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Submitted to: Symposium Series
Publication Type: Abstract Only Publication Acceptance Date: 5/5/2005 Publication Date: 6/1/2005 Citation: Volk, G.M., Caspersen, A.M. Ultrastructural changes in mint meristems during the cryopreservation process. In: Symposium Series Meristems, June 2-5, 2005, Ames, Iowa. p. 21. Interpretive Summary: Cryopreservation techniques can be used to preserve specific plant genotypes. One millimeter shoot tips are excised and treated with cryoprotectant solutions to remove excess water and introduce solutes that promote glass formation during the cooling process. Shoot tips exposed to liquid nitrogen temperatures are diluted into concentrated sucrose solutions upon thawing. Shoot tips recover and form green shoots in tissue culture within a few weeks. Mint shoot tips exposed to each step of the cryopreservation procedure were embedded in resin, sectioned, and observed using transmission electron microscopy. We demonstrate that despite the significant water loss that occurs during cryoprotective treatments, meristem cells remain intact. These cells are primarily damaged during the warming and dilution treatments of the cryopreservation procedure. We believe that membrane rupture upon warming and cryoprotectant toxicity may be significant sources of damage during cryopreservation. We believe that membrane rupture upon warming and cryoprotectant toxicity may be significant sources of damage during cryopreservation. Technical Abstract: Cryopreservation techniques can be used to preserve specific plant genotypes. One millimeter shoot tips are excised and treated with cryoprotectant solutions to remove excess water and introduce solutes that promote glass formation during the cooling process. Shoot tips exposed to liquid nitrogen temperatures are diluted into concentrated sucrose solutions upon thawing. Shoot tips recover and form green shoots in tissue culture within a few weeks. Mint shoot tips exposed to each step of the cryopreservation procedure were embedded in resin, sectioned, and observed using transmission electron microscopy. Shoot tip water content during the cryopreservation process was determined using differential scanning calorimetry. Plasmolysis within meristematic cells was minimal, despite the significant loss of water during cryoprotectant treatments. Permeable cryoprotectants such as dimethyl sulfoxide and ethylene glycol replaced most of the free water within the cells. Significant damage was not observed after cryoprotectant treatment and liquid nitrogen exposure; however, shoot tips cooled to liquid nitrogen temperatures, and subsequently warmed and diluted in 1.2 M sucrose were plasmolyzed and appeared to have damaged membranes. Our data suggest that the loss of water during cryoprotectant treatments may not be the primary source of damage during the cryopreservation procedure. We believe that membrane rupture upon warming and cryoprotectant toxicity may be significant sources of damage during cryopreservation. |
