Proline: A Novel Cryoprotectant for the Freeze Preservation of Cultured Cells of Zea mays L

Abstract

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...

Trehalose as Cryoprotectant for the Freeze Preservation of Carrot and Tobacco Cells

“…Successful recovery of viable tissues usually requires the use of a cryoprotectant like DMSO which itself can be very toxic. The use of naturally occurring compounds like proline (12) or trehalose which may not be toxic would be advantageous.…”

Peteca rind pitting as influenced by proline content in winter lemon albedo

“…1977). Carpenter and Crowe (1988, cited by Kurt and Hanson, 1997) indicate that this amino acid is essential for stabilizing cell structures, presenting an osmoprotector and cryoprotector activity in fruits tissues, as well as cell protection against oxygen reactives and free radicals (Withers and King, 1979;Alia and Mohanty, 1997). This is important if we consider that damage by "peteca" has been associated to the action of oxygen reactives and a lack of oxygen diffusion in the cells (Nobel, 1999, cited by Storey andTreeby, 2002).…”

Problèmes posés par la conservation des souches cellulaires et des organes cultivésin vitro

“…a/ Traitement cryoprotecteur (conditionnement ou prétraitement) -Préculture : Des suspensions cellulaires en phase exponentielle de croissance ou en fin de phase de latence sont cultivées sur un milieu enrichi en mannitol (Withers et King, 1980 ;Maddox et coll., 1983), en proline (Withers et King, 1979) ou en sorbitol (Weber et coll., 198:J ;Maddox et coll., 1983 ;Chen et coll., 1984a), pendant des durées allant de quelques heures à quelques jours. Cette préculture n'est pas toujours né-cessaire (Sala et coll., 1979 ;Dougal!…”