Cytotoxicity and genotoxicity of zingiberene on different neuron cell lines in vitro

The main objective of this study is to investigate the cytotoxic, genotoxic and antioxidant properties of zingiberene (ZBN) in an in vitro rat brain cell culture study. The cytotoxic effect was determined against the rat neuron and N2a neuroblastoma (N2a-NB) cell lines using the 3,(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, while the antioxidant activity was assessed using the total antioxidant capacity (TAC) and total oxidative stress (TOS) assays. The effects on DNA damage were also evaluated in this study by the single cell gel electrophoresis assay. The results indicated that ZBN has an anti-proliferative activity suppressing the proliferation of N2a-NB cells at concentrations over 50 mg L⁻¹ and neuron cells at concentrations over 150 mg L⁻¹. In addition, ZBN treatments at higher doses (≤50 mg L⁻¹) led to increases of TOS levels in N2a-NB cell cultures. However 25 mg L⁻¹ of ZBN treatment caused increases of TAC levels in cultured neuron and N2a-NB cell cultures while ZBN at doses of 10–400 mg L⁻¹ did not increase the number of total damage score in both cell lines. This study clearly indicates that ZBN has a significant potential to be used as a natural anticancer agent in cultured N2a-NBs.     

Temporal changes of soil physic‐chemical properties at different soil depths during larch afforestation by multivariate analysis of covariance

Soil physic-chemical properties differ at different depths; however, differences in afforestation-induced temporal changes at different soil depths are seldom reported. By examining 19 parameters, the temporal changes and their interactions with soil depth in a large chronosequence dataset (159 plots; 636 profiles; 2544 samples) of larch plantations were checked by multivariate analysis of covariance (MANCOVA). No linear temporal changes were found in 9 parameters (N, K, N:P, available forms of N, P, K and ratios of N: available N, P: available P and K: available K), while marked linear changes were found in the rest 10 parameters. Four of them showed divergent temporal changes between surface and deep soils. At surface soils, changing rates were 262.1 g·kg⁻¹·year⁻¹ for SOM, 438.9 mg·g⁻¹·year⁻¹ for C:P, 5.3 mg·g⁻¹·year⁻¹ for C:K, and −3.23 mg·cm⁻³·year⁻¹ for bulk density, while contrary tendencies were found in deeper soils. These divergences resulted in much moderated or no changes in the overall 80-cm soil profile. The other six parameters showed significant temporal changes for overall 0–80-cm soil profile (P: −4.10 mg·kg⁻¹·year⁻¹; pH: −0.0061 unit·year⁻¹; C:N: 167.1 mg·g⁻¹·year⁻¹; K:P: 371.5 mg·g⁻¹ year⁻¹; N:K: −0.242 mg·g⁻¹·year⁻¹; EC: 0.169 μS·cm⁻¹·year⁻¹), but without significant differences at different soil depths (P > 0.05). Our findings highlight the importance of deep soils in studying physic-chemical changes of soil properties, and the temporal changes occurred in both surface and deep soils should be fully considered for forest management and soil nutrient balance.     

Brassinosteroid-mediated evaluation of antioxidant system and nitrogen metabolism in two contrasting cultivars of Vigna radiata under different levels of nickel

The role of 28-homobrassinolide (HBL) in countering nickel-induced oxidative damage through overexpression of antioxidant enzymes and proline in Vigna radiata has been investigated. Two varieties of V. radiata, one sensitive to Ni (PDM-139) and the other tolerant to Ni (T-44), were sown in the soil fed with different levels (0, 50, 100 or 150 mg kg⁻¹) of Ni, and at 29-day stage, foliage of plants was applied with deionized water (control), 10⁻⁸ or 10⁻⁶ M of HBL. The plants were sampled at 45-day stage of growth to assess various physiological as well as biochemical characteristics. The remaining plants were allowed to grow up to maturity to study the yield characteristics. The growth traits, leghemoglobin, nitrogen and carbohydrate content in the nodules, leaf chlorophyll content, photosynthesis efficiency, leaf water potential, activities of nitrate reductase, carbonic anhydrase and nitrogenase decreased proportionately with the increasing concentrations of nickel, whereas electrolyte leakage, various antioxidant enzymes viz. catalase, peroxidase and superoxide dismutase and accumulation of proline increased at 45-day stage. However, the exogenously applied HBL to the nickel-stressed or non-stressed plants improved growth, nodulation and photosynthesis and further enhanced the various antioxidant enzymes viz. catalase, peroxidase and superoxide dismutase and accumulation of proline. The deleterious impact of Ni on the plants was concentration dependent where HBL applied to the foliage induced overexpression of antioxidant enzyme and accumulation of proline (osmolyte) which could have conferred tolerance to Ni up to 100 mg kg⁻¹, resulting in improved growth, nodulation, photosynthesis and yield attributes.     

Effect of heat treatment around the fruit set region on growth and yield of watermelon [Citrullus lanatus (Thunb.) Matsum. and Nakai]

The present experiment was aimed to study the effect of imposing modulated temperature treatments 14 °C and 18 °C, around the fruiting region of watermelon plants, and to estimate the economic feasibility of the temperature treatments based on energy consumption for heating. Watermelon cultivar ‘Sambok-gul’ was selected and sown on perlite beds in a plastic house under controlled conditions at Watermelon Farm, Jeongeup-Jeonbuk, longitude 35° 31′ 47.51N, 126° 48′48.84E, altitude 37 m during the early spring season (2010–2011). The findings revealed that the temperature treatment at 18 °C caused significant increase in weight (2.0 kg plant⁻¹), fruit weight (8.3 kg plant⁻¹), soluble solid content (11.5 %), and fruit set rate (96.5 %) at harvest stage. Higher contents of Ca²⁺ and Mg²⁺ ions were observed in the 1st upper leaf of the fruit set node (79.3 mg L⁻¹) and the 1st lower leaf of the fruit set node (12.0 mg L⁻¹), respectively at 14 °C. The power consumption and extra costs of the temperature treatment 18 °C were suggested as affordable and in range of a farmer’s budget (41.14 USD/22 days). Hence, it was concluded that modulating temperature treatments could be utilized successfully to optimize the temperature range for enhancing the fruit yield and quality in the winter watermelon crops.     

Foliar spray of brassinosteroid enhances yield and quality of Solanum lycopersicum under cadmium stress

The presence of cadmium in the soil above a particular level is proposed to check not only plant growth but also productivity and fruit quality. Therefore, in the present study investigations are directed to evaluate the effect of four levels of cadmium (3, 6, 9, 12 mg kg⁻¹) in interaction with two analogs of brassinosteroids on the growth, fruit yield and quality of tomato. Under greenhouse conditions plants were analyzed for antioxidant system activity and photosynthetic assimilation efficiency. Cd stressed plants exhibited poor growth and biological yield. The metal also had a negative impact on the antioxidant system of the resulting fruits. However, the follow up application of BRs (10⁻⁸ M) neutralized the damaging effects of the metal on the plants.     

In vitro propagation of female Ephedra foliata Boiss. & Kotschy ex Boiss.: an endemic and threatened Gymnosperm of the Thar Desert

Ephedra foliata Boiss. & Kotschy ex Boiss., (family – Ephedraceae), is an ecologically and economically important threatened Gymnosperm of the Indian Thar Desert. A method for micropropagation of E. foliata using nodal explant of mature female plant has been developed. Maximum bud-break (90 %) of the explant was obtained on MS medium supplemented with 1.5 mg l⁻¹ of benzyl adenine (BA) + additives. Explant produces 5.3 ± 0.40 shoots from single node with 3.25 ± 0.29 cm length. The multiplication of shoots in culture was affected by salt composition of media, types and concentrations of plant growth regulators (PGR’s) and their interactions, time of transfer of the cultures. Maximum number of shoots (26.3 ± 0.82 per culture vessel) were regenerated on MS medium modified by reducing the concentration of nitrates to half supplemented with 200 mg l⁻¹ ammonium sulphate {(NH₄) ₂SO₄} (MMS3) + BA (0.25 mg l⁻¹), Kinetin (Kin; 0.25 mg l⁻¹), Indole-3-acetic acid (IAA; 0.1 mg l⁻¹) and additives. The in vitro produced shoots rooted under ex vitro on soilrite moistened with one-fourth strength of MS macro salts in screw cap bottles by treating the shoot base (s) with 500 mg l⁻¹ of Indole-3-butyric acid (IBA) for 5 min. The micropropagated plants were hardened in the green house. The described protocol can be applicable for (i) large scale plant production (ii) establishment of plants in natural habitat and (iii) germplasm conservation of this endemic Gymnosperm of arid regions.     

Isolation and Characterization of Antidermatophytic Bioactive Molecules from Piper longum L. Leaves

Piper longum L. (Piperaceae) commonly known as “long pepper” is a well known medicinal plant in ayurveda. Different parts of this plant, such as root, seed, fruit, whole plant etc. are used traditionally in various ailments. Here we have investigated the antidermatophytic activity of sequentially extracted petroleum ether, chloroform, methanol and water extracts from P. longum leaf against Trichophytonmentagrophytes, T. rubrum, T. tonsurans, Microsporum fulvum and M. gypseum. Better activity of chloroform and methanol extracts was observed. The chloroform extract was selected for further study and the MIC value was recorded as 5.0 mg ml⁻¹ against the test organisms. In the chloroform extract, tannins and phenolic compounds were detected. Further activity-guided fractionation of chloroform extract by silica gel column chromatography yielded nine major fractions. Among these, fraction-1, 4, 5 and 7 showed higher antidermatophytic activity. Fraction-4 on further purification by repeated column chromatography yielded a potential antidermatophytic fraction showing MIC value of 0.625 mg ml⁻¹ against T. mentagrophytes and T. rubrum as determined by broth microdilution method. The major compounds were identified as 1,2-benzenedicarboxylic acid, bis(2-ethylhexyl) ester (C₂₄H₃₈O₄] (41.45 %), 2,2-dimethoxybutane (C₆H₁₄O₂] (13.6 %) and β-myrcene (C₁₀H₁₆) (6.75 %) based on GC–MS data.     

Fat Oxidation,Fitness and Skeletal Muscle Expression of Oxidative/Lipid Metabolism Genes in South Asians: Implications for Insulin Resistance?

Background

South Asians are more insulin resistant than Europeans, which cannot be fully explained by differences in adiposity. We investigated whether differences in oxidative capacity and capacity for fatty acid utilisation in South Asians might contribute, using a range of whole-body and skeletal muscle measures.

Methodology/Principal Findings

Twenty men of South Asian ethnic origin and 20 age and BMI-matched men of white European descent underwent exercise and metabolic testing and provided a muscle biopsy to determine expression of oxidative and lipid metabolism genes and of insulin signalling proteins. In analyses adjusted for age, BMI, fat mass and physical activity, South Asians, compared to Europeans, exhibited; reduced insulin sensitivity by 26% (p = 0.010); lower VO_2max (40.6±6.6 vs 52.4±5.7 ml.kg⁻¹.min⁻¹, p = 0.001); and reduced fat oxidation during submaximal exercise at the same relative (3.77±2.02 vs 6.55±2.60 mg.kg⁻¹.min⁻¹ at 55% VO_2max, p = 0.013), and absolute (3.46±2.20 vs 6.00±1.93 mg.kg⁻¹.min⁻¹ at 25 ml O₂.kg⁻¹.min⁻¹, p = 0.021), exercise intensities. South Asians exhibited significantly higher skeletal muscle gene expression of CPT1A and FASN and significantly lower skeletal muscle protein expression of PI3K and PKB Ser473 phosphorylation. Fat oxidation during submaximal exercise and VO_2max both correlated significantly with insulin sensitivity index and PKB Ser473 phosphorylation, with VO_2max or fat oxidation during exercise explaining 10–13% of the variance in insulin sensitivity index, independent of age, body composition and physical activity.

Conclusions/Significance

These data indicate that reduced oxidative capacity and capacity for fatty acid utilisation at the whole body level are key features of the insulin resistant phenotype observed in South Asians, but that this is not the consequence of reduced skeletal muscle expression of oxidative and lipid metabolism genes.     

Antioxidants and ROS scavenging ability in ten Darjeeling tea clones may serve as markers for selection of potentially adapted clones against abiotic stress

Ten Darjeeling tea clones (BT15/263, RR17/144, B777, T253, B157, Sundaram, HV39, AV2, K1/1 and TTV1) were collected from the experimental garden of Darjeeling Tea Research and Development Centre, Kurseong. Total phenol, flavonoids and two antioxidating enzymes (peroxidase and superoxide dismutase) were estimated. The total phenol ranged between 241 and 28 GAE mg g⁻¹ of leaf dry weight. The highest amount obtained in four clones, B15/263 (241.47), RR17/144 (221.2), B777 (154.54) and B157 (140.23 mg g⁻¹). Flavonoids were estimated as Catechin equivalent (CE) and ranged between 56.88 and 20.81 CE mg g⁻¹ leaf dry weight. Higher amounts occurred in BT15/263 (56.88 mg g⁻¹), B777 (56.69) and RR17/144 (48.63). Antioxidant activities were measured following DPPH and ABTS free radicle scavenging procedures and the results were well according to total polyphenol content among the clones (in total phenols, ranges of correlation in DPPH assay were r² = 0.990–0.989, p ≤ 0.05; in flavonoids r² = 0.954, p ≤ 0.01–0.987, p ≤ 0.05). Similarly, ABTS percent scavenging results were quiet significant. The IC₅₀ values were determined for both DPPH and ABTS assay. PAGE expressions of isoforms in two antioxidative enzymes and quantification of them also varied much among the investigated clones. The incidence of total phenols, flavonoids, PRX and SOD and ROS scavenging assay in in-situ condition, might be used as biochemical markers towards the superior adaptability against abiotic stress. In the present work, four clones (B15/263, B777, RR17/144 and B157) would be designated as comparatively better suited to the predicted abiotic stress.     

Apigenin inhibits osteoblastogenesis and osteoclastogenesis and prevents bone loss in ovariectomized mice

Polyphenol have been reported to have physiological effects with respect to alleviating diseases such as osteoporosis and osteopetrosis. We recently reported that the olive polyphenol hydroxytyrosol accelerates bone formation both in vivo and in vitro. The present study was designed to evaluate the in vivo and in vitro effects of apigenin (4′,5,7-trihydroxyflavone), one of the major polyphenols in olives and parsley, on bone formation by using cultured osteoblasts and osteoclasts and ovariectomized (OVX) mice, respectively. Apigenin markedly inhibited cell proliferation and indices of osteoblast differentiation, such as collagen production, alkaline phosphatase activity, and calcium deposition in osteoblastic MC3T3-E1 cells at concentrations of 1–10 μM. At 10 μM, apigenin completely inhibited the formation of multinucleated osteoclasts from mouse splenic cells. Moreover, injection of apigenin at 10 mg kg⁻¹ body weight significantly suppressed trabecular bone loss in the femurs of OVX mice. Our findings indicate that apigenin may have critical effects on bone maintenance in vivo.     

Larvicidal activity of novel anthraquinone analogues and their molecular docking studies

To investigate the larvicidal activities of novel anthraquinones (1a-1k) against Culex quinquefasciatus mosquito larvae. Novel anthraquinones (1a-1k) derivatives were synthesis via condensation method. The compounds were confirmed through FT-IR spectroscopy, ¹H & ¹³C NMR spectrum, and mass spectral studies. The larvicidal activity of compound 1c was highly active LD₅₀ 20.92 µg/mL against Culex quinquefasciatus compared standard permethrin with LD₅₀ 25.49 µg/mL. Molecular docking studies were carried out for compound 1c against Odorant-binding protein of Culex quinquefasciatus. The compound 1c (−9.8 Kcal/mol) was a potent larvicide with more binding energy than control permethrin (−9.7 Kcal/mol). Therefore, compound (1c) may be more significant inhibitors of mosquito larvicidal.     

In vitro medicinal potentials of Bryum capillare,a moss sample,from Turkey

In this study was conducted the in vitro antimicrobial, antibiofilm, antioxidant, antigenotoxic and anticancer activities investigations on the moss Bryum capillare Hedw (BC). Antimicrobial and antibiofilm activity were tested by MIC and microplate biofilm methods on antibiotic resistant bacteria. While the antioxidant activity of the extract was evaluated by DPPH, metal chelating, plasma lipid peroxidation and total phenolic content, the antigenotoxicity and cytotoxicity were established by Comet test and the WST-1 Cell proliferation assay kit respectively. The MIC values were found to be ≥ 125 µg.mL⁻¹ and a biofilm inhibition of 3–5% against only S. epidermidis was observed. Total phenolic compounds were determined as 23.26 mg/g. The results of DPPH assay, chelating and plasma lipid peroxidation activity were found to be 15%, 3% and 4% respectively. The extract was observed to decrease the affect of H₂O₂ that cause DNA damage. The BC was also determined 60 ± 5% anticancer activity against SKBR 3 and 76 ± 5% anticancer activity against HeLa cells, where this concentration had only 18 ± 5% cytotoxicity against MCF-12A cells. Also, these results have indicated the potential of Bryum capillare for the first time in novel natural compounds search.     

Micropropagtion of Terminalia bellerica from nodal explants of mature tree and assessment of genetic fidelity using ISSR and RAPD markers

The present study reports an efficient in vitro micropropagation protocol for a medicinally important tree, Terminalia bellerica Roxb. from nodal segments of a 30 years old tree. Nodal segments taken from the mature tree in March-April and cultured on half strength MS medium gave the best shoot bud proliferation response. Combinations of serial transfer technique (ST) and incorporation of antioxidants (AO) [polyvinylpyrrolidone, PVP (50 mg l⁻¹) + ascorbic acid (100 mg l⁻¹) + citric acid (10 mg l⁻¹)] in the culture medium aided to minimize browning and improve explant survival during shoot bud induction. Highest multiplication of shoots was achieved on medium supplemented with 6-benzyladenine (BA, 8.8 μM) and α-naphthalene acetic acid (NAA, 2.6 μM) in addition to antioxidants. Shoot elongation was obtained on MS medium containing BA (4.4 μM) + phloroglucinol (PG, 3.9 μM). Elongated shoots were transferred to half strength MS medium containing indole-3-butyric acid (IBA, 2.5 μM) for root development. The acclimatization of plantlets was carried out under greenhouse conditions. The genetic fidelity of the regenerated plants was checked using inter simple sequence repeats (ISSR) and randomly amplified polymorphic DNA (RAPD) analysis. Comparison of the bands among the regenerants and mother plant confirmed true-to-type clonal plants.     

Murine liver response to Allium sativum treatment during infection induced-trypanosomiasis

Hepatic injury induced by trypanosomiasis is one of the major health problems not only to human but also to wild and domestic animals. This study aimed to evaluate the hepatoprotective role of Allium sativum extract (ASE) against Trypanosoma evansi infection in mice. Animals were divided into 4 groups. Group I received only saline while group II received ASE (20 mg/Kg). Animals of group III and group IV were infected with T. evansi. The latter group was treated with ASE. The infrared spectroscopic analysis of A. sativum extract exhibited bands between 3700 cm⁻¹ and 599 cm⁻¹. On day 4 post T. evansi infection, ASE decreased the parasitemia by about 15 fold. Also, ASE regulated the number of erythrocytes and leucocytes and the hemoglobin content. In addition, the histopathological damage was reduced after treatment with ASE. Moreover, the oxidant and the antioxidant markers (glutathione, malondialdehyde and catalase) were regulated in the infected-treated animals. Collectively, the results proved the protective role of ASE against T. evansi infection in mice.     

Salt-induced modulation in growth,photosynthesis and antioxidant system in two varieties of Brassica juncea

The present study was carried out to examine salt-induced modulation in growth, photosynthetic characteristics and antioxidant system in two cultivars of Brassica juncea Czern and Coss varieties (Varuna and RH-30). The surface sterilized seeds of these varieties were sown in the soil amended with different levels (2.8, 4.2 or 5.6 dsm⁻¹) of sodium chloride under a simple randomized block design. The salt treatment significantly decreased growth, net photosynthetic rate and its related attributes, chlorophyll fluorescence, SPAD value of chlorophyll, leaf carbonic anhydrase activity and leaf water potential, whereas electrolyte leakage, proline content, and activity of catalase, peroxidase and superoxide dismutase enzymes increased in both the varieties at 30 d stage of growth. The variety Varuna was found more resistant than RH-30 to the salt stress and possessed higher values for growth, photosynthetic attributes and antioxidant enzymes. Out of the graded concentrations (2.8, 4.2 or 5.6 dsm⁻¹) of sodium chloride, 2.8 sm⁻¹ was least toxic and 5.6 dsm⁻¹ was most harmful. The variation in the responses of these two varieties to salt stress is attributed to their differential photosynthetic traits, SPAD chlorophyll value and antioxidant capacity, which can be used as potential markers for screening mustard plants for salt tolerance.     

Role of nitrogen and magnesium for growth,yield and nutritional quality of radish

Nitrogen (N) affects all levels of plant function from metabolism to resource allocation, growth, and development and Magnesium (Mg) is a macronutrient that is necessary to both plant growth and health. Radish (Raphanus sativus L.) occupies an important position in the production and consumption of vegetables globally, but there are still many problems and challenges in its nutrient management. A pot trial was conducted to investigate the effects of nitrogen and magnesium fertilizers on radish during the year 2018–2019. Nitrogen and magnesium was applied at three rates (0, 0.200, and 0.300 g N kg⁻¹ soil) and (0, 0.050, and 0.100 g Mg kg⁻¹ soil) respectively. The experiment was laid out in a completely randomized design (CRD) and each treatment was replicated three times. Growth, yield and quality indicators of radish (plant height, root length, shoot length, plant weight, total soluble sugar, ascorbic acid, total soluble protein, crude fiber, etc.) were studied. The results indicated that different rates of nitrogen and magnesium fertilizer not only influence the growth dynamics and yields but also enhances radish quality. The results revealed that the growth, yield and nutrient contents of radish were increased at a range of 0.00 g N. kg⁻¹ soil to 0.300 g N. kg⁻¹ soil and 0.00 g Mg. kg⁻¹ soil to 0.050 g Mg. kg⁻¹ soil and then decreased gradually at a level of 0.100 g Mg. kg⁻¹ soil. In contrast, the crude fiber contents in radish decreased significantly with increasing nitrogen and magnesium level but increased significantly at Mg₂ level (0.050 g Mg. kg⁻¹ soil). The current study produced helpful results for increasing radish quality, decreasing production costs, and diminishing underground water contamination.     

Thermoreversible Nasal In situ Gel of Venlafaxine Hydrochloride: Formulation, Characterization, and Pharmacodynamic Evaluation

In order to improve the bioavailability of the antidepressant drug, venlafaxine hydrochloride, in situ mucoadhesive thermoreversible gel, was formulated using Lutrol F127 (18%) as a thermo gelling polymer. Mucoadhesion was modulated by trying carbopol 934, PVP K30, HPMC K4M, sodium alginate, tamarind seed gum, and carrageenan as mucoadhesive polymers. Results revealed that as the concentration of mucoadhesive polymer increased the mucoadhesive strength increased but gelation temperature decreased. Formulation was optimized on the basis of clarity, pH, gelation temperature, mucoadhesive strength, gel strength, viscosity, drug content, diffusion through sheep nasal mucosa, histopathological evaluation of mucosa, and pharmacodynamic study in rats. Final formulation T5 containing 18% Lutrol F127 and 0.3% PVP K30 was considered as an optimized formulation. T5 released 97.86 ± 0.073% drug in 150 min with a flux of 0.1545 mg cm⁻² min⁻¹ and gelation temperature 31.17 ± 0.30°C. Histopathological evaluation of nasal mucosa revealed that T5 formulation was safe for nasal administration as it caused no damage to nasal epithelium. From the results of pharmacodynamic study, mainly forced swim test (FST), it was concluded that venlafaxine hydrochloride was more effective as an antidepressant by nasal route as in situ gel nasal drops in comparison to oral administration of equivalent dose.Key words: lutrol F127, mucoadhesive, nasal in situ gel, thermoreversible, venlafaxine HCl     

The influence of Agrobacterium rhizogenes on induction of hairy roots and ß-carboline alkaloids production in Tribulus terrestris L.

We have developed an efficient transformation system for Tribulus terrestris L., an important medicinal plant, using Agrobacterium rhizogenes strains AR15834 and GMI9534 to generate hairy roots. Hairy roots were formed directly from the cut edges of leaf explants 10–14 days after inoculation with the Agrobacterium with highest frequency transformation being 49 %, which was achieved using Agrobacterium rhizogenes AR15834 on hormone-free MS medium after 28 days inoculation. PCR analysis showed that rolB genes of Ri plasmid of A. rhizogenes were integrated and expressed into the genome of transformed hairy roots. Isolated transgenic hairy roots grew rapidly on MS medium supplemented with indole-3-butyric acid. They showed characteristics of transformed roots such as fast growth and high lateral branching in comparison with untransformed roots. Isolated control and transgenic hairy roots grown in liquid medium containing IBA were analyzed to detect ß-carboline alkaloids by High Performance Thin Layer Chromatograghy (HPTLC). Harmine content was estimated to be 1.7 μg g⁻¹ of the dried weight of transgenic hairy root cultures at the end of 50 days of culturing. The transformed roots induced by AR15834 strain, spontaneously, dedifferentiated as callus on MS medium without hormone. Optimum callus induction and shoot regeneration of transformed roots in vitro was achieved on MS medium containing 0.4 mg L⁻¹ naphthaleneacetic acid and 2 mg L⁻¹ 6-benzylaminopurine (BAP) after 50 days. The main objective of this investigation was to establish hairy roots in this plant by using A. rhizogenes to synthesize secondary products at levels comparable to the wild-type roots.     

Somatic embryogenesis and in vitro plant regeneration of Bacopa monnieri (Linn.) Wettst., a potential medicinal water hyssop plant

Bacopa monnieri (Linn.) Wettst. commonly known as waterhyssop, Brahmi plant, traditionally used for memory enhancement, nerve tonic, epilepsy, central nervous system (CNS), antidepressant, anxiety, blood pressure and antioxidant activities. Due to pharmaceutical demands its lost natural habitat. At this juncture we describe a resourceful protocol for micropropagation of water hyssop plant. Surface sterilized leaf and nodal explants were inoculated on basal MS semi-solid medium added with PGRs; auxins, cytokinins. Highest calli formation from leaf explants was obtained on NAA (2.5 mg⁻¹) and showed (94.22%) accompanied via 2,4-D showed (2.5 mg⁻¹; 82.43%), maximum calli formation in nodal explants was obtained on 2,4-D showed (2.5 mg⁻¹; 71.14%) followed by NAA (2.5 mg⁻¹) showed (62.15%), in internodes explants uppermost calli formation was obtained from 2,4-D showed (2.5 mg⁻¹; 65.21%) followed by NAA (2.5 mg⁻¹) showed (52.14%). The maximum somatic embryogenic callus, calli induction and formation (84%) was observed on 2,4-D + KIN (2.0 + 1.5 mg⁻¹) amended solid medium. Uppermost shoot formation was observed in combination of IAA + BAP (1.0 + 1.0 mg⁻¹) showed (78.54%) shoot formation followed by IBA (2.0 mg⁻¹) alone showed (75.37%). The maximum shoot elongation was noticed from NAA + BAP (3.0 + 3.0 mg⁻¹) with 21.21 cm followed by NAA (2.0 mg⁻¹) showed (15.22 cm) although, chief root formation was obtained from IBA (2.0 mg⁻¹) with 83.75% root formation along higher number of roots (47.43%) per shoot. Followed by IAA (2.0 mg⁻¹) showed root induction (73.43%) and no of roots (38.54%) per shoot. In hardening under pot condition plants survivability (100%) was observed under glass house conditions, the present in vitro PTC techniques is extremely significant to gratifying its natural conservation.     

Formulation of a protein-free medium based on IPL-41 for the sustained growth of Drosophila melanogaster S2 cells

An animal protein-free medium was developed for Drosophila melanogaster S2 (S2AcGPV2) cells genetically modified to produce the rabies virus G glycoprotein (GPV). IPL-41, used as a basal medium, was supplemented with yeastolate, carbohydrates, amino acids and lipids aiming initially to reduce and further to eliminate the need of fetal bovine serum. The S2AcGPV2 cells were fully capable of growing in serum-free supplemented IPL-41 medium containing 6 g L⁻¹ yeastolate ultrafiltrate, 10 g L⁻¹ glucose, 3.5 g L⁻¹ glutamine, 0.5 g L⁻¹ fructose, 2 g L⁻¹ lactose, 0.6 g L⁻¹ tyrosine, 1.48 g L⁻¹ methionine and 1% (v/v) lipid emulsion, reaching 19 × 10⁶ cells mL⁻¹. Maximum specific growth rate and cell productivity were 0.025 h⁻¹ and 0.57 × 10⁵ cells mL⁻¹ h⁻¹, respectively. Glucose and lactose were consumed during cell culture, but not fructose. Lactate concentration generally decreased during cell culture, while ammonium concentration reached 167 mg L⁻¹, however, without noticeable deleterious effects on cell growth. GPV concentration values achieved were, however, modest in the proposed medium formulation.