Ability of leaf mesophyll to retain potassium correlates with salinity tolerance in wheat and barley

This work investigated the importance of the ability of leaf mesophyll cells to control K⁺ flux across the plasma membrane as a trait conferring tissue tolerance mechanism in plants grown under saline conditions. Four wheat (Triticum aestivum and Triticum turgidum) and four barley (Hordeum vulgare) genotypes contrasting in their salinity tolerance were grown under glasshouse conditions. Seven to 10‐day‐old leaves were excised, and net K⁺ and H⁺ fluxes were measured from either epidermal or mesophyll cells upon acute 100 mM treatment (mimicking plant failure to restrict Na⁺ delivery to the shoot) using non‐invasive microelectrode ion flux estimation (the MIFE) system. To enable net ion flux measurements from leaf epidermal cells, removal of epicuticular waxes was trialed with organic solvents. A series of methodological experiments was conducted to test the efficiency of different methods of wax removal, and the impact of experimental procedures on cell viability, in order to optimize the method. A strong positive correlation was found between plants' ability to retain K⁺ in salt‐treated leaves and their salinity tolerance, in both wheat and especially barley. The observed effects were related to the ionic but not osmotic component of salt stress. Pharmacological experiments have suggested that voltage‐gated K⁺‐permeable channels mediate K⁺ retention in leaf mesophyll upon elevated NaCl levels in the apoplast. It is concluded that MIFE measurements of NaCl‐induced K⁺ fluxes from leaf mesophyll may be used as an efficient screening tool for breeding in cereals for salinity tissue tolerance.     

Genotypic difference in salinity tolerance in quinoa is determined by differential control of xylem Na loading and stomatal density

Quinoa is regarded as a highly salt tolerant halophyte crop, of great potential for cultivation on saline areas around the world. Fourteen quinoa genotypes of different geographical origin, differing in salinity tolerance, were grown under greenhouse conditions. Salinity treatment started on 10 day old seedlings. Six weeks after the treatment commenced, leaf sap Na and K content and osmolality, stomatal density, chlorophyll fluorescence characteristics, and xylem sap Na and K composition were measured. Responses to salinity differed greatly among the varieties. All cultivars had substantially increased K⁺ concentrations in the leaf sap, but the most tolerant cultivars had lower xylem Na⁺ content at the time of sampling. Most tolerant cultivars had lowest leaf sap osmolality. All varieties reduced stomata density when grown under saline conditions. All varieties clustered into two groups (includers and excluders) depending on their strategy of handling Na⁺ under saline conditions. Under control (non-saline) conditions, a strong positive correlation was observed between salinity tolerance and plants ability to accumulate Na⁺ in the shoot. Increased leaf sap K⁺, controlled Na⁺ loading to the xylem, and reduced stomata density are important physiological traits contributing to genotypic differences in salinity tolerance in quinoa, a halophyte species from Chenopodium family.     

Comparison of salt tolerance and osmotic adjustment of low-sodium and high-sodium subspecies of the C4 halophyte, Atriplex canescens

The relationship between Na⁺ accumulation and salt tolerance was tested by comparing subspecies of the halophyte, Atriplex canescens (fourwing saltbush), that differed markedly in Na⁺ content and Na:K ratios. Above ground tissues of one low-sodium and two high-sodium subspecies were compared with respect to cation accumulation, osmotic adjustment and growth along a salinity gradient in greenhouse trials. Plants of each subspecies were grown for 80 d on 2.2, 180, 540 and 720 mol m^?3 NaCl. At harvest, A. canescens ssp. canescens had significantly lower Na⁺ levels, higher K⁺ levels and lower Na:K ratios in leaf and stem tissues than A. canescens ssp. macropoda and linearis over the salinity range (P < 0.05 or 0.01). Na:K ratios in leaves of the latter two, high-sodium, subspecies were approximately 2 on the lowest salinity treatment and ranged from 5 to 10 on the more saline solutions. By contrast, Na:K ratios in leaves of the low-sodium subspecies canescens, were only 0.4 on the lowest salinity and ranged narrowly from 1.7 to 2.3 at higher salinities. However, despite different patterns of Na⁺ and K⁺ accumulation, all three subspecies exhibited equally high salt tolerance and had similar osmotic pressures in their leaves or stems over the salinity range. Contrary to expectations, high salt tolerance was not necessarily dependent on high levels of Na⁺ accumulation in this species.     

Salinity tolerance of Aegilops cylindrica genotypes collected from hyper-saline shores of Uremia Salt Lake using physiological traits and SSR markers

Uremia Salt Lake, in North West Iran, has a hyper-saline water. A rare highly salinity-tolerant grass species, Aegilops cylindrica grows along its shores. Salinity tolerance of 44 genotypes of Ae. cylindrica, mainly collected from the Lake, was evaluated under control and 400 mM NaCl conditions using the physiological traits of plant height, dry weight, proline content, Na⁺ and K⁺ concentrations as well as K⁺/Na⁺ ratio. To evaluate the association between microsatellite (EST-SSR and SSR) markers and salinity tolerance, 35 primer pairs were used. Results showed a significant variation in the 44 genotypes studied in terms of their traits except for proline content. Ten most salinity-tolerant genotypes were identified based on their ability to survive, to produce the highest dry weight, and to sustain the least leaf Na⁺ concentration under salinity stress. The very high negative correlation found between Na⁺ concentration and salinity tolerance revealed the importance of individual or a combination of Na⁺ exclusion and excretion mechanisms contributing to the hyper-salinity tolerance of these genotypes. Clustering analysis based on marker data divided the 44 studied genotypes into two groups that were consistent with their saline and non-saline geographical areas. Results of molecular markers showed that four microsatellite markers (Xgwm312, Xwmc170, Xgwm291 and Xgwm410) generated a distinguished banding pattern in ten most salinity-tolerant genotypes. These results supported previous reports on their linkage with Na⁺ exclusion genes (HKT1;5 and HKT1;4) in wheat, which provided further evidence of usefulness of both genes and the linked markers to the salinity tolerance of the halophytic grass family species.     

Exploration of relationships between physiological parameters and growth performance of rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.) seedlings under salinity stress using multivariate analysis

Knowledge of relationships between physiological parameters and growth performance of seedlings and respective genotypic differences would permit selection of salt tolerance at early growth stages. The goals of this study were to investigate the relationships between physiological parameters and growth performance and quantify the respective genotypic differences using multivariate analysis.. Plants of thirty-one genotypes were grown in sand tanks in a greenhouse and irrigated with Yoshida nutrient solution. Two salinity treatments were imposed at 0.9 dSm^–1 (control) and 6.4 dSm^–1 with sodium chloride and calcium chloride (~ 6: 1 molar ratio). Seedlings were sampled 34 days after planting (7^th to 8^th leaf stage). The characters of Na⁺, K⁺, Ca²⁺, K-Na selectivity (SK,Na) and Na-Ca selectivity (S_Na,Ca) were measured as physiological parameters. The characters of tiller number, leaf area, plant height and shoot dry weight were measured as growth performance. Under salinity stress, S_K,Na increased whereas S_Na,Ca decreased compared to the controls. Canonical correlation analysis indicates a strong relationship between physiological parameters and growth performance. Tiller number is a desirable parameter among the growth parameters analyzed to predict seedling growth under salinity stress. Genotypes grouped into four clusters based on ion contents and ion selectivity using Wards minimum-variance cluster analysis. S_K,Na and shoot Na⁺ content contributed the most to the cluster formation. Similarly, genotypes grouped into four clusters based on growth performance. Ge notypes were classified into three categories based on ion cluster rankings: Category 1 with high S_K,Na and low shoot Na⁺ content; Category 2 with intermediate S_K,Na and shoot Na⁺ content; Category 3 with low S_K,Na and high shoot Na⁺ content. The classification of the genotypes into Categories 1 and 3 based on their high or low S_K,Na was generally consistent with their growth performance under salt stress. In contrast, ion selectivity was a less dominant mechanism controlling salt tolerance in Category 2 with intermediate S_K,Na. It was concluded that ion selectivity was a relatively dominant mechanism controlling salt tolerance among rice genotypes although multiple mechanisms may be involved under moderate salt stress. The results also provide the first example of the effectiveness of cluster analysis for physiological responses to salinity stress.     

Enhancement of the salt tolerance of Triticum turgidum L. by the Kna1 locus transferred from the Triticum aestivum L. chromosome 4D by homoeologous recombination

Durum wheat, Triticum turgidum L. (2n= 4x=28, genome formula AABB) is inferior to bread wheat, T. aestivum L. (2n=6x=42, genome formula AABBDD), in the ability to exclude Na⁺ under salt strees, in the ratio of the accumulated K⁺ to Na⁺ in the leaves under salt stress, and in tolerance of salt stress. Previous work showed that chromosome 4D has a major effect on Na⁺ and K⁺ accumulation in the leaves of bread wheat. The 4D chromosome was recombined with chromosome 4B in the genetic background of durum wheat. The recombinants showed that Na⁺ exclusion and enhanced K⁺/Na⁺ ratio in the shoots were controlled by a single locus, Kna1, in the long arm of chromosome 4D. The recombinant families were grown in the field under non-saline conditions and two levels of salinity to determine whether Kna1 confers salt tolerance. Under salt stress, the Kna1 families had higher K⁺/Na⁺ ratios in the flag leaves and higher yields of grain and biomass than the Kna1 ^- families and the parental cultivars. Kna1 is, therefore, one of the factors responsible for the higher salt tolerance of bread wheat relative to durum wheat. The present work provides conceptual evidence that tolerance of salt stress can be transferred between species in the tribe Triticeae.     

Evaluation of salinity tolerance and analysis of allelic function of <Emphasis Type="Italic">HvHKT1</Emphasis> and <Emphasis Type="Italic">HvHKT2</Emphasis> in Tibetan wild barley

Tibetan wild barley is rich in genetic diversity with potential allelic variation useful for salinity-tolerant improvement of the crop. The objectives of this study were to evaluate salinity tolerance and analysis of the allelic function of HvHKT1 and HvHKT2 in Tibetan wild barley. Salinity tolerance of 189 Tibetan wild barley accessions was evaluated in terms of reduced dry biomass under salinity stress. In addition, Na⁺ and K⁺ concentrations of 48 representative accessions differing in salinity tolerance were determined. Furthermore, the allelic and functional diversity of HvHKT1 and HvHKT2 was determined by association analysis as well as gene expression assay. There was a wide variation among wild barley genotypes in salt tolerance, with some accessions being higher in tolerance than cultivated barley CM 72, and salinity tolerance was significantly associated with K⁺/Na⁺ ratio. Association analysis revealed that HvHKT1 and HvHKT2 mainly control Na⁺ and K⁺ transporting under salinity stress, respectively, which was validated by further analysis of gene expression. The present results indicated that Tibetan wild barley offers elite alleles of HvHKT1 and HvHKT2 conferring salinity tolerance.     

The expression of salt tolerance from Triticum tauschii in hexaploid wheat

Summary Accessions of Triticum tauschii (Coss.) Schmal. (D genome donor to hexaploid wheat) vary in salt tolerance and in the rate that Na⁺ accumulates in leaves. The aim of this study was to determine whether these differences in salt tolerance and leaf Na⁺ concentration would be expressed in hexaploid wheat. Synthetic hexaploids were produced from five T. tauschii accessions varying in salt tolerance and two salt-sensitive T. turgidum cultivars. The degree of salt tolerance of the hexaploids was evaluated as the grain yield per plant in 150 mol m^-3 NaCl relative to grain yield in 1 mol m^-3 NaCl (control). Sodium concentration in leaf 5 was measured after the leaf was fully expanded. The salt tolerance of the genotypes correlated negatively with the concentration of Na⁺ in leaf 5. The salt tolerance of the synthetic hexaploids was greater than the tetraploid parents primarily due to the maintenance of kernel weight under saline conditions. Synthetic hexaploids varied in salt tolerance with the source of their D genome which demonstrates that genes for salt tolerance from the diploid are expressed at the hexaploid level.     

How much sodium accumulation is necessary for salt tolerance in subspecies of the halophyte Atriplex canescens?

Two sympatric subspecies of the xerohalophyte Atriplex canescens Pursh. (Nutt.) were compared for 84 d in outdoor salinity trials in their native coastal desert environment in Sonora, Mexico. Subspecies linearis grows naturally on sea water in the high intertidal zone of estuaries while subspecies canescens grows on dunes. In lysimeter pot experiments, ssp. linearis exhibited 50% growth reduction when the mean root zone salinity reached 1160 mol m⁻³ NaCl compared to just 760 mol m⁻³ for ssp. canescens. When irrigated with sea water in a flood plot, ssp. linearis had 50% higher growth rates than ssp. canescens. The specialization of ssp. linearis for a saline environment was associated with greater net transport of Na⁺ from root to shoot, greater Na⁺ accumulation in the leaves and a higher Na:K ratio in the leaves compared to ssp. canescens. On the other hand, the two subspecies achieved approximately the same degree of osmotic adjustment in the leaves, equal to two to three times the external salinity, and had similar water use efficiencies. Even at relatively low salinities, both subspecies accumulated larger quantities of Na⁺ for osmotic adjustment than K⁺. The results suggest that breeding for Na⁺ accumulation rather than exclusion might be the more effective strategy for improving salt tolerance of conventional crop plants.     

Multi-locus genome-wide association studies reveal novel genomic regions associated with vegetative stage salt tolerance in bread wheat (Triticum aestivum L.)

Soil salinity is one of the typical abiotic stresses affecting sustainability of wheat production worldwide. In the present study, we performed a 35 K SNP genotyping assay on association panel of 135 diverse wheat genotypes evaluated for vegetative stage tolerance in hydroponics. Association analyses using five multi-locus GWAS models revealed 42 reliable QTNs for 10 salt tolerance associated traits. Among these 42 reliable QTNs, 9, 17 and 16 QTNs were associated with physiological, biomass and shoot ionic traits respectively. Novel major QTNs were identified for chlorophyll content, shoot fresh weight, seedling total biomass, Na⁺ and K⁺ concentration and Na⁺/K⁺ ratio in shoots. Further, 10 major QTNs showed significant effect on the corresponding salt tolerance traits. Gene ontology analysis of the associated genomic regions identified 58 candidate genes. The information generated in this study will be of potential value for improvement of salt tolerance of wheat cultivars using marker assisted selection.     

Sodium exclusion QTL associated with improved seedling growth in bread wheat under salinity stress

Worldwide, dryland salinity is a major limitation to crop production. Breeding for salinity tolerance could be an effective way of improving yield and yield stability on saline-sodic soils of dryland agriculture. However, this requires a good understanding of inheritance of this quantitative trait. In the present study, a doubled-haploid bread wheat population (Berkut/Krichauff) was grown in supported hydroponics to identify quantitative trait loci (QTL) associated with salinity tolerance traits commonly reported in the literature (leaf symptoms, tiller number, seedling biomass, chlorophyll content, and shoot Na⁺ and K⁺ concentrations), understand the relationships amongst these traits, and determine their genetic value for marker-assisted selection. There was considerable segregation within the population for all traits measured. With a genetic map of 527 SSR-, DArT- and gene-based markers, a total of 40 QTL were detected for all seven traits. For the first time in a cereal species, a QTL interval for Na⁺ exclusion (wPt-3114-wmc170) was associated with an increase (10%) in seedling biomass. Of the five QTL identified for Na⁺ exclusion, two were co-located with seedling biomass (2A and 6A). The 2A QTL appears to coincide with the previously reported Na⁺ exclusion locus in durum wheat that hosts one active HKT1;4 (Nax1) and one inactive HKT1;4 gene. Using these sequences as template for primer design enabled mapping of at least three HKT1;4 genes onto chromosome 2AL in bread wheat, suggesting that bread wheat carries more HKT1;4 gene family members than durum wheat. However, the combined effects of all Na⁺ exclusion loci only accounted for 18% of the variation in seedling biomass under salinity stress indicating that there were other mechanisms of salinity tolerance operative at the seedling stage in this population. Na⁺ and K⁺ accumulation appear under separate genetic control. The molecular markers wmc170 (2A) and cfd080 (6A) are expected to facilitate breeding for salinity tolerance in bread wheat, the latter being associated with seedling vigour.     

Comparative physiological analysis in the tolerance to salinity and drought individual and combination in two cotton genotypes with contrasting salt tolerance

Soil salinity and drought are the two most common and frequently co‐occurring abiotic stresses limiting cotton growth and productivity. However, physiological mechanisms of tolerance to such condition remain elusive. Greenhouse pot experiments were performed to study genotypic differences in response to single drought (4% soil moisture; D) and salinity (200 mM NaCl; S) stress and combined stresses (D + S) using two cotton genotypes Zhongmian 23 (salt‐tolerant) and Zhongmian 41 (salt‐sensitive). Our results showed that drought and salinity stresses, alone or in combination, caused significant reduction in plant growth, chlorophyll content and photosynthesis in the two cotton genotypes, with the largest impact visible under combined stress. Interestingly, Zhongmian 23 was more tolerant than Zhongmian 41 under the three stresses and displayed higher plant dry weight, photosynthesis and antioxidant enzymes activities such as superoxide dismutase (SOD), peroxidase (POD) catalase (CAT) and ascorbate peroxidase (APX) activities compared to control, while those parameters were significantly decreased in salt‐stresses Zhongmian 41 compared to control. Moreover, Na⁺/K⁺‐ATPase activity was more enhanced in Zhongmian 23 than in Zhongmian 41 under salinity stress. However, under single drought stress and D + S stress no significant differences were observed between the two genotypes. No significant differences were detected in Ca²⁺/Mg²⁺‐ATPase activity in Zhongmian 41, while in Zhongmian 23 it was increased under salinity stress. Furthermore, Zhongmian 23 accumulated more soluble sugar, glycine‐betaine and K⁺, but less Na⁺ under the three stresses compared with Zhongmian 41. Obvious changes in leaf and root tips cell ultrastructure was observed in the two cotton genotypes. However, Zhongmian 23 was less affected than Zhongmian 41 especially under salinity stress. These results give a novel insight into the mechanisms of single and combined effects of drought and salinity stresses on cotton genotypes.     

Effect of salt stress on photosynthesis and physiological parameters of three contrasting barley genotypes

In order to understand the physiological traits important in conferring salt tolerance in three barley genotypes, this study was performed under field conditions with three water salinity levels (2, 10, and 18 dS m–1). High salinity decreased net photosynthetic rate, transpiration rate, and stomatal conductance, K⁺ concentration, K⁺:Na⁺ ratio, and grain yield, but increased electrolyte leakage and Na⁺ content. Under 10 and 18 dS m–1 salinity, Khatam (salt-tolerant) had the maximum stomatal conductance, K⁺, K⁺:Na⁺ ratio, and the grain yield, and a minimum Na⁺ content and electrolyte leakage, whereas Morocco (salt-sensitive) had the lowest net photosynthetic rate, stomatal conductance, K⁺ content, K⁺:Na⁺ ratio, and grain yield, and the highest Na⁺ content and electrolyte leakage. This study showed that tolerant genotypes of barley may avoid Na⁺ accumulation in aboveground parts, facilitating a higher photosynthetic rate and higher grain yield.     

Salt Tolerance in Aquatic Macrophytes: Ionic Relation and Interaction

Effects of seawater salinity (SWS) and pure NaCl on the intracellular contents of Na⁺, K⁺, Mg²⁺, Ca²⁺, chlorophylls (Chl) and carotenoids (Car) were studied in three submerged aquatic macrophytes, Hydrilla verticillata, Najas indica and Najas gramenia, which differed in their tolerance to salinity. NaCl resulted in significant increase in Chl/Car ratio in the salt-sensitive H. verticillata and moderately salt-tolerant N. indica, but not in the salt-tolerant N. gramenia. SWS treatment did not result in any significant change in the ratio. The intracellular content of Na⁺ increased significantly in all the test plants upon exposure to both NaCl and SWS. The content of K⁺ decreased significantly in these plants upon salinity treatment, except in N. gramenia. The contents of Ca²⁺ and Mg²⁺ decreased significantly upon NaCl treatment and remained unchanged or increased upon SWS treatment. No relationship between salt tolerance and K⁺/Na⁺ ratio was observed. The maintenance of a minimal level of K⁺ was observed to be the most probable requirement of salt tolerance in aquatic macrophytes.     

Natural variation of salinity response,population structure and candidate genes associated with salinity tolerance in perennial ryegrass accessions

Natural variation in salinity response, effects of population structure on growth and physiological traits and gene–trait association were examined in 56 global collections of diverse perennial ryegrass (Lolium perenne L.) accessions. Three population structure groups were identified with 66 simple sequence repeat markers, which on average accounted for 9 and 11% of phenotypic variation for the control and salinity treatment at 300 mm NaCl. Group 1 (10 accessions) had greater plant height, leaf dry weight and water content, chlorophyll index, K⁺ concentration and K⁺/Na⁺ than group 2 (39 accessions) and group 3 (7 accessions) under salinity stress, while group 3 had higher Na⁺ than groups 1 and 2. Eighty‐seven single nucleotide polymorphisms were detected from four partial candidate genes encoding aquaporin and Na⁺/H⁺ antiporter in both plasma and tonoplast membranes. Overall, rapid decay of linkage disequilibrium was observed within 500 bp. Significant associations were found between the putative LpTIP1 and Na⁺ for the control and between the putative LpNHX1 and K⁺/Na⁺ under the control and salinity treatments after controlling population structure. These results indicate that population structure influenced phenotypic traits, and allelic variation in LpNHX1 may affect salinity tolerance of perennial ryegrass.     

Novel screening protocol for precise phenotyping of salt-tolerance at reproductive stage in rice

The present study reports an unequivocal and improved protocol for efficient screening of salt tolerance at flowering stage in rice, which can aid phenotyping of population for subsequent identification of QTLs associated with salinity stress, particularly at reproductive stage. To validate the new method, the selection criteria, level and time of imposition of stress; plant growth medium were standardized using three rice genotypes. The setup was established with a piezometer placed in a perforated pot for continuous monitoring of soil EC and pH throughout the period of study. Further, fertilizer enriched soil was partially substituted by gravels for stabilization and maintaining the uniformity of soil EC in pots without hindering its buffering capacity. The protocol including modified medium (Soil:Stone, 4:1) at 8 dS m^?1 salinity level was validated using seven different genotypes possessing differential salt sensitivity. Based on the important selection traits such as high stability index for plant yield, harvest index and number of grains/panicle and also high K⁺ concentration and low Na⁺– K⁺ ratio in flag leaf at grain filling stage were validated and employed in the evaluation of a mapping population in the modified screening medium. The method was found significantly efficient for easy maintenance of desired level of soil salinity and identification of genotypes tolerant to salinity at reproductive stage.     

Agronomical and physiological characterization of salinity tolerance in a commercial tomato hybrid

The salt tolerance of the commercial F1 tomato hybrid (Lycopersicon esculentum Mill) Radja (GC-793) has been agronomically and physiologically evaluated under greenhouse conditions, using a control (nutrient solution), a moderate (70 mM NaCl added to the nutrient solution) and a high salt level (140 mM NaCl), applied for 130 days. The results show that Radja is a Na⁺-excluder genotype, tolerant to moderate salinity. Fruit yield was reduced by 16% and 60% and the shoot biomass by 30% and more than 75% under moderate and high salinities, respectively. At 90 days of salt treatment (DST), the mature leaves feeding the 4th truss at fruiting accumulated little Na⁺ (178 mmol kg^-1 DW). At this time, the sucrose concentration in these leaves even increased with moderate salinity and the amino acid proline was not accumulated under salt conditions as compared to control. At 130 DST, Na⁺ was accumulated mainly by the roots in proportion to the salt level applied, while in leaves appreciable amounts were found only at high salinity (452 mmol kg^-1 DW). In the leaves, Cl^- was always accumulated in proportion to the salt level and in a very much greater amounts than Na⁺ (until 1640 mmol kg^-1 DW). The sucrose content was reduced in all plants by salinity, and was distributed preferentially toward the distal stem and peduncle of a truss at fruiting under moderate salinity, and toward the basal stem and root at high salinity. Moreover, proline was accumulated in different organs of the plant only at high salinity, coinciding with Na⁺ accumulation in leaves. Attempts are made to find a clear relationship between physiological behaviour triggered by stress and the agronomical behaviour, in order to assess the validity of physiological traits used for salt-tolerance selection and breeding in tomato.     

Calcium requirement of wheat in saline and non-saline conditions

Supplemental calcium (Ca²⁺) is used in hydroponic studies on salinity to lessen the potential for Ca²⁺ deficiency. However, the Ca²⁺ concentration and the sodium (Na⁺): Ca²⁺ ratio used vary considerably. The implications of using a wide range of Na⁺: Ca²⁺ ratios for studies of salinity tolerance in wheat are not known. Also, despite the risk of development of Ca²⁺ deficiency under salinity stress, there are few reliable reports on the critical level of Ca²⁺ which can be used to diagnose Ca²⁺ deficiency in wheat. Two experiments were conducted to examine Ca²⁺ requirements of wheat under saline and non-saline conditions and to derive a critical level for Ca²⁺. Four bread wheat genotypes (Triticum aestivum L.) and a durum wheat genotype [Triticum turgidum subsp. durum) (Desf.) Husn.] with known differences in salinity tolerance were grown at 100 mM NaCl for four weeks with varying levels of external Ca²⁺ which resulted in Na⁺:Ca²⁺ ratios of 30, 20, 15, 5 and 2. The critical Ca²⁺ concentration was defined in a second experiment by growing the same wheat genotypes at seven levels of Ca²⁺ (0.05, 0.1, 0.2, 0.5, 1, 2 and 10 mM) under non-saline conditions. When grown at 100 mM NaCl salinity tolerance was greatest when the Na⁺:Ca²⁺ ratio ranged from 5 to 15. Growing plants at lower or higher Na⁺:Ca²⁺ ratios induced nutrient imbalances and additional osmotic stress which reduced the growth of plants. Transient Ca²⁺ deficiency occurred at high Na⁺:Ca²⁺ ratios and low Mg²⁺ occurred at the lowest Na⁺:Ca²⁺ ratio. Adding NaCl raised the tissue Na⁺ concentration and reduced the Ca²⁺ concentration and the most appropriate Na⁺:Ca²⁺ ratio in the solution was that which resulted in tissue Ca²⁺ concentrations similar to those of non-salinised plants. The critical level of Ca²⁺ in the youngest fully emerged leaf blades was 15–23 mmol kg^-1 DW (600–900 mg kg^-1 DW).