Download LABORATORY 9 - Transpiration LABORATORY 9

LABORATORY 9 - Transpiration
LABORATORY 9: TRANSPIRATION
OVERVIEW
In this laboratory you will apply what you learned about water potential from Laboratory 1b (Water Potential II)
to the movement of water within a plant. You will study the organization of the plant stem as it relates to these
processes by observing sections of fresh tissue.
pp. 757-760 6th ed. Campbell, Reece
OBJECTIVES
Before you begin this lab you should fully understand the following definitions:
- Transpiration: The process by which plants lose water from their surface by evaporation
- Stomata: A microscopic pore surrounded by guard cells in the epidermis of leaves and stems that allows gas
exchange between the environment and the interior of the plant
- Cohesion: The binding together of like molecules, often by hydrogen bonds
- Adhesion: The clinging of one substance to another
- Water Potential
At the completion of this lab you should be able to:
- describe how differences in water potential affect the transport of water from roots to stems to leaves
- relate transpiration to the overall process of water transport in plants
- discuss the importance of the properties of water--including hydrogen bonding, adhesion, and cohesion--to
the transport of water in plants
- quantitatively demonstrate the effects of different environmental conditions on the rate of transpiration
- identify the vascular tissues of the plant stem and describe their functions
INTRODUCTION
The amount of water needed daily by plants for growth and maintenance of tissues is small in comparison to the
amount that is lost through the process of transpiration and guttation (the loss of liquids from the ends of
vascular tissue at the margins of leaves). If the water that is lost from aerial plant parts is not replaced by water
transported up from plant roots, the plant will wilt and eventually die.
The transport of water from the roots in the xylem tissue is governed by differences in water potential. These
differences account for water movement, not only from cell to cell, but over long distances within the plant. In a root,
minerals transported from the soil accumulate in the xylem vessels of the vascular tissues of the stele. This, in addition
to the negative pressure (tension) in the xylem tissues, lowers the water potential of the xylem. Thus, water will move
into the xylem by osmosis, forcing fluid up the xylem vessels. This upward movement results in root pressure, but this
pressure can only move water a short distance up the xylem. Rather than being pushed up
from below by root pressure, the water and dissolved minerals in the xylem (xylem sap) are pulled upward as a result
of transpiration.
The stomatal openings of a leaf open into the air spaces that surround the mesophyll cells of the leaf. The moist
air in these spaces has a higher water potential than the air outside the leaf and water tends to evaporate
from the leaf surface moving from an area of higher water potential to an area of lower water potential. The
moisture in the air spaces is replaced by water from the mesophyll cells, lowering their water potential (solute in
the mesophyll cells becomes more concentrated when less water is present; recall that increasing solute
concentration lowers water potential). Water will then move into the mesophyll cells by osmosis from
surrounding cells with higher water potential, including those of the xylem. The gradient in water potential
between the xylem and the air outside the leaf that is caused by transpiration results in the transpirational pull of
water from the xylem into the leaf mesophyll cells and eventually into the leaf spaces. Cohesion of water
molecules to one another due to hydrogen bonding formation causes this pull to be transmitted throughout the
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LABORATORY 9 - Transpiration
column of water in the xylem, all the way from the leaves to the roots. Adhesion of water molecules to the walls of the
xylem cells aids in offsetting the effects of gravity.
EXERCISE 9A: TRANSPIRATION
MATERIALS
~ 0.1 mL graduated pipette inserted into a 16-inch piece of Tygon® tubing
~ ring stand with two clamps (at least one thermometer clamp)
~ plant (suggested species: Phaseolus vulgaris (bean plant))
~ petroleum jelly
~ electronic balance
~ razor blade
~ water bath (small bowl)
~ fa n
~ floodlight (100 watts)
~ spray bottle filled with water
TIPS:
1. Make sure no air bubbles are in the Tygon® tubing. If there are, start over and refill the tubing using the
Pasteur Pipette or eye dropper.
2. When moving the cut stem from the water bath to the tubing, try to expose it to air as little as possible.
3. Be patient when waiting for the potometer to equilibrate. If you start when the water level is above the zero
mark, it will be difficult to get accurate readings, and thus invalidate the final data.
ALTERNATIVE PROCEDURE FOR FILLING THE POTOMETER
1. Submerge the tubing and pipette in a shallow tray of water. Draw water through the tubing until all bubbles
are eliminated.
2. Carefully cut your plant stem under water. This step is very important, because no air bubbles must be
introduced into the xylem.
3. While your plant and tubing are submerged, insert the freshly cut stem into the open end of the tubing.
4. Bend the tubing upward into a "U" and use the clamp on a ring stand to hold both the pipette and the tubing.
PROCEDURE
1. Obtain a ring stand with two clamps.
2. Obtain a 0.1-mL pipette that has been inserted into a 16-inch piece
of Tygon® (clear plastic) tubing.
3. Use one of the clamps on the ring stand to hold the pipette end. It
is easiest if you clamp around the section where the pipette and tubing
meet. See the setup to the right.
4. Using a Pasteur Pipette or an eye dropper, fill the open end of the
tubing with water until the water reaches the line marked zero (0) or a little
bit above.
5. Bend the tubing upward into a "U" and use the other clamp on the
ring stand to hold the pipette.
6. Fill a small bowl with water and place the cut stem of the plant in
the water. Recut the stem under water so that the plant has not been
exposed to air.
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LABORATORY 9 - Transpiration
7. Bring the bowl up to the tubing and transfer the cut stem into the open end. Use petroleum jelly to make an air
tight seal surrounding the stem. Make sure that the end of the bean stem is immersed into water within the
Tygon® tubing. Do not put the petroleum jelly on the cut end of the stem.
8. Expose the plant in the Tygon® tubing to one of the following treatments (you will be assigned two treatments
by your teacher):
a. room conditions
b. floodlight - 100 watts (1 meter from the plant)
c. fan (1 meter from the plant)
d. mist (mist the leaves with water and cover with a transparent plastic bag; leave the bottom of the bag
open)
9. Let the potometer equilibrate for 10 minutes.
10. If the water line is above zero, move the side of the tubing without the plant up above the level of the opposite
side. Then put petroleum jelly around the plant and Tygon® tubing.
11. Read the level of water in the pipette at the beginning of your experiment (time zero) and record the finding
in Table 9.1.
12. Continue to record readings every 3 minutes for 30 minutes and record the data.
Table 9.1 Potometer Readings
treatment Time (min)
0
Reading
(mL)
Reading
(mL)
3
6
9
12
15
18
21
24
27
13. Calculate the total leaf surface area by: (You should do BOTH ways for EACH trial)
ENTER ALL DATA/CALCULATIONS IN TABLE 9.2.
(a) Leaf Mass Method: At the end of your experiment, cut the leaves off your bean plant and weigh them.
Remember to blot off all excess water. (as usual...SHOW all calculations)
Calculate the total leaf surface area for your plant using the following formula:
Total Mass of Leaves (grams ) Leaf Surface Area (m 2 )
160 g / m 2
Table 9.2 Calculating Leaf Surface Area
treatment
Method (a) - [Leaf Mass]
(NOTE: this calculation is valid
only if using Phaseolus vulgaris)
Method (b) - [Leaf Trace]
** Use the calculated averages in determining "individual water loss" in Table 9.3.
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Average (m2)
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LABORATORY 9 - Transpiration
(b) Leaf Trace Method:
Arrange all the cut-off leaves (from ONE trial) on a piece of graph paper; trace around your leaves. Count
all of the grids that are completely within the tracing and estimate the number of grids that lie partially
within the tracing. The grid has been constructed so that a square of four blocks equals 1 cm2.
[NOTE: you should include the drawing in your report] (you may need an additional piece of paper)
Table 9.3 Individual Water Loss in mL/m2
treatment
0
Water Loss
(mL)
Water loss
per m2
Water Loss
(mL)
Water loss
per m2
3
6
Time (minutes)
9
12
15
18
21
24
0
0
0
0
(you may be able to simply transfer your data from Table 9.1; the goal is to start the readings at 0)
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LABORATORY 9 - Transpiration
14. Record the averages of the class data for each treatment in Table 9.4.
Table 9.4 Class Average Cumulative Water Loss in mL/m2
Time/
0
3
6
9
12
15
18
21
24
27
30
Treatment
room
0
light
0fa n
0mist
0
15. Graph:
(1) your data (2) Table
9.4. (3) ideal data
You may need to convert the data to scientific notation. All numbers must be reported to the same power of ten for
graphing purposes.
a. What is the independent variable? ___________________ [label the horizontal (X) axis]
b. What is the dependent variable? ____________________ [label the vertical (Y) axis]
Title: ________________________________________________
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LABORATORY 9 - Transpiration
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LABORATORY 9 - Transpiration
Title: ________________________________________________
Title: ________________________________________________
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LABORATORY 9 - Transpiration
ANALYSIS OF RESULTS
1. Calculate the rate of water loss per minute of the treatments by taking the slope of the best-fit line on (1) the
class average graph; (2) ideal data graph:
Condition
Room
from Class Average graph
from Ideal Data graph
Fan
Light
Mist
2. Explain why each of the conditions (based on ideal data) causes an increase or decrease in transpiration
compared to the control.
CONDITION
Fan
EFFECT
REASON
Light
Mist
3. How did each condition affect the gradient of water potential from stem to leaf in the test plant?
4. What is the advantage of closed stomata when water is in short supply? What are the disadvantages?
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LABORATORY 9 - Transpiration
5. Describe several adaptations that enable plants to reduce water loss from their leaves. Include both structural
and physical adaptations.
6. Why did you need to calculate leaf surface area in tabulating your results?.
FOR ERROR ANALYSIS:
Compare your individual/class results and the ideal results (given at the end of the lab by your teacher). If they differ,
give possible reasons for these differences.
EXERCISE 9B: STRUCTURE OF THE STEM
MATERIALS
~ nut and bolt microtome
~ razor blade
~ bean stem (same bean plant from part A)
~ melted paraffin
~ small petri dishes for: (1) 50% ethanol; (2) toluidine blue O stain; (3) dH2O
~ microscope slide and coverslips
~ 50% glycerol
~ light microscope
PROCEDURE
1. Obtain a nut and bolt microtome.
2. Turn the nut until it is almost at the end of the bolt, forming a small "cup."
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LABORATORY 9 - Transpiration
3. Using a new, single-edge razor blade, cut a short piece of bean stem (approximately 5mm -- slightly longer
than the depth of the "cup" in the nut) from the base of your plant. Make two cuts so that both ends are freshly cut.
Make sure that this portion of the stem is free of petroleum jelly .
4. Stand the stem up on its end in the opening of the nut and carefully pour melted paraffin into the nut until it
fills the opening, completely covering the stem. This assembly will allow you to hold your stem upright and cut thin
slices.
5. Hold the head of the bolt flat on the table with one hand. Holding the razor blade in your other hand, remove
the excess wax on top by slicing down to the nut. This technique will keep your fingers out of the way of the razor
blade (see Figure 9.2).
Figure 9.2: Using the Nut-and-Bolt Microtome
Side View
Top View
6. Twist the bolt just a little, so a very thin core of paraffin and stem sticks up above the surface of the nut.
7. Slice with the razor blade, and cut this section down to the nut. Use as much of the razor as possible by
starting on one end and sliding down to the other with each slice.
8. Put the slice in a petri dish or watch glass containing 50% ethanol.
9. Twist the bolt a bit more to get another slice. Remember: you are trying to get the thinnest possible slice. It is
better to get part of a thin slice than a thick slice that is entirely round. Slice 8-10 pieces.
10. Leave the sections in the 50% ethanol for 5 minutes. Free the plant tissue from the paraffin if necessary.
11. Using the forceps, move the sections to a dish of toluidine blue O stain and leave them there for 1 to 2
minutes.
12. Rinse the sections in a petri dish of distilled water.
13. Mount the sections in a drop of 50% glycerin on a microscope slide.
14. Add a coverslip and observe the sections using a compound microscope.
15. Make a drawing of your section.
Identify and label the cell and tissue types described on the following page.
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LABORATORY 9 - Transpiration
Cell Types:
Parenchyma. The most abundant cell type is parenchyma. These cells are relatively unspecialized and retain
their protoplasts through their existence. They have primary cell walls. They make up the mesophyll of leaves
(where most of the photosynthetic activity takes place), the flesh of fruits, the pith of stems, and the root and
stem cortex. Many parenchyma cells are used for food storage, mainly starch. Starch, you will recall, is a
polymer of glucose. Starch forms grains within the parenchyma cells. These grains can be seen inside the cells. Take a
look at your sections again and label the parenchyma cells in your drawing.
Sclerenchyma. Fibers are an elongated type of sclerenchyma cell. They have thick secondary cell walls, often
lignified, and the protoplasts die at maturity. Fibers may be found in leaves, stems, and fruits. Usually they are
in bundles, serving a support function, and often are associated with vascular tissue. Check your stem cross
section for fibers. They will be found just outside the vascular bundle, their thick walls stained bright blue.
Collenchyma. Many young stems and leaves contain collenchyma cells for support. These cells are living at
maturity and characteristically have primary cell walls that are thickened at the corners. Locate collenchyma cells in
your cross sections.
Cell Tissues:
Xylem. Xylem is a tissue composed of several different cell types. Xylem is the water-conducting tissue that
conveys water and minerals from the soil through the plant. The cells that actually carry the water were
misnamed "tracheary elements" in the seventeenth century--"trachea" means air duct--and the name was never
corrected when the truth was discovered. The earliest xylem cells to evolve were fiberlike with thick lignified
secondary walls, arranged with overlapping ends, with a series of membrane-covered "pits" for passing water from
one cell to the next. These are the tracheids. Vessel elements evolved later, first appearing in flowering
plants, and are larger in diameter, have holes rather than pits, and offer less resistance to water flow than
tracheids. Both vessel elements and tracheids are dead at maturity. Xylem tissue may also contain parenchyma cells
and fibers. Look at your cross-sections and label the xylem in your drawing.
Phloem. Phloem is a tissue that distributes the carbohydrate products of photosynthesis throughout the plant.
This is achieved in flowering plants by the sieve tube members, which have primary cell walls and living
protoplasts at maturity but lack nuclei. Companion cells are associated with sieve tube members. These cells
have nuclei and play an important role in transfer of substances from cell to cell. Phloem may also contain
parenchyma cells and fibers. Look at your cross-section. The phloem is located outside the xylem. This
aggregation of xylem and phloem is called the vascular bundle. The arrangement of vascular tissue in the bean stem
is typical of the primary growth pattern in dicots. Monocots have different arrangements, but the cell and tissue types
involved are the same.
Epidermis. The epidermis is the outermost layer of cells that serves as a covering for the above-ground plant
parts. Some epidermal tissues are covered with a layer of cutin which prevents water loss. The specialized guard
cells of the epidermis open and close the stomates. Locate the epidermis on your stem section.
Drawings (Don't forget to include the magnification and to label specialized parts)
[see pp.679, 687 (5th ed.) in "Biology" by Campbell, Reece & Mitchell for helpful diagrams]
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LABORATORY 9 - Transpiration
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