- Email: [email protected]
Genetic relationship between transgressive segregations and genetic distance based on SSR markers in Cicer species
S38
Oral presentations / Current Opinion in Biotechnology 24S (2013) S28–S47
Nanobiotechnology Poly(lactic acid)-based fructose-dioxygen biofuel cell
microarray analysis. This study represent a comprehensive expression profile for CRC and indicates that there is significant difference of transcriptome profile between colon cancer and normal cells.
Jaroslav Filip, Jan Tkac Slovak Academy of Sciences, Institute of Chemistry, Center for Glycomics, Bratislava, Slovakia E-mail address: jaroslav.fi[email protected] (J. Filip). In enzymatic biofuel cells (BFCs), organic substrates are enzymatically oxidized at an anode surface, which, if connected to a cathodic depolarization reduction, allows to turn chemical energy of the biofuel into electricity. To immobilize a sufficient amount of anodic and cathodic biocatalysts onto electrode surfaces is crucial for high power outputs of biofuel cells; a diverse range of nanomaterials is used to fulfil this task. In this work, electrode nanointerface was prepared from cheap carbonaceous nanoparticles Ketjen Black (KB) dispersed in polylactic acid (PLA) matrix. The interface allowed adsorption of fructose dehydrogenase (FDH) and bilirubin oxidase (BOD), thus a fructose-dioxygen BFC could be prepared and tested. PLA was dissolved in dimethylformamide and mixed with KB. This mixture was pippetted on a glassy carbon electrode (GCE) surface and dried. Consequent incubation with enzyme solutions resulted in GCE/KB-PLA/FDH bioanode and GCE/KB-PLA/BOD biocathode which were combined into a BFC, that is, dipped into an air-bubbled acetate buffer solution, pH 6, containing fructose. Some parameters were optimized such as optimal KB concentration and amount of KB-PLA applied to electrode. Power density of 57 W cm−2 and open circuit potential of 680 mV of PLA-based BFC obtained was comparable with a previous concept based on chitosan, thus potential applications of BFCs were broadened using another biodegradable, unexpensive and renewable matrix (PLA).
http://dx.doi.org/10.1016/j.copbio.2013.05.075 Plant Biotechnology A novel method for gene transfer to durum wheat (Triticum durum Desf.) via Agrobacterium tumefaciens Mustafa Yıldız 1 , Behrouz Alizadeh 2 , Mehdi Taher 2 , Murat Aycan 2 1
University of Ankara, Faculty of Agriculture, Department of Field Crops, 06110 Diskapi, Ankara, Turkey 2 University of Ankara, Graduate School of Natural and Applied Sciences, Department of Field Crops, 06110 Diskapi, Ankara, Turkey E-mail address: [email protected] (M. Yıldız).
Transcriptome profiling of colorectal cancer
Agrobacterium-mediated transformation has been widely used for the introduction of foreign genes into plants. In the current study, transgenic plants were recovered from inoculated mature embryos of durum wheat cv. ‘C¸akmak 79’. Agrobacterium tumefaciens strain GV2260 harboring plasmid p35S GUS-INT was used for inoculation. Mature seeds were surface-sterilized with 70% (v/v) ethanol for 5 min, washed several times with sterile distilled water, immersed in commercial bleach (containing 5% sodium hypochlorite) for 25 min, and rinsed 7 times with sterile distilled water. Mature embryos from imbibed and dehulled seeds were aseptically excised slightly with a scalpel. Then, the embryos were again imbibed in 50 ml sterile distilled water containing 50 l bacterial solution for 24, 48 and 72 hours at 21◦ C in a rotary shaker (120 rpm) for inoculation. After inoculation, embryos were transferred to germination medium in Magenta vessels supplemented with 100 mg l−1 kanamycin and 500 mg l−1 augmentin. Three weeks after, putative transgenic plants were transferred to pots. Plants were irrigated with 50 ml water containing 100 mg l−1 kanamycin at 2-day intervals during 14 days. At the end of the study, high frequency transgenic wheat plants were obtained.
Nevin Belder 1 , Berna Savas¸ 2 , Arzu Ensari 2 , Mehmet Ayhan Kuzu 2 , Hilal Özda˘g 1
http://dx.doi.org/10.1016/j.copbio.2013.05.076
http://dx.doi.org/10.1016/j.copbio.2013.05.074 Omic Sciences
1
Ankara University Biotechnology Institute, Ankara, Turkey Ankara University, School of Medicine, Ankara, Turkey E-mail address: [email protected] (N. Belder). 2
Colorectal cancer (CRC) is the third most commonly diagnosed cancer in the world after lung and breast cancer. The identification of dysregulation in cancer transcriptome through genome-wide tanscriptome profiling would provide valuable information for molecular mechanism of CRC. The aim of this study is to detect cancer-specific gene expression alterations and evaluate the biological significance of the disease specific transcriptome profile. Fifty tumor and matched normal formalin-fixed, paraffinembedded (FFPE) samples were used in this study. We optimized a workflow for FFPE tissue from deparaffinization to microarray hybridization. Samples were studied using modified RNeasy FFPE kit and X3P Array-Ovation FFPE WTA System combination. Microarray data was analyzed by dChip software. DAVID software was used to gene set enrichment and functional annotation analysis. Gene expression analysis revealed a total of 191 altered genes with 65 upregulated and 126 downregulated genes in tumor cells compared with paired normal colon cells. These genes belong to different functional groups such as metabolism, cellular adhesion, transporters, signaling and immune system. Our study provide a robust workflow for future high-throughput transcriptional analysis by optimizing FFPE tissue protocol from RNA isolation to
Genetic relationship between transgressive segregations and genetic distance based on SSR markers in Cicer species Cengiz Toker, Cengiz Ikten, Fatma Oncu Ceylan, Esra Bolucek, Bulent Uzun Faculty of Agriculture, Akdeniz University, TR-07070 Antalya, Turkey E-mail address: [email protected] (C. Toker). This is the first report on genetic relationships between transgressive (fruitful) segregations and genetic distance based on molecular data in Cicer species including C. arietinum L., C. echinospermum P.H. Davis and C. reticulatum Ladiz. Genetic relationships between hybrid vigor or fruitful segregations and genetic distance based on molecular markers have been widely used in vegetables and in many economically important plant species. The objectives of this study were: first, to evaluate of relationship between hybrid vigor (heterosis and heterobeltiosis) in F1s and genetic distance among Cicer species, and secondly, for assessment of correlations between transgressive segregations in F2-3s and genetic distance among Cicer species based on SSR markers. Three Cicer species including two accessions of C. reticulatum, one accession of C. echinospermum and three accessions of the cultivated chickpea were crossed. Genetic distance among Cicer species was predicted using SSR markers. A significant correlation between
Oral presentations / Current Opinion in Biotechnology 24S (2013) S28–S47
the genetic distance of Cicer species and hybrid vigor for yield and yield criteria was observed in F1s. Similarly a significant relationship between genetic distance and transgressive segregations for yield and yield components was found in F2-3s. The hybrid vigor for yield and yield components in F1s was higher in interspecific crosses than those of intraspecific crosses. Results indicated that fruitful segregations in F2-3s were higher in interspecific crosses than those of intraspecific ones. Results have proven that genetic differences among Cicer species based on SSR markers have been powerful predictor for hybrid vigor in F1s and fruitful segregations in F2-3s. http://dx.doi.org/10.1016/j.copbio.2013.05.077 PC2 containment glasshouse facilities for horticultural trees and vines at PFR Mount Albert, Auckland Monica Anisoara Dragulescu, Ngaire Markwick, Roger Helens, Andrew Gleave Plant and Food Research Institute, Mount Albert, Auckland, New Zealand E-mail address: [email protected] (M.A. Dragulescu). PC2 containment glasshouse facilities for horticultural trees and vines at PFR Mount Albert, Auckland, New Zealand’s horticultural sector combines natural advantages with human innovation and world-leading technology to produce and export a variety of premium fruit and vegetable products. Apples and kiwifruit are New Zealand’s major horticultural exports, and maintaining a position of global leadership in these crops depends upon the development of new premier cultivars. Functional genomics, using transgenic apple trees and kiwifruit vines, is being used to link alleles definitively with traits. Establishing the allele/trait association is a critical factor in ‘faster breeding’ approaches to developing new and novel cultivars, whether they are generated ultimately through genetic engineering (GE) or the application of marker-assisted selection in conventional breeding. Carrying out functional genomics with apples and kiwifruit requires optimum growing conditions to ensure flowering and fruiting, and currently in New Zealand, this must be carried out in Physical Containment level 2 (PC2) facilities. Recently PFR commissioned a new facility designed specifically to provide such conditions for tree and vine growth whilst meeting these physical containment requirements. The facility includes two large growing areas, with high light intensity and sophisticated mechanical ventilation, misting/fogging and shading systems. These features facilitate control of the temperature, humidity and vapour pressure deficit levels to emulate orchard conditions as closely as is possible in a glassenclosed space, like ‘A Field in a Box’. Details on the design, features and operation of this PC2 glasshouse facility will be presented. http://dx.doi.org/10.1016/j.copbio.2013.05.078
S39
Bulblets regeneration from in vitro improved leaf of Fritillaria imperialis L. and F. persica L. Suleyman Kizil 1 , Khalid Mahmood Khawar 2 , Ugur Sesiz 1 1
Department of Field Crops, Faculty of Agriculture, Dicle University, Diyarbakir, Turkey 2 Department of Field Crops, Faculty of Agriculture, Ankara University, Ankara, Turkey E-mail address: [email protected] (S. Kizil). Fritillaria imperialis and F. ersica are used as ornamental plants. Propagation of these plants instead of gathering them from the nature has been a current issue in Turkey. When speed and multiplication rate are considered, traditional production methods are inadequate. The application of in vitro propagation techniques offers possibility of producing large number of uniform plants for further field culture. This study was carried out on shoot tip, middle portions and the lower portion of leaf explant (portion of lamina in between leaf sheath and middle portion of the leaf blade) obtained from germinated seedlings of Fritillaria imperialis and F. persica on MS medium in Petri dishes cultured at 4◦ C in the dark. The study aimed to determine the regeneration competency of the three explants of to regenerate bulblets on MS medium containing various concentrations of BAP–NAA. The results showed that the leaf tip, middle portion of the leaf and lower portion of the leaf were competent and induced callus, shoots and mean number of shoots per explant variably. However, lower portion of the leaf explant was most competent to regenerate new bulblets. This suggests that MS medium containing various concentration of BAP–NAA were suitable for propagation of both species. The best regeneration was achieved on MS medium containing 1 mg/l BAP–0.1 mg/l NAA from the lower portion of the leaves used as explant. This protocol could be effectively used for propagation of the species and has possibility of producing large numbers of uniform plants for nurseries and field culture. http://dx.doi.org/10.1016/j.copbio.2013.05.079 Molecular diagnosis of peanut witches’-broom group (16SrII) phytoplasma infecting sesame in Turkey Cengiz Ikten 1 , Engin Yol 2 , Mursel Catal 1 , Rustem Ustun 2 , Bulent Uzun 2 1
Department of Plant Protection, Faculty of Agriculture, Akdeniz University, TR-07058 Antalya, Turkey 2 Department of Field Crops, Faculty of Agriculture, Akdeniz University, TR-07058 Antalya, Turkey E-mail address: [email protected] (B. Uzun). Severe shoot proliferation, small leaves and leaf rolling resembling phytoplasma disease symptoms were observed in sesame (Sesamum indicum L.) plants growing in the fields of Antalya province in Turkey. In order to investigate the possibility of phytoplasma infection, total DNA was isolated from leaf tissues of infected and healthy sesame plants and 16S rRNA gene was amplified using phytoplasma universal primers P1/P7 and R16F2n/R16R2 in direct and nested PCR amplifications. Amplicons of expected 1.8 kb and 1.2 kb, respectively, were obtained from infected sesame samples. No amplification was observed in samples from healthy sesame plants used as negative controls. Amplicons of the nested PCR were sequenced and analyzed by NCBI BLAST tool. Analysis of the 1118 bp amplicons showed that the sequence shared 99.0% sequence homology with that of the peanut witches’-broom phytoplasma reference strain (GenBank accession: L33765.1). This relationship was further supported by virtual restriction frag-
Oral presentations / Current Opinion in Biotechnology 24S (2013) S28–S47
Nanobiotechnology Poly(lactic acid)-based fructose-dioxygen biofuel cell
microarray analysis. This study represent a comprehensive expression profile for CRC and indicates that there is significant difference of transcriptome profile between colon cancer and normal cells.
Jaroslav Filip, Jan Tkac Slovak Academy of Sciences, Institute of Chemistry, Center for Glycomics, Bratislava, Slovakia E-mail address: jaroslav.fi[email protected] (J. Filip). In enzymatic biofuel cells (BFCs), organic substrates are enzymatically oxidized at an anode surface, which, if connected to a cathodic depolarization reduction, allows to turn chemical energy of the biofuel into electricity. To immobilize a sufficient amount of anodic and cathodic biocatalysts onto electrode surfaces is crucial for high power outputs of biofuel cells; a diverse range of nanomaterials is used to fulfil this task. In this work, electrode nanointerface was prepared from cheap carbonaceous nanoparticles Ketjen Black (KB) dispersed in polylactic acid (PLA) matrix. The interface allowed adsorption of fructose dehydrogenase (FDH) and bilirubin oxidase (BOD), thus a fructose-dioxygen BFC could be prepared and tested. PLA was dissolved in dimethylformamide and mixed with KB. This mixture was pippetted on a glassy carbon electrode (GCE) surface and dried. Consequent incubation with enzyme solutions resulted in GCE/KB-PLA/FDH bioanode and GCE/KB-PLA/BOD biocathode which were combined into a BFC, that is, dipped into an air-bubbled acetate buffer solution, pH 6, containing fructose. Some parameters were optimized such as optimal KB concentration and amount of KB-PLA applied to electrode. Power density of 57 W cm−2 and open circuit potential of 680 mV of PLA-based BFC obtained was comparable with a previous concept based on chitosan, thus potential applications of BFCs were broadened using another biodegradable, unexpensive and renewable matrix (PLA).
http://dx.doi.org/10.1016/j.copbio.2013.05.075 Plant Biotechnology A novel method for gene transfer to durum wheat (Triticum durum Desf.) via Agrobacterium tumefaciens Mustafa Yıldız 1 , Behrouz Alizadeh 2 , Mehdi Taher 2 , Murat Aycan 2 1
University of Ankara, Faculty of Agriculture, Department of Field Crops, 06110 Diskapi, Ankara, Turkey 2 University of Ankara, Graduate School of Natural and Applied Sciences, Department of Field Crops, 06110 Diskapi, Ankara, Turkey E-mail address: [email protected] (M. Yıldız).
Transcriptome profiling of colorectal cancer
Agrobacterium-mediated transformation has been widely used for the introduction of foreign genes into plants. In the current study, transgenic plants were recovered from inoculated mature embryos of durum wheat cv. ‘C¸akmak 79’. Agrobacterium tumefaciens strain GV2260 harboring plasmid p35S GUS-INT was used for inoculation. Mature seeds were surface-sterilized with 70% (v/v) ethanol for 5 min, washed several times with sterile distilled water, immersed in commercial bleach (containing 5% sodium hypochlorite) for 25 min, and rinsed 7 times with sterile distilled water. Mature embryos from imbibed and dehulled seeds were aseptically excised slightly with a scalpel. Then, the embryos were again imbibed in 50 ml sterile distilled water containing 50 l bacterial solution for 24, 48 and 72 hours at 21◦ C in a rotary shaker (120 rpm) for inoculation. After inoculation, embryos were transferred to germination medium in Magenta vessels supplemented with 100 mg l−1 kanamycin and 500 mg l−1 augmentin. Three weeks after, putative transgenic plants were transferred to pots. Plants were irrigated with 50 ml water containing 100 mg l−1 kanamycin at 2-day intervals during 14 days. At the end of the study, high frequency transgenic wheat plants were obtained.
Nevin Belder 1 , Berna Savas¸ 2 , Arzu Ensari 2 , Mehmet Ayhan Kuzu 2 , Hilal Özda˘g 1
http://dx.doi.org/10.1016/j.copbio.2013.05.076
http://dx.doi.org/10.1016/j.copbio.2013.05.074 Omic Sciences
1
Ankara University Biotechnology Institute, Ankara, Turkey Ankara University, School of Medicine, Ankara, Turkey E-mail address: [email protected] (N. Belder). 2
Colorectal cancer (CRC) is the third most commonly diagnosed cancer in the world after lung and breast cancer. The identification of dysregulation in cancer transcriptome through genome-wide tanscriptome profiling would provide valuable information for molecular mechanism of CRC. The aim of this study is to detect cancer-specific gene expression alterations and evaluate the biological significance of the disease specific transcriptome profile. Fifty tumor and matched normal formalin-fixed, paraffinembedded (FFPE) samples were used in this study. We optimized a workflow for FFPE tissue from deparaffinization to microarray hybridization. Samples were studied using modified RNeasy FFPE kit and X3P Array-Ovation FFPE WTA System combination. Microarray data was analyzed by dChip software. DAVID software was used to gene set enrichment and functional annotation analysis. Gene expression analysis revealed a total of 191 altered genes with 65 upregulated and 126 downregulated genes in tumor cells compared with paired normal colon cells. These genes belong to different functional groups such as metabolism, cellular adhesion, transporters, signaling and immune system. Our study provide a robust workflow for future high-throughput transcriptional analysis by optimizing FFPE tissue protocol from RNA isolation to
Genetic relationship between transgressive segregations and genetic distance based on SSR markers in Cicer species Cengiz Toker, Cengiz Ikten, Fatma Oncu Ceylan, Esra Bolucek, Bulent Uzun Faculty of Agriculture, Akdeniz University, TR-07070 Antalya, Turkey E-mail address: [email protected] (C. Toker). This is the first report on genetic relationships between transgressive (fruitful) segregations and genetic distance based on molecular data in Cicer species including C. arietinum L., C. echinospermum P.H. Davis and C. reticulatum Ladiz. Genetic relationships between hybrid vigor or fruitful segregations and genetic distance based on molecular markers have been widely used in vegetables and in many economically important plant species. The objectives of this study were: first, to evaluate of relationship between hybrid vigor (heterosis and heterobeltiosis) in F1s and genetic distance among Cicer species, and secondly, for assessment of correlations between transgressive segregations in F2-3s and genetic distance among Cicer species based on SSR markers. Three Cicer species including two accessions of C. reticulatum, one accession of C. echinospermum and three accessions of the cultivated chickpea were crossed. Genetic distance among Cicer species was predicted using SSR markers. A significant correlation between
Oral presentations / Current Opinion in Biotechnology 24S (2013) S28–S47
the genetic distance of Cicer species and hybrid vigor for yield and yield criteria was observed in F1s. Similarly a significant relationship between genetic distance and transgressive segregations for yield and yield components was found in F2-3s. The hybrid vigor for yield and yield components in F1s was higher in interspecific crosses than those of intraspecific crosses. Results indicated that fruitful segregations in F2-3s were higher in interspecific crosses than those of intraspecific ones. Results have proven that genetic differences among Cicer species based on SSR markers have been powerful predictor for hybrid vigor in F1s and fruitful segregations in F2-3s. http://dx.doi.org/10.1016/j.copbio.2013.05.077 PC2 containment glasshouse facilities for horticultural trees and vines at PFR Mount Albert, Auckland Monica Anisoara Dragulescu, Ngaire Markwick, Roger Helens, Andrew Gleave Plant and Food Research Institute, Mount Albert, Auckland, New Zealand E-mail address: [email protected] (M.A. Dragulescu). PC2 containment glasshouse facilities for horticultural trees and vines at PFR Mount Albert, Auckland, New Zealand’s horticultural sector combines natural advantages with human innovation and world-leading technology to produce and export a variety of premium fruit and vegetable products. Apples and kiwifruit are New Zealand’s major horticultural exports, and maintaining a position of global leadership in these crops depends upon the development of new premier cultivars. Functional genomics, using transgenic apple trees and kiwifruit vines, is being used to link alleles definitively with traits. Establishing the allele/trait association is a critical factor in ‘faster breeding’ approaches to developing new and novel cultivars, whether they are generated ultimately through genetic engineering (GE) or the application of marker-assisted selection in conventional breeding. Carrying out functional genomics with apples and kiwifruit requires optimum growing conditions to ensure flowering and fruiting, and currently in New Zealand, this must be carried out in Physical Containment level 2 (PC2) facilities. Recently PFR commissioned a new facility designed specifically to provide such conditions for tree and vine growth whilst meeting these physical containment requirements. The facility includes two large growing areas, with high light intensity and sophisticated mechanical ventilation, misting/fogging and shading systems. These features facilitate control of the temperature, humidity and vapour pressure deficit levels to emulate orchard conditions as closely as is possible in a glassenclosed space, like ‘A Field in a Box’. Details on the design, features and operation of this PC2 glasshouse facility will be presented. http://dx.doi.org/10.1016/j.copbio.2013.05.078
S39
Bulblets regeneration from in vitro improved leaf of Fritillaria imperialis L. and F. persica L. Suleyman Kizil 1 , Khalid Mahmood Khawar 2 , Ugur Sesiz 1 1
Department of Field Crops, Faculty of Agriculture, Dicle University, Diyarbakir, Turkey 2 Department of Field Crops, Faculty of Agriculture, Ankara University, Ankara, Turkey E-mail address: [email protected] (S. Kizil). Fritillaria imperialis and F. ersica are used as ornamental plants. Propagation of these plants instead of gathering them from the nature has been a current issue in Turkey. When speed and multiplication rate are considered, traditional production methods are inadequate. The application of in vitro propagation techniques offers possibility of producing large number of uniform plants for further field culture. This study was carried out on shoot tip, middle portions and the lower portion of leaf explant (portion of lamina in between leaf sheath and middle portion of the leaf blade) obtained from germinated seedlings of Fritillaria imperialis and F. persica on MS medium in Petri dishes cultured at 4◦ C in the dark. The study aimed to determine the regeneration competency of the three explants of to regenerate bulblets on MS medium containing various concentrations of BAP–NAA. The results showed that the leaf tip, middle portion of the leaf and lower portion of the leaf were competent and induced callus, shoots and mean number of shoots per explant variably. However, lower portion of the leaf explant was most competent to regenerate new bulblets. This suggests that MS medium containing various concentration of BAP–NAA were suitable for propagation of both species. The best regeneration was achieved on MS medium containing 1 mg/l BAP–0.1 mg/l NAA from the lower portion of the leaves used as explant. This protocol could be effectively used for propagation of the species and has possibility of producing large numbers of uniform plants for nurseries and field culture. http://dx.doi.org/10.1016/j.copbio.2013.05.079 Molecular diagnosis of peanut witches’-broom group (16SrII) phytoplasma infecting sesame in Turkey Cengiz Ikten 1 , Engin Yol 2 , Mursel Catal 1 , Rustem Ustun 2 , Bulent Uzun 2 1
Department of Plant Protection, Faculty of Agriculture, Akdeniz University, TR-07058 Antalya, Turkey 2 Department of Field Crops, Faculty of Agriculture, Akdeniz University, TR-07058 Antalya, Turkey E-mail address: [email protected] (B. Uzun). Severe shoot proliferation, small leaves and leaf rolling resembling phytoplasma disease symptoms were observed in sesame (Sesamum indicum L.) plants growing in the fields of Antalya province in Turkey. In order to investigate the possibility of phytoplasma infection, total DNA was isolated from leaf tissues of infected and healthy sesame plants and 16S rRNA gene was amplified using phytoplasma universal primers P1/P7 and R16F2n/R16R2 in direct and nested PCR amplifications. Amplicons of expected 1.8 kb and 1.2 kb, respectively, were obtained from infected sesame samples. No amplification was observed in samples from healthy sesame plants used as negative controls. Amplicons of the nested PCR were sequenced and analyzed by NCBI BLAST tool. Analysis of the 1118 bp amplicons showed that the sequence shared 99.0% sequence homology with that of the peanut witches’-broom phytoplasma reference strain (GenBank accession: L33765.1). This relationship was further supported by virtual restriction frag-