Open Access Original Research Article

In vitro Screening of Some Indian Medicinal Plants for Their Activity against Extended Spectrum Beta Lactamases

Anupma Malik, Sheema Bai, Rahul Taneja, Sunita Dalal

Biotechnology Journal International, Page 1-10
DOI: 10.9734/BBJ/2015/18406

Context: Extended Spectrum Beta Lactamases (ESBLs) play an important role in pathogenesis of various infections by enabling the bacterial species to be resistant to β-lactam antibiotics including extended-spectrum cephalosporins. Plants were selected on the basis of their traditional applications. 

Objective: Our investigation screens and evaluates15 Indian medicinal plants for antimicrobial efficacy and synergistic potential against ESBLs producing bacteria.

Materials and Methods: 6 bacterial strains were screened for their ability to release ESBLs. Plant extracts in methanol and aqueous solvents were screened for their effect on ESBLs producing bacteria. Antimicrobial-linked ESBLs inhibition activity and minimum inhibitory concentration (MIC) of extracts were evaluated by agar well diffusion and microdilution method. Synergistic interactions between plant extracts exhibiting good antimicrobial activity and extended spectrum cephalosporins were explored by Checkerboard method.

Results: Three strains were detected as ESBL positive. The results of susceptibility assay clearly showed strong ESBLs inhibitory effect of Crinum latifolium, Oroxylem indicum, Punica granatum, Sapindus emarginatus and Terminalia chebula and their MIC values ranged from 1.87-30 mg/ml. In vitro interactions between plant extracts and antibiotics cefotaxime and ceftaizidime evaluated in terms of fractional inhibitory concentration (FIC) indices indicated synergism.

Discussion and Conclusion: Higher resistance of ESBLs positive strains to β-lactam antibiotics encourages us to search the novel ESBLs inhibitors. Maximum 10-fold decline in the MIC of antibiotics cefotaxime and ceftaizidime was achieved in combination with plant extracts. Owing to enormous clinical significance of ESBL-producing organisms coupled with limited therapeutic options, the results revealed by present study are of paramount importance.

Open Access Original Research Article

In vitro Plantlet Regeneration of Four Local Garlic (Allium sativum) Accessions of Bangladesh

Sayed Raihanul Haider, Mohammad Rashed Hossain, Shanjida Rahman, Shirin Sultana, Tamanna Quddus, Moutoshi Chakraborti, Aunamika Hoque, Md Hasan Shahriar, Md Ashraful Haque

Biotechnology Journal International, Page 1-12
DOI: 10.9734/BBJ/2015/18619

Aims: The genetic improvement of garlic can be achieved by biotechnological manipulations as breeding in this vegetatively propagated crop is limited. The current research was conducted with a view to develop an efficient in vitro regeneration protocol for four local garlic accessions namely, G121, G122, G123 and G124.

Place, Duration and Design of Study: The experiment was conducted in the Tissue Culture Laboratory of the Department of Genetics and Plant Breeding, Bangladesh Agricultural University during the period from June 2013 to June 2014 using three-factorial experimental design.

Methodology: The root tips, basal disc and leaf base were cultured in MS medium supplemented with 2, 4-Dichlorophenoxyacetic acid (2, 4-D) alone, and with both 2, 4-D and 6-Benzylaminopurine (BAP) together for callus induction and the later for subsequent sub-culturing and proliferation of callus. MS medium supplemented with 1-Naphthaleneacetic acid (NAA) and BAP was used for plantlet regeneration.

Results: The percentage of callus induction increased with the increase in the concentration of 2,4-D, starting from 0.5 mg L-1 till 2.0 mg L-1 and declined with further increase in the concentration of 2,4-D. The MS medium supplemented with 2,4-D and BAP showed higher percentage of callus induction and callus  proliferation compared to that of with 2,4-D alone. The highest percentage of callus induction was observed in the genotype G124 from the explant basal disc (85%) and in the genotype G121 from the explant leaf base (80%) with 2.0 mg L-1 2,4-D and 2.0 mg L-1 BAP. MS medium supplemented with 2 mg L-1 2,4-D + 0.5 mg L-1 BAP showed highest percentage of callus proliferation (90%) in almost all the genotypes. The highest percentage of plantlet regeneration were observed in the genotype G124 for the explants basal disc (63.33%) in MS medium supplemented with 2 mg L-1 NAA + 1 mg L-1 BAP. The survival rate of the plantlets after acclimatization varied from 40% (in G123) to 70% (in G121).

Conclusion: The optimized protocol of plant regeneration from local garlic accessions will be useful for any future garlic improvement programs using biotechnological means.

Open Access Original Research Article

Isolation, Molecular Characterisation of Polyhydroxyalkanoate Producing Novel Bacillus sp., skm7T from a Polluted Pond Water

K. Chaitanya, S. K. Mahmood, M. Mohammed Idris

Biotechnology Journal International, Page 1-9
DOI: 10.9734/BBJ/2015/17999

Bio-plastics are natural biopolymers that are synthesized and catabolised by various organisms and these materials do not cause toxic effects in the host and have certain advantages over petroleum-derived plastics. The current emphasis on sustainability, eco-efficiency and green chemistry has led to intensive search for renewable and environmentally friendly resources. Thus, sustainable development is recognized to be essential for the growth of the economy and industrial productivity. A polyhydroxyalkanoate (PHA) producing Gram-positive, rod-shaped, motile bacterium was isolated from the polluted pond water. Strain SKM7T grew at 15–40ºC and pH 5.0–8.5 and in the presence of 0–1.5% (w/v) NaCl. The strain was catalase-positive and oxidase-positive. Antimicrobial activities were studied. The DNA G+C content was 53 mol%. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain is a member of the genus Bacillus and is most closely related to B. aryabhattai B8W22(T) (99.01%), B. megaterium IAM 13418(T)  (98.86%), B. flexus IFO 15715(T) (97.80%). The sequence of the 16S rDNA gene of strain SKM-7 was determined as 1554 bp and deposited in the EMBL under accession no. LM655314. The major isoprenoid quinine was MK-7 and an unidentified glycolipid was identified. The major fatty acid are dominated by saturated iso and anteiso (anteisoC14:0, iso-C15:0, anteisoC15:0, anteisoC16::0). The phenotypic and genotypic properties clearly indicate that strain represents a novel species of the genus Bacillus. The strain produced PHA in mineral medium consisting of glucose and nitrogenous substances. The type strain is SKM7T (=KCTC 33686T).

Open Access Original Research Article

Histo-architechtural Evaluation of Conventional Versus Two Rapid Microwave Processing Techniques

Tobias Peter Pwajok Choji, Anthony Ajuluchukwu Ngokere, Samuel Ifedioranma Ogenyi, Peterside Rinle Kumbish

Biotechnology Journal International, Page 1-19
DOI: 10.9734/BBJ/2015/18948

Aims: This work aims to reduce the time of tissue processing, exclude xylene from tissue processing as well as to reduce the total quantity of reagents used per cycle of paraffin wax processing technique.

Study Design: Harvesting and fixation of tissues. Grossing into triplicates, processing using three different techniques. Staining and grading of sections.

Place and Duration of Study: Apparently healthy rabbits from the animal house, National Veterinary research Institute, Vom, Jos, Nigeria, between August and December, 2014.

Methodology: Two apparently healthy rabbits were sacrificed and the Trachea, Lungs, Heart, Liver, Kidney, Stomach, Skin, Brain, and the Spleen were harvested and fixed in 10% buffered formalin for three days. They were grossed into triplicates, labeled and processed using the conventional, microwave without vacuum and microwave with vacuum respectively. They were sectioned and stained simultaneously using the haematoxylin and eosin staining technique as well as the Gordon and Sweet’s method. They were graded as excellent if they permitted microscopy, fair if not very good but can permit microscopy and poor if they cannot permit microscopy at all.

Results: There is a drastic reduction in the duration of processing as well as the quantity of reagents used in the microwave techniques as compared to the conventional method. Xylene was completely eliminated in the microwave techniques. Tissue histo-architechture, special features as well as silver impregnation were clearly demonstrated without significant differences. Dye uptake as well as section thickness were comparable among the three techniques.

Conclusion: Same-day turn-around is possible in histology, with reduced reagent consumption and elimination of xylene, without compromising section quality, dye uptake or ability to reduce silver to its metallic form. This will result to quick diagnosis hence quick intervention at a cheaper rate to both laboratories and clients. It is a good innovation in forensic and diagnostic histopathology and should be encouraged. Its compatibility with histochemical, immunohistochemical and molecular techniques should be evaluated to give a wider application.

Open Access Original Research Article

Development of Maize Haploid Inducer Lines and Doubled Haploid Lines in Pakistan

Khunsa Khakwani, Muhammad Rafiq Dogar, Muhammad Ahsan, Amir Hussain, Muhammad Asif, Ahsan Raza Malhi, Muhammad Altaf

Biotechnology Journal International, Page 1-7
DOI: 10.9734/BBJ/2015/18394

The work on maize doubled haploid development has started at the Maize Research Station, Ayub Agricultural Research Institute, Faisalabad, Pakistan during 2010 in collaboration with University of Agriculture Faisalabad, Pakistan. The aim of current study was to develop locally adopted maize haploid inducer lines utilizing cheap and easy source of Stock6 and indeterminate gametophyte mutant ig1 gene lines imported from Maize genetic Coop Stock Centre. The lines were later utilized to produce doubled haploid inbred lines. The environmental conditions of Faisalabad are extreme in nature. In spring crop during pollination period temperature may reach up to 45°C. Therefore, locally adopted haploid inducers are needed. Best performing local inbred lines were screened having branched heavy tassel, bold seed, good pollen shedding ability and vigor. These lines were utilized as female donor parent while imported lines as recurrent pollen parent in back cross breeding program until BC4 generation. These lines were selfed twice until BC4F2 and evaluated for haploid induction rate (HIR). Lines with high HIR were further intercrossed to achieve maximum transgressive segregation. Mass selection for the adaptation traits was exercised for individual F2 plants followed by ear-to-row plantation of selected progeny. Four best haploid inducer lines with HIR up to 5% having very good tassel size, height and heat stress tolerance were selected in 2014. Haploid seeds collected in different induction crosses until 2012, were used for colchicine doubling treatment. The doubling percentage of the haploid plants was very low 0.15% and out of 1000 treated seedling 15 survived and only 5 reached up to maturity, where only one D0 cob was harvested. This was successfully selfed and was grown in three different locations for next two seasons and there was no segregation in the successive generations. Developing countries that cannot afford costly haploid inducer lines can work on Stock6 and other cheap sources available free of cost and can develop their own haploid inducer lines well adapted to their own climatic conditions.