The role of arbuscular mycorrhizal fungi in alleviating salt stress in Medicago sativa L. var. icon

Medicago sativa L. is the most important forage crop in arid and semi-arid areas, where increased salinity is a major factor limiting plant growth and crop productivity. The role of arbuscular mycorrhizal (AM) fungus Glomus viscosum H.T. Nicolson strain A6 in protecting alfalfa plants from salt stress, induced by sodium chloride (NaCl), was studied in two ways. Firstly, the root systems of 3-month old M. sativa plants, both mycorrhizal (AM+) and non-mycorrhizal (non-AM) (M. sativa L. var. icon), were placed in solutions of increasing salt concentrations (0, 50, 100, 150, 200 mM NaCl) to study the wilting response. G. viscosum improved the tolerance to salinity stress and the benefit was expressed in terms of the time required to reach the T4 stage in the wilting experiment. Secondly, to evaluate the ability of the Glomus-alfalfa symbiosis to tolerate salt, a pot experiment was set up in a glasshouse in which 3-month old alfalfa plants (M. sativa var. icon) were grown in a peat substratum at three salinity levels (0, 100, 150 mM NaCl). The AM symbiosis stimulated plant height, leaf area, root density, fresh and dry plant weight under saline conditions. Furthermore, proline accumulation was higher in mycorrhizal M. sativa plants than in non-mycorrhizal plants under conditions of salt stress. These and other results indicated that the micropropagated selected clone of M. sativa var. icon, when in symbiosis with G. viscosum H.T. Nicolson strain A6, exhibited better growth and physiological activities under saline conditions than non-AM plants. The AM+ plants also had lower sodium and chloride concentrations in tissues than non-AM plants.     

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.     

Analysis of antioxidant enzyme activity during germination of alfalfa under salt and drought stresses

To understand the adaptability of alfalfa (Medicago sativa L.) to environmental stresses, we analyzed the activity of several antioxidant enzymes, including superoxide dismutase (SOD), peroxidase (POD), ascorbate peroxidase (APX), and catalase (CAT), in alfalfa shoots and roots subjected to salt and drought stresses during germination. The germination rate of six alfalfa cultivars was comparatively studied under 200 mM NaCl or 35% PEG treatment. Alfalfa Xinmu No. 1 and Northstar varieties were selected as stress-tolerant and -sensitive cultivars, respectively, and were used for further characterization. After NaCl or PEG treatment, Xinmu No. 1 showed enhanced seedling growth, compared with Northstar. Xinmu No. 1 also exhibited low levels of hydrogen peroxide (H₂O₂) production and lipid peroxidation, compared with Northstar. In addition, Xinmu No. 1 showed higher enzymatic activity of SOD, APX, CAT, and POD in its shoots and roots than Northstar. These results seem to indicate that Xinmu No. 1 cultivar's tolerance to salt or drought stresses during germination is associated with enhanced activity of antioxidant enzymes. This study highlights the importance of antioxidant enzymes in the establishment of alfalfa seedlings under drought and salinity conditions typical of desertification.     

<i>In vitro</i> Evaluation of Seed Germination in Twelve Alfalfa Cultivars under Salt Stress

Alfalfa (Medicago sativa L.), when exposed to abiotic stress such as salinity, suffers significant losses in yield and productivity. The present study evaluated the salinity tolerance of 12 alfalfa cultivars in vitro using five concentrations of sodium chloride (NaCl), ranging from 0 to 250 mmol L⁻¹ . The results obtained in the current study revealed that the Saudi cultivars, Kasimi and Hassawi, and the German cultivar (Berlin) had the highest salinity tolerance in terms of germination percentage (GP), corrected germination rate index (CGRI), days to reach 50% germination (GT50), and ability to form cotyledonary and true leaves. Under mmol L⁻¹ NaCl, the Saudi cultivar Kasimi cultivar showed GP, CGRI, and GT50 of 55.20%, 123.15, and 3.77 days, respectively. Similarly, the German cultivar (Berlin) showed GP, CGRI, and GT50 of 50.06%, 86.61, and 5.17 days, respectively. These findings might reveal a pivotal aspect in salt tolerance in alfalfa. Our results will help to select salt-tolerant alfalfa cultivars that could thrive in arid and semi-arid areas with salinity problems.     

Messenger RNA induction in cellular salt tolerance of Alfalfa (Medicago sativa)

A salt tolerant alfalfaMedicago sativa L. cell line (HG2-N1) has been selected for growth in 171 mM NaCl. The salt tolerance characteristic is stable and is retained after growth in absence of salt selection for two months.In vitro translation was used to compare mRNA composition from the salt tolerant HG2-N1 and parent salt sensitive HG2 cell lines grown in the presence and absence of 171 mM NaCl. The results suggest that the mRNA composition differs between HG2-N1 and HG2 in a number of RNA species. The salt tolerant HG2-N1 shows both increases and decreases in specific polypeptides as compared to HG2. Many of the enhanced polypeptide bands from mRNA in the salt tolerant HG2-N1 variant appear to be constitutively expressed, since they can be detected from HG2-N1 cells grown in presence and absence of NaCl, but the expression of a few bands may depend on the presence of added NaCl. Most enhanced polypeptides, which are detected from mRNA in the salt tolerant variant HG2-N1 (grown on NaCl) are different from polypeptide bands enhanced in the salt sensitive HG2 line as a result of 24 hour salt stress. Similar results were obtained from two dimensional analysis ofin vivo labeled polypeptides. At least one isolated cDNA clone shows selective expression of mRNA in salt tolerant cells grown in NaCl. These results indicate that adaptive mechanisms for salt tolerance may differ in some aspects from acute stress mechanisms.     

Enhanced tolerance to salinity following cellular acclimation to increasing NaCl levels in Medicago truncatula

Salinity is a major abiotic stress that limits plant productivity. Plants respond to salinity by switching on a coordinated set of physiological and molecular responses that can result in acclimation. Medicago truncatula is an important model legume species, thus understanding salt stress responses and acclimation in this species is of both fundamental and applied interest. The aim of this work was to test whether acclimation could enhance NaCl tolerance in calli of M. truncatula. A new protocol is described incorporating multi-step up acclimation over 0–350 mM exogenous NaCl. By the end of the experiment, calli were tolerant to 150 mM and competent for embryogenesis at 100 mM NaCl. Positive and negative linear relationships between Na⁺ and K⁺ uptake and exogenous NaCl concentration intercepted at 160 mM suggesting a Na⁺/K⁺ homeostasis. Proline level peaked at 100/150 mM whilst highest osmolarity and lowest water content occurred at 250/350 mM NaCl. The concentration of water soluble sugars was positively related to 0–250 mM NaCl whilst callus growth and embryogenesis occurred regardless of endoreduplication. Expression of genes linked to growth (WEE1), in vitro embryogenesis (SERK), salt tolerance (SOS1), proline synthesis (P5CS) and ploidy level (CCS52 and WEE1) peaked at 100/150 mM NaCl. Hence, these genes and various physiological traits except sugar levels, served as useful markers of NaCl tolerance. To our knowledge, this is the first report of a multi-step acclimation conferring tolerance to 150 mM NaCl in leaf-derived calli of M. truncatula.     

Growth attributes,biochemical modulations,antioxidant enzymatic metabolism and yield in Brassica napus varieties for salinity tolerance

Improvement in salinity tolerance of plants is of immense significance as salt stress particularly threatens the productivity of agricultural crops. This study was designed to assess the tolerance level of six Brassica napus varieties (Super, Sandal, Faisal, CON-111, AC Excel and Punjab) under different levels of salinity (0, 50, 100, 150 & 200 mM) with three replications under CRD. Salt induced osmotic stress curtailed the plant growth attributes, photosynthetic pigments and disturbed ionic homeostasis (K⁺, Na⁺, Ca^2+, Cl^-) but least disturbance as compared to control was found in Super and Sandal cultivars. Punjab canola and AC Excel canola cultivars were least tolerant to salinity because these displayed greater decline in all growth and biochemical attributes. Plants subjected to NaCl induced stress exhibited considerable decline in all attributes under study with proline as exception. Antioxidants (CAT, SOD & POD) showed an obvious change in Canola plants under stress, but greatest decline was displayed at 200 mM NaCl level in all six cultivars. Over all these attributes presented a comparatively stable trend in super and sandal cultivars. This shows presence of physiological resilience and metabolic capacity in these two cultivars to tackle salinity. Similarly, all yield attributes displayed adverse behavior under 150 mM & 200 mM salinity stress. Our results demonstrated that Super and Sandal cultivars of Brassica napus exhibit good performance in salinity tolerance and can be good option for cultivation in salt affected areas.     

Organic substances responsible for salt tolerance inEruca sativa

Responses of a salt tolerant and a normal population of an oilseed crop,Eruca sativa Mill. were assessed after four weeks growth in sand culture salinized with 0 (control), 100, 200, or 300 mol m^?3 NaCl. The salt tolerant plants produced significantly greater dry biomass than the normal population. The populations did not differ significantly in leaf osmotic potential, relative water content and leaf soluble proteins. However, the tolerant population accumulated significantly greater amounts of soluble sugars, proline and free amino acids in the leaves compared with the non-tolerant population. It is established that leaf soluble sugars, proline, and free amino acids are important components of salt tolerance inEruca sativa.     

Antioxidative response in different sorghum species under short-term salinity stress

Seedlings of sorghum varieties (M35-1, a drought tolerant species; SPV-839, a drought sensitive one) differing in their drought tolerance were subjected to 150 mM NaCl stress for a short duration of time (up to 72 h). Both the varieties failed to exhibit efficient ion exclusion mechanism like that of salt tolerant species, but in turn resulted in higher accumulation of Na⁺ and Cl⁻ ions over a period of 72 h salt stress. In addition, accumulation of calcium, potassium and proline in seedlings of sorghum varieties was moderate to short-term NaCl stress. The modulation of antioxidant components significantly diverged between the two varieties during seed germination, further the efficiency of antioxidant scavenging system is maintained during short-term NaCl treatments. In comparison to tolerant variety, the sensitive variety depicted higher SOD activity under control and salinity treatments but specific activity of catalase was significantly reduced. In contrast, drought tolerant variety exhibited efficient hydrogen peroxide scavenging mechanisms with higher catalase and GST activities under control and salt stress conditions, but not in the sensitive one. In conclusion, our comparative studies indicate that drought tolerant and susceptible varieties of sorghum induce efficient differential oxidative components of enzymatic machinery for scavenging ROS thereby alleviating the oxidative stress generated by salt stress during seedling growth.     

Selection for NaCl tolerance in cell cultures of Medicago sativa and recovery of plants from a NaCl-tolerant cell line

Medicago sativa lines with a high incidence of regeneration were established as suspension cultures and used to select for NaCl tolerant lines. Attempts were then made to regenerate plants from these lines. Regeneration was severely depressed in NaCl tolerant calli and the only plants that were successfully regenerated were from one callus of M. sativa cv. Regen S which grew in 62.5 mM NaCl. Plants from this callus, and new calli derived from the recovered plants, have shown a tolerance to NaCl comparable to calli and plants from the initial seed stock rather than an improved level of tolerance.     

Characterization of salt tolerant alfalfa (Medicago sativa L.) plants regenerated from salt tolerant cell lines

Salt tolerant cell lines have been selected from Medicago sativa, by a single step selection process on tissue culture medium containing 1% NaCl. Plants regenerated from these lines show improved salt tolerance compared to parent plants. The regenerated plants are vigorous, have flowered and are self fertile. The cellular salt tolerance characteristic can be passaged through the regenerated plants, since callus cultures initiated from immature ovaries of the salt tolerant regenerated plants are salt tolerant without additional selection on 1% NaCl. Several of these second generation callus cultures have been regenerated to produce vigorous plants which maintain the salt tolerance characteristic. The tolerance phenotype appears dominant in seeds obtained from self fertilization of the tolerant plants. The regenerated salt tolerant plants are therefore a valuable source as genotypes in plant breeding for salt tolerance and isolation, identification and manipulation of genes which confer salt tolerance in alfalfa.Abbreviations SH Schenk and Hildebrandt medium - 2,4-D 2,4-dichlorophenoxyacetic acid     

Physiological responses to salt stress of T2 alfalfa progenies carrying a transgene for betaine aldehyde dehydrogenase

Glycinebetaine is an important quaternary ammonium compound generated in response to salt and other osmotic stresses in many organisms. Its synthesis requires the catalysis of betaine aldehyde dehydrogenase encoded by a Betaine Aldehyde Dehydrogenase (BADH) gene that converts betaine aldehyde into glycinebetaine in some halotolerant plants. In this study, a BADH gene was over expressed in transgenic alfalfa (Medicago sativa L) plants using Agrobacterium-mediated transformation. Transgenic alfalfa plants grown under 9‰ NaCl grew well; while non-transgenic control plants turned yellowish in color, wilted, and eventually died. Polymerase chain reaction (PCR) and Northern blot hybridization analyses demonstrated that the BADH gene was transferred into the T2 generation and segregated in a Mendelian fashion. Transgenic alfalfa plants expressing BADH showed significantly higher BADH enzyme activity and betaine contents when grown under 6‰ NaCl. Moreover, proline content in T2 lines were higher while electrolyte leakage and malonaldehyde content were lower in T2 lines compared with non-transgenic plants. These findings indicated that transgenic plants expressing BADH transgene exhibited higher salt tolerance than non-transgenic plants.     

Arabidopsis sos1 mutant in a salt-tolerant accession revealed an importance of salt acclimation ability in plant salt tolerance

An analysis of the salinity tolerance of 354 Arabidopsis thaliana accessions showed that some accessions were more tolerant to salt shock than the reference accession, Col-0, when transferred from 0 to 225 mM NaCl. In addition, several accessions, including Zu-0, showed marked acquired salt tolerance after exposure to moderate salt stress. It is likely therefore that Arabidopsis plants have at least two types of tolerance, salt shock tolerance and acquired salt tolerance. To evaluate a role of well-known salt shock tolerant gene SOS1 in acquired salt tolerance, we isolated a sos1 mutant from ion-beam-mutagenized Zu-0 seedlings. The mutant showed severe growth inhibition under salt shock stress owing to a single base deletion in the SOS1 gene and was even more salt sensitive than Col-0. Nevertheless, it was able to survive after acclimation on 100 mM NaCl for 7 d followed by 750 mM sorbitol for 20 d, whereas Col-0 became chlorotic under the same conditions. We propose that genes for salt acclimation ability are different from genes for salt shock tolerance and play an important role in the acquisition of salt or osmotic tolerance.     

Growth, nitrogen fixation and ion distribution in Medicago truncatula subjected to salt stress

This study compared the growth, nodulation, N₂ fixation, and ion distribution in three Medicago truncatula lines, in response to salt in nutrient solution. Two local lines (TN8.20 and TN6.18) and a reference line (Jemalong 6) were inoculated with a reference strain Sinorhizobium meliloti 2011, a very tolerant strain to salinity (700 mM NaCl) and grown in a controlled glasshouse with or without 75 mM NaCl. A genotypic variation in tolerance to salt was found: TN6.18 was the most sensitive line whereas TN8.20 was the most tolerant. The relative tolerance of TN8.20 was concomitant with the lowest leaf Na⁺ concentration and the highest nodule biomass production. However, nodule efficiency (amount of nitrogen fixed per g dry weight nodule) decreased in all lines. Results suggest that the tolerance to salt seems to depend on the host plant ability to protect its leaves against an excessive Na⁺ (and Cl^?) accumulation, and its ability to maintain the development of an abundant nodular system, which in turn determines an important rate of nitrogen fixation and allows the plants to conserve their growth potentialities. The loss of the nodular efficiency under salt stress seems to be compensated by a large nodule biomass.     

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     

Differential Histone Acetylation in Alfalfa (Medicago sativa) Due to Growth in NaCl : Responses in Salt Stressed and Salt Tolerant Callus Cultures

The steady state distribution of histone variant proteins and their modifications by acetylation were characterized in wild type and salinity stress adapted alfalfa (Medicago sativa). Isotopic labeling detected dynamic acetylation at four sites in the histone H3 variants and five sites in histones H4 and H2B. Histone variant H3.2 was the most highly acetylated histone with 25% higher steady state acetylation and a two- to threefold higher acetylation labeling than histone H3.1. Histone phosphorylation was limited to histone variants H1.A, H1.B, and H1.C and to histone H2A.3, which was also acetylated. Histone variant composition was unaffected by cellular exposure to NaCl. Histone acetylation was qualitatively similar in salt-tolerant and salt-sensitive cells under normal growth conditions. However, short term salt stress in salt sensitive cells or continued growth at 1% NaCl in salt tolerant cells led to major increases in the multiacetylated forms of histone H4 and the two variants of histone H3. These changes were more pronounced in the diploid than in the tetraploid alfalfa strains. The increase in multiacetylation of core histones serves as an in vivo reporter suggesting an altered intranuclear ionic environment in the presence of salt. It may also represent an adaptive response in chromatin structure to permit chromatin function in a more saline intranuclear environment.