Current trends in chloroplast genome research

Chloroplast is an important cellular organelle of autotrophs which has an independent, circular, doublestranded DNA molecule termed as chloroplast genome. The chloroplast DNA (cpDNA) contains essential genes for its maintenance and operation. Several components of the photosystems andproteins involved in biosynthetic pathways are also encoded by the chloroplast genome. Exploring the genetic repository of this organelle is vital due to its conserved nature, small size, persistent gene organization and promising ability for transgenic expression. Therefore, cpDNA sequence information has been instrumental in phylogenetic studies and molecular taxonomy of plants. Chloroplast genome sequencing efforts have being initiated with conventional cloning and chain-termination sequencing technologies. Dedicated databases such as CGDB and GOBASE among others have been established as more and more complete cpDNA sequences are being reported. Presently, elegant molecular biologytechniques including shotgun sequencing, rolling circle amplification (RCA), Amplification, Sequencing and Annotation of Plasteome (ASAP) and Next generation sequencing are being used to accelerate data output. Owing to many fold increase in submission of cpDNA sequences in nucleotide databases, challenges of in-depth data analysis stimulated the emergence of devoted annotation, assembling and phylogenetic software. Recently, reported bioinformatics software for chloroplast genome studiescomprise of DOGMA for annotation, SCAN-SE, ARAGON and PREP suit for RNA analyses and CG viewer for circular map construction/comparative analysis. Faster algorithms for gene-order based phylogenetic reconstruction and bootstrap analysis have attracted the attention of research community. Current trends in sequencing strategies and bioinformatics with reference to chloroplast genomes hold great potential to illuminate more hidden corners of this ancient cell organelle

Host plant selection and oviposition behaviour of whitefly Bemisia tabaci (Gennadius) in a mono and simulated polyculture crop habitat

The host plant selection, oviposition behaviour and survivorship of whitefly (Bemisia tabaci (Gennadius) was evaluated in green house. Three host plants cultivars namely: Brinjal (Solanum malagna), chilli (Capsicum annuum) and tomato (Solanum lycopersicum) were placed in a multiple crop habitat at 34 - 36°C, 70 - 80% relative humidity (RH) in a normal day light. There was a variation of morphological characteristic among host plants (smooth and thick trichome leaves) where all developmental stages of pest were given free choice of foraging. Although the host plants genus C. annum and S. lycopersicum were also the potential host of B. tabaci but in the presence of S. malagna, the attack rate remains minimum on both the host plants. The feeding and egg laying was significantly higher on S. malagna L. leaves as compare to other two host plants in the open arena. S malagna L. was also preferred when pest was tested in confined cages for free choice probing on capsicum and S. malagna L. There was no significant but a slight difference in survivorship of all developmental stages of whitefly when Brinjal and Chilli and then Brinjal and tomato from two different experimental arenas were compared. There was no host plant varietal effect on the overall developmental time from egg eclosion to the adult. The leaves of S. malagna with thick trichomes were chosen significantly more higher for egg laying compared to other host plants. The morphological characters and plant architecture contribute to higher densities of adult whitefly (wf) compared to new leaves. No symptoms of viral infection has been observed on the chilli with Brinjal, whereas, the same variety of chilli was reasonably infected with virus in chilli monocrop arena.Key words: Multiple cropping, host plant selection, oviposition fecundity, intrinsic rate of increase

Comparative genomics of an endophytic Pseudomonas putida isolated from mango orchard.

This is the final version of the article. Available from the publisher via the DOI in this record.We analyzed the genome sequence of an endophytic bacterial strain Pseudomonas putida TJI51 isolated from mango bark tissues. Next generation DNA sequencing and short read de novo assembly generated the 5,805,096 bp draft genome of P. putida TJI51. Out of 6,036 protein coding genes in P. putida TJI51 sequences, 4,367 (72%) were annotated with functional specifications, while the remaining encoded hypothetical proteins. Comparative genome sequence analysis revealed that the P. putida TJI51genome contains several regions, not identified in so far sequenced P. putida genomes. Some of these regions were predicted to encode enzymes, including acetylornithine deacetylase, betaine aldehyde dehydrogenase, aldehyde dehydrogenase, benzoylformate decarboxylase, hydroxyacylglutathione hydrolase, and uroporphyrinogen decarboxylase. The genome of P. putida TJI51 contained three nonribosomal peptide synthetase gene clusters. Genome sequence analysis of P. putidaTJI51 identified this bacterium as an endophytic resident. The endophytic fitness might be linked with alginate, which facilitates bacterial colonization in plant tissues. Genome sequence analysis shed light on the presence of a diverse spectrum of metabolic activities and adaptation of this isolate to various niches.This research was financially supported by the Higher Education Commission, Islamabad Pakistan

Optimization of in vitro multiplication for exotic banana (Musa spp.) in Pakistan

The present attempt aimed to optimize micropropagation protocols supplemented with different concentrations and combinations of benzylaminopurine (BAP) (0, 2, 4, 6 mg L-1) and indole acetic acid (IAA) (0.5 and 1.0 mg L-1). Exotic banana (Musa spp) genotypes GCTCV-215 (AAA), ‘Yangambi’ Yangambi Km-5 (AAA) and FHIA-23 (AAAA) were used in research work. Experiments were conducted at Plant Tissue Culture Laboratory of Nuclear Institute of Agriculture (NIA), Tando Jam. Data collected for in vitro shoot consists of the following parameters: days for bud initiation, rate of shoot proliferation (%), number of multiple shoots, shoot length (cm) and fresh mass of shoot (g). Significant (p≤ 0.05) variations were observed for varieties, treatments and varieties x treatment for all the parameters. Synergistic effects of BAP and IAA were observed in GCTCV-215 and Yangambi Km-5. Out of various treatments, best concentration for multiple shoot in short period of time for GCTCV-215 and Yangambi Km-5 was found in 4.0 mg /l BAP + 0.5 mg L-1 IAA. Maximum fresh mass of shoot observed at same concentration and combination of BAP and IAA and for shoot length combination of 4.0 mg L-1 BAP with 1.0 mg L-1 IAA was found to be most suitable for GCTCV-215 and Yangambi Km-5. FHIA-23, show better performance in MS medium supplemented with only BAP at concentration 4.0 mg/L-1. After development of root, in vitro plantlets were shifted from growth room to green house in polythene bags containing garden soil and humus mixture in ratio (1:1).Keywords: Micropropagation efficiency, exotic musa genotype, growth regulators

Biomass expansion factors of Olea ferruginea (Royle) in sub tropical forests of Pakistan

Wood biomass gives information about total productivity of the forest as well as individual tree. Olea ferruginea (Royle) which is small and evergreen is widely distributed in native sub tropical forests of Pakistan and extensively used as fuelwood domestically. This study was carried out in the sub tropical forests of Pakistan at 33° 38’ north and 73° 00’ east latitude and longitude, respectively, and at an elevation of 917 m. Trees with exploitable diameter were selected randomly from the entire forest.Destructive sampling techniques were used for measuring biomass (kgm-3) in all the tree components. For this purpose, 5 trees were felled and the biomass of each component of the tree including main stem, branches, leaves, twigs and roots were estimated separately using volume, weight and density. The generic data of wood density (kgm-3) was used to determine the biomass (kg). The study showed that average contribution of stem portion of the tree was 49.01% of the total tree biomass, and branches showed 31.17%, leaves 1.98%, twigs 1.05% and roots 16.65% of the total tree biomass. So, it was found that the major part of the total tree biomass was present in the stem portion of O. ferruginea. Totalvolume of the tree was also found to be dependent on the diameter of the tree. Mean volume of the tree was 0.475 ± 0.07 m3. The prepared  biomass expansion factor will be helpful in estimating productivity, carbon stocks and yield of the forest.Key words: Biomass, biomass expansion factor, tree volume, Olea ferruginea

Isolation and screening of alkaline protease producing bacteria and physio-chemical characterization of the enzyme

Soil samples from different habitats including tanneries, soap industries, garden soil and soil compost were screened for the presence of alkalophilic Bacillus isolates capable of producing alkaline protease in large quantities. One hundred and eighteen (118) isolates were found having proteolytic activity on skim milk agar plates. Isolates forming larger zones, as a result of casein hydrolysis were further studied for quantitative production of extracellular alkaline protease activity in the shake flask studies. Isolate CEMB10370 gave maximum activity. Time course studies indicated that strain CEMB10370 had the highest protease activity (380 APU/mL) after 48 h of fermentation. The wild type enzyme was biochemically characterized. The enzyme exhibits optimal activity at 50°C and pH 11.5. The protease enzyme was completely inhibited by phenylmethylsulfonyl (PMSF, serine protease inhibitor) and its isoelectric point was ~9.5. The enzyme was purified by ion-exchange chromatography using CMSepharose column as a ~29 Kilo Dalton (kDa) protein.Key words: Alkaline protease, alkalophilic ,Bacillus subtili

Development of RAPD based markers for wheat rust resistance gene cluster (Lr37-Sr38-Yr17) derived from Triticum ventricosum L.

Rust diseases are the major cause of low yield of wheat in Pakistan. Wheat breeders all over the world as well as in Pakistan are deriving rust resistance genes from alien species like Triticum ventricosumand introducing them in common wheat (Triticum aestivum). One such example is the introgression of rust resistance gene cluster Lr37-Sr38-Yr17 derived from T. ventricosum chromosome 2NS into thecommon wheat. A basic prerequisite to introduce alien rust resistance gene (like those present on 2NS segment) in locally adapted varieties is availability of a suitable marker system which can be used tokeep track of presence of newly added gene in the old background. In this present study, one hundred and fifty Randomly Amplified Polymorphic DNA (RAPD) primers were used to detect polymorphismbetween two near isogenic lines NILs (Anza and Anza+2NS) of wheat and to develop RAPD based molecular markers for rust resistance gene cluster derived from T. ventricosum. Polymerase chainreactions were carried out using standard protocols. All the amplification products were in the range of 250 to 1000 bp. Thirteen molecular markers (RAPDs) out of a total of 150 (approximately 8.6%) were developed for rust resistance gene cluster Lr37-Sr38-Yr17 and recommendations have been made to utilize these markers in Pakistani wheat breeding programs aimed at establishing rust resistantgermplasm

Absence of Both IL-7 and IL-15 Severely Impairs the Development of CD8+ T Cell Response against Toxoplasma gondii

CD8+ T cells play an essential role in the protection against both acute as well as chronic Toxoplasma gondii infection. Although the role of IL-15 has been reported to be important for the development of long-term CD8+ T cell immunity against the pathogen, the simultaneous roles played by both IL-15 and related γ-chain family cytokine IL-7 in the generation of this response during acute phase of infection has not been described. We demonstrate that while lack of IL-7 or IL-15 alone has minimal impact on splenic CD8+ T cell maturation or effector function development during acute Toxoplasmosis, absence of both IL-7 and IL-15 only in the context of infection severely down-regulates the development of a potent CD8+ T cell response. This impairment is characterized by reduction in CD44 expression, IFN-γ production, proliferation and cytotoxicity. However, attenuated maturation and decreased effector functions in these mice are essentially downstream consequences of reduced number of antigen-specific CD8+ T cells. Interestingly, the absence of both cytokines did not impair initial CD8+ T cell generation but affected their survival and differentiation into memory phenotype IL-7Rαhi cells. Significantly lack of both cytokines severely affected expression of Bcl-2, an anti-apoptotic protein, but minimally affected proliferation. The overarching role played by these cytokines in eliciting a potent CD8+ T cell immunity against T. gondii infection is further evidenced by poor survival and high parasite burden in anti IL-7 treated IL-15−/− mice. These studies demonstrate that the two cytokines, IL-7 and IL-15, are exclusively important for the development of protective CD8+ T cell immune response against T. gondii. To the best of our knowledge this synergism between IL-7 and IL-15 in generating an optimal CD8+ T cell immunity against intracellular parasite or any other infectious disease model has not been previously reported

Fabrication of Microbicidal Silver Nanoparticles: Green Synthesis and Implications in the Containment of Bacterial Biofilm on Orthodontal Appliances

Among various metal-based nanoparticles, silver nanoparticles (AgNPs) manifest superior inhibitory effects against several microorganisms. In fact, the AgNP-based treatment has been reported to inhibit both sensitive and resistant isolates of bacteria and other disease-causing microbes with equal propensity. Keeping this fact into consideration, we executed bio-mediated synthesis of AgNPs employing extract of flower and various other parts (such as bud and leaf) of the Hibiscus rosa-sinensis plant. The physicochemical characterization of as-synthesized AgNPs was executed employing transmission electron microscopy (TEM), dynamic light scattering (DLS), zeta potential, Fourier transform infrared (FTIR) spectroscopy, and UV-Vis spectroscopy, etc. The as-synthesized AgNPs demonstrated strong antimicrobial activity against both Gram-positive and Gram-negative bacteria with equal propensity. The as-synthesized AgNPs successfully inhibited Streptococcus mutans (S. mutans), one of the main causative bacteria responsible for dental caries. Considering the fact that orthodontic appliances facilitate infliction of the oral cavity with a range of microbes including S. mutans, we determined the growth inhibitory and anti-adherence activities of AgNPs on orthodontic appliances. We performed microbiological assays employing AgNPs adsorbed onto the surface of nickel–titanium (Ni-Ti) orthodontic wires. A topographic analysis of the decontaminated Ni-Ti orthodontic wires was performed by scanning electron microscopy. In addition to antimicrobial and anti-biofilm activities against oral S. mutans, the as-fabricated AgNPs demonstrated significant inhibitory and anti-biofilm properties against other biofilm-forming bacteria such as Escherichia coli and Listeria monocytogenes

Novel microwell-based spectrophotometric assay for determination of atorvastatin calcium in its pharmaceutical formulations

The formation of a colored charge-transfer (CT) complex between atorvastatin calcium (ATR-Ca) as a n-electron donor and 2, 3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ) as a π-electron acceptor was investigated, for the first time. The spectral characteristics of the CT complex have been described, and the reaction mechanism has been proved by computational molecular modeling. The reaction was employed in the development of a novel microwell-based spectrophotometric assay for determination of ATR-Ca in its pharmaceutical formulations. The proposed assay was carried out in 96-microwell plates. The absorbance of the colored-CT complex was measured at 460 nm by microwell-plate absorbance reader. The optimum conditions of the reaction and the analytical procedures of the assay were established. Under the optimum conditions, linear relationship with good correlation coefficient (0.9995) was found between the absorbance and the concentration of ATR-Ca in the range of 10-150 μg/well. The limits of detection and quantitation were 5.3 and 15.8 μg/well, respectively. No interference was observed from the additives that are present in the pharmaceutical formulation or from the drugs that are co-formulated with ATR-Ca in its combined formulations. The assay was successfully applied to the analysis of ATR-Ca in its pharmaceutical dosage forms with good accuracy and precision. The assay described herein has great practical value in the routine analysis of ATR-Ca in quality control laboratories, as it has high throughput property, consumes minimum volume of organic solvent thus it offers the reduction in the exposures of the analysts to the toxic effects of organic solvents, and reduction in the analysis cost by 50-fold. Although the proposed assay was validated for ATR-Ca, however, the same methodology could be used for any electron-donating analyte for which a CT reaction can be performed