Research Journal of Biotechnology

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Vol. 7(3) August 2012

Seed Germination and Development of Serapias vomeracea (Burm.fil.) Briq. ssp. orientalis Greuter in Tissue Culture

Gumus Cevdet 1* and Ellialtıoglu Sebnem 2

Serapias vomeracea (Burm. fil.) Briq. ssp. orientalis Greuter plants were obtained via asymbiotic germination under in vitro conditions. For the germination of seeds, different doses of gibberellic acid (GA3) (0, 0.1 and 0.5 mg/L) were added into a basic nutrient media consisting of half-strength MS (½ MS), Van Waes and DeBergh (VW and DB), Knudson C and non-inorganic nitrogen Knudson C (KC-N). Cultures were incubated in darkness for the first 3 months and thereafter transferred to a light/dark photoperiod of 16:8. At the end of the third month, protocorm rates were identified. They were subcultured every 4 weeks and ½ MS medium was used as a transfer medium. The highest germination rate, protocorm rate and plant growth rate were obtained from the Knudson C medium. The germination rate and number of plantlets were affected negatively by adding GA3.

Phenotype Characteristics of Spotted Leaf 1 Mutant in Rice

Lee Kyeong Eun, Saief Saifullah Ahmed and Kang Sang Gu *

Rice spotted leaf 1 (spl1) exhibits spontaneous cell death on the leaves in the absence of any pathogenic infection. We studied the phenotypic characteristics of the spl1 mutant as compared to those of the spl3, spl4, spl5 and spl6 mutants. The inheritance pattern of spl1 mutant was controlled by single recessive gene. Small brown lesions on the leaves of spl1 were initiated at the tillering stage and formed parallel lines on the leaves at the reproductive stage. Chlorophyll contents in the leaves of spl1 mutant were drastically reduced in the 60-day-old leaves. Furthermore, the mesophyll chloroplasts were severely damaged with disrupted tylakoid membrane in the lesion spot area. The transcripts for transketolase, RuBisCO-activase small isoform, RuBisCO-L and protein disulfide isomerase in the lesion spotted leaves were reduced comparing to those in green non-spotted leaves of the spl1 mutant. This investigation demonstrated indirectly that the chloroplast disruption is an exclusive phenotype of spl1 mutant in the typical spotted leaf mutant group in rice.

Assessment of Genetic Diversity in Three Cultivars of Syzygium samarangense grown in Malaysia by uussiinngg Morphological and Physiological Parameters

Moneruzzaman K.M.,* Alebidi A. I. and Al-Saif A. M.

The present study was conducted to estimate the genetic relationship between the three cultivars of wax jambu (Syzygium samarangense) Giant the ‘Green’, Masam manis ‘Pink’ and Jambu madu ‘Red’ cultivars grown under the agro climate conditions of Banting, Selangor, Malaysia. Various morphological characteristics like tree height, canopy width, shape, foliage density, color and phyllotaxy, leaf length, wide, shape, orientation, drymatter, moister percentage and flagrant, flowering time, color, bud/ cluster, panicle length, number of anthers and carpels per flower, filament and style size and inflorescence position, fruit shape, size, surface, weight, maturity time, dropping and yield, seed number and juice percentage were taken into account and variation in the characteristics has been discussed. Large variability has been observed in all morphological and physiological characteristics of three cultivars and that was due to their different genetic makeup.

Transcriptional analysis of the ρ-coumarate 3-hydroxylase (C3H) gene from Hibiscus cannabinus L. during developmental stages in various tissues and in response to abiotic stresses

BoSung Choi1, Si-Yong Kang2, Hyeun-Jong Bae3, Hyoun-Sub Lim4, Woo-Suk Bang5* and Hanhong Bae1*

Kenaf (Hibiscus cannabinus L.) is a fast growing herbaceous plant and has characteristics useful for biomass production. We cloned a full-length of the gene putatively encoding ρ-coumarate 3-hydroxylase in kenaf (HcC3H) and analyzed its expression during developmental stages in various tissues and organs as well as in response to abiotic stresses. C3H is one of the membrane-associated cytochrome P450 enzymes, involved in lignin biosynthesis. The full-length HcC3H consists of a 1,533 bp open reading frame (ORF) encoding 510 amino acids. The deduced amino acids of HcC3H showed 89, 89 and 79% amino acid identity with the C3H of Populus trichocarpa, Populus alba gradidentata and Arabidopsis thaliana respectively with the conserved heme-binding domain (FXXGXRXCXG). According to quantitative real-time reverse transcription PCR (QPCR) analysis, HcC3H was expressed in all kenaf tissues and organs, with the highest expression in stem tissue. The effects of abiotic stresses on HcC3H expression were examined in stems of 3-week-old kenaf. HcC3H expression was induced in response to all treatments applied (wound, SA, NaCl, cold, H2O2, ABA and MeJA). The highest transcript level of HcC3H was detected at late time points (24 or 48 h) after treatments with NaCl, cold, H2O2, ABA and MeJA, while the highest transcript level was detected at an early time point (6 h) after wound and SA treatments. In summary, the C3H ortholog may encode the C3H protein involved in lignin biosynthesis in Hibiscus cannabinus and were expressed in all tissues and organs as well as in response to various treatments.

Cytotoxicity of Gold-nanorods to Human HEp-2 and Canine MDCK Cells

Xu Dan1,3 , Zhang Yinan2, Wang Yili3, Jun Yu1 and Chen Yu2*

Despite great potentials in biomedical sensing, imaging and therapy, the cytotoxicity of gold nanorods (GNRs) remains to be controversial. Here we address this issue via studying the cytotoxicity of gold GNRs of different aspect ratios to two types of cells: human tumor HEp-2 cells and canine MDCK cells of ‘normal epithelium’. It is found that all types of GNRs displayed minimal cytotoxicity at ~10-12 M to both cell-lines in MTT assays. However, cytotoxicity increased accordingly when dosages increased and HEp-2 cells were in general more susceptible than MDCK cells to GNRs. Time-lapse study revealed that majority cell death occurred within one hour. In contrast, free CTAB causes cell death in a time- and dose-dependent manner. This suggests that the cytotoxicity of GNRs arises from their intrinsic property rather than from free CTAB. Finally, GNRs induce apoptosis, the programmed death, which may be exploited further for tumor treatments.

Antibacterial properties of linolenic sophorolipid and its chemically esterified methyl ester form

Gupta Reetika, Uma S. Kumar and Prabhune Asmita*

The antibacterial activity of Linolenic SL mixture (containing 80% lactone) and its methyl ester derivative against Gram positive (B. subtilis) and Gram negative (E. coli and P. aeruginosa) bacteria is reported here. Bacterial cultures were treated with increasing concentrations of Linolenic SL mixture and its methyl ester derivative and antibacterial activity was checked at different time-intervals (2, 4 and 6 hrs) using standard dilution micromethod and spread plate method. Decrease in bacterial colonies was observed with increase in concentrations of compounds as well as incubation time but the level of effectiveness varies with the compound and bacteria. The minimum inhibitory concentrations (MIC) of Linolenic SL mixture (LNNSL, containing 80% lactone) against B. subtilis, E. coli and P. aeruginosa were found to be 20, 10 and 10 μg ml-1 respectively. The MIC values of methyl ester form (LNNSLME) against B. subtilis, E. coli and P. aeruginosa were determined to be >20, 20 and 20 μg ml-1 respectively. The results suggest that Linolenic SL mixture (containing 80% lactone) as compared to its methyl ester derivative showed good antibacterial activity towards both the Gram positive and Gram negative bacteria and were found to be more potent against Gram negative bacteria.

High Level Production of Extracellular Protease from Bacillus licheniformis ATCC 12759 in Submerged Fermentation

Akcan Nurullah

Production of extracellular protease by Bacillus licheniformis ATCC 12759 in submerged fermentation was the aim of this study. Various nutrients belonging to three categories, carbon, nitrogen and amino acid sources, were investigated in terms of their effect on the production of extracellular protease by Bacillus licheniformis ATCC 12759. Comparison with the control in media containing the all simple sugars resulted in a decrease in proteolytic activity, while there was significant increase in enzyme yield in the case of the supplementation complex carbon source such as wheat flour and rice flour. Urea, soy flour and sodium nitrate are the best organic and inorganic sources respectively. Among the amino acid sources tested, L-phenylalanine, L-cysteine, Glysine, L-valine, L-tyrosine, L-isoleusine, L-tryptophane and Glutamic acid favored the production respectively. FeSO4, ZnSO4 and CuSO4 inhibited protease production. Maximum protease production (10738.2±44.2 U/mg) was obtained in a medium containing 0.1% MgSO4 in 24 h 37 oC.

Tolerance and Biodegradation of Benzene, Toluene, Ethylbenzene and Xylenes (BTEX) by a Metal Acclimatized Bacterial Consortium Culture

Fellie E. A.,1 Sannasi P., *2 Wong K. K.,1 Salmijah S.,1 and Kader J.3

This investigation reports the tolerance and biodegradation of benzene, toluene, ethylbenzene and xylene isomers (BTEX) by a heavy metal-adapted environmental bacterial consortium, known as consortium culture (CC). Higher tolerance was observed with benzene (IC50 value up to 191.25 mg/L), followed by toluene (IC50 = 139.67 mg/L), xylene (IC50 = 97.04 mg/L) and ethylbenzene (IC50 =96.99 mg/L). Significant decrease (p < 0.05) in the specific growth rate (μ), however was observed as the concentrations of each individual BTEX were increased from 10 mg/L to 500 mg/L. Growth of CC was completely inhibited at 250 mg/L ethylbenzene and 500 mg/L xylene. Toxicity followed the trend: BT>X>E.

A simple, fast anesthesia technology and the influence of volatile anesthetics on the nematodes Caenorhabditis elegans

Qiu Hong Niu,1,2* Fengli Hui, Siliang Huang1, Tao Ke1, Ke Qin Zhang2, Xiaowei Huang2 and Lin Zhang1,2

This study describes a simple, fast method to anaesthetize nematodes. The method provides a cost-effective and efficient way to make nematodes immobile long for 30~40 minutes followed by recovering full motion. It is convenient to study fine characterizations of nematodes while immobile. Moreover, we synthetically evaluate influence of nine different volatile anesthetics against nematodes and the remarkable anesthetics may serve as probes to understand consciousness using nematode Caenorhabditis elegans as anesthetic endpoints.

Vibrio detection in market Seafood samples of Kuwait bbyy biochemical (API 20E) strips and its evaluation against 16s rDNA-based molecular methods

Al-Mouqati S.,2 Azad I.S.,1* Al-Baijan D.2 and Benhaji A.2

Seafood Vibrio is one of the causes of food-borne infections in Kuwait. Surveillance of food borne diseases is one of the high priority tasks in the public health agenda worldwide. This study was conducted over a period of two years from 2008-2010. Three fish markets in Kuwait were selected for seafood sampling, at quarterly intervals, for six quarters and a total of 238 seafood samples were collected. The seafood collections included important food fishes and shellfishes such as; grouper, silver pomfret, bluefin bream and shrimp. Bacterial isolates from the skin swabs, the muscle extracts and kidney were analyzed for the identity using classical biochemical assays on API 20E and compared with the results obtained on the 16s rDNA amplification. The samples were plated on triptone citrate bile salt sucrose (TCBS) agar for a presumptive vibrio diagnostic. Prevalence of Vibrio in the seafood samples was found to be 77.99 ± 7.48 % (presumptive Vibrio positivity or PVP). The PVP was highly and positively correlated with the surface Vibrio contamination (r = 0.63). A sample of 45 randomly-selected isolates analyzed using API 20E and the analytical probability index (API) so obtained showed that 43.75 % of the samples had confirmatory Vibrio levels. The vibrio positive random samples selected after API 20E analyses were found to amplify the vibrio specific 625 bp 16s rDNA gene.

Secondary metbolites and antioxidant activity of Vitex agnus castus

Maltas Esra * and Yildiz Salih

The antioxidant activity of methanolic extract of Vitex agnus castus was evaluated by β-carotene–linoleate bleaching assay, DPPH radical scavenging assay and ferric reducing antioxidant power assay. The highest antioxidant activity was observed as 83.69±0.54 % by DPPH radical scavenging assay. Hovewer, several phenolics and fatty acids of the extract were analysed. Data showed that the extract had high amount of caffeic acid with 0.466±0.021 mg/g and eriodictyol with 0.414±0.041 mg/g as phenolics and linoleic acid with 42.92 % as fatty acid. Results suggested that Vitex agnus castus may be important in nutraceuticals, bio-pharmaceuticals and food additives.

The effect of 2,,4-dichlorophenoxyacetic Acid and 6- benzylaminopurine on Callus Induction and Plant Regeneration of Allamanda cathartica – A Valuable Medicinal Plant

Wong K.F.* and Taha R.M.

Allamanda cathartica is a potential medicinal plant which starts to achieve awareness in society due to its value to treat various types of diseases, especially in the treatment of jaundice, malaria and cancer. In the present study, the effects of 2,4-dichlorophenoxyacetic acid (2,4-D) and 6-benzylaminopurine (BAP) on the callus induction from the leaf and stem explants were investigated. Plant regeneration from the nodal explants was achieved. Surface sterilization by mercuric chloride (HgCl2) was utilized in order to surface sterilize the leaf (0.1%), stem and nodal (0.2%) explants. The leaf and stem explants were cultured on full-strength Murashige and Skoog (MS) medium supplemented with different concentrations of 2,4-D alone (0.5 and 1.0 mg/L) or in combinations of 2,4- D (0.5, 1.0 and 1.5 mg/L) with BAP (0.5, 1.0 and 1.5 mg/L). In the study of plant regeneration, the nodal explants were cultured on MS medium supplemented with BAP at 1.0, 3.0 or 5.0 mg/L for shoot multiplication. MS basal medium was used as a control and also used for shoot elongation. All the cultures were incubated under a photoperiod of 16 hours light and 8 hours darkness. For callus induction, the leaf and stem explants cultured on 1.0 mg/L 2,4-D and 1.0 mg/L BAP gave the best callus response (100%) with yellow-white, greenish friable callus (0.0707 0.0549 g with callus initiated after 6 days) and brown-white, greenish friable callus (0.0207 0.0009 g with callus initiated after 5 days), respectively. For shoot multiplication, MS supplemented with 5 mg/L BAP gave the best response (100%) with multiple shoots formed (6 0.6 shoots per explant) from the nodal explants. In the plant growth regulator (PGR)-free medium, the elongated shoots were developed (1.01 0.07 cm) with white hairy roots.

Batchwisee Biosorption of Sn2+ Ions by using Chemically treated Banana Peel

Muhammad Aqeel Ashraf1*, Mohd. Jamil Maah1 and Ismail Yusoff2

Biosorption has attracted attention as a cost-effective means for the treatment of metal-bearing wastewater. While using novel biosorbents, optimization of operating conditions becomes more important for the efficiency of the process. In this study, a novel biosorbent i.e. banana peel was used for biosorption of Sn2+ from aqueous media. The effect of various parameters, such as contacts time of solution with biosorbent, temperature, pH, biosorbent dose and agitation speed were studied. A comparative study of modification of biosorbent using acid and base was also performed. It has been found that acid treated banana peel has greater biosorption capacity as compared to untreated and base treated peel. Langmuir and Freundlich isotherms were also employed to evaluate maximum biosorption capacity of untreated and chemically treated banana peel for Sn2+. This research work is of great significance with respect to practical wastewater treatment by biosorption.

Purification and Characterization of Protease Enzyme from Bacillus Cereus

Kavitha B.1* and Thankamani V.2

The aim and objective of this study was to purify and characterize thermostable alkaline protease from Bacillus cereus. The purified enzyme showed optimum activity at pH 9.0, temperature 500C, period of incubation one hour, enzyme concentration 420 μg.ml-1 and substrate concentration 20 mg. ml-1 of casein. The protease enzyme was inhibited by PMSF and HgCl2 and Mn++ showed 20-30 times increase of enzyme activity than other divalent cations. The protease enzyme in B.cereus was completely inhibited by EDTA indicating that it might be a metalloprotease. In this present study we purified a 45 kDa protein from the culture broth of B, cereus cultivated at 370C. The enzyme could be effectively used in different industries.

Improvement of Rooting in Semi-hardwood Nandina domestica Stem Cuttings as influenced by Exogenous IBA and NAA Auxins

Negahdar Naser 1, Hashemabadi Davood 1*, Kaviani Behzad2 and Zarchini Mohammad2

Here, we present a strategy for propagation of Nandina domestica, a semi-hardwood ornamental and medicinal plant, in presence of indole-3-butyric acid (IBA) and naphthalene acetic acid (NAA) which enables to stimulate the adventitious roots on stem cuttings. 1000 mg.L-1 IBA + 1000 mg.L-1 NAA significantly enhanced rooting of cuttings and subsequent growth.

Review paper: Microbial Biosensors and Bioelectronics

Somayeh Dolatabadi* and Manjulakumari D.

A microbial biosensor consists of a transducer in conjunction with immobilized viable or non-viable microbial cells. Non-viable cells obtained after permeabilization or whole cells containing peri-plasmic enzymes have mostly been used as an economical substitute for enzymes. Viable cells make use of the respiratory and metabolic functions of the cell, while the analyte to be monitored is either a substrate or an inhibitor of these processes. Bioluminescence-based microbial biosensors have also been developed using genetically engineered microorganisms constructed by fusing the lux gene with an inducible gene promoter for toxicity and bioavailability testing.

Review paper: Naturally Derived Antioxidants in Poultry Nutrition

Christaki E.

An important field of research today is the control of biological damages such as oxidative stress and oxidative rancidity caused by free radicals. Natural antioxidants can be used because of their health benefits against oxidative stress which is involved in the pathology of several diseases in living organisms. Moreover, antioxidants have been used in the food industry to prevent oxidative rancidity which represents one of the major causes of food deterioration. Poultry meat is particular susceptible to lipid oxidation due to its high content in polyunsaturated fatty acids. Over the last several years much attention has been focused on herbs, spices, plants and especially aromatic plants like oregano, rosemary, thyme and saffron which are traditionally used in folk medicine. These plants have many bioactive compounds and particularly polyphenols which are known for their antioxidative capacity. Therefore, they have been used in poultry diets as a simple and convenient strategy to introduce natural antioxidants into meat. Nowadays, the consumer demand for natural products has created an interest in the use of plant derived antioxidants in poultry nutrition.

Review paper: Overview of Genetic Manipulation in Plant Carotenoid Biosynthesis Pathway

Babu Merlene Ann and Gothandam K.M.*

Carotenoids, the naturally occurring organic pigments are isoprenoids attributed with many health promoting and aesthetic values. Several epidemiological studies have projected its role in the prevention of onset of chronic diseases like cardiovascular diseases and certain cancers due to its anti-oxidant activity. Carotenoids such as beta carotene act as Vitamin A precursor that aids in the prevention of Vitamin A deficiency. Animals are unable to synthesize carotenoids de novo. Thus they depend on dietary sources to procure these health benefactors. The rapid increase in the population and their ever increasing demand of food resources is a major challenge faced at present as land resources dedicated to agriculture is not par with the requirements of the society. Biotechnology tools play a central position in this scenario to present transgenic plants with enhanced yield and nutritional quality. In this context, genetic engineering approaches towards the enhancement of carotenoids in crop plants will open new avenues in addressing food security problems.