Research Journal of Biotechnology

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Vol. 7(4) November 2012

Cloning of Acyl-ACP thioesterase FatA from Arachis hypogaea L. and its expression in Escherichia coli

Chen G., Peng Z.Y., Xuan N., Zhang Y. and Bi Y.P.*

In this study, a full-length cDNA of the acyl-ACP thioesterase, AhFatA, was cloned from developing seeds of Arachis hypogaea L. by 3’-RACE. Sequence analysis showed that the open reading frame encodes a peptide of 372 amino acids and showed 50-70% identity with FatA from other plants. Real-time quantitative PCR analysis revealed that AhFatA was expressed in all tissues of A. hypogaea L., but most strongly in the immature seeds harvested at 60 days after pegging. Heterologous expression of AhFatA in Escherichia coli affected bacterial growth and changed the fatty acid profiles of the membrane lipid, resulting in directed accumulation towards palmitoleic acid and oleic acid. These results indicate that AhFatA is at least partially responsible for determining the high palmitoleic acid and oleic acid composition of E. coli.

HPLC Assessment of Production of 6-Aminopenicillanic Acid by Free and Alginate immobilized locally isolated Soil Bacteria

Dolui A. K.*, Bisht Asha1 and Kumar A.

In the present study different bacterial samples were isolated from the soil of different places of Dibrugarh and incubated in the presence of penicillin G (2mg/ml) for biotransformation to 6- Aminopenicillanic acid (6-APA) which was detected by TLC. Two gram positive bacterial samples designated as AKDD-3 and AKDD-8 produced 6-APA. Assessment of production of 6-APA after incubation in penicillin G by two selected samples separately in free and alginate immobilized state was done by HPLC analysis. Biotransformation to 6-APA by immobilized cells was successful after 6 days also.

Comparative antioxidant activity of non-enzymatic and enzymatic extracts of Curcuma caesia Roxb. -an important medicinal plant

Dhal Yogamaya 1, Deo Bandita 1 and Sahu R. K. 2*

Curcuma caesia Roxb. belongs to family Zingiberaceae and shows active antioxidant enzymes like catalase, guaicol peroxidase, glutathione peroxidase and superoxide dismutase which have various biological activities. Both the enzymatic and crude extracts of the rhizome of this plant have been analyzed for their free radical-scavenging activity in different vitro systems, e.g. DPPH radical scavenging activity, hydroxyl radical-scavenging activity. The free radical scavenging activities were compared with a standard antioxidant, ascorbic acid. The DPPH radical scavenging activity was found to be 65.32 ± 0.29 with 625μg/ml of the crude extracts and 31.2± 0.02 with 200μg/ml of the enzymatic extracts respectively. The maximum antioxidant activity was found in catalase, superoxide dismutase and glutathione peroxidase enzyme respectively where as the guaiacol peroxidase shows poor antioxidant activity. The hydroxyl radical scavenging activity was found to be 39.26±0.04 with 50μg/ml of the crude extracts and 26.07±0.01 with 125μg/ml of the enzymatic extracts respectively. Therefore, it is suggested that C. caesia Roxb. could be a potential source of natural antioxidant that could have great importance as therapeutic agent in preventing or slowing down the progressive ageing and age associated oxidative stress related degenerative diseases.

Bacterial and Fungal Burden in Boar Semen

Ciornei Ş.G.1, Roşca P.1* and Drugociu D.2

The aim of this study was to evaluate the bacterial and fungal burden in boar semen samples, before and after their dilution. These were analyzed over a hundred ejaculates from 20 boars living in two farms located in Northeast Romania. The samples were microbiologically tested, both quantitatively and qualitatively, immediately after the harvest and after 12 hours of preservation at 17°C, using current laboratory methods. A current semen analysis for sperm concentration and motility was also performed. For the raw semen, the total viable aerobic count indicated an average of 65.59x103 CFU/mL for the samples collected in farm 1 and 99.52x103 CFU/mL for those collected in farm 2. A total number of 15 bacterial and 9 fungal genera have been identified. After the dilution of all ejaculates, the average total viable aerobic count dropped to 0.21x103 CFU/mL in samples from farm 1 and 0.78x103 CFU/mL in samples from farm 2 respectively. This obvious decrease was due to the semen extenders that contain antibacterial, but not antifungal agents. Particularly important is the fact that after 12 hours of preservation, the fungal burden in diluted semen increased, the yeasts belonging to Candida parapsilosis and other Candida species being identified in more than 90% of samples. All semen extenders contain glucose which can be used by yeasts for their development and multiplication during storage at 17°C for 5-7 days, with possible negative effects on semen quality and fecundity.

Water Deficit-Induced Oxidative Stress in Leaves of Garden Orach (Atriplex hortensis)

Sai Kachout S. 1*, Karray Bouraoui N. 1, Jaffel K. 1, Rejeb M.N. 2, Leclerc J.C. 3 and Ouerghi Z. 1

Drought stress is considered as a restricting factor for plant products. Plants differ in their ability to tolerate water stress. Atriplex hortensis is a xero-halophyte, C3 shrub. This study examined the influence of drought on antioxidant activity, growth and content of carotenoids and anthocyanins of the A. hortensis leaves using two varieties: green orach (var. purpurea) and red orach (var. rubra). To study the enzymatic response of the Atriplex plants against water deficit, antioxidant enzymes in the leaves were analyzed, such as superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX) and glutathione reductase (GR). We observed that under water stress both varieties decreased the activity of SOD in leaf tissue, especially in plants grown in severe water deficit. In the exposure to moderate and mild drought stress, more reactive oxygen species might promote the increase in activities of antioxidant enzymes such as CAT and APX, as found in leaves of the Atriplex plant. Regarding GR, a significant decrease was observed only in A. hortensis var. purpurea, this antioxidant could be important in protecting against oxidative stress triggered by water deficit. In this report we examined the effect of water stress on both carotenoids and anthocyanins content. In particular, water deficit decreased content of carotenoids. It also decreased the concentration of anthocyanin pigments in leaves of both Atriplex. Result show that water stress decreased growth of two varieties significantly in this study. Drought effects on enzyme activities in leaves vary widely, depending on the plant varieties; however, in var. rubra, we observed a much better capacity to preserve the photosynthetic apparatus against overproduction of ROS.

Production and characterization of α-amylase isolated from Aspergillus niger by Solid State FFeerrmmeennttaattiioonn

Take Ajaykumar M.1* and Kharat Rajhans G.2

Enzymes have their own importance in food science. In order to fulfill the increasing demand of α amylases at the level of industries, cost effective procedures should be applied in α-amylase production. α- amylase was produced by Aspergillus niger using the techniques of solid state fermentation. Solid state fermentation techniques are gaining importance in commercial processes to produce a wide variety of enzymes mainly from fungal origin. Among the carbon source tested, wheat bran was found to be most suitable for α amylase production. It may be due to its high carbohydrate content and its buffering capacity. The wheat bran medium was optimized for amylase production. The kinetic properties of amylase produced were studied. It was observed that the amylase produced has optimum pH at 4.0. The enzyme showed maximum activity at 30°C. The activation energy and Michaelis Menten constant (Km) were calculated by taking variations in substrate concentration and reaction temperature.

Water Deficit-Induced Oxidative Stress in Leaves of Garden Orach (Atriplex hortensis)

Kachout S. Sai 1*, Bouraoui N. Karray 1, Jaffel K. 1, Rejeb M.N. 2, Leclerc J.C. 3 and Ouerghi Z. 1

Drought stress is considered as a restricting factor for plant products. Plants differ in their ability to tolerate water stress. Atriplex hortensis is a xero-halophyte, C3 shrub. This study examined the influence of drought on antioxidant activity, growth and content of carotenoids and anthocyanins of the A. hortensis leaves using two varieties: green orach (var. purpurea) and red orach (var. rubra). To study the enzymatic response of the Atriplex plants against water deficit, antioxidant enzymes in the leaves were analyzed, such as superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX) and glutathione reductase (GR). We observed that under water stress both varieties decreased the activity of SOD in leaf tissue, especially in plants grown in severe water deficit. In the exposure to moderate and mild drought stress, more reactive oxygen species might promote the increase in activities of antioxidant enzymes such as CAT and APX, as found in leaves of the Atriplex plant. Regarding GR, a significant decrease was observed only in A. hortensis var. purpurea, this antioxidant could be important in protecting against oxidative stress triggered by water deficit. In this report we examined the effect of water stress on both carotenoids and anthocyanins content. In particular, water deficit decreased content of carotenoids. It also decreased the concentration of anthocyanin pigments in leaves of both Atriplex. Result show that water stress decreased growth of two varieties significantly in this study. Drought effects on enzyme activities in leaves vary widely, depending on the plant varieties; however, in var. rubra, we observed a much better capacity to preserve the photosynthetic apparatus against overproduction of ROS.

In Vitro Propagation and Assessment of Plant Genetic Fidelity by RAPD and SSR Markers in Gerbera

Ghani Minerva *, Kumar Surinder and Thakur Manisha

Department of Biotechnology Department, University of Horticulture and Forestry, Solan (H.P.), INDIA *minervaghani@yahoo.in

In the present study, an attempt was made to develop an efficient technique to regenerate plants from petiole explants of Gerbera jamesonii cv.`Harley’ in the year 2009. The explant segments (0.5-1 cm) were cultured on Murashige and Skoog (MS) medium supplemented with 4 mg/l BA + 1 mg/l TDZ. The growth regulator free medium was ineffective in producing shoots on the petiole explant. The regenerated shoots were multiplied on MS medium supplemented 1 mg/l BA + 0.5 mg/l IAA and 4% sucrose. The shoots were separated and the individual shoot was rooted with 4 mg/l IBA. The rooted plantlets were hardened with 90% survival success after six months of transfer in the pots. The genetic fidelity of the plants was assessed using RAPD and SSR markers. 10 RAPD and 10 SSR primers used, produced clear, reproducible and scorable bands. All banding profiles from micropropagated plants were monomorphic and similar to those of the mother plant. A similarity matrix revealed that the pair-wise value between the mother and the in vitro raised plants was 1, indicating 100% similarity which confirmed the true-to-type nature of the in vitro-raised plants.

Optimization of Nutritional Factors for Extracellular Amylase Production from Bacillus cereus MCM B-326 using Response Surface Methodology

Zambare V.P., Nilegaonkar S.S. * and Kanekar P.P.

The optimization of nutritional factors and their concentrations for the amylase production by Bacillus cereus MCM B-326 in submerged fermentation was carried out using response surface methodology (RSM) based on the central composite design (CCD). The design contains a total of 20 experimental trials containing starch, soybean meal and CaCO3 as model factors for three levels. The mutual interaction between these variables resulted into 1.36 fold increase in amylase activity as compared to the mean predicted response at zero level of all variables. Amylase from B. cereus has approximate molecular weight of 40 kDa with optimum activity at pH 7.0 and temperature 30°C.

Isolation and Characterization of Perchlorate respiring Strains of Proteobacterium and Pseudomonas aeruginosa species from Perchlorate enriched effluent sludge

Muruganandam L.* and Anoop Raj J.R.

Perchlorate is a toxicant when discharged into the environment creates serious implications on human beings. In order to carry out perchlorate remediation, microbial screening has been carried out for the identification of perchlorate degrading isolates from enriched culture media. Two strains were isolated and named ARJR SMBS and LMN SMBS. These are the first perchlorate respiring bacteria (PRBs) which come under Gamma Proteobacteria outside of the phylogenetic groupings of known PRBs. The secondary structure of the bacterial 16S rDNA and restriction site analysis was predicted using Genbee and NEB cutter software. The results show that strain ARJR SMBS contains GC:AT in the ratio of 55:45 with free energy of structure -338.9 kkal/mol whereas the strain LMN SMBS contains GC:AT in the ratio of 54:46 with free energy of structure -323.5 kkal/mol. Phylogenetic tree analyses of 16s rRNA gene sequences revealed that strain ARJR SMBS belongs to the class of gamma Proteobacteria and strain LMN SMBS belongs to pseudomonas species. Growth curve and perchlorate degradation efficiency were studied and strain ARJR SMBS was found to have better degradation efficiency compared to strain LMN SMBS. This study can be summarized as mild growth inhibitory effect of perchlorate with potential perchlorate degrading isolates ARJR SMBS and LMN SMBS. Probably in future, strain ARJR SMBS would be relevant for the design of perchlorate remediation systems.

Extraction and Deproteinization of Cucumber Polysaccharide with High Scavenging Ability toward Superoxide Anion

Huang Gangliang *, Cheng Fei and Ding Xiang

Preparation and antioxidant activity of polysaccharide from cucumber (Cucumis saticus L.) were investigated. The crude polysaccharide was prepared by the method of hot-water extraction. The percentages of deproteinization and polysaccharide loss were compared as indexes using the trichloroacetic acid (TCA) method and CaCl2 method respectively. The infrared (IR) spectra analysis and content analysis showed that the TCA method exhibited the higher percentage of deproteinization and a little higher percentage of polysaccharide loss than the CaCl2 method. The scavenging ability of cucumber polysaccharide toward superoxide anions was evaluated by means of chemiluminescence (CL). The results showed that the cucumber polysaccharide had high scavenging ability toward superoxide anion.

In Vitro Plant Regeneration of Cochlospermum Religiosum through Shoot Tip Culture

Sasikala A.* and Savithramma N.

Shoot induction in Cochlospermum religiosum was achieved from shoot tip explants on Murashige and Skoog’s (MS) medium fortified with 2 mg 1-1 BAP. The shoots were sub cultured after two weeks on same concentration of BAP for multiple shoot induction. After four weeks the shoots were excised and sub cultured for rooting on half strength M.S. medium fortified with BAP (0.5 mg l-1 ) and IAA (2 mg l -1 ). The in vitro generated plantlets were successfully acclimatized in pots containing vermiculite and soil (1:3) with the survival rate of 50 to 60%.

Gellan Gum a novel polysaccharide matrix for Immobilization of thermo-tolerant yeast cells with Invertase activity: factorial design and rheological studies

Karandikar Sulabha2 and Prabhune Asmita1*

The microbial extracellular polysaccharide gellan gum has been explored for its application in whole cell immobilization. Preliminary evaluation of the physicochemical properties of gellan gum in the presence of monovalent cation tetramethyl ammonium chloride (TMACl) was carried out. Gellan gum immobilized cells demonstrated better rheological properties than gellan gum. Attempts have been made to reveal the evaluation of gellan gum as a matrix to immobilize a thermotolerant yeast Kluyveromyces marxianus NCYC 2675. A 32 factorial design was used to study the simultaneous effect of two variables. The effect of the polymer concentration and TMACl concentration on various dependent variables like gelling temperature, mean particle size and enzyme activity of the yeast cells was studied. A statistical model with a significant interaction term was obtained to predict the results. Further, optimized immobilized system was evaluated with significant storage stability at 10ºC and enhanced reusability with maximum conversion of > 90 % when cross-linked with glutaraldehyde.

Obtaining Deoxyribonucleases from Soil Bacterium belonging to Aeromonas Species

Kamble K. D.* and Gadakh P.V.

In search of novel chemotherapeutic compounds bacterial cells are directly lysed and the DNA is expressed without any attempt of culturing. Fastidious microorganism predominate the routinely employed laboratory conditions, still other bacteria inspite of the natural potential for industrial or chemotherapeutic applications may escape culturing without any special growth requirement. Our attempt was to characterize a novel extracellular de-oxyribonuclease producing bacterium from soil; as soil is the richest source of microorganisms. The bacterium characterized is an efficient DNase producing strain obtained from farm soil of Western India; a typical subtropical soil wherein temperature ranges from 30°C to 35°C. Morphological, physiological and biochemical properties match to the genus Aeromonas, however 16S rRNA sequence analysis of the strain also showed closest match to Aeromonas species and few uncultured bacteria (>99.1%).

In Vitro Micropropagation of Different species of Citrus

Kumar Gaurav1* and Srivastava Richa 2

In vitro approaches became necessary for overcoming the hurdles of cultivation of Citrus, a commercially important fruit. The present study deals with establishment of protocol for micropropagation of six different species of Citrus viz.Citrus sinensis, Citrus jambhiri, Citrus limonia, Carrizo citrange, Citrus limon and Citrus pectinifera via callus induction and regeneration. Leaf segments, epicotyl, shoot apex and axillary bud segments excised from in vitro raised seedlings were used as explants. The Explants were cultured on Murashige and Skoog medium (MS) containing 30 g/L sucrose and 8 g/L agar supplemented with different concentrations and combinations of different phyto-hormones; 6-benzylaminopurine (BAP), naphthalene acetic acid (NAA), 2,4-dichloro phenoxy acetic acid (2,4 D). The maximum callus induction was observed from axillary bud of Citrus limonia observed on MS medium supplemented with 2,4 D (3 mg/L) followed by MS medium supplemented with BAP (3 mg/L). Maximum shoot regeneration response (75%) was observed on MS medium supplemented with BAP (3 mg/L) in Carrizo citrange. The in vitro regeneration system developed in this study can be used for effective propagation of selected genotypes and can be an ideal source of homogenous material for regeneration of genetically modified plants.

Penicillium expansum SK 16 as a novel inulinase-producing strain isolated from decompoosed Dahlia tubers

Maria Rosa Vela Sebastiao Fernandes, Consolate Nsabimana and Bo Jiang*

The objective of this research was to isolate from decayed Dahlia tubers a new fungal strain with a high ability to produce inulinase. In fact, three different fungi have been screened and isolated based on their capacity to grow on a simple culture medium containing inulin. The three strains were analyzed for inulinase activity and one of them showed the best results. Based on the morphological, physiological and biochemical characteristics, this strain was identified as Penicillium expansum SK 16 by China Center Type Culture Collection (CCTCC). The gene sequence for Penicillium expansum SK 16 has been deposited to the NCBI GenBank database under the accession number GU561988.

Establishment and analysis of in vitro fast-growing normal root culture of Taverniera cuneifolia (Roth) Arn: a substitute for commercial liquorice

Jamdhade Vijay C.,* Balkhande Shrivardhan C. and Surwase Babasaheb S.

Taverniera cuneifolia (Roth) Arn. is popularly known as Indian liquorice. Roots of this plant are used as a substitute for the commercial liquorice-Glycyrrhiza glabra. The roots of G. glabra are rich in bioactivities like antiulcer, anti-inflammatory, antibacterial, antimalarial, antithrombic, antidiuretic, antitherro sclerotic, antifungal, estrogenic, antiallergic, antidi-abetic and antimutagenic activities. T. cuneifolia possesses considerable in vivo anti-inflammatory, protective activity from EMS induced toxicity in Salmonella typhumurium, inhibition of Agrobacterium induced tumors and antifungal activity. Fast-growing normal root culture of Taverniera cuneifolia was established from leaf explants obtained from in vitro raised seedlings in ½ MS. Explants were cultured on half-strength MS medium supplemented with different concentrations of indole-3-acetic acid (IAA), indole-3-butyric acid (IBA), naphthalene acetic acid (NAA), and Coconut water (CW). Leaf explants incubated in total darkness in half-strength MS medium supplemented with 2mg/l IBA+10% CW (Coconut water) favored induction of roots with highest biomass of roots (14 gm/ explant on fresh wt basis) after six weeks. However, supplement of NAA and IAA to half strength MS medium did not enhance the root production. HPLC analysis of in vitro fast-growing normal roots of Taverniera cuneifolia confirmed the presence of a sweetening principle Glycyrrhizin.

Anthelmintic activity of rhizome extract of Acorus calamus L. in comparison with beta and alpha asarone

Asha Devi S., Ganjewala Deepak and Subramanian Babu*

The study was done to evaluate the anthelmintic activity of rhizome extract of Acorus calamus in comparison with commercial beta and alpha asarone against earthworms. Different concentrations of asarone (1-5 mg/mL) and rhizome extract (2-10 mg/mL) were studied. The evaluation parameters involved the determination of time of paralysis and time of death of the worms. Piperazine citrate and alpendazole was used as standard at 10 mg/mL concentration and saline as control. The results were dose dependent and higher activity was observed in beta asarone at 5 mg/mL concentration compared to alpha asarone and rhizome extract.

Efficacy of Maximum Likelihood Method and longer Sequences in Phylogenetic Analysis

Shanker Asheesh and Sharma Vinay *

The usefulness of any molecular phylogenetic method depends on the availability of tools to infer accurate trees. Different methods proposed to reconstruct the trees include distance, parsimony and maximum likelihood method. To check the efficacy of tree construction methods we analyzed rRNA (5S, 16S and 23S) and protein sequence data from chloroplast genomes. Additionally effect of small and longer sequences in tree construction was tested. Three methods were used to construct the trees from chloroplast sequences. The most accurate tree obtained from 23S rRNA sequences confirms the utility of longer sequences in phylogenetic analysis. Moreover the maximum likelihood method was found to outperform distance and parsimony methods in generating accurate tree topology. Our analysis also demonstrates the use of appropriate model of sequence evolution during tree construction.

Isolation and Characterization of Thiosulphate utilizing Delftia sp. from bulk polymetallic concentrate leachate

Dave S.R.*, Varjani S.J. and Tipre D.R.

Polymetallic concentrate leachate sample showed extreme pH, redox potential, total dissolved solids (TDS) and conductivity. From this extreme ecosystem, screening of thiosulphate utilizers led to isolation of 9 distinct thiosulphate utilizing organisms. Among the isolates, gram negative short rod was selected for study. Cultural, morphological and biochemical characters and antibiotic sensitivity of the isolate were studied. After 72 h of incubation thiosulphate utilized was 93.34%, which resulted in increase of medium pH from 4.5 to 7.0. This indicates that this organism can be used to control the acidity generated by acid mine drainage (AMD). It was observed that the isolate showed optimum growth in well aerated condition. The isolate grew from pH 1 to 7 and salt concentration from 1% to 7%. The growth of the isolate was studied in presence of copper, zinc and lead. The concentrations of CuSO4, ZnSO4 and Pb(NO3)2 selected were 0.5% to 2.5%, 1.0% to 10.0%, and 0.5% to 2.0% respectively. On the basis of 16S rRNA gene sequencing the isolate was identified as Delftia sp. and gene sequence was deposited in GenBank under the accession number HQ398998 as Delftia sp.SRSTH5.

Protective efficacy of Moringa oleifera during aflatoxin exposure in broilers

Umaya Suganthi Rajendran *, Parvatham Raju and Vandana Thimmaiah

A study was conducted to evaluate the efficacy of Moringa oleifera leaves (MOL) in protecting against the toxic effects of aflatoxin in broilers. A total of 120 one day old Ross broiler chicks were divided into four groups: G1: Control (basal diet); G2: Basal diet +3mg/kg feed M.oleifera leaves (MOL); G3:AFB1 (100μg/kg feed); AFB1+ MOL (3mg/kg feed) for 42 days. The addition of MOL to 100ppb AFB1 containing diet significantly reduced the adverse effects of aflatoxin on blood biochemical parameters and liver lipid peroxidation and antioxidant status in broilers. These results suggest that MOL can be beneficial in broilers for protection of aflatoxin toxicity at the level above.

Statistical optimization of process parameters Influencing the biotransformation of limonin by Pseudomonas putida G7

Malik Meenakshi, Ganguli Abhijit and Ghosh Moushumi*

Response surface methodology (RSM) was used to optimize the cultural parameters for the enhanced limonin biotransformation by Pseudomonas putida G7. First, screening of significant variables [temperature, pH, incubation time and (NH4)2SO4 (1gm/L)] which enhances limonin biotransformation was done using Plackett-Burman (PB) design, among the 11 variables. Secondly, 24 factorial central composite design (CCD) and RSM were applied to develop a mathematical model for the detection of the optimum concentration of the significant variables and confirm its authenticity experimentally. A regression analysis of the experimental values was done to determine second order polynomial, in order to understand the relationship between the limonin biotransformation and significant variables. The optimum values for the critical variables were obtained as temperature 300C, pH 8.0 and inocubation time 34 hours and (NH4)2 SO4 1gm/L with a predicted and experiemental value of maximum limonin biotransformation upto 68.88% and 70.3 % respectively. The regression coefficient R2 (0.94) ensures the adequate integrity of the model. The enhanced limonin biotransformation ability of P.putida G7 by optimizing the cultural parameters using statistical tools suggests a suitable method to enhance the debittering of citrus juices.

Identification of host plant resistant to Dolichos Yellow Mosaic Virus (DYMV) in Dolichos bean (Lablab purpureus)

Singh P. K.1, Ashish Kumar1,2*, Rai N. 1 and Singh D. V. 3

Three hundred Dolichos bean (Lablab purpureus) genotypes were screened against Dolichos Yellow Mosaic Virus (DYMV) disease. Initial screening was done under field conditions where disease incidence was calculated for each genotype. Subsequently, selfed progenies of 34 symptomless lines were challenged by sap inoculation under field conditions, out of which only three genotypes, viz.VRSEM-894, VRSEM-887 and VRSEM-860 did not show any symptoms. Using root stalk of susceptible genotype (Ankur Goldy), these three putative symptomless genotypes were further challenged by grafting. The resistant reactions of VRSEM-894, VRSEM-887 and VRSEM-860 were confirmed as even after 60 days of successful grafting, no viral symptom appeared on all the grafted plants of these genotypes. When subjected to PCR amplification with DYMV coat protein gene specific primer, these three symptomless genotypes did not show any amplification, suggesting that there was no infection of Dolichos Yellow Mosaic Virus in those genotypes.

In Vitro Plant Regeneration of Cochlospermum Religiosum through Shoot Tip Culture

Sasikala A.* and Savithramma N.

Shoot induction in Cochlospermum religiosum was achieved from shoot tip explants on Murashige and Skoog’s (MS) medium fortified with 2 mg 1-1 BAP. The shoots were sub cultured after two weeks on same concentration of BAP for multiple shoot induction. After four weeks the shoots were excised and sub cultured for rooting on half strength M.S. medium fortified with BAP (0.5 mg l-1 ) and IAA (2 mg l -1 ). The in vitro generated plantlets were successfully acclimatized in pots containing vermiculite and soil (1:3) with the survival rate of 50 to 60%.

In vitro cytotoxic activity of methanolic extract of stem bark ooff Cassia fistula L.

Mathew Linu1* and Shashidhar Shankar2

As part of a permanent screening programme, which considers the search for plants and natural products with anticancer properties, the plants are subjected to bioscreening assay testing for cytotoxity. Another crucial component of pre-clinical oncology drug development is the study and monitoring of cell death in tumour and normal tissues. Therefore, methanolic extract stem bark of cassia fistula L was tested for in vitro cytotoxicity and apoptogenic potential by MTT assay, DAPI assay, mitosensor assay and caspase assay.

Influence of herbal greens, Murraya koenigii, Coriandrum sativum and Menthe arvensis on growth performance of the freshwater prawn Macrobrachium rosenbergii post larvae

Saravana Bhavan P.,* Manickam N. and Radhakrishnan S.

In this study, evaluation of growth promoting ability of curry leaves (Murraya koenigii), coriander leaves (Coriandrum sativum) and mint leaves (Menthe arvensis) on Macrobrachium rosenbergii post larvae (PL) was performed in a sustainable manner. Feeds were formulated with soya bean meal, groundnut oil cake, cow gram, wheat bran and maize bran as basal ingredients. Tapioca flour and egg albumin were used as binding agents. Sunflower oil was added as lipid source. Vitamin B-complex with vitamin-C and a pinch of salt were also mixed. Diets with incorporation of M. koenigii, C. sativum and M. arvensis at 3%, 7% and 10% of each were served as experimental feeds. Diet without incorporation of these greens was served as control. These feeds were fed to M. rosenbergii PL in a triplicate feeding trial conducted for a period of 45 days (PL30-75) under laboratory condition. The growth performance in terms of morphometric data, nutritional indices (weight gain, specific growth rate and survival rate except condition factor), energy utilization parameters (feeding rate, absorption rate, conversion rate, ammonia excretion rate and metabolic rate), activities of digestive enzymes (protease, amylase and lipase), concentrations of biochemical constituents (total protein and amino acid except carbohydrate and lipid) and levels of non-enzymatic antioxidants (vitamin C and E), particularly in 7% of these greens incorporated feeds fed PL groups were all found to be significantly (P<0.1; P<0.05) increased/ improved when compared with control. Among the three greens tested, M. koenigii incorporated feed produced the best growth performance followed by C. sativum and M. arvensis. Among three concentrations tested in each green, change was in the order of 7% incorporations > 10% > 3%. It is suggested that these greens up to 7% can be incorporated in aqua feed formulations for sustainable development of Macrobrachium culture.

Small Molecule Inhibitors of PTP1B and TCPTP

Sekhar Reddy M.V.V.V. 1, Chakshusmathi G. 1 and Lakshmi Narasu M. 2

Fragment based drug design is a new approach in identifying the initial chemical starting point for drug discovery programs. Fragment based drug design allows screening of substantially fewer compounds usually hundred - thousand compounds. It identifies fragments which bind specifically but with low affinity in the range of 0.1-10mM. The small sized fragments make the subsequent optimization relatively easier to build the molecule by exploring the chemical space in the binding pocket. The virtual screening study of small molecule compounds from a chemical library was carried out and few molecules were selected as inhibitors of PTP1B/TCPTP. These molecules were tested by in-vitro biochemical assay and were inhibiting both PTP1B and TCPTP. One of the compounds AU- 008 was found selective to PTP1B over TCPTP by 3 fold.

Enhanced production of gymnemic acid using HR bioelicitor extracted from Xanthomonas spp.

Subathra Devi C. *, Nandi Ipsita, Mohana Srinivasan V. and Sriramkalyan P.

Use of Gymnema sylvestre, commonly known as periploca of woods, an Indian medicinal woody climber has increased recently due to the pharmaceutical potential of gymnemic acids, found in its leaves. Gymnemic acids have been reported to effect a natural treatment for diabetes. This study developed a novel cell culture system for in vitro growth and production of this species, suggesting a possible technology for large scale production of gymnemic acids. Leaf explants grown in Murashige and Skoog salts supplemented with IAA 1.5mg/l and BA 0.5mg/l gave maximum percentage of callus formation compared to other treatments evaluated. The growth rate and gymnemic acid accumulation in the callus suspension culture was determined. The HR protein from Xanthomonas spp. was used as an elicitor for the production of gymnemic acid. When compared to non-elicited cultures, two fold increase of gymnemic acids yield in elicited cultures was observed. The quantification of gymnemic acid was done using HPLC. The total gynemagenin after 21st day of incubation was 30.2389mg/100ml.

Variation in Morphological Parameters and Andrographolide Content in Andrographis paniculata (Burm.f.) Nees collected from Different Provenances of Chhattisgarh

Minz Preeti Lata and Koche Vijaya*

Kalmegh (Andrographis paniculata (Burm.f.) Nees) leaves constitute a high amount of vigorous compounds, namely diterpenes lactones, signifying that it could be useful in the prevention of several diseases. It is used in different traditional systems of medicine and exhibits anti-inflammatory, anti-HIV, antibacterial, antioxidantantidibietic, anticarcinoge- nic, antipyretic, hepatoprotective, nematocidal and various other activities. The present study was planned to determine the morphological parameters and variation of andrographolide contents to acquire superior genotypes collected from five different provenances (Ambikapur, Jagdalpur, Kawardha, Korba and Raigarh) of Chhattisgarh. The average amount of dry weight per samples was between 10.0 to 43.0 g. The total andrographolide content varied from 0.08-0.5% in the dried samples of A. paniculata. The variation in growth parameters and andrographolide content among the plants collected from different provenances were statistically significant. Quantification of andrographolide was determined by HPLC which revealed that the variation in the andrographolide content is highest in the Korba provenances than the plants from the other provenances. The results indicated that these populations are potentially important sources for breeding, improvement of genotypes and best sources for obtaining higher drug yield.

Molecular cloning, characterization and functional validation of TPS1 gene isolated from S. cerevisiae

Varalaxmi Yellisetty 1, Ananda Reddy L. 2, Yadav Sushil Kumar 1 and Maheswari Mandapaka1*

A new sequence of trehalose-6-phosphate synthase (TPS1) gene was cloned from strain MTCC No. 174 of Saccharomyces cerevisiae. Sequence analysis and putative protein characterization revealed its homology with the TPS1 gene sequences in many eukaryotic and prokaryotic organisms. The TPS1 protein was expressed in E.coli and purified. This sequence was used as an exotic gene to construct a plant expression vector pCAMBIA1303TPS1 and functionally validated in tobacco. Transient GUS expression assays and molecular characterization using PCR, RT-PCR and Southern confirmed the integration and expression of TPS1 gene. Phenotypic alterations such as lancet shaped leaves and increase in root growth were observed in TPS1 transgenics of tobacco. Seed (T1) germination as well as root and shoot growth of seedlings was higher in transgenics of tobacco when challenged with 200mM NaCl. These results clearly demonstrate the isolation, cloning, expression and functional validation of TPS1 gene.

Protective role of Ghrelin in lipopolysaccharide-induced kidney dysfunction syndrome in rat

Cheraghi Javad 1*, Hashemi Ehsan 2, Daliri Morteza 2, Abdollahipoor Ehsan 3, Hafezi Mohamadreza 4 , Taati Mjid 5 , Zendehdel Morteza 5 and Hooshmandfar Reza 1

Ghrelin is produced in the stomach and acts on growth hormone (GH) secretagogue receptors in hypothalamic regions to potently increase food intake and body weight, with additional effects on blood glucose levels and inflammation. In this study we investigate the role of ghrelin in endotoxemia caused by lipopolysaccharide (LPS) in rats. Fifty young matured male wistar rats were used. Rats were divided as per treatment into 4 and kept 2 as control group. Animals received a final concentration of 20mg/kg B.W LPS and 65μg/kg B.W ghrelin alone and or together at various time intervals by route of intraperitoneal injection. A saline-treated and ghrelin-treated animal served as controls. Biochemical and histopathological examination of blood before sacrificing the animals revealed endotoxemia associated with LPS with a significant rise in the serum levels of creatinine, urea, uric acid, and BUN (p<0.05) leading to renal dysfunction where administration of ghrelin significantly decreased serum levels of uric acid, creatinine, urea, and BUN (p<0.05) and counteracted these alterations induced by LPS.These findings indicate that ghrelin may play role to prevent endotoxemia i.e. lipopolysaccharide-induced kidney dysfunction syndrome in rats.

Antioxidant activity and Inhibitory potential of few Indian medicinal plants against key hyperglycemic enzymes

Majumdar Mala

This study evaluated the in vitro antioxidant potential and inhibition of α-amylase and α-glucosidase activities of methanolic extracts of leaves of few Indian medicinal plants. The antioxidant activities of the samples were measured by two different methods while the Folin– Ciocalteu reagent assay was used to estimate the phenolic contents of extracts. Methanolic extracts from dried leaves of P. emblica, C. cajan, C. indica and F. pratensis were prepared. Antioxidant activities were estimated by DPPH radical scavenging and iron chelating methods. The inhibition of carbohydrate-hydrolysing key enzymes e.g. α-amylase and α-glucosidase activities were assayed. Acarbose was used as reference compound in both the assays. Experimental results are presented as the mean± (SEM) of triplicates. Following order C.indica ≥ C. cajan >F.pratensis > P.emblica was observed with both DPPH and iron chelating methods. Observed α amylase inhibitory effect was F.pratensis (74.9%), C.indica(59.52%), P.emblica (55.33%) and C.cajan (48.74%) at a concentration of 200μg/ml. C.indica (86.9%) exhibited highest α-glucosidase inhibition followed by C.cajan(70.23%), F.pratensis(26.19%) and P.emblica(5.95%). The inhibition of these enzymes can lead to lower postprandial blood glucose. Based on the results, C. indica exhibiting high antioxidant and enzyme inhibition should be further investigated to identify bioactive compounds.

Induction of Systemic Resistance by Mixtures of Rhizobacterial Isolates against Pythium aphanidermatum

Boominathan U. and Sivakumaar P.K.*

The efficacy of seven Rhizosbacterial isolates was tested for their ability to inhibit the growth of Pythium aphanidermatum, the causal agent of turmeric rhizome rot. In vitro studies revealed that Pseudomonas flurocense (PF25) and Bacillus megaterium (BM29) showed the highest inhibition of Mycelial growth (72.4%; 76.2%) of P. aphanidermatum. Both the antagonists were compatible with each other and they were tested alone and together in vivo for the control of P.aphanidermatum. Besides, the induction of defense- related enzymes such as peroxide (PO), polyphenoloxidase (PPO), phenylalanine ammonia-lyase (PAL), B1, 3-glucanase and the accumulation of phenolics in turmeric rhizomes due to the application of bioagents were also studied. Combined application of Rhizobacteria and challenge inoculated with P. aphanidermatum recorded maximum induction of defense- related enzymes and accumulation of phenolics compared with individual application. This study suggests that the increased induction of defense – related enzymes (4 to 5 fold) and phenolic content (6 fold) due to combination treatment of bioagents might have involved in the reduction of rhizome- rot incidence and in turn increased the plant growth and yield of turmeric.

Microwave-assisted extraction and in vitro antioxidant activity of Monascus mycelium polysaccharides

Wang Pengrong, Jiang Donghua*, Dong Xiameng, Ji Hao and Panpan Chen

Response surface methodology (RSM) was used to optimize the microwave power, extraction time and ratio of water to raw material to obtain a high polysaccharides yield from Monascus mycelium (MMP) by microwave-assisted extraction technique. The optimum extraction conditions were as follows: microwave power 440 w, extraction time 3.6 min and ratio of water to raw material 28 ml/g. Under these conditions, the experimental yield was 11.82%, well matched with the predicted models with the coefficients of determination (R2) of 0.9949. Furthermore, in vitro antioxidant activities of MMP were investigated including superoxide radical, hydroxyl radical and 2, 2-diphenyl-1-picrylhydrazyl (DPPH) radical. The antioxidant results showed the MMP exhibited strong hydroxyl radical (73.26% at 100 mg/L) scavenging activity, moderate superoxide anion radicals (83.02% at 0.875 mg/ml) and DPPH radicals (92.33% at 3 mg/ml) scavenging activities.

Distribution of virulence genes in A. hydrophila and A. sobria in L. rohita (Rohu Fish) in N.E India

Sharma Indu¹* and Kumar A.2

A multiplex PCR assay was designed to amplify the Aeromonas hydrophila and A. sobria for hemolysin and aerolysin genes. The assay was used to evaluate 137 fish samples (Labio rohita) procured from retail shops (Meghalaya, India) of which 18 Aeromonas isolates were isolated. It was observed that 3 isolates of A. hydrophila (16.6%) were ahh1 positive which included 15 (83.3%) of the A. hydrophila isolates, 2 (11.1%) A. sobria isolates and 1 (5.5%) A. caviae isolate were examined. Amplicons for AH-aerA primer set were produced only by A. hydrophila isolates [1 of 18 (5.5%)]. Fourteen of the 18 Aeromonas isolates (77.7%) were asa1 positive. Majority of the isolates produced haemolysin. Out of the 18 isolates of Aeromonas spp. isolated, 12 (66.6%) of them exhibited haemolytic activity.

Production of Naringinase by a new soil isolate of Serratia Sp.: Effect of different carbon aanndd nitrogen sources

Pavithra M.,* Prasanna D. Belur and Saidutta M.B.

Four strains of Naringin degrading bacteria were isolated and tested for naringinase activity. All the four isolates showed extracellular naringinase activity. The one which showed consistently good activity in three different media was selected (2 U/L) and identified by phenotypic characterization as Serratia Sp. In shake-flask trials, effect of various carbon and nitrogen sources was studied. Among all the carbon sources, glucose enhanced the naringinase production. Peptone supplemented with ammonium nitrate was found to be favourable. Maximum of 9.2 U/L naringinase activity was achieved in the medium comprising naringin, glucose, peptone, ammonium nitrate and salts.

Antimicrobial, Phytochemical and Antioxidant assay of Bauhinia variegata (Kachnaar) extracts against some pathogenic microbes

Dhaka Divya and Punia Anita*

Phytomedicines are cost effective, safe and renewable herbal medicine so these have been used as alternative medicine for ancient time. Phytoconstituents present in these medicinal plants are responsible for antimicrobial and antioxidant activity which consequently enhance immune function. The various organic extracts of leaves of Bauhinia variegata were prepared. The antimicrobial activities of extracts were evaluated and compared by Agar well diffusion method against human pathogens such as Escherichia coli, Streptococcus mutans, Staphylococcus aureus, Candida albicans, Pseudomonas aeruginosa. The propanol extract showed wide range of activity on these pathogens following by butanol and acetone. Hexane and ethyl acetate (MIC-1mg/ml) extract were most effective against E. coli followed by acetone and chloroform. Bioactive compounds revealed by phytochemical screening were saponins, tannins, flavonoids, cardiac glycosides and steroids. Free radical scavenging capacity was evaluated by reducing power assay which demonstrated a correlation between concentration of extract and antioxidant potential.

Genetic variation in ecoraces of eri silkworm Philosamia ricini (Lepidoptera: Saturniidae) using RAPD technique

Ray Priti Pragyan* and Rao T.V.

The genetic diversity and phylogenetic relationships of 150 individual Eri silkworm Philosamia ricini (Hutton; Lepidoptera : Saturniidae) ecoraces namely Borduar, Mendipathar and Titabar reared largely by farmers of North Eastern India, particularly states of Assam and Meghalaya were studied using RAPD analysis. Polymorphisms within and between ecoraces were assayed using 60 random primers and182 loci were amplified ranging from 350bp to 2500bp. The percentage of polymorphic loci was found to be 35.22%, 17.3% and 16.2%, for Borduar, Mendipathar and Titabar ecoraces respectively. The total genetic diversity was 0.231, total genetic differentiation was 0.61 and the total gene flow among the ecoraces was calculated to be 0.319. Genetically, the Borduar ecorace was more closely related to Mendipathar ecorace than to the Titabar ecorace. The higher genetic variability (82%) between the ecoraces using RAPD markers, suggest that these ecoraces have been differentiated into separate gene pools. These different gene pools should be conserved separately and maintained without any intermixing. Genetically distinct genotypes identified in the present study could be potential sources of germplasm for Eri silkworm improvement.

Comprehensive evaluation of target genes conferring salinity stress tolerance to Debaryomyces hansenii by transcriptomic analysis

Chao Hsiu-fung1 and Yen Yung-fu2*

Debaryomyces hansenii is one of the most salt-tolerant species of yeast and has become a model organism for studying salinity tolerance mechanisms. This study examines genomic responses to NaCl using yeast DNA microarrays to indentify the specific D. hansenii genes involved in salinity response. Expression profiles reveal that exposure to 2.5 M NaCl for 24 min induced changes in gene expression in D. hansenii. Among the 10.8% of D. hansenii genes showing statistically significant differences in expression, salt increased expression levels by at least two-fold in 186 genes and decreased expression levels two-fold in 85 genes. This study classifies differentially expressed genes according to their expression ratio and their function and compares them to a control using web-based ErmineJ and GOEAST. Validation studies for SSB, FRS1, PRR1, MLH3, PH085, DAK2, YCK1 and SSA using real-time PCR, confirmed the patterns of gene expression observed in microarray experiments. One of the salt-induced genes, SSA, is orthologous to the Saccharomyces cerevisiae SSA encoded HSP70.

Agrobacterium rhizogenes – mediated hairy root induction of Momordica charantia Linn. and the detection of charantin, a potent hypoglycaemic agent in hairy roots

Swarna J. and Ravindhran R.*

Momordica charantia L. cultivated in India is widely used as vegetable as well as in ayurvedic and unani system of medicines for the treatment of many diseases. The fruit of the plant possesses various medicinal properties like antimicrobial, antihelminthic, anticancerous, antimutagenic, antitumo urous, antifertility, antidiabetic and abortifacient activities. Valuable secondary metabolites are produced by Agrobacterium rhizogenes-mediated hairy root induction. Hairy root production in Momordica charantia to produce charantin, an important hypoglycaemic component, was attempted for the first time. Transformation frequency in leaf and cotyledon explants was found to be 37.5 % and 25 % respectively. Hairy roots (3.7 cm in length) were observed from co-cultivated leaf explants after a period of three weeks. Charantin was detected in hairy roots and compared with fruit and leaf samples by performing thin layer chromatography.

Herbicides (2, 4-Dichlorophenoxy Acetic Acid, Isoproturon) and its interaction with Triticum aestivum L. alone and in combination

Kumar Sanjay 1*, Singh Atul Kumar 2 and Prasad Ramasare 3*

The effect of two herbicides, singly or in combination, was studied on the three wheat varieties (HUW 234, HUW 468 and HUW 533). The wheat seeds were grown for 72h in different concentrations 0, 50, 100, 200, 400, 800, 1200 ppm) of herbicides at normal laboratory temperature (25±2°C) and light condition. The parameters like seedling morphology, seed germination percent and root length were studied and the parameters were analyzed statistically by SPSS ver.16. The results indicated that both herbicides were able to induce bulbous structure in wheat seedlings at 400-1200 ppm. The seed germination percent and root length (cm) gradually decreased from lower to higher concentrations. The overall result showed that both herbicides had dose dependant effects on seedlings of wheat varieties. Further, the herbicides 2, 4-Dichlorophenoxy acetic acid (2, 4-D), isoproturon (IPU) and their combinations (2, 4-D+IPU) were used to study their phyto-morphological effects on wheat. The morphological parameters were taken as leaf area (cm2), internode length (cm), shoot length (cm), shoot biomass (FWg-1plant-1), shoot biomass (DWg-1plant-1) and seed weight (g inflorescense-1). The different concentrations applied singly or in combinations, as expected, showed phyto-depressive effects on morphological parameters studied.

Review Paper: Production of protease Enzyme using various sources

Kiran kumari S.P*., Satyavani Y., Chandana Lakshmi M.V.V. and Sridevi V.

Enzymes are proteins that catalyze various biochemical reactions. There are three types of enzymes present generally which help to sustain life and they are metabolic enzymes, digestive enzymes and food enzymes. Among these enzymes, protease is one of the digestive enzymes which split proteins into their monomers. Proteases are the single class of enzymes which occupy a pivotal position with respect to their applications in both physiological and commercial fields. Proteases are physiologically necessary for living organisms and are ubiquitous, being found in a wide diversity of sources such as plant, animals and microorganisms. Among these sources, microbial proteases represent excellent sources of enzyme owing to their susceptibility to genetic manipulation. Microbial proteases account for approximately 40% of the total worldwide enzyme sales as they possess almost all the characteristics desired for biotechnological applications. By using process parameters it will be very easy to produce protease in large scale or modify the applied parameters in the best way to produce high amount of protease. This study deals with various productions of protease both in solid-state and submerged fermentation.

Distribution of virulence genes in A. hydrophila and A. sobria in L. rohita (Rohu Fish) in N.E India

Sharma Indu¹* and Kumar A.2

A multiplex PCR assay was designed to amplify the Aeromonas hydrophila and A. sobria for hemolysin and aerolysin genes. The assay was used to evaluate 137 fish samples (Labio rohita) procured from retail shops (Meghalaya, India) of which 18 Aeromonas isolates were isolated. It was observed that 3 isolates of A. hydrophila (16.6%) were ahh1 positive which included 15 (83.3%) of the A. hydrophila isolates, 2 (11.1%) A. sobria isolates and 1 (5.5%) A. caviae isolate were examined. Amplicons for AH-aerA primer set were produced only by A. hydrophila isolates [1 of 18 (5.5%)]. Fourteen of the 18 Aeromonas isolates (77.7%) were asa1 positive. Majority of the isolates produced haemolysin. Out of the 18 isolates of Aeromonas spp. isolated, 12 (66.6%) of them exhibited haemolytic activity.