Rapid in vitro propagation of Eclipta alba (L.) Hassk. through high frequency axillary shoot proliferation

An efficient protocol has been developed for rapid in vitro propagation of Eclipta alba L. (Asteraceae) through axillary bud multiplication. Murashige and Skoog (MS) medium supplemented with BA (10 M) was found to be most effective in breaking bud dormancy. An average number of 23 ± 0.57 shoots per explant was recorded after 30 days. Culture of node segments on fresh medium with lower concentration of BA (2 M) enhanced the multiplication rate. A maximum of 79 ± 1.90 mean number of shoots were obtained after three subcultures without any decline in multiplication rate. The regenerated microshoots showed the most efficient rooting on half strength MS medium augmented with 0.5 M IBA. Plantlets went through a hardening phase prior to ex vitro transfer and established in earthen pots containing garden soil; survival of about 90%. The established plants were uniform and exhibited morphological characters identical to mother plants.     

In vitro propagation of Cuphea procumbens Orteg. and Evaluation of genetic fidelity in plantlets using RAPD marker

An efficient, rapid and reproducible plant regeneration protocol was successfully developed for Cuphea procumbens Orteg. using cotyledonary node explants excised from 15?days old aseptic seedlings. A range of cytokinins were investigated for multiple shoot regeneration. Of the three cytokinins, 6-benzyladenine (BA), Kinetin (Kin) and 2-isopentenyl adenine (2-iP) evaluated as supplement to Murashige and Skoog (MS) medium, BA at a concentration of 2.5???M was effective in inducing multiple shoots. The highest number of multiple shoots (9.33?±?0.60) and maximum average shoot length (4.16?±?0.44?cm) was standardized on MS medium supplemented with 2.5???M BA alongwith 0.5???M NAA. Addition of 200?mg/l Casein hydrolysate (CH) to the shoot induction medium enhanced the growth of regenerants. Rooting of in vitro regenerated shoots was best achieved on 1/2 strength MS medium. The in vitro raised plantlets with well developed shoots and roots were hardened, successfully established in earthen pots containing garden soil and maintained in greenhouse with 80% survival rate. Randomly Amplified Polymorphic DNA (RAPD) markers were used to evaluate the genetic stability among in vitro regenerated progenies. All RAPD profiles from the micropropagated plants were monomorphic and similar to control plant. These results suggests that the culture conditions used for the axillary bud proliferation are appropriate for clonal propagation of this medicinally important plant as they do not appear to interfere with genetic integrity of in vitro regenerated plants. The described method can be successfully employed for large-scale multiplication and in vitro conservation of C. procumbens.     

Evaluation of antioxidant and anticancer activities of chemical constituents of the Saururus chinensis root extracts

Evaluation of antioxidant and anticancer activities were screened by various Saururus chinensis root extracts. Four solvents (ethyl acetate, methanol, ethanol, and water) extracts were investigated for their total flavonoids, phenol contents and their antioxidant activity of DPPH (2,2-diphenyl-1-picrylhydrazyl), NO (nitric oxide), H₂O₂ (hydrogen peroxide), ABTS 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonicacid)diammonium assays, FRAP (ferric reducing ability of plasma) assays and anticancer activity. The total phenolic and flavonoid content of extracts were determined by using FC (Folin–Ciocalteu) and AlCl₃ colorimetric assay method. Total flavonoid content in these plants ranged from 24.7 to 72.1 mg g^?1 and amount of free phenolic compounds was between 11.2 and 67.1 mg g^?1 extract. The all extracts have significant levels of phenolics and flavonoids content. Anticancer activity was screened for MCF-7 breast cancer cell line. Ethanol extract shows significant of antioxidant activity and water extract shows significant of anticancer activity compared with standard (BHT) butylated hydroxy toluene. These ethanol and water extracts could be considered as a natural source for using antioxidant, and anticancer agents compared to commercial available synthetic drugs.     

Regulation of Primary Metabolism in Response to Low Oxygen Availability as Revealed by Carbon and Nitrogen Isotope Redistribution

Based on enzyme activity assays and metabolic responses to waterlogging of the legume Lotus japonicus, it was previously suggested that, during hypoxia, the tricarboxylic acid cycle switches to a noncyclic operation mode. Hypotheses were postulated to explain the alternative metabolic pathways involved, but as yet, a direct analysis of the relative redistribution of label through the corresponding pathways was not made. Here, we describe the use of stable isotope-labeling experiments for studying metabolism under hypoxia using wild-type roots of the crop legume soybean (Glycine max). [¹³C]Pyruvate labeling was performed to compare metabolism through the tricarboxylic acid cycle, fermentation, alanine metabolism, and the γ-aminobutyric acid shunt, while [¹³C]glutamate and [¹⁵N]ammonium labeling were performed to address the metabolism via glutamate to succinate. Following these labelings, the time course for the redistribution of the ¹³C/¹⁵N label throughout the metabolic network was evaluated with gas chromatography-time of flight-mass spectrometry. Our combined labeling data suggest the inhibition of the tricarboxylic acid cycle enzyme succinate dehydrogenase, also known as complex II of the mitochondrial electron transport chain, providing support for the bifurcation of the cycle and the down-regulation of the rate of respiration measured during hypoxic stress. Moreover, up-regulation of the γ-aminobutyric acid shunt and alanine metabolism explained the accumulation of succinate and alanine during hypoxia.Plants are sessile, unable to relocate when exposed to diverse environmental and seasonal stimuli, and hence must be able to respond rapidly to survive stress conditions. Flooding or waterlogging of the soil is a common environmental condition that can greatly affect crop production and quality by blocking the entry of oxygen into the soil so that roots and other belowground organs cannot maintain respiration. In recent decades, the number of extreme floodings has strongly increased, which is especially tragic because most arable land worldwide is located in regions that are threatened by regular flooding events (Voesenek and Bailey-Serres, 2015).In plant heterotrophic tissues, respiratory metabolism is composed of various pathways, including glycolysis, the mitochondrial tricarboxylic acid cycle, and the mitochondrial electron transport chain. Under normal conditions, the conversion of Glc to pyruvate in the cytosol involves an initial input of ATP and produces the reduced cofactor NADH. The reactions of the tricarboxylic acid cycle occur within the mitochondrial matrix and lead to the complete oxidation of pyruvate, moving electrons from organic acids to the oxidized redox cofactors NAD⁺ and FAD, forming the reducing equivalents NADH and FADH₂ and concomitantly releasing carbon dioxide (Tovar-Méndez et al., 2003; Millar et al., 2011). Finally, the reduced cofactors generated during glycolysis and the tricarboxylic acid cycle are subsequently oxidized by the mitochondrial electron transport chain to fuel ATP synthesis by a process known as oxidative phosphorylation (Fernie et al., 2004; Plaxton and Podesta, 2006). The tricarboxylic acid cycle turnover rate depends greatly on the rate of NADH reoxidation by the mitochondrial electron transport chain and on the cellular rate of ATP utilization (Plaxton and Podesta, 2006). Besides supporting ATP synthesis, the reactions of the tricarboxylic acid cycle also contribute to the production of key metabolic intermediates for use in many other fundamental biosynthetic processes elsewhere in the cell (Fernie et al., 2004; Sweetlove et al., 2010; van Dongen et al., 2011; Araújo et al., 2012). Nevertheless, the control and regulation of the carbon flux through the tricarboxylic acid cycle are still poorly understood in plants, and noncyclic modes have been described to operate under certain circumstances (Rocha et al., 2010; Sweetlove et al., 2010; Araújo et al., 2012).Upon hypoxia, respiratory energy (ATP) production via oxidative phosphorylation by the mitochondrial electron transport chain goes down. To compensate for this, the glycolytic flux increases and Glc is consumed faster in an attempt to produce ATP via the glycolytic pathway, a process known as the Pasteur effect. To survive short-term hypoxia during flooding or waterlogging, plants must generate sufficient ATP and regenerate NADP⁺ and NAD⁺, which are required for glycolysis (Narsai et al., 2011; van Dongen et al., 2011). In addition to the accumulation of ethanol and lactate in oxygen-deprived plant tissues, metabolites such as Ala, succinate, and γ-aminobutyric acid (GABA) have also been shown to accumulate (Sousa and Sodek, 2003; Kreuzwieser et al., 2009; van Dongen et al., 2009; Rocha et al., 2010; Zabalza et al., 2011), although hardly anything is known about the fate of these products of hypoxic metabolism. However, the relative abundance of these products of hypoxic metabolism varies between plant species, genotypes, and tissues and can change throughout the course of oxygen limitation stress as well (Narsai et al., 2011).A model describing metabolic changes during hypoxia has been described previously for waterlogged roots of the highly flood-tolerant model crop legume Lotus japonicus (Rocha et al., 2010): upon waterlogging, the rate of pyruvate production is enhanced due to the activation of glycolysis (Pasteur effect) and the concomitant production of ATP via substrate-level phosphorylation. At the same time, the fermentation pathway is activated with the accumulation of lactate via lactate dehydrogenase and ethanol via two subsequent reactions catalyzed by pyruvate decarboxylase and alcohol dehydrogenase (Tadege et al., 1999). The amount of pyruvate produced can be reduced via alanine aminotransferease (AlaAT), which catalyzes the reversible reaction interconverting pyruvate and Glu to Ala and 2-oxoglutarate (2OG). Concomitantly, 2OG was suggested to reenter the tricarboxylic acid cycle to be used to produce another ATP and also succinate, which accumulates in the cell (Rocha et al., 2010). This Ala pathway provides a means for the role of Ala accumulation during hypoxia via reorganization of the tricarboxylic acid cycle. Furthermore, given that the use of this strategy prevents pyruvate accumulation, the continued operation of glycolysis during waterlogging can occur.It should be noted, however, that measurements of metabolite levels alone do not provide information about the actual activity of the metabolic pathways involved. Furthermore, the previous studies did not reveal which enzymes of the tricarboxylic acid cycle change their activity that leads to reorganization of the tricarboxylic acid cycle. To overcome this, analysis of metabolism using isotope-labeled substrates has proven to be essential for understanding the control and regulation of metabolic networks, and it has often been observed that significant changes in carbon flow are sometimes associated with only small adjustments in metabolite abundance (Schwender et al., 2004; Ratcliffe and Shachar-Hill, 2006). Metabolomics studies that require extensive metabolite labeling utilize uniformly labeled stable isotope tracers. Alternatively, detailed analysis of central carbon metabolism can make use of positional labeling as well. Following the extraction of labeled metabolites, the ¹³C label redistribution is measured usually with NMR or gas chromatography-mass spectrometry methods (Jorge et al., 2015). Schwender and Ohlrogge (2002) used both labeling approaches to investigate embryo development in Brassica napus seeds. While uniformly labeled [¹³C₆]Glc and [¹³C₁₂]Suc were applied to determine the metabolic flux through the major pathways of carbon metabolism, positionally labeled [1,2-¹³C]Glc was used to specifically outline the glycolytic/oxidative pentose phosphate pathway network during embryo development (Schwender and Ohlrogge, 2002). Gas chromatography-mass spectrometry analysis was used in this study to evaluate the ¹³C enrichment and isotopomer composition. In earlier studies of hypoxic metabolism, positionally labeled [1-¹³C]Glc was used to specifically investigate energy metabolism and pH regulation in hypoxic maize (Zea mays) root tips (Roberts et al., 1992; Edwards et al., 1998).In this study, we performed stable isotope labeling experiments using wild-type soybean (Glycine max) roots in order to better understand the dynamics of metabolism in operation in plant cells under hypoxic conditions. For this, we used fully labeled ¹³C and ¹⁵N tracers rather than positional labeling, as this allowed us to cover a broad view of the central carbon and nitrogen metabolic network. The labeling pattern of metabolites was subsequently measured with gas chromatography-time of flight-mass spectrometry (GC-TOF-MS). Our studies confirm the activity of Ala metabolism while revealing the parallel activity of the GABA shunt. The results provide evidence that the bifurcation of the tricarboxylic acid cycle results from the inhibition of the tricarboxylic acid cycle enzyme succinate dehydrogenase (SDH), also known as complex II of the mitochondrial electron transport chain (mETC).     

Unique Characteristics of Recombinant Hybrid Manganese Superoxide Dismutase from <Emphasis Type="Italic">Staphylococcus equorum and S. saprophyticus</Emphasis>

A recombinant hybrid of manganese dependent-superoxide dismutase of Staphylococcus equorum and S. saprophyticus has successfully been overexpressed in Escherichia coli BL21(DE3), purified, and characterized. The recombinant enzyme suffered from degradation and aggregation upon storage at ?20 °C, but not at room temperature nor in cold. Chromatographic analysis in a size exclusion column suggested the occurrence of dimeric form, which has been reported to contribute in maintaining the stability of the enzyme. Effect of monovalent (Na⁺, K⁺), divalent (Ca²⁺, Mg²⁺), multivalent (Mn^2+/4+, Zn^2+/4+) cations and anions (Cl^?, SO₄ ^2?) to the enzyme stability or dimeric state depended on type of cation or anion, its concentration, and pH. However, tremendous effect was observed with 50 mM ZnSO₄, in which thermostability of both the dimer and monomer was increased. Similar situation was not observed with MnSO₄, and its presence was detrimental at 200 mM. Finally, chelating agent appeared to destabilize the dimer around neutral pH and dissociate it at basic pH. The monomer remained stable upon addition of ethylene diamine tetraacetic acid. Here we reported unique characteristics and stability of manganese dependent-superoxide dismutase from S. equorum/saprophyticus.     

Direct plant regeneration from encapsulated nodal segments of Vitex negundo

An efficient protocol for encapsulation of nodal segments of Vitex negundo L. has been developed for the production of non-embryogenic synthetic seeds. The encapsulations of nodal segments were significantly affected by the concentrations of sodium alginate and calcium chloride. A 3 % Na₂-alginate with 100 mM CaCl₂ has been found to be optimum concentration for the production of uniform synthetic seed. For germination, the synseeds were cultured on Murashige and Skoog (MS) basal medium supplemented with kinetin (KIN) and α-naphthalene acetic acid (NAA) either singly or in various combinations. MS medium containing 2.5 μM KIN in combination with 1.0 μM NAA was found to be the optimum for maximum (92.6 ± 3.71 %) plantlet conversion frequency. Well developed regenerated plantlets were hardened, acclimatized and established in field, where they grew well without any detectable variation.     

Two new protease-inhibiting glycosphingolipids from Buddleja crispa

Crispins A (1) and B (2), two new glycosphingolipids, were isolated from the whole plant Buddleja crispa, along with three known compounds: alpha-amyrin, linoleic acid, and stigmasterol. Their structures were elucidated by chemical and spectroscopic techniques. Both 1 and 2 showed significant inhibitory activity against alpha-chymotrypsin in a concentration-dependent manner.