A fine-structural study of the freeze-preservation of plant tissue cultures

Summary

Suspension culture cells of sycamore (Acer pseudoplatanus L.) and carrot(Daucus carota L.) were frozen to ultralow temperatures either rapidly (⩾ 100 °C s⁻¹), or slowly at controlled rates of 1 or 2 °C min⁻¹ in the presence and in the absence of cryoprotective compounds. When examined by the conventional freeze-etch procedure, the appearance of the frozen cells correlated closely with that of unfrozen control material examined by thin sectioning after glutaraldehyde-osmium fixation. Cells frozen rapidly or slowly in the absence of cryoprotectants suffered damage by gross intracellular ice formation. Rapid freezing, in the presence of cryoprotectants at levels used in freeze-preservation protocols, caused intracellular ice formation, but the ice crystals size was sufficiently small to avoid compression or rupture of organelles. Cells frozen by the above procedures cannot be recovered in a viable state and this is considered largely to be due to intracellular ice formation which, where not directly damaging during freezing, undergoes disruptive recrystallization during thawing. Slow freezing in the presence of cryoprotectants was associated with a reduction in cell volume by dehydration, reduced intracellular ice formation and good