title: Cryopreservation of protocorm-like body-derived shoot tips of Calanthe davidii by droplet-vitrification.
abstract: BACKGROUND
  Evaluation of the conservation status of Calanthe plants in China indicated that 18 species were endangered or critically endangered. Comprehensive conservation solutions including ex situ methods, are urgently required to protect Calanthe species in China.
  
  
  OBJECTIVE
  The study aims to develop a simple and efficient cryopreservation protocol using droplet-vitrification for Calanthe davidii.
  
  
  METHODS
  Protocorm-like bodies (PLBs) were induced from nodal sections of in vitro shoots, and their proliferation was promoted by a thin cell layer culture procedure. Shoot tips excised from three leaf-stage PLBs were used in cryopreservation experiments. Key factors of the droplet-vitrification procedure including sucrose preculture, treatment with PVS2 solution and post-rewarming culture conditions were optimized to achieve a high level of regeneration.
  
  
  RESULTS
  When the optimized procedure was applied, 77.8 ± 3.9% of cryopreserved shoot tips withstood liquid nitrogen exposure and regenerated into new PLBs.
  
  
  CONCLUSIONS
  These results highlighted the importance of post-rewarming osmo-conditioning for regeneration of cryopreserved shoot tips.
authors.affiliation: Germplasm Bank of Wild Species, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming, China.
authors.forename: Liang
authors.initials: L
authors.lastname: Lin
authors.affiliation: Germplasm Bank of Wild Species, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming, China.
authors.forename: Qian
authors.initials: Q
authors.lastname: Xu
authors.affiliation: Germplasm Bank of Wild Species, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming, China.
authors.forename: Junchao
authors.initials: J
authors.lastname: Ma
authors.affiliation: Germplasm Bank of Wild Species, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming, China.
authors.forename: Bin
authors.initials: B
authors.lastname: Yuan
authors.affiliation: Germplasm Bank of Wild Species, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming, China. weiqili@mail.kib.ac.cn.
authors.forename: Weiqi Li
authors.initials: WL
authors.lastname: Li
mesh_terms: D002681:China; D003247:Conservation of Natural Resources; D015925:Cryopreservation / Q000379:methods*; D003451:Cryoprotective Agents / Q000494:pharmacology; D003470:Culture Media; D056727:Endangered Species; D029595:Orchidaceae / Q000187:drug effects / Q000502:physiology*; D009994:Osmolar Concentration; D018520:Plant Shoots / Q000187:drug effects / Q000502:physiology*; D012038:Regeneration / Q000502:physiology*; D013395:Sucrose / Q000494:pharmacology; D058989:Vitrification
chemical_list: D003451:Cryoprotective Agents; D003470:Culture Media; D013395:Sucrose