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.     

Abscisic Acid Stimulated Osmotic Adjustment and Its Involvement in Adaptation of Tobacco Cells to NaCl

Osmotic adjustment of cultured tobacco (Nicotiana tabacum L. var Wisconsin 38) cells was stimulated by 10 micromolar (±) abscisic acid (ABA) during adaptation to water deficit imposed by various solutes including NaCl, KCl, K₂SO₄, Na₂SO₄, sucrose, mannitol, or glucose. The maximum difference in cell osmotic potential (Ψπ) caused by ABA treatment during adaptation to 171 millimolar NaCl was about 6 to 7 bar. The cell Ψπ differences elicited by ABA were not due to growth inhibition since ABA stimulated growth of cells in the presence of 171 millimolar NaCl. ABA caused a cell Ψπ difference of about 1 to 2 bar in medium without added NaCl. Intracellular concentrations of Na⁺, K⁺, Cl⁻, free amino acids, or organic acids could not account for the Ψπ differences induced by ABA in NaCl treated cells. However, since growth of NaCl treated cells is more rapid in the presence of ABA than in its absence, greater accumulation of Na⁺, K⁺, and Cl⁻ was necessary for ion pool maintenance. Higher intracellular sucrose and reducing sugar concentrations could account for the majority of the greater osmotic adjustment of ABA treated cells. More rapid accumulation of proline associated with ABA treatment was highly correlated with the effects of ABA on cell Ψπ. These and other data indicate that the role of ABA in accelerating salt adaptation is not mediated by simply stimulating osmotic adjustment.     

Aspects of Salt Tolerance in a NaCl-Selected Stable Cell Line of Citrus sinensis

A NaCl-tolerant cell line which was selected from ovular callus of `Shamouti' orange (Citrus sinensis L. Osbeck) proved to be a true cell line variant. This conclusion is based on the following observations. (a) Cells which have been removed from the selection pressure for at least four passages retain the same NaCl tolerance as do cells which are kept constantly on 0.2 molar NaCl. (b) Na⁺ and Cl⁻ uptake are considerably lower in salt-tolerant cells (R-10) than in salt-sensitive cells (L-5) at a given external NaCl concentration. (c) Growth of salt-tolerant cells is markedly suppressed upon replacement of NaCl by KCl, whereas the growth of salt-sensitive cells is only slightly affected. Accumulation of K⁺ and Cl⁻ accompanies the inhibition of growth. Experiments carried out with sodium and potassium sulfate suggest that the toxic effect is due to the accumulated Cl⁻. (d) Removal of Ca²⁺ from the growth medium severely inhibits the growth of salt-tolerant cells in the presence of NaCl, while it has a minor effect on growth of salt-sensitive cells in the presence of NaCl. (e) Electron micrographs show that the salt-tolerant cells have very big vacuoles when exposed to salt, while the size of the vacuoles of the salt-sensitive cells does not change.     

Comparative Toxicities of Salts on Microbial Processes in Soil

Soil salinization is a growing threat to global agriculture and carbon sequestration, but to date it remains unclear how microbial processes will respond. We studied the acute response to salt exposure of a range of anabolic and catabolic microbial processes, including bacterial (leucine incorporation) and fungal (acetate incorporation into ergosterol) growth rates, respiration, and gross N mineralization and nitrification rates. To distinguish effects of specific ions from those of overall ionic strength, we compared the addition of four salts frequently associated with soil salinization (NaCl, KCl, Na₂SO₄, and K₂SO₄) to a nonsaline soil. To compare the tolerance of different microbial processes to salt and to interrelate the toxicity of different salts, concentration-response relationships were established. Growth-based measurements revealed that fungi were more resistant to salt exposure than bacteria. Effects by salt on C and N mineralization were indistinguishable, and in contrast to previous studies, nitrification was not found to be more sensitive to salt exposure than other microbial processes. The ion-specific toxicity of certain salts could be observed only for respiration, which was less inhibited by salts containing SO₄²⁻ than Cl⁻ salts, in contrast to the microbial growth assessments. This suggested that the inhibition of microbial growth was explained solely by total ionic strength, while ion-specific toxicity also should be considered for effects on microbial decomposition. This difference resulted in an apparent reduction of microbial growth efficiency in response to exposure to SO₄²⁻ salts but not to Cl⁻ salts; no evidence was found to distinguish K⁺ and Na⁺ salts.     

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.     

Effects of Catchment and Riparian Landscape Setting on Water Chemistry and Seasonal Evolution of Water Quality in the Upper Han River Basin,China

Six-year (2005–2010) evolution of water chemistry (Cl⁻, NO₃ ⁻, SO₄ ²⁻, HCO₃ ⁻, Na⁺, K⁺, Ca²⁺ and Mg²⁺) and their interactions with morphological properties (i.e., slope and area), land cover, and hydrological seasonality were examined to identify controlling factors and processes governing patterns of stream water quality in the upper Han River, China. Correlation analysis and stepwise multiple regression models revealed significant correlations between ions (i.e., Cl⁻, SO₄ ²⁻, Na⁺ and K⁺) and land cover (i.e., vegetation and bare land) over the entire catchment in both high- and low-flow periods, and in the buffer zone the correlation was much more stronger in the low-flow period. Catchment with steeper slope (>15°) was negatively correlated with major ions, largely due to multicollinearity of basin characteristics. Land cover within the buffer zone explained slightly less of major elements than at catchment scale in the rainy season, whereas in the dry season, land cover along the river networks in particular this within 100 m riparian zone much better explained major elements rather than this over the entire catchment. Anthropogenic land uses (i.e., urban and agriculture) however could not explain water chemical variables, albeit EC, TDS, anthropogenic markers (Cl⁻, NO₃ ⁻, SO₄ ²), Na⁺, K⁺ and Ca²⁺ significantly increased during 2005–2010, which was corroborated by principal component analyses (PCA) that indicated anthropogenic inputs. Observations demonstrated much higher solute concentrations in the industrial-polluted river. Our results suggested that seasonal evolution of water quality in combined with spatial analysis at multiple scales should be a vital part of identifying the controls on spatio-temporal patterns of water quality.     

Patterns of ion distribution in selected NaCl tolerant and normal lines of four grass species

Selected NaCl tolerant and unselected control lines ofHolcus lanatus L.,Lolium perenne L.,Dactylis glomerata L., andFestuca rubra L. were grown in sand culture at 0, 100, 200, 250, and/or 300 ml m^-3NaCl for seven weeks. The tolerant lines of all four species produced significantly greater both shoot and root dry matter at all NaCl treatments compared with the unselected control lines. Na⁺, K⁺, Cl^-, Ca²⁺, and Mg²⁺ contents of leaf, stalk, and roots of each species were determined. The tolerant lines ofH. lanatus contained less Na⁺ and less Ca²⁺ but higher K⁺ in shoots, compared with the unselected line. By contrast theL. perenne tolerant line had higher Na⁺ and Cl^- contents at 250, and 300 mol m^-3 NaCl in shoots than the unselected line suggesting a halophytic nature of the tolerant line.D. glomerata accumulated greater quantities of ions compared with the other species examined. The tolerant line contained significantly less Cl^- but more K⁺ in its shoots than the unselected line. Na⁺, Cl^-, and K⁺ contents in the shoots of the tolerant line ofF.rubra were higher than in the unselected line shoots. Therefore selection for NaCl tolerance may provide useful material for examining the basis of tolerance.     

Salt tolerance inNitellopsis obtusa

Summary The mechanism of salt tolerance was studied using isolated internodal cells of the charophyteNitellopsis obtusa grown in fresh water. When 100 mM NaCl was added to artificial pond water (0.1 mM each of NaCl, KC1, CaCl₂), no cell survived for more than one day. Within the first 30 minutes, membrane potential (E_m) depolarized and membrane resistance (R_m) decreased markedly. Simultaneously, cytoplasmic Na⁺ increased and K⁺ decreased greatly. At steady state the increase in Na⁺ content was roughly equal to the decrease in K⁺ content. The Cl^– content of the cytoplasm did not change. These results suggest that Na⁺ enters the cytoplasm by exchange with cytoplasmic K⁺. Both the entry of Na⁺ and the exit of K⁺ are assumed to be passive and the latter being caused by membrane depolarization. Vacuolar K⁺, Na⁺, and Cl^– remained virtually constant, suggesting that rapid influx of Na⁺ from the cytoplasm did not occur.In 100 mM NaCl containing 10 mM CaCl₂, membrane depolarization, membrane resistance decrease and changes in cytoplasmic [Na⁺] and [K⁺] did not occur, and cells survived for many days. When cells treated with 100 mM NaCl were transferred within 1 hour to 100 mM NaCl containing 10 mM CaCl₂, Em decreased, Rm increased, cytoplasmic Na⁺ and K⁺ returned to their initial levels, and cells survived. Two possible mechanisms for the role of Ca²⁺ in salt tolerance inNitellopsis are discussed; one a reduction in plasmalemma permeability to Na⁺ and the other a stimulation of active Na⁺-extrusion.     

The High Salt Requirement of the Moderate Halophile Chromohalobacter salexigens DSM3043 Can Be Met Not Only by NaCl but by Other Ions

The growth rate of Chromohalobacter salexigens DSM 3043 can be stimulated in media containing 0.3 M NaCl by a 0.7 M concentration of other salts of Na⁺, K⁺, Rb⁺, or NH₄⁺, Cl⁻, Br⁻, NO₃⁻, or SO₄²⁻ ions. To our knowledge, growth rate stimulation by a general high ion concentration has not been reported for any organism previously.     

Selection and characterization of sodium chloride-tolerant callus of Glycine max (L.) Merr cv. Acme

Callus cultures were initiated from soybean (Glycine max (L.) Merr cv. Acme) cotyledons onMiller's basal medium supplemented with 2 mg L^–1NAA and 0.5 mg L^–1 kinetin. Growing cells wereexposed to increasing concentrations of NaCl in themedium. A concentration of 100 mM NaCl completelyinhibited callus growth. After incubation for 28 d,cells which could tolerate this concentration of NaClgrew to form cell colonies. A NaCl-tolerant line wasobtained through continuous subculturing on 100 mMNaCl. Salt tolerance in this culture was characterizedby an altered growth behavior, reduced cell volume, and accumulation of Na⁺, Cl^–, proline and sugars when grown under salt stress, as well as on normal media. These characteristics, which proved tobe stable after the culture was transferred to asalt-free medium, is commonly associated with halophytes. Presented data suggest that this salt tolerance is the result of a shift towards a halophytic behavior.     

Effects of Helminthosporium carbonum Toxin on Absorption of Solutes by Corn Roots

Susceptible corn roots exposed to the host-selective toxin of Helminthosporium carbonum took up and retained more NO₃⁻, Na⁺, Cl⁻, 3-o-methylglucose, and leucine than did control roots. Stimulatory effects on uptake were more pronounced with freshly cut roots than with roots that were washed and aged. Solutes were accumulated against a concentration gradient, and toxin-treated tissues developed a steeper gradient than did control tissues. Toxin affected both the low and high affinity uptake systems for Na⁺ and Cl⁻. Toxin did not affect uptake of Na₂⁻, K⁺, Ca²⁺, phosphate ion (H₂PO₄⁻ and HPO₄⁻), SO₄⁻, and glutamic acid. No toxin-induced leakage of any solute tested was detected within 5 to 6 hr after initial exposure to toxin. The data suggest that toxin from H. carbonum does not cause the general plasma membrane derangement caused by other host-selective toxins. Instead, H. carbonum toxin may cause specific changes in characteristics of the plasmalemma, which result in increased uptake of certain solutes.     

Physiological Control of Chloride Transport in Chara corallina: II. THE ROLE OF CHLORIDE AS A VACUOLAR OSMOTICUM

The extent to which Cl⁻ is replaceable as the major anionic constituent of the vacuole of Chara corallina was investigated. It was found that external Cl⁻ is not essential in order for nongrowing cells to increase internal osmotic pressure. After growth of cells in low (9 micromolar) Cl⁻, the vacuolar Cl⁻ concentration is one-half that of cells grown at normal external Cl⁻ concentration (850 micromolar). In contrast, both internal osmotic pressure and total concentration of the major cations, K⁺ and Na⁺, in the same cells were found to be only slightly sensitive to the external Cl⁻ concentration. Thus, it is proposed that, at limiting external Cl⁻ concentration, the cell is able to transport or synthesize another anion for vacuolar use rather than utilize a neutral solute.     

Role of Internal Potassium in Maintaining Growth of Cultured Citrus Cells on Increasing NaCl and CaCl(2) Concentrations

Shamouti orange (Citrus sinensis L. Osbeck) salt-tolerant cells were grown under low water potential conditions induced by polyethylene glycol (PEG), NaCl, and CaCl₂. On the basis of equal osmotic potentials, PEG was the least inhibitory, NaCl next, and CaCl₂ the most inhibitory. The relation between growth capacity and ion content can be summarized as follows. (a) Internal K⁺ concentration was a major factor which changed in the presence of PEG, NaCl, and CaCl₂ and probably played a key role in determining growth capacity. (b) Internal concentrations of Na⁺, Ca²⁺, or Cl⁻ could not be directly correlated with growth. (C) Internal Mg²⁺ concentration could be significant only in the presence of high external Ca²⁺ concentrations. (d) The contribution of nitrate and phosphate to the internal osmoticum was negligible. The ratio of external (Ca²⁺)/(Na⁺)² concentration is crucial for growth. Ratios above 0.5 × 10⁻⁴ per millimolar gave maximal protection from adverse effects of NaCl. Growth capacity was found to be determined by the combination of (Ca²⁺)/(Na⁺)² ratio and the absolute external concentration of NaCl. However, a correlation between internal K⁺ concentration and growth capacity seemed independent of external NaCl concentration.     

Relationship between coupled Na+−K+−Cl− transport and water absorption across the seawater eel intestine

Summary Simultaneous measurements of net ion and water fluxes were made in the stripped intestine of the seawater eel, and the relationship between Na⁺, K⁺, Cl^– and water transport were examined in the presence of mucosal KCl and serosal NaCl Ringer (standard condition). When Cl^– was removed from both sides of the intestine, net K⁺ flux from mucosa to serosa was reduced, accompanied by complete blockage of water absorption. Since it has been shown that net Cl^– and water fluxes depend on K⁺ transport under the standard condition (Ando 1983), the interdependence of K⁺ and Cl^– transport suggests the existence of a coupled KCl transport system, while the parallelism between the net Cl^– and water fluxes suggests that water absorption is linked to the coupled KCl transport. The coupled KCl and water transport were inhibited by treatment with ouabain or with Na⁺-free Ringer solutions, suggesting the existence of a Na⁺-dependent KCl transport system and linkage of water absorption to the coupled Na⁺–K⁺–Cl^– transport. Since ouabain blocked the active Na⁺–K⁺–Cl^– transport almost completely, the permeability coefficients for K⁺ and Na⁺ through the paracellular shunt pathway were estimated as P_K=0.076 and P_Na=0.058 cm/h, and P_Cl was calculated as 0.005 cm/h. Although Na⁺-independent K⁺ and Cl^tt- fluxes were observed again in the present study, these fluxes were not inhibited by CN^–, ouabain or diuretics, and evoked even after blocking the Na⁺–K⁺–Cl^– transport completely with ouabain. These results indicate that the Na⁺-independent K⁺ and Cl^– fluxes are distinct from the active Na⁺–K⁺–Cl^– transport and are not themselves active.     

Isolated Thellungiella shoots do not require roots to survive NaCl and Na2SO4 salt stresses

Shoots of Thellungiella derived by micropropagation were used to estimate the plants'' salt tolerance and ability to regulate Na⁺ uptake. Two species with differing salt tolerances were studied: Thellungiella salsuginea (halophilla), which is less tolerant, and Thellungiella botschantzevii, which is more tolerant. Although the shoots of neither ecotype survived at 700 mM NaCl or 200 mM Na₂SO₄, micropropagated shoots of T. botschantzevii were more tolerant to Na₂SO₄ (10–100 mM) and NaCl (100–300 mM). In the absence of roots, Na₂SO₄ salinity reduced shoot growth more dramatically than NaCl salinity. Plantlets of both species were able to adapt to salt stress even when they did not form roots. First, there was no significant correlation between Na⁺ accumulation in shoots and Na⁺ concentration in the growth media. Second, K⁺ concentrations in the shoots exposed to different salt concentrations were maintained at equivalent levels to control plants grown in medium without NaCl or Na₂SO₄. These results suggest that isolated shoots of Thellungiella possess their own mechanisms for enabling salt tolerance, which contribute to salt tolerance in intact plants.Key words: Thellungiella salsuginea, Thellungiella botschantzevii, salt tolerance, isolated shoots, growth, rhizogenesis, ion accumulation     

Cation pretreatment effects on nitrate uptake, xylem exudate, and malate levels in wheat seedlings

Week-old wheat seedlings absorbed at least 40% NO₃⁻ from NaNO₃ when preloaded with K⁺ than when preloaded with Na⁺ or Ca²⁺. Cultures of Triticum vulgare L. cv. Arthur were grown for 5 days on 0.2 mm CaSO₄, pretreated for 48 hours with either 1 mm CaSO₄, K₂SO₄, or Na₂SO₄, and then transferred to 1 mm NaNO₃. All solutions contained 0.2 mm CaSO₄. Shoots of K⁺-preloaded plants accumulated three times more NO₃⁻ than shoots of the other two treatments. Initially, the K⁺-preloaded plants contained 10-fold more malate than either Na⁺- or Ca²⁺-preloaded seedlings. During the 48-hour treatment with NaNO₃, malate in both roots and shoots of the K⁺-preloaded seedlings decreased. Seedlings preloaded with K⁺ reduced 25% more NO₃⁻ than those preloaded with either Na⁺ or Ca²⁺. These experiments indicate that K⁺ enhanced NO₃⁻ uptake and reduction even though the absorption of K⁺ and NO₃⁻ were separated in time. Xylem exudate of K⁺-pretreated plants contained roughly equivalent concentrations of K⁺ and NO₃⁻, but exudate from Na⁺ and Ca²⁺-pretreated plants contained two to four times more NO₃⁻ than K⁺. Therefore K⁺ is not an obligatory counterion for NO₃⁻ transport in xylem.     

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.     

Response of sugarcane to different types of salt stress

Summary Due to climatic conditions and prevailing water regime the yield and sucrose recovery in sugarcane are high in South Western India. However, excessive irrigation, poor drainage and luxuriant use of fertilizers have resulted in conversion of large fertile areas into saline lands. The salinity is due to the excess of Na⁺, Ca⁺⁺, Mg⁺⁺, SO₄ ^– and Cl^– ions. Individual salts of NaCl, Na₂SO₄, MgCl₂ and MgSO₄ were employed in culture experiments to study salt stress effect on sugarcane variety Co 740. It was observed that sulphate salinity was more toxic to sugarcane than the chloride one. Sulphate salts caused more inhibition of growth, chlorophyll synthesis, PEPCase activity, decreased the uptake of K⁺ and Ca⁺⁺ ions but stimulated nitrate reductase. The stress did not result in proline accumulation in the sugarcane cultivar Co 740. The degree of toxicity of different ions in decreasing order in sugarcane cultivar Co 740 is SO₄ ^–>Na⁺>Cl^–>Mg⁺⁺.     

Inhibition of anion transport in corn root protoplasts

The effects of several amino-reactive disulfonic stilbene derivatives and N-(4-azido-2-nitrophenyl)-2-aminoethylsulfonate on Cl⁻, SO₄²⁻, and inorganic phosphate (Pi) uptake in protoplasts isolated from corn root tissue were studied. 4-Acetamido-4′-isothiocyano-2,2′-stilbenedisulfonic acid, 4,4′-diisothiocyano-2,2′-stilbenedisulfonic acid, 4,4′-diamino-2,2′-stilbenedisulfonic acid, and NAP-taurine inhibited Cl⁻ and SO₄²⁻ but not Pi and K⁺ uptake in corn root protoplasts; whereas mersalyl inhibited Pi but not Cl⁻ or SO₄²⁻ uptake. The rate of uptake of all anions decreased with increasing external pH. In addition, these reagents markedly inhibited plasmalemma ATPase activity isolated from corn root tissue. Excised root segments were less sensitive to Cl⁻ and SO₄²⁻ transport inhibitors.     

The Effect of Different Salts of Sodium and Potassium on the Accumulation of Glycinebetaine in Atriplex Prostrata

Atriplex prostrata was grown for one month in nutrient solutions with NaCl, KCl, Na₂SO₄, and K₂SO₄ (at osmotic potentials of 0, –0.75, –1.00, and –1.50 MPa). Plants treated with K₂SO₄ had less glycinebetaine at –1.0 and –1.50 MPa than those treated with Na⁺ salts, probably due to the inhibitory effects of K⁺ on glycinebetaine accumulation.