Facilitation by Haloxylon persicum Shrubs Enhances Density and Richness of Soil Seed Bank of Annual Plants in a Hyper-Arid Ecosystem.

The soil seed bank is an essential functional component of plant communities. In arid ecosystems, the island-like distribution of shrubs influences the spatial distribution of the soil seed bank. Very little is known about seed banks in deserts of the Middle East. The present study aimed to evaluate the facilitative effects of Haloxylon persicum shrubs on the soil seed bank of annual plants in a sandy desert region in northwestern Saudi Arabia during two consecutive growing seasons (2017-2018 and 2018-2019) with contrasting rainfall. A total of 480 soil samples at 12 stands were collected from two microhabitats, under shrubs and in open areas, soon after the two growing seasons. The germinable seed bank of annual plants was estimated by controlled seedling emergence method. Shrubs significantly facilitated the accumulation of seed bank beneath their canopies after the two growing seasons. In both microhabitats, the size and species richness of soil seed bank were significantly greater after the wet growing season (2018-2019) than following the dry season (2017-2018). The facilitative effects of shrubs were greater following the moister growing season than after the dry season. The effect of shrubs on seed bank-annual vegetation similarity varied between growing seasons, being greater in shrub interspaces than beneath shrub canopies for the dry growing season, while during the wet season, the similarity of the seed bank with standing annual vegetation was greater in sub-canopy microhabitat than in bare soil.

Impact of petroleum industry on goats in Saudi Arabia: heavy metal accumulation, oxidative stress, and tissue injury.

Heavy metals (HMs) constitute a group of persistent toxic pollutants, and the petroleum industry is one of the sources of these metals. This study aimed to evaluate the levels of lead (Pb), cadmium (Cd), nickel (Ni), and vanadium (V) in Plantago ovata and milk and tissues of domestic goats in the eastern region of Saudi Arabia. Plant samples and blood, milk, muscle, liver, and kidney samples were collected from domestic goats and the levels of Pb, Cd, V, and Ni were determined. Liver and kidney tissue injury, oxidative stress, and expression of pro-inflammatory and apoptosis markers were evaluated. Pb, Cd, V, and Ni were increased in Plantago ovata as well as in milk, blood, muscle, liver, and kidney of goats collected from the polluted site. Aminotransferases, creatinine, and urea were increased in serum, and histopathological changes were observed in the liver and kidney of goats at the oil extraction site. Malondialdehyde and the expression levels of pro-inflammatory cytokines, Bax, and caspase-3 were increased, whereas cellular antioxidants and Bcl-2 were decreased in liver and kidney of goats at the polluted site. In conclusion, petroleum industry caused liver and kidney injury, oxidative stress, and upregulated pro-inflammatory and apoptosis markers in goats. These findings highlight the negative impact of petroleum industry on the environment and call attention to the assessment of its effect on the health of nearby communities.

Eco-physiological response and genotoxicity induced by crude petroleum oil in the potential phytoremediator Vinca rosea L.

BACKGROUND: Phytoremediation is determined as an emerging green technology suitable for the safe remediation and restoration of polluted terrestrial and aquatic environments. In this study, the assessment of an ornamental plant, Vinca rosea L., as a phytoremediator of crude oil in polluted soils was conducted. In an open greenhouse experiment, plants were raised in sandy-clayey soils treated with 1, 3, 5, and 7% oil by weight. The experiment was conducted over 5 months. RESULTS: Total petroleum hydrocarbon (TPH) degradation percentage by V. rosea after a 5-month growth period ranged from 86.83 ± 0.44% to 59.05% ± 0.45% in soil treated with 1 and 7%, respectively. Plants raised in polluted soils demonstrated a dramatic reduction in germination rates, in addition to growth inhibition outcomes shown from decreased plant height. An increase in branching was observed with an increase in oil pollution percentages. Moreover, the phytomass allocated to the leaves was higher, while the phytomass witnessed lower values for fine roots, flowering and fruiting when compared to the controls. Apart from the apparent morphological changes, there was a decrease in chlorophyll a/b ratio, which was inversely proportional to the oil pollution level. The contents of carotenoids, tannins, phenolics, flavonoids, and antioxidant capacity were elevated directly with an increase in oil pollution level. The start codon-targeted (SCoT) polymorphisms and inter-simple sequence repeat (ISSR) primers showed the molecular variations between the control and plants raised in polluted soils. The genetic similarity and genomic DNA stability were negatively affected by increased levels of crude oil pollution. CONCLUSIONS: The ability of V. rosea to degrade TPH and balance the increased or decreased plant functional traits at the macro and micro levels of plant structure in response to crude oil pollution supports the use of the species for phytoremediation of crude oil-polluted sites. The genotoxic effects of crude oil on V. rosea still require further investigation. Further studies are required to demonstrate the mechanism of phenolic, flavonoid, and antioxidant compounds in the protection of plants against crude oil pollution stress. Testing different molecular markers and studying the differentially expressed genes will help understand the behavior of genetic polymorphism and stress-resistant genes in response to crude oil pollution.

Heavy Metal Accumulation, Tissue Injury, Oxidative Stress, and Inflammation in Dromedary Camels Living near Petroleum Industry Sites in Saudi Arabia.

The petroleum industry can impact the environment and human health. Heavy metals (HMs), including lead (Pb), cadmium (Cd), nickel (Ni), and vanadium (V), are toxic pollutants found in petroleum that can cause several severe diseases. This study investigated the impact of the oil industry on the Arabian camel (Camelus dromedarius) in the eastern region of Saudi Arabia, pointing to HMs accumulation, tissue injury, redox imbalance, inflammation, and apoptosis. Soil and camel samples (milk, blood, muscle, liver, and kidney) were collected from a site near an oil industry field and another two sites to analyze HMs. Pb, Cd, Ni, and V were increased in the soil and in the camel's milk, blood, muscle, liver, and kidney at the polluted site. Serum aminotransferases, urea, and creatinine were elevated, and histopathological alterations were observed in the liver and kidney of camels at the oil industry site. Hepatic and renal lipid peroxidation, pro-inflammatory cytokines, Bax, and caspase-3 were increased, whereas cellular antioxidants and Bcl-2 declined in camels at the oil extraction site. In conclusion, the oil industry caused soil and tissue accumulation of HMs, liver and kidney injury, oxidative stress, and apoptosis in camels living close to the oil extraction site. These findings pinpoint the negative impact of the oil industry on the environment, animal, and human health.

Effect of Visnagin on Altered Steroidogenesis and Spermatogenesis, and Testicular Injury Induced by the Heavy Metal Lead.

BACKGROUND: Lead (Pb) is an environmental pollutant causing serious health problems, including impairment of reproduction. Visnagin (VIS) is a furanochromone with promising antioxidant and anti-inflammatory effects; however, its protective efficacy against Pb toxicity has not been investigated. OBJECTIVE: This study evaluated the protective effect of VIS on Pb reproductive toxicity, impaired steroidogenesis and spermatogenesis, oxidative stress and inflammation. METHODS: Rats received VIS (30 or 60 mg/kg) and 50 mg/kg lead acetate for 3 weeks and blood and testes samples were collected. RESULTS: Pb intoxication impaired the pituitary-testicular axis (PTA) manifested by the decreased serum levels of gonadotropins and testosterone. Pb decreased sperm count, motility and viability, increased sperm abnormalities, and downregulated the steroidogenesis markers StAR, CYP17A1, 3ß-HSD and 17ß-HSD in the testis of rats. VIS significantly increased serum gonadotropins and testosterone, alleviated sperm parameters and upregulated steroidogenesis. In addition, VIS decreased pro-inflammatory cytokines, testicular lipid peroxidation and DNA fragmentation, downregulated Bax, and enhanced antioxidants and Bcl-2. CONCLUSION: These results demonstrate the protective effect of VIS against Pb reproductive toxicity in rats. VIS improved serum gonadotropins and testosterone, enhanced steroidogenesis and spermatogenesis, and attenuated oxidative injury, inflammation and apoptosis. Therefore, VIS is a promising candidate for the protection against Pb-induced reproduction impairment.

Facilitation Effects of Haloxylon salicornicum Shrubs on Associated Understory Annuals, and a Modified "Stress-Gradient" Hypothesis for Droughty Times.

Perennial shrub-annual plant interactions play key roles in desert regions influencing the structure and dynamics of plant communities there. In the present study, carried out in northwestern Saudi Arabia, we examined the effect of Haloxylon salicornicum shrubs on their associated understory annual species across four consecutive growing seasons, along with a record of the seasonal rainfall patterns. We measured density and species richness of all the annual species in permanent quadrats located beneath individual shrubs, as well as in the spaces between shrubs. During wet growing season H. salicornicum shrubs significantly enhanced the density and species richness of sub-canopy species, whereas in the relatively dry seasons they exerted negative effects on the associated species. In all growing seasons, the presence of shrubs was associated with enhanced soil properties, including increased organic carbon content, silt + clay, and levels of nutrients (N, P and K). Shrubs improved soil moisture content beneath their canopies in the wet growing season, while in the dry seasons they had negative effects on water availability. Differences in effects of H. salicornicum on understory plants between growing seasons seem due to the temporal changes in the impact of shrubs on water availability. Our results suggest the facilitative effects of shrubs on sub-canopy annuals in arid ecosystems may switch to negative effects with increasing drought stress. We discuss the study in light of recent refinements of the well-known "stress-gradient hypothesis".

Impact of Transgenic Arabidopsis thaliana Plants on Herbicide Isoproturon Phytoremediation through Expressing Human Cytochrome P450-1A2.

The excessive use of herbicides is a major cause of many environmental problems. The use of isoproturon herbicide as a weed controller has been a common practice globally. Phytoremediation technology can help in cleaning up polluted areas. In this paper the ability of CYP1A2 transgenic A. thaliana plants in the phytoremediation of isoproturon herbicides has been investigated. We tested the capability of P450-1A2 overexpression on the detoxification and degradation of isoproturon. We explored the toxic effect of isoproturon on the plant phenotypic characteristics, including the primary root length, rosette diameter, and fresh, dry weight for transgenic and wild type A. thaliana. The results revealed that no morphological changes appeared on CYP1A2 transgenic plants with a high tolerance to isoproturon herbicide applications either via foliar spraying or supplementation of the growth medium. Deleterious effects were observed on the morphological characteristics of plants of the wild type grown in soil under different treatments with isoproturon. The transgenic A. thaliana plants exhibited a vigorous growth even at high doses of isoproturon treatments. In contrast, the growth of the wild type was significantly impaired with doses above 50 µM isoproturon. The transgenic A. thaliana plants expressing P450-1A2 were able to metabolize the phenylurea herbicide isoproturon. Therefore, this method can be determined as a potential bioremediation agent.

Functional Traits Plasticity of the Invasive Herb Argemone ochroleuca Sweet in Different Arid Habitats.

Understanding the strategies and mechanisms of invasive species could guide their control and management especially in arid ecosystems. This study compares the vegetative and reproductive functional traits of the invasive Mexican poppy (Argemone ochroleuca), in seven habitat types, in southwestern Saudi Arabia. The results showed that the aboveground phenological attributes such as plant height, leaf area, and leaf dry mass attained the highest values in the wadi channels, whereas these attributes attained the lowest values in the mountain ranges. Maximum specific leaf area, root parameters, and all reproductive traits were recorded in the abandoned fields. In contrast to all other habitats, populations from abandoned fields had a greater investment of resources in belowground structures, while the population growing in the wadi channels and mountain ranges habitat allocated more energy to vegetative parts. The plasticity in vegetative and reproductive resource allocation in A. ochroleuca is an important mechanism in determining its colonizing ability in different habitat types and expanding the distribution range. The present data of the functional traits of A. ochroleuca agree with the resource fluctuation hypothesis, where the plant flourished in the abandoned fields that attained the highest values of organic matter and nutrients. Therefore, the restoration of these disturbed habitats could improve the resistance toward invasion by this noxious weed.

Monitoring the Efficiency of Rhazya stricta L. Plants in Phytoremediation of Heavy Metal-Contaminated Soil.

Heavy metal-contaminated soil constitutes many environmental concerns. The toxic nature of heavy metals poses serious threats to human health and the ecosystem. Decontamination of the polluted soil by phytoremediation is of fundamental importance. Vegetation is an appealing and cost-effective green technology for the large-scale phytoremediation of polluted soils. In this paper, a greenhouse experiment was carried out to test the potential of Rhazya stricta as a heavy metal phytoremediator in polluted soil. Plants were grown for three months in pots filled with soils treated with the heavy metals Cd, Pb, Cu, and Zn at rates of 10, 50, and 100 mg/kg. The bioaccumulation factor (BCF) and translocation factor (TF) were calculated to detect the ability of R. stricta to accumulate and transfer heavy metals from soil to plant organs. The results showed that under increasing levels of soil pollution, the bioconcentration of Cd and Zn heavy metals showed the highest values in plant roots followed by leaves, whereas in the case of Pb and Cu, roots showed the highest values followed by stems. Heavy metals accumulation was higher in roots than in stems and leaves. The BCF of Zn reached the highest values in roots and stems for 10 mg/kg soil treatment, followed by the BCFs of Cd, Cu, and Pb. The TF for the different heavy metal pollutants' concentrations was less than unity, suggesting that the plants remediate pollutants by phytostabilization. The TF values ranged from higher to lower were in the order Zn > Cu > Cd > Pb. The rapid growth of R. stricta and its tolerance of heavy metals, as well as its ability to absorb and accumulate metals within the plant, recommends its use in the phytoremediation of slightly polluted soils in arid lands by limiting the heavy metals transport.

Green Technology for Remediation of Water Polluted with Petroleum Crude Oil: Using of Eichhornia crassipes (Mart.) Solms Combined with Magnetic Nanoparticles Capped with Myrrh Resources of Saudi Arabia.

Crude oil pollution of water bodies is a worldwide problem that affects water ecosystems and is detrimental to human health and the diversity of living organisms. The objective of this study was to assess the ability of water hyacinth (Eichhornia crassipes (Mart.) Solms) combined with the presence of magnetic nanoparticles capped with natural products based on Myrrh to treat fresh water contaminated by crude petroleum oil. Magnetic nanoparticles based on magnetite capped with Myrrh extracts were prepared, characterized, and used to adsorb heavy components of the crude oil. The hydrophobic hexane and ether Myrrh extracts were isolated and used as capping for magnetite nanoparticles. The chemical structures, morphologies, particle sizes, and magnetic characteristics of the magnetic nanoparticles were investigated. The adsorption efficiencies of the magnetic nanoparticles show a greater efficiency to adsorb more than 95% of the heavy crude oil components. Offsets of Water hyacinth were raised in bowls containing Nile River fresh water under open greenhouse conditions, and subjected to varying crude oil contamination treatments of 0.5, 1, 2, 3, and 5 mL/L for one month. Plants were harvested and separated into shoots and roots, oven dried at 65 °C, and grounded into powder for further analysis of sulphur and total aromatic and saturated hydrocarbons, as well as individual aromatic constituents. The pigments of chlorophylls and carotenoids were measured spectrophotometrically in fresh plant leaves. The results indicated that the bioaccumulation of sulphur in plant tissues increased with the increased level of oil contamination. Water analysis showed significant reduction in polyaromatic hydrocarbons. The increase of crude oil contamination resulted in a decrease of chlorophylls and carotenoid content of the plant tissues. The results indicate that the water hyacinth can be used for remediation of water slightly polluted by crude petroleum oil. The presence of magnetite nanoparticles capped with Myrrh resources improved the remediation of water highly polluted by petroleum crude oil.

Impact of off-road vehicles on soil and vegetation in a desert rangeland in Saudi Arabia.

Off-road vehicle driving is considered as main contributor to land degradation in arid regions. This study examined the impact of off-road vehicles (ORV) on soil and vegetation in a natural recreational desert meadow of Raudhat Khuraim, Saudi Arabia. Vegetation canopy cover and plant height away from road tracks were assessed. Also, species density and canopy cover, bare ground cover and soil attributes were assessed in four microhabitats; tracks, inter-tracks, verges, and away from vehicle tracks (undisturbed natural areas). Results show that the cover of forbs and grasses was negatively associated with distance from road verges. It was observed that the height of woody species responded negatively to distance away from tracks. Cover of native species decreased under verge, inter-track and track microhabitats giving more opportunity for weeds to flourish. Bare ground was highest (60.7%) in tracks. ORV impact on soil bulk density was clear with an increase of 38% under tracks compared to soils of undisturbed natural vegetation and a similar decrease in porosity was observed. On the other hand, soil electrical conductivity was significantly higher (5.45 mS cm-1) under disturbance compared to 1.32 mS cm-1 in undisturbed natural vegetation. Organic matter and nitrogen were not affected significantly by ORV disturbance. The results emphasize that managing off-road vehicle driving is essential for conserving native vegetation.

Chemical ingredients and antioxidant activities of underutilized wild fruits.

This study evaluated nutritional values, bioactive constituents and antioxidant activities of the five wild underutilized fruits in the mountains of southwest Saudi Arabia (Coccinia grandis (L.) Voigt, Diospyros mespiliformis Hochst. Ex A.Dc., Cissus rotundifolius (L.), Ephedra foeminea Forssk., and Grewia villosa Willd.). The moisture content, crude fibers, total protein, total lipids, total hydrolyzable carbohydrate, total soluble sugars, and total free amino acids were analyzed. The results showed varying amounts among fruits of the five study species. In addition, the mineral composition, amino acid content, phenolic compounds, vitamins, and antioxidant activity were assessed. The highest content of total phenolic and total tannins was measured in D. mespiliformis (20.69 mg/g and 3.84 mg/g) and the lowest in E. foeminea (10.83 mg/g and 1.44 mg/g), respectively. The methanol extract (1 mg/ml) of D. mespiliformis displayed higher percentage of DPPH radical scavenging activity (87.36%). The methanol extract (1 mg/ml) of C. grandis exhibited the highest effect of total antioxidant activity and hydrogen peroxide scavenging activity (71.53%). The sufficient nutritional and antioxidant value of these wild fruits provide healthy food source for the local residents, much the same as many cultivated fruits and vegetables.

Molecular and phytochemical analysis of wild type and olive cultivars grown under Saudi Arabian environment.

This study aimed to assess genetic variability at molecular and phytochemical levels among the four most commonly grown olive cultivars and the wild-type olive of Saudi Arabia. Sixty-six and 80 amplicons were generated from 9 random amplified polymorphic DNA (RAPD) and inter simple sequence repeats (ISSR) primers, each, producing an average of 95.9 and 86.44% polymorphism for the two markers, respectively. The PIC values were 82.2% for the RAPD and 85.4% for the ISSR markers and the discrimination power for both the markers was 11.1%. The UPGMA cluster analysis based on the RAPD and ISSR data resulted in the aggregation of cultivars and wild accession with a good bootstrapping value according to their origin. Furthermore, a total of 199 compounds were identified in the cultivars based on peak area, retention time, and molecular formula using GC-MS analyses of methanolic and ethanolic extracts. These compounds were classified according to their chemical class; most of them were fatty acids, alcoholic compounds, carboxylic acids, aldehydes, heterocyclic compounds, ketones, alkanes, and phenols. Genetic and phytochemical distances were significantly correlated, based on the Mantel test. The Saudi wild accession also had high numbers of fatty acids and their esters, and can be used in breeding programs for generating new genotypes with interesting characters.

Phytoremediation of the organic Xenobiotic simazine by p450-1a2 transgenic Arabidopsis thaliana plants.

The potential use of human P450-transgenic plants for phytoremediation of pesticide contaminated soils was tested in laboratory and greenhouse experiments. The transgenic P450 CYP1A2 gene Arabidopsis thaliana plants metabolize number of herbicides, insecticides and industrial chemicals. The P450 isozymes CYP1A2 expressed in A. thaliana were examined regarding the herbicide simazine (SIM). Transgenic A. thaliana plants expressing CYP1A2 gene showed significant resistance to SIM supplemented either in plant growth medium or sprayed on foliar parts. The results showed that SIM produces harmful effect on both rosette diameter and primary root length of the wild type (WT) plants. In transgenic A. thaliana lines, the rosette diameter and primary root length were not affected by SIM concentrations used in this experiment. The results indicate that CYP1A2 can be used as a selectable marker for plant transformation, allowing efficient selection of transgenic lines in growth medium and/or in soil-grown plants. The transgenic A. thaliana plants exhibited a healthy growth using doses of up to 250 µmol SIM treatments, while the non-transgenic A. thaliana plants were severely damaged with doses above 50 µmol SIM treatments. The transgenic A. thaliana plants can be used as phytoremediator of environmental SIM contaminants.

Effects of open grazing and livestock exclusion on floristic composition and diversity in natural ecosystem of Western Saudi Arabia.

Livestock grazing is one of the main causes of rangeland degradation in Saudi Arabia. Fencing to exclude grazers is one of the main management practices used to restore vegetation and conserve biodiversity. The main objectives of this study were to investigate the changes in plant diversity and abundance, floristic composition and plant groups of the major life forms in response to thirty-five years of grazing exclosure in western Saudi Arabia. These vegetation attributes and palatability were compared in 30 sampling stands located in the excluded and grazed sites. Our results showed that livestock exclusion significantly increased covers, density and species richness of annuals, grasses, perennial forbs, shrubs and trees. Exclosure enhanced the abundance and richness of palatable species and depressed the development of weedy species. About 66.7% of the recorded species at the excluded site were highly palatable compared to 34.5% at the grazed site. In contrary, about 55.2% unpalatable species were found in the grazed site compared to 25.8% in the protected site. Jaccard's similarity index between the excluded and grazed sites showed lower values of 0.39%, 0.40% and 0.31% at levels of families, genus and species, respectively. The results suggest that establishing livestock exclusion may be a useful sustainable management tool for vegetation restoration and conservation of plant diversity in degraded rangelands of arid regions.

Diversity of active constituents in Cichorium endivia and Cynara cornigera extracts.

The present study attempts to explore the phytochemical constituents of different extracts from Cynara cornigera and Cichorium endivia plant materials. The two species studied are native in Egypt. Five different solvents, viz., aqueous, methylene chloride, petroleum ether, ethyl acetate, and n-butanol were used. Phytochemical analysis revealed the presence of phenols, flavonoids, sterols (stigmasterol and beta-sitosterol), terpenes (α-amyrin, ursolic and oleanolic acid), and hydrocarbons (n-alkane), the latter found in low amount. The ethyl acetate and water extracts of C. cornigera root showed lower mass fractions of phenolic compounds ranged from 20 to 81 g/100 g, and higher amounts in ethyl acetate extract of the inflorescences and butanol extract of the root where values ranged from 195 to 399 g/100 g. The ß-sitosterol and stigmasterol were present in all plant extracts. Oleanolic and ursolic acids were detected in roots, leaves and inflorescences of C. cornigera and in C. endivia shoot. The ethyl acetate extracts from C. cornigera leaf and inflorescence attained higher chemical diversity than the other extracts. Alternatively, sterols and triterpenes were the major constituents. The high chemical diversity of active constituents justifies the future potential use of the two species at commercial level.

Major constituents of Boswellia carteri resin exhibit cyclooxygenase enzyme inhibition and antiproliferative activity.

Aromatic gum from Boswellia carteri (olibanum oleogum) has long been used in Egyptian traditional medicine. Cyclooxygenase-1 (COX-1) enzyme inhibitory assay guided purification of the extracts of this resin resulted in five bioactive compounds, 3alpha-O-acetyl-8,24-dien-tirucallic acid (1), verticilla-4(20),7,11-triene (2), cembrene A (3), incensole acetate (4), and incensole (5). The pure isolates were investigated for their inhibitory effects on COX-1 and -2 enzymes and human tumor cell lines Hep-G2, MCF-7 and RAW 264.7. Compounds 1-5 inhibited COX-2 enzyme by 39.0, 32.7, 60.0, 46.3, and 49.8%, respectively. Furthermore, compound 2 showed an inhibitory concentration of 50% (IC50) at 9 microg/mL against Hep-G2 tumor cell line. This is the first report of COX-1 and -2 enzyme and tumor cell proliferation inhibitory effects of compounds 1 and 2.

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.

Molecular and biochemical characterization in Rauvolfia tetraphylla plantlets grown from synthetic seeds following in vitro cold storage.

Synseed technology is one of the most important applications of plant biotechnology for in vitro conservation and regeneration of medicinal and aromatic plants. In the present investigation, synseeds of Rauvolfia tetraphylla were produced using in vitro-proliferated shoots upon complexation of 3 % sodium alginate and 100 mM CaCl(2). The encapsulated buds were stored at 4, 8, 12, and 16 °C and high conversion was observed in synseeds stored at 4 °C for 4 weeks. The effect of different medium strength on in vitro conversion response of synseed was evaluated and the maximum conversion (80.6 %) into plantlets was recorded on half-strength woody plant medium supplemented with 7.5 µM 6-benzyladenine and 2.5 µM α-naphthalene acetic acid after 8 weeks of culture. Plantlets with well-developed shoot and roots were hardened and successfully transplanted in field condition. After 4 weeks of transfer to ex vitro conditions, the performance of synseed-derived plantlets was evaluated on the basis of some physiological and biochemical parameters and compared with the in vivo-grown plants. Short-term storage of synthetic seeds at low temperature had no negative impact on physiological and biochemical profile of the plants that survived the storage process. Furthermore, clonal fidelity of synseed-derived plantlets was also assessed and compared with mother plant using rapid amplified polymorphic DNA and inter-simple sequence repeats analysis. No changes in molecular profiles were found among the regenerated plantlets and comparable to mother plant, which confirm the genetic stability among clones. This synseed protocol could be useful for in vitro clonal multiplication, conservation, and short-term storage and exchange of germplasm of this antihypertensive drug-producing plant.

An efficient and reproducible method for in vitro clonal multiplication of Rauvolfia tetraphylla L. and evaluation of genetic stability using DNA-based markers.

An efficient protocol is described for the rapid in vitro clonal propagation of an endangered medicinal plant, Rauvolfia tetraphylla L., through high frequency shoot induction from nodal explants collected from young shoots of a field grown plant. Effects of growth regulators [6-benzyladenine (BA), kinetin (Kin) 2iP, or α-naphthalene acetic acid (NAA)], carbohydrates, different medium [Murashige and Skoog (MS), Woody Plant Medium (WPM), Gamborg medium (B5), Linsmier and Skoog medium (LS)], and various pH levels on in vitro morphogenesis were investigated. The highest frequency of shoot regeneration (90 %) and maximum number of shoot (35.4 ± 2.3) per explant were observed on WPM medium supplemented with 7.5 µM BA, 2.5 µM NAA, and 30 g/l sucrose at pH 5.8. Well-developed shoots, 4-5 cm in length, were successfully rooted ex vitro at 90 % by a 30-min pulse treatment with 150 µM IBA prior to their transfer in planting substrates. The survival rate of transplantation reached 90 % when transferred to field condition. Genetic stability of micropropagated plantlets was assessed and compared with mother plant using Random Amplified Polymorphic DNA and Inter Simple Sequence Repeats markers. No variation was observed in DNA fingerprinting patterns among the micropropagated plants, which were similar to that of the donor plant illustrating their genetic uniformity and clonal fidelity. This confirms that clonal propagation of this plant using axillary shoot buds can be used for commercial exploitation of the selected genotype where a high degree of fidelity is an essential prerequisite. The work contributed to a better in vitro regeneration and clonal mass multiplication of R. tetraphylla and to develop a strategy for the germplasm conservation of this endangered medicinal plant.