Download Laboratory 1 - Vascular Plant Anatomy

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Laboratory 1 - Vascular Plant Anatomy
One of the major distinctions between the study of morphology and that of anatomy is that cells
themselves do not organize the plant. Rather, the organization of the plant determines that of its cells.
Therefore, cell form and expression does not determine plant structure. Instead, plant structural
demands determine how their component parts need to be organized and then cells are formed
accordingly. This underlies the classical distinction between the two closely allied areas of
morphology and anatomy, yet remain as distinct as the theories of light--whether it is a particle or a
wave. Both approaches have demonstrable utility. In order to conduct morphology, we must know
how plants are anatomically organized to understand how the plant is formed.
I.
Cell Types: Organization and Relationships
There are only five (5) major cell types in vascular plants: parenchyma, collenchyma, sclerenchyma
(fibers and sclereids), xylem (tracheids and vessel members) and phloem (sieve cells and sieve tube
members). These are in turn organized into tissues, such as epidermis, cortex, pith; in some cases
(e.g., xylem and phloem) the name of the cell and tissue is essentially the same. Some cells are
specialized within their immediate cellular environment (ie., vascular parenchyma, etc). Cell
organization is summarized in Gifford and Foster, pp. 36-42). Sachs' method (Gifford and Foster, p.
34-36) is used here to identify the general plant organization into dermal, ground and vascular
systems.
A.
Ground Tissues
The ground tissues are principally parenchyma. Parenchyma cells are particularly important in the
plant: they make up most of the living plant and most of the developmental potential of the plant
resides in these cells. These cells have only thin, primary cell walls and are located in many regions
of the plant. Obtain a thin cross section of a "stalk" of celery. Did you notice what organ it represents
in the plant? Now, prepare and observe the wet mount using a compound microscope. The large
cells with nuclei and relatively thin cell walls are parenchyma. How can you tell that they are alive?
Keep this slide for the next section on collenchyma.
B.
Support Tissues
Collenchyma and sclerenchyma form the two major support tissues. Collenchyma is living cell type
with thick, pearlly cell walls. It is located near the periphery of the plant and remains living during
function, depending on turgor pressure to remain strongly supportive. Re-examine the celery section
and look for these distinctive structures. How do they look from outside of the plant? Where are they
located in the petiole and in relation to the vasculature? What happens to the plant when collenchyma
cells lose their turgor? Do the cells differ in length or width when the tissue loses turgor? (Retain the
celery for further observation of vasculature in Section C.)
Sclerenchyma is typically dead at maturity, with thick, irreversible lignified cell walls. These are
often centrally located in vascular tissue or mixed in other areas of the plant. Obtain a cross section
of Hoya stem. Where are the sclerenchyma cells typically located? What is their general shape?
What clues can you obtain from their staining color and what does it indicate? Obtain and observe a
slide of Linum. Where are these cells located in the stem? Guess their shape in three-dimensions.
Cells that are less than 10 times longer than wide are known as sclereids and are typically separate or
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in small groups, particularly in ground tissues. The longer cells are known as fibers and are
commonly grouped together near or within the vasculature. What type of cells are found in Hoya?
What type in Linum? Would the dried tissues of this plant be flexible or resistent?
C.
Vascular Tissues
Xylem and phloem form two cell types found in vasculature. Xylem forms a dead pipeline with
lignified wall thickenings. Cells that will become xylem "line up" during their formation, develop
necessary structural cell wall supports for water conduction, form inter-cellular pores, and then
undergo pre-programmed cell lysis to become water conductive cells. There are two types of cells:
tracheids and vessel tube members. Phloem cells are food-conductive cells that remain alive during
function; however, at maturity they lose their nucleus and rely on surrounding cells for protein
synthesis and metabolic needs. These also "line up" during their formation, develop necessary cell
wall structures, and develop inter-phloem cell pores. Identify phloem and xylem cells within the
vasculature of the celery petiole. How can you tell the difference? Which is on the inside of the
petiole (adaxial)? Outside (abaxial)?
The order of primary xylem maturation may be determined in cross sections or in longitudinal
section. The first to mature is protoxylem, which is smallest in cross sectional diameter. Next to
mature is metaxylem, which may be significantly larger. Identify these in celery. Did you see the
structure of any of the secondary xylem wall thickenings?
Phloem is not easy to see under the best of conditions. Try to identify some based on cell wall
characteristics, cell size and relationships with companion cells. If you do not identify any, find
someone who did and look at their preparation, so that you may convince yourself that this tissue
exists.
II.
Anatomical Relationships
A.
Organization of the Stem
The angiosperm stem is a highly variable organ that we will have an opportunity to examine at a later
date. At this time, only the most general features are to be stressed: epidermis, cortex, vascular
bundles and pith. Not all stems have all of these features. The lower vascular plants will have
essentially no secondary growth, for example. Obtain a prepared slide of a young stem of
Pelargonium in cross section. Identify all of these tissues and the basic cell types. What is the
function of each? Is there any secondary growth?
B.
Organization of the Root
The root consists of the following tissues: epidermis, cortex and vasculature. Secondary growth is
rare in the lower vascular plants. In monocots, there may also be a pith in the root, but this tissue is
rarely seen in vascular plants in general. Examine a root of Ranunculus. Did you find all the the
tissues? This is a dicot, so if you saw a pith, you should check again.
III.
Technical Data Acquisition
Several basic techniques are used to gather the data used by morphologists in their scientific
interpretations. Four of the most common techniques include the following, each of which is
available on a demonstration basis:
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A.
Maceration
Cells are separated from one another to determine the size and shape of individual cells. Typically,
the cells are chemically fixed to retain their shape and size, and then are dissociated by acids, bases or
enzymes, depending on how resistent the material is. One of the most common is a xylem cell
preparation. Observe the native form of the dired cells and observe some under a microscope at the
side table.
B.
Sectioning
Sectioning of material may be accomplished using freehand sections with a razor blade or with a
microtome. The material may be fixed or frozen, and is typically embedded in a support matrix to
facilitate sectioning. The use of chemical solvents replaces water (and other soluble elements) of the
biological material) with either paraffin wax or plastic resin. Using wax, sections as thin as 1
micrometer (1/1000 millimeter) can be made by the most skillful technicians. Plastic embedding is
necessary to make thinner sections (as thin as 30 nanometers [30/1,000,000 mm]), like those required
for transmission electron microscopy. This is followed by staining to give constrast to the object to
be studied. A wide variety of stains are used and several journals are devoted to development of new
staining techniques. Sectioning is advantageous in determining cellular arrangements, where higher
resolution is required; however, three-dimensional reconstruction may be ultimately required to "put
it back together." Interpretation of three-dimensional data from sections is an acquired ability.
C.
Clearing
Clearing involves the extraction of all of the opaque materials of the plant in order to visualize the
object of interest. Clearings render the organ transparent, although a stain like safranin or others is
usually added to contrast the vasculature. Leaf skeletonization is done by simply continuing the
process and beating the leaf until only the xylem remains. Internal tissues, such as those within the
ovule, are usually observed by using special optics (phase contrast or interference contrast
microscopy) to see unstained soft tissues. Clearings retain all of the three dimensional information, in
situ, but do not always provide the degree of resolution needed to answer all of the questions that
could conceivably be posed about an organ.
D.
Surficial techniques
Occasionally, observing the surface of the plant alone is more important morphologically than seeing
the internal organization. The emergence of primordial organs, in particular, may be facilitated by
examining just the surface layers. The easiest method includes the use of a simple dissecting
microscope. The usefulness of this may be extended by using off-axis illumination or stains to
increase visibility of specific structures. Crystal violet, for example, may render external cell walls
more visible, if applied briefly to the surface.
For greater resolution and depth of focus, the scanning electron microscope is used to visualize
surfaces. Different techniques may be used for tissue preparation. Chemical fixation is usually
followed by chemical dehydration and critical point drying--a process whereby the phase change
between liquid and gas is done with specific temperature and pressure requirements so that a phase
boundary or drying front does not pass through the object. A relatively new development in this area
is cryoSEM observation, where a quickly specimen is frozen and kept at liquid nitrogen temperatures
throughout observation, thus eliminating many of the drying artifacts of other techniques.
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Laboratory 2 - Vascular Plant Reproductive Morphology
Although the mosses and liverworts are not true vascular plants, they display patterns of reproduction
which are exactly similar to land plants and which will present us with a starting point for our study
of reproductive morphology. Using bryophytes for this lab also provides an excellent perspective for
us to make future comparisons on the evolutionary reduction of the gametophyte.
I.
Nature and Ontogeny of the Archegonium
A.
Marchantia
Orient yourself by studying the mature archegoniophore in the liverwort Marchantia. What is the
nuclear condition (ploidy level) of the leafy body of the liverwort? Of the archegoniophore?
Archegonia are formed underneath the umbrella-like splash platform. Study a microscope slide of the
archegonia of Marchantia. First examine a mature archegonium and locate the neck cells, neck canal
cells, ventral canal cell and the egg. Which portion of this mature archegonium constitutes the
venter? After you have found all of these, begin looking for all phases in the development of the
archegonium. Can you find evidence of the primary cover cell and inner cell? How about the
primary ventral cell and the primary canal cell? Describe and draw the ontogeny of an archegonium
and list the cells that comprise the axial row.
B.
Mnium
The moss genus Mnium possesses archegonia which are considered by some to be among the most
primitive types of archegonia in the "Embryophyta". Notice that the segmentation which was obvious
in Marchantia is missing in Mnium. Locate the archegonial stalk, the venter and the axial row. As
you did for Marchantia, locate the neck cells and the neck canal cells. Study the slide and locate
formative stages in the development of the archegonium. Can you be sure that what you have
identified as primary cover cell and central cell are not just early precursors of the archegonial stalk?
What characters lead you to believe that the Mnium archegonium is primitive?
II.
Nature and Ontogeny of the Antheridium
A.
Mnium
In contrast to most vascular plants, the antheridium of Mnium is formed externally and not embedded
in gametophytic tissue. Examine a slide of the genus Mnium and locate the jacket cells and the
spermatogenous tissue. During the ontogeny of this type of antheridium, the initial division between
the jacket cell and primary spermatogenous cell occurs after formation of the initial antheridial stalk.
In this respect, how does the antheridium of Mnium resemble the archegonia? Try to locate a
developing antheridium at approxiamately the 8-16 cell stage to see if you can observe the initial
segmentation of the jacket and spermatogenous cells. Where is the operculum?
B.
Marchantia
The antheridia of Marchantia are borne within antheridial chambers embedded within the tissues of
the antheridiophore. Obtain a slide of an antheridial splash platform and study the antheridia. Locate
the spermatogenous tissue and the jacket cells. Did you notice how the immature antheridia are
attached to the base of the chamber wall early in their ontogeny? Diagram the stages of antheridial
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development. Do you notice the very regular arrangements of the spermatocytes? What happens to
this regularity as the sperm are produced?
III.
Embryo
A.
Marchantia
The early development of the sporophyte in Marchantia is a good example of an exoscopic embryo.
In embryos which are exoscopic, the initial division of the zygote yields an apical pole and a basal
pole. In higher plants, whole organ systems can be traced back to certain initial divisions within the
zygote. Observe a slide of the young embryo of Marchantia. Notice that the archegonium itself is
covered by a sheath of tissue which protects the archegonium and the developing embryo. Can you
detect which groups of cells arose from the initial division? Obtain a slide of older sporophytes of
Marchantia and notice that almost the entire sporophyte is one large sporangium that contains
sporogenous cells.
IV.
Sporophyte and Sporangium
A.
Mature Sporophyte of Psilotum
We will study the sporophyte of Psilotum in greater detail later. For now, observe the living
specimen of Psilotum nudum in the lab and make a rough sketch of its organography. Where are the
leaves, if any? Pay particular attention to the large three-lobed sporangia which are located laterally
along the shoot. What type of branching pattern does the sporophyte exhibit?
B.
Mature Sporangium of Psilotum
In most vascular plants, the sporangium is a eusporangium which is characterized by its initiation
from a superficial patch of initials. At maturity the sporangium of Psilotum is actually a three-lobed
structure called a synangium which is composed of three fused sporangia. Obtain a slide of the
sporangium of Psilotum. Observe the segmentation of the sporangium into wall cells and
sporogenous cells. If the sporangium is mature, numerous spores will be present. Is their nutritive
tissue present? Can you tell whether the tapetum is plasmodial or secretory in nature?
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Laboratory 3 - Shoot Longitudinal Symmetry
I.
Internodal Growth and Plant Form
Variations in internodal and longitudinal thickening growth during ontogeny of the shoot.
The shoot system of a higher plant is not simply a series of equal metameric units similar to a
tapeworm, but each stem unit differs from the preceding section in length and thickness and this can
be demonstrated in any dicot plant. In this case, we are using a tomato plant (which should be saved
for part II). Using a ruler, measure the length of each internode from the very base of the shoot (if
possible from the first node of the epicotyl) to the summit of the axis. Is the internode distance
constant during the life of this shoot? Next, measure the thickness of the internode along the length
of the shoot axis. What is the relationship between growth in thickness in the stem region and growth
in length?
II.
Thickening Growth in Shoot Systems
A.
Effects of primary versus secondary thickening on external form of the shoot axis
Using the same plant as for the previous part, take a razor blade and split the plant along its length.
Measure the internal features of the stem now, including the xylem, cortex, and pith. Using Toluidine
blue O will aid you in identifying the xylem. Determine whether the increase in diameter of the shoot
is due to increased xylem production from a vascular cambium, or from primary growth alone.
Notice that the shape of the pith is obconic. Why is this obconic form not evident in the shoots of
these dicotyledons?
B.
Comparison of primary versus secondary thickening in two gymnospermous shoots
Observe microscope slides of a cross section of the axis of the cycad Zamia and the stem of Pinus.
Compare the diameters of the respective axes and determine the amount of secondary xylem
produced in each shoot. Which axis has expanded primarily by secondary growth and which by
primary thickening processes? Compare slides of the shoot apical meristems of these two genera.
Note that in the case of the cycad shoot, the apical meristem is very large. The additional expansion
of the pith immediately beneath the apical meristem proper is responsible for the massive increase in
shoot diameter. This contrasts with the situation in Pinus where the apical meristem is relatively
small and the young axis is correspondingly small in diameter.
C.
Primary thickening growth in herbaceous plants
To demonstrate primary thickening growth, someone in class will make median longitudinal sections
of two dicotyledonous plants for class inspection. Draw these stems and their attached apices and
determine the region of thickening growth in these succulent species.
III.
Different Expressions of Internodal Growth
For each of the greenhouse species designated, make a drawing of the habit of the shoot system.
Examine in each case how differences in the degree of internodal elongation have resulted in
differences in plant form.
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Laboratory 4 - Shoot Lateral Symmetry
I.
Lateral Symmetry of Shoots
A.
Radially symmetrical shoots
Obtain shoots of a living plant with spiral phyllotaxis as an example of shoots with a radial symmetry.
(There should be a few to select from. Note that leaves can be found in equidistant positions around
the circumference of the shoot axis. With a selected plant, make a x-section of the terminal bud
which illustrates the radial symmetry of leaf initiation in this shoot. On one demonstration plant,
someone should peel off all the leaves until the initiation of the youngest leaves at the shoot apex are
exposed. Determine the phyllotactic formula by counting the leaves upward around the stem to the
next superimposed leaf in the sequence. Phyllotaxis = # of concentric spirals needed to repeat the
pattern of leaf initiation divided by number of leaves in the complete cycle.
B.
Bilaterally symmetrical (plagiotropic) shoots
1.
Platyclades
Platyclades are flattened shoots that are segmented into definite nodes and internodes. Study the
displayed platyclades from the greenhouse. What is the symmetry of these shoots and their
orientation with respect to the main axis of the plant? Do all shoots of these plants show a similar
organization and symmetry, or are there only certain branches on the plant that assume the platyclade
configuration? If present, what is their morphology? How is the vascular system organized? How
similar is it to a typical stem?
2.
Phylloclades
Phylloclades are flattened branches that are not differentiated into marked nodes and internodes and
hence resemble leaves to a remarkable degree. Ruscus aculeatus (Liliaceae) is an excellent example
of phylloclades. Study the displayed illustrations of aerial shoots of and the nature of the
phylloclades which are the ultimate lateral branches in this shoot system. Again, how would you
determine that these are branches and not just leaves? How would these look in cross section—
particularly with respect to the organization of the vascular bundles? In what respects do
phylloclades differ from platyclades? Draw the structure of this organ.
C.
Dorsiventral shoots
Observe the gross habit of the dorsiventral rhizome of Iris, Acorus or whatever other examples are
available, and examine the morphology of their subterranean shoot system. What is the direction of
growth of this shoot during the vegetative period of development? Is there any change in the
direction of growth with the onset of flowering? What types of symmetry are related to the respective
growth habits of the plant during vegetative versus reproductive shoot development? Observe a
prepared slide of a transection of the rhizome of Acorus and make an outline drawing of this shoot.
Can you identify the dorsal and ventral sides? Can you see any leaf or root traces? What features of
the form of the rhizome are responsible for its dorsiventral organization? Is the vascular system
arranged in the same bilateral arrangement as the rhizome body?
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D.
Anisophylly as an expression of lateral symmetry in shoot systems
Anisophylly refers to unequal development of leaves around the circumference of the stem. Two
types of anisophylly can be recognized in seed plants and are illustrated here by coniferous shoots.
1.
Lateral anisophylly
In lateral anisophylly the shoot actually has a radial symmetry but the plagiotropic orientation of the
shoot induces a dorsiventrally and, correspondingly, an unequal development of leaves on the dorsal
versus the ventral sides of the shoot. If the particular shoot in question could be induced to grow
orthotropically, that is at right angles to a substrate, it would be radially symmetrical and isophyllous.
Observe the ultimate shoots of the specimens of Taxus available in the laboratory. Notice that all the
leaves lie in essentially one plane. Make a transverse section with a razor blade through the terminal
bud, place the sections on a slide and observe them on the microscope. What is the lateral symmetry
of the shoot tip? What is the actual phyllotaxis? Note that the apparent distachous, two-ranked
appearance of the leaves is due to the fact that the leaves on the underside of the shoot are
differentially developed; those on the ventral side are longer and have become twisted so that they lie
in about the same plane as those of the upper and lateral surfaces. Verify this feature by removing
several of the leaves and keeping track of their relative positions.
2.
Habitual anisophylly
In habitual anisophylly the shoots display unequal leaf development despite the position of the shoot;
it is what has been called an autonomous anisophylly. Determine the actual differences in leaf
structure in relation to position for shoots of Thuja orientale available in the laboratory. Make
transverse sections as well as observing the differences in leaf morphology with the dissecting
microscope. With what particular type of shoot symmetry is anisophylly associated?
II.
Heterophylly During Transition to Flowering
Heterophylly is the phenomenon in which leaves on various portions of the shoot system display
markedly different shapes. Anisophylly is a lateral form of heterophylly seen in shoot systems.
Longitudinal expressions of heterophylly, however, are much more common. The most marked
expression of heterophylly is displayed as the shoot undergoes a transition to flowering. Obtain
shoots of Coleus and examine carefully the nature of leaves on the vegetative and reproductive
portions of the shoot. Is the transition gradual or abrupt? Diagram vegetative and floral leaves. Is
there a noticeable difference in lateral symmetry between these two forms?
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Laboratory 5 - Vegetative Morphology of Lycopodium and Selaginella
I.
Lycopodium
A.
Organography of the shoot system of Lycopodium
Observe the following specimens of Lycopodium, as available:
Living specimens:
Lycopodium lucidulum
L. caroliniana?
Herbarium specimens:
L. annotinum
L. cernuum
L. clavatum
L. complanatum
L. lucidulum
L. obscurum
Preserved specimens:
Lycopodium alopecuroides
L. annotinum
L. complanatum
L. fastigatum
L. inundatum
L. lucidulum
L. obscurum
L. pachystachyon
L. pithyoides
L. reflexum
L. serratum
L. varicum
For each of the above specimens, make a schematic diagram of the branching system. Assuming that
the most primitive branching system within the genus Lycopodium is dichotomous, arrange the
species in a typological sequence indicating the probable lines of evolution within the genus
Lycopodium. Is it possible to determine evolutionary sequences from typological sequences such as
are shown by Lycopodium? In which species in this sequence does over-topping appear? In which
species is the genus pseudomonopodial? What is the difference between pseudomonopodial and
monopodial? If the branching is pseudomonopodial is this true for lateral branches as well?
B.
Anatomy of stem and rhizome
Obtain a prepared slide of the rhizome or stem of some species of Lycopodium. What is the
difference between stem and rhizome? Draw a cross section of the axis. Note the epidermis, cortex,
and endodermis. Can you determine the exact position of the endodermis? Is a Casparian strip
present? What is the function of the Casparian strip in Lycopodium? Diagram the morphology of the
plectostele locate the position of the xylem, phloem, protoxylem and metaxylem. What type of xylem
development is this? Compare your slide with that of another species of Lycopodium. Is the
plectostele as well defined? Is there any relationship between the presence of a haplostele, actinostele
and plectostele and its position within a given plant? If there are leaves present on the outside of the
axis, retain this prepared slide for reference for leaf structure.
C.
Anatomy of the leaf
Remove a leaf from living material and examine it under the dissecting scope. Describe the venation
of the leaf and any surface qualities it displays. Is a petiole present? Obtain a slide of the leaf in
transverse section (present on many stem cross sections) and examine its internal anatomy. How
many veins are present? Where is the xylem and phloem? Is the mesophyll segmented into zones as
in many angiospermous leaves? Describe the cuticle. If you could not locate leaf traces in the stem,
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go back and obtain another slide until you can see the leaf trace within the stem. Better yet, make
your own slide using fresh material and a razor blade.
D.
Position and anatomy of the root
Roots of Lycopodium arise near the shoot apex and do not immediately exit. The roots will often
traverse the length of the stem. Obtain a prepared slide showing this phenomenon, or refer to Fig. 95A, if a slide is not available. How many roots are present within the stem? If living material is
available, obtain a portion of the rhizome. Make a transverse section across the axis at the point
where the root exits and then try to trace the root through the stem by systematically following the
root in cross section. How far does the root extend? Do roots also arise in aerial portions of the
plant? Obtain a prepared slide of the root of Lycopodium (you may have to share) and study it. Is the
xylem maturation endarch or exarch? What is the position of the protoxylem and metaxylem?
Where is the phloem? Examine living material to determine whether there is any indication of
secondary roots.
E.
Production of gemmae
Occasionally, some species of Lycopodium will undergo vegetative reproduction by means of
gemmae. Lycopodium lucidulum and L. selago are particularly known for this feature. Can you find
gemmae on the living material available? The insertion of gemmae on a fertile stem tip can be seen
in one prepared slide of L. lucidulum. Examine this demonstration slide closely as it shows not only
the structure of the gemma but also the shoot apex. Sketch the shoot apex. Does the apex grow by a
group of initials or by a single tetrahedral apical cell? Examine the leaf on this slide and determine
whether the leaf grows by a group of initials or a single apical cell. Can you see the differentiation of
cells into protoderm, ground meristem, and procambium? What will each of these tissues eventually
become?
II.
Selaginella
A.
Organography of the shoot
The shoots of various genera of Selaginella are all pseudomonopodial with varying degrees of
elaboration of dichotomous branching systems. Study the organography and branching patterns of the
following as available:
Living specimens:
Selaginella apoda
S. braunii
S. kraussiana
S. lepidophylla
S. peruvianum
S. uncinata
Preserved specimens:
S. arencola
S. douglassii
Herbarium specimens:
Selaginella anceps
S. arenicola
S. articulata
S. cuspidata
S. diffusa
S. exalta
S. flabellata
S. pallescens
S. rupestris
S. selaginoides
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These specimens give you some idea of the diversity within the genus Selaginella. The beautiful,
large, leafy forms are chiefly tropical, but arid-adapted species are also admirably adapted for their
environment. Notice the Oklahoma native, Selaginella peruviana. (Other Oklahoma natives include
S. arenicola var. apoda, S. riddellii, S. densa, S. underwoodii, and S. rupestris.) For each of the
displayed species above, ask the following:
Is the branching primarily dichotomous or
pseudomonopodial? Are the lateral branches also pseudomonopodial? Is the leaf arrangement tworanked or four-ranked? How pronounced is the anisophylly?
B.
Arrangement of leaves
Obtain a short section of a living shoot of Selaginella kraussiana or S. braunii and examine it under
the dissecting microscope. In how many ranks are the leaves arranged? What is the phyllotaxis of
the leaves? In the laboratory dealing with anisophylly, we studied lateral and habitual anisophylly.
Which type is demonstrated by this species? Is this the common case in Selaginella species? You
might wish to remove specimens from the other species for comparison. What are the evolutionary
trends in this genus?
C.
Anatomy of the stem and rhizome
The axis of Selaginella is characterized by a number of vascular bundles which run in a hollow
section in the center of the stem. Obtain a prepared slide of the stem or rhizome of Selaginella and
observe its structure. Locate and diagram the position of the meristeles. Is the maturation of the
xylem endarch or exarch? Locate the trabeculae. Where is the Casparian strip usually found in
Selaginella? Is there a discrete endodermis? The pseudomonopodial habit of Selaginella is easy to
see on the living and preserved specimens. Examine a median longitudinal section of the shoot apex
of Selaginella. Is this pattern of branching evident at initiation? How close to the apex does the
anisotomous nature of the branching become evident? How do trabeculae arise?
D.
Anatomy of the rhizophore
Obtain or observe rhizophores of Selaginella kraussiana or S. braunii. Where do they arise along the
shoot systems? Can you locate roots at the terminal end of the rhizophore? Is there any indication of
the so-called angle-meristem? Is it possible to induce rhizophores by chemical means? Obtain a
slide showing a cross-section of the rhizophore. Does the internal structure of the rhizophore
resemble a stem or a root? You might wish to refer to Fig. 9-19 for general organography and Fig. 922B for its anatomy.
E.
Anatomy of the leaf and ligule
Obtain a short section of the shoot of Selaginella kraussiana and observe it under the dissecting
microscope. Can you locate the ligule? Obtain a prepared slide of a cross section of Selaginella
showing the position and structure of the ligule. Does Lycopodium have ligules? Do ligules in
Selaginella occur on both vegetative and reproductive leaves?
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Laboratory 6 - Reproductive Morphology of Lycopodium and Selaginella
I.
Lycopodium
A.
Morphology of Lycopodium strobili and subgenera
1.
Subgenus Huperzia (old Urostachya)
Observe herbarium and preserved specimens of the following species of Lycopodium, subgenus
Huperzia (ref. Gifford and Foster, p. 123), as available:
Lycopodium lucidulum
L. selago
L. reflexum
L. alopecuroides
All of these species show strobili which are not compact and merely represent fertile areas of the
stem. Arrange these specimens in typological order, if possible, starting with the least specialized
strobilar organization and proceeding to the most specialized. What vegetative features do these
urostachyoid Lycopodium types have in common? Obtain pickled material of L. lucidulum if fresh
strobili are not available. Locate the sporangium, the sporophyll and the shoot apex. Is a ligule
present? Obtain and observe a prepared slide of the strobilus of L. lucidulum or L. selago. Describe
the shape of the sporangium. Is the sporangium located on a stalk? Is the line of dehiscence obvious?
Is it longitudinal or transverse? Carefully remove a single sporophyll from a plant and observe it
under the dissecting microscope. Is the morphology of the sporophyll the same as that of a vegetative
leaf? Is the phyllotaxis of the sporophylls similar to that of the vegetative leaf? Is the genus
Lycopodium heterosporous or homosporous?
2.
Subgenera Lycopodium, Diphasiastrum and Lycopodiella (old Rhopalostachya)
Observe herbarium and preserved specimens of the following species of Lycopodium as available:
L. annotinum
L. cernuum
L. clavatum
L. complanatum
L. inundatum
L. obscurum
L. pachystachyon
These species exhibit strobili which are very distinct and not merely fertile, little-differentiated areas
of the shoot. The most primitive of these are those in which the condensation of the sporophyll
section is not completed and thus a distinct stalk and strobilus are not differentiated. Arrange the
above specimens n a typological sequence from the most primitive to most advanced. Now compare
the most primitive of this group with the most advanced of the Huperzia species. How close is the
intergradation? Examine all the true strobilate species and determine how they differ vegetatively
from the Huperzia species. Obtain a strobilus of one of these "rhopalostachyoids" that has been
preserved. Make a longitudinal section along the length of the strobilus. (You might also wish to
observe a prepared slide of one of these species!) Are the sporangia dorsal or ventral with respect to
13
the sporophyll? Is this adaxial or abaxial? Turn your free-hand section over so that the flat side is
down and remove a sporophyll to expose a sporangium. Draw the sporangium, noting its shape. Can
you observe the possible line of dehiscence in this sporangium? Is the sporangium stalked? Save this
strobilus for use later in the laboratory.
The three genera in this group are Lycopodium, Diphasiastrum and Lycopodiella, according to
Gifford and Foster, p. 123. Morphologically, how do these subgenera differ? Which is most
advanced? Which is most primitive? Are there also gametophytic differences? Which species above
belong to which subgenera?
B.
Anatomy of the strobilus, sporangia and spores
1.
Structure of the strobilus
In section A we learned that the strobilus is a condensed fertile shoot or portion of a shoot. Obtain a
prepared slide of a longitudinal section of the strobilus and locate a sporophyll with its associated
sporangium. Is the sporophyll vascularized? Describe the shape of the sporophyll. In your species,
is there an abaxial extension of the sporophpyll above which covers the sporangium? Does this
extension occur in all Lycopodiums? Does the type of dehiscence change with the presence or
absence of an abaxial flap? Is the sporangium stalked? Are the spores all similar (homosporous)?
Are spores similar between different sporangia? Is the tapetum plasmodial or cellular? Draw the
structure of the sporangium noting wall layer, spores and stalk.
2.
Ontogeny of the sporangium
Observe a demonstration slide of Lycopodium (m.l.s.) showing the development of the eusporangium.
Beside this prepared slide is a photograph indicating sporangial ontogeny, also shown in Fig. 9-9.
Can you tell if the sporangium arises from a single cell or patch of surface initials? How many layers
of cells are involved in this ontogeny? At what point are the sporogenous cells derived? From what
group of cells is the stalk derived? How many wall layers are present at maturity and are all of these
present at all stages of ontogeny? In the slide of the Lycopodium species you observed, in the bottom
row of sporophylls, can you see the entire sequence of early sporangial development? Diagram this
sequence as it demonstrates a good, representative example of eusporangiate ontogeny.
3.
Spores
Study the ontogeny of spore development in a prepared slide of Lycopodium. After you have an idea
of spore development, scrape some spores out of a strobilus. Prepare a wet mount slide of these
spores and observe them under the compound microscope. Can you locate the triradiate ridge? Why
do spores like these have triradiate ridges? (SEM of a spore and its organization in a tetrad is shown
in Fig. 9-11, p. 118 in Gifford and Foster. Does this ridge serve any purpose in the life cycle? Is the
outer layer of the spore sculptured?
C.
The gametophyte: antheridia and archegonia.
Since Lycopodium is homosporous, the antheridia and archegonia are frequently located on the same
gametophyte. Obtain a prepared slide of the gametophyte of Lycopodium. Is it endosporic or
exosporic? Draw its basic structure. Locate the area containing the endophytic fungus. What is its
function? Do all of the Lycopodium species have this? How has the presence of the fungus modified
the morphology of the gametophyte? Locate the antheridia and examine their distribution on the
gametophyte body. Draw their general structure. Try to find archegonia. If you cannot see
archegonia on your slide, observe them on a demonstration slide at the front of the room. As
14
archegonia go, it's not too swift. Consult Gifford and Foster for a description of the above-ground
type of gametophyte. Which is considered to be advanced, the subterannean or the above-ground
gametophyte?
D.
The embryo
The embryo of Lycopodium is endoscopic--that is, it contains its embryo within gametophytic tissue
until, by shear size, the young sporophyte breaks out of the gametophyte. Lycopodium also has a
suspensor which acts to push the embryo into the gametophytic tissue. Polarity is established very
early in embryo development. It is usually easy to ascertain which areas of the embryo will develop
into root, stem, etc. Observe the demonstration slide and photograph. Locate those areas of the
embryo which are the presumptive root, foot, leaf, and apex. Is it possible to observe the suspensor?
What differences are observable between sporophyte development in the above-ground and belowground gametophytes.
E.
The monotypic genus Phylloglossum
If you enjoy rare and esoteric information, undoubtedly the specimen of Phylloglossum drummondii
will be of interest to you. Although the condition of the specimen is poor, a strobilus, stem and tuber
are present. Gifford and Foster briefly mention this reduced genus (pp. 105-107, Fig. 9-1) as a
member of the Lycopodiales. Is it considered to be more primitive, intermediate or more advanced
than the genus Lycopodium? Sections of the tuber are available in the room. How is it organized?
Does it have a haplostele, actinostele or plectostele? Where is this genus found as a native? Is it
homosporous or heterosporous?
II.
Selaginella
A.
Organography of the strobilus
All members of Selaginella have sporophylls condensed into distinct strobili. Observe the following
specimens with strobili, as available:
Selaginella arenicola
S. densa
S. plumosa
S. lepidophylla
S. oaxaca
S. epirrhizos
S. eurynota
S. anceps
For each of the above species, note the position of the strobilus. Is it terminal or lateral? Drawing
upon knowledge from previous lab exercises, to what extent is the habitual anisophylly of the
vegetative body reflected in the arrangement of sporophylls? Is the arrangement still opposite and
decussate? Obtain a pickled strobilus or two of Selaginella arenicola or from one of the other living
species. Under the dissecting scope, make a careful dissection of the strobilus. Pull off a sporophyll
and notice the ligule on the adaxial surface. Remove a sporangium and determine wheher its
dehiscence is longitudinal or transverse. Is this a microsporangium or megasporangium? Is
heterangy present? Can you tell from the outside? What is the segmentation of sporangia in the
sporangium--longitudinal or transverse? Are both micro- and megasporangia found in the same
strobilus?
15
B.
Anatomy of the strobilus
Obtain a slide of a longitudinal section of a strobilus of Selaginella. Locate the sporophyll, ligule
and both microsporangia and megasporangia. Is the segmentation of sporangia transverse or
longitudinal? Notice the approximate size and shape of micro- and megasporangia. Is the tapetum
cellular or plasmodial? Notice the wall of the sporangia. Does it contain thin areas which will
become the dehiscence line? To what extent does the abaxial portion overlap the subjacent
sporangium?
C.
Ontogeny of the sporangium
Observe a prepared slide showing the ontogeny of sporangia of Selaginella. How many wall layers
are present early in ontogeny? What happens to these layers? Can you identify the tapetal layer of
the wall? Is the sporangium stalked early in development? Is it possible to tell at what point the
sporangia will differentiate? Does the ligule mature before or after the sporangium? What is the
function of the ligule?
D.
The gametophyte, gametangia and germination
Selaginella is heterosporous. The endosporic gametophytes are not retained within the sporangium
but are liberated. We have no material of the microgametophyte or antheridia of Selaginella, please
refer to Gifford and Foster, pages 139-140 for a fuller explanation of this process. Obtain prepared
slides of the megagametophyte showing some stage of archegonial development. Notice that the
megagametophyte breaks out of the spore wall only in response to archegonial development. Can you
locate any rhizoids? Observe the three stages on demonstration which show the embryo. Can you
discern any embryonic structure such as is shown in Gifford and Foster, Figure 9-13? The embryo, as
in Lycopodium, is endoscopic with a suspensor. Notice the young sporophyte as it begins to
germinate. Now obtain a prepared slide showing the sporophyte at a later stage of development.
Locate the shoot apex, the leaves and the root. Can you discern the meristeles at this early stage of
development?
16
Laboratory 7 - Lycophyta: Vegetative and Reproductive Morphology of Isoetes
I.
General Organography of Isoetes and Stylites
Examine the following living, herbarium and preserved specimens as available:
Isoetes melanopoda
I. storkii
I. triquetra
Compare the diversity found in these species of Isoetes with the diversity in Lycopodium and
Selaginella. What is it about Isoetes which could cause this lack of organographic diversity? The
name "Isoetes" comes from a Greek word meaning "leek". To what extent does Isoetes resemble an
onion? Examine the available photographs of Stylites. Compare the corm size between Stylites and
Isoetes. Examine the diagrams from the article by Rauh and Falk. What mode of branching is
expressed by the stem of members of the Isoetes? To what extent is this branching exhibited in fossil
members such as Pleuromeia and Nathorstiana?
II.
Anatomy of the Corm
A.
Obtain living specimens of Isoetes
Ninety percent of the confusion in understanding the morphology of Isoetes is in deciphering the
spatial arrangements of the cambia, furrows and meristems. Place one under the dissecting scope and
carefully dissect off the leaves and roots one by one noting the arrangement of leaves and roots and
their position on the corm. Be sure to remove all the decaying brown material. Observe the corm.
Locate the lobes and the furrow. Where is the apical meristem? Do roots arise in the furrow? Where
is the furrow (sagittal) plane? Where is the frontal plane? Which plane is the transverse plane? Take
one corm (for the class) and make a frontal section with a razor blade. Examine this section closely
under the dissecting microscope. Locate the apical meristem and the xylem core. Sketch this
arrangement for future reference. Locate the area of the basal meristem. Can you see root traces?
Leaf traces? Take the second corm and make a sagittal section. Again locate the apical meristem,
xylem core and basal meristem. Describe the morphology of the xylem core and locate the area of the
lateral meristem and prismatic layer.
B.
Obtain prepared slide of a longitudinal section of the corm of Isoetes
First, observe this slide under the dissection scope and place next to it a section of live material,
comparing structural similarities. Now, observe the prepared slide under the compound microscope.
Locate the shoot apex. Where are the lateral and basal meristems? Which tissues compose the
prismatic layer? Where is the primary xylem? Locate the leaf traces if any are present. Locate the
root producing meristem and the root primordia. Where is the secondary phloem? Describe the
action, morphology and derivatives of the lateral cambium. Why could Isoetes be called a "steady
state plant"? Obtain a prepared slide showing a transverse section of the corm. Locate the tissues of
the lateral cambium, prismatic layer, xylem and phloem.
17
III.
Anatomy of the leaf and root
A.
Leaf
Observe one of the leaves you have removed from the corm of Isoetes. How much of the leaf is
bifacial and how much is unifacial? Make a longitudinal section. Describe the morphology of the
internal structure. Where are the septa (diaphrams)? Describe both the longitudinal and lateral
segmentation of the leaf. Are the leaves fertile at this stage? Is there a distinct difference between
sterile and fertile leaves in Isoetes as in Lycopodium or Selaginella? Obtain a prepared slide showing
the leaf of Isoetes in cross section. (Look among your various corm sections for this.) How many
chambers are present? Where are the vascular tissues located? What is the function of the
chambering of the leaf?
B.
Root
1.
Anatomy
Carefully analyze the morphology of the root system of Isoetes. Describe the morphology of the
branching system of the root. Is it dichotomous? Locate the root cap. Obtain a prepared slide
showing the root in transverse section. Diagram the root's internal structure labeling epidermis,
cortex, internal cavity and vascular cylinder. Diagram the endodermis, xylem and phloem. Isn't this
strange? Obtain a prepared slide of the rootlet of Stigmaria ficoides, an extinct member of the
Lepidodendrales from the early Carboniferous. Examine this specimen under the dissecting scope.
Notice that the basic morphological pattern of Stigmaria is very similar to that of Isoetes. Examine
the vascular system of Stigmaria under the compound microscope. What does this say about the
morphological nature of the root in Isoetes?
2.
Rhizotaxis
Obtain a corm of Isoetes and remove all but 1/8 inch of the leaves and roots. On a piece of graph
paper, draw a line representing the furrow and then place a dot representing the place of emergence of
a root from the stem. Compare the resulting diagram with Figures 17 and 18 from the article by
Paolillo. How do they compare? Are roots in Isoetes arranged irregularly or do they follow a definite
rhizotaxis? Calculate the number of roots possible for your plant by calculating
x = yz (z + 1)
where x = total number of roots
y = number of lobes
z = series on each lobe flank
How does this method correlate with your actual observations?
IV.
Reproductive Morphology
A.
Microsporophyll
Obtain a prepared slide showing the microsporophyll of Isoetes. Locate the microsporangium on the
adaxial side of the sporophyll. Locate the velum and determine its origin. Locate the ligule and
determine were the foot is. Is a sheath or glossopodium present? Is the ligule in Isoetes in the same
morphological position as that of Selaginella? Examine the microsporangium. Is it eusporangiate?
18
Locate the trabeculae and the tapetum. Are the trabecula similar to those found in Selaginella?
Examine the microspores. Are they tetrahedral or bifacial? From what types of cells do the
trabeculae arise? The tapetum? The spores?
B.
Megasporophyll
Obtain a prepared slide of a megasporophpyll of Isoetes and examine it under the dissecting
microscope. Locate the ligule, velum, and sporangium. How many megaspores are present in the
megasporangium?
C.
Gametophyte and embryo
Gametophyte development is similar to Selaginella. The embryo, although endoscopic, does not have
a suspensor. We do not have very much (or good) material showing embryogenesis in Isoetes.
Please examine photographs showing these processes. Notice that the embryo of Isoetes in general
resembles that of Lycopodium and Selaginella, although the specific ontogeny of parts differs.
19
Laboratory 8 - Sphenophyta: Vegetative and Reproductive Morphology of Equisetum
I.
Organography of the shoot of Equisetum
The organography of Equisetum follows three basic patterns of distribution of reproductive and
vegetative shoots. What are these basic organographic ground plans? Did you differentiate between
leaves and stems? Observe living, herbarium and preserved specimens of the following, as available:
Equisetum arvense
E. fluviatile
E. giganteum
E. hyamale
E. laevigatum
E. palustre
E. pratense
E. prealtum
E. scirpiodes
E. sylvaticum
For each of the above, indicate which represent primative, intermediate, and advanced organographic
patterns. Note the place where each was collected and see if the morphological advancement is
correlated with geographic trends.
II.
Vegetative body of Equisetum
A.
Structure of the plant
Observe the living specimen of Equisetum. Locate the leaves. How are they organized along the
stem? Does Equisetum demonstrate longitudinal symmetry? Graph the node length vs. number if
you remain unconvinced. To what extent is branching evident in the aerial shoot? In the plant as a
whole? Observe the herbarium specimen of the incredible Equisetum giganteum. Notice the origin
of lateral branches of the aerial shoots. Where do these branches arise? How are the buds located in
relation to the leaves of the same whorl? Notice the distinctive ribbing of the stem. What is meant by
articulation and jointing of the stem?
B.
Anatomy of rhizome and aerial stem
Obtain a prepared slide showing a transverse section of the rhizome of Equisetum. For comparison,
obtain a slide of the comparable aerial shoot of this species. The stem of Equisetum varies from
species to species. We have numerous examples and if you have time you should compare some of
these species and determine the nature of its variability. Compare the fertile, sterile and rhizomatous
stems. How do these differ? How are they similar?
Locate the following: pith cavity, vallecular canals, carinal canals, primary xylem, primary phloem,
cortex and epidermis. How are the vallecular canals and pith cavity formed? How does the
arrangement of vascular tissue compare with members of the Lycophyta? What type of arrangement
is present in Equisetum? What type(s) of endodermis is present in E. hyamale? What difference can
be noticed between the rhizome and the aerial shoots? What functional reason could account for this?
How does each node develop? How does the apex itself develop -- from a group of initials or a single
tetrahedral apical cell? Where are the stomata located along the stem?
20
C.
Leaves and nodal anatomy
Observe a cross section of the stem of Equisetum taken at a node. It should be possible to identify
leaves, leaf traces and vascular bundles. The organization of the stem of Equisetum at the node is a
particularly interesting problem. Did you find that the vallecular and carinal canals are continuous or
discontinuous at the node? How about the pith cavity? Slides are available which detail this anatomy
from one end of the node to the other. Did you notice how many vascular traces pass into the leaves?
Is this anatomy typical for a microphyllous plant? Is this unusual organization possibly reflective of
the evolutionary origin of the leaf in the Sphenophytes? How so?
D.
Anatomy of the root
As in all members of the Lycophyta, the root which is formed during embryogenesis never develops
into a functional organ. Instead, roots are formed endogenously in the rhizome at the node. Observe
a transverse section of a root of Equisetum. Locate the single large metaxylem element in the center
of the root. In larger roots, the xylem is diarch or triarch. How is the apex organized?
III.
Reproductive morphology
A.
Organography of the strobilus
Obtain a preserved specimen of a strobilus of Equisetum. Locate the annulus. Make a longitudinal
section of the cone and observe the orientation of the sporangiophores. Remove a single
sporangiophore. How many sporangia are found on a single sporangiophore? What is meant by the
term peltate? Although the sporangium of Equisetum is initiated by a single surface initial, it is still
considered to be a eusporangium. Why is this so? What is the morphological nature of the
sporangiophore?
B.
Anatomy of the strobilus
Observe a prepared slide of a longitudinal section of the strobilus of Equisetum. (Once again a
variety are available in the lab.) Locate the main axis of the strobilus and the sporangiophore. Locate
the spores and associated elators. Is Equisetum homosporous? Heterosporous? Anisosporous?
(What is the difference?) How many wall layers are present at maturity? Where is the tapetum and
how does it act in association with the periplasmodium? What is a periplasmodium?
C.
Anatomy of spores and elators
Obtain a prepared slide showing the mature spores and associated elators. Notice that the spores are
round at maturity. Does this indicate that they are not the product of an initially tetrahedral division
of the spore mother cells? What is the function of the elators? What is meant by the term
hygroscopic? How does the sporangial wall break? Are all of the spores the same size? If not, does
this represent heterospory or anisospory? (What is the difference?!)
D.
Gametophyte and embryogenesis
Equisetum spores are notoriously short-lived. Although their gametophytes, at up to 1 inch are quite
aggressive among the fern allies, the spores will usually germinate for only about a week. Observe a
prepared slide of whole mounts of fixed Equisetum gametophytes. Can you see any gametangia? In
the various forms of gametophytes that you see in your slide, can you sense a developmental pattern
21
in gametophyte formation? Did you see any bisexual gametophytes? Why is there a controversy
about this and does it relate to possible anisospory? (See Gifford and Foster pg. 192 for a summary.)
Sectioned gametangia are also available.
Observe the embryo of Equisetum. The embryo in the sphenophytes is truly exoscopic. Is a
suspensor present? From which initial cells do the root, shoot and foot originate? If the slides are not
adequate to answer these questions, Fig. 10-19 in Gifford and Foster illustrates the basic pattern of
development well. How soon is an apical cell organized?
22
Laboratory 9 - Paleobotany of Lower Vascular Plant Groups
I. Lycophyta
A. Herbaceous representatives
1. Lycopodites
The genus Lycopodites includes eligulate lycopods with helically-arranged microphylls (either
isophyllous or anisophyllous) and disperse or compact strobili similar in organography to
Lycopodium. Observe the specimen from the Carboniferous of Kentucky and determine the mode of
preservation. Is anatomical deta1il present? How can this genus be distinguished from terminal axes
of the arborescent lycopods? Specimens such as this one represent much of the known morphological
diversity of the living genus Lycopodium are are found from the early Carboniferous to the present.
2. Ligulate forms
The extinct genus Selaginellites represents bisporangiate strobili similar to the living genus
Selaginella and have been found connected to the stem genus Paurodendron. Although
representatives of these genera are not available in this laboratory, it is important to note similarities
in the presence of ligules, meristelar organization of the stem and the presence of a hollow chamber in
the center of the stem. Representatives of the form genus Isoetites, also unrepresented here, consist
of sporophylls which strongly resemble those of the living genus Isoetes. The oldest specimens
apparently date back to the Triassic. What implications would this have for the postulated ancestry of
Isoetes from Pleuromeia and Nathorstiana?
B. Arborescent representatives
The arborescent lycopods were tall, heterosporous, ligulate plants which apparently dominated the
coal-forming swamps of the early Carboniferous. In gross organography, these plants possessed a
number of unique features. The most conspicuous of these is the leaf cushions which bore the long
microphylls found attached to axes of Lepidodrendron, Sigillaria, and Lepidophloios, among other
genera. The organization of the leaf cushions on the stem after extensive secondary growth suggests
that leaf cushions were capable of continued growth throughout the life of the plant. Observe the
organization of the leaf cushions of Lepidodendron both externally by observing compressed
specimens and internally by studying coal ball peels. What is the organization of the parichnos?
What is their function? Observe the location of the ligule. Is the function of the ligule any less
enigmatic in fossil lycopods? Examine the compressed terminal axes of Lepidodendron and note the
external similarity to Lycopodites.
The internal anatomy of the arborescent lycopods was marked by extensive secondary growth,
producing large amounts of secondary growth visible to the exterior of the protostelic center of the
stem. Although phloem has been observed in such stems, it is minute in comparison with the xylem.
This has been taken as an indication that the vascular cambium of these plants were typically
unifacial--producing xylem, but no phloem. The wood of lycopods is distinctive because of the
presence of fimbrils between the scalariform thickenings, which are also known as Williamson
striations. Rays were poorly developed as evident in the coal ball peels before you. Examine the
specimens available. What degree of development did the cork cambium express?
23
The underground portion of the stem is also entirely dichotomous, and originates from a root stock
known as Stigmaria, a commonly found root compression genus. The roots as in Isoetes bore roots in
a definite rhizotaxis, with the rootlets in extinct species commonly similar in structure to the living
genus. In these extinct species, it is even more evident that it is possible that the roots of these
arborescent forms arose in a manner similar to stems, and perhaps possessed an entirely different
evolutionary history than did the roots of derivatives of the Rhyniophytes. Observe the rootlets of
Stigmaria on display and the pictures of several spectacular stumps in your textbook and elsewhere.
The reproduction of the tree lycopods is particularly fascinating since it appears to reflect many trends
encountered in achieving the seed habit. The vast majority of the lycopods were monosporic,
possessing either microspores or megaspores. An exception to this is Barinophyton which has
identifiable spores of both morphotypes present in the sporangia. Although in all cases, the strobilus
was organized into sporophylls bearing sporangia, the extent of dimorphism between sterile
microphylls and the sporangium-bearing sphorophylls was variable. Also variable was the degree of
reduction of the number of megaspores produced in the megasporangium. The most extreme cases of
reduction in spore numbers in the female occurred iyn Lepidocarpon, where only a single, large
functional megaspore remained after megasporogenesis. Whether the megaspoe was capable of
fertilization while on the tree is not well-known, but it is evident that the resulting megagametophyte
was similar in many ways to previously discussed heterosporous lycopods. The presence of a line of
dehiscence near the apex of the megaspore and the obvious lack of true integuments differentiate
these structures from seeds; however, the trends are evident and striking. A trend for protection of
the megasporangium in these lycopods relied primarily on the elaboration of the laminae of the
sporophyll and reached various degrees of completion and complexity.
24
Laboratory 12 - The Eusporangiate Ferns
The ferns are typically segregated two general groups: eusporangiate and leptosporangiate ferns. The
extant eusporangiate ferns are placed in two orders differing widely in their morphology: the
Ophioglossales and Marattiales.
I.
Ophioglossales - Botrychium
A.
Organography
Observe herbarium specimens of the following species of Botrychium as available:
Botrychium biternatum
B. dissectum
B. lunaria
B. virginianum
Identify the sterile and fertile portions of each specimen. Make a careful observation of how the
sporangia are borne on the shoot. The most advanced members of the genus are generally considered
to be those that show a reduced level of branching within the aerial portions. Place each of the above
specimens into a typological series leading from the seemingly most primitive species to those which
appear most advanced. Observe a number of specimens of Botrychium virginianum to familiarize
yourself with the amount of variability occurs within this single species.
What two morphological theories have attempted to account for the unique aerial organography of
Botrychium and other Ophioglossales? Which seems the most probable based on the available
evidence?
B.
Botrychium stem and rhizome
Observe a slide showing a bud of Botrychium and observe the special demonstration slide of a
median longitudinal section of the apex. The apical bud of Botrychium is unusual because it contains
the numerous primordial buds for leaves that will not mature for several years. Locate the leaf
primordia, and if possible, identify the shoot apex and apical cell. (If you really see the apical cell,
please share it!)
Next, observe a prepared slide showing a transverse section of the rhizome. Notice that the primary
vascular tissues are arranged into an ectophloic siphonostele--that is, a continuous cylinder of phloem
outside the xylem. A pith is located in the center. You will probably not see a ring of vascular tissue
because of the presence of leaf gaps. To familiarize yourself with this, attempt to diagram what your
slide shows in a three-dimensional drawing. In many cases, the rhizome of Botrychium possesses a
reasonable amount of secondary growth. Locate the secondary xylem, secondary phloem and, if
possible, periderm. If you have any additional time, you might like to examine a cross section of the
rhizome of Helminostachys. How different is this from Botrychium?
25
C.
Botrychium sporangia
Observe a pickled specimen of the fertile shoot of Botrychium and place it under the microscope for
observation (don't chop it up as there isn't a lot of material). Notice that the eusporangia are separate
and appear stalked and are not associated with strobili. Notice the large number of sporangia on the
fertile shoot (pinna). Obtain a prepared slide showing sections of the eusporangia. From how many
cells does this sporangium arise? How many wall layers are present in the mature sporangium? Is
Botrychium homosporous or heterosporous? About how many spores are produced per sporangia, but
please don't count them (about 2,000)?
D.
Botrychium gametophytes and embryogenesis
Within the Ophioglossales, gametophyte development is strictly exosporic. Observe the organization
of the archegonium and young embryo in the prepared slide on demonstration. Is it possible to locate
archegonia or antheridia? Do they occur on the same plant at the same time? How big is the
archegonium? How about the embryo? You might like to refer to photographs of the subterranean
tuberous gametophyte in Bierhorst to orient yourself. How does embryo development occur within
the genus Botrychium? Is it endoscopic or exoscopic? For a precise answer to this question, examine
Bierhorst, p. 148. Nowhere else within a single order is there so much variability of embryo
development as is found in the Ophioglossales.
II.
Ophioglossales - Ophioglossum
A.
Organography
Observe herbarium specimens of the following species of Ophioglossum as available:
Ophioglossum crotalophoroides
O. engelmanii
O. vulgatum
Notice that the general organography of Ophioglossum is structurally similar to that of Botrychium
but outwardly displays a greater simplicity in both its sterile and fertile areas. In what ways does the
external organography of Ophioglossum seem to be more advanced than that found in Botrychium?
A spike? Venation patterns? Gifford and Foster contend that the larger epiphytic species of
Ophioglossum are more primitive than the smaller, inconspicuous types. What do you think?
B.
Ophioglossum stem, root and stipe
Obtain a prepared slide showing the transectional anatomy of the above organs (there are several, but
please share). Observe the rhizome first and notice the ectophloic siphonostele, as is present in
Botrychium. Is secondary growth present? Next, examine the anatomy of the stipe. Notice the
discrete vascular bundles. These will later divide, some branching out into the fertile segment of the
shoot system and some into the sterile segment.
C.
Ophioglossum fertile spike and sporangia
Obtain preserved material showing the fertile spike of Ophioglossum and observe it under the
dissecting microscope. Notice that, in contrast to Botrychium, the eusporangia are embedded within
the fertile spike tissue. Obtain a longitudinal section of the fertile spike (a synangium?) and draw the
26
details of sporangial structure, especially noting, if possible, how each sporangium receives its own
vascular bundle. What might this indicate about the phylogeny of Ophioglossum?
III. Marattiales
The Marattiales include all of the fern-like plants that produce eusporangia on the abaxial face of the
leaf. The Marattiales have a number of features in common with the Ophioglossales--including
eusporangia, similar ontogeny of archegonia and antheridia and the late differentiation of embryonic
organs. Because the vascular system is so complicated--a polycyclic dictyostele with commissural
strands--it would be best to forego the vascular system at this time and concentrate on the unique
reproductive morphology.
A.
Organography
Observe the following herbarium specimens of these members of the Marattiales as available:
Angiopteris evecta
Danaea crispa
Marattia salicina
For each of the above, note the fern-like foliage and also the amount of dissection of each individual
frond. For each of the above, sketch the nature of the sporangia, noting whether they are separate,
slightly fused, or totally synangiate. What is the method of dehiscence for each type (see Gifford and
Foster, Fig. 12-15). On which surface of the leaf are the reproductive structures found. Observe the
herbarium specimens and photographs available in the lab. Notice the hard, thick stipules which are
present at the base of the leaves. Certain genera of Marattiales can reach more than a meter in height.
B.
Synangial organization
Obtain longitudinal prepared slides showing the structure of the synangia in Angiopteris,
Christensenia, Danaea and Marattia. How many wall layers are present? What is the morphological
origin of the wall of the synangia in Marattia? Are the spores homosporous or heterosporous? What
type of gametophyte will eventually be formed? What will the initial polarity of the embryo be?
What characteristics link the Marattiales with Ophioglossales? Which with the Filicales?
27
Laboratory 13 - Leptosporangiate Ferns: Vegetative Morphology
I.
Organography
Observe living specimens of the following genera:
Adiantum capillis-veneris
Asplenium nidis
Azolla sp.
Davallia sp.
Lygodium sp.
Marsilea vestita
Nephrolepsis exaltata
Platycerium bifurcatum
Polypodium sp.
Salvinia sp.
and herbarium sheets as available. For each of the above, observe the organography of the plant as a
whole, locating the rhizome, frond, stipe, rachis. For each determine whether the frond is once
pinnate, twice pinnate or pinnatifid. Notice the degree of morphological variability present in basic
plant organization in these Filicalean (and heterosporous) representatives. In fact, only the
angiosperms are a more morphologically diverse group among land plants. To what extent is the
morphological expression of these plants indicative of their habitat? Are ferns restricted to moist
areas?
II.
Stelar Anatomy
Nowhere else in the plant kingdom is there such a wealth of variability of the primary plant form than
within the ferns, and this is reflected particularly well in the primary vasculature. As you examine the
diversity of stelar types assembled today, be aware that stelar organization is a three-dimensional task,
and that this is a very difficult task to study from simple cross sectional views.
Protostelic Organization
Examine the following prepared microscope slides in order:
Gleichenia - rhizome
Lygodium - rhizome X-section
Trichomanes - rhizome (Hymenophyllaceae)
Notice that each of the above genera express a protostelic organization of the vascular tissue. In
Lygodium the vascular tissues alone seem to occupy the center of the stem, but in Trichomanes, some
parenchyma seems to be present. In Gleichenia, the presence of parenchyma within the xylem is
better expressed, and is an excellent example of the so-called vitalized or medullated protostele.
Notice the large number of cells which have not matured into tracheids and remain as parenchyma.
To what extent does this stelar arrangement support the "transformation" or "stelar" origin of the pith?
Consult the phylogenetic chart of the ferns in Gifford and Foster (Fig. 13-46) and determine whether
these protostelic genera are primitive or advanced members of the Filicales.
Ectophloic Siphonostele
28
Obtain slides of the relatively primitive Filicalean genus Osmunda which show an ectophloic
siphonostele which has a large number of leaf gaps making this almost a eustele. Locate the xylem,
phloem, and leaf gaps. Notice that the phloem seems to occupy the leaf gap position causing the
vascular tissue to appear as a group of amphicribal bundles.
Amphiphloic Siphonostele
An amphiphloic siphonostele is a vascular cylinder surrounding a pith in which the cylinder of xylem
is surrounded both to the outside and to inside by separate cylinders of phloem. Obtain prepared
slides of Adiantum sp. and Dicksonia sp. Notice in both of these genera, the leaf gaps do not overlap
so the cylindrical nature of the stele is obvious. Locate the xylem, phloem, and pith. Where is the
leaf gap? Obtain a prepared slide of transverse sections of the genus Matonia showing a fantastic
complex solenostele with concentric amphiphloic cylinders. Where does the leaf trace appear? Is the
leaf trace also complex? To what extent is the so-called "polystelic" organization of the vasculature
in Matonia a misnomer?
Dissected Solenostele (Dictyostele)
A dictyostele is a solenostele (amphiphloic siphonostele) which has been seemingly broken up into
separate amphicribal bundles. Also, there is a separate type of dictyostele known as a dissected
dictyostele which is characterized by having additional gaps not associated with the divergence of leaf
traces and also by usually having two traces associated with a single gap. It is very difficult to discern
a simple dictyostele from a dissected dictyostele in cross section. Most ferns are characterized by the
possession of some type of dictyostele. Obtain prepared slides of the following:
Polypodium
Pteris
Woodwardia
Notice that Polypodium and Woodwardia have their dictyostelic elements (dissected in both) arranged
in a ring around the outside of the stem circumference. In these vascular segments, locate the areas of
xylem and phloem. The genus Pteris shows a vascular organization which represents a great level of
sophistication in the Filicales. In Pteris the dictyostele is now dissected to the point in which discrete
meristeles may be located, each with its own endodermis. Locate each meristele and note differences
in the organization of each meristele. Notice that the entire stele is no longer radially arranged as in
Polypodium, but has taken on a dorsiventral aspect.
III.
Roots
Observe the roots of Pteris as an example of the organization of the filicalean root. Locate the
epidermis, cortex, and stele. To what extent is the complexity of the rhizome expressed in the
structure of the root? Examine living material and determine where the roots arise and how they
originate. How extensive is xylem development in roots. Is the maturation of the xylem endarch or
exarch? Endarch, mesarch, or exarch in the rhizome?
IV.
Venation
Make a cursory examination of the vascular system of the leaves or pinnae of some of the herbarium
sheets observed in the first section. To what extent is the dichotomous venation pattern expressed in
the photosynthetic organs of ferns? Obtain a prepared microscope slide showing clearings of pinnae
with open dichotomous venation patterns.
29
Laboratory 14 - Leptosporangiate Ferns: Reproductive Morphology
I.
Gametophyte and Gametangia
Obtain preserved specimens of the gametophytes of a typical fern. Notice the apical notch. Are there
antheridia and archegonia present? Observe the segmentation of the gametangia if they are present.
Obtain prepared slides of both archegoniate and antheridial gametangia. Although the ferns are
generally homosporous there is frequently a time delay in formation of either male or female
gametangia. What is the biological significance of this discoordinate development? First observe the
mature antheridia. Locate if possible, the opercular cell, first ring cell, and second ring cell. Can you
locate the primary spermatogenous cells or the sperms themselves? Where are the antheridia
positioned on the gametophyte? Locate the neck cells, neck canal cells and, if possible, the ventral
canal cell and the egg. What position do the archegonia occupy? Is this always a consistent feature?
Do filicalean gametophytes have an endophytic fungus? What are some other shapes which are
manifested in fern gametophytes?
II.
Sporangium
Observe living sporangia on a portion of a leaf of Polypodium. Notice the lack of an indusium
covering the sorus. Can you detect the single stalk and annulus of each sporangium? Have any of the
sporangia dehisced? If so, what is the direction and method of dehiscence? Obtain a prepared slide
of the sorus of Cyrtomium. Locate the base, stalk and umbrella of the indusium. What function is
served by the indusium? Locate the sporangium. Is the maturation of the sporangia simple, gradate,
or mixed? For each sporangium, locate the stalk, capsule, annulus, spores, and if possible, the
stomium. Is it possible to determine the developmental stages involved in the development of the
leptosporangium from this slide? How do leptosporangia differ from eusporangia in development
and mature structure? How is the tapetum organized within the leptosporangium? From what cell
initials is it derived? Can you locate the tapetum in the Cyrtomium sporangium? Locate a
sporangium showing a primary sporogenous cell and another showing the four or eight cell stages.
How many spores are ultimately produced by a typical leptosporangium?
III.
Soral Position and Associated Indusial Structures
Perhaps the most interesting facet in the study of the Filicales lies in the supposed trends of evolution
of soral position and the formation of the indusium. These trends are the subject of much controversy
and are entirely speculative, so be forewarned as you examine them. It does serve a purpose though
to illustrate morphological diversity within some framework, and that is why the concept of phyletic
slide is emphasized here.
Davalloid line
Within the typological series which terminates in the genus Davallia and related types there is a
progressive overtopping and outgrowth in margins. Consult the attached handout and phylogenetic
chart in Gifford and Foster to trace the typological progression. Observe the following:
Davallia - living material, prepared slide
Dicksonia sp. - prepared slide
Lygodium sp. - herbarium sheet, prepared slide*
Nephrolepsis sp. - living material, prepared slide
How has the concept of phyletic slide been utilized to explain this series?
30
Pteroid Line
The line of supposed evolution leading from the marginal sporangia of Lygodium to the false
indusium of Pteris and Adiantum is said to result from the phyletic slide of the sporangium to the
adaxial surface of the pinna and a downward folding of the pinna margin. Trace this typological
series with the following examples:
Adiantum - slide, living material, herbarium material
Cheilanthes - herbarium specimen
Dicksonia - studied previously
Lygodium - studied previously
Pellea - prepared slide
Pteridium aquilinum - prepared slide*
Dryopterid Line
The most complex line of evolution within the ferns seems to be the Dryopterid line which has
seemingly given rise to number of diverse forms. The initial soral position is abaxial rather than
marginal and is best examined in the genus Gleichenia. The initial indusium appears cup-shaped and
appears to be derived from a scale-like indusium. Various types of soral and indusial types are also
derived including the vein-following coenosorus of Asplenium and the naked sorus of Polypodium.
Use the following examples:
Anthyrium - herbarium specimen
Asplenium - prepared slide
Cyathea - prepared slide*
Cyrtomium falcatum - prepared slide*
Cystopteris - prepared slide
Dryopteris - prepared slide
Gleichenia - prepared slide*
Polystichum - prepared slide
Woodsia - prepared slide
Gymnogrammoid Line
The Gymnogrammoid line is presumed to have begun from the primitive group related (or actually) to
Osmunda with marginal sporangia. A phyletic slide results in abaxial sporangia and a restriction to
the more marginal position yield the Gymnogramma or Cryptogramma type. Observe these:
Cryptogramma - prepared slides
Osmunda - prepared slides, herbarium sheets
Blechnoid and Onocleoid Line
Many members of this line of evolution are dimorphic in that certain whole fronds are fertile while
others are sterile. The protective indusia of the Blechnum-type is presumed to have arisen de novo
from initially reflexed leaf margins. Observe the following:
Gleichenia - observed previously
Onoclea sensibilis - prepared slide*, herbarium sheets
Hymenophylloid Line
As the name implies the Hymenophylloid ferns have thin, membrane-like leaves. In fact, the "filmyferns" are only one-leaf cell thick, usually with marginal, occasionally trumpet-like indusia. Observe:
Hymenophyllum - prepared slide*, preserved material
Schizea - herbarium sheet, preserved material
Trichomanes crispum - prepared slides, preserved material
31
Laboratory 15 - Leptosporangiate Ferns: Transitional Groups and Heterosporous Ferns
I.
Osmundales
Although the systematic position of the Osmundales remains somewhat unclear, much evidence
suggests that the order is an early offshoot of the filicalean line and that modern Osmundales
represent a phylogenetic dead-end -- possibly somewhere between the eusporangiate, and truly
leptosporangiate ferns. The group is well-represented in the fossil record from the upper Jurassic to
present. Observe the organography of the herbarium specimens of Osmunda in the laboratory. What
is the morphological nature of the structure bearing the sporangia? Does the structure represent a
pinnule, pinna, or frond? What does the degree of variability in fertile frond organization suggest
about the evolutionary position of members of the Osmundales? Of the group in general? Does the
vegetative structure of the plant resemble the typical filicalean fern? Obtain a prepared slide of the
rhizome of Osmunda and examine the organization of the vasculature. What type of stelar
organization does this display? How does the organization of the vasculature differ from that of the
typical eustele? What is the position of the phloem? Now examine the structure of the unusual
sporangium of Osmunda, looking especially at the number of wall layers composing the sporangium
and the numbers of spores produced. Is this typical of a leptosporangiate fern? What is the
organization of the annulus? How does this sporangium resemble a leptosporangium? How does it
resemble a eusporangium?
II.
Psilotales
One of the most perplexing questions in plant morphology concerns the systematic position of the
Psilotales. Among morphologists, Bierhorst is perhaps the major proponent of the idea that these
represent primitive ferns; however, other morphologists have remained quite skeptical. Certainly, a
comparison between Psilotum and any advanced filicalean fern would lead to the conclusion that they
are not closely related. Come to your own conclusions on this matter by examining the living
material of Psilotum nudum and preserved materials of Tmesipteris, the only other genus in the order.
How do these species resemble each other? How do they differ? Observe cross sections of the
rhizome or aerial stem of Psilotum. What type of stelar organization does this represent? Is this
specimen exarch, mesarch, or endarch? Is there an endophytic fungus present? Now observe the
reproductive organography. Does the mode of insertion of the sporangia differ in Psilotum and
Tmesipteris? Are there significant differences in the organization of their synangia? Observe a cross
section of each. What is the organization of the sporangium? Did you notice the unusual tapetal
tissues? How does the sporangium of Psilotum dehisce? Is an annulus present? Does it function?
III.
Marsileales
Observe the living specimen of Marsilea vestita. This order of aquatic, heterosporous ferns is
characterized by bisporangiate sori located within the sporocarp. The plant is organized into an
underground rhizome and floating or emergent leaves. For each of the above, locate the frond,
rhizome, and fertile pinnae (sporocarps). Briefly examine a cross section of the rhizome of Marsilea
to note the conspicuous aerenchyma within the submerged stem. Then turn your attention to the
sporocarps in living and preserved material. Consider the morphological nature of the sporocarps.
Do they represent an entire simple pinna, a compound pinna, or entire frond? What is considered to
be the morphological nature of the two bumps on the sporocarp? Examine closely the positional
relationships of the sporocarp and frond. Now obtain a living sporocarp, scarify the outer wall of the
mature sporocarp and immerse it in water. What happens? How does the gelatinous tube arise
32
during dehiscence? What is the arrangement of the sori on the gelatinous tube? Observe a single
sorus and examine it closely. Notice both the microsporangia and megasporangia. Are they located
on the same sorus? First observe the megasporangium. How many megaspores are produced in each
megasporangium? Did you notice the organization of the megaspore into a resistant walled structure
with an expanded apical protrusion at the apex? Did you see the sperm lake and gelatinous coat
located around the megasporangium and at its apex? Maturation of the megasporangium takes
approximately 22-24 hr. Obtain a sorus of a sporocarp which germinated two days ago. Can you
identify the archegonium or early stages in embryo development? Are sperm present?
Now obtain a prepared slide of a longitudinal section of a young sporocarp of Marsilea and locate the
developing microsporangia and megasporangia. Notice the chambering around each sporangial
group--what does each of these chambers represent? Notice the arrangement of micro- and
megasporangia on the receptacle. are the megasporangia consistently borne at the apex? It may be
necessary to look at a number of slides in order to appreciate the developmental sequence of each of
these types of sporangia. Where is the gelatinous tube located within the sporocarp? Now observe
the structure of the newly released sperm cells. How much time is required after sporocarp opening is
required before the release of the sperms? Are these multiflagellate sperms more complex than others
in the homosporous ferns? Next, observe the young embryos. How old are these? Notice the young
first leaf, and if possible, the sheath (or calyptra), rhizoids, foot, and root. How does the duration of
embryonic development compare with that of the homosporous ferns? With that of the angiosperms?
IV.
Salviniales
The Salviniales include the most highly reduced of the heterosporous ferns and possess functionally
monosporangiate sporocarps. Obtain living and preserved material of the following:
Azolla sp.
Salvinia sp.
For each describe and draw its organography. What are the most obvious adaptations of these plants
to the floating habit? Did you notice the "egg-beater" trichomes on the Salvinia? Note especially the
phyllotaxis, presence or absence of a rhizome, dorsiventral lobing of the leaf. Is there an evident
branching pattern? What is the morphological nature of the structures which appear to be roots in
Salvinia? In Azolla? Do the roots originate from the same place in both genera?
Search the living and preserved material for the presence of sporocarps. Note the position of the
sporocarps. How does the insertion and numbers of sporocarps per frond compare with that in the
Marsileales? The Salviniales differ from the Marsileales in that each sporocarp itself is indusial in
nature and contains only megasporangia or microsporangia at maturity. The smaller mature
sporocarps contain a single megasporangium with a single megaspore, whereas the larger sporocarps
contain numerous microsporangia. Obtain a prepared slide of a longitudinal section of a sporocarp of
Salvinia or Azolla. A gradation in maturity of the sporocarps will be evident in the section, with a
number of developmental stages evident. Find the organization of the mature microsporangium and
determine the location of massulae and glochidia, if present. What is the origin of the massulae?
Would the separation of male and female sporocarps represent a selective advantage over the
bisporangiate reproductive system of the Marsileales?
33
Laboratory 16 - Comparative Morphology of Seeds and Germination Strategies
I.
Morphology of seeds
The evolution of the seed habit may represent the last major adaptation of land plants to the terrestrial
environment--a modification that permits them to reproduce in the absence of free water in the
environment. Your task today is primarily to examine the general organization of seeds and
seedlings, with particular emphasis on organography and external morphology. Embryological details
of the gymnosperms and angiosperms will be covered in detail later.
First, obtain living material of Capsella bursa-pastoris and dissect it to understand the organization
of the ovules within the ovary. Now observe prepared slides of longitudinal sections of nearly mature
seeds of Capsella to see the orientation and location of the embryo. Note that the curved shape of the
embryo conforms to the curvature of the amphitropous ovule. Locate the two cotyledons, shoot apex,
the hypocotyl-root axis, and the root apex of the radicle. Following germination, what will be the fate
of each of these regions of the embryo? What is its nutritional source during germination and
thereafter?
The various seeds available in the lab were chosen to represent a number of different strategies for
food storage within the seed and germination modes. Observe the organization of the ovule, noting
the characteristic structures of seeds including the micropyle, hilum, placenta, and funiculus. Cut
some of the seeds in half to determine the organization of the embryo. Compare the location of seed
food stores of the available seeds. What types of food storage products are present in each of the
seeds? How does the embryo finally break these down for nutrition during germination and soon
afterwards? What is the ultimate source of these nutritional food reserves? Which are the
gametophytic tissues?
II.
Modes of germination
Now, observe seedlings of the various seed types and compare their general organography. Locate
the hypocotyl, root apex, epicotyl, shoot apex and cotyledons. Germination is usually described as
conforming to one of three general patterns--hypogeal, mesogeal and epigeal, based on the degree of
hypocotyl elongation which occurs during seedling development and whether it contributes directly to
above ground growth. What is the general location of the cotyledon and embryo axis relative to the
seed coat? How is seed germination triggered in the various plants? In the lab, try to identify
examples of the different modes of germination discussed in class. For certain specialized modes
(like germination in orchids) examine the course of development in pictures. Notice the apparent
universality of the so-called cotyledonary "hook" which seems to protect the emerging embryo. This
morphology is consistent in many, if not all, of the seed plant embryos observed to date. Its
development, however, still remains a subject of research since the hook is not fixed in position.
III.
Evolution of the seed habit
Available in the laboratory are petrographic slides of examples of seed fern seeds from the
Carboniferous for examination. Observe and draw the ovule of either Lagenostoma physoides or L.
ovoides and label the tissues constituting the integument, nucellus, and gametophyte. In addition to
these layers, which are evident in extant plants, there are also two structures which appear to be
restricted to this group of fossil seed ferns: the lagenostome--a mound of tissue just below the pollen
chamber and the cupule--a layer of tissue located around the periphery of the ovule. The morphology
34
of the cupule is better seen in specimens of compressed seed (see one of the available texts). Of the
two major groups of seed ferns (the Lyginopteridales and Medullosales), the largest seeds were
produced by members of the Medullosales, including the genera Pachytesta (a genus for petrified
seeds) and Trigonocarpus (a genus for compressed seeds). Seeds of these Pteridosperms are
primitively large. Measuring two to three inches in length, these are among the largest observed in
the plant kingdom.
During the last twenty years, much has been learned concerning the biology of seed function in these
plants. Even the presence of a "pollination droplet" and the occurrence of male gametes in the ovule
have been described in fossil specimens.
The morphology of the male gametes and
microgametophytes strongly resembles that of cycads. Look at photographs of these specimens in
Taylor's book on paleobotany, and compare the sperms to those of extant plants in Gifford and
Foster's text. Inexplicably, there seems to be an entire absence of pteridospermous embryos in even
well-preserved material. Do you have any explanations as to why the embryos may not have been
preserved? At what part of the life history might these seeds have been shed? Is there any hope of
improving our data on this important phase of the pteridospermous life history?
35
Laboratory 17 - Cycadales and Ginkgoales: Vegetative Morphology
I.
Cycadales
A.
General Organography
Cycads represent among the most primitive of the seed-bearing plants on both vegetative and
reproductive grounds. Observe living specimens of the following:
Ceratozamia mexicana
Cycas circinalis
Cycas revoluta
Dioon spinulosum
Stangeria paradoxa
Zamia floridana
(We do not have living specimens of the other genera at present, for example: Bowenia,
Encephalartos, Microcycas, Macrozamia.) For each of the above examine the large frond-like
leaves, noting how the leaves form a crown at the top of the stem. Locate the leaf bases (sometimes
called cataphylls) remaining as armaments on the trunk. What general phyllotaxis is exemplified by
these cycads? What type of vernation is shown by each pinna? Do cycads ever produce sizable
lateral branches? Locate the apogeotropous roots growing upward out of the soil. What is their
significance?
B.
Stem Anatomy
Obtain a large prepared slide of a transverse section of the genus Zamia. Locate the pith, vascular
cylinder, secondary xylem, phloem, cortex girdling leaf traces and epidermis. Is this stem pycnoxylic
or manoxylic? Notice that the rays within the secondary xylem are quite wide. Have you been able
to follow the course of one of the leaf traces? Do leaf gaps occur in the primary xylem of the stem?
C.
Anatomy of a Leaflet
Obtain a prepared slide showing a transverse section of the leaflet of Zamia. Locate the upper
epidermis, hypodermis, mesophyll, vascular bundle, xylem, phloem and bundle sheath extension. Is
this type of vascular system characteristic of all cycads? Compare the venation patterns of the various
living species which you have examined in part IA. Especially notice the leaflets of Cycas revoluta
which has only a single midrib and which lacks vascularization of the wings of the lamina.
D.
Roots
Did you noice the general organography of the root system when you examined the plants previously?
Obtain prepared slides of transverse sections of both Cycas and Zamia roots, as available. What
pattern of xylem is manifested in these species? Do any of the living specimens produce aerial roots
with endophytic algae. If so, make a free-hand section of the root and locate the area of algal
infection.
36
II.
Ginkgoales
A.
General Organography
Observe the living specimen of the maidenhair tree Ginkgo biloba which may just be beginning to
produce this year's foliage. We will have to take a walk to do this though, because it is located
outside the President's office. Observe the pattern of irregular branching which is customary in
Ginkgo. Pay particular attention to the dimorphic pattern of branching and sketch the morphology of
the short shoot system. Are leaves borne on both the long and short "spur" shoots? Make a
longitudinal section of one of the short shoots obtained from a living tree. Locate the leaf primordia.
Examine each individual leaf and note the amount of lobing and the general shape. What type of
venation pattern is expressed in these leaves?
B.
Anatomy of the Stem
Obtain a prepared slide showing the cross sectional anatomy of the manoxylic short shoot of Ginkgo.
At older stages in stem morphology the stem is occupied largely by secondary xylem. At this early
stage of development the stem of Ginkgo looks almost like a mature section of the Zamia stem you
saw earlier in lab. Can the concept of neoteny be used to explain the manoxylic wood of Zamia? In
Ginkgo locate the pith, secondary xylem, secondary phloem, and cortex. Obtain a section of a twig
from a long shoot of Ginkgo. Notice that the secondary xylem is well expressed in the long shoot and
pycnoxylic development is amply manifested.
C.
Anatomy of the Leaf
Ginkgo seems unique among gymnosperms in showing a peculiar seasonal heterophylly. Leaves
produced early in the season originate during the previous season, overwinter, and are rapidly
exposed and develop as soon as the bud breaks in the spring. The late leaves are those leaves which
are formed during the spring and summer but whose primordia are not present in the over-wintering
bud. Obtain herbarium specimens showing this phenomenon. Sketch a typical early leaf and a
typical late leaf and other manifestations of this heterophylly. Obtain a preared slide of a cross
section of the leaf of Ginkgo. Identify the component cells and tissues of the leaf.
III.
Morphology of the Cycad Cone
In the next lab we will begin to examine the morphology of the ovule and seed; however, it seems
appropriate to examine the organization of the strobilar axis and its modified leaves at this time.
Whatever you don't finish today you can finish next time.
A.
Comparative Morphology of the Microsporangiate Strobilus
Study and draw the structure of the microsporangiate strobili of the various cycads in the lab. What is
the phyllotaxis of the strobilus? Does it differ radically from that of the main axis? Obtain individual
microsporophylls of C. revoluta and other species. Draw the structure of the microsporophylls and
the pattern of distribution of microsporangia. Is the microsporophyll homologous with a foliage leaf?
What evidence would you cite in support of this viewpoint? How has this organ been modified in
relation to the differentiation of the strobilus?
37
B.
Comparative Morphology of the Megasporangiate Strobilus
For each of the following species as available, draw the gross structure of the megasporangiate
strobilus and indicate their normal position on the shoot apex.
Cycas circinalis
Cycas revoluta
Dioon spinulosum
Ceratozamia mexicana
Zamia floridana
Is there any indication that megasporophylls may also represent modified foliar organs? Arrange the
above sporophylls in a typological series of what you believe to be the direction of specialization of
cone appendages and strobilus differentiation.
38
Laboratory 18 - Cycadales and Ginkgoales: Reproductive Morphology
I.
Cycadales
A.
Microstrobilus, Microspores and Microgametogenesis
Observe the longitudinal section of a mature cone of Zamia available in the lab. Locate the cone axis
and the microsporophylls with microsporangia on the abaxial surface of the microsporophyll. Obtain
a prepared slide of the microsporangium of Zamia in transverse section. Scan the slide of Zamia for
evidence of microgametophyte development. Can you identify a prothallial cell, tube cell or
generative cell? If not, examine illustrations of these developmental stages in Gifford and Foster or
Bierhorst for labelled illustrations.
B.
Megaspores, Megagametogenesis, and the Ovule
The formation of the seed requires that the functional megaspore is retained within the
megasporangium. The single functional megaspore enlarges within the nucellus and undergoes a
period of free-nuclear division. Obtain a prepared slide of Zamia ovules at the free-nuclear stage.
Locate the integument, nucellus, megaspore wall, free nuclear portion and cellular portion of the
young megagametophyte. After the centripetal cell wall formation has taken place, the archegonia
begin to form--often several per ovule. Obtain prepared slides showing a median section of an
archegonium of Zamia. Locate the neck cells and the huge central cell. At maturity the central cell
divides to produce a small distal, ventral canal cell and a large egg. In fact, at maturity the entire
archegonium is almost a single large egg. Locate the micropyle and the nucellus on your slide of the
Zamia ovule. Locate the archegonial chamber and the archegonial jacket. What is the source of the
archegonial jacket cells?
C.
Fertilization and Embryogenesis
Obtain a prepared slide showing a longitudinal section of the nucellus containing
microgametopohytes approaching the archegonium. Are the sperm of Zamia motile? What is their
mechanism of movement and what is the fate of the sperm cytoplasm during the process of syngamy?
Locate the pollen chamber and the archegonial chamber. Now observe the early proembryonic phase
of development illustrated in Zamia. Identify the cells of the embryo proper and the suspensor. What
is the polarity of the embryo? Does its polarity in the ovule bear any relationship to its source of its
nutrition? Do you have any suggestion as to the role of the suspensor in embryo development? What
is the function of the gametophytic tissue (the so-called gymnosperm "endosperm") at this stage? At
what stage is the ovule referred to as a seed? Now take some of the preserved seeds and crack them
roughly in half. Within many of these will be beautiful embryos at a variety of developmental stages.
In these embryos try to determine the developmental stage in which you find the embryo, and relate it
to the embryo development patterns in Gifford and Foster. If you find a mature embryo identify the
root and shoot apices, the cotyledons and the suspensors. How many cotyledons are there. Now
examine the seedlings of Zamia. To which mode of germination strategy do these most closely
adhere? How old would you expect these embryos be to reach this stage of development? How long
does seed development take in totality?
39
II.
Ginkgoales
A.
External Morphology of Male and Female Strobili
Observe the living specimen of the maidenhair tree Ginkgo biloba which is just beginning to produce
this year's microsporangiate strobili. Carefully dissect apart this cone and note in particular the mode
of attachment of the pendant microsporangia and their morphology. Is there any evidence that these
are, in fact, borne on microsporophylls?
Where is the dehiscence line located on the
microsporangium and what posture do the microsporangia assume at dehiscence time? Obtain female
reproductive shoots for observation in comparison with the male shoots. What are the relative
positions of these reproductive structure in the short shoot? Is this the same in female and male trees
of Ginkgo? Does the formation of the reproductive shoot (a determinite shoot) end the growth of an
apex in Ginkgo? Why or why not?
B.
Microsporangium and Microspores
Obtain a prepared slide showing a longitudinal section of the male strobilus of Ginkgo. Notice the
large wall cells, the cellular tapetum and the microspore. Microgametogenesis occurs in a manner
similar to that of cycads. Try to locate early stages of this process in your slide. Compare these to the
microspores obtained from the 'near' President's tree. At what developmental stage are these?
C.
Megasporangium and Megagametogenesis
Remove the short shoots and peduncles bearing ovules from the preserved material of a female tree of
Ginkgo. Make a drawing of the ovuliferous shoot under a dissecting microscope. What is the
position of the ovuliferous shoot on the plant? To what other plant organ do you think this is
homologous? Identify the ovules at the distal end and the collar-like growth surrounding the base of
each ovule. Does the examination of fossil Ginkgophytes provide any more lucid interpretation of the
morphological nature of the ovule-bearing axis of Ginkgo? Obtain slides showing early stages in
ovule development of Ginkgo. Identify the integument, nucellus of the ovule, and then the female
reproductive cells: including the megaspore mother cell, megaspores or megagametophyte,
depending on the developmental stage obtained. As you observe later stages of development, notice
the organization of the mature unfertilized ovule, including, if you are lucky, the massive
megagametophyte, the pollen and archegonial chambers and the cells of the archegonium.
D.
Embryogenesis and Structure of the Mature Seed
Obtain prepared slides showing developmental stages near the time of fertilization in Ginkgo. Are
there any microgametophytes evident in the nucellus, approaching the archegonial chamber? Have
you been able to observe any stages in the early embryogenesis? Are the sperms of Ginkgo
flagellated and motile as are those of the cycads? Cut a longitudinal section through one of the
preserved seeds of Ginkgo to reveal a mature embryo within the seed coat. Draw and label the
various structures surrounding the embryo, including the integuments, nucellus, megagametophyte,
and possibly even the archegonial jacket, if the embryo is very immature. Notice the position of the
suspensor, shoot and root apices. What is the typical orientation of embryo development in seed
plants in general? Now obtain prepared slides showing the proembryo and embryonic stages of
development in Ginkgo. How many generations are present within a single seed? Are all of these
layers present here? Does the fleshy outer covering of the seed of Ginkgo provide any obvious
advantage to the seed? How does this covering differ from that of an angiosperm in structure and
morphology?
40
Laboratory 19 - Coniferophyta: Vegetative Morphology
I.
Coniferales
A.
General Organography
Observe living and preserved specimens of the following conifers:
Araucaria heterophylla (greenhouse)
Calocedrus sp. (on campus)
Cedrus lebani (on campus)
Cupressus arizonica (on campus)
Juniperus scopulorum (on campus)
Libocedrus decurrens (on campus)
Pinus banksiana (on campus)
Pinus sylvestris (on campus)
Podocarpus sp. (preserved and dried)
Sequoia sempervirens (in greenhouse)
Taxodium distachum (on campus)
Thuja orientalis (on campus)
Carefully observe the organization of the coniferous shoot. Particularly note where the lateral
branches form and where the buds originate on the stems. Are the buds always associated with
leaves? How does the organization of representatives of the Coniferales compare to Ginkgo biloba
observed last week? Notice the diverse leaf forms present in the Coniferales. Which are considered
primitive? Which advanced? Carefully observe the leaves of Pinus, which are bound together in
fascicles and dissect one under a dissecting microscope. What is the morphological nature of the
fascicle? Observe a longitudinal section of the fascicle of P. laricio as is shown in Gifford and
Foster, Fig. 17-8.
B.
Anatomy of the Stem
Obtain a prepared slide of a cross section of the young stem of Pinus. Identify the cork, cork
cambium, phloem, vascular cambium, secondary xylem and annual rings. Distinguish between the
primary and secondary xylem tissues. Does this represent manoxylic or pycnoxylic development?
Are the resin ducts of this species oriented longitudinally, radially or in both directions? Are the resin
ducts of this species associated with the rays? Now observe a longitudinal section of a pine stem.
Observe in particular the pitting on tracheid walls. Notice the pattern of tracheid wall deposition in
the primary and secondary vasculature. How does the organization of the vasculature compare that
in the stem of Ginkgo?
C.
Leaf
Obtain a prepared slide of a cross section of the leaf of Pinus. Note the layers comprising the leaf and
the extent of each layer, and particularly the organization of the vascular tissue. Notice the number of
traces present in the leaf. Present near the edge of the vasculature you may be able to see cells with
pits near the endodermis layer. These cells form the so-called transfusion cells. What is the extent of
primary and secondary growth in the leaf? Leaves of Pinus may be retained from 3 to 27 years. In
bristlecone pine, Pinus aristida, which retains its leaves for 27 years, there is a functional vascular
cambium that produces largely phloem cells. How similar is this to the leaves we have examined
previously? Now examine a cross section of the leaves of Podocarpus, a conifer with strap-shaped
leaves. How are similar to the leaves of Pinus? How do they differ?
41
Laboratory 20 - Coniferophyta: Reproductive Morphology
I.
Coniferales
A.
Structure and Morphology of the Megasporangiate Strobilus
Study the morphology of the megasporangiate cones of the following conifer species available in the
laboratory. There is an excellent collection of conifer cones available in class. Pay particular
attention to only one specimen of each genus -- just scan the other specimens to get an idea about
variability in the genus. Identify the ovuliferous scale and bract. What structural patterns do these
cones have in common and in what respects do they vary? Is there a relationship between the
phyllotaxis of the cone scale and the patterns of phyllotaxis of the parent vegetative shoot?
Araucariaceae
Araucaria bidwillii
Araucaria cunninghamii
Araucaria excelsa (in greenhouse)
Agathis brownii
Agathis sp.
Cupressaceae
Actinostrobus pyramidalus
Callitris canescens
Callitris macleayana
Callitris preissii
Callitris propinqua
Callitris rhomboidea
Callitris tasmanica
Callitris verrucosa
Cupressus arizonica (on campus)
Cupressus macrocarpa
Juniperus scopulorum (on campus)
Juniperus virginianum (on campus)
Libocedrus decurrens (on campus)
Thuja orientalis (on campus)
Taxodiaceae
Cryptomeria japonica
Cunninghamia lanceolata
Sequoia sempervirens (in greenhouse)
Sequoiadendron gigantaea
Taxodium distachum (on campus)
Pinaceae
Abies balsamea
Cedrus deodora (on campus)
Cedrus atlantica
Picea abies
Picea contorta
Picea englemannii
42
Picea pungens
Pinus aristida
Pinus contorta
Pinus edulis
Pinus monticula
Pinus sabiniana
Pseudotsuga mensizeii
Pseudotsuga taxifolia
B.
Morphology of the Bract-Ovuliferous Scale Complex
For each of the following representatives of conifer families obtain an ovuliferous scale (in a nondestructive manner, if possible!). Draw the form of the ovuliferous scale, determine the number of
ovules per ovuliferous scale and draw the relationship of the ovuliferous scale to the subtending bract.
What are the main trends in structural specialization that appear to have evolved? For the
Taxodiaceae and Pinaceae, make a longitudinal section through the cone axis to further clarify the
organization of the bract-scale complex.
C.
Megagametogenesis, Fertilization, and Embryogenesis of Pinus
The life cycle of Pinus is in many ways representative of conifer embryogenesis. In one respect,
however, Pinus is atypical. In early embryogenesis, the embryo splits into four to eight embryos by
cleavage embryogenesis. Obtain a prepared slide showing the megaspore mother cell and the
structure of the immature ovule. Locate the cone axis, bract, and ovuliferous scale and the
integument. Identify the nucellus and locate the large megasporocyte. What is the nuclear condition
of this cell? Of the nucellus?
Now observe the following demonstration slides of sexual reproduction. Cooperation is important to
facilitate these observations because of the volume of material. When you see a stage, let others see it
as well! Study these slides and reconstruct the course of embryogenesis in diagrammatic form. The
following slides are available for observation:
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
D.
Early free nuclear megagametophyte
Later free nuclear megagametophyte
Mature megagametophyte and archegonium
Entry of the pollen tube into the archegonium; structure of the pollen tube
Fertilization: the outlines of the male and female nuclei are evident
Condensation and intermingling of the chromosomes
Zygote
First zygotic division
Two nucleate proembryo
Basal nuclei (late four nucleate division)
16 nucleate proembryo with elongating suspensors
Proembryo in megagametophyte
Proembryo whole mount, showing polyembryony
Mature embryo
Seed of Pinus
Obtain mature seeds of Pinus edule available in the laboratory. Make a longitudinal section through
the seed and draw the structure of the seed as seen from one cut surface. Illustrate the integument and
its constituent layer as well as the remains of the nucellus, megagametophyte and embryo. Are there
43
any vascular elements in the ovule of Pinus? How does this condition compare and contrast with
seeds of cycads and Ginkgo?
II.
Taxales
For each of the following genera, observe the female strobilus and note its organization. Select just a
single genus per family to examine.
Podocarpaceae
Dacridium cupressinum
Podocarpus clatus
Podocarpus falcatus
Podocarpus macrophyllus
Podocarpus neriifolius
Taxaceae
Taxus baccata
Taxus canadensis
Torreya californica
Torreya taxifolia
Note the fleshy covering of the ovule. How do these relate to the outside of the seed? How many
seeds are located inside the aril (Taxaceae) or epimatium (Podocarpaceae)?
III.
Morphology of the Conifer Microstrobilus
A.
External Structure of the Male Strobilus
In contrast to the complex structure that characterizes the female strobilus, the male strobilus is
simple: it is composed of simple microsporangia borne on microsporophylls with no scale or
subtending bract. Observe and dissect the male strobili of Pinus or Picea and observe them under the
dissecting microscope. Describe the morphology of the microsporophyll. What is the position of the
sporangium on the sporophyll? How are the strobili organized along the shoot? Obtain a prepared
slide showing the male cone cluster in Pinus. Locate the shoot axis, cone axis, microsporophylls,
microsporangia, and developing microspores. Obtain a prepare slide of a Pinus male cone in
transverse section and identify the morphological components.
B.
Pollen Grains
Obtain a prepared slide of the pollen grain of a conifer. Locate the wall of the pollen grain and also
the two large wings, characteristic of many of the conifers. Scan the slide and find a pollen grain with
two prothallial cells and the generative nucleus with its inclusive tube nucleus and generative nucleus.
What is the nuclear condition of each of the cells of the male gametophyte?
44
Laboratory 23 - Gnetophyta: Vegetative and Reproductive Morphology
I.
Vegetative Morphology
A.
General Organography
The study of the morphology of the Gnetales has historically been colored by the politics of the
regions in which these genera thrive. Perhaps the best understood of these is the North American
genus Ephedra. Examine the general organography of the following specimens of Ephedra as
represented by herbarium specimens and pickled material:
Ephedra antisyphiletica
E. distachya
E. intermedia
E. gerardina
Where are the leaves and how are they arranged? What is the pattern of branching in each species?
In what ways are the leaves of Ephedra and Equisetum similar? How do they differ?
For each of Gnetum and Welwitschia examine drawings and photographs available in the laboratory.
What is the major distinguishing feature of each of these genera? Can you account for the bizarre
organography of the genus Welwitschia? What is the morphological interpretation of the large white
"scale bodies" that are evident at the summit of the shoot of this plant?
B.
Anatomy of the Stem and Wood
Obtain a prepared slide showing a cross-section of a young stem of Ephedra, still in the primary
stages of its growth. Identify all the cellular and tissue components, especially the vascular bundles,
pith, large cortex, and thick epidermis. Now obtain and examine an older twig of Ephedra in cross
and longitudinal section. Is it possible to distinguish between the tracheids and vessel elements in
these stems? What are the major differences between tracheids and vessel elements? These
differences are evident in macerated tissue of Ephedra, as illustrated in Fig. 18-10 of Gifford and
Foster. Can you distinguish between the primary and secondary xylem in longitudinal section? Do
any other non-angiospermous plants have vessel elements in their life cycles? Examine the
demonstration slides of Gnetum wood. How is it similar to Ephedra? How does it differ?
II.
Reproductive Morphology
A.
Ephedra
Obtain a preserved androstrobilus of Ephedra and identify the perianth, subtending bract and
microsporophylls. What is the phyllotactic pattern of the strobilus? Of the inflorescence as a whole?
What is the position of attachment of each strobilar part? Now, obtain a prepared slide of the male
strobilus and identify the same features. Why is this considered to be a compound strobilus? Locate
the thin perianth and the enclosed sporophylls. How do the sporophylls differ from those in the
angiospermous stamen? Observe the pollen grains of Ephedra in this slide and in SEM in Fig. 18-15
of Gifford and Foster. Pollen grains such as those of Ephedra and Welwitschia have been found as
early in the fossil record as the Permian; however, there is no megafossil evidence (unfortunately) for
this fascinating group.
45
The gynostrobilus or female strobilus of Ephedra consists of from five to seven pairs of decussate
sterile bracts which subtend two ovules borne on short branches in the axils of the uppermost bracts.
Obtain a single gynostrobilus and locate the sterile bracts, the ovule, its surrounding involucre and the
elongated integumentary tube which constitutes the micropyle of Ephedra. By what means can this
be distinguished as a compound strobilus? Obtain a prepared slide showing the ovule of Ephedra.
Can you identify the archegonium, embryo, and developmental stage that is shown? Do other
members of the Gnetales possess an archegonium? Now observe and sketch some of the stages of
early embryology in this rare and beautiful series of slides of Ephedra. Can you determine the fate of
the first sperm cell fusion? Of the second?
B.
Gnetum and Welwitschia
We are most fortunate that we have any material available on these fascinating plants, meager though
it may be. Observe the organization of the inflorescence of Gnetum. This is obviously a female
structure, since it has seeds. Observe the illustrations in Bierhorst and Chamberlain to see the
organization of the male inflorescence. Also, observe the male strobilus of Welwitschia available in
lab. Why is this considered a compound strobilus? Is there any external evidence to support this
conclusion? Refer to texts for the structure of the female strobilus and the embryology of both.
Marten's book entitled "Les Gnetophytes" (1970) is an excellent source for published interpretations
of these plants (especially if you can read enough French to understand the figure captions). The
embryology of these plants is bizarre and the reports, contradictory. Their reproduction remains as
perhaps the most major basic biological problem of our time and one that has not received nearly
enough research interest.
46
Laboratory 24 - Magnoliophyta: Vegetative Morphology I
Comparative Morphology of Lateral Appendages and Shoots
I.
Protective adaptations
A.
Thorns
1. Thorns are derived from highly modified shoots, which are usually axillary in position. They may
be either branched or unbranched and may or may not have any apparent lateral foliar appendages.
An example of a fairly simple thorn is that of Bougainvillea spectabilis. Examine a branch of this
plant and determine how these thorns are borne on the axis and how they develop on the shoot. Can
you justify the classification of these spiny projections as modified shoots through serial homology.
With what shoot structure is this thorn homologous?
2. Now examine Carissa grandiflora (natal plum, Apocynaceae) and see how the thorns relate to the
shoot. How does the organography of these thorns resemble Bougainvillea? How do they differ?
Closely observe the branched thorn. Can you identify nodes and internodes on the thorn? Now look
carefully at the base of the thorn and note the presence and distribution of accessory buds. These
buds are capable of forming new leaves later in the season. Some will remain dormant for the life of
the plant.
3. Thorn shoots may also be flattened. Observe a photograph of Colletia cruciata (Fig. 435, vol. 1 of
Troll, 1935) and determine what morphological features of this structure allow you to decide on its
morphological nature. If you observe this photograph carefully, you should be able to see the leaves
and the development of these interesting and unusual appendages.
B.
Spines
1. Spines are pointed structures derived either from whole leaves or various parts of the leaf. Any
projection which arises from a leaf part, cataphyll, or reduced part thereof may be properly termed a
spine. In many cases the entire leaf itself may become sclerified (it is usually non-photosynthetic)
and may assume the function of a spine. One good example whole leaf replacement with a spine is
Larreya tridentata, commonly known as creosote. Where do the foliage leaves emerge in relation to
the spines? Another example is Asparagus sprengeri, which is horticulturally promoted as a "fern"
because of its complex and filiform leaflet morphology. Numerous spines are present on this plant.
Notice the pointed structures on the underside of the stems. Are these spines? How about the
"lamina" of these leaflets? Notice that in all of these cases, the leaf is usually readily identifiable by
its relationship with axillary buds and/or shoots.
2. Members of the family Cactaceae are well-armed with spines, but in this case, the main leaf is not
involved. An entire evolution of the family is represented by primitive genera such as Pereskia and
Pereskiopsis (which have fairly large, but seasonally deciduous leaves), followed by intermediate
forms such as the standard cylindrical and flattened cacti (where the leaf is quite temporary and peglike) and finally the unarmed, tropical epiphytes like Rhipsalis and Epiphyllum. To understand the
origin of the spines in the cacti, it is useful to examine a stem of Pereskia closely. Can you account
for the origin of the spines? Are they associated with an axis? If so, what is their homology?
Glochids are small, needle-like trichomes which are also associated with the areole. Do these share
the same homology?
47
3. Members of the Euphorbiaceae outwardly appear similar to the Cactaceae, but differ in the parts of
the leaf they employ as armaments. A number of members of the Euphorbiaceae are present in class.
Observe their stems and notice the arrangement of the spines on the axis. What is their relationship
to the leaf? Are the armaments always paired? Look to the upper part of the plant to see if the
development of these spines helps you to refine your ideas about the morphological nature of these
spines.
C.
Prickles
There are also morphological entities which arise as simple eruptions of epidermal and sub-epidermal
tissue, which have no homology to either stems or leaves. Examples of this are on display, including
Chorisia speciosa (floss-silk tree, Bombacaceae) and Erythrina crista-galli (Fabaceae). Can you
follow these emergences to their inception on the stem? Do they bear any relationship to the
arrangement of leaves or stems? In Chorisia, notice that basal portions of the plant possess more
emergences in odd orientations than could possibly be answered by either a stem or leaf origin.
Presumably, this is the result of continuing production of emergences throughout the history of the
plant. This is also the case for the common rose (Rosa sp.), which is typically quite well armed.
Examine these structure on the available specimens. In some cases, these structures are
morphologically regarded as epidermal emergences, or "prickles," while others may have a separate
origin and represent true thorns. Outwardly, the two structures resemble one another, but their
internal structure may differ and they are not homologous.
There are several specimens on which you may try your knowledge of pointly parts. These are
Pachypodium lamerei (Madagascar palm, Pachypodiaceae), which is fairly easy, and Ponicerus
trifoliata (trifoliate orange, Rutaceae) and Douryalis caffra (Flacourtiaceae), which are similar but
fairly difficult to determine. What is the nature of their armaments? Do these represent the reduction
of whole leaves or simply modifications of leaf parts? Observe these plants closely and try to come
to your own conclusions about the morphological nature of these spine-like projections.
II.
Runners or stolons
Runners or stolons are above-ground lateral branches that have widely separated nodes with highly
reduced leaves. Usually associated with an axillary bud, these modified stems are particularly
important in vegetative reproduction, such as in Frageria, the strawberry. Observe the growth habit
of Chlorophytum cosmosum, the spider plant, which has an erect, orthotropic shoot system and a
well-developed, lax, or plagiotropic runner. Note periodic resumption of the orthotropous habit is
accompanied by a growth curvature at the transitional nodes. In what position do roots form? How
can the new orthotropic axis be regarded in a morphological sense?
III.
Storage stems
A.
Rhizomes
Rhizomes are horizontal axes travelling at or below the surface of the ground and commonly contain
storage reserves. Several examples of rhizomes are readily available. A common over-wintering
rhizome is typified by Iris, Sanseviera, Amaryllis or Acorus, which we have looked at before. The
important morphological characteristics of the rhizome, namely the highly reduced leaf scales, welldeveloped internal storage materials, and infrequent branching, are typical of the rhizome. The
rhizomes of Johnson grass, a common weed here, and water lilies are particularly aggressive and
quick successful in vegetative reproduction.
48
B.
Tubers
Tubers are modified underground storage shoots. The most frequent example is, of course, that of
Solanum tuberosum, which produces numerous tubers called potatoes, which are available for
inspection in the lab today. Carefully examine their external features and origin. How can you prove
that it is a shoot? Locate its connection to the axis of the parent plant as well as the terminal bud at
the opposite end of the tuber. Notice the structure of its buds ("eyes") and their arrangement on the
tuber. What do the eyes represent morphologically? Locate the reduced scale leaves on the tuber. If
you are not entirely convinced, examine some preserved young tubers of this plant available in the
lab.
C.
Bulbs
Bulbs are orthotropic shoot systems with short internodes and thickened leaf bases that are modified
for storage. Although the bulb is a shoot, it is typically located underground and may be covered by
one or more layers of scale-like dead leaves. The classical example, of course, is Allium cepa, the
common onion. Examine a mature bulb, and if someone has not already done so, make a median
longitudinal section. If you cut through the center of the shoot, you may be able to see the so-called
"renewal bud". Distinguish the stem from the leaves at this time and determine its method of
branching. Does the terminal bud form the aerial portion of the plant during the next cycle of
growth? On a different note, you should be able to determine the botanical basis for onion "rings".
49
Laboratory 25 - Magnoliophyta: Vegetative Morphology II
I.
Succulent shoots
A.
Primary structural modifications
The majority of Cactaceae have shoots in which the leaf is reduced to the lower leaf region or leaf
base and the upper leaf is abscised early in development or shed so early in development that it is no
longer apparent on the mature leaf base. Some forms show a reduction in the leaf base (the so-called
podarium) as a derived characteristic (e.g. the epiphytic tropical forms). It is possible to observe all
stages of morphological diversification in the Cactaceae in our greenhouse from leafy forms like
Pereskea, to more traditional cacti like Opuntia and Mammilaria, to highly derived epiphytic forms
like Rhipsalis and Epiphyllum. Observe these in the greenhouse and draw one or two of the examples
of each degree of morphological specialization.
The podaria of cacti represent enhanced leaf bases that have been modified for photosynthesis. The
areoles represent the fate of a short, axillary shoot. Bud scales have been modified to form spines,
whereas other cataphylls within the series have finely divided leaves forming lignified cilia known as
glochids. For Pereskea, Opuntia and Mammilaria, examine these structures and compare the
immature and mature forms. Which of these have typical parastichys? Orthostichys? How is the
succulence of these stems achieved--through primary or secondary growth?
Apparent parallel evolution toward succulent stems has occurred in the Euphorbiaceae and
Asclepiadaceae. Observe the specimens available in the greenhouse. Compare the organs that have
been modified in these plants. How is the general similarity in form achieved in these plants
compared to the Cactaceae? How do they differ?
B.
Secondary structural modifications
Some desert species develop large stems for the specific purpose of storing water through almost
exclusively secondary growth. Examples are found in diverse families, but are characterized by the
following characteristics: increase in bark production, increased parenchyma produced in the wood
and typically decreased leaf surface area. Examine the available representatives.
2.
Succulent leaves
Many succulent plants do not resort to large complex stems as organs of water storage, but retain
much of their water within succulent leaf tissues. The Liliaceae (lily family), Crassulaceae (jade plant
family) and the Aizoaceae (ice plant family) are families in which leaf succulents are quite common.
Examine the representatives of these families closely. Section a representative of one of these
examples and observe it as a wet mount. Where is the photosynthetic surface located? Vasculature?
Is the internal organization of the leaf also an adaptation to xeric climates?
A particularly interesting adaptation to the internalization of the photosynthetic surface in leaves is
the development of window leaves, where the upper surface of a succulent leaf contains unpigmented
areas that allow light to penetrate. This is regarded as an example of an equifacial leaf because both
adaxial and abaxial leaf surfaces participate in photosynthesis. Representatives come from a variety
of families. Supposedly light is then scattered internally to the lower leaf surface. Observe the
available specimens. Make a cross section of one leaf passing through both window areas and the
lower surface of the leaf, and then observe the specimen as a wet mount.
50
Laboratory 26 - Magnoliophyta: Vegetative Morphology III
I.
Dicotyledonous leaves
A.
General organization of the leaf
In dicotyledonous angiosperms, all of the different leaf structures are expressed to a greater or lesser
degree: the Oberblatt (upper leaf zone), Unterblatt (lower leaf zone), and Niederblätter (transitional
leaves). By this time in the semester, usually all of the overwintering buds have opened and the
Niederblätter are difficult to find. Hopefully, you have seen them during the early spring and you can
recall their form vaguely. From the lowermost leaves of the overwintering buds to the innermost
foliage leaves, a transitional series of leaf forms can be observed. The buds tend to be leathery, nonphotosynthetic and may have some suberized cork on their outer surface (which gives the wood-like
appearance to budscales in overwintering buds of trees and shrubs). The next series may be larger
and may have a greatly reduced form of the upper leaf, from a small recognizable leaf to, at
minimum, a precursor tip (Preläufespitze). Examples of these transitional leaves are also shown in
Troll's Vergeichende Morphologie der Höheren Pflanzen (pp 1320-2, 1338-9). Which of the leaves
would properly be termed cataphylls? prophylls? Notice how the expression of each of these leaves
changes in the bud. How does their function differ? How can these various components be
distinguished?
Observe the general organization of overwintering buds. Identify the terminal bud of the previous
year's growth on an available winter stem. How does the size of the terminal bud compare in size to
axillary buds of the present year's growth? Does the terminal bud unfurl to produce leaves? As you
may realize, the unfurling of leaves in the spring is only an expansion of leaves held dormant in the
bud from the previous season (or before), which have just gained expression at this time. Can you
identify terminal bud scale scars? leaf scars? How is a year's growth delimited in these twigs? Will
the growth from the terminal bud represent prolepsis or syllepsis?
B.
Ontogeny of simple leaves
Now examine any of the numerous examples of simple leaf formation from selected specimens in the
greenhouse. Observe, in general terms, which region of the leaf expands and reaches its mature form
first. Also, note the general orientation of maturation on the axis. Is it acropetal? A careful
dissection of the shoot apex will show the degree of development of the various leaves. When is the
form of these leaves initiated?
C.
Ontogeny of compound leaves
Now, observe the origin of a pinnately compound leaf. First, note the differences in general
organography from the simple leaf. From which segment of the leaf do the leaflets arise? Now,
carefully dissect the shoot apex until all the newest, initiated leaves are evident. Draw the
organization of the smallest of these leaves and in particular note the early organization of the leaf,
leaflet primordia, and the order of their initiation. How does this compare to the simple leaves
studied previously? How would you account for the formation of palmately compound leaves, using
this as a basis for comparison? How would you compare this to peltate leaves? For comparison, you
may like to examine the immature leaves of either Hypocotyl or Tropaeolum (nasturtium). What type
of leaf development do these resemble?
51
D. Phyllodes
Most leaves are clearly bifacial, with a consistent organization of the lamina into a dorsal and ventral
face, vasculature with a crescentic configuration, and a generally bilaterally symmetrical organization.
Two forms of unifacial leaf challenge this pattern: the unifacial leaf of such monocotyledonous
plants (described later) and the phyllode of such dicotyledonous plants as Acacia and Eucalyptus.
Examine available specimens as examples of phyllodate leaves. Can you identify any traditional,
juvenile leaves? How do phyllodate leaves develop? How is their vasculature organized? Oriented?
E. Carnivorous plants
Among the most spectacular examples of carnivorous plants are Dionaea muscipula, Venus' flytrap,
and the several forms of Drosera, the sundew. Observe the Venus' flytrap first. What constitutes the
upper and lower leaf zones? How is the trap set? How is it triggered? How is the insect
immobilized? How is it digested? Please feel free to experiment, but leave enough traps open so
everyone can see how they close. (Traps take alot longer to open then they do to close!) Now
observe the sundew. How does the this plant lure and trap its prey? How is the insect digested then?
What constitutes the upper and lower leaf zones here?
The patterns of leaf morphology in the insectivorous pitcher plants have been a constant source of
interest on the part of morphologists since this is the closest approach in leaf morphology to that of
the carpel. Examine examples of these various forms of pitcher plant as available in living specimens
and in books, as exemplified by the genera Cephalotus, Darlingtonia, Heliamphora, Napenthes and
Sarracenia, all of which possess epiascidiate leaves. Which of these have their own digestive
enzymes? Which rely on symbionts to digest insects?
II.
Monocotyledonous patterns
A.
A basic pattern of organization
Monocots rarely express the degree of development of the Oberblatt and Niederblatt typical of the
dicots. An extreme example in the development of the Oberblatt in monocots is Sansieveria
cylindrica, which consists of a large bilaterally symmetrical lower leaf zone and large cylindrical
upper leaf zone. An intermediate form available for observation is Sansieveria trifasciate, mother-inlaw plant, which has a much more reduced upper leaf zone. The typical monocot is represented by
strap-shaped, bilaterally-symmetrical, bifacial leaves like those of the lilies and orchids. Draw one of
these typical bifacial leaves as a representative specimen. Take cross-sections at various levels of the
leaf to determine the organization and orientation of the leaf vasculature. Why is this considered to
represent a bifacial leaf.
Now make some cursory observations of the organization of a typical grass leaf. Identify the
Oberblatt, Unterblatt, and distinctive ligule. What is the morphological nature of the ligule? Do
grass leaves express the same type of determinate growth pattern characteristic of other angiosperms?
How do grass leaves continue to elongate, once initiated?
B.
Patterns of compound and complex leaf development
The Unterblatt is expressed to extreme degrees where the upper regions of the lower leaf become
extremely flattened to the extent that they simulate well-developed laminae. Observe an archetype of
this pattern in Monostera deliciosa. Note that in this plant, laminar development is evident, along
with secondary formation of deep lobes with approach becoming leaflets. This is indicative of the
52
formation of compound leaves by schizogeny in monocots, in marked contrast to their independent
formation in dicots.
Palms have a characteristic leaf form which differs from all others. In the bud, a pleated laminar
surface forms early in development, and only later does either the upper or lower surface degenerate
to form the separated leaflets. The leaflets remain connected at the tips by structures known as the
reins. As the leaf unfurls, the palm leaf splits laterally at the middle, with the split progressing
upward to the reins and downward to the petiole. At the completion of unfurling, the reins are
discarded.
C.
Unifacial leaves
The monocot analog to the phyllode is the unifacial leaf of such monocots as Acorus and Iris, which
have an ontogenetically later determined form of unifacial development. Examine the leaf of Iris
carefully to identify the unifacial and bifacial elements of the leaf present. Take progressive crosssections at different levels of the leaf. Diagram the organization of the vasculature in the unifacial
segments of the leaf. How does this differ from the phyllode in its mature form? How does the
development of this unifacial leaf differ from that of the phyllodate plants?
53
Laboratory 27 - Magnoliophyta: Vegetative Morphology IV
The primary organization of monocots and dicots is dramatically different. Dicots have a well
developed primary root. Additional orders of branching in dicots usually originate directly from the
primary root. Monocots, however, have a rather short-lived primary root, with most roots originating
from the base of the stem and additional roots erupting from the stem at nodes. Additional orders of
branching in monocots emerge from the adventitious root system. What is the normal pattern of root
production in the various plant groups that we have looked at over the course of the semester--is it
more similar to dicots or monocots or is it mixed for different characteristics?
1.
General organization
Observe the organization of the primary (tap) root in any dicot. What relationship does the tap root
have to the radical during seed germination? How many orders of branching do you observe? How
do the laterals originate in dicots and monocots--from the root apex or distant from the apex?
Superficially (exogenously) or subsuperficially (endogenously)?
Now, observe the fibrous organization of the roots of a monocot. Can you identify the radical in the
structure of the mature monocot plant? How many orders of branching can you identify? Does this
differ from the dicot? Where are root hairs expected to occur? What is their function and
morphological nature?
2.
Specialized root organizations
A.
Contractile roots
Contractile roots are those which become shorter with age, pulling the plant downward into the
anchoring matrix in which its secondaries are located. Perhaps the most familiar example is
Taraxacum officinale (the dandelion). Observe the young and older roots. How is the contractile
behavior of these roots indicated on the surface. Make a longitudinal section of one of the contracting
roots and observe it as a wet mount in the compound microscope. What tissues are involved in this
behavior? Although this example shows strong contractile behavior in the tap root, there also are
monocots with contractile roots.
B.
Aerial roots
Aerial roots in orchids are highly differentiated organs that play a role not only in binding the plant to
its support matrix but also in obtaining water and nutrition. Observe the aerial roots of some
epiphytic orchids. Note that the surface of some of these roots is covered with a highly modified
water-retaining material known as velamen--part of the highly modified multiseriate epidermis.
Slides of this material are available for observation. Once water is retained on the surface, it is
rapidly absorbed by subjacent layers of the root.
C.
Prop and stilt roots
Prop roots are those that are ramified to support the plant. A classical example is that of Rhizophora
mangle, the mangrove, which grows in the intertidal zone on seashores and in estuaries. This plant
has developed the remarkable adaptation of premature germination of the seeds on the plant, resulting
in the primary root elongating to 10-15 cm before release from the plant. When the seed is shed, the
primary root is dropped into the mud, establishing the apex above water. This root continues to
branch to stabilize the growing plant, producing an extensive system of prop roots. Observe the
54
living specimen, noting the mode of branching and origin of the initial branch of the root and its suborders.
Stilt roots originate regularly from the node and are typical of monocots. Observe a corn plant to
determine the origin and general organization of the stilt roots. Are these absorptive organs or strictly
architectural in function in this plant?
D.
Root nodules
The formation of stable root nodules in legumes is the result of a stable symbiosis of modified short
laterals that have become infected with nitrogen-fixing bacteria, frequently of the genus Rhizobium.
This provides organic nutrients and a protective matrix for the bacteria and enhanced ability to
accumulate mineral nutrients (particularly nitrogen) for the legume--obviously the basis for a very
successful symbiotic relationship. Observe the organization of the roots in these representatives:
Coronilla varia and Trifolium pratens, both common weeds, and other examples as available. Where
is the bacterium located within the nodules? What is the morphological basis of the nodule (what
organ forms the root nodule)?
55
Laboratory 28 - Magnoliophyta: Reproductive Morphology I
I.
Flowers
The flower may be regarded as a highly modified bisexual strobilus. Modifications in its form are
related to the transmission of pollen and the mode of pollination largely determines the form of the
flower. All organs of the flower have been drastically modified during the course of evolution, but
the outer whorls better retain their leaf-like characteristics. For today's lab, observe as many flowers
as you can from the fresh material available from outside and the greenhouse. As you dissect these
flowers, consider how their form and function inter-relate and try to observe as many floral
reductions, fusions and modifications as possible.
A.
Calyx and Corolla
The most leaf-like organs in the flower are the calyx and corolla. How many traces are present? Are
traces associated with a gap or interruption of the vasculature? When organs from one floral series
fuse, this is called connation and the organs are said to be connate. When organs from different floral
series fuse, this is called adnation and the organs are said to be adnate. When fusion occurs at the
time the organ is initiated, it is congenital; when it is usually fused at a later stage of development
after initiation, it is postgenital. Two spectacularly different flowers to observe are orchids, grasses
and Euphorbias. Identify the sepals and petals on these different flowers. Could you clearly
differentiate between monocots and dicots? Did you find any sepals or petals in Euphorbia flowers?
B.
Androecium
The androecium consists of one cycle or more of stamens, each consisting of a filament and anther
sac (morphologically termed, the microsporangium). Although its leaf-like nature is suppressed in
most angiosperms, horticulturally-derived plants frequently have sterile stamens, known as
staminodes that replace elements of the androecium with petal-like appendages. The staminode can
be distinguished by the presence of an abortive anther at the tip of the organ; the edges of the filament
are elongated into laminae! The androecial series is frequently fused to preceding series, and
numerous terms are applied to different arrangements--some of which are evident in only a single
family.
The primitively leaf-like nature is also evident in Drimys, water lilies (Nymphaea) and Magnolia,
where the microsporangia are lateral and extend the length of the filament. Microsporangia in the
flowering plants are typically simple, but as they mature, the septum between parallel anther sacs
breaks down, resulting in a functionally synangiate microsporangium at anthesis.
C.
Gynoecium
The least leaf-like series in the flower is the gynoecium, which truly appears leaf-like in the rarest of
circumstances--the most commonly available example perhaps being the pea pod or bean, which is a
fruit formed by a single carpel (= simple pistil). The carpel is the unit of organization in the female
parts, but is so frequently fused into a compound organ that students sometimes get confused on what
a carpel looks like and what it represents within the gynoecium. Without some experience, this is not
usually immediately evident. The pistil is differentiated into a stigma--the pollen receptive area, a
style--the pollen tube transmitting tract and an ovary--the region of the pistil containing the ovules.
56
As viewed by one major theory, the carpel is clearly represents a derived form of the megasporophyll
obtained by a folding of the primitive seed leaf. Unfortunately, this is evident in very few plants, the
most notable of them being in the woody Ranales, in the families Winteraceae and Degenariaceae-families also noteworthy in lacking vessels. Observe fixed or fresh flowers of Drimys, a member of
the Winteraceae. What is the phyllotaxis of the floral parts of this plant? How many sepals? petals?
anthers? carpels? Is there any fusion of floral parts evident in this flower? Can you see the
conduplicate suture in this plant? the stigmatic crest? What is the path of pollen tubes in this plant?
Observing more complicated plants, it is possible to follow the means by which the carpels fused to
give rise to the compound pistil. Usually, the attachment of the ovules to the interior of the carpel-known as the placentation gives a clue. Placentation is usually termed axile, parietal, basal or free
central. Axile placentation results when the carpels fuse near their sutures; parietal placentation
results when carpels fuse in the opposite orientation. Basal and free-central placentation involve the
elimination of septae between carpels and is a derived characteristic. Laminar placentation, seen in
genera like Drimys, is quite primitive and present in few plants. In a compound pistil, its multiple
nature is usually reflected in the organization of the stigma (# of lobes = number of carpels), the
symmetry of the style and the organization and vasculature of the ovary. In compound ovaries with
septae, each locule corresponds to one carpel.
The location of the ovary relative to the sterile cycles determines whether it is hypogynous, with a
superior ovary, perigynous with a fused hypanthium or epigynous, displaying an inferior ovary.
Examine floral representatives available to determine their pattern of organization, placentation and
means of carpel fusion. When you examine a representative of the epigynous ovary, determine what
floral organs are fused in order to form the hypanthium. Are these connate or adnate? Congenitally
or postgenitally fused?
II.
Inflorescence types
Inflorescence types determine the appearance of the fertile area of the plant to potential pollinators
and therefore may be as important as floral morphology in certain pollination strategies. These types
are defined traditionally as being either determinate or indeterminate (closed and open types,
respectively) based on the rapidity with which the floral apex disappears and is used up and whether
the stem forms a terminal flower. Determinate inflorescences may terminate more quickly resulting
in an immediate and showy display, whereas indeterminate inflorecences may bloom for weeks,
presenting flowers at all stages of development at a given time.
A.
Determinate types
These end in the formation of a flower and are classified as closed types. The main types of
determinate inflorescences are classified as solitary, clusters or cymes.
B.
Indeterminate types
These do not terminate in flowers and are classified as open types. The main types are racemes,
spikes, catkins, heads, corymbs, umbels and panicles. These are terms that are useful for
identification of plants, but do not have any implied morphological relationship. Examine available
specimens and classify them.
57
MAJOR INFLORESCENCE TYPES
1.
Inflorescence determinate.................................................................................................. 2
Inflorescence indeterminate .............................................................................................. 6
2.
Inflorescence of only one flower (solitary) ....................................................................... 3
Inflorescence of more than one flower .............................................................................. 4
3.
Inflorescence terminates a major axis ........................................................ scapose solitary
Inflorescence terminates a non-major axis ................................................. axillary solitary
4.
Inflorescence terminates a major axis ............................................................................... 5
Inflorescence terminates a non-major axis ............................................................... cluster
5.
Branching in more than one direction ........................................................................ cyme
Branching one-sided....................................................................................scorpioid cyme
6.
Secondary axis absent ....................................................................................................... 7
Secondary axis present .................................................................................................... 13
7.
Inflorescence forms linear series ....................................................................................... 8
Inflorescence forms circular series .................................................................................. 12
8.
Peduncle present...................................................................................................... raceme
Peduncle reduced (inflorescence sessile) .......................................................................... 9
9.
Axis erect and slender ..................................................................................................... 10
Axis not erect or slender ................................................................................................. 11
10.
Flowers not reduced ................................................................................................... spike
Flowers reduced ..................................................................................................... spikelet
11.
Axis not fleshy .......................................................................................................... catkin
Axis fleshy ............................................................................................................... spadix
12.
Inflorescence sessile .................................................................................................... head
Inflorescence pedunculate ....................................................................................... corymb
13.
Inflorescence whorled on primary axis ........................................................................... 14
Inflorescence not whorled on primary axis ..................................................................... 15
14.
Tertiary axis absent ................................................................................................... umbel
Tertiary axis present ................................................................................ compound umbel
15.
Secondary axis determinate ....................................................................... paniculate cyme
Secondary axis indeterminate.......................................................................................... 16
16.
Branches all on one side ......................................................................... secondary panicle
Branches in more than one direction ....................................................................... panicle
58
Laboratory 29 - Magnoliophyta: Reproductive Morphology II
I.
Microsporogenesis and microgametogenesis
A.
Pollen tube growth
Growing pollen tubes is simple if you are using bicellular pollen. Physiologically, it is possible to
grow many pollen grains on a fairly simple Brewbaker-Kwack medium (0.01% H3BO3, 0.03%
Ca(NO3)2·4H2O, 0.02% MgSO4·7H2O and 0.01% KNO3) with from 0% to 20% sucrose added.
Obtain a slide and place a small drop of pollen growth solution on it. Then sprinkle some fresh
pollen on the drop. Wait for a minute or two for the pollen to settle into the solution and hydrate.
Then, VERY CAREFULLY add a cover slip. (Hydrated pollen grains break easily!) Now, to prevent
the slide from drying, either put it in a humid chamber or seal the edges of the cover slip by applying
liquid Vaseline to the outside edges of the cover slip. (A warm paper clip is an ideal applicator, if
used carefully.) Pollen tubes should form within 10-30 minutes. These will be visible using a
compound microscope or high magnification with a dissecting microscope. For showing rapid
growth, pollen of Tradescantia and Impatiens is excellent. To show the movement of the generative
cell down the pollen tube, Amaryllis is ideal. As these elongate, can you see the vegetative nucleus?
the generative cell? a linkage between these structures? When are the sperm cells formed?
B.
Pollen grain formation
A developmental sequence of light and electron micrographs is available on demonstration. Each of
the following developmental stages in the formation of the mature pollen, typified by Plumbago
zeylanica. The following stages are represented:
1.
2.
3.
4.
5.
6.
Microsporocyte in prophase I. Notice the number of microsporocytes, their
organization,
and
their
coenocytic
organization
during
early
microsporogenesis. The fine structural organization of the meiotic cytoplasm
and organelles appears nearly as drastic as those events occurring in the
nucleus.
Cytokinesis occurs simultaneously in all four of the microspores in the
dicotyledonous pattern of microsporogenesis, shown here.
Cytokinesis is followed by enclosure of the microspores within a callosic
special wall which is presumed to regulate the entry of certain external
metabolites, including nucleic acids. This is also the stage at which the
carbohydrate skeleton of the exine is laid down.
Expansion and vacuolization of the microspores accompanies the formation
and differentiation of the pollen wall. At this stage, the nucleus has a small
nucleolus and is centrally located in the cytoplasm. The tapetum actively
secretes sporopollenin precursors at this stage. Microspores have not
completed inflation.
Post-meiotic mitosis results in the formation of a separate vegetative cell with a
vegetative nucleus and a lenticular generative cell attached to the intine wall of
the pollen. Vacuolization continues until full volume of the pollen grains is
reached. The tapetum begins degeneration.
The generative cell next separates from the intine to become a free cell. At this
stage, the pollen reaches full inflation, morphogenesis of the generative cell
into a spindle-shaped cell occurs, and it comes to lie next to the vegetative
nucleus.
59
7.
At the transition to maturity, the two sperm cells are formed. Vacuoles are
replaced by cytoplasm at this stage. The cytoplasm contains many is secretory
vesicles (polysaccharide vesicles used in pollen tube growth), numerous
mitochondria, plastids, and either starch or lipid bodies as a storage material.
The storage material in pollen grains appears to be consistent with pollination
strategy. Insect-borne pollen tends to contain lipid bodies; wind-borne pollen
tends to contain starch grains.
In angiosperms, the mature pollen may be either bicellular, containing a generative cell, or tricellular,
containing fully formed sperm cells. This characteristic varies by genus and family in the
angiosperms. (In Populus species, including cottonwood, there is the transitional condition-sometimes both bicellular and tricellular pollen may occur in the same anther. The formation of
sperm cells prior to germination is considered to be the derived characteristic. Compatibility systems
and pollen physiology relate to the time of sperm cells formation relative to anthesis. See table.
C.
Pollen wall micromorphology
Pollen wall morphology consists of two layers: the intine, which is primary cell wall, and the exine,
which contains the extremely resistant compound sporopollenin. The exine provides protection
against desiccation and physical deformation; however, the pollen tube emerges from and is
continuous with the intine. Therefore, one or more apertures are present. Observe scanning electron
micrographs of selected pollen grains, observing apertures, sculpturing and diversity. These are
extras, so you may take some souvenirs to keep, if you wish.
Functionally, the pollen must be protected by a resistant cell wall, but must respond as a naked cell to
recognition stimuli. One response to this evolutionary pressure has been the diversification of pollen
wall sculpturing to retain superficial glycoproteins and lipids on the grain. These proteins and lipids
form a sort of pseudomembrane on the pollen that activate imbibition when deposited on the
appropriate stigma. Remarkably different sculpturing patterns are present on different pollen grains.
Presumably, these have occurred in response to evolutionary pressures from a variety of directions-most notably for efficient dispersal, germination and recognition. Cell wall organization and cellular
condition of the pollen is systematically significant in some plant groups.
D.
Internal organization of the male germ unit
The male germ cells (either the sperm or generative cells, depending on the pollen grain and stage of
development) are typically associated with the vegetative nucleus prior to germination and during
pollen tube growth. This association has been termed the male germ unit. Examples are in the lab.
Although the exact form of the MGU varies from one species to the next, this association may be
important in delivering the two gametes into the embryo sac at the same time, preventing
heterofertilization (a condition in which sperm cells from different pollen tubes fertilize the egg and
central cell.) The two sperm cells are linked by retaining a common cell wall, as a result of
generative cell cytokinesis and both are contained within an internal vegetative cell plasma
membrane. A second physical association occurs between at least one of the sperm cells and the
vegetative nucleus. This forms a more or less intricate arrangement of complementary surfaces that is
maintained up to the time of fertilization. The grasses are exceptionally interesting in this regard,
because their association is severed early in development, but re-established just prior to fertilization.
This argues strongly for the importance of the MGU. This association appears to occur in the vast
majority of angiosperms.
60
II.
Megasporogenesis and megagametogenesis
A.
Megagametophyte development
The vast majority of flowering plants (over 70% of the families) have monosporic development, in
which the embryo sac is genetically homogeneous and all cells within are derived from a single
megaspore, the others aborting (see Fig. 20-13). On demonstration are slides representing these
events from initiation of the megasporocyte to maturity in both Zea mays, corn, and Cucurbita, the
cucumber. The captions explain the events in greater detail and, at least in corn, illustrate how
callose wall patterns may determine the pattern or abortion between the four megaspores. Note that
in corn, the micropylar dyad cell may begin to abort prior to the completion of meiosis, so that in this
case, sometimes only three cells are formed as a result.
The other types of embryo sac formation require the participation of either two megaspores (bisporic
development) or four megaspores (tetrasporic development), resulting in genetically heterogeneous
nuclei within the megagametophyte (see Fig. 20-15, p. 584, for the diversity of different types). The
majority of these variant forms are tetrasporic. One form of tetrasporic embryo sac development that
is particularly striking is that of lily, which displays Fritillaria- type embryo sac development. The
events of that type of embryo sac development are discussed in the text (p. 587).
B.
Organization and variability in the mature megagametophyte
A small display shows the general organization of the mature megagametophyte and a major variant
of this type in the synergid-lacking megagametophyte of Plumbago.
III.
Embryogenesis
A.
Fertilization
The function of the mature embryo sac is a complex process that can be divided into four stages:
1.
2.
3.
4.
Progamic phase - A preparative stage in which the male and female
gametophytes are slowly modified prior to arrival of the pollen tube. This
includes, for example, the degeneration of the receptive synergid
Gamete discharge - Arrival and discharge of the pollen tube, constituting
sperm deposition
Gametic fusion - fusion of plasma membranes of the gametes, transmitting the
nuclei of the male gametes into the egg cell and central cell
Nuclear fusion - fusion of the nuclear envelopes of the male and female
gametic nuclei
Demonstrations are available on each facet of the process. The exhibit centers on results using
Plumbago zeylanica and Populus deltoides, two plants in which these processes have been clearly
elucidated. Several alternative models are also shown in another figure on display.
B.
Embryogenesis
After the formation of the zygote and primary endosperm nucleus, considerable development is
usually required to organize the mature seed. Endosperm development, which creates the nutritive
material for the embryo, usually precedes the division of the zygote and further embryogenesis. The
61
processes of embryogenesis and endosperm development are illustrated in Capsella bursa-pastoris,
shepard's purse, which is the classical plant for this research (and is similar to Arabidopsis in its
pattern of developement). Slides are available illustrating the organization of the young embryo,
including:
1.
2.
3.
4.
globular embryo stage - the first stage in the formation of a three-dimensional
cell division system producing tissue thickness in the embryo. Note the long
suspensor and unusual basal cell, which is found in many members of the
Brassicaceae. What is the function of the suspensor?
heart-shaped stage - differentiation of the early cotyledons. This stage also
shows the first conspicuous evidence of a protoderm. What is the
developmental potential of that layer?
torpedo stage - elongation of the embryo. The embryo becomes more
complicated at this stage and the apices are becoming organized.
maturational stage - growth of the embryo is completed within the sclerified
ovular integuments, forming the seed.
One caveat--Capsella has a curved megagametophyte (ana-amphitropous), which is a highly
restricted characteristic in angiosperms. The events are given in detail for Capsella on pages 602-606
of Gifford and Foster.
A remarkable reduction of the embryo is shown in the embryo of Monotropa uniflora, the parasitic
Indian pipe. Embryos of this plant are only two cells at maturity. In angiosperms, there is wide
variation in the organization of the embryo in different groups.
As a last exercise on the organization of embryos, examine the mature embryo of Zea mays, as a
representative of the monocots. The embryo of corn is particularly precocious compared to most
flowering plants. Observe a prepared slide and identify the scutella, shoot apex, root apex, epiblast,
coleoptile, coleorhiza and young foliage leaves. What constitutes the cotyledon? Is there any
evidence that a second cotyledon was ever present in monocots?
62
COMPARISON OF BICELLULAR AND TRICELLULAR POLLEN GRAINS
Those released with generative cells at anthesis are known as bicellular pollen grains, while those
containing sperm cells at anthesis are tricellular.
Characteristic:
Tricellular
Bicellular
Sperm formed:
Yes
No
Duration of growth:
Short
(< 10 hr)
Long
(12 hr or longer)
Growth rate:
Rapid at stigma
slower near end of
growth
Slower growth initially
faster after sperm
formation
Mitochondria:
Extremely active
Sluggish
Mitochondrial longevity:
Short
Normal
Incompatibility system:
Sporophytic
Gametophytic
Rejection site:
On stigma (lack of
hydration) or early
in stylar growth
In upper, mid and
lower style usually
prior to sperm
formation
Means of rejection:
Inhibition at stigma
imbibition fails
tube aborts/bursts.
Specific recognition
gene regulation
Callose walls thicken
growth slowly ceases
tube dies in situ.
Growth inhibition /
starvation
63
Name: ____________________________
BOTANY 5264 - COMPARATIVE MORPHOLOGY OF VASCULAR PLANTS
Midterm Exam # 1 Thursday, February XX, 20XX
1. ____________________________
26. ____________________________
2. ____________________________
27. ____________________________
3. ____________________________
28. ____________________________
4. ____________________________
29. ____________________________
5. ____________________________
30. ____________________________
6. ____________________________
31. ____________________________
7. ____________________________
32. ____________________________
8. ____________________________
33. ____________________________
9. ____________________________
34. ____________________________
10. ____________________________
35. ____________________________
11. ____________________________
36. ____________________________
12. ____________________________
37. ____________________________
13. ____________________________
38. ____________________________
14. ____________________________
39. ____________________________
15. ____________________________
40. ____________________________
16. ____________________________
41. ____________________________
17. ____________________________
42. ____________________________
18. ____________________________
43. ____________________________
19. ____________________________
44. ____________________________
20. ____________________________
45. ____________________________
21. ____________________________
46. ____________________________
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47. ____________________________
23. ____________________________
48. ____________________________
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49. ____________________________
25. ____________________________
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64
Name: ____________________________
BOTANY 5264 - COMPARATIVE MORPHOLOGY OF VASCULAR PLANTS
Midterm Lecture Exam # 1 Tuesday, February XX, 20XX
(15) 1.
Each of the genera below have had central roles in elucidating the evolutionary
relationships of land plant groups. For each, give a brief description of its morphology, organography, and
salient points of its anatomy. Then explain how it is linked to understanding the plant group(s) to which
each is associated.
(a) Asteroxylon
(b) Ibyka
(c) Renalia
(30)
(20)
2.
3.
Complete TWO of the following three comparisons:
(a)
Compare and contrast strobilus organization and structure in the primitive,
urostachyoid (Huperzia) and advanced, rhopalostachyoid (Lycopodiella,
Diphasiastrum, Lycopodium) members of the genus Lycopodium.
(b)
Sketch and explain the anatomy and mode of secondary growth in the genus
Isoetes. Why is this unusual compared to other higher plants?
(c)
Explain the importance of anisospory in Equisetum in relation to the evolution of
heterospory in the sphenophyte line.
Define and explain the botanical significance of the following terms:
(a) form genus
(b) protoxylem lacuna
(c) foot
(d) distachous
(e) actinostele
(f) lateral anisophylly
(g) serial homology
(15)
4.
From which group (or groups) did the vascular land plants originate? (1) Explain the
evidence for this origin. (2) What modifications of their structure are required in order
to obtain a typical primitive land plant. (3) When did land plants originate (geological
period and approximate age)?
(20)
5.
Construct and defend an evolutionary scheme among six hypothetical species of
Equisetum. First, draw and "name" your "species". Second, diagram evolutionary
relationships among the species. Third, explain how this exercise relates to the concept
of typology.
65
Name: ____________________________
BOTANY 5264 - COMPARATIVE MORPHOLOGY OF VASCULAR PLANTS
Midterm Lecture Exam # 2 Thursday, April X, 20XX
(20)
1.
Answer TWO of the following three questions:
(a)
Outline the major evolutionary changes that would have been required to have
converted a heterosporous free-sporing plant like Selaginella to the seed habit as
it occurs in the gymnosperms.
(b)
Sketch and discuss the fossil evidence regarding the origin of the integument in
the gymnosperm ovule. Of what significance is the cupule among the fossil
plants studied and how did it originate?
(c)
Outline the various types of siphonosteles discussed in class (with sketches)
indicate their relationships, and discuss which types are believed to be the most
advanced.
(15)
2.
(a) Construct and defend an evolutionary scheme among the extant (living) orders of
ferns. Present all important evidence available to support your scheme. (b) Discuss
whether you believe that the ferns are a monophyletic or polyphyletic group and provide
support for your interpretation.
(20)
3.
Define and explain the botanical significance of the following terms:
(a) compound strobilus
(b) Calamites
(c) hypogeal germination
(d) coenosorus
(e) pycnoxylic
(f) telome theory
(g) Lagenostoma
(30)
(20)
4.
5.
Complete TWO of the following three comparisons:
(a)
Compare and contrast the vegetative and reproductive organization of the orders
Pinales and Taxales in the Coniferophyta.
(b)
Compare and contrast the female strobilus of Cycas with that of Pinus. Are
these two structures entirely homologous? Why or why not?
(c)
Compare and contrast sexual reproduction and fertilization in Marsilea with that
of any member of the Filicales.
Outline the sequence of reproductive events in either Pinus OR the cycads from
initiation of the seed (megaspore mother cell) through fertilization to the mature seed.
Which did you select (cycad or pine)? How much time does this process take?
66
Name: ____________________________
BOTANY 5264 - COMPARATIVE MORPHOLOGY OF VASCULAR PLANTS
Final Lecture Examination Tuesday, May X, 20XX
(20)
(20)
1.
2.
Answer ONE of the following three questions:
(a)
In the lab, we observed a variety of transitional leaf forms present in winter
buds. Morphologically, what do these leaves represent and how do these relate
to the various leaf zonation concepts that we discussed in class.
(b)
Based on morphological trends in the angiosperms, reconstruct a "primitive"
angiosperm. Please discuss the major floral and vegetative morphological
characteristics expected in this flowering plant.
(c)
Discuss the major growth habits and morphological patterns of adaptation to the
aquatic environment.
Define and explain the botanical significance of the following terms:
(a) hemiepiphyte
(b) fractals in nature
(c) double fertilization
(d) orthostichy
(e) atactostele
(f) Oberblatt
(g) prophyll
(40)
3.
Complete TWO of the following three comparisons:
(a)
Compare and contrast double fertilization in Ephedra with double fertilization in
the angiosperms. What does this mean in an evolutionary sense?
(b)
Construct a typology accounting for the vascular organization of the stem and
root of monocots and dicots starting with a common ancestor.
(c)
Compare and contrast female gametophyte formation in a typical monosporic
angiosperm with that of a tetrasporic one. Please label fully. What "types" did
you draw?