Research Journal of Biotechnology

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Research Journal of Biotechnology

Agrobacterium tumefaciens-Mediated Transformation as an efficient tool for insertional mutagenesis of Aspergillus clavatus

Han Xiaofei and Jiang Donghua

Many transformation methods that introduce DNA into filamentous fungi have been developed. One of these methods used frequently was Agrobacterium tumefaciens-mediated transformation (ATMT). In this study, an efficient genetic transformation system of the filamentous fungi Aspergillus clavatus that was first used in the transformation was established. The transformation was mediated by A. tumefaciens strain AGL-2 that contained binary T-DNA vector (pKD1) with hygromycin B resistance selection gene (hph) and green fluorescent protein (GFP) marker gene and conidia were used as the recipients. Afterwards, the transformation system was optimized and the optimum conditions were achieved. Transformation efficiency was reached 300-400 transformants per 105 conidia under the optimal conditions. With this protocol, lots of random-insertion mutants were obtained and several of them were validated by analysis of the GFP gene expression and Southern blotting. Abundant mutants altered morphologic phenotypes in colonies, conidia and hyphae formations of A. clavatus provided possible to study the relationships between the functions and the interrupted genes.

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Molecular characterization of Associative N2 fixing bacterium isolated from the roots of Finger millet (Eleusine coracana L.) and its interaction effects with microbial consortia on growth and yield

- Santosha Gowda G.B. and Earanna N.

An associative N2 fixing bacterium was isolated from the roots of finger millet (Ragi) using LGI-P medium and purified Genomic DNA was isolated, amplified using 16S rRNA primers and sequenced. Based on the sequence homology available at NCBI (National Center for Biotechnological Information) GenBank, the bacterium was identified as Gluconacetobacter diazotrophicus. Further, its efficacy was tested for improving growth and yield of finger millet by inoculating the bacterium either singly or in combination with Bacillus megaterium (PO4 solubilizer) and Glomus fasciculatum (arbuscular mycorrhiza) to finger millet under glass house conditions. The consortia of G. diazotrophicus + B. megaterium + G. fasciculatum inoculation showed significant increase in plant height, number of leaves, number of tillers, shoot and root biomass, grain yield compared to single inoculation treatments. The un-inoculated plants (control) produced least growth and yield. Thus, this study confirmed the efficacy of the isolate (G. diazotrophicus) and its interaction with beneficial microflora to improve growth and yield of the crop.

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Synthesis and Characterization of Needle like Hydroxyapatite Nanoparticles using Casein as a friendly matrix

Sahba Reyhaneh, Mirabdullah Seyed Sadjadi, Nazanin Farhadyar, Ali Akbar Sajjadi and Babak Sadeghi

Collaidal calcium phosphate-based nanocomposites are nontoxic and biodegradable and can be used as attractive candidate for bioimaging and therapeutic drug delivery applications. To this end we attempted for the synthesis and characterization of needle like nanohydroxyapatite (nHA) at room temperature via inverse micro emulsion method by using different values of casein micelles as a matrix. Characterization of the samples was carried out by using X-ray diffraction (XRD) analysis, Scanning and Transmission Electron Microscopy (SEM, TEM) and Fourier transform infrared spectroscopy (FT-IR). The results revealed that casein protein is a best template agent in the preparation and growth of the HA nanoparticles influencing their size and morphology.

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Diversity assessment of hulled barley (Hordeum vulgare L.) accessions by agro-morphological traits and SSR markers

Kumar Pawan, Tikle A.N., Verma R.P.S. and Malik Rekha

Barley accessions from ICARDA, Lebanon were grown in Indian condition at ICARDA-IRP (Indian Research Platform), Amlaha, (M.P.) and analyzed for morphological and genetic variability using nine agro-morphological traits and molecular markers (SSR). Total 94 alleles were scored for 24 genotypes with 37 microsatellite markers. The number of alleles ranged from 2 to 4 with an average of 2.54 alleles per locus. The band fragment size varied from 100 bp to 455 bp with PIC values ranging from 0.153 to 0.707. On comparing molecular variability across linkage groups, chromosome 3H with PIC and mean allele values 0.577 and 2.67 were observed to be most variable. The clusters obtained from NTSYS grouped ICARDA accessions in three major groups in accordance to their ancestry. These exotic accessions evaluated can be further used for primary or secondary introduction in Indian breeding program.

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In vitro conservation of Ceropegia juncea, an endemic species of Western Ghats

Saraswathy M., Kalimuthu K., Chinnadurai V. and Prabakaran R.

Micropropagation of Ceropegia juncea, an endemic medicinal plant of Asclepiadaceae has been investigated through nodal explants. The shoots were induced from nodal explant on Murashige and Skoog (MS) medium containing BAP alone or BAP with KIN or TDZ. Maximum number of shoots are 4 in initiation and 30 during subculture with 91.66 shoots sprouting frequency observed on the medium containing BAP (4.44 µM) along with TDZ (1.135 µM). Regenerated shoots were rooted on the same medium BAP (4.44 µM +TDZ (1.135 µM) with 93.33 % of rooting and 5.5 mean number of root and 6.05 mean root length. The plantlets were hardened in shade house and successfully transferred to the pot with a survival percentage of 92. This one step less complicated protocol is useful for germplasm conservation, mass multiplication and extraction of active principles from in vitro raised plants without disturbing the wild population.

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Comparative Analysis of Anatomic Structure and Enzyme Activities of Fruit Rinds in Crack-resistant and Crack-prone Watermelon Lines

Gao Meiling, Guo Yu and Li Wenbin

Fruit cracking is an economically important issue in breeding and production of watermelon, Citrullus lanatus. The physiological and genetic basis of watermelon fruit cracking is not well understood. In the present study, we compared the fruit pericarp anatomic structure and enzyme activities in two mini-watermelon inbred lines: the crack resistant line K2 with thick pericarp (~1.0 cm) and the crack prone line L1 with thin pericarp(~0.6cm). Microscopic structures of the two lines were examined by paraffin sectioning of the fruit pericarps that were sampled 21, 24, 27 and 30 days after pollination. The stone cells in the crack-resistant K2 were smaller and more round-shaped than those in the crack-susceptible line L1. An obvious transition from the small to big cells was observed in the crack-resistant line. Variation in the degree of pericarp structure of the crack-resistant was more obvious than of the crack-susceptible lines at late fruit development. Fruit-cracking resistance was associated with the variation and arrangement of pericarp structure in the process of mini-watermelon maturity. At the same time, the fruit pericarps that were sampled 30 days after self-pollination were observed for the activities of four enzymes. The activities of the pectinase, cellulose and peroxidase (POD) in the thin pericarp L1 were higher than those in the crack-resistant line K2. Cracking tendency and pectinase activity in the pericarp were positively and significantly correlated whereas pericarp thickness and pectinase activity were negatively correlated. Information obtained the present study is useful in understanding the mechanisms of cracking resistance in watermelon.

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Grain Color Development and Segregation of Blue Wheat

Diddugodage Chamila Jeewani and Wang Zhong Hua

Anthocyanins are one of the most valuable antioxidants which showed the fabulous importance to human health. Blue grained wheat has been identified as high anthocyanin containing cereal compared to other colored wheat varieties. Little is known about the inheritance of the blue grain traits including their genome location. In this experiment, we observed the grain color development and the segregation of blue wheat. The blue color development of wheat grains started at the mid grain development stage of 20 days post anthesis (dpa) and completed within 26 dpa. Segregation pattern of blue wheat was studied using an F2 population derived from blue and white wheat and it was found that the blue color is dominant to white color. The segregation did not follow the simple Mendalian inheritance. The different color intensities of blue wheat grains may be due to genetic dosage effect and the effect of minor genes. The main reason for the huge variation in the segregation of blue and white seeds in the same spike of the blue population is the effect of xenia. Several other genetic and environmental factors also affect for the blue trait in wheat. These findings will be helpful to identify the genetic basis of blue aleurone traits which will be utilized for further improvement of the blue wheat crop.

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Purification, Characterization of a Novel Lactobacillin LA1 produced by Lactobacillus rhamnosus ZRX01

Ran Junjian, Liang Xinhong, Zhao Ruixiang, Zhao Lili and Li Gang

A strain presenting antimicrobial activity against pathogens was isolated from fermentation sauerkraut. The isolated strain was tentatively identified as Lactobacillus rhamnosus ZRX01 based on biochemical tests and partial 16S rRNA sequence similarity. A novel bacteriocin named “lactobacillin LA1” produced by L. rhamnosus ZRX01 was purified using a series of steps consisting of ammonium sulfate precipitation, ion exchange, gel ultra-filtration and desalting. As revealed by SDS-PAGE, lactobacillin LA1 was homogeneous, showing an approximate molecular weight of 5800 Da. It was confirmed as having a molecular mass of 5812.4 Da by mass spectrometry. Lactobacillin LA1 exhibited broad-spectrum antimicrobial activities and had a good hear stability and pH changes in the range of 2.6 to 9.0. However, its activity was abolished upon proteinase K, trypsin and neutral protease treatments. The results obtained are adding a new variety of antimicrobial substance produced by Lactobacillus rhamnosus.

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Cellular tumor antigen p53 in the Dastarcus helophoroides (Fairmaire) (Coleoptera: Bothrideridae): Expression in Different Development Stages, Tissues and High Tempreature Treatments

Thiquynhtrang Vu, Thanh Pham and Menglou Li

The cellular tumor antigen p53 is an important key element which responds to diverse cellular stresses to regulate expression of target genes thereby inducing cell cycle arrest, apoptosis, senescence, DNA repair, or changes in metabolism. We identified Dh-p53 gene from Dastarcus helophoroides and found that it is highly homologous to p53 genes in other insects in which Dh-p53 had a full length of 1319 bp and contained 1035 bp open reading frame that encoded 344 amino acids. The Dh-p53 gene expression levels had significant differences among development stage, tissue distribution as well as Dh-p53 mRNA transcription levels were over-expression in both all development stages (adult, pupae, larvae) after high temperature treatment. Increased expression of the Dh-p53 gene after these treatments suggested that the gene was involved to control cell cycle progression in response to cellular stress like temperature-sensitive.

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Gasification Potential of Garden Tea (Camellia Sinensis) Waste using Downdraft Gasifier

Kumar Sonu and Pranav P.K.

Garden tea waste (GTW) is the small and thin branch of tea plant obtained during pruning. The gasification potential of GTW was studied using downdraft gasifier. The collected garden tea waste (GTW) was cut into small pieces and physical, proximate and ultimate properties were measured before gasification. Equilibrium modelling was also carried out to predict the gasification behaviour with respect to equivalence ratio. The gasification was conducted at four air flow rates and gas was analysed in terms of calorific value and compositions. It was observed that the GTW was having all properties for good gasification. The combustible gases in producer gas are hydrogen, carbon monoxide and methane. The total combustible gases composition was found maximum 29.50 % at 0.42 equivalence ratio out of which CO and H2 were 17.40 and 10.20 %, respectively. The potential of combustible gases in GTW was observed maximum 0.71 Nm3/kg with the calorific value of 4.2 MJ/Nm3. Maximum carbon conversion efficiency and dry gas yield were observed 99.50 % and 3.38 Nm3/kg respectively. The cold gas efficiency of producer gas was found maximum 60.51% at 0.42 equivalence ratio.

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Physico-chemical characteristics and phytoplankton biodiversity in Sulur lake of Coimbatore, South India

Manickam N., Saravana Bhavan P., Santhanam P., Bhuvaneswari R. and Chitrarasu P.

Phytoplankton is playing an important role in maintaining the global carbon cycle. All through photosynthesis, phytoplankton uses carbon and returns to oxygen in water and atmosphere. The present study dealt the physico-chemical characteristics and phytoplankton diversity in freshwater perennial lake of Sulur (Lat. 11º01'46.11" N and Long. 77°07'08.52" E), Coimbatore District, South India, for the period of one year from March 2011 - February 2012. Totally 50 species of phytoplankton (14 species of cyanophyceae, 19 species of chlorophyceae, 14 species of bacillariophyceae and 3 species of euglenophyceae) were recorded from the Sulur lake. The overall phytoplankton species composition was caused by chlorophyceae (49%) followed by cyanophyceae (24%), bacillariophyceae (23%) and euglenophyceae (4%). Phytoplankton population was positively correlated with various physico-chemical parameters, such as atmospheric and water temperature, pH, salinity, electrical conductivity, total dissolved solids and dissolved oxygen in the lake water. Population density of phytoplankton was higher in Sulur Lake which might be due to higher nutrients and favorable water quality condition. Some phytoplankton species recorded, in this study were registered as pollution indicators producing an odor compound that impact water quality. The study revealed that the Sulur lake possesses appreciable quantum of phytoplankton and it can be used for fish culture with continuous monitoring of physico-chemical parameters.

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