title: Why is intracellular ice lethal? A microscopical study showing evidence of programmed cell death in cryo-exposed embryonic axes of recalcitrant seeds of Acer saccharinum.
abstract: OBJECTIVE
  Conservation of the genetic diversity afforded by recalcitrant seeds is achieved by cryopreservation, in which excised embryonic axes (or, where possible, embryos) are treated and stored at temperatures lower than -180 °C using liquid nitrogen. It has previously been shown that intracellular ice forms in rapidly cooled embryonic axes of Acer saccharinum (silver maple) but this is not necessarily lethal when ice crystals are small. This study seeks to understand the nature and extent of damage from intracellular ice, and the course of recovery and regrowth in surviving tissues.
  
  
  METHODS
  Embryonic axes of A. saccharinum, not subjected to dehydration or cryoprotection treatments (water content was 1·9 g H2O g(-1) dry mass), were cooled to liquid nitrogen temperatures using two methods: plunging into nitrogen slush to achieve a cooling rate of 97 °C s(-1) or programmed cooling at 3·3 °C s(-1). Samples were thawed rapidly (177 °C s(-1)) and cell structure was examined microscopically immediately, and at intervals up to 72 h in vitro. Survival was assessed after 4 weeks in vitro. Axes were processed conventionally for optical microscopy and ultrastructural examination.
  
  
  RESULTS
  Immediately following thaw after cryogenic exposure, cells from axes did not show signs of damage at an ultrastructural level. Signs that cells had been damaged were apparent after several hours of in vitro culture and appeared as autophagic decomposition. In surviving tissues, dead cells were sloughed off and pockets of living cells were the origin of regrowth. In roots, regrowth occurred from the ground meristem and procambium, not the distal meristem, which became lethally damaged. Regrowth of shoots occurred from isolated pockets of surviving cells of peripheral and pith meristems. The size of these pockets may determine the possibility for, the extent of and the vigour of regrowth.
  
  
  CONCLUSIONS
  Autophagic degradation and ultimately autolysis of cells following cryo-exposure and formation of small (0·2-0·4 µm) intracellular ice crystals challenges current ideas that ice causes immediate physical damage to cells. Instead, freezing stress may induce a signal for programmed cell death (PCD). Cells that form more ice crystals during cooling have faster PCD responses.
authors: Plant Germplasm Conservation Research, School of Life Sciences, University of KwaZulu-Natal (Westville Campus), Durban, 4001 South Africa, National Centre for Nanostructured Materials, Council for Scientific and Industrial Research, 1 Meiring Naude Rd, Brummeria, Pretoria, 0002 South Africa and USDA-ARS, National Center for Genetic Resources Preservation, 1111 South Mason Street, Fort Collins, CO 80521, USA Plant Germplasm Conservation Research, School of Life Sciences, University of KwaZulu-Natal (Westville Campus), Durban, 4001 South Africa, National Centre for Nanostructured Materials, Council for Scientific and Industrial Research, 1 Meiring Naude Rd, Brummeria, Pretoria, 0002 South Africa and USDA-ARS, National Center for Genetic Resources Preservation, 1111 South Mason Street, Fort Collins, CO 80521, USA.
authors: James
authors: J
authors: Wesley-Smith
authors: Plant Germplasm Conservation Research, School of Life Sciences, University of KwaZulu-Natal (Westville Campus), Durban, 4001 South Africa, National Centre for Nanostructured Materials, Council for Scientific and Industrial Research, 1 Meiring Naude Rd, Brummeria, Pretoria, 0002 South Africa and USDA-ARS, National Center for Genetic Resources Preservation, 1111 South Mason Street, Fort Collins, CO 80521, USA Christina.Walters@ARS.USDA.GOV.
authors: Christina
authors: C
authors: Walters
authors: Plant Germplasm Conservation Research, School of Life Sciences, University of KwaZulu-Natal (Westville Campus), Durban, 4001 South Africa, National Centre for Nanostructured Materials, Council for Scientific and Industrial Research, 1 Meiring Naude Rd, Brummeria, Pretoria, 0002 South Africa and USDA-ARS, National Center for Genetic Resources Preservation, 1111 South Mason Street, Fort Collins, CO 80521, USA.
authors: N W
authors: NW
authors: Pammenter
authors: Plant Germplasm Conservation Research, School of Life Sciences, University of KwaZulu-Natal (Westville Campus), Durban, 4001 South Africa, National Centre for Nanostructured Materials, Council for Scientific and Industrial Research, 1 Meiring Naude Rd, Brummeria, Pretoria, 0002 South Africa and USDA-ARS, National Center for Genetic Resources Preservation, 1111 South Mason Street, Fort Collins, CO 80521, USA Christina.Walters@ARS.USDA.GOV.
authors: Patricia
authors: P
authors: Berjak
mesh_terms: D031002:Acer / Q000166:cytology / Q000196:embryology* / Q000254:growth & development / Q000648:ultrastructure; D017209:Apoptosis; D002470:Cell Survival; D015925:Cryopreservation; D018525:Germination; D007053:Ice; D042541:Intracellular Space / Q000378:metabolism*; D008853:Microscopy / Q000379:methods*; D012639:Seeds / Q000166:cytology* / Q000648:ultrastructure
chemical_list: D007053:Ice
keywords: Acer saccharinum; Aceraceae; TEM; autophagy; cooling rate; cryopreservation; embryonic axes; intracellular ice; light microscopy; mechanical stress; programmed cell death; recalcitrant seed; silver maple; transmission electron microscopy