Physiological and biochemical parameters for evaluation and clustering of rice cultivars differing in salt tolerance at seedling stage

Salinity tolerance levels and physiological changes were evaluated for twelve rice cultivars, including four white rice and eight black glutinous rice cultivars, during their seedling stage in response to salinity stress at 100 mM NaCl. All the rice cultivars evaluated showed an apparent decrease in growth characteristics and chlorophyll accumulation under salinity stress. By contrast an increase in proline, hydrogen peroxide, peroxidase (POX) activity and anthocyanins were observed for all cultivars. The K⁺/Na⁺ ratios evaluated for all rice cultivars were noted to be highly correlated with the salinity scores thus indicating that the K⁺/Na⁺ ratio serves as a reliable indicator of salt stress tolerance in rice. Principal component analysis (PCA) based on physiological salt tolerance indexes could clearly distinguish rice cultivars into 4 salt tolerance clusters. Noteworthy, in comparison to the salt-sensitive ones, rice cultivars that possessed higher degrees of salt tolerance displayed more enhanced activity of catalase (CAT), a smaller increase in anthocyanin, hydrogen peroxide and proline content but a smaller drop in the K⁺/Na⁺ ratio and chlorophyll accumulation.     

The effect of NaCl on proline accumulation in potato seedlings and calli

The effects of salt stress were studied on the accumulation and metabolism of proline and its correlation with Na⁺ and K⁺ content in shoots and callus tissue of four potato cultivars, viz., Agria, Kennebec (relatively salt tolerant), Diamant and Ajax (relatively salt sensitive). Na⁺ and proline contents increased in all cultivars under salt stress. However, K⁺ and protein contents decreased in response to NaCl treatments. The activities of enzymes involved in proline metabolism, Δ¹-pyrroline-5-carboxylate synthetase (P5CS) and proline dehydrogenase (ProDH) increased and decreased, respectively, in response to elevated NaCl concentrations. The changes of P5CS and ProDH activities in more salt sensitive cultivars (Diamant, Ajax) were more than those in the tolerant ones. Then the stimulation of synthesis in combination with a partially increase of protein proteolysis, a decrease in proline utilization and inhibition of oxidation resulted in high proline contents in seedlings and calli under salt stress. In callus tissue, reduced growth and cell size may be partially responsible for high proline accumulation in response to high NaCl levels. However, although the basic proline contents in the seedlings of more salt tolerant cultivars were higher than the sensitive ones, a clear relationship was not generally observed between accumulation of proline and salt tolerance in potato.     

Enhancement of antioxidant enzyme activities in rice callus by ascorbic acid under salinity stress

Ascorbic acid (AsA) is naturally occurring compound with antioxidant activity and plays a pivotal role in plant cell adaptation to salinity stress. The objective of this work was to assess the influence of exogenous AsA on the embryogenic callus of indica rice (Oryza sativa L.) cv. MRQ74 cultivated under saline conditions. NaCl (200 mM) decreased callus fresh and dry masses, relative growth rate, and K⁺ and Ca⁺² content, and increased Na⁺ content and Na⁺/K⁺ ratio. Application of AsA (0.5 or 1 mM) alleviated these effects of salinity. Activities of peroxidase, catalase, superoxide dismutase, as well as content of proline increased due to the NaCl treatment, and these parameters were mostly further increased by 0.5 mM AsA. Thus, AsA can increase callus tolerance to NaCl stress.     

Evaluation of NaCl Tolerance in the Callus Cultures of Suaeda nudiflora Moq.

Salt tolerance was studied in the callus cultures of Suaeda nudiflora Moq. a dicotyledonous succulent halophyte. Growth was significantly inhibited at 50, 100, 150 and 200 mM NaCl. Inorganic ions and proline accumulated in response to salinity. Ion accumulation pattern reflected the utilization of Na⁺ as an osmoticum. Na⁺/K⁺ ratio rose steadily as a function of external NaCl concentration. Salt stress enhanced the activity of peroxidase, whereas it decreased activities of superoxide dismutase and catalase. This revised version was published online in July 2006 with corrections to the Cover Date.     

Physiological and proteomic analysis of salinity tolerance of the halotolerant cyanobacterium <Emphasis Type="Italic">Anabaena</Emphasis> sp

The halotolerant cyanobacterium Anabaena sp was grown under NaCl concentration of 0, 170 and 515 mM and physiological and proteomic analysis was performed. At 515 mM NaCl the cyanobacterium showed reduced photosynthetic activities and significant increase in soluble sugar content, proline and SOD activity. On the other hand Anabaena sp grown at 170 mM NaCl showed optimal growth, photosynthetic activities and comparatively low soluble sugar content, proline accumulation and SOD activity. The intracellular Na⁺ content of the cells increased both at 170 and 515 mM NaCl. In contrast, the K⁺ content of the cyanobacterium Anabaena sp remained stable in response to growth at identical concentration of NaCl. While cells grown at 170 mM NaCl showed highest intracellular K⁺/Na⁺ ratio, salinity level of 515 mM NaCl resulted in reduced ratio of K⁺/Na⁺. Proteomic analysis revealed 50 salt-responsive proteins in the cyanobacterium Anabaena sp under salt treatment compared with control. Ten protein spots were subjected to MALDI-TOF–MS/MS analysis and the identified proteins are involved in photosynthesis, protein folding, cell organization and energy metabolism. Differential expression of proteins related to photosynthesis, energy metabolism was observed in Anabaena sp grown at 170 mM NaCl. At 170 mM NaCl increased expression of photosynthesis related proteins and effective osmotic adjustment through increased antioxidant enzymes and modulation of intracellular ions contributed to better salinity tolerance and optimal growth. On the contrary, increased intracellular Na⁺ content coupled with down regulation of photosynthetic and energy related proteins resulted in reduced growth at 515 mM NaCl. Therefore reduced growth at 515 mM NaCl could be due to accumulation of Na⁺ ions and requirement to maintain higher organic osmolytes and antioxidants which is energy intensive. The results thus show that the basis of salt tolerance is different when the halotolerant cyanobacterium Anabaena sp is grown under low and high salinity levels.     

Growth rate, water relations and ion accumulation of soybean callus lines differing in salinity tolerance under salinity stress and its subsequent relief

A selected Glycine max (L.) salt-tolerant calluscell line (R100) was significantly more tolerant to salt than a salt-sensitiveline (S100) during exposure to salt stress. Growth (Fresh and Dry weights) ofthe R100 cell line declined significantly at NaCl concentrations greater than 75mM, while growth of the S100 cell line was already impaired at 25mM NaCl. Levels of Na⁺ and Cl^– inthe callus were elevated as the salt concentration increased, whileK⁺, Ca²⁺ and Mg²⁺ levels weremarkedly reduced. The lower s reduction and Na⁺accumulation found in the S100 callus corresponded with the higher callusdehydration during salinity. Calli grown on Miller's basal medium weresupplied with 100 mM NaCl for 12 days and then supplied with mediumwithout NaCl to relieve salinity stress. The Na⁺ andCl^– content decreased in both R100 and S100 cell lines duringthe first 24 h and reached normal levels four days after transferto the normal medium. This lower concentration was maintained until the end ofthe experiment. Concurrently, the K⁺ content andK⁺/Na⁺ ratio increased sharply and reached theirhighest levels within 24 h in both salt-sensitive and salt-tolerantcell lines. These data suggest that the inhibitory effects of salinization ongrowth and accumulation of potentially toxic ions (Na⁺,Cl^–) can be readily reversed when salinity is relieved.     

Comparative salt tolerance analysis between Arabidopsis thaliana and Thellungiella halophila, with special emphasis on K(+)/Na(+) selectivity and proline accumulation

The eco-physiology of salt tolerance, with an emphasis on K⁺ nutrition and proline accumulation, was investigated in the halophyte Thellungiella halophila and in both wild type and eskimo-1 mutant of the glycophyte Arabidopsis thaliana, which differ in their proline accumulation capacity. Plants cultivated in inert sand were challenged for 3 weeks with up to 500 mM NaCl. Low salinity significantly decreased A. thaliana growth, whereas growth restriction was significant only at salt concentrations equal to or exceeding 300 mM NaCl in T. halophila. Na⁺ content generally increased with the amount of salt added in the culture medium in both species, but T. halophila showed an ability to control Na⁺ accumulation in shoots. The analysis of the relationship between water and Na⁺ contents suggested an apoplastic sodium accumulation in both species; this trait was more pronounced in A. thaliana than in T. halophila. The better NaCl tolerance in the latter was associated with a better K⁺ supply, resulting in higher K⁺/Na⁺ ratios. It was also noteworthy that, despite highly accumulating proline, the A. thaliana eskimo-1 mutant was the most salt-sensitive species. Taken together, our findings indicate that salt tolerance may be partly linked to the plants’ ability to control Na⁺ influx and to ensure appropriate K⁺ nutrition, but is not linked to proline accumulation.     

Effects of iso-osmotic NaCl and mannitol on growth, proline content, and antioxidant defense in Mammillaria gracilis Pfeiff. in vitro-grown cultures

Effects of iso-osmotic concentrations of NaCl and mannitol were studied in Mammilaria gracilis (Cactaceae) in both calli and tumors grown in vitro. In both tissues, relative growth rates were reduced under osmotic stress, which were accompanied by a decrease in both tissue water and K⁺ content. However, growth was inhibited to a lesser extent after exposure to NaCl, when accumulation of Na⁺ ions was observed. In calli, only salinity increased proline content, whereas with tumors proline accumulated after both osmotic stresses. Osmotic stresses also induced oxidative damage in both cactus tissues, although higher oxidative injury was caused by mannitol in calli and by salt in tumors. Low iso-osmotic concentrations of NaCl (75 mM) and mannitol (150 mM) increased peroxidase, ascorbate peroxidase, and esterase activities, whereas elevated catalase activity was recorded only after mannitol treatment in both tissues. High osmotic stress generally decreased enzymatic activities. However, in calli, esterase activity increased in response to high salinity, whereas ascorbate peroxidase activity was enhanced after high mannitol stress. In conclusion, both in vitro-grown cactus tissues were found to be sensitive to osmotic stress caused by either mannitol or NaCl, but accumulation of Na⁺ ions in response to salt somewhat contributed to osmotic adjustment. However, more prominent oxidative damage induced by NaCl compared to mannitol in tumor could be related to ion toxicity. The mechanisms that mediate responses to salt- and mannitol-induced osmotic stresses differed and were dependent on tissue type.     

Salt tolerance in rice in vitro: Implication of accumulation of Na+, K+ and proline

The implication of accumulation of both inorganic (Na⁺, K⁺) and organic (proline) solutes were evaluated in unadapted and NaCl-adapted callus of a salt-sensitive (Basmati 370) and a salt-tolerant (SR-26B) cultivar of rice (Oryza sativa L.) after a NaCl shock. Accumulation of Na⁺,K⁺ and/or proline in callus was co relatable and the relative presence of these components in tissues after shock treatment was found to be important factors to support differential regrowth capacities of the shock treated calluses. Presence or retention of K⁺ in rice callus was a key factor for salt tolerance as it was observed to be positively correlated with growth in both the varieties. The results indicated that K⁺ was the first candidate to counteract the negative water potential of outside milieu, while proline was probably the last metabolic device that rice calluses opted for when exposed to salt stress.     

Dynamics of Na+, K+, and Proline Accumulation in Salttreated Vigna sinensis (L) and Phaseolus aureus (L)

The dynamics of Na⁺, K⁺, and proline accumulation in various organs of non nodulated Vigna sinensis and Phaseolus aureus was followed during their acclimation to two levels of salinities for a period of 35 days and was correlated to the vegetative growth of the two species. The rate of Na⁺ and K⁺ absorption is at a maximum during the first 15 to 20 days of culture. K⁺ absorption is not completely inhibited even at 100 mM NaCl although the endogenous Na⁺ largely surpasses that of K⁺ in certain organs. Low salinity rather accelerates K⁺ absorption in both species. The relative growth rates (RGR) correlate with the rate of Na⁺ and K⁺ accumulation. At low salinity (10 mM NaCl), the RGR of V. sinensis is greater than that of P. aureus. However, at high salinity (100 mM NaCl) the RGR is the same for both species. The growth of the younger parts of the two species is not arrested by salt treatment. Very high accumulation of Na⁺ is avoided in organs with less vacuolated tissues. At no time does the endogenous K : Na ratio in these organs fall below 1.0. Certain organs, especially the roots, hypocotyls, and the lower parts of the stems are capable of storing large quantities of Na⁺. In V. sinensis, the accumulated Na⁺ and K⁺ are evenly distributed among the various organs while in P. aureus they are rather concentrated in the roots. External salinity creates water deficiency in the younger plant parts and as a consequence, proline accumulates especially in the youngest aerial organs - more in P. aureus than in V. sinensis. The accumulation of this amino acid in both the species is dependent on time and correlates directly, not only with the water deficit, but also with the K⁺ contents. In contrast, it does not seem to depend directly on the endogenous Na⁺ content. The relative salt tolerance of the two species and the possible role of K⁺, Na⁺ and proline in the osmotic adjustments of the two species under saline conditions are discussed.     

Effects of salt stress in relation to osmotic adjustment on sugarcane (<Emphasis Type="Italic">Saccharum officinarum</Emphasis> L.) callus cultures

In vitro responses of embryogenic sugarcane (Saccharum officinarum L.; cv. CoC-671) calli stressed with different levels of NaCl (0.0, 42.8, 85.6, 128.3, 171.1, 213.9 or 256.7 mM) were studied. The results showed that a significant decrease in callus growth and cell viability occurred with ≥85.6 mM NaCl. Higher amounts of free proline and glycine betaine were accumulated in NaCl-stressed calli. Although the leached and retained Na⁺ contents increased, the retained K⁺ content decreased with increasing levels of NaCl. Such a mechanism implies that sugarcane can be considered as a Na⁺-excluder. The accumulation of salt ions and osmolytes could play an important role in osmotic adjustment in sugarcane cells under salt stress.     

Effect of sodium and calcium chlorides,abscisic acid and proline on callus cultures ofArachis hypogaea L.

Callus cultures ofArachis hypogaea L. cv. JL-24 adapted to 200 mM NaCl (otherwise lethal to cells) were used for the study. Calli grew slowly when transferred to 250 mM NaCl, but the growth was enhanced when ABA was included in the medium. ABA induced increase in growth of callus was not accompanied by corresponding increase in internal free proline levels. 0.5 mM of CaCl₂ ameliorated the negative effect of NaCl indicating that cells require a specific Ca²⁺/Na⁺ ratio for their growth. Proline content also increased at this ratio thereby suggesting that increase in growth at 0.5 mM Ca²⁺ may be due to an increase in proline content. However, exogenous proline did not increase the growth of callus (adapted to 200 mM), and higher concentrations even inhibited the growth. This shows that proline is not required for growth or adaptation of cells to salt stress, but is produced as a consequence of stress.     

Simultaneous application of salicylic acid and calcium improves salt tolerance in two contrasting tomato (Solanum lycopersicum) cultivars

Soil salinity is one of the most important environmental factors responsible for serious agricultural problems. Tomato salt tolerance may be improved by genetic selection and by the use of adapted physiological tools. The aim of this study was to investigate the impact of exogenous application of salicylic acid (SA 0.01 mM) and calcium sulphate (CaSO₄ 5 mM), singly or in combination, on plant growth, photosynthetic pigments, nutritional behaviour and some metabolic parameters (total chlorophyll, carotenoids, soluble sugars, proline and lipid peroxidation) of two tomato cultivars (cv. Super Marmande and cv. Red River) exposed to salt stress (100 mM NaCl). Application of 100 mM NaCl reduced plant growth, total chlorophyll and carotenoid contents. Salt stress also induced an accumulation of Na⁺, a decrease in K⁺ and Ca^2 + concentration and root sugar level, an increase in malondialdehyde (MDA) and proline concentration. Deleterious impact of salinity was related to modification in ion content rather than modification in the plant water status. Exogenous application of SA or Ca alone improved plant behaviour in the presence of NaCl. Nevertheless, the best results in terms of growth, photosynthetic pigment concentrations and mineral nutrition (limitation of Na⁺ accumulation and maintenance of K⁺ and Ca^2 + content) were obtained in response to the combined SA + Ca treatment. Although the involved physiological parameters varied depending on the considered cultivar, our results suggest that Ca^2 + and SA may interact to reduce the stress experienced by the plant in the presence of NaCl.     

Effect of salinity on osmotic adjustment,proline accumulation and possible role of ornithine-δ-aminotransferase in proline biosynthesis in <Emphasis Type="Italic">Cakile maritima</Emphasis>

The short time response to salt stress was studied in Cakile maritima. Plants were exposed to different salt concentrations (0, 100, 200 and 400 mM NaCl) and harvested after 4, 24, 72 and 168 h of treatment. Before harvesting plants, tissue hydration, osmotic potential, inorganic and organic solute contents, and ornithine-δ-aminotransferase activity were measured. Plants of C. maritima maintained turgor and tissue hydration at low osmotic potential mainly at 400 mM NaCl. The results showed that, in leaves and stems, Na⁺ content increased significantly after the first 4 h of treatment. However, in roots, the increase of Na⁺ content remained relatively unchanged with increasing salt. The K⁺ content decreased sharply at 200 and 400 mM NaCl with treatment duration. This decrease was more pronounced in roots. The content of proline and amino acids increased with increasing salinity and treatment duration. These results indicated that the accumulation of inorganic and organic compounds was a central adaptive mechanism by which C. maritima maintained intracellular ionic balance under saline conditions. However, their percentage contribution to total osmotic adjustment varies from organ to organ; for example, Na⁺ accumulation mainly contributes in osmotic adjustment of stem tissue (60%). Proline contribution to osmotic adjustment reached 36% in roots. In all organs, proline as well as δ-OAT activity increased with salt concentration and treatment duration. Under normal growth conditions, δ-OAT is mainly involved in the mobilization of nitrogen required for plant growth. However, the highly significant positive correlation between proline and δ-OAT activity under salt-stress conditions suggests that ornithine pathway contributed to proline synthesis.     

Counteraction of Salinity Stress on Wheat Plants by Grain Soaking in Ascorbic Acid, Thiamin or Sodium Salicylate

The interactive effects of salinity stress (40, 80, 120 and 160 mM NaCl) and ascorbic acid (0.6 mM), thiamin (0.3 mM) or sodium salicylate (0.6 mM) were studied in wheat (Triticum aestivum L.). The contents of cellulose, lignin of either shoots or roots, pectin of root and soluble sugars of shoots were lowered with the rise of NaCl concentration. On the other hand, the contents of hemicellulose and soluble sugars of roots, starch and soluble proteins of shoots, proline of either shoots or roots, and amino acids of roots were raised. Also, increasing NaCl concentration in the culture media increased Na⁺ and Ca²⁺ accumulation and gradually lowered K⁺ and Mg²⁺ concentration in different organs of wheat plant. Grain soaking in ascorbic acid, thiamin or sodium salicylate could counteract the adverse effects of NaCl salinity on the seedlings of wheat plant by suppression of salt stress induced accumulation of proline.     

Effect of nitric oxide signaling in bacterial-treated soybean plant under salt stress

To understand protective roles of nitric oxide against salt stress, the effects of exogenous sodium nitroprusside on activities of lipoxygenase, peroxidase, phenylalanine ammonialyase, catalase, superoxide dismutase enzymes, proline accumulation, and distribution of sodium in soybean plants under salt were determined. Application of sodium nitroprusside + bacterium enhanced plant growth-promotion characteristics, activities of different enzymes, and proline accumulation in the presence of sodium nitroprusside under salt stress. Treatment with NaCl at 200 mM and sodium nitroprusside (0.1 mM) reduced Na⁺ levels but increased K⁺ levels in leaves in comparison with the NaCl-treated plants. Correspondingly, the plants treated with exogenous sodium nitroprusside and NaCl maintained a lower ratio of [Na⁺]/[K⁺] in NaCl-stressed plants.     

Cation co-tolerance phenomenon in cell cultures of Oryza sativa adapted to LiCl and NaCl

Cell lines of Oryza sativa L. (cv. Taipei-309) were adapted to 30 mM LiCl and 150 mM NaCl. Both adapted lines were considerably more tolerant than non adapted line when grown on 200, 250 and 300 mM NaCl and 30 mM LiCl stresses. The tolerance of LiCl-adapted line to NaCl (150 to 300 mM) and the tolerance of NaCl-adapted cells line to LiCl (30 mM) indicated that there was a cross-adaptation towards alkali metals (Na⁺ and Li⁺) not the Cl^–. Na⁺ and K⁺ contents of all lines which increased with increasing medium salinity but to a different degree. The increase in Na⁺ and K⁺ content in NaCl-adapted and non-adapted lines were comparable, while LiCl-adapted line accumulated significantly lower Na⁺and higher K⁺ content. Proline content of all lines increased with the increase in NaCl-stress but the magnitude of increase was much higher in the LiCl-adapted than other lines. The differential response of adapted lines to NaCl stress in accumulating proline and maintaining the ionic contents reveals that adapted lines have evolved different features of adaptation to cope with NaCl stress.     

Effect of varying NaCl doses on flavonoid production in suspension cells of Ginkgo biloba: relationship to chlorophyll fluorescence,ion homeostasis,antioxidant system and ultrastructure

Ginkgo suspension cells were used to investigate the mechanism that governs the shift between primary and secondary metabolism under NaCl elicitation. The production of three flavonol glycosides, chlorophyll fluorescence, ion content, the antioxidant system, and the cellular ultrastructure in the presence of NaCl doses from 5 to 175 mM were examined. At low salt doses (5–50 mM), cell growth and flavonol glycosides accumulation were stimulated without damaging cell structure or inducing oxidative stress by maintaining high K⁺ and chlorophyll content. At moderate salt doses (75–125 mM), the cells could withstand the salt stress without an impact on survival by changing internal cellular structure, maintaining high levels of K⁺ and Ca²⁺ and increasing anti-oxidative enzyme activities rather than flavonol glycosides to counteract the inhibition of the photosystem II, the accumulation of Na⁺ and hydrogen peroxide (H₂O₂) in the cells. This allowed cells to divert their metabolism from growth to defense-related pathways and tolerate NaCl stress. At higher salinity (150–175 mM), the cellular structure was damaged, and the high Na⁺ and low K⁺ content led to osmotic stress, and therefore, the stimulation of peroxidase (POD) and catalase (CAT) was not enough to cope with high H₂O₂ accumulation. The high production of flavonol glycosides may be a response of elicitation stimulation to serious damage at 175 mM NaCl. In conclusion, the use of 175 mM NaCl may be desirable for the induction of flavonol glycoside production in Ginkgo suspension cells.     

Exogenous Putrescine Reduces Sodium and Chloride Accumulation in NaCl-Treated Calli of the Salt-Sensitive Rice Cultivar I Kong Pao

In order to gain information on the putative involvement of polyamines (PAs) in the response of rice cells to salinity, mature embryo-derived calli issued from the salt-sensitive cultivar I Kong Pao were exposed for 3 months to the simultaneous presence of NaCl (0, 150 and 300 mM) and exogenous polyamines (putrescine (Put): 1 and 10 mM; spermidine (Spd): 1 and 10 mM; spermine (Spm): 1 mM). Callus growth, endogenous PAs, Na⁺, K⁺ and Cl⁻ concentrations were quantified and analysed in relation to cell viability based on 2,3,5-triphenytetrazolium chloride (TTC) reduction. All exogenous PAs were efficiently absorbed from the external medium. Exogenous Put 1 mM clearly stimulated growth of salt-stressed calli in relation to a decrease in both Na⁺ and Cl⁻ accumulation. In contrast, Spd 10 mM and Spm 1 mM exacerbated the deleterious impact of NaCl on callus growth and induced a decrease in K⁺ concentration. While Put helped in the maintenance of cell viability, Spd 10 mM and Spm 1 mM decreased cell viability, mainly in relation to an inhibition of the alternative respiratory pathway. It is proposed that Put may assume positive functions in salt stress resistance in rice.     

Selection of callus cultures of sugarcane (Saccharum sp.) tolerant to NaCl and their response to salt stress

Stable callus cultures tolerant to NaCl (68 mM) were developed from salt-sensitive sugarcane cultivar CP65-357 by in vitro selection process. The accumulation of both inorganic (Na⁺, Cl⁻ and K⁺) and organic (proline and soluble sugars) solutes was determined in selected and non-selected calli after a NaCl shock in order to evaluate their implication in in vitro salt tolerance of the selected lines. Both salt-tolerant and non-selected calli showed similar relative fresh weight growth in the absence of NaCl. No growth reduction was observed in salt-tolerant calli while a significant reduction about 32% was observed in nonselected ones when both were cultivated on 68 mM NaCl. Accumulation of Na⁺ was similar in both salt-tolerant and non-selected calli in the presence of NaCl. Accumulation of Cl⁻ was lower in NaCl-tolerant than in non-selected calli while proline and soluble sugars were more accumulated in salt-tolerant than in non-selected calli when both were exposed to salt. K⁺ level decreased more severely in non-selected calli than in NaCl-tolerant ones after NaCl shock. The results indicated that K⁺ and Cl⁻ may play a key role in in vitro salt-tolerance in sugarcance cell lines obtained by in vitro selection and that organic solutes could contribute mainly to counteract the negative water potential of the outside medium.