Arabidopsis / Triphysaria well plate assay

 

1)      Sterilize Arabidopsis seeds (70% EtOH for 5 min, 25% bleach + 0.1% Triton for 10 min.) Plate on 1/4 MS 0.4% Phytagar germination plates. Put directly in 16C or store at 2C for up to one month.

2)      Sterilize Triphysaria seeds (70% EtOH for 10 min, 50% bleach + 0.2% Triton for 30 min.) Plate on 1/4 Hoagland's 0.6% Phytagar germination plates. Put in 2C for 7 to 30 days.

3)      Make 1/4 Hoagland's media in culture bottles. Autoclave 45 min liquid. Cool bottles in hot water bath. Once cooled, shake bottles to get all Phytagar in solution. In hood, pipette 2 ml media into each well of a 24 well plate using sterile disposable 25 ml pipettes.

4)      Germinate Arabidopsis and Triphysaria seeds at 16C 10 to 14 days before transferring to well plates.

5)      Using a sterile transfer pipette, add 3 drops of sterile water to each well. Using sterile forceps, add 2 Triphysaria plants and 1 Arabidopsis plant to each well.

6)      Wrap sides of well plates with one layer of micropore tape. Store plates in clear plastic containers in 16C growth room.

7)      After 3 weeks, examine the bottom of the wells for haustoria. Continue scoring plates once a week for 5 weeks or until all of the wells have haustoria.

8)      After 5 weeks, transplant non-inducing Arabidopsis plants. Fill a 4 inch square pot with sunshine mix and soak with 1X Hoagland's solution. Make a small hole in the soil with forceps. Carefully remove Arabidopsis plant from well with forceps and push roots into hole in soil. Pat down soil around plant and spray base of plant with water to remove air pockets in soil. Put plant in a flat and cover with a plastic lid. Grow in 22C growth chamber with cover on for one week. Remove cover and grow to seed. Water every 1 to 3 days with 1X Hoagland's solution.

Note: Assay may be changed to determine intraspecific interactions by adding two Triphysaria and no Arabidopsis in step 5. Assay may also be used to study autohaustoria by using 48 well plates, adding 1 ml media per well and 1 Triphysaria per well. In addition, the assay may be used to study other hosts by substituting a different host in step 5.