Gene flow amongst AA genome rice species Oryza sativa (cultivated rice) and 0. rufipogon and 0. nivara (wild relatives) is known to occur in rice agro-ecosystems in many Asian countries. Gene flow leads to the formation of hybrid plants that may coexist with parental species. These hybrids (especially 'weedy rice'- plants that are very similar to cultivars but possessing undesirable traits) are a threat to rice production as they may reduce cultivated rice yields, both qualitatively and quantitatively. The precise origins of 'weedy' rice (WR) genotypes are unknown but two main hypotheses have been proposed: endoferality (by segregation and selection of weedy traits from existing heterozygous cultivars in areas where no wild rice exists) and exoferality via gene flow between cultivated and wild rice species. With a continuous growing world population especially in Asia, food demand has to be satisfied with an increase in rice production, playing a key role.

Particle bombardment of immature scutella was used to generate multiple lines of transgenic wheat with either PHY A, PHYB or PHYC from Arabidopsis or PHY A from oat.;Responses of segregating transgenic seedlings to cFR light were used as a screen for biologically active lines. The 'short coleoptile' phenotype under cFR was shown to segregate with the oat PHYA transgene and rtPCR detection of transcript. All of the transgenic lines containing the Arabidopsis PHYA and several oat PHYA lines demonstrated phenotypes indistinct to wild-type, possibly demonstrating transcriptional silencing.;Biologically active lines with Mendelian segregation ratios of 3:1 were selected for further analysis and homozygous individuals for each identified by PCR. Southern analysis of selected lines demonstrated that all originated from a distinct transformation event and harboured at least a single intact copy of the transgene. The complexity of gene integration was related to observed gene dosage and silencing effects.;Adult transgenic plants under white light with high R:FR ratio, demonstrated an increased chlorophyll content, a reduced production of superfluous tillers, a reduction in sensitivity to shortened day-length and an increased developmental rate leading to a reduction in days to heading.;The transgenic lines demonstrated an altered response to shade. In addition to the phenotypes observed under white light, plant height was significantly reduced and harvest index increased compared to the nulls, these responses were distinct to the those observed under white light and suggest an altered response to reduced R:FR.;The results suggest that oat PHYA is biologically active in transgenic wheat and that over-expressing PHYA alters the responses of etiolated seedlings to cFR, the light grown morphology and development of adult plants, and the responses of the plants to shade.

Mobile DNA sequences - transposable elements (TEs) that amplify and move within genomes represent a high proportion of the DNA in most eukaryotes. The present study aimed to define TE nature, structure and abundance in two contrasting groups of diploid and polyploids crop genera, Brassica (dicotyledon) and Musa (monocotyledon). Rather than starting with known TE sequences, a sequence-data driven approach was used, comparing homologous and homoeologous BAC pairs. Over ~100 kb regions, any stretch of sequence was characterized that was inserted or deleted in the evolutionary time since divergence of the two BAC genomic sequences. Almost all the sequences were indeed TEs, representatives of existing and a few novel superfamilies. Polymorphisms due to activity were measured by PCR with flanking primers in 40 (Brassica) or 96 (Musa) accessions, and some families were localized on chromosomes by fluorescent in situ hybridization. Autonomous and non-autonomous TEs were found; class I retrotransposons like Copia and Gypsy (LTR) predominated in both genera, while SINEs and LINEs (Non-LTR) were abundant in Brassica genomes. Large retrotransposon derivatives (LARDs) were in both genera, with a very few terminal-repeat in miniature (TRIM) elements. Class II DNA transposons included CACTA, hAT, Harbinger, Mariner and Mutator like MITEs in Brassica, while CACTA and Mutator were uncommon in Musa. Among miniature inverted-repeat transposable elements (MITEs), Stowaway, Tourist, and Mutator-like MITEs were abundant with several novel families identified and characterized. In diploid and allopolyploid Brassica species, A- or C-genome specific elements were found while others were more active. PCR enabled accession identification and phylogenetic reconstruction in Brassica and Musa. As well as known element families, few novel types of TEs were identified, including several variable, short elements with characteristic structural features. The analysis provides insight into the nature and diversity of TEs as an important genomic component; results are useful for genome annotation and understanding evolution and variation within these crops and the associated pool of wild germplasm.

Transformation of the chloroplast genome is a technique that allows for the production of recombinant proteins in plants. Chloroplast transformation allows the very high expression of a transgene while limiting the gene flow to other species. In addition, multiple genes can be transferred in one transformation event and no gene silencing as been shown so far. Despite these advantages, little work has been directed at assessing the commercial feasibility of using the chloroplast as a means of expressing high-value proteins. In this thesis, a new expression system was developed to allow the very high expression of transgenes in the chloroplast under contained conditions. Tobacco plants were transformed with a tobacco chloroplast vector expressing green fluorescent \ protein. Cell suspensions were induced from these leaves. Using a bioreactor, I was able to demonstrate that coupling temporary immersion with a hormonal shift triggered the rapid production of plant tissue whilst retaining a high level of expression of green fluorescent protein. Another aspect of this thesis was to assess the potential of several fusion tags to improve the solubility and purification of various target proteins in transformed tobacco chloroplasts. The main proteins studied were a mannanase from coffee, which is involved in the detergent, pulp and, more recently, the bioethanol industry, as well as alpha defensin 1 peptide, which has potential therapeutic value in the treatment of several diseases such as HIV and Herpes. N- and C-terminal constructions were created with oleosin, dehydrin, fibrillin, maltose-binding protein and glutathione-S-transferase as tags. Constructs were first evaluated in Escherichia coli before being integrated into the tobacco plastome. Apart from oleo sin, all fusion proteins were successfully expressed in transplastomic tobacco. My work has identified dehydrin, GST and MBP as promising affinity-tags to be used in chloroplast transformation experiments. Finally, I describe the development of experimental tools and procedures for the transformation of the chloroplast genome of coffee, which is one of the world's major cash crops. For this, coffee-specific vectors were created and direct somatic embryogenesis employed to propagate transformed tissue.

It has been shown that under high light (HL) conditions, Arabidopsis bundle sheath cell (BSC) , chloroplasts accumulate hydrogen peroxide (H202) as part of a retrograde signalling network associated with the induction of HL-responsive ASCORBATE PEROXIDASE2 (APX2). It was postulated that this network is mediated by both the "core" abscisic acid (ABA) signalling pathway and G protein signalling and involves the heterotrimeric Ga subunit (GP AI), type 2C protein phosphatases (PP2Cs), SNFI-related kinases 2 (SnRK2s) and respiratory burst oxidase homo logs (RBOHs). The main objective of this study was to determine to what extent these two pathways were implicated in the regulation of APX2 and establish whether the same pathway regulates the expression of other HL-responsive genes in Arabidopsis. Finally, the project looked at identifying if GPAI and SnRK2.6 were integrated into the same HL, ABA-directed signalling pathway. Screening of null mutants under HL showed that GP Al was responsible for the negative regulation of APX2 and extracellular H202 together with ABI2. It was predicted that RBOH F acts upstream of RBOH D where it may interact with SnRK2.6 and in turn regulates ROS production from RBOH D. Screening of the F3 population from the cross between ost1-1 and gpa1-4 under HL indicated that both GPAI and OSTI do not feature on the same signalling pathway. A microarray analysis of HL- responsive gene overlaps between PP2C and SnRK2 mutants revealed that only a proportion of genes regulated by ABA and HL were regulated by this pathway. The promoters of these genes contained both heat shock elements (HSEs) and ABA-responsive elements (ABREs). This highlighted the fact that only a few genes conformed to the same signalling that occurs for APX2. In summary, ABA-directed signalling may control three separate pathways that are regulated by HSFs, ABFs/ AREBS and genes that respond indirectly to HL via other regulatory pathways.

No description available.

To investigate the role of extracytoplasmic function sigma factors in the Rhizobium-legume symbiosis, the genome sequence of Rhizobium leguminosarum bivoar viciae 3841 was analysed using bioinformatics approaches. In total, twenty sigma factors were identified, including sixteen extracytoplasmic function sigma factors. Twelve of the sixteen ECF sigma factors appeared to be co-transcribed with a regulatory gene. The role of the ECF sigma factors in responding to legume produced compounds was investigated by growing strain 3841 containing various ecf reporter plasmids with pea root exudate. Three ECF sigma factors showed different expression patterns when grown with root exudate compared to minimal medium. One of the three sigma factor aenes. ecfR, which was induced by pea root exudates, was investigated further and was shown to be expressed in pea infection threads. Over-expression of ecfR was lethal to R. leguminosarum 3841, but co-expression with the adjacent gene, asfR, prevented the lethal effect.

No description available.

No description available.

No description available.