Materials Included In Kit

Additional Materials Required

Prelab Preparation

1. Use forceps or a dissecting needle to remove the cotton plugs from the pipets.
2. Use scissors or a knife to cut the tubing into 16-inch lengths.
3. Begin germinating bean seeds at least two weeks in advance.
   1. Soak the red bean seeds in warm water for one hour prior to planting.
   2. Place peat pellets into tray and add warm water to expand them to 1–1½ inches tall.
   3. Gently pour off the excess water.
   4. Pull the netting open on top of the peat pellets.
   5. Sow 2–3 seeds per pellet by pushing them under the top layer of peat.
   6. Cover the tray with the clear cover and place the tray in a warm location. Note: The optimal soil temperature for the germination of bean seeds is 65–85 °F. If the seeds start to mold, remove the cover for a day to allow better air circulation around the seeds. If the mold continues, remove the seeds and begin new plants.
   7. When the seeds begin to sprout, remove the cover and place the tray in a sunny location or under a grow light.
   8. Rotate the tray ¼ turn daily and water when the peat pellets turn light brown.

Safety Precautions

The scalpel is a sharp object; care must be taken when cutting with the scalpel, always cut away from the body and away from others. Although the materials in this lab activity are nonhazardous, follow normal safety precautions. Remind students to wash hands thoroughly with soap and water before leaving the laboratory. Please review current Safety Data Sheets for additional safety, handling and disposal information.

Disposal

Please consult your current Flinn Scientific Catalog/Reference Manual for general guidelines and specific procedures, and review all federal, state and local regulations that may apply, before proceeding. Scalpels may be disposed of according to Flinn Biological Waste Disposal Method V, sharps and broken glass. All other materials in this laboratory may be disposed of using Flinn Biological Waste Disposal Method VI, in the regular trash.

Lab Hints

• Enough materials are provided in this kit for 24 students working in groups of four or for 6 groups of students. The laboratory work for this experiment can reasonably be completed in one 50-minute lab period with proper preparation. The most important element for success in an inquiry-based activity is student preparation. Sufficient class time should be allotted before lab to think through the measurements that must be made and how the experiment should be conducted. The Prelab section contains leading questions to stimulate group discussion during this phase of the laboratory.
• To ensure a safe lab environment, it is essential that the teacher provide a mechanism for checking the students’ proposed procedures and their understanding of the necessary safety precautions to conduct the experiment.
• Some teachers tell us that for an inquiry-based lab, they require the students to submit their proposed procedures a week before the lab. The teachers then check the procedures and return the proofed copies to the students the day before the lab. This ensures that students are prepared and that teachers have time to supervise the actual lab activity, not proof the procedures, during lab time.
• Possible environmental conditions to test include:
  1. Gentle breeze—Place a fan at least 1 meter from the plant, on low speed.
  2. High humidity—Mist leaves with water and cover with a transparent plastic bag leaving the bottom of the bag open.
  3. Strong light (one of the following)
     1. Place a 1-L beaker filled with tap water between a 150-W flood lamp and the area in which students will place their samples. The water serves as a heat sink for the light so that the leaves do not become heated.
     2. Turn on a bright overhead projector and point the light directly at the area in which students will place their samples. The overhead projector should be located close to the plant area to ensure a very bright light source.
     3. Turn on a bright overhead projector and place the potometer on the transparency area of the overhead projector.

Teacher Tips

• Assembling a bubble-free potometer takes practice; ensure that more than two plants are available for each student group.
• Using a blunt-end syringe, add one drop of food coloring to the top of the water in the pipet once the potometer is assembled. The drop of dye will help the students read the amount of water lost during the activity.
• Discuss with students how scientists cope with faulty data.

Answers to Prelab Questions

1. Calculate the transpiration rate for a red kidney bean seedling exposed to room conditions (as a control) based upon the following data:
   1. Water lost in 30 minutes as determined using a potometer = 1.26 mL
   2. Mass of all leaves = 1.200 g
   3. Mass of a 1 cm² section of the leaves = 0.010 g
      {10933_PreLabAnswers_Equation_5}
2. Calculate the transpiration rate for a red kidney bean seedling exposed to a gentle breeze based upon the following data:
   1. Water lost in 30 minutes as determined using a potometer = 9.75 mL
   2. Mass of all leaves = 1.100 g
   3. Mass of a 1 cm² section of the leaves = 0.010 g
      {10933_PreLabAnswers_Equation_6}
3. Determine which environmental condition your group would like to test. After seeking approval from your teacher you will be designing a controlled experiment to determine the effect the environmental change has on the transpiration rate for red kidney bean plants. Consider the following questions:
   1. The independent variable in an experiment is the variable that is changed by the experimenter, while the dependent variable responds to (depends on) changes in the independent variable. Choose the dependent and independent variables for your proposed experiment.
   2. What other variables will affect the results in this experiment? How can these variables be controlled? For example, how will you control the environmental condition surrounding the plants? Do not forget to control the effect on other lab groups.
   3. What measurements must be made for each seedling? How will these be made?
   4. Read the Safety Precautions, Materials and Procedure sections. Write a step-by-step procedure for the experiment, including the safety precautions that must be followed for your specific experiment.
   5. Review your experimental plan with your teacher before you proceed.

Answers to Questions

1. Calculate the transpiration rate for each of your red kidney bean seedlings used in the experiment.

   Answers will vary. Sample results follow.

   {10933_Answers_Table_1}
2. Write a paragraph explaining why the treatment caused an increase or decrease in transpiration compared with the room conditions. Include in this paragraph a discussion of the possible errors involved in the experiment and their affect on the results.

   Answers will vary. Generalized results follow for three possible environmental conditions.

   1. Gentle Breeze: An increase in wind speed results in an increase in the rate of leaf water loss because the water evaporates more rapidly off the leaf creating lower water potential in the surrounding air and therefore a greater rate of transpiration. A strong breeze may cause the stomata to close, reducing the rate of transpiration.
   2. High Humidity: Increased humidity in the air surrounding the leaf decreases the water potential gradient between the saturated air in the leaf air spaces and the air surrounding the leaf, resulting in a decreased rate of leaf water loss.
   3. Strong Light: Absorption of light results in an increase in leaf temperature; since the rate of water evaporation increases as the temperature increases, the increase in leaf temperature results in an increased rate of leaf water loss.
3. Why was it necessary to calculate the leaf surface area using an indirect method, such as the gravimetric method presented in this laboratory?

   The mathematical formula required to directly determine the leaf surface area is not as simple as calculating the area of a circle or cube. Scientists must often use indirect methods in their experiments and gravimetry is a very common method.

4. Explain the role transpiration (and therefore plants) plays in the water cycle.

   Transpiration acts to move ground water into the atmosphere where it reacts with and influences climate conditions, eventually producing precipitation which becomes ground water thereby beginning the water cycle again.

5. What is the advantage of closed stomata to a plant when water is in short supply? What are the disadvantages?

   The advantage to closing stomata when water is in short supply is that the closed stomata do not lose water as rapidly, preventing wilting and eventually death. The disadvantage to closed stomata is that the amount of carbon dioxide available for photosynthesis is limited and the plant cannot grow.

References

Biology: Lab Manual; College Entrance Examination Board: 2001.

Introduction

In actively growing plants, tissues (leaves and root tips) can contain 80–90% water while the woody parts may be 45–60% water. What role does water play in plants? Why do plants need so much water to stay alive?

Concepts

• Potometer
• Transpiration
• Water potential

Background

Plants use water as a medium for transporting nutrients within the plant, to provide hydrostatic support and evaporative cooling and as a reactant in biochemical processes. Water has many unique chemical properties that make it an excellent solvent. Water is a polar compound that can dissolve both ionic and polar substances, such as minerals and carbohydrates, respectively. Once dissolved, these nutrients are then transported throughout the plant. Water also acts as a reactant in many chemical reactions in the plant, including photosynthesis. During photosynthesis, water serves as the source of electrons for the conversion of carbon dioxide (CO₂) into glucose. However, only a small amount of water is needed for the actual electron transfer step in photosynthesis. Most of the water needed by a plant for photosynthesis is used to keep the cell surfaces moist so that the cells can absorb carbon dioxide gas from the atmosphere. Carbon dioxide is very soluble in water, whether it is found inside a cell (cytoplasm) or outside of the cell.

Another important function of water is that it maintains turgidity (or pressure) in plant tissue. Water literally “inflates” leaves, giving structure and support to leaves. Turgidity is also necessary for cell growth and enlargement. New cells expand in size by creating more cytoplasm, which is composed mostly of water. In fact, the loss of water is easy to see when a plant wilts. Wilting is actually the dehydration of plant cells. A plant that suffers from long term dehydration stops growing and eventually dies.

Water must follow the laws of thermodynamics. Consequently, water always moves from regions of high energy to regions of low energy. In a plant this means that water flows from regions of high water potential to regions of low water potential. This occurs through the processes of osmosis, root pressure, and adhesion and cohesion of water molecules. Water is transported from cell to cell within the plant because of differences in water potential within the plant.

Plants need water around their roots consistently because they constantly lose water through their leaves via transpiration and guttation. Transpiration is the loss of water by evaporation from the leaves and is the main method for pulling water from the roots to the leaves. Guttation is the appearance of drops of sap on the tips or edges of leaves.

Transpiration begins with evaporation of water through the stomata (singular: stoma or stomate). Stomata are tiny openings (pores) used for the absorption of CO₂ for photosynthesis and oxygen (O₂) for cell respiration (see Figure 1). Thousands of stomata occur on the underside of a typical dicot or on the upper surface of a plant whose leaves float on water. Each stoma is formed by a pair of specialized cells known as guard cells, which are responsible for regulating the size of the pore’s opening. By adjusting the size of the opening, the guard cells control the rate of CO₂ and O₂ uptake and the loss of water by the leaf. In this way, by regulating the diffusion of CO₂ into the cells, the guard cells also control the rate of photosynthesis in the leaf. The guard cells swell when they are full of water, opening the stoma into air spaces that surround the middle layer of leaf cells. This middle layer of cells is covered with a thin layer of water from the xylem. The water coating the cells evaporates due to the lower water potential in the outside air. New water molecules then move into the cells by osmosis from the xylem.

As each water molecule moves from the xylem onto the middle layer of cells, it exerts a pull on the column of water molecules in the xylem, from the leaves to the roots (see Figure 2). This transpiration pull is caused by the cohesion and adhesion of water molecules within the column of xylem cells. The upward transpiration pull on the fluid in the xylem causes negative pressure to form in the xylem, creating a need to replenish the water inside the xylem. This need is transmitted all the way from the leaves to the roots, causes water to move inward from the soil, through the root hairs, and into the xylem. If the moisture content in the middle layer of the leaf is equal to or less than the moisture level of the air outside the leaf, the guard cells will lose their water, and the cells will “deflate” and close. Transpiration rates differ due to variations in the environmental conditions such as humidity, air currents, temperature and amount of light experienced by the plant.

Experiment Overview

The purpose of the inquiry-based experiment is to design and carry out a procedure to determine the change in the transpiration rate due to a change in the environmental conditions surrounding a plant. In this laboratory, the rate of transpiration will be measured using a potometer.

Materials

Prelab Questions

A potometer is used to determine the transpiration rate for a plant. Potometers range from simple laboratory made devices to elaborate, expensive instrumentation used in research institutions. Potometers simply measure the amount of water lost through the leaves of a plant during an experiment. The transpiration rate is the amount of water lost per minute per square meter of leaf surface area (mL/min/m²). In addition to the amount of water lost per minute as determined using the potometer it is necessary to calculate the total surface area of the leaves on the plant. There are several methods that may be used to accomplish this. The simplest method involves weighing just the leaves, followed by cutting and weighing a 1 cm² area from a leaf and calculating the total surface area. The directions for this procedure follow:

1. Use scissors to cut all the leaves off the plant and blot them dry.
2. On a balance, mass the leaves to the nearest thousandth of a gram (0.001 g).
3. Use a ruler and scalpel to cut a 1 cm² section out of one leaf.
4. Mass the 1 cm² section to the nearest thousandth of a gram (0.001 g) on the balance.
5. Calculate the mass per square meter of leaf by multiplying the 1 cm² section’s mass by 10,000. Note: There are 10,000 cm² per m².
   {10933_PreLab_Equation_1}
6. Calculate the leaf surface area by dividing the total mass of the leaves by the mass per square meter.
   {10933_PreLab_Equation_2}
7. Determine the amount of water lost per minute (mL/min) by dividing the volume of water lost (mL) as observed on the potometer by the amount of time for this amount to be transpired (min).
   {10933_PreLab_Equation_3}
8. Finally, divide the water lost per minute, Equation 3, by the leaf surface area, Equation 2.
   {10933_PreLab_Equation_4}

1. Calculate the transpiration rate for a red kidney bean seedling exposed to room conditions based upon the following data:
   1. Water lost in 30 minutes as determined using a potometer = 1.26 mL
   2. Mass of all leaves = 1.200 g
   3. Mass of a 1 cm² section of the leaves = 0.010 g
2. Calculate the transpiration rate for a red kidney bean seedling exposed to a gentle breeze based upon the following data:
   1. Water lost in 30 minutes as determined using a potometer = 9.75 mL
   2. Mass of all leaves = 1.100 g
   3. Mass of a 1 cm² section of the leaves = 0.010 g
3. Determine which environmental condition your group would like to test. After seeking approval from your teacher, you will be designing a controlled experiment to determine the effect the environmental change has on the transpiration rate for red kidney bean plants. Consider the following questions:
   1. The independent variable in an experiment is the variable that is changed by the experimenter, while the dependent variable responds to (depends on) changes in the independent variable. Choose the dependent and independent variables for your proposed experiment.
   2. What other variables will affect the results in this experiment? How can these variables be controlled? For example, how will you control the environmental condition surrounding the plants? Do not forget to control the effect on other lab groups.
   3. What measurements must be made for each seedling? How will these be made?
   4. Read the Safety Precautions, Materials and Procedure sections. Write a step-by-step procedure for the experiment, including the safety precautions that must be followed for your specific experiment.
   5. Review your experimental plan with your teacher before you proceed.

Safety Precautions

Scalpels are sharp instruments; use caution when cutting, always cut away from your body and away from others. Light sources may be hot, do not touch the lamp or lamp housing. Electrical outlets have the potential to shock if not used properly. Ensure the area surrounding electrical equipment and hands are dry. Follow all normal safety precautions. Wash hands thoroughly with soap and water before leaving the laboratory.

Procedure

Construct a Potometer

1. Place two test tube clamps on opposite sides of the support stand (see Figure 3).
   {10933_Procedure_Figure_3}
2. Gently place the tapered tip of the 0.5-mL pipet into the hole of a #3 stopper. Slide the stopper up until it is at about the 0.25-mL mark.
3. Place the tapered tip of the 0.5-mL pipet into the piece of clear plastic tubing.
4. Place the free end of the tubing through the hole of a second stopper. Slide the stopper up until it is about 2" from the end of the tubing.
5. Place the plant into a small plastic bag, leaving the base of the stem sticking outside the bag (see Figure 4).
   {10933_Procedure_Figure_4}
6. Complete the next three steps quickly.
   1. Use a scalpel to cut the plant stem from the roots just above the surface of the soil.
   2. Wrap a rubber band around the bag, creating a moderately tight seal to keep water off of the leaves of the plant in steps 7 through 10. The rubber band will be used later to create a seal around the plant stem.
   3. Place the plant stem into the pan of water.
7. Submerge the tubing and the pipet in a pan of water. Use a syringe to draw water through the tubing until all the air bubbles are eliminated. Note: Air bubbles will stop the water from entering the stem of the plant.
8. Use the scalpel to create a new cut on the plant stem while it is under water. Note: This step must be done under water. It is very important that no air bubbles be introduced into the xylem.
9. While the plant and tubing are submerged, insert the freshly cut stem into the open end of the tubing.
10. Have one group member hold the plant leaves out of the water while a second member completes the following steps.
    1. Secure the rubber band around the tubing with the stem inside.
    2. Place a generous amount of petroleum jelly around the top of the tubing and the stem junction to seal the opening. Note: Do not put petroleum jelly on the end of the stem because it will interfere with osmosis.
11. Bend the tubing upward into a “U.” Use the clamps on the support stand to hold both of the stoppers (see Figure 5). Note: Do not allow any air bubbles into the potometer. If an air bubble appears, quickly immerse the pipet, tubing, and part of the stem (but not the leaves) into the pan of water. Use the syringe to flush the air bubble from the potometer. Note: If an air bubble touches the cut end of the stem, then the stem must be cut again under water.
    {10933_Procedure_Figure_5}
12. Remove the bag from the leaves and let the potometer equilibrate for 10 minutes before beginning the experiment. Make sure to read the level of water in the pipet as the zero time value before exposing the plant to your experimental conditions.
13. Consult your instructor for appropriate disposal procedures.

Student Worksheet PDF

10933_Student1.pdf