<Emphasis Type="Italic">In vitro</Emphasis> selection of NaCl-tolerant callus lines and regeneration of plantlets in a bamboo (<Emphasis Type="Italic">Dendrocalamus strictus</Emphasis> Nees)

Summary Sodium chloride-tolerant plantlets of Dendrocalamus strictus were regenerated successfully from NaCl-tolerant embryogenic callus via somatic embryogenesis. The selection of embryogenic callus tolerant to 100 mM NaCl was made by exposing the callus to increasing (0–200 mM) concentrations of NaCl in Murashige and Skoog medium having 3% (w/v) sucrose, 0.8% (w/v) agar, 3.0 mg l⁻¹ (13.6 μM) 2,4-dichlorophenoxyacetic acid (2,4-D), and 0.5mg l⁻¹ (2.3μM) kinetin (callus initiation medium). The tolerance of the selected embryogenic callus to 100 mM NaCl was stable through three successive transfers on NaCl-free callus initiation medium. The tolerant embryogenic callus had high levels of Na⁺, sugar, free amino acids, and proline but a slight decline was recorded in K⁺ level. The stable 100 mM NaCl-tolerant embryogenic callus differentiated somatic embryos on maintenance medium [MS medium +3% sucrose +0.8% agar +2.0 mg l⁻¹ (9.0 μM) 2,4-D+0.5 mg l⁻¹ (2.3 μM) kinetin] supplemented with different (0–200 mM) concentrations of NaCl. About 39% of mature somatic embryos tolerant to 100 mM NaCl germinated and converted into plantlets in germination medium [half-strength MS+2% sucrose+0.02 mg l⁻¹ (0.1 μM) α-naphthaleneacetic acid +0.1 mg l⁻¹ (0.49 μM) indole-3-butyric acid] containing 100 mM NaCl. Of these plantlets about 31% established well on transplantation into a garden soil and sand (1:1) mixture containing 0.2% (w/w) NaCl.     

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.     

Hormones-induced modifications in the response of wheat flag leaf to NaCl

A field experiment was carried out to investigate the effects of presoaking the wheat grains (Triticum aestivum L.) in 33 or 66 mM NaCl and indolyl-3-acetic acid (IAA at 50 g m⁻³), gibberellic acid (GA₃ at 100 g m⁻³) or kinetin (100 g m⁻³) on some tolerance criteria in wheat flag leaf at different stages of development. At various stages of flag leaf development pretreatment with 33 or 66 mM NaCl decreased degree of succulence (particularly 66 mM), relative growth rate, net assimilation rate, relative water content, K⁺ content and K⁺/Na⁺ ratio and at the same time induced accumulation of abscisic acid and Na⁺. In the majority of cases grain pretreatment with GA₃ or kinetin and to a lesser extent with IAA alleviated either partially or completely the deleterious effect of salinity on the above mentioned parameters.     

Growth and ion relations in response to combined salinity and waterlogging in the perennial forage legumes Lotus corniculatus and Lotus tenuis

Lotus tenuis (Wadst. & Kit.) is a perennial legume widely grown for pasture in the flood-prone and salt affected Pampa region of Argentina. The physiology of salt and waterlogging tolerance in L. tenuis (four cultivars) was evaluated, and compared with Lotus corniculatus (three cultivars); the most widely cultivated Lotus species. Overall, L. tenuis cultivars accumulated less Na⁺ and Cl⁻, and more K⁺ in shoots than L. corniculatus cultivars, when exposed to 200 mM NaCl for 28 days in aerated or in stagnant solutions. Root porosity was higher in L. tenuis cultivars due to greater aerenchyma formation. In a NaCl dose–response experiment (0–400 mM NaCl in aerated solution), L. tenuis (cv. Chaja) accumulated half as much Cl⁻ in its shoots than L. corniculatus (cv. San Gabriel) at all external NaCl concentrations, and about 30% less shoot Na⁺ in treatments above 250 mM NaCl. Ion distributions in shoots were determined for plants at 200 mM NaCl. L. tenuis (cv. Chaja) again accumulated about half as much Cl⁻ in old leaves, young leaves and stems, compared with concentrations in L. corniculatus (cv. San Gabriel). There were not, however, significant differences between the two species for Na⁺ concentrations in the various shoot tissues. The higher root porosity, and maintenance of lower shoot Cl⁻ and Na⁺ concentrations in L. tenuis, compared with L. corniculatus, contributes to the greater tolerance to combined salt and waterlogging stress in L. tenuis. Moreover, significant variation for tolerance to combined salinity and waterlogging stress was identified within both L. tenuis and L. corniculatus.     

Salt resistance of chickpea genotypes in solutions salinized with NaCl or Na2SO4

Summary To assess the potential for developing a salt resistant cultivar of chickpea (Cicer arietinum L.) 160 genotypes were screened for percent survival after 9 weeks in greenhouse solution cultures, with 50 mM NaCl or 25 mM Na₂SO₄. All plants grew well in the sulfate treatment but only cv. L-550 survived the chloride treatment. Salt damage appeared and developed slowly. To check these apparent effects of cultivar and kind of anion, three genotypes including cv. L-550 were then grown in solutions with isoosmotic NaCl or Na₂SO₄ at three levels (−0.044, −0.088, and −0.132 MPa), and in a separate experiment cv. L-550 was grown with NaCl and Na₂SO₄ at four levels: 10, 20, 30 and 50 mM Na. Salt composition affected shoot weight less than salt level or cultivar did. Shoot dry weight was only slightly less in chloride treatments than in isoosmotic sulfate, and for the least sensitive cultivar (L-550) this held only at the highest salt level, corresponding to that in the screening trial. Further, sensitivity to sulfate and to chloride was equal when sodium concentrations in shoots were equal, regardless of anion compositions of media. Shoot Na concentration was a useful negative indicator of growth under salt stress regardles of cultivar, and may be a useful tolerance indicator also for other species that neither accumulate nor efficiently exclude Na.     

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.     

Accumulation of inorganic and organic osmolytes and their role in osmotic adjustment in NaCl-stressed vetiver grass seedlings

The accumulation of inorganic and organic osmolytes and their role in osmotic adjustment were investigated in roots and leaves of vetiver grass (Vetiveria zizanioides) seedlings stressed with 100, 200, and 300 mM NaCl for 9 days. The results showed that, although the contents of inorganic (K⁺, Na⁺, Ca²⁺, Mg²⁺, Cl⁻, NO₃⁻, SO₄²⁻ and H₂PO₃⁻)) and organic (soluble sugar, organic acids, and free amino acids) osmolytes all increased with NaCl concentration, the contribution of inorganic ions (mainly Na⁺, K⁺, and Cl⁻) to osmotic adjustment was higher (71.50–80.56% of total) than that of organic solutes (19.43–28.50%). The contribution of inorganic ions increased and that of organic solutes decreased in roots with the enhanced NaCl concentration, whereas the case in leaves was opposite. On the other hand, the osmotic adjustment was only effective for vetiver grass seedlings under moderate saline stress (less than 200 mM NaCl).     

Tolerance of submerged germinating rice to 50–200 mM NaCl in aerated solution

This paper concerns tolerance to 50–200 mM NaCl of submerged rice (Oryza sativa cv. Amaroo) during germination and the first 138–186 h of development in aerated solution. Rice was able to germinate and the seedlings even tolerated exposure to 200 mM NaCl, albeit with severe growth restrictions. After return to 0.3 mM NaCl, growth increased, indicating that even at 200 mM NaCl there was no irreparable injury. Osmotic adjustment was achieved by using Na⁺ and Cl^– as the major osmotica. At 200 mM NaCl commenced at sowing, the shoot Na⁺ and Cl^– concentrations between 50–110 h were about 210 and 260 mM, respectively, i.e. above the external concentration. Thus, there was a high tissue tolerance to NaCl. The internal concentrations declined subsequently, concurrent with a decline in growth. At 50–200 mM NaCl, the contributions from ions to π_sap were 81–92% in roots and 62–74% in shoots. The assessed turgor pressures at 200 mM NaCl were 0.33 MPa in shoots and 0.15 MPa in roots, compared to 0.62 and 0.43 MPa at 0.3 mM NaCl. In the General Discussion section, we compare the different responses of submerged seedlings to the responses of transpiring rice plants, reported in the literature, and suggest that the submerged system is useful to evaluate effects of NaCl on turgor pressure and particularly to establish whether there are specific effects of Na⁺ and Cl^– in tissues.     

Relative importance of Na+, Cl−, and abscisic acid in NaCl induced inhibition of root growth of rice seedlings

The relative importance of endogenous abscisic acid (ABA), as well as Na⁺ and Cl^– in NaCl-induced responses related to growth in roots of rice seedlings were investigated. The increase in ammonium, proline and H₂O₂ levels, and cell wall peroxidase (POD) activity has been shown to be related to NaCl-inhibited root growth of rice seedlings. Increasing concentrations of NaCl from 50 to 150 mM progressively decreased root growth and increased both Na⁺ and Cl^–. Treatment with NaCl in the presence of 4,4-diisothiocyano-2,2-disulfonic acid (DIDS, a nonpermeating amino-reactive disulfonic acid known to inhibit the uptake of Cl^–) had less Cl^– level in roots than that in the absence of DIDS, but did not affect the levels of Na⁺, and responses related to growth in roots. Treatment with 50 mM Na-gluconate (the anion of which is not permeable to membrane) had similar Na⁺ level in roots as that with 100 mM NaCl. It was found that treatment with 50 mM Na-gluconate effected growth reduction and growth-related responses in roots in the same way as 100 mM NaCl. All these results suggest that Cl^– is not required for NaCl-induced responses in root of rice seedlings. Endogenous ABA level showed no increase in roots of rice seedlings exposed to 150 mM NaCl. It is unlikely that ABA is associated with NaCl-inhibited root growth of rice seedlings.     

Growth characteristic and ion contents of rice callus under the influence of NaCl and hydroxyproline

Calli of salt tolerant (Bhoora rata) and salt susceptible (GR₁₁) rice varieties were cultured on Linsmaeir and Skoog’s medium containing LD₅₀ concentration of NaCl (200 mM) and hydroxyproline (10 mM). Growth rate of callus and Na⁺, K⁺, Cl⁻, Mg⁺², and Ca⁺² contents of the cultured rice tissues were determined at the end of 0, 2, 4 and 6 weeks of incubation. Hydroxyproline resistant calli of both rice varieties when cultured on Linsmaeir and Skoog’s medium containing both NaCl and hydroxyproline showed increased dry weight and enhanced intracellular levels of K⁺, Mg⁺² and Ca⁺². The accumulation of Na⁺ and Cl⁻ ions was less in the hydroxyproline resistant calli.     

Salinity effect on plant growth and leaf demography of the mangrove, Avicennia germinans L.

We assessed the effect of salinity on plant growth and leaf expansion rates, as well as the leaf life span and the dynamics of leaf production and mortality in seedlings of Avicennia germinans L. grown at 0, 170, 430, 680, and 940 mol m⁻³ NaCl. The relative growth rates (RGR) after 27 weeks reached a maximum (10.4 mg g⁻¹ d⁻¹) in 170 mol m⁻³ NaCl and decreased by 47 and 44% in plants grown at 680 and 940 mol m⁻³ NaCl. The relative leaf expansion rate (RLER) was maximal at 170 mol m⁻³ NaCl (120 cm m⁻² d⁻¹) and decreased by 57 and 52% in plants grown at 680 and 940 mol m⁻³ NaCl, respectively. In the same manner as RGR and RLER, the leaf production (P) and leaf death (D) decreased in 81 and 67% when salinity increased from 170 to 940 mol m⁻³ NaCl, respectively. Since the decrease in P with salinity was more pronounced than the decrease in D, the net accumulation of leaves per plant decreased with salinity. Additionally, an evident increase in annual mortality rates (λ) and death probability was observed with salinity. Leaf half-life (t _0.5) was 425 days in plants grown at 0 mol m⁻³ NaCl, and decreased to 75 days at 940 mol m⁻³ NaCl. Thus, increasing salinity caused an increase in mortality rate whereas production of new leaves and leaf longevity decreased and, finally, the leaf area was reduced.     

Ability of multicellular salt glands in <Emphasis Type="Italic">Tamarix</Emphasis> species to secrete Na<Superscript>+</Superscript> and K<Superscript>+</Superscript> selectively

The present study aimed to determine the mechanism of cation-selective secretion by multicellular salt glands. Using a hydroponic culture system, the secretion and accumulation of Na⁺ and K⁺ in Tamarix ramosissima and T. laxa under different salt stresses (NaCl, KCl and NaCl+KCl) were studied. Additionally, the effects of salt gland inhibitors (orthovanadate, Ba²⁺, ouabain, tetraethylammonium (TEA) and verapamil) on Na⁺ and K⁺ secretion and accumulation were examined. Treatment with NaCl (at 0–200 mmol L⁻¹ levels) significantly increased Na⁺ secretion, whereas KCl treatment (at 0–200 mmol L⁻¹ levels) significantly increased K⁺ secretion. The ratio of secretion to accumulation of Na⁺ was higher than that of K⁺. The changes in Na⁺ and K⁺ secretion differed after adding different ions into the single-salt solutions. Addition of NaCl to the KCl solution (at 100 mmol L⁻¹ level, respectively) led to a significant decrease in K⁺ secretion rate, whereas addition of KCl to the NaCl solution (at 100 mmol L⁻¹ level, respectively) had little impact on the Na⁺ secretion rate. These results indicated that Na+ secretion in Tamarix was highly selective. In addition, Na⁺ secretion was significantly inhibited by orthovanadate, ouabain, TEA and verapamil, and K⁺ secretion was significantly inhibited by ouabain, TEA and verapamil. The different impacts of orthovanadate on Na⁺ and K⁺ secretion might be the primary cause for the different Na⁺ and K⁺ secretion abilities of multicellular salt glands in Tamarix.     

Kinetics of K-Cl Cotransport in Frog Erythrocyte Membrane: Effect of External Sodium

In frog red blood cells, K-Cl cotransport (i.e., the difference between ouabain-resistant K fluxes in Cl and NO₃) has been shown to mediate a large fraction of the total K⁺ transport. In the present study, Cl⁻-dependent and Cl⁻-independent K⁺ fluxes via frog erythrocyte membranes were investigated as a function of external and internal K⁺ ([K⁺] _e and [K⁺] _i ) concentration. The dependence of ouabain-resistant Cl⁻-dependent K⁺ (⁸⁶Rb) influx on [K⁺] _e over the range 0–20 mm fitted the Michaelis-Menten equation, with an apparent affinity (K _m ) of 8.2 ± 1.3 mm and maximal velocity (V _max ) of 10.4 ± 1.6 mmol/l cells/hr under isotonic conditions. Hypotonic stimulation of the Cl⁻-dependent K⁺ influx increased both K _m (12.8 ± 1.7 mm, P < 0.05) and V _max (20.2 ± 2.9 mmol/l/hr, P < 0.001). Raising [K⁺] _e above 20 mm in isotonic media significantly reduced the Cl⁻-dependent K⁺ influx due to a reciprocal decrease of the external Na⁺ ([Na⁺] _e ) concentration below 50 mm. Replacing [Na⁺] _e by NMDG⁺ markedly decreased V _max (3.2 ± 0.7 mmol/l/hr, P < 0.001) and increased K _m (15.7 ± 2.1 mm, P < 0.03) of Cl⁻-dependent K⁺ influx. Moreover, NMDG⁺ Cl substitution for NaCl in isotonic and hypotonic media containing 10 mm RbCl significantly reduced both Rb⁺ uptake and K⁺ loss from red cells. Cell swelling did not affect the Na⁺-dependent changes in Rb⁺ uptake and K⁺ loss. In a nominally K⁺(Rb⁺)-free medium, net K⁺ loss was reduced after lowering [Na⁺] _e below 50 mm. These results indicate that over 50 mm [Na⁺] _e is required for complete activation of the K-Cl cotransporter. In nystatin-pretreated cells with various intracellular K⁺, Cl⁻-dependent K⁺ loss in K⁺-free media was a linear function of [K⁺] _i , with a rate constant of 0.11 ± 0.01 and 0.18 ± 0.008 hr⁻¹ (P < 0.001) in isotonic and hypotonic media, respectively. Thus K-Cl cotransport in frog erythrocytes exhibits a strong asymmetry with respect to transported K⁺ ions. The residual, ouabain-resistant K⁺ fluxes in NO₃ were only 5–10% of the total and were well fitted to linear regressions. The rate constants for the residual influxes were not different from those for K⁺ effluxes in isotonic (∼0.014 hr⁻¹) and hypotonic (∼0.022 hr⁻¹) media, but cell swelling resulted in a significant increase in the rate constants. Received: 19 November 1998/Revised: 23 August 1999     

Single-Channel Analysis of a Delayed Rectifier K+ Channel in Xenopus Myelinated Nerve

Single-channel properties of a delayed rectifier voltage-gated K⁺ channel (I-type) were investigated in peripheral myelinated axons from Xenopus laevis. Channels activated between −60 and −40 mV with a potential of half-maximal activation, E₅₀, at −47.5 mV. Averaged single-channel currents activated with a time delay at all membrane potentials tested. Time to half-maximal activation decreased from 80 to 1.6 msec between −60 and +40 mV. The channel inactivated monoexponentially with a time constant of 10.9 sec at −40 mV. The time constant of deactivation was 126 msec at −80 mV and 16.9 msec at −110 mV. In symmetrical 105 mm K⁺, the single-channel conductance (γ) was 22 and 13 pS at negative and positive membrane potentials, respectively, at 13–15°C. In Na⁺-rich solution with 2.5 mm extracellular K⁺γ was 7 pS and the reversal potential was negative to −80 mV, indicating a high selectivity for K⁺ over Na⁺. γ depended on extracellular K⁺ concentration (K _D = 19.6 mm) and temperature (Q ₁₀= 1.45). External tetraethylammonium (TEA) reduced the apparent single-channel current amplitude at all potentials tested with a half-maximal inhibiting concentration (IC₅₀) of 0.6 mm. Open probability of the channel, but not single-channel current amplitude was decreased by extracellular dendrotoxin (DTX, IC₅₀= 6.8 nm) and mast cell degranulating peptide (MCDP, IC₅₀= 41.9 nm). In Ringer solution the membrane potential of macroscopic I-channel patches was about −65 mV and depolarized under TEA and DTX. It is concluded that besides their activation during action potentials, I-channels may also stabilize the resting membrane potential. Received: 2 June 1995/Revised: 13 October 1995     

Effects of salinity on growth and compatible solutes of callus induced from <Emphasis Type="Italic">Populus euphratica</Emphasis>

Summary The present study aimed to evaluate the response to salinity of Populus euphratica, which is more salt-resistant than other poplar cultivars, at the cellular level. To this purpose, callus was induced from shoot segments of P. euphratica on Murashige and Skoog (MS) medium supplemented with 0.5 mg l⁻¹ (2.2 μM) 6-benzyladenine (BA) and 0.5 mg l⁻¹ (2.7 μM 1-naphthaleneacetic acid (NAA). Callus was transferred to MS medium supplemented with 0.25 mg l⁻¹ (1.1 μM) BA and 0.5 mg l⁻¹ NAA. The relative growth rate of callus reached a maximum in the presence of 50 mmol l⁻¹ NaCl and growth was inhibited with increasing NaCl concentrations. Examination of the changes of osmotic substances under salt stress showed that accumulation of proline, glycine betaine, and total soluble sugars increased with increasing salt concentrations. The results indicate that the response of the callus of P. euphratica to salt stress is similar to that of the whole plant.     

H+ ATPase and Cl− Interaction in Regulation of MDCK Cell pH

MDCK cells display several acid-base transport systems found in intercalated cells, such as Na⁺-H⁺ exchange, H⁺–K⁺ ATPase and Cl⁻/HCO⁻ ₃ exchange. In this work we studied the functional activity of a vacuolar H⁺-ATPase in MDCK cells and its chloride dependence. We measured intracellular pH (pHi) in monolayers grown on glass cover slips utilizing the pH sensitive probe BCECF. To analyze the functional activity of the H⁺ transporters we observed the intracellular alkalinization in response to an acute acid load due to a 20 mm NH⁺ ₄ pulse, and calculated the initial rate of pHi recovery (dpHi/dt). The cells have a basal pHi of 7.17 ± 0.01 (n= 23) and control dpHi/dt of 0.121 ± 0.006 (n= 23) pHi units/min. This pHi recovery rate is markedly decreased when Na⁺ was removed, to 0.069 ± 0.004 (n= 16). It was further reduced to 0.042 ± 0.005 (n= 12) when concanamycin 4.6 × 10⁻⁸ m (a specific inhibitor of the vacuolar H⁺-ATPase) was added to the zero Na⁺ solution. When using a solution with zero Na⁺, low K⁺ (0.5 mm) plus concanamycin, pHi recovery fell again, significantly, to 0.023 ± 0.006 (n= 14) as expected in the presence of a H⁺–K⁺-ATPase. This result was confirmed by the use of 5 × 10⁻⁵ m Schering 28080. The Na⁺ independent pHi recovery was significantly reduced from 0.069 ± 0.004 to 0.042 ± 0.004 (n= 12) when NPPB 10⁻⁵ m (a specific blocker of Cl⁻ channels in renal tubules) was utilized. When the cells were preincubated in 0 Cl⁻/normal Na⁺ solution for 8 min. before the ammonium pulse, the pHi recovery fell from 0.069 ± 0.004 to 0.041 ± 0.007 (n= 12) in a Na⁺ and Cl⁻ free solution. From these results we conclude that: (i) MDCK cells have two Na⁺-independent mechanisms of pHi recovery, a concanamycin sensitive H⁺-ATPase and a K⁺ dependent, Schering 28080 sensitive H⁺–K⁺ ATPase; and, (ii) pHi recovery in Na⁺-free medium depends on the presence of a chloride current which can be blocked by NPPB and impaired by preincubation in Cl⁻–free medium. This finding supports a role for chloride in the function of the H⁺ ATPase, which might be electrical shunting or a biochemical interaction. Received: 24 October 1997/Revised: 19 February 1998     

Cellular and whole plant responses ofVigna radiata to NaCl stress

The effect of different NaCl regimes was examined on the growth and ion accumulation in whole plants and callus cultures ofVigna radiata. Whole plants grown in sand culture were watered with Hoagland's solution supplemented with 0–350 mol m⁻³ of NaCl. Callus cultures were initiated from leaves of 7-d old seedlings of the same seed stock and grown in modified PC-L2 medium containing the same levels of NaCl as in Hoagland's solution. Callus showed the same tolerance to salt as did the whole plant suggesting thatV. radiata appears to have a mechanism(s) for salt tolerance which operates at the cellular level. Ion analysis of whole plant showed that root sodium concentrations of the tolerant cultivar G-65 was much higher while shoot sodium was much less than those of salt sensitive cultivar ML-1. Callus cultures of cv. G-65 also accumulated higher Na⁺ levels. Thus, the greater salt tolerance of cv. G-65 was associated with the control of sodium accumulation at the shoot or cellular level. Communicated by J. POSPíŠILOVá     

Highly-efficient somatic embryogenesis from cell suspension cultures of phalaenopsis orchids by adjusting carbohydrate sources

Summary The influences of various carbohydrate sources, dried yeast (DY), and 6-benzylaminopurine (BA) were estimated on growth and development of shoot tip-derived suspension cells of phalaenopsis orchid. Among the carbohydrates tested on Doriataenopsis cultured on gelled medium, glucose at 58.4 mM gave the highest efficiency of protocorm-like body (PLB) formation. Maltose and sorbitol only induced PLB formation without callus proliferation. Sucrose induced comparable callus proliferation to glucose but without PLB formation. In contrast, fructose resulted in half the amount of callus proliferation as occurred with glucose. Lactose was an inadequate carbon source as neither PLB formation nor callus proliferation occurred. DY enhanced cell proliferation at 0.1–1gl⁻¹ but inhibited both cell proliferation and PLB formation at 10gl⁻¹. Low BA (0.4 μM) slightly increased callus proliferation but inhibited PLB formation. Only one treatment, sucrose and 1 gl⁻¹ DY, yielded a small number of plants. For suspension cultures of Phalaenopsis Snow Parade and P. Wedding Promenade, PLB formation was most efficiently induced by sucrose at 29.2 mM for P. Snow Parade and 14.6 mM glucose for P. Wedding Promenade. Histological observation revealed that cells in suspension culture developed into plants through the same developmental proess as germinating seeds.     

Comparison of the effects of NaCl and KCl at the roots on seedling growth,cell death and the size,frequency and secretion rate of salt glands in leaves of <Emphasis Type="Italic">Limonium sinense</Emphasis>

Twenty days’ exposure to 50 or 100 mM NaCl in the rooting medium substantially increased fresh and dry weights of seedling shoots of the recretohalophyte Limonium sinense while 200 or 300 mM were increasingly inhibitory. KCl treatment was only slightly stimulating (50 mM) or strongly inhibitory (100–300 mM). Lesser effects on leaf area were also seen. Diameter of foliar salt glands was significantly larger than that of controls in 100 and 200 mM NaCl with the effect being reversed at higher concentrations. Gland enlargement was also observed in the presence of 100 mM KCl, while larger concentrations reduced gland size. Generally, gland diameter was larger in the presence of NaCl than in KCl. NaCl and KCl also increased gland number per leaf and secretion rate per gland. At 100 and 200 mM NaCl or KCl, Na⁺ secretion per leaf from NaCl-treated plants exceeded K⁺ secretion rate from KCl-treated plants while at 200 mM, Na⁺ secretion per gland was significantly higher for Na⁺ than for K⁺. Evidence of cell death in leaves of salt-treated plants using Evans blue staining indicates that release of cell contents through loss of membrane integrity contributed to the secretion values. We conclude that the greater tolerance of L. sinenseto to NaCl compared to KCl is linked to the more effective secretion of Na⁺ than of K⁺ and, in turn, to a greater stimulation of salt gland formation and activity and larger gland diameter.     

Na<Superscript>+</Superscript>/H<Superscript>+</Superscript> exchange activity in the alkaliphile halotolerant cyanobacterium <Emphasis Type="Italic">Aphanothece halophytica</Emphasis>

The activity of Na⁺/H⁺ exchanger to remove toxic Na⁺ is important for growth of organisms under high salinity. In this study, the halotolerant cyanobacterium Aphanothece halophytica was shown to possess Na⁺/H⁺ exchange activity since exogenously added Na⁺ could dissipate a pre-formed pH gradient, and decrease extracellular pH. Kinetic analysis yielded apparent K _m (Na⁺) and V _max of 20.7 ± 3.1 mM and 3,333 ± 370 nmol H⁺ min⁻¹ mg⁻¹, respectively. For cells grown under salt-stress condition, the apparent K _m (Na⁺) and V _max was 18.3 ± 3.5 mM and 3,703 ± 350 nmol H⁺ min⁻¹ mg⁻¹, respectively. Three cations with decreasing efficiency namely Li⁺, Ca²⁺, and K⁺ were also able to dissipate pH gradient. Only marginal exchange activity was observed for Mg²⁺. The exchange activity was strongly inhibited by Na⁺-gradient dissipators, monensin, and sodium ionophore as well as by CCCP, a protonophore. A. halophytica showed high Na⁺/H⁺ exchange activity at neutral and alkaline pH up to pH 10. Cells grown at pH 7.6 under high salinity exhibited higher Na⁺/H⁺ exchange activity than those grown under low salinity during 15 days of growth suggesting a role of Na⁺/H⁺ exchanger for salt tolerance in A. halophytica. Cells grown at alkaline pH of 9.0 also exhibited a progressive increase of Na⁺/H⁺ exchange activity during 15 days of growth.