Selection scheme used at GABI-Kat for resistance to sulfadiazine


Sterilisation of A.thaliana seed

  1. incubate seed in sterilisation solution (see table below - 1 ml for up to ~2500 seed) for 7 min, shake gently
  2. spin down seeds
  3. aspirate supernatant
  4. add ~1 ml sterile water (sterile work bench), mix and spin down again
  5. repeat wash 5 times
  6. after the last wash, transfer suspended seed to a Petri dish


"Plating" of seed

  1. place single seed on a Petri dish containing MS-Agar containing sulfadiazine
  2. include seed of accession Columbia as a negative control
  3. seal plate with Parafilm and incubate for 4-5 days at 4 to 8 C (cold room or fridge)
  4. transfer plate to a light room / growth chamber (long day conditions, 18 - 20 C)
  5. after 4 to 5 days, you may determine the germination rate
  6. after 14 days, a clear difference between resistant and sensitive plants should be visible (all yellow plantlets which did not form real first leaves are considered to be sensitive)

Note: in case of a fungal infection of the germinating seedlings, we use MS plate medium without sucrose. Alternatively, we sow single seeds without sterilisation directly on soil. 


MS plate medium (1 l)

  • 4.3 g MS salts (Duchefa M-0221)
  • 30 g Sucrose (saccharose)
  • 0.1 g Myo-Inositol
  • 0.5 g MES
  1. fill up to 1 l with water, adjust pH to 5.7 with KOH, distribute into 2 bottels containing 4.5 g Agar each, autoclave
  2. allow to cool to about 50 C, add the 4 vitamins from stock solutions (0.5 ml each per 0.5 l) and sul solution (0.5 ml per 0.5 l)


Vitamin/Antibiotic stocks

  • Biotin: 100 mg per 100 ml
  • Nicotinic acid: 100 mg per 100 ml
  • Pyridoxine: 100 mg per 100 ml
  • Thiamin: 2.0 g per 100 ml
  • Sulfadiazin: 75 mg per 10 ml (Sodium salt of sulfadiazin: 4-amino-N-[2-pyrimidinyl]benzene-sulfonamide-Na, Sigma S-6387)
Note: Water is used as solvent for the preparation of all stock solutions.


Sterilisation solution

  • 20 ml DanKlorix (Colgate-Palmolive GmbH)
  • 40 ml water
  • 2.5 ml N-lauryl-sarcosine-sol. (20%) 


Note: Please read the protocol critically and let us know if there are unclear descriptions.