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Cryopreservation of Thymus lotocephalus by droplet vitrification: effect of cold hardening
Áreas de investigación
  • Biodiversidad
Thymus lotocephalus G. López & R. Morales is a rare aromatic species endemic to the Algarve region (South of Portugal). In order to fight the extinction of this species and contribute to its conservation, our group has been developing ex situ strategies, such as in vitro propagation and seed cryopreservation (1, 2). Now we intend to implement a cryopreservation protocol for shoot tips of T. lotocephalus as an alternative method for long-term preservation. Droplet vitrification was the method tested for shoot tip cryopreservation and in this study we set out to evaluate the response of shoot tips after cold-hardening of in vitro donor plants. Apices were excised from in vitro-grown shoots of T. lotocephalus maintained at a constant temperature of 25ºC during the first two weeks and then transferred to constant 10ºC or alternate 25/-1ºC for further two weeks. After excision, shoot tips were pre-cultured on semi-solid Murashige and Skoog medium containing 0.3 M sucrose for one day. Shoot tips were placed in loading solution (0.4 M sucrose + 2 M glycerol) for 20 min, followed by dehydration in PVS2 solution at 0 ºC for 60 min, placed on aluminum foil strips and then immersed in liquid nitrogen. Rewarming took place in unloading solution (1.2 M sucrose) for 20 min at room temperature. Four weeks after rewarming, cryopreserved apices excised from cold-hardened shoots presented higher survival percentages, 96.7 ± 3.3 and 93.3 ± 4.2% for 10ºC and 25/-1ºC, respectively, than the control, 86.7 ± 6.7%. This tendency was maintained eight weeks after rewarming, as the survival and regrowth percentages were higher for cold-hardened shoot tips. The best result, 73.3 ± 9.9%, for both survival and regrowth percentages, was obtained in cryopreserved shoot tips excised from in vitro-grown shoots maintained at 10ºC. According to these results, cold-hardening of in vitro donor plants improves the survival and regrowth of cryopreserved shoot tips of T. lotocephalus by droplet-vitrification. Acknowledgments: Coelho N and Gonçalves S acknowledge a grant from Portuguese Science and Technology Foundation (FCT, grants SFRH/BD/63501/2009 and SFRH/BPD/31534/2006, respectively). References 1. Coelho N, Gonçalves S, González-Benito ME and Romano A (2012) Plant Growth Regulation 66, 69-74. 2. Coelho N, Gonçalves S, González-Benito ME and Romano A (2012) Scientia Horticulturae 145, 84-86.
Nombre congreso
Annual Meetinf of the Society of Low Temperature Biology
Tipo de participación
Lugar del congreso
Londres (Reio Unido)
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Título de las actas
Cryoletters (34)
Esta actividad pertenece a memorias de investigación
  • Autor: N. Coelho (Universidad de Algarve)
  • Autor: M. Elena Gonzalez Benito (UPM)
  • Autor: S. Gonsalves (Universidad de Algarve)
  • Autor: A. Romano (Universidad de Algarve)
Grupos de investigación, Departamentos, Centros e Institutos de I+D+i relacionados
  • Creador: Grupo de Investigación: Biodiversidad y conservación de recursos fitogenéticos
  • Departamento: Biología Vegetal
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