Arabidopsis Thaliana Root Exudate

Arabidopsis thaliana root exudate

1. Seed 100 sterilized seeds on a autoclaved Whatman paper filter disc placed on top of a MS medium plate. Use at least 3-5 plates for each technical replicate of the same treatment to have enough root exudate to perform successive analysis steps or to discard eventually contaminated plates.

2. Incubate plates in a growth chamber for 1 month. (discard eventually contaminated plates)

3. Carefully remove the paper disc from the plate and avoid that plants standing on the disc fall down

4. Put the paper disc with the adhered plants in a sterile petri dish filled with 20 ml of sterile H2O (this step is needed to avoid nutrient contamination of the root exudates)

5. Wait 20 minutes, remove the water and replace it with 20 ml of new sterile H2O

6. Repeat step 5 for 4 additional times to make sure that all residual nutrients are gone (fundamental!!)

7. Carefully put the paper disc with the adhered plants into a new sterile petri dish filled with 5 ml of H2O.

8. Parafilm the plate and incubate it for additional 2 days in a growth chamber. (discard eventually contaminated plates)

9. Recover the root exudate and pool those from a single technical replicate.

10. Filter sterilize the exudates through 0.2 µ nylon filters

11. Freeze the exudates in 15 ml falcon tubes containing each a volume of approximately 5 ml (write down on each tube the exact volume of root exudate added …. In order to know in how much volume to resuspend them after lyophilization) and lyophilize them.

MS Medium plates

Murashige and Skoog 4.4 g

Sucrose 30 g

H2O up to 1L

Adjust pH to 5.8 with KOH

Add Agar Plant 8 g

Autoclave

Seed sterilization

Put the desired amount of seeds (100 seeds=2 mg approx) in an epphendorf tube

Add 1 ml of 95% etoh and vortex gently for 1 minute

Remove supernatant

Add 1ml of sodium hypochlorite 50% and vortex...