Embryogenic callus formation,growth and regeneration in callus and suspension cultures of Miscanthus x ogiformis Honda Giganteus' as affected by proline

The effects of proline additions to culture systems of Miscanthus x ogiformis Honda Giganteus' were investigated. Proline was added in concentrations of 0, 12.5, 25, 50, 100 or 300 mM to the callus induction and suspension culture media containing either Murashige and Skoog or N6 basal salts and 22.6 μM 2,4-dichlorophenoxyacetic acid. Shoot apices and leaves from in vitro-propagated shoots, and immature inflorescences from greenhouse-grown plants were used as explants for callus induction and formation. Suspension cultures initiated from embryogenic callus of immature inflorescences were used to test the effect of proline in suspension cultures. The proline additions affected the formation of embryogenic callus and the growth of suspension cultures. Improvements depended on the proline concentration and the basal salts of the medium. Addition of 12.5 to 50 mM proline to callus induction medium with Murashige and Skoog salts increased embryogenic callus formation on shoot apices and leaf explants while proline had no effect on embryogenic callus formation in medium with N6 salts. Increased growth with increasing proline concentration was obtained in suspension aggregates grown in medium with N6 salts, whereas proline only increased growth of suspension aggregates grown in medium with Murashige and Skoog salts at concentrations of 12.5 or 25 mM. A stimulating effect of proline on plant regeneration was observed in short-term cultures of callus as well as in long-term cultures of suspension aggregates. An optimum proline concentration for plant regeneration was found at 12.5 mM. This revised version was published online in June 2006 with corrections to the Cover Date.     

Glycogen synthetase and the control of glycogen synthesis in the cellular slime mould Dictyostelium discoideum during the growth (myxamoebal) phase

1. Myxamoebae of the cellular slime mould Dictyostelium discoideum Ax-2 that are grown in axenic medium containing 86mm-glucose have seven times the glycogen content of the same myxamoebae grown in the same medium but lacking added carbohydrate. 2. During the transition from the exponential to the stationary phase of growth in axenic medium containing glucose myxamoebae preferentially synthesize glycogen and can have as much as three times the glycogen content during the stationary phase as they have during the exponential phase of growth. 3. The rate of glycogen degradation by myxamoebae is, under all conditions of growth, small compared with the rate of glycogen accumulation and the changes in glycogen content thus reflect altered rates of glycogen synthesis. 4. There is no correlation between the rate of glycogen synthesis by myxamoebae and the glycogen synthetase content of the myxamoebae. 5. The activity of glycogen synthetase of D. discoideum is inhibited by a physiological concentration of ATP and this inhibition is overcome by glucose 6-phosphate. Both effects are especially marked at physiological concentrations of UDP-glucose. 6. The rate of glycogen accumulation by myxamoebae growing exponentially in axenic media can be satisfactorily accounted for in terms of the known intracellular concentrations of glucose 6-phosphate, UDP-glucose and glycogen synthetase. The rate-limiting factors controlling glycogen synthesis by the myxamoebae are apparently the substrate (UDP-glucose) and effector (glucose 6-phosphate and ATP) concentrations rather than the amount of the enzyme.     

Characteristics of the heterotrophic bacterial populations in hypersaline environments of different salt concentrations

Solar salterns, based on a multi-pond system, give a discontinuous gradient of salt concentrations. The heterotrophic bacterial populations of ponds containing from 10% salt to saturation have been studied. Saltern samples were spread on agar plates containing different media for halophilic bacteria and one medium made with water of the pond plus nutrients. Replica plating was done to determine the salt range for growth of the colonies. We studied 150 strains to determine the salt spectra of growth, the morphology, and nutrient requirements. The following conclusions were reached: (a) In salt concentrations above 10% (total salts), most bacteria are halophilic and few are halotolerant; (b) the two types of halophilic bacteria, moderate and extreme, show different distributions; in these ponds a narrow overlap exists between 25% and 32% salts with moderate halophiles predominating below this interval and extreme halophiles above it; (c) the populations of moderate halophiles are highly heterogeneous, and the salt concentration of their habitat affects their taxonomic composition, salt range for growth, and nutrient requirements. The population composition of extreme halophiles is less affected by the salt concentrations at which these bacteria are found.     

Physiological effects of Na2SO4 and NaCl on callus cultures of Brassica campestris (Chinese cabbage)

Hypocotyl-derived callus cultures of Brassica campestris L. ssp. pekinensis cv. Kim-jung (Chinese cabbage) were grown on Murashige and Skoog medium containing no additional salt, NaCl or Na₂SO₄. Na₂SO₄ was more than twice as inhibitory in comparison to the same concentration of NaCl when growth and fresh:dry weight ratios of established callus were measured. Levels of protein, starch, sucrose and α-amino nitrogen were not significantly altered in salt-grown callus. Concentrations of reducing sugars and chlorophyll were 2–3 times greater in callus grown on either salt. Proline concentration increased 15–20 fold on the highest levels of salt. Final concentrations (reached in 20–24 days) were closely correlated to the initial Na⁺ concentration of the medium, regardless of salt type. The osmotic potential in callus transferred to NaCl or Na₂SO₄ reached a maximum negative value after 16 days. For both salts, subsequent increases were correlated to increases in fresh:dry weight and growth. On both salts, turgor remained relatively constant (0. 6–0.75 MPa). Changes in Na⁺, K⁺, Mg²⁺ and Ca²⁺ content were correlated to initial Na⁺ concentration in the medium, not salt type. Accumulation of Na⁺ was accompanied by loss of K⁺ and Mg²⁺. Six to seven times less sulfate was measured in callus grown on Na₂SO₄ than chloride in callus grown on similar concentrations of NaCl.     

Method for the simultaneous establishment of many axenic cultures of Paramecium

A method is described for the simultaneous treatment of 42 (or more) stocks of Paramecium, and their adaptation to growth in axenic culture. Samples of dense cultures of these ciliates growing with Enterobacter aerogenes are rendered bacteria-free by migration through 2 sets of tubes containing Adaptation Medium (Peters' salts solution, stigmaterol, vitamins, and autoclaved E. aerogenes). The 2nd set of tubes contains Adaptation Medium plus antibiotics. Bacteria-free samples containing approximately 100 animals are then transferred to test tubes containing Adaptation Medium without antibiotics. This medium also serves as a growth medium. It supports indefinite growth of all Paramecium stocks tested. After adaptation to this medium, the ciliattes can be grown in the axenic medium developed by Soldo, Godoy & van Wagtendonk. On a single trial at least half of the stocks can be expected to produce axenic cultures within 5 to 10 days by these procedures. The method has been applied successfully to several of the species of the Paramecium aurelia complex, to all syngens of Paramecium multimicronucleatum, to several stocks of Paramecium jenningsi, and to 1 stock of Paramecium caudatum and Paramecium calkinsi. A modification of the method also works for Didinium nasutum.     

NaCl-tolerant plants of Poncirus trifoliata regenerated from tolerant cell lines

Summary Salt-tolerant cell lines of citrus rootstock (Poncirus trifoliata cv Pomeroy) were selected by subculturing embryo-derived calli on media containing sublethal concentrations of NaCl (5 and 10 g/l). Selected lines showed a normal growth in the presence of salt at the concentrations used for selection, and salt tolerance persisted after a passage on a salt-free media. Their K⁺ and Ca²⁺ content remained higher than in control cells for increasing NaCl concentration in the medium, suggesting a modification of cell membrane permeability as the main cause of NaCl tolerance. Shoots and plants regenerated from selected cell lines showed improved growth and salt tolerance. Calli induced from these plants tolerated a salt concentration of 10 g/l, indicating the persistance of the selected trait.     

Phosphate Metabolism in the Cyanobacterium Anabaena doliolum Under Salt Stress

In the present study, we have investigated the effects of NaCl concentrations on the growth and phosphate metabolism of an Anabaena doliolum strain isolated from a paddy field, in order to determine the possible effects of salinization. Growth rate, chlorophyll content, and protein content decreased with increasing salt concentration in the growth medium, while carbohydrate concentration increased. Phosphate content and phosphate uptake rate decreased. There was an increase in total alkaline phosphatase activity, with an approximately 7-fold increase in extracellular activity compensating for an approximately 3-fold decrease in cell-bound activity. NaCl effects on protein and chlorophyll concentrations were greater in P-deficient medium, while presence or absence of P in the medium had little effect on cellular carbohydrate concentrations. It is concluded that growth in high salt likely leads to reduced phosphate uptake in A. doliolum.     

In vitro characterization of salt stress effects and the selection of salt tolerant plants in rice (Oryza sativa L.)

Summary The response of plant cells to salt stress was studied on embryo derived calli of rice (Oryza sativa L.) in order to identify cellular phenotypes associated with the stress. The feasability of selecting salt tolerant callus and its subsequent regeneration to plants was also studied. Callus was grown on agar-solidified media containing 0%, 1% and 2% (w/v) NaCl for 24 days. Parameters such as fresh weight, dry weight, soluble protein and proline content were measured. The callus growth decreased markedly with increasing NaCl concentration in the medium. The proline content was enhanced several fold in salt stressed calli. A prolonged exposure of callus to the salt environment led to discolouration and arrested growth in the majority of the calli and only a small number of callus cells maintained healthy and stable growth. These variants were subcultured every three weeks for a period of four months onto medium containing 1% NaCl to identify tolerant lines. At the end of the third cell passage, the tolerant calli were transferred to regeneration medium to regenerate plants. The regeneration frequency in the salt-selected lines was enhanced when compared to unselected lines.     

Effect of Light Intensity on Adaptation of Dunaliella to Very High Salt Concentrations

Light intensity was found to have a strong effect on the adaptationto high salt concentrations of a green microalga, Dunaliellaparva, normally grown at low and medium salt concentrations.At high light intensities (200 µmol m^–2 s^–1)the cell glycerol content increased in parallel with an increasein external salt concentration; protein synthesis and cell divisioncontinued with no period of arrest. At low light intensitiesno glycerol synthesis occurred as the external salt concentrationwas raised; protein synthesis and cell division were equallyarrested. The importance of high light intensity during theinitial phase of increase of salt concentration was demonstratedand was found to be a requirement for protein synthesis andcell division. In experiments designed to discover the effectof light intensity on cells growing in media in which the saltconcentration was kept constant, it was confirmed that the lightintensity needed for growth increases as the salt concentrationof the medium is increased. Key words: Dunaliella, salt concentration, light intensity, growth rates     

Method for the Simultaneous Establishment of Many Axenic Cultures of Paramecium*†

SYNOPSIS. A method is described for the simultaneous treatment of 42 (or more) stocks of Paramecium, and their adaptation to growth in axenic culture. Samples of dense cultures of these ciliates growing with Enterobacter aerogenes are rendered bacteria-free by migration through 2 sets of tubes containing Adaptation Medium (Peters' salts solution, stigmasterol, vitamins, and autoclaved E. aerogenes). The 2nd set of tubes contains Adaptation Medium plus antibiotics. Bacteria-free samples containing ～ 100 animals are then transferred to test tubes containing Adaptation Medium without antibiotics. This medium also serves as a growth medium. It supports indefinite growth of all Paramecium stocks tested. After adaptation to this medium, the ciliates can be grown in the axenic medium developed by Soldo, Godoy & van Wagtendonk. On a single trial at least half of the stocks can be expected to produce axenic cultures within 5 to 10 days by these procedures. The method has been applied successfully to several of the species of the Paramecium aurelia complex, to all syngens of Paramecium multimicronucleatum, to several stocks of Paramecium jenningsi, and to 1 stock each of Paramecium caudatum and Paramecium calkinsi. A modification of the method also works for Didinium nasutum.     

An Immobilization Antigen in Tetrahymena pyriformis Expressed Under Conditions of High Salt Stress*

SYNOPSIS. Cells of Tetrahymena pyriformis syngen 1 exposed to 200 mM NaC1 without previous exposure to a saline medium die within minutes. Lines that have been adapted to higher salt concentrations by growing in axenic peptone medium with 100 mM NaC1 are able to grow in previously intolerable concentrations. Acclimation to higher salt concentrations is accompanied by a transformation from the H serotype, expressed at room temperature in the standard peptone medium, to a new serotype, St. Complete transformation occurs at concentrations approaching the LD₅₀ for nonadapted cells, and the time required for the transformation is 3–4 days. The reverse transformation, St to H, involves a time interval during which the cells are refractory to both anti-St and anti-H. Among 12 of the 13 inbred families of T. pyriformis syngen 1, 2 different high salt serotypes are found. One of these, Sta, is found among families A, Al, A3, B, D, D1, E, and F. The other, Stc, occurs in families C, C1, C2, C3, and possibly B2.     

Changes in the hydrophobic-hydrophilic cell surface character of Halomonas elongata in response to NaCl.

Phase-partitioning studies of the euryhaline bacterium Halomonas elongata demonstrated that the hydrophobic-hydrophilic nature of the cell surface changed as the bacterium grew in different NaCl concentrations. Mid-log-phase cells grown in a high (3.4 M) NaCl concentration were more hydrophilic than were cells grown in a low (0.05 M) NaCl concentration. Mid-log-phase cells from defined medium containing 3.4 M NaCl normally produced a hydrophobicity reading of only 14 (hexadecane hydrophobicity = 100), while corresponding cells from defined medium containing 0.05M NaCl gave a hydrophobicity reading of 90. Compared with cells grown in low salt concentrations, cells grown in high salt concentrations were more hydrophilic at all stages of growth. Rapid suspension of log-phase cells grown in 1.37 M NaCl into a 0.05 or 3.4 M NaCl solution produced no detectable rapid changes in surface hydrophobicity. These data suggest that as H. elongata adapts to different NaCl concentrations, it alters the affinity of its outermost cell surface to water.     

Salt Stress-induced Responses in Growth and Metabolism in Callus Cultures and Differentiating In Vitro Shoots of Indian Ginseng (Withania somnifera Dunal)

In vitro-grown shoots and calli of Withania somnifera, an important medicinal plant, were exposed to various types of salts under in vitro culture conditions. Membrane permeability, lipid peroxidation, and the antioxidant system increased in shoots as well as in unorganized callus tissues under all the three concentrations of KCl, NaCl, KNO₃, NaNO₃, and CaCl₂. The growth responses of shoots and callus cultures under various salt treatments revealed that the tissue could grow better under NaCl and KNO₃ compared to other salts and the in vitro shoots appeared healthy at 50?mM concentration of NaCl and KNO_3. The activity of antioxidant enzymes such as catalase (CAT), ascorbate peroxidase, guaiacol peroxidase, lipoxygenase, polyphenol oxidase, and glutathione reductase increased under salt treatments, especially at higher concentrations. The greatest activity increase was recorded for peroxidases, whereas CAT was the least responsive. Only two isoforms, Mn-superoxide dismutase (Mn-SOD) and Fe-SOD, could be visualized in callus tissue while Cu/Zn-SOD was absent. Diaphorase 4 was totally missing in callus tissue and was detected only in shoots. Phenolics accumulated at all the concentrations of the salts tested as an induced protective response. The higher concentration of CaCl₂ produced maximum increases in antioxidants and enzymatic activities compared to other salts. Thus, for W. somnifera the presence of excess calcium in the growing medium is most deleterious compared to other salts. Results also suggest that the nonenzymatic and enzymatic antioxidant systems of both the tissues played a primary role in combating the imposed salt stress.     

Calcium and Salt Toleration by Bean Plants

The role of calcium in the salt relations of the bean plant, Phaseolus vulgaris, was examined. Brittle wax bush bean plants were cultured in nutrient solutions containing 50 mM NaCl. In the absence of added calcium the plants showed a general breakdown of the roots. A low concentration of calcium in the nutrient solution (0.1 mM) prevented this. Without added calcium the plants absorbed and translocated sodium at such a rate that high concentrations of it built up in the leaves within two days. With increasing concentrations of calcium in the nutrient solution the leaves contained progressively less sodium, and at 3 mM CaSO₄ the concentrations of sodium in the leaves was equal to that of the control plants grown without addition of salt. Even after both roots and stems had reached a high concentration of sodium, the leaves of plants grown in the presence of adequate concentrations of calcium contained little sodium.     

Salt stress in cultured rice cells: effects of proline and abscisic acid

Abstract. The presence of 1 and 10 mol m⁻³ proline in media containing 100 and 200 mol m⁻³ of NaCl, had little effect on the growth of salt-adapted callus of rice. However, in such callus proline accumulation was stimulated by 10 mol m⁻³ proline in the presence of 100 mol m⁻³ NaCl. On the other hand, with 100 mol m⁻³ NaCl, both 1 and 10 mol m⁻³ proline significantly increased both the growth and proline content of salt-unadapted callus. On replacing NaCl with KCl (100 and 200 mol m⁻³), growth of saltadapted as well as unadapted callus was inhibited, but the presence of 10 mol m⁻³ proline had an ameliorating effect. Abscisic acid (ABA) supressed the growth of both salt-adapted and unadapted callus of rice in the absence of salt stress. ABA inhibited the growth of callus adapted to and grown in 100 and 200 mol m⁻³ of NaCl or when it was replaced by equimolar concentrations of KCl. Growth of 100 mol m⁻³ NaCl adapted cells was inhibited when they were transferred to a medium containing 200 mol m⁻³ of NaCl, but in the presence of ABA it was stimulated. ABA increased the growth of unadapted cells when subjected to different salts. Also, ABA accelerated the adaptation of cells exposed to salt but not to water deficits imposed by nonionic solutes.     

In Vitro Selection for Salt Tolerance in Rice

In six cultivars of rice (Oryza sativa L.), Pusa Basmati 1, Basmati 370, Type III, Pant Dhan 4, CSR 10 and Pokkali, embryogenic callus growth, plant regeneration, and proline and total protein contents were studied under salt stress (on agar solidified media containing 0, 0.5, 1.0, 1.5 and 2.0 % NaCl). Four weeks after inoculation the callus fresh mass decreased with increasing salt concentration in all the six cultivars. The regeneration frequency in salt stressed callus was also lower as compared to control. 15 d and 30 d after inoculation proline content increased several fold whereas total protein content decreased markedly with increase in salt concentration.     

Plant regeneration by organogenesis in Glycine max

A procedure for the regeneration of fertile plants by organogenesis from tissue cultures of soybeans, Glycine max is described. Seeds were germinated on reduced inorganic salt MS medium containing 5M BA. Cotyledonary nodes were excised and cultured on the same medium. Presence of BA in the medium during seed germination and culture of nodal explants was required for multiple shoot and shoot-bud formation. Histological analyses established the de novo nature of shoot regeneration. Separate reduction of the concentration of inorganic salts or substitution of sucrose for fructose during culture had minimal effects on the regeneration response. Conversely, if the BA was reduced, the inhibition response could not be overcome by increased salt concentration or altered carbon source.Abbreviations BA benzyladenine - IAA indoleacetic acid - SAS secondary axillary shoots - MS Murashige and Skoog (1962) medium     

In vitro selection of salt tolerant variants following <Superscript>60</Superscript>Co gamma irradiation of long-term callus cultures of <Emphasis Type="Italic">Zoysia matrella</Emphasis> [L.] Merr.

To study growth in the presence of NaCl, in vitro plantlets regenerated from callus of manilagrass (Zoysia matrella [L.] Merr.) were cultured on regeneration medium supplemented with or without 0.3 M NaCl. The results indicated that growth was significantly inhibited by NaCl, with the leaves becoming relatively shorter and thicker. The differences of in vitro plantlets grown under NaCl stress provided specific criteria for the selection of salt tolerant variants. The 6-year maintained calli were treated with different doses (0, 5, 10, 20, 40, 80, 100, 150, 200, 250, and 300 Gy) of ₆₀Co γ rays. Regeneration rate and regeneration capacity of the calli were highest after treatment with 20 Gy ⁶⁰Co γ rays, 27.08 and 91.67% respectively. When the irradiation dose was increased to 100 Gy, 10.42% of the calli developed shoots, but at 150 Gy, both regeneration capacity and regeneration rate declined significantly, and no shoot was observed after 6 weeks of regeneration. Therefore, 100–150 Gy is the most appropriate irradiation span for inducing somaclonal variation. The irradiated calli were selected in vitro for NaCl tolerance. Five NaCl tolerant variant lines, Ze1, Fv1, Te1, Tw1, Fr1, were selected on subculture medium supplemented with 0.35 M NaCl, then transferred to regeneration medium containing 0.25 M NaCl, and grown in a greenhouse. The dark green colour index (DGCI) was used to identify the amount of injury caused by NaCl treatment. This was significantly higher in four of the lines, Ze1, Fv1, Te1, Fr1 (30.88, 31.17, 30.45 and 37.70%, respectively) compared to the control line (CK), which was regenerated from calli subcultured monthly (27.39%), indicating that watering with NaCl caused less injury in these four lines. These lines had lower proline contents than CK under salt stress. The superoxide dismutase (SOD) activity was higher in Ze1 under control condition and its peroxidase (POD) activity increased significantly under salt stress. With Fr1 catalase (CAT) activity was higher under salt stress. The higher activity of these antioxidant enzymes may contribute to the enhanced salt tolerance of the four plant lines.