cytokinin preparation

This method describes the isolation of cytokinins from snapdragon tissue material as published by Okubo H et al. 1991.
  1. Collect fresh plant material (from 2 to 20 g).
  2. Lyophilize material
  3. Ground
  4. extract with 80% cold methanol at 5 degrees Celsius for 24h by shaking in the dark
  5. centrifuge
  6. filtrate
  7. reduce in vacuo at 37 degrees Celsius to aqueous solution
  8. extract with ethylacetate at pH 3.0
  9. adjust to pH 2.0 with HCl
  10. pass through a DOWEX 50 W column and elute with 5% NH4OH
  11. concentrate the eluate in vacuo
  12. extract four times with n-butanol at pH 7.0
  13. evaporate the n-butanol extract to dryness
  14. adjust to 5% methanol with distilled water
  15. pass final extract through a C18 cartridge (Waters Associates)
  16. wash with 90% methanol
  17. evaporate to dryness