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Hasancebi S.,Scientific and Technological Research Council of Turkey | Turgut Kara N.,Istanbul University | Cakir O.,Istanbul University | Ari S.,Istanbul University | Ari S.,Research and Application Center for Biotechnology and Genetic Engineering
Turkish Journal of Botany | Year: 2011

Efficient micropropagation and root culture protocols were developed for the endemic Astragalus chrysochlorus Boiss. & Kotschy. A high frequency of shoot formation (100%) and maximum multiplication (13 shoots per hypocotyls explant) were achieved on Murashige and Skoog (MS) media supplemented with 0.5 mg/L trans-Zeatin riboside (ZR). By using single regenerated hypocotyl explants, rooting was achieved at a rate of 93% on MS medium containing 2% sucrose without growth regulators. High frequency callus initiation and growth were achieved when single hypocotyls explants were inoculated on MS medium supplemented with 0.5 mg/L 2,4-Dichlorophenoxyacetic acid. Plant regeneration through indirect organogenesis was achieved on MS medium supplemented with 0.5 mg/L ZR. Root cultures were successsfully established in liquid MS medium containing 0.5 mg/L α-Naphthaleneacetic acid. The optimised in vitro propagation, callus culture, and root culture protocols offer the possibility to use cell/root culture techniques for vegetative propagation and secondary metabolism studies on Astragalus L. species. © Tübi̇tak.


Gurel F.,IstanbulUniversity | Gurel F.,Research and Application Center for Biotechnology and Genetic Engineering | Arican E.,IstanbulUniversity | Gozukirmizi N.,IstanbulUniversity | Ari S.,IstanbulUniversity
Scientific Research and Essays | Year: 2011

Genetically modified (GM) crops are the plant varieties carrying single or multiple transgenes in their genomes modified by recombinant DNA technology. Detection of transgenic elements associated with GM crops is an important issue for their traceability in food chain and also for the risk assessments related to environment and transgene introduction into human diet. A number of methods have been developed for screening GM crop products with the aim of increasing reliability and molecular sensitivity. This review article is focused on the published methods which are mainly based on PCR and DNA hybridizations as well as biosensors as a recently utilized technology. DNA hybridization methods including probe immobilization on solid surfaces and subsequent hybridization by target DNA are variously depended on surface types, probe labeling conditions or some modifications such as the use of peptide nucleic acids (PNA). Quantitative real-time PCR (qRT-PCR) is the most routinely used and compatible method for quantification which is a crucial issue in GMO content analyses. Finally, biosensors represent more advanced assays with high detection sensitivity provided by specific transducers sense DNA hybridization events. Progress in technical implementations related to GM crop analyses will contribute not only to environmental safety but also to guarantee global market functioning and the consumer rights to choose. © 2011 Academic Journals.


Gurel F.,Istanbul University | Gurel F.,Research and Application Center for Biotechnology and Genetic Engineering | Ucarl C.,Istanbul University | Tufan F.,Istanbul University | Kalaskar D.M.,University College London
Applied Biochemistry and Biotechnology | Year: 2015

A major limitation of transforming barley tissues by Agrobacterium tumefaciens is the low frequency of T-DNA transfer due to recalcitrance of barley as a host. The effect of extracellular cellulose and lectin on Agrobacterium transformation efficiency was investigated in this study. Barley callus cultures were transformed with the AGL1 strain containing the vector pBI121 in the presence of 10 mg mL−1 cellulose or 0.001, 0.05 and 0.1 mg mL−1 lectin. Addition of cellulose significantly (P ≤ 0.05) increased the number of GUS spots by 50 % compared to standard conditions in the presence of only 200 μM acetosyringone (AS). Frequency of G418-resistant aggregates on the surfaces of callus cultures was 29 and 71.5 %, following AS and AS + cellulose treatments, respectively, after 4 weeks of selection. Presence of 0.05 or 0.1 mg mL−1 lectin also increased the number of GUS spots and frequency of G418-resistant cells in the selection period, but the increase in blue spots was not significant. We examined the effect of lectin and cellulose on bacterial attachment to callus tissues. Both cellulose and lectin were found to have a significant positive effect on the numbers of bacteria attached to barley callus. Epifluorescence microscopy revealed that Agrobacterium cells had accumulated in the scaffolds of irregular fibrous cellulose with a mean particle size of 200 μm. Expression of nptII in transformed callus lines confirmed the stable transformation of the gene. Our study showed for the first time the binding of Agrobacterium cells to fibrous cellulose and also demonstrated how polysaccharides and glycoproteins can be used to improve T-DNA transfer in monocotyledon transformation procedures. © 2015, Springer Science+Business Media New York.


Turgut-Kara N.,Istanbul University | Ari S.,Istanbul University | Ari S.,Research and Application Center for Biotechnology and Genetic Engineering
Plant OMICS | Year: 2011

In this study, elicitor-inducible cytochrome P450 biosynthesis in Astragalus chrysochlorus was investigated for further analysis on phenylpropanoid metabolism. In order to analyse cytochrome P450s under yeast extract elicited conditions, we used non-radioactive P450 targeted differential display method. The P450 targeted differential display of mRNA technique was performed with upstream primers based on the conserved heme-binding region [PFG] of P450s, as a result 56 clearly differential bands were revealed; 37 of the bands were correctly analysed, and one of the PCR products was contained the P450 fingerprint. This sequence has been confirmed to be up-regulated and subsequently cloned and sequenced. Homology analysis of the 400 bp long sequence revealed that 81 % similarity with cinnamate 4-hydroxylase in the manner of amino acid. Quantitative real-time-PCR analysis showed that putative C4H gene was up-regulated 13,24-fold by 6h yeast extract treatment unlike untreated control. 1338 bp long cDNA fragment (Accession no: GQ844863) of A. chrysochlorus C4H (AcC4H) has been obtained by PCR with degenerate primers. Bioinformatics analyses revealed that putative AcC4H (1338 bp) was highly similar (95 %) to trans-cinnamate 4-monooxygenase (EC 1.14.13.11). As a result, we have isolated a putative C4H fragment from A. chrysochlorus suspension cells under yeast extract elicited conditions. This knowledge will use for obtaining whole C4H sequence, and to manupulate phenylpropanoid metabolic pathway of this medicinal plant.


Ari S.,Istanbul University | Ari S.,Research and Application Center for Biotechnology and Genetic Engineering | Cakir O.,Istanbul University | Turgut-Kara N.,Istanbul University
Acta Physiologiae Plantarum | Year: 2010

Selenium (Se) plays an indispensable role in human nutrition and has been implicated to have important health benefits, including being a cancer preventative agent. Selected members of the genus Astragalus (Fabaceae) are known for their ability to accumulate high levels of selenium, mainly in the form of methyl-selenocysteine (MeSeCys). The Se-hyperaccumulator Astragalus bisulcatus metabolizes >90% of the accumulated Se into MeSeCys in young shoot tissue. Selenocysteine methyltransferase (SMT) catalyzes the methylation of SeCys to yield MeSeCys. In this study, we aimed to investigate selenium accumulation ability of Astragalus chrysochlorus. For this reason, A. chrysochlorus plants were cultured in Murashige and Skoog medium containing 1, 5, 25 or 75 ppm sodium selenate. Both shoot and root length decreased significantly when plants exposed more than 5 ppm sodium selenate. Dried plant materials were analysed with ICP-MS in the terms of Se and S accumulation. Regarding the calculated discrimination coefficients (DCi) value (0.95) A. chrysochlorus was evaluated as a secondary Se accumulator plant. Putative SMT fragments were amplified by using the primers which were designed according to conserved region of Astragalus bisulcatus, Camelia sinensis and Brassica oleracea SMT genes. Reverse transcription PCR demonstrated that 595 bp fragment was expressed (Accession no: GQ844862), and a database search indicated that the similarity between A. bisulcatus SMT nucleotide sequence and the putative AcSMT fragment was 92%. The putative AcSMT gene represents a single copy sequence in Astragalus chrysoclorus genome. © 2010 Franciszek Górski Institute of Plant Physiology, Polish Academy of Sciences, Kraków.


Cakir O.,Istanbul University | Ari S.,Istanbul University | Ari S.,Research and Application Center for Biotechnology and Genetic Engineering
Plant OMICS | Year: 2013

Selenium (Se) is a dietary essenStial trace element in human nutrition and has been implicated in health, including cancer preventation. Methylselenocysteine (MeSeCys) is an effective chemopreventative compound. Astragalus sp. are known to accumulate Se with the majority of the selenoamino acids in the form of MeSeCys. The aim of this study was the cloning and charactherization of a cDNA encoding selenocysteine methyltransferase (SMT), the key enzyme responsible for MeSeCys formation, from Astragalus chrysochlorus using specific primers. Our results showed that Astragalus chrysochlorus SMT (AchSMT) is a 1020 bp (base pair) cDNA (GQ844862) with an open reading frame predicted to encode a 339 amino acid, 36.94 kDa protein. The predicted amino acid sequence of AchSMT (AEI53593) revealed 97% identity with A. bisulcatus selenocysteine methyltransferase (AbSMT). Bioinformatic analysis revealed that AchSMT lacks chloroplast and mitochondrial targeting sequences. The AchSMT possess a possible zinc-binding motif (GGCC) and a conserved cysteine residue upstream of this motif. AchSMT was expressed in Escherichia coli, then confirmed by DNA sequence analysis, western blotting and enzyme activity. Expression of AchSMT correlated with the presence of SMT enzyme activity in cell extracts. In A. chrysochlrous plants, analysis of AchSMT gene expression in response to selenate treatments showed that the AchSMT was constitutively expressed. The isolation of SMT gene will result in further studies for overproduction of valuable metabolite, methylselenocysteine.


Arican G.O.,Istanbul University | Cakir O.,Istanbul University | Arican E.,Istanbul University | Kara N.T.,Istanbul University | And 3 more authors.
African Journal of Biotechnology | Year: 2012

Geven (Astragalus L.) root extract is used for asthma, diarrhea, and cancer therapy in Chinese Medicine. Liquid Astragalus root extracts are traditionally used in Anatolia for leukemia and wound healing. 439 species of this plant, which has 3000 species in the world, grow in Turkey and 204 of these are endemic. In this study, molecular mechanism of apoptotic and cytotoxic effects of root ethyl acetate extract of Astragalus chrysochlorus on HeLa cells was investigated. For this purpose, 1, 0.5, 0.15 and 0.1 mg/ml concentrations of Astragalus root extract were applied to HeLa cell cultures for 24, 48 and 72 h. Cell kinetic parameters as proliferation rate (MTT assay), Mitotic Index and apoptotic index were used for experimental cytotoxicity assay of root extract. Furthermore, effects of root extract on gene expression rates which take place in apoptosis mechanism as Bcl-2, Bax, Bak, Bcl-x, Bik, Mcl-1, Bfl-1 were determined by reverse transcription-polymerase chain reaction (RT-PCR) technique. As a result of investigated parameters, root extract of Astragalus chrysochlorus is found to have cytotoxic effect on HeLa cell cultures. This cytotoxic effect which appeared in our experiments achieved statistically significant decrease on proliferation rate of HeLa cells depending on application time and concentration. Also mitotic index and apoptotic index presented statistically significant increase on particular concentrations and hours (p < 0.05). Likewise, it is determined on molecular basis that root extract causes significant changes on especially antiapoptotic genes. © 2011 Academic Journals.


Meric S.,Istanbul University | Cakir O.,Istanbul University | Turgut-Kara N.,Istanbul University | Ari S.,Istanbul University | Ari S.,Research and Application Center for Biotechnology and Genetic Engineering
Genetics and Molecular Research | Year: 2014

Despite the controversy about genetically modified (GM) plants, they are still incrementally cultivated. In recent years, many food and feed products produced by genetic engineering technology have appeared on store shelves. Controlling the production and legal presentation of GM crops are very important for the environment and human health, especially in terms of long-term consumption. In this study, 11 kinds of feed obtained from different regions of Turkey were used for genetic analysis based on foreign gene determination. All samples were screened by conventional polymerase chain reaction (PCR) technique for widely used genetic elements; cauliflower mosaic virus 35S promoter (CaMV35S promoter), and nopaline synthase terminator (T-NOS) sequences for GM plants. After determination of GM plant-containing samples, nested PCR and conventional PCR analysis were performed to find out whether the samples contained Bt176 or GTS-40-3-2 for maize and soy, respectively. As a result of PCR-based GM plant analysis, all samples were found to be transgenic. Both 35S- and NOS-containing feed samples or potentially Bt176-containing samples, in other words, were analyzed with Bt176 insect resistant cryIAb gene- specific primers via nested PCR. Eventually, none of them were found Bt176-positive. On the other hand, when we applied conventional PCR to the same samples with the herbicide resistance CTP4-EPSPS construct-specific primers for transgenic soy variety GTS-40-3-2, we found that all samples were positive for GTS-40-3-2. © FUNPEC-RP.


Turgut-Kara N.,Istanbul University | Ari S.,Istanbul University | Ari S.,Research and Application Center for Biotechnology and Genetic Engineering
African Journal of Biotechnology | Year: 2010

Somatic embryo tissues of Astragalus chrysochlorus were transformed with the β-glucuronidase (GUS) and neomycin phosphotransferase II (npt II) genes by electroporation. The effect of electric field strength was tested for transient expression. It was found that 1000 V/cm and 200 μs and 1 pulse was the optimum combination to transform embryo tissues (expression level was 61.5%). Electroporated somatic embryo tissues were positive for GUS expression and PCR analysis for the genes GUS and npt II. After PCR analysis, we found that the efficiency of the somatic embryos with transient GUS expression by electroporation was 48%. © 2010 Academic Journals.


Mandaci M.,Istanbul University | Cakir O.,Istanbul University | Turgut-Kara N.,Istanbul University | Meric S.,Istanbul University | And 2 more authors.
Food Science and Technology | Year: 2015

PCR-based technique for GMO detection is the most reliable choice because of its high sensitivity and specificity. As a candidate of the European Union, Turkey must comply with the rules for launching into the market, traceability, and labeling of GMOs as established by EU legislation. Therefore, the objective of this study is to assess soybean products in the Turkish market to verify compliance with legislation using qualitative Polymerase Chain Reaction (PCR) assay to detect the presence of GM soybean and to quantify its amount of GM soybean in the samples tested positive using real-time PCR. DNA extracted by the modified CTAB method was properly used for PCR amplification of food materials. The amplification of a 118 bp DNA fragment of the lectin gene from soybean by PCR was successfully achieved in all samples. The GMO screening was based on the detection of 35S promoter and NOS terminator sequences. The GM positive samples were subjected to detection of Roundup ReadyTM soybean (RR) using quantitative real-time PCR. It was found that 100% of the tested food samples contained less than 0.1 per cent of EPSPS gene. © 2014, Sociedade Brasileira de Ciencia e Tecnologia de Alimentos, SBCTA. All rights reserved.

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