Cryopreservation of Thymus lotocephalus shoot tips and assessment of genetic stability.

ABSTRACT

BACKGROUND: Thymus lotocephalus is a rare endemic species from the Algarve, Portugal, and is legally protected by Portuguese and European legislation. OBJECTIVE: The aim is to develop a cryopreservation protocol for T. lotocephalus shoot tips, as an alternative approach for the long-term conservation of this species. METHODS: Several methods (droplet-vitrification, vitrification and encapsulation-dehydration) were tested. Conditions regarding the subculture period, cold-hardening and preculture were optimized. Cryopreserved shoot tips were also assessed for their genetic stability using RAPD markers. RESULTS: Droplet-vitrification presented the best results. The best regrowth of cryopreserved shoot tips obtained eight weeks after rewarming was 67%. This was accomplished with four weeks subculture period of in vitro-donor plants at 25 degree C, preculture of excised shoot tips for one day on MS medium containing 0.3 M sucrose, treatment in PVS2 for 60 min, and MS supplemented with 0.2 mg per L zeatin as recovery medium. The assessment using RAPD markers observed variation at a low frequency and shoots regenerated from cryopreserved apices showed normal development compared to the regular in vitro-grown shoots. CONCLUSION: Droplet-vitrification is thus a viable method for the cryopreservation of T. lotocephalus shoot tips.