Comparative effect of calcium and EDTA on arsenic uptake and physiological attributes of Pisum sativum

In this study, we determined the effect of ethylenediaminetetraacetic acid (EDTA) and calcium (Ca) on arsenic (As) uptake and toxicity to Pisum sativum. Plants were treated with three levels of As (25, 125, and 250 µM) in the presence and absence of three levels of Ca (1, 5, and 10 mM) and EDTA (25, 125, and 250 µM). Exposure to As caused an overproduction of hydrogen peroxide (H₂O₂) in roots and leaves, which induced lipid peroxidation and decreased pigment contents. Application of both Ca and EDTA significantly reduced As accumulation by pea, Ca being more effective in reducing As accumulation. Both Ca and EDTA enhanced As-induced H₂O₂ production, but reduced lipid peroxidation. In the case of pigment contents, EDTA significantly reduced pigment contents, whereas Ca significantly enhanced pigment contents compared to As alone. The effect of As treatment in the presence and absence of EDTA and Ca was more pronounced in younger leaves compared to older leaves. The effect of amendments varied greatly with their applied levels, as well as type and age of plant organs. Importantly, due to possible precipitation of Ca-As compounds, the soils with higher levels of Ca ions are likely to be less prone to food chain contamination.     

Methyl jasmonate alleviates arsenic-induced oxidative damage and modulates the ascorbate–glutathione cycle in oilseed rape roots

Methyl jasmonate (MJ) is an important plant growth regulator, involves in various physiological processes of plants. In the present study, role of MJ in tolerance to oilseed rape (Brassica napus L.) roots under arsenic (As) stress was investigated. The treatments were comprised of three MJ doses (0, 0.1, and 1 µM) and two levels of As (0 and 200 µM). Arsenic stress resulted in oxidative damage as evidenced by decreased root growth and enhanced reactive oxygen species and lipid peroxidation. However, plants treated with MJ decreased the H₂O₂ and O₂ ^·? contents in roots and have higher antioxidant activities. Importantly, results showed that MJ enhanced the redox states of AsA and GSH, and the related enzymes involved in the AsA–GSH cycle. Moreover, MJ also induced the secondary metabolites related enzymes (PAL and PPO) activities, under As stress. PAL and PPO expression was further increased by MJ application in the roots of B. napus under As stress. MJ also reduced the total As content compared with As alone treated plants. These findings suggest the role of MJ in mitigation of the As-induced oxidative damage by regulating AsA and GSH redox states and by reducing As uptake in both cultivars.     

Copper phytotoxicity affects root elongation and iron nutrition in durum wheat (Triticum turgidum durum L.)

This work investigated how copper (Cu) phytotoxicity affected iron (Fe) nutrition and root elongation in hydroponically grown durum wheat (Triticum turgidum durum L., cv Acalou) in order to establish the critical level of Cu concentration in roots above which significant Cu phytotoxicity occurs. This was assessed at two levels of Fe supply (2 and 100 μM). Severe symptoms of Cu phytotoxicity were observed at Cu²⁺ concentration above 1 μM, i.e. interveinal chlorosis symptoms and global root growth alteration. Total root Cu concentration of about 100, 150 and 250–300 mg kg^?1 corresponded to 10%, 25% and 50% reduction in root elongation, respectively. Copper and Fe concentrations as well as amounts of Cu and Fe accumulated in shoots varied inversely which suggested an antagonism between Cu and Fe leading to Fe deficiency. In addition, the root-induced release of complexing compounds increased significantly with increasing Cu concentration in nutrient solution and was positively correlated with Cu uptake without significant difference between the two Fe treatments (high and low Fe supply). This work suggests that total root Cu concentration might be a simple, sensitive indicator of Cu rhizotoxicity. It also indicated that Cu phytotoxicity which may have resulted in Fe deficiency and significant increase in root-induced release of complexing compounds (presumably phytosiderophores) was independent of the level of Fe supply provided that the threshold values of phytotoxicity were based on the free Cu-ion concentration.     

Role of glutathione and glutathione S-transferase in lead tolerance and bioaccumulation by Dodonaea viscosa (L.) Jacq

Glutathione (GSH) plays a central role in the plant tolerance against the toxic effects of metals. It is a key antioxidant and acts as a cofactor for glutathione S-transferase (GST). The main objective of this study was to determine the Pb tolerance and bioaccumulation by Dodonaea viscosa (L.) Jacq. and their relation to GSH production and GST activity. The relationship between the Pb tolerance and bioaccumulation by D. viscosa and the effect of the exposure time on the GSH production or the GST activity was assessed in trials with perlite under different Pb treatments. D. viscosa showed a remarkable tolerance to Pb [half-inhibitory concentration (IC₅₀) = 2,797 mg kg^?1] and accumulated up to 11,428 mg Pb kg^?1 in dry roots with a limited translocation to shoots without any signs of phytotoxicity after 105 days of exposure. The stress caused by the fast Pb uptake rate (489 mg kg^?1 day^?1) during the first 10 days of exposure was strongly correlated to increased GSH contents (~1.3-fold) and GST activities (~3.6-fold) in both shoots and roots. The results indicate that the Pb stress triggered a defense mechanism that involved increased contents of GSH and GST activities, suggesting that both variables are involved in the tolerance of D. viscosa against Pb toxicity.     

Selected bioavailability assays to test the efficacy of amendment-induced immobilization of lead in soils

Lead immobilization in 10 soils contaminated with Pb from different origin was examined using lime (CaCO₃), a mix of cyclonic ash and steelshots (CA+ST), and a North Carolina phosphate rock. The immobilization efficacy of the three amendments was evaluated using single (CaCl₂solution) and sequential (BCR method) chemical extractions in tandem with a microbiological Pb biosensor (BIOMET), a Pb phytotoxicity test, Pb plant uptake, and a Physiologically Based Extraction Test (PBET) mimicking Pb bioavailability in the human gastro-intestinal tract. The results demonstrated the necessity of using a diverse suite of bioavailability methodology when in situ metal immobilization is assessed. Sequential (BCR) extractions and PBET analysis indicated that PR was the most effective amendment. PR however, proved ineffective in totally preventing Pb phytotoxicity and Pb uptake on all soils tested. On the contrary, CA+ST and lime decreased BIOMET Pb, Pb phytotoxicity, and Pb uptake to a far greater extent than did PR. BIOMET detectable Pb and Pb uptake, however, were strongly related to Pb in soluble or exchangeable soil fractions (i.e., CaCl₂ extractable). By combining these fractions with the acid-extractable Pb, accomplished by using acetic acid extractant, the recently developed BCR sequential extraction scheme appeared to have lost some valuable information on judging Pb bioavailability. The data show that different amendments do not behave consistently across different soils with different sources of contamination. Different indices for measuring Pb bioavailability are also not necessarily consistent within particular soil and amendment combinations.     

Salicylic acid accumulation as a result of Cu,Zn, Cd and Pb interactions in common reed (<Emphasis Type="Italic">Phragmites australis</Emphasis>) growing in natural ecosystems

Common reed (Phragmites australis (Cav.) Trin. ex Steud.) plants were harvested from four natural water ecosystems of the Bogdanka river catchment (Poznań, Poland) four times throughout the 2014 vegetative season. Over the year, average metal contents followed different decreasing trends according to the analyzed tissue: Zn > Cu ≈ Pb > Cd (rhizomes) and Zn > Pb > Cu > Cd (leaves), and mean translocation ratios (leaves/rhizomes) were found as follows: 0.93, 0.70, 0.65, 0.40 for Zn, Pb, Cd and Cu, respectively. Metal content increased gradually during the growing season, and in the case of Cu, Cd and Pb exceeded the upper limit of average concentration detected in plants from natural ecosystems. However, the content of salicylic acid did not follow the increase of metal accumulation. In rhizomes, the highest production of the metabolite was observed in May and reached 324 ng g^?1 fresh weight (FW) (mean value). Afterwards, a significant drop to 50 ng g^?1 FW was observed. Simultaneously, the highest values of total salicylic acid in P. australis leaves were observed in July and accompanied the intensive development of the aboveground biomass of the plant (11.3 µg g^?1 FW–mean value). Subsequently, its content in leaves showed a significant decrease down to 2.1 µg g^?1 FW in November. Multivariate regression analysis revealed significant interactions between analyzed metals influencing the plant response to metal-derived stress. Cu and Zn showed antagonistic properties considering their uptake and the induction of salicylic acid biosynthesis, whereas non-essential metals (Pb and Cd) acted similarly and stimulated the formation of salicylic acid glucoside.     

Role of Exogenous Nitric Oxide in Alleviating Iron Deficiency Stress of Peanut Seedlings (<Emphasis Type="Italic">Arachis hypogaea</Emphasis> L.)

A greenhouse hydroponic experiment was performed to evaluate how peanut seedlings (Arachis hypogaea L.) responded to iron (Fe) deficiency stress in the presence of sodium nitroprusside (SNP), a nitric oxide (NO) donor. The results showed that Fe deficiency inhibited peanut plant growth, decreased chlorophyll and active Fe concentrations, and dramatically disturbed ion balance. The addition of 50, 100, 250, and 500 µM SNP, significantly promoted the absorption of Fe in the cell wall, cell organelles, and soluble fractions, increased the concentrations of active Fe and chlorophyll in peanut plants, and alleviated the excess absorption of manganese (Mn) and copper (Cu) induced by Fe deficiency. In addition, SNP also significantly increased the activities of superoxide dismutase, peroxidase, and catalase, which is beneficial to inhibit the accumulation of malondialdehyde and reactive oxygen species. Addition of 250 µM SNP had the most significant alleviating effect against Fe-deficiency stress, and after 15 days of treatment, the plants with the 250 µM SNP treatment achieved comparable NO levels with those grown under optimal nutrition conditions. However, the effects of SNP were reversed by addition of hemoglobin (Hb, a NO scavenger). These results suggest that NO released from SNP decomposition was responsible for the effect of SNP-induced alleviation on Fe deficiency.     

Physiological responses of peanut seedlings to exposure to low or high cadmium concentration and the alleviating effect of exogenous nitric oxide to high cadmium concentration stress

Abstract

The physiological responses of peanut seedlings exposed to low (5 µM) or high (200 µM) cadmium (Cd) concentration and the ability of sodium nitroprusside (SNP, a donor of NO) to reverse the harmful effects of Cd on peanut (Arachis hypogaea L.) were studied. Changes in plant growth parameters, chlorophyll content, antioxidant system, nutrient contents and Cd accumulation were investigated. The results showed that SNP and 5 µM Cd improved plant growth and chlorophyll content. Furthermore, antioxidative system was up-regulated, and as a result, the production rate of superoxide radical (O₂^??) was reduced. Moreover, the absorption of nutrient elements was not impacted, and Cd toxicity was not observed. However, 200 µM Cd had negative effects on the above measured parameters and dramatically increased the accumulation of Cd in all the plant organs. In the 200 µM Cd treatment, addition of 250 µM SNP stimulated plant growth and increased chlorophyll content. It also enhanced the regulation of antioxidative system and reduced the production rate of O₂^?? and malondialdehyde (MDA) content. Besides, SNP supply enhanced the absorption of nutrient elements and restrained the absorption and transport of Cd.     

Lignan accumulation in two-phase cultures of <Emphasis Type="Italic">Taxus</Emphasis> x <Emphasis Type="Italic">media</Emphasis> hairy roots

The biosynthetic potential for six lignans accumulation in two lines of Taxus x media hairy roots was investigated. The cultures of KT and ATMA hairy root lines were supplemented with precursors: coniferyl alcohol (CA 1, 10 or 100 µM) and/or l-phenylalanine (100 µM PHEN) and/or methyl jasmonate (100 µM MeJa). Moreover the two-phase in vitro cultures supported with perfluorodecalin (PFD) as a gas carrier and in situ extrahent were used. The hairy root lines differed in lignan production profiles. In the control untreated cultures KT roots did not accumulate secoisolariciresinol and lariciresinol while ATMA roots did not accumulate matairesinol. In ATMA roots the treatment with CA (1 or 10 µM) resulted in the production of lariciresinol and secoisolariciresinol whereas solely lariciresinol was present after 100 µM CA application. Elicitation with 1 µM CA and MeJa yielded with hydroxymatairesinol aglyca and lariciresinol glucosides with their highest content 37.88 and 3.19 µg/g DW, respectively. The stimulatory effect of simultaneous treatment with 1 µM CA, PHEN and MeJa on lignan production was observed when the cultures were supplemented with PFD-aerated or degassed. In ATMA root cultures these applied conditions were the most favourable for matairesinol content which amounted to 199.86 and 160.25 µg/g DW in PFD-aerated and PFD-degassed supported cultures, respectively. In KT root cultures solely, hydroxymatairesinol and coniferin/CA content was enhanced with their highest yield 59.29 and 134.60 µg/g DW in PFD-aerated and PFD-degassed cultures, respectively.     

Effects of arsenate on tobacco hairy root and seedling growth,and its removal

Arsenic (As) is a highly toxic environmental contaminant to which most living organisms are exposed. Plants have evolved several mechanisms to cope with this toxic metalloid; however, these mechanisms are only partially understood. The response of plants to As phytotoxicity is highly complex, with considerable variation among species. In this study, arsenate (As⁺⁵) effects on germination and early root development of tobacco (Nicotiana tabacum) seedlings were investigated. Also, As⁺⁵ tolerance and removal efficiency of tobacco hairy roots (HRs) and seedlings were assessed and compared. Total seed germination capacity was not affected by 10 to 200 μM As⁺⁵, while primary root length and root branching were reduced by As⁺⁵ concentrations that were at or above 100 μM. Both systems were able to tolerate As⁺⁵ concentrations of 10 μM since no growth inhibition was detected. For higher As⁺⁵ concentrations, phytotoxicity increased, but it was mitigated by higher phosphate (Pi) availability. Under the studied conditions, As⁺⁵ removal efficiency of HRs greatly exceeded that of seedlings. Further, tobacco HRs were able to accumulate As in their tissues. These results justify further investigations on As tolerance and detoxification mechanisms in tobacco, an easy-to-transform crop species with high biomass, which could allow evaluation of the possible application of wild type or alternatively transgenic tobacco plants for As phytoextraction.     

Efficient in vitro propagation of <Emphasis Type="Italic">Artemisia nilagirica</Emphasis> var. <Emphasis Type="Italic">nilagirica</Emphasis> (Indian wormwood) and assessment of genetic fidelity of micropropagated plants

A reliable protocol has been established for in vitro propagation of Artemisia nilagirica var. nilagirica (Indian wormwood), a valuable medicinal plant from India. A highly proliferating organogenic callus was obtained on Murashige and Skoog (MS) medium supplemented with 2.5 µM IAA when nodal explants were cultured on MS medium supplemented with various growth regulators. Further, highest regeneration frequency (83.3 %) of adventitious shoots was observed, when the callus was sub-cultured on MS medium supplemented with 6-benzylaminopurine (BAP; 2.5 µM) along with 7.5 µM 2-isopentenyl adenine (2-iP). An optimal of 10.16 ± 2.24 shoots were regenerated on medium supplemented with 2.5 µM BAP + 7.5 µM 2-iP. Quarter strength MS medium supplemented with 10 µM IBA was effective for rooting of the shoots. Ex-vitro plants were normal and were established successfully. Cytological and molecular marker studies showed that regenerated plants showed genetic stability in micro-propagated plants.     

Lead accumulation and distribution in maize seedlings: Relevance to biomass production and metal phytoextraction

Among trace metals, lead is a highly toxic contaminant, being hazardous to humans and animals. Application of maize plants for phytoremediation of polluted soils and waters has recently been of particular interest. The aim of this work is to investigate the Pb-phytoextraction potential of the maize cv. Tzariza used widely in Eastern European agriculture. Maize seedlings were exposed in a nutrient solution to 1–10000 µM of Pb²⁺ for 21 days. Lead accumulated mostly in conductive tissues and shoots at 0.1 mM and higher concentrations of Pb in growth medium. Pb at concentrations of 1 and 10 mM caused an increase in the superoxide anion level and the catalase activity in maize leaves. Lead ions were tolerable to maize seedlings within a concentration range up to 1000 µM of Pb^2+. The levels of lead in the nutrient solution above 1 mM resulted in inhibition of the growth of axial organs, decrease in leaf area, inhibition of water absorption, and reduction in accumulation of biomass. Theoretical considerations indicate that in the temperate climates of the phytoremediation with maize may allow annual removal up to 90 kg of Pb per km², depending on the initial level of soil contamination.     

Fractionation,Source Identification and Risk Assessments for Heavy Metals in Soils near a Small-Scale Industrial Area (Çanakkale-Turkey)

Contamination of soils by heavy metals due to urbanization increases various environmental concerns. The objective of this research was to determine the potential sources of heavy metals in agricultural soils in the vicinity of a small-scale industrial area and to assess their environmental impacts. Soil samples were obtained from 15 different locations near a small industrial area in the Çanakkale province of Turkey. Heavy metal (Cd, Co, Cu, Ni, Pb, Zn) contents of soil samples were determined with four different geochemical fractions via a sequential extraction procedure. The results revealed that pseudo-total heavy metal concentrations were ordered in decreasing order as Zn > Pb > Cu > Ni > Co > Cd. Considering the results, Cd (1.95 ± 0.12 µg/g), Pb (39.21 ± 2.14 µg/g) and Zn (64.99 ± 8.16 µg/g) values were above the normal values specified for agricultural lands. The findings obtained from sequential extraction procedure showed that Cd (78%) and Pb (65%) existed mostly in mobile phases. Such mobile phases originated mostly from anthropogenic sources. These findings were also supported by chemometric analyses. Risk assessments pointed out that while Pb and Zn have moderate risks on the environment, Cd creates high risks.     

The Role of Ca<Superscript>2+</Superscript> Imbalance in the Induction of Acute Oxidative Stress and Cytotoxicity in Cultured Rat Cerebellar Granule Cells Challenged with Tetrabromobisphenol A

Using primary cultures of rat cerebellar granule cells (CGC) we examined the role of calcium transients induced by tetrabromobisphenol A (TBBPA) in triggering oxidative stress and cytotoxicity. CGC were exposed for 30 min to 10 or 25 µM TBBPA. Changes in intracellular calcium concentration ([Ca²⁺]_i), in the production of reactive oxygen species (ROS), and in the potential of mitochondria (?Ψm) were measured fluorometrically during the exposure. The intracellular glutathione (GSH) and catalase activity were determined after the incubation; cell viability was evaluated 24 h later. TBBPA concentration-dependently increased [Ca²⁺]_i and ROS production, and reduced GSH content, catalase activity, ?Ψm and neuronal viability. The combination of NMDA and ryanodine receptor antagonists, MK-801 and bastadin 12 with ryanodine, respectively, prevented Ca²⁺ transients and partially reduced cytotoxicity induced by TBBPA at both concentrations. The antagonists also completely inhibited oxidative stress and depolarization of mitochondria evoked by 10 µM TBBPA, whereas these effects were only partially reduced in the 25 µM TBBPA treatment. Free radical scavengers prevented TBBPA-induced development of oxidative stress and improved CGC viability without having any effect on the rises in Ca²⁺ and drop in ?Ψm. The co-administration of scavengers with NMDA and ryanodine receptor antagonists provided almost complete neuroprotection. These results indicate that Ca²⁺ imbalance and oxidative stress both mediate acute toxicity of TBBPA in CGC. At 10 µM TBBPA Ca²⁺ imbalance is a primary event, inducing oxidative stress, depolarization of mitochondria and cytotoxicity, whilst at a concentration of 25 µM TBBPA an additional Ca²⁺-independent portion of oxidative stress and cytotoxicity emerges.     

Comparative Effects of Hydrogen Sulfide-Releasing Compounds on [<Superscript>3</Superscript>H]D-Aspartate Release from Bovine Isolated Retinae

We investigated the pharmacological actions of a slow-releasing H₂S donor, GYY 4137; a substrate for the biosynthesis of H₂S, l-cysteine and its precursor, N-acetylcysteine on potassium (K⁺; 50 mM)-evoked [³H]D-aspartate release from bovine isolated retinae using the Superfusion Method. GYY 4137 (10 nM–10 µM), l-cysteine (100 nM–10 µM) and N-acetylcysteine (10 µM–1 mM) elicited a concentration-dependent decrease in K⁺-evoked [³H]D-aspartate release from isolated bovine retinae without affecting basal tritium efflux. At equimolar concentration of 10 µM, the rank order of activity was as follows: l-cysteine?>?GYY 4137?>?N-acetylcysteine. A dual inhibitor of the biosynthetic enzymes for H₂S, cystathionine β-synthase (CBS) and cystathionine γ-lyase (CSE), amino-oxyacetic acid (AOA; 3 mM) reversed the inhibitory responses caused by GYY 4137, l-cysteine and N-acetylcysteine on K⁺-evoked [³H]D-aspartate release. Glibenclamide (300 µM), an inhibitor of K_ATP channels blocked the inhibitory action of GYY 4137 and l-cysteine but not that elicited by N-acetylcysteine on K⁺-induced [³H]D-aspartate release. The inhibitory effect of GYY 4137 and l-cysteine on K⁺-evoked [³H]D-aspartate release was reversed by the non-specific inhibitor of nitric oxide synthase (NOS), l-NAME (300 µM). Furthermore, a specific inhibitor of inducible NOS (iNOS), aminoguanidine (10 µM) blocked the inhibitory action of l-cysteine on K⁺-evoked [³H]D-aspartate release. We conclude that both donors and substrates for H₂S production can inhibit amino acid neurotransmission in bovine isolated retinae, an effect that is dependent, at least in part, upon the intramural biosynthesis of this gas, and on the activity of K_ATP channels and NO synthase.     

High salinity helps the halophyte <Emphasis Type="Italic">Sesuvium portulacastrum</Emphasis> in defense against Cd toxicity by maintaining redox balance and photosynthesis

Main conclusion

NaCl alleviates Cd toxicity in Sesvium portulacastrum by maintaining plant water status and redox balance, protecting chloroplasts structure and inducing some potential Cd ²⁺ chelators as GSH and proline. It has been demonstrated that NaCl alleviates Cd-induced growth inhibition in the halophyte Sesuvium portulacastrum. However, the processes that mediate this effect are still unclear. In this work we combined physiological, biochemical and ultrastructural studies to highlight the effects of salt on the redox balance and photosynthesis in Cd-stressed plants. Seedlings were exposed to different Cd concentrations (0, 25 and 50 µM Cd) combined with low (0.09 mM) (LS), or high (200 mM) NaCl (HS) in hydroponic culture. Plant–water relations, photosynthesis rate, leaf gas exchange, chlorophyll fluorescence, chloroplast ultrastructure, and proline and glutathione concentrations were analyzed after 1 month of treatment. In addition, the endogenous levels of stress-related hormones were determined in plants subjected to 25 µM Cd combined with both NaCl concentrations. In plants with low salt supply (LS), Cd reduced growth, induced plant dehydration, disrupted chloroplast structure and functioning, decreased net CO₂ assimilation rate (A) and transpiration rate (E), inhibited the maximum potential quantum efficiency (Fv/Fm) and the quantum yield efficiency (Φ _PSII) of PSII, and enhanced the non-photochemical quenching (NPQ). The addition of 200 mM NaCl (HS) to the Cd-containing medium culture significantly mitigated Cd phytotoxicity. Hence, even at similar internal Cd concentrations, HS-Cd plants were less affected by Cd than LS-Cd ones. Hence, 200 mM NaCl significantly alleviates Cd-induced toxicity symptoms, growth inhibition, and photosynthesis disturbances. The cell ultrastructure was better preserved in HS-Cd plants but affected in LS-Cd plants. The HS-Cd plants showed also higher concentrations of reduced glutathione (GSH), proline and jasmonic acid (JA) than the LS-Cd plants. However, under LS-Cd conditions, plants maintained higher concentration of salicylic acid (SA) and abscisic acid (ABA) than the HS-Cd ones. We conclude that in S. portulacastrum alleviation of Cd toxicity by NaCl is related to the modification of GSH and proline contents as well as stress hormone levels thus protecting redox balance and photosynthesis.

     

Cadmium and lead interactive effects on oxidative stress and antioxidative responses in rice seedlings

Interactive effects of two heavy metal pollutants Cd and Pb in the growth medium were examined on their uptake, production of reactive oxygen species (ROS), induction of oxidative stress and antioxidative defence responses in Indica rice (Oryza sativa L.) seedlings. When rice seedlings in sand culture were exposed to 150 μM Cd (NO₃)₂ or 600 μM Pb (CH₃COO)₂ individually or in combination for 8–16 days, a significant reduction in root/shoot length, fresh weight, relative water content, photosynthetic pigments and increased production of ROS (O₂˙^? and H₂O₂) was observed. Both Cd and Pb were readily taken up by rice roots and localisation of absorbed metals was greater in roots than in shoots. When present together in the growth medium, uptake of both the metals Cd and Pb declined by 25–40 %. Scanning electron microscope (SEM) imaging of leaf stomata revealed that Pb caused more distortion in the shape of guard cells than Cd. Dithizone staining of roots showed localisation of absorbed Cd on root hairs and epidermal cells. Both Cd and Pb caused increased lipid peroxidation, protein carbonylation, decline in protein thiol and increase in non-protein thiol. The level of reduced forms of non-enzymic antioxidants glutathione (GSH) and ascorbate (AsA) and their redox ratios (GSH/AsA) declined, whereas the activities of antioxidative enzymes superoxide dismutase (SOD) and guaiacol peroxidase (GPX) increased in metal treated seedlings compared to controls. In-gel activity staining also revealed increased intensities of SOD and GPX isoforms with metal treatments. Catalase (CAT) activity increased during early days (8 days) of metal exposure and declined by 16 days. Results suggest that oxidative stress is an important component in expression of Cd and Pb toxicities in rice, though uptake of both metals gets reduced considerably when present together in the medium.     

Epibrassinolide Application Regulates Some Key Physio-biochemical Attributes As Well As Oxidative Defense System in Maize Plants Grown Under Saline Stress

It was aimed to investigate the ameliorative effect of exogenously applied 24-epibrassinolide (EBR) on some key growth parameters and mineral elements in two salt-stressed maize (PR 32T83 and PR 34N24) cultivars. A factorial experiment was designed with two electrical permeability (EC) levels (1.1 and 8.0 dS/m) and two levels (1.5 and 2.0 µM) of EBR supplied as a seed treatment, foliar spray, or both in combination. The foliar application of EBR was done once a week during the experiment. After 42 days of these treatments, the plants were harvested to assess growth, water relations, and oxidative and antioxidative systems. Salt stress markedly reduced plant fresh and dry weights, maximum fluorescence yield of PS-II, chlorophyll contents, leaf water potential, and leaf K and Ca, but it increased membrane permeability, the activities of superoxide dismutase (SOD; EC 1.15.1.1), peroxidase (POD; EC. 1.11.1.7), and catalase (CAT; EC. 1.11.1.6) enzymes, and the contents of proline and glycine betaine, leaf sap osmotic pressure, lipid peroxidation, hydrogen peroxide, and leaf Na and Cl. However, both seed treatment and foliar application of EBR to the maize plants exposed to saline conditions enhanced key growth attributes, water relations, and the activities of various antioxidant enzymes as well as the levels of proline, but they reduced electrolyte leakage, and H₂O₂ and MDA contents. Saline stress reduced leaf N, Ca²⁺, K⁺, and P contents as compared to those in the non-stressed plants. Both seed treatment and foliar application of EBR reduced Na⁺ and Cl^? concentrations, but increased those of N, Ca²⁺, K⁺, and P. Foliar application of EBR was more effective in increasing nutrient levels of plants grown at the high saline regime compared to the seed treatment of EBR. The study clearly indicates that both seed treatment and foliar application of EBR at the rate of 2.0 µM can overcome the detrimental effect of salinity stress on maize growth, which was found to be significantly linked to reduced concentrations of Na, Cl, MDA, and H₂O₂ as well as EL and increased activities of key antioxidant enzymes in the maize plants.     

High-frequency clonal propagation of <Emphasis Type="Italic">Curcuma angustifolia</Emphasis> ensuring genetic fidelity of micropropagated plants

A high-frequency clonal propagation protocol was developed for Curcuma angustifolia Roxb., a high valued traditional medicinal plant. Axillary bud explants of C. angustifolia were explanted on Murashige and Skoog (MS) medium fortified with 4.4–22.2 µM 6-benzyladenine (BA), 2.9–5.7 µM indole-3-acetic acid (IAA), 2.3–23.2 µM kinetin (Kin), 2.7–5.4 µM naphthalene acetic acid (NAA) and 67.8-271.5 µM adenine sulphate (Ads) in different combinations. The maximum number of shoots per explants (14.1?±?0.55) and roots per shoot (7.6?±?0.47) was achieved on media containing 13.3 µM BA, 5.7 µM IAA and 135.7 µM Ads. Stability in phytomedicinal yield potential of micropropagated plants was assessed through GC–MS and HPTLC. Gas chromatogram of essential oil of conventional and micropropagated plants of C. angustifolia had similar essential oil profile. HPTLC analysis of rhizome extracts of in vitro and field grown plants revealed no significant differences in the fingerprint pattern and in curcumin content. Genetic integrity of in vitro and field grown derived plants were evaluated with inter-simple sequence repeat (ISSR) primers and flow cytometry using Glycine max as an internal standard. A total of 1260 well resolved bands were generated by 12 ISSR primers showing monomorphic banding patterns across all plants analyzed. The mean 2C DNA content of conventionally and micropropagated plant was estimated to be 2.26 pg and 2.31 pg, respectively. As no somaclonal variations were detected in tissue culture plantlets, the present micropropagation protocol could be applied for in vitro conservation and large-scale production of C. angustifolia.