Plantlet regeneration from a NaCl-selected salt-tolerant callus culture of Shamouti orange (Citrus sinensis L. Osbeck)

Plantlets were regenerated from a selected salt-tolerant cell line of Shamouti orange (Citrus sinensis L. Osbeck). Embryogenesis was carried out both in the presence and absence of NaCl, yielding green and white globular embryos, respectively. Greening could be induced subsequently and normal heart shape embryo development was obtained. Plantlet formation required exposure to kinetin prior to the introduction of the root-inducing hormone naphthalene acetic acid. This system differs from the designed protocol for plant regeneration from the salt-sensitive, i.e., unselected callus. It is concluded that NaCl interferes with the regeneration process, with embryogenesis and/or embryo development into plantlets. Its presence during callus growth probably changes the balance of the phytohormones which is later manifested in plant regeneration. Citrus salt-tolerant callus yields salt-tolerant embryos. Salt-tolerant calli derived from regenerated plantlets indicate acquisition of salt tolerance on the whole plant level.     

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.     

Development of NaCl-tolerant line in Chrysanthemum morifolium Ramat. through shoot organogenesis of selected callus line

Plants were regenerated successfully through shoot organogenesis of a NaCl-selected callus line of Chrysanthemum morifolium Ramat. cv. Maghi Yellow (a salt sensitive cultivar), developed through stepwise increase in NaCl concentration (0-100mM) in the MS medium. The stepwise increase in NaCl concentration from a relatively low level to cytotoxic level was found to be a better way to isolate NaCl-tolerant callus line, since direct transfer of callus to high saline medium was detrimental to callus survival and growth. The selected callus line exhibited significant increase in superoxide dismutase (EC 1.15.1.1), ascorbate peroxidase (EC 1.11.1.11) and glutathione reductase (EC 1.6.4.2) activities compared to control callus (grown in medium devoid of NaCl). Stability of salt tolerance character of the selected callus line was checked by growing the calli in NaCl-free medium for 3 consecutive months followed by re-exposure to higher salinity stress (120mM NaCl). Among different growth regulator treatments, a combination of 5mgl(-1) TDZ (Thidiazuron) along with 0.25mgl(-1) NAA and 0.5mgl(-1) GA(3) was found to be the most effective for shoot organogenesis in selected callus line. The regeneration potential of the NaCl-tolerant callus ranged from 20.8% to 0% against 62.4% to 0% in control callus line. Under elevated stress condition (medium supplemented with 250mM NaCl), selected calli derived regenerants (S1 plants) exhibited significantly higher SOD and APX activities over both PC (positive control: control callus derived plants grown on MS medium devoid of NaCl) and NC (negative control: control callus derived plants subjected to 250mM NaCl stress) plants. In addition, the NC plants showed stunted growth, delayed root initiation, and had lesser number of roots as compared to S1 plants. Based on growth performance and antioxidant capacity, the S1 plants could be considered as NaCl-tolerant line showing all positive adaptive features towards the salinity stress. Further study on agronomic performance of these S1 plants under saline soil condition need to be undertaken to check the genetic stability of the induced salt-tolerance.     

Tissue culture evaluation of NaCl tolerance in Medicago species: Cellular versus whole plant response

Tissue culture responses to three levels of NaCl (0, 85mM and 170 mM) were evaluated in several Medicago species including: M. dzhawakhetica, M. marina, M. rhodopea, M. rupestris, M. sativa (alfalfa) and M. suffruticosa. The whole plant responses of the same genotypes were evaluated in half-strength Hoagland's solution containing 0, 51.5, and 103 mM NaCl. One or more genotypes of M. dzhawakhetica, M. rhodopea, M. rupestris, and M. sativa exhibited in vitro NaCl tolerance at 85 mM. In addition, one genotype each of M. dzhawakhetica, M. rhodopea, and M. sativa was tolerant of 170 mM NaCl. However, all of the genotypes that demonstrated NaCl tolerance in vitro were NaCl sensitive at the whole plant level. Conversely, M. marina the only species exhibiting whole plant NaCl tolerance, had the most NaCl sensitive genotypes at the in vitro level. Although an in vitro NaCl tolerance mechanism which confers whole plant NaCl tolerance was not observed, a potential NaCl tolerance germplasm source, M. marina, was identified.     

<Emphasis Type="Italic">In vitro</Emphasis> selection of salt tolerant cell lines in <Emphasis Type="Italic">Solanum tuberosum</Emphasis> L.

Cell lines able to grow on media containing 50, 100, 150 or 200 mM NaCl were established from potato callus cultures by direct recurrent selection or gradual selection. In callus subjected to direct selection only small clusters of cells survived on medium with 150 or 200 mM NaCl, whereas on 100 mM small cell portions appear necrotic. When cell lines were obtained by successive subcultures on media with increased concentrations of NaCl, salt-tolerant calli were more compact and developed a greenish colour free from necrotic areas. The response of calli lines grown on media with NaCl was compared to control line. The NaCl-tolerant calli showed a decrease in relative growth rate and water content, with higher reductions in the 150 mM tolerant callus. Lipid peroxidation was increased in 50 mM and 100 mM NaCl-tolerant calli, while in 150 mM tolerant callus remained similar to 100 mM values. There was a significant increase in ascorbic acid content in 100 mM and 150 mM NaCl-tolerant calli as compared to the 50 mM, that was two-fold the value found in the control. Also, the contents of soluble and insoluble proteins increased in salt-tolerant lines. SDS-PAGE of soluble proteins showed the synthesis of specific polypeptides in the presence of NaCl in culture medium and the synthesis of a new polypeptide.     

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

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

Behaviour of antioxidant defense system in the adaptive response to salt stress in Helianthus annuus L. cells

A relationship between the antioxidant defense system and salt tolerance in two types of sunflower calli differing in salt sensitivity was studied. No reduction in growth occurred in the NaCl-salt-adapted cell line (T) when grown on 175 mM NaCl but growth of the salt-stressed cell line (S) was reduced by 83%. Lipid peroxidation and protein oxidation increased during acute stress of salt stressed cells at 14 and 28 d of the experiment, while salt-adapted calli (T) remained similar to non-shocked (C) values. The antioxidant defense system of callus adapted to growth under NaCl responded differently to 175 mM of salt compared with the corresponding controls under shock treatment. Salt-adapted and salt-stressed calli showed a similar pattern in GSH content at day 14 but at day 28 in S calli, GSH content was increased 100% over the non-shocked calli, while T calli returned to the initial values. In the salt-stressed calli, a general decrease in all the antioxidant enzymes studied (except for glutathione reductase and dehydroascorbate reductase activities) was observed at day 28. Except for catalase, the antioxidant enzymes were elevated constitutively in adapted calli as compared to stressed cells, when both were grown in the absence of NaCl (time 0), and remained unaltered until 28 d after the beginning of the experiment. These results suggest the involvement of an enzymatic antioxidant defense system in the adaptive response to salt stress in Helianthus annuus L. cells.     

Effet du Chlorure de Sodium sur la Croissance et le Potentiel Osmotique de Cals Normaux et Habitués de Betterave Sucrière

NaCl (0 to 274 mM) was added to the culture media of normal and habituated (auxin and cytokinin independent) sugarbeet calli and its effect on growth (estimated by the increase of dry and organic matters), water content and osmotic potential was tested. Growth of normal callus was stimulated by 68 mM NaCl after a lag period of two weeks. This callus tolerated up to 137 mM NaCl without growth reduction and maintained its hydric status by readjustment of its osmotic potential in 24 h. NaCl quantities under 34 mM stimulated growth of the habituated callus from the 3rd day on; higher NaCl concentrations (68 to 274 mM) inhibited growth or were lethal. NaCl sensitivity of this habituated callus was not due to its inability to adjust its osmotic potential: this adjustment occurred from the 4th h of culture whatever the media. From the 3rd day on, however, this callus presented a water deficit which depended on NaCl concentration. It is suggested that the lowering of osmotic potential corresponds to an important water loss in relation to changes in membrane permeability. This study finally shows that mechanisms of salt tolerance may have developed at the cellular level. Lower growth and lower salt tolerance of the habituated callus need further investigation in relation to cell structure and hormone autonomy.      

Selection and characterization of mannitol-tolerant callus lines of Vigna radiata (L.) Wilczek

An osmotically (mannitol) tolerant callus line of Vigna radiata (L.) Wilczek has been isolated from callus cultures grown on modified PC-L2 medium supplemented with increasing concentrations of mannitol. The tolerance was stable and retained after growth in the absence of mannitol selection for 2 months. The growth of the tolerant line, in the presence of mannitol (540 mol m^-3) was comparable to that of a sensitive callus line growing in the absence of mannitol. This line not only grew well on media containing up to 720 mol m^-3 mannitol, but also required 450 mol m^-3 mannitol for its optimal growth. Osmotically tolerant callus also showed increased tolerance to NaCl (0–250 mol m^-3) stress as compared to sensitive callus. Accumulation of Na⁺ was lower, and the level of K⁺ was more stable in osmotically tolerant than in sensitive calli, when both were exposed to salt. The free proline content of both tolerant and sensitive calli increased on media supplemented with mannitol or NaCl. However, the proline content of sensitive callus was higher than in tolerant callus in the presence of same concentrations of mannitol or NaCl.Abbreviations NAA -naphthaleneacetic acid - 2,4-d 2,4-dichlorophenoxyacetic acid - BAP 6-benzylaminopurine     

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.     

Salinity-induced Physiological Modification in the Callus from Halophyte Nitraria tangutorum Bobr.

Little is known about the physiological adaptation mechanisms of the desert halophyte Nitraria tangutorum Bobr. to the environment. In this study, callus from Nitraria tangutorum Bobr. was used to investigate physiological responses to salinity and the regulatory function of nitric oxide (NO) on catalase (CAT) activity. Increased dry weight and soluble proteins were observed in the callus exposed to lower salinity (50 and 100 mM NaCl), whereas 200 mM NaCl led to significant decreases of these two growth parameters, and the levels of proline and soluble carbohydrates also were enhanced under NaCl treatment. In addition, short-term stress from 50 mM NaCl and the application of lower sodium nitroprusside (SNP, a NO donor) concentration resulted in decreased levels of malondialdehyde (MDA). In contrast, higher concentrations of NaCl and SNP induced significant oxidative damage in Nitraria tangutorum Bobr. callus. Analysis based on the fluorescent probe DAF-FM DA revealed that NaCl and SNP treatment led to enhanced levels of NO in the callus cells. Moreover, the NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (c-PTIO) reduced endogenous NO concentrations and abolished the enhancement in dry weight and the decrease in MDA level under 50-mM-NaCl treatment. CAT activity increased under salt stress, and the 50-mM-NaCl effect was alleviated by treatment with c-PTIO or the nitric oxide synthase inhibitor N^ω-nitro-l-arginine. We suggest that Nitraria tangutorum Bobr. callus exhibited tolerance to lower-salinity stress. We also showed that increased NO generation in response to salinity might be associated with regulation of growth, protection against oxidative damage, and excitation of CAT activity in Nitraria tangutorum Bobr. callus under salt stress.     

Salt tolerance in cultured cells of Spartina pectinata

Suspension cultures with cell doubling times of ca. 2 days were developed from the halophytic grass Spartina pectinata. Maximum rates of exponential growth measured by direct cell counts and by total culture packed-cell-volume were not significantly reduced by NaCl up to 200 mM but dropped beyond this point. In contrast, total cell production over a one week culture cycle, by both measures, was reduced in a roughly linear fashion between 0 and 500 mM NaCl. The pattern of growth in relation to NaCl is very similar to that of previously described cell suspensions derived from another halophyte, Distichlis spicata. In the field the latter is much more salt tolerant. The basis for the whole plant differences is not clear. They do not appear to reflect effectiveness of cell based salt tolerance or the presence of salt glands, which are reported here for the first time in S. pectinata and are found on the leaves of both species.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid     

In vitro selection for salt tolerant lines in Lycopersicon peruvianum

A salt-tolerant callus line of Lycopersicon peruvianum has been obtained by exposing the cells, in suspension cultures and then in callus, to increasing concentrations of NaCl (50–350mM). This selected line grew better than the nonselected line at all levels of NaCl. Moreover, this selected line grew better in media containing salt than in those without it. It retained its tolerance after subculture for 3 passages (3 months) on salt-free medium. The growth of the selected line in mannitol was similar to that of the nonselected line, which suggested that the superiority of the selected line under salt stress was not due to osmotic stress tolerance. The ions SO ₄ ^–– and K⁺ were highly toxic to L. peruvianum root callus, while Na⁺, Mg⁺⁺ and Cl^– were less toxic.     

Selection of salt tolerant plants of Nicotiana tabacum L. through in vitro and its biochemical characterization

Sodium chloride tolerant organogenic callus lines of Nicotiana tabacum were developed in vitro on Murashige and Skoog [16] medium supplemented with BA, IAA and different concentration of NaCl. The maximum shoot bud regeneration was achieved from both tolerant and non-tolerant calluses on MS medium supplemented with 1.0 mg/l BA, 0.1 mg/l IAA with or without NaCl within 4 weeks of culture. Standard growth parameters such as fresh weight and dry weight of organogenic callus, growth tolerant index and enzyme activity (peroxidase and catalase) were used as indicators of salt tolerance. The growth tolerance index in the 4-week after the beginning of treatments yielded significant differences among the non-tolerant and tolerant organogenic callus lines. The regenerated shoots were rooted on half-strength MS basal salts supplemented with 2% sucrose but devoid of growth regulator. The regenerated plants from tolerant callus lines were capable of growing in vitro in presence of 175 mM NaCl. SDS-PAGE profile showed that the progenies derived from tolerant sources were tolerant to salt. This investigation may help in the selection and characterization of salt tolerance in plant improvement programme.     

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.     

Increased 5-enolpyruvylshikimic acid 3-phosphate synthase activity in a glyphosate-tolerant variant strain of tomato cells

A glyphosate-tolerant variant of cultured tomato cells (Lycopersicon esculentum × L. peruvianum hybrid) was isolated via a single-step selection. Growth of the variant in suspension culture was essentially unaffected by 10 mM glyphosate, 100 times the concentration needed to significantly reduce the growth rate of wild type cells. When treated with glyphosate, variant cells accumulated much less shikimic acid than did the wild type cells. In analyses of 5-enolpyruvyl-shikimic acid 3-phosphate (EPSP) synthase activity in two separate experiments, the variant cells had 8 and 13 times higher specific activity than the wild type cells. The enzyme activities from the two types of cells were equally inhibited by glyphosate. These results suggest that the glyphosate tolerance of the variant results from overaccumulation of a glyphosate-sensitive EPSP synthase. Attempts to regenerate fertile plants from the variant cells were unsuccessful, but abnormal shoots were regenerated and callus from leaves of these shoots retained the tolerance to glyphosate.     

NaCl-Dependent growth, ion content and regeneration of calluses initiated from the mangrove plant,Bruguiera sexangula

Calluses initiated from leaves and seedlings of the mangrove,Bruguiera sexangula, were isolated from the original tissues and subcultured. Effects of NaCl on growth and ion content of each callus were measured. The growth rate of calluses derived from leaves (leaf callus) gradually decreased as the NaCl concentration in the medium increased, while that of calluses derived from seedlings (seedling callus) was highest in the medium containing 100 mM NaCl. Concentrations of Na and Cl in both calluses increased with increasing the NaCl concentration in the culture medium. The concentration of K of leaf calluses greatly decreased at 300 mM NaCl, while the K concentration of seedling calluses decreased only slightly and remained relatively high even in the presence of 300 mM NaCl. Transient treatment of leaf calluses with media containing high concentrations of NaCl frequently induced regeneration of adventitious tissues.     

Salt stress enhanced antioxidant response in callus of three halophytes (Salsola baryosma, Trianthema triquetra, Zygophyllum simplex) of Thar Desert

Effects of salinity on growth, protein content, proline, catalase and antioxidant enzyme activity in callus of three halophytes of the Thar Desert; Salsola baryosma, Trianthema triquetra and Zygophyllum simplex were evaluated. Callus tissues were cultured on Murashige and Skoog’s medium containing different concentrations of NaCl (50, 100 and 200 mM). Increase in dry weight and soluble proteins were observed in the callus exposed to lower salinity (50 and 100 mM NaCl) in all the three species, whereas on the medium containing 200 mM NaCl, significant decrease in these two growth parameters was recorded. Under the salinity stress maximum proline accumulation was found in S. baryosma with parallel increase in soluble sugars. Among the three species, T. triquetra callus showed maximum CAT activity with 50 and 100 mM NaCl treatment, whereas the enzyme activity decreased at 200 mM NaCl treatment in all three species. The antioxidant potential steadily elevated under salt treatment in all the above three species using 1, 1-diphenyl-2-picrylhydrazyl (DPPH) and ferric reducing antioxidant potential (FRAP) assay. Whereas, superoxide dismutase (SOD) quenching were recorded maximum at low (50 and 100 mM) concentrations in all the three species. However, T. triquetra callus showed maximum total phenolic content (TPC) 15 mg GAE g^?1 with the elevated concentration of NaCl up to 200 mM, and S. baryosma callus showed lower TPC as compared to both species. A significant correlation between antioxidant capacity and TPC was observed indicating that phenolic compounds are the major contributors to the antioxidant potential in these halophyte species. FRAP and DPPH activity of Z. simplex showed maximum correlation (R = 0.992), as compared to other two species. We can conclude that all the three species exhibit a protection mechanism by sustaining growth parameters and antioxidant capacity. Due to high antioxidant property of all these species, the plant extracts may be included in nutraceutical formulations.