Selection, characterization and regeneration of hydroxyproline-resistant cell lines of Solanum tuberosum: Tolerance to NaCl and freezing stress

Sixty-seven hydroxyproline-resistant (hyp^r) cell lines were selected from cell suspensions of a diploid potato ( Solanum tuberosum L., clone H²578) after plating on 5 and 10 m M hydroxyproline (hyp). Resistant colonies were obtained with a spontaneous frequency of 2.9×10⁻⁶. No clear influence could be shown from treatment with N-ethyl-N-nitrosourea (10 or 50 μ M ). Ninety % of the variant lines contained more proline than the wild type when cells were grown away from hyp for 1 month. Total free amino acid content was increased 2.2 to 6.8 times. When the lines were grown for another 2–5 months on non-selective medium, the content of proline and other amino acids and hyp resistance decreased. After this period the values were, however, still substantially higher than in the wild type. When tested for growth on media with other amino acid analogues (azetidine-2-carboxylic acid and dehydroproline, analogues of proline; aminoethyl-cysteine, analogue of lysine and 3-fluorotyrosine, analogue of tyrosine) and on media with inhibitory concentrations of lysine + threonine. lines H4a and H4b4 were cross resistant to these compounds. When tested on media with inhibitory NaCl concentrations, variant lines H2a, H4a, and H6 showed better tolerance than the wild type. One variant cell line (H4a) was successfully regenerated into plants. Preliminary results showed an increased frost tolerance in the leaves of these plants (−4.5°C compared to −3°C for the wild type), accompanied by a higher leaf proline content. Callus initiated from leaves of the regenerated clones was more resistant to hyp than wild type callus, indicating that the variant trait might be due to a mutation.     

Effect of cold hardening, wilting and exogenously applied proline on leaf proline content and frost tolerance of several genotypes of Solanum

Frost tolerance and leaf proline content were examined in a number of potato hybrids selected for frost tolerance and in the cv. Astarte before and after hardening. Cold hardening (2°C for 20 days) in a dry environment (50/90% relative humidity, day/night) resulted in decreased water content, increased proline content and increased frost tolerance of the leaves of all genotypes. Frost tolerance before and after hardening was positively related to leaf proline content, but not to leaf water content. Drought stress alone, imposed by wilting excised leaves for 4 days, resulted in an accumulation of proline comparable to that after hardening in a dry environment, but the increase in frost tolerance was smaller. Cold hardening in a humid environment (90% relative humidity continuously) only caused a minor accumulation of proline and a small increase in frost tolerance, but the increase in frost tolerance was high in relation to the amount of proline accumulated. Proline, exogenously applied to one of the genotypes, was accumulated in the leaves of shoot cultures, resulting in an increase in frost tolerance. A possible role of proline in frost tolerance is discussed.     

Frost tolerance in cell cultures of potato

Different methods of freezing and of estimating frost damage in cell cultures of Solanum tuberosum L. and a number of wild Solanum species were compared. Frost-killing temperatures (FKT, i.e. the temperature resulting in 50% of the maximum possible frost damage) in leaves of these species were -6°C ( S. acaule ), 5°C ( S. me-gistacrotobum ), -4.5°C ( S. commersonii ) and -3°C ( S. polytrichon and S. tubero-sum ) No appreciable species differences were found in FKT when cells were submerged in either buffer or medium and frozen. However, differences did exist when cells were frozen in a non-submerged condition: S. acaule and S. commersonii callus were more sensitive to frost than suspensions, whereas suspensions of the other species were the most sensitive. Measurement of freezing damage by either electrolyte leakage or by 2,3,5-triphenyltetrazolium chloride viability assays revealed similar FKT values. Cell cultures of S. acaule showed better frost tolerance than S. tuberosum (FKT values were -4.5 to -6°C and -2.5°C. respectively), however, frost tolerance of S. megistacrolobum and S. commersonii was only poorly expressed at the cell level (FKT values were between -2 and -3°C). Variant cell lines previously selected for resistance to the amino acid analogues hydroxyprotine, aminoethylcysteine and 5-methyhryptophan appeared to be more tolerant to frost than the wild type S. tuberosum clone.     

Evaluation of Abiotic Stress Resistance in Mutated Populations of Cauliflower (Brassica oleracea var. botrytis)

An efficient in vitro screening method has been developed for cauliflower to create NEU and NMU induced mutant lines selected on hydroxyproline containing medium. Mutant lines and control plants were sub-cultured many times on maintenance medium and stored at 5°C for 2 years and then tested for salt and hydroxyproline resistance as in vitro and in vivo plants. In vitro shoot tips were also sub-cultured to media containing hydroxyproline and NaCl for 28 days and then assessed for their leaf proline content. Non-acclimated and acclimated in vivo plants were also assessed for resistance to freezing. Populations of control and selected lines were created by mass pollination and subsequently tested for their NaCl and frost resistance. Control plants had little or no NaCl or hydroxyproline resistance whilst selected plants showed varying degrees of resistance. In vitro and in vivo responses of selected lines were correlated. Leaf proline content was increased markedly in the mutant lines and the greatest proline contents occurred following NaCl stress with the most respondent line having 100-fold levels compared to the controls. Both non-acclimated and acclimated selected lines showed improved frost resistance over controls. Improvements in frost resistance were heritable but improvements in NaCl resistance were not. The results clearly demonstrated that NaCl, frost and hydroxyproline resistance were stable traits over repeated in vitro sub-cultures and prolonged low temperature storage. A complete range of mutants with single, double or triple resistance traits were produced. The level of resistance however was not necessarily correlated with the level of proline and some lines showed resistance without elevated proline. It is concluded that elevated proline is not essential for improved resistance to abiotic stress in cauliflower, but where it does occur it does improve resistance.     

Effect of nitrogen supply on frost resistance, nitrogen metabolism and carbohydrate content in white clover (Trifolium repens)

Effects of mineral nitrogen (2, 4, 6 and 8 m M NH₄NO₃) and nodulation with Rhizobium on frost hardiness in seedlings of white clover ( Trifolium repens ) have been studied. Seedlings of a population from Bodø (67°N lat.) were grown in Leonard jars under controlled conditions in a phytotron. For induction of frost hardening, plants were first exposed to 12 h photoperiod conditions for 2 weeks at 18°C, then for 2 weeks at 6°C and finally for 2 weeks at 0.5°C. Frost hardiness after treatments at 6 and 0.5°C was significantly enhanced by increasing nitrogen supply and was positively correlated with total nitrogen content of the stolons. Frost hardiness of nodulated plants correlated to the tissue nitrogen concentration. Content of soluble proteins in stolons decreased during hardening at 6°C but did not change during treatment at 0.5°C. There were minor changes in total amount of free amino acids during hardening. Both absolute and relative amounts of proline and arginine increased, and those of asparagine decreased during hardening. Absolute amounts of all free amino acids increased with increasing nitrogen supply, but the changes during hardening were similar in all treatments. There was a significant increase in the content of soluble carbohydrates during hardening. However, this increase was inversely related to nitrogen supply.     

AN INCREASE OF ORNITHINE δ-AMINOTRANSFERASE-MEDIATED PROLINE SYNTHESIS IN RELATION TO HIGH-TEMPERATURE INJURY IN GRACILARIA TENUISTIPITATA (GIGARTINALES, RHODOPHYTA)

Changes of proline biosynthesis in relation to high-temperature (35° C) injury were investigated in Gracilaria tenuistipitata var. liui Zhang et Xia. On exposure to 35° C, the specific growth rate decreased after 5 days while free proline levels increased gradually after 2 days and reached the maximal level on days 4–6 but decreased at day 7. The repair ability of thalli treated at 35° C by measuring the growth rate after transfer to 25° C for another 5 days decreased in thalli that had been grown at 35° C for more than 2 days, and the extent increased as treatment at 35° C was prolonged. After 4 days of treatment at 35° C, the activities of both ornithine δ-aminotransferase (δ-OAT; EC 2.6.11.3) and Δ¹-pyrroline-5-carboxylate reductase (P5CR; EC 1.5.1.2) increased, but that of γ-glutamyl kinase (γ-GK; EC 2.7.2.11) remained unchanged, and that of glutamate-5-semialdehyde dehydrogenase (GSAd; EC 1.4.1.3) decreased. The application of 10 μM gabaculine, an irreversible inhibitor of δ-OAT, at 35° C recovered the growth ability but inhibited the increase of both δ-OAT activity and free proline level; its effects were reversed by 1 mM proline. G. saliconia, which is relatively tolerant to high temperature, showed a decrease of both δ-OAT activity and free proline level at 35° C. It seems that a stimulation of proline synthesis from the ornithine pathway via an increase in both δ-OAT and P5CR activities might be associated with high-temperature injury in G. tenuistipitata.     

Hardening, abscisic acid, proline and freezing resistance in two winter wheat varieties

Two varieties of winter wheat ( Triticum aestivum L.) differing in freezing resistance ("Holme" from Sweden, freezing resistant, and "Amandus" from Germany, less freezing resistant) were hardened for five weeks by gradually reducing the day/night temperature from 20°C/15°C during the first week to 2° C/0° C during the fifth week and the photoperiod from 15 to 9 h. This treatment increased the freezing resistance of both varieties in comparison to unhardened control plants. Hardening caused an increase in osmolarity of cell sap and in the levels of proline and abscisic acid (ABA). Increase in osmolarity preceded the increase in ABA level, and proline levels increased later than ABA levels. Holme had higher values of osmolarity as well as higher levels of ABA and proline. but the differences between the two varieties were significant only for proline. Since the pressure potential remained constant or increased slightly during the hardening period, it is suggested that the accumulation of ABA is due to the hardening process and not to simple water stress caused by cold-induced inhibition of water uptake by the root.
Spraying hardened plants with 10⁻⁴ M ABA 24 h before a freezing test increased freezing resistance in both varieties, but did not obliterate the differences in freezing resistance between the two varieties. Spraying hardened plants with an aqueous proline solution (10%, w/v) was without effect on freezing resistance. It is concluded that the hardening procedure causes an accumulation of ABA in winter wheat leaves and that ABA is involved in the chain of events leading to freezing resistance.     

Genotype-specific differences in chilling tolerance of maize in relation to chilling-induced changes in water status and abscisic acid accumulation

Four inbred maize lines differing in chilling tolerance were used to study changes in water status and abscisic acid (ABA) levels before, during and after a chilling period. Seedlings were raised in fertilized soil at 24/22°C (day/night), 70% relative humidity. and a 12-h photoperiod with 200 μmol m⁻² s⁻¹ from fluorescent tubes. At an age of 2 weeks the plants were conditioned at 14/12°C for 4 days and then chilled for 5 days at 5/3°C. The other conditions (relative humidity, quantum flux, photoperiod) were unchanged. After the chilling period the plants were transferred to the original conditions for recovery. The third leaves were used to study changes in leaf necrosis, ion efflux, transpiration, water status and ABA accumulation. Pronounced differences in chilling tolerance between the 4 lines as estimated by necrotic leaf areas, ion efflux and whole plant survival were observed. Conditioning significantly increased tolerance against chilling at 5/3°C in all genotypes. The genotypes with low chilling tolerance had lower water and osmotic potentials than the more tolerant genotypes during a chilling period at 5/3°C. These differences were related to higher transpiration rates and lower diffusive resistance values of the more susceptible lines. During chilling stress at 5/3°C ABA levels were quadrupled. Only a small rise was measurable during conditioning at 14/12°C. However, conditioning enhanced the rise of ABA during subsequent chilling. ABA accumulation in the two lines with a higher chilling tolerance was triggered at a higher leaf water potential and reached higher levels than in the less tolerant lines. We conclude that chilling tolerance in maize is related to the ability for fast and pronounced formation of ABA as a protective agent against chilling injury.     

Adaptational change in proline and water content of Staphylococcus aureus after alteration of environmental salt concentration

Adaptation of Staphylococcus aureus to a change in salinity was studied by estimating the intracellular content of water and proline after alteration of the salt concentration of the culture medium. The intracellular water content of S. aureus cultured in normal broth was 1.70 g/g (dry weight). After transfer to 1.8 M NaCl-containing broth, the water content decreased to 0.80 g/g (dry weight) within 1 min. After changing the salt concentration of the medium, intracellular free proline (assumed to be one of the osmoregulators in S. aureus) increased gradually from 0 to 1,400 mumol/g (dry weight) during 30 min of incubation at 37 degrees C. The water content rose to 0.88 g/g (dry weight) in 30 min. Proline was not taken up at 0 to 4 degrees C, suggesting that the process was one of active transport. The salt tolerance of S. aureus, therefore, appears to occur initially by dehydration of the cell after transfer from a medium of low salinity to one of high salinity and then by accumulation of proline, which carries water into the cell with it.     

Adaptational change in proline and water content of Staphylococcus aureus after alteration of environmental salt concentration.

Adaptation of Staphylococcus aureus to a change in salinity was studied by estimating the intracellular content of water and proline after alteration of the salt concentration of the culture medium. The intracellular water content of S. aureus cultured in normal broth was 1.70 g/g (dry weight). After transfer to 1.8 M NaCl-containing broth, the water content decreased to 0.80 g/g (dry weight) within 1 min. After changing the salt concentration of the medium, intracellular free proline (assumed to be one of the osmoregulators in S. aureus) increased gradually from 0 to 1,400 mumol/g (dry weight) during 30 min of incubation at 37 degrees C. The water content rose to 0.88 g/g (dry weight) in 30 min. Proline was not taken up at 0 to 4 degrees C, suggesting that the process was one of active transport. The salt tolerance of S. aureus, therefore, appears to occur initially by dehydration of the cell after transfer from a medium of low salinity to one of high salinity and then by accumulation of proline, which carries water into the cell with it.     

Growth, proline accumulation and water relations of NaCl-selected and non-selected callus lines of Dactylis glomerata L

Sodium chloride-tolerant calli were selected from leaf-derived embryogenic calli of Dactylis glomerata L. on agar solidified medium supplemented with 200 mM NaCl, a concentration lethal to non-selected calli. Growth characteristics, water relations and proline accumulation pattern were compared in selected and non-selected lines. The objective was to gain an understanding of the mechanism(s) of tolerance in the NaCl-tolerant line. Growth in the selected line, as expressed in terms of tolerance index (ratio of fresh wt. on NaCl medium:fresh wt. on NaCl free medium x 100), was greater than that of the non-selected line at all levels of NaCl between 50 and 300 mM. There was no significant difference in proline accumulation in the selected and non-selected lines. Maintenance of turgor by osmotic adjustment was observed in the non-selected line despite decreased growth. In contrast, the selected line lost either the need or the ability to adjust osmotically. There was little or no increase in symplastic osmolality in the selected line when exposed to NaCl. Presumably, selection was made for a salt-excluding tissue that has lost the ability to accumulate solutes and adjust turgor with NaCl stress.     

The interrelationship of growth and frost tolerance in winter rye

The reduction in growth of winter cereals that occurs in the fall is thought to be required for the development of frost resistance. In the present study, the interrelationship of freezing tolerance and growth was examined by raising winter rye ( Secale cereale cv. Puma) plants at 20/16°C (day/night) and at 5/3°C under 8-, 16- and 24-h daylengths to vary growth rates and frost tolerance. Temperature and irradiance were quantified as thermal time, photothermal time and photosynthetic photon flux and examined by multiple linear regression in order to determine their effects on growth and frost tolerance of rye shoots. At low temperature, both growth and frost tolerance were markedly influenced by daylength and irradiance. Plants grown at 5/3°C with a short daylength accumulated shoot dry weight and increased frost tolerance at a greater rate per unit photothermal time or photon flux than plants grown at longer daylengths. Moreover, 5/3°C plants grown with a 16-h day grew more slowly and were less frost tolerant than plants grown with a 24-h day. We conclude that the interrelationship between growth and frost tolerance is a quantitative one. Frost tolerance is induced only by low temperature, but the development of forst tolerance is dependent upon both irradiance, which affects the amount of photoassimilate available, and daylength, which may affect the partitioning of photoassimilates between growth and frost tolerance.     

In vitro selection of salinity tolerant variants from triploid bermudagrass (<Emphasis Type="Italic">Cynodon transvaalensis</Emphasis> × <Emphasis Type="Italic">C. dactylon</Emphasis>) and their physiological responses to salt and drought stress

A protocol was established for in vitro selection of salinity tolerant somaclonal variations from suspension cultured calli of triploid bermudagrass cv. TifEagle. To induce somaclonal variations the calli were subcultured for 18 months and were then subject to three-round selections for salt-tolerant calli by placing on solid medium containing 0.3 M NaCl for 10 days followed by a recovery for 2 weeks. The surviving calli were regenerated on regeneration medium containing 0.1 M NaCl. Three somaclonal variant lines (2, 71, and 77) were obtained and analyzed. The selected somaclonal lines showed higher relative growth and less injury than TifEagle under salt stress, indicating that they increased salt tolerance. In addition, they had higher relative water content and lower electrolyte leakage than TifEagle after withholding irrigation, indicating that they also increased drought tolerance. The three somaclonal variant lines had higher proline content than TifEagle under normal growth condition. The line 71 had a higher K⁺/Na⁺ ratio, whereas the lines 2 and 77 had higher CAT activity under control and salt stress conditions, indicating that different mechanisms for salt tolerance might exist in these three lines.     

Induction of freezing tolerance in potato (Solanum commersonü) suspension cultured cells

The induction of freezing tolerance by abscisic acid (ABA) or cold treatment in suspension cultured cells of Solanum commersonii was studied. Both ABA (50–100 μ M ) at 23°C and low temperature (4°C) increased freezing tolerance in cultured Solanum commersonii cells from a LT₅₀ (freezing temperature at which 50% cells were killed) of —5°C (control) to —11.5°C in 2 days. Cold-induced freezing tolerance reached its maximum at 2 days and remained constant throughout the cold acclimation period of 11 days. The freezing tolerance induced by ABA, however, showed a rapid decline 2 to 5 days after initiation of ABA treatments. Addition of ABA (100 μ M ) to the culture medium at the inception of low temperature treatment did not enhance freezing tolerance of the cells beyond the level attainable by either treatment singly. Poly(A⁺)-RNA was isolated from the respective treatments, translated in a rabbit reticulocyte lysate cell free system, and the translation products were resolved by two dimensional polyacrylamide gel electrophoresis (ID-PAGE). Analysis of the in vitro translated products revealed changes in the abundance of approximately 26 products (encoding for polypeptides with M, of 14 to 69 kDa and pl of 4.90 to 6.60) in ABA-treated cells 12 h after treatment, and 20 (encoding for polypeptides with M_r of 12 to 69 kDa, with pl of 4.80 to 6.42) in cells exposed to 4°C for 12 h. There were only 5 novel translation products observed when the ABA-treated cells reached the highest level of freezing tolerance (2 days after the initiation of ABA treatment). Changes in translatable RNA populations during the induction of freezing tolerance in cells treated with either ABA or low temperature are discussed.     

The effect of salt stress and abscisic acid on proline production,chlorophyll content and growth of <Emphasis Type="Italic">in vitro</Emphasis> propagated shoots of <Emphasis Type="Italic">Eucalyptus camaldulensis</Emphasis>

Three clones, selected for their variation in salt tolerance, were examined regarding their growth and physiological responses on exposure to salt (NaCl) and abscisic acid (ABA) in vitro. The shoot proline levels significantly increased in two salt tolerant clones when exposed to 100 mM NaCl in the shoot multiplication medium. In contrast, proline in a salt sensitive clone did not change in comparison to the control treatment. When 10 M ABA was included in the medium all clones had an increase in proline regardless of whether they were salt tolerant or salt sensitive, linking proline production to the stress hormone ABA. Callus production was so variable that it was not possible to produce callus of consistent texture, colour and growth for all three clones. For the two clones where consistent growth was achievable, both the salt tolerant and salt sensitive clones increased proline production when exposed to salt. This response, however, was greater in the salt tolerant clone. Other parameters examined were growth (dry weight) and shoot chlorophyll content. These characteristics did not correlate with the salt tolerance of the clones, with similar weights being produced on non salt and salt media and similar chlorophyll in both salt sensitive and salt tolerant clones regardless of the medium in which they were grown. The production of proline is considered with regard to selection for differences in salt tolerance in vitro.     

Selection and characterization of an L-thiazolidine-4-carboxylic acid resistant callus culture of Vigna radiata (L.) Wilczek var. radiata

Callus cultures were established from seedling root tips of mungbean (Vigna radiata (L.) Wilczek var. radiata) cv. K 851. The growing calli were exposed to increasing concentrations of thioproline — an analog of proline, in the medium. A concentration of 3.0 mM thioproline completely inhibited the growth of the cells. However, after 25 days incubation 5 cell clones were obtained which could grow on this concentration of thioproline. Out of them one vigorously growing cell clone was further characterized. This selected clone contained higher endogenous levels of free proline (5 fold) and K⁺ (1.5 fold) and exhibited elevated tolerance, not only to thioproline but also to exogenously applied NaCl in the growth medium, as compared to the normal sensitive callus cells. Higher endogenous levels of free proline and K⁺ appear to impart dual resistance to thioproline and NaCl to the selected cell strain.     

Frost resistance of winter rape leaves as related to the changes in water potential and growth capability

Differential thermal analysis indicated that the frost resistance of winter rape leaves ( Brassica napus L. var. oleifera L. cv. Gòrczanski), collected from plants grown in the cold (5/2°C), relies mainly on their ability to supercool to −9 to −11°C, i.e. consists in freezing avoidance. Initiation of ice formation in the cold-acclimated leaves resulted in the death of more than 50% of the cells as determined with a conductivity method. The development of freezing tolerance appeared to be an attribute of the second stage of plant hardening and was induced by the exposure of plants to a slightly subzero temperature (−5°C) for 18 h. Such a treatment brought about a sudden and persistent water potential decrease in the leaves, despite the fact that they had reabsorbed water from the medium prior to water potential measurements. Water potential changes were associated with a higher growth capability of the leaves as checked by determinations of disk area increments. It is suggested that the increased frost tolerance of the cold-grown winter rape leaves, subjected to subfreezing temperature, is related to the decreased water potential of the tissue caused by changes in turgor and/or in osmotic pressures of the cells.     

Physiological changes in gentian axillary buds during two-step preculturing with sucrose that conferred high levels of tolerance to desiccation and cryopreservation

BACKGROUND AND AIMS: Induction of dehydration tolerance is a key to achieving high survival rates in cryopreservation of plant specimens. It has been reported previously that two-step preculturing with sucrose effectively increased desiccation tolerance in axillary buds of gentian (Gentiana scabra), which allow the buds to survive cryopreservation. This study is aimed at characterizing each step of this preculturing and to elucidate physiological changes induced during this preculturing. METHODS: In standard two-step preculture, excised gentian axillary buds were incubated for 11 d on MS medium with 0.1 m sucrose at 25 degrees C (first step: mild osmotic stress was given) and the subsequent incubation on MS medium with 0.4 m and 0.7 m sucrose for 1 d each (second step). The levels of abscisic acid (ABA), proline and soluble sugars in gentian buds during the preculture were determined. Effects of various combinations of two-step preculturing and of exogenous ABA and proline were studied. KEY RESULTS: During the first preculture step, there was a transient increase in ABA content peaking on day 4, which declined to a background level at the end of the first and second step preculturing. Proline level increased steadily during the first preculture step and increased further in the second preculture step. Incubating buds with medium containing proline, instead of the two-step preculturing, did not allow them to survive desiccation. Incubating buds with ABA instead of 0.1 m sucrose-preculturing effectively increased desiccation tolerance only when it was followed by the second preculture step. Fluridone, an ABA synthesis inhibitor included in the two-step preculture medium, reduced desiccation tolerance of the buds. The normal first-step preculture increased the levels of soluble sugars 2.4-fold, especially sucrose and raffinose. Buds treated with the second preculture step had greatly increased sucrose levels. CONCLUSIONS: These observations lead to the hypothesis that the first preculture step involves ABA-mediated cellular changes and the second step induces loading of sucrose in the gentian buds.     

Cadmium tolerance in tobacco cell culture and its relevance to temperature stress

Growth of unselected tobacco (Nicotiana tabacum W38) cell suspension cultures was reduced by 50–200 M cadmium (Cd) in the culture medium and cells were killed by 400 M Cd. Tolerance to Cd was increased either by using rapidly growing cells or by culturing cells at higher densities. Cell lines tolerant to 2 mM Cd were established by progressively elevating levels of Cd in the culture medium. The Cd tolerance was not due to differences in uptake between unselected and Cd-tolerant cell lines, and the tolerance to Cd was not lost during long term culture in the absence of Cd. Cd-tolerant cells also showed higher tolerance to heat shock (37.5°C, 2–8 hours) and cold treatments (4°C, 1–7 days) than the unselected cells.     

Effect of Temperature, Light, Nutrients and Dehardening on Abscisic Acid Induced Cold Hardiness in Bromus inermis Leyss Suspension Cultured Cells

The effects of culture conditions on abscisic acid (ABA)-inducedfreezing tolerance were determined in smooth bromegrass Bromusinermis Leyss cv. Manchar) cell suspension cultures. Bromegrasscultures initiated with 2 g fr wt of cells achieved maximumfreezing tolerances (greater than –32?C) at 25 to 30?Cin the presence of 75 to 100 µM ABA. High levels of freezingtolerance induced by ABA were correlated with high growth ratesat 25 and 30?C. In control cells, incubation at 10?C inducedoptimum levels of hardiness with minimal growth. Prolonged exposure(6 weeks) of cells to 3?C, with or without ABA, increased freezingtolerance only by several degrees. Exogenous ABA concentrationsgreater than 100 µM were not inhibitory to growth. Repeatedexposure to ABA, however, retarded growth and made the cellstolerant to temperatures below –40?C. Removal of ABA fromthe medium resulted in dehardening of the cells both at 25 and3?C. Nitrogen had a marginal effect on ABA-induced hardeningat 25?C, but inhibited age-dependent hardening of control cellcultures. Light had no effect on the freezing tolerance of culturedcells. Addition of 10% sucrose, 30 min prior to freezing, tobromegrass cells treated with ABA for 4 days increased freezingtolerance more than 15?C. These observations are discussed inrelation to the contrasting behaviour of the low temperatureand photoperiod dependent cold acclimation of plants (Received July 14, 1989; Accepted October 23, 1989)