In this thesis, the potential of barley anther culture as a method for the rapid and efficient production of homozygous lines has been investigated. The substitution of sucrose by maltose in the anther culture medium led to a substantial improvement in the frequency of green plant regeneration. High concentrations (6-12%) of maltose gave greater number of green plants than low (1-3%) concentrations. In addition, plant regeneration was predominantly via an embryogenic route rather than an intermediate callus phase. Three commercially important spring barley cultivars, Tweed, Tyne and Natasha were considered. All three responded on a maltose-based medium, although to differing extents. The genetic stability of anther culture-derived lines produced by this method was assessed by karyotype analysis, and by using a range of biochemical and molecular markers. Low levels of variation were detected and the relative stability of the system may be attributed to the embryogenic mode of regeneration attained on a medium containing maltose. Despite the improvements obtained using the maltose protocol, response to anther culture was shown to be largely dependent on genotype. An investigation of the transmission of anther culture responsiveness into F1 hybrids produced from <i>H.vulgare</i> x <i>H.spontaneum</i> crosses showed that responsiveness was dominant to non-responsiveness. A high degree of heterosis for anther culture response was also observed in F1 progenies. An investigation of anther culture response in the wheat/barley disomic chromosome addition lines showed that addition of a single pair of barley chromosomes could not positively modify anther culture response in an unresponsive wheat background. The implications of these results are discussed in relation to the application of anther culture technology to barley breeding and genetics.

Understanding the genetic basis of quantitative traits represents a huge goal in modern molecular and evolutionary biology. Here, the natural genetics of the genus <i>Antirrhinum, </i>within which separate species can be successfully interbred, are used to investigate differences in a range of morphological characteristics. The two species used in the study, <i>Antirrhinum majus </i>and <i>Antirrhinum molle,</i> have become adapted to very diverse environments and consequently exhibit large variance in a wide range of traits. A large-scale FI mutant screen, from a cross between a transposon-active <i>A. majus </i>line and <i>A. molle,</i> isolated segregating mutations for flower size, flower colour, trichome density and branching in self-pollinated F2 populations. Amplified Fragment Length Polymorphism analysis of the F2 and the use of molecular maps have shown the mutations generally correspond to known Quantitative Trait Loci, and the roles of genes linked to these regions are discussed. The technique sheds some light on the molecular and evolutionary mechanisms underpinning diversity in <i>Antirrhinum </i>and has implications for the use of transposon-tagging in locating QTL in other plant systems.

Significant differences were observed among 79 geographically diverse <i>Arabidopsis </i>accessions in response to the wheat powdery mildew pathogen, <i>Blumeria graminis </i>f.sp. <i>tritici </i>(<i>Bgt</i>) and the wheat leaf rust pathogen <i>Puccinia triticina </i>(<i>Ptr</i>). In response to <i>Bgt </i>genotypes classified into two major classes based on the degree of compatibility, Wc-1 an accession from Germany expressed significantly high frequency of penetration. Interestingly, in response to <i>Ptr</i>, a high frequency of guard cell death and sub-stomata vesicle formation (SVF) was observed on Wa-1, an accession from Poland. Attempted <i>Ptr </i>infection induced the production of reaction oxygen intermediates (ROI), nitric oxide, salicylic acid (SA) and camalexin. The expression of SA, jasmonic acid and ROI-dependent genes were also detected. Multiple small-to-medium effect quantitative trait loci (QTL) were identified that govern the expression of NMR in <i>Arabidopsis </i>against <i>Ptr.</i> In response to <i>Bgt,</i> a leaf collapse phenotype was observed in L<i>er</i> when it was pre-treated with Cytochalasin E, an inhibitor of actin microfilament polymerization. Whereas, Col did not express a similar phenotype. This reaction showed a complicated genetic basis with the involvement of several genes. Our genetic analysis revealed two major QTLs on chromosomes one and three with the existence of episatsis effects. A role for <i>ASYMMETRIC LEAVES1 (AS1) </i>in plant immunity has recently been identified. My experiments showed a conserved regulatory function for <i>NSPHAN</i>, an orthologue of <i>ASI </i>gene in <i>Nicotiana sylvestris </i>when challenged with host and nonhost pathogens. This regulatory gene action remained consistent when the <i>as1 </i>mutant was coupled with key Arabidopsis defence related mutants.

In plants and animals, cell identity is maintained by epigenetic functions that prevent changes in cell type specific transcription programs, for example, in the control of homeotic gene expression patterns that specify regional fate during development. Central to the cell memory machinery are the Polycomb-Group (Pc-G) genes. The <i>Arabidopsis </i>(Pc-G) genes <i>FERTILISATION-INDEPENDENT ENDOSPERM (FIE), FERTILISATION INDEPENDENT SEED 2 (FIS2) </i>and <i>MEDEA (MEA) </i>are required for ovule and seed development. These three genes confer similar mutant phenotypes in both unfertilized embryo sacs and developing seeds; that is, fertilisation-independent endosperm proliferation and the abortion of fertilised seeds that carry a maternally inherited mutant <i>mea, fis2</i> or <i>fie</i> allele. I have shown that MEA and FIE, and MEA and FIS2 interact in yeast-two-hybrid, and that the interaction of MEA and FIE is mediated by the amino-terminal region of MEA, whilst interaction of MEA and FIS2 requires the FIS2 C-terminal VEFS box. Furthermore, <i>FIE</i> and <i>MEA</i> RNA expression over-laps <i>in vivo</i> and the proteins co-localise within the nucleus <i>in planta. </i>I conclude that the similarity of the <i>fie, fis2</i>, and <i>mea</i> phenotypes reflects the interaction of their gene products as part of a multiprotein complex. FIE, MEA and FIS2 do not possess specific DNA binding activity, and it remains unclear how the FIE complex is directed to its target genes. Yeast-two-hybrid assays were used to identify a novel FIE interacting protein: RAP-1, a member of the basic helix loop helix (bHLH) family of transcription factors. Molecular and genetic studies were used to characterise its interaction with FIE. <i>FIE</i> is expressed in both embryo and endosperm, but predominantly affects endosperm development. In order to establish if <i>FIE</i> is needed in both, I have used a strategy for targeted expression of <i>FIE</i> to the embryo and endosperm regions in a <i>fie</i> background, and observed a partial rescue of <i>fie.</i>

In <i>Antirrhinum majus </i>(Scrophulariaceae), floral dorso-ventral asymmetry (monosymmetry) is controlled by two genes belonging to the TCP gene family, <i>CYCLOIDEA </i>and <i>DICHOTOMA. </i>My<i> </i>project was to investigate if putative orthologs of <i>CYC </i>and <i>DICH </i>have a conserved role in <i>Schizanthus wisotonensis </i>(Solanaceae), a species also bearing monosymmetrical flowers. To do so, TCP genes from <i>S. wisotonensis </i>were isolated and their expression pattern was characterised in both a wild type and a mutant plant with decreased dorso-ventral asymmetry. In the first chapter of results, a thorough morphological description of floral development in <i>S. wisotonensis </i>was carried out using the support of Scanning Electron Microscopy and quantitative analysis. The development of wild type <i>S. wisotonensis </i>was then compared to that of the mutant. In the second chapter of results, the cloning of 6 TCP genes in <i>S. wisotonensis is </i>reported <i>(SCHCYCI-6). To </i>identify the putative orthologs of <i>CYC, </i>a phylogenetic analysis was carried out using sequence information from other angiosperm TCP genes. This study showed that <i>SCHCYCl, 2 </i>and <i>3 </i>are the most likely candidates to control dorso-ventral asymmetry in <i>S. wisotonensis.</i> Finally, in the third chapter of results, the study of expression patterns obtained for <i>SCHCYCI-3 </i>using RNA <i>in situ </i>hybridisation in both the wild type and the mutant is reported. The results obtained from the morphological description and the study of gene expression patterns suggest that (i) the establishment of dorso-ventral asymmetry in <i>S. wisotonensis </i>follows a different dynamic to that in <i>A. majus (ii) SCHCYCI </i>may have a function related to that of <i>CYCLOIDEA </i>and <i>DICHOTOMA </i>but unlike for <i>A. majus, </i>it is likely to be restricted to the androecium (iii) <i>CYC</i>-like genes (i.e. <i>SCHCYC2-3) </i>may have a function in the development of the inflorescence where they are expressed in overlapping domains.

The mechanisms regulating cell-layer organisation in developing plant organs are fundamental to plant growth, but remain largely un-investigated. In order to understand the signalling pathways potentially involved in this process, the receptor kinase-encoding <i>Arabidopsis Crinkly 4 (ACR4</i>) gene was studied. <i>ACR4</i> expression is restricted to the L1/outside cell-layer of most meristems and organ primordial, including those of the ovule integuments. Mutant analysis shows that <i>ACR4</i> is required for regulation of cellular organisation during the development of sepal margins an ovule integument outgrowth. <i>ACR4</i> encodes a protein that in ovules, and possibly other tissues, is abundant in anticlinal and the inner periclinal plasma membrane of “outside” cells. It is proposed that ACR4 may be involved in maintaining L1 cell-layer integrity in aerial organs by receiving and transmitting signals from neighbouring L1 cells and/or from underlying cell layers. In order to discover additional components involved in this process a mutagenesis screen based on the <i>acr4</i> line was carried out. Potential enhancers of the <i>acr4</i> phenotype were identified and selected for future analysis. In order to further investigate the molecular mechanism of ACR4 function a comprehensive functional dissection of the ACR4 protein was carried out, based on the ability of deletion derivatives to complement in mutant phenotype. This has permitted identification of functionally important domains of ACR4 and the formulation of a functional model for ACR4 as a partially redundant component of a developmentally crucial signalling pathway involved in the maintenance of L1-layer integrity throughout <i>Arabidopsis </i>development.

This project was a collaboration between groups within the <i>Arabidopsis</i> research community and the crop-based research community through which the best resources of both could be utilised to study aspects of resistance to biotrophic pathogens. An objective was to work towards transferring information between models and crop plant. Activation tagged Arabidopsis lines were screened for disease susceptibility to <i>Pseudomonas</i> <i>syringae</i> pv <i>tomato</i> (DC3000). Two mutant lines, named activated disease susceptibility (ads), were confirmed to be susceptible and candidate gene(s) affected by the activation tag were identified. On the other hand, functional analysis of genes in crop species, which currently lack sequenced genomes, relies on post-transcriptional gene silencing (PTGS) approaches that require sequence information prior to experimentation. Suppression subtractive hybridisation isolated a cathepsin B ESTs from P. infestans challenged potato undergoing resistance gene­-mediated hypersensitive response (HR). N. benthamiana is mode for studying the HR, is a close relative of potato and therefore an established virus-induced gene silencing system was utilised to investigate the role of cathepsin B. Silencing of cathepsin B resulted in suppression of the <i>Erwinia</i> <i>amylovora</i>-induced HR which was confirmed using mammalian cathepsin B inhibitors. A final aim was the development of a PTGS system in <i>Solanum tuberosum</i> to allow functional investigation for current and future defence-related genes identified from models and crop plant species. A PVX-based VIGS system was successfully developed for cultivated, tetraploid potato cultivars and for wild diploid Solanum species.

The mechanisms by which genetic mutation translates to morphological variation between species are poorly understood, but there is increasing support for the involvement of homeobox genes in generating morphological novelty. Homologues of a <i>knotted</i>-like homeobox gene, that is involved in determining meristem identity in model plants, were isolated from different species of <i>Streptocarpus </i>which show marked morphological variation, and these were named <i>Sknox1 </i>genes. Crosses were made between two species with different form, which suggested that their differences were determined by two loci, consistent with previous reports. To test whether <i>Sknox1</i> was involved in generating morphological differences between species of <i>Streptocarpus</i>, its expression was analysed in three species with different growth form. This showed conventional expression in a species with caulescent form, but novel patterns of expression in that in species with one-leafed or rosette-like form. Differences in expression of <i>Sknox1</i> therefore correlate with interspecific differences in form. Genetic analysis demonstrated that <i>Sknox1</i> did not directly cause morphological differences between species. <i>Sknox1</i> introns showed substantial variation between species. Intron sequences showed evidence of concerned evolution and sometimes contained a repetitive element that may have arisen by gene conversion.

The genetic control of floral symmetry in the Leguminosae and the genetic basis for the apparent reversal to radial symmetry in <i>Cadia </i>were investigated using a candidate gene approach. In the model organism <i>Antirrhinum majus </i>(snapdragon, Lamiales), two paralogous genes <i>CYCLOIDEA (CYC)</i> and <i>DICHOTOMA (DICH)</i> determine dorsal (adaxial) floral identity and play a crucial role in the establishment of zygomorphy. The orthologue of <i>CYCIDICH </i>in <i>Arabidopsis thaliana TCP1 </i>also has adaxial expression in the early stages of floral development. <i>CYC</i>-like genes may therefore be good candidates for the control of dorso-ventral floral symmetry in lineages outside of <i>Antirrhinum</i>. Using a phylogenetic approach, homologous of <i>CYCITCP1 </i>were identified in legume taxa from the major clades of the Papilionoideae, as well as from subfamilies Caesalpinioideae and Mimosoideae. LEGCYC genes have duplicated prior to the evolution of the Papilionoideae and form three main groups (LEGCYC1A, LEGCYC1B and LEGCYC2). Within these major gene groups, the precise relationships of paralogues between species from the main clades of the Papilionoideae was difficult to determine because of the rapid rate of sequence evolution outside of the conserved TCP and R domains characterise of <i>CYC-</i>like genes. Nevertheless, the phylogenetic framework enabled the identified of orthologous gene pairs in the radially symmetrical papilionoid taxa <i>Cadia purpurea </i>and in a closely related species, <i>Lupinus nanus</i>, with typical zygomorphic flowers. LEGCYC1A and LEGCYC1B expression in <i>L. nanus </i>was restricted to the adaxial part of the floral meristem and was maintained throughout flower development. This pattern is very similar to <i>Antirrhinum CYC</i> and suggests these genes are important for the development of bilateral symmetry in legumes.

No description available.