Research Journal of Biotechnology

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Research Journal of Biotechnology





L-2-aminooxy-3-phenylpropionic acid (AOPP) promoting sshhoooott formation from hypocotyl explants of ggplant

Tanaka H.,1 Ueda H.,1 Mitsukuri K.,1,2 Tezuka T.,1 Shiozaki S.1 and Oda M.1*

The effects of L-2-aminooxy-3-phenylpropionic acid (AOPP) on bud and shoot formation were investigated in hypocotyl explants of eggplant. Application of AOPP to shoot induction medium (SIM) containing 4.4 μM N6-benzyladenine (BA) promoted adventitious bud formation, which occurred one week earlier than in the 0 μM AOPP control treatment. After eight weeks of culture, the number of adventitious buds per explant treated with 0, 2, 10, 50 and 250 μM AOPP was 8.4, 16.1, 14.3, 17.6 and 14.6 respectively. Shoot development was promoted by treatment with 10 μM AOPP, but 50 and 250 μM AOPP inhibited shoot development compared with the control. After eight weeks of culture, the number of shoots obtained per explant treated with 0, 2, 10, 50 and 250 μM AOPP was 1.0, 1.5, 4.2, 0.3 and 0.9 respectively. Rooting from the obtained shoots was not significantly different among all media. These results indicate that AOPP promotes adventitious bud and shoot regeneration from eggplant hypocotyl explants.

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Identification of the RAPD Marker linked to Powdery Mildew Resistant Gene(s) in Black Gram by using Bulk Segregant Analysis

Srivastava Priya, Pandey Anjana* and Singh Major

Black gram leads to loss of yield production due to wide spread of powdery mildew, which is the most prevalent fungal pathogen of black gram. It can be effectively controlled by incorporation of genetic resistance into susceptible species. In present study PCR- based technique RAPD has been used followed by bulked segregant analysis to identify two Random Amplified Polymorphic DNA marker linked to a gene for resistance to powdery mildew in Vigna mungo. In present work, F2 population was derived from a cross between two different species- BL-20, a highly susceptible line and other species VS, a highly resistant line against pathogen. Phenotypic data were collected by screening the F2 population of RILs (Recombinant Inbred Lines) according to the presence of infections by pathogen. By using BSA (Bulked segregant analysis), a polymorphic loci was observed along with hybrid and parental lines and a RAPD primer was identified as a molecular marker for resistance gene. A total of 80 arbitrary decamer oligonucleotide primers were used for RAPD analysis. Primer OPA-13, producing one RAPD marker OPA-13420, linked to the powdery mildew resistant gene. Linkage analysis was carried out using marker OPA-13420 on 120 individuals of F2 progenies from the crossing between BL-20 × Vs. The results demonstrated that the genetic distance between OPA-13420 and powdery mildew resistant gene was 5.83 cM.

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Design and Implementation of a Clinical Data Classifier: A Supervised Learning Approach

Jacob Gracia * and Ramani Geetha R.

Research in the sphere of artificial intelligence and its application in medicine have witnessed a remarkable advancement in the recent past initiating a renaissance in the field of bio-informatics, medicine and computer science. In this paper, our aim is to present a new methodology to formulate and evaluate a rule-based Clinical Data Classifier (CDC) that is designed to diagnose diverse ailments and classify DNA sequences. The proposed methodology utilizes computational approaches to prepare and process clinical patient records followed by the application of supervised machine learning techniques to generate classification rules. We record the performance and evaluation results of twenty supervised machine learning techniques on diverse clinical datasets comprising of more than 11, 000 patient records. The design is implemented as a Java-based clinical classifier with a user-friendly interface. The classification accuracy obtained with designed methodology in the training phase is 100% while the implemented CDC reports an accuracy ranging from 55% to 98% spanning varied clinical ailments. Our results prove that the C4.5 Decision Tree Algorithm and the Random Tree Classification Algorithm are the most efficient in classifying clinical patient records of varied nature.

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A plate assay method for isolation of bacteria having potent Sodium Doedecyl Sulfate (SDS) degrading ability

Chaturvedi Venkatesh

A plate assay which is based on the formation of halo zones by degradation of Sodium Dodecyl sulfate (SDS) in petri plates containing the complex of SDS and the cationic dye methylene blue has been formulated. This plate assay is simple, specific, reliable and rapid for isolation of corresponding SDS degrading microorganisms. It has potential for increasing the efficacy of screening of microorganisms capable of degrading SDS. This method is far more efficient than the earlier method for detection of SDS degrading bacteria, where SDS degrading bacteria formed halo zones at low temperatures as SDS crystallizes at this temperature.

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Production of alpha Amylase by Candida tropicalis SSK 01 strain Soil Isolate using Vegetable Waste Liquor Media and its Stability towards Metals, Surfactants and other Inhibitors

Namasivayam S. K. R.1* and Sivasubramanian S. 2

In the present study, Candida tropicalis SSK 01 strain local soil isolate isolated from petroleum contaminated soil produced α- amylase in mixed vegetable waste liquor media under controlled batch condition. Candida tropicalis SSK 01 strain was identified based on cultural and molecular characteristics. Based on 18S rRNA analysis, this strain was most closely related to Candida tropicalis (98.7 % similarity). The media supported growth and amylase production. Amylase production was observed during 12th hour. The enzyme stability was evaluated with heavy metals such as chromium, zinc and nickel, surfactant tween 20, EDTA, H2O2 and glycine at the concentration of 1,2,3,4 and 5 M. The enzyme retains its activity in all the tested concentration of heavy metals, surfactants and inhibitors. The enzyme was highly thermo and alkaline stable and maximum enzyme activity was observed at 80ºC and pH 9.0.

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Isolation, Sequence Identification and Tissue Expression Profile of a Novel SShheeeepp gene-PGK1

Yu Lei1 and Liu Yonggang2*

The full-length cDNA sequences of one sheep gene, PGK1, was amplified using the rapid amplification of cDNA ends (RACE) method based on one sheep EST sequence which was highly homologous to the coding sequence of human PGK1 gene. Sequence prediction analysis revealed that the open reading frame of this gene encodes a protein of 417 amino acids that has high homology with the phosphoglycerate kinase 1 (PGK1) of ten species—cattle (998%), human (97%), rhesus monkey (97%), rabbit (97%), pig (96%), dog (96%), horse (96%), giant panda (95%),mouse (95%), rat (95%)—so that it can be defined as sheep PGK1 gene. This novel sheep gene was assigned to GeneID: 100216440. The phylogenetic analysis revealed that the sheep PGK1 gene has a closer genetic relationship with the PGK1 gene of cattle. Tissue expression analysis indicated that the sheep PGK1 gene is differentially expressed in detected tissues including spleen, muscle, skin, kidney, lung, liver, fat and heart.

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Validation of root trait associated gene OsRAA1 to improve abiotic stress tolerance by overexpression in tobacco

Nethra P. 1* and Nataraja karaba N. 2

Plants are frequently exposed to abiotic stress which is a most serious problem in agriculture. To overcome these problems, plants have evolved certain definite strategies to survive and grow under stressful conditions. The strategy such as water mining with the help of a good root system is considered as an important drought adaptive mechanism. Therefore understanding the root biology and the molecular genetics of root development was a major task. Hence attempts were made to clone OsRAA1 gene into a Gateway binary vector and overexpress in tobacco. The expression and the integration of the transformed OsRAA1 lines were confirmed and advanced to the next generation. Under drought, the transgenic plants showed increased tolerance as evidenced by relative water content, membrane integrity, chlorophyll stability, cell viability, osmotic adjustment. These data suggest that overexpression of OsRAA1 can give tolerance to abiotic stress, indicating its significance in stress tolerance.

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Thin layer drying behavior of industrial tomato bye-products in a convective dryer at low temperatures

Celma Antonio Ruiz1, Cuadros Francisco2, Lopez-Rodríguez Fernando3 and Redrado Aida4*

The present work is mainly focused on the study of the thin layer drying behavior of industrial tomato by-products, peels and seeds, experimentally in a convective dryer. The drying experiments were conducted at inlet temperatures of drying air of 25 ºC, 35 ºC and 45 ºC and at an airflow rate of 1 m/s and 1.3 m/s. The drying rate was found to increase with temperature and velocity, hence reducing the total drying time. In particular, as drying temperature was raised from 25 ºC to 45 ºC, the time period needed to reduce the moisture content of the sample from 194.46 wt% down to 11.53 wt% (dry basis) was observed to decrease from more than 580 min to 295 min (1 m/s) and from 460 min to 150 min (1.3 m/s). Using a non-linear regression (Marquart´s method) together with a multiple regression analysis, a mathematical model for the thin-layer convective drying process of industrial tomato residues was proposed. The values of the diffusivity coefficients at each temperature were obtained using Fick´s second law of diffusion and varied from 3.238 x 10-9 m2/s to 1.245 x 10-8 m2/s over the temperature and velocity range. The temperature dependence of the effective diffusivity coefficient was described following an Arrhenius-type relationship. The activation energy for the moisture diffusion was determined as 23.14 kJ/mol and 43.36 kJ/mol, for airflow rates of 1 m/s and 1.3 m/s respectively.

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Chicken waste meal as an alternative for fishmeal for better survival and growth of the freshwater prawn Macrobrachium rosenbergii post larvae

Muralisankar T. and Saravana Bhavan P.*

The present study was conducted to assess the suitability of chicken waste meal as an alternative protein source for fishmeal in sustainable nursery maintenance of Macrobrachium rosenbergii post larvae (PL). Three types of feeds with 40% optimum protein were formulated by using fishmeal (FM), chicken waste meal (CWM) and soya bean meal (SBM) independently with other basal ingredients, such as green gram and ground nut oilcake. Cod liver oil was used as lipid source. Egg albumin and tapioca flour were used as binding agents. Vitamin B-complex with vitamin-C and a pinch of salt were also mixed. M. rosenbergii PL-20 (0.9-1.14 cm length and 0.12-0.16 g weight) was subjected to feeding trail with these feeds for a period of 60 days. The overall growth performance was found to be in the following order: FM > CWM > SBM. The differences recoded11 in survival rate, weight gain and food conversion ratio; colorific energy utilized through feeding, absorption, conversion, ammonia excretion and metabolism; concentrations of total protein, amino acid, carbohydrate, lipid, ash and moisture between FM and CWM including feeds fed PL groups were not statistically significant. However, the differences obtained for these parameters between FM and SBM, and between CWM and SBM were found to be significant (P<0.05). The staining intensity of polypeptide bands resolved between 116-14 kDa as well was found to be appreciable in PL groups fed with FM and CWM when compared with SBM. Therefore, CWM was equally good and nutritious as FM and the superior quality of these two meals are attributed to their protein and lipid contents. Hence, FM can be replaced by CWM as an ideal protein source in low cost feed formulation for sustainable nursery maintenance of M. rosenbergii PL.

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Expressional Quantitation of Transcription Factor Genes under Charcoal Rot Disease Manifestation in Sorghum bicolor using qRT-PCR

Patil Ayyanagouda1* and Fakrudin Bashashab2

Charcoal rot is a soil borne disease caused by Macrophomina phaseolina. This disease is influenced by drought stress in terminal stages of the crop growth. Since plants are continually exposed to a vast array of pathogens and generally succeed in defending themselves through the process of induction of defense gene products, the understanding of genes which show the regulation for resistance would be of greater importance in disease management. In plants regulation of disease resistance is multi-factorial and includes regulatory proteins known as transcription factors (TFs), which form the early responsive gene and directly regulate the expression of defense-related genes. In the present study, resistant (E36-1) and susceptible (SPV 86) variety for charcoal rot disease were chosen to study the differential gene expression of transcription factor genes through qRT-PCR. Out of 185 TF genes selected based on their direct or indirect involvement in biotic and abiotic stress signaling, 121 TF genes were up-regulated in resistant and susceptible genotypes at 75 and 90 days after Sowing (DAS). A set of 43 genes largely belonging to the families BHLH (9 TF genes), PHD (10 TF genes) and NAC (9 TF genes) families failed to express in situation tested. The genes coding for WRKY related protein (PTSb00328.1 and PTSb00331.1) were found significantly up regulated in their expression at 75 and 90 DAS in resistant cultivar compared to susceptible ones, indicating the possible role in charcoal rot resistance. The approach described here has allowed the identification of TF genes involved in the response of M. phaseolina infection to the sorghum plant.These key transcription factor genes involved in plant defense can be used for engineering increased resistance to plant pathogens.

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Isolation, identification and evaluation of a novel cellulose degrading fungus Emericella nidulans

Trivedi Minal J.,1 Goyal N. R. 2 and Dave S. R.3*

Cellulose degrading fungus isolated from garden soil was identified as Emericella nidulans based on morphology and DNA sequence analysis. It showed 99% similarity score with four other strains of Emericella nidulans having BLAST score ranging between 929 to 931 bits. Solubilisation of carboxy methyl cellulose resulted in production of 951.03μg/ml free sugar whereas 91.8 μg/ml sugar with paper pulp as substrate. Optimum pH and temperature for biotransformation of cellulose was found to be 4 and 30oC by Emericella nidulans. Sugar production increases as increase in substrate concentration. The isolate showed about 14.3 fold higher CMCase activity as compared to FPase activity. Isolated fungus Emericella nidulansi was found to be efficient cellulose degrader both on CMC and paper pulp.

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Use of comet assay in the study of DNA break in blood and gill cells of rohu (Labeo rohita) after an exposure to furadan, a carbamate pesticide

Mohanty G.,1 Mohanty J.,2 Garnayak S.K.,2 Rath S.K.3* and Dutta S.K.4

Labeo rohita (rohu) fingerlings were exposed to 0.002, 0.004 and 0.02ppm of Furadan, a carbamate pesticide for 96h. Samplings were done at 24, 48, 72 and 96h. The study was intended to assess tissue specific genotoxic effects produced by furadan on two different tissue systems in fish. Results showed low baseline DNA break in blood cells followed by gill in control group whereas more DNA breaks in gill followed by blood cells in treated individuals. Concentration -dependent DNA breaks (AU) showed a linear and positive relationship (r2 = >0.7) in both the tissues. Time response curves showed a significant rise in AU up to 48h followed by a fall at 96h in blood. In gill inclination in 0.002 and 0.02ppm treated fishes continued up to 72h followed with a plunge at 96h. The results allowed us to advocate that rohu is a potential biomonitoring animal, comet assay can be used in DNA break studies and response to pollutants in multicellular animals is often tissue specific.

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Studies on a thermostable α-amylase from Geobacillus sp.: Optimization of culture conditions for production of the extracellular α-amylase

Nair Roopa and Sabharwal Sushma*

Geobacillus sp. is a gram positive thermophilic organism that grows optimally at 60° C. The present work describes the optimization of the culture conditions for the production of extracellular α- amylase from the Geobacillus sp. The best carbon source was found to be starch (1 %) that induced the production of α-amylase by Geobacillus sp. The use of organic nitrogen such as yeast and peptone favoured the production of α- amylase compared to inorganic nitrogen sources. Micro nutrients like di-potassium hydrogen phosphate, lithium sulphate, ferric chloride and calcium chloride (anhydrous) were also found to enhance the production of α-amylase. Effect of different agricultural wastes (oil cakes) on α-amylase production was evaluated. Mixed agricultural waste was found to be more effective in enhancing the production of α-amylase than the synthetic substrates.

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Report of Funneliformis mosseae (Nicol. & Gerd.) Gerd. and Trappe from Rangeland Soil of Saudi Arabia

Al-Qarawi A. A., Mridha M. A. U. * and Dhar P. P.

Funneliformis mosseae (=Glomus mosseae) is one of the most important arbuscular mycorrhizal fungi (AMF), widely distributed throughout the world and associated with plants of different regions of the world including rangeland. We have frequently encountered F. mosseae during our ongoing survey of AMF from rangeland plants collected from different habitats of Saudi Arabia. As there was no systematic record of this fungus from Saudi Arabia, so we have recorded the occurrence of this fungus with many different types of hosts obtained from rangeland plants of Saudi Arabia and a taxonomic description of the fungus is mentioned in this communication from a large number of spore samples.

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