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Phyllosphere protocols

Creating Sterile Agar Plates

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

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

1. Add 4.31 g of Murashige and Skoog (MS) basal salt mixture and 0.5 g of 2-(N-Morpholino) ethanesulfonic acid (MES) to a beaker containing 0.8 L of distilled water and stir to dissolve. Add distilled water to final volume of 1 L. Check and adjust pH to 5.7 using 1M KOH.

2. Divide the media into two 1 L glass bottles, 500 mL in each. Add 5 g of agar granulated per bottle. Keep the lid loose.

3. Autoclave for 20 min at 121°C, 15 psi with a magnetic stirring device in the bottle.

4. Place the bottles on a stir plate at low speed, and allow the agar medium to cool to 45-50°C (until the container can be held with bare hands).

5. Starting from this step, perform all the steps in sterile conditions in a laminar flow hood. Add (optional) 1-2% sucrose and 1 mL Gamborg’s Vitamin Solution, stirring to evenly dissolve. Optional sucrose and vitamins should be added after autoclaving and only after the agar media cools, because vitamins are thermo-labile and 15-25% of the sucrose may be hydrolyzed to glucose and fructose at elevated temperatures. Plants grow more vigorously and quickly on media containing 1-2% of sucrose, however, fungal and bacterial contamination must be rigorously avoided by seed sterilization. Note that germination of some mutants might be delayed on sucrose-containing media.

6. Label the bottom of Petri plates with identification number or name, including the date.

7. Pour enough media into plates to cover approximately half of the depth of the plate.

8. Allow the plates to cool at room temperature for about an hour to allow the agar to solidify. If the plates are not to be used immediately, wrap them in plastic and store at 4°C (refrigerator temperature). Covered plates, boxes, or tubes with solidified agar can be stored for several weeks at 4°C in a container that prevents desiccation.

NOTE: Our lab will likely be using Magnenta GA-7 plant culture boxes rather than petri dishes - if any changes to this protocol are necessary due to this difference, it will be addressed in Version 2 of this protocol.

Sterilization and Germination Protocol for Arabidopsis thaliana Seeds in Gnotobiotic Experiments

Version 1: Based on ABRC's Seed Handling FAQ for Seed Handling

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. 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.

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