Elucidation of the metabolic and structural basis for the cryopreservationrecalcitrance of Vaucheria sessilis

Freezing injury in Vaucheria sessilis was attributed to three modes of dama ge. Primary direct injury, due to intracellular ice nucleation and the move ment of ice fronts within the filament. This resulted in gross disruption o f major organelles including the mitochondria, nuclei, chloroplasts, and en doplasmic reticulum. Dehydration-induced injury caused by a rise in solute concentration during freezing, Injury due to other freeze-induced stresses including disruption of intracellular organelles by extracellular ice crush ing and "turbulent" displacement and mixing of organelles between -17.4 and -26 degrees C. Ln addition, the alga suffered disruption to normal metabol ism, manifested as an inhibition of photosynthetic oxygen evolution on cryo protectant addition and cooling, with a total loss of photosynthetic capaci ty in freeze/thawed samples. Furthermore, there was evidence of oxidative s tress-induced injuries with a significant (P < 0.004) increase in products of lipid peroxidation when V. sessilis was exposed to stresses associated w ith sectioning, cryoprotectant exposure and freezing. However, despite some damage, V. sessilis retained full metabolic capabilities post-supercooling , suggesting that it may ultimately be possible to cryopreserve this organi sm.