Grow Plants in Microgravity with an Arduino Clinostat

Build Your Arduino Clinostat

1. Make the following connections on the breadboard (Figure 4).
   1. Connect the Arduino's 5V pin to the power bus (+) on the breadboard.
   2. Connect the Arduino's GND pin to the (-) bus on the breadboard.
   3. Put the potentiometer in the breadboard. Make sure the three pins are in three different breadboard rows.
      1. Connect one of the potentiometer's outer pins to the power bus.
      2. Connect the other outer pin to the ground bus.
      3. Connect the middle pin to Arduino analog input pin A0.
   
   Figure 4. Breadboard diagram for the clinostat circuit.
2. Connect the servo motor to the circuit. Your servo motor has three wires. Check your servo motor's documentation to confirm the color coding for the wires. The servo shown in Figure 4 has the following connections. Your servo may be different.
   1. Orange: signal (Arduino pin 8)
   2. Red: power (breadboard power bus)
   3. Brown: ground (breadboard ground bus)
3. Open the Arduino IDE.
4. Plug your Arduino into your computer with the USB cable.
5. Make sure you have the correct board and port selected under Tools.
6. Copy and paste the following code into the IDE, save it, and upload the file to your Arduino.

// code to control continuous rotation servo with potentiometer

#include <Servo.h>

Servo myservo; // create servo object to control a servo

int pot;    // variable for potentiometer analog reading, 0-123
int speed;  // variable for servo speed, 0-180.
            // servo is STOPPED at speed = 90
            // full speed in either direction at 0 or 180. 
            // this is NOT the servo's angular position

void setup() {
  // setup code that only runs once
  myservo.attach(8); // use pin 8 to control the servo
}

void loop() {
  // code that loops repeatedly
  pot = analogRead(A0);                // read analog voltage from potentiometer
  speed = map(pot, 0, 1023, 0, 180);   // convert analog reading 0-1023 to speed 0-180
  myservo.write(speed);                // send control signal to servo
}

7. Turn the potentiometer. You should be able to use it to control the motor's speed. The motor should stop when the potentiometer is approximately in the middle. If your motor does not work, carefully double check all of your wiring.
8. Turn the motor off for now.
9. Build a frame or support for your clinostat. The frame should support the motor so it is mounted horizontally, high enough in the air that it can rotate a petri dish without the petri dish hitting the ground. Figure 5 shows an example.
10. Make sure you press the servo horn onto the motor's shaft, as shown in Figure 6. You will use this to attach the petri dish.

Figure 5. A support frame for the clinostat made from scrap wood. The motor is attached to the tall piece of wood with double-sided foam tape.

Figure 6. Servo horn attached to the motor shaft.

Prepare Your Petri Dishes

How to Prepare Agar Plates for Seed Germination Experiments

https://www.youtube.com/watch?v=YfQXpzi8qno

1. Note: it is best to do this experiment when you will be around to observe the seeds as they germinate. You do not want to miss a good observation period while you are at school or asleep. For example, you could prepare the seeds in the evening, let them start to germinate overnight, and then observe root development throughout the following day. The seeds we tested had ~1 cm roots after about 24 hours and developed shoots after about 36 hours, but your results may vary. Germination time can vary due to many factors like humidity and exposure to light.
2. Print the seed placement template.
3. Use a permanent marker to draw an arrow on the back side of four petri dishes. This arrow will indicate the "down" direction when you initially plant the seeds (Figure 7).
4. Label the four petri dishes (write on the back side): 1. Constant gravity, 2. Rotated 90 degrees, 3. Clinostat, 4. Extra.
   
   Figure 7. Labeled petri dishes.
5. Measure 1.5 g of agar-agar. Stir it into 100 mL of tap water in a pot.
6. Boil the agar-agar solution until it is clear (approximately 1–2 minutes). Stir it while boiling to prevent lumps from forming (Figure 8).
   
   
   Figure 8. Top: the agar-agar solution will be cloudy when you first start to boil it. Bottom: the solution should become clear within a couple minutes.
7. Wait for five minutes for the solution to cool down.
8. Fill the four petri dishes about halfway with the agar-agar solution. Make sure the complete bottom surface of each petri dish is covered (Figure 9).
   
   
   Figure 9. Top: Petri dishes filled with the agar-agar solution. Bottom: a side view of one of the petri dishes, showing the depth of the solution.
9. Cover each petri dish with a lid.
10. Wait until the agar-agar has become solid. This may take anywhere from a few minutes up to half an hour. You can test it by poking it with the tweezers.
11. If there is condensation on the inside of the lids, gently wipe it off with a paper towel, or remove the lids briefly so it can evaporate.
12. One at a time, place each petri dish on the seed placement template. The arrow you drew on the petri dish should point in the same direction as the arrow on the template.
13. Use tweezers to gently embed nine cress seeds in the agar-agar. The seeds should only be partially submerged in the agar-agar, so they still have access to oxygen. The micropyle on each seed should point downward, in the direction of the arrow. When it germinates, the seed will split at the micropyle. Figure 10 shows a close-up of a seed with the micropyle labeled. Figure 11 shows a completed petri dish with nine embedded seeds. Note that cress seeds are very small. If you have difficulty seeing the micropyle, use a magnifying glass.
14. Repeat steps 12–13 for each petri dish.
    
    Figure 10. Close-up picture of a cress seed with an arrow pointing to the micropyle.
    
    
    
    
    Figure 11. Petri dish with nine cress seeds embedded in the agar-agar.
15. Wait 10 minutes for the seeds to absorb some water in their outer layer and stick to the agar-agar.
16. Try standing one of the petri dishes vertically to make sure the seeds do not fall out. If any of the seeds fall out, put them back into their place and wait longer before continuing.
17. Use transparent tape to attach the petri dish lids. Only use a few pieces of tape, so the seeds can still get oxygen. Do not seal the entire perimeter with tape.
18. You are now ready to begin your experiment. You should have four petri dishes prepared, with a total of 36 seeds.

Run the Experiment

This section contains instructions to conduct a basic experiment to examine the effect of gravity on germination and root development in cress seeds. However, there are many other experiments you can do with your clinostat. See the Variations section for other experiment ideas.

For the basic experiment, you will use three of your petri dishes. You will keep one in a constant vertical orientation. You will start one out vertically, then rotate it once by 90 degrees partway through the experiment. You will attach the third petri dish to the clinostat so it rotates continuously. The fourth petri dish serves as a backup in case you drop or damage any of the others.

1. Use double-sided foam tape to mount two of the petri dishes vertically to a support like a wood block or cardboard box. The arrows you drew on the petri dishes should point down (Figure 12).
   
   
   Figure 12. Petri dishes mounted vertically to a support.
2. Use double-sided foam tape to mount the third petri dish to the servo horn. Make sure the petri dish is centered on the servo horn (Figure 13).
3. Immediately turn the motor on to the slowest speed you can achieve before it stops. Note that you do not want the petri dish to spin too fast, or the seeds may experience centrifugal effects (they are "flung" toward the outside of the petri dish). If it spins too slow, the seeds may experience gravitropic effects. A speed of approximately 10–15 RPM, easily achievable with common servo motors available for Arduino, should be sufficient.
4. Count the number of revolutions the petri dish makes in one minute. Write this number down (you can try varying the motor's speed for future experiments).
   
   Figure 13. Petri dish mounted to clinostat.
5. Since light can also affect your seeds' germination and growth, make sure your petri dishes are all facing in the same direction. Ideally, your petri dishes will all face a window so they get natural light. If you use another source of light, make sure it shines on all the petri dishes in the same direction.
6. Write down the time you started your experiment. If you have a camera available, take a picture of each petri dish (it is OK to briefly stop the motor to take a picture, but make sure you immediately start it again).
7. It is time to watch and wait! How long it takes your seeds to germinate may depend on several factors, like the humidity of the air and exposure to light. If you started the experiment at night, go to bed and check on the plants first thing in the morning. If you started the experiment in the morning, check in every hour or so until you start to see roots develop.
8. After the plants have started to develop roots, check in more frequently (every 30–60 minutes). Take pictures of each petri dish if possible. You can write the date and time on a sticky note and include it in the picture to make it easier to track the seeds' growth later.
9. After most of the seeds have developed roots that are at least 1 cm long, rotate one of the fixed petri dishes by 90 degrees (so the "gravity" arrow now points sideways), draw a new arrow in the new "down" direction, label this arrow "after rotation," and re-mount it to the support.
10. After most of the seeds have developed roots that are at least a few centimeters long, stop the motor, remove all the petri dishes from the tape, and remove their lids.
11. Examine each petri dish.
    1. How many seeds germinated in each petri dish?
    2. Look at your pictures. When did seeds start to germinate in each dish?
    3. Measure the root length of each seed. You can do this by cutting a piece of string, laying it along the path of the root (curving if necessary), then measuring the string. You can also use an image analysis program (see the United Nations reference in the Bibliography and instructions for using a program called ImageJ).
    4. What do you notice about the orientation of the roots in each petri dish? Does the orientation seem related to the initial orientation of the seeds and/or the direction of gravity?
12. Based on your analysis of the petri dishes, can you draw any conclusions about the effects of microgravity on seed germination?