Bot 410 Lab - "Simple" Tissues
       Parenchyma - Collenchyma & Sclerenchyma  

    This lab is designed to give you information on the primary non-vascular tissues. These are relatively simple compared to xylem and phloem. However, we will see that there is a considerable amount of variation within these tissues. In addition, you will observe the major components of the protoplast that are visible with the light microscope.

    Study cell shape, contents, and wall structure, the relation of cells to one another for intact tissues, the presence of intercellular connections via pits, and the presence or absence of intercellular spaces.

    The cell walls and air spaces constitute the Apoplast.

    The Plasmalemma and all within it constitute the Symplast.

    These are Extremely Important concepts, which must be appreciated to understand Plant Physiology!

    Within the Symplast, look for the cytoplasm, nuclei, chloroplasts, other plastids, crystals, and vacuoles colored with anthocyanins. Use polarizing filters to locate starch grains and crystals. Also use polarizers and stains to study cell wall organization and composition.

    PARENCHYMA

    Cortex of Pereskia stem:

  1. Observe free-hand cross sections, and mount in water.
    The Cortex occurs between the epidermis and the vascular tissues. It contains some Collenchyma near the epidermis and Parenchyma near the vascular tissues.

  2. Stain cross sections with Toluidine Blue. What colors are visible in parenchyma cell walls? What does this indicate in terms of cell wall composition?

The Parenchyma consists of relatively large, thin-walled cells.
The cells are arranged loosely, that is, there are intercellular spaces among them. The protoplasts of these cells contain chloroplasts. Some of these cells may have amyloplasts and crystals. Pereskia is a member of the cactus family. It has spines but it also has normal leaves. Its flowers are extremely beautiful like those of most Cactaceae.

 

Young leaf of Elodea.   

  1. Mount an entire leaf in a drop of water. Study cells in
    the region halfway between middle and margin. These leaves are only two cells thick, except at the midrib, and there is little tissue differentiation. This is good material to demonstrate cytoplasmic streaming. The general term Chlorenchyma is used to describe Photosynthetic Parenchyma regardless of its location.

  2. Use the condenser iris to observe the cytoplasm.

  3. Observe demo with phase contrast optics to study the cytoplasm.

Observe Macerated Pith of Begonia (prepared by boiling in dilute KOH): Note numerous faces of individual cells. What term is used to describe cells which have this shape?

Parenchyma cell with Isodiameteric Shape seen in a section

Whole Isodiametric Parenchyma Cell
              

 

Aerenchyma & Stellate Parenchyma

A strikingly different shape of parenchyma cells is illustrated by stellate parenchyma. These are branched and adjacent cells are connected with each other by means of the branches. Parenchyma composed of branched cells is highly lacunose; that is, it has a large volume of intercellular space. The spongy layer in leaves can have branched cells with large intercellular spaces.

The term Aerenchyma is often used to describe parenchyma, which has large air spaces.

  1. Locate Stellate Parenchyma cells in petioles and midribs of Canna (ali’iope) leaves. Cut hand sections and examine with a dissecting scope before observing with a compound microscope. Do these have 3D branching?

  2. Examine the Parenchyma in Papyrus (Cyperus papyrus) stems by making transverse sections. Find the Aerenchyma with a dissecting scope and examine with a compound microscope. What is the shape of the individual cells which comprise the Aerenchyma? Are they branched in 2D or 3D?

  3. Stain these with IKI and look for starch containing Amyloplasts


Cyperus papyrus stem stained with IKI


Aerenchyma in Cyperus javanicus


Cyperus papyrus


Cyperus laevigatus
(makaloa)

 

Aerenchyma can be found in the stems of other members of the Cyperaceae, like C. laevigatus (makaloa). Makaloa stems are smooth and resilient. The Aerenchyma is like foam rubber on a microscopic scale. Makaloa stems were used to make fine sleeping mats by ancient Hawaiians and the qualities of their Aerenchyma contributed to their utility.


Sample from a makaloa mat: Note the Air Spaces (A)


Canna Flowers

 

Pineapple leaves also contain stellate parenchyma. What functions are suggested by the 3-dimensional shape of these cells?


Canna Petiole Cross Section


Stellate Parenchyma Cells

 

Endosperm Cells:

The parenchyma cells you have examined thus far have relatively thin walls, but there are also thick-walled parenchyma cells.

  1. Examine the demonstration slide of persimmon or palm (niu) endosperm. This material will also show fine lines traversing the thick walls from cell lumen to cell lumen. These lines are pits, which connect the symplast of adjacent cells.

  2. Examine fresh sections of unroasted coffee "beans" or palm fruits to observe the thick walls and their pits.

    What is the basic component of cellulose?

    Other than the typical function of cell walls what might the function of these thick walls be?


Persimmon Endosperm


Endosperm with Large Pits

 

You have already observed cytoplasm and chloroplasts. Other protoplast components include several more types of plastids, vacuoles, and various kinds of crystals.

Chromoplasts and Pigment Bodies.

These may be yellow, red, and orange colored plastids, and similarly colored crystal-like bodies. The latter are called pigment bodies because there is some question whether they may be classified as plastids.

  1. Observe chromoplasts and pigment bodies in free-hand sections of bell pepper fruits, various flower petals, and the root of carrot. Chloroplasts are green Chromoplasts!


Chromoplasts from Red Pepper (Capsicum)


Chromoplasts from Flower Petals


Red Bell Pepper Fruit


The color of Alamanda Flowers is due to Chromoplasts

 

Leucoplasts

Leucoplasts are mysterious and difficult to demonstrate without special techniques.

  1. Observe these in cells of the lower epidermis from leaves of Rhoeo, or Zebrina. These are best seen in epidermal peels. This will be demonstrated for you. Amyloplasts are filled with starch, which sometimes occupies the entire organelle. They are also regarded as Leucoplasts because they lack color.

  2. Observe thin free-hand sections of Papyrus and stain with IKI. We will have a demo of potato amyloplasts.

  3. Observe an unstained specimen and use the polarizers.

  4. Observe Demo slide of Potato (Solanum tuberosum) tuber and locate the Amyloplasts


Leucoplasts clustered around the Nucleus of a Parenchyma Cell stained with Toluidine Blue


Unstained potato Amyloplasts


Commercial slide of Potato Amyloplasts


Amyloplasts from Canna seen with normal illumination


Amyloplasts from Canna seen with crossed Polarizers


Amyloplasts stained with IKI

 TaroAmylo.jpg (117678 bytes)
Amyloplasts from Taro (Kalo) Unstained
Find the Nucleus!

 

"Statoliths" are amyloplasts, which contain many large multifaceted starch grains, similar to those above. Their function may be related to gravity perception.

  1. Study a Demo of  thin longitudinal sections of Hibiscus petiole or stem, stained with IKI. The statolith-containing cells occur just outside the vascular bundles. Their starch grains are much larger than other starch grains in the stem.

  2. Statoliths may also be seen in corn root tips.

 

Non-cytoplasmic Contents

  1. Make slides of Rhoeo or Zebrina epidermis (see above for Leucoplasts). These demonstrate vacuoles, which contain anthocyanin.

  2. The pigmentation in many flower petals, like Erithrina (wiliwili), is also contained in vacuoles. This is best observed by looking at fresh cross sections of the petals. How can you tell if the color is due to chromoplasts or vacuolar pigments?


Anthocyanins in a surface view of Epidermal Cells


Zebrina Leaves: The Anthocyanins are on the lower surface of the Leaves. I wonder what they are doing down there????

 

Crystals

Crystals occur in Vacuoles.

  1. Observe star-like (Druses) in Begonia or Pereskia stems. Druse crystals are very common. The other commonly observed crystals are spear-like Raphides. Both are birefringent (bright) in polarized light. They probably deter herbivory and are more abundant in plants that grow in dry environments.

  2. Observe Raphides in sections of taro kalo (taro) leaves, petioles of Heliconia, or stems of Impatiens, Dieffenbachia and Pereskia.


Druse Crystal seen with Bright Field Illumination


Druse Crystals seen with Crossed Polarizers.


Raphides in an isolated Vacuole seen with crossed Polarizers


Raphides on the Loose!!!!

 

  1. Observe Demo of Prismatic tetragonal crystals in the
    outer, dry scales of Allium cepa (onion) bulbs. These scales have been soaked in alcohol to remove the air.

  2. Try your polarizers on these!!!!!

 

The function of these crystals is relatively uncertain. They seem to be more abundant in plants, which grow in arid and xeric environments. They are all composed of Calcium oxalate, which causes animal epithelial cells to swell. Consequently, they should deter herbivory.

 

COLLENCHYMA

Collenchyma is closely related to parenchyma. However, the plastids are not well differentiated in collenchyma while they are well differentiated and obvious in parenchyma.

Collenchyma always occurs just beneath the epidermis, while parenchyma occurs throughout the plant.

Collenchyma cell walls are unevenly thickened. When the thickening occurs at the corners where cells are joined it is called angular. Lamellar collenchyma has thickenings on their tangential walls, which are parallel with the surface.

Lignin is usually not present in collenchyma.

  1. Locate Collenchyma in hand sections of hau (Hibiscus tiliaceus) stem, Celery or Water Lily (Nymphaea) petioles.

  2. Determine cell shape by

    a] observing cross sections and a

    b] demo of a longitudinal section.

  3. Mount fresh sections in water.

  4. After examining them, stain with Toluidine Blue and then examine again.

  5. What does the pink color of the cell walls indicate?

  6. Observe free-hand cross sections of celery petioles:. The cell wall thickenings are in the corners where adjacent cells meet. Lamination in the wall may be discernible. It results from a centripetal deposition of wall layers differing chemically and physically from one another. What type of Collenchyma is this??????


Unstained Collenchyma in hau Stem


Collenchyma from Coleus stained with Toluidine Blue


Unstained Collenchyma in Celery


Unstained Collenchyma in Celery
CollenLSTB-2-250.jpg (74050 bytes)

Longitudinal Section through Collenchyma from Celery Stained with Toluidine Blue:

Note the degree of cell elongation compared to the Parenchyma cells on the far left side of the picture. The thickness of Collenchyma cell walls is also illustrated. The pink color indicate the presence of Pectins & absence of Lignin.

 

  1. Observe (prepared slides) of Sambucus stems.

  2. The thickenings are chiefly on the tangential walls. Tangential in these case means walls oriented parallel to the surface of the structure.

  3. What type of Collenchyma is this?

 

SCLERENCHYMA

The distinction between parenchyma, collenchyma and sclerenchyma is largely based on the wall structure.

Parenchyma cell walls are usually thin and primary.

Sclerenchyma cells have a thick secondary wall that is formed inside the primary wall. Secondary walls are those, which develop after a cell, has ceased to enlarge.

Collenchyma cells have secondary wall thickenings but these are uneven in their distribution. Furthermore, the cellulose fibrils in Collenchyma are not as highly organized or tightly bound as in Sclerenchyma.

Finally, Sclerenchyma cells can be found in many locations throughout the plant body but Collenchyma cells are always just beneath the Epidermis.

Sclerenchyma cells are usually classified into Sclereids or Fibers on the basis of form as well as the abundance and type of pitting.

Sclereids are generally shorter than fibers and their walls show more abundant pitting. The pits are often branched (ramiform). Walls of sclerenchyma cells are usually lignified and, therefore, stain red with safranin or phloroglucinol-hydrochloric acid. They often show concentric laminations, which indicate different periods of wall synthesis. Sclereids vary in shape and occur in all parts of the plant.

Fibers tend to be highly elongated cells with tapering ends, and they often occur in bundles. There are few pits in the walls of fibers. The pits, when present, are usually simple and unbranched.

In studying Sclerenchyma observe their (1) overall shape; (2) wall structure; (3) pits; (4) staining reactions to Phloroglucinol & Toluidine Blue; (5) appearance with crossed polarizers.

Sclereids

  1. Study cross sections from the stem of Hoya (wax plant) or Begonia. Sclereids occur between the cortex and the vascular region, and in the pith. They resemble parenchyma cells in shape but have thick walls. A comparison of the sclereids with the adjacent parenchyma cells illustrates the two extremes in the variation of plant cell walls

  2. Stain transverse (cross sections) sections with Toluidine Blue. Use older stems for lignified sclereids. The parenchyma cells have thin primary, nonlignified walls. The sclereids have a thick lignified secondary wall deposited inside the thin primary wall. The secondary wall obscures the primary wall and shows concentric lamination because it is deposited in successive layers. It also shows prominent canal-like pits. To observe details of the pits, focus up and down while examining them. The primary walls of adjacent sclereids, and the middle lamella are tightly joined and obscured by lignin deposition. Lignin makes the cell walls very strong and waterproof.

Cross Section of Hoya Stem stained with Toluidine Blue: The sclereids occur in a unicellular band in the outer part of the stem.


Solitary Sclereid on Hoya Stem stained with Toluidine Blue
Various Wall Layers in Sclerenchyma

  1. Examine demos of partly macerated leaves of Monstera, Olive (Olea), Osmanthus, or bulb scales of garlic (Allium sativum). Tease these apart in a drop of water on slide, using dissecting needles.


Part of a Garlic Bulb Scale

  1. Examine cross sections of Podocarpus leaves and locate the large sclereids with polarizing filters!



Cross section of Podocarpus Leaf


Sclereid from Podocarpus Leaf


Cluster of Brachysclereids


Solitary Brachysclereid

  1. Mash a small piece of pear (Pyrus) flesh and

  2. mount it in phloroglucinol-HCl.

  3. These sclereids are stone cells or brachysclereids. Note the ramiform pits.

  4. Examine the partly macerated seed coats of peas and beans. Brachysclerids, Macrosclereids and Osteosclereids are present.

  5. Observe the sclereids in the olive leaf. They occur in the mesophyll and are long and fiber-like Trichosclereids. Tricho means hair!!!


Partly Dissected leaf showing Trichosclereids


Trichosclereids from Olive Leaf

AstroSclerComp250.jpg (10006 bytes)

  1. Observe Macrosclereids in Osmanthus   
    leaves. These are columnar and ramified
    at each end.

  2. Look for Astrosclereids in fresh sections of Nymphaea (water lily) leaves.

 

Fibers

Like sclereids, fibers may be found in various parts of the plant. Fibers are particularly common near the phloem (phloem or bast fibers) and the xylem (xylem fibers). In monocots fibers often enclose vascular bundles (fibrovascular bundles) or appear as strands that are independent of vascular tissues.

The best commercial fibers are usually associated with the phloem. This includes hau (Hibiscus tiliaceus) and wauke (Broussonetia papyrifera) plus Cannabis. Coarse fibers can be obtained from monocot leaves like uki uki grass (Dianella sandwichensis) and Agave. Agave was grown in Hawaii but was uneconomical. Some of these plants have escaped cultivation and can be found in nature. They are slow growing but once established, they may be difficult to eradicate. This could present a problem for native species if they can't compete with Agave.

  1. Observe free-hand cross and longitudinal sections of stems of hau (Hibiscus tiliaceus).

  2. Mount in phloroglucinol-HCl.

  3. Deploy your polarizers!

The phloem fibers of this plant were used by ancient Hawaiians for making rope.


Unstained hau fibers from an Hawaiian artifact


Hau fibers stained with Phloroglucinol & viewed with crossed polarizers

  1. Using prepared slides, compare fibers Linen (Linum) and Hemp (Cannabis).

  2. Note the fact that the linen fibers do not stain for lignin. Lignin makes the fibers brittle and it discolors them as well.

  3. Cannabis fibers contain some lignin but not as much as heavily lignified walls.

  4. Examine a DEMO of a fiber from wood. This will provide some information on the overall shape and length of wood fibers. We will encounter these again so don't worry about the details just yet.


Cross sections of flax stem that show the phloem or "bast" fibers which are green.

In this case the green color indicates the absence of Lignin.


Cannabis stem cross section


Highly Magnified Fibers

WB00677_.gif (630 bytes)   Fiber from Oak wood

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