For High School Teachers
 
 
  From DNA to Organism: A Study in DNA Function for the High School Biology Classroom
 
 

Tips for growing Arapbidopsis

           Obtain your seed stocks from www.arabidopsis.org. The Web site is fairly easy to navigate and has numerous resources related to Arabidopsis. The stocks that are suggested in Module 1 are ones that I have tested and had luck with. Feel free to browse the list of seed stocks that they have and choose other mutant plants. They list a price for seed stocks but were more than happy to donate the small amounts that I needed. Just give them a call and let them know that you are a high school teacher.

           The biggest problem that I ran into trying to grow the Arabidopsis was the size of the seeds (think dust!). Once I got over the initial shock, I was able to develop some simple tricks to get the seeds to germinate and grow. Some useful resources for growing Arabidopsis are listed on the Arabidopsis project Web site www.arabidopsis.org (Click “about ABRC,” then “plant and seed handling.”).

 

     Growing Arabidopsis in soil

    1. Obtain plastic pots for growing plants in. I have had the best luck with watering the plants from below (i.e. a wick system or plastic growth trays with pots). You can either obtain these from local garden stores or from scientific supply companies (Fisher, etc.) Another, more cost-effective, approach would be to use the bottle biology idea from www.fastplants.org.   They have plans for growing Fastplants (similar to Arabidopsis) in old film canisters which would work just as well for these purposes.  
    2. Wet the soil before filling the pots. The soil should be wet enough to drip when you squeeze a handful of it but not so wet that it drips on its own. It may take a little testing to get this right.  
    3. Fill the containers to 1 cm below the rim with loosely packed damp soil.   Do not pack down the soil.
    4. Pour out the seeds on a sheet of white paper (this makes them easier to see and work with).
    5. Using a wet paint brush, carefully place the desired number of seed on the surface of the soil. Arabidopsis seeds need light to germinate so do not bury them. Space the seeds so there is about 1– 2 cm between seeds.   Don’t worry about overcrowding yet. When the plants grow, you can always pull out and discard or transplant the extra seedlings.
    6. If you are planting more than one type of plant (i.e., wild type and mutant plants), plant the different varieties in different plant trays so that they can be separated. This will limit the chances of cross pollination when they begin to produce flower and help you to maintain your own seed stocks.
    7. Cover the top of the containers with clear plastic wrap to keep the moisture in.
    8. Poke a few small holes in the wrap with a toothpick or other small device to allow for air to enter and leave the container.
    9. Place the seeds in the refrigerator for 48– 96 hours (over two nights is suggested). Cold shocking the seeds helps to get them to germinate.
    10. After the cold shock period, place the plants under plant grow lights or in a sunny location. Seeds should germinate in 3– 5 days depending on conditions (temp, etc.).
    11. When the majority of the seeds have germinated you can pull the plastic wrap from your pots and begin to care for your plants as listed below.

     Care for the plants

    1. Check the pots each day to make sure that they are still damp. Again best to maintain a constant level of water in a reservoir under the pots and let the water wick up into the pots.
    2. After a few days of growth, begin to thin out the seedlings. Each plant should have 3– 5cm of space to be able to grow. With a little bit of care, you can pick the extra plants from one pot and transfer them to other pots to fill in for seeds that did not germinate.
    3. It will take about 2– 3 weeks for the plants to reach full size and should begin to flower soon after.

     Growing Arabidopsis in agar

    1. Take your seeds and place them into a 1.5ml tube with 500ml of 30% bleach solution. Soak the seeds in the bleach for 5 minutes, shaking the tube occasionally. This will help to kill off any bacteria that may be on the seeds. The media that the plants will be growing on is also be good for bacterial growth, so it is important that the seeds be clean before we plant them.
    2. Spin the seeds in the centrifuge at low speed for 30 seconds and pipette off the liquid. Be careful not to pipette off the seeds!
    3. Add 200ml of sterile water to the tube, gently shake, centrifuge on low for 30 seconds and pipette off the liquid again.
    4. Repeat step 3 (washing with water) twice more, pipetting off the water after each step.
    5. Add 100ml of 0.1% agarose solution to the tube.
    6. Obtain a dish with the appropriate growth media in it.
    7. Use a 1000ml pipette to pipette out the seeds and the 0.1% agarose solution.
    8. Gently pipette the desired number of seeds on the surface of the agar. Try to space the seeds evenly on the dish.
    9. Replace the cover the dish and place the dish in the refrigerator overnight.
    10. The next day place the plants under plant grow lights.
    11. Check and measure your plants every day for growth.

      Seed collection

    1. When the plants begin to produce seeds, harvesting the seeds will allow you to create a stock of your own seeds.
    2. Wait for the seed pods to dry out (better to wait longer rather than rush this) and carefully pull them off of the plants. I have had luck collecting the seeds by cutting the whole seed stalk off the plant and removing the seeds from the pods by gently rubbing them between gloved fingers so that they fall onto a clean sheet of white paper.
    3. The hardest part of collecting the seeds was separating the seeds from the pods, flower parts and leaves. The best approach I have found is placing the whole mix on a sheet of paper, angling the paper and gently tapping the mix to the bottom. The smallest pieces (seeds) tend to fall faster than the bigger pieces (pods, flowers, etc.). Let the seeds fall onto a new sheet of paper and then discard the chaff on the paper. After a few transfers from sheet to sheet, you should have a fairly pure seed collection. You can use a dry paint brush to clean out the remaining debris.
    4. Place your seed collection into an appropriate storage container. I have been using 1.5 or 2ml tubes to keep my seeds and they seem to work well.
    5. Make sure to label the tubes with the type of seed and the date collected.