Soil salinity negatively affects agricultural production in Turkey by decreasing the yield and quality. Direct introduction of stress related genes by genetic engineering is one of the most rapid approaches to develop stress tolerant crops. In this study, TaNHX1 gene was isolated from bread wheat and three different local wheat cultivars were transformed with overexpression vectors containing TaNHX1 gene by using Agrobacterium-mediated and particle bombardment gene transfer techniques. Immature embryo and inflorescence of Triticum durum cv. Kiziltan-91 and Triticum aestivum cv. Y&uuml / regir-89 and mature embryo of Triticum durum cv. Mirzabey-2000 were used as an explant. In this manner, totally 8960 and 5650 explants were used during particle bombardment and Agrobacterium-mediated transformation, respectively. Moreover, leaves of Nicotiana tabacum cv. Petit Havana were transformed by TaSTR gene to develop salt resistant transgenic tobacco plants by using Agrobacterium-mediated transformation. Stable expression and inheritance of the transgenes was confirmed by both genetic and molecular analyses. T1 progeny showed segregation of the transgenes in a typical Mendelian fashion in most of the plants. Expression of TaSTRG in tobacco was evaluated by physiological and biochemical analysis, such as germination test, root length and MDA analysis. In addition to the nuclear transformation, chloroplast transformation of tobacco was performed with Xyl10B gene responsible for the synthesis of hyperthermostable xylanase enzyme. Stable integration of transgenes and homoplasmy were confirmed with PCR and Southern blotting.

Abstract Transcription factors (TFs) play an indispensable role in cell biology as they are responsible for regulating gene activity. TFs act by binding to specific DNA sequences in a gene promoter resulting in either activation or repression of transcription of the target gene. TFs interact with target genes through a DNA binding domain which is often highly conserved and can be used to classify TFs into families. Dof (DNA binding with one finger) TFs are classified by the presence of a highly conserved bi-functional Dof domain. Characterisation of various Dof proteins has identified essential regulatory roles in plant-specific processes. This includes roles in carbon metabolism in maize, seed development and germination in Arabidopsis and cereal crops and circadian responses in Arabidopsis and rice. Despite the important role of Dof proteins in plant growth and development, this family has been studied to a limited extent in wheat with only two Dof proteins reported to date. Therefore, the aim of this thesis is to identify and initiate characterisation of the Dof TF family in wheat (Triticum aestivum) and investigate the potential role of members of this family in wheat productivity-associated physiological processes. Thirty-one Dof genes were identified after extensive searching of available Triticum aestivum ESTs and contig assembly. Phylogenetic analysis grouped these 31 genes into four clades. Extensive gene expression profiling of the TaDof family was undertaken and revealed that the majority of TaDof members were constitutively expressed in major vegetative organs with a few displaying a grain-predominant expression pattern. The TaDof family appears to be enriched with light-responsive or drought down-regulated members, suggesting that the role of this family is predominantly in growth-related processes. To further investigate their role in growth-related physiological processes, two Dof genes, TaDof4 and TaDof5, were selected for more detailed characterisation. TaDof5 was identified to be similar to the Cycling Dof Factors from Arabidopsis and the recently identified rice Dof daily fluctuations genes which are involved in the photoperiodic regulation of flowering time. TaDof5 gene expression was diurnally regulated, had strong expression in the stem and head and a peak in expression level at anthesis. Using publicly available Affymetrix data, correlation analysis suggested co-expression of TaDof5 with a number of circadian-regulated genes associated with flowering. Extensive analysis of the DNA-binding specificity of TaDof5 revealed a preferred binding motif of 5’GAAAAAGTGC. The binding of TaDof5 to DNA requires two (A/T)AAAG(T/C) core motifs in adjacent positions. The binding sequence of TaDof5 was identified in the promoter of one of the TaDof5 co-expressed genes in wheat and subsequent analysis showed that TaDof5 was capable of binding to this promoter region with high affinity. These data suggest that TaDof5 may be involved in photoperiod responses associated with flowering time. TaDof4 was among a large number of previously identified growth-related genes expressed at significantly higher levels in wheat cultivars and progeny lines with high transpiration efficiency (TE) (the amount of biomass produced per unit of water transpired). Field trials were undertaken with the parents and progeny from a cross between Quarrion (high TE) and Genaro 81 (low TE) and demonstrated that the high TE progeny lines had improved early vegetative growth. TaDof4 was therefore characterised further for a role related to biomass production and/or contribution to the TE trait. Expression profiling showed that TaDof4 was consistently expressed at higher levels in the lines with high TE, constitutively expressed in major vegetative organs, drought down-regulated and sucrose up-regulated. Over-expression of TaDof4 identified one line with significantly improved biomass. DNA binding specificity analysis demonstrated that TaDof4 binds to the AAAG(T/C) core target motif essential for Dof DNA binding. These results suggest that TaDof4 is potentially associated with growth-related processes in wheat. In conclusion this study has made the following achievements: (1) identified 31 TaDof family members in Triticum aestivum, (2) used phylogenetic analysis and expression profiling to infer potential functional roles for wheat TaDof family members, (3) further characterised TaDof5 to reveal a potential role in photoperiod responses associated with flowering and elucidated its DNA-binding specificity, (4) further characterised TaDof4 to suggest a potential role in growth-related processes. These results provide fundamental molecular information that increases our understanding of the diverse biological roles of the TaDof family, particularly in growth-related physiological processes in wheat.

Doctor of Philosophy / Plant Pathology / John P. Fellers / Harold N. Trick / Wheat streak mosaic virus (WSMV) and Triticum mosaic virus (TriMV) are two viruses affecting wheat in the Great Plains region of the United States. Genetic resistance is severely limited, requiring management methods focusing on the deployment of resistant varieties and various cultural practices. Evaluation of resistance is complicated by the lack of a standard rating scale. The objective of this work was to develop new avenues to mitigate these challenges. A standardized virus symptom rating scale was developed using historical Kansas rating scales, and validated using multiple wheat populations. Two independent RNA interference (RNAi) expression vectors targeting portions of viral coat protein (CP) of WSMV and TriMV were previously transformed into wheat. T₂ plants and beyond were evaluated using PCR, reverse transcription-PCR and bioassays in which plants were challenged with their respective virus. These lines were evaluated for resistance through the T₆ generation. Crosses were made with the susceptible winter wheat cultivars, ‘Overley’ and ‘Karl 92.’ Real-time PCR results show viral titer was up to 20-fold lower in the T₆ transgenic lines, the F₁, and the BC₁F₁ compared to control plants. This provides evidence that this RNAi silencing method is stable in wheat over multiple generations. WSMV and TriMV use host eukaryotic initiation factors (eIF) in order to facilitate replication of their genomes. Previously created RNAi expression vectors were derived from the sequences of the wheat genes eIF(iso)4E-2 and eIF4G. Evaluation of these lines began in the T₁ generation. Resistance has been demonstrated in three lines of eIF(iso)4E-2 and four lines of eIF4G, derived by single seed descent. T₆ progeny co-infected with WSMV and TriMV continue to be resistant. Crosses have been performed with the winter wheat ‘Karl 92’ and three Kansas elite lines, KS030887K-6, KS09H19-2-3, and KS10HW78-1-1. RNAi construct effectiveness was evaluated using real-time PCR. Results show up to 18-fold reduction in viral titer in the transgenic lines, the F₁, and the BC₁F₁ in comparison to control plants. This research provides the first evidence that a single host transgene can provide resistance to multiple viruses and has great potential benefits to both breeders and producers.