Angiosperm means covered seed. The flower is the most important adaptation for the sexual reproduction of plants. The hallmark of angiosperm reproduction is the Carpel.

The carpel is a highly modified leaf which bears Ovules. To grasp this, imagine a pea pod. The peas are inside. Carefully slice along one margin with a knife, and unfold the fruit. Voila!!!! What do you see??? A leaf!!!! A Carpel!!! Now, imagine what would happen if you fused several of these together!!! The Carpels constitute the Gynoecium.

The flower also has an Androecium (Stamens), a Corolla (Petals) and a Calyx (Sepals). These also play roles in sexual reproduction.

One of the goals of this lab is to understand the basic organs which are found in flowers.

We saw that the Ovules of Gymnosperms were exposed to the atmosphere even if they were tucked away in a crevice. The angiosperm ovule is housed within the ovary wall (Pericarp). This provides an extra measure of protection for ovule and seed development. It may also provide adaptations which help in seed dispersal.

One goal for the lab is to link flower structure to fruit type.

We saw the tremendous reduction in complexity with the Gametophytes of Gymnosperms. Further simplification occurs with Angiosperms.

One of the major goals of this lab is to understand the basic outcomes for the  gametogenesis of angiosperms.

We will have several simple flowers for you to dissect.

Make sure you find all of the reproductive structures as well as the Sepals and Petals.

Note any instances of Coalescence or Adnation.

Fill out the Table for Floral Traits

Table for Floral Traits

Determine whether the Gynoecium is composed of many individual Carpels, one Carpel or several fused Carpels

Is the Ovary Superior or Inferior?

Make a cross section through the Ovary to locate the Ovules.

Identify the Pericarp (Ovary Wall)

Androecium

Locate the Stamens

Locate the Anthers & Filaments

Examine cross sections of Lilium Flower Buds & Locate the following

Sepals        

Petals       

Anthers       

Carpels   

Ovules

How many Carpels do you observe?

Megasporogenesis

The scheme that I gave you for the lecture is a lie!!!! Megasporogenesis in Lilium is rather complex and is a bad choice in terms of learning about this. I  actually used the Polygonum type. I don't want to go into the Lilium type, so we will put out the slides you need to examine in order to understand what is called Monosporic Development. This refers to the fact that only one Megaspore survives and it produces the Megagametophyte (Embryo Sac).

Ovules are produced in the Ovary of the Carpel. One cell, near the Micropyle, becomes enlarged and ultimately produces four Megaspores. The enlarged cell is called the Megasporocyte. Cell that produces Megaspores. The tissue from which it came is called the Nucellus.The Megasporocyte produces four haploid Megaspores. Three of these die. The "functional" Megaspore enters Mitosis and produces Eight Haploid Nuclei. This is followed by Cell Formation. This multicellular structure is the Megagametophyte or Embryo Sac. Three cells develop near the Micropyle. This is the Egg Apparatus and consists of the Egg and two Synergids. Three similar cells form at the opposite pole of the Megagametophyte and are called the Antipodals. The remaining two nuclei form a large Central Cell. Following Double Fertilization, the Egg forms the Zygote and the Central Cell becomes the Endosperm. The Endosperm is consequently 3N in this case.

It is Most Important that you understand the structure and function of the Micro and Megagametophytes. I will not ask you to recite the steps that take place. However, you will find it easier to learn this if you work through these processes.

Observe Demo Slide of a Mature Ovule with Embryo Sac.

Locate

Integuments

Micropyle

Embryo Sac

Region of Egg Apparatus

Central Cell (How many Nuclei are there in the Central Cell)

Microsporogenesis

Sporogenous cells occur in the Pollen Sacs of the Anther. These are called Microsporocytes. These undergo meiosis and produce four haploid Microspores. Each Microspore undergoes Mitosis such that there are two nuclei in one cell. One of the nuclei divides again to produce the nuclei of each Sperm. When cell formation is complete there is one large cell (Tube Cell) and two smaller cells called Sperm. The Sperm cells float in the cytoplasm of the Tube Cell. The Microgametophyte is known as a Pollen Grain! When Pollen lands on the Stigma of a receptive carpel it germinates. The Tube Cell produces the Pollen Tube and the Sperm are carried forward as the tube elongates. The Tube Nucleus is usually near the tip of the Pollen Tube and the Sperm are in a more basal position. The Pollen Tube may cover a relatively enormous distance to reach the Egg. The silk strands on corn cobs are the Stigmas and Styles of individual Carpels.

Observe Mature Pollen Grains (Microgametophyte)

Locate

Tube Cell

Generative Cell or Sperm

Microscopic Flower Buds

Observe Commercial Slides of Various Flower Buds and Locate the Floral Parts.

Observe Cross-Sections of Bean (Phaseolus) Flower Buds.

Locate                         

Ovary Wall

Ovule         

Placenta                                                               

Three Veins

Observe DEMO Longitudinal section of a Bean Carpel.

Locate

Receptacle

Pericarp

Ovule

Observe DEMO of a Mature Bean Fruit with Seed &    

Locate

Ovary Wall

Seed Coat (Testa)

Cotyledons (C)

Funuculus

The Embryo & Seed

The mature ovule is a seed. We do not have enough time to look at seed structure in depth but you should observe Demo Slides showing Sagittaria, Lilium & Capsella seeds with Embryos.

Fresh Fruit (Optional)

The Mature Ovary is the Fruit. We will have a small number of fruits for you to examine. Try to locate any residual floral structures that might help you determine whether the fruit is Simple, Aggregate  Multiple, Accessory.

Additional Demos

Lilium Young Flower Bud (Longitudinal Section)

Lilium Early Ovule (Find the Megasporocyte a.k.a. Megaspore Mother Cell)

Lilium Pollen (Whole Mount)

Lilium Pollen with Pollen Tubes

Lilium Stigma with Pollen Tubes (The Style is hollow and has a secretory Epidermis)

Top of Sexual Reproductio Lab

  Bot 201 -Anthophyta Lab - Secondary Growth in Dicot Stems 

Vascular Bundle & interfascicular zone	Active Vascular Cambium	Induction of cambial activity in the interfascicular region

Slide C:

Two-three year old stems (slide C) exhibit considerable secondary growth.

Vascular Bundles in a 3 year-old stem

Pith in a 3 year-old stem

Results of secondary growth on the stem fibers	Effects of secondary growth on the phloem

The epidermis is still intact except in a few places where lentils are forming from the phellogen. Since the interfascicular cambium is composed only of ray initials, the vascular bundles remain distinct, and are separated by wide rays of parenchyma.

The xylem shows two growth layers.

Many pith cells are crushed, as a consequence both of movement of the vine and of inward pressure of developing xylem tissue.

The fact that many vines have isolated vascular bundles or wide areas of parenchyma in the xylem suggests that this might have functional significance. Considering the manner in which vines grow, can you imagine what function this tissue organization may have?

Slide D:

Older stems (slide D) clearly show periderm formation. In Aristolochia the periderm develops first in isolated vertical strips. Thus, as seen in cross sections, parts of the stem's circumference have an intact epidermis while other parts have a well developed periderm with several layers of cork cells (phellem), cork cambium (phellogen) and a relatively wide phelloderm.

The Phellogen (Cork Cambium) produces Phellem (Cork) to the outside and Phellogen towards the Inside.

The Phellogen can be located by following files of vacuolate Phellem & Phellogen cells towards the centrally located meristematic cells.

Remember that meristematic cells are densely cytoplasmic and lack prominent vacuoles.

The phelloderm is usually not prominent and may consist of a single layer of parenchyma. Aristolochia has an uncharacteristically large phelloderm.

Note the lenticels in the periderm. These are areas of hyperactive cell division compared to the rest of the phellogen. Cells in the lenticils have less suberin and are loosely aggregated. This allows for gas exchange. Other parts of the periderm are relatively impervious to gases.

The xylem shows several growth layers. As the individual wedges of vascular tissues increase in size, new rays develop within them.The pith and some of the inner parts of the interfascicular areas are almost completely crushed.

Coleus Stems

The development of secondary growth in stems is readily seen by making a series of cross sections from a Coleus stem.

The large primary bundles are conveniently located in the corners of its square stem. By taking sections from more mature parts of the stem the pattern of development is readily discerned!

Examine the series of photos below, then observe fresh sections.

Study a series fresh sections of Coleus or another related plant stained with Phloroglucinol or Toluidine Blue.

Compare fresh sections with a commercial slide of Coleus stem.

Developmental Sequence of Vascular Differentiation in Coleus

Coleus and other mints (Lamiaceae)  have large primary vascular bundles that occur in each corner of the stem. The bundles have Bast Fibers, Phloem and Xylem. A Vascular Cambium is present between the xylem and phloem. It divides periclinally to produce files of cells in two directions. Cells displaced towards the interior become xylem. Those displaced towards the periphery become phloem. Cambial activity spreads to the interfascicular parenchyma. This spreads laterally until a complete ring of cambium encompasses the stem and unites the bundles.

Growth from the vascular cambium increases the girth or diameter of the stem. The vascular cambium of Coleus usually produces more xylem than phloem. The thick-walled xylem cells accumulate like loaves of bread which enlarge as they mature and have a hard crust after they are baked. These files of sturdy cells create pressure towards the inside and outside of the plant. The thin-walled cells of the pith and phloem are crushed as the pressure increases. The vascular cambium replenishes the phloem. However, the amount of active phloem may be limited to a small zone. Fibers may be present in the secondary phloem. These protect the sieve elements and allow for the expansion of the active phloem.

 

Coleus stem with some Secondary Growth: Large Vascular Bundles (VBs) are found in the corners. Small bundles are located in the Interfascicular Region. The Vascular Cambium has produced secondary vascular tissues in the VBs and has spread across the Interfascicular Region where it has has produced noticeable amounts of Secondary Xylem. Coleus Stem showing the Interfascicular region after some Secondary growth. Locate the small Vascular Bundles. Note the uniform appearance of the Secondary Xylem. The linear files of cells testify to the presence of a Lateral meristem like the Vascular Cambium.

One Corner of a Coleus Stem with some Secondary Growth: This shows one of the four  large Vascular Bundle and the adjoining Interfascicular Region with its Secondary Xylem.

Periderm Development

The Phellogen (Cork Cambium) is another lateral meristem. It usually develops from subepidermal cells in stems. It divides periclinally to produce rows of cork (Phellem) cells towards the exterior. Cork cells are dead at maturity and have suberized walls.

Early stages in Periderm Development

Suberin is a lot like cutin. It protects the stem because It is impervious to water and most pathogens, and provides some insulation. It stains positively with Phloroglucinol.

Look in the outer cortex of Coleus for these early stages of periderm formation. Periderm forms the outer bark. We will have more to say about this later.

Study unstained sections of Eucalyptus bark and observe the layers of Phellem (Cork).

Stain with Phloroglucinol which stains Suberin as well as Lignin.

What accounts for the Layers that you see in the Periderm of Eucalyptus?

These questions are really getting to be a major annoyance! Can't a guy or gal enjoy some microscopy without all of these nagging questions!

We can study the transition from primary to secondary growth by studying Cotton (Gossypium tomentosm) and/or hau (Hibiscus tiliaceus)

Hawaiian cotton is indigenous to these islands but is probably part of a larger taxon that is widely distributed in the Pacific. It does not produce the finest fibers but it has important traits that have been used in cotton breeding programs. This supports the concept of population/species preservation in that valuable genes may exist in natural populations. If wild populations are destroyed, the beneficial genes are also destroyed.

Study cross sections and stain with Phloroglucinol or Toluidine blue.

Locate the youngest internodes on stems of either species.

Examine more mature internodes which have a brown exterior and treat as above.

Locate the following:

Dermal Layer (Epidermis – Periderm (Phellogen [Cork Cambium] – Phellem [Cork])

Cortex (Collenchyma – Sclerenchyma – Parenchyma)

Vascular Tissue (Phloem Fibers – Phloem – Vascular Cambium – Xylem

Interfascicular Region (Parenchyma – Vascular Cambium)

Pith (Parenchyma – Other Cell Types)

Study the primary Vascular Bundles. There may already be some secondary growth but evidence of the primary bundles is usually present.

Primary xylem will jut into the pith due to the production of secondary xylem.

Secondary xylem is usually more linear in its organization and may have a distinctly different cell composition compared to primary xylem.

Phloem fibers can also mark the location of primary vascular bundles.

Woody dicot: Tilia (basswood) stem.

Tilia sp.

Tilia flowers

Tilia fruits

Tilia americana is the model plant that is routinely studied
for the stem anatomy of woody angiosperms. Its characteristics encompass a good sample of anatomical features for this kind of plant.

Compare this with the hau stem sections!  

Study commercial slides of stems in several
stages of development.

Work from the outside towards the inside of the stem!

The following details may be observed:

In younger stages the epidermis is present.

In older stems it is replaced by a periderm containing several layers of tannin-filled cork cells. Tannins are brown and impart a dark color to the Cork. The smallest periderm cells that contain  protoplasts with nuclei are the innermost cells in each radial file of cork cells. These constitute the phellogen or cork cambium.

The Cortex is composed of collenchyma and parenchyma. The large cells with red-staining contents are mucilage cells.

The Vascular Tissues form a continuous ring.

The Phloem in older stems appears to be divided into
two kinds of wedges with opposite orientations.

The wedge that points towards the outside contains sieve-tube members, companion cells, phloem parenchyma and phloem fibers.

The fibers occur in tangential bands (parallel to the surface) alternating with bands that contain the rest of the phloem components.

The outermost fiber bundles are the primary phloem fibers, the others arose from the vascular cambium and are part of the secondary phloem.

The other Wedge is principally composed of
parenchyma. These are dilated phloem rays.
Dilated means broadened or expanded.

In addition to the dilated rays, there are narrow undilated rays.

Note the continuity of the rays from the phloem to the xylem.

The stems in slides labeled "young stem" are just starting to form secondary xylem. 

In older sections from one to several increments of
secondary xylem occur, and the primary xylem is next to the pith.

The secondary xylem of Tilia contains vessel elements (widest cells), tracheids, fibers (narrow cells with relatively thick walls), and xylem parenchyma (small dark cells).

The Pith contains thick-walled cells with dark globules of tannin.

The large "spaces" in the pith, which may be filled with red-staining material, are mucilage ducts.

Top of Secondary Growth Lab