tissue culture

This is the method for tissue culture of Antirrhinum majus as described in Atkinson NJ et al. 1988.
  1. Wash seeds with 70% (v/v) ethanol.
  2. Wash seeds with 2.5% (v/v) household bleach.
  3. Place 10 seeds in 25 X 150mm Pyrex tubes containing 15ml hormone-free culture medium with 1% sucrose.
  4. Cap with autoclave tape.
  5. Incubate at 25 degrees C with 16h daylight.
  6. After 24 days remove shoot tip just below the cotyledones and just above the root.
  7. Place four segments each in 90mm Petri dish, containing
    1. 0.25 mg per liter naphtoxyacetic acid (NOA)
    2. 10% coconut milk (CM) prepared according to Dodds JH et al. 1985 but only heated to 60 degrees for 30 minutes.
  8. Seal with Parafilm (Amer. Can. Co.)
  9. Subculture at 24 day intervals.
  10. Remove regenerated shoots longer than 4mm.
  11. Place shoot tips base down in 25 X 150 mm Pyrex tubes with 15ml of 1mg per liter benzylaminopurine (BAP).