BIOL 3601: PLANT DIVERSITY
DR. JULIE R. ETTERSON
FALL 2013
Lab 13
Seedless Vascular Plants
DOMAIN Eukarya
KINGDOM Plante
PHYLA Lycophyta
Psilotophyta
Sphenophyta
Pterophyta
Lycophyta
Lycopodiophyta
A club moss
Pteridophyta
A whisk fern
As their name implies, the vascular plants, unlike the bryophytes,
are characterized by the presence of the vascular tissues xylem
and phloem. Their sporophytes, the dominant generation of
vascular plants, have greater reproductive potential than the
bryophytes because they can branch profusely and produce
multiple sporangia. In contrast, each bryophyte sporophyte,
which is dependent upon the dominant gametophyte, is
unbranched and produces a single sporangium.
Pteridophyta
A fern
The vascular plants can be divided artificially into two major
groups, the seedless vascular plants, and the seed plants. There
are four major phyla of seedless vascular plants: The
Psilotophyta, the Lycophyta, the Sphenophyta, and the Pterophyta. The first three phyla, often
referred to as the "fern allies," have few living representatives although they are well
represented in the fossil record.
EXERCISE 1:
Phylum Lycophyta
Genera: Isoetes: Quillwort
Lycopodiaceae: Club mosses
Selaginella: Resurrection plant
The living representatives of the Lycophyta are all relatively small plants, with true roots, true
stems, and true leaves. Among the fossil members of this phyla, however, are woody, treelike
forms, which numbered among the dominant plants of the coal-forming forests of the
Carboniferous periods. Most gametophytes of the Lycopodiaceae are subterranean, and hence
they are difficult to find.
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BIOL 3601: PLANT DIVERSITY
DR. JULIE R. ETTERSON
FALL 2013
Isoetes – Quillwort
Quillworts are the nearest living relatives of the ancient tree Lycophytes of the Carboniferous
period. These plants tend to be aquatic but can grow in vernal pools. The sporophyte of
Isoetes consists of a short, fleshy underground stem bearing quill-like microphylls on its upper
surface and roots on its lower surface. In Isoetes, the leaves are attached to a corm like
structure (a fleshy stem) that is difficult to interpret morphologically. Dichotomously branching
roots arise from the lower portion of the stem.
Examine living specimen of Isoetes.
Are these leaves microphylls or megaphylls?
Where are the sporangia?
Examine the prepared slide of a longitudinal section of an Isoetes rhizophore. Draw what you
see.
Is this species homosporous or heterosporous?
Label the microsporangium and megasporangium on your diagram.
How do you know which is which?
Lycopodium – club moss
Examine a dried herbarium specimen of Lycopodium on your lab table. Review the life cycle
of Lycopodium clavatum.
Are the leaves microphylls or megaphylls?
Where are the sporangia?
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BIOL 3601: PLANT DIVERSITY
DR. JULIE R. ETTERSON
FALL 2013
What is the sporangium-bearing leaf called?
Examine a prepared slide of a longitudinal section of a Lycopodium gametophyte. Draw what
you see. Label the antheridia. If you look at the prepared slide with the orange dot on it, you
will also see archegonia.
Examine a prepared slide of a cross section of a Lycopodium strobilus. Draw what you see.
Sellaginella – Resurrection Plant
Review the lifecycle handout of Selaginella.
Following the directions given below, you should be able to determine whether this species is
homosporous or heterosporous? Collect a piece of branch that has very young strobili at the
tip. You can tell because the shape of the branch changes where the strobili begin. Examine
the branch tip under the dissecting microscope. Probe around under the sporophylls (leaf-like
structures enveloping the sporangia. Dissect out several sporangia and examine them side-byside under the dissecting scope. Rupture the sporangia with a dissecting needle. Draw what
you see.
Can you see more than one kind of sporangia?
What size and number of spores are produced in the megasporangium?
What size and number of spores are produced in the yellow microsporangium?
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BIOL 3601: PLANT DIVERSITY
DR. JULIE R. ETTERSON
FALL 2013
View the prepared slide of a Selaginella strobilis and draw what you see. Label the
megasporangium, megaspores, microsporangium, and microspores.
EXERCISE 2:
Phylum Psilotophyta: The Whisk Ferns
The Psilotales are represented by only two living genera, Psilotum and Tmesipteris, both of
which have very simple sporophytes. We will study a living plant and examine prepared slides
of species from the genus Psilotum. Review the lifecycle handout and examine the illustrations
of Psilotum gametophytes on the center bench.
LIVING PLANT
Find the Psilotum specimen on the center bench. Diagram the branching pattern of the aerial
portion of the plant body.
What is this branching pattern called?
Does this plant have leaves? How about its ancestors?
Can you find the sporangia? Draw a picture that shows where the sporangia are borne.
Psilotum is homosporous. Is the gametophyte uni- or bisexual?
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BIOL 3601: PLANT DIVERSITY
DR. JULIE R. ETTERSON
FALL 2013
Compare the prepared slides showing a cross section of a Psilotum stem and rhizome. What
kind of stele does Psilotum have? Draw what you see.
Does the structure of the below-ground vascular structure differ much from the stem
vascular structure?
Examine the prepared slide of a longitudinal section of a Psilotum sporangia. Draw what you
see.
Is this species homosporous or heterosporous. How can you tell?
EXERCISE 3.
Phylum Sphenophyta: The horsetails
Although once a very abundant and diverse group of plants, the Equisetales today are
represented by a single herbaceous genus Equisetum. Examine the living and herbarium
specimens of Equisetum. The sporophyte of Equisetum differs from that of the other fern allies
in having jointed and ribbed stems with the leaves arranged in whorls at the nodes.
Are these leaves microphylls or megaphylls?
Equisetum is homosporous and produces green, free-living, bisexual gametophytes. Examine
the illustrations and or specimens of Equisetum gametophytes provided. Review the life cycle
of Equisetum.
Where does photosynthesis take place in the Equisetum sporophyte?
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BIOL 3601: PLANT DIVERSITY
DR. JULIE R. ETTERSON
FALL 2013
Under the dissecting microscope, examine the cones or strobili,
on dried the specimens provided on your table. Draw what you
see.
The strobili of Equisetum differ from those of Lycopodium and
Selaginella in that the sporangia are borne on umbrella-like
structures called sporangiophores, rather than on the sporophylls.
Compare the prepared slides of a cross section through an Equisetum stem and root. Label
the vascular bundles.
What kind of stele do you see?
Examine the prepared slide of the Equisetum gametophyte. Draw what you
see and label the antheridia and archegonia.
EXERCISE 4:
Phylum Pterophyta The ferns
The ferns are by far the largest group of seedless vascular plants accounting for about 2/3 of
the approximately 11,000 species.
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BIOL 3601: PLANT DIVERSITY
DR. JULIE R. ETTERSON
FALL 2013
The Gametophyte
Most ferns produce only one kind of spore and are therefore homosporous. The gametophytic
generation, commonly referred to as the prothallus, is heart-shaped and typically bisexual.
Examine the fern gametophytes growing at the base of the plant provided.
Do any of these gametophytes have young sporophytes associated with them yet?
Examine the prepared slide of fern prothallium whole mount. Draw what you see and label the
antheridia and archegonia.
The sporophyte
Examine the potted angiosperm on the center bench. Carefully look at the mossy edge of the
soil near the rim of the pot. Can you find fern gametophytes? Can you find a gametophyte
with a sporophyte attached?
Obtain a herbarium sheet with a fern specimen (sporophyte) attached. Identify the frond (leaf),
the rhizome (underground stem), and the roots. Most fern fronds are compound; that is, the
blade, or lamina, is divided into leaflets, or pinnae, which are attached to a rachis. The rachis is
an extension of the petiole, the stalk like part of the frond below the pinnae.
Are the leaves of ferns microphylls or megaphylls?
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BIOL 3601: PLANT DIVERSITY
DR. JULIE R. ETTERSON
FALL 2013
Among the ferns, only the water ferns are heterosporous, that is, produce two types of spores.
There are two water ferns on the center bench: Marsilea and Salviniales. Examine the spores
that have been released from the Marsilea species.
Draw what you see:
EXERCISE 5.
Chemotaxis in ferns
Chemotaxis is a widespread biological phenomenon that occurs in essentially every form of life,
from single cells to multicellular organisms. Chemotaxis involves the ability by specific cell types
to recognize the presence of a chemical gradient and to respond either positively or negatively
to that gradient. Familiar examples of chemotaxis are the movement of bacteria toward or
away from a particular chemical (e.g., a food source) and the nearly universal ability for single
celled gametes to locate one another in order to accomplish fertilization.
Chemotaxis is important for fertilization of lower plants that depend upon swimming sperm to
find the egg. In this exercise we will examine 12-day old C Fern™ gametophytes. The
gametophytes of this species has two distinct sexual types, small thumb shaped males and
larger heart shaped hermaphrodites. While the males contain only antheridia (male sex
organs), hermaphrodites contain both antheridia and archegonia (female sex organs).
Antheridia are composed of a few outer cells that enclose 16 sperm at maturity. Archegonia
consist of a short neck that protrudes from the surface of the gametophyte directly behind the
actively growing meristem region located in the notch of the heart. An egg is located at the
base of each archegonial neck.
By adding water to a mature culture, it is possible to observe the release of thousands of
swimming sperm (mostly from males). The sperm are positively attracted to receptive
archegonia. How do sperm know where the archegonia (and eggs) are? Sperm are attracted to
chemical substances that are contained in a small drop of liquid that is discharged from the
necks of receptive archegonia. This attraction is an example of positive chemotaxis. One sperm
eventually succeeds in fertilizing each egg.
Today we will use a simple technique to obtain a suspension of sperm from 12-day-old C-fern
gametophytes. The C-fern is a special rapid cycling fern that is used in biotechnology. We will
test five different test substances to determine whether their chemical structure resembles the
natural chemoattractant produced by archegonia.
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BIOL 3601: PLANT DIVERSITY
DR. JULIE R. ETTERSON
FALL 2013
Procedures:
1. Using the razor blade, carefully sharpen 6 of the wooden toothpicks provided so that one of
the ends has a very fine point. Do not touch the sharpened end with your fingers. Carefully lay
the toothpicks down on a clean surface.
2. Use the pen to make from 1 to 5 small dots along the side of the toothpicks near the
unsharpened ends, so that each toothpick has a unique marking (i.e., 1, 2, 3, 4, 5) and one is
kept unmarked. Take the toothpick with one dot and dip the sharpened end of it into the vial
containing test solution # 1. Place it, sharp end up, in the toothpick
holder (foam block).
Repeat with the remaining 4 test solutions, leaving the final unmarked toothpick dry.
Which of the toothpicks will serve as a control? Why is the one you chose appropriate to be
used as the control?
3. Obtain a depression slide and place one drop of the Sperm Release Buffer (SRB) in the
central depression.
4. Unscrew the light on your dissecting microscope so that you can shine the light on the
gametophytes from the side. You may want to have two dissecting microscopes set up side by
side – one for picking out male gametophytes and one for observing sperm.
5. Now obtain a petri dish containing mature C Fern™ gametophytes. Open the dish and
observe it under a stereomicroscope using transmitted (bottom) illumination. Note the two
types of gametophytes that are present smaller, thumb shaped males that have many bumps
on them, and larger, heart shaped hermaphrodites (See diagram on the chalk board). Take the
dissecting needle and carefully pick up males only and transfer them to the drop in the
concavity slide. Be careful not to damage or wound the gametophytes during transfer. If one is
wounded, discard it. Transfer a total of 7-10 male gametophytes to the drop. It is not necessary
to submerge them completely, only to place them within the drop of buffer.
6. Place the slide on either the lid or bottom of an empty petri dish, edges up, and observe it
under the dissecting under low magnification (12x or higher). The use of the petri dish will keep
the slide and sperm suspension cooler and provide a clearer view. In a few minutes, usually less
than five, sperm should begin to be released from antheridia. Adjust the illumination on the
stereomicroscope to provide the best contrast for viewing the sperm.
7. After a large number of sperm are released (about 3-5 min), begin testing the response to
the test solutions as follows:
a. Using 12-20x magnification, carefully focus on the TOP surface of the drop of sperm
suspension, in an area free of male gametophytes.
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BIOL 3601: PLANT DIVERSITY
DR. JULIE R. ETTERSON
FALL 2013
b. Take a test toothpick and, while looking through the microscope, gently and briefly
touch the sharpened end of the toothpick to the surface of the drop. Do not stick
the toothpick fully into the drop only touch the surface briefly.
c. Observe what happens, if anything, during the next minute, and record your
observations in Table 1.
d. After you have made your observations, repeat the procedure with the remaining
toothpicks. If necessary, use the dissecting needle to stir the sperm suspension and
redistribute sperm after each of the tests. This is typically needed only after a strong
chermotactic response is observed.
e. For comparison of the relative strength of the chemo attractants, you can test more
than one substance side by side. Briefly touch the pair of tips to the drop of
suspension as before. Observe the two points of contact and compare the
responses.
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BIOL 3601: PLANT DIVERSITY
DR. JULIE R. ETTERSON
FALL 2013
Results:
You may have observed a very weak response by the sperm to the control toothpick. How
could this be explained, and does it invalidate the experiment?
Which of the test chemicals elicited the strongest response?
The chemical structure of each substance is shown on the on center bench in the lab. Can you
relate the biological reactivity of the sperm to the chemical structure of the test substances?
Table 1. Chemotactic response of sperm to test substances.
Test
Substance
Intensity
(low, medium, high)
Swarming response
Duration
(short, medium, long)
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