Research Journal of Biotechnology

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Vol. 13(8) August 2018

In Vitro Regeneration in Pomegranate (Punica granatum L.) cv. Bhagwa using Double Nodal Segments

Guranna Prabhuling* and Hoolageri Huchesh

Page No: 1-10

Abstract: India is the second largest pomegranate producer in the world but the productivity is lower mainly due to non-availability of healthy and disease free planting material. Conventional methods of propagation of pomegranate through stem cutting are time consuming and tedious as they require about one year to raise the saplings. The present study was carried out with the objective to standardize efficient and reproducible protocol for direct regeneration of plantlets in pomegranate cv. “Bhagwa”. Different explants, duration of mercuric chloride treatment, different antioxidants and growth regulators were tried on in vitro regeneration.

The current season twigs containing shoot tip as well as different region internodes were collected from healthy mother plant. Among the various treatments, surface sterilization of double nodal explants containing IIIrd + IVth nodes with HgCl2 0.10% for 3 minutes resulted in maximum establishment aseptic culture (55%), lowest contamination (15%) and lesser intensity of browning (+) when incubated onto MS medium containing BAP 1 mg/l + AgNO3 1 mg/l + activated charcoal 2000 mg/l.

Shoot proliferation was found superior when aseptic cultures were transferred onto the MS medium containing ancymidol 0.02 mg/l + AgNO3 1 mg/l + activated charcoal 500 mg/l with 5 shoot/explants, 4.97 cm shoot length and 18.23 number of leaves/shoot. Finally, regenerated microshoots were transferred onto the full and half strength MS medium supplemented with NAA and IBA, 1-5 mg /l along with activated charcoal 200 mg/l. Highest rooting was observed on half strength MS medium supplemented with IBA 2 mg/l + AgNO3 1 mg/l + activated charcoal 200 mg/l, which took 22 days for rooting with 48% rooting, 5.50 primary root/shoot and 4.32 cm root length. Later the rooted plantlets were successfully hardened under polytunnel with 75 % survival rate in potting mixture containing sterilized coco peat, perlite and sand (1:1:1 v/v).

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High-milk genotype Arvana breed camels raised in the Caspian lowlands and Mangistau peninsula: identification and certification according to the profile alleles

Adilbekova Elmira*, Alybaev Nuradin, Esimova Anar and Аbuov Galymzhan

Page No: 11-15

Abstract: The subject for study is high-milk genotype Arvana breed camels raised in the Caspian lowlands (LLP “Zhana-Tan”) and Mangistau peninsula (LLP “Taushyk”). Arvana breed has been carried out using DNA technology in identification and certification of high-milk genotypes. Milk productivity of high-milk camel populations has been studied. Arvana breed camels genetic diversity analysis has been carried out using heterozygosis indicator and accidental inbreeding values as criteria for two populations. Interpopulation differences between camel populations by 8 microsatellite loci have been obtained by population of LLP “Taushyk”. Its average allele number is 11.13, heterozygosis is 0.7207, inbreeding is 0.076. The singularity of the population is that 21 private alleles are in 7 loci, this is distinctive feature of the population and speaks for genetic differences of these populations.

The distinctive feature of LLP “Zhana-Tan” population is presence of private alleles in 5 loci. According to this population, the average allele number is 9.75, heterozygosis is 0.6531, inbreeding is 0.097, this is indicative of genetic differences between these populations. “Animal database” module in the created system holds data on genotypic animals. Currently, there is information about 204 animals in this system and genetic certification of high-valued genotypes is reliable part of zootechnic accounting in breeding farms.

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Lectin histochemical examination of the mucosal changes in the urinary bladder of rats in cyclophosphamide-induced hemorrhagic cystitis

Arikketh Devi*, Joel P. Joseph, Arul Jothi K.N. and Harishankar M.K.

Page No: 16-21

Abstract: Cyclophosphamide (CYP) is an alkylating agent widely used as an anti-neoplastic drug to treat cancer. Hemorrhagic cystitis (HC) is one of the toxicities caused by prolonged cyclophosphamide (CYP) treatment. We investigated the mucosal changes in the urinary bladder of rats with cyclophosphamide (CYP) induced hemorrhagic cystitis by Lectin histochemistry. In this work, twelve Wistar strain male albino rats were used. The animals were divided into two groups – control group (Group I) and the CYP treated group (Group II).

The control group was injected with sterile distilled water and the test group was treated with 30mg/kg of CYP daily for 10 days. The tissue sections were stained to observe for the induction of Hemmorhagic Cystitis. Bladder sections of all the animals were stained with four biotinylated lectins namely, Peanut agglutinin (PNA), Datura stramonium (DSL), Wheat germ agglutinin (WGA) and Maackia amurensis (MAL-II). The lectin histochemical staining of the urinary bladder of rats with CYP induced hemorrhagic cystitis was characteristically different from the control group rats and varied according to the nominal sugar specificities of the lectins.

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Formulation development and characterization of polymeric nanoparticles of antituberculosis drug Isoniazid

Srivastava Saurabh* and Ahmad Irfan

Page No: 22-26

Abstract: The objective of this study was to develop nanoparticles of Isoniazid, enhancement of bioavailability and reduction in side effects as an antituberculosis drug. Nanoparticles were prepared by Ionotropic gelation method using chitosan as polymer and tripolyphosphate crosslinking agent. Nanoparticles were characterized for particle size, surface morphology, entrapment efficiency, zeta potential and in- vitro drug release. The optimized formulation exhibited a particle size of 412.9±0.28 nm; drug entrapment efficiency of 67.40 ± 0.59% and the in-vitro release was 91.87% extended up to 8 hrs. Stability study at 5±10C and room temperature (250C) showed that there was no significant alteration in physical appearance, size, shape, drug content and in-vitro drug release after 45 days.

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Characterization of morphological variation in mitotic images of five allium species from Nagaland (India)

Kumar Sanjay* and Thonger Tsipila

Page No: 27-61

Abstract: The Allium species (A. chinense, A. tuberosum, A. hookeri, A. ascalonicum and A. sativum) were collected from the different regions of the Nagaland (India). The species were maintained in the departmental garden and root tips were processed according to the conventional root tip squash technique for the collection of different mitotic stages for 24 h under the microscope. The recorded stages and their variations with their characteristics were reported in the present study.

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Transcriptome profiling of tea (Camellia sinensis (L) O. Kuntze) during Pestalotiopsis theae infection revealed stress/disease responsive genes

Senthilkumar P.*, Purushothaman N., Suganthi M. and Mandal A.K.A.*

Page No: 62-71

Abstract: Tea is an important agricultural commodity in many Asian, African and South American countries. Tea is cultivated for its tender leaf and any disease of leaf seriously affects the production of tea. Among the leaf diseases, grey blight caused by Pestalotiopsis theae is one of the most important and devastating diseases in tea. This problem persists from a long time, even though genetic improvement strategies are not so successful in tea due to lack of cloned genes from this plant. To overcome this problem, we have attempted this study. An enriched cDNA library during grey blight disease infection in tea has been constructed. The cDNA library contained about 1200 clones. One thousand colonies were picked up and screened by colony PCR to find out colonies with ≥ 250 bp insert. Colony PCR confirmed 480 clones with insert size above 250 bp.

Plasmids were extracted from all these colonies and sequenced. 320 sequences were identified as unique. Among these sequences, 67 sequences have a high degree of sequence similarity with genes from other species which were related to disease resistance/stress tolerance. 53 sequences have a high degree of sequence similarity with hypothetical proteins/ribosomal proteins while 200 sequences have no significant match in the database and are considered as novel. The cDNA library specific to grey blight disease infection yielded many important disease resistance genes and genes involving signal transduction pathways in the form of ESTs and partial CDS. Based on the sequence information, these genes can be isolated in full length and utilized in future crop improvement programs like developing increased disease tolerant/resistant varieties.

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DNA extraction and molecular characterization of Acacia pseudoeburnea – An endemic species

Dhakad Ashok Kumar*, Chandra Anup, Barthwal Santan, Thakur Ajay and Rawat Janhvi Mishra

Page No: 72-78

Abstract: Acacia pseudoeburnea J.R. Drumm. ex Dunn belonging to the family Leguminosae is an important plant of Indian semiarid ecosystem. The species is endemic to North-Western India with ambiguity of its identification and speciation to Acacia eburnea. Lately its distribution and occurrence are shrinking, so there is need of genetic characterization and germplasm conservation. Study conducted on its molecular characterization yielded a new and rapid DNA extraction protocol which was compatible with other extraction protocols and produced quality gDNA with concentration of 12.9-44.1 (ng/µl) with a purity of 1.71-1.86 in term of optical density.

Isolated DNA was suitable for the PCR amplification with universal plastid region of rbcL gene. Neighbor-Joining tree was constructed with consensus sequence and closely related ten sequences retrieved from NCBI GenBank through BLAST search. Search results revealed that the rbcL sequence of A. pseudoeburnea showed high similarity with Acacia nilotica based on nucleotide homology and phylogenetic analysis. This suggests that A. pseudoeburnea is genetically similar to Acacia nilotica based on DNA sequence of rbcL gene, although study is still needed for further research.

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Screening and optimization of polyhydroxyalkanoate (PHA) produced by Bacillus drentensis VIT-SSR3 isolated from industrial effluent contaminated soil

Soruba Evangeline and Sridharan T.B.*

Page No: 79-84

Abstract: Petroplastics have caused a negative impact on the environment and hence this has led to the search for bioplastics with superior material properties. Unlike petroplastics, Polyhydroxyalknoates (PHAs) biopolymers were synthesized by a vast genus of microorganisms intracellularly as carbon and energy reserve materials. The aim of the study was to isolate novel bacteria capable of accumulating polyhydroxyalkanoate (PHA) from an industrial effluent contaminated soil environment. All the isolates obtained were screened for PHA production by Sudan Black B staining and Nile Blue A staining, of which one isolate VIT-SSR3 was found to have potential PHA accumulation capacity.

The biochemical and phenotypic analysis identified the isolate as Bacillus drentensis VIT-SSR3. The maximum PHA content of (0.186±0.003) g/L was obtained when glucose was used as the carbon source and the maximum PHA yield of (33.08±0.30) % was obtained when grown on ammonium sulphate as the nitrogen source. The optimum pH and temperature for maximum PHA production were determined to be 7 and 35°C respectively. The produced biopolymer was analyzed using Fourier Transform Infrared Spectroscopy (FT-IR) and Scanning Electron Microscopy (SEM).

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Genetic variation analysis in Aegle marmelos L. plants using RAPD Molecular Markers

Soni Pravesh* and Koche Vijaya

Page No: 85-89

Abstract:The present study evaluated genetic variability of superior bael genotypes collected from different parts of Chhattisgarh, India based on the morphological features and random amplified polymorphic DNA (RAPD) markers. Genomic DNA extracted from leaf material using cetyl trimethyl ammonium bromide (CTAB) method was subjected to polymerase chain reaction (PCR) with 24 polymorphic primers. A total of 450 polymorphic loci were identified with mean value of 18.75 bands per primer and overall 41% polymorphism. Application of unweighted pair group method using arithmetic average cluster analysis generated three genotypic groups. The genotypes collected from Raipur (E) and Dhamtari (F) were most similar genotypes whereas genotypes collected from Kolkatta (G) and (H) were extreme divergent.

The clusters based on molecular data were not in agreement with the morphological traits in most of the cases as low level of correlation was observed between the classification methods based on fruit characteristics and RAPD markers. The bael genotypes were found to have considerable genetic variability, demonstrating the importance of RAPD markers to analyze each genotype in a collection in order to efficiently maintain the germplasm collection for genetic improvement of bael.

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Phytochemical screening, in vitro Antibacterial and Antibiofilm activities of Alstonia scholaris leaf extract against several biofilm-forming bacteria

Abinaya M. and Gayathri M.*

Page No: 90-101

Abstract: The present study was to evaluate the antibacterial and antibiofilm activities of various solvent extracts of Alstonia scholaris leaf against biofilm forming bacterial pathogens. The petroleum ether, methanol, acetone and chloroform extracts were examined for phytochemical analysis, agar well diffusion, minimum inhibitory concentration, biofilm inhibitory effects, growth curve and light microscope analysis performed against the biofilm forming bacteria. The total phenolic and flavonoid content were determined by the spectrophotometric method. The quantitative analysis of GC-MS was further examined.

Phytochemical screening of various extracts shows the presence of different phytoconstituent. Methanol extracts showed the highest phenolic and flavonoid content than the other extracts. The antibacterial and antibiofilm activities for extracts have been performed against four bacterial pathogens. Almost 90% of inhibited bacterial biofilm formation was concentration-dependent of various extracts at 1mg/ml. Growth curve analysis revealed that all the solvent extracts had no effect on the growth of bacterial pathogens at 1mg/ml concentration when compared to the control. Microscopic analysis of various extracts against the bacterial strains significantly reduced the biofilm formation when compared to the control. The preliminary studies revealed that the methanol extracts of Alstonia scholaris leaf acquire the antibiofilm activity against bacterial pathogen by interrupting the synthesis of bacterial cell wall and inhibition of biofilm formation.

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Characterization of the cellulolytic enzyme produced by Bacillus endophyticus N2 isolated from the fermented yellow water of the traditional Chinese liquor fermentation process

Zhang Qing* and Yuan Chunhong

Page No: 102-109

Abstract: Cellulase-producing bacteria were isolated from fermented yellow water which is the by-product of the traditional Chinese liquor fermentation process. In this study, 12 strains of cellulolytic bacteria were screened and the best cellulase producer strain N2 was identified as Bacillus endophyticus using morphological, physiological and biochemical characterization and 16S rRNA gene analysis. Optimization of the culture conditions and fermentation medium for maximum cellulase production of Bacillus endophyticus N2 was carried out.

The optimum conditions for four cellulolytic enzymes production were 370C at pH of 7.0 with 6% (w/v) wheat bran as carbon source and 2% (w/v) ammonium sulfate as nitrgon source. The maximum production of CMCase (153.36 U/mL), avicelase (17.64 U/mL), β-glucosidase (126 U/mL) and FPase (30.48 U/mL) were obtained after incubation 72 h with inoculum size of 4% (v/v).

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Evaluation of AChE enzyme inhibition potential of Indian Medicinal Herbs Ficus hispida, Morinda tinctoria, Sapindus emarginatus and their significance in Alzheimer’s disease therapy

Sivaraman Dhanasekaran

Page No: 110-115

Abstract: Alzheimer’s disease (AD) is a progressive neurodegenerative disorder primarily manifesting as a loss of memory, senile dementia, intra neuronal neurofibrillary tangle formation. Cerebral parenchyma deposition of the β-amyloid protein in the form of amyloid plaques is the most stereotypic cognitive and neuropathological hallmarks of AD. It is evident through several studies that AD patient brain has higher level of acetyl cholinesterase (AChE) enzyme activity which in turn depletes the biologically significant neurotransmitter called acetylcholine and further leads to cognitive impairment. As acetylcholinesterase (AChE) inhibitors are an important therapeutic strategy in AD, efforts are being made in search of new molecules with anti-AChE activity.

The main aim of the present investigation is to screen the Indian medicinal herbs such as Ficus hispida, Morinda tinctoria, Sapindus emarginatus for possible AChE enzyme inhibition property using a colorimetric method in the presence or absence of the extracts along with Physostigmine as a positive control.

Results of the study have shown that among all three extracts examined for AChE enzyme inhibition assay, the EEMT showed the strongest inhibition activity with 76.46± 9.85 % followed by the second-best activity exerted by EEFH with 51.89± 8.51% and EESE with highest percentage inhibition of 44.22± 3.89% when compared with standard Physostigmine with 94.09± 3.08 % inhibition. From the results of the present investigation, it was concluded that all three extracts have promising AChE enzyme inhibition property in which the EEMT has revealed significantly higher level of activity when compared to EEFH and EESE. Hence these medicinal plants may act as a promising candidate for rendering useful phytocomponents that may be effective in treating neurodegenerative disorders like AD.

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Therapeutic Perspectives of Dyslipidemia and Associated Complications: Phytochemical Alternatives for Statins

Pandey Ankit, Rao Ankita, Devi A. and Arul Jothi K.N.*

Page No: 116-124

Abstract: World Health Organization estimated cardiovascular disease as the major cause of mortality in recent decades. Dyslipidemia or hyperlipidemia is the root cause of most of the cardiovascular diseases like atherosclerosis, stroke and myocardial infarction. Different factors like age, genetic and life style contribute to the development of these conditions worldwide. Considering the therapeutic perspective, most countries prescribe statin-based drugs for dyslipidemic and cardiovascular patients.

In this review we discuss the mechanism of statin action, the prescribed dosage levels in different countries, the variants of statins and the adverse effects of statins in the patients. This review also focuses on therapeutic approaches using different phytochemicals for dyslipidemia conditions without any adverse effects and also describes how phytochemicals are advantageous over statins.

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