Abdulrahman A. Alatar

Phytotoxic hazards of NiO-nanoparticles in tomato: A study on mechanism of cell death

Nickel oxide nanoparticles (NiO-NPs) in the concentration range of 0.025-2.0mg/ml were examined for the induction of oxidative stress, mitochondrial dysfunction, apoptosis/necrosis in tomato seedling roots, as an in vivo model for nanotoxicity assessment in plants. Compared to the control, catalase (CAT), glutathione (GSH), superoxide dismutase (SOD) and lipid peroxidation (LPO) in 2.0mg/ml NiO-NPs treatments exhibited 6.8, 3.7, 1.7 and 2.6-fold higher activities of antioxidative enzymes. At 2.0mg/ml, 122% and 125.4% increase in intracellular reactive oxygen species (ROS) and mitochondrial membrane potential (ΔΨm) of seedling roots confirmed the oxidative stress and mitochondrial dysfunction. Comet assay exhibited a significant increase in the number of apoptotic (21.8%) and necrotic (24.0%) cells in 2.0mg/ml treatment groups vis-á-vis in control 7% apoptotic and 9.6% of necrotic cells were observed. Flow cytometric analysis revealed 65.7% of apoptotic/necrotic cell populations and 2.14-fold higher caspase-3 like protease activity were recorded in 2.0mg/ml treatment groups. Ultrastructure analysis revealed NiO-NPs translocation, nuclear condensation, abundance in peroxisomes and degenerated mitochondrial cristae. The dissolution of Ni ions from NiO-NPs signifies its potential to induce cell death presumably by Ni ions, triggering the mitochondrial dependent intrinsic apoptotic pathway.

Assessment of Genetic Fidelity in Rauvolfia serpentina Plantlets Grown from Synthetic (Encapsulated) Seeds Following in Vitro Storage at 4 °C

An efficient method was developed for plant regeneration and establishment from alginate encapsulated synthetic seeds of Rauvolfia serpentina. Synthetic seeds were produced using in vitro proliferated microshoots upon complexation of 3% sodium alginate prepared in Llyod and McCown woody plant medium (WPM) and 100 mM calcium chloride. Re-growth ability of encapsulated nodal segments was evaluated after storage at 4 °C for 0, 1, 2, 4, 6 and 8 weeks and compared with non-encapsulated buds. Effects of different media viz; Murashige and Skoog medium; Lloyd and McCown woody Plant medium, Gamborg’s B5 medium and Schenk and Hildebrandt medium was also investigated for conversion into plantlets. The maximum frequency of conversion into plantlets from encapsulated nodal segments stored at 4 °C for 4 weeks was achieved on woody plant medium supplement with 5.0 μM BA and 1.0 μM NAA. Rooting in plantlets was achieved in half-strength Murashige and Skoog liquid medium containing 0.5 μM indole-3-acetic acid (IAA) on filter paper bridges. Plantlets obtained from stored synseeds were hardened, established successfully ex vitro and were morphologically similar to each other as well as their mother plant. The genetic fidelity of Rauvolfia clones raised from synthetic seeds following four weeks of storage at 4 °C were assessed by using random amplified polymorphic DNA (RAPD) and inter-simple sequence repeat (ISSR) markers. All the RAPD and ISSR profiles from generated plantlets were monomorphic and comparable to the mother plant, which confirms the genetic stability among the clones. This synseed protocol could be useful for establishing a particular system for conservation, short-term storage and production of genetically identical and stable plants before it is released for commercial purposes.

Antigenic role of the adaptive immune response to d-ribose glycated LDL in diabetes, atherosclerosis and diabetes atherosclerotic patients.

AIMS Glycation of proteins leads to the formation of advanced glycation end products (AGEs, which have significant role in the pathophysiology of diabetes complications. d-ribose appears to be the most reactive among the naturally occurring sugars and contribute significantly to the generation of AGEs. Glycation also results in the generation of free radicals causing structural modification which leads to the generation of neoantigenic epitopes. The aim of the present study was to investigate whether LDL modification results in auto-antibodies formation against its glycated conformer in diabetes and atherosclerosis patients. METHODS The binding characteristics of circulating auto-antibodies in patients against native and modified LDL were assessed. T2D (n=105), ATH (n=106) and T2D-ATH patients (n=72) were examined by direct binding ELISA as well as inhibition ELISA, compared with healthy age-matched controls (n=50). Furthermore, ketoamine moieties, HMF and carbonyl content were also estimated in these patients and healthy subjects. KEY FINDINGS High degree of specific binding was observed by 41.91% of T2D, 54.72% of ATH and 70.83% T2D-ATH patients sera towards d-ribose glycated LDL, in comparison to its native analog (P<0.05). Normal human sera showed negligible binding with either antigen. Competitive inhibition ELISA reiterates the direct binding results. The higher concentration of HMF, ketoamine and carbonyl content was observed in patients sera than healthy subjects. SIGNIFICANCE LDL glycation results in structural perturbation causing generation of neoantigenic epitopes that are better antigens for antibodies in T2D, ATH and T2D-ATH patients where T2D-ATH subjects showed higher prevalence in auto-antibodies against ribosylated LDL.

Thidiazuron induced efficient in vitro multiplication and ex vitro conservation of Rauvolfia serpentina – a potent antihypertensive drug producing plant

An efficient system for in vitro propagation of the endangered medicinal plant Rauvolfia serpentina has been developed. Proliferation of shoots was achieved from nodal segment explants, excised from a field grown plants on Murashige and Skoog (MS) medium supplemented with thidiazuron (TDZ) (0.1–2.5 μmol/L) although with low regeneration response and few number of shoots per explant. Greater number of shoots was achieved from nodal explants pretreated with higher concentrations of TDZ (5–100 μmol/L) in liquid MS medium for different time periods (4, 8, 12 and 16 days), followed by their transfer on a growth regulator-free medium. The highest response in terms of percent regeneration (90%), average number of shoots/explant (23.17 ± 2.15) and maximum shoot length (5.3 cm  ±  0.83 cm) were achieved by pretreating the nodal explants with 50 μmol/L TDZ for 8 days. Rooting was achieved in MS medium supplemented with 0.5 μmol/L indole-3-butyric acid with an average of 5.50 ± 0.92 roots per microshoot. The rooted plantlets were transferred to soil, where 90% of them grew well and exhibited a normal development. Chlorophyll a and b, carotenoids, net photosynthetic rate and antioxidative enzymes, viz., superoxide dismutase and catalase activities were measured in leaf tissues during ex vitro acclimatization at 0, 7, 14, 21 and 28 days. Most of the activities showed a decreasing trend but subsequently increased after 7 days of acclimatization. These changes suggested the adjustment of the in vitro propagated plantlets to ex vitro conditions.

Soil characteristics influence the radionuclide uptake of different plant species

The key point of food plant agriculture is how to regulate the harmonious relationship between the soil and the plant environment. This study deals with radionuclide uptake by two food plant and two fruit tree species in relation to the geochemical characteristics of the soil. Uranium and thorium content was determined in coastal black sand and inland cultivated soils. Four commonly cultivated species Eruca sativa, Lycopersicon esculentum, Psidium guajava and Mangifera indica were investigated. Physical and chemical properties of the soil were analysed in relation to uranium and thorium uptake by plants. The results revealed the ability of plants to accumulate uranium and thorium in their edible portions. The absorbed radionuclides were positively correlated with their concentrations in the soil and the geochemical characteristics of the soil. The transfer of radioactive elements from soil to plant is a complex process that can be regulated by controlling the geochemical characteristics of the soil, including pH, clay, silt and organic matter content that reduce the bioavailability of soil radionuclides to plants, and in turn reduce the risks of biota and human exposure to radionuclide contamination.

An efficient system for in vitro multiplication of Ocimum basilicum through node culture

An efficient in vitro micropropagation system was developed for direct shoot growth of Ocimum basilicum , an important medicinal plant, using nodal explants. The excised nodes were cultured on Murashige and Skoog (MS) medium containing two plant growth regulators (6-benzyladenine and 2isopentanyl adenine) with various combinations and concentrations for the study of shoot induction. Addition of L-glutamine was essential to induce sprouting of axillary buds. The best condition for shoot growth was with 6-benzyladenine (BA) 10.0 µM + L-glutamine 30 mg/L in MS medium. The optimum shoot formation frequency was 100% and about 13.4 ± 1.80 shoots were obtained from each explant after 8 weeks of culture. Shoots (more than 4 cm) were rooted most effectively in 5.0 µM indole-3-butyric acid (IBA) supplemented with half-strength MS medium. The plantlets thus obtained hardened off and were transferred to natural soil, where they grew well and attained sexual maturity.

Hazards of low dose flame-retardants (BDE-47 and BDE-32): Influence on transcriptome regulation and cell death in human liver cells.

We have evaluated the in vitro low dose hepatotoxic effects of two flame-retardants (BDE-47 and BDE-32) in HepG2 cells. Both congeners declined the viability of cells in MTT and NRU cell viability assays. Higher level of intracellular reactive oxygen species (ROS) and dysfunction of mitochondrial membrane potential (ΔΨm) were observed in the treated cells. Comet assay data confirmed the DNA damaging potential of both congeners. BDE-47 exposure results in the appearance of subG1 apoptotic peak (30.1%) at 100 nM, while BDE-32 arrested the cells in G2/M phase. Among the set of 84 genes, BDE-47 induces downregulation of majority of mRNA transcripts, whilst BDE-32 showed differential expression of transcripts in HepG2. The ultrastructural analysis revealed mitochondrial swelling and degeneration of cristae in BDE-47 and BDE-32 treated cells. Overall our data demonstrated the hepatotoxic potential of both congeners via alteration of vital cellular pathways.

The battle of two ions: Ca2+ signalling against Na+ stress

Soil salinity adversely affects plant growth, crop yield and the composition of ecosystems. Salinity stress impacts plants by combined effects of Na+ toxicity and osmotic perturbation. Plants have evolved elaborate mechanisms to counteract the detrimental consequences of salinity. Here we reflect on recent advances in our understanding of plant salt tolerance mechanisms. We discuss the embedding of the salt tolerance-mediating SOS pathway in plant hormonal and developmental adaptation. Moreover, we review newly accumulating evidence indicating a crucial role of a transpiration-dependent salinity tolerance pathway, that is centred around the function of the NADPH oxidase RBOHF and its role in endodermal and Casparian strip differentiation. Together, these data suggest a unifying and coordinating role for Ca2+ signalling in combating salinity stress at the cellular and organismal level.

ISSR marker‐based detection of genomic stability in Cassia occidentalis L. plantlets derived from somatic embryogenesis

An efficient in vitro technology has been designed for mass multiplication of Cassia occidentalis (coffee senna) through somatic embryogenesis. Genetic stability of both regenerants and mother plant was evaluated. Embryogenic calli were produced on Murashige and Skoog (MS) medium supplemented with 20.0 μM 2,4‐dichlorophenoxy acetic acid (2,4‐D). Induction of somatic embryos occurred after transfer of calli to medium with reduced concentration of 2,4‐D (10.0 μM) fortified with 1.0 μM abscisic acid (ABA). Subculturing of these embryos onto the maturation medium (1.5 μM 6‐benzyladenine + 1.0 μM ABA + 0.3 μM α‐naphthalene acetic acid) favored progression of the embryos through torpedo, heart‐shaped, and cotyledonary stages; one‐half MS medium was considered the best for conversion of cotyledonary stage embryos to young plantlets. The plantlets were acclimatized to autoclaved soil rite, after which they were transferred to the green house. Among the survived plantlets, 10 plants for each primer were randomly selected for inter‐simple sequence repeat (ISSR) analysis. Of the 10 primers tested, 5 produced reproducible and monomorphic bands, 2 led to minor variation with the appearance of unique bands, and the remaining 3 did not show any banding pattern. The majority of the regenerants had similar characteristics to the donor plant, suggesting genetic homogeneity of in vitro raised plants.

Production of Plant Secondary Metabolites: Examples, Tips and Suggestions for Biotechnologists

Plants are sessile organisms and, in order to defend themselves against exogenous (a)biotic constraints, they synthesize an array of secondary metabolites which have important physiological and ecological effects. Plant secondary metabolites can be classified into four major classes: terpenoids, phenolic compounds, alkaloids and sulphur-containing compounds. These phytochemicals can be antimicrobial, act as attractants/repellents, or as deterrents against herbivores. The synthesis of such a rich variety of phytochemicals is also observed in undifferentiated plant cells under laboratory conditions and can be further induced with elicitors or by feeding precursors. In this review, we discuss the recent literature on the production of representatives of three plant secondary metabolite classes: artemisinin (a sesquiterpene), lignans (phenolic compounds) and caffeine (an alkaloid). Their respective production in well-known plants, i.e., Artemisia, Coffea arabica L., as well as neglected species, like the fibre-producing plant Urtica dioica L., will be surveyed. The production of artemisinin and caffeine in heterologous hosts will also be discussed. Additionally, metabolic engineering strategies to increase the bioactivity and stability of plant secondary metabolites will be surveyed, by focusing on glycosyltransferases (GTs). We end our review by proposing strategies to enhance the production of plant secondary metabolites in cell cultures by inducing cell wall modifications with chemicals/drugs, or with altered concentrations of the micronutrient boron and the quasi-essential element silicon.