Plant Physiol. (1979) 64, 675-678 0032-0889/79/64/0675/04/$00.50/0

Proline: A Novel Cryoprotectant for the Freeze Preservation of Cultured Cells of Zea mays L. Received for publication March 1, 1979 and in revised form June 5, 1979

LYNDSEY A. WITHERS' AND PATRICK J. KING Friedrich Miescher-Institut, Postfach 273, CH-4002 Basel, Switzerland2

ABSTRACT sient low postthaw viabilities may furthermore involve strict, undesired selection for specific cell types. Proline is an effective cryoprotectant for the storage of cultured cells of The cytotoxicity of conventional cryoprotectants, especially Zea mays L. in liquid N2. Increased freeze tolerance can be achieved by glycerol, manifested as a rapid decline ofhigh immediate postthaw pregrowth for 3 to 4 days in medium containing proline. Cells cryoprotected viabilities (20), prompts the search for other compounds. Proline, with proline have an increased recovery potential when compared with cells which has been implicated in protection against salt stress in cryoprotected with dimethylsulfoxide and glycerol. They also show a halophytes (17, 18) and cold and desiccation stress in a wide range reduced postthaw viability loss and greater tolerance of a range ofpostthaw of plants (2, 12), is an ideal candidate for natural cryoprotection culture conditions. It is suggested that the mechanism of action of proline since it has a very high solubility, is neutral, exerts a high osmotic may be similar to that in its putative role of conferring protection against pressure, and is nontoxic at high concentrations (3, 17). Exoge- natural stresses. It may be protecting the cell against solution effects nously applied proline can confer stress resistance to plant tissues caused by dehydration during freezing. These findings are discussed in (17, see also 6), for example, tolerance ofcooling to -7 C has been relation to other freeze tolerance enhancing treatments. reported (8). However, at such a temperature intracellular freezing may be avoided by supercooling. It is essential, for the purposes of genome storage, that tolerance of freezing to ultralow temper- atures be demonstrated. In the present study, proline has been used as both a cryopro- tectant and pregrowth additive (23) for the freeze preservation of Freeze preservation in LN3 as a means of genome storage can cultured cells ofmaize. It will be demonstrated that this compound now be applied to a very limited range of plant tissue cultures. is highly effective in both modes of use and is superior in a The reasons for the present limitations are not fully understood number of respects to conventional cryoprotectants. (1, 20, 24). In biological systems, the two major sources of cryoinjury are MATERIALS AND METHODS ice damage and solution effects, the latter caused by the excessive A suspension culture of Zea mays L. (a cell line derived from concentration of intracellular solutes (9, 10). Protection against cultivar B73 [inbred] and kindly supplied by Dr. Ingo Potrykus) cryoinjury may be achieved by either freezing slowly to induce was maintained in exponential growth under the conditions de- dehydration by extracellular freezing, thereby minimizing intra- scribed by Potrykus et aL (13). Cells were harvested for freezing cellular ice formation, or alternatively by freezing and thawing 3 or 4 days after subculturing. Additionally, a cell suspension was very rapidly to prevent dehydration while maintaining a small, pregrown for 3 or 4 days in medium supplemented with 5 or 10%1o innocuous, ice crystal size. The addition of cryoprotective com- were reduce in either or In (w/v) L-proline. Cryoprotectants prepared using analytical pounds may cryoinjury rapid slow freezing. grades of L-proline, glycerol and DMSO, at double the desired the majority offreeze preservation protocols applied to plant cells, concentrations in culture medium, and sterilized by filtration. The cryoprotectants (usually DMSO and/or glycerol) are used in solutions and cell were chilled on ice at a rate cryoprotectant suspensions combination with freezing slow (1, 24). The high water and added to an volume of content of cells results in an enhanced to the cryoprotectants gradually equal plant susceptibility cell suspension in four aliquots over a period of 1 h. After 1 further cryoinjury. At the level of dehydration required to avoid solution hour, the cells were dispensed into sterile polypropylene ampoules effect stresses, it is still normally necessary to thaw very rapidly to ml into each 2-ml and then minimize ice recrystallization damage (1, 20, 24). (1 ampoule) transferred to a controlled Recovery of cultures after thawing is dependent upon both a freezing apparatus (24). A modified stepwise freezing program high and further was employed (20): cooling at 1 C min- to -30 C, followed by postthaw viability survival through a recovery holding at -30 C for 30 to 40 min, then plunging the specimens period preceding renewal of normal metabolic functions and cell into LN. The rate was monitored a division (15, 20). However, cryoprotectant toxicity and rehydra- cooling using resistance and thermometer inserted into a dummy specimen. Supercooling of tion/deplasmolysis injury upon thawing cryoprotectant re- the specimen during the initial period of slow cooling was avoided moval contribute at present to viability losses which may delay or by agitation of the ampoules to induce freezing of the suspending often preclude recovery (21). Given the heterogeneity of plant medium. After storage in LN for up to 14 days, the cell suspensions cultures, the recovery of cultures from populations showing tran- were thawed rapidly (within about 3 min) by agitation of the ampoules in warm (40 C) water. Viability and capacity for re- 'Present address: Botanical Laboratories, University of Leicester, growth were estimated by FDA staining (20). Recovery growth Leicester, LEI 7RH, United Kingdom. was initiated by one of the following procedures: 2Reprint requests to be directed to this address. 1. Spreading the cell suspension onto 5 ml of culture medium 'Abbreviations: LN: liquid nitrogen; DMSO: dimethylsulfoxide; FDA: solidified with agar in a 5-cm diameter Petri dish (a) directly after fluorescein diacetate. thawing, (b) as (a) but draining off the liquid medium, (c) after 675 676 WITHERS AND KING Plant Physiol. Vol. 64, 1979 washing cells with sterile water, (d) after washing with fresh liquid medium, (e) after washing with medium plus lo0 (w/v) proline. 2. Dispersing the cell suspension into 10 ml of culture medium in an Erlenmeyer flask and rotary shaking. 3. Dispensing the cell suspension as hanging droplets onto a Petri dish lid. 4. Dispensing the cell suspension into a 5-cm diameter Petri dish. 5. Plating after mixing the cell suspension with 5 ml agar medium. Cryoprotectant-free control preparations were frozen, thawed, and returned to culture in parallel with the cryoprotectant-treated preparations. Additionally, unfrozen control preparations of chilled and cryoprotectant-treated cells were returned to culture, inoculated at the same or one-tenth the cell density ofexperimental 6 0 2 preparations. time (days) FIG. 1. Recovery of cells after cryoprotection with proline or other RESULTS compounds and regrowth under various conditions. Per cent viability (FDA staining) is shown against days after thawing. Cryoprotectants: Proline Concentration. A suitable level of proline was estab- proline (@-), DMSO (A-A), DMS0 + proline (C-0), and DMSO lished for comparison with the conventional cryoprotectants. Be- + glycerol (E A). Regrowth conditions: (a) spreading direct onto agar tween 2.5 and 25% (w/v) proline, cryoprotection increased with medium; (b) spreading followed by draining of old medium; (c) spreading concentration but there was a loss of after washing cells with water, (d) spreading after washing with fresh pronounced viability by medium; (e) spreading after washing with medium plus 10%3o proline. Points excessive plasmolysis prior to freezing when using 25% proline in Figures I through 3 are the mean of two estimates, each of two replicate (25-30%o mortality). Proline at 1O0%o gave a minimum of plasmolysis samples. damage with a satisfactory level of cryoprotection and was adopted for the following experiments. Comparison of Proline with DMSO and Glycerol Using Various 100~ ~~ Regrowth Conditions. Freezing cells without any prior cryopro- tectant treatment consistently resulted in a zero viability response bc1W/. immediately after thawing, and consequently no recovery growth. 00 The cryoprotection afforded by proline alone was compared with DMSO alone and mixtures of DMSO + proline (each agent at a concentration of 10%) using the standard freezing program. The performance of cells in terms of their per cent viability over a 7-day period after thawing in regrowth conditions I (a-e) is shown in Figure 1. In all regrowth conditions, treatments involving proline as the sole cryoprotectant were most effective. Rapid, useful recovery occurred when proline-treated cells were spread 10 20 30 onto agar medium together with the original cryoprotectant-con- time (days) taining medium (Fig. la). Removal of this medium (Fig. lb) FIG. 2. Recovery of cells after cryoprotection with proline or DMSO delayed recovery growth, perhaps by depletion of essential factors + glycerol and regrowth under various conditions. Per cent viability (FDA released into the medium during freezing and thawing (21). The staining) is shown against days after thawing. Cryoprotectants: proline other regrowth conditions were all deleterious (Fig. 1, c-e). Wash- ( ), DMSO + glycerol (A). Regrowth conditions: spreading on agar ing with water caused the greatest initial loss of viability and cells medium ( ,dispersing in liquid medium and shaking (---), droplet treated with DMSO + glycerol exhibited the greatest sensitivity to culture ( **. Affow indicates the addition of fresh medium to droplets. washing. The general order of effectiveness of the cryoprotectants was: by resuspending in liquid medium (method 2) directly after thaw- Proline > DMSO + glycerol = DMSO + proline > DMSO alone ing (Fig. 2). In droplet culture (or regrowth in dishes; methods 3 and 4) their viability was initially stable but declined unless fresh The freezing protocols using proline alone or DMSO + glycerol medium was added. However, regrowth by plating into agar and spreading onto agar without washing (method la) were medium (method 5) failed with both cryoprotectant treatments; repeated several times. The immediate postthaw viability counts no colony development was recorded. showed some variability, but those of the DMSO + glycerol- Throughout, all unfrozen cryoprotected control preparations treated cells were consistently higher (x2). These, however, de- recovered growth upon returning to culture under all regrowth creased rapidly, yielding recovering cultures only when the via- conditions. A 10-fold dilution delayed recovery except in prepa- bility loss was checked in the days following thawing. Recovery rations treated with DMSO + glycerol where recovery was accel- sometimes failed and often was slow, with a reduced plating erated. All of these controls recovered at a more rapid rate than efficiency. experimental treatments, even when in the latter cases the imme- The DMSO + glycerol-treated cells failed to recover in any diate postthaw viability count was in excess of 109'o and viability regrowth conditions other than on agar, even when initial post- did not fall below this level during the postthaw period. thaw viabilities were very high (Fig. 2). In complete contrast, Pregrowth in Proline-supplemented Medium. Cells grown in the proline-treated cells were much more tolerant of the range of presence of 5% or 1001o proline for 3 to 4 days were frozen with or postthaw treatments applied. When spread onto agar, they often without further cryoprotectant treatment, thawed, and regrown by maintained the immediate postthaw viability levels before em- spreading onto agar medium. Viability counts for the postthaw barking upon regrowth, and could be transferred to suspension period are given in Figure 3. Pregrowth in proline markedly culture in liquid medium after about 14 days (Fig. 2). Despite an improved all aspects of recovery growth compared to use of initial viability loss, proline-treated cells also could be recovered proline for cryoprotection only. The improvements were sustained Plant Physiol. Vol. 64, 1979 FREEZE PRESERVATION USING PROLINE 677 1001 possible that when applied as a cryoprotectant shortly before freezing, proline acts as a nontoxic intracellular (and possibly extracytoplasmic) solute, protecting the cell against the denaturing effects of hyperosmolality induced by dehydration during slow freezing. The enhanced recovery potential of proline-treated cells sug- 5- gests that either there is a reduced level of latent injury, a protec- tion against postthaw deplasmolysis effects, e.g. by membrane stabilization (6), or that proline has an active role in recovery metabolism as proposed by Blum and Ebercon (2). The pregrowth effect of proline is particularly significant since it is the first report of freeze preservation of a suspension culture 0o0 ~5 10 5 time (days) in a suspending medium which can also support growth. In other FIG. 3. Recovery of cells after pregrowth in proline and cryoprotection reports (e.g. 23), further cryoprotection was necessary before freez- in proline or DMSO + glycerol. Per cent viability (FDA staining) is shown ing since the pregrowth supplement was inadequate as a cryopro- against days after thawing. Regrowth in every case was by spreading onto tectant, yet effective levels of conventional cryoprotectants would agar medium. be toxic in longer term, pregrowth application (1). Symbol The efficacy of pregrowth in proline may simply be due to Pregrowth Freezing enhanced uptake during the prolonged period of exposure. From 0... 0 10%o prol. the findings reported here and from further with 0e...0 10% prol. 10%o prol. experimentation ---- larger-celled species L. and Petunia *0 5% prol. 10% prol. (Acer pseudoplatanus hybrida 10% prol. L.; Withers and King, unpublished observations) it would appear --A A- 5% prol. DMSO + gly. that pregrowth effects upon cell size and cytoplasm to vacuole A...A 10% prol. DMSO + gly. ratio are important contributors to the over-all cryoprotective 0----0 5% prol. effect. Hydroxyproline has been shown to inhibit cell elongation (5). following pregrowth in l1o proline, even without addition of Additionally, incorporation of exogenously supplied proline into further proline as cryoprotectant. The use of DMSO + glycerol as cell wall hydroxyproline may affect cell wall flexibility and tensile cryoprotectant after pregrowth in proline was markedly delete- strength (see 7). These factors, with the suggested membrane- rious. stabilizing effect of proline (6), may lead to cell wall and mem- Growth in medium supplemented with 10o (w/v) proline for 3 brane modifications occurring during pregrowth, which contribute to 4 days produced minor changes in cell appearance, in particular to tolerance of the forces of dehydration, deformation, and com- an increase in cytoplasm to vacuole ratio. Cells grown to stationary pression suffered by cells during slow freezing (22). phase in presence of proline did not undergo normal cell expan- "Cold hardening" by pregrowth at low temperatures (14, 16), sion. pregrowth in medium of enhanced osmosity to reduce cell volume Composite Freezing Protocol Using Proline. The following and increase the cytoplasm to vacuole ratio (23, 24), and desicca- protocol, now in routine use for cultures of Z. mays L. and other tion (21, see also 24), have led to marked improvements in recovery species, has been developed to incorporate the findings reported of some frozen plant tissue cultures. All of these pretreatments above. An actively dividing cell suspension is subcultured into may induce intracellular accumulation of proline by analogy with medium supplemented with 10%1o (w/v) proline. A cryoprotectant established effects (2, 12, 17, 18). Alteration of the level of satu- solution of 20%1o proline in sterile medium is prepared. After 3 or ration of membrane fatty acids during cold hardening may be the 4 days of pregrowth in the presence of proline, chilled cryoprotec- critical factor leading to cold tolerance in some cases but not all tant is added in four aliquots over a period of 1 h, to an equal evidence points to this (19). It is possible that the hardening volume of chilled cell suspension. * * After 1 h on ice, the cells are pretreatments are producing both effects, membrane changes and dispensed into ampoules and cooled at a rate of about 1 C min-' proline accumulation, and that the former increases membrane to -30 C, held at that temperature for 30 to 40 min, and then fluidity at low temperatures to facilitate dehydration, the latter transferred to LN. To thaw, the ampoules are agitated in water at protecting against the consequent solute concentration. No com- 40 C. Growth is reinitiated by spreading the contents of each plete explanation can be offered in these terms regarding the case ampoule onto 5 ml of agar culture medium. Freezing can be of cryoprotection being increased by cold pretreatments before carried out without further addition of proline by following the rapid freezing (16). procedure from ** using cells pregrown in the presence of proline The work of Heber et al. (6) indicates that proline may be only for 3 or 4 days. one of a range of amino acids which can afford protection. Further, studies of the responses of plants to natural stresses DISCUSSION implicate other compounds, particularly glycine betaine, in an osmotically protective role (18). Our own unpublished work would The use of proline as cryoprotectant coupled with pregrowth in confirm the possibility of their being a group of protective organic proline-supplemented medium significantly enhanced the recov- solutes. We have found that y-amino butyric acid, hydroxyproline, ery of maize cells after freezing in LN when compared with use of and aspartate can confer cryoprotection upon maize cells. A the conventional cryoprotectants, DMSO and glycerol. Immediate mixture of proline at 10%7o with DMSO and glycerol each at 5% is postthaw viabilities were more predictably maintained with pro- extremely effective, giving very high postthaw viability levels and line and recovery growth rapidly ensued under a variety of rapid recovery. Glycine betaine in combination (but not alone) regrowth conditions. However, the drop in viability to the low would also appear to have some cryoprotectant properties. This levels recorded in some instances still indicates a risk ofphenotypic work will, upon completion, be published separately. or genotypic selection and the factors underlying this variation in The preliminary results reported here indicate that through a postthaw survival merit further study. different approach to cellular cryoprotection, improvements may The precise mechanism of cryoprotection by proline remains to be made in the range of application of cryopreservation to genome be established, although from its suggested role in protection storage. The necessary compromise between solution effects and against natural stresses, certain speculations can be made. It is ice damage may be more easily achieved by "natural" protection 678 WITHERS ALND KING Plant Physiol. Vol. 64, 1979 against the former under freezing conditions which prevent the 71: 345-355 10. MERYMAN HT, RI WILLIAMS. MSJ DOUGLAS 1977 Freezing injury from "solution effects" and latter. The finding that the underlying mechanisms are not nec- its prevention by natural or artificial cryoprotection. Cryobiology 14: 287-302 essarily confined to monocotyledonous plants (3, 4, 18) or even 11. MURPHY DJI 1977 Metabolic and tissue changes associated with changes in the freezing only to plants (11, see also 17, 18), suggests that a potential tolerance of the bivalve mollusc Modiolus demissus. J Exp Biol 69: 1-12 application may be found in all branches of cryobiology. 12. PAQUIN R 1977 Effet de basses temperatures sur la resistance au gel de la luzerne (Medicago media Pers.) et son contenu en proline libre. Physiol Veg 15: 657-665 Acknowledgments- The authors are grateful to Professor H. Smith for allowing the senior author 13. POTRYKUS 1, CT HARMS, H LORZ 1979 Callus formation from cell culture protoplasts of corn the facilities of the Botanical Laboratories, University of Leicester. continuing an arrangement (Zea mays L.). Theor Appl Genet 54: 207-214 made with the late Professor H. E. Street. We are further grateful to colleagues at the Friedrich 14. SAKAI A, Y SUGAWARA 1973 Survival of poplar callus at super-low temperatures after cold Miescher Institut for helpful discussion. acclimation. Plant Cell Physiol 14: 1201-1204 15. SALA F. R CELLA, F ROLLO 1979 Freeze-preservation of rice cells. Physiol Plant 45: 170-176 16. SEIBERT M, PJ WETHERBEE 1977 Increased survival and differentiation of frozen plant organ LITERATURE CITED cultures through cold treatment. Plant Physiol 59: 1043-1046 17. STEWART GR, JA LEE 1974 The role of proline accumulation in halophytes. Planta 120: 279- 1. BAJA] YPS, J REINERT 1977 Cryobiology of plant cell cultures and the establishment of gene 289 banks. In I Reinert, YPS Bajaj, eds, Applied and Fundamental Aspects of Plant Cell. Tissue 18. STOREY R, N AHMAD, RG WYN JONES 1977 Taxonomic and ecological aspects of the and Organ Culture. Springer-Verlag, Berlin. pp 757-777 distribution of glycine betaine and related compounds in plants. Oecologia 27: 319-332 2. BLUM A, A EBERCON 1976 Genotypic responses in Sorghum to drought stress. III. Free proline 19. WILLEMOT C, HJ HoPE, RJ WILLIAMS, R MICHAUD 1977 Changes in fatty acid composition of accumulation and drought resistance. Crop Sci 16: 428-431 winter wheat during frost hardening. Cryobiology 14: 87-93 2. BROWN LM, JA HELLEBUST 1978 Sorbitol and proline as intracellular osmotic solutes in the 20. WITHERS LA 1978 Freeze-preservation ofcultured cells and tissues. In TA Thorpe, ed, Frontiers green alga Stichococcus bacillaris. Can J Bot 56: 676-679 of Plant Tissue Culture 1978. Proc 4th Int Congr Plant Tissue and Cell Culture. IAPTC/ 4. CHu TM, D ASPINALL, LG PALEG 1974 Stress metabolism. Part 6. Temperature stress and the Calgary University Press, pp. 297-306 accumulation of proline in barley and radish. Aust J Plant Physiol 1: 87-97 21. WITHERS LA 1979 Freeze preservation of somatic embryos and clonal plantlets of carrot 5. CLELAND R 1967 Inhibition of cell elongation in Avena coleoptile by hydroxyproline. Plant carota Plant 63: 460-467 Physiol 42: 271-274 (Daucus L.). Physiol 22. WITHERS LA, MR DAN'EY 1978 A fine-structural study of the freeze-preservation of plant tissue 6. HEBER U. L TYANKOVA, KA SANTARIUS 1971 Stabilization and inactivation of biologial cultures. I. The frozen state. Protoplasma 94: 207-219 membranes during freezing in the presence of amino acids. Biochim. Biophys Acta 241: 23. WITHERS LA, HE STREET 1978 Freeze-preservation of cultured plant cells. III. The pregrowth 578-592 phase. Physiol Plant 39: 171-178 7. LAMPORT DTA 1970 Cell wall metabolism. Annu Rev Plant Physiol 21: 235-271 24. WITHERS LA, HE STREET 1978 Freeze-preservation of plant cell cultures. In W Barz, E 8. LEDDET C, J SCHAEVERBEKE 1975 Action de la proline sur la resistance au gel des tissus de Reinhard. MH Zenk, eds. Plant Tissue Culture and its Bio-Technological Application. topinambour maintenus en survie. CR Acad Sci Paris Ser D 280: 2849-2852 Springer-Verlag, Berlin, pp 226-244 9. MAZUR P, SP LEIBO, EHR CHU 1972 A two-factor hypothesis of freezing injury. Exp Cell Res