Research Journal of Biotechnology

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Vol. 11(3) March 2016

Improvement of Agrobacterium-mediated transformation efficiency of maize (Zea mays L.) genotype Hi-II by Optimizing Infection and Regeneration Conditions

You Xu, Wen Ren, Ya Liu and Jiuran Zhao

Improving Agrobacterium tumefaciens mediated transformation of maize would be beneficial for maize transgenic research. Although, maize genotype Hi-II is used in various maize transformation research efforts, improvements in the transformation frequency of this genotype are still needed. In the present study, the immature maize embryos were given heat-shock pre-treatment prior to Agrobacterium-mediated transformation. About 9% increase in the transformation frequency was achieved when the embryos were subjected to 42 °C for 3 min or 38°C for 9 min. It was also found that the use of 100 mg/L Casein Hydrolysate (CH) accelerated somatic embryogenesis in the initial period of regeneration. The combined use of 0.5 mg/L Naphthaleneacetic acid (NAA) and 2.0 mg/L Multi-effect triazole (MET) at different regenerate stage could effectively enhance root development and seedlings vigour to improve the survival rate of the transformed plantlets. Through integrating several methods can effectively improve the Hi-II genetic transformation efficiency.

Feeding of phytase over-expressed Lactobacillus decreases fecal phosphate in chicken

Li Qingzhi and Shi Yang

Phytic acid is the main phosphate pool in plant seeds which are widely used as chicken feedstuff. However, as monogastric animal, chicken can only utilize phytic acid very inefficiently because of lacking effective phytase. The current situation is mineral phosphate supplemented in feedstuff increasing cost and organic phosphate in phytic acid excreted in feces leading to environmental pollution. Probiotic lactobacilli, especially L. acidophilus were innate isolate of health chicken gut microbiota. Except their health-promoting effect, using them as hosts for delivery and secret the appA gene into chicken digestive tract is attractive. The study validated the applicability of this novel strategy with the recombinant L. acidophilus/ pTRKH2P that can secret the Eschericha coli appA encoded phytase under the promoter region and signal peptide sequence of lactococcal usp45 gene. In this study, we report the engineering of the recombinant strain L. acidophilus/pTRKH2P, which secrets active phytase and can degrade phytic acid in maize. Feeding of this construct decreased fecal phosphate in chicks compared to control strain that harboring empty vector. Such a strategy improved the utilization of phosphate in phytic acid and decreased phosphate excretion at the same time.

Effects of Green Light Intensity on Shade Avoidance Symptoms and Chlorophyll Degradation in Rice Seedlings

Chen Chang-Chang, Huang Wen-Dar and Yang Chi-Ming

Our objectives in this study were to investigate morphological traits and dynamics of chlorophyll (Chl) degradation intermediates (chlorophyllide, Chlide; pheophytin, Phe; pheophorbide, Pho) in leaves of rice (Oryza sativa L.) seedlings under increasing green light intensity. Seedlings of Taichung Native 1 (TCN1) were grown under equal intensities (40 μmol m-2 s-1) of red and blue light with four levels of green light intensity (0, 20, 40 and 60 μmol m-2 s-1). Light emitting diodes (LED) were used to control lighting treatments. Sheaths of rice seedling leaves elongated and leaves grew erectly under red and blue light with increasing green light intensity. These morphological traits are known as shade avoidance symptoms (SAS). Increasing green light intensity resulted in decreases in total chlorophyll, the effective quantum yield of photosystem II (ФPSII) and Phe/Chlide ratios and increases in non-photochemical quenching (NPQ) and Chlide levels. These results indicated that green light induced SAS and mediated Chl degradation routes in rice seedlings.

DNA based Genetic Diversity and Relationship Analysis in Indian Domesticated Capsicum spp.

Dutta Deka Sharmila, Dadlani Malavika, Sharma Ramendranath and Talukdar Akshay

Cultivated genotypes of Capsicum annum are reported to have close genetic association because of their sharing of common ancestors. In the present investigation focus was given on the application of molecular markers in efficient utilization for the conservation of plant genetic recourses, with insights into the genome fingerprinting, genetic diversity and relationships analysis of Capsicum genotypes. In the present study genetic structure and divergence of sixty capsicum accessions were analyzed with 20 RAPD, 34 SSR and 15 ISSR markers. Suitable markers were identified to study the diversity among the capsicum genotypes. These molecular markers grouped sixty genotypes into six clusters based on genetic similarities. Significant polymorphism information content (PIC) values of RAPD (0.42), ISSR (0.68) and SSR (0.80) profiles were obtained. The results illustrated the potential of RAPD and ISSR marker systems to distinguish the capsicum genotypes. The present communication can be planned for efficient application of RAPD, ISSR and SSR markers for the conservation and identification of capsicum annum germplasm.

Antioxidant and Antidiabetic properties of Abelmoschus esculentus extract – an in vitro assay

Babu Thabraz Ahmed and Sekar Ashok Kumar

Diabetes mellitus is a leading cause of morbitity and mortality worldwide. There has been a constant search for natural compounds that possess anti-diabetic effect with little or no side effects unlike the synthetic compounds. The purpose of this investigation is to determine the antioxidant potential and in vitro anti-diabetic activities of Abelmoschus esculentus. The antioxidant activity of Abelmoschus esculentus extracts was determined by total antioxidant activities like DPPH, ferrous ion chelating activity and inhibition of β- carotene bleaching. At 0.125 to 2.0 mg/mL, the scavenging activities of aqueous, ethanol and methanol extracts on DPPH radical ranged from 10.2 to 80.2%, 11.5 to 90.6% and 12.8 to 93.6%, respectively. The strongest chelating effect 68.5% was obtained from methanol whereas ethanol shows 65.6% at 1.0 mg/mL. At this concentration, the lowest ferrous ion chelating effect was exhibited by aqueous extract (62.4%). Abelmoschus esculentus efficiently inhibits both α-amylase and α-glucosidase enzymes in vitro in a dose dependent manner. These results revealed that methanolic extracts of Abelmoschus esculentus have better antioxidant and antidiabetic activity than ethanol and aqueous extracts which support the medicinal properties of Abelmoschus esculentus.

Development of PCR based SCAR markers for rapid authentication of Boerhavia errecta, B. diffusa and their adulterant Trianthema species

Kumar Vinay

Boerhavia diffusa (B. diffusa), also known as Punarnava, is an indigenous plant and an important component in traditional Indian medicine. The accurate identification and collection of this medicinal herb is vital to enhance the drug’s efficacy and biosafety. In this study, DNA based molecular technique has been applied to identify and distinguish B. diffusa from its closely-related species B. errecta and their widely used adulterant Trianthema species. A protocol for extraction of total genomic DNA was optimized and found that 4% CTAB produced better quality and quantity of DNA for fresh leaves. A total 60 numbers of RAPD primers of OPN, OPF and OPQ series 1-20 were screened for the identification of polymorphic, reproducible and species specific markers among the three species. Among them 8 primers namely, OPN 5, 7, 8, 9, 10, 15, 16 and OPQ1 were found to produce polymorphic band in any of the three species. A total of six species specific bands (two of each species) were cloned and sequenced and from their nucleotide sequences, species specific SCAR markers developed. B. errecta specific SCAR marker, BEN-07-1 and BEN-07-2 produced 408bp and 487bp amplicons exclusively in B. errecta whereas Trianthema specific marker TMM-01 produced 206bp amplicon in Trianthema and no amplicons were obtained in B. diffusa and B. errecta. The markers developed are efficient and reliable in authenticating the B. errecta, B. diffusa and its adulterant Trianthema. These markers could be used as molecular pharmacognostic tool in quality control of raw drug.

Correlation Studies on the Role of Phospholipids and Cholesterol with Morphologically Normal Sperm Cells for Diagnosis of Human Male Infertility

Ranganathan Parameswari, Sundaram Vickram, Balasundaram Sridharan and Manian Ramesh Pathy

Evaluation of phospholipids and cholesterol plays an important role in morphological function of sperm cells. The major objective of this study was to compare and correlate the phospholipids and cholesterol levels in seminal plasma and sperm cells of fertile and infertile men. Phospholipids and cholesterol concentration in the semen samples of 25 fertile and 25 infertile men were used for this study. There is a significant relationship between morphology of sperm with phospholipids concentration in infertile sperm subjects, (P < 0.0001, spearman r = 0.97), also a negative correlation between morphology of sperm with cholesterol concentration in infertile subjects, (P < 0.01, spearman r = -0.04). These results suggested that phospholipids were important for maintaining the normal morphology of sperm and cholesterol was found to have no effect on the morphology of sperm in fertile and infertile men.

Effects of calcium on mitochondrial function and antioxidant defense in roots of apple rootstock (Malus baccata Borkh.) under rapid changes in temperature

Su Hong, Ma Huaiyu and Lyu Deguo

Calcium, an important second messenger, is involved in plant temperature stress response. In order to explore the effect of calcium signaling on mitochondrial function in response to rapid changes in temperature, the seedlings of Malus baccata Borkh. were used as a research subject and treated with distilled water (control), calcium chloride (CaCl2), calmodulin antagonist and trifluoperazine (TFP) before temperature treatment. In this study, rapid changes in temperature caused mitochondrial dysfunction and accumulation of reactive oxygen species which finally resulted in lipid peroxidation and decline in root vitality. Exogenous application of CaCl2 improved calmodulin concentration effectively alleviated mitochondrial dysfunction and oxidative damage triggered by rapid changes in temperature and raised the activities of alternative (Alt) pathway and antioxidant enzymes which ultimately improved root vitality. The TFP treatment reversed the effects of CaCl2. This study provides the evidence of the important and protective roles of exogenous calcium in M. baccata response to rapid changes in temperature, partially via enhancing the calcium signal transduction and activating the antioxidants and Alt pathway. Calcium (e.g., CaCl2) application on M. baccata in northern China should be conducted in early spring to mitigate damages induced by rapid changes in temperature.

In Vitro Genotoxicity Evaluation of Combinational Pesticide Chlorpyrifos 50% + Cypermethrin 5% EC

Rajini A. and Revathy K.

In vitro screening was carried out to evaluate mutagenic and cytogenotoxic (inhibitory) potential of Chlorpyrifos 50% + cypermethrin 5% EC. It is extensively used in controlling pests in soil and foliage, household and used as animal ectoparasiticide in animal house and controlling adult mosquitos, larvae in public health. The objective of this study is to extend the knowledge about the effects of the pesticide in virtue of genotoxic potentials. AMES assay was performed using five tester strains of Salmonella typhimurium. It is non mutagenic at the concentrations of 9.75, 19, 39, 78 and 156 µg/plate tested. In cultured human peripheral blood lymphocytes following three treatment regimes 4 hour with and without S9 activation and a continuous treatment without S9 activation. Cytotoxicity in terms of hemolysis and decreased cellularity was observed at higher concentrations of 2, 1 and 0.5 µg/mL of 4 h exposure of with and without S9 and in continuous exposure 2, 1, 0.5 and 0.25 µg/mL tested. No significant increase in numerical and structural aberrations was found compared to the negative controls at all treatment regimes. Hence Chlorpyrifos 50% + cypermethrin 5% EC are adjudged to be non genotoxic with Invitro chromosomal aberration assay.

Expression differences of pBD-1, pBD-2 and pBD-3 genes in tissues from Wujin and Yuedawu pigs

An Qingcong and Guo Rongfu

Defensins are useful materials to animal health for their antibacterial function. In this experiment, the differential expression of three porcine β-defensin genes(pBD-1, pBD-2 and pBD-3) in different tissues from Wujin and Yuedawu pigs were determined by qRT-PCR. Results revealed that significant expression differences of pBD-1, pBD-2 and pBD-3 genes were found at P<0.05 or P<0.01 level among some tissues for both Wujin and Yuedawu pigs. Our results also indicated that the expression of pBD-1, pBD-2 and pBD-3 genes of Yuedawu pigs in all detected tissues are extensively higher than those of Wujin pigs. For pBD-1 gene, significant expression differences were found in heart, liver, lung, kidney, ileum, skin, muscle, pancreas, testis and ovary (P<0.05 or P<0.01) tissues. For pBD-2 gene, significant expression differences were found in heart, lung, kidney, jejunum, duodenum, muscle and ovary (P<0.05 or P<0.01) tissues. For pBD-3 gene, significant differences were found in heart, liver, lung, kidney, ileum, duodenum, skin, muscle, pancreas, testis and ovary (P<0.05 or P<0.01) tissues. These data suggested that the expression of porcine pBD-1, pBD-2 and pBD-3 genes has breed and tissue specificity.

Genetic Diversity Analysis in Some Marine Fish Species of Gujarat Coast through Morphological and Molecular Markers

Yusufzai S. I., Padhiyar Shital M., Lende Smit, Tomar Rukam S., Thummar Vibha D., Thakkar Jalpa R., Rathod Visha M., Kheni Jasmin V., Kothari Vishal, Parakhia M. V. and Golakiya B. A.

The investigation was carried out to explore the morphological and molecular characterization in 25 species of fish available at the coastal belt of Veraval, Gujarat, INDIA. For morphological characterization, total 15 different characters were studied which showed significant variation for all the traits studies. The total length of fish varied from 96cm to 528cm while the other traits like standard length (73-368cm), fork length (89-384cm), head length (8 to 127cm), pre orbital length (4-92cm), eye diameter(3-16cm), post orbital length (10-64cm), 1st dorsal fin base (11-329cm), inter dorsal space(4-49cm), 2nd dorsal fin base (3-181cm), length of caudal peduncle (5-99cm), depth of body (28-132cm), length of pectoral fin (19-149cm) anal fin base (11-211cm) and pelvic fin (4-69cm) also showed sufficient range to distinguish the fishes into different groups. Molecular characterization using fifty four Inter Simple Sequence Repeats (ISSRs) primers yielded 459 fragments with an average of 8.5 fragments/primer. The overall grouping pattern of cluster analysis clearly showed the similarity and variability among each species. These DNA based markers along with morphological markers can be used for taxonomic issues, breeding program, studying genetic variability, gene tagging and also for genetic-diversity assessment, cultivar fingerprinting and phylogenetic studies.

Genetic aspects of F1 crossbred between Black Bedouin and Damascus goats

Al-Atiyat Raed M.

The crossbreeding strategy is the least expensive and most widely used in order to increase yields. A first generation cross of Damascus and Black Bedouin goats in Jordan was assessed for their genetic aspects at DNA level using 13 microsatellite markers. All the microsatellites were polymorphic, with 3-9 alleles for the three breeds. The mean number of alleles per locus was 4.31, 5.77 and 4.54 in Damascus, Black Bedouin goats and F1 crossbred respectively. The heterozygosity varied from 0.20 to 0.9, with a mean of 0.746, 0.707 and 0.785 in Damascus, Black Bedouin goats and F1 crossbred respectively. The inbreeding coefficient (Fis) values were mostly negative for the Crossbreds with an average of -0.158. The differentiation coefficient (Fst) results suggest that the F1 crossbred was closely related to Damascus than Black Bedouin goats. Furthermore, the structure analysis showed heterogeneous genotypes reflecting the assumption that interactions among favorable alleles belonging to the two parental breeds expressed the heterosis. Summing up, the resulted genetic aspects of F1 crossbred could support increasing heterogeneity, but prospects for determining the best directional crosses remain questionable.

Removal of Staphylococcus aureus from Various Surfaces using Bacteriophage SA11

Kim Young Deuk, Jo Yunyeol and Myung Heejoon

Methicillin-resistant Staphylococcus aureus (MRSA) is a bacterium responsible for several difficult-to-treat infections in human. The bacteria are frequently found on surfaces of skin or medical devices. A safe and efficient method other than antibiotics to remove these bacteria would be useful. Thus, phage application against surface-contaminated S. aureus needs to be tested. Bacteriophage SA11 was previously shown to infect S. aureus. We showed that it could infect 7 laboratory strains and 11 antibiotic-resistant strains of S. aureus. Its latent period was 20 minutes and burst size was 79. The bacteria were readily removed from porcine skinwhen treated with SA11. The phage also effectively removed persisters appearing after antibiotic treatment. The removal was in a dose- and time-dependent manner. Preformed biofilm on either polystyrene surfaces or glass surfaces by S. aureus was effectively removed when phage SA11 was added. The phage also efficiently inhibited biofilm formation by the bacteria. Bacteriophage SA11 treatment was effective for removal of surface-contaminated S. aureus including antibiotic-resistant strains and persisters.

DNA barcoding identifies the component species in the powder formulations of plant derived raw drugs sold in retail market in India

Rex Arunraj, Moorthy Abiramavalli and Pasupathi Rathinasabapathi

Plant derived raw drugs are marketed for oral consumption in various formulations. Adulteration in the species specific medicinal principle of plant endangers life. Therefore, the authenticity of the raw drug is crucial. We have used two chloroplast loci ribulose-bisphosphate carboxylase gene (rbcL) and intergenic spacer trnH-psbA to authenticate 11 orally consumed raw drugs. Barcodes rbcL and trnH-psbA in combination with analytical tools including BLAST, phylogeny and the DNA distinguisher sequences (DNA-BAR) authenticated 63.63% of the powdered raw drugs to its species and 18.18% to its genera. Barcode rbcL show high PCR and sequencing efficiency while trnH-psbA shows higher discriminatory power. This study widens the scope of quality control in plant processed raw drugs in various formulations, species dependent efficacy of traditional herbal medicine and trade and commercialization of plant derived raw drugs.

Isolation, screening and characterization of PGPR isolated from rhizospheric soils of Pigeonpea

Tiwari Ashish, Devi Shikha, Singh Nand Kumar and Sharma Shivesh

Plant Growth Promoting Rhizobacteria (PGPR) is group of bacteria that colonize roots of plant and help in plant growth and disease suppression by various direct and indirect mechanisms. PGPRs are recognized as efficient soil microbes as bio-fertilizers for enhancing the growth of several crops and controlling soil borne pathogens. Hence, the present investigation was carried out to isolate, screen and characterize the PGPR from the rhizosphere soil of pigeonpea growing in different areas of Bijarkala and Kumarganj. A total of nine bacteria were isolated with the occurrence percentage of 1.92% to 98.1% from pigeonpea rhizosphere here and characterized for various plant growth promoting activities. In the present study, six isolates produced indole-3-acetic acid in the range of 56 to 97µg/ml and only single isolate (PN13) displayed phosphate solubilizing activity in Pikovskaya agar. In addition, six isolates were found to be positive for siderophore production and also showed antifungal activity against Fusarium udum. Catalase production was shown by almost all the isolates and production of HCN was detected in only single isolate i.e. PN11.Out of nine isolates, seven isolates were exhibited in vitro plant growth promotion activities and indicated that these isolates may be exploited as biofertilizers and microbial inoculants for pigeon pea crop as they enhanced plant growth via diverse mechanisms and offered an attractive strategy to replace synthetic fertilizers and pesticides.

Screening for porcine Sn gene-specific amiRNAs

Song Hongqin, Jiang Cuicui, Wang Juan and Sun Huaichang

In order to construct a specific artificial miRNA (amiRNA) expression vector targeting the porcine sialoadhesin (Sn) gene and to screen for effective amiRNAs, the 1147-2280 bp sequence of the porcine Sn cDNA was amplified by RT-PCR and inserted into the eukaryotic expression vector pEGFP-N1 to construct the reporter vector pSn-EGFP for amiRNA screening. Ten amiRNAs targeting the porcine Sn gene were designed and the corresponding recombinant interfering plasmids (pcDNA5-miRSn-1-10) were constructed. NIH-3T3 cells were co-transfected with the reporter vector and the interfering vectors and the expression of the sequence-specific amiRNAs was assessed by poly(A)-tailed RT-PCR. Fluorescent microscopy and FACS analysis revealed that all ten Sn gene-specific amiRNAs expressed from the vectors inhibit Sn-EGFP fusion protein levels. Of these ten amiRNAs, the highest inhibition efficacies were observed for amiRSn3, amiRSn8 and amiRSn9 which inhibited Sn protein expression by 95.37%, 91.27% and 84.7% respectively. These findings suggest that effective amiRNAs identified by this screening can be used to construct adenovirus amiRNA expression vectors targeting the Sn gene.

Antibiotic with different antibacterial spectrum changed intestinal microflora structure and reduced immune response of Lingnan yellow broiler

Wang Jinbo, Qi Lili and Han Fei

The effects of antibiotics with different antibacterial spectrum on the intestinal flora structure and immune function of Lingnan yellow broiler were explored. This trial was designed with single factor completely randomized block. During the trial period of four weeks, three kinds of antibiotics with different antibacterial spectrum were added in the feed. 240 healthy one-day-old broilers were randomly divided into 4 groups. The results showed that the daily weight gain could be improved by all the antibiotics; however the feed conversion ratio had not been affected. The antibiotics resulted in the change of intestinal flora structure. The levels of serum IgM were reduced by the three kinds of antibiotics. The secretion of intestinal sIgA was inhibited by the antibiotics. The levels of IL-1β, IL-6 and TNF-α mRNA were reduced by all the antibiotics. The inhibition induced by lincomycin was mediated by TLR2; the colistin sulfate induced decrease of inflammatory cytokine expression mediated by TLR4, while zinc bacitracin suppressed the inflammatory response by down-regulating the expression of both TLR2 and TLR4. In conclusion, the antibiotics improved the growth performance, changed the intestinal microflora, attenuated the stimulation of the bacteria to the intestinal mucosa and decreased the intestinal inflammatory response of broilers.

The Uniformity and Diversity of the Ribozyme Ribonuclease P: A Review

Singh Anupama

Ribonuclease P is a ribozyme involved in tRNA processing that is present in all cells and organelles that synthesize tRNA. RNase P holoenzymes have been demonstrated to be ribonucleoprotein complexes of an essential RNA subunit and one or more protein components, varying from one in bacteria to at least four in archaea and nine to ten in eukarya. However, the catalytic activity resides with the RNA subunit in all the three domains of life. The increase in protein content is apparently a response to the increased complexity of the cellular environment in the more evolutionarily advanced organisms. The present review outlines the recent advances in our understanding of subunit composition in the three domains of life.

Review on the Antimicrobial and Anticancer Properties of Noble Metal Nanoparticles Synthesized using Variety of Plant Extracts: A Green Strategic Approach

Kishore S. and Radhika P.

This review offers a summary of silver/gold nanoparticles (Ag/Au NPs) preparation via the green strategic route which has an advantage over other conventional methods involving chemicals that result in environmental toxicity. Many parameters influence the synthesis of NPs production particularly concentration of salt and extract, pH, temperature and time. Green synthesis results revealed that Ag/Au NPs show different morphologies and sizes. Eco-friendly bioorganisms include plant extracts that contain proteins, which expectedly act as both reducing and capping agents, thereby forming stable and consistent Ag/Au NPs.

The prepared NPs were characterized with the help of UV-VIS Absorption Spectroscopy, FTIR Spectroscopy, Scanning Electron Microscopy (SEM), with Energy Dispersive X-ray (EDX), Transmission Electron Microscopy (TEM), Atomic Force Microscopy (AFM) and X-ray Diffraction (XRD) analysis. Both the Ag and Au NPs were modified through biocompatible capping agents who exhibited elevated antimicrobial activity. The Ag NP interaction with the cell membranes of bacteria is representative of the mechanism of bactericidal activity. The potential to induce genes linked with cell cycle progression, DNA degradation and apoptosis in human cells at non-cytotoxic doses has been revealed by extensive anti-cancer research.