title: 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.
  
  
  CONCLUSIONS
  Droplet-vitrification is thus a viable method for the cryopreservation of T. lotocephalus shoot tips.
authors.affiliation: IBB/CGB, Faculty of Sciences and Technology, University of Algarve, Campus de Gambelas, Faro, Portugal.
authors.forename: Natacha
authors.initials: N
authors.lastname: Coelho
authors.affiliation: Departamento de Biologia Vegetal, Universidad Politecnica de Madrid, Ciudad Universitaria, Madrid, Spain.
authors.forename: María Elena
authors.initials: ME
authors.lastname: González-Benito
authors.affiliation: Departamento de Biologia Vegetal, Universidad Politecnica de Madrid, Ciudad Universitaria, Madrid, Spain.
authors.forename: Carmen
authors.initials: C
authors.lastname: Martín
authors.affiliation: IBB/CGB, Faculty of Sciences and Technology, University of Algarve, Campus de Gambelas, Faro, Portugal. aromano@ualg.pt.
authors.forename: Anabela
authors.initials: A
authors.lastname: Romano
mesh_terms: D000222:Adaptation, Physiological; D003247:Conservation of Natural Resources; D015925:Cryopreservation; D003451:Cryoprotective Agents / Q000494:pharmacology; D003470:Culture Media; D018744:DNA, Plant / Q000235:genetics*; D003890:Desiccation; D056727:Endangered Species; D018745:Genome, Plant; D042822:Genomic Instability; D010937:Plant Growth Regulators / Q000494:pharmacology; D018520:Plant Shoots / Q000187:drug effects / Q000502:physiology*; D011174:Portugal; D019105:Random Amplified Polymorphic DNA Technique; D013395:Sucrose / Q000494:pharmacology; D046930:Thymus Plant / Q000187:drug effects / Q000502:physiology*; D058989:Vitrification; D015026:Zeatin / Q000494:pharmacology
chemical_list: D003451:Cryoprotective Agents; D003470:Culture Media; D018744:DNA, Plant; D010937:Plant Growth Regulators; D013395:Sucrose; D015026:Zeatin