title: Intracellular ice and cell survival in cryo-exposed embryonic axes of recalcitrant seeds of Acer saccharinum: an ultrastructural study of factors affecting cell and ice structures.
abstract: OBJECTIVE
  Cryopreservation is the only long-term conservation strategy available for germplasm of recalcitrant-seeded species. Efforts to cryopreserve this form of germplasm are hampered by potentially lethal intracellular freezing events; thus, it is important to understand the relationships among cryo-exposure techniques, water content, structure and survival.
  
  
  METHODS
  Undried embryonic axes of Acer saccharinum and those rapidly dried to two different water contents were cooled at three rates and re-warmed at two rates. Ultrastructural observations were carried out on radicle and shoot tips prepared by freeze-fracture and freeze-substitution to assess immediate (i.e. pre-thaw) responses to cooling treatments. Survival of axes was assessed in vitro.
  
  
  RESULTS
  Intracellular ice formation was not necessarily lethal. Embryo cells survived when crystal diameter was between 0·2 and 0·4 µm and fewer than 20 crystals were distributed per μm(2) in the cytoplasm. Ice was not uniformly distributed within the cells. In fully hydrated axes cooled at an intermediate rate, the interiors of many organelles were apparently ice-free; this may have prevented the disruption of vital intracellular machinery. Intracytoplasmic ice formation did not apparently impact the integrity of the plasmalemma. The maximum number of ice crystals was far greater in shoot apices, which were more sensitive than radicles to cryo-exposure.
  
  
  CONCLUSIONS
  The findings challenge the accepted paradigm that intracellular ice formation is always lethal, as the results show that cells can survive intracellular ice if crystals are small and localized in the cytoplasm. Further understanding of the interactions among water content, cooling rate, cell structure and ice structure is required to optimize cryopreservation treatments without undue reliance on empirical approaches.
authors.affiliation: Plant Germplasm Conservation Research, School of Life Sciences, University of KwaZulu-Natal (Westville Campus), Durban, 4001 South Africa.
authors.forename: James
authors.initials: J
authors.lastname: Wesley-Smith
authors.forename: Patricia
authors.initials: P
authors.lastname: Berjak
authors.forename: N W
authors.initials: NW
authors.lastname: Pammenter
authors.forename: Christina
authors.initials: C
authors.lastname: Walters
mesh_terms: D031002:Acer / Q000196:embryology / Q000502:physiology / Q000648:ultrastructure*; D002470:Cell Survival; D015925:Cryopreservation / Q000379:methods*; D003593:Cytoplasm / Q000648:ultrastructure; D005615:Freezing / Q000009:adverse effects; D007053:Ice / Q000009:adverse effects*; D008854:Microscopy, Electron; D012639:Seeds / Q000196:embryology / Q000502:physiology / Q000648:ultrastructure*; D014867:Water / Q000502:physiology*
chemical_list: D007053:Ice; D014867:Water
keywords: Acer saccharinum; cooling rate; cryopreservation; electron microscopy; embryonic axes; freeze-fracture; freeze-substitution; ice crystal distribution; ice crystal size; recalcitrant seeds; warming rate; water content