Freeze Preservation of Cultured Plant Cells. III. The Pregrowth Phase

There is an inverse relationship between cell size and capacity to survive the freeze‐preservation protocol. Pregrowth of cell suspensions in media rendered more negative in water potential by addition of mannitol enhances the survival capacity of Acer pseudoplatanus and Capsicum annuum cells but this effect can only partially be explained in terms of the associated reduction in mean cell size. Studies with cell suspensions of Daucus carota indicate the importance for successful freeze‐preservation of the stage in the growth cycle of suspensions propagated in batch culture; highest survival was recorded for cells taken at lag phase or early exponential phase. Regrowth of recovered cells depends upon the establishment of an appropriate inoculum density of cells which have retained the capacity to divide. The dividing cells only achieve a growth rate equal to that of untreated cells after a number of cell generations. A proportion of the recovered cells giving an initial positive fluorescein diacetate reaction lose this capacity rapidly (within 24 h), others lose the capacity more slowly and others, in which the positive reaction persists, are incapable of division. These observations indicate that different levels of injury are inflicted by the freeze‐preservation protocol and that only in a proportion of the cells is the injury reparable or compatible with growth by cell division.