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

Suspension culture cells of sycamore (Acer pseudoplatanus L.) and carrot (Daucus carota L.) were frozen to ultralow temperatures under rapid (⩾ 100 °C s−1) and slow, controlled (1 or 2 °C min−1) rates, in the presence and absence of cryoprotective compounds. After storage at −196 °C, cells were recovered by thawing either slowly, in air at room temperature (ca. 20 °C min−1) or rapidly, in a water bath at 40 °C (ca. 100 °C min−1). The ultrastructure of the thawed cells was examined by thin-sectioning and compared with unfrozen controls and cells examined in the frozen state. Cells frozen rapidly, in the presence of cryoprotectants, or frozen slowly in their absence, suffered serious ultrastructural damage and a total loss of viability. Carrot cells frozen at a rate of 2 °C min−1 in the presence of cryoprotectants and thawed at either rate, yielded up to 70% of viable cells. The recovered aggregates of carrot cells comprised some centrally located, seriously damaged cells and, at the periphery, groups of cells with a high electron opacity neighbouring well preserved cells, showing little ultrastructural modification compared with unfrozen controls. The highest rate of survival of sycamore cells (ca. 30%) was observed when they were frozen at a rate of 1 °C min−1 and thawed rapidly. In all recoverd cells of sycamore some ultrastructural modifications were evident. These included: dilation of mitochondria, plastids, golgi and ER cisternae and the nuclear envelope, decrease in polysomes, increase in nuclear and cytoplasmic microfilaments and changes in nuclear and nucleolar granularity. The probable causes and timing of the ultrastructural changes and their effects on the potential for regrowth of the recovered cells are discussed.