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.     

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.     

Some morphogenic effects of sodium sulfate on tobacco callus

Callus cultures of Nicotiana tabacum L cv. Wisconsin 38 were initiated and grown on shoot-forming (SF) and callus proliferation (CP) medium with or without Na₂SO₄. Two cultures were maintained on SF medium with 0, 0.75, 1 or 1.5% Na₂SO₄ for 2.5 and 3.5 years. In the older culture only callus grown on salt formed shoots throughout the maintenance period, while in the younger culture the control responded best and Na₂SO₄ was inhibitory. Callus from the older culture which had been grown on salt continued to form shoots in the absence of salt. Na₂SO₄ caused adventitious shoot formation in three cultures on CP medium. These shoots were present for 7 subcultures after removal of Na₂SO₄; but established, control callus, did not form shoots when transferred to Na₂SO₄. Callus initiated and maintained on NaCl or mannitol showed a slight increase in shoot initiation. On NaCl, Na₂SO₄ or mannitol, the tissue osmotic potential became more negative and proline concentration increased.     

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     

Effect of sodium and calcium chlorides,abscisic acid and proline on callus cultures ofArachis hypogaea L.

Callus cultures ofArachis hypogaea L. cv. JL-24 adapted to 200 mM NaCl (otherwise lethal to cells) were used for the study. Calli grew slowly when transferred to 250 mM NaCl, but the growth was enhanced when ABA was included in the medium. ABA induced increase in growth of callus was not accompanied by corresponding increase in internal free proline levels. 0.5 mM of CaCl₂ ameliorated the negative effect of NaCl indicating that cells require a specific Ca²⁺/Na⁺ ratio for their growth. Proline content also increased at this ratio thereby suggesting that increase in growth at 0.5 mM Ca²⁺ may be due to an increase in proline content. However, exogenous proline did not increase the growth of callus (adapted to 200 mM), and higher concentrations even inhibited the growth. This shows that proline is not required for growth or adaptation of cells to salt stress, but is produced as a consequence of stress.     

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.     

Optimization of Ionic and Chelated Iron and Its Interaction with Disodium Ethylenediaminetetraacetic Acid for Enhancement of Plant Regeneration in Rice (Oryza sativa L)

Interactive effects of Fe₂(SO₄)3 and Na₂EDTA on callus induction and plant regeneration in indica rice (Oryza sativa cv Pusa Basmati-1) was investigated. Callus induction and subsequent plant regeneration from seed was obtained on MS medium supplemented with 11.31 µM 2,4-D and 2.68 µM NAA + 8.87 µM BAP respectively. Both the callus induction and the plant regeneration media were supplemented with different levels of Fe₂(SO₄)₃, Na₂EDTA and their combinations. Callus induction, its morphogenic potential, average number of regenerated plants as well as the appearance of the regenerants was influenced by the levels of Fe₂(SO₄)₃ and Na₂EDTA. Embryogenic callus could be induced in the presence of Fe₂(SO₄)₃ / Na₂EDTA. Iron was essential for plant regeneration. Non-chelated iron could induce callus formation as well as plant regeneration, yet the chelated form was more suitable. A higher level of Na₂EDTA in the regeneration medium was detrimental. Differential requirement of Fe₂(SO₄)₃ at induction and regeneration level was observed. Method of medium preparation affected the regeneration response. Nearly three fold increase in the number of regenerated plants was achieved with 0.05 mM Fe₂(SO₄)₃ + 0.1 mM Na₂EDTA at callus induction and 0.1 mM Fe₂(SO₄)₃+ 0.1 mM Na₂EDTA at plant regeneration and standard method of autoclaving.     

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.     

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

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

Callus culture from hypocotyls of Kosteletzkya virginica (L.) seedlings

It was shown that callus established from Kosteletzkya virginica (L.) Presl. (Malvaceae) can grow in salinities higher than 200 mM NaCl if previously accomodated stepwise. Callus lines developed from seedlings of different harvests or of the same harvest at different times, all showed the same pattern of growth and sensitiviy to salinity. The absorption of Na⁺ into the callus increased with increasing external NaCl concentration. In the callus, Na⁺ was apparently distributed outside and inside a cellular membrane (possibly the plasmalemma). This membrane was, apparently, capable of regulating the Na⁺ concentration in the protoplast. Outside this membrane Na⁺ accumulated to concentrations higher than in the external growth medium. Exogenously supplied proline or glycine-betaine did not affect the growth of the callus. Externally applied ABA stimulated growth under saline conditions and increased the accumulation of proline. Growth and proline content were positively correlated in callus exposed to salinity, but in the presence of ABA they were negatively correlated. ABA was involved in both growth and proline accumulation, but there was no clear relationship between these two effects. Both ABA and proline, if added to the growth medium, improved the appearence of the callus.Abbreviations ABA abscisic acid - B₅ Gamborg's medium - BA benzylalanine - 2,4-d 2,4-dichlorophenoxy acetic acid - FW fresh weight - G B₅ medium without growth regulators - GH B₅ medium supplemented with growth regulators - NAA naphthalene acetic acid - PGR plant growth regulators - Q _T total amount of a certain ion in the tissue - Q _s amount of the ion that has leaked out - QAC Quaternarty Ammonium Compounds - RGR mean relative growth rate - W₁ and W₂ fresh weight at times t₁ and t₂     

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.     

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     

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.     

Cell wall synthesis and salt (saline) sensitivity in cultured colt cherry (Prunus avium × pseudocerasus) protoplasts

In order to investigate the cellular basis of salt tolerance, Colt cherry (Prunus avium ×pseudocerasus) protoplasts from mesophyll tissues and root cell suspension cultures were cultured in the presence of NaCl, KCl or Na₂SO₄, at normalities of 25, 50, 100 or 200 mN for each salt and with or without 2,6-dichlorobenzonitrile, an inhibitor of cell wall synthesis. Results showed that the acquisition of salt tolerance was concomitant with the onset of cell wall regeneration, with protoplasts exhibiting a greater salt tolerance than cells.Abbreviations DBN 2,6-dichlorobenzonitrile - FDA fluorescein diacetate     

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.     

Steroidal alkaloids in tissue cultures and regenerated plants of Solanum dulcamara

Callus and cell suspension cultures were established with shoots of the soladulcidine variety of the bittersweet Solanum dulcamara L. Plantlets were regenerated from undifferentiated callus. From mixotrophic callus as well as mixotrophic suspension cultures soladulicidine, solasodine and the corresponding neutral spirostanes tigogenin and diosgenin were isolated and identified by thin layer chromatography and mass spectrometry. Total alkaloid concentrations were about 0.2 mg/g dry weight (callus) and 0.1 mg/g dry weight (green suspension cultures). In the heterotrophic cell line only the neutral sapogenins could be detected. Alkaloid accumulation in callus of Solanum dulcamara could be enhanced by the induction of organogenesis. The shoots of the regenerated plants from the mixotrophic callus contained soladulcidine (1.6 mg/g dry weight) and tigogenin. Thus, in concentration and composition the regenerated plants equalled the source plant.Abbreviations 2.4-D 2.4-dichlorophenoxyacetic acid - NAA naphthylacetic acid - HPLC high performance liquid chromatography - TLC thin layer chromatography     

Ion‐dependent metabolic responses of Vicia faba L. to salt stress

Salt‐affected farmlands are increasingly burdened by chlorides, carbonates, and sulfates of sodium, calcium, and magnesium. Intriguingly, the underlying physiological processes are studied almost always under NaCl stress. Two faba bean cultivars were subjected to low‐ and high‐salt treatments of NaCl, Na₂SO₄, and KCl. Assimilation rate and leaf water vapor conductance were reduced to approximately 25–30% without biomass reduction after 7 days salt stress, but this did not cause severe carbon shortage. The equimolar treatments of Na⁺, K⁺, and Cl^? showed comparable accumulation patterns in leaves and roots, except for SO₄^2? which did not accumulate. To gain a detailed understanding of the effects caused by the tested ion combinations, we performed nontargeted gas chromatography–mass spectrometry‐based metabolite profiling. Metabolic responses to various salts were in part highly linearly correlated, but only a few metabolite responses were common to all salts and in both cultivars. At high salt concentrations, only myo‐inositol, allantoin, and glycerophosphoglycerol were highly significantly increased in roots under all tested conditions. We discovered several metabolic responses that were preferentially associated with the presence of Na⁺, K⁺, or Cl^?. For example, increases of leaf proline and decreases of leaf fumaric acid and malic acid were apparently associated with Cl^? accumulation.     

The effect of gelling agents on plaunotol accumulation in callus cultures ofCroton sublyratus Kurz

Callus cultures established from the leaves ofCroton sublyratus Kurz (Euphorbiaceae) contained little or no plaunotol on various media with different hormone combinations. Plaunotol accumulation was observed in calli which were cultured in media with increasing concentrations of gelling agents, especially gellan gum and agarose. The accumulation was observed within three weeks after transfer to the medium and was accompanied by chlorophyll increase, tracheid development and slow growth. Light and increasing concentration of gelling agents were observed to be indispensable for plaunotol accumulation in the callus.Abbreviations MS Murashige and Skoog (1962) basal medium - NAA 1-naphthaleneacetic acid - BA 6-benzylaminopurine     

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.     

Effects of Na2SO4, K2SO4 and KCl on Growth and Ion Uptake of Callus Cultures of Vaccinium corymbosum L. cv. Blue Crop

Non-selected and Na₂SO-, K₂SO₄- or KCl-selected callus culturesof Vaccinium corymbosum L. cv. Blue Crop were grown on mediasupplemented with 0, 25 and 50 mM Na₂SO₄ (non-selected and Na₂SO(-selectedonly), 0, 25 and 50mMK₂SO₄ (non-selected and K₂SO₄-selectedonly) or 0, 50 and 100 mM KCl (non-selected and KCl-selectedonly). On all media, growth of selected callus (on a fresh-weightor dry-weight basis) was greater than that of non-selected callus,and selected callus grew optimally on the level and type ofsalt on which it was selected. Selected callus was friable andmaintained a higher f. wt:d. wt ratio. Tissue water potentialin selected callus was more negative than in non-selected callus. Flame photometry and chloridometry showed Na⁺, K⁺ and Cl^–accumulated in callus to concentrations equal to or greaterthan the initial concentration in the medium. Turbidometry showedthat tissue SO₄^2- concentration was lower than the concentrationin the medium. In most cases selected callus accumulated moreNa⁺, K^sup, SO₄^2– or Cl^– than non-selected callus.Vacuolar ion concentration was measured by electronprobe X-raymicroanalysis, and on most media selected callus had highervacuolar ion concentrations than non-selected callus. SO₄^2–and Cl^– were accumulated in the vacuoles at concentrationshigher than the external medium, but vacuolar Na⁺ concentrationdid not reach external concentration on Na₂SO₄ and on potassiumsalts was maintained between 12 and 17 mM. Vacuolar K⁺ concentration(approx. 142–191 mM on no salt) decreased on Na₂SO₄ andincreased on K₂SO₄ and KCl. There was no precise correlation between total or specific ionaccumulation (Na⁺, K⁺, SO₄^2– and Cl^– and fresh-weightyield. Results suggest that selection results in adaptationin response to decreased water potential of the medium. Vaccinium corymbosum, blueberry, electronprobe X-ray microanalysis, callus, in vitro selection, salt tolerance, KCl, K₂SO₄, Na₂SO₄