Sterilization and Germination Protocol for ''Arabidopsis thaliana'' Seeds in Gnotobiotic Experiments

Version 1: Adapted from ABRC's Seed Handling FAQ for Seed Handling

Citation: ABRC Seed Handling FAQ PDF [1], accessed Sept. 23, 2018.

1. Surface sterilize seeds in microcentrifuge tubes by soaking for 20 min in 50% household bleach with the addition of 0.05% Tween®20 detergent.
2. Remove all bleach residue by rinsing 5-7 times with sterile distilled water.
   1. Add 1 mL of sterile distilled water to epitube and invert once; remove water and continue 5 more times.
3. For planting of individual seeds at low density, adhere one seed to the tip of a pipette using suction, then release seed onto the agar in desired location. For planting seeds at higher densities, mix seeds in sterile distilled water (or 0.1% cooled top agar), pour onto plate, and immediately swirl to achieve even distribution. Use a sterile pipet tip to adjust the distribution and remove excess water. Allow the water or top agar to dry slightly before placing lid onto plate.
4. Seal with Micropore paper tape to prevent desiccation, while allowing slight aeration.
5. Place the plates at 4°C for 3 days. This cold treatment, also called stratification, will improve the rate and synchrony of germination. The use of an extended cold treatment of approximately 7 days is especially important for freshly harvested seeds, which have more pronounced dormancy. An extended cold treatment is also necessary for certain natural accessions (e.g., Dobra-1, Don-0, Altai-5, Anz-0, Cen-0, WestKar-4). Cold treatment of dry seeds is usually not effective in breaking dormancy.
   1. NOTE: Experiments will be conducted that will determine what amount of cold stratification leads to maximum synchrony in seed germination time for Col-0 Arabidopsis thaliana seeds.

Version 2

NOTE: This version was created after we practiced pipetting seeds with a glycerol/water mixture, and after we determined the amount of cold stratification that is ideal for germination synchrony.

Citation: ABRC Seed Handling FAQ PDF [2], accessed Sept. 23, 2018.

1. Surface sterilize seeds in microcentrifuge tubes by soaking for 20 min in 50% household bleach with the addition of 0.05% Tween®20 detergent.
2. Remove all bleach residue by rinsing 5-7 times with sterile distilled water.
   1. Add 1 mL of sterile distilled water to epitube and invert once; remove water and continue 5 more times.
3. Create a 1:1 solution of sterile glycerol (99% concentration) and sterile milliQ water by combining 200 uL of each in an epitube and carefully vortexing to mix. Add this solution to the sterilized seeds, pipet up and down to mix, and then pipet the solution drop by drop onto the surface of the growing medium until enough seeds are placed on the medium's surface.
4. Place the lid back on the grow box. Repeat the above step until all grow boxes are planted.
5. Place the boxes at 4°C for 3 days. This cold treatment, also called stratification, will improve the rate and synchrony of germination. The use of an extended cold treatment of approximately 7 days is especially important for freshly harvested seeds, which have more pronounced dormancy. An extended cold treatment is also necessary for certain natural accessions (e.g., Dobra-1, Don-0, Altai-5, Anz-0, Cen-0, WestKar-4). Cold treatment of dry seeds is usually not effective in breaking dormancy.
   1. NOTE: We determined that a time period of 7 days was sufficient to ideally cold-stratify our Col-0 Arabidopsis thaliana seeds. However, for experiments where germination synchrony is not very needed, cold stratification for 3 days is fine.

Version 3

NOTE: This version was created after we practiced sterilizing seeds and pipetting them onto MS media on 10/31/2018.

Citation: ABRC Seed Handling FAQ PDF [3], accessed Sept. 23, 2018.

1. Surface sterilize seeds in microcentrifuge tubes by soaking for 20 min in 800 mL of 50% household bleach with the addition of 200 mL of 0.05% Tween®20 detergent.
   1. In our first sterilization we accidentally used 0.5% Tween20, and so the results of this more-concentrated Tween20 on overall seed sterilization will be noted in later lab entries.
2. Remove all bleach residue by rinsing 5-7 times with sterile distilled water.
   1. Add 1 mL of sterile distilled water to epitube and invert once; remove water and continue 5 more times.
   2. If planting seeds that are not to be sterilized but that will be planted into sterile conditions, skip the above steps and begin at the step following this one.
3. Create a 1:1 solution of sterile glycerol (99% concentration) and sterile milliQ water by combining 200 uL of each in an epitube and carefully vortexing to mix. Add this solution to the sterilized seeds along with an equal volume of sterile MilliQ water, pipet up and down to mix, and then pipet the solution drop by drop onto the surface of the growing medium until enough seeds are placed on the medium's surface.
   1. One method of seed placement is to place 4 individual seeds on the MS media equidistant from both the edges of the grow-box and the other seeds.
4. Place the lid back on the grow box. Repeat the above step until all grow boxes are planted.
5. Place the boxes at 4°C for 3 days. This cold treatment, also called stratification, will improve the rate and synchrony of germination. The use of an extended cold treatment of approximately 7 days is especially important for freshly harvested seeds, which have more pronounced dormancy. An extended cold treatment is also necessary for certain natural accessions (e.g., Dobra-1, Don-0, Altai-5, Anz-0, Cen-0, WestKar-4). Cold treatment of dry seeds is usually not effective in breaking dormancy.
   1. NOTE: We determined that a time period of 7 days was sufficient to ideally cold-stratify our Col-0 Arabidopsis thaliana seeds. However, for experiments where germination synchrony is not very needed, cold stratification for 3 days is fine.

Version 4

NOTE: This version was created after talking with David Higgins (Haverford Faculty) about the best ways to pipet Arabidopsis seeds.

Citation: ABRC Seed Handling FAQ PDF [4], accessed Sept. 23, 2018.

1. Obtain approximately 5 glass pasteur pipettes and a few rubber bulbs - place in a foil packet and autoclave to sterilize.
   1. If possible, pasteur pipettes with a filter are the best option as they reduce the risk of contamination between the rubber bulb and the pipet.
2. Surface sterilize seeds in microcentrifuge tubes by soaking for 20 min in 800 uL of 50% household bleach with the addition of 200 uL of 0.05% Tween®20 detergent.
   1. To create 200 uL of 0.05% Tween20 from concentrated Tween20, add 5 uL of concentrated Tween20 to 995 uL of sterile MilliQ water, vortex, and then add 20 uL of that mixture to 180 uL of sterile MilliQ water.
3. Remove all bleach residue by rinsing 5-7 times with sterile distilled water.
   1. Add 1 mL of sterile distilled water to epitube and invert once; remove water and continue 5 more times.
   2. If planting seeds that are not to be sterilized but that will be planted into sterile conditions, skip the above steps and begin at the step following this one.
4. Create a 1:1 solution of sterile glycerol (99% concentration) and sterile milliQ water by combining 200 uL of each in an epitube and carefully vortexing to mix. Add this solution to the sterilized seeds, pipet up and down to mix, and then pipet the solution using a pasteur pipet to drop only one seed onto the surface of the growing medium for each grow-box.
5. Place the lid back on the grow box. Repeat the above step until all grow boxes are planted.
6. Place the boxes at 4°C for 7 days. This cold treatment, also called stratification, will improve the rate and synchrony of germination.
   1. NOTE: We determined that a time period of 7 days was sufficient to ideally cold-stratify our Col-0 Arabidopsis thaliana seeds. However, for experiments where germination synchrony is not very needed, cold stratification for 3 days is fine.

Version 5

NOTE: This version was created to cut out the step using glycerol solution as a vehicle for pipetting seeds.

Citation: ABRC Seed Handling FAQ PDF [5], accessed Sept. 23, 2018.

1. Obtain approximately 5 glass pasteur pipettes and a few rubber bulbs - place in a foil packet and autoclave to sterilize.
   1. If possible, pasteur pipettes with a filter are the best option as they reduce the risk of contamination between the rubber bulb and the pipet.
2. Surface sterilize seeds in microcentrifuge tubes by soaking for 20 min in 800 uL of 50% household bleach with the addition of 200 uL of 0.05% Tween®20 detergent.
   1. To create 200 uL of 0.05% Tween20 from concentrated Tween20, add 5 uL of concentrated Tween20 to 995 uL of sterile MilliQ water, vortex, and then add 20 uL of that mixture to 180 uL of sterile MilliQ water.
3. Remove all bleach residue by rinsing 5-7 times with sterile distilled water.
   1. Add 1 mL of sterile distilled water to epitube and invert once; remove water and continue 5 more times.
   2. If planting seeds that are not to be sterilized but that will be planted into sterile conditions, skip the above steps and begin at the step following this one.
4. Add 1 mL sterile milliQ water to the sterilized seeds, pipet up and down to mix, and then pipet the solution using a pasteur pipet to drop only one seed onto the surface of the growing medium for each grow-box.
5. Place the lid back on the grow box. Repeat the above step until all grow boxes are planted.
6. Place the boxes at 4°C for 7 days. This cold treatment, also called stratification, will improve the rate and synchrony of germination.
   1. NOTE: We determined that a time period of 7 days was sufficient to ideally cold-stratify our Col-0 Arabidopsis thaliana seeds. However, for experiments where germination synchrony is not very needed, cold stratification for 3 days is fine.

Version 6

Citation: ABRC Seed Handling FAQ PDF [6], accessed Sept. 23, 2018.

1. Obtain approximately 5 glass pasteur pipettes and a few rubber bulbs - place in a foil packet and autoclave to sterilize.
   1. If possible, pasteur pipettes with a filter are the best option as they reduce the risk of contamination between the rubber bulb and the pipet.
2. Surface sterilize seeds in microcentrifuge tubes by soaking for 20 min in 800 uL of 50% household bleach with the addition of 200 uL of 0.05% Tween®20 detergent.

Note: This was changed from Hope Ebert's original protocol based on the previous versions

1. 1. To create 200 uL of 0.05% Tween20 from concentrated Tween20, add 5 uL of concentrated Tween20 to 995 uL of sterile MilliQ water, vortex, and then add 20 uL of that mixture to 180 uL of sterile MilliQ water.
2. Remove all bleach residue by rinsing 5-7 times with sterile distilled water.
   1. Add 1 mL of sterile distilled water to epitube and invert once; remove water and continue 5 more times.
   2. If planting seeds that are not to be sterilized but that will be planted into sterile conditions, skip the above steps and begin at the step following this one.
3. Add 1 mL sterile milliQ water to the sterilized seeds, pipet up and down to mix, and then pipet the solution using a pasteur pipet to drop only one seed onto the surface of the growing medium for each grow-box.
4. Place the lid back on the grow box. Repeat the above step until all grow boxes are planted.
5. Place the boxes at 4°C for 7 days. This cold treatment, also called stratification, will improve the rate and synchrony of germination.
   1. NOTE: We determined that a time period of 7 days was sufficient to ideally cold-stratify our Col-0 Arabidopsis thaliana seeds. However, for experiments where germination synchrony is not very needed, cold stratification for 3 days is fine.

Note for future research: Although we determined that a 50/50 solution of bleach is effective at sterilizing seeds without impacting healthy A. thaliana growth, further experiments testing the effects of less amounts of bleach in the sterilizing solution would be helpful for optimizing the gnotobiotic system.

Version 7

Note: This is a updated protocol based on growing the Arabidopsis thaliana in soil and modifying the previous versions of this protocol. This protocol goes through both the preparation of the boxes, the sterilization steps, and the best practices to plant the seeds evenly.

The original procedure is based off of the ABRC Seed Handling FAQ sheet.

Citation: ABRC Seed Handling FAQ PDF [7], accessed Sept. 23, 2018.

Creating Sterile Soil Boxes

Day 1

1. Put Scotts® Premium Topsoil in a large plastic container.
2. Shake the container to bring the large chunks to the top and break up the largest pieces with a shovel.
3. Measure out 65 g of soil into each box.
4. Secure the lids and mark the tray to be autoclaved.
5. Autoclave the boxes.

Sterilizing the Seeds

Day 2

1. Obtain approximately 5 glass pasteur pipettes and a few rubber bulbs - place in a foil packet and autoclave to sterilize.
   1. If possible, pasteur pipettes with a filter are the best option as they reduce the risk of contamination between the rubber bulb and the pipet.
2. Surface sterilize seeds in microcentrifuge tubes by soaking for 20 min in 800 uL of 50% household bleach with the addition of 200 uL of 0.05% Tween®20 detergent.
   1. To create 200 uL of 0.05% Tween20 from concentrated Tween20, add 5 uL of concentrated Tween20 to 995 uL of sterile MilliQ water, vortex, and then add 20 uL of that mixture to 180 uL of sterile MilliQ water.
3. Remove all bleach residue by rinsing 5-7 times with sterile distilled water.
   1. Add 1 mL of sterile distilled water to epitube and invert once; remove water and continue 5 more times.

Planting the Seeds

Note: Perform this in sterile conditions.

1. Add 1 mL sterile milliQ water to the sterilized seeds, pipet up and down to mix, and then pipet the solution using a pasteur pipet to drop only one seed onto the surface of the growing medium for each grow-box.
2. Place the lid back on the grow box. Repeat the above step until all grow boxes are planted.
3. Place the boxes at 4°C for 7 days. This cold treatment, also called stratification, will improve the rate and synchrony of germination.

NOTE: We determined that a time period of 7 days was sufficient to ideally cold-stratify our Col-0 Arabidopsis thaliana seeds. However, for experiments where germination synchrony is not very needed, cold stratification for 3 days is fine.