Research Journal of Biotechnology

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Research Journal of Biotechnology

Morphological, Biochemical and Differential Gene Expression in Leaves of YVMV Resistant and Susceptible Lines of Okra using RT-PCR

Meena Rakesh Kumar, Chatteerji Tanushree and Thakur Sanket

Development of cultivars resistant to yellow mosaic virus disease has long been a major breeding objective since this disease affects the production and productivity of many crops including okra. Functional genomics tools can play a major role in genetic improvement for resistance to biotic stresses in crop plants. This study covered morphological, pathological and biochemical characterization of YVMV resistant and susceptible lines of okra and expression profiles of randomly selected 5 PR and 5 NBS-LRR genes involved in defence for disease among okra lines for the first time examined using RT-PCR. Furthermore, we investigated correlations among gene expression levels, yellow vein mosaic disease severity and some biochemical parameters (Phenol, chlorophyll and sugar content) in the visible lesion area of inoculated plants. In correlation studies, it was observed that phenol content and total chlorophyll content were positively related to each other while total soluble sugar content was negatively correlated with other both the traits. PR-10, PR-13, PR-20 and PR-24 gene transcripts were found to show strong induction in RT-PCR with up regulation and PR-24 showed highest up regulation with up to 3.5 folds up regulation in resistant line as compared to susceptible line while PR-26 showed down regulation. Similarly, with NBS-LRR proteins, NBS-LRR-5, NBS-LRR-8 and NBS-LRR-16 showed up regulation while NBS-LRR-12 and NBS-LRR-15 were found down regulated in YVMV infection. Further characterization of these genes may show that they have potential application for development of okra lines with improved yellow mosaic resistance in okra.

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Effect of Dietary Crude Fiber Level on the Development of Reproductive System in Rabbits

Cristina Simeanu, Simeanu D. and Doliş M.G.

The development of animal body in general and that of the reproductive system in particular are influenced by many factors such as the species, age, breed, gender of the animals and of course, the nutritional aspects existing naturally or provided by the farmer in terms of levels and quality. Taking into account, these factors as well as that the domestic rabbits (Oryctolagus cuniculus domesticus) are non-ruminant herbivores consuming feed reach in crude fiber, the goal of this study was to reveal the effect exerted by the dietary level of crude fiber on the development and functioning of the reproductive systems of these animals. 8 groups of rabbits were organized (4 groups o males and 4 groups of females); one control group and three experimental ones were onset for each gender. The grouping was carried on when younglings have been weaned. Then, the rabbits were raised till turned 120 days old. The mixed feed given to the studied rabbits contained between 137.4 g/kg DM and 274.3 g/kg DM crude fiber. Domestic rabbits feeding (males and females) using combined feed presenting different levels of the crude fiber significantly affected the development of the reproductive system especially in males compared with the females.

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Bioremediation of OCDF-contaminated soils: batch, bioreactor and gene studies

Lin J.L. and Kao C.M.

In this study, effectiveness of using Pseudomonas mendocina NSYSU (P. mendocina NSYSU) on the bioremediation of octachlorinated dibenzofuran (OCDF)-polluted soils was evaluated through batch and bioreactor experiments under aerobic conditions. The goals of the research were to assess: (1) Feasibility of biodegradation of OCDF by indigenous soil bacteria and isolated bacterial strain (P. mendocina NSYSU) from OCDF-polluted soils, (2) Existence of functional genes for the aerobic biodegradation of OCDF, (3) Variations in functional genes in P. mendocina NSYSU during the aerobic biodegradation of OCDF and (4) Effectiveness of OCDF biodegradation in a bioreactor system. Results show that P. mendocina NSYSU was able to degrade OCDF through the aerobic co-metabolic mechanisms with the addition of carbon substrates. Up to 62% of OCDF was removed after a 50-day operation with carbon substrate (glucose) supplement. Results indicate that primary substrate supplement is required for the enhancement of aerobic biodegradation of OCDF. Results from the gene analyses show that two intradiol and extradiol ring-cleavage dioxygenase genes (Pmen_0474 and Pmen_2526) were identified. Increase in gene concentration was observed with the supplement by carbon substrate. The populations of Pmen_0474 and Pmen_2526 increased from 6.1×103 to 4.5×106 and from 7.6×103 to 4.8×106 gene copy/L after 50 days of incubation respectively. The detected specific genes played important roles in OCDF biodegradation under aerobic conditions. Results from the bioreactor study show that up to 65% of OCDF could be degraded after a 60-day operational period with substrate addition. Results reveal that an aerobic bioremediation system using P. mendocina NSYSU as the inocula and glucose as the carbon substrate would be a cost-effective system to remediate OCDF-polluted soils.

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Species-specific PCR to detection of potentially toxigenic Aspergillus in wheat and wheat flour

Mahmoud Mohamed A. and Abd-El-Aziz Abeer R. M.

Aspergilli are one of the most dangerous aflatoxin-producing fungi that contaminate food, beverages and feed. In this work, a sensitive and highly specific PCR protocol was established to detect toxigenic Aspergilli present in retail samples of wheat and wheat flour collected from Riyadh in Saudi Arabia. Occurrence of toxigenic Aspergillus species was higher in wheat than in wheat flour samples (50% and 25%, respectively). Wheat showed also the highest percentage of co-occurrence of two different species (18.75%) in comparison with wheat flour (6.25%). The most common Aspergilli were Aspergillus flavus (34.38%) followed by Aspergillus niger (34.3%) and Aspergillus ochraceus (25%). Aspergillus fumigatus and Aspergillus terreus were detected at 12.5%. A. parasiticus was detected at lower frequency (9.4%). Four Aspergillus species A. flavus, A. ochraceus, A. fumigatus, A. niger were: detected in wheat and wheat flour. A. parasiticus was detected only in wheat also, A. terreus was detected in wheat flour.

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Protective role of Heptamethoxy flavone on hyperglycemic retinopathy in zebrafish

Swarnalatha Y. and Thippabathani Jayakrishna

The current study is aimed to prove the protective role of HMF on hyperglycaemic retinopathy and serum glucose levels in zebrafish. The crude alcoholic extract of the Sphaeranthus amaranthodies was subjected to column chromatography, the collected fractions was qualitatively analyzed and the fractions 10 and 11 were subjected to NMR. Antioxidant enzyme levels and serum glucose levels were studied; H and E staining was performed to stain retina. The serum glucose levels of zebrafish were higher after inducing hyperglycaemic than control group for 30min after the treatment. However, treatment with HMF significantly lowered the serum glucose levels in post treated groups and was dose dependent. The levels of SOD, CAT and GSH were more or less similar in the retina of HMF treated fishes and the normal fishes. Significant difference in retinas of HMF treated and hyperglycaemic fishes and there was a decreased thickness in inner plexiform layer and ganglion layer in HMF treated fishes. Based on the above results, HMF isolated from sphaeranthus amaranthoides is capable of protection against hyperglycemic activity.

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Evaluation of non-target effect of potential fungal biopesticidal metabolites obtained from Nomuraea rileyi (F.) Samson on soil and plant growth parameters

R. S. Arvind Bharani and S. Karthick Raja Namasivayam

Biocontrol agents based on fungi play a pivotal role in insect pest management against economic imperative pests. Microbial control which makes use of naturally occurring microbes to control weeds, pathogens and pests, is less detrimental to non-target organisms and the environment than the chemical pesticides. Among the fungi, Nomuraea rileyi (Farlow) Samson is extensively used in agriculture area as bio-pesticidal agents. In the present study, the non-target effect of fungal bio-pesticidal metabolites extracted from Nomuraea rileyi (F.) Samson was studied under field conditions. Pesticidal metabolites were extracted using dichloromethane and methanol. The extracted pesticidal metabolites were characterized by Gas chromatography- Mass spectroscopy (GC-MS) which revealed the presence of pesticidal compounds. Field experiments were conducted on groundnut field (TMV-7), Rajakoil, Vellore District, Tamilnadu, India. Randomized block design was followed under recommended irrigation methods except chemical pesticides application. The treated plots showed no changes in all the tested soil and plant growth parameters at all the tested time intervals. The present study will suggest the possible utilization of pesticidal metabolites extracted from Nomuraea rileyi as an effective strategy of pest control under field condition with less or complete absence of non-target effect.

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Probioticated plaintain pith juice using Lactobacillus plantarum

Nithya Priya S. and Ramalingam P.

The aim of the present investigation was to develop a probioticated plaintain pith juice by optimizing the concentration of prebiotic (inulin), stevia (Stevia rebudiana) and inoculum Lactobacillus plantarum by one factor at a time (OFAT), single factorial analysis. The probiotic strain L. plantarum proved its ability and suitability to ferment plaintain pith juice. Plaintain pith juice fermented with 2% prebiotic had a pH 3.39, acidity 0.342%, total sugars172 μg/mL, reducing sugars 258.7 μg/mL and microbial viability of 8.1X 107CFU/mL respectively. Plaintain pith juice with 3% stevia had a pH 3.76, acidity 0.38%, total sugars 214 μg/mL, reducing sugars 254.2μg/mL and microbial viability of 2.8X 108 CFU/mL respectively. The juice with 3% inoculum size had a pH 3.82, acidity 0.41%, total sugars 191.3μg/mL, reducing sugars 249.4μg/mL and microbial viability of 8.4X 107CFU/mL respectively. Maximum growth of the probiotic was obtained at the end of 48 h of fermentation for 2% prebiotic, 3% stevia and 3% inoculum size. The results clearly indicated the effectiveness of the inoculums to make use of the plaintain pith juice for its survival.

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Bioprospecting endophytic fungus Colletotrichum sp. isolated from Artocarpus heterophyllus for anticancer activity

Swetha Sunkar, Sibitha V., C. Valli Nachiyar, Prakash P. and Renugadevi K.

Exploring endophytes is always an interesting realm of research as they are known to be a repository of various novel compounds. The present study was undertaken to isolate endophytic fungi from Artocarpus heterophyllus and to study the potential of the fungus for various bioactivities. The isolated endophytic fungus was identified using ITS sequencing and identified as Colletotrichum sp. Large scale cultivation was carried out and the secondary metabolites were extracted using the solvent ethyl acetate. The crude extract was checked for antibacterial, antioxidant activity and anticancer activity. The cell free supernatant was used for the benign synthesis of TiO2 nanoparticles which were further characterized by various instrumental analyses namely XRD, FTIR and SEM. When tested for bioactivities, the crude extract displayed moderate antimicrobial and antioxidant activities but on the other hand, the extract showed appreciable anticancer activity against Hep2 cells. Secondly, the fungus was able to synthesize TiO2 nanoparticles extracellularly whose sizes were in the range of 70 – 100 nm with potential anticancer activity. The results demonstrate that the isolated endophytic fungus was found to be promising producer of anticancer agents with significant potential to impede the proliferation of cancer cells. This organism was also able to bring about the reduction of metal to nanosize under benign conditions there by pronouncing the use of microbes the field of nanomaterial synthesis.

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Optimization of bacteriocin production in Lactobacillus plantarum DFR 4 using a simple food grade growth medium

Divya, Raj Xavier Janifer and Karna Venkata Ramana

Food spoilage and foodborne pathogens and the occurrence of human illness all over the world are major concern in the food industry. Application of lactic acid bacteria and their metabolites for food preservation is generally recognized as safe (GRAS) in comparison with chemical preservatives. Lactobacillus plantarum DFR 4 was isolated from raw cabbage sample showing bacteriocin production. Production of bacteriocin using simple and food grade medium is found to be essential for food application as well as down-stream processing. A simple medium was developed to address these aspects, the medium developed in the study consisted of 3.50 % yeast extract and 3.50 % dextrose using response surface methodology (RSM) and the desirability of the model (two factors with interactions) was found to be 92 % for bacteriocin production. The Analysis of variance (ANOVA) was calculated using the regression equation Z = + 25.82983 - 2.40294x -7.08995y + 2.42500xy, where the R2 value was 0.93, the maximum zone of inhibition (Z) obtained was 23.30±0.173 mm and lack of fit was found to be not significant for the model. The Fischer’s F test value of 40.34 and p < 0.05 demonstrated that the polynomial model is significant. The isolate Lactobacillus plantarum DFR 4 was able to utilize the nutrients present in the medium developed for bacteriocin production at the initial pH 7.0, however, the pH noticed at the end of the fermentation was 3.50 and the optimal temperature was maintained at 35˚C. The bacteriocin was partially purified using cold acetone precipitation showing antimicrobial activity against indicator organisms such as Listeria monocytogenes, Staphylococcus aureus, Escherichia coli, Bacillus cereus and Salmonella typhi etc.

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Cloning of plantaricin structural gene plnE derived from metagenome of TsoKar a Brackish Water lake of Ladakh region

Gupta Vishnu Kumar, Devi Tishu, Verma Vijeshwar and Rasool Shafaq

Plantaricins are small bioactive peptides produced by Lactobacillus plantarum strains that exhibit significant antimicrobial activity against microorganisms. In this study, the metagenomic DNA was isolated from TsoKar Brackish water sample. Plantaricin gene-specific primers were used to amplify plnE structural gene from TsoKar water metagenome. The amplified product of 700bp was cloned in pUC18 T tailed vector and then transformed into Escherichia coli DH5α. The plnE metagenomic library of TsoKar water was constructed and about 7000 clones were screened for antimicrobial activity. Amplification of plantaricin plnE gene from the water metagenome of TsoKar confirmed the presence of plantericin in brackish water.

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Repeated batch fermentation for the synthesis of L-Glutaminase by an immobilised Pseudomonas aeruginosa KRS7

Ravi Shankar K. and Mahalakshmi R.

L-Glutaminase, a therapeutically and industrially important enzyme was produced using Pseudomonas aeruginosa KRS7 by means of novel immobilization process. The new immobilization method offer several advantages over conventional system such as high specific activity for the target product. It was found that entrapment using Ca-alginate gave the best immobilized catalyst which was then further coated with chitosan to improve its mechanical strength. The combined entrapment-encapsulation immobilization technique was compared with that of bio flocculation process and the maximum L-Glutaminase was observed in entrapment-encapsulation immobilization at 35 o C and at pH 8. Further the impact of process parameters was optimized. Optimization process reveals the maximum L-Glutaminase activity of 250 U/ml when D-Glucose was used as a carbon source and 330 U/ml, when yeast extract was used as a nitrogen source. Repeated batch fermentation was carried out and the reusability of the immobilized beads was maintained constant for about 6 cycles. The enzyme was partially purified by ATPS and 78.52 U/mg specific activity was found after purification. Incorporation of silver nanoparticles to the ATPS system reduced the specific activity to 17.12 U/mg.

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Molecular markers as a tool for sex detection in Malabar tamarind [Garcinia cambogia (Gaertn.) Desr.]

Tharachand Choppa and Immanuel Selvaraj Chinnadurai

Garcinia cambogia (Gaertn.) Desr. also called as kodampuli or Malabar tamarind is beneficial as it contains Hydroxycitric acid (HCA). HCA, the primary constituent of the fruit rind has commercial and pharmaceutical applications. Sex of the seedlings cannot be distinguished morphologically. This study is an attempt to detect Random Amplified Polymorphic DNA (RAPD) markers for male and female populations of Malabar tamarind. The leaves of already identified male and female plants were collected from National Bureau of Plant Genetic Resources (NBPGR) Regional station, Thrissur, India. Out of thirty primers used for screening, seven primers produced clear and unambiguous polymorphic fragments. Two primers namely OPA 3 and OPA 11 were found to differentiate male and female accessions. RAPD primer OPA3 produced a unique band of 900 bp in all five male accessions which is absent in female accessions. RAPD primer OPA 11 produced 390 bp amplicon in all seven female accessions whereas this fragment is completely absent in male accessions. Therefore, the result of this study helps in recognizing the male and female seedlings at early stages. This could enable the farmers to identify female plants for having higher yield and also help in saving time and resources.

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Study on the Interaction Response of Biomasses on the Fluidized Bed Co-Gasification: A Statistical Approach

Ramarao M. and Vivekanandan S.

The objective of this work was to assess the combined effects of the blending ratio in a fluidized gasification process where the focus was to quantify the relationships between the response variables and vital operating factors. With a view to the shortcomings of the classical “one factor-at-a-time” method in identification of the effect of experimental factors and their interactions, a statistical design of the experiment based on response surface methodology (RSM) was used. The response variables used in this work were gasification efficiency, tar yield and carbon conversion with different bed materials such as silica and limestone. Experiments were conducted over a temperature range of 700-900°C using equivalent to 0.35. All the response variables were successfully fitted to either a two-factor interaction or quadratic model. Using RSM, the effects of individual operating factors and their interactions were categorically determined which were not otherwise possible by the classical design of experiment methodology. Using the resultant response variable correlations, gas efficiency was optimized as a function of the different blending ratios and bed materials respectively. The full potentiality of wheat husk, rice husk and coconut shell for fluidized gasification was successfully investigated via RSM. The results of this work, however, are only applicable to fluidized bed gasification updraft systems.

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