General Biology Laboratory

NTU Life Science

Experiment 1. The Light Microscope

Objectives and principles

The naked human eye is similar to a compound lens. However due to limitations
of the structure it can only resolve two lines 2 mm apart from a distance of 25 cm.
Beyond this resolving power, a light microscope is needed.
The function of a light microscope is to magnify and revolve the image of a small
object, that is, to make the image bigger and provide more details. Section staining
and special microscopic techniques such as phase-contrast, differential interference
contrast, dark field or polarization are able to enhance the image contrast.
Theoretically the oil immersion lens can increase the resolving power of an optical
microscope to 0.2 µm. However, many factors such as the quality of lens materials,
light source, specimen preparation and the use of a microscope may decrease the
resolving power.
The aims of this experiment is to introduce the structure, the use, and also the
maintenance of a light microscope.

The parts and components of a light microscope (Fig. 1-1)

1. Eyepiece / ocular: The eyepiece comes with three magnification levels (5X, 10X,
15X). A pointer and an ocular micrometer can be inserted into the eyepiece. With
a binocular microscope, the interpupillary distance and diopter can be adjusted.
2. Eyepiece tube: The eyepiece tube is mounted between the eyepieces and the
objective lenses.
3. Revolving nosepiece: The revolving nosepiece under the eyepiece tube holds
objective lenses and can be rotated to change magnification power easily.
4. Objective lens: Five magnification levels (4X, 10X, 20X, 40X, and 100X) are
available.
5. Stage: The stage is the platform for holding a microscopic slide. The hole in the
center allows light passing through.
6. Condenser: The condenser is located beneath the stage and above the light
source. It gathers and focuses the light on to the specimen. An iris diaphragm in
the condenser can adjust the amount of light a specimen receives.
7. Illuminator: The illuminator with tungsten or halogen lamp is located on the base.
The power and the intensity of light is controlled by the same knob. For the
2
 microscope without a built-in illuminator, a mirror with both convex and concave
side is beneath the condenser and served to receive and reflect light to the
condenser.
8. Arm: The arm connects the eyepiece tube and the base, it is the part to be held
when carrying a microscope.
9. Focusing knob: Focusing knobs for adjusting the focus of the image are located on
both sides of the eyepiece tube. The coarse focusing knob is bigger than the fine
one. Rotating the knob clockwise brings the stage down, and counterclockwise
brings the stage up.
10. Base: The base is the bottom of the microscope and is what the microscope stands
on.

Fig. 1-1 Light microscope

3
Instructions of using of a light microscope
1. Hold the arm with one hand and the base with the other, carry the microscope to
the table in front of your seat, then adjust the height of the chair.
2. Turn the revolving nosepiece so that the lowest power object lens is clicked into
position.
3. Turn on the power and adjust the light.
4. Place the microscope slide on the stage and fasten it with the stage clips, then
adjust the position of the slide so that the specimen is in the pathway of the light.
5. Look through the eyepiece and use the coarse focusing knob to bring the stage
upward until the image appears.
6. Adjust the interpupillary distance by rotating the eyepieces appropriately until you
can see only one circle of light with both eyes open. Then adjust diopter, first close
the eye over the eyepiece with the diopter adjustment and focus the microscope
using the fine focusing knob so that the open eye sees the image in focus. Next,
switch eyes and focus on the image by turning the diopter adjustment only. Now
with both eyes open, the image should be clear with both eyes.
7. Adjust the light intensity and the iris diaphragm to regulate resolution, contrast
and depth of field.
8. Once you have focused the specimen on the lowest power (4X objective), center
the specimen in your field of view, then switch to a higher magnification objective
successively. Remember to only use the fine focus knob to bring the image into
focus.
9. Switch to the lowest power objective before you change the microscope slide to
avoid scratching the lens and cracking the slide.
10. Before using 100X objective, first focus the image on 40X objective, then move the
objective away and place one drop of immersion oil on the coverslip to cover the
observed specimen. Switch to 100X objective and let the oil fill the gap between
objective lens and the slide. Readjust the condenser and diaphragm to get
adequate light and to turn the fine focusing knob to get focused.

Precautions
1. Be sure to hold the arm of microscope tightly with one hand and support the base
with the other hand. Place the microscope on the table with care.
2. Adjust the height of chair so that you can sit straight while using the microscope.
Get used to observe the microscopic image with both eyes open.
3. Use the revolving nosepiece to switch to a higher magnification objective carefully.
Use only the fine focus knob when using a higher magnification objective.

4
4. Use only lens paper to clean the lenses. Do not touch the lenses with fingers and
any other kind of paper.
5. After using the microscope, turn the stage all the way down to the lowest position,
take off the slide, and switch to the lowest magnification objective (4X).

The magnification and the instruction for using micrometers

1. The total magnification of the microscope is the product of the magnifying power
of the objective and that of the eyepiece.
2. An ocular micrometer inserted in the eyepiece is necessary for measuring the
actual size of the sample (Fig. 1-2a).
3. A stage micrometer is a microscope slide with a finely divided scale marked on the
surface. The scale is 1 mm and consists 100 divisions, i.e., 0.01mm (10 m) for
each division (Fig. 1-2b).
4. Rotate the ocular micrometer to make it parallel to the stage micrometer, and
then align the zero lines of both scales by moving the stage micrometer (Fig. 1-2c).
Scanning the scales and find a point where two lines align. The actual size of each
division in the ocular micrometer is calculated as:

number of divisions in the stage micrometer

10μm x
umber of divisions in the ocular micrometer

Fig. 1-2 Calibration of microscopic ocular micrometer

5
Stereomicroscope (Fig. 1-3)
1. Plug in the power cord and turn on the light using the switch at the back of the
base. Then use the light control panel to choose the incident, oblique or
transmitted light as you need.
2. Adjust the interpupillary distance by making the two eyepiece tubes closer or
farther.
3. Place the samples on the dissecting stage and observe it with a lower
magnification by rotating the magnifying knob, then turn the focusing knob to
bring the image into focus.
4. Change to higher magnifications the same way describe above.

Fig. 1-3 Steromicroscope

6
Materials
1. Egeria densa (水蘊草) 8. Xylene or ether and chloroform
2. Forceps mixture of 1:9
3. Dropper 9. Ocular micrometer
4. Dissecting needle 10. Stage micrometer
5. Absorbent paper 11. Cover glass
6. Lens paper 12. Slide
7. Immersion oil

Methods
1. Label the parts of the microscope in the Fig. 1-1 and Fig. 1-3. And use a light
microscope to observe leaves of Egeria densa.
2. Describe the features of a light microscope and a stereomicroscope in their use
and function.
3. Briefly describe how to use and maintain a light microscope.
4. Calculate the average size of a cell and a chloroplast of Egeria densa.

Answers of the figures

Fig. 1-1. j. Base

a. Ocular (Eyepiece)
b. Eyepieces tube Fig. 1-3.
c. Revolving nosepiece a. Ocular (Eyepiece)
d. Objective b. Magnifying knob
e. Arm c. Focusing knob
f. Slide clip d. Objective
g. Condenser e. Dissecting stage
h. Focusing knob f. Base
i. Illuminator

7
Experiment 2. The Plant cell

Intoduction
A simple unicellular organism consists of only one cell. In contrast, a multicellular
organism consists of many cells which are derived from cell divisions and
differentiation of one single fertilized egg. As the increases in cell number and cell size
during development, the cells become specialized in both structure and function and
work together to form tissues, organs, and systems.
The cell wall is located outside the cell membrane and gives the shape to a cell.
In hypotonic solutions, a plant cell gains water which increases turgor pressure.
However, in hypertonic solutions, a plant cell loses water and plasmolysis occurs. The
protoplast, a plant cell had its cell wall removed, is round in shape. It is similar to an
animal cell and is an excellent material for research in cell biology.
With a light microscope, we can see that the protoplast is surrounded by the cell
wall. The protoplast is consisted of plasmalemma, cytoplasm, and nucleus. The
plasmalemma can be observed during the plasmolysis process. One to more nucleoli
are embedded in the karyoplasm. Cytoplasm contains plastids, mitochondria,
vacuoles, and cytosol. The vacuole of a meristematic cell is small. A mature cell is
usually occupied with a big central vacuole which pushes the cytoplasm to the margin
of the cell. In general, ergastic substances such as crystals, starch grains, and other
non-crystal substances are stored in vacuoles and plastids. Cytoplasmic steaming
assists the movement of the organelles in the cell. With an electron microscope, Golgi
body, endoplasmic reticulum, microbody, ribosome, nuclear envelope, tonoplast,
plasmodesma, and cytoskeketon can be seen.
In this experiment, we will observe cells of several plants and also their contents.

Materials
1. Plant materials
1) Leaf of Egeria densa (水蘊草)
2) Leaf of Rhoeo discolor (紫背萬年青)
3) Scale leaf and root of Allium cepa (洋蔥)
4) Fruit of Lycopersicon esculentum (番茄)

8
 5) Fruit of Capsicum annuum (辣椒)
6) Tuber of Solanum tuberosum (馬鈴薯)
7) Endosperm of Oryza sativa (稻米)
8) Pulp of Musa sapientum (香蕉)
9) Leaf of Ficus elastica (印度橡膠樹)
10) Stem of Tradescantia virginiana (紫鴨跖草)
1) Petiole of Begonia sp. (秋海棠)
2. Solutions
1) Salt water
2) Methylene blue; 0.02% Eosin solution; 0.01% Neutral red
3) Iodine solution

Observations
5. Green plant cells (Fig. 2-1)
Take a young leaf of Egeria densa and make a wet mount slide by placing the
sample on a microscopic slide, adding a drop of water, and covering it with a
coverslip. Use a light microscope for observation, start with a low power objective
to observe the shape and the arrangement of leaf cells. Compare the similarity
and difference between cells at the margin and cells in the interior. Switch to a
higher power objective, observe the details of a cell including the cell wall, the
cytoplasm, chloroplasts, the nucleus, and vacuoles. Observe the cytoplasmic
streaming, is there any specific direction for the streaming. How many layers of
cells are there in the leaf of Egeria densa. What is the structure that gives the leaf
green color?

Fig. 2-1 A mesophyllous cell of Egeria sp.

9
6. Non-green plant cells
In general, the epidermal cells do not contain chloroplasts.
1) Rhoeo discolor
(1) Use a razor blade or/and a pair of forceps to take a piece of lower
epidermis of the Rhoeo discolor leaf and make a wet mount slide. Be sure
to let the surface side face up. Observe the cell types and the cell
arrangement at a lower magnification and the cell contents at a higher
one. What cause the cells purple? And where are they stored in?
(2) Replace water with salt water when making a wet mount slide and observe
the changes of the cells. What phenomenon is it? How does this
phenomenon happen?

2) Allium cepa (Fig. 2-2)

(1) Fresh epidermis: Use a razor blade to cut the inner epidermis of a scale
leaf to small squares and take a piece of epidermal tissue with a pair of
forceps, and prepare a wet mount slide. Examine the shape and the
arrangement of epidermal cells with a low power objective, and then
switch to a higher power objective for the translucent cell wall, grey
cytoplasm, nucleus, and vacuoles. Is there any plastid in the cells?
(2) Stained epidermis: Observe the cells stained with methylene blue, eosin,
and neutral red. What structures become clearer after stained with each of
the three dyes?

Fig. 2-2. An epidermal cell of Allium sp.

10
7. Root tip cells
Using the permanent slide of longitudinal root sections of Allium cepa to observe
the shape and the arrangement of root cells with a low power objective. What
regions can you see dividing cells? Then use a higher power objective to examine
the cellular structures including the nucleus (nuclear envelope, nucleolus, and
nucleoplasm), and cytoplasm. Can you find vacuoles? If yes, are the vacuoles small
or big? What is the ratio of the nucleus to the cell?

8. Chromoplasts
Cut a thin section of mature tomato or pepper with a razor blade and make a wet
mount slide. Use a light microscope to observe the shape of cells and
chromoplasts. Chromoplasts are developed from chloroplasts and are inside the
cytoplasm instead of vacuoles. Chromoplasts in the mature tomato could have
disintegrated into yellow or orange granules or pieces with irregular shape.

9. Ergastic substances
1) Starch grains (Fig. 2-4)
Use a pair of forceps to take a tiny amount of tissue from potato tuber, rice
endosperm, and banana pulp, then make a wet mount slide for each sample.
Use a light microscope to observe the shape of starch grains, the hilum and
the concentric rings. Do starch grains of different plants have the same shape?
What happens to the starch grains when applying a drop of iodine solution
under the coverslip?

Fig. 2-4 Starch grains

11
 2) Crystals (Fig. 2-3)
Make freehand sections and wet mount slides for the leaf of Ficus elastica,
stem of Tradescantia virginiana, and petiole of Begonia. Find the cells with
crystals using a light microscope and observe the morphology of various
crystals. Are the shape and the size of the cell containing crystals the same as
the neighboring cells? What is the composition of different kinds of crystals?

Fig. 2-3 Crystals

10. Ultrastructure of the plant cell (Fig. 2-5)

Look at the transmission electron microscope micrograph of a plant cell, and label
the structures in figures.

Fig. 2-5. Ultrastructure of a plant cell

12
Questions
1. Look at the ultrastructural micrograph of the plant cell carefully. Compare the size
of major organelles. What organelles have single unit membrane? And what
organelles have double unit membrane?
2. What kinds of ergastic substances do plant cells have? Where are these ergastic
substances located?
Exposure Egeria immersed in water with sunlight can accelerate the
cytoplasmic streaming when compared with the control without sunlight
exposure. Explain why. What is the function of the cytoplasmic
streaming?

Answers of the figures

Fig. 2-1. d. Cytoplasm c. Smooth ER

a. Karyoplasm e. Nucleus d. Golgi body
b. Nucleolus e. Nuclear pore
c. Cell wall Fig. 2-3. f. Nucleolus
d. Tonoplast a. Cystolith g. Chromatin
e. Cytoplasm b. Druse h. Nuclear envelope
f. Plasmalemma c. Prismatic crystal i. Mitochondrion
g. Vacuole d. Raphides j. Chloroplast
h. Chloroplasts k. Plastid
i. Middle lamella Fig. 2-4. l. Microtubule
a. Hilum m. Central vacuole
Fig. 2-2. n. Microfilament
a. Cell wall Fig. 2-5. o. Plasmalemma(Plasma
b. Tonoplast a. Ribosome membrane)
c. Vacuole b. Rough ER p. Cell wall

13
Experiment 5. Roots

Introduction
The function of roots is to absorb water and nutrients, to anchor the plant, and to
transport and store foods. To obtain the best benefits from farming, cultivation,
irrigation, and fertilization, it is necessary to understand the distribution of roots in the
soil and also the inner structure of the roots.
1. Root systems
1) Taproot system
The primary root which is originated from the radicle develops to the taproot.
From the taproot arise smaller lateral roots (secondary roots), which in turn
produce even smaller lateral roots (tertiary roots). Most dicotyledonous plants
and gymnosperms produce taproot system.
2) Fibrous root system
The primary root which is originated from the radicle stop growing early, and
many adventitious roots of about equal diameter arise from the base of the
stem. Most monocotyledons plants have fibrous root system.

2. Inner structures of roots

1) Longitudinal section of roots
The root cap is at the tip of the root and protects the meristematic tissue.
Inside the root cap is apical meristem. The meristematic cells are able to
produce new cells and they are small, tightly packed, and of dense cytoplasm.
The region of elongation is behind the apical meristem. The cells in this region
grow very quickly and push the root tip through the soil. Behind the region of
elongation is the region of differentiation or maturation in which the
epidermis, cortex, and stele are distinguishable. This region is also called the
root hair zone because root hairs protrude from the epidermal cell to promote
water absorption.
2) Transverse section of roots
Structures of a root in the zone of differentiation in the transverse section are
14
as follows:
(1) Epidermis
The epidermis of a root is composed of one layer of parenchyma cells in
most plants. Some epidermal cells outgrowth to form root hairs.
(2) Cortex
The cortex is located between the epidermis and the stele. It consists of
several layers of parenchyma cells with obvious intercellular spaces. The
innermost layer of the cortex is the endodermis. The endodermal cells are
tightly packed and may have a band of suberized material (Casparian strip)
on the radial and transverse walls.
(3) Stele
a. Pericycle
The pericycle is the outermost layer of the stele and is right next to the
endodermis. It consists of one to more layers of parenchyma cells which
are able to divide and produce lateral roots.
b. Xylem
The xylem of a dicot root is in the center of the root and is radially
distributed. It is composed of tracheids, vessel elements, and parenchyma
cells. Protoxylem is the part that matures first in the xylem. Cells in
protoxylem are small and are located at the outer edge, i.e., the tip of the
xylem ray. Metaxylem matures later than protoxylem, cells in metaxylem
are bigger and are located in the central part of xylem.
c. Phloem
The phloem of a dicot root is distributed between the xylem tissues.
Phloem is composed of sieve tubes, companion cells, and parenchyma
cells.
In roots of a perennial dicot plant, undifferentiated procambium exists
between xylem and phloem. Procambium can divide and differentiate to
vascular cambium that gives rise to secondary xylem and phloem. In
monocot roots, instead of xylem, pith which is made of parenchyma cells is
located in the center of the root.

15
3. Modified roots
1) Storage root
The storage root is able to store a lot amount of nutrients, such as roots of
carrots and sweet potatoes.
2) Aerial root
Aerial roots are adventitious roots that expose to the air, such as roots of
banyans (Ficus sp.) and orchids.
3) Prop root
Prop roots are adventitious roots that support the plants, such as the
adventitious roots of corns. The aerial roots of banyans grow vertically
downwards, penetrate the soil and become trunks for support.
4) Parasitic root
Parasitic roots are the roots that penetrate into the host tissue and absorb
nutrients from their host plants, such as the haustoria of dodder.
5) Mycorrhiza
A mycorrhiza is a symbiotic association between a fungus and roots of a
vascular plant, for example, the mycorrhiza formed by roots of a pine tree or
an oak tree with its associated fungus.
6) Root Nodule
Root nodules form on the roots of plants (primarily Fabaceae) that associate
with symbiotic nitrogen-fixing bacteria.

Materials
1. Root systems of spinach and rice
2. Permanent slides
1) longitudinal sections of onion (Allium cepa) roots
2) transverse sections of buttercup (Rannuculus sp.) roots
3) transverse sections of corn (Zea mays) roots
4) transverse sections of coleus (Coleus sp.) roots
3. Various of modified roots

16
Observations and questions
3. Observe the root systems of spinach and rice (Fig. 5-1). Try to compare the
differences between them. What is the root part that develops to the main root of
spinach? What kind of root dose the corn root system consist of?

Fig. 5-1. Root system

4. Observe the longitudinal section of onion roots (Fig. 5-2). Which part of the root
cap is younger? And which part is older? Is it easy to observe cell divisions in the
meristematic tissue? What mitotic phases have you observed? What are the
morphological differences among the cells of the epidermis, the cortex, and the
stele? Have you seen root hairs? Are they unicellular or multicellular?

Fig. 5-2. Longitudinal section of root of Allium cepa

17
5. Observe the transverse section of buttercup roots (Fig. 5-3). Make a rough drawing
and label the relative position of each tissue. Then make a detailed drawing of the
stele and label the tissues and cells of the stele. Find the protoxylem in the root
section. What kind of cells are located in the center of the root? Describe the
function of the pericycle. How many arches (strand) are there in the xylem of the
sample you observe? Have you seen plastids in the parenchyma cells of the
cortex? If you have, what kind of plastid is it?

Fig. 5-3. C.S. of root of Ranunculus sp

6. Observe the transverse section of corn roots (Fig. 5-4). Make a rough drawing and
label each tissues. What are the features of the endodermis? What kind of
meristematic tissue does the pith in the center originate from? What are the
differences between the corn root and the buttercup root in anatomy?

18
 Fig. 5-4. C.S. of root of Zea mays

7. Observe the transverse section of coleus roots. What kind of tissue does the
lateral root originate from? Does the periclinal division or anticlinal division occur
first during its development?

8. Observe various modified roots.

19
Answers of the figures

Fig. 5-1. Fig. 5-3. c. Xylem

a. Adventitious root a. Epidermis d. Pith
A & B: Tap root system b. Cortex e. Endodermis
C & D: Fibrous root system c. Stele f. Epidermis
d. Endodermis g. Cortex
Fig. 5-2. e. Pericycle h. Metaxylem
a. Root hairs f. Protoxylem i. Phloem
b. Region of g. Metaxylem j. Pith
differentiation h. Phloem
c. Lateral root i. Procambium
primordium j. Cortex
d. Region of elongation
e. Apical meristem Fig. 5-4.
f. Root cap a. Epidermis
b. Cortex

20
Experiment 6. Stems

Introduction
The stem is the main aboveground axis of the plant. The functions of stems are to
support the plant, and to transport water and nutrients. The shoot apical meristem is
able to produce new cells, and thus forms leaves, branches, and flowers for vegetative
and reproductive growth.
1. The morphology of stems
The nodes and internodes are the most obvious parts of a stems or a shoot. A bud
is an undeveloped shoot, leaf, or flower. According to their location, buds are
classified as the terminal bud, lateral bud, axillary bud, and adventitious bud. The
leaf scar is the mark left on a stem surface after a leaf falls. The lenticels are visible
as raised spots scattered on the surface of a stem, through which gaseous
exchange occurs.
2. Inner structures of stems
1) Longitudinal section of shoot apical meristem
The shoot apex is a meristematic tissue. The bulges on two sides of the apex
are leaf primordia. The bud primordium is located in the leaf axial.
2) Transverse section of stems
Dicot stems
(1) Epidermis
The epidermis is the outermost layer of the stem; it is composed of one
layer of cells. The outer wall of an epidermal cell is thicker; the radial wall is
getting thinner from outside to inside; and the inner wall is thin. The
thickened outer wall is covered with cuticle and provides protection for the
plant. Stomata and trichomes are common on younger stems.
(2) Cortex
The cortex is located between the epidermis and vascular tissues. It
consists of parenchyma and collenchyma cells. Some of the parenchyma
cells differentiate into secretory cells.
(3) Vascular tissue
21
 The vascular tissue in dicot stems is composed of xylem, phloem and
procambium or vascular cambium. The vascular tissue is organized within
vascular bundles which are arranged in a ring in the cross section of stems.
a. Xylem
The xylem is located in the centripetal position of a vascular bundle. It
is composed of vessel elements, tracheids, fibers and parenchymal
cells. Protoxylem is located in the innermost part of xylem, and
metaxylem is next to procambium or vascular cambium.
b. Vascular cambium
The vascular cambium is originated from undifferentiated parenchyma
cells of the procambium and the interfascicular tissue which connects
the procambium of two neighbor vascular bundles. Between xylem and
phloem is procambium which is composed of meristematic cells that
give rise to phloem outwards and xylem inwards. The interfascicular
tissue is located between vascular bundles.
c. Phloem
The phloem is in the centrifugal position of a vascular bundle. It is
composed of sieve tubes, companion cells, phloem fibers, and
parenchymal cells.
(4) Pith and pith rays
The pith which is in the center of a stem is composed of parenchyma cells
that serve to store nutrients. During the second growth, the pith is often
compressed. The pith rays are parenchyma cells between vascular bundles
which appear as radiating lines from the pith to cortex. Their function is to
transport water and nutrients radially.

Monocot stems
(1) Epidermis
The epidermis is the outermost layer of the monocot stem and is
composed of one layer of cells.
(2) Ground tissue

22
 The ground tissue is distributed among the vascular tissues. It is composed
of parenchyma cells. Some plants have pith cavity in the center of the
stem.
(3) Vascular tissue
Vascular bundles are scattered throughout the ground tissue. Each vascular
bundle is often surrounded by sclerenchyma cells and consists of xylem
and phloem.
a. Xylem
The xylem is located in the centripetal position of the vascular bundle.
Both protoxylem and metaxylem are obvious, and they are composed
of tracheids and vessel elements.
b. Phloem
The phloem is in the centrifugal position of the vascular bundle. The
protophloem is usually crushed and damaged. The metaphloem is
obvious and consists of sieve tubes and companion cells.

3. Secondary growth of stems

In perennial woody plants, increases of new secondary vascular tissues produced
by vascular cambium each year cause the stem thickening. The amount of
secondary xylem produced each growing season is more than that of secondary
phloem. The secondary phloem does not thicken because it is often pushed
outward and damaged by inner new formed tissues. The secondary xylem
produced earlier in the growing season is called the early wood, which is
characterized by light-colored large thin-walled cells. The late wood is produced
later in the growing season, and is characterized by dark-colored small thick-walled
cells. The growth ring or annular ring is made of many concentric rings of
alternately arranged early wood and late wood.

4. Modified stems
1) Rhizomes
A rhizome is a modified underground stem of a plant, it is a cylindrical stem

23
 with nodes and internodes, such as stems of lotus and iris.
2) Tuber
A tuber is a short and thickened underground stem with nodes and
internodes. The eyes (a group of buds) grows on the nodes, such as potatoes.
3) Corm
A corm is a short and swollen underground stem, such as stems of water
chestnut and gladiolus.
4) Bulb
A bulb is a compressed stem made of layers of thick scale leaves, such as onion
and garlic.
5) Stolon
A stolon is a thin root runs on the ground or just under the ground. It is a good
propagation method by giving rise to new plants at nodes, such as stems of
strawberry.
6) Cladode
A cladode is a flattened stem resembling and functioning as a leaf, such as
stems of asparagus and asparagus fern.
7) Stem tendril
A stem tendril is a thin, wiry, leafless and spirally coiled branch that functions
to attach a climbing plant to its support, such as tendrils of grape vines.

Materials
1. Young shoot of Formosan sweet gum (Liquidambar formosana, 楓香)
2. Permanent slides
1) longitudinal sections of coleus (Coleus sp., 彩葉草) shoot apex.
2) transverse sections of sunflower (Helianthus annus, 向日葵) stems
3) transverse sections of corn (Zea mays, 玉米) stems
4) transverse sections of panicum (Panicum sp., 鋪地黍) stems
5) transverse sections of one-year old and older pine (Pinus sp., 松) stems
3. Various modified stems

observations and questions

1. Take a young shoot of Formosan sweet gum. Observe the arrangement of buds
and leaves on the shoot, and also the morphology of lenticels on the stem surface.
24
 Is the bud of sweet gum covered by scales? What is the name of this type of bud?
2. Use a light microscope to observe the longitudinal section of coleus shoot apex.
Make a rough drawing and label the tissues. How to distinguish the bud
primordium form the leaf primordium?
3. Observe the transverse section of sunflower stems, and label the tissues in Fig. 6-
1. What kinds of cells is the cortex composed of? Where is the location of the
protoxylem and the metaxylem in a vascular bundle? How does the arrangement
of protoxylem and metaxylem differ from that of the dicot root? What kinds of
cells can you find in the xylem? And in the phloem? What characteristics does an
undifferentiated procambial cell possess?

Fig. 6 -1. C.S. of stem of Helianthus annus

25
4. Observe the transverse section of corn stems, and label the tissues in Fig. 6-2.
How are the vascular bundles distributed in the ground tissue? Are the cells of
vascular bundle sheath parenchyma or sclerenchyma cells? What are the
differences between cells of protoxylem and of metaxylem? Is there vascular
cambium in the vascular bundle of corm stem? What characteristics does a sieve
tube member and a companion cell of phloem possess?

Fig. 6-2. C.S. of stem of Zea mays

26
5. Observe the transverse section of panicum stems, and label the tissues in Fig. 6-3.
Compare the observation with that of the corm stems.

Fig. 6-3. C.S. of stem of Panicum sp.

27
6. Observe the older pine stems (Fig. 6-4) with a low power objective. How to
distinguish among early woods, late woods, and secondary phloem? What are the
destinies of primary phloem, primary xylem, cortex, and pith after the secondary
growth begins?

Fig. 6-4. C.S.of stem of Pinus sp.

7. Observe the characteristics of various modified stems.

Answers of the figures

Fig. 6-1. b. Ground tissue e. Pith lacuna

a. Epidermis c. Vascular bundle f. Companion cell
b. Cortex d. Bundle sheath g. Sieve-tube member
c. Vascular bundle e. Companion cell h. Metaxylem
d. Pith f. Sieve-tube member i. Protoxylem lacuna
e. Interfascicular tissue g. Metaxylem j. Sclerenchyma
f. Phloem h. Protoxylem
g. Procambium i. Protoxylem lacuna Fig. 6-4.
h. Metaxylem a. Resin duct
i. Protoxylem Fig. 6-3. b. Phloem
j. Pith a. Epidermis c. Cambium
b. Sclerenchyma d. Secondary xylem
Fig. 6-2. c. Vascular bundle e. Pith
a. Epidermis d. Ground tissue f. Annual ring

28
Experiment 7. Leaves

Introduction
Leaves are important vegetative organs of advanced plants, they are the sites where
photosynthesis, respiration, and guttation take place. The inner structure of leaves is
closely related to these physiological process.
1. The morphology of leaves
The shape and the size of leaves are different among different plant species so the
morphological characteristics are often used for classification.
In magnoliids and eudicots, the leaf commonly consists of three parts: blade,
petiole, and stipule. A leaf with all three parts is a perfect leaf, and one that lacks
one or more parts is an imperfect leaf. The leaves are either simple or compound.
A simple leaf has a single blade which attaches to the petiole; and a compound
leaf has many leaflet that attach to the petiole (Fig. 7-1 C-F). Compound leaves are

Fig. 7-1. Various kinds of leaves

29
classified into pinnately and palmately compound leaf based on the position that
the leaflets attach to the stipule. A pinnately compound leaf resembles the
structure of a feather, with the leaflets arranged on both side of the rachis. And a
palmately compound leaf looks like a palm and has leaflets that radiate from a
single point at the distal end of the petiole.

In most monocots and certain eudicot, the leaf consists of sheath, blade, and
ligule. Some species also have auricle (Fig. 7-1A, B). The sheath is at the base of
the blade and wraps entirely around the stem. The blade is narrow and long in
general, and has a major vein in the center and many parallel minor veins on
lateral sides. The ligule is a transparent membrane structure at the junction of the
sheath and blade. The auricle looks like a small ear, and is the outgrowth at the
base of blade. Conifers of gymnosperms have needle-like and scaly leaves.
The arrangement of leaves on the stem is called the phyllotaxy. There are three
types of phyllotaxy: alternate, opposite, and whorled. The alternate phyllotaxy has
one leaf at each node and can be subdivided to spiral and distichous alternate.
The opposite phyllotaxy has two opposite leaves at each node, and can be
subdivided to distichous and decussate opposite. The whorled phyllotaxy has
three or more leaves that arranged in a whorl at each node.

Fig. 7-1. Various kinds of leaves

30
2. Anatomy of leaves
Transverse section of leaves
Dicot leaves
1) Epidermis
The epidermis of a leaf has a single layer or multiple layers of cells. The outer
cell wall that contacts the environment is covered with cuticle. The cuticle
prevents the excessive loss of water during the process of evaporation. There
is no chloroplast in epidermal cells and no intercellular space between them.
The epidermis has pores which are called stomata for regulating gas exchange.
Each stoma is surrounded by a pair of kidney shaped guard cells with
chloroplasts. The guard cell has a thicker inner wall which is next to the pore.
Some of the epidermal cells are specialized to form trichomes which provide
protection and prevent water loss.
2) Mesophyll
The mesophyll is located beneath the epidermal tissue. There are two types of
mesophyll based on the shape and the arrangement of cells:
(1) Palisade mesophyll
The palisade mesophyll cells are columnar and stacked closely together in
a layer beneath the upper epidermis. These cells contain many
chloroplasts and are the main sites for photosynthesis.
(2) Spongy mesophyll
The spongy mesophyll is located beneath the palisade mesophyll. The cells
are irregular in shape and arranged loosely with large spaces.
3) Vascular tissue
The vascular tissue in the leaf are called veins. The vascular tissue of the stem
or branch runs through the petiole to the mesophyll. The major vein of the leaf
is called midrib, and from which arise numerous lateral veins. Each vein is
surrounded by a bundle sheath. The major vein is composed of xylem near the
upper epidermis and phloem near the lower epidermis. However, the distal
part of a lateral vein possesses only xylem with tracheids but no phloem.
4) Monocot leaves

31
 The inner structure of a monocot leaf is similar to that of a dicot leaf. But there
is no differentiation of palisade and mesophyll for most monocot species. The
monocot leaves usually contain numerous sclerenchyma cells. Most
angiosperm leaves are bifacial structures with distinct adaxial (upper surface)
and abaxial (lower surface) identities. However, some monocots develop
unifacial leaves with only an encircling adaxial or abaxial epidermis.

3. Modified leaves
1) Bud scale
A bud scale is sessile (without petiole) and covered with trichomes. It
functions to protect the shoot apex and young leaves, such as the bud scale of
azalea.
2) Spine
A spine is the woody modified leaf of plants that functions to prevent water
loss, such as the spine of cactus.
3) Leaf tendril
A leaf tendril is a green threadlike structure modified from a leaf which helps
in climbing around the support, such as the lead tendril of pea.
4) Storage leaf
A storage leaf is a leaf modified to store foods, such as the scale leaf in the
bulb of an onion or lily.
5) Insectivorous leaf
An insectivorous leaf is a leaf of carnivorous plants modified to capture and
digest insects.
6) Reproductive leaf
A reproductive leaf is a leaf modified for reproduction. Tiny plants are form at
the edge of the reproductive leaf, such as the reproductive leaf in Bryophyllum.
7) Phyllode
A phyllode is a flattened petiole or leaf rachis that resembles and functions as
a leaf. First leaves of Acacia seedling are pinnately compound leaves, but other
leaves are phyllodes with only petioles.

32
Materials
1. Fresh shoots
Liquidambar formosana (楓香) Citrus maxima (柚子)
Ficus microcarpa (榕) Hedychium coronarium (野薑花)
Chionanthus retusus (流蘇) Gladiolus sp. (劍蘭)
Palimora alstonia (黑板樹) Iris sp. (鳶尾)
Murraya paniculata(月橘) Pinus luchuensis (琉球松)
Delonix regia (鳳凰木)
Schefflera arboricola (鵝掌藤)

2. Permanent slides
6) transverse sections of rose (Rosa sp.) leaves
7) transverse sections of corn (Zea mays) leaves
8) transverse sections of sword lily (Gladiolus sp.) leaves
9) transverse sections of pine (Pinus sp.) leaves
3. Various modified leaves

Observation and questions

1. Morphology of leaves
1) Distinguish the perfect leaf from the imperfect leaf by observing leaves of
Liquidambar formosana (楓香) ,Ficus sp. (榕), and Chionanthus retusus (流蘇).
2) Determine the phyllotaxy of Liquidambar formosana (楓香), Chionanthus
retusus (流蘇), and Palimora alstonia (黑板樹), explain how you do that.
3) Distinguish the pinnately from the palmately compound leaf by observing
leaves of Murraya paniculata (月橘), Delonix regia (鳳凰木), Schefflera
arboricola (鵝掌藤), and Citrus maxima (柚子). For the pinnately compound
leaves, determine the number of divisions.
4) Distinguish the parts of a monocot leaf by observing leaves of Hedychium
coronarium (野薑花).
5) Observe the morphology of a unifacial leaf by observing leaves of Gladiolus sp.
(劍蘭) or Iris sp. (鳶尾)
6) Observe the location and the morphology of the needle-like and scaly leaves
of Pinus luchuensis (琉球松).

2. Anatomy of leaves
1) Observe the inner structure of the rose leaf in cross section using a light
microscope, and label the structures in Fig. 7-2. How many layers of cells are
there in the palisade mesophyll? Are there stomata in the upper epidermis?

33
 How to recognize the cells in the xylem and in the phloem? The mesophyll is
usually called chlorenchyma, explain why.

Fig. 7- 2. C.S. of leaf of Rosa sp.

2) Observe the slide of the corn leaf (Fig.). How does the mesophyll differentiate?
Locate the bundle sheath cells. Are there chloroplasts in the bundle sheath
cells? How to distinguish the adaxial from the abaxial epidermis?

Fig. C.S. of leaf of Zea mays

34
3) Observe the slide of sword lily leaf cross section, and label the structures in
Fig. 7-3. Are there distinguishable upper epidermis and lower epidermis? How
are the veins distributed in the leaf? How does the mesophyll differentiate?
Are there any cells or tissues other than parenchyma cells? What are they?

Fig. 7- 3. C.S. of leaf of Gladiolus sp.

4) Observe the slide of the pine leaf cross section, and label the structures in Fig.
7-4. What are the characteristics of the epidermal cells in the pine leaf?
Describe the morphology of the mesophyll cells. What name is the cell layer
that surrounds the vascular tissue (vein)? And what are their features?

35
Fig. 7- 4. C.S. of leaf of Pinus sp.

36
Answers of the figures

Fig. 7-1. c. Midrib f. Epidermis

a. Blade d. Upper epidermis g. Hypodermal cell
b. Node e. Druse h. Secretory cell
c. Internode f. Xylem i. Resin canal
d. Blade g. Phloem j. Guard cell
e. Ligule h. Lower epidermis k. Lobed parenchyma
f. Auricles (Mesophyll)
g. Sheath Fig. 7-3. l. Endodermis
h. Blade a. Undifferentiated m. Xylem
i. Petiole mesophyll n. Phloem
j. Leaflet b. Epidermis
k. Petiole c. Xylem Fig. C.S. of leaf of Zea
l. Leaflet d. Phloem mays
m. Rachis e. Sclerenchyma a. Upper epidermis
n. Petiole Fig. 7-4. b. Mesophyll
o. Stipule a. Upper epidermis c. Bundle sheath cell
b. Resin duct d. Xylem
Fig. 7-2. c. Transfusion tissue e. Phloem
a. Vein d. Endodermis f. Lower
b. Mesophyll e. Lower epidermis

37
Experiment 14. Seed germination and -amylase

Introduction
Food reserves are synthesized and stored during the seed development, and are
used for early postgerminative growth of seedlings. Cereal crops store a lot of starch
grains in their grains. After seed germination, the starch is broken down to glucose by
-amylase and transformed to sucrose before transported to the growing shoot and
root. -amylase is the first enzyme that catalyzes the hydrolysis of the whole starch
grain. It is synthesized in the scutellum of embryo and the aleurone layer after the
seed imbibition. Then, -amylase is secreted to the endosperm and digest the starch
grains.
Iodine reacts with starch to produce a deep blue-black color. Using the iodine
test and thin-layered starch technique, we are able to observe the presence and the
synthesized sites of -amylase in the grain during the seed germination.

Materials and preparation before the experiment

1. Materials
1) Corn grains
2) Petri dish, razor blade, and forceps
3) Potato starch, agar, iodine solution
2. Preparation before the experiment
1) Seed germination
(1) At 0.5, 1, 2, and 3 days before the experiment, put corn grains in running
water for 16 hours. Plant them on moist vermiculite or in absorbent paper
and place them in the dark for seed germination.
2) Making starch agar plates
(1) Prepare 50 mM Na-Acetate buffer (pH 5.5, with 10 mM CaCl2).
(2) Add 0.3 g starch to 100 ml Na-Acetate buffer, and heat for starch
gelatinization.
(3) Add 1 g agar powder per 100 ml buffer with starch, heat until the agar
dissolves.
(4) Mix the agar solution thoroughly and let it cool down to 50℃. Pour 10 ml
starch agar in each petri dish and then swirl it slowly so that the starch agar
covers the bottom completely. Place the petri dishes horizontally and allow
the plates to cool and solidified.
38
 3) Making Iodine solution
Dissolve 1.2 g I2 and 12 g KI in 1 litter 0.1N HCl.

Methods
1) Take a starch plate for each group. Divide the plate to 4 areas for seeds of 4
stages by drawing a cross with a marker on the bottom of the petri dish.
2) Take one germinated seedling of 0.5, 1, 2, and 3 days each. Wash the
vermiculite off and remove the young shoot and root from the grain.
3) Dry the grain with a piece of absorbent paper. Cut the grain longitudinally with
a sharp razor blade.
4) Place the cutting surface of the two half grains toward the starch agar with
care, and press them gently with a pair of forceps so that the cutting surface
has good contact with the starch agar. Notice that do not move the grain once
the grain contacts the starch agar.
5) Wait for 10-15 minutes, and pour proper amount of iodine solution to just
cover the agar plate. Remove the grains immediately, and whirl the petri dish
so that the iodine solution covers the whole plate.
6) Soon after the iodine solution covers the plate, discard the iodine solution to
avoid overstaining.
7) Observe the contact areas between the grains and the starch agar. Draw and
compare the location of the transparent zones in the grains at the four stages
of seed germination.

Fig. 14-1 The staining results of amylase reaction of corn seeds on thin-layered
starch agar plates (DAI: day after imbibition)

39
Experiment 15. Starch phosphorylase

Introduction
Starch phosphorylase catalyzes the degradation of starch that produces glucose-1-
phosphate (G-1-P), and also catalyzes the reverse reaction that synthesizes starch
from G-1-P. In general, small starch molecules or oligosaccharides that works as a
primer is needed for starch phosphorylase to catalyze the reaction of starch synthesis.
(Glucose)n + G-1-P ↹ (Glucose)n+1 +Pi
The objective of this experiment is to understand the properties of starch
phosphorylase by observing the effects of environmental factors on the activity of
starch phosphorylase using the iodine test.

Materials and preparation

1. Materials
1) Potato
2) 96 wells microplate, dropper, and test tube rack
3) 0.01 N NaF; 0.01 M glucose-1-Phosphate, 0.2 M KH2PO4, 0.2% starch solution
4) Iodine solution (2 g I2 and 20 g KI in 1 litter 0.1 N HCl)
2. Preparation of the crude enzyme extract
(1) Prepare the crude enzyme extract at 0-4℃ to keep the enzyme activity.
(2) Take 2 to 4 potatoes depending on the size, clean and peel the potato skin off,
and cut them to small pieces. Homogenize the potato pieces with 500 ml ice-
cold 0.01 N NaF by using a blender at high speed. (NaF inhibits activity of
phosphatase and thus prevents the degradation of G-1-P to glucose and
phosphate (Pi).)
(3) Filter the enzyme extract through cheesecloth of 4 layers to remove the cell
debris, collect the enzyme extract and place it in an ice bath.
(4) Centrifuge at 10,000 g (or GSA rotor, 8k rpm) for 10 minutes, collect the clear
extract and keep it on ice.
(5) Heat 100 ml clear extract for 1 minute with a microwave, or boil it in a water
bath for 5 minute.

40
Methods
1. Take a test tube rack and 12 test tubes for each group. Label the test tubes from 1
to 12. Add 5 ml iodine solution into the 12th tube, and add solutions to the other
tubes following the instruction of table 15-1. Examine the height of reaction
solution of all the 11 tubes to make sure that the total amount of reactants for
each tube is correct.
2. Prepare a 96 wells microplate, 12 droppers, a piece of white paper, and a few
tissue paper. Put the white paper under the microplate to make the observation
easier.
3. Add 2 ml heated enzyme extract into the 7th and 11th test tubes, and 2 ml fresh
enzyme extract each to the other tubes. Put a dropper in each tube and mix well
immediately with the individual dropper and set up the timer to count up.
4. Start from the 1st tube, put a drop of reaction solution to the 1st well and add a
drop of iodine solution in it, and then observe if the black-blue starch-iodine
reaction happens or not. Present the degree of blue color with the number of
positive sign (+). Repeat the above procedure for the rest 10 tubes and record
their result.
5. Repeat to test and record the result of the 11 tubes every 5 minutes until 45
minutes.
6. After finishing all the tests, examine the pH value by dipping the pH test stripe into
the mixture of the 2nd, 5th, 6th, 8th, 9th, and 10th tubes, respectively.
7. Add 1 ml iodine solution in each tube which has about 5 ml reaction solution and
mix well. Compare the degree of blue color which indicates the amount of starch.
8. Recycle the iodine solution to the bottle labelled “waste iodine solution”.

Table 15-1
no. of test tube
1 2 3 4 5 6 7 8 9 10 11
reagent
glucose solution 3 ̶ ̶ ̶ ̶ ̶ ̶ ̶ ̶ ̶ ̶
G-1-P ̶ 3 3 3 3 3 3 ̶ ̶ ̶ ̶
starch solution ★ ★ ̶ ★ ★ ★ ★ 3 3 3 3
KH2PO4 solution ̶ ̶ ̶
1 ̶ ̶ ̶ 1 1 1 1
0.05N NaOH ̶ ̶ ̶ ̶ 0.5 ̶ ̶ ̶ 0.5 ̶ ̶
0.05N HCl ̶ ̶ ̶ ̶ ̶ 0.5 ̶ ̶ ̶ 0.5 ̶
distilled H2O 1.5 1.5 1.5 0.5 1 1 1.5 0.5 ̶ ̶ 0.5
fresh enzyme extract 2 2 2 2 2 2 ̶ 2 2 2 ̶
heated enzyme extract ̶ ̶ ̶ ̶ ̶ ̶ 2 ̶ ̶ ̶ 2

41
Discussion
Study the properties of starch phosphorylase by comparing the results of the
following test tubes.
1. Speculate about what the reactants are of starch phosphorylase by comparing the
results of the 1st, 2nd, and 8th tubes.
2. Explain the importance of the primer by comparing the results of the 2nd and 3rd
tubes.
3. Explain the effect of KH2PO4 by comparing the result of the 2nd and 3rd tubes.
4. Explain the effect of pH by comparing the results of the 2nd, 5th, and 6th tubes and
those of the 8th, 9th, and 10th tubes.
5. Explain the influence of heating to the enzyme activity by comparing the results of
the 2nd and 7th tubes and those of the 8th and 11th tubes.
6. Discuss the fact that this enzyme extract is able to catalyze both the forward and
reverse reactions by by comparing the results of the 2nd and 8

42
Experiment 18. Photosynthesis – The extraction, separation, and

absorption spectrum of plant pigments

Introduction
Photosynthesis is the process that green plants convert CO2 and H2O into sugars
by using solar energy. The process contains photoreaction and Calvin cycle; the overall
equation is as follows:

The photoreaction takes place in the grana of the chloroplast. The pigment
systems transport the absorbed solar energy to the reaction center, and then
transformed to assimilatory power (NADPH and ATP) through the electron transport
chain. The Calvin cycle takes place in the stroma of the chloroplast. Using NADPH and
ATP produced in the photoreaction as the energy, a series of enzymatic reactions
assimilates CO2 and generates sugars and other important metabolic intermediates.
Green plants contain many specific pigments, such as chlorophyll a, chlorophyll b, and
carotenoids. The chlorophyll a and b absorb light most strongly in the red (600-700
nm) and blue (400-500 nm) regions of the visible spectrum; the carotenoids absorb
mainly in the blue region. In general, green light is the least absorbed and the most
reflected, so most leaves are green. This is also the reason why the blue light and red
light can increase the efficiency of photosynthesis.
After being absorbed by the pigments, the possible pathways of the energy flow
include: (1) photochemical reaction: the energy is transferred to NADPH and ATP via
the electron transport chain. (2) the energy is emitted in forms of fluorescence,
phosphorescence, or heat. (3) the energy is transferred to other molecules, such as
oxygen molecules or carotenoids. The energy absorbed by pigments in the plant body
mostly is emitted in the form of heat or enters the pathway of the photochemical
reaction. However, the energy absorbed by the pigments which are extracted with
organic solvents can only be emitted in the forms of fluorescence and heat.
The objectives of this experiment are to: (1) extract the plant pigments with organic
solvents; (2) separate the pigments by using thin layer chromatography technique;
43
and (3) establish the absorption spectrum of the photosynthetic pigments using the
spectrophotometer.

Materials and preparation

1. quick dried spinach leaves at a high temperature with an oven; fresh spinach
leaves
2. 80% acetone
3. developing solvent (acetone: ether: n-Hexane = 2: 3: 6)
4. TLC plate
5. spectrophotometer (Thermo spectronic, Helios ε)
6. capillary
7. mortar, pestle, filter paper, funnel, beaker, and graduated cylinder
8. cuvette

Methods
1. Extraction of plant pigments
1) Take 1 g of dried spinach leaf, tear the leaf into tiny pieces and put them in a
mortar and grind them into powder with a pestle. Add 10 ml 80% acetone and
continue grinding. Filter the pigment extract with a funnel and filter paper,
collect the extract with a beaker or a vial. Add 5 ml acetone for further
extraction, collect the pigment extract of 15 ml.
2) Place the pigment extract against the black background or illuminate the
pigment extract with a flashlight, observe the red fluorescence emitted by the
chlorophylls that absorb light.

2. Separation of plant pigments

1) Take a stripe of TLC plate (20cm * 2 cm). Use a pencil to draw a line about 3
cm from the bottom of the plate. Be aware and avoid scrapping the coating of
the plate! With a capillary apply the pigment extract onto the plate along the
line, and repeat the procedure after the pigments dry out. Repeat at least 10
times or until the line turns dark green.
2) Place 5 ml developing solvent into a 50 ml or 100 ml graduated cylinder. The
height of the developing solvent should be lower than the pigment line of the
TLC plate.
3) Place the TLC plate with pigments into the graduated cylinder, cover the top of
the graduated cylinder with plastic wrap to seal the system.
4) The developing solvent moves upward due to the capillary action of the TLC
plate and thus separate the pigments with different solubility in the solvent.
44
 5) Remove the TLC plate from the graduated cylinder after the pigments are
separated and mark the position of the solvent front immediately. After the
developing solvent dries out, mark the position of each pigment and calculate
their Rf values.
Rf= distance of the pigment travels / distance of the solvent front travels

3. Establishment of the absorption spectrum of photosynthetic pigments

1) Use of Spectrophotometer：Turn the power of the spectrophotometer on and
let it warn up for 30 minutes or longer to stabilize the machine. Adjust the
tested wavelength, zero the spectrophotometer by placing a cuvette
containing only 80% acetone (the blank) into the cuvette chamber and press
the “ZERO” button. Change to the cuvette containing the pigment extract and
read the value of the absorbance or optical density (O.D). Re-zero the
spectrophotometer whenever the wavelength is changed.
2) Take 0.1 g of fresh spinach leaf (avoid the midrib and large vein) into a mortar
and grind it to paste with a pestle. Add 5 ml 80% acetone and continue
grinding the paste until no debris can be seen. Filter the pigment extract with a
funnel and filter paper, collect the extract with a graduated cylinder. Let the
mortar contact with the pestle when pouring the extract to the funnel. Use a
little 80% acetone to wash the remained pigment extract out of the mortar
and pestle and collect it. Extract the pigment on the filter paper by adding the
acetone drop by drop with a dropper until no pigment is remained on the
filter. Keep the volume of the total pigment extract down to 20 ml or less.
Measure the volume of the pigment extract.
3) Measure the O. D. of the pigment extract at 430 nm, the optimal O.D. is
between 0.8 to 1.2. Dilute the extract with 80% acetone if the concentration of
the pigment is too high. Record the final volume of the pigment extract after
dilution. Re-do the extraction if the concentration is too low.
4) Start from the wavelength of 400 nm, measure and record the O.D. of the
pigment extract every 10 nm until 700 nm.
5) Fine the wavelength of the peaks, and measure the O. D. values of 5 nm below
and above the wavelength.
6) Plot the absorption spectrum for the pigment extract by placing the
wavelength on x-axis and absorbance (O. D.) on y-axis.
7) Calculate the contents of chlorophylls: Measure the O. D. values at the
wavelength of 645 nm, 663 nm, and 652 nm. Calculate the total chlorophyll
content, chlorophyll a, and chlorophyll b per gram of spinach leaf (mg/g) using
the following equations.

45
 O. D.: optical density, absorbance
V: the total volume of the pigment extract in 80% acetone
W: the weight of the spinach leaf

Reference
Amon, D. 1949. Copper enzymes in isolated chloroplasts Polyphenoloxidase in Beta
vulgaris. Plant Physiol 24: 1-15

46
Experiment 19. Photosynthesis – Inner structures of C3, C4, and

CAM plants

Introduction
Based on the photosynthetic mechanisms, plants are categorized to three types:
C3, C4, and Crassulaceous Acid Metabolism (CAM).
1. C3 plants fix CO2 during the day. The first stable product of CO2 fixation is a three-
carbon compound, 3-PGA. The rate of photorespiration of the C3 plant is high
under high temperature and high light intensity and thus reduces the productivity
of the plant. The C3 plants examples include: peanut, tobacco, soybean, and rice.
2. C4 plants fix CO2 during the day. The CO2 absorbed by the mesophyll is fixed to a
four-carbon compound, oxaloacetic acid (OAA). OAA is then converted to malic
acid or aspartic acid and transported to the adjacent bundle-sheath cells where
CO2 is released and enters the C3 pathway to produce sugars. The rate of
photorespiration of the C4 plant is very low under high temperature and high light
intensity so that productivity of the C4 plant is high. The C4 plants examples
include maize and sugarcane.
3. CAM plants fix CO2 in two steps which occur at separate times but within the same
cell (mesophyll). During the night, the stomata in the leaves of CAM plants open
and collect CO2 which is fixed to four-carbon compounds (the same as in C4
plants). These compounds are converted to malic acid and stored in the vacuole.
During the day under the light, the stomata are closed and the malic acid leaves
the vacuole and releases CO2 in the cytosol. Then CO2 enters the C3 pathway to
produce sugars. Most succulent plants are CAM plants such as Bryophyllum, cacti,
and orchids.
The C4 plants often possess a specialized leaf anatomy called kranz anatomy. Their
vascular bundles are surrounded by a ring of large bundle sheath cells which contain
many chloroplasts and fully developed organelles. And the bundle sheath cells are
surrounded by a ring of radiate mesophyll cells. The C3 plants do not have this
characteristic.

47
Materials
Permanent slides of leaf cross section of C3 (rose), C4 (maize), and CAM (aloe) plants.

observations
Observe the leaf structures of the C3 (rose), C4 (maize), and CAM (aloe) plants with
permanent slides and a light microscope. Draw their leaf structures and describe the
similarities and differences among them.

48
Experiment 4. Mitosis &Meiosis

Introduction
Mitosis (asexual division) is the nuclear division of a somatic cell (Fig. 4-1). The main
goal of mitosis is to produce two genetically identical new cells. Before entering
mitosis, cells are in the stage of interphase. The individual chromosome in the nucleus
is not visible during interphases, but one or more nucleoli can be seen. The duration
of interphase is long and DNA synthesis occurs in the nucleus. Once the nuclear
division begins, the process of mitosis is divided to four phases:
1. Prophase
The loosely packed chromatin fibers coils into visible thread-like chromosomes
gradually. The nucleoli disappear and the nuclear envelope breaks down. The
rodlike chromosomes which consist of two sister chromatids can be observed later
in this phase.
2. Metaphase
The chromosomes are getting shorter and thicker. Each chromosome is composed
of two sister chromatids which are jointed at the centromere. With the act of the
spindle fibers, chromosomes move to the center of the cell and are aligned along
the equatorial plane.
3. Anaphase
The chromosome splits longitudinally at the centromere to separate the two sister
chromatids and form two independent daughter chromosomes. Then the
daughter chromosomes are pulled by the spindle to the opposite poles of the cell.
4. Telophase
Two groups of daughter chromosomes have moved to the opposite poles.
Chromosomes unwind to be thread-like, the nucleoli and nuclear envelope
reappear. The nuclei restore to the appearance during interphase.

Cytokinesis is preceded by nuclear division for most cells. In animal cells, a cleavage
furrow forms and then advances inwards to separate the two daughter cells. In plant
cells, vesicles rich in pectin appears in the center and fused to form cell plate. The cell
49
plate develops into middle lamella; then cellulose is deposited on either side of the
middle lamella to from the primary wall and two complete daughter cells with the
same chromosome set are formed.

Fig. 4-1. Mitosis

50
Meiosis occurs when germ cells generate gametes. It is also called sexual division and
has two rounds of cell division (Fig. 4-2). The process of meiosis is similar in animals
and plants, also in female and male. Take the microsporogenesis of Zea mays as an
example to describe the process of meiosis that a diploid microsporocyte produces
four haploid microspores as follows.
1. Prophase I can be divided into five sub-phases
1) Leptonema
Chromosomes are very long and not visible. Nucleoli are visible in the nucleus.
2) Zygonema
Thread-like homologous chromosomes begin to pair.
3) Pachynema
Homologous chromosomes finish pairing and form bivalents. Chromosomes
continue thickening and shortening. Crossover occurs in this phase.
Observable characteristics of the chromosome include Chromomeres,
centromere, heterochromatin, and euchromatin. The nucleolus is attached to
the nucleolar organizer center of the chromosome.
4) Diplonema
The two homologous chromosomes of each bivalent begin to repel one
another but remain attached by chiasmata. Chromosomes keep thickening and
shortening.
5) Diakinesis
Chromosomes condense further. The homologous chromosomes of each
bivalent continue to repel one another and move the chiasmata to the end of
the bivalent. The nucleoli and the nuclear envelope disappear at the end of
diakinesis.
2. Metaphase I
The chromosomes are fully condensed. The bivalents are aligned along the
equatorial plane.
3. Anaphase I
The two chromosomes of each bivalent separate and begin to move toward the
opposite poles of the cell

51
4. Telophase I
The two groups of chromosomes reach the opposite poles of the cell and a
daughter nucleus forms at each pole. Cell membrane and cell wall are produced
between the two nuclei and finally two daughter cells are formed. The number of
chromosomes of the daughter cell reduces to half because of the pairing and
separation of homologous chromosomes. The whole set of one maternal
chromosomes is unlikely to reach the same pole because that the homologous
chromosomes follow the principle of independent assortment to separate and
distribute to the two poles. Besides, the crossover that happens in the pachynema
stage results in genetic recombination. So the genetic composition of the two
daughter cells formed in the first division is not exactly the same, and neither the
same as their parental cells.
5. Interkinesis
The stage before the secondary division. The nature of the secondary division is
the same as that of mitosis. The number of chromosomes keeps unchanged.
6. Prophase II
The two chromatids of each chromosome are clearly visible because of the
shortening and thickening of chromosomes.
7. Metaphase II
The chromosomes are aligned along the equatorial plane in the center of the cell.
8. Anaphase II
The chromosome splits longitudinally at the centromere to separate the two sister
chromatids and form two independent daughter chromosomes. And the daughter
chromosomes move to the opposite poles of the cell.
9. Telophase II
Four daughter nuclei form in each microsporocyte, they were separated by new
cell walls to form four haploid microspores.

52
 Fig. 4-2. Meiosis

Materials
3. Permanent slides
1) Longitudinal sections and smear slides of Allium cepa (洋蔥) root
2) Meiosis of Zea mays microsporocyte.
4. Preparation of mitotic slides of root tip cells of Allium cepa: red onion bulbs, 2 mM
8-hydroxyquinoline, 95% alcohol: glacial acetic acid (3:1), 1N HCl, leuco-basic
fuchsin, 1% pectinase, 45% acetic acid
5. Molecular models of DNA.

53
Methods
9. Observation with permanent slides
1) Observe the four stages of mitosis of onion root tip cells with high power
objectives.
2) Observe the stages of meiosis of Zea mays microsporocyte with high power
objectives.
10. Observation with fresh made mitotic slides of of Allium sp
1) Preparation of the root materials
(1) Growing onion roots
Grow red onion roots with a suitable jar. Trim old roots of onion bulbs with
a razor blade and then put the bulbs on the mouth of a jar that is filled
with water. Make sure that the very bottom of bulbs is immersed in water,
check every day and add water in need. Collect the roots of 2-3 cm before
the following treatment.
(2) Chromosome condensation and fixation
Treat the roots with 2 mM 8-hydroxyquinoline at 18-20℃ for 4-5 hours.
Discard the fluid and replace it with 95% alcohol: glacial acetic acid (3:1)
solution for fixation at room temperature overnight. Put the bottle with
roots and fixative into a freezer for storage before further process.
(3) Depurination
Rinse root tip samples with distilled water for three minutes twice, then
treat them with 1N HCl at 60℃ for 8 minutes. Rinse root tip samples with
distilled water for three minutes twice.
(4) Staining and cell separation
Discard water and let the sample as dry as possible, then add leuco-basic
fuchsin just enough to cover the sample for staining. Place the samples in
the dark for 30 minutes. Discard the stain solution and add 1% pectinase to
separate the cells.
2) Making the mitotic slides
Place the stained root tip on the microscopic slide, add a drop of 45% acetic
acid, break the root tip into fine pieces, and cover it with a coverslip. Flip the
microscopic slide over and put it onto three layers of filter paper, and then use
the blunt end of a chopstick to press on the area with root tip samples to
separate the chromosomes
3) Observation
Use a light microscope to observe the morphology of chromosomes and count
the number of chromosomes.

54
11. Molecular models of DNA
Make an assigned sequence of DNA using the molecular models of DNA.

Answers of the figures

Fig. 4-1. Fig. 4-2.

a. Nucleus a. Homologous chromosomes
b. Nucleolus b. Nucleus
c. Chromosome c. Spindle fiber
d. Spindle fiber d. Cell plate
e. Centromere
f. Cell plate

55
Experiment 31. Analysis of artificial grassland community

composition in NTU campus

Introduction
The objectives of the plant community investigation are to: (1) compare the
similarities and differences among different plant community and categorize them; (2)
study the relationship between the plant community and the environment by
comparing the distribution and the variation of the community with the gradient
changes of the environment.; and (3) analysis the structure of the plant community
and its dynamic change.
The parameters are important in plant community investigation, including the density,
frequency, coverage, dominance, height, and weight, etc. The type and the amount of
parameters used in the investigation depend on the objective of the investigation and
the availability of labor and material resources. Generally, unless for the specific need
such as the forest stock volume calculation, the labor-intensive complete enumeration
(每木調查) is not necessary. The common sampling methods are: (1) the quadrat
method (樣區法), (2) the transect method (樣線法), (3) the point-quadrat method
(點樣法), and (4) the point-centered quarter method (點四分法).
With the point-quadrat method, satisfied results are often obtained in grassland
community investigations. There are three situations for plant community in a
quadrat: the plant is of full coverage, non-coverage, and partial coverage in the
quadrat; however, the plant is only of fully coverage and non-coverage when a
quadrat is minimized to a point. So the ration of the number of sampling points that a
certain plant appears to the number of total sampling points is able to represent the
coverage of the plant. The coverage is one of the important bases of the importance
value.
The objective of this experiment is to study the influence of environmental factors to
the composition of grassland community.

Sampling sites
The grassland in front of the Si-Liang building (the open area and the shade area) or
the library, or along the Little-coconut Boulevard.

Materials
Plastic ropes of 4 meters, soft plastic ruler longer than 1 meter, chopsticks, and record
paper

56
Methods
1. According to the objective of your experiment, choose two or more grasslands of
different environment as the studied grasslands (stratified sampling method).
2. Select a quadrat site on the studied grassland (purposive or selective sampling
method). Set the square quadrat of 1 m * 1 m with the plastic rope and 4
chopsticks.
3. Systematically choose 100 sampling points by dividing the quadrat into 10 * 10
grids (systematic sampling method). Avoid letting the sampling points fall on the
edge of the quadrat by starting from the corner and setting the first sampling
point 5 cm from the edge.
4. Insert a chopstick to the first sampling point and record all the grass species that
are contact with the chopstick and their frequency of contact. Finish the 100
sampling points.
5. Analyze the coverage (C), frequency (F), relative coverage (RC), relative frequency
and (RF), and important value (IV) of each grass species using the following
equations.
𝐶𝑜𝑣𝑒𝑟𝑎𝑔 𝑜𝑓 𝑠𝑝𝑒𝑐𝑖𝑒𝑠 𝐴 (𝐶)
total times of A appears in all sampling sites
=
total sampling points (100 points ∗ n sites)

number of sampling sites that A appears

𝐹𝑟𝑒𝑞𝑢𝑒𝑛𝑐𝑦 𝑜𝑓 𝑠𝑝𝑒𝑐𝑖𝑒𝑠 𝐴 (𝐹) =
total sampling sites (n sites)

Coverage of A
𝑅𝑒𝑙𝑎𝑡𝑖𝑣𝑒 𝐶𝑜𝑣𝑒𝑎𝑔𝑒 𝑜𝑓 𝑠𝑝𝑒𝑐𝑖𝑒𝑠 𝐴 (𝑅𝐶) =
total Coverage of all species

Frequency of A
𝑅𝑒𝑙𝑎𝑡𝑖𝑣𝑒 𝐹𝑟𝑒𝑞𝑢𝑒𝑛𝑐𝑦 𝑜𝑓 𝑠𝑝𝑒𝑐𝑖𝑒𝑠 𝐴 (𝑅𝐹) =
total Frequency of all species

Importace Value (IV) = Relative coverage (RC) + Relative Frequency (RF)

Questions
1. What are the main grass species in the studied grassland?
2. List the 10 grass species in order with the largest parameters of the important
value, coverage, and frequency respectively.
3. How to determine the dominant species in a plant community?
4. Describe the stratified, random, and systematic sampling method.

57
58
 Recording sheet

Sampling site:____________ date:_________ Investigators:_______________

Plant / times of appearance Plant / times of appearance

1 51

2 52

3 53

4 54

5 55

6 56

7 57

8 58

9 59

10 60

11 61

12 62

13 63

14 64

15 65

16 66

17 67

18 68

19 69

20 70

21 71

22 72

23 73

24 74
59
25 75

26 76

27 77

28 78

29 79

30 80

31 81

32 82

33 83

34 84

35 85

36 86

37 87

38 88

39 89

40 90

41 91

42 92

43 93

44 94

45 95

46 96

47 97

48 98

49 99

50 100

60
Experiment 23 Bacteria

Introduction
Bacteria are tiny microorganisms that are visible with light microscope but not
with naked eyes. The unit of length used for the size of bacteria is micromter. All
bacteria were catergorized to the Kindom Monera previously. But based on the
analysis of 16S rRNA sequence, bacteria are catergorized into Eubacteria and Archaea.
Prokaryotes are unicellular organisms that lack nuclear menbrane and unit membrane
bounded organelles. Spindle fibers are not formed during the cell division. Bacteria
reproduce mainly by binary fission which is very rapid. A new generation is produced
in a few minutes to a few hours. In the solid medium the daugher generations of a
bacterium are together to form a colony. Procaryotes are thought to be the earliest
organisms on the Earth. There are numerous species of bacteria which exist in solis,
waters, airs, and the inside or the outside of other organisms. Bacteria are also found
in the extreme environments (too hot, too cold, too acidic, and too basic) where some
eukaryotes cannot survive.
The main forms of bacteria are coccus, bacillus, and spirillum. There are some
exceptions such as square bacteria, amorphous bacteria, and filamentous
actinobacteria that look like fungi. Bacteria have cell walls composed of mainly
peptidoglycan, except mycoplasma and L-form bacteria. Some bacteria have
endospores, caspules, flagella, and pilus.
Bacteria is closely related to human beings. Some are harmful to people directly
or indirectly, but some are greatly beneficial to people. Rescently, scientists use
bacteria as materials in the researches of physiology, biochemistry, and genetics.
Hopely, these researches can help us to explain some phenomena of life in nature.
The bacteriology has bacome an essential knowledge of scientific researches.
The objective of this experiment is to determine the susceptibility of a bacterium
against different antibiotic agents.

Materials
1. Suspension of Escherichia coli (大腸桿菌) and Bacillus subtilis (枯草桿菌)
2. Agar plates
3. Antibiotics: Penicillin, Polymyxin B, and Streptomycin
4. Sterile cotton swabs, and parafilm

Methods
1. The antibiotic susceptibility test is operated in the laminar flow hood.

61
2. On the back of the plate, divide the plate into three sectors with a marker, and
label the name of the bacterium.
3. Swab the bacterial suspension on the agar plate evenly.
4. Place each of the three antibiotic discs on the surface in the middle of each sector,
then seal the agar plate with the parafilm.
5. Put the plates in an incubator overnight.
6. Observe the formation of inhibition zones and measure their diameters.

Questions
1. Recently, most of the winners of the Nobel Prizes in Chemistry and in Medicine
use bacteria as their research materials. What are the advantages of using bacteria
as research materials?
2. In the experiment of bacterial distribution, what does the appearance of bacteria
colonies mean except for proving the existence of bacteria in the environment?
3. Besides coccus, bacillus, and spirillum, are there any other forms of bacteria?
Please take an example.
4. What is the principle of using an immersion objective?

62
Experiment 24 Algae (1)

Introduction
Algae are a diverse group of aquatic photosynthetic organisms that belong to
several different biological groups. Algae differ from plants in several ways: (1) The
haploid individuals of unicellular algae are gametes themselves. (2) The gametangium
of some multicellular algae is a unicellular structure. (3) The gametangium of the
other algae is a multicellular structure and each individual cell produces one gamete.
Algae belong to the Monera and Ptotista of five kingdoms. Most algae are
capable of performing photosynthesis and thus produce oxygen. The majority of algae
are small in size except the large algae such as brown algae. About 34,000 algae
species are already known, and their classification is bases on: (1) the existence of the
nuclear membrane, (2) the types of photosynthetic pigments, (3) the types of
photosynthetic products for storage, (4) the composition of the cell wall, (5) the
number, position, and morphology of the flagella, (6) the morphology of the algae,
and (7) the molecular features of nuclear acid and protein.
The classification of algae has been changed dramatically because of the progress
in algae researches and the discovery of numerous new algae species in the past
decades. A recognized classification is listed in table 24-1. Among the algae,
Cyanophyta and Prochlorophyta belong to Prokaryonta, the others belong to
Eukaronta.

Table 24-1 The classification of algae

Division Chinese English species
1. Cyanophyta 藍綠藻門 blue-green algae; cyanobacteria 2,000
2. Prochlorophyta 原綠藻門 prochlorophytes 3
3. Chlorophyta 綠藻門 green algae 17,000
4. Charophyta 輪藻門 Stoneworts 250
5. Euglenophyta 裸藻門 Euglenoids 900
6. Cryptophyta 隱藻門 Cryptomonoids 200
7. Haptophyta 定鞭藻門 Haptophytes 300
8. Dinophyta 渦鞭毛藻門 Dinoflagellates 4,000
9. Chrysophyta 金黃藻門 golden algae 1,200
10. Bacillariophyta 矽藻門 Diatoms 12,000
11. Phaeophyta 褐藻門 brown algae 1,200
12. Rhodophyta 紅藻門 red algae 6,000
13. Glaucophyta 共生藻門 Glaucophytes 13

63
 Like plants, algae play a role as a primary producer. Algae are the main food
sources of the primary consumers in aquatic ecosystems. The shape and the size of
algae vary a lot among species. The size of the smaller algae are only 5-10 m both in
length and in width such as unicellular Chlamydomonas sp. (單胞藻); however, the
length of the larger ones can be up to several dozens of meters such as kelps.
The objective of this and next experiment is to study the morphology of algae by
observing typical algae species of each division with both naked eyes and/or light
microscopes and recording their characteristics.

Materials
1. Permanent microscopic slides
1) Cyanophyta: Anabaena sp. (念珠藻)
2) Chlorophyta: Desmids (鼓藻類), Spirogyra sp. (水綿)
3) Charophyta: Chara sp. (輪藻)
2. Dried sample: Ulva sp.(石蓴)
3. Fresh water samples containing algae of Cyanophyta, Chlorophyta, Charophyta,
and Euglenophyta
4. Azolla sp. (滿江紅) containing Anabaena sp.

Observations
1. Cyanophyta
1) Chroococcus sp. (色球藻)and Gloeocapsa sp. (藍鼓藻)(Fig. 24-1)
Prepare wet mount slides of Chroococcus sp. and Gloeocapsa sp. that grow on
the soil surface. Observe them with a high power objective. What color is
Chroococcus sp. and Gloeocapsa sp.? Is there any organelle? Observe the
nearly transparent gelatinous sheath outside the cells. Each daughter cell
forms new gelatinous sheath after cell division. All the daughter cells are
surrounded by the original gelatinous sheath, this makes them look like a
colony. How are the daughter cells released?

Fig. 24-1 Gloeocapsa sp. Fig. 24-2 Anabaena sp.

64
 2) Anabaena sp. (Fig. 24-2)
On the microscopic slide press the leaf of Azolla sp. to break the leaf tissue and
release Anabaena sp. Cover with a coverslip and observe Anabaena sp. with a
high power objective. Find the multicellular filament of Anabaena sp. What is
the shape and the color of vegetaive cells of Anabaena. Is there any organelle
in the cell? Find heterocysts which are round and thick-walled, and are bigger
than vegetative cells. The function of heterocysts is related to nitrogen
fixation. Try to find another specialized cells, akinetes, which are also thick-
walled and are darker and bigger than heterocysts. Akinetes persist in harsh
conditions, and grow into new algae filament when favorable environment
returns. If you cannot find them in the fresh sample, please try the
permemant slide.

2. Chlorophyta
1) Desmid：The cell of unicellular desmids consists of two half cells, named
semi-cell, the connecting region is known as the isthmus. The nucleus lies in
the isthmus. Each hemicell contains chloroplasts, what is the shape of the
chloroplast? Is there any pyrenoid in the chloroplast? Representative of
desmids are Closterium sp. (鼓藻), Closterium sp. (新月藻) and Staurastrum
sp. (角星鼓藻) which grow in freshwater.

2) Pediastrum sp. (星盤藻) (Fig. 24-3)

Find Pediastrum sp. from the water samples with a light microscope. How
many cells does a colony consist of? Is there any rule for the number? What is
the color of the cell? Is there any organelle?

Fig. 24-3 Pediastrum sp. Fig. 24-4 Spirogyra sp.

65
 3) Spirogyra sp. (Fig. 24-4)
Find Spirogyra sp. from the water sample with a light microscope. What is the
shape of the chloroplast? The name of Spirogyra is named after the shape of
chloroplast. Several pyrenoids, opague structures, are scattered in the
chloroplast. The pyrenoid functions as the enzymatic center of starch
synthesis. Each cell contains a nucleus. Is there any vacuole in the cell?
Spirogyra sp. reproduces asexually by fragmentation and sexually by
conjungation. Observe the process of conjugation from the permanent slide of
Spirogyra sp. with a light microscope. The contents of one cell passes through
the conjugation tube and fuse with the contents of anonother cell to form a
diploid zygote. These two cells may be in the same filament or in two different
filaments. Is the morphology of the zygote the same as a vegetative cell?

4) Ulva sp.(Fig. 24-5)

Observe the dried specimen of Ulva sp. which is a thin membranous green
algae growing from a discoid holdfast. The membrane is only two layer thick
which increases the surface area and thus increase the light interception.

Fig. 24-5 Ulva sp.

3. Charophyta (Fig. 24-9 ~ 24-12)

Unlike the form of other algae, members of Charophyta have whorled branches as
well as the differentiation of nodes and internodes. With the fresh sample or/and
the permenant slide of Chara sp., observe the morphology and the location of the
reproductive structures, the antheridium and oogonium.

Fig. 24-9~12 Chara sp.; Leaves on axis; Antheridium; Segment with oogonium

66
4. Euglenophyta
Observe Euglena sp. (Fig. 24-13) from the water samples. Observe the way of their
movement. The menbers of Euglenophyta do not have cell wall. However, having
chloroplasts classified them as algae. All euglenoids have two flagella, one
flagellum is very short, while the other is relatively long, and often easily visible
with light microscopy. Have you seen the flagellum located at the anterior end?
The stigma or eyespot near the anterior end of the cell could be related to the
phototaxis of Euglena sp.

Fig. 24-13 Euglena sp.

Answers of the figures

Fig. 24-1. Fig. 24-4. Fig. 24-12.

a. Cell wall a. Gamete a. Crown cell
b. Common sheath b. Conjugation tube b. Appendages
c. Chloroplast c. Tube cell
Fig.24-2. d. Pyrenoid d. Antheridium
a. Heterocyst
b. Akinete Fig. 24-10. Fig. 24-13.
c. Vegetative cell a. Appendage a. Chloroplast
b. Node b. Nucleus
Fig. 24-3. c. Paramylon
a. Spines Fig. 24-11. d. Flagellum
b. Pyrenoid a. Antheridium e. Periplast
b. Shield cell

67
Experiment 25 Algae (2)

Introduction
This experiment continues the observation of algae including the members of
Dinophyta (dinoflagellates), Bacillariophyta (diatoms), Phaeophyta (brown algae) , and,
Rhodophyta (red algae). Most of these algae grow in marine environments. Some red
algae and brown algae are edible and used for producing agar and of economic
impotance. The diatomaceous earth, fossilized remains of diatoms, is used much in
industrial products such as the reflective paints for road sign boards.

Materials
1. Permanent microscopic slides
4) Dinophyta (渦鞭毛藻門): Ceratium sp. (角甲藻)；Peridinium sp. (甲藻)
5) Bacillariophyta (矽藻門): mixed diatoms
6) Phaeophyta (褐藻門): Laminaria sp. (昆布)
2. Dried samples
1) Phaeophyta (褐藻門): Fucus sp. (石衣藻), Sargassum sp. (馬尾藻), Laminaria
sp. (昆布)
2) Rhodophyta (紅藻門): Gelidium amansii (石花菜) and Porphyra sp. (紫菜)
3. Fresh water samples containing algae

Observations
1. Dinophyta
Observe the Peridinium sp. (甲藻) and Ceratium sp. (角甲藻) from the permenant
slides (Fig. 25-10, 25-11). What are the characteristics of their cell wall? Observe
them with different focuses and draw their three-dimensional structures.

Fig 25-10 Peridinium sp. Fig. 25-11 Ceratium sp.

68
 Dinoflagellates are important phytoplankton, and most of them live in marine
water. The red tide happens when the marine dinoflagellates bloom and thus turn
the water red and with an awful odor. The dead dinoflagellates are decomposed
by bacteria and thus produce toxic substances and deplete oxygen in the water.
Besides, some dinoflagellates secrete toxin and thus cause the deaths of
numberous marine animals. The overgrowth of freshwater dinoflagellates causes
the algae bloom such as the algae bloom happened in Techi Reservoir.

2. Bacillariophyta
Observe the morphology of diatoms from the water samples (Fig. 25-12). Find the
yellowish and boat-shaped diatoms. They may slide slowly in the water, do they
have flagella? Each diatom is comosed of a upper epitheca and a lower hypotheca
that fit together like a small box or a petri dish. The cell walls of diatoms are rich in
silica and with various beautiful patterns which is one of the the main
characteristics for classification. Observe the girdle band. What is the function of
the girdle band? Diatoms grow in marine and fresh water. After they dies, the silica
frusules sink to the bottom and form the diatomaceous earth. Observe the cell
wall patterns from the permanent slide of the mixed diatoms. Distinguish the
pennate diatoms from the centric diatoms.

Fig. 25-12 Different kinds and views of diatoms

69
3. Phaeophyta
1) Fucus sp. (石衣藻): Observe the dried specimen. Fucus sp. is a marine
species that attatchs to the rock. Find the holdfast. The thallus of Fucus sp. is
dichotomous branched, flattened and with a distinct midrib. The round air
bladders along both sides of the midrinb help the thallus to float in the water.
(Fig. 25-3)

Fig. 25-3 Fucus sp. Fig. 25-4 Sargassum sp.

2) Sargassum sp. (馬尾藻) (Fig. 25-4): Observe the dried or fluid-preserved

specimen. The form of Sargassum sp. is highly specialized. Try to find the
holdfast, stipe, and lamina (blade) which resemble the root, stem, and leaf of
the plant, repectively. Also find the air bladders. What are the differences
between Sargassum sp. and Fucus sp. in morphology?

3) Laminaria sp. (昆布): Observe the dried seaweed specimen. The edible kelps
are algae of the genus Laminaria. They are large algae which length ranges
from a few meters to several tens of meters. The body of kelps differentiates
into the holdfast, stipe, and lamina (Fig. 25-7). Kelps grow by intercalary
growth and the meristematic region is loacterd between the lamina and the
stipe. Can these brown algae perform photosynthesis? The kelp body is the
sporophyte which produce zoospores. Examine the permenant slids of
Laminaria sp. and find the zoosporangia, where are they? What is their
function? Do the other cells of the lamina differentiate (Fig. 25-8, 25-9)?

70
 Fig. 25-7 Laminaria sp. Fig. 25-8 C.S. of fiertile area of Laminaria sp.

Fig. 25-9 C.S. of blade of Laminaria sp.

4. Rhodophyta
Gelidium amansii (石花菜)
Observe the branching body of Gelidium amansii from the dried specimen.
The agar extracted from Gelidium is used as an ingredient of jellies and other
desserts.
Porphyra sp. (紫菜)
The membranous body of Porphyra sp. is similar to that of Ulva sp. Take a
small piece of nori to make a wet mount slide and observe the structure
under the light microscope.

71
Answers of the figures

Fig. 25-3. Fig. 25-8. c. Flagella

a. Air bladder a. Zoospore
b. Receptacle b. Mucilage layer Fig. 25-11.
c. Midrib c. Zoosporangium a. Groove
d. Stipe d. Paraphysis
e. Holdfast Fig. 25-12.
Fig. 25-9. a. Epitheca
Fig. 25-4. a. Photosynthetic tissue b. Hypotheca
a. Air bladder b. Cortex c. Oil drop
c. Medulla d. Irregular plastids
Fig. 25-7. e. Nucleus
a. Blade Fig. 25-10. f. Girdle band
b. Stipe a. Cell wall
c. Holdfast b. Groove

72
Experiment 26 Fungi

Introduction
Fungi (s. fungus) are eukaryotic organisms without chlorophylls and store
nutrient as glycogen. The fungal structure is simple without the differentiation of
roots, stems and leaves. The vegetative part of a fungus is composed of a single cell or
a mass of thread-like structure, hyphae (s. hypha). Many hyphae get together to form
the mycelium (pl. mycelia).
Fungal species distribute widely. They may vary in habits and nutrient needs,
however they all need to absorb organic substances form other source to maintain
their growth and reproduction, i.e., heterotrophy. These nutrient source could be
dead or living organisms. Fungi are classified as saprobe or parasite depending on the
way of living. Are fungi plants? One needs to understand the debates and rules out
the evidences to answer this question.
Either saprobe or parasite, harmful or beneficial fungi, they all influence human
survival or civilization directly or indirectly. Athlete's foot, antibiotics, vitamins, beer,
bread, cheese, and soy sauce are all related to fungi. Most fungi are saprobe and play
an important role as decomposer in the ecosystem just like bacteria.
Fungi are everywhere, they live in the land, the fresh water, and the marine.
Most of the known fungal species produce spores which germinate to form new
individuals under favorite environment. The number of spores is tremendous, for
example, each mature mushroom sold in the market can release up to 100 million
spores per hour and lasts for 5 to 6 days. Once the spores germinate and the hyphae
penetrate into the cells and tissues of animals or plants, they might cause damage and
rot.
In this experiment, we will observe the spores and the spore formation of the
representative species of different taxa of fungi, and to distinguish the similarities and
differences among these fungi. There are two types of spores: asexual spores and
sexual spores. Asexual spores are formed by mitotic cell division of a cell or hypha.
They are haploid and the number of chromosomes is the same as that of the mother
cells or hyphae. The sexual spores are formed by the meiotic cell division of a diploid
nucleus into four haploid nuclei.

Materials: dried samples, fresh cultures, and/or permanent microscopic slides

1. Kingdom Protista (原生生物界)
1) 卵菌門 (Oomycota)：Achlya sp. (綿黴菌)、Saprolegnia sp. (水黴菌)

73
2. Kingdom Fungi (真菌界)
1) Zygomycota (接合菌門)：Mucor sp. (毛黴菌)、Rhizopus sp. (黑黴菌)
2) Ascomycota (子囊菌門)：Peziza sp. (盤菌)、Saccharomyces sp. (酵母菌)、
Sordaria fimicola (囊生殼菌)
3) Basidiomycota (擔子菌門)： Agaricus bisporus (洋菇)、Coprinus sp. (墨水
菇)、Lentinus edodes (香菇)、Puccinia graminis (禾柄銹菌)
4) Deuteromycota；Imperfect fungi (不完全菌門)：Aspergillus sp. (麴菌)、
Penicillium sp. (青黴菌)

Observations
1. 卵菌門 (Oomycota)
The members of Oomycota are aquatic. Observe fresh culture of Achlya sp.
(綿黴菌) and/or Saprolegnia sp. (水黴菌) under a lower power of a microscope.
The zoosporangia grow and swell at the tip of a hypha and releases asexual
zoospores when they mature. Find the zoosporangia and observe their shape.
Each zoospores has two flagella and swim fast. Find, observe, and describe the
zoosporangium that is releasing zoospores. What are the characteristics of the
hyphae. Achlya sp. (綿黴菌) and Saprolegnia sp. (水黴菌) live in the water and
their hyphae grow radially on the organic substance of dead insects or other
organisms. The released zoospores will find an organic material and germinate to
form a mycelium.
During sexual reproduction, the hypha forms branches and an oogonium (pl.
oogonia) or an antheridium (s. antheridia) is developed at the top of each branch.
Find the swollen spherical structure, oogonium, at the lower power. How many
eggs in an oogonium? When the antheridium contact with the oogonium, the
sperm nuclei enter the oogonium through the fertilization tube and fuse with the
egg to form oospores. What is the role of the oospores in reproduction of
oomycetes? And how does an oospore germinate to form a mycelium?

Fig. 26-1. Asexual spores of Saprolegnia sp.

74
 Fig. 26-2.Sexual spores formation of Saprolegnia sp.

Fig. 26-3. Asexual spores of Achlya sp.

2. 接合菌門 (Zygomycota)
Observe the black (bread) mold grown in the bread or agar plate at the lower
power. Do not open the lid of the agar plate. Spherical sporangia with numerous
spores are formed during asexual reproduction. When the sporangium matures
and collapses, sporangiospores are released and the columella is exposed.
Observe the sporangia, the color of sporangiospores and its developing site,
columella, and the sporangiophores.
Observe the black mold on the plate with naked eyes, in the center of the
plate there is a darker linear zone, where the sexual reproductive occurs. With a
stereo microscope find the dark, spherical zygosporangium with a zygospore. Most
black molds are heterothallic. During sexual reproduction, hyphae of two mating
types form lateral branches when they are close to each other. The tip of each
lateral branch is septate and forms a gametangium. The zygosporngium is formed
when the two gametangia fuse together.
Observe zygosporangia of different developing stages in the slide of sexual
reproduction at the lower and the higher power. A mature zygosporangium
contains a zygospore with many diploid nuclei. The zygosporangium germinates to
another sporangium with many haploid spores. When does the meiosis happen?

Fig. 26-4. Rhizopus stolonifer

A. Proliferation by stolon
B. Group of sporangiophores
C. Mature sporangium
D. Sporangiospores
E. Early stage in
zygosporangium formation
F. Mature zygospore

75
 Fig. 26-5. Stages of zygosporogenesis in Rhizopus sp.

3. 子囊菌門 (Ascomycota)
Fungi of Ascomycota have septate hyphae. The sexual spores, ascospores, are
developed in the ascus (pl. asci). The fungi reproduce rapidly by asexual spores,
conidium; conidiospore.

i. Budding of yeast
Prepare a fresh mount of yeast culture, observe at a higher power and record
their form. What are the buds on some cells for? Yeasts are unicellular
organisms, Budding is a simple, fast asexual reproduction.

Fig. 26-6. Saccharomyces sp.

ii. Ascospores of Peziza sp.

The ascocarp of Peziza sp. is called the apothecium. Hymenium which is at the
top layer of the apothecium is where the asci are produced. Observe the asci
and ascospores from the slide of ascocarp section. How many ascospores are
there in an ascus? Ascospores are sexual spores which are produced through
meiosis. How is the number of the ascospores determined?

A. B
Fig. 26-9. Peziza sp. A. Apothecium B. Asci

76
 iii. Ascospores of Sordaria (殼菌)
Examine the Sordaria fimicola grown on the agar plate. The black dots on the
plate are their ascocarps which are called perithecia (s. perithecium). There is
an opening at one end of the perithecium. Examine the section of an ascocarp
and observe the shape of an ascus, the site of asci attach. There are 8
ascospores in each ascus.

Fig. 26-11. Sordaria fimicola

4. 擔子菌門 (Basidiomycota)
Hyphae of Basidiomycota are septate. Sexual spores, basidiospores, are
meiotic products of the basidium. Basidia are borne in the basidiocarp for most
Basidiomycota. Mushrooms are fruiting bodies, i.e., basidiocarps. Some fungi of
Basidiomycota produce asexual spores in their life cycle, e.g., Puccinia graminis (禾
柄銹菌), but some do not, e.g., Agaricus bisporus (洋菇).
a. Mushrooms (菇蕈)
Some mushroom are of dietary value, such as Agaricus bisporus (洋菇)
and Lentinus edodes (香菇), but some are poisonous. So never eat a
mushroom without confirm its species.
Observe the morphology of mushrooms and distinguish their parts
including the pileus (蕈蓋), stipe (蕈柄) , annulus (蕈環) , gill (蕈褶) , and
velum (蕈膜). Some mushrooms have a volva (蕈托) at the base of the stipe.
The mushrooms we eat are basidiocarps, and most of their mycelia are still
growing in the soil (compost). Why the hyphal growth are necessary for the
production of compost (堆肥).
The shape of gills and the color of spores are important for mushroom
identification so the spore print technique are developed. Spore prints are
made by cutting stipe off, place the pileus on a sheet of paper with the gill side
down, and wait for mature spores to fall on the paper to form spore prints.
To find out where the basidiospores are borne, observe the margin of the

77
 gills form the prepared slide of Coprinus sp. (墨水菇) or a fresh mount
sections at a higher power. Four basidiospores are borne on each basidium.
Each basidiospore is supported by a sterigma.

Fig. 26-12. Mushroom of Amanita sp.

Fig. 26-13. Mushroom of Agaricus sp.

b. Puccinia graminis (禾柄銹菌)

Observe the section of uredinium and telium of Puccinia graminis
parasiting on leaves of Triticum sp. Find hyphae in the plant tissue and observe
where the spores are borne. What is the shape of spores? How many spores
are there at the tip of each hypha? In the life cycle of Puccinia graminis, two
hosts are involved, i.e., Triticum sp and Berberis sp; five types of spores are
produced. Rust-colored urediniospores are produced in the Triticum leaves
during the spring and summer, and dark-colored teliospores are also produced
in the Triticum leaves but during the end of summer and autumn.
Basidiospores are produced during the sexual reproduction and are dispersed
to Berberis leaves where spermatia and aeciospores are formed.

78
 Fig. 26-10. Life cycle of Puccinia graminis

5. 不完全菌門 (Deuteromycota；Imperfect fungi)

Sexual spores have not been found for fungi of Deuteromycota. They
reproduce by asexual conidiospores mainly, and are thus called imperfect fungi.
Conidiospores are borne on the conidiophores. Some fungi of Deuteromycota are
highly economically valued, i.g., antibiotic producing fungi; some are pathogens
that cause diseases of plant and animal including human beings.
Observe molded orange or other fruits inside the container. The green-
colored mold on the orange peel is Penicillium sp. (青黴菌), the spores are green.
What is the color of the mycelium? Observe the conidiospores and erect
conidiophores of Penicillium sp. (青黴菌) and Aspergillus sp. (麴菌) from the

79
 permanent slide. What colors are their conidiospores? Are the conidiospores
produced earlier located at the bottom or the top? How do you know? Where can
you find similar molds? Why do they grow there? How to prove your hypothesis?

Fig.26-7. Penicillium sp. Fig. 26-8. Aspergillus sp.

6. 菌根 (mycorrhiza)
Mycorrhiza is the symbion of a soil-born fungus and the root of a vascular
plant. The fungus is responsible for absorbing water and mineral nutrients for the
root. Observe the mycorrhiza in the petri-dish by a stereo microscope. The hyphae
are forked, short, and well-branched. They form sheath and cover the root.
Depend on the visibility of root hairs, they are classified as ectotrophic mycorrhiza
(with visible root hairs) (外生菌根) and endotrophic mycorrhiza (without visible
root hairs)(內生菌根). The hyphae of ectotrophic mycorrhiza grow in the cortex
extracellularly and form a sheathe around the whole root. However the hyphae of
endotrophic mycorrhiza do not form sheath. They grow in the cortex intercellularly
and form vesicles and arbuscules inside cells. Find them from the permanent
slides.

Fig. 26-14. Mycorrhizae of Pinus sp.

Questions
5. Please list the advantages and disadvantages of fungi?
6. What are the effective methods for preventing or inhibiting the growth of harmful
fungi?

80
Answers of the figures

Fig. 26-1 e. Teliospores

a. Zoospore Fig. 26-6. f. Basidiospore
b. Zoosporangium a. Bud
Fig. 26-11.
Fig. 26-2. Fig. 26-7. a. Ascus
a. Antheridium a. Conidiospore b. Ascospore
b. Oospore b. Sterigma c. Perithecium
c. Oogonium c. Conidiophore
Fig. 26-12.
Fig. 26-3 Fig.26-8. a. Pileus
a. Zoospore a. Conidiospore b. Gills
b. Zoosporangium b. Sterigma c. Annulus
c. Conidiophore d. Stipe
Fig. 26-4. e. Volva
a. Rhizoid Fig. 26-9.
b. Stolon a. Hymenium Fig. 26-13.
c. Sporangiophore b. Ascospore a. Pileus
d. Sporangia c. Paraphysis b. Gills
e. Spore d. Ascus c. Stipe
f. Gametangia d. Gills
g. Zygosporangium Fig. 26-10.
a. Spermatium Fig. 26-14.
Fig. 26-5. b. Receptive hypha a. Mycorrhiza
a. Zygosporangium c. Aeciospores
b. Suspensor d. Urediniospore

81
Experiment 27. Lichens

Introduction
A lichen is a symbiosis of an alga (mostly green algae and cyanobacteria) and a
fungus (mostly Ascomycota and rarely Basidiomycota). The symbiotic relationship
keeps in harmony: the alga contains chlorophylls and produce organic nutrients for
the fungus; and the fungus may play the role of protecting the alga. However, the
researches about the growth of lichens found that the relationship between the alga
and the fungus could be a coordinated controlled parasitism, i.e., the fungus is a
parasite on the alga.
Lichens are wildly distributed and are diverse in their form which is determined
by the fungus. Lichens can grow but slowly in extreme environments that are harsh to
other organisms, such as bold rocks. The lichens are classified into three categories
according their morphology: crustose lichen (殼狀地衣), fruticose lichen (莖狀地衣),
and foliose lichen (葉狀地衣). Lichens can be indicator species of pollution and are of
potential values in medical and chemical industries.

Materials
1. Dried samples of crustose lichen (殼狀地衣), fruticose lichen (莖狀地衣), and
foliose lichen (葉狀地衣)
2. Permanent slide of thallus cross section of foliose lichen (葉狀地衣)
3. Permanent slide of ascocarp cross section of foliose lichen (葉狀地衣)

Methods
1. Observe the morphology, the color, and the structure of crustose lichen (殼狀地
衣), fruticose lichen (莖狀地衣), and foliose lichen (葉狀地衣) (Fig. 27-2)

A________________ B________________ C________________

Fig.27-2. Growth forms of lichens

2. Soften the thallus lichen with water, place a piece of thallus between two
microscopic slides and crash the sample. Take a small piece of sample, make a
82
 fresh mount sample, and observe with a microscope. At the higher power, what is
the shape of the alga? Green or blue-colored? How is the alga distributed? Is the
hypha septate?

3. Observe the cross section of lichen thallus with a microscope, identify the
following structures:
a. Cortical layers are the uppermost and the lowermost layers, they are
composed of compact hyphae with gelatinous materials. The cell structure in
the cortical layer is not clear. Some lower cortical layers have rhizoid, but some
have not.
b. Algal layer: Algal cells are almost completely surrounded by hyphae and are
confined between the upper cortex and the medulla. Depends on species, the
algal layer could be apparent or unapparent. The hyphae usually have
haustoria to absorb nutrient from algal cells.
c. Medulla: Medulla occupies most area of the thallus. The volume of hyphae are
huge and they are slightly gelatinized. The medulla is a loosely arranged layer
of interlaced hyphae and is the main food and water storage area.

4. Observe the cross section of the ascocarp from the permanent slide of lichen with
a microscope (Fig. 27-1). Find the apothecium and spores. What type are the
spores? Are these spores responsible for reproducing lichen individuals?

Fig.27-1. Section of an apothecium of lichen

Questions
1. Give a reasonable and complete definition for the lichen.

Answers of the figures

Fig. 27-1. (p.107) d. Lower cortex Fig. 27-2. (p.107)
a. Upper cortex e. Ascospore a. Fruticose lichen
b. Algae f. Ascus b. Foliose lichen
c. Medulla c. Crustose lichen

83
Experiment 32. Hydrophytes

Introduction
Water is necessary in the life history of early terrestrial plants such as
bryophytes. As the evolution of vascular tissues (water transduction) and stomata,
plants possess various water-preserving structures to prevent excessive water loss and
reproduce continuously in different environments. However, some terrestrial
angiosperm species and ferns returned to aquatic environment. For example, water
hyacinth (布袋蓮, Eichhornia crassipes) lives in the water, however, their ancestors
(other monocotyledons) are terrestrial. These plants re-adapt the aquatic life
physiologically and morphologically. They do not need to prevent water loss and are
not short of water. However, gas exchange and excessive water become the problems
of their life. Moreover, new adaptation in reproduction strategy is needed for
flowering plants. For example, to complete the whole generation in water via
pollination and seed dispersal by water.
Hydrophytes in Taiwan are classified as emergent (挺水), floating-leaved (浮葉),
submerged (沉水), and free floating hydrophytes (浮水). They are distributed in the
seaside, rivers, swamps, and lakes in the whole island. Many habitats of hydrophytes
are developed and disappear due to the disturbances and destructions by human
beings, especially in Taoyuan (桃園) and Yilan (宜蘭) where are famous of ponds.
Furthermore, with the competition of dominant foreign hydrophytes such as water
hyacinth and water cabbage (大萍, Pistia stratiotes), endemic hydrophytes are
endangered. To learn more about the hydrophytes in Taiwan, we will 1) observe leaf
morphology and stomatal structure of hydrophytes, and 2) investigate the ecological
habit and evolutional adaptation of hydrophytes in this experiment. We will focus on
floating-leaved, free floating, and ubmerged hydrophytes.
Convergent evolution is a common biological phenomenon, it is the process in
which organisms that are not closely related independently evolve similar features
(phenotypes). The fin of a whale and the fin of a fish is an example of convergent
evolution, neither both whale and fish have a common ancestor, nor does their
common ancestor have a fin. Hydrophyte are species with similar functions but
without evolutional relationship. The ecological trait “hydrophytic” has evolved many
times in different lineage so the “hydrophytes” is a type of convergent evolution.

Materials
水韭 (Isoetes sp.，水韭科)
田字草 (Marsilea minuta，蘋科)

84
 槐葉蘋 (Salvinia sp.，槐葉蘋科)
滿江紅 (Azolla sp.，滿江紅科)
睡蓮 (Nymphaea sp.，睡蓮科)
臺灣萍蓬草 (Nuphar shimadai，睡蓮科)
小莕菜 (Nymphoides coreana，龍膽科)
菱 (Trapa bispinosa，菱科)
狸藻 (Utricularia spp.，狸藻科)
金魚藻 (Ceratophyllum demersum，金魚藻科)
布袋蓮 (Eichhornia crassipes，雨久花科)
浮萍/水萍 (Spirodela polyrhiza，浮萍科)
大萍 (Pistia stratiotes，天南星科)

Methods
5. Observe the cell arrangement in leaf using free hand made cross sections.
6. Observe the distribution of stomata on leaf surface.
7. Observe flowers and fruits of provided materials as fresh materials, specimens,
and pictures.

85
Questions
5. List the name of the plants and their environments: emergent (挺水), floating-
leaved (浮葉), submerged (沉水), or free floating hydrophytes (浮水).
6. What plants have both submersed and floating (and/or) emergent leaves? What
are the morphological differences between these leaves?
7. What traits of convergent evolution do these hydrophytes have?
8. Speculate the reproductive strategies of these hydrophytes by observing
structures of their flowers and fruits. Explain the ways they adapt to the aquatic
environment.

References
1. 李松柏，1999。台中縣的濕地與水生植物。台中縣自然生態保育協會。
2. 林春吉，2002。臺灣水生植物 (1)、(2)。田野影像出版社。
3. 陳玉峰，1990。墾丁國家公國海岸植被。墾丁國家公園管理處。
4. 黃淑芳、楊國禎，1991。夢幻湖傳奇。陽明山國家公園管理處。
5. 黃朝慶、李松柏，1999。臺灣珍稀水生植物。清水鎮牛罵文化協會。
6. Judd, W. S. et al. 2002. Plant systematics – a phylogenetic approach, 2nd ed.
Sinauer Associates, Inc., MA, USA.

86
Experiment 8. Flowers

Introduction
Flowers are reproductive organs of angiosperms. Flowers of different species have
the same basic structure though they could be morphologically different. A flower is a
modified branch from the view of developmental morphology. Various appendages
evolved from leaves attached to this shortened branch which apical meristem
disappear and stop growing. This is different from the strobilus of conifers which
apical meristem is degenerated but re-regrows sometimes.
For a solitary flower, the flower attaches to the peduncle (花柄) and bracts (苞片)
grow on the base of the peduncle. In a compound inflorescence, the flower attaches
to the pedicel (花梗) which further attaches to the peduncle. Bracts grow at the base
of the peduncle and the pedicel. The bract at the pedicel may be a smaller leaf. In a
more complex inflorescence, the axis is called the floral axis. Floral parts grow at the
tip of the pedicel and the swollen tip is called receptacle (花托).

4. Structure of flower
A flower consists of four floral parts: sepal, petal, stamen, and pistil from outside
to inside.
1) Sepal (萼片): Collectively the sepals are called calyx (花萼), the outermost
whorl of a flower. Sepals are green in general and shaped like small leaves.
They protect the structures inside the flower bud before opening.
2) Petal (花瓣): Collectively petals are called corolla (花冠) which is next to the
calyx. Usually petals are colorful in flowers of insect pollination, they protect
stamen and pistils in the flower bud. Calyx and corolla are called perianth (花
被) collectively.
3) Stamen (雄蕊): Number of stamens varies among species. Collectively they are
called androecium (雄花器) which is interior to corolla. A stamen is composed
of a filament (花絲) and anthers (花藥). The length of filaments varies among
species. The anther is at the tip of the filament and is a swollen sac structure
that produces pollens.
4) Pistil (雌蕊): Collectively the pistils are called gynoecium (雌花器). The
gynoecium is located in the center of a flower and is composed of carpels (心
皮). A flower may have (1) one carpel, such as Fabaceae (豆科); (2) many
carpels that are separated (apocarpy; 離生心皮), such as Magnoliaceae (木蘭
科); and (3) many carpels that join to each other (syncarpy; 合生心皮), such

87
 as Liliaceae (百合科).
5) Bract (苞片): Except terminal solitary flowers, lateral flowers are developed
from the leaf axil. Leaves at the base of a solitary flower is usually normal,
however, leaves at the base of a flower in an inflorescence usually shrink and
surround the whole flower bud and are called bracts. A flower or a few flowers
may be surrounded by many bracts, i.e., involucre (總苞). For example, the
heads of Asteraceae (菊科)and female flowers of Fagaceae (殼斗科) are
surrounded by involucres.

5. Morphology of flowers
5) Regular flower (actinomorphic flower) (整齊花): Both whorls of sepals and
petals are actinomorphically arranged. Each sepal and each petal of a whorl is
similar in shape and size, e.g., Hibiscus rosasinensis (朱槿).
6) Irregular flower (zygomorphic flower) (不整齊花): The calyx and especially the
corolla is not radially arranged. Each sepal and each petal of a whorl is
dissimilar in shape and size, e.g., pea (Pisum sativum; 豌豆). The corolla of
pea is butterfly-like with the biggest standard (旗瓣), two wings (翼瓣) at both
sides, and a keel (龍骨瓣) fused by two petals surrounding stamens and pistils.
Flowers of pea have diadelphous stamens (二體雄蕊). In a flower there are
ten stamens which are separated into two groups: the filaments of nine
stamens are united and the other one is independent. The pistil has a carpel
with a locule, and a stigma at the tip of the style.

6. Structure of stamen and pistil

1) Stamen (雄蕊): The stamen is composed of a filament and an anther, the
differentiation of these two parts are not obvious in some plants such as
Magnoliaceae (木蘭科). The anther has two anther lobes (花藥瓣); each lobe
has two pollen sacs (花粉囊) with the connective (藥隔) in between. The wall
of the pollen sac consists of the epidermis, endothecium (內殼), middle layer,
and tapetum (營養層). The pollen sac produces microspores (小孢子) that
develop into pollen (花粉).

Fig. 8-1. C. S. of anther of Lilium sp.

88
 Fig. 8-2. Microspore, pollen and germination of a pollen grain of Lilium sp.

2) Pistil (雌蕊): Gynoecium is composed of pistils. Each pistil consists of a swollen

basal ovary (子房) with ovules (胚珠) , an apical style (花柱) (or styles) which
may be absent, and one or more stigmas (柱頭), the tissue receptive to pollen
grains. Ovules of the carpel attach to the surface of the ovary wall (placenta;
胎座) with funiculus (珠柄). Each ovule has two layers of integument (珠被)
surrounding the nucellus (珠心) which has a megasporocyte (大孢子母細胞).
The megasporocyte forms megaspores through meiosis. The megaspores
develop to form the megagametophyte (大配子體; 雌配子體). The mature
megagametophyte with 7 cells and 8 nuclei is called the embryo sac (胚囊).

F ig. 8-3. A. C. S. of ovary of Lilium sp. B. L. S. of ovule of Lilium sp.

7. The position of floral parts on the receptacle

7) Superior ovary (子房上位); hypogynous flower (下位花): A superior ovary is
one with sepals, petals, and stamens, and/or hypanthium(冠萼筒) attached at
the base of the ovary. The term hypogynous is used for sepals, petals, and
stamens attached at base of a superior ovary.
8) Superior ovary (子房上位); Perigynous flower (週位花): Perigynous denotes a
hypanthium attached at the base of a superior ovary.
9) Inferior ovary (子房下位); Epigynous flower (上位花): An inferior ovary
position has sepals, petals, stamens, and/or hypanthium attached at the ovary
apex. Epigynous refers to the sepals, petals, and stamens attached at apex of
an inferior ovary.
89
8. Inflorescence (花序)
An inflorescence is a group or cluster of flowers. No typical leaves grow on an
inflorescence. The way of branching and the blooming order are different among
inflorescences.
A. Indeterminate inflorescence (無限花序): The tip of the indeterminate
inflorescence has no flower on it and can keep growing. Or the tip has flowers
that open the latest. The indeterminate inflorescence keeps growing until it is
short of nutrients.
1) Raceme (總狀花序): Flowers with pedicels are alternately arranged on the
central axis which is able to elongate, such as the inflorescence of Cardamine
sp. (碎米薺), Cassia fistula (阿勃勒), Cymbidium sp. (報歲蘭), Elaeocarpus
serratus (錫蘭橄欖), and Barbarea orthocera (山芥菜)
2) Compound raceme (複總狀花序) ; Panicle (圓錐花序): A compound raceme
(panicle) is like a branched raceme, defined as an indeterminate inflorescence
having several branched axes bearing pedicellate flowers. Examples are
Eriobotrya sp. (山枇杷) and Raphanus sativus (蘿蔔).
3) Spike (穗狀花序): a racemose inflorescence, unbranched, the flowers sessile,
such as Plantago sp. (車前草).
4) Compound spike (複穗狀花序): A compound spike has spikelets(小穗)
arranged on the main axis of spike alternatively, such as Arenga sp. (山棕),
Triticum sp. (小麥) and Oryza sativa sp. (稻).
5) Spadix (佛焰花序): a spike inflorescence with congested flowers and a stout,
often succulent axis, often more or less surrounded by a spathe (佛焰苞).
Examples are the Arum family (天南星科植物), such as Alocasia sp. (海芋)
and Anthurium sp. (火鶴), and the Palm family (棕櫚科植物), such as
Roystonea sp. (大王椰子), Chrysalidocarpus sp. (黃椰子)and Livistona sp. (蒲
葵).
6) Umbel (繖形花序): a racemose inflorescence, all the individual flower stalks
arising in a cluster at the top of the peduncle and of about equal length, such
as Allium sp. (孤挺花) and Crinum sp. (文珠蘭).
7) Compound umbel (複繖形花序): a compound umbel is an umbel of umbels,
such as Daucus carota (胡蘿蔔)、Apium sp. (芹菜 and 茴香 included).
8) Head (頭狀花序): an inflorescence with sessile flowers aggregated into a
dense cluster, sometimes made up of disc and ray flowers (管狀花 and 舌狀
花), as Compositae (菊科).
9) Hypanthodium (隱頭花序): An inflorescence with enlarged receptacle
90
 enclosing flowers inside, with a small opening. Examples are Ficus sp. (無花果
and 薜荔 included)
10) Corymb (繖房花序): a racemose inflorescence in which the pedicels of the
lower flowers are longer than those of the flowers above, bringing all flowers
to about the same level. Examples are Prunus sp. (山櫻花) and Pyrus sp. (梨).
11) Catkin (柔荑花序): A unisexual, typically male spike or elongate axis that falls
as a unit after flowering or fruiting, e.g. Salix sp. (柳樹) and Populus sp. (楊樹).

91
 B. Determinate inflorescence (有限花序): An inflorescence in which the terminal
flower matures first, maturating from apex to base.
1) Cyme (聚繖花序): An inflorescence consists of a single terminal flower and
two, opposite lateral flowers, e.g., Caryophyllaceae (石竹科).
2) Compound cyme (複聚繖花序): The axis terminates in a flower, lateral floral
branches bearing cymules develop below the terminal flower, e.g., Murraya
paniculata (七里香), Oxalis martiana (紫花酢漿草) and Dianthus sp. (康乃馨)

C. Mixed inflorescence (混合花序): An inflorescence has both indeterminate

inflorescence and determinate inflorescence, e.g., the axis of a thyrse (聚繖圓
錐花序) is indeterminate and the branches are determinate, e.g., Koelreuteria
sp. (欒樹) or Melia sp. (楝樹).

Materials and observations

1. Structure of flower
Dissect and examine the floral parts (calyx, corolla, androecium, and gynoecium)
of the flower of Lilium sp. (百合花) or Rhododendron sp. (杜鵑花). Drawing the
longitudinal section and label each part. Does the flower have all four parts?

2. Morphology of flowers: Irregular flower

Examine and draw the morphology of the calyx, corolla (standard , wing, and keel),
androecium, and gynoecium of flowers of Pisum sativum (豌豆) 或 Phaseolus
vulgaris (菜豆). Label the parts on your drawing.

3. Structure of stamen and pistil

1) Stamen: Observe the cellular structure of anther in the cross section of anther
of Lilium sp. using a microscope. Draw the anther structure briefly and label
the following structures: epidermis, endothecium, middle layer, tapetum,
92
 pollen, connective, and vascular bundle.

2) Pistil: Observe the cross section of the ovary of Lilium sp. Draw the structure
briefly and label the following structures: ovary wall, septum, placenta, ovule,
integument, micropyle, nucellus, embryo sac, and funiculus.

4. Inflorescence
A. Indeterminate inflorescence (無限花序): Draw the branching of an
indeterminate inflorescence of any kind and indicate the flowering order.
B. Determinate inflorescence (有限花序): Draw the branching of a determinate
inflorescence of any kind and indicate the flowering order.
C. Mixed inflorescence (混合花序): Examine the inflorescence of Koelreuteria sp.
(欒樹) or Melia sp. (楝樹). Examine and draw the branching, indicate the part
of indeterminate and determinate inflorescence.

Questions
1. What are the features that show the floral parts are modified from leaves?
2. What are the differences between anemophilous flowers (風媒花) and
entomophilous flowers (蟲媒花)?
3. What advantages in evolution and adaptation for the flowers angiosperm?

Answers of the figures

Fig. 8-1. d. Sperms e. Ovule

a. Connective e. Tube nucleus f. Megagametophyte
b. Endothecium g. Outer integument
Fig. 8-3. h. Nucellus
Fig. 8-2. a. Integument i. Inner integument
a. Pollen wall b. Ovary locule j. Funiculus
b. Tube cell c. Axile k. Micropyle
c. Generative cell d. Ovary wall

93
Experiment 9. Fruits

Introduction
Learning the basic structure of fruits and the diversity is the objective of this
experiment. After fertilization, the ovary develops into the fruit, that is, the basic
structure of the fruit is a mature swelling ovary. Usually the ovary contains seeds.
However, some drupes are developed by parthenocarpy (單性結果). Thus seeds are
not the essential part of fruits. Besides, the tissue outside the ovary such as receptacle
and other floral parts may develop with the ovary to form accessory fruit (假果).
The basic structure of fruit contains three layers. The outer most layer is the
exocarp (外果皮) which includes the outer epidermis and the cortex connecting to
the epidermis. The inner most layer is the endocarp(內果皮) which includes the inner
epidermis and the tissue attaching to the inner epidermis. The tissue between the
exocarp and the endocarp is the mesocarp(中果皮) which contains the cortex and
vascular tissue. These three tissues collectively are called pericarp, that is, the whole
tissue of the fruit. The thickness of the three layer differs among different fruits, and
the three layers may not be separated completely. Based on the number of ovary and
the structure of pericarp, fruits can be classified as:

1. Simple fruit (單果)

A simple fruit is developed from a single ovary and can be classified as dry fruits
and fleshy fruits.
(1) Dry fruits (乾果): The pericarp is dry at maturity.
1) Dehiscent fruits (裂果): The pericarp dehisces at maturity.
1) Legume (莢果): Bauhinia sp. (洋紫荊), Glycine max (大豆)
2) Loment (節莢果): Desmodium triflorum (蠅翼草), Mimosa sp. (含羞草)

94
 3) Capsule (蒴果)
i. Septicidal dehiscence (間裂) : Rhododendron sp. (杜鵑花)
ii. Loculicidal dehiscence (背裂) : Malvaceae (錦葵科), such as Hibiscus
sp.(山芙蓉,黃槿)
iii. Circumscissile dehiscence (蓋裂) : Plantago sp. (車前草),
Portulaca sp. (馬齒莧)
iv. Poricidal dehiscence (孔裂) : Papaver sp. (罌粟,虞美人)

4) Silique (長角果) : Cardamine sp. (碎米薺) , Rorippa sp. (山芥菜)

5) Silicle (短角果) : Capsella sp. (薺菜) , Lepidium sp. (獨行菜)
6) Follicle (蓇葖果): Sterculia sp. (蘋婆) , Illicium sp. (八角茴香)
7) Schizocarp (離果) : Daucus sp. (胡蘿蔔) , Apium sp. (茴香)

2) Indehiscent fruits (閉果): The pericarp does not dehisce at maturity.

1) Caryopsis (穎果) : Oryza sativa(稻穀), Zea mays (玉米), Triticum sp.(小
麥)
2) Utricle (胞果) : Amaranthus sp. (莧屬) , Chenopodium sp. (藜屬)
3) Achene (瘦果) : Helianthus annus (向日葵) , Clematis sp. (鐵線蓮)
4) Samara (翅果) : Acer sp. (楓樹) , Pterocarpus sp. (紫檀)
5) Nut (堅果) : Quercus sp. (櫟樹) , Castanea sp. (板栗)

95
 (2) Fleshy fruits (肉果): The pericarp is soft at maturity.
1) Berry (漿果): tomato(番茄), grape(葡萄)
2) Hesperidium (柑果) : citrus (柑橘), pomelo(柚子), orange(柳橙)
3) Pepo (瓜果) : watermelon (西瓜), melon (甜瓜)
4) Drupe (核果) : peach (桃子)、ceylon-olive elaeocarpus(錫蘭橄欖)。
5) Pome(仁果): A fruit that consists of ovary, receptacle and other floral
parts, such as apple(蘋果), loquat (枇杷 Eriobotrya japonica).

2. Aggregate fruits (聚生果)

An aggregate fruit is developed from the schizocarpic carpels of a flower together
with its receptacle, e.g., Fragaria sp. (草莓), Duchesnes sp. (蛇莓), and Rubus sp.
(懸鉤子)
3. Multiple fruits (多花果)
A multiple fruit is developed from the many flowers of the whole inflorescence. It
may also contain the tissue of the receptacle or floral axis, e.g., mulberries(桑葚),
figs(無花果), and pineapples(鳳梨).

96
Materials and Methods
1. Prepare various fruits representing different fruit types. Label each parts and
speculate their possible dispersal
2. Draw the outer morphology and the longitudinal section of each fruit type, e.g.,
dehiscent fruit, indehiscent fruit, fleshy fruit, aggregate fruits, and multiple fruit.

Questions
1. Explain the meanings of parthenocarpy (單性結果) and parthenogenesis (孤雌生
殖), also point out their functions in evolution and adaptation.
2. What are the edible parts of the following fruit?
1. Watermelon (西瓜), 2. Apple (蘋果), 3. Lichee (荔枝), 4. Strawberry (草莓)
3. What is the edible part of Hibiscus sabdariffa (洛神葵)
4. What are the roles of mature fruits of each type in plant life?

97
 Key of fruits
1. the fruit is developed from many closely arranged flowers……………… Multiple fruit
1. the fruit is developed from a single flower
2. it is developed from many schizocarpic carpels……………………………. Aggregate fruit
2. it is developed from many syncarpic carpels or a single carpel….… Simple fruit
3. with fleshy pericarp, usually indehiscence (fleshy fruits)
4. the pericarp is homogenous and juicy, many carpels……………. Berry
4. the pericarp is not homogenous, with different degree of
hardness
5. with hard exocarp and soft endocarp
6. with septate, several locules………………………………………… Hesperidium
6. without septate, without divided locules…………………….. Pepo
5. with soft exocarp and hard endocarp
7. with a single carpel, a single seed………………………………… Drupe
7. with several carpels, several seeds………………………………. Pome
3. with dry pericarp (dry fruit)
8. the pericarp is not dehisces at maturity (indehiscent fruit)
9. the pericarp extends and is wing-shaped…………………………. Samara
9. the pericarp is not wing-shaped
10. the pericarp is thin
11. the pericarp is not separated from seedcoat………… Caryopsis
11. the pericarp is separated from seedcoat………………. Utricle
10. the pericarp is thick and hard
12. small fruit, with a single carpel…………………………….. Achene
12. large fruit, with several syncarpic carpels……………… Nut
8. the pericarp dehisces at maturity (dehiscent fruit)
13. with a single carpel
14. longitudinal dehiscence
15. dehisce along a suture………………………………………….. Follicle
15. dehisce along two sutures……………………………………. Legume
14. transversal dehisce along the node……………………………. Loment
13. with two to several carpels
16. the fruit splits into mericarps……………………………………… Schizocarp
16. the fruit does not split into mericarp
17. the fruit dehisces transversely
18. the fruit dehisces transversely into two parts, Circumscissile
the upper one looks like a lid……………………………. dehiscence
18. the top of the fruit has a pore………………………… Poricidal
dehiscence
17. the fruit dehisces longitudinal
19. with three or more carpels, without pseudo-
septate
20. the fruit splits between carpels………………… Septicidal
dehiscence
20. the fruit splits along the dorsal central line of Loculicidal
each carpel…………………………………………… dehiscence
19. with two carpels, with pseudo-septate
21. the fruit is long and with many seeds…..…… Silique
21. the fruit is short and with few seeds…………. Silicle

98
 Experiment 10. Seeds and seedlings

Introduction
The objective of this experiment is to learn the basic structure of seeds, the
germination process, and the seedling types. The basic structure of angiosperm seeds
is different from that of gymnosperms. The angiosperm seed has seed coat (種皮),
embryo (胚), and endosperm (胚乳). The seed often has two layers of seed coat
because the integument of an ovary is a two-layered structure. The embryo is a young
seedling developed from a fertilized egg. It contains plumule (胚芽), cotyledon (子葉)
(1 or 2), hypocotyl (下胚軸), and radicle (胚根). The gymnosperm seeds may have 2
to 10 cotyledons. One sperm nucleus fertilizes with the polar nucleus and forms the
endosperm mother cell. The cell subsequently divides and develops into the
endosperm whose chromosome is usually 3n or more. The endosperm is rich in
nutrients which is used for the embryo growth when germination. For seeds of some
plants, the nutrient of the endosperm is transferred to the cotyledons and the
endosperm degenerates and dries out. And they are called seeds without endosperm.
Seeds are classified as:

1. Mature dicotyledonous seeds: based on the exist of endosperm

1) Seeds with endosperm: Ricinus communis (蓖麻) (Fig. 10-1), Passiflora
edulis (百香果)
2) Seeds without endosperm: Arachis hypogaea (花生), Pisum sp. (豌豆), and
Phaseolus sp. (菜豆)

2. Mature monocotyledonous seeds: all seeds have endosperm, e.g., Zea mays (玉
米) (Fig. 10-2), Allium fistulosum (蔥), and Lilium sp. (百合)

3. Seed type based on germination

A. Dicotyledonous seeds
1) Epigeous seed(子葉出土型種子) (Fig. 10-3): the cotyledon is raised
aboveground and performs photosynthesis when germination. The
examples are beans, Ricinus communis, passion fruit and most
dicotyledonous herbaceous plants.
2) Hypogeous seed (子葉不出土型種子) (Fig. 10-4): the cotyledon
remains in the seed when germination, e.g., pea and citrus.
B. Monocotyledonous seeds
99
 1) Epigeous seed (Fig. 10-6): For seeds with normal cotyledon such as
onion and lily, the cotyledon is raised aboveground and forms a hook.
2) Hypogeous seed (Fig. 10-5): For seeds with scutellum (吸片)specified
from cotyledon such as seeds of poaceae(禾本科), cyperacea(莎草科)
and palm family(棕櫚科植物), the cotyledon remains in the seed and
absorbs nutrient from endosperm. Take the corn seed as an example,
the embryo has a coleoptile protecting the young shoot and a coleorhiza
protecting the young root. Its cotyledon is buried in the endosperm and
turns into scutellum. After germination, the coleorhiza emerges first and
then the young root. Adventitious roots grow out from the hypocotyl.
Both the young root and Adventitious roots are temporary roots. After
the emerge of the coleoptile, the first true leaf emerges. Adventitious
roots can also grow out from the node at the base of the true leaf.
Adventitious roots that develop at the second to the tenth nodes
become permanent roots. The part between the cotyledon and the first
node is the mesocotyl (中胚軸).

Materials
1. Seeds of Ricinus communis or passion fruit
2. Beans
3. Seeds of corn or onion

Methods
1. Observe the structure of seeds: Peel the seed carefully, observe the structure of
the seed coat, endosperm, and embryo. Draw seed sections of each type, and
label the structures.
2. Observe the seedlings: Plant seeds in the wet sand two weeks before the class.
Observe the structure of seedlings in detail. Draw briefly the seedlings of
represent species such as Ricinus communis, pea, and corn. Draw the line of the
soil surface and label each structure.

Questions
1. What are seeds with embryo and seeds without embryo? What are the
differences between them?
2. What are the differences between seeds of angiosperms from gymnosperms?
3. During germination, the young stem of dicotyledonous plant forms hypocotyl
hook underground and it becomes straight after emerging aboveground. What
are the functions of this phenomenon in adaptation?
100
4. The cotyledon of poaceae and palm family turns into the scutellum. Pleased
describe the physiological change when the scutellum resorbs the nutrient from
the endosperm.
5. What is the crown root (冠根)?

Fig. 10-1. The seed of Ricinus communis

a. The external feature of seed
b. L.S. of seed showing embryo and endosperm
c. L.S. seed showing a cotyledon

Fig. 10-2. L.S. of fruit of Zea mays

101
 Fig. 10-3. Epigeous seed

Fig. 10-4. Hypogeous seed

Fig. 10-5. Germination of the corn seed

102
 Fig. 10-6. Seed germination of Allium cepa

Answers of the figures

Fig. 10-1. (p.53) d. Coleoptile c. First foliage leaf

a. Endosperm e. First leaves d. Adventitious root
b. Cotyledon f. Shoot apex e. Radicle
c. Seed coat g. Radicle
d. Shoot apex h. Root cap Fig. 10-6. (p.55)
e. Caruncle i. Coleorhiza a. Cotyledon
j. Caryopsis coat b. Seed coat
Fig. 10-2. (p.53) c. Plumule
a. Endosperm Fig. 10-5. (p.55) d. Primary root
b. Aleurone layer a. Coleoptile
c. Scutellum b. Radicle

103
Experiment 20. Measurement of the water potential of the plant

tissue by the falling-drop method

Introduction
The objective of this experiment is to learn how to measure the water potential of the
plant tissue by the falling-drop method.

4. Measurement of the water potential

Water moves from soil to plant and to air (soil-plant-air continuum) follows the
rule that water moves from high water potential to low. When the plant tissue has
a higher water potential than the environment, net water moves out of the plant
tissue. In contrary, net water moves into the plant tissue where the water
potential is lower. When the plant tissue is immersed in a solution of the same
water potential, the amount of water moves into and out of the plant tissue
remains static balanced thus no net water movement occurs. In this situation, the
water potential of the plant tissue is equal to the osmotic potential of the solution
which can be calculated by knowing the solute concentration. The falling-drop
method is aimed to find the isotonic solution which has the same water potential
as the plant tissue.

5. Determination of the isotonic solution

When the plant tissue is immersed in a hypertonic solution, water molecules move
from the plant tissue to the solution. After a period of time, the concentration of
the solution deceases as the water molecules increase. Place a drop of the current
solution to the original solution, the drop will rise up gradually for its lower
density. In contrary, when the plant tissue is immersed in a hypotonic solution,
water molecules move from the solution to the plant tissue and the concentration
(density) of the solution increase, thus the drop falls down gradually. When
immersed in the isotonic solution, the movement of water into and out of the
plant tissue is balanced thus the drop neither rises up nor falls down but diffuses

104
 in the original solution. Observing the movement of the drop of the solution leads
us to find out the isotonic solution of the plant tissue.

Materials
1. Potatoes
2. Test tubes, droppers
3. Sucrose solution
4. 0.4% Methylene blue (0.4 g methylene in 100 ml (0.2M) sucrose solution)

Method
1. Prepare a series of sucrose solution: .01M, 0.15M, 0.2M, 0.25M, 0.3M, 0.35M,
and 0.4M
2. Put 7 test tubes in the rake. Prepare the test series by adding 5 ml sucrose solution
of each concentration into each test tube.
3. Repeat the above preparation for the control series.
4. Prepare 7 pieces of potato sticks of the same size using a drill. Roll the potato
sticks on a piece of tissue paper to dry their surface, and then put one potato stick
into each tube of the test series. Make sure that the potato sticks are immersed in
the sucrose solution.
5. Wait for at least 60 minutes and take the potato sticks out of the tubes. Add one
drop of methylene blue into the solution and mixed thoroughly with a dropper.
6. Draw some stained solution with a dropper, insert the dropper into the middle of
its control tube slowly, release one drop of the stained solution and pull the
dropper out of the tube. Observe the movement of this blue drop with a piece of
white paper as the background and record the results.
7. Find out the concentration of the isotonic solution. Calculate the water potential
of the potato tissue by using the following formula:
= s + p
s = - icRT
= water potential; s= osmotic potential; p= pressure potential;
i= dissociation constant; c= concentration (M);
R= 0.00832 LMPa mol-1K-1 ; T= absolute temperature (K);

Qusestions
1. What are the matters you need to pay attention when cutting the plant tissue to
small pieces and placing them into the test tubes, respectively?
2. Why not adding more ethylene blue to darken the solution for easier recognition?

105
106