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Achillea millefolium belonging to the Astreaceae family is an endangered medicinal plant of Jordan and of its neighboring countries. As an alternative to seed propagation, an efficient micropropagation of A. millefolium and its subsequent rooting were developed as an option for its in vitro conservation. A maximum of 5.9 shoots per microshoot were obtained on Murashige and Skoog agar medium supplemented with 0.9 mg L^-1 of 6-Benzyl Amino Purine (BAP). The effect of different types and concentrations of auxins were tested, i.e. IBA (Indole-3-Butyric-Acid), IAA (Indole-3-Acetic-Acid) or Naphthalene Acetic Acid (NAA). Maximum root number (20.8 roots ex-plant^-1) was obtained from media containing 1.2 mg L^-1 of IBA. A survival of 70% was obtained when rooted explants were acclimatized in vivo in equal portions of perlite and peat soil. In vitro, A. millefolium shoots were successfully stored for up to 32 weeks on MS medium supplemented with different concentrations of either sucrose, glucose or fructose, at 24±2°C. After 32 weeks past, 88.6% of the shoots survived on the medium supplemented with 3% sucrose. Moreover, 85.3% of the shoots were able to re-grow when stored under light conditions. Cryopreservation through vitrification was successfully achieved (80% re-growth) when shoot tips precultured on a medium supplemented with 0.4M sorbitol and 0.1M sucrose for 1 day, followed by loading shoot tips with concentrated plant vitrification solution 2 (PVS2) for 20 minutes, then being dehydrated with PVS2 for 60 minutes at 0°C prior to storage in Liquid Nitrogen (LN).

Keywords: Achillea millefolium, Liquid nitrogen, Long-term conservation, Shoot tips, Vitrification

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