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.
   1. 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.