Influence of salinity on axillary bud cultures of six lowland tropical varieties of potato (Solanum tuberosum)

Axillary bud cultures of six low-land tropical varieties of potato Solanum tuberosum (L. T. 2, 5, 6, 7, 8 and 9) were studied for their response to salinity stress using crude sea salt. Concentrations of 0.8% and above severely inhibited the growth, while decreased growth was observed between 0.4% and 0.6% salt in all the varieties, with occasional shoot tip necrosis and compound leaf formation. A lower concentration of salt (0.2%) was beneficial and increased shoot weight in most varieties. Protein content showed a positive correlation with proline levels, indicating that proline accumulation was due to new synthesis rather than breakdown of existing proteins. Soluble carbohydrates increased in the stem with increasing salt. Roots showed an increase in sodium content while shoots showed increased potassium levels, with increasing salt concentrations. There was a positive correlation between dry weight yields and K/Na ratio. It is apparent that the synthesis of proline, accumulation of soluble carbohydrates as well as increase in potassium and sodium content were all used to adjust the physiology in response to salt stress by the different varieties of potato, with varying degrees, in the present study.     

In vitro shoot multiplication of eastern white cedar (Thuja occidentalis)

Summary A protocol for clonal propagation of eastern white cedar (Thuja occidentalis L.) was enhanced by optimizing the shoot multiplication stage using unbranched in vitro-produced shoots. This was achieved by careful selection of different medium components. An optimum range of 10 to 14 axillary shoots was obtained when shoots were cultured on half-strength Quiorin and LePoivre medium containing 10μM filter-sterilized zeatin for 3 wk. Transfer of the treated shoots to cytokinin-free medium containing 0.05% activated charcoal improved both the number and quality of the axillary shoots produced. Maximum axillary bud induction was also accomplished when shoots were pulsed in 1 mM liquid, filter-sterilized zeatin for 3 h, and then transferred to half-strength Quiorin and LePoivre, charcoal-containing medium. Inclusion of 4% sucrose improved the number of axillary shoots obtained. Half strength of the major salts produced an optimum response. Shoots obtained from different cultures (1 to 5 yr old) responded similarly to the applied cytokinin; however, newly induced shoots (4 mo. old) gave a significantly higher response.     

In vitro propagation of cashew from young trees

Summary Regeneration of cashew (Anacardium occidentale L.) from shoot explants of young grafts of mature tree origin is described. Establishment of shoot cultures was affected by season of collection, source, and type of explant. Explants from young grafts established better than those collected from field trees, and nodal cuttings regenerated better than shoot tips. Maximum percentage bud break and minimum contamination was noticed when shoots were collected in dry months (January to May). Pre-conditioning of stock plants by hormonal spray with 6-benzyladenine (BA) and gibberellic acid (GA₃) and brief presoaking of shoots in BA had no significant effect on culture establishment. MS (Murashige and Skoog, 1962) medium with half-strength major nutrients, 2.74 mM l-glutamine, 87.6 mM sucrose, and 2.25 gl⁻¹ phytagel was ideal for culture initiation. Inclusion of 0.1% polyvinylpyrrolidone (PVP-360) in the media reduced phenolic exudation. Solidified media was superior to liquid medium. Sucrose/glucose as energy source was found essential in the medium and had significant effect on percentage bud break and shoot development. A repeatable axillary shoot-bud induction was obtained on the above basal medium containing thidiazuron (TDZ) alone and in combination with BA. TDZ at 0.45 μM was best for axillary shoot-bud proliferation (4.5 buds per shoot) with maximum response (100%). Bud elongation could be stimulated in multiple shoots on medium containing 116.8 mM sucrose. In vitro rooting on auxin media and pulsing microshoots in 10 mM naphthalenaacetic acid (NAA) was ineffective. Rooting inability was, however, overcome by a micrografting procedure.     

Differential growth of some grapevine varieties in Syria in response to salt <Emphasis Type="Italic">in vitro</Emphasis>

Summary Growth sensitivity of four local grapevine (Vitis vinifera) varieties, Ashlamesh, Helwani, Kassofee, and Khoudeiry, were evaluated for salt. They were cultured on DSD1 medium until rooting stage, then they were transferred to a liquid DSD1 medium containing 0, 10, 20, 30, 40, 80, 120, or 150 mM NaCl for 30 d. The shoot length and leaf number of Ashlamesh, Helwani, and Kassofee were significantly increased at 10 and/or 30 mM NaCl, whereas, 150 mM NaCl decreased shoot length of all varieties except Kassofee. The presence of NaCl at 80 mM or higher concentrations decreased the chlorophyll content and root number of all varieties, while 30 mM NaCl increased root number of Kassofec.     

Rice callus growth, proline content and activity of proline and IAA oxidase under the influence of hydroxyproline and NaCl

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, proline content and activity of proline and IAA oxidases of the cultured 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 hydroxyproline and NaCl showed increased dry weight and proline content as compared to NaCl stressed calli. The levels of proline and IAA oxidases were also low in the hydroxyproline resistant calli.     

Response of seeds ofLupinus termis andVicia faba to the interactive effect of salinity and ascorbic acid or pyridoxine

The interactive effect of salinity and presoaking in ascorbic acid or phyridoxine on germination, seedling growth, and some relevant metabolic changes ofLupinus termis andVicia faba seeds were studied. Germination studies indicated that broad bean tolerated NaCl salinity up to 240mM NaCl and lupin to 200mM NaCl. The lengths of roots and shoots and their water content, as well as dry matter yield, remained more or less unchanged up to the level of 80mM NaCl. Salinity induced marked progressive increases of carbohydrates and proline in broad bean and soluble protein in lupin seedlings, irrespective of the salinity level used. The other organic solutes (soluble protein in broad bean and carbohydrates in lupin seedlings) remained more or less unchanged at low and moderate levels of NaCl. However, under the higher salinity levels, in lupin the losses in carbohydrates were accompanied by increases in soluble protein, whereas in broad bean an opposite effect was obtained. The level of 40mM NaCl had a pronounced stimulatory effect on the all the variables studied. Presoaking seeds in either ascorbic acid or pyridoxine counteracted the adverse effects of salinity on germination and seedling growth as well as on some metabolic mechanisms of lupin and broad bean plants. The importance of these processes to the salinity tolerance of broad bean and lupin have been discussed.     

On the role of exogenous gibberellin and cytokinin in the correlation between the leaf and its axillary bud in hortensia (Hydrangea opuloides C. KOCH)

The role of exogenously supplied gibberellin (GA₃) and cytokinin (benzyladenin -BA) in the correlation between the mature leaf and its axillary bud was investigated in one-node segments ofHydrangea. When both leaves were left on the segments, then both GA and BA were able to determine the dominance between axillary buds, that means that the bud treated with the corresponding growth regulator grew more vigorously. When one of the leaves was removed, the bud belonging to the removed leaf grew more vigorously, but GA applied onto the axillary bud belonging to the remaining leaf caused a complete correlation reversal: the bud belonging to the remaining leaf grew more vigorously. On the contrary, the application of BA onto the bud of the remaining leaf resulted in only insignificantly stimulated growth of the bud belonging to this leaf.     

Proline and betaine provide protection to antioxidant and methylglyoxal detoxification systems during cold stress in <Emphasis Type="Italic">Camellia sinensis</Emphasis> (L.) O. Kuntze

The aim of this study was to monitor the influence of proline and betaine exposure on antioxidant and methylglyoxal (MG) detoxification system during cold stress in Camellia sinensis (L.) O. Kuntze. Cold stress enhanced MG and lipid peroxidation levels in tea bud (youngest topmost leaf). This increase was resisted upon the exposure of tea bud to proline and betaine. Exposure of tea bud with proline and betaine also help in maintaining thiol/disulfide ratio during cold stress. Proline exposure enhanced glutathione-S-transferase and glutathione reductase (GR) activity, while betaine exposure increased only GR activity during cold stress. Furthermore, effect of proline/betaine was studied on glyoxalase pathway enzymes that are involved in MG detoxification and comprise of two enzymes glyoxalase I and glyoxalase II. Both proline and betaine showed protective effect on glyoxalase I and activating effect on glyoxalase II during cold stress in tea bud. This investigation, therefore, suggest that proline and betaine might provide protection to cold stress in tea by regulating MG and lipid peroxidation formation as well as by activating or protecting some of antioxidant and glyoxalase pathway enzymes.     

In vitro micropropagation of the macarronesian evergreen treePersea indica (L.) K. Spreng

Summary Shoot propagation ofPersea indica (L.) K. Spreng was achieved using seedling axillary buds cultured on MS (Murashige and Skoog, 1962) medium with 1 mg/l (2.8 μM) N⁶-benzyladenine (BA). Forty percent of the obtained shoots did not elongate, but showed bud proliferation, which was maximal (three axillary buds per shoot) at the end of the seventh subculture. Sixty percent of the shoots elongated, did not show bud proliferation, and formed calluses at their base. Successful rooting (84.6%) was achieved dipping the base of each elongated shoot in 3 g/l (16.11 mM) indolebutyric acid (IBA) for 1–2 s, and transferring to half strength MS medium without growth regulators. These shoots presented an acclimatization success of 100%. Results suggest that micropropagated elongated shoots ofP. indica can be adequately used in reforestation programs.     

Evidence for an interaction between basal medium and plant growth regulatiors during adventitious or axillary shoot formation of cauliflower

Summary Leafy shoots of cauliflower were produced from hypocotyl segments, curd branches (debarassed of existing meristems) and intact curd pieces submitted to various basal media. Adventitious shoot production and axillary bud break exhibited very different nutritive requirements. In each case, an interaction between the plant growth regulators and the composition of the basal medium was discovered. Propagation from curd pieces was dependent on the interaction between plant growth regulators and high sucrose-to-macronutrients ratio in the medium. A medium containing 168 mM sucrose, supplemented with 2.5 μM N⁶-benzyladenine and 5 μM naphthaleneacetic acid allowed the production of numerous shoots without any hyperhydric symptoms.     

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.     

Leaf position-dependent changes in proline,pyrroline-5-carboxylate reductase activity and water relations under salt-stress in genetically stable salt-tolerant somaclones ofBrassica juncea L.

The magnitude of the effect of salt stress on proline content, pyrroline-5-carboxylate (P5C) reductase activity and water relations was found to be leaf position dependent in an advance generation (R4) of twoBrassica juncea L. somaclones (SR-2 and SR-3) selected in vitro for NaCl-tolerance and the parent cv. Prakash. Free proline content and P5C reductase activity increased with increase in salt stress in all the lines but at different rates; the maximum increase being in the SR-3 derived somaclonal line. At 100 mM NaCl, SR-3 showed a nearly 19 fold increase in proline content compared to a 4–5 fold increase in the other two genotypes. The proline level and P5C reductase activity of the first (youngest) leaf was higher than in the other leaves and decreased linearly with increase in age of the leaf in all the lines. The relationship between relative water content and osmotic potential of the leaves at different positions also varied. The results indicate that a significant effect of salt may appear non-significant if the position of the leaves is not taken into account while sampling.     

Proline metabolism in response to nitrogen deficiency in French Bean plants (Phaseolus vulgaris L. cv Strike)

The objective of the present work was to determine the impact ofnitrogen deficiency on proline metabolism in French Bean plants(Phaseolus vulgaris L. cv. Strike). The nitrogen wasapplied to the nutrient solution in the form of NH₄NO₃ at1.45 mM (N1), 2.90mM (N2) and 5.80mM (N3, optimal level). Our results indicateNdeficiency is characterised by a decline in proline accumulation both in theroot and leaves, fundamentally because proline degradation is encouraged by thestimulation of the enzyme proline dehydrogenase. By contrast, under conditionsof adequate N (N3), proline levels rise due to the action of ornithine,suggesting predominance of the ornithine pathway over the glutamine pathway, inaddition to the inhibition of proline dehydrogenase activity.     

In vitro Propagation and Isozyme Polymorphism of the Medicinal Plant Hypericum brasiliense

Responses of 20 d-old plants of two Brassica napus L. cultivars Dunkeld and Cyclon to NaCl salinized soil [electrical conductivity 2.4 (control), 4.0, 8.0 or 12.0 dS m⁻¹] were examined. The salt tolerant line Dunkeld had significantly higher fresh and dry masses of shoots, and seed yield than salt sensitive line Cyclon in all salinities. The effect of salt stress on reduction in total leaf soluble sugars was markedly greater in Dunkeld as compared to that in Cyclon. No effect of salt stress was observed on leaf soluble proteins but there was a slight increase in total free amino acids of both cultivars. Leaf proline content increased markedly in both cultivars and Dunkeld had greater proline content than Cyclon at all salinities. Salt stress had no significant effect on seed oil content and erucic acid content of seed oil, however, content of glucosinolates in the seed meal increased and Cyclon had greater content of glucosinolates than Dunkeld. This revised version was published online in July 2006 with corrections to the Cover Date.     

ROLE OF PROLINE ACCUMULATION IN RESPONSE TO TOXIC COPPER IN CHLORELLA SP. (CHLOROPHYCEAE) CELLS

The green alga Chlorella sp. (strain 2350) was found to accumulate proline, up to a maximum of 0.027 fmol·cell⁻¹, under stressful concentrations of cupric ions. The function of the accumulated proline was studied with respect to its effect on copper (Cu) uptake. By induction with salt stress, cells with various levels of intracellular proline were used for this study. It is shown that the amount of Cu taken up by the cells was reduced when the intracellular proline levels were enhanced. When proline was exogenously supplied prior to Cu treatment, the adsorption of Cu was markedly reduced. When exogenous proline was supplied after Cu treatment, it resulted in a remarkable desorption of the adsorbed Cu immediately after the addition of proline. The results of the present study indicate that proline may exert some action on the cell surface and suggest that one function of accumulated proline is to reduce the uptake of metal ions. The accumulation of proline may be related to a tolerance mechanism for dealing with Cu stress.     

Effect of potassium on the tolerance to PEG-induced water stress of two white clover varieties (Trifolium repens L.)

The response to water stress was studied on white clover grown hydroponically. Two varieties (Crau and Huia) were both subjected to a moderate and a more-severe stress, induced by polyethylene glycol (10 and 20% respectively), in the presence of a nutrient solution poor in potassium (K₁=0.005 mM), or abundantly supplied (K₂=5mM). Dawn water potential and nitrogen fixation (acetylene reduction activity) decreased with the increasing stress. Conversely, the stomatal resistance increased whenosmoticum was added. Crau had a lower stomatal resistance to the deficit, than did Huia. In relation with the K supply, treatment K₂ confirmed the superiority of Crau. Crau also showed greater nodule mass and number than Huia. The data show relationships between dawn water potential, stomatal resistance and nitrogen fixation activity.     

Hypersensitivity of an Arabidopsis sugar signaling mutant toward exogenous proline application

In transgenic Arabidopsis a patatin class I promoter from potato is regulated by sugars and proline (Pro), thus integrating signals derived from carbon and nitrogen metabolism. In both cases a signaling cascade involving protein phosphatases is involved in induction. Other endogenous genes are also regulated by both Pro and carbohydrates. Chalcone synthase (CHS) gene expression is induced by both, whereas the Pro biosynthetic Delta(1)-pyrroline-5-carboxylate synthetase (P5CS) is induced by high Suc concentrations but repressed by Pro, and Pro dehydrogenase (ProDH) is inversely regulated. The mutant rsr1-1, impaired in sugar dependent induction of the patatin promoter, is hypersensitive to low levels of external Pro and develops autofluorescence and necroses. Toxicity of Pro can be ameliorated by salt stress and exogenously supplied metabolizable carbohydrates. The rsr1-1 mutant shows a reduced response regarding sugar induction of CHS and P5CS expression. ProDH expression is de-repressed in the mutant but still down-regulated by sugar. Pro toxicity seems to be mediated by the degradation intermediate Delta(1)-pyrroline-5-carboxylate. Induction of the patatin promoter by carbohydrates and Pro, together with the Pro hypersensitivity of the mutant rsr1-1, demonstrate a new link between carbon/nitrogen and stress responses.     

Selection for salt tolerance in vitro using microspore-derived embryos ofBrassica napus cv. topas,and the characterization of putative tolerant plants

Microspore-derived embryos ofBrassica napus cv. Topas that survived salt stress, were obtained after selection against otherwise lethal doses (0.6 and 0.7%) of NaCl after mutagen treatment. A total of 10 salt-surviving embryos were obtained out of a possible 834 000 embryos that were mutagenized. One embryo out of a possible 845 000 obtained from nonmutagenized controls survived but failed to develop into a plant. Visual assessment after salt stress indicated that both the putative salt-tolerant plants and plants from control seeds behaved similarly. However, based on individual characteristics related to salt tolerance, one of the lines (PST-2) accumulated less sodium and retained more potassium, and hence was able to maintain a more favorable Na:K ratio as compared to the controls under salt stress. Also chlorophylla fluorescence induction and quenching signals indicated a high energetic state of the thylakoid membranes in PST-2 under salt stress. The other putative salt-tolerant line (PST-1) had a higher background level of proline that may have enabled it to survive salt stress during initial screening, although its later performance was no better than the control plants.     

Establishment of Salt Stress Tolerant Rice Plants Through Step Up NaCl Treatment In Vitro

Establishment of salt tolerant rice plants was examined by single step or step up NaCl treatments of shoot bud clumps in vitro, and variation among in vitro salt tolerant plants were examined by rapid amplified polymorphic DNA (RAPD). Shoot bud clumps were necrotic, stubbed or dead when subjected to single step treatment with 1.5 or 2.0 % NaCl. Conversely all the clumps could grow vigorously when subjected to step up salt treatment with 0.5, 1.0, 1.5 and 2.0 % NaCl at 3 week intervals and 2 % NaCl tolerant plants were established. RAPD revealed shoot bud clumps with and without different NaCl treatments, seedlings from field and grown in vitro, and regenerants from callus were genetically close to one another. Conversely, callus cultures were genetically isolated. Growth under different salt stress conditions was not correlated with the genetic variation, suggesting that 2.0 % NaCl tolerant plants might not result from genetic mutation but were due to adaptation of plants by step up NaCl treatment in vitro.     

Effects of NaCl stress on proline and cation accumulation in salt sensitive and tolerant turfgrasses

Summary Concentrations of proline, sodium and potassium in shoot tissues of five turfgrass species were measured following exposure to 170 mM NaCl salinity stress. Salt tolerant ‘Fults’ alkaligrass and ‘Dawson’ red fescue restricted the accumulation of Na-ions to significatnly low levels compared to the salt sensitive Kentucky bluegrasses (‘Adelphi’ and ‘Ram I’) and ‘Jamestown’ red fescue. Accumulation of proline began in all species within 24 h of initiation of salt stress but at a more rapid rate and higher overall concentration for ‘Fults’ alkaligrass. Proline levels were variable and too low in relation to sodium accumulations to have any significant osmoregulatory role in salt tolerance among all cultivars tested with the possible exception of alkaligrass.