Dissertationen: „Genetic transformation“

7

Soloki, Mahmod. "Genetic transformation of grape somatic embryos." Thesis, University of Nottingham, 1997. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.387659.

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8

Faria, Maria José Sparça Salles de. "Red raspberry transformation using agrobacterium." Thesis, McGill University, 1993. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=69522.

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Regeneration and transformation protocols for 'Comet' red raspberry were optimized with the purpose of making the Agrobacterium-mediated gene transfer system efficient for this crop. Adventitious shoot regeneration from leaf discs was improved using explants 10 mm in diameter and transferring to fresh medium at the fourth week of incubation. Additions of liquid medium to solid medium during incubation decreased regeneration and attempts to release the suppressive influence of larger shoots on initials (apical dominance) did not succeed. The presence of claforan did not affect shoot regeneration, but inoculations with Agrobacterium and the presence of kanamycin decreased regeneration moderately or considerably, respectively. The threshold for kanamycin concentration for screening for kanamycin resistant transformed raspberry tissue was 30 to 40 mg l$ sp{-1}.$ The best co-incubation interval between wild-type Agrobacterium and 'Comet' leaf discs ranged from 2 days for highly virulent strains to 3 or more days for moderate to low virulent strains. Among several wild-type strains, C58 was chosen as the most appropriate partially because a disarmed form was commercially available for use as a non-oncogenic vector for transformation of red raspberry. The binary plasmid pBI121 containing the marker genes NPTII and GUS encoding kanamycin resistance and $ beta$-glucuronidase activity, respectively, was successfully introduced into the Agrobacterium strain LBA4404, which is a disarmed C58 derivative. Transformation of 'Comet' red raspberry was apparently achieved by inoculating leaf disc explants with LBA4404 containing pBI121. The probable integration and expression of the foreign genes into the plant cells were confirmed by screening for kanamycin resistance, GUS assays and Southern blot analyses. This transformation system appears to be effective and may be useful in further studies on red raspberry for both introduction of genes for desirable agronomic traits and basic studies of gene expression.

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9

Robson, Julia. "The construction of an expression vector for the transformation of the grape chloroplast genome." Thesis, Stellenbosch : Stellenbosch University, 2003. http://hdl.handle.net/10019.1/53621.

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Thesis (MSc)--University of Stellenbosch, 2003. ENGLISH ABSTRACT: The genetic information of plants is found in the nucleus, the mitochondria, and the plastids. The DNA of plastids is comprised of multiple copies of a double-stranded, circular, prokaryoticallyderived genome of -150 kb. The genome equivalents of plastid organelles in higher plant cells are an attractive target for genetic engineering as high protein expression levels are readily obtained due to the high genome copy number per organelle. The resultant proteins are contained within the plastid organelle and the corresponding transgenes are inherited, in most crop plants, uniparentally, preventing pollen transmission of DNA. Plastid transformation involves the uniform modification of all the plastid genome copies, a process facilitated by homologous recombination and the non-Mendelian segregation of plastids upon cell division. The plastid genomes are in a continuous state of inter- and intra-molecular exchange due to their common genetic complement. This enables the site-specific integration of any piece of DNA flanked by plastid targeting sequences, via homologous recombination. The attainment of homoplasmy, where all genomes are transformed, requires the inclusion of a plastid-specific selectable marker. Selective pressure favouring the propagation of the transformed genome copies, as well as the random segregation of plastids upon cell division, make it feasible to acquire uniformity and hence genetic stability. From this, a complete transplastomie line is obtained where all plastid genome copies present are transgenic, having eliminated all wild-type genome copies. The prokaryotic nature of the chloroplast genetic system enables expression of multiple proteins from polycistronic mRNAs, allowing the introduction of entire operons in a single transformation. Expression cassettes in vectors thus include single regulatory elements of plastid origin, and harbour genes encoding selectable and screenable markers, as well as one or more genes of interest. Each coding region is preceded by an appropriate translation control region to ensure efficient translation from the polycistronic mRNA. The function of a plastid transformation vector is to enable transfer and stable integration of foreign genes into the chloroplast genomes of higher plants. The expression vector constructed in this research is specific for the transformation of the grape chloroplast genome. Vitis vinifera L., from the family, Vitaceae, is the choice species for the production of wine and therefore our target for plastid transformation. All chloroplast derived regulatory elements and sequences included in the vector thus originated from this species. AFRIKAANSE OPSOMMING: Die genetiese inligting van plante word gevind in die kern, die mitochondria, en die plastiede. Die DNA van plastiede bestaan uit veelvuldige kopieë van 'n ~ 150 kb dubbelstring, sirkulêre genoom van prokariotiese oorsprong. Die genoomekwivalente van plastiede in hoër plante is 'n aantreklike teiken vir genetiese manipulering, aangesien die hoë genoom kopiegetal per organel dit moontlik maak om gereeld hoë vlakke van proteïenuitdrukking te verkry. Hierdie proteïene word tot die plastied beperk, en die ooreenstemmende transgene word in die meeste plante sitoplasmies oorgeërf, sonder die oordrag van DNA deur die stuifmeel. Plastied transformasie behels die uniforme modifikasie van al die plastied genoomkopieë, 'n proses wat deur homoloë rekombinasie en die nie-Mendeliese segregasie van plastiede tydens seldeling gefasiliteer word. As gevolg van die gemeenskaplike genetiese komplement, vind aanhoudende interen intra-molekulêre uitruiling van plastiedgenome plaas. Dit maak die setel-spesifieke integrasie, via homoloë rekombinasie, van enige stuk DNA wat deur plastied teikenvolgordes begrens word, moontlik. Vir die verkrying van homoplasmie, waar alle genome getransformeer is, word die insluiting van 'n plastiedspesifieke selekteerbare merker benodig. Seleksiedruk wat die vermeerdering van die getransformeerde genoomkopieë bevoordeel, en die lukrake segregasie van plastiede tydens seldeling, maak dit moontlik om genetiese stabiliteit en uniformiteit van die genoom te verkry. Dit kan op sy beurt tot die verkryging van 'n volledige transplastomiese lyn lei, waar alle aanwesige plastiedgenome transgenies is, en wilde tipe genoomkopieë geëlimineer is. Die prokariotiese aard van die chloroplas genetiese sisteem maak die uitdrukking van veelvuldige proteïene vanaf polisistroniese mRNAs moontlik, wat die toevoeging van volledige operons in 'n enkele transformasie toelaat. Uitdrukkingskassette in vektore bevat dus enkel regulatoriese elemente van plastied oorsprong, gene wat kodeer vir selekteerbare en sifbare merkers, asook een of meer gene van belang (teikengene). Voor elke koderingsstreek, is daar ook 'n toepaslike translasie beheerstreek om doeltreffende translasie vanaf die polisistroniese mRNA te verseker. Die funksie van 'n plastied transformasie vektor is om die oordrag en stabiele integrasie van transgene in chloroplasgenome van hoër plante moontlik te maak. Die uitdrukkingsvektor wat in hierdie studie gekonstrueer is, is spesifiek vir die transformasie van die druif chloroplasgenoom. Vitis vinifera L., van die familie Vitaceae, is die voorkeur species vir die produksie van wyn, en daarom die teiken vir plastied transformasie. Alle chloroplast-afgeleide regulatoriese elemente en volgordes wat in hierdie vektor ingesluit is, het huloorsprong vanaf VUis vinifera L.

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10

Cook, Marisa Anne. "Replicons derived from endogenously isolated plasmids used to classify plasmids occurring in marine sediment bacteria." Thesis, Georgia Institute of Technology, 2001. http://hdl.handle.net/1853/25736.

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11

Stummer, Belinda E. "Micropropagation and genetic transformation of 'verticordia grandis' /." Adelaide : Thesis (Ph.D.)--University of Adelaide, Departments of Plant and and Soil Sciences, 1993. http://web4.library.adelaide.edu.au/theses/09PH/09phs934.pdf.

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12

Singh, S. "Somatic embryogenesis and genetic transformation in peanut." Thesis(Ph.D.), CSIR-National Chemical Laboratory, Pune, 2008. http://dspace.ncl.res.in:8080/xmlui/handle/20.500.12252/2710.

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13

Wu, Jiang. "Transformation of Rhizoctonia solani." Thesis, Wu, Jiang (2003) Transformation of Rhizoctonia solani. PhD thesis, Murdoch University, 2003. https://researchrepository.murdoch.edu.au/id/eprint/417/.

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The aim of this study was to develop a genetic transformation system for the plant pathogenic fungus Rhizoctonia solani (teliomorph, Thanatephprus cucumeris Frank [Donk]). The availability of a transformation system would allow us to study gene exchange, epidemiology, and to use techniques such as gene disruption or gene silencing to investigate the role of fungal enzymes in pathogenesis. The approach adopted was to use Agrobacterium tumefaciens to transform the fungus as reports in the literature suggested that this was the most efficient and easiest method to use. As a preliminary test, Fusarium oxysporum was transformed using a binary vector (pBINAN) containing a hygromycin resistance gene under control of an ascomycete promoter and terminator. Hygromycin resistant transformants were obtained after co-incubation of fungal conidia with the bacterium. The presence of the transgene was confirmed by analysis of DNA. The number of transformants depended on the genetic background of the A tumefaciens. Strains AGLO or AGRO gave higher numbers of transformants compared to LBA4404. No transformants were obtained when the hygromycin gene was under control of a basidiomycete (pBINHL1), or a plant (CaMV35S) promoter. Since the basidiomycete promoter used in pBINHL1 originates from Ustilago maydis, the vector was tested by transformation of Ustilago cynodontis. Stable transformants of U cynodontis were obtained with this vector. A series of experiments were carried out on transformation of R. solani mycelium. Both the protoplast and the Agrobacterium transformation methods were tested. Parameters affecting protoplast production and regeneration were examined. Protoplast production varied with the age of the mycelium, with the osmotic stabilizer used, and with time of treatment with protoplasting enzymes. Regeneration of protoplasts was also affected by the osmotic stabilizer and the growth medium. Transformation of several isolates from different anastomosis groups (AG) was attempted by inducing protoplasts to take up DNA using polyethyleneglycol. Two plasmids were used; (1) pAN7-1 containing the resistance gene under control of an ascomycete promoter, and (2) pHL-1 in which the resistance gene is under control of a basidiomycete promoter. No transformants were obtained. Attempts were then made to transform mycelium and protoplasts using A tumefaciens. The experiments used both mycelium and protoplasts as the recipient. A number of small resistant colonies were obtained using binary plasmid (pBINHL1) in which mycelium was transformed with the resistance gene was driven by the basidiomycete promoter. On transfer to fresh medium these colonies would grow to about 2cm diameter, and then stop growing. On a second transfer to fresh medium they failed to show any growth. No resistant colonies were obtained from A. tumefaciens transformation of protoplasts. To improve transformation efficiency, a vector was constructed in which the hygromycin resistance gene was fused to an R solani laccase promoter sequence. No resistant colonies were obtained using this vector. Further experiments were carried out using a hygromycin resistance gene specially modified for expression in basidiomycetes by the insertion of artificial introns in the 5' and 3' untranslated regions, and a number of AT to CG conversions in the coding region. Most of the recipient isolates gave transformants with the unstable resistance phenotype. However, one AG 6 isolate gave transformants with a stable resistance phenotype. Of six transformants recovered from this isolate, five were shown by PCR and southern blotting to contain the transgene. In four of these transformants the resistance phenotype was stable in the absence of selection.

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14

Wu, Jiang. "Transformation of Rhizoctonia solani." Wu, Jiang (2003) Transformation of Rhizoctonia solani. PhD thesis, Murdoch University, 2003. http://researchrepository.murdoch.edu.au/417/.

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The aim of this study was to develop a genetic transformation system for the plant pathogenic fungus Rhizoctonia solani (teliomorph, Thanatephprus cucumeris Frank [Donk]). The availability of a transformation system would allow us to study gene exchange, epidemiology, and to use techniques such as gene disruption or gene silencing to investigate the role of fungal enzymes in pathogenesis. The approach adopted was to use Agrobacterium tumefaciens to transform the fungus as reports in the literature suggested that this was the most efficient and easiest method to use. As a preliminary test, Fusarium oxysporum was transformed using a binary vector (pBINAN) containing a hygromycin resistance gene under control of an ascomycete promoter and terminator. Hygromycin resistant transformants were obtained after co-incubation of fungal conidia with the bacterium. The presence of the transgene was confirmed by analysis of DNA. The number of transformants depended on the genetic background of the A tumefaciens. Strains AGLO or AGRO gave higher numbers of transformants compared to LBA4404. No transformants were obtained when the hygromycin gene was under control of a basidiomycete (pBINHL1), or a plant (CaMV35S) promoter. Since the basidiomycete promoter used in pBINHL1 originates from Ustilago maydis, the vector was tested by transformation of Ustilago cynodontis. Stable transformants of U cynodontis were obtained with this vector. A series of experiments were carried out on transformation of R. solani mycelium. Both the protoplast and the Agrobacterium transformation methods were tested. Parameters affecting protoplast production and regeneration were examined. Protoplast production varied with the age of the mycelium, with the osmotic stabilizer used, and with time of treatment with protoplasting enzymes. Regeneration of protoplasts was also affected by the osmotic stabilizer and the growth medium. Transformation of several isolates from different anastomosis groups (AG) was attempted by inducing protoplasts to take up DNA using polyethyleneglycol. Two plasmids were used; (1) pAN7-1 containing the resistance gene under control of an ascomycete promoter, and (2) pHL-1 in which the resistance gene is under control of a basidiomycete promoter. No transformants were obtained. Attempts were then made to transform mycelium and protoplasts using A tumefaciens. The experiments used both mycelium and protoplasts as the recipient. A number of small resistant colonies were obtained using binary plasmid (pBINHL1) in which mycelium was transformed with the resistance gene was driven by the basidiomycete promoter. On transfer to fresh medium these colonies would grow to about 2cm diameter, and then stop growing. On a second transfer to fresh medium they failed to show any growth. No resistant colonies were obtained from A. tumefaciens transformation of protoplasts. To improve transformation efficiency, a vector was constructed in which the hygromycin resistance gene was fused to an R solani laccase promoter sequence. No resistant colonies were obtained using this vector. Further experiments were carried out using a hygromycin resistance gene specially modified for expression in basidiomycetes by the insertion of artificial introns in the 5' and 3' untranslated regions, and a number of AT to CG conversions in the coding region. Most of the recipient isolates gave transformants with the unstable resistance phenotype. However, one AG 6 isolate gave transformants with a stable resistance phenotype. Of six transformants recovered from this isolate, five were shown by PCR and southern blotting to contain the transgene. In four of these transformants the resistance phenotype was stable in the absence of selection.

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15

Williamson, Phillip C. "A Novel Mechanism for Site-Directed Mutagenesis of Large Catabolic Plasmids Using Natural Transformation." Thesis, University of North Texas, 2001. https://digital.library.unt.edu/ark:/67531/metadc2828/.

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Natural transformation is the process by which cells take up DNA from the surrounding medium under physiological conditions, altering the genotype in a heritable fashion. This occurs without chemical or physical treatment of the cells. Certain Acinetobacter strains exhibit a strong tendency to incorporate homologous DNA into their chromosomes by natural transformation. Transformation in Acinetobacter exhibits several unique properties that indicate this system's superiority as a model for transformation studies or studies which benefit from the use of transformation as an experimental method of gene manipulation. Pseudomonas putida is the natural host of TOL plasmids, ranging between 50 kbp and 300 kbp in size and encoding genes for the catabolism of toluene, meta-toluate, and xylene. These very large, single-copy plasmids are difficult to isolate, manipulate, or modify in vitro. In this study, the TOL plasmid pDKR1 was introduced into Acinetobacter calcoaceticus strains and genetically engineered utilizing natural transformation as part of the process. Following engineering by transformation, the recombinant DNA molecule was returned to the native genetic background of the original host P. putida strain. Specific parameters for the successful manipulation of large plasmids by natural transformation in Acinetobacter were identified and are outlined. The effects of growth phase, total transforming DNA concentration, transforming DNA conformation, and gene dosage on transformation efficiency are presented. Addition of Acinetobacter plasmid DNA sequences to the manipulated constructs did not have an effect on transformation rates. Results suggest that a broadly applicable and efficient method to carry out site-directed genetic manipulations of large plasmids has been identified. The ability to easily reintroduce the recombinant DNA molecules back into the original host organism was maintained.

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16

Morris, Alison Claire. "Genetic transformation of the mosquito Aedes aegypti using a transposable genetic element." Thesis, University of Liverpool, 1989. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.279703.

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17

au, jiangwu@central murdoch edu, and Jiang Wu. "Transformation of Rhizoctonia solani." Murdoch University, 2003. http://wwwlib.murdoch.edu.au/adt/browse/view/adt-MU20050728.141653.

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The aim of this study was to develop a genetic transformation system for the plant pathogenic fungus Rhizoctonia solani (teliomorph, Thanatephprus cucumeris Frank [Donk]). The availability of a transformation system would allow us to study gene exchange, epidemiology, and to use techniques such as gene disruption or gene silencing to investigate the role of fungal enzymes in pathogenesis. The approach adopted was to use Agrobacterium tumefaciens to transform the fungus as reports in the literature suggested that this was the most efficient and easiest method to use. As a preliminary test, Fusarium oxysporum was transformed using a binary vector (pBINAN) containing a hygromycin resistance gene under control of an ascomycete promoter and terminator. Hygromycin resistant transformants were obtained after co-incubation of fungal conidia with the bacterium. The presence of the transgene was confirmed by analysis of DNA. The number of transformants depended on the genetic background of the A tumefaciens. Strains AGLO or AGRO gave higher numbers of transformants compared to LBA4404. No transformants were obtained when the hygromycin gene was under control of a basidiomycete (pBINHL1), or a plant (CaMV35S) promoter. Since the basidiomycete promoter used in pBINHL1 originates from Ustilago maydis, the vector was tested by transformation of Ustilago cynodontis. Stable transformants of U cynodontis were obtained with this vector. A series of experiments were carried out on transformation of R. solani mycelium. Both the protoplast and the Agrobacterium transformation methods were tested. Parameters affecting protoplast production and regeneration were examined. Protoplast production varied with the age of the mycelium, with the osmotic stabilizer used, and with time of treatment with protoplasting enzymes. Regeneration of protoplasts was also affected by the osmotic stabilizer and the growth medium. Transformation of several isolates from different anastomosis groups (AG) was attempted by inducing protoplasts to take up DNA using polyethyleneglycol. Two plasmids were used; (1) pAN7-1 containing the resistance gene under control of an ascomycete promoter, and (2) pHL-1 in which the resistance gene is under control of a basidiomycete promoter. No transformants were obtained. Attempts were then made to transform mycelium and protoplasts using A tumefaciens. The experiments used both mycelium and protoplasts as the recipient. A number of small resistant colonies were obtained using binary plasmid (pBINHL1) in which mycelium was transformed with the resistance gene was driven by the basidiomycete promoter. On transfer to fresh medium these colonies would grow to about 2cm diameter, and then stop growing. On a second transfer to fresh medium they failed to show any growth. No resistant colonies were obtained from A. tumefaciens transformation of protoplasts. To improve transformation efficiency, a vector was constructed in which the hygromycin resistance gene was fused to an R solani laccase promoter sequence. No resistant colonies were obtained using this vector. Further experiments were carried out using a hygromycin resistance gene specially modified for expression in basidiomycetes by the insertion of artificial introns in the 5 and 3 untranslated regions, and a number of AT to CG conversions in the coding region. Most of the recipient isolates gave transformants with the unstable resistance phenotype. However, one AG 6 isolate gave transformants with a stable resistance phenotype. Of six transformants recovered from this isolate, five were shown by PCR and southern blotting to contain the transgene. In four of these transformants the resistance phenotype was stable in the absence of selection.

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18

Wang, Tsung-Tsan 1959. "Transformant system and gene expression of yeast Schwanniomyces occidentalis." Thesis, McGill University, 1999. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=35955.

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Schwanniomyces occidentalis (Debaryomyces occidentalis ) is able to grow rapidly with high cell mass on cheap starch as a carbon source, produce strong amylolytic enzymes extracellularly and secrete large proteins without hyper-glycosylation and measurable extracellular proteases. Schw. occidentalis thus has a high potential as, a useful alternative to Saccharomyces cerevisiae in the production of heterologous proteins. However, the molecular study of Schw. occidentalis has been very limited due to the insufficient transformation system and lack of gene expression information. A new transformation system of Schw. occidentalis has been developed. This system was based on vector YEp13 ( LEU2) and a stable leu auxotrophic mutant, Schw. occidentalis DW88, obtained by treating the yeast with 1-methyl-3-nitro-1-nitrosoguanidine. The transformation efficiency of YEp13 by spheroplast-mediating method was 103 transformants/mug DNA. The 2-mum replicon is proposed to be responsible for YEp13 replication in Schw. occidentalis. The YEp13 stability in Schw. occidentalis was low, but it kept its structure in the yeast, suggesting that Schw. occidentalis DW88 does not modify foreign DNA. After analysis of 14 cloned Schw. occidentalis genes and comparison of associated genes from both Schw. occidentalis and S. cerevisiae, 25 codons were arbitrarily chosen as putative preferred codons for Schw. occidentalis. They are similar to those of S. cerevisiae, except for TTA for leucine, and AAA for lysine. Codon Bias Index (CBI), a criterion to evaluate gene expression, is calculated from preferred codons. A computer program (PCBI) which reads a gene containing introns was developed to quickly calculate CBI. Schw. occidentalis DWSS should be a good host to produce and secrete heterologous proteins and the putative preferred codons and program PCBI can facilitate molecular study of Schw. occidentalis. (Abstract shortened by UMI.)

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19

Zhou, Chen, and 周辰. "Genome-informed studies on Penicillium marneffei: horizontal gene transfer survey and differentialsecretomics." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2008. http://hub.hku.hk/bib/B41633672.

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20

Iglesias, Victor Alejandro. "Genetic transformation studies in wheat using particle bombardment /." [S.l.] : [s.n.], 1994. http://e-collection.ethbib.ethz.ch/show?type=diss&nr=10628.

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21

Jiang, Liwen. "Somatic embryogenesis and genetic transformation in douglas-fir." Thesis, University of British Columbia, 1991. http://hdl.handle.net/2429/29882.

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Cell division was obtained from cultured microspores of Douglas-fir on medium supplemented with auxin, cytokinin, and sucrose, but without medium salts. Embryogenic callus was initiated from excised mature and immature zygotic embryos of Douglas-fir on media supplemented with cytokinin and auxin. Precotyledonary embryos produced most of the embryogenic calli in the cultures. Secondary embryogenic callus production, and subsequent subculturing, were required for the establishment of stable embryogenic callus lines for both mature and immature zygotic embryos. Somatic embryos at the precotyledonary stage were obtained in high frequency when Douglas-fir embryogenic callus was transferred onto hormone-free medium supplemented with 1% activated charcoal, while some cotyledonary somatic embryos were obtained from hormone-free medium supplemented with low ABA levels (0-10 uM). The level of ABA in the maturation medium significantly affected the quality of the somatic embryos produced. Cell suspensions were established from embryogenic calli and have been maintained for over one year. Protoplasts were isolated from suspension, cell colonies and calli were regenerated from protoplasts. GUS and CAT genes were successfully introduced into protoplasts of Douglas-fir via electroporation, and their transient expression was obtained 2-4 days after electroporation. The results so far indicate that the production of somatic embryos via embryogenesis in vitro is obtainable, and the application of direct gene transfer via electroporation for genetic engineering of trees in this species is promising. Forestry, Faculty of Graduate

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22

Travella, Silvia. "Improving and understanding the barley genetic transformation process." Thesis, University of East Anglia, 2000. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.365058.

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23

Tan, Chia Lock. "Tissue culture and genetic transformation of Theobroma cacao." Thesis, University of Nottingham, 2000. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.310835.

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24

Deljou, Ali. "Tissue culture and genetic transformation in potato breeding." Thesis, University of Nottingham, 1997. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.339661.

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25

Owuama, C. I. "Genetic transformation of Saccharomyces cerevisiae with chimaeric plasmids." Thesis, University of Liverpool, 1987. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.381362.

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26

Sparrow, Penelope Amelia Claire. "Plant morphogenesis and genetic transformation of horticultural brassicas." Thesis, Open University, 2002. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.252364.

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27

Tolonen, Andrew Carl. "Prochlorococcus genetic transformation and genomics of nitrogen metabolism." Thesis, Massachusetts Institute of Technology, 2005. http://hdl.handle.net/1721.1/43721.

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Thesis (Ph. D.)--Joint Program in Biological Oceanography (Massachusetts Institute of Technology, Dept. of Biology; and the Woods Hole Oceanographic Institution), 2005. Includes bibliographical references. Prochlorococcus, a unicellular cyanobacterium, is the most abundant phytoplankton in the oligotrophic, oceanic gyres where major plant nutrients such as nitrogen (N) and phosphorus (P) are at nanomolar concentrations. Nitrogen availability controls primary productivity in many of these regions. The cellular mechanisms that Prochlorococcus uses to acquire and metabolize nitrogen are thus central to its ecology. One of the goals of this thesis was to investigate how two Prochlorococcus strains responded on a physiological and genetic level to changes in ambient nitrogen. We characterized the N-starvation response of Prochlorococcus MED4 and MIT9313 by quantifying changes in global mRNA expression, chlorophyll fluorescence, and Fv/Fm along a time-series of increasing N starvation. In addition to efficiently scavenging ambient nitrogen, Prochlorococcus strains are hypothesized to niche-partition the water column by utilizing different N sources. We thus studied the global mRNA expression profiles of these two Prochlorococcus strains on different N sources. The recent sequencing of a number of Prochlorococcus genomes has revealed that nearly half of Prochlorococcus genes are of unknown function. (cont.) Genetic methods such as reporter gene assays and tagged mutagenesis are critical tools for unveiling the function of these genes. As the basis for such approaches, another goal of this thesis was to find conditions by which interspecific conjugation with Escherichia coli could be used to transfer plasmid DNA into Prochlorococcus MIT9313. Following conjugation, E. coli were removed from the Prochlorococcus cultures by infection with E. coli phage T7. We applied these methods to show that an RSF1010-derived plasmid will replicate in Prochlorococcus MIT9313. When this plasmid was modified to contain green fluorescent protein (GFP) we detected its expression in Prochlorococcus by Western blot and cellular fluorescence. Further, we applied these conjugation methods to show that Tn5 will transpose in vivo in Prochlorococcus. Collectively, these methods provide a means to experimentally alter the expression of genes in the Prochlorococcus cell. by Andrew Carl Tolonen. Ph.D.

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Mohandas, Kavitha. "Genetic transformation of the entomopathogenic deuteromycete, Metarhizium flavoviride." Thesis, University of Bath, 1998. https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.245885.

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29

Korban, Martine. "Agrobacterium-mediated transformation of common bean (Phaseolus vulgaris L.)." Thesis, McGill University, 1994. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=41644.

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Regeneration and shoot multiplication of common bean (Phaseolus vulgaris L. 'ICA Pijao') from half-cotyledonary nodes was achieved on modified Murashige and Skoog (1962) basal medium amended with 5 $ mu$M 6-benzylaminopurine. Histological studies confirmed the adventitious origin of the regenerated buds. Shoots were rooted ex vitro and developed into morphologically normal plants compared with seed-grown controls. The relative susceptibility of bean tissues to infection by a collection of wild-type Agrobacterium strains was tested. Positive transformation events were evaluated based on morphological and biochemical changes observed following Agrobacterium infection. The A. tumefaciens strain C58 was particularly virulent on greenhouse-grown plants, in vitro-derived stem sections, half-cotyledonary nodes and seedlings. A sensitive and rapid method was developed to detect opines using thin layer chromatography. Transient $ beta$-glucuronidase (GUS) gene expression was detected in 'ICA Pijao' bean buds regenerated from half-cotyledonary nodes following Agrobacterium-mediated gene transfer with the binary vector pGV1040 or p35SGUSINT. Four out of eight putative transformants contained the chimeric GUSINT gene following polymerase chain reaction (PCR) analysis. This was confirmed by Southern analysis of blotted PCR gels. However, there was no stable integration of the GUSINT gene as none of the R1 progeny showed an amplified GUSINT fragment with PCR.

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Becker, Eric Christian. "Recognition of oriT at the termination of conjugal transfer by MobA, the R1162 DNA strand transferase." Access restricted to users with UT Austin EID Full text (PDF) from UMI/Dissertation Abstracts International, 2002. http://wwwlib.umi.com/cr/utexas/fullcit?p3077405.

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Van, Eeden C. (Christiaan). "The construction of gene silencing transformation vectors for the introduction of multiple-virus resistance in grapevines." Thesis, Stellenbosch : Stellenbosch University, 2004. http://hdl.handle.net/10019.1/53764.

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Thesis (MSc)--University of Stellenbosch, 2004. ENGLISH ABSTRACT: Viruses are some of the most important pathogens of grapevines. There are no effective chemical treatments, and no grapevine- or other natural resistance genes have been discovered against grapevine infecting viruses. The primary method of grapevine virus control is prevention by biological indexing and molecular- and serological screening of rootstocks and scions before propagation. Due to the spread of grapevine viruses through insect vectors, and in the case of GRSPaV the absence of serological screening, these methods of virus control are not always effective. In the past several methods, from cross-protection to pathogen derived resistance (PDR), have been applied to induce plant virus resistance, but with inconsistent results. In recent years the application of post-transcriptional gene silencing (PTGS), a naturally occurring plant defense mechanism, to induce targeted virus resistance has achieved great success. The Waterhouse research group has designed plant transformation vectors that facilitate specific virus resistance through PTGS. The primary focus of this study was the production of virus specific transformation vectors for the introduction of grapevine virus resistance. The Waterhouse system has been successfully utilised for the construction of three transformation vectors with the pHannibal vector as backbone. Each vector contains homologous virus coat protein (CP) gene segments, cloned in a complementary conformation upstream and downstream of an intron sequence. The primary vector (pHann-SAScon) contains complementary CP gene segments of both GRSPaV and GLRaV-3 and was designed for the introduction of multiple-virus resistance. For the construction of the primary vector the GRSPaV CP gene was isolated from RSP infected grapevines. A clone of the GLRaV-3 CP gene was acquired. The second vector (pHann- LR3CPsas) contains complementary CP gene segments of GLRaV-3. The third vector (pHann-LR2CPsas) contains complementary CP gene segments of GLRaV-2. The cassette containing the complementary CP gene segments of both GRSPaV and GLRaV-3 was cloned into pART27 (pART27-HSAScon), and used to transform N tabacum cv. Petit Havana (SRI), through A. tumefaciens mediated transformation. Unfortunately potential transformants failed to regenerate on rooting media; hence no molecular tests were performed to confirm transformation. Once successful transformants are generated, infection with a recombinant virus vector (consisting of PYX, the GFP gene as screenable marker and the complementary CP gene segments of both GRSPaV and GLRaV-3) will be used to test for the efficacy of the vectors to induce resistance. A secondary aim was added to this project when a need was identified within the South African viticulture industry for GRSPaV specific antibodies to be used in serological screening. To facilitate future serological detection of GRSPaV, the CP gene was isolated and expressed with a bacterial expression system (pETI4b) within the E. coli BL2I(DE3)pLysS cell line. The expressed protein will be used to generate GRSPaV CP specific antibodies. AFRIKAANSE OPSOMMING: Virusse is van die belangrikste patogene by wingerd. Daar bestaan geen effektiewe chemiese beheer nie, en geen wingerd- of ander natuurlike weerstandsgene teen wingerdvirusse is al ontdek nie. Die primêre metode van beheer t.o.v. wingerdvirusse is voorkoming deur biologiese indeksering, en molekulêre- en serologiese toetsing van onderstokke en entlote voor verspreiding. As gevolg van die verspreiding van wingerdvirusse deur insekvektore, en in die geval van GRSPa V die tekort aan serologiese toetsing, is dié metodes van virusbeheer nie altyd effektief nie. In die verlede is metodes soos kruis-beskerming en patogeen-afgeleide weerstand (PDR) gebruik om virusweerstand te induseer, maar met inkonsekwente resultate. In onlangse jare is post-transkripsionele geenonderdrukking (PTGS), 'n natuurlike plantbeskermingsmeganisme, met groot sukses toegepas om geteikende virusweerstand te induseer. Die Waterhouse-navorsingsgroep het planttransformasievektore ontwerp wat spesifieke virusweerstand induseer d.m.v. PTGS. Die vervaardiging van virus spesifieke tranformasievektore vir die indusering van wingerdvirusweerstand was die primêre doelwit van hierdie studie. Die Waterhouse-sisteem was gebruik vir die konstruksie van drie transformasievektore, met die pHannibal vektor as basis. Elke vektor bevat homoloë virus kapsiedproteïen (CP) geensegmente, gekloneer in 'n komplementêre vorm stroom-op en stroom-af van 'n intronvolgorde. Die primêre vektor (pHann-SAScon) bevat komplementêre CP geensegmente van beide GRSPaV en GLRaV-3, en was ontwerp vir die indusering van veelvoudige-virusweerstand. Die CP-geen van GRSPa V was vanuit RSP-geïnfekteerde wingerd geïsoleer, vir die konstruksie van die primêre vektor. 'n Kloon van die GLRa V-3 CP-geen was verkry. Die tweede vektor (pHann-LR3CPsas) bevat komplementêre CP geensegmente van GLRaV-3. Die derde vektor (pHann-LR2CPsas) bevat komplementêre CP geensegmente van GLRa V-2. Die kasset bestaande uit die komplementêre CP geensegmente van beide GRSPaV en GLRaV-3, was gekloneer in pART27 (pART27-HSAScon), en gebruik om N tabacum cv. Petit Havana (SRI) te transformeer d.m.v. A. tumefaciens bemiddelde transformasie. Ongelukkig het potensiële transformante nie geregenereer op bewortelingsmedia nie; gevolglik was geen molekulêre toetse gedoen om transformasie te bevestig nie. Na suksesvolle transformante gegenereer is, sal infeksie met 'n rekombinante-virusvektor (bestaande uit PYX, die GFP geen as waarneembare merker en die komplementêre CP geensegmente van beide GRSPa V en GLRa V-3) gebruik word om die effektiwiteit van die vektore as weerstandsinduseerders te toets. 'n Sekondêre doelwit is by die projek gevoeg toe 'n behoefte aan GRSPaV spesifieke teenliggame binne die Suid-Afrikaanse wynbedryf geïdentifiseer is, vir gebruik in serologiese toetsing. Om toekomstige serologiese toetsing van GRSPa V te bemiddel, was die CP-geen geïsoleer en in 'n bakteriële uitdrukkingsisteem (PETI4b) uitgedruk, in die E. coli BL21(DE3)pLysS sellyn. Die uitgedrukte proteïne sal gebruik word vir die vervaardiging van GRSPa V CP spesifieke antiliggame.

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梁子明 and Tze-ming Leung. "Immunological studies of a phosphorylcholine-specific antibody using gene transfer techniques." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 1994. http://hub.hku.hk/bib/B31233740.

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Tiengtum, Pimol. "Towards the genetic manipulation of flower colour in Petunia and Curcuma." Thesis, University of Nottingham, 2002. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.269714.

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34

Suso, Henri-Pierre. "Development of a system for the genetic transformation of white lupin (Lupinus albus)." Thesis, University of Reading, 2001. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.271196.

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Leung, Tze-ming. "Immunological studies of a phosphorylcholine-specific antibody using gene transfer techniques /." [Hong Kong] : University of Hong Kong, 1994. http://sunzi.lib.hku.hk/hkuto/record.jsp?B13762680.

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Husnain, Tayyab. "Transformation studies in the forage legume Onobrychis viciifolia." Thesis, University of Nottingham, 1990. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.258236.

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37

Limberis, Maria. "A lentiviral gene transfer vector for the treatment of cystic fibrosis airway disease." Title page, synopsis and list of contents only, 2002. http://web4.library.adelaide.edu.au/theses/09PH/09phl735.pdf.

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"16th September 2002." Accompanying CD contains 2 MPEG clips with accompanying text, and a copy in PDF format of: Recovery of airway cystic fibrosis transmembrane conductance regulator function in mice with cystic fibrosis after single-dose lentivirus-mediated gene transfer / M. Limberis ... [et al.], published in Human gene therapy vol. 13 (2002). Bibliography: leaves xxix-li. This thesis focuses on modulating the physical barriers of the airway epithelium with mild detergents, so as to enhance gene transfer by a HIV-1 based lentivirus vector in vivo. The efficiency of the gene transfer was evaluated in the nasal airway of C57B1/6 mice using the Lac Z marker gene. This demonstration of lentivirus-mediated in vivo recovery of CFTR function in CF airway epithelium illustrated the potential of combining a pre-conditioning of the airway surface with a simple and brief HIV-1 based gene transfer vector exposure to produce therapeutic gene expression in the intact airway.

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Cheng, Wai-sheung, and 鄭偉嫦. "PTEN-PKB in endometriosis and related malignant transformation." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2005. http://hub.hku.hk/bib/B45009910.

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Zhang, Peng. "Studies on cassava (Manihot esculenta Crantz) transformation: towards genetic improvement /." [S.l.] : [s.n.], 2000. http://e-collection.ethbib.ethz.ch/show?type=diss&nr=13962.

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40

Kridel, Robert. "The genetic basis of transformation and progression in follicular lymphoma." Thesis, University of British Columbia, 2016. http://hdl.handle.net/2429/57547.

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The clonal evolution theory of cancer has been recognized for decades and follows principles of Darwinian selection, in which there is selection of the fittest clones in an ecosystem that is fundamentally heterogeneous and undergoes selective pressure. Follicular lymphoma (FL) emerges as a prototypical disease in which to study clonal evolution. It is the most common indolent lymphoma and, although the median overall survival largely surpasses 10 years, patients almost invariably experience progressive disease. Furthermore, a subset of FL patients is at risk of early lymphoma-related death due to rapid progression or transformation to aggressive lymphoma. Yet, the clonal dynamics and the landscape of genomic alterations underlying progression and transformation remain to be uncovered. Herein, we applied whole genome sequencing to a discovery set of transformed, progressed and non-progressed FL cases, re-constructed clonal phylogenies, and interrogated a larger set of transformed FLs and clinical extremes by capture-based targeted sequencing. Moreover, we applied the Lymph2Cx cell-of-origin assay to determine whether molecular subtypes can be defined in transformed follicular lymphoma (TFL) by gene-expression profiling. We discovered that transformation is typically the result of drastic clonal shifts during which TFL-specific clones rapidly outcompete indolent clones. In a subset of cases, these aggressive clones can be found at low levels (< 1% of tumour cells) at diagnosis. In contrast, primary progression generally results from the outgrowth of subclones that are readily detectable at diagnosis, suggesting that the genomic features conferring treatment resistance can be detected prior to initial treatment using low-pass sequencing technology. In addition, we identified discrete gene mutations that are associated with early progression and transformation, and uncovered that a subset of TFLs (16%) have an activated B-cell phenotype and are enriched for mutations in CD79B and BCL10. In summary, we found striking contrast in clonal trajectories between distinct clinical scenarios and described novel associations of gene mutations with transformation and progression. Our findings have translational relevance as they suggest that early progression, and to a lesser extent transformation, can potentially be predicted at diagnosis and thus provide a rationale for novel therapeutic approaches in TFL. Medicine, Faculty of Pathology and Laboratory Medicine, Department of Graduate

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Lee, Sun K. "Genetic transformation of broccoli and promoter tagging in Brassica species." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1996. http://www.collectionscanada.ca/obj/s4/f2/dsk3/ftp04/nq24027.pdf.

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42

Triggs, Heidi M. "Haploid production and genetic transformation of wheat (Triticum aestivum L.)." Thesis, University of Nottingham, 1998. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.243689.

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43

Drake, Pascal M. W. "Approaches to the genetic transformation of Sitka spruce (Picea sitchensis)." Thesis, University of Nottingham, 1996. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.320788.

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44

Moore, Ian Robert. "Development of a strategy for genetic transformation of plant mitochondria." Thesis, University of Edinburgh, 1989. http://hdl.handle.net/1842/11183.

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45

Silva, José Rodrigo da. "Improvement of chickpea rhizobia by genetic transformation with symbiosis genes." Doctoral thesis, Universidade de Évora, 2018. http://hdl.handle.net/10174/23169.

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Rhizobia are soil bacteria able to induce the formation of nodules in leguminous plants and convert atmospheric nitrogen into assimilable forms to these plants. Some Mesorhizobium species establish symbiosis with chickpea and can increase productivity of this culture. Rhizobia symbiosis genes, such as nod and nif, are involved in nodule development and nitrogen fixation. Nevertheless, genes involved in other molecular mechanisms, namely stress response may influence the symbiotic interaction plantrhizobia. The objective of this study was to evaluate the effects of overexpressing symbiotic and stress response genes in the symbiotic performance of chickpea Mesorhizobium. Mesorhizobium strains were transformed with pRKnifA, pRKnodD, pRKenvZ and pRKgroEL (expression vector pRK415 with nifA, nodD, envZ and groEL genes from M. mediterraneum UPM-Ca36T, respectively). From the four strains transformed with extra nifA copies, only V15-b was able to increase plant biomass, when compared to wild-type and empty vector strains. Among the four strains transformed with extra nodD copies, ST-2 and PMI-6 showed a higher symbiotic effectiveness compared to wild type and control strains. Additional copies of envZ led to in a higher symbiotic effectiveness when introduced in PMI-6 and EE-7. Evaluation of the symbiotic effectiveness of the four strains overexpressing groEL showed that only ST-2 improved, compared to wild-type and empty vector strains. For all these strains the rate of nodule formation was seen to be higher and further analysis of the infection process and nodule histological analysis were performed. Overall, this study shows that extra copies of a given gene may have different effects in the symbiotic effectiveness, depending on the modified strain. This study contributes to a better understanding of the nodulation and nitrogen fixation processes, namely regarding the contribution of non-symbiotic genes, especially envZ, which was to our knowledge for the first time reported to be involved in the rhizobia-legume symbiosis; Resumo: Melhoramento de rizóbios de grão-de-bico por transformação genética com genes simbióticos Rhizóbios são bactérias capazes de induzir a formação de nódulos em leguminosas e converter azoto atmosférico em formas assimiláveis por essas plantas. Algumas espécies de Mesorhizobium estabelecem simbioses com grão-de-bico e conseguem promover a produtividade desta cultura. Genes simbióticos, como nod e nif, estão envolvidos na formação dos nódulos e fixação de azoto. No entanto, genes envolvidos noutros mecanismos, nomeadamente a resposta ao stresse podem influenciar a interação simbiótica planta-rizóbio. O objetivo deste estudo foi avaliar a eficiência simbiótica de Mesorhizobium de grão-de-bico sobre-expressando genes simbióticos e de resposta ao stresse. Estirpes de Mesorhizobium foram transformadas com pRKnifA, pRKnodD, pRKenvZ e pRKgroEL (vetor de expressão pRK415 com nifA, nodD, envZ e groEL de M. mediterraneum UPM-Ca36T, respetivamente). Das quatro estirpes transformadas com cópias extras de nifA, apenas V15-b foi capaz de produzir um aumento na biomassa das plantas inoculadas, quando comparada às estirpes selvagem e com vetor vazio. Das quatro estirpes transformadas com cópias extras de nodD, ST- 2 e PMI-6 apresentaram maior eficiência simbiótica em comparação com as estirpes controlo. Cópias adicionais de envZ resultaram numa maior eficiência simbiótica quando introduzidas em PMI-6 e EE-7. A avaliação da eficiência simbiótica das quatro estirpes que sobre-expressam groEL mostrou que apenas a transformação de ST-2 levou a uma eficiência superior, em comparação com as estirpes selvagem e com vetor vazio. Para todas estas estirpes, a taxa de formação de nódulos também foi melhorada, pelo que análises do processo de infeção e da histologia dos nódulos foram efetuadas. Em geral, este estudo mostra que um gene introduzido pode ter efeitos diferentes na eficiência simbiótica, dependendo da estirpe modificada. Este estudo contribui para uma melhor compreensão dos processos de nodulação e fixação de azoto, nomeadamente a contribuição de genes não-simbióticos, especialmente envZ, que tanto quanto sabemos não foi previamente descrito como envolvido nestas simbioses.

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Burns, Kevin Andrew. "Genetic Detection of Neurogenesis and Astrocytic Transformation of Radial Glia." University of Cincinnati / OhioLINK, 2007. http://rave.ohiolink.edu/etdc/view?acc_num=ucin1195153606.

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47

Pavlova, Natalya Nickolayevna. "A Role for PVRL4-Driven Cell-Cell Interactions in Tumorigenesis." Thesis, Harvard University, 2012. http://dissertations.umi.com/gsas.harvard:10722.

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Deciphering genetic determinants of tumorigenesis is the greatest challenge and promise of the present-day era of biomedical research. As extensive tumor genome characterization efforts of the past decade had revealed, tumor genomes harbor multiple point mutations and gene copy number alterations. This exquisite complexity brings forth the challenge of distinguishing numerous incidental alterations from those that are functionally relevant to tumorigenesis. During the past decade, functional genetic screens have shown their utility in identifying genetic changes that functionally contribute to tumor-specific hallmarks and thus hold a great potential for identifying promising new targets for the rational design of successful anticancer therapies. A key hallmark of cancer cells is their ability to escape signals that govern homeostasis of normal tissue. In normal epithelia, growth and survival of cells is dictated by their physical anchorage to the extracellular matrix, and disruption of proper cell-matrix anchorage triggers cell death. Tumors of epithelial origin develop ways to subvert anoikis signals, which enables both their uncontrollable expansion at the primary site as well as metastatic colonization of distant organs. Understanding the genetic determinants of matrix-independent growth of cancer cells is a promising approach to identify potent and selective anticancer targets. In the work presented in this dissertation, we use an unbiased functional genetic screening approach to test a large set of eight thousand human genes to identify those that are involved in inducing and maintaining resistance of mammary epithelial cells to matrix detachment-induced cell death. We show that a cell adhesion molecule PVRL4 promotes cell survival in the absence of matrix anchorage in normal epithelial cells and in cancer cells. Our work reveals that PVRL4 promotes anchorage-independent growth by promoting cell-to-cell attachment and matrix-independent c-Src activation. PVRL4 is focally and frequently amplified in several types of solid tumors. Growth of orthotopically implanted tumors in vivo is inhibited by blocking PVRL4-driven cell-to-cell attachment with monoclonal antibodies, demonstrating a novel strategy for targeted therapy of cancer.

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Davis, Ashley Stuart. "Aspects of rice transformation using direct DNA uptake." Thesis, University of Nottingham, 1990. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.258367.

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49

Kim, Changhyeon. "Development of a Genetic Transformation System of Raspberry Cultivars for Gene Function Analysis." Thesis, North Dakota State University, 2018. https://hdl.handle.net/10365/29223.

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An Agrobacterium-mediated transformation system of purple raspberry ‘Amethyst’ was established after a series of experiments that determined the effect of genotype, inoculum density, and co-cultivation time on transformation. In this study, a plant regeneration protocol was established for ‘Joan J’ and ‘Polana’ (the regeneration protocol of ‘Amethyst’ was previously developed). Agrobacterium-mediated transformation was conducted for all three cultivars. The minimum killing level of hygromycin B and kanamycin was determined. Inoculum density and co-cultivation time were optimized. Polymerase chain reaction (PCR) verified a successful transformation of ‘Amethyst’ with the frequency of 3.3 ~ 4.4 % when leaves were infected with Agrobacterium EHA105 at the cell density of OD600 0.3 and co-cultivated for 3 days in the medium with 25.0 mg∙l-1 kanamycin. Transgenic lines with the PtFIT gene were hydroponically grown under iron sufficiency or deficiency. The real-time quantitative PCR verified the gene expression in response to iron sufficiency and deficiency conditions.

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Canseco-Sedano, Rodolfo. "Factors affecting the efficiency of gene transfer in mice." Diss., This resource online, 1992. http://scholar.lib.vt.edu/theses/available/etd-03172010-020810/.

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Dissertationen zum Thema „Genetic transformation“

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