In vitro germination of Triphysaria

Seed sterilization:

Place approximately 200 seeds in a 15ml polypropylene culture tube and add 5 ml of 70% ethanol.  Allow seeds to soak in ethanol for 10 minutes with occasional mixing.

Remove ethanol with a sterile Pasteur pipette and discard.

Add 5 ml of household bleach (sodium hypochlorite 5.25%) and 5 ml of a 0.02% Triton X-100 (Sigma Aldrich, St. Louis, MO) detergent solution to make a sterilization solution of 50% (v/v) bleach and 0.01% (v/v) detergent.  Cap the tube tightly and, if necessary, seal the cap with parafilm to prevent leakage.

Place the tube horizontally on a shaker for 30 min at approximately 20 rpm to ensure contact of seeds with the sterilization solution.  Longer incubations in the sterilization solution will dramatically decrease germination and may kill all of the seeds.

After 30 minutes, carefully decant bleach/detergent mix into the sink and watch for escaping seeds.  Rinse seeds a minimum of 10 times with sterile water by filling the tube with sterile water and decanting. Continue rinsing if you still smell bleach.

Suspend seeds in approximately 10 ml of either sterile water or 0.1% Noble agar.  Plate immediately. 

Plating seeds:

After cleaning the laminar flow hood with 70% ethanol, arrange agar plates, Pasteur pipettes, and tubes containing sterilized seeds in hood.  Wear clean gloves, sprayed with 70% ethanol. Use a pipette to remove about half of the seeds (~100) from a tube and place them on the agar surface of a plate. *Care must be taken to avoid contaminating the agar—do not let the pipet tip touch any non-sterile surface and obtain a new pipette for each sample.  When plating seeds, avoid breathing/sneezing on the agar plates and depending on type of flow hood, keep hands/arms out of air flowing towards the plates.

When plating seeds that are suspended in sterile water, remove all water from the plate with a sterile pipette and ensure that seeds are well-spaced on the surface of the agar.  When plating seeds suspended in 0.1% Phytagar, ensure that seeds are well-spaced. Allow the plates to dry for approximately 20 minutes before sealing with parafilm.

Place plates in the 4°C cold room for 1-2 days, up to 2 weeks (optional cold treatment to increase the rate and synchronicity of germination). 

Move plates to the 16°C growth chamber room (fluorescent lighting—12hr day length) to stimulate germination.  Seeds will begin to germinate within one week and most will germinate within 10 days.