Effect of sodium sulfate on in vitro organogenesis of tobacco callus

Callus of tobacco (Nicotiana tabacum L. cv. Wisconsin 38) was grown on callus-proliferating (CP) and shoot-forming (SF) media with elevated sodium sulfate (Na₂SO₄) concentrations either in the light or dark for more than one year. An increase in Na₂SO₄ concentration resulted in a decrease in callus growth index, an increase in percent dry weight of callus tissues grown on both media, and a decrease in both number of calli forming shoots and number of shoots per callus in SF medium. The CP callus grown in the light spontaneously began to form shoots after the 5th monthly transfer, and spontaneous root formation occured after the 16th transfer in the presence of 0.75 and 1.0% Na₂SO₄. Both water () and osmotic (_s) potentials of the callus increased with increasing Na₂SO₄ concentration; and callus exhibited greater and _s in the light than dark for both CP and SF media.     

Characterization of Growth, Water Relations, and Proline Accumulation in Sodium Sulfate Tolerant Callus of Brassica napus L. cv Westar (Canola)

Unselected and sodium sulfate tolerant callus cultures of Brassica napus L. cv Westar were grown on media supplemented with mannitol, NaCl, or Na₂SO₄. In all cases, growth of tolerant callus, measured on a fresh weight or dry weight basis, was greater than that of unselected callus, which was also subject to necrosis on high levels of salt. Tissue water potential became more negative in both unselected and tolerant callus grown in the presence of mannitol or Na₂SO₄. Water potentials in unselected callus were more negative than those of the tolerant tissues; but over a range of Na₂SO₄ concentrations both cultures displayed osmotic adjustment, maintaining relatively constant turgor. Proline accumulation in both unselected and tolerant callus was low (15 to 20 micromoles per gram dry weight) in the absence of stress, but increased on media supplemented with mannitol, NaCl, or Na₂SO₄. Increases in proline concentration were approximately linear in tolerant callus, reaching a maximum of 130 to 175 micromoles per gram dry weight. In unselected callus, concentrations were higher, reaching 390 to 520 micromoles per gram dry weight. Proline accumulation was correlated with inhibition of growth, and there was a negative correlation between proline concentration and culture age for tolerant callus.     

Retention of shoot regeneration capacity of tobacco callus by Na2SO4

Callus of tobacco (Nicotiana tabacum L. cv. Wisconsin 38) was grown in the light on shoot-forming medium in the presence of Na₂SO₄ for over a year. An increase in Na₂SO₄ concentration resulted in decreasing callus growth, decreasing percentage of calli producing shoots and number of shoots per callus, and increasing callus percent dry weight. Regeneration of shoots from callus grown in the absence of Na₂SO₄ began to decline after 14 months in culture, and shoot regeneration capacity was completely lost after 18 months. In contrast, 18-month old callus continuously grown in the presence of Na₂SO₄ retained the ability to form shoots. The highest percent of callus pieces that formed shoots and the maximum number of shoots per callus occurred at 70 mM (1%) Na₂SO₄. All plants arising from the 18-month old callus were polyploid. Both 9- and 18-month old callus exhibited more negative water and osmotic potentials in the presence of increasing Na₂SO₄ concentration.Abbreviations water potential - _S osmotic potential - CGI callus growth index     

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     

Stable sodium sulfate tolerance inBrassica napus cv. Westar callus cultures

Summary Sodium-sulfate-tolerant callus ofBrassica napus cv. Westar, selected on medium containing 105 mM Na₂SO₄, was maintained on medium without the salt to test for stability of tolerance. Tolerance to Na₂SO₄ was retained even after 18 subcultures on no salt. This tissue also showed tolerance to K₂SO₄, NaCl, and KCl. However, with the exception of callus grown on KCl fresh weight yields were less than that of tolerant callus maintained continuously on Na₂SO₄. Tolerant callus maintained on no salt had a mixture of the compact morphology of unselected callus and the friable morphology of tolerant callus. Both callus types expressed salt tolerance. Sucrose, reducing sugars and proline concentrations were measured in unselected callus, tolerant callus maintained continuously on Na₂SO₄, and tolerant callus maintained on no salt. Sucrose levels were similar in all cases. Whether maintained on or off Na₂SO₄., tolerant callus had reducing sugars levels three to fou times greater than unselected callus. Tolerant callus maintained on no salt had twice the amount of proline found in the unselected callus. Tolerant callus maintained in the absence of salt had an ash content, sodium concentration, and potassium concentration significantly lower than that of unselected callus. Supported by the Natural Sciences and Engineering Research Council of Canada Strategic Grant nos. G 0949 and G 1642 to T. A. Thorpe, D. M. Reid, and E.C. Yeung.     

Proline accumulation and sodium sulfate tolerance in callus cultures of Brassica napus L. cv. Westar

Callus cultures of Brassica napus L. cv. Westar were selected which contained 5 – 6 times more proline than unselected callus. Callus pieces from these cultures were able to survive much better after subculture to medium containing 105 mM Na₂SO₄ than unselected callus, or unselected callus cultured on exogenous proline before or during transfer to the salt. Exogenous proline was rapidly absorbed. In unselected callus there was a peak in proline accumulation ca. 2 days after transfer to Na₂SO₄, followed by a decline. In contrast proline accumulation in tolerant callus was linear with time, reaching maximum levels at 8 days. Proline levels induced by exposure to salt were maintained in the absence of stress.Abbreviations DW Dry weight - FW Fresh weight     

Differential Na2SO4 tolerance in tobacco plants regenerated from Na2SO4-grown callus

Abstract The regenerated shoots from sodium sulphate (Na₂SO₄) grown callus of tobacco (Nicotiana tabacum L. cv. Wisconsin 38) were evaluated for Na₂SO₄ tolerance based on shoot proliferation and rooting in vitro, and seed germination in vivo in response to Na₂SO₄. An increase in Na₂SO₄ concentration resulted in significantly decreasing shoot fresh weight, number of shoots, shoot length and leaf size, and increasing per cent shoot dry weight of both control and Na₂SO₄-grown cultures. In rooting, shoots of Na₂SO₄-grown cultures exhibited the highest per cent rooting (85%) in the presence of 1% w/v Na₂SO₄. However, per cent rooting, root number per rooted cutting and root fresh weight decreased significantly with increasing Na₂SO₄ concentration when shoots were transferred to the medium in the absence of Na₂SO₄ for 4-monthly passages. Following acclimatization of the rooted shoots of Na₂SO₄-grown cultures, phenotypic variation was observed during growth and development. There were 13.2% sterile plants. Fertile plants were sorted into normal (N), tolerant (T), and sensitive (S) categories and the respective percentages of plants were 31.6, 44.7 and 10.5, based on per cent germination, germination velocity index and seedling survival to Na₂SO₄. The response of N, T and S types to Na₂SO₄ in subsequent shoot proliferation was similar to that of seed germination.     

Isolated Thellungiella shoots do not require roots to survive NaCl and Na2SO4 salt stresses

Shoots of Thellungiella derived by micropropagation were used to estimate the plants'' salt tolerance and ability to regulate Na⁺ uptake. Two species with differing salt tolerances were studied: Thellungiella salsuginea (halophilla), which is less tolerant, and Thellungiella botschantzevii, which is more tolerant. Although the shoots of neither ecotype survived at 700 mM NaCl or 200 mM Na₂SO₄, micropropagated shoots of T. botschantzevii were more tolerant to Na₂SO₄ (10–100 mM) and NaCl (100–300 mM). In the absence of roots, Na₂SO₄ salinity reduced shoot growth more dramatically than NaCl salinity. Plantlets of both species were able to adapt to salt stress even when they did not form roots. First, there was no significant correlation between Na⁺ accumulation in shoots and Na⁺ concentration in the growth media. Second, K⁺ concentrations in the shoots exposed to different salt concentrations were maintained at equivalent levels to control plants grown in medium without NaCl or Na₂SO₄. These results suggest that isolated shoots of Thellungiella possess their own mechanisms for enabling salt tolerance, which contribute to salt tolerance in intact plants.Key words: Thellungiella salsuginea, Thellungiella botschantzevii, salt tolerance, isolated shoots, growth, rhizogenesis, ion accumulation     

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.     

Development of callus and suspension cultures of potato resistant to NaCl and mannitol and their response to stress

Callus and suspension cultures adapted to various concentrations of NaCl or mannitol were developed from the cultivated potato Solanum tuberosum cv. Desire. Growth of the calli was less inhibited by mannitol than by iso-osmotic concentrations of NaCl. Reduction of growth by both NaCl and mannitol was considerably lower in osmotically adapted calli than in non-adapted ones. Salt-adapted suspension cultures that grew in the medium to which they had been originally adapted had a shorter lag in growth as well as a shorter time required to achieve the maximum growth, as compared with non-adapted cells. Suspension cultures adapted to NaCl concentrations higher than 150 mM were obtained only after preadaptation to osmotic stress. Adaptation of these cells was found to be stable. Accumulation of Na⁺ was lower and level of K⁺ was more stable in osmotically adapted than in non-adapted calli, when both were exposed to salt. Potassium level in NaCl-adapted calli exposed to saline medium was lower than that in non-adapted calli in standard medium. The maximum of Cl^– and Na⁺ accumulation was reached at higher external salt concentration in salt-adapted than in non-adapted suspension cultures. In both callus and suspension cultures, Cl^– accumulated more than Na⁺. Potassium level decreased more in non-adapted than in NaCl-adapted suspension cultures. The decrease of osmotic potential in osmotically adapted calli exposed to mannitol and in salt-adapted calli and suspension cultures exposed to salt was correlated to the increase of the external concentration. Such a correlation was not found in osmotically adapted calli exposed to salt. Non-electrolytes were found to be the main contributors to the decrease is osmotic potential in both callus and suspension cultures.     

Disruption of <Emphasis Type="Italic">RCI2A</Emphasis> leads to over-accumulation of Na<Superscript>+</Superscript> and increased salt sensitivity in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> plants

For plant salt tolerance, it is important to regulate the uptake and accumulation of Na⁺ ions. The yeast pmp3 mutant which lacks PMP3 gene accumulates excess Na⁺ ions in the cell and shows increased Na⁺ sensitivity. Although the function of PMP3 is not fully understood, it is proposed that PMP3 contributes to the restriction of Na⁺ uptake and consequently salt tolerance in yeasts. In this paper, we have investigated whether the lack of RCI2A gene, homologous to PMP3 gene, causes a salt sensitive phenotype in Arabidopsis (Arabidopsis thaliana (L.) Heynh.) plants; and to thereby indicate the physiological role of RCI2A in higher plants. Two T-DNA insertional mutants of RCI2A were identified. Although the growth of rci2a mutants was comparable with that of wild type under normal conditions, high NaCl treatment caused increased accumulation of Na⁺ and more reduction of the growth of roots and shoots of rci2a mutants than that of wild type. Undifferentiated callus cultures regenerated from rci2a mutants also accumulated more Na⁺ than that from wild type under high NaCl treatment. Furthermore, when wild-type and rci2a plants were treated with NaCl, NaNO₃, Na₂SO₄, KCl, KNO₃, K₂SO₄ or LiCl, the rci2a mutants showed more reduction of shoot growth than wild type. Under treatments of tetramethylammonium chloride, CaCl₂, MgCl₂, mannitol or sorbitol, the growth reduction was comparable between wild-type and rci2a plants. These results suggested that RCI2A plays a role directly or indirectly for avoiding over-accumulation of excess Na⁺ and K⁺ ions in plants, and contributes to salt tolerance.     

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.     

Culture surface and exogenous putrescine-altered shoot growth pattern in mannitol- and cadmium chloride-pretreated callus of <Emphasis Type="BoldItalic">Cymbidium</Emphasis> Via del Playa “Yvonne”

Evidence for the link between the surface of culture medium and abnormal shoot formation in the presence of exogenous putrescine is presented. Little is known about the role of putrescine in rapid shoot regeneration in the callus of orchids pretreated with mannitol and cadmium chloride (CdCl₂). Ultrastructural analysis of putrescine-altered CdCl₂- and mannitol-treated callus after 4 wk of culture showed the presence of numerous oil droplets, rough endoplasmic reticulum, and dictyosomes. Callus pretreated with mannitol and CdCl₂ but without exogenous putrescine showed the presence of amyloplasts, condensed mitochondria, and active vacuole within 12 h of pretreatment. This study not only establishes a new method to achieve rapid regeneration of Cymbidium callus but also describes different types of shoots on the basis of its contact with the surface of the culture medium. Histologically, two types of cells in the apical meristem are defined. One type of cells has compromised volume which was lost upon division and is represented by small deeply stained cells of the shoots regenerated from the upper portion of the callus and is considered normal. The other type is represented by highly vacuolated and less deeply stained cells of the shoots regenerated from the lower portion of the callus in contact with the medium and is considered abnormal in this study. To avoid such abnormalities, frequent subculture of callus halves which were obtained by a median transverse cut before the regenerants (PLBs) were converted to shoots is recommended. This new methodology was successful in achieving rapid regeneration of uniform shoots from callus.     

Plant regeneration from tissue cultures of Persian buttercup (Ranunculus asiaticus L.)

Different plant explants of Persian buttercup (Ranunculus asiaticus L.) were screened for callus induction and adventitious shoot regeneration on different media to establish totipotent cultures. Murashige & Skoog (MS) medium was used, supplemented with different concentrations of the following growth regulators: kinetin, benzyladenine (BA), naphthaleneacetic acid (NAA) and indoleacetic acid (IAA). Callus was induced and adventitious buds regenerated only from cotyledonary explants after 4–5 weeks. Subculture of the regenerated buds on the same basal medium in presence of gibberellic acid (GA₃) and BA produced well-organized shoots. Rooting was obtained by transferring shoots to growth regulator-free MS medium. A high rate of shoot multiplication has been achieved on medium with high concentration of kinetin and long-day photoperiod. Finally the plants were successfully transferred to soil and grown in a greenhouse.     

Uptake of inorganic ions and compatible solutes in cultured mangrove cells during salt stress

Summary Callus of the mangrove plant, Sonneratia alba J. Smith, established from pistils of flower buds were cultured on solid Murashige and Skoog medium supplemented with 0 to 500 mM NaCl. Maximum growth was observed with 50 mM NaCl, and net growth of callus occurred for concentrations up to 200 mM NaCl. At 500 mM NaCl, growth of callus was completely inhibited, although a part of the tissue was still alive after 30 d. Cellular levels of Na⁺ and Cl⁻ were greatly increased by the treatment with NaCl. Uptake of K⁺ was also enhanced and was accompanied by increasing levels of Na⁺ and Cl⁻ so that the Na⁺/K⁺ ratio was almost constant (4.1–4.2) in callus grown with 50–200 mM NaCl. Levels of Mg²⁺ and Ca²⁺ were not changed significantly with 50–200 mM NaCl, whereas levels of free NH ₄ ⁺ , NO ₃ ⁻ and SO ₄ ²⁻ ions, which are convertible to organic compounds, were lowest in callus grown with 50 mM NaCl. The rate of conversion of ¹⁵NH ₄ ⁺ into macromolecules during 30 d culture with 0–100 mM NaCl did not vary greatly, but 200 mM NaCl reduced the biosynthesis of macromolecules from this ion. The highest rate of conversion of ¹⁵NO ₃ ⁻ into macromolecules was observed at 50 mM NaCl. Identification of compatible solutes with NMR-spectroscopy indicated that mannitol is the compatible solute for intact plants of Sonneratia alba, but no accumulation of mannitol was found in calluses, not even in those grown at high concentrations of NaCl.     

Increased NaCl-tolerance in wheat (Triticum aestivum L. andT. durum desf.) through in vitro selection

Summary Callus cultures were initiated from immature embryos of oneTriticum aestivum and threeT. durum cultivars. Growing morphogenic calli were exposed to different concentrations of NaCl (0, 0.3, 0.5, and 0.7%) added to the culture medium during two subsequent subcultures (4 wk each). The growth rate of the calli was determined by the relative fresh weight callus growth (RFWCG). The callus growth of all investigated genotypes was slightly changed in the presence of 0.3 and 0.5% NaCl, but strongly inhibited by 0.7% NaCl. Selected NaCl-tolerant clones were isolated and plants were regenerated on MS-based regeneration medium without NaCl. The regeneration capacity of the selected calli was highly reduced compared to the control. The highest number of regenerants was scored for cv. Gladiator (T. aestivum). All regenerated plants were morphologically normal and many developed to maturity and set seeds. Seedlings from the R₁ generation of selected and control plants were treated with 0.5% NaCl in vivo in liquid cultures for 6 wk. Salt tolerance of the progenies of selected plants appeared in all cultivars, but those derived from calli grown on medium with 0.7% NaCl showed the highest survival rate.T. aestivum showed higher tolerance to NaCl salinity thanT. durum.     

Effects of NaCl and Na2SO4 on red-osier dogwood (Cornus stolonifera Michx) seedlings

Sodium chloride and sodium sulfate are commonly present in extraction tailings waters produced as a result of surface mining and affect plants on reclaimed areas. Red-osier dogwood (Cornus stolonifera Michx) seedlings were demonstrated to be relatively resistant to these high salinity oil sands tailings waters. The objectives of this study were to compare the effects of Na₂SO₄ and NaCl, on growth, tissue ion content, water relations and gas exchange in red-osier dogwood (Cornus stolonifera Michx) seedlings. In the present study, red-osier dogwood seedlings were grown in aerated half-strength modified Hoagland's mineral solution containing 0, 25, 50 or 100 mM of NaCl or Na₂SO₄. After four weeks of treatment, plant dry weights decreased and the amount of Na⁺ in plant tissues increased with increasing salt concentration. Na⁺ tissue content was higher in plants treated with NaCl than Na₂SO₄ and it was greater in roots than shoots. However, Cl^– concentration in the NaCl treated plants was higher in shoots than in roots. The decrease in stomatal conductance and photosynthetic rates observed in presence of salts is likely to contribute to the growth reduction. Our results suggest that red-osier dogwood is able to control the transport of Na⁺ from roots to shoots when external concentrations are 50 mM or less.     

Growth, proline accumulation and ion content in sodium chloride-stressed callus of date palm

Summary Growth and physiological responses of date palm. Phoenix dactylifera L. cv. Barhee, callus to salinity stress were examined. Callus induced from shoot tips of offshoots was cultured on Murashige and Skoog medium supplemented with NaCl at concentrations ranging from 0 to 225 mM, in consective increments of 25 mM. Data obtained after 6 wk of exposure to salt have shown a significant increase in callus proliferation in response to 25 mM NaCl the lowest level tested, beyond which callus weight decreased. At 125 mM NaCl and higher, callus growth was nearly completely inhibited. Physiological studies on callus exposed to salt stress have shown an increase in proline accumulation in response to increased salinity. Proline accumulation was correlated to callus growth inhibition. Furthermore, increasing the concentration of NaCl in the culture medium generally resulted in a steady increase in Na⁺ and reduction in K⁺ concentrations. However, at 25 mM NaCl, the only level at which callus growth was significantly enhanced, an increase in K⁺ content was noted, in comparison to the NaCl free control. In response to increasing external NaCl level, the Na⁺/K⁺ ratio increased The Na⁺/K⁺ ratio was positively correlated to proline accumulation and hence callus growth inhibition. This study provides, an understanding of the response of date palm callus to salinity, which is important for future studies aimed at developing strategies for selecting and characterizing somaclonal variants tolerant to salt stress.     

Rubidium chloride tolerant callus cultures of rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.) accumulate more potassium and cross tolerate to other salts

Callus cultures from salt tolerant (CSR-10) and susceptible (Swarnadhan) varieties of Oryza sativa L. were established in Murashige and Skoog’s (MS) medium containing lethal concentrations (50 mM) of rubidium chloride (RbCl) as a selective agent. While 95–100% cells were viable in callus cultures grown without RbCl, viability was 75% in 50 mM RbCl selected cultures. Growth of RbCl selected calli in presence of salt was comparable to that of callus grown without it. Cells tolerant to RbCl showed more vacuoles and accumulated more K⁺ in comparison with their corresponding controls. Suspension cultures were established and uptake of ⁸⁶Rb⁺ was measured at 10 and 20 min intervals, which revealed a linear relationship between the absorption of K⁺ and time. Callus cultures (560-day-old) tolerant to 50 mM RbCl regenerated shoots with 35–40% frequencies in both the varieties, but the same age-old callus grown in the medium devoid of RbCl did not show any organogenesis. Callus cultures that are tolerant to 50 mM RbCl when exposed to 25 mM LiCl, 50 mM NaCl, 50 mM KCl and 25 mM CsCl also exhibited cross tolerance in both the varieties. This is the first time that a callus line of rice resistant to RbCl was raised and shown to accumulate a major cation K⁺ and also an increased influx of it.     

Efficient callus initiation from leaf of mangrove plant,Bruguiera sexangula in amino acid medium: Effect of NaCl on callus initiation

Experimental conditions for efficient callus initiation from mangrove plants were investigated. As a source explant, leaf ofBruguiera sexangula was used. Mangrove plant is one of the most famous woody plants which can grow at the salty area. The initiated callus can be a suitable material for the investigation of salt tolerant mechanisms of mangrove plants. Leaf pieces cultured in an Amino Acid medium supplemented with 2 μM 2,4-dichlorophenoxyacetic acid and 2 μMN-(2-chloro-4-pyridyl)-N′-phenylurea at 30 C developed calluses. Microscopic observation suggested that the callus was initiated from the tissue in the vascular bundles in the leaf. We also examined the effect of NaCl on callus initiation and short-term culture of the calluses on the leaves. Callus initiation rate decreased with increasing NaCl concentration higher than 100 mM in the culture media. The medium containing 100 mM NaCl produced the largest callus on the leaf, compared with higher or lower concentrations of NaCl.