Proline is an effective cryoprotectant for the storage of cultured cells of Zea mays L. in liquid N2. Increased freeze tolerance can be achieved by pregrowth for 3 to 4 days in medium containing proline. Cells cryoprotected with proline have an increased recovery potential when compared with cells cryoprotected with dimethylsulfoxide and glycerol. They also show a reduced postthaw viability loss and greater tolerance of a range of postthaw culture conditions. It is suggested that the mechanism of action of proline may be similar to that in its putative role of conferring protection against natural stresses. It may be protecting the cell against solution effects caused by dehydration during freezing. These findings are discussed in relation to other freeze tolerance enhancing treatments.Freeze preservation in LN3 as a means of genome storage can now be applied to a very limited range of plant tissue cultures. The reasons for the present limitations are not fully understood (1,20,24).In biological systems, the two major sources of cryoinjury are ice damage and solution effects, the latter caused by the excessive concentration of intracellular solutes (9, 10). Protection against cryoinjury may be achieved by either freezing slowly to induce dehydration by extracellular freezing, thereby minimizing intracellular ice formation, or alternatively by freezing and thawing very rapidly to prevent dehydration while maintaining a small, innocuous, ice crystal size. The addition of cryoprotective compounds may reduce cryoinjury in either rapid or slow freezing. In the majority of freeze preservation protocols applied to plant cells, cryoprotectants (usually DMSO and/or glycerol) are used in combination with freezing at a slow rate (1, 24). The high water content of plant cells results in an enhanced susceptibility to cryoinjury. At the level of dehydration required to avoid solution effect stresses, it is still normally necessary to thaw very rapidly to minimize ice recrystallization damage (1,20,24).Recovery of cultures after thawing is dependent upon both a high postthaw viability and further survival through a recovery period preceding renewal of normal metabolic functions and cell division (15,20 In the present study, proline has been used as both a cryoprotectant and pregrowth additive (23) for the freeze preservation of cultured cells of maize. It will be demonstrated that this compound is highly effective in both modes of use and is superior in a number of respects to conventional cryoprotectants. MATERIALS AND METHODSA suspension culture of Zea mays L. (a cell line derived from cultivar B73 [inbred] and kindly supplied by Dr. Ingo Potrykus) was maintained in exponential growth under the conditions described by Potrykus et aL (13). Cells were harvested for freezing 3 or 4 days after subculturing. Additionally, a cell suspension was pregrown for 3 or 4 days in medium supplemented with 5 or 10%1o (w/v) L-proline. Cryoprotectants were prepared using analytical grades of L-proline, glycerol and DMSO, at double th...

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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.

Cryopreservation, that is, the viable storage of cells at the temperature of liquid nitrogen (-196°C), has wide relevance in many areas of pure and applied biology. Examples of its very successful use can be found in the storage of microbes and of semen (1). More recently, attention has been given to the development of cryopreservation methods for embryos, including those of humans, tissues, and organs for transplantation and blood components. In the context of plant research, realization of the potential applications of cryopreservation has been somewhat latent. However, several clear areas for application can be identified that involve the need to store exact genotypes in a stable state.