Aluminum resistance in wheat involves maintenance of leaf Ca<Superscript>2+</Superscript> and Mg<Superscript>2+</Superscript> content,decreased lipid peroxidation and Al accumulation,and low photosystem II excitation pressure

The phytotoxic aluminum species (Al³⁺) is considered as the primary factor limiting crop productivity in over 40 % of world’s arable land that is acidic. We evaluated the responses of two wheat cultivars (Triticum aestivum L.) with differential Al resistance, cv. Yecora E (Al-resistant) and cv. Dio (Al-sensitive), exposed to 0, 37, 74 and 148 μM Al for 14 days in hydroponic culture at pH 4.5. With increasing Al concentration, leaf Ca²⁺ and Mg²⁺ content decreased, as well as the effective quantum yield of photosystem II (PSII) photochemistry (Φ _PSII ), while a gradual increase in leaf membrane lipid peroxidation, Al accumulation, photoinhibition (estimated as F _v /F _m ), and PSII excitation pressure (1 ? q _p ) occurred. However, the Al-resistant cultivar with lower Al accumulation, retained larger concentrations of Ca²⁺ and Mg²⁺ in the leaves and kept a larger fraction of the PSII reaction centres (RCs) in an open configuration, i.e. a higher ratio of oxidized to reduced quinone A (Q_A), than plants of the Al-sensitive cultivar. Four times higher Al concentration in the nutrient solution was required for Al-resistant plants (148 μM Al) than for Al-sensitive (37 μM Al), in order to establish the same closed RCs. Yet, the decline in photosynthetic efficiency in the cultivar Dio was not only due to closure of PSII RCs but also to a decrease in the quantum yield of the open RCs. We suggest that Al³⁺ toxicity may be mediated by nutrient deficiency and oxidative stress, and that Al-resistance of the wheat cultivar Yecora E, may be due at least partially, from the decreased Al accumulation that resulted to decreased reactive oxygen species (ROS) formation. However, under equal internal Al accumulation (exposure Al concentration: Dio 74 μM, Yecora E 148 μM) that resulted to the same oxidative stress, the reduced PSII excitation pressure and the better PSII functioning of the Al-resistant cultivar was probably due to the larger concentrations of Ca²⁺ and Mg²⁺ in the leaves. We propose that the different sensitivities of wheat cultivars to Al³⁺ toxicity can be correlated to differences in the redox state of Q_A. Thus, chlorophyll fluorescence measurements can be a promising tool for rapid screening of Al resistance in wheat cultivars.     

Cadmium tolerance in <Emphasis Type="Italic">Schinus molle</Emphasis> trees is modulated by enhanced leaf anatomy and photosynthesis

Key message

Low concentrations of cadmium cause anatomical responses in leaf chlorenchyma enhancing Schinus molle photosynthesis and tolerance.

Abstract

This work is aimed to evaluate the effects of cadmium (Cd) on leaf anatomy and photosynthesis in Schinus molle, a species that can cope with harsh environments. Seven-month-old S. molle plants were exposed over 90 days to varying Cd concentrations (0, 10, 20, 50, 125 or 250 µM using Cd(NO₃)₂ as the Cd source). The plants were placed in vases containing washed sand and vermiculite as the substrate and nutrient solution. Throughout the experiment, the substrate was maintained at field capacity, and the nutrient solution was replaced at 15-day intervals. After 90 days, leaves were collected and processed for anatomical analysis using typical plant microtechniques. In addition, plant growth, photosynthesis, chlorophyll content and A/Ci curve were evaluated using an infrared gas analyzer. S. molle growth was not affected by Cd. Lower Cd concentrations (10 and 20 µM) resulted in greater net photosynthesis, stomatal conductance and density, Vcmax, Jmax and mesophyll thickness. However, Cd concentrations of 50 µM or greater resulted in a reduction of most of the evaluated characteristics to levels close to control. All of the tested Cd concentrations resulted in reduced chlorophyll content and stomatal size. Therefore, the effect of Cd in a tolerant species such as S. molle is concentration dependent, and at low Cd concentrations, these plants can cope with the toxicity by adjusting leaf structure and function.

     

A two [4Fe-4S]-cluster-containing ferredoxin as an alternative electron donor for 2-hydroxyglutaryl-CoA dehydratase from<Emphasis Type="Italic"> Acidaminococcus fermentans</Emphasis>

The key step in the fermentation of glutamate by Acidaminococcus fermentans is a reversible syn-elimination of water from (R)-2-hydroxyglutaryl-CoA to (E)-glutaconyl-CoA catalyzed by 2-hydroxyglutaryl-CoA dehydratase, a two-component enzyme system. The actual dehydration is mediated by component D, which contains 1.0 [4Fe-4S]²⁺ cluster, 1.0 reduced riboflavin-5′-phosphate and about 0.1 molybdenum (VI) per heterodimer. The enzyme has to be activated by the extremely oxygen-sensitive [4Fe-4S]^1+/2+-cluster-containing homodimeric component A, which generates Mo(V) by an ATP/Mg²⁺-induced one-electron transfer. Previous experiments established that the hydroquinone state of a flavodoxin (m=14.6 kDa) isolated from A. fermentans served as one-electron donor of component A, whereby the blue semiquinone is formed. Here we describe the isolation and characterization of an alternative electron donor from the same organism, a two [4Fe-4S]^1+/2+-cluster-containing ferredoxin (m=5.6 kDa) closely related to that from Clostridium acidiurici. The protein was purified to homogeneity and almost completely sequenced; the magnetically interacting [4Fe-4S] clusters were characterized by EPR and Mössbauer spectroscopy. The redox potentials of the ferredoxin were determined as ?405 mV and ?340 mV. Growth experiments with A. fermentans in the presence of different iron concentrations in the medium (7–45 μM) showed that flavodoxin is the dominant electron donor protein under iron-limiting conditions. Its concentration continuously decreased from 3.5 μmol/g protein at 7 μM Fe to 0.02 μmol/g at 45 μM Fe. In contrast, the concentration of ferredoxin increased stepwise from about 0.2 μmol/g at 7–13 μM Fe to 1.1±0.1 μmol/g at 17–45 μM Fe.     

Efficient direct plant regeneration from immature leaf roll explants of sugarcane (<Emphasis Type="Italic">Saccharum officinarum</Emphasis> L.) using polyamines and assessment of genetic fidelity by SCoT markers

A rapid and efficient method for in vitro direct plant regeneration from immature leaf roll explants of Saccharum officinarum L. (sugarcane) cv. Co 86032 was developed by the application of exogenous polyamines (PA). The effect of explant source from apical meristems and pre-culture of explants in the dark on shoot regeneration was studied. Adventitious shoot regeneration occurred on the proximal regions of immature leaf roll explants when pre-incubated in the dark for 2 wk and the regeneration response was decreased from the middle to distal end. A higher number of direct shoots (130 primary shoots explant^?1) and multiple shoots (657 secondary shoots explant^?1), were obtained with a combination of spermidine (103.27 μM), spermine (49.42 μM), and putrescine (31.04 μM) along with plant growth regulators. Shoot induction was increased up to twofold and multiplication was increased up to threefold in the medium supplemented with PA. Profuse rooting was observed in putrescine (93.12 μM), spermidine (68.84 μM), and spermine (24.71 μM), with mean number of 57 roots. A twofold increase in the number of roots was observed in medium supplemented with PA with respect to control cultures, which facilitated the successful transplantation and acclimatization process of in vitro propagated sugarcane plants. Histology and scanning electron microscopy analyses supported adventitious direct shoot regeneration from immature leaf roll explants. The genetic stability of in vitro regenerated plants was confirmed using start codon targeted polymorphism marker system.     

<Emphasis Type="Italic">In vitro</Emphasis> regeneration of <Emphasis Type="Italic">Artemisia abrotanum</Emphasis> L. by means of somatic organogenesis

The goal of this project was to regenerate Artemisia abrotanum L., Southern wormwood, by means of organogenesis from leaves. In vitro plant propagation may greatly support the molecular characterization of the medicinal qualities of A. abrotanum. Young, intact leaves were excised from mature plants and surface sterilized. Abundant callus growth, as well as shoot formation, was produced on an MS medium supplemented with 4.44 μM BA and 0.54 μM or 0.81 μM NAA. Shoots, with some residual callus, rooted equally well in MS media with 0.49 μM IBA, 0.54 μM NAA, or without hormones. Rooted plants were best acclimated in potting soil.     

Effects of zinc on the production of alcohol by <Emphasis Type="Italic">Clostridium carboxidivorans</Emphasis> P7 using model syngas

Renewable energy, including biofuels such as ethanol and butanol from syngas bioconversed by Clostridium carboxidivorans P7, has been drawing extensive attention due to the fossil energy depletion and global eco-environmental issues. Effects of zinc on the growth and metabolites of C. carboxidivorans P7 were investigated with model syngas as the carbon source. The cell concentration was doubled, the ethanol content increased 3.02-fold and the butanol content increased 7.60-fold, the hexanol content increased 44.00-fold in the medium with 280 μM Zn²⁺, when comparing with those in the control medium [Zn²⁺, (7 μM)]. Studies of the genes expression involved in the carbon fixation as well as acid and alcohol production in the medium with 280 μM Zn²⁺ indicated that fdhII was up-regulated on the second day, acs A, fdhII, bdh35 and bdh50 were up-regulated on the third day and bdh35, acsB, fdhI, fdhIII, fdhIV, buk, bdh10, bdh35, bdh40 and bdh50 were up-regulated on the fourth day. The results indicated that the increased Zn²⁺ content increased the alcohol production through increase in the gene expression of the carbon fixation and alcohol dehydrogenase.     

Diversity of low-molecular weight organic acids synthesized by <Emphasis Type="Italic">Salix</Emphasis> growing in soils characterized by different Cu,Pb and Zn concentrations

The aim of the study was to evaluate the biosynthesis and exudation of 10 low-molecular weight organic acids (LMWOAs) into the rhizosphere with a simultaneous analysis of the acid contents in the roots and leaves of 9 Salix taxa growing on two experimental areas, differing in their concentrations of copper (Cu), lead (Pb) and zinc (Zn) in the soil (Area 1—low, Area 2—high concentration). The obtained results reveal a significant difference in the phytoextraction of the tested Salix taxa for the analysed metals in both areas. The highest contents of Cu, Pb and Zn were observed for all Salix collected from Area 2, especially in S. × smithiana roots (116 ± 8.76, 87.84 ± 7.30 and 203.42 ± 14.62 mg kg^?1 DW, respectively). The results obtained in Area 2 also revealed acidification of the rhizosphere and a higher concentration of acids, mainly oxalic, malic, malonic, acetic and citric acids. Contents of oxalic, malic, acetic and citric acids increased in the roots of Salix taxa from Area 2, while in the leaves formic and succinic acids were also present. S. × smithiana was the taxon with the highest concentration of acids in the rhizosphere and roots (73.48 ± 6.77 and 49.79 ± 2.65 μM 100 g^?1 DW, respectively), while in leaves a higher content was observed for S. alba and S. viminalis ‘PR’ taxa (78.12 ± 3.95 and 71.12 ± 3.75 μM 100 g^?1 DW, respectively).     

Cd<Superscript>2+</Superscript> extrusion by P-type Cd<Superscript>2+</Superscript>-ATPase of <Emphasis Type="Italic">Staphylococcus aureus</Emphasis> 17810R via energy-dependent Cd<Superscript>2+</Superscript>/H<Superscript>+</Superscript> exchange mechanism

Cd²⁺ is highly toxic to Staphylococcus aureus since it blocks dithiols in cytoplasmic 2-oxoglutarate dehydrogenase complex (ODHC) participating in energy conservation process. However, S. aureus 17810R is Cd²⁺-resistant due to possession of cadA-coded Cd²⁺ efflux system, recognized here as P-type Cd²⁺-ATPase. This Cd²⁺ pump utilizing cellular energy—ATP, ?μ _H ⁺ (electrochemical proton potential) and respiratory protons, extrudes Cd²⁺ from cytoplasm to protect dithiols in ODHC, but the mechanism of Cd²⁺ extrusion remains unknown. Here we propose that two Cd²⁺ taken up by strain 17810R via Mn²⁺ uniporter down membrane potential (?ψ) generated during glutamate oxidation in 100 mM phosphate buffer (high P_iB) are trapped probably by high affinity sites in cytoplasmic domain of Cd²⁺-ATPase, forming SCdS. This stops Cd²⁺ transport towards dithiols in ODHC, allowing undisturbed NADH production, its oxidation and energy conservation, while ATP could change orientation of SCdS towards facing transmembrane channel. Now, increased number of P_i-dependent protons pumped electrogenically via respiratory chain and countertransported through the channel down ?ψ, extrude two trapped cytoplasmic Cd²⁺, which move to low affinity sites, being then extruded into extracellular space via ?ψ-dependent Cd²⁺/H⁺ exchange. In 1 mM phosphate buffer (low P_iB), external Cd²⁺ competing with decreased number of P_i-dependent protons, binds to ψ_s of Cd²⁺-ATPase channel, enters cytoplasm through the channel down ?ψ via Cd²⁺/Cd²⁺ exchange and blocks dithiols in ODHC. However, Mg²⁺ pretreatment preventing external Cd²⁺ countertransport through the channel down ?ψ, allowed undisturbed NADH production, its oxidation and extrusion of two cytoplasmic Cd²⁺ via Cd²⁺/H⁺ exchange, despite low P_iB.     

The influence of flask sealing on <Emphasis Type="Italic">in vitro</Emphasis> morphogenesis of eggplant (<Emphasis Type="Italic">Solanum melongena</Emphasis> L.)

The influence of flask sealing on eggplant morphogenic responses and morpho-anatomical characteristics was evaluated. Eggplant seeds from the cultivar Embu were disinfected and inoculated in MS medium supplemented with B₅ vitamins, 0.55 mM myo-inositol, 2% (w/v) sucrose, and 0.65% (w/v) agar. NAA (53.7 μM) and IAA (0.57 μM) were added to the medium to elicit morphogenic responses from cotyledon and hypocotyl explants via somatic embryogenesis and organogenesis, respectively. The plates were sealed with Micropore® 3M, Parafilm®, or polyvinyl chloride (PVC) film. The effect of glass vessel capping on morphogenesis was also evaluated for shoot apexes inoculated on medium containing half-strength MS where the capping consisted of polypropylene lids with or without two vents (0.45-μm MilliSeal® air vent) and PVC film. Leaf histological analysis and leaf bleaching from each treatment were performed. No significant differences were observed in the number of embryos and root primordia in media containing either 53.7 μM NAA or 0.57 μM IAA. However, embryogenic calli fresh weight was higher for PVC and Parafilm®. Morphogenesis from the shoot apex was influenced, except the plant height. Plants maintained in glass flasks capped with vented lids showed more vigorous growth and differentiated anatomical structures compared to plants under other treatments. This treatment resulted in more expanded leaves, wider stems, and higher dry and fresh weights. In all treatments, the number of stomata was higher in the abaxial surfaces of leaves. Our results indicate that the flasks with vents provided air exchange beneficial for plant morphogenesis.     

TDZ-induced plant regeneration in <Emphasis Type="Italic">Brassica oleracea</Emphasis> L. var. <Emphasis Type="Italic">botrytis</Emphasis>: effect of antioxidative enzyme activity and genetic stability in regenerated plantlets

The effects of various combinations of plant growth regulators on regeneration potential from seedling-derived leaf tissues of Brassica oleracea L. var. botrytis were evaluated. Callus was induced from 2-wk-old leaf explants. The explants were incubated on Gamborg’s (MSB₅) medium. The maximum frequency of callus induction (85.56%) was recorded on MSB₅ medium supplemented with 9.1 μM thidiazuron (TDZ) and 0.5 μM α-naphthaleneacetic acid (NAA). Optimum shoot induction (54.44%) was obtained on MSB₅ medium supplemented with 4.5 μM TDZ and 0.5 μM NAA. The maximum number of shoots per explant (5.33) was recorded on MSB₅ medium with 4.5 μM TDZ and 0.5 μM NAA, whereas the maximum shoot length (4.86 cm) was recorded for shoots cultured on MSB₅ medium supplemented with 4.5 μM TDZ and 5.7 μM gibberellic acid (GA₃). However, optimum root induction (71.11%) occurred on half-strength Murashige and Skoog basal medium supplemented with 4.9 μM indole-3 butyric acid (IBA). Studies on the antioxidant activity of superoxide dismutase, ascorbate peroxidase, and peroxidase in seedlings, callus, regenerated shoots, and regenerated plantlets cultured on 4.5 μM TDZ and 0.5 μM NAA medium revealed the roles of these key antioxidative enzymes in callus induction and regeneration. The genetic stability of the regenerated plantlets was assessed using inter simple sequence repeat primers. The monomorphic amplification products confirmed true-to-type in vitro regenerated plants. This in vitro regeneration method can be useful in the large-scale production of genetically uniform plants, for genetic transformation, and conservation of elite germplasm of plant species.     

<Emphasis Type="Italic">In vitro</Emphasis> sucrose concentration influences microtuber production and diosgenin content in white yam (<Emphasis Type="Italic">Dioscorea rotundata</Emphasis> Poir)

Dioscorea spp. is an important food crop in many countries and the source of the phytochemical diosgenin. Efficient microtuber production could provide source materials for farm-planting stock, for food markets, and for the production of high-diosgenin-producing cultivars. The first step in this study was optimizing the plant growth regulators for plantlet production, followed by a study of the effects of sucrose concentration on microtuber induction and diosgenin production. Significantly, more shoots (3.5) were produced at 4.65 μM (1 mg L^?1) kinetin (KIN), longer shoots (4.1 cm) were obtained at 2.46 μM (0.5 mg L^?1) indole-3-butyric acid (IBA), and root number (3.9) was significantly higher at 5.38 μM (1 mg L^?1) naphthalene acetic acid (NAA) than in other treatments. Increased sucrose concentrations in the optimized growth medium with 4.65 μM KIN and 5.38 μM NAA had significant effects on microtuber production (p < 0.01) and diosgenin content (p < 0.05). The most microtubers (6.2) were obtained with 100 g L^?1 sucrose, while those on 80 g L^?1 sucrose were the heaviest (0.7 g) and longest (7.4 mm). Microtubers formed in medium with 80 g L^?1 sucrose had significantly higher diosgenin content (3.64% [w/w]) than those in other sucrose treatments (< 2%) and was similar to that of field-grown parent tubers (3.79%). This result indicates an important role for sucrose in both microtuber growth and diosgenin production. Medium containing 4.65 μM KIN and 5.38 μM NAA is recommended for plantlet production, and medium containing 80 g L^?1 sucrose is recommended for microtuber and diosgenin production.     

Effect of cadmium uptake and heat stress on root ultrastructure, membrane damage and antioxidative response in rice seedlings

Excess cadmium (Cd²⁺) in the soil environment is taken up by plants and can cause phytotoxicity. Elevated temperatures also lead to deleterious effects on plants. Plants are very often exposed to a combination of stresses rather than a single stress. The effect of Cd²⁺ and heat stress (HS) on the growth, root ultrastructure, lipid peroxidation (MDA), hydrogen peroxide accumulation and the activities of antioxidant enzymes peroxidase (POD), catalase (CAT) and ascorbate peroxidase (APX) of rice roots from sensitive cv. DR-92 and tolerant cv. Bh-1 were investigated at 10 and 20 day of growth under controlled conditions. At day 10 under all Cd²⁺ treatments, the Cd²⁺ content between the two rice cultivars were almost similar. Application of 500 μM Cd²⁺ significantly increased metal concentrations at day 20 in the roots of rice seedlings resulting in a maximum accumulation of 44.25 μg Cd²⁺ g^-1 dry wt in cv. DR-92 and 30 μg Cd²⁺ g^-1 dry wt in cv. Bh-1 with a ~25 % decline in Relative Growth Index (RGI) in cv. DR-92. TEM studies revealed slight disorganization with cell wall ingrowths in root tissues from cv. DR-92 grown in 100 μM Cd²⁺ + HS. Uptake and accumulation of Cd²⁺ increased upon heat treatment in parenchyma, vacuoles and vascular cylinder of root tissues. Peroxidase primarily located in cell walls, the intensity being higher in sensitive cv. DR-92. Under Cd²⁺ stress alone, plants of sensitive cv. DR-92 significantly increased the H₂O₂ and MDA levels together with increased activities of the enzymes POD, CAT and APX at day 10 but remained almost stable at day 20. A strong increase in MDA levels was noted at day 20 in tolerant cv. Bh-1. Cd²⁺ + HS treatments in tolerant cv.Bh-1 led to a decreased H₂O₂ and MDA levels and decreased activities of the enzymes POD, CAT and APX. Results suggest stimulation of root antioxidant system under combination of two stresses and that heat stress seem to have a direct protective role by mitigating the effect of mild Cd²⁺ toxicity largely by enhanced Cd²⁺-MT formation contributing thereby towards the management of Cd²⁺ toxicity at cellular level that confers Cd²⁺ tolerance to rice cv. Bh-1.     

The aromatic cytokinin <Emphasis Type="Italic">meta-</Emphasis>topolin promotes in vitro propagation,shoot quality and micrografting in <Emphasis Type="Italic">Corylus colurna</Emphasis> L.

The role of 4.1 or 8.2 μM meta-topolin (mT) on shoot multiplication, rooting and ex vitro acclimatization of micropropagated Corylus colurna L., a promising non-suckering rootstock for hazelnut (Corylus avellana L.), was examined in comparison to N⁶-benzyladenine (BA), the most used cytokinin in tissue culture of Corylus spp. The influence of 8.2 μM mT and BA on photosynthetic pigments content and antioxidant enzymes activity, catalase (CAT) and guaiacol peroxidase (POD), in regenerated shoots, and on the preparation of the rootstock for micrografting was also evaluated. The highest shoot multiplication was recorded on medium containing 8.2 μM mT and an overall positive effect of mT on growth and quality of micropropagated shoots was found. The highest chlorophyll a content (1.236 mg g^?1 fresh weight, FW) and chlorophyll a/b ratio (2.48), and the lowest total carotenoids content (0.292 mg g^?1 FW) and CAT activity (25.8 μmol min^?1 mg^?1 protein) were detected after 8.2 μM mT application, while no significant differences were found in chlorophyll b content and POD activity between the two cytokinins. The best rhizogenesis response (98% for 4.1 μM and 100% for 8.2 μM mT) and ex vitro acclimatization competence (higher than 78%) were exhibited from shoots multiplied on mT. Furthermore, the multiplication of rootstock on mT allowed obtaining the highest (70%) response of successful micrografting. The present findings provide the first evidence of the successful applicability of mT in C. colurna tissue culture and development of micrografted plantlets.     

Interspecific variation in extracellular polysaccharide content and colony formation of <Emphasis Type="Italic">Microcystis</Emphasis> spp. cultured under different light intensities and temperatures

Single cells of five different Microcystis species (M. ichthyoblabe, M. viridis, M. flos-aquae, M. wesenbergii, and M. aeruginosa) were batch-cultured at different temperatures and light intensities: (a) 25 °C and 50 μmol photons m^?2 s^?1 (control culture); (b) 25 °C and 10 μmol photons m^?2 s^?1; and (c) 15 °C and 50 μmol photons m^?2 s^?1. The extracellular polysaccharide content was significantly higher in treatments b and c than in the control treatment. All Microcystis species existed as single cells under the control treatment but formed colonies in treatments b and c. All of the colonies were irregular with indistinct margins. M. ichthyoblabe, M. viridis, M. flos-aquae, and M. wesenbergii formed colonies with similar morphologies and their cells were loosely aggregated. In contrast, M. aeruginosa formed denser colonies with no distinct holes. The colony morphologies differed from the classic morphology of M. ichthyoblabe field-grown colonies but resembled that of small colonies found in Lake Taihu (Yangtze Delta Plain, China) during early spring. This indicates that field- and laboratory-grown colonies are governed by similar formation processes. We suggest that in laboratory and field environments, M. ichthyoblabe (or M. flos-aquae) colonies are representative of small colonies formed from single Microcystis cells, whereas the morphology of older colonies evolves to resemble M. wesenbergii and M. aeruginosa colonies.     

Tolerance and potential for bioaccumulation of <Emphasis Type="Italic">Alternanthera tenella</Emphasis> Colla to cadmium under in vitro conditions

High concentrations of cadmium (Cd) in the environment can threaten the local biota and one of its main sources is anthropic activities such as zinc (Zn) mining. Some plant species are able to tolerate high Cd concentrations, using anatomical and physiological strategies to avoid the absorption or accumulation of this element in their biomass. The in vitro assessment of these strategies is an efficient way to control variables external to the experiment. We aimed to investigate the anatomical and physiological changes in Alternanthera tenella exposed to Cd and its potential for accumulation in controlled microenvironmental conditions. We evaluated changes in the leaf and root anatomy, antioxidant system, and biomass of A. tenella grown in a culture medium containing increasing Cd concentrations (0, 50, 100, and 150 μM), in the presence of 1500 μM Zn. Alternanthera tenella was able to accumulate Cd and Zn and these elements competed for absorption by the species. Increase in Cd in the medium led to a progressive thickening of the root tissues, which was also observed on the leaves, albeit only at concentrations below 100 μM Cd. The concentration of 150 μM Cd was toxic to the leaf tissue and stimulated the formation of hydrogen peroxide, interfering with the antioxidant system and reducing plant biomass and the chlorophyll levels. Therefore, in vitro cultivated A. tenella can accumulate Cd and tolerate up to 100 μM Cd by modifying its anatomy and physiology in order to cope with Cd stress.     

<Emphasis Type="Italic">In vitro</Emphasis> growth profile and comparative leaf anatomy of the C<Subscript>3</Subscript>–C<Subscript>4</Subscript> intermediate plant <Emphasis Type="Italic">Mollugo nudicaulis</Emphasis> Lam.

Mollugo nudicaulis Lam., commonly known as John’s folly or naked-stem carpetweed, is an ephemeral species of tropical regions. The plant is ideal to study the eco-physiological adaptations of C₃–C₄ intermediate plants. In the present report, in vitro growth profiling of the plant and comparative leaf anatomy under in vitro and ex vitro conditions were studied. In vitro propagation of the plant was carried out on Murashige and Skoog (MS) basal medium augmented with additives and solidified with 0.8% (w/v) agar-agar or 0.16% (w/v) Phytagel?. The concentration of plant growth regulators (PGRs) in the basal medium was optimized for callus induction, callus proliferation, shoot regeneration, and in vitro rooting. The optimum callus induction was obtained from M. nudicaulis seedling hypocotyls. The highest regeneration induction of about 88% or nearly 41 shoots with about 142 leaves per culture vessel was observed from friable callus on MS basal medium solidified with Phytagel? and containing 4.44 μM 6-benzylaminopurine, 4.65 μM kinetin, 2.69 μM naphthaleneacetic acid, and 0.91 μM thidiazuron. In leaf anatomy, differences related to photosynthetic tissue organization were observed in leaves of in vitro and ex vitro plants, which indicated that changes in the environment affected the anatomy of subsequent leaves in plants. This is the first report of an efficient micropropagation protocol for M. nudicaulis, using an indirect organogenesis method. Efforts were made to optimize the concentrations of various PGRs and organic compounds for in vitro growth of regenerated shoots.     

Effects of Combined Abiotic Stresses on Growth,Trace Element Accumulation,and Phytohormone Regulation in Two Halophytic Species

Trace element contamination of lands is a serious environmental problem that limits yield and threatens human health. To study the combined effect of high salinity and toxic levels of trace elements on halophytes, the performance of two marsh species, Atriplex halimus and Suaeda fruticosa, grown for 1 month with an irrigation solution supplemented with 200 mM NaCl and 400 μM Cd²⁺ or 400 μM Cu²⁺ was evaluated. The effect of the combined stress conditions on hormone signaling was also assessed. Biomass production and chlorophyll content decreased under Cd²⁺ stress in both species, whereas Cu²⁺ had a lower impact on plant performance. The different plant sensibilities to the two trace elements assayed indicate that each metal has a different effect on plants. Furthermore, the deleterious effect of metal toxicity was alleviated when NaCl was added to the irrigation solution, demonstrating that NaCl improves plant performance and tolerance of halophytic species to cope with trace element intoxication. Results show that both species accumulated important quantities of Cd²⁺ and Cu²⁺ in roots (Cd²⁺: 2,690–3,130 μg g^?1 DW and Cu²⁺: 2,070–2,770 μg g^?1 DW); this finding allows us to classify these species among the hyperaccumulator plants. Cd²⁺ and Cu²⁺ differently affected endogenous phytohormone contents in both species. Data suggest an essential involvement of roots on the regulation of tolerance to trace elements. Therefore, indole-3-acetic acid levels increased in roots of both species irrigated with high levels of Cd²⁺, which suggests that the auxin may stimulate root promotion and growth under these stress conditions. Other compounds, classically considered as “stress hormones” showed very different patterns of accumulation. Whereas, salicylic acid (SA) levels in roots and leaves increased in response to Cd²⁺, root contents of jasmonic acid (JA), and abscisic acid (ABA) decreased. In leaves, the rambling pattern of accumulation observed for JA and ABA suggested the lack of a specific role in regulation against trace element toxicity. Together, data suggest that SA could act as a specific signal that detects trace element toxicity, whereas JA and ABA promote general responses against abiotic stress.     

Analysis of the effect of plant growth regulators and organic elicitors on antibacterial activity of <Emphasis Type="Italic">Eucomis autumnalis</Emphasis> and <Emphasis Type="Italic">Drimia robusta</Emphasis> ex vitro-grown biomass

The effects of plant growth regulators (PGRs) and organic elicitors (OEs) on in vitro propagation of Eucomis autumnalis was established. Three-year-old ex vitro grown plants from organogenesis of E. autumnalis and somatic embryogenesis (previously reported protocol) of Drimia robusta were investigated for antibacterial activity. In vitro propagation from leaf explants of E. autumnalis was established using different PGRs and OE treatments for mass propagation, biomass production and bioactivity analysis to supplement the use of wild plant material. Prolific shoots (16.0?±?0.94 shoots per explant) were obtained with MS (Murashige and Skoog in Physiol Plant 15:473–497, 1962) medium containing 100 mg l^?1 haemoglobin (HB), 10 µM benzyladenine (BA) and 2 µM naphthaleneacetic acid (NAA). The shoots were rooted effectively with a combination of 2.5 µM indole-3-acetic acid and 5.0 µM indole-3-butyric acid. The plantlets were successfully acclimatized in a vermiculite-soil mixture (1:1 v/v) in the greenhouse. Three-year-old ex vitro-grown E. autumnalis and D. robusta plants derived via organogenesis and somatic embryogenesis respectively exhibited antibacterial activity and varied with PGR and OE treatments, plant parts and bacteria. The leaves of E. autumnalis ex vitro-derived from a combination of HB, BA and NAA followed by the individual treatments of BA and HB gave the best antibacterial activities (<?1 mg ml^?1: minimum inhibitory concentration from 0.098 to 0.78 mg ml^?1) against all tested pathogenic bacteria (Bacillus subtilis, Enterococcus faecalis, Micrococcus luteus, Staphylococcus aureus, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa). The bulbs of D. robusta ex vitro-derived from solid culture with 10 µM picloram, 1 µM thidiazuron and 20 µM glutamine exhibited good antibacterial activity against E. faecalis, M. luteus and S. aureus when compared with other treatments and mother plants. The ex vitro-grown E. autumnalis and D. robusta biomass produced with PGRs along with OE treatments confirmed a good potent bioresource and can be used as antibacterial agents. The in vitro plant regeneration of E. autumnalis and D. robusta protocols and ex vitro plants could be used for conservation strategies, bioactivity and traditional medicinal use.     

Genotype-dependent effects of phosphorus supply on physiological and biochemical responses to Al-stress in cultivated and Tibetan wild barley

Aluminium (Al) toxicity and phosphorus (P) deficiency often co-exist in acidic soils and limit plant growth and crop production. To investigate the alleviating effects of different levels of phosphorus on Al stress, greenhouse hydroponic experiments were conducted using two contrasting Tibetan wild barley genotypes XZ16 and XZ61 of Al tolerant and sensitive, respectively, and Al tolerant cv. Dayton. The results showed that Al stress induced reduction in P accumulation in plants; and stem and leaf P concentrations of the three genotypes, except of XZ16 under HP + Al (100 µM Al with high level of 360 µM P) which was close to the control level. XZ16 recorded significantly higher P accumulation in plants, compared with XZ61 and Dayton, and P concentrations in leaves under Al stress, and in stems under NP + Al (100 µM Al with normal level of 180 µM P) and HP + Al. Meanwhile, H⁺-, Ca²⁺Mg²⁺-, and Total- ATPase activities in XZ16 and Dayton under Al stress were markedly higher than in XZ61. Normal or high level of P under Al stress could relieve Al stress as enhanced plant biomass, with increased photosystem II photochemistry (Fv/Fm) and P content, relative to the low level of 90 µM P. Compared with XZ61, addition of high P concentration for XZ16 significantly increased the values of Gs and Tr, with higher root GPX and H⁺-ATPase activities, and such nutrient elements as P, Mg and Ca in stems and leaves, and induced more malate secretion, but less MDA accumulation.