The Procedure

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The corn and barley plants.

  

Working together to set up aquaria.

   

  

The settings for the CO2.

  

The aquarium set up.

The experiment in progress.

  

The second day observations.

   

  

This is how much the corn grew in three days.

  

The data being gathered.

Materials:

4 Aquaria Aquarium tubing

Masking tape

4 Thermometers – 1 per "system"

CO2 canister with regulator

Dissolved O2 probe or kit Marking pen

8 pots of C3 plant species - barley

pH paper or probe

8 pots of C4 plant species - corn

Fluorescent light source

Razor blade

Slides and cover slips

"Y" tube connectors x 3

1 Aquarium pump with two outlets

4 plexiglass covers

Water

Microscope

4 Beakers – at least 600 mL

Vaseline/sealant

2 Reptile heaters

Clear nail polish

Permanent red and blue markers

Prepared C3 and C4 cross section leaf slides

4 air stones for aquarium

    

 

Procedure:
  1. Label each aquarium as follows:

    2. A - Room temperature/High CO2 + air

    B - Increased temperature/High CO2 + air

    C - Room temperature/ air

    D - Increased temperature/ air

  2. Label each pot of plants as follows:

    3. A - Corn 1 A - Corn 2 A - Barley 1 A - Barley 2

    B - Corn 1 B - Corn 2 B - Barley 1 B - Barley 2

    C - Corn 1 C - Corn 2 C - Barley 1 C - Barley 2

    D - Corn 1 D - Corn 2 D - Barley 1 D - Barley 2

  3. Make initial leaf measurements as follows:

Leaf length

Use a permanent marker to make a dot on each leaf of a plant close to the area of emergence. After 6 days, measure the growth of each leaf (including new leaves) and record the length from the area of emergence to the dot. Add the total growth and divide by 6 to find the average growth. Record.

Leaf number

  1. Count the number of leaves per plant at the beginning and at the end of the experiment. Record.
  2. Place the two 2 pots of each plant species in each aquarium.
  3. In each "system" put a beaker with 500 mL water close to each plant.
  4. To equilibrate the water with the gases, aerate the water in the beaker before taking initial measurements and proceeding with the experiment. To do this, connect the air pump to one end of a length of tubing and an air stone on the other end. Place the air stone in the water and aerate for at least two hours.
  5. To introduce the desired atmospheric and CO2 gases, connect the purchased CO2 cylinder, tubing, and pump as in the Bubble Method described below.
  6. To obtain temperatures higher than room temperature in B and D, attach a reptile heater to the bottom of those labeled aquaria, below the plants.
  7. Place a thermometer in each aquarium and make initial temperature measurements as described below. Record.
  8. Cover the system then seal with vaseline/sealant. If using an aquarium hood, use masking tape to seal all openings to eliminate any exchange of gases during the treatment. If using a piece of plexiglass, cut the plexiglass to fit the top of the aquarium. Place a bead of vaseline around the top of the aquarium. Place the plexiglass on top of this bead.
  9. Put all aquaria under constant irradiance using fluorescent lights.

Methods of Measurements:

It is important to emphasize that all parameters except those that are tested for their effects on the experiment should remain as constant as possible for all treatments.

  1. The temperature inside each aquarium should be measured by placing a thermometer in each aquarium for the duration of the experiment.
  2. The pH of the water will be used to calculate the CO2 concentration in the
  3. atmosphere inside the aquaria. The pH of the water in the beakers may be measured using: a) a pH meter. Make sure water is stirred and that the beaker is sealed to limit atmospheric exchange as much as possible. b) pH paper. Use pH paper that will give a measure of change in the pH by approximately 1 unit pH.
  4. Light is measured with a spherical radiometer. Make the measurements of light close to the four plants. If a radiometer is not available be sure to use the same light set up for each aquarium. Use new bulbs with the same wattage.
  5. Oxygen can be measured in two ways: a) Dissolved oxygen meter. Be sure to stir water as you measure and to keep the container sealed as much as possible to outside air. b) Kit for measuring dissolved oxygen
  6. Leaf number and size: Count leaf number on the first and last days of the experiment.
  7. Calculate the leaf growth rate. Subtract the initial length(To) from the final length(Tf) of each leaf [ Tf – To = leaf growth]. Find the average leaf growth and divide this by the number of days(d)of the experiment. [ Ave / d ] This will give you the average leaf growth rate (cm/d).
  8. Microscopic observation: Obtain a small sample of leaf from each plant and prepare them for microscopic observations employing the following method:
    1. Apply a thin layer of clear nail polish 1 cm2 to the underside of the leaf. Allow nail polish to dry.
    2. Place scotch tape over polished area, pull leaf away from tape.
    3. Place tape (sticky side up) on microscope slide.
  9. Place cover slip on top.
  10. Examine and draw differences in morphology between the species. This may be done once to see major differences between the two plants or at the beginning and at the end to compare the changes in stomates during growth.
  11. Remove and section leaf samples. Make cross section and longitudinal samples. Place one drop of water on sample and cover with cover slip. Examine and draw differences in morphology between the two species. This can be done at the beginning of the experiment and as a final observation.
  12. After six days open the aquaria. Immediately upon opening the aquaria, take water samples from each beaker. Be sure to keep sample covered as you measure dissolved O2, CO2, and pH before the water has time to change temperatures and interact with external atmosphere.

Bubble Method

In order to increase the CO2 concentration 100X, three percent of the gases bubbled by the two-outlet aquarium pump entering the closed system must be determined by using the bubble method.

Put a drop of liquid soap and three drops of water into the plastic tubing to be connected to one outlet of the air pump. Clamp the other outlet shut. Connect a graduated pipette onto the plastic tubing. Plug the pump and find the rate (cm/min) of air flow through the tube using a soap bubble; see Figure A1. Using the same connection, remove the air pump and attach the CO2 cylinder. Before, allowing the CO2 gas through the tubing, place a clamp on the side closest to the cylinder and away from the water and soap. Turn on the CO2 and adjust the clamp so the gas moves at 3% of the air rate A2. (back to procedure)

Figure A1

Figure A2

Air rate X .03 = CO2 rate

 

After the CO2 rate has been determined, rinse the soap completely from the tubing without disturbing the adjusted clamp. All four treatments will be set up as in Figure B.

Figure B

  (back to procedure)

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The Woodrow Wilson National Fellowship Foundation 
CN 5281, Princeton NJ 08543-5281 - Tel:(609)452-7007 - Fax:(609)452-0066 
Technical contact: lpt@woodrow.org