Slime Mold,
Physarum.

Slime molds produce spore-bearing fruiting bodies in response to environmental changes such as
shortening days, increased light, or dryness. Observe a demonstration plate of Physarum with fruiting
bodies.

Figure 10-74. Lycogala.

Figure 10-75. Fuligo.

Figure 10-76. Arcyria.

a. Fruiting Body Diversity

Sketch the fruiting bodies formed by each of the slime mold genera on display, emphasizing
differences in form and size.

Figure 10-77. Stemonitis Fruiting Body Cross Section.

b. Stemonitis

5
Observe the prepared slide of the fruiting body of Stemonitis to see its internal structure. You
should be able to distinguish the short stalk, a solid central core that extends through the
sporangium (columella), thread-like capillitia that help keep the spores from sticking to one
another, and the spores themselves. You may be able to see remnants of the sporangium wall
(peridium). Label Figure 10 -77. Be sure you understand the correspondence between this
microscopic view and the Stemonitis fruiting body.

c. Dictydium
Examine the prepared slide of the fruiting body of Dictydium for a contrasting kind of fruiting body.
Note the distinctive flower-like shape.
Slime mold spores germinate to form swarm cells. These may either be cells that swim with
flagella and therefore depend on the presence of a film of water or cells that move over the
surface like an amoeba. In either case, eventually the swarm cells fuse to form a zygote that
eventually grows into a plasmodium with nuclei that proliferate by mitotic divisions. If available,
observe the demonstration slide showing spore germination.

Figure 10-78. Dictydium Fruiting Body.

Figure 10-79. Saprolegnia Sporangia and Zoospores (Asexual).

B. Egg Fungi
Egg fungi are water- and soil-inhabiting fungi. Some of the more advanced species are obligate
parasites of terrestrial angiosperms and, as such, spend their entire life cycle in their hosts. The
purely aquatic egg fungi are of little economic importance. A few parasitize fish, aquatic plants,
freshwater algae, and other aquatic fungi. The morphological structures that characterize this fungal
grouping are a zoosporangium (containing asexual zoospores with two flagella) and sexual spores
called oospores. The mycelium is coenocytic (non-septate).

1. The Genus Saprolegnia

Members of this genus are mostly saprophytic. A few species parasitize fish. They are commonly
called water molds because of their abundance in clear waters.
(a) Observe a living culture of Saprolegnia on demonstration under the dissecting microscope. Note
the filamentous growth pattern. You may be able to see zoosporangia developing from the tips of
hyphae. If the zoosporangia are mature, zoospores will be escaping. After a short period the
zoospores will encyst and then reemerge as a secondary zoospore or germinate into mycelium. The
prepared slide will show asexual reproduction. Label Figure 10 -79.

Figure 10-80. Saprolegnia Sexual Structures.

(b) Examine a prepared slide showing the sexual stage of Saprolegnia. The oogonia are round
structures which have oospheres (unfertilized eggs) inside. Slender branches may surround the
oogonia. These are the male structures (antheridia). Fertilization tubes from the antheridia
penetrate the oospheres and thick-walled oospores result. The living cultures will also show the
sexual stage. Figure 10 -80 is a drawing of sexual reproduction typical of Saprolegnia. Saprolegnia
is an example of a homothallic fungus which means that the organism is self-fertile (able to reproduce
sexually without a partner).

2. The Genus Phytophthora

The genus is well known to plant pathologists. Phytophthora infestans is the causal organism of the
late blight disease of potato
and tomato. We will examine
Figure 10-81. Phytophthora Zoosporangium with Zoospores.
the species P. palmivora, which

Figure 10-82. Chains of Albugo Sporangia on Leaf (Asexual).

causes bud rot of coconut and various stem rots of household plants. Plate cultures of the organism
have been grown in the dark for a few days. This induces the formation of the asexual stage
(zoosporangia) of the fungus. Sterile water has been added to the plate and the surface has been
gently scraped.
a. Add a single drop of fluid from the plate to a depression slide. Observe the zoosporangia under
100X or 200X without a cover slip. You should be able to see zoospores developing in the
zoosporangia in about 5 minutes. Spores that have been released from the sporangia will be actively
swimming. Label Figure 10 -81.

3. The Genus Albugo, parasite of flowering plants.

This organism is an example of an obligate plant parasite. Albugo causes a plant disease commonly
known as white rust on radish, spinach and cabbage.

9
(a) Examine a prepared slide that shows the asexual stage of the organism. Note the chain-like
arrangement of sporangia, which rupture through the epidermis and create a white crust on the host
surface. These sporangia will eventually germinate to produce zoospores. Label Figure 10 -82.

(b) Sexual reproduction of Albugo results in the formation of oospores. Examine a prepared slide of
an infected leaf that contains dark-red oospores. Note the warty wall. Oospores develop by
thickening of the oogonium wall after fertilization. Germination of the oospore results in the formation
of zoospores. A swimming spore is of obvious benefit to an aquatic organism, but in this pathogen of
terrestrial plants, it requires a film of water for successful spread. Albugo and other pathogenic egg
fungi tend to spread well in very wet weather. Label Figure 10 -83.

C. Protistan Algae
1. Diatoms

Figure 10-83. Sexual Structures of Albugo: Oogonium, Antheridium, Oospore.

10

Diatoms have silica cell

walls that endure after the
cell has died. These cell
walls overlap or fit
together like Petri dish
halves and may bear
beautiful patterns.
Figure 10-84. Diatom Structure: Valve and Girdle Views.
Diatoms may well be
more important to aquatic
food chains than much
more conspicuous plants
such as seaweed.
Diatoms are often the most abundant phytoplankton [suspended aquatic plants] in both fresh and
salt water. They serve as food for many zooplankton [suspended aquatic animals], and represent
the base of many significant aquatic food chains. Centuries of accumulation of empty diatom cell
walls, excreted by the many diatom feeders of the oceans, form diatomaceous earth, which is
remarkable useful to human technology, as a filter, insulator, and fine-textured abrasive. Coloration
from the accessory pigment fucoxanthin may be present. Diatoms store food as a chemically
distinctive starch, chrysolaminarin, and also as oils, which contribute to their buoyancy.
(a) Examine the prepared slide and see if you can detect the two valves of a diatom. Try to
distinguish the girdle (side) view from the valve (top) view (Figure 10 -84).
(b) Examine a small moistened portion of diatomaceous earth and note the many types of diatom cell
walls present.
(c) Look at fresh plankton and see the many forms of algae and animals present. Sketch the shape of
the species that seem most abundant:
Figure 10-85. Diatom Shapes. (Sketch.)

Note the large percentage of circular forms

among the marine diatoms (diatomaceous earth)
relative to the fresh water diatoms.

2. Genus Vaucheria (Water Felt)

Vaucheria is an example of a yellow-green alga. Some taxonomists classify yellow-green algae with
the diatoms in the Chrysophyta, others put them in a separate division, the Xanthophyta. The
common name of Vaucheria is water felt. It is widespread in fresh water and on moist soils, which are

11

Figure 10-86. Filament and Sexual Structures of Vaucheria: Antheridium, Oogonium (Becomes an
Oospore).

more similar as habitats for protists than you might expect. The filaments have no cross walls
(aseptate) and contain many chloroplasts and small nuclei. The gametangia (oogonia and
antheridia) resemble those of egg fungi. Like diatoms, yellow-green algae store food as
chrysolaminarin.
Make a wet mount of the fresh or preserved material and note the lack of cross walls in the filaments.
Nuclei and chloroplasts should be visible. In the prepared slides look for the oogonia and antheridia
and label Figure 10 -86. Can you find the eggs? The fertilized egg will become an oospore and
eventually can produce a new individual. Asexual reproduction is by large multiflagellate zoospores
produced in a terminal zoosporangium.

3. Dinoflagellates

12
Dinoflagellates (see Figure 10 -87) are almost as abundant and significant phytoplankton as diatoms,
but more predominantly marine. Some species undergo population explosions producing "red tides"
in which accumulated toxins can kill millions of fish. Dinoflagellates are often bizarre in shape but
have the common characteristic of a girdling flagellum and a trailing flagellum. In the spirit of
protistan diversity, some lack chlorophyll and are heterotrophs! Dinoflagellate cytology (cell structure)
differs from other eukaryotic organisms: Their cell walls consist of cellulose plates inside the plasma
membrane. Their chromosomes do not disappear between cell divisions, and nuclear division is
somewhat bacterium-like. Dinoflagellates are usually classified in their own division, Pyrrophyta.

Figure 10-87. Dinoflagellates.

13

Figure 10-88. Euglena.

Examine the prepared slide on demonstration and other information as available. Dinoflagellate study
material is far more difficult to obtain, and dinoflagellate structure more difficult to observe than that of
diatoms, despite comparable biological importance.

4. Euglenoids
Euglenoids occur mostly in fresh water. They may rightfully be claimed by zoologists because only
about 1/3 of the genera are photosynthetic, none have cell walls, and most have flagella and an
eyespot.
Examine the demonstration slide of stained Euglena. Look for the flagellum, eye spot, nucleus and
chloroplasts. Living material may be available for study. Label Figure 10 -88.

14

KEY WORDS
protist
plasmodium (pl. plasmodia)
zoosporangium (pl. zoosporangia)
zoospore
oospore
oogonium (pl. oogonia)
antheridium (pl. antheridia)
late blight
white rust
silica
phytoplankton
zooplankton
diatomaceous earth
fucoxanthin
chrysolaminarin
filament
gametangium (pl. gametangia)
red tide
girdling flagellum
trailing flagellum
eyespot

15

(blank page)

Laboratory 12

Figure 11-89. Protococcus.

Green Algae
Brown and Red Algae
I. Green Algae
Introduction
Green algae include a wide range of growth forms, reproductive patterns, and habitat preferences.
Most green algae are fresh water plants but some species may be found in a variety of other sites,
including soil, bark, as part of lichens, on snow, and in salt water. Green algae may be motile with
flagella or non-motile (without flagella). Many species of green algae are unicellular, many are
filamentous, some form colonies, and some are complex multicellular plants with specialized
tissues and division of labor. Reproduction may be asexual (fission, zoospores, fragmentation) or
sexual with isogametes or heterogametes. Isogametes are physically identical, whereas
heterogametes differ, for example sperm and eggs. It is thought the green algae gave rise to the first
primitive land plants. What these diverse green algae have in common is the same specific food
chemistry as most land plants. They have chlorophyll A and B as primary pigments, carotenes and
xanthophylls as accessory pigments, and store food as ordinary starch.

Exercises
A. Non-Motile Unicellular Green Algae
1. Protococcus (Pleurococcus)
This alga is normally found on bark and stone. Specimens for a wet mount may be obtained from the
bark sample by scraping. Look under microscope at the cells in a wet mount. Now add iodine
solution so that more cell parts may be distinguished. Label Figure 11 -89.

2. Chlorella
This small round green alga occurs frequently as a contaminant in growth media. It is often
mentioned as a possible source of protein and oxygen on long manned-space flights. Look for the
cup-shaped chloroplast on the prepared slide and label Figure 11 -90. Make a wet mount of
available specimens but under a low powered microscope details may be lacking.

Figure 11-90. Chlorella.

3. Desmids
These green algae are important as freshwater phytoplankton. Morphologically varied, the single
cells have mirror-image halves with a central nucleus. Note the many diverse shapes and forms in
the prepared slide. On Figure 11 -91, label the central nucleus, the single two-lobed chloroplast and
the

Figure 11-91. Assorted Desmids.

symmetrical halves of the cell. Make a wet mount from specimens, if available.

B. Motile Unicellular AlgaeChlamydomonas

4
These algae have two flagella and are motile. Their cells may have pyrenoids (for starch synthesis)
and an orange colored eye-spot at the anterior end. If fresh material is available, carefully introduce
single drops of two different two mating types (+ and -). The strains will mate and join to form a
zygote. After observing the mating you may use an iodine solution to kill and stain the cells. Label
Figure 11 -92.

C. Filamentous Algae
1. Ulothrix
If available use living specimens, otherwise use preserved. Make a wet mount and observe Ulothrix
under the microscope. The filaments are unbranched and a holdfast may be present. Study an
individual cell looking for the nucleus, collar-shaped chloroplast, and pyrenoids (spots of starch
accumulation on the chloroplast). Cells containing isogametes (32-64 small cells) and zoospores (216 larger cells) may be present. Look at the prepared slides and label Figure 11 -93.

Figure 11-92. Chlamydomonas.

Figure 11-93. Ulothrix showing sexual and asexual reproduction.

2. Spirogyra

Figure 11-94. Spirogyra. a. Vegetative Cells. b. Sequence of Changes

during Conjugation (Usually Occur More or Less Simultaneously).

6
These algae lack holdfasts. Observe the spiral shaped chloroplasts with pyrenoids. Sexual
reproduction is by conjugation, in which entire cell contents migrate through conjugation tubes into
cells from an adjacent filament. The receiving cell develops into a persistent zygospore. Look for the
conjugation tubes and zygospores in the fresh or preserved material and/or the prepared microscope
slides. Label Figure 11 -94.

3. Oedogonium
Look for the two different sexual structures in this green alga. The oogonium is a larger than normal
cell containing an egg and maturing into an oospore. The antheridia are notably smaller cells which
occur in groups and produce swimming sperm cells. Both should be present on the prepared slides
and should be labeled on Figure 11 -95. Make a wet mount and look for the reproductive parts of the
filament.

Figure 11-95. Oedogonium. a. Filament with antheridium

(male reproductive structure). b. Filament with
oogonium (female reproductive structure).

D. Colonial Algae
1. Hydrodictyon (water net)
Water net is colonial and becomes large enough to be seen with the unaided eye. Under the
microscope, examine the individual cells of the colony for chloroplasts and numerous nuclei. Label
Figure 11 -96.

Figure 11-96. Hydrodictyon.

Figure 11-97. Volvox. a. Small Colony. b. Mature Colony with Daughter Colonies inside.
c. Cellular Detail of Colony Surface.

2. Volvox
These hollow spheres are made up of many individual cells, some of which can be specialized for
sexual reproduction. Look at fresh or preserved material and at the prepared slides. Small daughter
colonies will be present within some of the larger colonies. Label Figure 11 -97.

3. Acetabularia (mermaid's looking glass, Venus mirror, etc.)

These green algae occur in shallow warm seawater such as found in Florida and the Caribbean. It is
an organism made up of a huge single cell with one nucleus that eventually produces isogametes in
the parasol-shaped cap. Observe the specimen on demonstration.

E. Multicellular Green Algae

1. Marine algae:
Ulva (sea lettuce)

10
These algae have a life cycle in which almost identical diploid and haploid plants alternate with each
other. It is used for human food mainly as a condiment. Observe the preserved material.

2. Freshwater algae:
a. Chara (stonewort)
These freshwater algae are abundant in some local lakes. They impart a bitter taste to the water,
The plants are often encrusted with lime deposits, and are largely free of the microscopic flora
and fauna that occur abundantly on most submerged macroscopic plants. Look at the
demonstration plants and slides of the oogonium and antheridium.
b. Cladophora
Locally abundant in the Great Lakes, Cladophora species are filamentous and branched. Their
life cycle is similar to that of Ulva. Cladophora establishment is sometimes an indication of
nutrient pollution of a lake. Observe plants on demonstration.

11

II. Brown and Red Algae

Introduction.
Brown and red algae are mostly macroscopic seaweeds. Brown algae tend to be more abundant in
cooler waters, on intertidal rocks and in relatively shallow waters. The brown coloration comes from
the pigment fucoxanthin. The stored food is laminarin, a starch which is distinctively different from
that of green algae and land plants. The brown algae include the largest algae such as kelps and
rock weeds.
Red algae tend to frequent warm tropical seas and may occur at depths as great as 200 meters in
especially clear water. A few species of red algae are found, surprisingly, in cold freshwater streams.
The red coloration comes from the pigment phycoerythrin but phycocyanin is also often present as in
the blue-green algae. Red algae store food as Floridean starch, another small but fundamental
difference from green algae and land plants. Some species of red algae even form coral-like reefs.
Some brown and red algae have extensive commercial value. Algin from brown algae and
carrageenin from red algae are used to stabilize the consistency of chocolate milk, dessert mixes,
shaving cream, cosmetics and many other products. Agar from the red alga species Chondris
crispus is essential to medical research as a culture substrate for microorganisms.

Exercises
A. Brown Algae
1. Fucus (rockweed)
Examine the preserved material and find the holdfast, stipe, dichotomously branched blade and air
bladders. At the end of blade branches are swollen tips, receptacles, which bear smaller dark
lumps. The smaller lumps (swellings on swellings) are the conceptacles, sexual structures which
contain oogonia and antheridia. The oogonia are usually oval and contain four large brown egg cells.
The antheridia are somewhat smaller and contain numerous very small, dark sperm cells. Examine
the prepared slide from your set, looking for antheridia and oogonia. Label Figure 11 -98.
Some Fucus species have conceptacles that produce both gametes, some are monoecious and
have male conceptacles and female conceptacles, and some are dioecious, which means an entire
plant is either male or female.
Which is shown on your slide?

Label Figure 11 -98

12

Figure 11-98. Fucus. a. General View, Showing Holdfast, Blade, Receptacles and Conceptacles.
b. Conceptacle. c. Detail of Antheridia containing Sperm. d. Detail of Oogonium with 4 Eggs.

13

Figure 11-100. Sargassum.

Figure 11-99. Nereocystis.

2. Kelps
Kelps include many of the largest seaweeds. Examine Nereocystis, Sargassum, Postelsia and
Laminaria, looking for similarities and differences in morphology. Look for holdfasts, stipe, blade and
air bladders. Label Figure 11 -99, Figure 11 -100, Figure 11 -101 and Figure 11 -102.

14

Figure 11-101. Postelsia.

Figure 11-102. Laminaria.

15

Figure 11-103. Batrachospermum.

B. Red Algae
1. Batrachospermum
Examine the preserved material and the demonstration slide. These freshwater red algae are found
in relatively unpolluted cold water streams in the Great Lakes Region. See Figure 11 -103.

2. Corallina
Coral forming alga. See Figure 11 -104
Figure 11-104. Corallina.

3. Porphyra
These red algae are used for human consumption.

16

4. Other Red Algae

Assorted red algae to note variation in color, size and shape. See Figure 11 -105.

C. Algal Products
Note products produced from brown algae (algin) and red algae (carrageenin and agar and various
packaged foods).

Figure 11-105. Bonnemaisonia. A Representative Red Alga.

17

KEY WORDS
unicellular
filamentous
colonial
complex multicellular plants
division of labor
isogamete
heterogamete
carotene
xanthophyll
pyrenoid
conjugation tube
laminarin
Floridean starch
coral
algin
carrageenin
agar
holdfast
stipe
blade
air bladder
receptacle
conceptacle
monoecious
dioecious
rockweed
kelp

Laboratory 13
Lichens
Bryophytes:
Liverworts
Hornworts
Mosses
Introduction
The organisms featured in this laboratory have similar life styles, but differ enormously in
classification. Lichens, mosses, hornworts, and liverworts are primarily photosynthetic land plants,
none with the ability to grow tall. All are limited to small size by their ineffective conduction of food
and water. Consequently, they are mostly found either on the ground underfoot, on rocks, or growing
on the stems or branches of trees.
Mosses, hornworts, and liverworts are collectively referred to as bryophytes. Traditionally, they have
been classified together, but evidence has been accumulating that suggests that their relationship is
probably not great.
Despite a similarity in habitat, however, lichens are fundamentally different from any bryophyte.
Though looking and functioning like a single plant, each lichen is composed of two radically different
organisms, an alga and a fungus. Growing together symbiotically as a lichen they produce an entity
which is entirely unlike either one growing separately.

I. Lichens
The algal component of a lichen is either a green alga or a cyanobacteriium. Lichen algae are usually
one of just a few species, the most common being Trebouxia, a one-celled green alga. The lichen
fungus, in contrast, might be any one of thousands of fungus species, but almost always a sac
fungus.
The lichen thallus usually consists of several indentifiable layers, which vary somewhat depending
upon the growth form. Three principal growth forms are recognized in lichens: (1) foliose, flat and
leaf-like, with lobes, (2) fruticose, branched and either upright like miniature shrubs or hanging and
similar to Spanish moss, and (3) crustose, tightly appressed, almost like a coat of paint on the
substrate. The thallus usually has a fairly dense upper or outer cortex composed of fungal hyphae
covering an algal layer crisscrossed with fungus hyphae, and then a loose fungus layer. In foliose
lichens an lower cortex is present next to the substrate, with rhizines or other attaching structures that
anchor the plant. Lichens produce acids that may etch the substrate beneath.
If sexual structures are present, they are those of the fungus, usually either apothecia or perithecia.
The spores they release germinate to produce hyphae, which must serendipitously come in contact
with the correct alga to produce a new lichen. Soredia are small pieces of lichen produced on the
thallus containing fungal hyphae and algal cells. The soredia may be wind or water borne to produce
new lichens. Fragmentation of the thallus may also produce new lichens.
Lichens may be used for dyes (orcein, litmus, and cudbear), in medicines (salves or antibiotics), as a
food source for reindeer and occasionally as human food in times of famine. Lichens are good air
pollution indicators, being vulnerable to acid rain and heavy metals. Lichens also act as soil builders
and binders.

Exercises
A. Foliose Lichens
1. Physcia (star lichen)
This lichen is common on the bark of trees and on twigs. Unlike many of the other foliose lichens on
display, Physcia has conspicuous fruiting structures, apothecia, on the upper surface. Soredia may or
may not be present depending upon the species of Physcia on display. Notice the difference in
texture and color between dry and wet lichens. Make a cross section of an apothecium to examine
the asci and spores.
Examine the
Figure 12-106. Foliose Lichen. a. Thallus, with Apothecia. b. Cross
prepared slide of
Section of Thallus Only.
the lichen thallus
and apothecia.
Label Figure 12
-106. Note the
upper and lower layers of dense fungus hyphae, the discrete attachment point, the alga layer (dark
round cells), and the loose fungus hyphal layer.

Figure 12 -107 shows a cross section through an apothecium. Label the thallus, an apothecium, an
ascus, and ascospores.

2. Other Foliose Lichens:

Rock tripe (edible), lung lichen, Parmelia, star lichen, and other foliose lichens are on display.

B. Fruticose Lichens
Fruticose lichens typically have an upright, tubular thallus with both a well-defined outer and inner
surface of more densely packed fungus hyphae. Label Figure 12 -108. Note the outer and inner
layers of dense fungus hyphae sandwiching the algae layer (dark round cells).

Figure 12-107. Physia. Cross Section of Apothecium and Thallus.

Figure 12-108. Fruticose Lichen. a. Growth Form. b. Cross-Section.

1. The Genus Cladonia.

The most common fruticose lichens are species of Cladonia, which are often found on soil and dead
wood. British soldiers and reindeer moss are in the genus Cladonia. Note the red fruiting structures
of British soldiers.

2. Other Fruticose Lichens

Examine fruticose lichens on demonstration: Awl lichen, pixie cup, ladder lichen, old mans beard and
others.

C. Crustose
Lichens

Figure 12-109. Crustose Lichen. a. Growth Form. b. Cross-Section.

5
These lichens are often easily overlooked on their rock, soil or wood substrate. Apothecia, if present,
are likely to be the only features that stand above the substrate. Crustose lichens have a discrete
upper surface, but the hyphae of their lower surface collectively adhere to the substrate (Figure 12
-109).

D. Lichen diversity
Look at other species of lichens on display, trying to assign each one to one of the three
growth forms: foliose, fruticose and crustose.

Figure 12-110. Detail of Lichen Soredium (Asexual).

E. Soredia
Soredia are small sand-like granules consisting of fungus hyphae surrounding a few alga cells. That
means they have everything needed to propagate a lichen. Soredia form on the surface of the lichen,
and break off easily, and can be carried far by wind or water. Look for soredia under the
demonstration microscope. See Figure 12 -110.

F. Lichen Color Test

Lichens produce acids that may be useful in identification of genera or species. Perform the lichen
color test that is on the demonstration bench. The KOH solution will react instantly with paretin in
Xanthoria to produce a dark purple color. Candelaria will not react with KOH.

G. Economic Uses of Lichens

Various examples of human uses of lichens are on display.

II. Bryophytes
Bryophytes are apparently of great geologic age (to Devonian?), but do not fossilize well for lack of
hard tissue. They have persisted and occur throughout the world in a wide range of habitats.
These are the first embryophytes to be studied in this course. In embryophytes, the zygote resulting
from gamete fusion develops, at least for a little while, within the female sex organ (archegonium or
embryo sac). The gamete producing structures themselves have a layer of sterile cells around the
eventual organs. The bryophyte life cycle consists of an alternation between a haploid gametophyte
plant with a diploid sporophyte plant in what is referred to as alternation of generations. In
bryophytes the sporophytes typically contain little chlorophyll and are dependent upon the free living
green gametophyte. The other members of the Plant Kingdom that we will study in the next few labs
are likely to have a dominant sporophyte generation. The sporophyte of bryophytes is produced after
a swimming sperm from the antheridium fertilize an egg in the archegonium. The spores that the
sporophyte generation eventually produces are haploid. With the exception of Sphagnum (peat
moss), bryophytes have little economic value except perhaps for erosion control and as a seedbed for
trees. Ecologically, they serve as habitat for small insects and many other forms of small animal life.
Peat moss generally makes up at least a portion of the peat that is used for fuel or in horticulture.
Peat moss is unique in being able to absorb great amounts of water in its dead leaf cells and the
ability to change the pH of the surrounding environment by cation exchange. Bogs in Wisconsin
typically contain from 5-15 species of Sphagnum, each occupying its own particular ecological niche.

Figure 12-111. Liverwort. a. Thallus. b. Detail of Gemma Cup.

Figure 12-112. Liverwort Thallus Cross Section.

Exercises
A. Liverworts
1. Marchantia, a Thallose Liverwort
a. Thallus
Examine the chlorophyllous gametophyte plant that is ribbon-like, dichotomously branched
(two-prong forking), and has an upper (dorsal) and lower (ventral) side. These should be
examined for branching, rhizoids and scales on the ventral surface, pores on the upper surface,
and if present, gemma cups. Make a wet mount of the unicellular rhizoids and use your
microscope to compare them with the multicellular scales. Both serve functions similar to roots
for absorption of water and anchorage to the substrate, but not as well. Label Figure 12 -111.

b. Thallus Detail

8
Observe on demonstration a cross section of the thallus for the air chambers, pores,
chlorenchyma (cells specialized for photosynthesis), scales and rhizoids. Label Figure 12
-112.
c. Gemma Cups
Observe on demonstration the longitudinal section of a gemma cup. The gemmae serve as
propagules for asexual reproduction.

Figure 12-113. Liverwort Antheridial Head. a. Overall Aspect. b. Cross Section.

d. Sexual Reproduction
Since Marchantia is dioecious it has both male plants which produce antheridia and sperm and
female plants which form archegonia and eggs. These sexual parts are in specialized structures.
Antheridia are borne by stalked, flat-topped antheridiophores and archegonia develop under
finger-like projections from the umbrella-like archegoniophores. 1) Examine specimens of male
and female plants for these structures. 2) Look at a prepared slide and note the antheridia in the
antheridia disc with stalk, a sterile jacket layer and sperms or spermatogenous tissue (Figure 12
-113).
Examine a prepared slide of an archegonial plant for the archegonia with neck, neck canal cells,
and egg (Figure 12 -114). These are typical parts of most bryophyte sex structures.
e. Sporophyte
After the egg is fertilized a sporophyte is produced and may be seen on the prepared slide. The
sporophyte (2N) of most bryophytes consists of a foot, a seta (or stalk), and the capsule or
sporangium. Within the capsule spores (N) are produced by meiosis. In the case of Marchantia
(and some other liverworts) additional sterile cells within the capsule, elaters, twist as they dry,
and aid in the dispersal of spores. Sometimes left on the capsule are remnants of the old
archegonium (calyptra). Label Figure 12 -115.

2. A Leafy Liverwort [not illustrated]

Look at preserved or fresh material of Porella or other leafy liverwort, as available. Note the leaf-like
structures on the gametophyte plant and that it is dorsi-ventral with an upper and lower side. The
"leaf" lacks the midrib (costa) found in most mosses. Rhizoids may also be seen on the ventral
surface. These plants will be dioecious and have male and female parts on separate plants. They
are not in specialized structures such as in the antheridiophore. The sporophytes on these plants are
very ephemeral persisting only for a short time while the spores are shed to produce new
gametophytes.

10

Figure 12-114. Liverwort Archegonial Head. a. Overall Aspect when Mature. b. Cross Section at
Time of Fertilization.

11

Figure 12-115. Liverwort Sporophyte Cross Section. Scales of the

Mother Gametophyte Surround it.

12

Figure 12-116. Hornwort.

B. Hornworts
Examine the demonstration material of Anthoceros. The gametophyte thallus resembles that of some
liverworts but differs in having only a single large chloroplast in each cell and in having pyrenoids.
Masses of the blue-green alga, Nostoc, often occupy chambers within the thallus. Gametophytes
have both sexual structures on the same plant. The sporophyte is an elongate, cylindrical structure
consisting of a foot and capsule. At the base of the capsule is a zone of cells which retain the ability
to divide, so that the capsule is continually renewed at the base and matures at the top. Within the
mature part of the capsule there is sporogenous tissue surrounding a sterile center or columella. At
maturity the spores are intermixed with elater-like structures and are released as the capsule splits.
On the capsule epidermis one can see stomates, normally considered structures confined to higher
plants. Label Figure 12 -116.

C. Mosses

13

Figure 12-117. Moss protonema and early development of a leafy plant.

1. True Mosses
The gametophyte plant usually starts out as thin strands called protonema. These appear much like
algal filaments but usually have slanted cross walls. They can arise from spores or almost any part of
a moss (leaves, individual cells, rhizoid, sporophyte, etc.) and are usually short-lived.
a. Protonema
Take a portion of the protonema, make a wet mount and examine under the microscope. The
bud-like structures on the protonema will eventually produce the mature gametophyte. Examine a
prepared slide of protonema with buds and a leafy plant. Find the appropriate material and label
Figure 12 -117.
b. Gemmae and Bulbils
Gemmae or bulbils, which function like resting spores or miniature bulbs, may be present on the
rhizoids as spherical brown multicellular structures. Examine a prepared slide showing these
bulbils.
c. Gametophyte
Obtain a gametophyte and note its symmetry, leaf-like structures (not true leaves), the stem-like
(not true stems), and rhizoids. Mosses may be dioecious or monoecious. The male plants of
some species may be identified by the broad antheridial heads (Polytrichumhair capped moss).
A narrow, pointed archegonial head identifies the female plant. Examine a male and female plant
and label Figure 12 -118.

14
d. Leaf
Remove a leaf from the gametophyte plant supplied and examine the shape, margin, thickness,
and cell arrangement. A costa or midrib may be present. No stomates are found in the
gametophytic tissue of bryophytes.

Figure 12-118. Polytrichum (Moss). a. Male Plant. b. Female Plant.

15
e. Antheridia
Prepared slides of antheridial heads should be studied under the microscope to find the
antheridium and the adjacent sterile structures called paraphyses. Note the stalk, sterile jacket
layer, and spermatogenous tissue or sperms. Label Figure 12 -119a.
f. Archegonia
A slide with an archegonial head should be examined to see the stalk, venter, neck canal cells,

Figure 12-119. Moss Sexual Structures, Cross Sections. a. Antheridial Head. b. Archegonial
Head.

and egg cell of the archegonium. Label Figure 12 -119b.

g. Sporophyte
The sporophyte of the moss consists of the foot, seta or stalk, and the capsule or sporangium on
which may be a lid or operculum. Under the operculum most mosses have a ring of toothlike
structures termed the peristome, which aid in spore dispersal. Dissect the capsule available and
look for these parts. Also a calyptra may be present as a hood or cover over the capsule. These
structures can usually be seen under the dissecting scope. Bryophyte stomates are only found on

16

Figure 12-120. Moss Sporophyte. a. Overall Aspect. b. Capsule Cross Section.

the capsule. A prepared microscope slide is available in your slide set for seeing greater detail.
Label Figure 12 -120.

2. Sphagnum Moss (Peat Moss)

Usually fresh, preserved, and dried material will be furnished for Sphagnum. Look at the round
capsules. In nature, they develop a pressure of several bars, which eventually blows the top away
with explosive force, much promoting spore dispersal. Note the gametophyte and the unique water
absorption caused by the unusual leaf structure. Green photosynthetic cells form a thin network,
interspersed with large empty dead cells with pores. Sphagnum mosses can absorb many times their
weight in water. Examine a Sphagnum leaf under the microscope. Its unusual morphology makes
Sphagnum ideal for holding water in horticultural purposes. In the past, it was even used for dressing
of wounds, since it is absorptive, and usually acid enough to be virtually bacteria free. Label Figure
12 -121.

17

3. Moss Diversity

Figure 12-121. Peat Moss. a. Overall Aspect, Leafy Gametophyte with Two Sporophytes and
Capsules. b. Leaf Surface View. b. Leaf Cross Section.

18
Other Mosses (such as Mnium) will be placed on demonstration along with other plants that are
commonly called mosses but are not (such as Spanish moss).

19

KEY WORDS
bryophyte

capsule

lichen

elater

symbiosis

calyptra

thallus

protonema

foliose

bulbil

fruticose

costa (pl. costae)

crustose

operculum (pl. opercula)

cortex

peristome

alga layer
soredium (pl. soredia)
lichen color test
embryophyte
egg
archegonium (pl. archegonia)
sperm
antheridium (pl. antheridia)
dichotomous branching
dorsal
ventral
gametophyte
sporophyte
alternation of generations
air chamber
pore
scale
rhizoid
gemma cup
gemma (pl. gemmae)
antheridiophore
archegoniophore
foot
seta

Laboratory 14
Fern Allies
Ferns
This laboratory features seedless vascular plants. Unlike bryophytes, vascular plants have highly
specialized water and food conducting systems, collectively forming a vascular system. Like
bryophytes, though, the sexual reproduction of these plants results in the production only of singlecelled spores, rather than continuing on the development of the complex structures known as seeds
that are produced by the plants treated in Laboratories 14 and 15. Our treatment of seedless
vascular plants includes ferns, which of course have large, complex leaves (megaphylls), as well as
plants often called fern allies, whose leaves (microphylls) are primitive at best, or even lacking
entirely. As seedless vascular plants, ferns and fern allies share the same general life cycle: The
dominant diploid plant produces spores by meiosis, which grow into an independent but small and
inconspicuous haploid plant, which, in turn produces sperm and egg cells which unite in the
production of a zygote, which develops into an embryo that matures into a diploid plant again. Club
mosses and a very few of the ferns are distinctive in having two kinds of spores (heterosporous)
rather than one (homosporous). These attract our interest, because the same pattern is found in the
reproductive development of seed plants

I. Fern Allies
Introduction
Each of these plant groups may be considered a surviving remnant of plants that were much more
important in the geological past. Fossils indicate that they were of especially great importance during
the coal forming Mesozoic time period.

Exercises
A. Whisk Fern
Psilotum is generally thought to be similar to the first vascular land plants. The appearance may be
fortuitous, for botanists are not convinced that Psilotum should really be classified with the fossil
general Rhynia and Psilophyton. Some even think they may represent primitive ferns! Psilotum has
no true leaves or roots, consisting of little more than stems. The underground stems are rhizomes
equipped with water and mineral
absorbing rhizoids. The green,
photosynthetic, above-ground stems are
distinguished from most other vascular
plants by dichotomous branching; that is,
each member of a branching pair tends
to be equal rather than being
distinguished as a primary stem and a
secondary branch. The leaf-like scales
contain no vascular tissue and are
considered anatomically to be stem
outgrowths. Sporangia are produced
terminally on vanishingly short branches.
Note Figure 13 -122.
Figure 13-122. Psilotum. a. Growth Form.
b. Dichotomous Branching and Sporangia. c. Detail
of Sporangium.

2
Look at the demonstration slide of the sporangia.
The gametophyte of Psilotum contrasts with that of ferns in typically being heterotrophic, lacking
chlorophyll and depending on a symbiotic fungus during its development. Despite being haploid, the
gametophyte looks quite a bit like the rhizome of the sporophyte. Psilotum has only one
contemporary relative, an epiphytic plant, Tmesipteris. (Epiphytes are plants that use other plants for
support. Neither Psilotum nor Tmesipteris can survive in climates with an extensive dry or cold
season.

B. Club Mosses
Unlike Psilotum, club mosses have real roots and much of their photosynthesis is accomplished with
leaves, albeit primitive ones (microphylls). Contemporary club mosses are all small and relatively
insignificant, but fossils suggest a history of tree sized club mosses of wide geographical distribution
and great ecological importance. There are five contemporary genera of club mosses, three of which
occur naturally in Wisconsin:

1. Lycopodiumclub mosses in the strict sense

Figure 13-123. Lycopodium. a. Growth Form. b. Cross Section of Strobilus.

3
Lycopodium species are mostly evergreen understory plants that look more or less like conifer
seedlings. The sporangia are produced at the base of modified leaves (sporophylls) usually clustered
in terminal structures called strobili, popularly known as cones. Examine these structures both in
the preserved material and prepared slides. Pay special attention to the appearance of spores for
comparison with Selaginella below. Label .
The gametophyte of some species of Lycopodium is lobed and green, while that of others is
subterranean, branching and dependent on a symbiotic fungus during its development.

Figure 13-124. Selaginella. a and b. Growth Form of Two Species. c. Cross Section Showing
Spore-Bearing Leaves (Sporophylls).

2. SelaginellaSpike Mosses
Selaginella species are often very moss-like in appearance, but have a vegetative structure much like
Lycopodium. An abundance of tree-sized spike mosses are also present in the fossil record. Most
contemporary spike mosses grow in relatively open sites. On display are typical spike mosses as well
as an atypical and remarkable species known as resurrection plant. Examine the living material of
Selaginella. If present, the sporophylls (leaves which bear sporangia) will contain both megaspores
and microspores, for Selaginella is heterosporous. In any event, you should be able to find both
spore types in the prepared slide. Label Figure 13 -124, identifying the microspores, megaspores,
and the microsporangia and megasporangia in which they are found.
Selaginella gametophytes develop within the spore wall! Microgametophytes develop in
microspores, producing antheridia and sperm. Megagametophytes develop in megaspores,
producing archegonia and eggs. Fertilization occurs by swimming sperm, as in many seed plants, the
embryo uses the megagamophyte tissue for its first growth. Unlike seed plants, once it is formed, the
megaspore receives no direct support from the parent plant, there is no dormant phase between the
maturing of the embryo and the development of a seedling. Spike mosses seem to represent a
bridge between spore and seed plants.

5
Figure 13-125. Isoetes.

3. IsoetesQuillworts
Quillworts are small plants with a very short fleshy underground stem topped by a crown of quill-like
leaves (see Figure 13 -125). The leaves seem too long to be considered microphylls, but are simpler
in structure and stem attachment than megaphylls. Quillworts are heterosporous. Quillworts are
reasonably common in the shallows of Wisconsin lakes.

C. EquisetumHorsetails or Scouring Rushes

Once quite abundant in the fossil record with many different genera, only one genus (Equisetum) still
exists. The sporophyte of Equisetum has jointed, ribbed, hollow photosynthetic stems with scale-like,
non-photosynthetic leaves. Both stem branches and leaves occur at the joints in whorls. Rhizomes
and roots are present. The stem may contain silica, which makes them rough to the touch and
produces the scouring ability. Note the variation in strobili (cones) of the specimens on display.
Some horsetails are dimorphic: some stems are vegetative only and others are non- green but have
strobili. Other plants have strobili on green stems. Label Figure 13 -126a.

6
The sporangia are in paired umbrella-like structures called sporangiophores usually bearing six
sporangia on their inner surface. These have been considered to be modified branches. Examine
microscope slides of the sporangiophore. Label Figure 13 -126b and Figure 13 -126c.
Equisetum is homosporous. The gametophyte is green and bisexual with antheridium and
archegonium. If available, look at the microscopic sexual structures present on the gametophytes.
The spores also have special structures called elaters which aid in their dispersal. Examine the
demonstration slide of the spores. Label Figure 13 -127.

D. Fossils
The plants which formed the great coal deposits during Carboniferous times (345 to 280 million years
ago) were mainly lycopods or horsetails. It is thought the prevailing environment favored the growth
of swamp forests whose fossilized remains became coal beds. Later when the climate became cooler
and drier, these coal forming plants became extinct with only a few species of small stature surviving
today. The lycopod fossils include Lepidodendron (reaching to 40 m high). Fossil horsetails include
Calamites and Sphenophyllum. Calamites reached to a height of 30 meters and 40 cm in diameter.
Coal ball fossils are a conglomeration of many types of plants together with some of the original
organic matter preserved.
Types of fossilization:
IMPRESSIONSOnly the impression may be left from the plant (or animal such as dinosaur tracks).
COMPRESSIONSOrganic matter may be preserved and sometimes can be removed intact from
the rock in which it occurs,

Figure 13-126. Equisetum. a. Growth Form. b. Strobilus. c. Sporangia on Sporangiophore.

Figure 13-127. Equisetum Spore with Elaters.

PETRIFACTIONInternal cellular material

is replaced with silica, iron pyrate, calcium
carbonate but cell walls may remain intact
such as found in petrified wood..

9
Observe the fossil specimens on demonstration. How can you distinguish lycopod from horsetail
fossils?

What parts of the plants are shown?

II. Ferns
Introduction
Ferns are the largest group of seedless, vascular plants with over 10,000 species, the majority of
which are found in the tropics. They differ from fern allies by having large, complex leaves
(megaphylls).

Exercises
A. Sporophyte

10

1. Vegetative Form.
Note the great diversity of ferns on display. The vegetative part of a fern sporophyte typically consists
of a horizontal underground rhizome from which arise clusters of fronds so large and divided that
each leaf appears to be a leafy stem. Growing down from the rhizome are true roots. Look closely at
the fronds of some of the fronds on the lab benches. Be sure you can identify the full extent of the
frond (leaf) which is divided into stipe (or petiole or leaf stalk) and blade. The blade may be divided
into pinnae (leaflets), which may be divided further into pinnules. Even additional levels of

Figure 13-128. Fern Leaves Illustrating Different Levels and Patterns of Compounding.

subdivision can occur. Leaves that have leaflets are said to be compound. See Figure 13 -128 for
examples.

11

The central axis of a leaf or leaflet is called the rachis. Fern fronds may also be completely or partly
dimorphic; that is, separated into reproductively fertile and vegetative leaves or leaflets. Find a fern
with young leaves to see the distinctive way in which they unfold. An expanding fern leaf is called a
crozier or fiddlehead. Look for a fern in which the rhizomes are above the ground surface, or dangle
off the sides of the pot. Label Figure 13 -129.

12

Figure 13-129. Fern (Sporophyte Generation).

13

2. Reproductive Structures
The fronds should have sori that are clumps of sporangia usually in distinct shapes. These sporangia

Figure 13-130. Fern Sorus Section (Leaf Section at Top).

may be covered with a lip of tissue called an indusium that is a specialized outgrowth from the leaf.
All ferns except water ferns are homosporous.

a. Sori
Look at the demonstration slide of a cross section of a sorus with an indusium. Label Figure 13
-130.

b. Sporangia

14
Examine a prepared slide showing sporangia and see the special cells that aid in spore dispersal
(annulus and lip cells). Label Figure 13 -131.

Figure 13-131. Fern Sporangium.

Do spores have a haploid or diploid chromosome number?

15

What shape are the spores?

These will be dispersed and eventually produce the prothallus or gametophyte.

3. Aquatic Ferns
Look at the displayed water ferns, which are heterosporous (Azolla, Salvinia and Marsilea).

16

B. Gametophyte
1. Prothallus
Most ferns are homosporous with spores that produce a gametophyte (prothallus) with both
antheridium and archegonium. Note the shape of the gametophyte. It lacks stems, roots and leaves.

Figure 13-132. Fern Prothallus (Gametophyte Generation).

Rhizoids should be present, however. Look for the sexual structures in the fresh material and in the
prepared slide. Label Figure 13 -132.
How thick is the prothallus?

17

Is it photosynthetic?

2. Gametangia
From the demonstration slide of a prothallus cross section, distinguish between the antheridium and
archegonium (Figure 13 -133a and Figure 13 -133b).
When a sperm fertilizes the egg in the archegonium, the first growth of the new sporophyte begins.
Label Figure 13 -133c.

18

19

20

Figure 13-133. a. Fern Prothallus and First Growth of Sporophyte. b. Fern Archegonium Detail. c.
Fern Antheridium Detail.

21
Do the antheridia and archegonia mature at the same time?

Do they resemble bryophyte organs?

3. Embryo
After fertilization and growth of the zygote, how long is the sporophyte dependent upon the
gametophyte?

C. Fern Identification
Use the fern key on the next page to differentiate the species that are on your identification list.

22

Key to Ferns on Common Plant Identification List

1a. Fertile (spore-bearing) fronds or pinnae, brownish, lacking green or photosynthetic tissue, sharply
differentiated in size and color from green sterile (vegetative) fronds or pinnae.
2a. Fertile frond entirely brownish and separate from sterile frond above ground, although both
arising from the same rhizome.
3a. Fertile fronds hard and smooth; veins of sterile fronds much branched and forming a
network of closed loops..................................................Onoclea sensibilis SENSITIVE FERN
3b. Fertile fronds soft and covered with reddish-brown wool; smallest veins of pinnae lobes of
sterile fronds forking and forming a "U" which ends at the margin
..............................................................................Osmunda cinnamomea CINNAMON FERN
2b. Fertile pinnae small, brownish, appearing in middle or at end of green frond.
4a. Entire frond twice compound; fertile pinnae at top third of frond
................................................................................................ Osmunda regalis ROYAL FERN
4b. Entire frond once compound; fertile pinnae at about the middle of the frond
...........................................................................Osmunda claytoniana INTERRUPTED FERN
1b. Fertile fronds or pinnae green and photosynthetic, similar in size, shape, and color (except for the
brownish sori) to the sterile fronds, or specimen lacking fertile fronds.
5a. Stipe (petiole) of frond forking into two recurving branches; sporangia along upper edge of
pinnules, covered by its reflexed margin..........................Adiantum pedatum MAIDENHAIR FERN
5b. Stipe straight, continuing as mid-rib or rachis of the blade.
6a. Fronds compound two or more times.
7a. Blades broadly triangular and horizontal; sori forming a continuous band around
margins of pinnules.................................................Pteridium aquilinum BRACKEN FERN
7b. Blades oblong and vertical; pinnules sterile (small and turning brown if fertile)
.......................................................................................... Osmunda regalis ROYAL FERN
6b. Fronds once compound or only deeply lobed.
8a. Blades usually more than 50 cm long and once compound with more than 12 pairs
of pinnae; smallest veins of pinnae lobes forking and forming a "U" which ends at
margin.
9a. Pinnae with small tufts of woolly hair at base
............................................................Osmunda cinnamomea CINNAMON FERN
9b. Pinnae smooth at base..............Osmunda claytoniana INTERRUPTED FERN
8b. Blades less than 50 cm long, deeply lobed or with 1-3 pairs of pinnae at base;
veins forming closed loops or U branches which do not reach margins of lobes.
10a. Fronds thick and green through winter; sori round, without indusia; veins
forming a "U"......................................Polypodium virginianum POLYPODY FERN
10b. Fronds thin, sensitive to light frost; sori absent from green fronds; veins
forming a network of closed loops................Onoclea sensibilis SENSITIVE FERN

23

KEY WORDS
vascular plant
vascular system
megaphyll
microphyll
heterosporous
homosporous
strobilus (cone)
rhizome
epiphyte
sporophyll
prothallus (young gametophyte)
megaspore
microspore
microgametophyte
megagametophyte
whorl
dimorphic
sporangiophore
frond
sorus (pl. sori)
stipe
petiole
pinnatifid
pinna (pl. pinnae)
pinnule
compound leaf
rachis
crozier (fiddlehead)
sorus (pl. sori)
indusium (pl. indusia)
annulus
lip cell
gametangium (pl. gametangia)

24

(blank page)

Laboratory 15
Gymnosperms
Introduction
Gymnosperms and angiosperms (flowering plants) are the only plants that produce seeds.
Gymnosperms include only about 750 species compared with about 200,000 species of angiosperms.
The economic and ecological importance of the gymnosperms, however, is much greater than the
small number of species would suggest, since they include pines, spruces, firs, redwoods, cedars,
hemlocks and other needle-leaf trees. These trees dominate much of the mountain and cool
temperate to boreal lands of the world and yield much of our lumber and pulpwood.
Gymnosperms differ from angiosperms in several significant ways:
(1) Seeds are produced "naked" on the surface of modified leaves or cone scales, instead of being
enclosed in fruits.
(2)

No flowers are produced.

(3) Only a single male gamete is required for the production of one seed, in contrast to the "double
fertilization" of angiosperms.
(4)

Nutrition for the developing embryo is supplied by a comparatively large gametophyte.

(5)

Most species have relatively primitive food and water conducting cells.

Gymnosperms include four living subgroups: (1) conifers, yews, and their relatives; (2) cycads;
(3) Ginkgo; and (4) Gnetum, Welwitschia, and Ephedra. These four subgroups differ from each other
in fundamental ways and are not considered to be closely related.

Exercises
A. Gymnosperm Life Cycle
Note that gymnosperms, like bryophytes, ferns, and fern allies, have alternation of diploid and haploid
generations. Diploid sporophytes produce spores by meiosis, which grow into haploid gametophytes,
which produce gametes that unite to form the zygote, which grows into the sporophyte.
Ferns and fern allies, as spore-dispersed vascular plants, seem to have two major disadvantages for
reproducing and spreading in their terrestrial habitats: (1) Spores themselves have virtually no
resources of stored food to give the gametophyte a good start. (2) The gametophyte itself is small,
simple, and either directly vulnerable to dry conditions or indirectly vulnerable because of its
dependence on a symbiotic fungus. There are many habitats in which the sporophyte stage can
apparently thrive, but the species does not become established because of gametophyte restrictions.
Gymnosperms and angiosperms, as seed plants, avoid the problem of gametophyte frailty by keeping
the megagametophyte within the sporophyte plant during its entire development. The gametophyte is
able to benefit from the water and food resources of its parent. Once the egg is fertilized, the parent
sporophyte continues to support the developing embryo. The resulting seed, in great contrast to a
spore, contains food reserves and a predeveloped sporophyte plant (the embryo).
Seed production is not without its problems. (1) Getting sperm from one gametophyte to another is
much simpler with very numerous, tiny plants on moist ground than from one large sporophyte to
another. The seed plant answer is a very reduced, airborne microgametophyte more popularly known
as pollen. (2) Seeds are expensive to produce. Spores can be produced in extraordinary numbers
with the same investment of materials as a relatively small number of seeds. Of course, any single
spore has a very small likelihood of surviving.
Examine the diagrams of the generalized seed plant life cycle on demonstration in lab.

2
Review the previous lab and note that the life cycles of gymnosperms and of Selaginella have much
in common. Notable differences include (1) Selaginella produces no pollen, and )2) in Selaginella any
food supplement from the parent sporophyte is part of the megaspore from the start.
Pine (Pinus) will be used to show details of a gymnosperm life cycle.

1. Pine Pollen Cones

a. Gross Anatomy
Microspores and the early stages of development of a microgametophyte occur in small pollen
cones borne at the tips of branches. These cones are most evident in early spring prior to the
release of pollen grains. They disintegrate after the pollen is shed. Examine a branch bearing
pollen cones and the pollen cone provided for your observations. Dissect out a single scale.
Note that the scale is narrow and bears two large sacs on the underside. Each sac is a
microsporangium. Younger microsporangia attached to scales near the apex of the cone may
not have split at this stage, but lower ones probably have split open. Find one bearing pollen
grains (yellow and about the size of salt grains) and make a wet mount of the pollen. Examine the
slide on your microscope. Note that each pollen grain consists of a central spherical chamber and
two lateral somewhat flattened spheres which are called wings. Label Figure 14 -134b and
Figure 14 -134c.
b. Detailed Anatomy and Development

Figure 14-134. Pine Cones: a. Pollen Cone. b. Young Seed Cone. c. Maturing Seed Cone. d.
Seed Cone at Time of Seed Dispersal.

Figure 14-135. Pine Pollen Production. a. Cluster of Pollen Cones. b. Microsporangia.

c. Individual Pollen Grain.

Examine the prepared slide of the pollen cone of pine in longitudinal section. Note the
arrangement of the scales along the central axis of the cone and that a microsporangium lies
below the scale to which it is attached. Look at various pollen grains; some will be sectioned at a
level to show two nuclei within the same central chamber of the pollen grain. These pollen grains
are young microgametophyte plants. Study the pine pollen grains in the microsporangia on the
pollen cone slide and label Figure 14 -135.
c. Developmental Sequence
(1) The adult pine, a sporophyte, produces young male cones near tips of branches.
(2) Spore mother cells (2n) in the developing sporangia undergo meiosis, and each mother cell
produces four microspores (1n).
(3) The nucleus (1n) of each microspore divides twice by mitosis producing a four celled young
microgametophyte (male gametophyte). The two small cells at one side are called prothallial
cells and represent the remnants of what would be the vegetative tissue of an independent
gametophyte. A larger cell, the generative cell, lies next to the prothallial cell. It will eventually
produce two sperm cells. The largest of the four cells is the tube cell. It will govern development
of the pollen tube.
(4) The young microgametophyte becomes dormant and it is now called a pollen grain. It is shed
into the wind as the sporangium splits open.

Figure 14-136. Germinating Pine Pollen.

d. Germinating Pine Pollen

Examine the prepared slide of germinating pine pollen. Note the emerging pollen tube
containing (at least eventually) four nuclei: two sperms near the center, plus a tube nucleus and
a stalk cell nucleus. Label Figure 14 -136.

2. Pine Seed Cones

The seed cones of pine require two or three years to complete their role in the life cycle which
includes meiosis and megaspore production, megagametophyte (female gametophyte) development,
nurturing the microgametophyte, and production of seed.
a. Gross Anatomy
Examine the demonstration material of a young first-year seed cone (also called an ovulate cone).
Note that it is a small spheroidal structure usually borne near the tip of a branch.
b. Detailed Anatomy

5
Examine the slide of an ovulate cone (seed cone) in longitudinal section. This cone is more
complex than the pollen cone. A spiraling series of short bracts arises from the cone axis, and a
longer ovuliferous scale extends outward from the cone axis above each bract. The ovuliferous
scale bears two outgrowths on its upper surface (Figure 14 -137). Each outgrowth is a young
ovule and initially it consists of a spheroid mass of 2n cells called the nucellus that slowly
becomes almost completely surrounded by a cup-shaped outgrowth from the scale called the
integument.
If there is an enlarged cell visible near the center of an ovules. It is either the megaspore
mother cell (2n) or the one of the four megaspores (1n) which is large and able to develop. The
other three megaspores have almost no cytoplasm, are hard to detect, and fail to develop. Label
Figure 14 -137.

Figure 14-137. a. Two Pine Ovules on a Seed Cone Scale. b. Section of Seed Cone Scale Showing
One Ovule (Above Right).

6
c. Gametophyte Development
Following meiosis, the single functional megaspore will begin to grow and develop as a
megagametophyte. Pollination on the surface of the ovule has normally occurred by this stage.
It consists of a passive and accidental transfer of the pollen grains (young, dormant
microgametophytes) by wind currents to the young ovulate cones where the grains are captured
by sticky droplets at the micropyle (the gap in the integument) of each ovule. During the next
year the megagametophytes, the microgametophytes, the integuments, and the ovulate cone
grow and develop.
d. Ovulate Cone Development
Examine a second year ovulate cone. Note the increase in size. Note that it is a considerable
distance from the branch tip, indicating a year's growth by the branch.
e. Ovule Development
Examine the prepared slides showing the pine ovule in free nuclear stage and the egg nuclei
stage. Early in the development of the megagametophyte, many of the cells of the rapidly
growing gametophyte havent yet formed cell walls. This is referred to as the free-nuclear stage.
Label Figure 14 -138a. At maturity the megagametophyte (1n) is a large structure composed of
more than one thousand cells. Two to five archegonia with eggs in them lie near the micropyle.
Label Figure 14 -138b. At this point, one year after pollination, the mature microgametophyte is
ready to release two sperm. If one fuses with an egg cell, fertilization is accomplished, and the
zygote will begin growing into an embryo (the future seedling). As the megagametophyte
continues to enlarge, it accumulates stored food for the seedling.

f. Seed Release
The ovulate cone may be mature and fully developed by the end of the second growing season.
Examine a mature pine cone. Note the two winged seeds above most of the scales. These
seeds are usually dispersed by the wind.

4. Pine Seeds and Seedlings

a. Seeds
Examine the winged pine seeds and study the prepared slide of a mature seed. Note the embryo
sporophyte in the center. Its two most conspicuous parts are the radicle or embryonic root
occupying the lower half, and the several cotyledons or embryonic seed leaves in the upper
portion. The embryo is embedded in the female gametophyte tissue that at this stage consists
mostly of stored food for the embryo. The seed coat, developed from the integuments of the
ovule, forms the dry, thin outer covering of the seed. (This seed coat may be absent on some
slides). Label Figure 14 -139.

Figure 14-138. a. Pine Ovule with Developing Megagametophyte. b. Pine Ovule

with Mature Megagametophyte.

Figure 14-139. Pine Seed. a. General Aspect. b. Cross Section.

Figure 14-140. Pine Seedling.

b. Seedlings
Observe the demonstration of pine or spruce seedlings. In an early stage of germination, the
seed coat may be carried above ground encasing the tip of the cotyledons that together form a
structure resembling a tiny bird cage. Later as the seed coat is shed and as the cotyledons
spread outward and downward the emerging stem shoot with small leaves begins to elongate.
Label Figure 14 -140.

B. Other Gymnosperms
Observe the display of other gymnosperms.
1. Cycads
Cycads superficially resemble palm trees. They are dioecious (some plants bear only pollen cones;
others only ovulate cones). Note the large, heavy cones and large leathery, but fern-like leaves.
Such cones and leaves require thick stems for support, and the necessity of maintaining a wide
growing tip may be responsible in part for their slow growth in height. Zamia is native to southern
Florida.

10

2. Ginkgo
Ginkgo biloba, the ginkgo or maidenhair tree, is the only living member of a distinctive group. Note
the heavy seed that superficially resembles a fruit such as an olive or a plum. A seed such as this
must rely on animal dispersal, but its foul odor repels many animals. Note also the unique fan-shaped
leaves with the forking or dichotomous venation.

3. Gnetum and Relatives

Gnetum includes tropical vines and shrubs resembling the dicots of the angiosperms. Ephedra,
called joint-fir or Mormon-tea, is a desert-dweller with leaves reduced to small scales. Welwitschia
resembles no other plant in the world. This African desert plant may live more than one thousand
years, but it bears only two leaves in its entire life from a stem that suggests a giant woody turnip.

C. Common Conifers
Examine the display of some of the more common species of conifers and yew of this area. You will
be required to recognize many of these genera and species.

D. Key to Some Common Native and Cultivated Gymnosperms of Wisconsin

Use the following key to identify specimens of unknown gymnosperms.

11
Key to Native and Commonly Cultivated Wisconsin Gymnosperms
1a. Leaves fan-shaped with many fine forking veins radiating from petiole, deciduous; seeds solitary,
fleshy, plum like, about 3 cm in diameter..................................................................Ginkgo biloba GINKGO
1b. Leaves needle-like, scale-like, or awl-shaped, evergreen or deciduous; seeds in cones, or if
surrounded by fleshy tissue, less than 2 cm in diameter.............................................................................2
2a. Leaves needle-shaped, narrow, parallel-sided, alternate or spirally arranged, or in clusters................3
2b. Leaves scale-like, pressed against twig, or awl-shaped (sharp slender triangles less than 1 cm long),
opposite, or in whorls of three................................................................................................................... 23
3a. Needles in fascicles of 2-5 which form a cylinder when drawn together...............................................4
3b. Needles attached singly to branches or numerous in a dense cluster on spur shoots.......................10
4a. Needles in fascicles of 5, relatively soft; cones 10-15 cm long, less than one-third as wide, with thin
scales...............................................................................................Pinus strobus EASTERN WHITE PINE
4b. Needles in fascicles of 2 or 3, relatively stiff; cones usually less than 10 cm long and at least half as
wide, with thick scales................................................................................................................................. 5
5a. Needles usually less than 10 cm long.................................................................................................. 6
5b. Needles more than 10 cm long............................................................................................................. 8
6a. Shrub or low tree with several trunks; needles not twisted or widely spreading; 2 and 3 year old twigs
stout, about 1 cm thick........................................................................................ Pinus mugo MUGHO PINE
6b. Tree normally with a single trunk; needles twisted or widely divergent; 2 and 3 year old twigs slender,
about 5 mm wide......................................................................................................................................... 7
7a. Needles 2-5 cm long, rigid, flat, slightly twisted, divergent at 60-90 degree angle; cones projecting
forward, often persisting for several years; bark of large branches grayish-black; native
........................................................................................................................ Pinus banksiana JACK PINE
7b. Needles 3-8 cm long, not rigid, strongly twisted and often meeting near the tip; cones reflexed,
dropping after maturity; bark of large branches orange; cultivated
.................................................................................................................... Pinus sylvestris SCOTCH PINE
8a. Needles slender and breaking cleanly when bent back; cone scales without spine; bark reddish-gray;
native................................................................................................................... Pinus resinosa RED PINE
8b. Needles relatively thick, not breaking cleanly, but held together by vascular bundles when bent back;
cone scales with small spines; bark grayish black; cultivated.....................................................................9
9a. Some needles in fascicles of 3; cones about 10 cm long
.......................................................................................................... Pinus ponderosa PONDEROSA PINE
9b. All needles in fascicles of 2; cones 6-8 cm long.......................................Pinus nigra AUSTRIAN PINE
10a. Needles soft, completely deciduous in autumn, spirally-arranged on lead shoots, but densely
crowded on the numerous spur shoots...................................................................................................... 11
10b. Needles firm, evergreen, attached singly on all branches.................................................................13
11a. Cones 1-2 cm long with 10-15 glabrous scales; needles 15-30 mm long; native
.............................................................................................................................. Larix laricina TAMARACK
11b. Cones 2-4 cm long with 40-60 pubescent scales; needles 20-40 mm long; cultivated......................12
12a. Cone scales recurved near tip; needles with 2 distinct white bands beneath
............................................................................................................. Larix kaempferi JAPANESE LARCH
12b. Cone scales straight; needles green on all sides...........................Larix decidua EUROPEAN LARCH
13a. Needles four-sided or diamond-shaped in cross-section, attached to small raised pegs..................14
13b. Needles flattened, not attached to raised pegs.................................................................................17

12
14a. Twigs finely pubescent; needles fragrant when crushed...................................................................15
14b. Twigs glabrous; needles emitting unpleasant odor when crushed.....................................................16
15a. Needles blunt at apex, 0.5-1.5 cm long; cones less than 3.5 cm long; branches not drooping; native
................................................................................................................... Picea mariana BLACK SPRUCE
15b. Needles pointed at apex, 1.5-2.5 cm long; cones 12-15 cm long; branches usually drooping;
cultivated.................................................................................................... Picea abies NORWAY SPRUCE
16a. Needles 1-2 cm long, spreading from twig; cones 3-5 cm long with scales bluntly rounded to straight
at tip; native.................................................................................................. Picea glauca WHITE SPRUCE
16b. Needles 2-3 cm long, projecting forward on twig; cones more than 6 cm long with scale wedgeshaped and wavy at tip; cultivated.........................................Picea pungens COLORADO BLUE SPRUCE
17a. Needles yellowish green beneath, with an abrupt tooth at tip and a petiole adhering 1-2 cm along
stem; seeds borne singly, surrounded by a fleshy cup..............................................................................18
17b. Needles green or whitish beneath, rounded to slightly pointed at apex and lacking a petiole
continuing downward along stem; seeds several in woody cones.............................................................19
18a. Needles 1-2 mm wide, pale or dull green, borne in horizontal plane; native
................................................................................................................. Taxus canadensis CANADA YEW
18b. Needles 2-3 mm wide, bright green above and yellowish below, some borne above horizontal plane;
cultivated................................................................................................. Taxus cuspidata JAPANESE YEW
19a. Needles often less than 2 cm long, whitish beneath, with narrow petioles (dropping quickly when
dry); cones less than 3 cm long....................................................Tsuga canadensis EASTERN HEMLOCK
19b. Needles more than 2 cm long, greenish beneath, without a well-marked petiole; cones more than 4
cm long...................................................................................................................................................... 20
20a. Lower edge of leaf scar raised slightly; cones drooping with papery three-pronged seed bracts
protruding beyond cone scales.........................................................Pseudotsuga menziesii DOUGLASFIR
20b. Leaf scar flush with twig; cones erect and disintegrating at maturity.................................................21
21a. Needles 4-7 cm. long, bluish-white; cones 8-13 cm long............................Abies concolor WHITE FIR
21b. Needles 1-3 cm long, green; cones 5-10 cm long.............................................................................22
22a. Needles with narrow whitish lines (of 4-5 rows of stomates) below; native
....................................................................................................................... Abies balsamea BALSAM FIR
22b. Needles with wide whitish lines (of 8-10 rows of stomates) below; cultivated
............................................................................................................................ Abies fraseri FRASER FIR
23a. Twigs and branches strongly flattened; leaves opposite, scale-like, those on the face of the twig flat,
those on the side doubled over; cones woody
.......................................................................................... Thuja occidentalis NORTHERN WHITE CEDAR
23b. Twigs rounded in cross-section; leaves in whorls of three, or opposite, awl-shaped or scale-like but
not differentiated; cones fleshy and berry-like........................................................................................... 24
24a. Leaves in whorls of three and all awl-shaped (sharply pointed narrow triangles)
................................................................................................... Juniperus communis COMMON JUNIPER
24b. At least some of the leaves opposite or appressed scales................................................................25
25a. Scale leaves blunt; awl-shaped leaves in two's and three's; cones brownish; cultivated
...................................................................................................... Juniperus chinensis CHINESE JUNIPER
25b. Scale leaves pointed; awl-shaped leaves opposite except on lead shoots; cones bluish, native and
cultivated................................................................................................................................................... 26
26a. Trees; cones 5-6 mm wide, on straight ascending stalks, bearing 1-2 seeds
.............................................................................................. Juniperus virginiana EASTERN RED CEDAR
26b. Creeping shrubs; cones 6-10 mm wide, on stalks curved downward, bearing 3-5 seeds
................................................................................................ Juniperus horizontalis CREEPING JUNIPER

13

KEY WORDS
seed
pollen
microgametophyte
pollen cone
microsporangium (pl. microsporangia)
prothallial cell
generative cell
pollen tube
tube nucleus
stalk cell nucleus
ovulate cone
seed cone
ovule
nucellus
integument
megaspore mother cell
megaspore
megagametophyte
pollination
micropyle
free nuclear stage
fertilization
embryo
radicle
cotyledon
seed leaf
seed coat
seed germination
cycad
conifer

Laboratory 16
Angiosperms and the Flower
Introduction
Angiosperms (flowering plants) constitute the largest subdivision of the plant kingdom. The group
includes an estimated 200,000 species and it comprises the most conspicuous part of the vegetation
in most of the land areas of the world except for those parts dominated by coniferous forests.
Angiosperms are the direct or indirect source of nearly all food for humans and higher animals. In
addition, angiosperms provide textile fibers, hardwood lumber and pulp, spices, drugs, and raw
materials for countless manufactured products.
Angiosperms can be distinguished from other plant groups by a combination of characteristics
including the following:

Figure 15-141. Flower Parts, Model Dicotyledon.

2
(1)

Seeds develop encased within a fruit.

(2)

Flowers are produced, although they are not necessarily colorful and conspicuous.

(3) A "double fertilization" involving two male gametes is normally required to produce the embryo
and the endosperm of one seed.
(4)

Most species have vessels in their water conducting tissue.

Angiosperms are divided into two major subgroups: dicots and monocots. Approximately threefourths of the species belong to the former subgroup, but both subgroups are large and diverse,
including many orders, families, and genera.

Exercises
A. Insect Pollinated Flowers
Insect-pollinated flowers are more familiar to most people than those pollinated by wind. They tend to
be larger, and to have brightly colored parts, petals typically, which attract the attention of insects. No
particular flower can be considered typical in every respect, so there is a flower model provided in lab
to help study flower parts at their simplest and most obvious (see Figure 15 -141).

1. Flowers with Separate Petals and Sepals

Examine, then dissect, the Flower Provided.
a. Flower Parts
The basal stalk of the flower, the pedicel, is very short. Two paired green modified leaves called
bracts are attached to the pedicel. Bracts are not considered real flower parts since they are
attached below the receptacle of the flower. The receptacle is the expanded base of the flower to
which the other flower parts are attached. Note that the ovary of the gladiola appears to be
sunken into the receptacle and lies below the other flower parts. It is described as an inferior
ovary, referring to its position below the other flower parts. A short tube consisting of the fused
base of sepals, petals, and stamens extends above the receptacle and ovary. The three sepals
and the three petals are quite similar in color and shape. They alternate with each other (sepal
petalsepal, etc.) in a circle around the flower; the sepals lie slightly outside the petals. Three

Figure 15-142. Gladiola Flower (One Sepal and Two Petals Removed and Ovary Sectioned).

Figure 15-143. Compound Pistil Evolution. a. Seed Leaf. b. Modification into Simple Pistil (One
Carpel). c. Compound Pistil with Three Carpels.

stamens are attached inside the base of the sepals and petals. Each stamen consists of a
narrow filament and an expanded anther. Pollen is produced in the two anther sacs. The style
of the pistil ascends through the center of the flower. It is capped by the three-lobed stigma.
Pollen grains adhere to the stigma and germinate here, with the pollen tubes eventually growing
down through the style to the ovary. Label Figure 15 -142. Look for similarities and differences
between the gladiola and the model flower.
b. Ovary Cross Section
Make a cross-section through the ovary of the gladiola flower. Note that the ovary contains three
cavities and three double rows of ovules. The pistil of the gladiola is composed of three carpels.
A sepal, a petal, a stamen, and a carpel each represent a modified leaf. A carpel represents a
modification of a leaf which bears ovules along its two lateral margins. It is rolled inward, fusing
along the two lateral margins, thereby creating a double row of ovules along this suture and
forming a hollow chamber or cavity. The fact that three carpels or modified leaves have been
fused into a single unit, a compound pistil, is indicated by the three double rows of seeds, the
three cavities, and also by the three stigma lobes atop the style. Label Figure 15 -143.

2. Flowers with Fused Petals or Sepals

Examine the flower of a snapdragon or a butter-and-eggs plant. (If recently-thawed frozen flowers are
used, place them in a dish of water to counteract their limpness.) These flowers are more highly
specialized to limit pollination to a few insects, primarily bees (bees are efficient pollinators, tending to
visit flowers of one species at a time). These flowers have a bilateral symmetry (a right side and a
left side) rather than nearly radial symmetry of the gladiola. Five green sepals are partially fused at
the base of the flower. The five petals are fused together toward the base. Two of these petals form
the top and the upper lip of the flower; the other three petals form the lower lip that includes the
landing area for the insect. Nectar is produced near the base of the petal tube in the snapdragon and

5
at the tip of a downward pointing projection of the petal tube that is called a nectar spur in the butterand-eggs.
Slit and open the petal tube. Note that the four stamens and the style arch along the upper side of the
petal tube in ideal position to deposit or remove pollen grains from hairs on the back of a bee as it
crawls toward the nectar.
Note that the ovary of the pistil lies inside the petal tube with the sepals, and petals attached to the
receptacle below it. Because it sits above the base of these other flower parts, it is referred to as a
superior ovary. Cut a cross-section through the ovary. Note that it consists of two chambers or
cavities; the pistil therefore is a compound pistil composed of two carpels. Label Figure 15 -144.

Figure 15-144. Snapdragon Flower, Longitudinal Section.

Figure 15-145. Grass Flowers. a. Spikelet with Glumes Spread to Expose Three Florets. b. Floret
Detail.

B. Wind-Pollinated Flowers
Wind-pollinated flowers are usually small, drab-green or brown, and without petals. Showy petals
have no attraction to the wind. Anthers and stigmas, however, are usually relatively large since
pollination by wind is hardly efficient.

1. Grass Flower
Examine the flowers and spikelets of oats. The flower of oats and other grasses is reduced to the
essential parts for pollination and seed production: three stamens, and a pistil of one ovary and two
feather-like stigmas. Each flower is enclosed by two scales, the larger lemma and the smaller palea,
forming a floret; two or three florets occur above two empty scales called glumes. The whole unit of
florets and paired glumes comprise a spikelet. See Figure 15 -145.

2. Other Wind-Pollinated Flowers.

Observe other examples of wind-pollinated flowers on demonstration. Remember that it is such
inconspicuous flowers that may cause hay fever, not showy, colorful flowers such as goldenrods and
roses that are pollinated by insects.

C. Composite Heads
Most of the approximately 25,000 species of the daisy or sunflower family, one of the two largest
families or angiosperms, have tiny flowers aggregated into a larger unit, a composite head, which
superficially resembles a large single flower. Examine the sunflower heads on demonstration. The
several whorls of small leaves on the lower half of the head are bracts. The small individual flowers,
or individual fruits in a head at a late stage of maturity, are attached to a common disk-shaped
receptacle. The individual flowers have an inferior ovary which will develop into a single-seeded fruit.
At the top on the rim of the ovary or fruit is the pappus: a group of scales here, but more commonly
hairs or bristles in other species. The pappus represents modified sepals. The five petals are united
into a short tube. Each of the flowers at the outermost edge of the head have the five fused petals
greatly elongated to project to the outside of the head thereby stimulating a single petal and helping to
make the head resemble a single large flower. The flowers of this outer circle are referred to as ray
flowers or as ligulate flowers; they are often sterile or contain pistils only. The remainder of the

7
Figure 15-146. Composite Head.

head is composed of disk or tubular flowers that have petal tubes terminating in five short lobes.
Five stamens arise inside the petal tube and are fused by their anthers around the style. The style
divides above the anthers to bear two stigmas. In less mature composite heads the tubular flowers in
the center may still be in a closed bud stage of development. Observe other examples of composite
heads and label Figure 15 -146.

D. Stamen and Pollen

8
The gametophyte stage of the alternation-of-generations life cycle is at its shortest and most reduced
in the angiosperms compared with other members of the Plant Kingdom. Study the sequence of
slides listed below showing this part of the angiosperm life cycle.
(1) Examine a slide of lily (Lilium) anthers containing microspore mother cells. Note that the
anthers consist of four sacs. All tissue, including the microspore mother cells, are diploid at this
stage.
(2) Examine the second slide in the sequence showing stages of meiosis in the microscope mother
cells. Meiosis in each microspore mother cell results in a set of four haploid microspores.
(3) Examine the slide of lily (Lilium) anther containing mature pollen grains. Each pollen grain is
derived from a microspore. It has a thickened outer wall with small spiny protrusions. By this stage
the haploid nucleus has divided once by mitosis to produce a young microgametophyte. One of the
two nuclei is called the tube nucleus. It is destined to govern growth of the pollen tube. The other
nucleus, the generative nucleus, will divide again to become the two sperms. When the pollen
grains are mature the Lilium anther splits lengthwise along the outer edge to permit the release of
the pollen. Label Figure 15 -147.
(4) Examine the slide showing a mixture of pollen grains. Note variations in size and wall texture.
The slide includes pine pollen, recognizable by the two lateral wings, and also pollen of a species in
which the four microspores failed to separate after meiosis, producing a tetrad of pollen grains.
Label Figure 15 -148.
(5) Observe a slide showing germinated pollen grains with emerging pollen tubes. Germination
normally takes place on a stigma and the pollen tube grows through the style to the ovary. After the
generative nucleus has divided to become sperm, the pollen grain and tube is regarded as a mature
microgametophyte (male gametophyte). Fresh pollen often can be induced to germinate in a sugar
solution. Make a wet mount and observe living germinating pollen if available.

E.

Figure 15-147. Lily Anther Cross Section. a. Youthful, Spore Mother Cells. b. Developing,
Meiosis. c. Mature, at Pollen Release.

Ovule and Megagametophyte Development

Figure 15-148. Lily Pollen Cross

Section.

10
Study the sequence of slides on demonstration showing the development of the ovule and the
megagametophyte (female megagametophyte). Examine slides showing the developing ovule. The
ovule grows from the inner wall of the ovary. It includes a stalk region called the funiculus and
spheroid mass of cells called the nucellus surrounded by two cup-shaped layers called integuments.
The gap between the integuments is called the micropyle.
A central cell within the nucellus enlarges and becomes the megaspore mother cell. Meiosis occurs
here and three of the four resulting megaspores die in most species. The remaining megaspore
usually divides three times to produce eight haploid nuclei. In the mature megagametophyte
(sometimes called the embryo sac) the eight nuclei are separated into a set of three, including the
egg nucleus, at one end near the micropyle, a set of three at the other end, and a pair in the middle,
the polar nuclei. At fertilization two sperm enter from the pollen tube. One sperm fuses with the egg
cell to form the zygote (which will grow into the seedling) and the other fuses with the polar nuclei to
form the endosperm. Label Figure 15 -149.

11

F. Angiosperm Diversity
Among the reasons for the angiosperms' domination of most of the land areas of the world are their

Figure 15-149. Lily Pistil Sections. a. Cross Section of Ovary. b. Longitudinal Section of
Pistil. c. Detail of Ovule at Time of Double Fertilization.

diversity and adaptability of growth forms. Angiosperms include trees, shrubs, vines and herbs;
annuals, biennials, and perennials; autotrophs, saprophytes, parasites and "carnivores," and forms

12
adapted to living in water, in deserts, and on tree branches. Observe the demonstrations of
angiosperm diversity

13

KEY WORDS
fruit

composite head

flower

pappus

double fertilization

ray flower (ligulate flower)

vessel

disk flower (tubular flower)

dicot (dicotyledon)

funiculus

monocot (monocotyledon)

embryo sac

pedicel
bract
receptacle
ovary
inferior ovary
sepal
petal
stamen
filament
anther
style
stigma
ovule
pistil
carpel
compound pistil
bilateral symmetry
radial symmetry
nectar
nectar spur
lemma
palea
floret
glume
spikelet

Laboratory 17
Seeds
Seed Germination
Fruits
I. Seeds.
Introduction
A seed is a matured ovule and its major functions include protecting the young sporophyte, the
embryo, during dispersal, allowing it to pass through a period of unfavorable environmental conditions
in a dormant stage, and providing stored food for the embryo until it can obtain its own food.
A fruit is a matured ovary and its major function is effective dispersal of the seed or seeds contained
within it. Another of the important reasons for the success of the angiosperms is their great diversity
of fruit types and strategies for dispersal compared to the gymnosperms which lack true fruits and
which rely primarily on wind for seed dispersal.

Exercises
A. Ovule and Seed Development
As noted in the preceding chapter dealing with angiosperms and their flowers, the base of a pistil is
called the ovary and it contains one or more ovules. Pollen grains germinate on the stigma of the
pistil following pollination, and the pollen tubes grow through the style and the micropyles (opening) of
the ovules. One pollen grain and pollen tube is required for each ovule destined to become a seed.
Two sperm enter the megagametophyte (or embryo sac) from the pollen tube. One sperm fuses with
the polar nuclei to initiate formation of the endosperm and the other sperm unites with the egg
nucleus to form the diploid zygote: the first cell of the new sporophyte plant.

1. Young Embryo Stage

Most parts of the ovule(s) enlarge over the next several days. The primary endosperm nucleus
divides repeatedly while a supply of stored food contributed by the parent plant is accumulated.
Meanwhile the one-celled zygote divides many times forming a young embryo seedling. Examine a
slide of a young seed of shepherds purse (Capsella) on demonstration and note that the embryo is
confined to the portion of the ovule near the micropyle. It consists of a narrow row of cells called the
suspensor, plus a wider embryonic root-stem axis. A lobe of tissue projects forward on each side of
the axis. These two lobes are young cotyledons. The remainder of the interior of the young seed is
only sparsely filled with endosperm.

2. Mature Embryo Stage

After a few more days the seed matures. The integuments harden to become the seed coat, the
endosperm becomes more dense containing more stored food, and in many species the cotyledons
begin to absorb the food from the endosperm. Examine a slide of a mature seed on demonstration
and find the parts of the seed. The portion of the embryo's seed-root axis below the cotyledons
consists of hypocotyl and the radicle; the portion above the cotyledons attachment is scarcely
developed at this stage but will eventually consist of the epicotyl (plumule). Note the suspensor
cells between the radicle and the seed coat which pushed the developing embryo up into the
endosperm. Note also the funiculus which is the connecting stalk between the ovule (or seed) and
the wall of the ovary (or fruit) through which food is passed to the ovule. Label Figure 16 -150.

Figure 16-150. Dicot Seed Development. a. Early Growth of Embryo. b. Mature Embryo.

B. Mature Seeds
1. Legume seeds
Examine a pea or a garden bean. Note the elliptical scar, called the hilum, which marks the former
point of attachment of the funiculus. A very small opening, the micropyle, occurs near it. It represents
the opening in the ovule through which the pollen tube had entered, and it will admit water as an initial
step in germination. Remove the seed coat. They have digested and absorbed the stored food from
the endosperm. Two cotyledons are found characteristically in dicot seeds. Locate the hypocotyl and
epicotyl portions of the embryo axis. The embryonic root region below the hypocotyl is the radicle.
The area above the cotyledon that bears tiny leaves may be called the plumule. Label Figure 16
-151.

Figure 16-152. Corn Fruit and Seed. a. External View.

b. Cross Section.

Figure 16-151. Bean. a. Fruit. b. External View of Seed. c. Cross Section of Seed.

2. Grain Seeds
Examine corn kernels and make a longitudinal cut through the flat side of one. Add iodine solution
(IKI) to stain the tissue. The majority of the interior of the seed consists of endosperm with the
embryo at the base and along one side of the seed. The largest part of the embryo is the scutellum,
which may be interpreted as the single cotyledon of this monocot seed. The embryo axis is offset to
the side, with the radicle surrounded by a conical sheath called a coleorhiza and the epicotyl by a
conical sheath called the coleoptile. Label Figure 16 -152. Compare your sectioned kernel with the
prepared slide on demonstration.

3. Castor Bean

4
Examine the castor bean seed and make a longitudinal cut if sectioned seeds are not provided. This
seed also contains considerable endosperm. The embryo lies near the middle of the seed and
consists of a short root-stem axis plus two large but very thin cotyledons. The pulpy knot at the
bottom of the seed is the caruncle. It aids in imbibing water prior to seed germination. Label Figure
16 -153.

Figure 16-153. Castor Bean. a. Fruit. b. Seed, External View. c and d. Seed Cross Sections.

C. Seed Diversity
Seeds vary greatly in size and shape. The tiny, almost microscopic, seeds of orchids lack endosperm
and are adapted for being carried by wind currents among tree branches where the majority of
orchids grow. The largest seeds are those of certain species of coconut which weigh up to 40 pounds
and are adapted to withstand long journeys on ocean currents and contain sufficient stored food to
establish a large seedling on storm-washed beaches. Another remarkable seed type is found among
mangroves. Mangroves represent several different groups of shrubs which grow at the ocean shore
with branches extending over the water. The seedling never becomes dormant, but grows through
the seed and fruit coat. The whole unit drops into the water and drifts with the currents; when it is
stranded at low tide the root takes hold quickly to anchor it before the high tide returns.
Seeds which are dispersed separately, leaving the fruit behind, tend to be lightweight, winged, or to
have hairs (such as in milkweed). Those that stay within the fruit for dispersal are more variable and
their shape is usually determined by the shape of the fruit and the manner in which the seeds are
packed together.

II. Seed Germination

Introduction
Nearly all seeds become dormant as the seeds and fruits approach maturity. In dormancy
physiological activities are greatly reduced, growth ceases, and the seed is substantially dehydrated.
Dormancy lasts through dispersal and until the seed germinates or loses its viability (dies). Seeds
vary greatly in their period of dormancy and viability, from a few days, to a century or more, depending
in part on the nature of the seed and in part on environmental conditions.

Exercises
A. Seed Dormancy and Conditions for Germination
The embryo within a dormant seed is alive (viable) but in a kind of suspended animation; it does not
grow and the rate of metabolic activities is very slow. A dormant seed may remain viable and still not
germinate and grow because of either external or internal conditions. A seed that is dormant due to
external factors will germinate if simply given the proper moisture, aeration, and growing temperature
required by the species. As an example, a garden pea seed is dormant in the hard, dry state, but
once it is watered in a well-aerated soil at a suitable temperature, it will germinate and grow.
On the other hand, the dormant seeds of some species will not germinate even if given suitable
external conditions; in these seeds internal factors prevent germination and growth. There are
several kinds of seed dormancy due to internal factors. In the simplest case, an embryo may be
immature; in time the embryo will mature and the seed will germinate (when the external factors of
water, air, and temperature are suitable, of course). Another easy example to understand is the
presence of a hard seed coat that prevents the entry of water and air into the seed. Dormancy in
these seeds is broken when the seed coat barrier is broken (scarification). In nature, the seed coat
may be broken by enzymes produced by soil microorganisms, by passing through the digestive tract
of an animal, by alternating low and high temperatures, or by the abrasive effects of being water- or
wind-borne across rocks. Fire is yet another natural scarifying agent. In the laboratory we can scarify
seeds with hard coats by damaging the seed coat with a knife, razor blade, file, by using sandpaper,
or by soaking the seeds in hot water or acid for short periods of time.
In other seeds, dormancy is due to the internal concentrations of natural chemical inhibitors. In some
of these seeds, the inhibitors may be present in the seed coat; rainwater is required to leach the
inhibitor from the seed coat. The amount of water required would depend on the water solubility of
the chemical and its concentration in the seed coat. In other cases, chemical inhibitors are present in
the embryonic tissues and are not leached out by water. Some of these seeds require a cold period
(the winter) before they will germinate when favorable external conditions return in the spring. Data
obtained with some seeds that require a cold period for germination (stratification) indicate that after

6
the cold requirement is met, the embryo is capable of producing enzymes which synthesize
germination stimulators (e.g. gibberelliins) and/or break down inhibitors. The most widespread,
naturally occurring chemical inhibitor in flowering plants is abscisic acid.
The length of time that a seed can remain viable and dormant varies greatly with the species and the
external conditions. The typical life span of wild seeds ranges from 10 to 50 years. However, the
percentage of germination may decrease dramatically with time. For example, Kentucky bluegrass
seeds have an initial germination rate of 91% (91 out of 100 seeds will germinate when the external
and internal conditions are suitable). After 30 years, some of these seeds are still viable, but only 1
out of every 100 seeds will germinate. Examples at the extremes may also be found. After only one
year, the germination rate of wild oat seeds drops from 70% to 9%. At the other extreme, arctic lupine
seeds frozen and deeply buried in lemming burrows in the Yukon have been dated as 10,000 years
old and have germinated when given proper external conditions.
In general, you may think of germination as taking place in four stages: (1) the seed imbibes (takes
up) water; the water hydrates the tissues and biomolecules (proteins, carbohydrates) of the embryo;
(2) enzymes are formed or activated; metabolic activity increases; (3) some embryo-manufactured
enzymes catalyze the breakdown of the endosperm into smaller food molecules; the embryo uses this
food for growth, and (4) the growing (elongating) radicle or embryonic root emerges from the seed
coat. Now observe the variety of seeds on demonstration.
Why do different species have different types of dormancy?

For the examples on demonstration, is dormancy controlled by environment, the plant itself, or both?

Examples of Seed Dormancy on Exhibit in the Lab:

(1) Seeds that germinate before dispersal from the parent plant (vivipary). The example here is the
mangrove plant.
(2) Dormancy due to a hard seed coat that requires mechanical or chemical scarification.
Examples are geranium, morning glory, and Mimosa (sensitive plant) seeds.
(3)

Dormancy due to an immature embryo. The example here is black ash (Fraxinus nigra).

(4) Dormancy due to chemical inhibitors in the seed coat or in the fruit tissue and juices that must
be leached out before germination will occur. Examples here are apple and tomato seeds.
(5)

Seeds that are inhibited from germinating by light. Examples are Phacelia and Amaranthus.

(6) Seeds that require light for germination. Examples are certain varieties of lettuce, celery and
tobacco.
(7) Seeds that require stratification (a cold, moist treatment) for germination. The examples on
display are Sitka spruce (Picea sitchensis) and black ash. Dormancy in black ash involves both an
immature embryo and a requirement for a stratification period of about 18 weeks.

Figure 16-154. Hypogeal Seed Germination, Dicot (Pea). a. Seed Cross Section. b. Seedling.

B. Hypogeal Germination
In the hypogeal pattern of germination and seedling emergence the cotyledon(s) remain(s) below
ground and within the seed coat, digesting, absorbing, and moving the stored food from the
endosperm to the growing regions. Typically, both the hypocotyl and epicotyl regions of the embryo
axis are fairly well formed at the time of germination. The radicle grows downward forming the root
and the epicotyl grows upward forming the stem with its leaves.

1. Dicot Seed and Seedling.

Examine the germinating pea seeds and young seedlings. Locate the cotyledons, the radicle and the
epicotyl regions of the seed and note their positions and development in the seedling. Label Figure
16 -154.

2. Monocot Seed and Seedling.

Figure 16-155. Hypogeal Seed Germination, Monocot (Corn). a. Seed Cross Section. b. Seedling.

Examine the corn seed and seedling. Corn (maize) and other grass seedlings are more complex than
most other monocots. The single cotyledon, the scutellum, remains within the kernel. The root
system not only has a small root developed from the radicle, but has additional roots, seminal roots,
which originate from a portion of the embryo axis within the seed. Note also that the embryonic
leaves of the epicotyl are protected in their passage through the soil by tubular sheath, the coleoptile,
which ceases elongating and separates at the apex shortly after it comes above the soil and is
exposed to light. Note the adventitious roots on the young corn seedling. Label .

C. Epigeal Germination
In the epigeal pattern of germination and seedling emergence, the cotyledon(s) is (are) carried above
ground by the elongating hypocotyl, which usually develops a hook or curve shortly after emergence
from the seed. The radicle extends downward and becomes the primary root while the hypocotyl
portion pushes its way through the soil pulling the cotyledon(s) above ground as it grows. Once
above ground, the hypocotyl straightens and the cotyledon(s) turn(s) green to begin carrying on
photosynthesis. Since the cotyledon(s) usually leave(s) the seed coat behind in the soil, or shed(s) it
before expanding, the cotyledon(s) must complete its (their) role of digesting and absorbing the stored
food from the endosperm by this stage. In many cases the stored food has been absorbed before
germination and the cotyledon(s) is (are) the largest part of the mature seed. Because of its role of
elevating the cotyledon(s) the hypocotyl is better developed than the epicotyl in early seedling stages.

Figure 16-156. Epigeal Seed Germination, Dicot (Bean). a. Seed Cross Section. b. Seedling.

1. Dicot Seed and Seedling.

Examine the germinating bean seed and seedling. Locate the cotyledons, the hypocotyl, radicle, and
epicotyl regions of the seed and note their positions and relative sizes in the seedling. Note the
differences between the cotyledons and the true leaves of the seedling. Label Figure 16 -156.

2. Monocot Seed and Seedling.

Examine the onion seedling. Locate the cotyledon and compare the seedling with the bean and with
corn.

10

III. Fruits
Introduction
The release of growth-regulating chemicals accompanying pollination and the development of ovules
into seeds stimulates the ovary of the flower to develop into a fruit. A true fruit is a matured ovary,
which is usually modified in some way for a role in dispersing the seed or seeds. Dispersal can be
accomplished in a variety of ways. Fruits are grouped in a classification system based in part on the
method of seed dispersal. Examine the examples on display and note the various fruit types.

A. Simple Fruits ( True Fruits).

Simple fruits of this type are developed from a single ovary that may be composed of one to several
carpels. The following are all simple fruits, and may be further subdivided into fleshy and dry fruits.

1. Fleshy Fruits.
The ovary wall of fleshy fruits enlarges with the accumulation of water, sugar, and usually more
organic acids. None of this stored food benefits the seedling. Instead, it is placed there as a "reward"
for an animal for transporting the fruit with its seed or seeds. The animal often eats the fruit down to
the seed, discarding the seeds, or it swallows the whole fruit, but the seeds are later excreted
undigested. The following are types of fleshy fruits:
(1) A drupe contains a single seed surrounded and protected by a bony or tough endocarp (inner
ovary wall) which discourages an animal from eating beyond this layer, or protects the seed from
being digested in the case of small drupes which are usually swallowed whole.
(2) A berry is a fleshy fruit with few to many seeds.
(3) A pepo is a berry with a tough, waxy, or leathery rind.
(4) A hesperidium is a berry with a separable leathery rind which contains oil glands.
(5) A pome is in part of "complex" or "false fruit" (see below) because the fleshy outer region is
composed at least in part of fleshy enlargement of the receptacle. The endocarp is papery or
bony and forms the "core."

2. Dry fruits.
Dry fruits usually rely on passive methods of dispersal such as being blown by wind, carried by
water, or by clinging to the fur of a mammal. Usually all of the stored food is placed in the seed for
use by the seedling. The following are dry fruits:
Single-seeded dry fruits are dispersed as a whole unit with the fruit coat intact surrounding the
seed.
(1) A nut is an exception to a basic characteristic of most dry fruits in that it contains stored food
(usually as starch, oil, or protein) in the enlarged wall as an inducement for a rodent or other
animal to disperse it. Rodents bury most of the nuts for later use as food. Since they fail to return
to many of these nuts, a portion of each year's nut crop is left to grow into a new plant.
(2) An achene is an unmodified dry fruit with a thin fruit coat.
(3) A samara is an achene with a wing-like extension of the fruit coat which aids in wind or water
dispersal.
(4) A caryopsis is an achene with the fruit coat and seed coat fused together totally.
Multi-seeded dry fruits almost always remain attached to the parent plant and split open at maturity.
The individual seeds are the dispersal units. If the whole fruit were to stay together during dispersal
many seeds would germinate in one spot and compete against each other.

11
(1) A follicle is derived from an ovary composed of only one carpel which splits open on its
suture.
(2) A legume is also derived from one carpel, but it splits along the suture and also along the
midrib. It is sometimes fleshy, especially when young.
(3) A capsule is derived from a compound pistil. It splits open on two or more lines.

B. Complex Fruits (False Fruits).

Complex fruits involve more than one ovary. The following are complex or false fruits (the term
"false fruit" is somewhat misleading, but it is commonly used in botany texts).

1. Aggregate Fruit
Aggregate fruits result from the fusion of several ovaries of a single flower. Examine the slide of the
longitudinal section through a raspberry.

2. Multiple Fruit
Multiple fruits result from fusion of parts of several flowers..

3. Accessory Fruit
Accessory fruits consist substantially of an enlarged receptacle or some other plant part in which the
true fruits, usually achenes, are embedded.
Fill in Table 16 -21.
Table 16-21. Fruit Types on Demonstration

Fruit Type
drupe
berry
pepo
hesperidium
pome
nut
achene
samara
caryopsis
follicle
legume
capsule
accessory
aggregate
multiple

Species

# of Carpels

Dispersal Mechanism

12

KEY WORDS
seed
fruit
polar nuclei (pl.)
endosperm
zygote
embryo
suspensor
cotyledon
hypocotyl
radicle
epicotyl
plumule
hilum
scutellum
coleorhiza
coleoptile
caruncle
dormant (adjective)
dormancy (noun)
viability
scarification
imbibe
stratification
hypogeal germination
epigeal germination
simple fruit (true fruit)
fleshy fruit
drupe
endocarp
berry
pepo
hesperidium
pome

13

dry fruit
nut
achene
samara
caryopsis
follicle
legume
capsule
complex fruit (false fruit)
aggregate fruit
multiple fruit
accessory fruit

Laboratory 18
Cell Types
Herbaceous Stems
Woody Stems
During the previous two labs, weve studied what could be called the reproductive anatomy of
flowering plants: flowers, fruits, and seeds. In this lab and the two that follow, well deal with
vegetative anatomy, the structure of the non-reproductive parts of the plant. Vascular plants are
generally considered to have three vegetative organs: roots, stems and leaves. The above ground
stems and leaves are collectively referred to as the shoot. Each organ consists of interacting groups
of cells known as tissues. Finally, the cells themselves are classified into a number of cell types.
All plant cells originate from unspecialized cells known as initials. With few exceptions, once a plant
cell has developed a particular form and function, it loses the ability to divide. That means that growth
of the plant depends on the continuing existence of initials and their continued division. Instead of
being scattered generally throughout the tissues of a plant, initials are typically confined to localized
areas called meristems.
We expect all vascular plants to have an apical meristem at the tip of every stem and root. (In
addition, there is an inactive apical meristem at the heart of every bud, which is activated if the bud
breaks dormancy and begins to grow into a branch.) Expansion and specialization of the cells
produced by apical meristems produces growth in length, or primary growth. The relatively soft,
young tissues that result from primary growth are referred to as herbaceous. Some stems such as
those of grasses have regions of primary growth at the base of individual stem sections, intercalary
meristems or basal meristems.
Many vascular plant stems and roots also have meristems specialized to produce growth in thickness.
These meristems consist of a hollow cylinder of initials some distance beneath the surface called a
lateral meristem or cambium. The thickening of stems and roots produced by the expansion and
specialization of the cells produce by a cambium is referred to as secondary growth.

Figure 17-157. Basic Cell Types, Cross Section. a. Parenchyma. b. Schlerenchyma.

c. Collenchyma.

I. Cell Types
The cells that develop from initials have a wide range of form and function, but most of them are
classified in only a few, somewhat variable, cell types.

A. Basic Cell Types

Observe and learn to differentiate between the three basic cell types: (1) Parenchyma are physically
unspecialized cells with a thin primary wall, usually stained green, blue or black in our material.
(2) Sclerenchyma are specialized cells with a moderately to extremely thick secondary wall.
Sclerenchyma usually function in rigid support and are typically stained red in our material. (3)
Collenchyma are relatively infrequently occurring cells with unevenly thickened primary walls.
Collenchyma function in flexible support, and are often stained green or black in our material. See .

B. Special Cell Types (On demonstration).

Uniquely specialized cells include sieve tube elements, vessel elements, tracheids, and fibers, all of
which develop from the same kinds of initials. Sieve tube elements are large and thin walled (and
usually green stained in our material). They appear to be empty, but In fact, are filled with cytoplasm
and serve as the major food conducting cells of the plant, though vessel elements may be pressed
into service when the sap rises in the spring. Sieve tube elements are arranged end to end in strands
(the sieve tubes themselves), with the walls between consecutive sieve tube elements forming a
porous sieve plate traversed by strands of cytoplasm (modified plasmodesmata). During
development, small companion cells divide off from the sieve tube elements, but retain cytoplasmic
connections to the parent cells. The companion cells essentially control the function of the cells to
which they are connected, for they retain the functional nucleus that the sieve tube elements lack.
Tracheids are non-living water conducting and supporting cells with thick secondary walls. Tracheids
are long and narrow with tapered ends that overlap. Instead of actual openings between overlapping
cells, there are paired, circular thin places in the adjacent secondary walls: bordered pits. Water
seeps from tracheid to tracheid through the bordered pits. Vessel elements are large non-living cells
with thick secondary walls (red stained in our material). They tend to be relatively short and have
actual openings (perforation plates) between consecutive elements so they comprise continuous
tubular vessels. Vessels conduct water more rapidly than aggregations of tracheids, but perhaps not
to so great a height. Their thick walls allow them to also contribute to support of the plant. Water
movement through tracheids is not as rapid as that through vessels, but it is sufficiently effective to
serve the tallest trees in the world. Fibers are essentially exaggerated tracheids (longer, thinner, nonliving, and tapered). They have extremely thick secondary cell walls with a tiny central opening, so
their function is support only.

Figure 17-158. Specialized Cell Types. a. Sieve Tube Elements (with Companion Cells). b. Vessel
Elements. c. Tracheids. d. Fibers.

II. Stems
A. Longitudinal Section
Observe a prepared slide of a longitudinal section of a Coleus shoot apex with a compound
microscope. Notice the change in cell size from the apical meristem down. Still further from the tip
the cells would be notably larger and differentiated into particular cell types. Identify the following
structures and label them in : apical meristem, leaf primordium, strand of procambium (the
developing vascular tissue), and axillary bud primordium. Primordium refers to a region of
unspecialized cells with a particular developmental future. An axil is the place just above where a leaf

a.
b.

Figure 17-159. Coleus Shoot Apex Longitudinal Section. a. General. b. Detail.

joins a stem.

b.

a.

d.

c.

Figure 17-160. Monocot Stem Cross Sections. a. Corn (Zea). b. Detail of Corn
Vascular Bundle. c. Wheat (Triticum). d. Detail of Wheat Vascular Bundle.

B. Monocot Stem Cross Section

1. Prepared Slides.
Observe the corn (Zea) stem cross section with an eye to comparing it to an herbaceous dicot stem
studied above. Note the arrangement of vascular bundles. With the help of the text below and the
instructor identify and label the following structures in a and b: epidermis, ground tissue, phloem
(sieve tube elements and companion cells), metaxylem, protoxylem and xylem parenchyma. The first
xylem to mature, while the stem is growing in length, is protoxylem. That which matures later, after
the stem is done elongating, is metaxylem. Differences in cell maturing may seem picky, but they
provide important clues for evolutionary relationships. The vascular bundle of the monocot looks like
a face. The forehead is composed of green stained sieve tube elements and companion cells. The
nose is a protoxylem vessel and the mouth is an air space left by pulling apart of the earliest formed
protoxylem. The eyes are metaxylem vessels. Xylem parenchyma are interspersed between the
protoxylem- and metaxylem.
Observe a prepared slide of a wheat (Triticum) stem cross section. In c and d identify and label the
same structures as you did in the corn sections.
What is the conspicuous difference between the corn and wheat stem cross sections?

2. Freehand Section
Make a thin free-hand section of a fresh monocot stem and stain with neutral red. You should be able
to see many of the same tissues that you saw on the prepared slide.

C. Woody Monocot Stem (Demonstrations)

1. Dracaena Stem Cross Section
Although most monocots have no secondary growth, a few do produce secondary tissue, in this case
from an atypical vascular cambium. The cambium itself is composed of a ring of thin-walled greenstained cells. Products of the cambium occur to the inside and consist of complete vascular bundles
composed of phloem (green stained) and xylem vessels (red stained). The xylem is difficult to
distinguish from the surrounding sclerified parenchyma that are also red-stained. Note that this
species also produces a well-developed periderm.

2. Palm and Bamboo Stems

Although palm stems may grow to be quite wide, their growth is considered to be primary only. No
vascular cambium is involved. The increase in width occurs in internodes just below the short apex
before the internodes elongate. Palms have no true wood (secondary xylem) but have enough
sclerified tissue to be hard and called "woody."

D. Herbaceous Dicot Stem Cross Section

1. Prepared Slide
Observe a prepared slide of a stem cross section of alfalfa (Medicago) starting with low power. With

a.

b.

Figure 17-161. Alfalfa (Medicago) Cross Section. a. Whole Section. b. Detail.

Note: Alfalfa is unusual in being square stemmed.

the help of the text below and your instructor, identify the epidermis, cortex, vascular bundles, and
pith. Label these tissues in .

The epidermis is the outermost single layer of cells. The cell features of the epidermis are unique to
the tissue. The cells bear an outer waxy layer (cuticle), and often have sinuous margins, much like
puzzle pieces. The end result is a tissue that forms a protective, waterproof seal except where it is
interrupted by closable pores called stomates. Cortex is the tissue just beneath the epidermis and
extends inward to the vascular bundles. It typically consists primarily of parenchyma, often
photosynthetic and containing chloroplasts. The innermost layer of cortex is sometimes composed of
large empty cells comprising a "starch sheath." Cortex is also the most likely place to find
collenchyma. If present, collenchyma add structural strength to the stem.
The vascular bundles are arranged in a ring between the cortex and the central tissue, pith. The
bundles contain the food and water conducting tissues, phloem and xylem, and also provide support.
Pith occupies the center of the stem and is composed of large parenchyma.
Increase magnification and study the vascular bundles in detail. Identify and label the following
tissues: bundle cap (may be absent), phloem, procambium (or vascular cambium in older stems),
xylem vessels and xylem parenchyma. The bundle cap is composed of sclerenchyma cells that
function mainly in support. Phloem is composed of sieve tube members and companion cells and
transports the products of photosynthesis (food). The vascular cambium is a single layer of
undifferentiated cells that separates xylem from phloem in each bundle, and later extends between
bundles forming a complete cylinder that runs the length of the stem. The initials that comprise the
cambium produce secondary growth that causes the stem to enlarge in diameter.

2. Freehand Section
Make several thin free-hand cross sections of a fresh herbaceous dicot stem. Your goal is to make
them literally only one cell thick (thin enough to read through). Stain these with neutral red, mount on
a clean slide in a drop of water and add a coverslip. Observe under the microscope. You should be
able to identify most of the tissues visible on the prepared slides.

D. Woody Stem Cross Section

Secondary xylem (wood!) is produced from the vascular cambium towards the center and secondary
phloem towards the outside. Xylem vessel elements are large sclerified cells that transport water.
The vessels occur in radial lines separated by small xylem parenchyma. Once secondary growth
begins, the stem is on its way to becoming woody instead of herbaceous.

1. Dicot
Observe the prepared slide of basswood (Tilia) stem cross section. With the help of the description
below and the instructor identify and label , identifying the following tissues (generally arranged from
center to outer edge, cell types are in brackets): pith [parenchyma], annual rings of secondary xylem
[vessel elements, tracheids and parenchyma], vascular ray, vascular cambium [initials, not yet
differentiated], phloem [fibers, sieve tube elements, companion cells, dilated phloem ray
[parenchyma], cortex [parenchyma, difficult to resolve in many woody stems because of cell crushing
and rearrangement], cork cambium [initials again] and cork [special cells, unique to this tissue].
Within each xylem ring, look for the large celled spring wood and lesser amount of small celled
summer wood that make each ring detectable. Which of the annual rings is oldest?

Look for rays traversing the xylem, vascular cambium, and phloem from central pith to cortex. How
many cells wide are the xylem rays?

What function might they serve?

What would these rays look like in three dimensions?

Can you see where they originate in the innermost annual ring? This beginning point is the boundary
between primary and secondary xylem. Wood is secondary xylem.
Bark, on the other hand is composed of several tissues, all those outside the vascular cambium: The
vascular cambium itself consists of inconspicuous cells, but is easy to locate because it marks the
pattern change from annual rings of wood to the phloem of the inner bark. Phloem is composed of
several kinds of cells. The cells with very thick, pink stained secondary walls are fibers. They add
strength to the inner bark. Interspersed with the fibers are patches of thin walled, sieve tube elements
and companion cells. Both stain blue or green. The sieve tube elements are large diameter, clearcentered, food conducting cells. Each has one or more small adjacent companion cells, which
contains the nucleus that is involved in sieve tube element functioning. Also conspicuous in
basswood phloem are wedges of large parenchyma cells known as dilated phloem rays. Somewhat
less conspicuous are the abundance of non-dilated phloem rays.

Figure 17-162. Woody Dicot Stem.

What would happen to the phloem as stem diameter growth continued if these cells did not develop?
(In many species they dont.)

9
The cortex is composed of parenchyma cells just as in the herbaceous stems, but new tissues have
developed that form the outer bark, notably a cork cambium and the cork tissue that it produces.
Cork takes over the function of the epidermis, which, trapped on the outside of an expanding cylinder,
is usually quickly sloughed off. Cork and cork cambium are collectively known as periderm.
Periderm can be distinguished from cortex because the cork cells are arranged in neat radial rows.
A slide showing periderm from a somewhat older stem is on demonstration. Be sure you can identify
the periderm and its components. The cork cambium (phellogen) is a band of narrow, thin-walled
blue or green staining cells. It produces cork (phellem), larger, brown- or red-stained cells to the
outside and sometimes a limited amount of phelloderm (larger green or blue cells) to the inside.
How can you distinguish between periderm and cortex?

What function does the periderm serve?

Differentiate between the precise meaning of cork and bark:

What part of the bark is living and what is dead?

2. Conifer
Observe the cross section of a pine (Pinus) stem in your slide set. Despite gymnosperms not being
flowering plants, most structure is the same as a woody dicot stem. The minor differences include
(1) The xylem has neither fibers nor vessels, only tracheids. (2) The phloem has nucleated sieve
cells without companion cells, considered a primitive condition. (3) The phloem also lacks fibers.
(4) Non-cellular tubes called resin ducts are scattered through the xylem, phloem and cortex. A last
difference is that essentially all conifers are essentially woody and not herbaceous. Find the
differences mentioned above, and reinforce your other knowledge of woody stem structure by labeling
.

10

b.

a.

11

c.

Figure 17-163. Conifer Stem. a. Entire Cross Section. b. Detail of Bark. c. Detail of Wood.

12

KEY WORDS
organ
shoot
tissue
cell type
initial
meristem
apical meristem
primary growth
herbaceous
lateral meristem
cambium
secondary growth
parenchyma
collenchyma
sclerenchyma
sieve tube element
sieve tube
sieve plate
companion cell
tracheid
bordered pit
vessel element
vessel
fiber
shoot apex
primordium
procambium
epidermis
cortex
starch sheath
pith
vascular bundle
phloem

13

bundle cap
xylem
protoxylem
metaxylem
vascular cambium
wood
annual ring
spring wood
summer wood
xylem ray
bark
dilated phloem ray
non-dilated phloem ray
periderm
cork cambium (phelloderm)
cork (phellem)
phelloderm
sieve cell
resin duct

18-i

Figure 18-1. Woody Dicot Twig, External Anatomy. a. General View. b. Detail of Leaf Scar and
Axillary Bud.

Laboratory 19
Woody Stems
Herbaceous Roots
Modified Roots and Stems
Introduction
In the last laboratory you studied stems. When the vascular cambium in dicots and gymnosperms
stems starts functioning, they develop the woody structure you will study more intensively today. Also
covered in todays lab are roots, and finally stems and roots that have become modified to take on
unusual functions.

I. Woody Stems, continued

Exercises
A. Winter Twigs
Woody plants in the winter condition can usually be identified to species by external morphological
characteristics of their twigs. Observe the twigs on demonstration, determine the leaf arrangement
(opposite, alternate, or whorled) and identify the following structures: terminal bud at the tip of
each main stem and branch, axillary or lateral buds on the sides of the branch, nodes (point of
attachment of leaves or scales to the stem), internodes (stem segment between two successive
nodes), lenticels (breaks in the bark that function in gas exchange), leaf scars (left where a former
leaf stalk detached), bundle scars (remains of vascular bundles in leaf scars), and terminal bud
scale scars (remains of previous year's terminal bud).
In most woody plants from seasonal climates, a terminal bud forms towards the end of every growing
season. Previous rings of terminal bud scale scars can therefore be used to determine the age of
woody seedlings or determine past twig growth rates. Use lab materials and your instructors help to
identify the structures listed. Label Figure 18-1.

B. Buds
1. Conifer Bud (Vegetative)
Using a sharp razor blade carefully remove the bud scales from an axillary or terminal bud of spruce
or fir. (Your instructor will demonstrate.) Observe with a dissecting microscope. The larger central
green dome is the shoot apex and the many smaller green surrounding bumps are the leaf primordia,
which will become needles when the bud grows into a twig. .

2. Dicot Buds (Vegetative or Mixed)

Carefully cut a lilac (Syringa) bud in half lengthwise. If it consists only of leaf-like parts, it is a
vegetative bud. If there are additional structures reminiscent of cauliflower or broccoli tips, it is a
mixed bud.

3. Flower Buds
The floral organs at their simplest are each considered to be modified leaves. Consequently, a
flower bud is somewhat analogous to a vegetative bud except that the leaf primordia have
undergone radical modification.

C. Wood Sections
Observe prepared slides of pine (Pinus), oak (Quercus) and basswood (Tilia) woods. These
represent the three major wood types; non-porous, ring-porous and diffuse-porous, respectively.
Gymnosperms (softwoods) are usually non-porous (lack vessels) whereas dicots (hardwoods) are
either ring porous or diffuse porous (have vessels in one pattern or the other). Note that the actual
hardness of the wood has no necessary bearing on whether a species is called hardwood or
softwood. To develop your understanding of the three dimensional structure of wood, try to identify all
three planes of sectioning: transverse or cross, tangential, and radial. Keys to distinguishing
among them include the pattern of the annual rings and rays, and the appearance of the vessel
elements, tracheids, and fibers. The ribbon-like rays appear as strips running across parts of a radial
section, but are cut at right angles by a tangential section and appear as narrow stacks of cells. In
Figure 18-2, Figure 18-3, and Figure 18-4 label the wood type, plane of section and the following
structures: rays, vessels, tracheids, and resin ducts. In oak wood, note the contrast between giant
vessel elements and normal sized ones, and the presence of giant rays and normal rays. From
the appearance of the thinner rays, did the big vessels elements expand before or after the
development of the rays?

a.

b.

Figure 18-2. Pine Wood Sections.

c.

a.

b.

Figure 18-3. Basswood Wood Sections.

c.

Figure 18-4. Oak Wood

Sections.

a.

b.

d.

c.

D. Wood Characteristics (Demonstrations)

1. Macroscopic Patterns
Identify all three planes of cuts (cross, tangential and radial) on woody stems and wood blocks. On
some wood you can see the xylem vessels ("pores") with the unaided eye. Identify each wood as ring
porous, nonporous or diffuse porous.

2. Tree Trunk
Study the cross cut tree trunk in some detail. Several locations in the wood are dated. How does one
determine the total age of such a tree or the year in which it was cut.

What clues do the annual rings give about weather patterns during the time when the tree was alive?

Locate the vascular cambium. Where are the epidermis and cortex?

Identify heartwood and sapwood. What do you think is involved in the conversion of heartwood to
sapwood?

6
Which of the rings still function in water conduction?

3. Knots
Examine the knots that are on display. What does a knot represent?

Differentiate between tight knots and loose knots. What is the explanation for the difference?

4. Lenticel
Look at the longitudinal section of a lenticel on microscopic display. Relate what you see to the
lenticels you saw on the woody twigs in part A above (Figure 18-1).

II. Herbaceous Roots

Roots contain most of the same tissues as stems, but with distinctive differences in arrangement and
appearance. As you examine roots, try to answer these questions:
How do stems of dicots differ from dicot roots?

How do stems of monocots differ from monocot roots?

Can you recognize at a glance dicot stems, monocot stems, dicot roots, and monocot roots?

7
Figure 18-5. Diagram of Root Longitudinal Section.

A. Longitudinal Section
Observe a prepared slide of an onion (Allium) root tip longitudinal section. As in the Coleus stem,
notice the change in cell size back from the apical meristem, but in this case both upwards towards
the older parts of the plant and downwards into the root cap. What do you think is the function of the
root cap?

Identify and label the root cap and root apical meristem (Figure 18-5). Also look for root hairs (not
shown in Figure 18-5 because they develop further from the tip).

B. Dicot Root Cross Section

Observe the prepared slide of Ranunculus (buttercup) root cross sections. The two sections differ in
age. Use the older one, in which many of the central cells are thick-walled and strongly stained, to
locate and identify tissues. Find the following tissues and label them on the mature root section in
Figure 18-6: cortex, endodermis, pericycle, phloem, procambium and xylem. The red-stained, starshaped tissue in the center of the root is xylem, and there is no pith. Between the xylem arms are

a.

patches of green-stained phloem. All the tissues inside the endodermis are collectively known as the
stele. Between the phloem and the central xylem cells are several rows of green-stained
procambium cells which may later form part of the vascular cambium. An erratically developed tissue,
pericycle, occurs just outside the phloem. It is most likely to be distinguishable at the ends of the
xylem arms, also called the poles of the xylem. Just outside the pericycle is the well-defined
innermost layer of the cortex, the endodermis. Some of the endodermal cells may be sclerified
(have thickened, red-stained cell walls). Others are green and thin-walled. Each endodermal cell has
a band of wax, the Casparian Strip, permeating the wall between it and the adjacent cell. It is
stained red in our material. See if you can use the Casparian Strip to detect the endodermis through
its complete circle in the root section. The Casparian Strip forces all the water that enters the stele to
pass through a living cell, thereby exerting control over what solutes are able to ascend to the rest of
the plant.. The cortex outside the endodermis consists of large parenchyma, typically functioning in
food storage. Note what a large part of the total cross section of the young root is cortex. As in
herbaceous stems, the outermost layer of cells is the epidermis. Unlike herbaceous stems, the root
epidermis is highly permeable to water.
Now look at the section in which many cells have not yet matured.
What are the differences between these two stages of development?

Note which cells have become sclerified in the older root. On which section is it easier to distinguish
between protoxylem and metaxylem?

b.

Figure 18-6. Buttercup (Ranunculus) Root. a. Cross Section. b. Detail of Stele.

10

C. Monocot Root Cross Section

Observe the prepared slide of a greenbrier (Smilax) root cross section (Figure 18-7). With the help of
wall charts, your text, the instructor, and your ingenuity, identify and label the following structures:
epidermis, cortex, endodermis, pericycle, phloem, (no procambium), xylem, and pith. Note especially
the differences between this root and the dicot root you just studied: (1) Monocot roots usually have a
pith; (2) monocot roots have many xylem arms; (how many were in the dicot you observed?); (3)

Figure 18-7. Greenbrier (Smilax) Root Cross Section.

11
monocot roots have no procambium between the xylem and phloem; (4) the endodermis is often
composed entirely of sclerenchyma in monocot roots; (5) in many monocot roots, the pericycle is
several cell layers thick.

12

D. Root Hairs
Place a germinated seedling of redtop (Agrostis) on a slide, add a drop of water and a coverslip.
Focus on the root in the region of the root hair zone. Note that each root hair is an extension of a
single epidermal cell. Root hairs very much increase the potential water and mineral absorbing
surface of a root. On this same slide also identify the root apical meristem and the root cap.

E. Branching of Roots
Stem branches develop from buds on the surface of a stem. Root branches, on the other hand, begin
their development well below the surface, usually from the pericycle on the perimeter of the stele. As
they grow out through the cortex, they have time to develop the root cap which will provide essential
protection when the root emerges and its tip is pushed through the soil. Observe the prepared slide
that shows a developing lateral root. Look closely for the exact tissue location of its origin. What is
the spatial relationship between the xylem of the existing root and the developing branch root?

Has the root cap formed yet on the young root tip?

Is this a monocot or dicot root? How can you tell?

F. Root Systems
Observe demonstrations of tap root and fibrous root systems. How do they differ?

Under what circumstances might a tap root system be more effective in getting water to a plant than a
fibrous root system?

13

III. Modified Roots and Stems

Introduction
So far, we have been dealing mostly with "typical" stems and roots. It is easy to form the impression
that such organs are about the same in all plants. Yet, if you consider the different demands of
contrasting environments, and the fact that vascular plants have been evolving for millions of years, it
is not surprising to learn that there is as much variation in the form of roots, stems, and leaves, as
there is among the flower types you've observed. This exercise concentrates on modifications that
are common to many species, "typical" modifications of "typical" structures.

Exercises
A. Modified Stems
Description of modified stems is confused by the fact that many terms are used differently by different
botanists. If you were to compare your text with those used by other lab sections, you would find
inconsistencies in stem terminology.

1. Corm
A corm is an underground stem that is fleshy, short, and thick, often to the point of being nearly
spherical. Two plants that produce corms are gladiolus and crocus.

2. Rhizomes and Stolons

A rhizome is an elongate horizontal underground stem. It functions in food storage, vegetative
reproduction, or both. Some botanists use rhizome and stolon interchangeably. Others restrict
rhizome to underground stems, and stolon to stems that trail along the surface of the ground, such as
strawberry runners. Cattails have under-water (and soil) rhizomes. Many grasses have underground
rhizomes. How can you distinguish a rhizome from a root?

3. Tuber
A tuber is a thickened portion of a rhizome. Good examples are Irish potatoes or Jerusalem
artichokes. From what you've learned about stem structure, how would you interpret the "eyes" of a
potato? How about the eyebrows?

14

B. Modified Roots
1. Haustorium
Observe the demonstration slide of dodder (Cuscuta), a non-photosynthetic parasitic angiosperm.
Note the cross-section of a stem from the host plant. The small structures penetrating the host are
the parasite's haustoria (modified roots, in this case). Note that the haustorium xylem has become
directly attached to the host xylem. The phloem of the host and parasite is also connected, but the
attachment is less obvious. Also observe the whole mounts. Be able to identify the parasite, the host,
and their parts.

2. Mycorrhizae
In many angiosperm and gymnosperm species a symbiotic relationship develops between the roots
and certain species of fungi. Both organisms benefit. This symbiotic association of fungi and roots is
called a mycorrhiza. The fungus hyphae may grow between the root cells (ectotrophic) or actually
penetrate them (endotrophic). On the demonstration slide identify the fungal hyphae within the
roots. Is this fungus endotrophic or ectotrophic?

What could be the consequence of planting pine trees (must have mycorrhizae to thrive) in prairie soil
where the symbiotic fungi do not occur? How can this problem be remedied?

3. Storage Roots
Many biennial and perennial plants, such as dandelion, radish and other root type vegetables,
accumulate food in their primary root systems. In some of these plants the enlarged portion is derived
partly from the stem and root. The upper portion of a carrot is derived from the stem. The dahlia and
sweet potato are derived from branch roots. When, during the year, would you expect storage roots
to reach their maximum food content?

When will they reach their minimum food content?

15

4. Adventitious Roots
Any plant part that arises at an unconventional place may be referred to as adventitious. A giant
grass like corn would blow over easily were it not for the prop roots that emerge from the sides of the
lower parts of the stem. Tropical hardwood trees sometimes put out adventitious roots high above the
ground to tap minerals accumulated in the debris developed by the eventual death of epiphytes (air
plants) which grow on their stems. Observe the adventitious roots that are on display.

KEY WORDS
opposite

root hair

alternate

stele

whorled

pericycle

terminal bud

xylem poles

axillary bud

endodermis

lateral bud

Casparian strip

node

tap root

internode

fibrous root

leaf scar

corm

(vascular) bundle scar

rhizome

terminal bud scale scar

stolon

vegetative bud

runner

mixed bud

tuber

non-porous

haustorium (pl. haustoria)

ring-porous

symbiotic

diffuse-porous

mycorrhiza (pl. mycorrhizae)

softwood

ectotrophic

hardwood

endotrophic

transverse (cross) section

tendril

tangential section

spine

radial section

thorn

giant vessel element

prickle

giant ray

adventitious root

heartwood

prop root

sapwood

epiphyte

tight knot
loose knot
root cap

Laboratory 20
Leaf Anatomy
Modified Leaves
I. Leaf Anatomy
Introduction
In studying the characteristics of stems and roots in previous laboratories, you have seen structures
specialized for physical support and for absorption and conduction of water and minerals. This lab
deals with the third vegetative plant organ typical of land plants: the leaf. Leaves have evolved as
plant organs that are (1) obviously adapted for light interception and (2) subtly adapted for carbon
dioxide uptake from the atmosphere for photosynthesis. Because of their large surface area and
because they have pores for gas exchange, leaves are very vulnerable to water loss. This
vulnerability results in a final adaptation found in all terrestrial vascular plant leaves: (3) features that
prevent excess water loss but at the same time reduce CO 2 uptake.
What is contradictory about a leaf preventing water loss? If the leaf were completely water tight, it
would also be completely air tight, and the carbon dioxide supply would be cut off. That would be
fatal! An ideal leaf would allow carbon dioxide to enter without allowing any water to escape. The
best scheme that evolution has produced, a system of controllable pores, stomates, falls far short of
that ideal. Stomate control of water loss will be dealt with extensively in the next lab, but even now it
is valuable to know a little about stomate response. Stomates tend to open when conditions are good
for photosynthesis. They tend to close when drying conditions are extreme.
The balance between photosynthetic opportunity and drought liability determines:
(1) Whether stomates are open or closed.
(2) How much water is lost from the plant.
(3) How much photosynthesis can occur.
An average plant is likely to take up at least 20 times as much water as it needs to supply its cells,
losing the excess to the atmosphere without any special benefit.

Exercises
A. Gross Leaf Anatomy and Plant Identification
As plant structures go, leaves are quite responsive to prevailing environmental conditions in their size
and form (see Exercise D). That somewhat limits their value for identifying plants. Flowers, in
contrast, tend to very consistent in their size and structure. Guides to plant identification typically
focus most on flower parts. Still, leaves are likely to be present on a plant throughout the growing
season, while flowers may be transitory, the fruits that grow from them quickly disseminated or eaten
by animals, or even completely absent if the plant is growing in an unfavorable site. The following
material summarizes leaf characteristics that are useful in plant identification.

1. Basic Leaf Structure.

Most plant leaves consist of a thin, flattened light interception surface, the blade; and an attaching
stalk, the petiole. In angiosperms, as shown in a previous lab, there is an axillary bud where the
leaf joins the stem. There may also be a pair of stipules present at the point of leaf attachment. In
some species, stipules are leaf-like; in others, papery or even spine-like. Most leaves also show
surface evidence of their internal plumbing as a distinctive vein pattern. Dicotyledon leaves typically
have a number of principal lateral veins diverging from a midrib. Monocotyledon leaves usually have
a number of main veins running parallel to the midrib, which itself may or may not be prominent.

Figure 19-165. Grass Leaves. a. General. b. Detail of Intersection with Stem.

Figure 19-164. Dicot Leaf.

Label the blade, petiole, stipules, midrib and side veins in Figure 19 -164. Does this leaf have a dicot
or a monocot vein pattern?

In many species, the blade is further subdivided into leaflets (see details below), which may
themselves look very much like individual leaves. Leaflets, however, have neither axillary buds nor
stipules. Both leaves and leaflets may be sessile (no petiole).

2. Graminoid (Grass-Like) Leaves

Grasses and many other monocots have leaves in which the lower portion is a sheath that completely
encircles the stem. The upper portion is likely to be a conventional blade, albeit a long and narrow
one. Leaf sheaths often overlap one another so that several concentric sheaths surround the lower
part of the stem. Study the labels in Figure 19 -165 and compare the illustration with the actual leaf
structure of grasses on display in the lab.

3. Using Leaf Characteristics in a dichotomous key.

Use the leaf key provided in lab to identify some of the greenhouse species on the lab tables.
Additional terminology useful in using leaves to identify plants can be found at the end of this chapter.

B. External Microanatomy of Leaves

Make a transparent acetate impression of a leaf surface by the procedure described below.
Collaborate with others so that you have results from both the upper and lower surface of both a dicot
and a monocot. Compare appearance of the surfaces: veins, epidermal cells, stomate numbers and
trichomes (hairs).
Procedure:
(1)

Remove one leaf from one of the plants provided.

(2) Put a few drops of acetone on a fragment of cellulose acetate, quickly spread the acetone
evenly over the surface, and allow it to evaporate.
(3) As soon as the liquid starts to turn filmy (15 to 60 seconds, depending on how much was initially
applied), apply the softened cellulose acetate surface to the leaf surface and firmly but carefully
hold in place for 60 seconds.
(4) Peel the acetate strip from the leaf, place it on a slide without using water, and examine under
the microscope. Close the diaphragm to deepen focus, and, if necessary, adjust the light coming
from the substage lamp to see maximum detail.
(5) Figure out what stomates look like, count the number which appear in your 400X microscope
field, and fill out Table 19 -22, combining your observations and those of other students working
with different surfaces.
Table 19-22. Leaf Surface Characteristics.

Surface
Bean Leaf

Trichomes (Hairs)
many / few / none

Stomates
many / few / none

Upper
Lower

Corn Leaf

Upper
Lower

Note: Stomate density and other leaf surface characteristics vary from place to place on the leaf and
from leaf to leaf on a plant. Typical stomate counts from a variety of plants are posted in lab.

C. Internal Anatomy of Leaves

1. Dicot, Monocot and Conifer Leaf Comparison
Collaborate with students in your vicinity by setting up THREE compound microscopes, one with a
lilac (Syringa) leaf slide, a second with a corn (Zea) leaf, and the third with a pine (Pinus) leaf.
Compare the slides at the same magnification and fill in Table 19 -23. The objective is to take a
close, comparative look at all three kinds of leaves, and considering the biological implications of their
differences. In some cases, the answers you enter in the table will depend on the slides you look at
and vary from group to group. Label Figure 19 -166, Figure 19 -167 and Figure 19 -168 with the
structures that are emphasized in the explanation that follows the figures.

Table 19-23. Leaf Comparison.

Lilac (Syringa)
Dicot

Corn (Zea)
Monocot

Thickness Rank
(Thickest to Thinnest)
Cross Sectional Shape (Describe
or Sketch)
Number of Epidermis-Like Layers
(Epidermis + Hypodermis)
Strong Difference between Upper
and Lower Halves of Leaf (Y/N)?
Shape of Mesophyll Cells
(Sketch)
Number of Vascular Bundles
Intercepted, Whole Leaf Width
(Scan and Count)
Stomates Sunken Below Surface
(Y/N)?
Bulliform Cells Present (Y/N)?
Number of Resin Canals Shown in
Cross Section
Cuticle Detectable (Y/N)?
[usually stains pink]

Pine (Pinus)
Gymnosperm

Figure 19-166. Lilac (Syringa) Leaf Cross Section.

Figure 19-167. Monocot Leaf Cross Section.

Figure 19-168. Pine (Pinus) Leaf Cross Section.

Interpretation
The three types of leaves you are comparing come from quite unrelated plant species, which also
happen to differ in their environmental tolerances. Lilac leaves are considered to be "normal"
dicotyledon leaves: a good example to learn for "typical" leaf structure. Corn, like many grasses of
tropical origin, is especially well adapted to rapid growth in warm, bright conditions. A pine leaf, finally,
is considered to be highly adapted to resist both drying and freezing. Unlike the lilac and corn leaves,
it will stay on the tree for at least part of two consecutive growing seasons, and survive the intervening
winter(s) unimpaired.
Lilac leaves are broad and thin and intercept light efficiently. Their extensive surface makes them
vulnerable to excess water loss. The loss is much reduced, however, by an epidermis of closely
packed cells covered with a transparent waxy cuticle. The thick pine leaf presents much less
evaporative surface for a particular amount of photosynthetic tissue, and is inherently less vulnerable
to water loss. This is especially important during the winter, when frozen xylem prevents water
replacement. Underlying and reinforcing the epidermis of pine leaves are one or two layers of
hypodermis. The combination of these extra layers and a well-developed cuticle result in pine
leaves having better control of water loss than lilac leaves. The corn leaf is intermediate in ability to
absorb light and intermediate in its ability to resist drying out. It survives frost no better than a lilac. A

6
distinctive feature of upper epidermis of a corn leaf is clusters of large, bulliform cells in the upper
epidermis. In many grasses, bulliform cells are involved in leaf folding or rolling to conserve water,
but corn leaves are too broad to show that response very well.
The photosynthetic mesophyll tissue occurs just inside the epidermis, often in several layers. Note
that in the lilac leaf the cells of the mesophyll are packed densely in the upper part of the leaf, and
loosely in the lower part. This is consistent with a leaf that is positioned horizontally to the ground,
with predictable upper and lower surfaces. The close packing and orderly arrangement of the upper
palisade mesophyll enhance light interception, perhaps at the expense of efficient diffusion of
carbon dioxide. The more loosely arranged spongy mesophyll beneath favors carbon dioxide
diffusion. Most of the stomates in the lilac leaf occur in the lower epidermis and provide passages
between the outside air and the spongy mesophyll.
Note that corn leaves lack mesophyll differentiation. This is in keeping with the less horizontal
orientation we expect in a grass leaf. Sun is likely to strike the relatively vertical corn leaf from either
side, depending on the time of day. Look for stomates in the corn leaf cross section. Above each
stomate there is likely to be an open chamber, which serves to improve the diffusion of carbon dioxide
through the mesophyll.
In lilac and corn leaves, the mesophyll occupies all the space between the upper and lower epidermis
except for the vascular bundles (veins) that traverse the leaf. In pine leaves, though, the mesophyll
extends only to the endodermis, which separates it from the central transfusion tissue, in which the
vascular bundles are embedded. Pine mesophyll cell packing is not easily inferred from a cross
section. Your slide may show densely packed mesophyll between the epidermal layers and the
endodermis as shown in Figure 19 -168, but it may show instead many air spaces and few cells. The
difference from one cross section to another results from bands of closely and loosely packed cells
alternating along the length of the leaf. Look for this variable distribution in the longitudinal pine
needle section displayed on the demonstration bench. The pine mesophyll cells themselves have
distinctive lobing and are likely to stain darkly in a prepared slide because of high resin content. Such
resin concentrations probably help increase freezing resistance. A last distinctive feature of the pine
leaf mesophyll is the presence of resin canals essentially identical to those found in the wood and
bark.
In lilac and corn leaves, vascular bundles range from very large, like the midrib, to a cylinder of just a
few cells near the end of a small vein. Look for that variation in your slides. In lilacs, all vascular
bundles except the largest and smallest are surrounded by a cylinder of thick-walled cells, the bundle
sheath. The bundle sheath adds strength to the lilac leaf, but not enough to completely support it.
Most of the rigid flatness of the leaf comes from the normal water pressure (turgidity) of the thinwalled parenchyma of the mesophyll. This becomes obvious during dry times when the leaves wilt.
Good sized lilac vascular bundles may be further anchored in place by bundle sheath extensions,
which extend to the epidermis. Bundle sheath extensions also anchor large veins in corn leaves.
Several smaller veins typically occur between each main vein, though. In many grasses, including the
corn you are observing, these smaller vascular bundles have a cellular arrangement called Kranz
(German for wreath) anatomy: each large cell of the bundle sheath is paired with one of the large
mesophyll cells. This pairing contributes to an especially efficient kind of photosynthesis covered later
in the course. The special bundle sheath of small corn veins contrasts with that of lilac, for its
principal function is physiological instead of physical.
Overall, a pine needle has a greater proportion of thick-walled cells than a lilac leaf or corn leaf, and is
less likely to show conspicuous wilting even if it loses a lot of water. In a lilac and corn leaves, the
stomates, with their paired guard cells, are right on the surface. In pines, each stomate is recessed
into the leaf surface, producing what are referred to as sunken stomates. Sunken stomates tend to
reduce water loss, because each water vapor molecule must follow a longer path to escape the leaf.
The price paid is in slower carbon dioxide uptake, also because of the longer diffusion path.

D. Variation in Leaf Development in Response to Environment

1. Aquatic/Terrestrial

7
Plants that grow in shallow water are faced with special requirements for efficient functioning. We
have seen that the leaf of a land plant is typically specialized for light interception, carbon dioxide
uptake, and insofar as possible, water retention. A leaf usually has little mineral absorption function,
because air is a far less reliable source of minerals than the soil.
An aquatic plant, in contrast, absorbs most of its minerals, as well as its carbon dioxide, through its
stems and leaves. Aquatic plant leaves are the principal organs of photosynthesis but of course they
dont need modifications for retaining water.
Observe on display the contrast in leaf development in plants that start their growth under water and
complete it in the atmosphere. The much-divided leaves represent a modification that facilitates
mineral and carbon dioxide uptake, though at the expense of the abilities to absorb light and to remain
flat. The leaves, or in some cases even the parts of leaves that develop in the air, have an anatomy
much more typical of the typical leaves covered previously. See the demonstration slides of floating
leaves of water lily and submerged leaves of Potomogeton. Especially note the cuticle development
and amount of air space within each leaf.
What sorts of anatomical contrasts would you expect between the upper and lower surfaces of a
floating leaf plant like water lily?

2. Sun-Shade LeavesInternal Anatomy

The differences between a sun leaf and a shade leaf are internal as well as external. Observe on
demonstration the differences in mesophyll development, cuticle thickness, stomate density, and vein
distribution between a sun leaf and a shade leaf of the same species.

3. Sun/Shade (Demonstration)
Less extreme variation in leaf anatomy is quite common in normal terrestrial plants that are tall
enough to be self-shading. On display are leaves taken from the shaded lower branches and from
the unshaded upper branches of Hill's oak.
If you wanted to experimentally stimulate an oak sapling to produce leaves with large blades and
small sinuses (open spaces between lobes), how should you modify its growing conditions?

II. Modified Leaves

Virtually any terrestrial species has leaves that are more or less specialized for water retention
against the evaporative demands of a dry atmosphere. (A variety of more extreme drought

8
adaptations of plants will be on display in the next lab). Many plants, however, have leaves
specialized for more unusual functions. Observe the specialized leaves on display in today's lab:

A. Tendrils
Tendrils are thread-like modifications of stems, leaves, or leaflets that help attach a plant to another
object. Both agricultural and weedy species in the pea family are likely to have leaves or leaflets
modified as tendrils. The value of tendrils to a forest vine is obvious in enabling the plant to grow up
to a better light environment. Could tendrils have similar value to meadow or pasture species?

B. Spines, Thorns, and Prickles

Spines are, technically speaking, modified leaves, but few of us are likely to be too concerned about
the origin of the plant part that is causing us pain. Real spines that have a leaf origin include those of
cacti, currents, and gooseberries. Similar structures of different origin include the thorns of
hawthorns (modified stems), and the prickles of blackberries and raspberries (stem epidermis
extension). Think about where you personally have encountered spiny plants: Swamps? Pastures?
Thickets? Deep shade under hardwoods?

C. Leaves as Agents of Reproduction

Some plants have notably succulent leaves (thick, water-filled). Typically, such leaves have the
capacity to form adventitious roots if they fall to the ground and eventually grow into whole new
plants. A still more extreme modification found in some species of the genus Kalanchoe is the direct
production of plantlets on the tips or edges of the leaves. Leaves that act as reproductive agents are
almost always thick enough to store water. In what sort of climates would you expect plants to
notably benefit from this sort of reproduction?

Is this reproduction sexual or asexual?

D. Colored Leaves.
Tropical forest-edge communities often include ground layer plants with brightly colored or patterned
leaves; for example, Coleus and Croton species. Since the brightest part of the leaf is often an area
without chlorophyll, it is intriguing to speculate about why such plants have evolved leaves that

9
actually have less photosynthetic surface. What competitive advantage would be gained by a plant
that has patterned colored leaves if it were growing in a dazzling sun-flecked habitat?

E. Water Shedding Leaves.

Species native to climates where rainfall is very great are likely to have leaves which have entire,
unlobed edges, and which terminate in an extended "drip tip." Such a leaf sheds water efficiently,
which may reduce the weight of water borne by the plant during a torrential downpour. Effective water
shedding may also keep the stomates from being blocked, so carbon dioxide uptake remains good.
What effect might efficient water shedding have on a leaf's resistance to pathogenic organisms like
fungi and bacteria?

F. Water and Mineral Trapping Leaves.

Species that grow in environments where water or minerals are difficult to obtain may have leaves
that are arranged to trap water instead of shedding it, and to catch airborne debris. Examples include
pineapples, which are native to dry habitats, epiphytes (plants which grow on other plants), whose
roots are aerial and do not reach the soil, and plants of semi-arid environments, like yuccas, agaves,
and aloes. In the mineral deficient bogs of northern Wisconsin are found even more extreme
modifications for mineral capture: the leaves of insect trapping pitcher plants and sundews. The
Venus fly-trap, present and even locally abundant on mineral deficient sandy soils in the Carolina
coastal plain, has the most spectacular leaf modification of all for supplementing its mineral supply.
Insect trapping plants cant grow in the dark any more than other plants can. What does this tell you
about what they actually get from the animals they trap?

G. Storage Leaves.
Food storage is typically associated with fleshy roots and stems, but leaves can be modified for food
storage, too. Plants like lilies and onions store food in bulbs. A bulb consists mostly of thickened leaf
sheathes, but is also likely to include some stem tissue at the base or in the center. Can you think of
any bulb-forming plants besides the examples above?

10

H. Poisonous Leaves.
A non-anatomical leaf modification is the production of substances that are poisonous to grazers or
even to other plants. Leaves that are highly toxic to man include those of dogbane (internal) and
poison ivy and poison sumac (external). Wisconsin species whose leaves are known or suspected to
contain substances toxic to other plants include black walnut trees, orange hawkweed, and bracken
fern. What kind of visual evidence would indicate the production of allelopathic (plant killing)
substances by a plant in nature?

I. Some Specialized Leaf Terminology

1. Arrangement

11
Alternate: attached singly (attached on alternating sides along the twig).
Opposite: attached to the twig in opposing pairs.
Whorled: attached in clusters of three or more.

Figure 19-169. Leaf Arrangement.

12

2. Compounding
Simple, if leaf consists of a single blade (remember from your study of twig anatomy, that there is
always an axillary bud where a true leaf joins the stem).
Compound, if leaf consists of two or more blades (leaflets) attached to a central axis without axillary
buds, the whole compound leaf attached to the twig with an axillary bud present.
Note: If the leaflets are further divided into smaller blades, the leaf is twice compound.

3. Venation
Net, if veins branch and re-branch into an elaborate network (dicotyledons).
Parallel, if all visible veins run side by side for the length of the leaf (monocotyledons).
Net-veined leaves tend to be broad; parallel veined leaves tend to be long and narrow but there are
plenty of exceptions.

13

Figure 19-170. Leaf Vein Pattern.

4. Overall Shape of Blades

Linear--very long and narrow.
Lanceolate--shaped like a spear point: quite narrow, but broadest towards the base and tapering
towards the tip.
Oblanceolate--as above, but widest beyond the middle so that the gradual taper is towards the leaf
stalk (petiole).
Ovate--egg shaped, but usually tapered towards the tip; broadest below the middle.
Obovate--as above, but widest beyond the middle.

Note: Many other descriptive terms are used to describe leaf shape; some obvious, like "triangular,"
and some not so obvious, like "cordate," which means heart-shaped, and "peltate," which means
circular.

14

5. Lobes
Both lobing and compounding can be either:
Palmate, if the lobes or leaflets converge on a single point, like the fingers to the palm of a hand, or
Pinnate, if the lobes or leaflets converge on different points along the midrib or main leaf axis.

Figure 19-171. Leaf Shape.

15

Figure 19-172. Lobed and Compound Leaves.

6. Serration
Entire leaves have no teeth.
Single toothed leaves have an evenly toothed margin.
Double toothed leaves have small teeth within larger teeth.
Toothed leaves are the norm in Wisconsin woody plants, but rare in the tropics.

16

Figure 19-173. Leaf Teeth.

7. Surface Texture
Glabrous leaves have no hairs.
Hairy leaves are described by almost countless numbers of terms, depending on the abundance,
density, and texture of the hairs. Gleason and Cronquist: Manual of Vascular Plants of Northeastern
United States and Canada, for instance, lists 33 different terms designating surface hairiness
variations!
Glaucous leaves are hairless, but have a gray or blue-green color from surface material that can be
easily wiped off.
Glands can also contribute to surface texture, often simply by secreting substances that are sticky to
the touch. The glands themselves may be directly visible or detectable only with a hand lens.

8. Size
The usefulness of leaf size in plant identification is reduced because of the dependence of size on
growing conditions. Size is useful, though, where expected differences are great, or where there are
few other obvious distinctions.

17

KEY WORDS
vegetative
carbon dioxide
stomate
blade
petiole
stipule
vein
midrib
leaflet
sessile
sheath
epidermis
cuticle
hypodermis
bulliform cell
mesophyll
palisade mesophyll
spongy mesophyll
vascular bundle
endodermis
transfusion tissue
bundle sheath
turgidity
bundle sheath extension
Kranz anatomy
sunken stomate
sun leaf
shade leaf
tendril
spine
thorn
prickle
succulent leaf

18

epiphyte
bulb
allelopathic
simple
compound
net-veined
parallel veined
linear
lanceolate
oblanceolate
ovate
obovate
palmate
pinnate
entire
single toothed
double toothed
glabrous
glaucous
gland

Laboratory 21
Diffusion and Osmosis
Plant Water Relations
Soils
Introduction
The next four laboratories will deal with plant physiology; that is, plant processes.

I. Diffusion and Osmosis

The relationship of a plant to its environment is greatly influenced by diffusion and osmosis. Gas
exchange between leaves and the atmosphere is by diffusion. Uptake of water and nutrients from the
soil is by diffusion and osmosis. The movement of many materials through the plant, into cells, out of
cells, and within cells is by diffusion. Because of distinctive and significant consequences of the
diffusion of water across biological membranes, we give it a special name, osmosis. The concepts
below are essential for your understanding of the subsequent experiments.
Kinetic Theory: Matter behaves as if its molecules are in constant motion. The rate of movement
increases with increased temperature.
Physical State: Molecules of a gas have no intrinsic bounds to their movement. Molecules of a liquid
move relative to one another but do not escape the liquid surface. Molecules of a solid hardly move
relative to one another.
Diffusion: The net movement of a material from an area of high chemical potential to an area of lower
chemical potential caused by the intrinsic movement of its molecules. Every material diffuses
according to differences in its own chemical potential, and independently of the chemical potentials of
other materials.
Chemical Potential: The ability to diffuse. It is influenced by energy, a function of temperature, and
concentration (the higher the concentration the higher chemical potential). Special Note: If two
adjacent sites are at the same temperature and pressure, materials diffuse between them from high to
low concentrations. If the chemical potential of a sample of low concentration is raised enough by
heat or pressure, though, the direction of diffusion could occur against the concentration difference.
Membranes: Cellular membranes are differentially permeable; that is, different materials pass
through at different rates. Generally, small molecules traverse a membrane easily, and larger ones
cannot pass.
Osmosis: The diffusion of water through a differentially permeable membrane.
Water Potential: The chemical potential of water; hence, the ability of water to diffuse. (1)
Temperature (kinetic energy), (2) concentration, and (3) pressure influence this.
(1) Temperature: The higher the temperature, the more rapid the molecular movement and the
greater the ability to diffuse.
(2) Concentration: The higher the purity of water the greater the ability to diffuse. Note that
dissolving a material in water will lower the proportion of water in the solution, and therefore lower
the water potential.
(3) Pressure: The higher the pressure the greater the molecular motion and the ability of water to
diffuse.
Megapascals: Measurement of water potential is confusing in several ways. The measurements
themselves are indirect. The scale of measurement essentially runs downward from zero! The
units of measurement are megapascals. Distilled water at room temperature and completely humid

2
room temperature air both have a water potential of zero. Decreases resulting from solutes, lowered
temperature or pressure, or absence of water result in increasingly negative values. A megapascal is
equal to 10 atmospheres or about 193 pounds/square inch.
Turgidity: The internal pressure of a cell produced primarily by the osmotic uptake of water. A turgid
cell is a full and firm cell. Any plant cell with a good supply of fresh water is turgid.
Wilting: Visible collapse of plant parts caused by loss of turgidity. Initially reversible, but permanent if
the following occurs.
Plasmolysis: The pulling away of the plasma membrane from the cell wall caused when water loss
is so great that the cell contents cannot fill the cell. Plasmolysis invariably results in death of the cell.

Exercises
A. Diffusion in Physical Systems
1. Simple Diffusion of Gas into Gas and Influence of Molecular Weight on the Rate of
Diffusion
In this demonstration ammonium hydroxide (NH 4OH) releases ammonia gas (NH3) at one end of the
tube. At the other end hydrochloric acid (HCl) evaporates into hydrogen chloride gas (HCl). When
NH3 and HCl meet, a white cloud of ammonium chloride (NH4Cl) will form. The atomic weights
involved are H = 1; N = 14; Cl = 35.5. Therefore, the molecular weight of NH 3 = 17 and HCl = 36.5.
Where does the NH4Cl cloud form?

What is the approximate rate of diffusion of NH 3 in cm/min? Of HCl?

Why does one substance diffuse faster than the other?

2. Simple Diffusion of Liquid into Liquid

The reagents used here include:
(1) Water containing an indicator (phenolphthalein). Phenolphthalein is red in a basic (alkaline) solution.
Note that the beginning solution is colorless.
(2) Alcoholic sodium hydroxide. This is an alkaline solution.
Procedure: Fill a test tube one-third full using reagent a. Pour an equal volume of reagent b onto the
top of reagent a very carefully to avoid excess mixing. Note the thickness of the pink band where the
solutions have mixed. Observe the experiment several times during the laboratory, noting the rate of
spread of the color.
From where does the pink color come?

What is the approximate rate of diffusion in mm/min?

How does the rate compare to that of the gas into gas demonstration? Why?

3. Osmosis
Observe the four osmometers. In all cases, the tube contains a high concentration of solutes
(sucrose) and the water in the beaker is distilled water (no solutes). The dialysis tubing covering the
lower opening is a differentially permeable membrane separating the syrup from the distilled water.
Water molecules can pass through but larger sugar molecules can not.
The osmometers are set up as follows:
(1) Full-strength syrup and distilled water at room temperature
(2) Full-strength syrup and distilled water in an ice bath
(3) Full-strength syrup and distilled water in a heated water bath
(4) Diluted syrup and distilled water at room temperature
Measure the rate of movement of the liquid level in the glass tube in the four osmometers.

4
What is the effect of concentration of solute on the movement of water?

What is the effect of temperature on the movement of water?

Will soil temperature influence water movement into a root?

What do you predict would happen if we could place increased pressure on the syrup or on the
water?

Explain when water movement into a turgid cell would stop. Think in terms of everything which
influences water potential.

B. Diffusion in Plants
1. Turgidity
a. Potato Pieces
Cut two small pieces of potato so they are equal in size. Place one into distilled water and the
other into a 10% salt solution. Observe and handle periodically until you notice a definite
difference in the two pieces.
Explain what has happened.

b. Celery Stalks
Observe the demonstration of celery in water and salt water.
Explain the results.

2. Plasmolysis
Repeat the experiment used in laboratory 2 to make the cell membrane visible by causing plasmolysis
in a cell. Working together with a partner, make two wet mounts of Elodea leaves on clean
microscope slides, one in a drop of water, and one in a drop of 10% salt solution. Compare the
appearance of the cells.
What is happening?

Why does it happen?

3. Imbibition

6
Observe the two blocks of wood. These were the same size before one of the blocks was soaked in
water. Compare the sizes of the two blocks.
Note which block is larger and explain why.

This is not an example of osmosis. Why not?

II. Plant Water Relations

Introduction
If you were interested in designing a system to dry out land as it was wetted from precipitation or
ground-water runoff, a typical vascular plant would not be a bad place to look for ideas. In fact, in
Buena Vista Marsh in the southern part of the county, willow trees have been planted specifically to
remove water from wet areas and make agriculture possible.
As we have seen in previous laboratories, a vascular plant combines 1) a branching and rebranching
root system for gathering water (and minerals); 2) a strong stem with highly specialized water and
mineral conducting channels; and 3) an enormous evaporating surface. One thing seems to be
missing though: a pump to push the water through the otherwise remarkable system. As
demonstrations in this laboratory should show, there is such a pump (root pressure). As other
material will demonstrate, however, much of the time it isn't even working, but water is moving
through the plant just the same.
Transpiration
Under most conditions, the uptake and loss of water by plants is by the process of transpiration. The
force which moves the water is simply the difference in water potential from the soil to the root to the
leaves to the air (see Error: Reference source not found). As mentioned before, fresh water at room
temperature has a water potential of zero. Soil, air, or tissues almost always have negative water
potentials, either from lack of water or because of solutes. The difference in water potential between
wet soil and the air on a sunny summer day can be well over 100 megapascals or almost 15,000
pounds/square inch: plenty of force to move water to the top of the tallest trees. What is a little harder
to believe is that the entire water column from the roots through the stem to the leaves is pulled
through the xylem cells by these water potential differences.

Figure 20-174. Water Potential and Transpiration (after Jensen, W. A. and Salisbury, F. B. 1984.
Botany, p. 275.).

8
Guttation
Many, but not all, vascular plants have a second way of moving water: guttation.
Table 20-24, on demonstration, summarizes some of the contrasts between transpiration and
guttation. In guttation, water is pushed through the plant by actual physical pressure generated in the
interior of the root. The pathway of water is the same, except in the leaf. Water loss in guttation is in
the form of liquid water extruded out onto the leaf surface where veins terminate in hydathodes. The
location of a hydathode on a leaf surface may be indicated by a tight cluster of stomates. Root
pressure is produced when active transport of minerals across the endodermis lowers the solute
potential of the interior of the root (stele), which lowers the water potential (see above), which
stimulates diffusion of water into the stele, which produces the pressure that pushes the guttation
stream up through the plants.

Exercises
A. Diameter of Water Conducting Cells
The smaller the enclosing tube, the more tension a water column can withstand before it breaks.
Circumstantial evidence suggests that a tree whose xylem consists mostly of large diameter vessel
elements (many hardwoods) can transpire water faster, but not to as great a height as a tree whose
xylem consists mostly of smaller diameter tracheids (conifers).
Procedure. Observe under a microscope a cross (transverse) section of the wood of a pine stem and
that of an oak stem. Estimate the diameter of the largest water conducting cells in each (tracheids in
the pine, vessel elements in the oak). Which are larger?

How much larger (half again, two times, three times, etc.)?

Relate your results to the fact that the largest pines include: ponderosa pine, 232 feet, and sugar pine,
246 feet, while the largest oaks include bur oak, 170 feet, white oak, 150 feet, and red oak, 150 feet.
Does this support the relationship between water column diameter and lifting force discussed above?
If so, how? If not, why not?

B. Conditions Favoring Transpiration

The flood lamp shining on the rubber tree provides a simulation of the sun's radiation. Electric
thermometer probes have been placed as described in the data table below.
Procedure.
(1) Turn on the electric thermometer and record the temperature at each point.
(2) Go through the following reasoning to figure out which leaf surface is subject to the greatest
evaporation stress:
(a) The air surrounding the leaf has about the same water content (at least until the plant
transpiration adds additional vapor).
(b) At a particular water content, the water potential of air is much more negative at high
temperatures.
(c) Therefore, the higher the temperature, the greater the evaporation stress.

Location

Temperature

Evaporation Stress
(greater, less)

Upper leaf surface

(Probe 1)
Air just above upper surface
(Probe 2)
Lower leaf surface
(Probe 3)
Air just below lower surface
(Probe 4)
Control (room air away from
lamp) (Probe 5)
Do the leaf temperatures observed here help explain the stomate differences observed in the
previous lab? Explain why or why not.

C. Conditions for Guttation

Note the tomato plants under the bell jars. Unlike the room air, the atmosphere in the bell jars is
saturated with water vapor. What is the water potential of the air under the bell jar?

How does the evaporation stress under the bell jar compare with room air?

10

Look closely at the leaves of the plants under the bell jars for signs of water being extruded at the
hydathodes by guttation. Look at the microscope slide to see the physical structure of a hydathode,
remembering that it is essentially a vein end.

D. Transpiration Estimation
1. Diffusive Resistance Meter
The meter measures the change in humidity in the sensor chamber. We can assume that leaf
surfaces with more open stomates should increase the humidity more than leaf surfaces with fewer
open stomates. Since the plants are in lab conditions favoring open stomates, we can assume
differences in transpiration rates are due to the number of stomates present.
If we would record the number of meter units moved during a 3 minute time interval, our expected
results are as follows, from greatest movement to least:
More transpiration from bean lower epidermis (>28,000 stomates/sq cm).
Less from bean upper epidermis (~ 4,000 stomates/sq cm).
More from corn lower epidermis (~ 6,800 stomates/sq cm).
Less from corn upper epidermis (~ 5,200/sq cm).
We suggest recording the data of the demonstration:

Plant

Surface

Time

Bean

Upper

3 min

Lower

3 min

Upper

3 min

Lower

3 min

Corn

Meter Units Moved

The basic principle of operation of the meter is that the sensor crystal, when dry, conducts very little
current, and the meter deflection is low. As the crystal gets wet (from transpired water vapor), its
conductivity increases and the meter deflection increases. The more sensitive Hum 2 setting will
probably work best. The measurements are most accurate between 20 and 80 on the meter. When it
gets to 80, you must dry the LiCl2 crystal down to below 20.
This instrument is relatively inexpensive, very portable, and consequently something we can use in
the plants normal habitat.
Caution: After use, be certain the crystal is dry and the meter is turned off!

2. Cobalt Chloride Disks

Cobalt chloride impregnated disks can be used as a really cheap, really crude substitute for the
diffusive resistance meter.. The disks remain blue as long as they are dry. Exposure to water vapor
turns them pale pink or white. By taping such disks to a leaf and timing the color change, one can
make crude comparative estimates of rates of water loss.

11

Figure 20-175. Transpiration Estimation with Cobalt Chloride Disks.

Procedure:
Use the same type of plant from which you obtained epidermal peels in the leaf lab.
(1) Remove a disk with tweezers from the container. Be sure to close the disk container
immediately to avoid uptake of water vapor from the air.
(2)

Fully moisturize the disk by breathing on it, and use it as a color reference in step 5.

(3) Working rapidly but carefully, and avoiding extensive finger contact, remove two more disks
from the container and apply them near the ends of a 5 cm strip of Scotch tape:
(4) Immediately affix the tape and disks to a plant leaf in such a way that one disk is in contact with
the upper leaf surface, and one is in contact with the lower leaf surface.
(5) Record the time required for each disk to change color in Table 20 -25. A quick change
indicates open stomates and transpiration of water from the plant. A slow change means either
closed stomates or no stomates on that surface.
(6)

Remove tape and disk from leaf when experiment has been completed.
Table 20-25. Transpiration Estimation.

Plant Species

Surface
Upper
Lower

Time for Color Change, Minutes

12

F. Drought Adaptations (Demonstration)

1. Drought Deciduousness
Plants that are native to climates with a notable dry season are sometimes drought deciduous. They
lose their leaves in response to drought, then leaf out again when moisture conditions are good. Our
native deciduous trees show a similar modification to avoid winter cold. Less obviously, they are also
avoiding potential water problems. What would happen to plant tissues if the water in the soil or in the
xylem were frozen, but the leaves still were losing water to the winter air?

2. Stem Succulents
Everyone is familiar with the succulent stems of cacti. Not so familiar are stem-succulents from
African and Asian deserts, where the cactus family does not occur. Species in the spurge and
milkweed families have evolved stem succulence in the Old World, and look remarkably like new
world cacti. Few stem succulents can survive hard freezes because of the tissue damage produced
by ice crystal formation, but there are species of small prickly pear that occur in Wisconsin. Their
shriveled appearance in the winter suggests that they avoid damage by drying out as winter
approaches.

3. Leaf Succulents
Many plant families include species with succulent leaves that are modified for water storage. Such
plants occur, of course, in dry climates, but they also are common in habitats like beaches, where
high concentrations of dissolved salts lower the soil water potential so much that plant water uptake
can occur only when the soil water is diluted after a rainstorm.
Both stem succulent and leaf succulent species typically have a Crassulacean acid metabolism
(CAM), which is a modified type of photosynthesis that works as follows:
Unlike normal plants, their stomates open at night, and carbon dioxide is chemically captured and
temporarily stored in the form of organic acids. In the daytime, the stomates close, water is
conserved, but photosynthesis still can proceed using carbon dioxide released from the accumulated
acids. Why don't all plants show this water-conserving type of photosynthesis?

The capacity of a leaf for accumulation of the organic acids seems to put an upper limit on growth
rate. Under favorable moisture conditions, plants with normal daytime gas exchange simply grow
faster than plants operating by CAM. Would you expect the leaf sap of a CAM species to have a
higher pH at sunset or at sunrise?

13

4. Root Succulents
Some plants face drought problems despite living in apparently moist environments. For example,
epiphytes, which are plants which grow on other plants, may develop water stress even in very moist
air simply because they are not rooted in soil. Because of this, epiphytes are typically more abundant
in humid environments. They are also likely to be either stem succulents, leaf succulents, or store
water in their roots. Plants that grow in especially droughty soils in otherwise adequate moisture
environments, like asparagus, may also have succulent roots. Succulent root species like spider
plants and asparagus ferns are good houseplants for people who forget to water.

5. Sclerophylly
Plants that grow in dry environments where replacement of drought-deciduous leaves would be slow
because of relatively low growing season temperatures, often have relatively small evergreen leaves
with especially well-developed epidermis and cuticle. We are all familiar with conifer needles with
such characteristics. Broad-leaf plants can also be sclerophyllous. Native examples include
partridgeberry and wintergreen of pine and oak woods; and cranberries, bog rosemary, bog laurel,
and Labrador tea of bogs. Drought in a bog? In the spring, open bogs combine high evaporation
stress on the shoots of plants with low moisture availability caused by cold or even frozen soils. Even
in the summer, the waterlogged soils of bogs are oxygen deficient, so plant root growth is poor, and
water uptake may be inefficient.

6. Malakophylly
A less conspicuous, but very common modification for dry environments, is possession of small, soft
leaves, which respond to drying out by developing high solute concentrations and very low water
potentials. Such plants can take up soil moisture that would be unavailable to normal plants. It is
very typical for such plants to essentially "abandon" their oldest leaves just before they dry out by
translocating valuable materials to areas of new growth. Examples include some of our goldenrod
species, and the sagebrush species of the arid west. These, and many other malakophyllous
species, are members of the composite family. Dandelions are in the composite family. Are they
malakophyllous?

7. Leaf Rolling
Grasses often respond to drought either by rolling or by folding their leaves in such a way that the
principal stomate bearing surfaces are protected from the evaporating influences of dry air. When
moister conditions return, the leaf will unroll or unfold. Special large bulliform cells ("bubble-form")
seem to be involved in such rolling or folding, either directly by enlarging or shrinking, or by acting as
water reservoirs that influence the enlarging or shrinking of more ordinary neighboring cells. Locate
the bulliform cells on the slide of a xerophytic leaf. Generally compare their numbers with ordinary
epidermis cells.

8. Fenestration
Some plants have their photosynthetic cells especially thoroughly enclosed in protective tissues, with
transparent "windows" which allow light to penetrate into the interior of the leaf. The most extreme
modifications occur in a few species in which the leaf is actually located below the soil surface, with
only the "window" exposed to evaporation.

9. High Albedo.
Since a warm leaf tends to lose more moisture than a cool leaf, it is not surprising to find that the
leaves of plants of dry, exposed places are often very light in color and have a high albedo (albedo
proportion of light reflected). Such plants reflect much of the light that would otherwise heat them up.

14
Obviously, their high reflectivity results in less energy available for photosynthesis. Native species
with this adaptation include pearly everlasting, pussytoes, and wormwood. The sagebrushes of the
west and some of the succulents on display are other examples. Note that plants of especially
favorable moisture sites, such as forest understories and even aquatic environments, are often deep
green, with a very low albedo.

10. Leaf Hairs

Leaf hairs may have many functions, including protection from insects, but leaf hairiness is often
simply a modification that reduces water loss. To understand why, we must understand that the rate
of evaporation from open stomates is inversely proportional to the distance between the moist air
inside the leaf and the drier air outside (the longer the distance, the slower the diffusion of water out of
the leaf). If a leaf surface is smooth and hairless, dry air can sweep close to the surface and carry off
water vapor. If the leaf surface is hairy, air movement near the leaf is slowed down. The result is a
build-up of water vapor in the still air just outside the stomates, and effectively longer distance from
the humid inside to the dry outside. Since the distance is very short to begin with, a layer of hairs can
produce a large proportional increase. What effect should the hairs have on rate of carbon dioxide
uptake?

11. Sunken Stomates

The sunken stomates of the pine leaf, and in many other drought-adapted leaves have an effect
comparable to leaf hairs, because of the increase in diffusing distance for the water vapor. On
demonstration is a cross-section of a resistant leaf in which the sunken stomates are further protected
from rapid water vapor removal by hairs in the sunken area that contains the stomates.

III. Soils
Introduction
Mineral uptake from the soil by plants is primarily by active transport into the root cells. Distribution
of minerals through the plant normally occurs via the transpiration stream. In the absence of a water
potential gradient minerals are transported during guttation.
The water and mineral holding capacities of a soil are likely to be quite similar. This is because both
water and minerals are retained in the soil by adsorption to the mineral or organic material. Clays,
by definition, consist of very small particles. Consequently, they have an enormous total surface area
and adsorb little water and small amounts of minerals.
Even though clay and organic soils have the highest water holding capacities, they may not provide
the most favorable moisture environments for plant growth. Clays may hold water so closely that
plants can't take it up. Many loams (soils with a mixture of silt, sand, and clay) actually hold more
available moisture than a clay soil, even though their total moisture capacity is lower. In this part of
the state, highly organic soils are confined to locations with a high water table. Consequently they are
often waterlogged, with the result that plant root growth is inhibited, and water uptake is poor!

15

Exercises
A. Soil Moisture Capacity
Equal volumes of dry (1) sand, (2) loam, (3) clay, and (4) very organic soils were placed in the
funnels, and equal volumes of water were added to each. Note how much water has been retained
by the soil and how much has percolated down into the graduated cylinders. Record your
observations in Table 20 -26. This simple demonstration is analogous to what happens to water in
the soil after a rain. The water potential of a soil at so called, field capacity, is variously estimated at
-.01 to -.03 mPa.
Table 20-26. Soil Moisture Capacity.

Water
Added, ml

Water in
Cylinder, ml

Water Retained
by Soil, ml

Sand
Loam
Clay
Organic
B. Nutrient Deficiencies
The weight of a normal plant is likely to be 70 to 80% water (woody plants less, succulent plants
more). In the dry matter that remains when the water is removed, carbon, hydrogen, and oxygen are
very much the most abundant elements. During the growth of a plant, these three elements are
obtained almost exclusively from carbon dioxide from the air and water from the soil. All the
remaining elements essential to plants are obtained primarily from the soil and distributed by
transpiration and guttation. These elements are typically referred to as minerals or nutrients. A
more or less arbitrary distinction is made between those elements that are needed in relatively large
amounts (macronutrients) and those that, while still essential, are needed in extremely small amounts
(micronutrients).
Table 20-27 (posted in lab) summarizes the minerals known to be needed for normal plant growth,
what they are needed for, the chemical form in which they are absorbed, and symptoms which
typically occur when they are lacking.
Observe the demonstration of plants that have been hydroponically grown. The control plants were
grown in a solution containing every mineral known to be necessary for plant growth. Each other
plant was grown in a solution that was identical except for the omission of the listed element.
Significance of appearance:
1. Size: Obviously, anything that affects a variety of plant processes or which is an important
structural part of plants is likely to significantly affect normal growth. If a mineral deficient plant is
close to normal size, one may assume that the missing element has a more specialized role.
2. Chlorosis: In some cases, chlorosis results because the missing element is actually part of the
chlorophyll molecule. In others, the missing element is essential to chlorophyll synthesis.
3. New or Old Growth: Both plants and animals have the capacity for removing some, not all, vital
materials from non-essential cells and tissues, and making them available elsewhere. If a mineral
deficiency shows first in the older parts of a plant, that mineral is said to be mobile, because the plant
has been able to translocate it (or more typically some of the chemicals in which it occurs) from old
parts to the site of new growth. Some minerals simply can't be transferred in such a way. Symptoms
of deficiency of a non-mobile mineral show first in new growth. Fill out Table 20 -28:

16
Table 20-28. Plant Nutrient Deficiencies.

Nutrient
Lacking

Size Relative
to Control

Leaf
Chlorosis?

Symptoms in New
or Old Growth?

C. Physiological Drought
Any mineral that a plant takes up from the soil must be in solution in the soil. Even minerals adsorbed
to soil particles must be released to the soil solution before they can be useful to the plant. If the
concentration is too high, however, the soil water potential will be lowered so much that the plant will
be unable to take up water for lack of the water potential gradient necessary for transpiration. In such
cases, the plant suffers from real water deficiency despite the presence of large amounts of water in
the soil.
Both over-fertilization and natural salinity can cause physiological drought. When making transplants,
it is wise to avoid adding fertilizer until the root system has been reestablished in the new soil.
The small sunflowers on demonstration have been transferred from their growth medium to salt
solutions of varying water potential. Observe the response of the plants in Table 20 -29.
The soil water potential popularly associated with the permanent wilting of crop plants is -1.5 mPa. At
what solute potential does the first serious wilting of the sunflowers occur?

Table 20-29. Physiological Drought.

CaCl2
(M)
0.00
0.01
0.02
0.03
0.05
0.10
0.20

Solution Water
Potential (mPa)

Water Loss
Detectable?

Plant
Appearance

17

D. Commercial Fertilizers
Most commercial fertilizers include on their label three numbers that express the percentage by
weight of nitrogen, phosphorous, and potassium, respectively. In addition, information is usually given
on the nature of the nitrogen source. If no information is given, then the nitrogen source is probably
nitrate, which is easily and rapidly taken up by most plants, but also rapidly washed out of the soil.
Soluble nitrogen includes nitrate, if present, and also "slow release" forms such as ammonium ions
and urea. Both of the latter become available to many plants only after being converted to nitrate by
soil bacteria, but both also tend to be retained longer in the soil than nitrates. Insoluble nitrogen
consists mostly of undecomposed protein. Most of it can be decomposed to soluble and usable forms
gradually by bacterial and fungal decay.
If fertilizer is being added to sandy soils that have poor mineral holding characteristics, slow release
forms of nitrogen are likely to be especially valuable. Not only is the nitrogen made available to the
plants gradually over a longer period of time, but less nitrate is likely to be leached into the ground
water, decreasing the likelihood of well contamination and surface water pollution.
Various commercial fertilizers are on display. Summarize information on their important nutrients in
Table 20 -30.
Table 20-30. Fertilizer Nutrient Composition.

Brand or Type

%N

%P

%K

Form of Nitrogen

E. House PlantsPreparation and Handling

For more information on how to propagate, care for and prepare soil for houseplants, see the
appendix.

18

KEY WORDS
kinetic theory

percolation

gas

field capacity

liquid

available moisture

solid

mineral

diffusion

macronutrient

chemical potential

micronutrient

differentially permeable membrane

hydroponic

osmosis

chlorosis

water potential

mobile minerals

megapascal

physiological drough

turgidity (turgor pressure)

wilting
plasmolysis
osmometer
imbibition
transpiration
guttation
hydathode
drought deciduous
stem succulent
leaf succulent
root succulent
Crassulacean acid metabolism (CAM)
sclerophylly
malakophylly
leaf rolling
bulliform cells
fenestration
high albedo
leaf hairs
sunken stomates
adsorption

Laboratory 22
Photosynthesis
Introduction
Most living organisms function either as primary producers or as consumers. Producers consume
some of the food they produce, but under normal circumstances, the rate of production exceeds the
rate of consumption. The producers are autotrophs (self-feeders), while the consumers are
heterotrophs. A few of the protista may live either way, depending on circumstances.
The overall accomplishment of photosynthesis may be summarized like this:
CO2 + H2O + [mineral ions] + light energy > food + O2 + H2O
We can emphasize the chemical balance by looking at the particular case of glucose production:
6 CO2 + 12 H2O + light energy > C6H12O6 + 6 O2 + 6 H2O
Review the structure of the chloroplast in Chapter 2 of this manual and the chloroplast model that is
on demonstration again. Consider the spatial progression from whole plant to leaf to parenchyma to
chloroplast to granum to thylakoid to pigment molecule (chlorophyll or accessory pigment).
The major events in the producing process of photosynthesis are:
Step 1: Absorption of light and transfer of energy to the electrons of the chloroplast pigments. This
occurs in the grana of the chloroplasts.
Step 2: Incorporation of that electron energy into the chemical compounds ATP and NADPH + H+.
These are not made from scratch, but by combining ADP with phosphate, and NADP + with electrons
and H+ respectively. These compounds are extremely reactive and are not well suited for storing
energy. This step occurs in the grana.
Step 3: Synthesis of enduring chemical compounds such as sugar. These products are made
primarily from CO2 and minerals and can be referred to collectively as food. The synthesis of these
compounds occurs in the chloroplast stroma and utilizes the energy carried by ATP and NADPH + H+
formed in step 2 above. The process of energy transfer degrades the ATP back to ADP and
phosphate, and the NADPH + H+ back to NADP+and H2O. Depending on the compounds produced
and on the plants specific situation, these chemicals can be used to 1) power the plants activities, 2)
built into cell structure, 3) exported to other parts of the plant, or 4) stored in place for reuse later.

Exercises
A. Tests for Photosynthetic Products: Starch and Sugar
1. Benedict's Test
Prepare three tubes as follows:
(1)

Add 5 ml of Benedict's solution to each test tube.

(2)

Then, to tube A add 2 mls of distilled water.

(3)

To tube B add 2 mls of a sugar (glucose) solution.

(4)

To tube C add 2 mls of a starch solution. Starch is a polymer of glucose molecules.

Label the three tubes and place them in water that is boiling. Observe for 2-3 minutes. A reddishbrown color (suspended precipitate) indicates the presence of sugar.

2
Observe the three tubes for several minutes. Does the starch solution begin to show a sugar test?
Why or why not?

2. Iodine Test for Starch

Place a pinch of starch on a slide. Add the iodine reagent (I 2KI). A blue color should develop.
Repeat, using a pinch of sugar. What happens?

The Benedict's and iodine tests are important for studying photosynthesis because photosynthesis
will produce sugar which can be stored in the starch polymer.

B. Requirement of Light for Photosynthesis

The following exercise uses the presence of starch as an indication of the occurrence of
photosynthesis. The leaf pigments must be removed in order to see the starch reaction with the
iodine reagent.
Exposed and Covered Leaves
Observe the demonstration. The control and test leaves were both exposed to light. The control leaf
should show a uniform distribution of starch. The test leaf was covered with a paper screen so that
some portion of it was not exposed to light. What is the distribution of starch in the test leaf?

Explain the results.

Is starch easily transferred from cell to cell?

3
Explain.

C. Photosynthesis Requires Chlorophyll

Control Leaf. A variegated Coleus leaf is used in this demonstration. Note the location of chlorophyll
and the area in which chlorophyll is absent.
Test Leaf. A variegated leaf was removed from a greenhouse grown Coleus plant. Since the leaf has
been exposed to light, photosynthesis has taken place. In order to extract the pigments, including
chlorophyll, the leaf was boiled in alcohol. The leaf was then placed in an iodine solution. Iodine and
starch combine to form a dark blue color. Photosynthesis produces glucose, and starch molecules
are made from glucose molecules. By observing the test leaf, determine the areas in which
photosynthesis occurred. What can you conclude?

Diagram the control and test leaves.

Figure 21-176. Chloroplast model.

Label the grana and stroma of the chloroplast in .

D. Separation of Photosynthetic Pigments by Paper Chromatography

Although simple in execution this experiment shows all the common photosynthetic pigments present
in the chloroplast. You should be able to separate chlorophylls a and b, carotene, and xanthophyll.
Isolating and purifying the samples of these pigments used later in these labs is much more difficult,
and the separation is not nearly as complete.
Procedure: Place a spinach leaf over a strip of filter paper. Using the end of the glass rod provided,
press hard on the leaf so the plant pigments are transferred to the filter paper strip about two inches
from the bottom of the strip.
Pour solvent into a test tube so it is about one inch deep. Place the filter paper into the solvent being
very careful to keep the green circle of pigments above the solvent. Cork the test tube to hold the
filter paper in place and to prevent evaporation of the solvent. The separation should be complete in
about 10 minutes.

5
Interpretation
Consider the role of the following in the separation of pigments.
(1)

Solubility of the pigment molecules in the solvent.

(2)

Affinity of the pigment molecules for the paper.

(3)

Size of the pigment molecules.

Can you explain why the separation occurred?

6
What pigments were present in the tissue extract?

What is the role of these pigments in a leaf?

E. Properties of Chlorophyll
1. The absorption of light
a. Pigment Color
Observe solutions of chloroplast pigments. Why are the solutions the color they appear to be?
That is, why is a green pigment green or a yellow pigment yellow?

b. Light Spectrum
A spectroscope is an instrument containing a prism that separates white light into the component
colors. Observe the spectrum of white light and record your observation.

7
c. Light Absorption by Pigments
Place the solution of chloroplast pigments (chlorophyll a, b, carotene and xanthophyll) and the
purified chlorophyll a, b, carotene and xanthophyll solution in front of the slit of the spectroscope.
What happens with each solution?

Is there a difference? Why?

d. Spectrophotometer Measurement of Light Transmission and Absorption

Use the Sequoia-Turner Model 340 Spectrophotometer to determine the percent transmission of
light by

chlorophyll a or b

carotene or xanthophyll

complete extract (all pigments present)

The solution in which the pigment is dissolved is the control or solvent blank. The blanks are
methanol for chlorophyll a and b; petroleum ether for carotene and the complete extract; and
diethyl ether for xanthophyll.
(1) Turn mode switch to TRANS. A 15-minute warm-up is needed.
(2) Set wavelength to 420 nm.
(3) [Stray light filter labeled 420-890 nm must be in place behind sample holder.]
(4) Insert blank into sample holder.
(5) Press and hold ZERO SET button while adjusting ZERO knob until display reads 00.0.
Release ZERO SET button. Zero is now set for all wavelengths.
(6) With the blank still in the sample holder, adjust 100%T COARSE knob to approximately 100.
Use FINE knob to make the setting exactly 100.0.
(7) Remove blank, insert pigment sample and record %T from the digital display.
(8) Replace blank, increase wavelength setting by 20 nm, and repeat steps 6 and 7 for each
wavelength setting until you have made measurements at 660 nm.
(9) If switching to a pigment sample that requires a different blank, return to step 4.
(10) Graph and explain the results.

Light Absorption by Photosynthetic Pigments

0

Inf*

10

1.00
0.71

30

0.52

40

0.40

50
60

0.30

70

0.22

80

0.16

90
100
420

Absorbance

Percent Transmission

20

0.10

440

460

480

500

520

540

560

580

Wavelength in Nanometers

600

620

640

0
0.05
660

0.00
* Infinite, or total absorbance

9
How do the spectrophotometer results relate to the wavelengths of light used in photosynthesis?

Explain the difference between transmission of light and absorption of light.

2. Fluorescence of Chlorophyll
Make the following observations by holding the test tube of chlorophyll close to the source of light
provided.
a. Transmitted Light
With the chlorophyll tube between you and the light, what color passes through the solution?

b. Fluorescence
Stand behind the light, with the light shining on the chlorophyll tube and away from you. What
color does the irradiated chlorophyll emit?

Use the knowledge that (1) chlorophyll captures light energy in excited electrons, (2) those electrons
have no place to go in a test tube of chlorophyll, and (3) energy loss can take the form of light
emission to explain these results.

10

F. Carbon Dioxide Use: Oxygen Release

Observe the demonstration in which some Elodea was placed in
(1) tap water
(2) boiled and then cooled water
(3) tap water enriched with CO2
Note that the water was boiled and then cooled before the plant was added. What does boiling do to
the water or the gases dissolved in the water?

Can you explain the results of this experiment?

How could you prove the gas collecting in the top of the test tube is oxygen?

G. Chloroplast Structure and Function in C3 and C4 Plants (A Brief Review)

Plants which make food solely by a universal series of reactions known as the Calvin cycle are called
C3 plants. This is because the first stable compound formed is a three-carbon compound
(phosphoglyceric acid, PGA). In the Calvin Cycle, carbon dioxide is essentially directly incorporated
into food. In these plants, the photoreactions take place in the grana and the sugar producing
reactions in the stroma of the same chloroplasts.
Food making efficiency of C3 plants is reduced by a phenomenon called photorespiration in which
oxygen competes with carbon dioxide in the food making reactions. To the extent that this occurs,
there is actually a loss in food content rather than a gain. It is not clear whether there is an overall
benefit resulting from photorespiration, but it certainly does reduce the amount of food that is made.
Photorespiration is particularly high when stomate closure reduces the carbon dioxide supply and
slows down oxygen export.

11
C4 plants have, in addition to the Calvin Cycle, a special set of reactions for capturing carbon dioxide
prior to actual food manufacture. The first stable compound formed is a four-carbon compound
(oxaloacetic acid). The captured carbon dioxide is then passed on to the Calvin Cycle in a manner
that avoids photorespiration. This accounts for the greater productivity of C4 plants.
In some, but not all, of these C4 species, there are two types of chloroplasts. One type is found in the
mesophyll of the leaf. It functions only in absorbing light and in trapping carbon dioxide by the extra
reactions. The four-carbon compound diffuses to the large, non-green, adjacent cells of the bundle
sheath where the carbon dioxide is transferred efficiently to the Calvin Cycle. Thus, the manufacture
of sugar takes place only in the bundle sheath cells, conveniently adjacent to the vascular tissue that
can carry it to other parts of the plant..
Observe the demonstration slides that show the anatomical differences between C3 and C4 plants.
Note that the C4 leaf has a sheath of cells around the vascular bundle which look different than the
other mesophyll cells. This is the wreath which was described in the leaf lab as Kranz anatomy.
Contrast the appearance of the corn mesophyll with that of a leaf from a C3 plant.

Do you think that cells that are biochemically distinct typically show visible differences?

Examples of C4 plants include maize, sorghum, sugar cane, Italian millet, crabgrass, and Bermuda
grass. Note that all of these are of tropical origin and that they are monocots. Some dicots such as
pigweed and Atriplex (a western weed) are C4 plants. Study the graphs of C3/C4 photosynthetic
response that are on display and be sure you understand their meaning.

12

KEY WORDS
primary producer
consumer
autotroph
heterotroph
chlorophyll
accessory pigment
ATP
NADPH + H+
granum (pl. grana)
ADP
phosphate
NADP+
Benedict's test
iodine test for starch
absorption spectrum
paper chromatography
spectroscope
spectrophotometer
solvent blank
fluorescence
Calvin Cycle
C3 plant
photorespiration
C4 plant

13

(blank page)

Laboratory 23

Figure 22-177. Mitochondria Structure.

Digestion and Respiration

Enzymes
I. Digestion and Respiration
Introduction
Living organisms obviously require a source of food. Autotrophs produce their own, heterotrophs
must supply their needs from some outside source. In times of abundance, organisms will store food
in forms such as starch, fats, or sometimes even proteins. Seeds are excellent food sources for
humans and other animals because of the large amounts of food they store for the use of the
developing seedling.
The term respiration is used to refer to the cellular processes that release energy from a food source
such as glucose. The process of digestion is the degradation (breakdown) of a large, often insoluble
form such as starch, into a material that can enter into respiration or be reassembled into other
compounds. When digested, carbohydrates yield sugars, proteins yield amino acids, and fats yield
fatty acids and glycerol.
Respiration is an oxidation process. A material such as glucose is oxidized by the removal of
hydrogens and their associated electrons. These are picked up by a hydrogen and electron
acceptor, usually NAD+, thereby converting it to NADH + H+ (the reduced form). Note that when the
food (glucose) is oxidized, the acceptor (NAD+) is reduced. The formation of NADH + H+takes place
both in the cytoplasm of the cell and in the matrix of the mitochondrion. See the model on
demonstration. Label Figure 22 -177.

2
The overall accomplishment of respiration is generally the reverse of photosynthesis and may be
summarized like this:
food + O2 > CO2 + H2O + mineral ions + chemical energy + heat
Focusing on the respiration of glucose, specifically:
C6H12O6 + 6 O2 + 36 ADP + 36 phosphate > 6 CO2 + 6 H2O + 36 ATP + heat
The part of aerobic respiration that yields most of the energy that is used in the production of ATP is
a series of electron transfers from molecule to molecule in the cristae of the mitochondria. Recall
that when light was absorbed in photosynthesis, the energy of electrons was increased. In the grand
scope of things, this is the energy that is released in respiration. Transparent beads on the inner
membrane of the mitochondrion model represent the electron transport system.
In the absence of molecular oxygen, respiratory oxidation is very incomplete. The energy yield is far
lower, and the products themselves may be somewhat toxic and difficult to get rid of. Respiration
without oxygen is referred to as anaerobic respiration or fermentation. Several different end
products can result, but this ethanol fermentation is especially common in plants:
C6H12O6 + 2 ADP + 2 phosphate > 2 C2H5OH + 2 CO2 + 2 ATP + heat
Note that the potential ATP yield of respiration is almost 20 times greater when oxygen is present.

Exercises
A. Stored Food: Starch
Prepare very thin slices of a raw and a boiled potato. Place the slice on a slide, add water and a
coverslip, and observe with your compound microscope. Look for starch granules within the cells.
Comment on the size and abundance of the starch granules. Also notice the successive layers of
starch deposited about the growth center (hilum) of the granule. Different types of starch granules will
have different arrangements of these markings or striations.
Now add a little dilute iodine (IKI) solution to the side of the coverslip and draw it through with filter
paper. Examine starch granules again. What happened to the appearance of the potato cells?

3
What happened to the appearance of the granules?

Figure 22-178. Starch Granules.

Label the parts of the starch granules in Figure 22 -178.

B. Amylase and Starch Digestion in Germinating Corn

Amylase is an enzyme that digests starch to glucose. The starch in a corn grain serves as a reserve
of food. Since starch is composed of many units of a simple sugar (glucose), digestion of starch
should free glucose. Test the germinated and ungerminated corn grains for glucose in the following
manner.

4
Tube A. Place a small amount of the ground germinated corn grains in a test tube. Add 5 ml of
Benedict's solution.
Tube B. Repeat the above procedure, using ungerminated corn grains.
Tube C. Add 1 ml of water to 1 ml of Benedict's solution. This will serve as the control.
Place the three test tubes in water that is boiling. Keep them there until you observe a color change
(usually 2 or 3 minutes). Observe the results.
Explain what has happened in both types of corn grains.

Why did this happen?

What would happen if you continued to boil the solutions for a very long period of time?

C. Respiration and Growth in The Presence and Absence of Oxygen Gas

Observe the demonstration of barley seeds growing in an environment with oxygen and in an
environment without oxygen. The seeds in the loosely capped container have oxygen gas available
for use in aerobic respiration. Oxygen has been chemically removed from the sealed container, so
seeds are restricted to anaerobic respiration. Knowing the difference in ATP production in aerobic
versus anaerobic respiration, which seeds should grow better?

Did they?

D. Production of Carbon Dioxide

1. Aerobic Respiration
Observe the respiration-train experiment on demonstration. It consists of five bottles with air moving
through in the direction indicated. The four bottles containing liquid were all completely clear at the
start of the experiment. The five bottles contain the following:
Bottle 1: Barium hydroxide solutionBa(OH)2
Bottle 2: Barium hydroxide solution
Bottle 3: Barium hydroxide solution
Bottle 4: Germinating peas
Bottle 5: Barium hydroxide solution
Barium hydroxide reacts with carbon dioxide as follows:
Ba(OH)2 + CO2 > BaCO3 + H2O
Barium carbonate (BaCO3) is insoluble in water and forms a white precipitate.
Explain the results.

What is the purpose of bottles 2 and 3?

Are there any other products formed in this process, and if so, what are they?

Is all the carbon dioxide produced a result of aerobic respiration?

2. Use of an Indicator to Show CO2 Production

Carbon dioxide in water will form carbonic acid (CO 2 + H2O > H2CO3). When this occurs, the pH
will be lowered due to the presence of the acid. The water reagent is pink in color because at the pH
of the solution the indicator phenol red is pink. If the pH is lowered, the indicator will change to a
yellowish color.
Fill each of three test tubes about one-fourth full with the phenol red solution. To the first tube, add
three or four living germinating seeds. To the second, add three or four killed germinating seeds. Use
the third tube as a control. It may take up to 15 minutes before the reaction is visible. Place in test
tube rack on your desk.
Explain the results.

3. Fermentation
Observe the fermentation tubes on demonstration. One tube contains Karo syrup and water, while
the other was prepared by adding yeast and Karo syrup to water. The tubes were completely full of
liquid at the start.
Explain the results.

How could you test to determine whether fermentation or aerobic respiration had occurred?

What gas is collecting in the tube?

What type(s) of respiration produce(s) this gas?

What food does the liquid contain?

What benefits does a yeast cell gain from fermentation?

Name a fermentation other than the alcoholic type.

E. Production of Heat During Respiration

Heat is released during respiration in all living cells. In this exercise, the amount produced by rapidly
respiring, germinating seeds is easily measured. Three thermos bottles are on demonstration and
contain the following materials:
Thermos 1: Germinating barley seeds
Thermos 2: Dead seeds (killed in formalin)
Thermos 3: Empty, to serve as a control

Results
Using the data from the temperature changes for each of the three thermos bottles, complete the
graph in Figure 22 -179. What effect will the growth of molds and bacteria have on the temperature?

Figure 22-179. Heat from Respiration.

40
T 35
E
M 30
P 25
C 20
15
1

TIME (days)

How does this information apply to putting wet hay into storage?

What accounts for the different results if stored hay is thoroughly dry?

9
How does this apply to a mulch pile?

II. Enzymes
Enzymes are proteins which act as a catalyst for (speed or promote) metabolic reactions. In order
for the enzyme to function, it must have the proper three-dimensional shape and chemical
configuration to match the physical and chemical shape of the substrate (the molecule whose
reaction it affects). Even the slightest alteration of internal configuration can result in a substantial or
complete loss of activity, even though the enzyme still consists of the same chemical parts. An
enzyme that has lost its catalytic nature is said to be denatured. Denaturing is almost never
reversible.

A. Effect of Extreme Temperatures on Enzyme Activity

Potatoes are rich in the enzyme tyrosinase. It catalyzes the breakdown of the amino acid tyrosine.
Catechol is also common in many plants, but its relevance here is that it is chemically similar to
tyrosine, and undergoes a color change in the presence of undenatured tyrosinase. The enzyme
preparations used for this experiment started out the same, but one has been frozen, then thawed,
and one has been boiled, then cooled. This enzyme oxidizes catechol to a colored compound. Use
the appearance of a darker color as a measure of enzyme activity.
Prepare the following tests in the test tubes provided:
(1) 5 ml of catechol + 5 ml of the fresh enzyme preparation
(2) 5 ml of catechol + 5 ml of the boiled enzyme preparation
(3) 5 ml of catechol + 5 ml of the frozen enzyme preparation
A control has been prepared in which 5 ml of distilled water was added to 5 ml of catechol. You must
compare the test results with the control results for this exercise to be meaningful. Describe
the resulting colors in Table 22 -31.

Results
Table 22-31. Effect of Extreme Temperatures on Enzyme Activity.

Fresh Enzyme

Boiled Enzyme

Frozen Enzyme

Control

10
Which enzyme preparation shows the most activity?

Does freezing denature the enzyme?

What does this tell you about the ability of plants to overwinter in a frozen soil?

Figure 22-180. Extracellular Enzyme Activity.

B. Extracellular Enzymes
Not all enzyme activities are confined within cells. Many fungi and bacteria secrete enzymes into their
immediate environment.

11
The Petri-dish that is on demonstration was filled with a starch-agar medium, the bacterium Bacillus
megaterium and the bacterium Staphylococcus aureus were added to a portion of the plate. Several
days later, iodine solution was added to test for starch. Recall that starch turns blue in the presence
of iodine. Make a diagram of the results of the staining with iodine in Figure 22 -180.
What type of reaction did the enzymes in this exercise carry out?

Of what value are extracellular enzymes to the organisms that form them?

C. Specificity of Enzymes as Shown by Carbohydrate Utilization

Four of the test tubes on demonstration contain the bacterium Salmonella in a nutrient broth medium.
Tube E contains only nutrient broth plus dye. There is only one kind of carbohydrate present in each
tube. The red color seen is due to the presence of an indicator dye (phenol red). In the presence of
acids, the dye color changes to yellow. The presence of a yellow color in the tube indicates that the
bacteria utilized the carbohydrate as a source of energy and released organic acids during its
breakdown. Enter your observations in Table 22 -32. (Refer back to Laboratory 7 for another
perspective on the same kind of experiment.)
Table 22-32. Carbohydrate Utilization by Bacteria.

Tube

Carbohydrate Present

Glucose (Dextrose)

Lactose

Maltose

Sucrose

Nutrient Broth + Dye (Control)

Which carbohydrates were utilized?

Which were not?

Dye Color

Any Gas
Produced?

12
How can these results be explained?

How could the nature of the organic acids produced be determined?

13

KEY WORDS
respiration
digestion
oxidation
starch
carbohydrates
sugars
proteins
amino acids
fatty acids
fats
glycerol
electron
electron acceptor
NAD+
NADH + H+
aerobic respiration
ATP
cristae
anaerobic respiration
fermentation
ethanol
amylase
respiration chain
fermentation tube
enzyme
catalyst
denature
extracellular enzyme (exoenzyme)
specificity
extracellular
aerobic
indicator

14

(blank page)

Laboratory 24
Control of Plant Growth
Introduction
The growth of any organism is controlled both by its genetic potential and by the environment in which
it is located. Plant growth and development can be described in terms of the results of the processes
of cell division, cell enlargement, and cell differentiation. There is general agreement that the
physiological processes are controlled by naturally occurring compounds called hormones. As a
result, plant growth discussions will include consideration of the various hormones, the impact of
environmental factors on the hormones, and the resultant influence on growth. The following
hormones will be studied in this laboratory section:

Auxins (IAA & IBA)

Abscisic Acid (ABA)

Cytokinins (CK)

Ethylene

Gibberellins (GA)

Some of the best-known plant responses involve growth of some plant part towards or away from a
stimulus such as gravity, light or even physical contact (as in a twining vine). Such directional
responses are collectively known as tropisms and they seem to typically be under hormone control.

Exercises
A. Gravitropism
1. Corn seeds between glass (Demonstration).
Observe the results.
How do stems react to gravity?

How do roots react to gravity?

Interpretation

Figure 23-181. Response of Plant Organs to IAA (after Thimann, K. V. 1937. Amer. J. Bot. 24:407412.)

Stem The stimulus of gravity is thought to be received within cells by structures that are large enough
to settle out toward the bottom of the cell. These large structures are called statoliths. Starch grains
in plastids (amyloplasts) are examples of statoliths. The statoliths influence auxin (IAA) distribution
by causing an accumulation of auxin on the bottom of the stem. This concentration of auxin
stimulates growth enough so that the bottom now grows faster than the top, resulting in an upward
curvature of the stem. Since it grows away from the stimulus of gravity, this is called a negative
gravitropism. Refer to Figure 23 -181.
Would the part of the stem exposed to an auxin concentration of 10 -6 grow faster or slower than one
with a concentration of 10-8?

Root The response of roots to gravity seems to be controlled by the ratio of IAA to the inhibitor
abscisic acid (ABA). Again, the statoliths influence the distribution of the hormones so that the
higher concentrations are on the bottom. Growth on the bottom of the root is inhibited compared to
growth on the top, causing the root to curve downward. This is a positive gravitropism.

2. Seedlings
Place a potted sunflower plant so that the stem is horizontal. Observe throughout the period. How
long does it take for the response to begin?

3
Explain the results.

B. Induced Curvature (Demonstration)

The explanation in Exercise A relies on an unequal distribution of hormone caused by the
environment (gravity). In this exercise, an unequal distribution was created by adding IAA, dissolved
in lanolin to keep it from evaporating away, to the tissues.
Lanolin paste containing IAA was added to one side of the coleoptile tip. Observe the coleoptile and
diagram the results. Explain what happened.

C. Phototropism
Observe the corn coleoptiles exposed to various colors of light and record the results.
Table 23-33. Phototropism Response by Color of Light.

Color of Light

Plant Response

Blue
Green
Red
Far-red
Which color(s) seem to cause the coleoptiles to grow toward light? Why?

Interpretation
In order for light to influence a process, it must be absorbed by some pigment(s) in the plant. Since
blue light is most influential in phototropism, some pigment must be present that absorbs light in that
area of the spectrum. Several carotenoid pigments and a B vitamin have absorption spectra that
match the action spectrum quite closely.
Refer to Figure 23-182. Action Spectrum for Phototropism, on display in lab, to see the results of
detailed investigation of phototropism in two very different organisms.

4
The light that is absorbed must somehow influence auxin distribution to give the observed response.
Explain what that distribution must be and why the plant grows the way it does.
What is the significance of phototropism?

D. The Phytochrome Pigment System.

Phytochrome is a pigment present in very small amounts in plant shoots. It forms a critical part of
the system that enables plants to respond to seasonal changes in day length. Plants which reliably
flower in spring or fall, or which leaf out or drop their leaves at almost the same calendar date every
year, use the phytochrome system as a mechanism to time the photoperiod (day length vs night
length). Light stimulates a conversion of phytochrome from an inactive form known as
phytochrome660 (P660) to an active form known as phytochrome730 (P730). In the dark, tissue levels of
P730 gradually decline and P660 gradually accumulates. The result is a system that responds not to the
length of the day but rather to the length of the night.
Synthesis P660 (inactive)

light

P730 (active)Breakdown

dark?

The subscripts refer to the wavelengths of light that are most active in stimulating conversion between
the two forms of phytochrome. Radiation with a wavelength of 660 nm is visible red, can be used in
photosynthesis, and is strongly absorbed by chlorophyll as you demonstrated in lab 21. Radiation
with a wavelength of 730 nm is not visible, has insufficient energy for photosynthesis, and is poorly
absorbed and strongly reflected by broad-leaved plants. It also specifically and rapidly drives P 730 to
the P660 form.
P660 (inactive)

660

730

P730 (active)

Whether or not the back conversion to the inactive form is significant in nature is a matter of
speculation.

1. Photoperiodism
Plants that reliably flower in the spring or in the fall may use the phytochrome system as a timing
mechanism. In the spring or fall, though, they may be responding to something else such as warming
of the soil and air. How can you distinguish between day length and temperature responses? Does
the phrase It was an early spring this year! give you a hint?

Plants that respond to day length in any of their responses are said to be photoperiodic. Collaborate
with other people at your end of the table in listing some plants that are probably photoperiodic:

2. Seed Germination
The seeds in this exercise were moistened, placed in darkness for one day, and then treated for two
days. Observe and record the percentage of germination of lettuce seeds in Table 23 -34.
Table 23-34. Germination of Light-Sensitive Lettuce Seeds.

Chemical Treatment
Light Treatment

Water

ABA

GA

Darkness
White light
Red light
Far-red light
Interpretation
Some types of seeds, such as this variety of lettuce, have their germination influenced by light. The
seed received the stimulus of light via phytochrome.
The phytochrome must then influence the production of germination promoting materials (such as
gibberellin) or of germination inhibiting materials (such as ABA). What hormone might be found in
seeds to keep them dormant?

Which would favor germination of these seeds, P 660 or P730?

E. Etiolation
Observe and compare the plants grown in the light and in the dark in terms of:
(1) stem length
(2) internode length
(3) color
(4) leaf size
(5) strength of stem
Plants with the characteristics of the dark-grown plants are said to be etiolated. What is the
significance of this type of growth for a seed that has been planted deep in the soil?

What about a plant growing in deep shade?

F. Ethylene and Epinasty of Tomato

Observe the three bell jars.
Jar 1. Control. Contains a tomato plant in normal air.
Jar 2. Ethylene added. This contains a tomato plant growing in air supplemented with
ethylene gas.
Jar 3. Ripening fruit added. This contains a tomato plant plus a ripening banana.
How do the tomato plants in jars 2 and 3 compare to that in jar 1?

7
How do the plants in jars 2 and 3 compare to each other?

What is the importance of the banana?

How do grocery suppliers control the ripening of bananas?

What does ethylene do to the normal pattern of growth?

G. Apical Dominance
Observe the pea plants on demonstration. Peas have compound leaves, and the distinction between
leaf and stem is critical to understanding the experiment, so be sure to look carefully. Note that some
of the plants in this experiment have not been treated, some have had their growing tip removed, and
some have been de-tipped but supplied with supplementary material. Record your observations in
Table 23 -35.
Table 23-35. Apical Dominance.

Treatment

Bud Development

Control (Intact Plant)

Tip Removal
Tip Removal Plus Lanolin
Tip Removal Plus Lanolin and IAA
Did the experiment work out as one would expect?

Interpretation
In many plants IAA produced in the apical meristem inhibits the growth of lateral buds below.
Removing the growing tip removes the source of inhibiting auxin. Artificially supplying the missing
hormone maintains the inhibition. Lanolin is merely a substance that holds the hormone in a
diffusable condition yet prevents the cut tip from drying out.
In some cases cytokinins will overcome the influence of IAA, thereby allowing lateral buds to grow,
though that isnt demonstrated by this particular experiment. Don't confuse apical dominance with
winter dormancy. Winter dormancy of buds is sometimes due to the presence of large amounts of
the inhibitor ABA (Abscisic Acid).

H. Rooting of Cuttings
Bean stems were cut above the level of the roots and placed in the following solutions:
(1) Distilled water
(2) Distilled water plus nutrients
(3) Distilled water, nutrients, and 0.1 mg IAA per liter.
(4) Distilled water, nutrients, and 1.0 mg IAA per liter.
Compare the four tests in terms of both number and length of roots. Count each little bump as a root.
These are actually root beginnings called primordia. Complete Table 23 -36.

9
What are the best conditions for root
initiation?

Table 23-36. Influence of Hormones and Nutrients

on Root Initiation.

Number of Roots

Length of Roots

1
2
3
4

For root elongation?

Discuss the value of this knowledge.

I. Selective Herbicides
Bean and corn seedlings were grown in each of the pots on demonstration and treated in the
following manner on the date listed on the pot:
Table 23-37. Effects of a Selective Herbicide.

Treatment

Flat

Distilled Water (Control)

Bean
Corn

Results

10

.05% 2,4-D

Bean
Corn

.2% 2,4-D

Bean
Corn

.5% 2,4-D

Bean
Corn

11
What selectivity is demonstrated by 2,4-D?

Why? (How is such selectivity achieved?)

Interpretation
Herbicide selectivity is controlled by factors such as:
(1) uptake into the plant
(2) movement within the plant
(3) detoxification or excretion from the plant
(4) Observe the pictures showing responses to various herbicides.

J. Turgor Movements
All plant movements studied so far in this lab are growth movements and slow. Whenever a plant
moves fast, its probably a turgor movement. Turgor movements typically involve sudden loss of
turgidity by strategically located cells called pulvini. The reversal of such movements is likely to be
much slower, since they will depend on osmosis to gradually restore the lost turgidity.

1. Mimosa
Touch one leaflet. Note the response. Touch the entire leaf. How does this response differ?

Locate the pulvini.

2. Venus fly trap

Observe the tactile hairs that must be touched to get a response.

3. Sundew
Observe the sundew demonstration.

12

KEY WORDS
cell division
cell enlargement
cell differentiation
tropism
gravitropism, positive and negative
statolith
auxin
IAA (indole acetic acid)
stimulate
abscissic acid (ABA)
inhibit
coleoptile
ethylene
abscisic acid (ABA)
coleoptile
phototropism, positive and negative
absorption spectrum
action spectrum
carotenoids
phytochrome
photoperiod
phytochrome660
phytochrome730
gibberellins
etiolation
ethylene
epinasty
apical dominance
cytokinins
winter dormancy
selective herbicide
2,4-D (2,4-dichlorophenoxyacetic acid)
turgor movement

13

pulvinus (pl. pulvini)

cytokinin (CK)
phytochrome

A-1

APPENDIX. Growing Plants

Growing Houseplants
Houseplants are vitally affected by the environment in which they live--factors that include containers,
soil, water, fertilizer, light, temperature, and pest control. Many plants thrive under a wide range of
conditions. The beginner should select hardy plants that are easy to grow with little care. then add
rarer, more difficult plants as one gains experience.

Pots
Potting and repotting plants concerns every owner of a greenhouse or window garden: unless it is
carried out correctly, the plants cannot make satisfactory progress. There are a variety of pots. For
best results, however, clay pots are still preferred by many growers because the porous nature of clay
allows air to reach the plant roots. Even painting a flowerpot is not recommended since paint clogs
the pores. Old pots must be cleaned thoroughly by washing them with a scrubbing brush in hot water.
There must be an opening in the bottom of the pot for drainage. "Crocks," pieces of broken
flowerpots, are placed over the opening on the bottom of the pot to prevent soil loss yet allow for
drainage.

Soil
The soil used for potting of most plants consists of two parts of loam and one part of peat moss or leaf
mold with a free scattering of coarse sand free of fine matter. The addition of bone meal at the rate of
1 lb. to each bushel of potting compost is usually advisable, and sometimes other fertilizers are
added. The ingredients of the potting compost must be mixed thoroughly. For seedlings, the
compost should be passed through a fine sieve; but for larger plants this process can be skipped.
Use only sterilized potting media to avoid soil borne insect problems.
The depth of the plant in the pot must be set so that the uppermost roots are covered with not less
than half an inch of soil; but space must be left at the top of the pot for watering.

When to pot
Bulbous, succulent, and slow growing plants do not need repotting for several years. In general, as
long as plants of the above mentioned types are prospering and can be provided adequate supplies
of nutrients by fertilizing them periodically (by top-dressing), repotting is unnecessary. Big plants in
large pots or tubs require repotting much less frequently than young specimens. Certain plants, for
instance calla lilies, benefit from being repotted annually. Yet others, especially annuals and plants
that are raised anew from cuttings each year are potted two or more times each season.

How to pot
Methods of potting vary slightly for different types of plants. A general rule is that when leafy plants in
active growth are potted, as much soil as possible must be retained about their roots and every care
must be taken to see that the roots suffer minimum disturbance and injury. When plants are in a
dormant state, it is practicable to prune back the tops more or less severely and thus reduce the
amount of top growth and foliage. This has two possible benefits: preventing a plant from exceeding
the space available to it, and decreasing the chance of transplant failure caused by excessive foliate
water loss. When moving into a larger pot, remove the plants with soil attached. You may remove old
soil from top of the ball and some along the side. At the same time prune off dead leaves and any
that are not healthy. Also you may prune roots some. Clean the plant of insects. Then place in a
new pot with new soil to fill up the pot and placing the ball so it is covered with 1/2 inch of soil. Be
sure to center your plant. Firm the soil around the plant.

A-2

Watering
Plants should be watered thoroughly a few hours before they are repotted, to make sure that the ball,
the mass of soil and roots, is moist throughout. When potting is finished, the soil should again be
watered, with the water applied in a fine spray. If using a brand new clay pot, water sufficiently
because the clay pot itself will absorb large amounts of water. Increase the frequency of subsequent
waterings until the roots have penetrated the new soil. After that, the soil should be allowed to
become moderately dry between waterings. Too frequent watering following potting is likely to sour
the soil and kill the roots by saturating the air spaces that are necessary for healthy roots. Do not
allow water to stand for long periods of time in a container under the pot. That would also prevent
proper aeration of the roots. When watering plants in the winter use lukewarm water to prevent
thermal shock. The important point to remember is that the soil in the pot should be watered often
enough to keep it moist and to maintain the plant in a vigorous growing condition, but infrequently
enough to allow good soil aeration.

Fertilizing
During seasons when plants are growing actively, fertilizer must be applied at frequent intervals to
keep the plants healthy and vigorous. Many plants, however, go through an inactive period each
year, typically corresponding to the short days of late fall and early winter. At this time, fertilizing
should be less frequent.

Growing Conditions
The amount of light needed depends upon the type of plants you are growing. Most plants need
more light than exists in a typically lighted room far from a window. At the other extreme, Many house
plants need to be protected from direct mid-day sun. The proper temperature for most house plants
is between 60 and 75 degrees for the best growth. Good air circulation is important too. Washing
the dust off leaves at frequent intervals will help the plants. Humidity for most plants should be
between 50 and 60%. Indoor winter air probably represents the greatest threat to survival of many
houseplants in our climate. A humidifier or frequent misting can help enormously. On the other hand,
persistently high humidities can promote plant disease.

Insect control
Insects can become a problem, especially when you accumulate a large number of plants in the same
room. If pesticide sprays are used to control insects, care should be taken to match the treatment to
the problem. The life cycle of the pest you are trying to control may call for a particular spraying
frequency. Some plants may be vulnerable to some pesticides. Finally, many pesticides are simply
unsafe for indoor use. Any use of pesticides should strictly follow label specifications.

Terrarium
To plant a terrarium, begin by placing a layer of either pebbles or broken bricks in the bottom to
ensure drainage. Cover this with moss or dead tree leaves to prevent the soil from washing through
and clogging the drainage passages. On top of this base, place some soil (an open, porous, gritty
mixture of topsoil, peat moss or humus and coarse sand or grit). Vary the depth of the soil in different
places to give a pretty landscape effect but do not let it be so deep anywhere that it detracts from the
decorative appearance of the finished terrarium. Small pieces of rock, pieces of cork, bark, etc, may
be introduced to suggest landscape features and, if desired, a "pool" made of a small mirror may be
featured.
When the general contours of the miniature landscape are established, planting may begin. A few
hours before planting is done, the plants to be used should be watered thoroughly. It is well to set the
bigger plants first, then the smaller ones, and finally creepers, mosses and other ground covers.
Do not crowd the plants unduly; remember that if all goes well they will grow and need more space.
After planting is completed, water gently with a fine spray and wash all leaves clean of soil and the
glass clean of smears. Be careful, when watering, not to flood the terrarium, especially if it is one that
does not have drainage holes.

A-3
Where natural light is deficient the plants contained in a terrarium will make much better growth and
will remain healthier if the terrarium is illuminated for 12-16 hours out of each 24 by artificial light.
Fluorescent units give considerable light without generating much heat and should be depended upon
as the chief source of illumination. The lights may be as close as 12 inches to the plants. Because
fluorescent light is somewhat deficient in red light, superior results may be had by supplementing it
with a smaller amount of light from ordinary incandescent bulbs.

Propagation
There are a variety of methods of propagating plants. Some of the more common ones are cuttings,
division, separations, grafting and budding and, of course, seeds.

Cuttings
Cuttings are one of the commonest, quickest, and easiest methods of increasing plants. The naming
of the various types of cuttings is based upon the parts of the plant from which they are formed and
upon the softness or firmness of the parts at the time the cuttings are made. It is possible to
propagate almost any cutting from any part of the plant as long as there are active living cells present,
but in practice some plants dont propagate well by cuttings. Its probably good to avoid those plants
that require special techniques until you are experienced enough to want a special challenge.
Cuttings are normally taken from (a) the stem or branch system, (b) the root system, or (c) the leaves.

Rooting Medium
(a) Water-plants root readily and can be transferred directly from water to potting soil. (b) For the
majority of cuttings, however, a well-aerated rooting medium, kept moist, is preferred, such as a
sharp gritty sand. Plants started in sand must be moved to a fertile soil once the cuttings are rooted.
(c) A mixture of sand and peat moss is an excellent rooting medium, particularly for plants that like
somewhat acid soil. (d) Horticultural grade vermiculite gives good aeration with better moisture
retention than sand. Cuttings that take a long time to root may be placed in a rooting medium of finely
chopped sphagnum moss or a mixture of sphagnum moss and sand in the proportion of 1 to 2. The
rooting medium should be firmed before planting the cuttings and then packed about the base of each
cutting.

Preventing Water Loss

Since cuttings begin with no roots, special efforts must be made to prevent them from drying out until
roots become established. To decrease water loss from the cuttings, remove some of the foliage and
all blossoms and provide shading, especially during the hottest part of the day. A simple safeguard is
providing a closed atmosphere with a glass or plastic lid supported 4-6 inches above the surface of
the medium, Be sure the medium is kept well-watered. Still better is a propagating case consisting of
a small wooden flat covered with clear polyethylene supported by a wire frame. Such a case can be
placed over a source of gentle heat or provided with inexpensive electric soil-heating cables that are
manufactured for that purpose. Such a propagating case provides moist and warm soil and air, all of
which promote the establishment of the cutting. This case should be placed where the plants can get
moderate light until they have rooted.

Preparation for Cuttings

Cuttings are best made with a razor blade or very sharp knife to minimize the chance for parasitic
fungi to invade. Cuttings should be well watered with a fine spray as soon as they are inserted.
Rooting can be quickened with root-inducing, substance (typically plant hormones)--by dipping the
base of the cutting in the powder or liquid. Strict sanitation of the cutting bed is extremely important in
order to prevent infections. Either throw away old media or sterilize it before re-use by using some
soil-sterilizing agent such as formaldehyde, Semesan or other mercury compound prepared for the
purpose. Note that soil-sterilizing chemicals often require special handling and disposal!

A-4

Cutting Sources
Stem cuttings of greenwood or soft-shoot plants are usually made from the terminal part of the stem;
hardwood stem cuttings often come from repeated sections of a young stem.
Leaf cuttings can be part or all of a leaf. If the petiole is not included, insert the bottom edge of the
cutting into the rooting medium. In the case of succulent leaves or stems only, let the cutting sit on a
dry surface for 24 hours before inserting in the rooting medium to decrease the chance of infection.
Leaf-bud cuttings are single leaves with a small piece of stem and the axillary bud. Bulb-cuttings are
obtained by quartering a large mature bulb with a sharp knife lengthwise. These can be subdivided
into more wedges, but be sure each section includes a part of the bottom or basal plate of the bulb.
Plant the bulb-cuttings in a vertical position.
Root cuttings consist simply of 1-2 inch lengths of stout roots. Plant root cuttings by inserting them in
an upright position in compost of half peat moss and half sand in early spring or in the fall.

Division
Division is one of the simplest methods or propagating plants. It is employed in increasing many hard
herbaceous perennials and rock garden plants, and a few shrubs as well as a good many pot plants.
Division consists of breaking up old plants or clumps into several rooted pieces, and replanting them
at once, or rooting them first, if necessary. The best and most vigorous pieces are found on the outer
parts of the older plants, and they alone should be chosen; the inner, old parts should not be used.

Separation
Separation refers to propagating plants by the use of naturally detachable vegetative parts such as
bulblets and other offsets, bulbils and plantlets. (Division is done by force in circumstances where
there are no natural lines of cleavage.) Most bulbs, corms, and tubers produce against or close to
themselves young bulbs (bulblets), young corms (cormels) or young tubers (tubercles). They should
be removed from the parent plant during the dormant season. They should be stored in a cool place
until the new growing season starts. Plantlets (tiny, easily detachable plants that develop on the
leaves or other-above ground parts of the parent plant) can be removed and planted when they reach
sufficient size. Start them in light, sandy soil in pots, flats, cold frames or outdoors, depending upon
the kind of plant.

Seeds
In order to be successful in raising plants from seeds, whether indoors or outside, one has to provide
adequate moisture, warmth and aeration. Of course, one must have viable seed, too. Under glass
one can better control the conditions that induce quick germination. Ideal germination temperatures
vary with different plants, but generally a temperature of 50-65 degrees is good. A good seed-sowing
compost consists of 2 parts (by bulk) of loam, 1 part of peat, and 1 part of sand to which are added 1
1/2 oz superphosphate and 3/4 oz ground limestone per bushel of soil. Timing is a consideration if
plants are to be started indoors, then transplanted outside. Plants started prematurely in the long
days of spring may be too large for transplanting when the danger of frost is over. If indoor-raised
seedlings are to be transplanted outdoors, it is a good idea to transfer their flats or pots outside in a
protected place for a couple of days before doing the actual transplanting.

Laboratory 25
Plant Genetics
Introduction
In the previous laboratory, you learned how to determine gamete and offspring ratios from various
crosses. In this lab, you will observe phenotypes in offspring populations and relate your
observations to expected results.
Two standard crosses for genetic analysis are monohybrid and dihybrid crosses. In this use of the
word, hybrid refers to a heterozygous condition. Therefore, a monohybrid is heterozygous for one
gene and a dihybrid is heterozygous for two.
You are familiar with dominance, but you will also see traits that are controlled by genes that exhibit
incomplete dominance. In this situation, the phenotype of the heterozygote is intermediate to that of
the homozygotes.
* Enter these numbers into the computer
spreadsheet.

Table 5-38. Count Summary, F2 Generation,

Monohybrid Dominant/Recessive Cross, Kernels.

Row

Purple

Yellow

1
2
3
4
5

Total
Counted

6
Total

Proportion

Exercises
A. Monohybrid Cross
1. Corn Kernels
You will be observing an ear of corn that was produced when a heterozygous female plant was
pollinated by a heterozygous male plant. The phenotype (purple vs. yellow kernels) is controlled by a
single gene that exhibits dominance. Count the number of purple and yellow kernels on six rows and
record your data .
Be sure to use the same rows for counting both purple and yellow kernels.

*Divide the number of kernels of a color by the total number of kernels.

Which allele is dominant, the one for purple or the one for yellow color?

Fill out , based on your observed results and those expected for a Yy x Yy cross. Base your
expected results on gamete genotypes and proportions and on what genes are dominant. You may
want to set up a Punnett square to organize your thinking.
Do the observations you entered in match your expectations?

How could you improve your chances of obtaining observed numbers that are very close to the
expected ones?

You can use a goodness of fit (chi-square, 2) test to determine whether the difference between
your observed and expected numbers are statistically significant.

(observed number expected number) 2

expected number

Calculations:

If your 2 value is greater than 3.841, then the difference between the observed and expected values
is large enough to support the conclusion that that the data do not support your hypothesis of a 3:1
Table 5-39. Analysis of Results, F2 Generation,
Monohybrid Dominant/Recessive Cross, Kernels.

Phenotype
Observed Number
Observed Proportion*
Expected Proportion**
Expected Number

Purple

Yellow

Total

ratio. A 2 value of < 3.841 indicates that a 3:1 ratio is an acceptable hypothesis. It does not,
however, rule out other hypotheses that may also be acceptable.

2. Corn seedlings
Observe the flat of corn seedlings that represent the offspring of a monohybrid cross. What trait are
you characterizing?

Which allele is dominant?

Table 5-40. Analysis of Results, F2 Generation,

Monohybrid Dominant/Recessive Cross, Corn Seedlings.

Phenotype

Green

White

Total

Observed Number
Observed Proportion
Expected Proportion
Expected Number

Count the number of seedlings of each phenotype and fill out as you did with the kernel phenotypes.

Did your observations match your expectations?

Use a 2 again test to determine whether your data support your hypothesized ratio.
Calculations:

Do you suppose the white seedlings, which lack chlorophyll and cannot photosynthesize, are able to
produce seeds?

If not, how is the allele for white color passed from one generation to another?

What were the genotypes and phenotypes of the grandparents of the seedlings in the flat?

3. Seedlings
Observe the flat of seedlings from a monohybrid cross. What phenotypes do you see? Look closely,
there should be three.

How do you suppose plant color in these soybean seedlings is genetically controlled?

Based on your observations and prediction of genetic control of plant color, fill out :
Table 5-41. Analysis of Results, F2 Generation,
Monohybrid Intermediate Inheritance.

Phenotype

Light
Green

Medium
Green

Dark
Green

Total

Observed Number
Observed Ratio
Expected Ratio
Expected Number
Test for goodness of fit with a 2 test. In this case, you will need to compare your reference value for
significant differences to 5.991 rather than 3.841. As the number of phenotypic classes increases, so
does the number that is used to compare with your 2 value.
Calculations:

B. Dihybrid Cross
1. Seeds
Observe a second ear of corn that was produced from a dihybrid cross. What are the two
characteristics that are exhibiting variation?

Again, count six rows and record your results in . Be very sure that the ear you use has shrunken,
raisin-like kernels. Kernels with slight depressions should be counted as plump.
Table 5-42. Count Summary, F2 Generation, Dihybrid Dominant/Recessive Cross, Corn Kernels.

Row

Purple
Plump

Purple
Shrunken

Yellow
Plump

Yellow
Shrunken

1
2
3
4
5

Total
Counted

6
Total

Proportion
* Enter these numbers into the computer spreadsheet
Which traits are dominant?

Fill out , based on your observed results and those expected for a YyPp x YyPp cross.
Table 5-43. Analysis of Results, F2 Generation,
Dihybrid Dominant/Recessive Cross, Corn Kernels.

Phenotype

Purple
Plump

Purple
Shrunken

Yellow
Plump

Observed Number
Observed Proportion
Expected Proportion
Expected Number
How closely do your observed and expected numbers match?

Yellow
Shrunken

Total

Calculate a 2 value and compare it to 7.815, which defines significance differences if there are four
phenotypic classes.
Calculations:

Do your data match more or less closely than those from the monohybrid cross?

Try to explain the difference in 2 results, if any, between the monohybrid and dihybrid experiments:

2. Seedlings
Observe the flat of seedlings segregating for two traits. List the traits involved. For example, the
traits involved in the previous exercise were purple kernels, yellow kernels, plump kernels, and
shrunken kernels.

Which traits are dominant?

Fill in .
Table 5-44. Analysis of Results, F2 Generation,
Dihybrid Dominant/Recessive Cross, Corn Seedlings.

Phenotype
Observed Number
Observed Ratio
Expected Ratio
Expected Number
Calculate a 2 value and compare it to 7.815.
Calculations:

On the average, we expect your observed numbers to deviate from expected more than they did with
the kernels. Suggest a reason for the increased deviation.

C. Molecular Genetics
1. Transcription and Translation
RNA is a nucleic acid that functions to transfer the information coded in the DNA of the genes to the
site where proteins are synthesized (ribosomes). Observe the models that demonstrate the
production of an RNA copy of a gene (transcription) and the translation of the RNA message into a
protein. How do proteins determine phenotype? This is not a casual question!

How do you suppose the allele for yellow tomato color differs from that for red color?

2. Genetic Engineering
Because the genetic code is essentially universal, it is possible to transfer a gene from one organism
to another and allow the recipient organism to produce the protein coded by the gene. Genes from
foreign organisms have been introduced into plant cells using bacterial pathogens, viruses, and even
microscopic shotguns. These techniques have resulted in plants expressing genes that protect them
against the virus, plants producing a bacterial toxin which kills insect pests, and plants that are
resistant to herbicides.
Bacteria are the workhorses of genetic engineering research. In addition to chromosomes, they carry
small circular pieces of DNA, called plasmids. It is possible to splice foreign DNA into plasmids and
then allow the bacteria to take them up. These bacteria will now express the foreign gene and make
the gene product.
Observe the demonstration of bacterial transformation. Chromosomal DNA from a bioluminescent
(light producing) bacterium (Vibrio fischeri), was cut into small pieces and the pieces were inserted
into plasmids. A bacterium that is not bioluminescent (Escherichia coli) was then allowed to take up
the plasmids. The blue colonies are E. coli bacteria that took up a plasmid in which no foreign (V.
fischeri) DNA had been inserted. The white colonies contain a plasmid that carries foreign genes.
Glowing colonies are expressing the genes for bioluminescence. Since E. coli is not naturally
bioluminescent, these colonies represent genetically engineered bacteria expressing a foreign gene.
Are there any blue glowing colonies? Why or why not?

Why don't all white colonies glow?

3. DNA Extraction
DNA molecules are relatively easy to extract from cells. In the following procedure, you will isolate
DNA from onion cells. First, you will free the DNA from the cells using chemical and mechanical
means. The liquid you extract will contain DNA dissolved in water. However, DNA is insoluble in
alcohol, so it can be precipitated out of solution by adding ice cold ethanol. Why do you suppose the
DNA precipitates better if the ethanol is cold?

Because DNA molecules are very long, they can be spooled up on a rod and pulled out of the alcohol.
If the DNA extraction procedure is very rough, the DNA may be sheared and will not be able to be
spooled.
(1)

Place approximately 12 g of onions and 50 ml of warm (60 C) homogenizing in a morter

(2)

Crush onion, in homogenizing medium, with a pestle to mechanically break the cell walls.

(3)

Incubate at 60 C for 15 minutes.

(4)

Filter into the beaker through 4 thicknesses of cheesecloth. Save the filtered liquid.

(5)

Place the extracted liquid on ice for 15 minutes.

(6)

Mix 20 ml of the extracted liquid and 40 ml of ice-cold 95% ethanol.

(7)

Place the mixture on ice for 2 minutes.

(8) Collect DNA by rolling a glass rod in the solution. Be sure to roll the rod in only one direction.
The DNA will appear as a slimy substance around the rod.
Describe the consistency and color of the DNA. Is it what you expected?

If you handle the DNA roughly during the extraction procedure and shear (break) it, how would your
results during the spooling step be different?

KEY WORDS
monohybrid
dihybrid
incomplete dominance
chi-square test (2)
RNA (ribonucleic acid)
transcription
translation
plasmid
bioluminescent

Progress Note
The laboratories up to here have dealt with techniques and characteristics relevant to most living
things, no matter what their classification. The next ten laboratories comprise a survey of particular
kinds of organisms which are more or less plant-like: The first labs focus on bacteria, then fungi, most
of which live lives devoted to decay or disease. Lab 10 deals with a potpourri of more or less plantlike organisms that almost defy classification. Labs 11 through 15 deal with a series of increasingly
familiar green plants.

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