Formation and Regeneration of Protoplasts of the Actinorhizal Nitrogen-Fixing Actinomycete Frankia

Procedures for forming and regenerating protoplasts of four Frankia strains are described. Cells obtained from growth medium containing 0.1% glycine were digested with lysozyme (250 μg/ml) in a medium containing 0.5 M sucrose, 5.0 mM CaCl₂, and 5.0 mM MgCl₂. Protoplasts were formed during 15 to 120 min of digestion at 25°C. Optimum conditions for protoplast regeneration involved placing protoplasts on a layer of complex growth medium containing 0.3 M sucrose, 5.0 mM CaCl₂, and 5.0 mM MgCl₂ which was overlaid with a layer of 0.8% low-melting-point agarose containing 0.5 M sucrose, 5.0 mM MgCl₂, and 5.0 mM CaCl₂. The maximum regeneration efficiency was 36.9% for strain CpI1, 1.3% for strain ACN1^AG, 27% for strain EAN1pec, and 20% for strain EuI1c.     

Increasing NaCl and CaCl<Subscript>2</Subscript> concentrations in the growth medium of quince leaves: I. Effects on somatic embryo and root regeneration

Summary The effects of increasing concentrations of NaCl and CaCl₂ on quince (Cydonia oblonga Mill. BA 29 clone) somatic embryogenesis and adventitious root regeneration were investigated. Leaves collected from in vitro-grown shoots were used as explants and induced for 2d in liquid Murashige and Skoog medium containing 11.3 μM 2,4-dichlorophenoxyacetic acid. Explants were then cultured on semisolid Murashige and Skoog medium enriched with 4.7 μM kinetin and 0.5 μM naphthaleneacetic acid under red light for 25 d and under white light for another 25 d. Two experiments were performed: in the first, NaCl was used at 0,25, 50, 100, and 200 mM in factorial combination with CaCl₂ at 3, 9, and 27 mM; in the second, NaCl was applied at 0, 5, 10, 20, 40, and 80 mM in combination with CaCl₂ at 0.3, 1.0, and 3.0 mM. Quince leaves revealed the capacity to regenerate somatic embryos and/or adventitious roots. Quantitative and qualitative regeneration from leaves was affected by NaCl treatments: increasing NaCl concentrations, in combination with CaCl₂ at 1 mM, led to an increase in the proportion of leaves producing somatic embryos only, and to a decrease of both leaves regenerating roots only and leaves simultaneously producing somatic embryos and adventitious roots. This suggests a beneficial effect of salt stress on the embryogenic process. The regeneration response decreased with increasing salt concentrations and was almost totally inhibited above 50 mM NaCl and 9 mM CaCl₂. The presence of CaCl₂ in the culture medium apparently mitigated the effects of salt stress, but only when NaCl was applied at 40 mM. NaCl at 5 mM, in the presence of 0.3 or 1 mM CaCl₂, was favorable both to somatic embryo and root production. No value of the ratio Na⁺/Ca²⁺ was found to be optimal for the regeneration processes.     

Effect of exogenous calcium on post-thaw growth recovery and subsequent plant regeneration of cryopreserved embryogenic calli of <Emphasis Type="Italic">Hevea brasiliensis</Emphasis> (Müll. Arg.)

A reliable cryopreservation technique was developed for friable embryogenic callus lines of Hevea brasiliensis. The study showed that reducing the CaCl₂ concentration of the pre-culture medium from 9 mM to 1 or 0 mM CaCl₂ before cryopreservation promoted post-thaw callus growth, 1 mM being the optimum CaCl₂ concentration for embryo regeneration. Post-thaw callus proliferation decreased in line with the increase of plated callus weight. The effect of cryopreservation was assessed on 39 independent lines showing that cryopreservation did not affect embryogenic and plant regeneration for a majority of lines. The decrease in CaCl₂ concentration of the pre-culture medium led to a drop in callus calcium content indicating a direct link between the CaCl₂ concentration of the pre-culture medium and the endogenous calcium content of the calli. It also highlighted the implication of tissue calcium content in cryotolerance. Callus water status and the different ways by which calcium could prevent cryoinjury is also discussed.     

Increasing NaCl and CaCl2 concentrations in the growth medium of quince leaves: II. Effects on shoot regeneration

Summary The effects of NaCl and CaCl₂ on shoot regeneration from quince (Cydonia oblonga BA L29 clone) leaves were investigated. Caulogenesis was induced on in vitro-grown leaves treated for 2d in liquid Murashige and Skoog (MS) medium with 11.3 μM 2,4-dichlorophenoxyacetic acid and cultured on MS gelled medium supplemented with 4.5 μM thidiazuron and 0.5 μM naphthaleneacetic acid. Three experiments were performed: in the first, we compared the effects of NaCl at 0, 25, 50, 100, and 200 mM in factorial combination with 3, 9, and 27 mM CaCl₂. In the second, NaCl was tested at 0, 5, 10, 20, 40, and 80 mM with CaCl₂ at 0.3, 1.0, and 3.0 mM. The third experiment was carried out with the same experimental design as the second one but replacing NaCl with Na₂SO₄. Shoot regeneration was evaluated after 50 d of culturing: 25 in darkness and 25 in white light. In the first experiment, shoot regeneration was very poor and was observed only at the lower salt concentrations. In the second experiment, the percentages of caulogenic leaves were much higher, but decreased with increasing NaCl concentration. The more pronounced negative effect of the highest NaCl concentrations appeared to be partly mitigated by CaCl₂ at 1 and 3 mM. The presence of 3 mM CaCl₂, in the experiment with Na₂SO₄, appeared to be even more effective in reducing the adverse effect of sodium stress on caulogenesis. This result was attributed to the lower Cl⁻ concentration in the growth medium, which resulted from replacing NaCl with Na₂SO₄. NaCl applied at low concentrations (5 and 10 mM) in combination with 3 mM CaCl₂ exerted a favorable effect on adventitious shoot regeneration. As regards the Na⁺ and Ca²⁺ interaction, when the Na⁺/Ca²⁺ ratio was below roughly 35 and 20, with NaCl and Na₂SO₄, respectively, at least 60% of leaves showed regenerating capacity, but optimal values of this ratio were not derived.     

Acid Metallophosphatase from the Ameba Amoeba proteus

Tartrate-resistant acid phosphatase (TR-AcPh) from the ameba Amoeba proteus is represented by 3 bands (electromorphs) revealed after disk-electrophoresis in PAAG, using 2-naphthylphosphate as substrate. The presence of 50 mmol/l MgCl₂ or CaCl₂ in the incubation mixture increases activities of all electromorphs of TR-AcPh, while of ZnCl₂, of two of them. The activity of the TR-AcPh electromorphs also rose after the 30-min incubation of the gels in MgCl₂, CaCl₂ or ZnCl₂ (10 and 100 mM) before gel staining. However, 1 M ZnCl₂, unlike 1 M CaCl₂ or 1 M MgCl₂, partly inactivated two out of three TR-AcPh electromorphs. The TR-AcPh electromorphs were inhibited by 1,10-phenanthroline (1,10-Ph), EDTA, and EGTA (all at a concentration of 5 mM) faster than by H₂O₂ (10 mM). The inactivation of the TR-AcPh electromorphs by the chelating agents did not depend (EGTA) or nearly did not depend (EDTA, 1,10-Ph) on their concentration (0.05, 0.5, and 5 mM). Out of 5 tested ions (Mg²⁺, Ca²⁺, Fe²⁺, Fe³⁺, and Zn²⁺), only Zn ions reactivated the TR-AcPh electromorphs inactivated by 1,10-Ph, EDTA or EGTA. The TR-AcPh electromorphs were reactivated worse after inactivation by EGTA than by EDTA or 1,10-Ph. It is suggested that the active site of TR-AcPh contains the zinc ion essential for catalytic activity of this enzyme, i.e., TR-AcPh of A. proteus is a metallophosphatase performing the phosphomonoesterase activity in acidic medium.     

An improved method of transformation in Pseudomonads

Summary A protocol for producing competent Pseuclomonas aeruginosa, Pseudomonas putida, and Xanthomonas maltophilia was adapted and modified from existing methods. Cells were incubated on ice for 30 minutes in buffered 100 mM MgCl₂ followed by 30 minutes in buffered 100 mM CaCl₂ prior to addition of DNA. The MgCl₂-CaCl₂ incubation increased transformation efficiency two to three times, compared with protocols which use incubation in either Mg²⁺ or Ca²⁺, but not both.     

A thioredoxin-activated fructose-1,6-bisphosphatase from the cyanobacterium Synechococcus 6301

Fructose-1,6-bisphosphatase was isolated from the cyanobacterium Synechococcus 6301 by acid precipitation, ammonium-sulfate fractionation, and Sephadex gel chromatography. The purified enzyme needed thiols and MgCl₂ for activity. The following K_m-values were obtained: a) for fructose-1,6-bisphosphate: 1.7 mM; b) for MgCl₂: 12.5 mM; c) for dithiocrythritol: 0,56 mM; d) for glutathione: 14 mM; e) for mercaptoethanol: 22 mM; f) for cysteine: 50 mM. Thioredoxin B isolated from this organism will activate this fructose-1,6-bisphosphatase. The K_m of thioredoxin B for this fructose-1,6-bisphosphatase was determined to be 1.7 M, endicotiy that thioredoxin might activate the fructose-1,6-bisphosphatase in Synechococcus in vivo.     

Halophilic and salts tolerant protoplast cultures of mangrove plants, Sonneratia alba and Avicennia alba

The effects of sea salts, NaCl, KCl, MgCl₂, MgSO₄, and CaCl₂, on the growth of protoplast cultures of two mangrove species, Sonneratia alba and Avicennia alba, were investigated using 96-well culture plates. Plants of these two species naturally grow at the seaward side of a mangrove forest. Cotyledon protoplasts of S. alba showed halophilic nature to NaCl, KCl, and MgCl₂ at low concentrations (10–50 mM) when cultured in Murashige and Skoog’s (MS) medium containing 0.6 M mannitol. CaCl₂ at a concentration higher than 25 mM was inhibitory to cell growth. On the other hand, in protoplast culture of A. alba suspension cells, which were induced from cotyledon tissues, in the modified amino acid (mAA) medium containing 1.2 M sorbitol, tolerance to NaCl, MgCl₂ and MgSO₄ were observed at a wide range of concentrations up to 400 mM. CaCl₂ was always inhibitory for cell divisions in A. alba, but stimulatory for spherical enlargement of cells. However, no difference in cell enlargement was observed among other salts. Similarity and difference in reactivity to salts between protoplasts and suspension cells from our previous studies were discussed in relation to the site of salt tolerance or halophilic adaptation within mangrove cells. For protoplast cultures, the site(s) for response of S. alba and A. alba are located in the cytoplasm and/or the cell membrane.     

Tentacle contraction and ultrastructure inDiscophrya collini: The response to cations

Summary Discophrya collini is a suctorian protozoan with contractile tentacles containing a microtubule-lined canal and microfilaments. The effects of a range of cations on tentacle contraction and ultrastructure have been determined. Treatment with 80 mM CaCl₂ and 95 mM MgCl₂ causes contraction to 28% and 57% of the control length respectively. Re-extension takes over 4 hours in the culture medium, but CaCl₂-treated tentacles are re-extended after a 5 minutes treatment with 10^–2 M EDTA or 5 × 10^–3 M EGTA. CuCl₂ causes a significant contraction at 10^–5 M (to 77%); LaCl₃ at 10^–4 M (to 65%); ZnCl₂ at 10^–2 M (to 65%), but BaCl₂, CoCl₂, MnCl₂, NiCl₂, and SrCl₂ cause significant changes only at 10^–1 M.The cytoplasm of CaCl₂-treated cells contains two forms of membraneous structures when viewed in TEM; that of MgCl₂-treated cells reveals granular areas of medium electron density. None of these features are seen in control cells. The microtubules of the tentacle canal appear to be intact upon its retraction into the cell with no change occurring in the numbers or relative positions of the microtubules. The tentacle cortex is wrinkled. It is suggested from this and previous work that tentacle contraction may be mediated by a microfilament-based mechanism, and that calcium may be involved.     

The rate constant for ATP hydrolysis by polymerized actin

Polymerized actin hydrolyzes bound ATP in a reaction that depends on the concentration of polymerized ATP-actin, not on the rate of incorporation of ATP-actin into the polymer. The apparent first order rate constant is about 0.07 s^?1 at 21°C in 50 mM KCl with 1 mM MgCl₂ or CaCl₂.     

Binding of fibrinogen to platelet integrin αIIbβ3 in solution as monitored by tracer sedimentation equilibrium

Fibrinogen showed essentially no binding (K_D>1 mM ) to platelet αIIbβ3 integrin in solution in the presence of Triton or octylglucoside above critical micellar concentrations. Under these conditions the integrin was an αβ monomer. After removal of the detergent from the Triton containing buffer (25 mM Tris/HCl;, 150 mM NaCl, 1 mM CaCl₂, 1 mM MgCl₂, pH 7.4) the integrin formed aggregates with hexamers as the most prominent species, as demonstrated by analytical ultracentrifugation and electron microscopy. Tracer sedimentation equilibrium experiments indicate that fibrinogen binds to the integrin aggregates, but with a surprisingly large K_D (at least 3 μM ). This value is 10- to 100-fold higher than values determined by solid phase assays or with integrins reconstituted onto lipid bilayers.     

The Decrease of Extracted Apoplast Protein in Soybean Root Tip by Aluminium Treatment

Aluminium effect on the mobility of apoplast protein in root tips was studied. Two-day seedlings of soybean (Glycine max. (L.) Merr. cv. Tsurunoko) were treated with 50 μM AlCl₃ for 2 h. Using infiltration method, the apoplast protein in root tips was extracted with 20 or 100 mM MgCl₂. When 20 mM MgCl₂ was used to collect weakly bound protein to apoplast, the amount of protein extracted was reduced to be about 20 % compared with that of control and the band of 97 kDa disappeared in SDS-PAGE gel. However, the 97 kDa protein could be extracted by 100 mM MgCl₂, which were used for extraction of more tightly bound protein to apoplast, and the amount was estimated to be the same as that of control. When the protein was further developed in two-dimensional electrophoresis, three spots were found between pI 6.4 and 6.5. This is the first report of an Al effect on the mobility of apoplast protein. This revised version was published online in July 2006 with corrections to the Cover Date.     

EFFECT OF SALINITY ON POLLEN I. POLLEN VIABILITY AS ALTERED BY INCREASING OSMOTIC PRESSURE WITH NaCl,MgCl2, and CaCl2

Petunia (Petunia hybrida Vilm. cv. ‘Snowstorm') plants were grown in saline solution (NaCl, MgCl₂, and/or CaCl₂) of 0, 1, 2, and 3 bars osmotic pressures. Pollen viability was tested by tetrazolium chloride staining and by germination (by the hanging drop method, using 15 % sucrose and 0.01 % boric acid as the nutrient medium, at 27 ± 1 C). Pollen viability decreased with increased salinity. Pollen from plants grown in single salt solutions of NaCl, MgCl₂, and CaCl₂ (each at 0, 1, 2, or 3 bars osmotic pressure) was germinated in base culture medium. Pollen viability decreased more with NaCl than with MgCl₂ or CaCl₂. In vitro studies of the effects of three salts, viz., NaCl, MgCl₂, and CaCl₂, on pollen germination and tube growth showed that NaCl inhibited germination and pollen tube growth more than did MgCl₂ or CaCl₂. MgCl₂ was least injurious, and even promoted tube growth at 0.5 and 0.75 bars osmotic pressure. Adding low concentrations of MgCl₂ reduced the toxic effect of NaCl and increased the percentage of germination. CaCl₂ reduced the effect of NaCl less than did MgCl₂. We conclude that specific ion effects were more important than osmotic pressure.     

Effect of CaCl2 on growth performance, photosynthetic efficiency and nitrogen assimilation of Cichorium intybus L. grown under NaCl stress

Pot culture experiments were conducted to assess the extent of growth, photosynthetic efficiency and nitrogen assimilation of chicory (Cichorium intybus L.) as affected by NaCl and CaCl₂ alone as well as in combination. Six treatments, i.e., 80 mM and 160 mM NaCl, 5 mM and 10 mM CaCl₂ and 80 mM + 10 mM and 160 mM + 10 mM of NaCl + CaCl₂ were given to the growing plants separately at three developmental stages, viz., the pre-flowering (30 DAS), flowering (120 DAS) and post-flowering (150 DAS) stages. Each NaCl treatment caused a significant reduction in total plant biomass, photosynthetic rate, stomatal conductance, total chlorophyll content, soluble protein content, NR activity and nitrogen content, although nitrate content increased. On the contrary CaCl₂ treatment gave a favorable effect, compared to the control. The effect of combined treatments was similar to that of NaCl but less in magnitude. Thus, the application of CaCl₂ may mitigate the adverse effect caused by NaCl.     

Evaluation of chemical methods for estimating available zinc and response of green gram (Phaseolus aureus roxb) to applied zinc in non-calcareous soils

Summary Studies were conducted in 22 non-calcareous soils (India) to evaluate various extractants,viz. (6N HCl, 0.1N HCl, EDTA (NH₄)₂CO₃, EDTA NH₄OAc, DTPA+CaCl₂ and 1M MgCl₂) to find critical levels of soil and plant Zn for green gram (Phaseolus aureus Roxb.). The order of extractability by the different extractants was 6N HCl>0.1N HCl>EDTA (NH₄)₂CO₃<EDTA NH₄OAc DTPA+CaCl₂>1M MgCl₂. Critical levels of 0.48 ppm DTPA × CaCl₂ extractable Zn, 0.80 ppm EDTA NH₄OAc extractable Zn, 0.70 ppm EDTA (NH₄)₂CO₃ extractable Zn, and 2.2 ppm 0.1N HCl extractable Zn were estimated for the soils tested. The critical Zn concentration in 6 weeks old plants was found to be 19 ppm. The 0.1N HCl method gave the best correlation (r=0.588^**) between extractable Zn and Bray's per cent yield, while with DTPA+CaCl₂, it was slightly low (r=0.542^**). The DTPA + CaCl₂ method gave significant (r=0.73^**) correlation with plant Zn concentration. The 0.1N HCl gave the higher correlation with Zn uptake (r=0.661^**) than DTPA (r=0.634^**) 6N HCl and 1M MgCl₂ method gave nonsignificant positive relationship with Bray's per cent yield. For noncalcareous soils apart from the common use of DTPA+CaCl₂, 0.1N HCl can also be used for predicting soil available Zn. The use of 0.1N HCl would be much cheaper than DTPA and other extractants used in the study.     

The effect of gamma radiation and N-ethyl-N-nitrosourea on cultured maize callus growth and plant regeneration

Regenerable maize calli of two inbred lines were exposed to 0 to 100 Gy of gamma rays or treated with 0 to 30 mM of N-ethyl-N-nitrosourea (ENU) to determine their effect on growth and plant regeneration capability. Both growth and plant regeneration capacity decreased with increasing levels of either gamma radiation or ENU; however, plant regeneration capacity was more sensitive to either agent than growth. The 50% inhibition dose (I₅₀) for callus growth (fresh-weight gain) was approximately 100 Gy of gamma radiation and 30 mM ENU. The I₅₀ for plant regeneration capacity of treated callus was approximately 25 Gy of gamma radiation and 2.5 mM ENU. The decrease in plant regeneration capacity correlated with a change in tissue composition of the treated callus from a hard, yellow and opaque tissue to a soft, grayish-yellow and translucent tissue. This change was quantified by measuring the reduction of MnO₄ ^- to MnO₂ (PR assay) by the callus. These results suggest that the effect of gamma radiation or ENU on plant regeneration capacity must be taken into consideration if these potentially mutagenic agents are to be used on maize callus cultures, for the purpose of producing useful mutations at a whole plant level. The data also suggest that the PR assay may be useful for predicting the actual plant regeneration capacity of maize callus.Abbreviations g f.w. gram fresh weight - ENU N-ethyl-N-nitrosourea - PR assay permanganate reduction assay - I₅₀ 50% inhibition dose     

Biosynthesis and function of trehalose inEctothiorhodospira halochloris

Trehalose-6-phosphate synthase, catalyzing the reaction between UDP-glucose and glucose 6-phosphate and forming trehalose 6-phosphate, was isolated and partially purified (30-fold) from the phototrophic, haloalkaliphilic bacteriumEctothiorhodospira halochloris. The activity is stabilized by 20mM MgCl₂, 50mM NaCe and 2M glycine betaine. The molecular weight was 63000.The enriched enzyme had a MgCl₂ optimum at 3–6mM, a pH optimum at 7.5 (in Tris-HCl buffer) and a temperature optimum at 50°C. The K_m-values were 1.5×10^–3M for UDP-glucose and 2×10^–3M for glucose 6-phosphate. The enzyme showed a salinity dependence with optimal concentrations between 100 and 300mM salt. Higher concentrations of salt resulted in a decrease in activity. In the presence of inhibitory salt concentrations the compatible solute glycine betaine had a protective effect with a maximum between 0.5 and 2.0M.     

Importance of myo-inositol,calcium, and ammonium for the viability and division of tomato (Lycopersicon esculentum) protoplasts

Plants from four cultivars of Lycopersicon esculentum were grown under different conditions, in controlled environment chambers. Low light intensity, long photoperiod (16 h), 25° C/17°C temperature alternance (day/night) were found to be the most convenient conditions for obtaining viable protoplasts. The use of myo-inositol as an osmoticum in the digestion medium and the adjustment of the pH to 6.5, instead of the usual 5.8, for this medium increased the yield of viable protoplasts and enhanced their stability. Under these conditions neither pretreatment (dark and cold treatments), nor preplasmolysis of leaf tissues, were required before protoplast isolation. The concentrations of ammonium nitrate, calcium chloride, myo-inositol, and sucrose were found to be critical for the success of protoplast culture. A medium containing 5 mM ammonium nitrate, 40 mM calcium chloride, 10 mg l^-1 adenine sulfate, 0.5% myo-inositol and 6% sucrose gave sustained protoplast divisions. Under these conditions, plating efficiency ranged from 5% for the cultivar Lukulus to 15% for the cultivar Golden Sunrise.Abbreviations BA benzylaminopurine - CaCl₂ calcium chloride, 2,4,-D-2,4-dichlorophenoxyacetic acid - EDTA ethylene diamine tetraacetic acid - KCl potassium chloride - MES-2-N morpholino ethane sulfonic acid - MgCl₂ magnesium chloride - NH₄NO₃ ammonium nitrate - NAA naphthalene acetic acid, p-protoplasts     

Purification and properties of extracellular phytase from Bacillus sp. KHU-10

Bacillus species producing a thermostable phytase was isolated from soil, boiled rice, and mezu (Korean traditinal koji). The activity of phytase increased markedly at the late stationary phase. An extracellular phytase from Bacillus sp. KHU-10 was purified to homogeneity by acetone precipitation and DEAE-Sepharose and phenyl-Sepharose column chromatographies. Its molecular weight was estimated to be 46 kDa on gel filtration and 44 kDa on SDS-polyacrylamide gel elctrophoresis. Its optimum pH and temperature for phytase activity were pH 6.5-8.5 and 40°C without 10 mM CaCl₂ and pH 6.0-9.5 and 60°C with 10 mM CaCl₂. About 50% of its original activity remained after incubation at 80°C or 10 min in the presence of 10 mM CaCl₂. The enzyme activity was fairly stable from pH 6.5 to 10.0. The enzyme had an isoelectric point of 6.8. As for substrate specificity, it was very specific for sodium phytate and showed no activity on other phosphate esters. The K _m value for sodium phytate was 50 M. Its activity was inhibited by EDTA and metal ions such as Ba²⁺, Cd²⁺, Co²⁺, Cr³⁺, Cu²⁺, Hg²⁺, and Mn²⁺ ions.     

Permeability of Bacteria to Inorganic Cations. A Comparison between a Psychrophilic Achromobacter Strain and Escherichia coli

The permeability of a psychrophilic Acbromobacter strain to the chlorides of Na, K, Mg and Ca was investigated with light-scattering technique. Comparisons, were made with cells of Escherichia coli B. Cells of both strains suspended in “water were plasmolyzed by 0.1 or 0.2 M solutions of MgCl₂ or CaCl₂ without subsequent deplasmolysis. NaCl or KCl also plasmolyzed the cells, but deplasmolysis followed.” When suspended in growth medium E. coli became completely de-plasmolyzed., whereas the psychrophile still excluded MgCl₂ and CaCl₂ to a great extent. The plasmolysis and deplasmolysis were reversible. Electron micrographs of the psychrophile exposed to CaCl₂ confirmed the presence of plasrnolysis.