Research Journal of Biotechnology

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Research Journal of Biotechnology

Genetic diversity analysis in coriander (Coriandrum sativum) genotypes through morphological and molecular characterization

Tomar Rukam S., Kulkarni G. U., Parakhia M. V., Thakkar J. R., Rathod V. M., Solanki R. K. and Golakiya B. A.

The coriander (Coriandrum sativum) is cross pollinated annual aromatic herb which belongs to Apiaceae family. The investigation was carried out to explore the morphological and molecular characterization in 25 genotypes of coriander. For morphological characterization, total 12 different characters were studied. Largest variation was exhibited by the character harvest index (36-75%), seed yield per plant (7-18 g), umbellets per plant (215-268) and seeds per umbel (20-29) while it was narrow in days to 50% flowering (27-33 days), plant height (75-80 cm), number of basal leaf (5-9) and longest basal leaf (5-7). Phenotypic Coefficient of Variability (PCV) was higher in magnitude than the Genotypic Coefficient of Variability (GCV) for all the characters except number of basal leaves. This indicated larger influence of environment for the expression of these characters. Total 38 Randomly Amplified Polymorphic DNA (RAPD) markers were used which yielded total 3721 fragments with average of 7.13 number of band. The polymorphism with RAPD primers ranged from 38 % to 100% with Jaccard’s similarity coefficient ranging from 88% to 56%. For Inter Simple Sequence Repeats (ISSR) total 28 primers were used which yielded 142 total fragments with average of 5.07 number of band. The overall grouping pattern of cluster analysis clearly showed the variability among each genotype. These DNA based markers along with morphological markers can be used for taxonomic issues, breeding program, studying genetic variability, gene tagging and also for genetic-diversity assessment, cultivar fingerprinting and phylogenetic studies.

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The effect of UV-C on the total antioxidant activity and phenolic content in hypericum scabroides raised under in vitro conditions

Namlı Süreyya¬ and Akcan Nurullah

Seeds of H. scabroides Robson and Poulter were germinated in vitro in Murashige and Skoog (MS) medium without hormones. The shoots thus obtained were then reproduced successfully in MS medium supplemented with 0.25 mg L-1 BAP (N6-benzylaminopurine). The shoots, into two groups of different periods of time (15, 30, 45, 60 min.) were exposed to UV-C as direct (covers of culture vessels are open) and indirect (covers of culture vessels are closed). After treatment, the antioxidant activities of the extracts obtained from H. scabroides were determined using the DPPH test method. The samples had lower activities than ascorbic acid (AA), butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT). In this work, extracts exposed for 15 min to direct UV-C had higher antioxidant activity (84.5 ± 0.4 I%) than extracts exposed for 30, 45, 60 min or control extracts (80.6 ± 0.7 I%). The results indicated that the control group extracts had a higher antioxidant activity than extracts exposed to 30, 45, 60 min or 15, 30, 45, 60 min of direct and indirect UV-C respectively. In the current study, brief UV-C exposure led to an increase in the amount of antioxidant activity of H. scabroides extracts, particularly for direct and indirect 15-min doses of UV-C. However, only the extracts exposed to15 min of direct UV-C had a higher response with respect to total phenolic content (837±2.1 μg/mg) than the control group (815±1.3 μg/mg).

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Investigation of inhibitory effects of triazole derivatives on the protease enzymes

Akin M., Nalbantoglu S., Arabaci G. and Saki N.

Triazoles constitute an important class of heterocyclic organic compounds because of their wide range of applications as pharmaceutical agents. They have common mode of action like inhibiting ergosterol synthesis, decreasing adhesion potential of pathogen cells to host tissues and morphogenetic transformation of yeasts to mycelial form. Fungicidal properties of azole agents have been known for many years, however, the investigation of inhibitiory effects of these molecules on various enzymes has increased in recent years. As being the most important group of industrial enzymes, proteases have received significant interest in the food, pharmaceutical and detergent industries. In this study, the inhibition effect of three triazole derivatives on serine (chymotrypsin and trypsin) and cysteine (papain) proteases was examined and IC50 values were determined. Also, the inhibition type for trypsin was established for each molecule separately.

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Effects of Dioryctria sylvestrella (Ratzeburg, 1840) on Basal Area Increment Loss of the young Brutian pine (Pinus brutia Ten.) Trees in the south-western of Turkey

Oğuzhan Sarıkaya and Yılmaz Çatal

Various factors play a role in the decrease of existence and efficiency of P. brutia forests in the Mediterranean region of Turkey. Insects are one of the most important of these factors. One of the most damaging species is the pine stem borer, Dioryctria sylvestrella (Ratzeburg 1840) (Lepidoptera: Pyralidae). Caterpillars cause great damage to saplings and young trees. In this study, the effect of D. sylvestrella on basal area increment was determined in a Pinus brutia plantations. For this aim, increment cores were obtained from sample trees. In the last four years (2009-2012) all groups (light, moderate, severe and control) were different statistically.

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Rcombinant expression, purification and immunogenecity evaluation of binding domain of botulinum neurotoxin type E in mice

Keshavarz Alikhani Hani, Ebrahimi Firouz and Hajizadeh Abbas

Botulinum neurotoxins (BoNTs) produced by Clostridium botulinum are the most toxic substances known. Considering toxoid vaccines incompetency, researches have recently turned to use recombinant vaccines against botulism. Studies have shown that the binding domains have a great importance in immunity against the disease of all serotypes. In this study the sequence of binding domain of botulinum neurotoxin type E was adopted from gene bank and corresponding gene was prepared as a synthetic construct and then transferred into E.coli BL21(DE3). It was evaluated for protein expression and confirmed by Western blotting technique. After protein purification by affinity chromatography, it was injected four times into mice and then the mice were challenged by active BoNT/E. Our results indicated that groups of mice challenged with 103LD50 of toxin completely survived and thus this recombinant protein is a good candidate for recombinant vaccine production against BoNT/E.

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Characterization of promoter of Terminal Flower1 (TFL1) gene of Arabidopsis

Kushwah Singh Neetu, Ahmad Israr and Ali Sajad

TFL1 is a shoot meristem identity gene required for maintaining overall architecture in Arabidopsis. TFL1 mRNA is expressed only in the L3 layer of the shoot apical meristem and its upregulation marks the vegetative SAM to switch to inflorescence identity. To explore in more detail the expression pattern of TFL1 in different tissues of Arabidopsis, we analyzed the TFL1 promoter using GUS reporter gene. In silico analysis of the 5′ upstream intergenic region of TFL1 revealed the presence of cis elements responsive to various hormones and light and binding sites for various root-specific transcription factors. A 2536 bp upstream intergenic region of TFL1 was PCR amplified and cloned to drive the GUS reporter gene in the pORE-R2 binary vector. Resulting construct pAtTFL1::GUS was used to generate transgenic Arabidopsis plants via floral dip method. Histochemical assay of stably transformed Arabidopsis plants for GUS expression showed that TFL1 promoter drives genes mainly in the root, inflorescence, stamen and ovule. Relative qRT-PCR of GUS and TFL1 showed that expression pattern of pAtTFL1::GUS is comparable to the native TFL1in various tissues but at a lower level compared with the TFL1suggesting cis elements located elsewhere enhance TFL1 expression. This study proves that besides maintaining SAM identity, TFL1 promoter has wider expression pattern.

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Molecular cloning and tissue expression analyses of two novel Capsicum annuum L. genes: GAL83 and PEX7

Wen Jin-fen, Huo Jin-long and Deng Ming-hua

In plants, many MYB or other transcriptional factors are involved in regulating anthocyanin biosynthesis. In this study, two transcription factor genes, GAL83 and PEX7, were isolated from Capsicum annuum L. and their complete coding sequences were amplified using the reverse transcriptase polymerase chain reaction (RT-PCR) based on the conserved sequence information of the Solanum lycopersicum and other plants. The nucleotide sequence analysis of these two genes revealed that C. annuum L. GAL83 gene encodes a protein of 286 amino acids that belongs to the E-set and AMPKBI superfamily. The tissue expression analysis indicated that the C. annuum L. GAL83 gene was highly expressed in pericarp, moderately expressed in stem and leaf, weakly expressed in placenta. There was no expression in root and seed. The PEX7 gene encodes a protein of 317 amino acids that belongs to the WD40 superfamily. The tissue expression analysis indicated that the C. annuum L. PEX7 gene was highly expressed in flower, moderately expressed in seed, weakly expressed in stem, pericarp and leaf. There was no expression in root and placenta. Our experiment established the primary foundation for further research on these two C. annuum L. genes.

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Investigation of hydrocolloidal nano-silver use in laying hens on egg quality, immune response and villus morphology

Naghizadeh M., Karimi Torshizi M. A. and Rahimi S.

This study was carried out to the efficacy of antibiotic growth promoter and hydrocolloidal nano-silver as dietary antimicrobial agents evaluating egg quality, immune response and villus morphology of layer hens. 72 layers (Lohmann LSL) were randomly allocated to four treatment groups in a randomized complete block design with 3 replications as follows: Control (no antimicrobial agent was provided neither in feed nor drinking water); Virginiamycin in feed (50 ppm); Nano-silver in drinking water (7.5 ppm) and Nano-silver in feed (15 ppm). The treatments lasted for 12weeks from 24to 36weeks of age and egg quality were determined through 12 weeks of experimental duration. At the end of experiment, 6 birds per treatment were sacrificed for morphometeric studies of small intestine mucosa. There were no differences between experimental groups for egg quality (P>0.05). Nano-silver in water significantly increased cellular immune response to DNCB and PHA compared to other groups (P<0.05). Nano-silver in feed and antibiotics reduced intestinal villus and height in duodenum (P<0.05). All the feed additives reduced the number of goblet cells compared to control group (P<0.05). Improvements were observed in cellular immune response to DNCB and PHA. Nano-silver mimics the antibiotic effects on small intestinal villus characteristics. Based on application route of nano-silver, there were divergent effects on some studied parameters (mainly, immunity and villus of upper intestine).

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Computer aided impact analysis of metacarpus verified by the biomechanical tests for improving composite limb guards

Yilmazcoban I. Kutay, Cilingir Ahmet C. and Kiratli-Yilmazcoban Nursel

In the development of equipment in sports or in security, new methods have to be applied for the daily activities. Initially ergonomic and biomechanics tests under in vitro conditions can be performed before in vivo tests and clinical research. These tests can be applied to muscles, tissues or bones etc. Several recent developments, including the mechanical tests of fresh whole bone specimens, offer the possibility to understand the behavior of the bones. Therefore, the aim of the present study is to determine the effects of mechanical tests on the lamb metacarpuses and impact tests of limb guards supported by the Finite Element Analysis (FEA) to improve durable composite limb guards. Easily found lamb metacarpuses are used for making an analogy between the mechanical behavior of lamb and human bones. Three point bending tests are conducted using three different compression speeds to assess and compare bones fracture properties. Computer controlled pendulum mechanism is used for the impact tests of the composite limb guards. To make an easy and various comparisons, dynamic impact computer simulations of the metacarpuses are calculated. For the composite limb guard layers, thickness and material type of the guards are evaluated by the results. Finally, durable and reliable composite limb guards for the sports or the security are obtained for determined conditions.

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Production of selected secondary metabolites in callus and shoot cultures of Ficus religiosa L. – a valuable medicinal plant.

Gill Anita Rani and Siwach Priyanka

Four different types of callus tissue were obtained from nodal explants of Ficus religiosa on conditions viz. FCt7 (2.0 mg/l TDZ+0.5 mg/l IAA), FCt13 (150 mg/l yeast extract+FCt7), FCt16 (100 mg/l casein hydrolysate+FCt7) and FCd1 (0.25 mg/l 2,4-D+2.0 mg/l IAA). The calli so obtained were subjected to biochemical analysis as well as shoot organogenesis. Callus raised on FCd1 medium was found richest in selected phytochemicals- natural phenols (211.2 mg/g), total flavonoids (98.07 mg/g), quercetin (6.47 µg/g) and myricetin (10.05 µg/g) (which was respectively 3.07 times, 2.6 times, 1.02 times and 5.52 times more than that in mother plant sample) but exhibited poor organogenic potential. While, callus from FCt16 medium exhibited comparatively low level of secondary metabolites but was highly organogenic; the other two types of calli (FCt7 and FCt13) exhibited a moderate response. The plants regenerated from the four different calli were found considerably rich in selected phytochemicals compared to seedling of the same age. Amongst these, plants differentiated from FCt13 callus exhibited unusually high amount of natural phenols (167.1 mg/g), total flavonoids (61.01 mg/g), quercetin (6.311 µg/g) and myricetin (3.90 µg/g), much higher to mother plant.

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A pilot Study on Screening and Simplified Fermentation of a Biosurfactant producing Strain with Seawater

Zhang X. S.

This study conducted a pilot study on screening of a salt resistant bacterial strain producing biosurfactant with seawater and simplified fermentation. Through blood NA plate method and shaking fermentation evaluation, one strain Z5 was screened out. With the sequencing analysis 16S rRNA gene and morphological, physiological and biochemical tests, it was identified as Pseudomonas aeruginosa. Chemical reaction tests, thin-layer chromatography and Fourier transform infrared spectrum tests showed that Z5 produced rhamnolipid by seawater fermentation. The biosurfactant production capacity with two kinds of media, the synthetic media and the seawater media, with two kinds of methods, shaking flask method and simplified fermentor method, was compared. In shaking flasks, Z5 could produce rhamnolipid in seawater adding only yeast extract and waste vegetable oil; the yield could reach 72.7% of synthetic media. With these methods, the production cost was lowered laying a solid foundation of industrialized production of rhamnolipid in coastal areas.

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