Thesis (Ph.D.)--McMaster University, 2005. / Supervisor: Dr. Christian Baron. Includes bibliographical references (leaves 94-101).

We have developed crown-gall resistant transgenic plants capable of suppressing Agrobacterium tumefaciens T-DNA oncogenes. Crown gall tumors result from overproduction of auxin and cytokinin in plant cells transformed by A. tumefaciens. High phytohormone levels result from expression of two auxin biosynthetic genes, tryptophan monooxygenase (iaaM) and indole acetamide hydrolase (iaaH), and isopentenyl transferase (ipt), which mediates cytokinin synthesis. Inactivation of ipt and either one of the two auxin biosynthesis genes prevents crown gall formation. To suppress T-DNA oncogene expression, we created transgenic tobacco that produce the corresponding untranslatable sense-strand RNAs. This phenomenon, called cosuppression, frequently blocks expression of transgenes in plants. Often, expression of an untranslatable sense-strand transgene elicits sequence-specific destruction of both the mutant mRNA and the corresponding wild-type mRNA. Here we show that cosuppression can block expression of A. tumefaciens T-DNA oncogenes, resulting in plants that are resistant to gall induction by certain strains of A. tumefaciens. / Graduation date: 1999

The RCg2 promoter was identified in a search for root-specific genes to combat the rice water weevil (RWW) but expressed at low frequency (~10%). Spatial expression of RCg2 was investigated using two reporter constructs YXA (RCg2-gus-ocs) and YXB (RCg2-gus-RCg2) that included 1.6 kb of the RCg2 5' sequence fused to the ?-glucuronidase (gus) coding region. YXB plants were generated via Agrobacterium-mediated transformation but only 8 of 158 plants analyzed showed strong GUS activity despite the presence of an intact construct. Reactivation of RCg2 gene in rice was investigated by treatment of R0 and R1 of YXB transgenic plants with 5-azacytidine. Reactivation of RCg2-gus was observed in some transgenic plants indicating different mechanisms involved in the gene silencing of the YXB lines. DNA methylation analysis, northern blotting, RT-PCR and small RNA analysis supported the conclusion that PTGS and TGS are present in the silenced plants. Promoter analysis in silico and using promoter deletion assays predicted that the RCg2 promoter contains a complex region that includes miRNA homologs, MITEs and repetitive sequences. The high frequency of promoter-related silencing suggests functional interactions of these elements of the transgene and the homologous endogenous gene. To identify key elements contributing to the root-preferential expression of RCg2 and the high frequency of silencing observed in transgenic (YXB) lines, several RCg2 promoter deletion constructs were designed. These include 5' deletions MC1, MC2, MC4, MC7 and MC8 and internal deletions MC5, MC11, MC12 and MC13. The frequency with which silencing was encountered in populations of the deletion mutants was used to characterize the effects of various promoter elements. Deletion of the region from -406 to -208 (compared MC11 to YXB, and MC13 to MC1) revealed that region contains a negative element. Among 36 independent transformants, 33% with MC11 expressed GUS and 85% with MC13 showed GUS expression. Comparing MC7 transgenic plants to MC1 revealed that the region ?888 to ?729 is another negative regulatory element, and comparing MC11 to MC12, the proportion of expression of transgenic plants indicated the region ?729 to ?406 is a positive regulatory element.

The RCg2 promoter was identified in a search for root-specific genes to combat the rice water weevil (RWW) but expressed at low frequency (~10%). Spatial expression of RCg2 was investigated using two reporter constructs YXA (RCg2-gus-ocs) and YXB (RCg2-gus-RCg2) that included 1.6 kb of the RCg2 5' sequence fused to the ?-glucuronidase (gus) coding region. YXB plants were generated via Agrobacterium-mediated transformation but only 8 of 158 plants analyzed showed strong GUS activity despite the presence of an intact construct. Reactivation of RCg2 gene in rice was investigated by treatment of R0 and R1 of YXB transgenic plants with 5-azacytidine. Reactivation of RCg2-gus was observed in some transgenic plants indicating different mechanisms involved in the gene silencing of the YXB lines. DNA methylation analysis, northern blotting, RT-PCR and small RNA analysis supported the conclusion that PTGS and TGS are present in the silenced plants. Promoter analysis in silico and using promoter deletion assays predicted that the RCg2 promoter contains a complex region that includes miRNA homologs, MITEs and repetitive sequences. The high frequency of promoter-related silencing suggests functional interactions of these elements of the transgene and the homologous endogenous gene. To identify key elements contributing to the root-preferential expression of RCg2 and the high frequency of silencing observed in transgenic (YXB) lines, several RCg2 promoter deletion constructs were designed. These include 5' deletions MC1, MC2, MC4, MC7 and MC8 and internal deletions MC5, MC11, MC12 and MC13. The frequency with which silencing was encountered in populations of the deletion mutants was used to characterize the effects of various promoter elements. Deletion of the region from -406 to -208 (compared MC11 to YXB, and MC13 to MC1) revealed that region contains a negative element. Among 36 independent transformants, 33% with MC11 expressed GUS and 85% with MC13 showed GUS expression. Comparing MC7 transgenic plants to MC1 revealed that the region ?888 to ?729 is another negative regulatory element, and comparing MC11 to MC12, the proportion of expression of transgenic plants indicated the region ?729 to ?406 is a positive regulatory element.

iv ABSTRACT GENETIC TRANSFORMATION OF LENTIL ( Lens culinaris M. cv.Sultan.1) WITH A TRANSCRIPTION FACTOR REGULATOR (MBF1c) AND ANALYSIS OF TRANSGENIC PLANTS KAM&Ccedil / I, Hamdi Ph.D., Biotechnology, Institute of Natural ad Applied Sciences Supervisor Prof. Dr. Meral Y&Uuml / CEL Co-Supervisor: Dr. Ufuk &Ccedil / elikkol AK&Ccedil / AY September 2011, 252 pages In this study, Agrobacterium mediated genetic transformation of lentil Sultan 1 cultivar with MBF1c and evaluation of transgenic plants was aimed. The study was initially based on optimized protocol with Agrobacterium tumefaciens KYRT1 strain and pTJK136 binary plasmid. Based on this protocol and transient marker gene expression in embryo apex, 15% stable transformation efficiency was aimed. However limited knowledge about pTJK136 and problem with curing KYRT1 leaded us to use Agrobacterium tumefaciens C58C1 strain and also to engineer an alternative binary plasmid / pPZP101. Hence, scope of this study became construction of a plant binary transformation vector and lentil transformation optimization with C58C1 strain.First plant transformation vector designed in this study was pPZP101ManA-MBF1c. Transformations with C58C1::pPZP101ManA-MBF1c were carried out with a reformulated co-cultivation media. Cotyledonary nodes were isolated from three days old lentil seedlings germinated with phytormone (BAP/TDZ) induction. Isolated nodes were either injured and pre-incubated in co-cultivation media or pre- incubated and then injured prior to transformation. Regeneration and necrosis behaviors of the transformed explants leaded us to the conclusion that explant preparation is the critical step of transformation. And data suggest that explants isolated from 2mg/l BAP, pre-incubated two days in co-cultivation media, injured and transformed performed significantly better scores for necrosis shoot regeneration and callus formation parameters. Transformed explants that survived in subsequent sub-cultures in mannose selection raised shoots. These shoots were grafted and regenerated into plantlets. The putative transgenic plantlets were screened for transgene with PCR. Initial amplification signals fainted and lost as grafts grew. In order to make a diagnosis of this fainting behavior the second plant transformation vector pPZP101ManA- GUSint-MBF1c was constructed and transient GUS expression analysis were made.

ABSTRACT GENETIC TRANSFORMATION OF LENTIL ( Lens culinaris M. cv.Sultan.1) WITH A TRANSCRIPTION FACTOR REGULATOR (MBF1c) AND ANALYSIS OF TRANSGENIC PLANTS KAM&Ccedil / I, Hamdi Ph.D., Biotechnology, Institute of Natural ad Applied Sciences Supervisor Prof. Dr. Meral Y&Uuml / CEL Co-Supervisor : Dr. Ufuk &Ccedil / elikkol AK&Ccedil / AY September 2011, 252 pages In this study, Agrobacterium mediated genetic transformation of lentil Sultan 1 cultivar with MBF1c and evaluation of transgenic plants was aimed. The study was initially based on optimized protocol with Agrobacterium tumefaciens KYRT1 strain and pTJK136 binary plasmid. Based on this protocol and transient marker gene expression in embryo apex, 15% stable transformation efficiency was aimed. However limited knowledge about pTJK136 and problem with curing KYRT1 leaded us to use Agrobacterium tumefaciens C58C1 strain and also to engineer an alternative binary plasmid / pPZP101. Hence, scope of this study became construction of a plant binary transformation vector and lentil transformation optimization with C58C1 strain. First plant transformation vector designed in this study was pPZP101ManA-MBF1c. Transformations with C58C1::pPZP101ManA-MBF1c were carried out with a reformulated co-cultivation media. Cotyledonary nodes were isolated from three days old lentil seedlings germinated with phytormone (BAP/TDZ) induction. Isolated nodes were either injured and pre-incubated in co-cultivation media or pre-incubated and then injured prior to transformation. Regeneration and necrosis behaviors of the transformed explants leaded us to the conclusion that explant preparation is the critical step of transformation. And data suggest that explants isolated from 2mg/l BAP, pre-incubated two days in co-cultivation media, injured and transformed performed significantly better scores for necrosis shoot regeneration and callus formation parameters. Transformed explants that survived in subsequent sub-cultures in mannose selection raised shoots. These shoots were grafted and regenerated into plantlets. The putative transgenic plantlets were screened for transgene with PCR. Initial amplification signals fainted and lost as grafts grew. In order to make a diagnosis of this fainting behavior the second plant transformation vector pPZP101ManA-GUSint-MBF1c was constructed and transient GUS expression analysis were made.

Human protein C (HPC) is a vitamin-K dependent plasma glycoprotein which is one of the major components regulating anticoagulation. HPC injection is a promising therapy for several diseases but a heterologous production system would be preferred over purifying HPC from human plasma because of its low concentration (4-5 $ mu$g/ml). A cDNA clone coding for HPC was inserted downstream of the CaMV 35S promoter and of a dimer of the CaMV 35S promoter. Tobacco plants were transformed using Agrobacterium and a binary vector strategy. Kanamycin resistant plants were regenerated and enzyme linked immunosorbent assay determined that HPC, in crude plant extracts, accounted for up to 0.03% of plant soluble proteins. HPC was found to be expressed by R$ sb1$ seedlings suggesting successful integration of the T-DNA into plant genome. A partial protein purification system was developed in order to enrich the protein mixture for HPC. HPC was found to bind tightly at pH 6.0 to Fast Flow Q Sepharose resin.

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.

In this study, optimization of a transformation and regeneration system via indirect organogenesis in cotyledonary petiole tissue of lentil (Lens culinaris Medik.) was investigated. Eight different medium types differing in their plant growth regulator compositions were employed to examine the callus induction potency of cotyledonary petiole. Except two, all other tested medium yielded more than 80% callus induction. Nine different medium types were studied to test the potencies of callus structures for shoot induction. Only the callus induced in medium H (1 mg/L Zeatin riboside + 1 mg/L Naphthalane acetic acid) yielded shoots at 8 to 40 % frequency. The most responsive medium was MS basal medium with no growth regulators. Also five and three different medium types were employed to examine callus induction potency of epicotyl tissues respectively. Each medium type yielded 90% callus induction. Only the callus induced in medium H yielded shoots At 6 to 26% frequency. Preliminary studies were carried out for somatic embryogenesis in cotyledonary petiole. Effects of salicylic acid on somatic embryogenesis were also investigated. Salicylic acid at 200&micro / M was found to enhance the percentage of somatic embryos by 25 % and reduce the necrosis 24 %. However none of the globular and heart shape embryos were able to regenerate. Transient GUS expression efficiencies of roots, shoot tips, and cotyledonary petioles were tested after Agrobacterium-mediated transformation. Transformation frequencies were 26, 74, and 38 % for cotyledonary petiole, shoot tips, and roots respectively.

Thesis (Ph.D.)--University of Melbourne, School of Forest and Ecosystem Science, 2006. / Typescript. Includes bibliographical references (leaves 184-216).