Wheat root exudate harvesting
From ActinoBase
Wheat root exudates and the recruitment of root microbes
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You will need:
Preparation of growth tubes
- 50ml falcon/centrifuge tubes
- 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
- Prepare as many tubes as plants required (1 batch of 50 tubes ~ ... mg final exudate).
- Drill a 10mm hole in each tube and rinse with water to remove any plastic debris.
- 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.
- Push the microcentrifuge tube into the hole of the 50ml falcon.
- Autoclave.
Growing of wheat plants
- Sterilise the wheat seeds.
- Spreadingly put the seeds onto a water agar plate and place into the fridge.
Week 1, Day 3
Growing of wheat plants
- Remove the water agar plates from the fridge and leave on the bench.
Week 1, Day 4
Growing of wheat plants
- Fill the falcon tubes with ~45ml 1/2 MS0 media.
- 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.
- Keep the growth tubes in a controlled environment room (...)
Week 2-4
Growing of wheat plants
- Keep the growth tubes topped up with 1/2 MS0 media.
Week 4, day 4
Growing of wheat plants
- Pour away the 1/2 MS0 and rinse the roots and tube with water (to remove any remaining salts).
- Fill the growth tubes with ~45ml fresh water (this will be the media which the exudates will be extracted from).
- Keep the growth tubes in a controlled environment room.
Week 5
Growing of wheat plants
- Keep the growth tubes topped up with sterile water.
Week 5, day 4
Exudate harvesting
- Pour the water from the growth tubes into a clean Duran bottle.
- Attach a sterile 0.22 micron Büchner filter to another sterile Duran bottle and fix to a vacuum pump.
- Pour the exudate solution into the Büchner filter and turn on the vacuum pump.
- 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
- Pierce a few holes in the lids of the frozen 50ml falcon tubes.
- Place the tubes into a freeze drier and leave over the weekend.
Week 6, day 1
Exudate harvesting
- The water should have evaporated and be left with some fluffy/web-like exudate.
- Resuspend the exudate in minimal sterile water so that it fits into a pre weighed (without lid) 4ml HPLC vile.
- Dry down your sample again (on a freeze drier or alternatively a Genevac) and weigh.
- Your sample can be resuspended at a known concentration. It is now ready for use.