title: Effect of growth phase on survival of bromegrass suspension cells following cryopreservation and abiotic stresses.
abstract: OBJECTIVE
  Cryopreservation is a practical method of preserving plant cell cultures and their genetic integrity. It has long been believed that cryopreservation of plant cell cultures is best performed with cells at the late lag or early exponential growth phase. At these stages the cells are small and non-vacuolated. This belief was based on studies using conventional slow prefreezing protocols and survival determined with fluorescein diacetate staining or 2,3,5-triphenyltetrazolium chloride assays. This classical issue was revisited here to determine the optimum growth phase for cryopreserving a bromegrass (Bromus inermis) suspension culture using more recently developed protocols and regrowth assays for determination of survival.
  
  
  METHODS
  Cells at different growth phases were cryopreserved using three protocols: slow prefreezing, rapid prefreezing and vitrification. Stage-dependent trends in cell osmolarity, water content and tolerance to freezing, heat and salt stresses were also determined. In all cases survival was assayed by regrowth of cells following the treatments.
  
  
  RESULTS
  Slow prefreezing and rapid prefreezing protocols resulted in higher cell survival compared with the vitrification method. For all the protocols used, the best regrowth was obtained using cells in the late exponential or early stationary phase, whereas lowest survival was obtained for cells in the late lag or early exponential phase. Cells at the late exponential phase were characterized by high water content and high osmolarity and were most tolerant to freezing, heat and salt stresses, whereas cells at the early exponential phase, characterized by low water content and low osmolarity, were least tolerant.
  
  
  CONCLUSIONS
  The results are contrary to the classical concept which utilizes cells in the late lag or early exponential growth phase for cryopreservation. The optimal growth phase for cryopreservation may depend upon the species or cell culture being cryopreserved and requires re-investigation for each cell culture. Stage-dependent survival following cryopreservation was proportionally correlated with the levels of abiotic stress tolerance in bromegrass cells.
authors.affiliation: Genetic Diversity Department, National Institute of Agrobiological Sciences, Kannondai 2-1-2, Tsukuba, Ibaraki, Japan 305-8602. isikawam@affrc.go.jp
authors.forename: Masaya
authors.initials: M
authors.lastname: Ishikawa
authors.forename: Mitsuteru
authors.initials: M
authors.lastname: Suzuki
authors.forename: Toshihide
authors.initials: T
authors.lastname: Nakamura
authors.forename: Tadashi
authors.initials: T
authors.lastname: Kishimoto
authors.forename: Albert J
authors.initials: AJ
authors.lastname: Robertson
authors.forename: Lawrence V
authors.initials: LV
authors.lastname: Gusta
mesh_terms: D031724:Bromus / Q000166:cytology* / Q000187:drug effects* / Q000254:growth & development; D018929:Cell Culture Techniques; D002470:Cell Survival / Q000187:drug effects; D015925:Cryopreservation / Q000379:methods*; D005615:Freezing; D006979:Hyperthermia, Induced; D009994:Osmolar Concentration; D012492:Salts / Q000494:pharmacology*; D013997:Time Factors; D014867:Water / Q000378:metabolism
chemical_list: D012492:Salts; D014867:Water