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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 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:
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, insitu, 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.
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 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 Psilotumnudum 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?
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.