Wheat root exudate harvesting

From ActinoBase


Wheat root exudates and the recruitment of root microbes

...

You will need:

Preparation of growth tubes

  • 50ml falcon/centrifuge tubes (1 tube = 1 plant)
  • Electric hand drill
  • 10mm drill bit
  • 2ml microcentifuge tubes
  • Dog nail cutters
  • Vermiculite (washed and autoclaved)

Growing of wheat plants

  • Wheat seeds
  • 70% ethanol
  • 3% bleach
  • dH2O
  • Sterile universal tube
  • Sterile tweezers
  • Water agar plates
  • 1/2 MS0 liquid media

Exudate harvesting

  • 2 1L duran bottles
  • 0.22 micron sterile Büchner filter/bottletop vaccum filter funnel
  • Vacuum pump
  • 50ml falcon/centrifuge tubes
  • Syringe needle or sharp pointed item
  • -80°C freezer
  • Freeze drier
  • dH2O
  • 4ml HPLC vile

Protocol:

Week 1, Day 1

Preparation of growth tubes

  1. Prepare as many tubes as plants required (1 batch of 50 tubes ~ ... mg final exudate).
  2. Drill a 10mm hole in each tube and rinse with water to remove any plastic debris.
  3. Using the dog nail cutters, cut the end of a 2ml microcentrifuge tube to create a ~0.5cm opening. Loosely fill the tube with the prepared vermiculite and close the lid.
  4. Push the microcentrifuge tube into the hole of the 50ml falcon.
  5. Autoclave.

Growing of wheat plants

  1. Sterilise the wheat seeds.
  2. Spreadingly put the seeds onto a water agar plate and place into the fridge.

Week 1, Day 3

Growing of wheat plants

  1. Remove the water agar plates from the fridge and leave on the bench.

Week 1, Day 4

Growing of wheat plants

  1. Fill the falcon tubes with ~45ml 1/2 MS0 media.
  2. When the seeds have sprouted two roots of ~0.5-1cm long, transfer the seed to the vermiculite tube so that the roots are pointing downwards into the vermiculite.
  3. Keep the growth tubes in a controlled environment room (...)

Week 2-4

Growing of wheat plants

  1. Keep the growth tubes topped up with 1/2 MS0 media.

Week 4, day 4

Growing of wheat plants

  1. Pour away the 1/2 MS0 and rinse the roots and tube with water (to remove any remaining salts).
  2. Fill the growth tubes with ~45ml fresh water (this will be the media which the exudates will be extracted from).
  3. Keep the growth tubes in a controlled environment room.

Week 5

Growing of wheat plants

  1. Keep the growth tubes topped up with sterile water.

Week 5, day 4

Exudate harvesting

  1. Pour the water from the growth tubes into a clean Duran bottle.
  2. Attach a sterile 0.22 micron Büchner filter to another sterile Duran bottle and fix to a vacuum pump.
  3. Pour the exudate solution into the Büchner filter and turn on the vacuum pump.
  4. Seperate the filtered exudate solution into clean 50ml falcon tubes (about 25-30ml each) and freeze at -80°C.

Week 5, day 5

Exudate harvesting

  1. Pierce a few holes in the lids of the frozen 50ml falcon tubes.
  2. Place the tubes into a freeze drier and leave over the weekend.

Week 6, day 1

Exudate harvesting

  1. The water should have evaporated and be left with some fluffy/web-like exudate.
  2. Resuspend the exudate in minimal sterile water so that it fits into a pre weighed (without lid) 4ml HPLC vile.
  3. Dry down your sample again (on a freeze drier or alternatively a Genevac) and weigh.
  4. Your sample can be resuspended at a known concentration. It is now ready for use.