The literature relating to ultrastructure studies of angiosperm embryos up to seed maturity is reviewed. Ultrastructural changes in the cotyledon of the embryo of Capsella bursa-pastoris from the torpedo stage of embryogenesis to the mature seed are described. A revised classification of the developmental stages of the embryo is devised which recognises 11 stages, of which the final 8 stages are examined in the present study: the earlier 3 stages have already been studied by others. Particular reference is made to the development of the chloroplast. The chloroplasts increase in number, size, starch content and lamellar complexity as the embryo develops up to the mature-green stage, after which starch disappears abruptly. The deterioration and disruption of chloroplast lamellae begin after mature-green stage and are completed in the ivory-white and dry cotyledons. Some organelles (Golgi bodies, endoplasmic reticulum and nucleus) are recognizable in the dry cotyledon while other organelles are poorly defined or missing. The cotyledon develops storage tissue with numerous oil bodies and protein bodies in each cell. A single type of protein grain (aleurone grain) is formed within vacuoles. Oil bodies begin to appear from torpedo stage onward and reach a maximum in the ivory-white cotyledon. Ribosomes are dispersed freely in the cytoplasm. Literature on the effect of light and darkness in chloroplast morphogenesis in higher plants is reviewed. Development of the etioplast is studied in the embryos and leaves of whole plants subjected to darkness. Etioplasts of the embryo in the ovule contain large crystalline prolamellar body after 3 days dark treatment. Plastids of leaves subjected to darkness do not form prolamellar bodies but instead an extensive development of very variable, irregularly-shaped, stroma lamellae with characteristic ring- or cup-shaped or looped lamellae. The extensive development of the abnormal lamellar system with such peculiar arrangement is hitherto unknown in the plastids of normal higher plants. The transformation of the etioplast into chloroplast under illumination is investigated utilising embryo culture techniques as it was not practicable to observe such development in embryos from whole plants. The problem of excessive accumulation of starch in plastids was encountered but solved by reducing the sucrose concentration in the growth medium to 0.5% from the 2% used by others. A 1% sucrose concentration in the growth medium gives overall maximum growth of the embryo. After 4 days in darkness the etioplasts of cultured embryos contain well-formed crystalline and concentric prolamellar bodies. Treatment with 3 hours of continuous illumination with white light of low intensity completes the transformation of etioplast into chloroplast. Preliminary studies on the effect of light of different wavelengths on the etioplast transformation are also presented. Etioplasts in cultured embryos show typical protuberances of the jacket which are regarded as peripheral reticulum. The possibility of the formation of mitochondria from such protuberances is noted. Some novel structures developed in plastids are reported. Two types of tubules, distinct from the tubules forming prolarnellar bodies, are produced in the embryo. They appear as rows of small circular tubules and a group of long parallel tubules. The production of tubules, possibly the result of a reaction to the changed environmental conditions, are regarded as a sign of degeneration rather than a normal stage of development. Another novel structure reported in the etioplasts consists of a complex network of intermingled lamellae. It is concluded that the use of embryo culture technique in relation to experimental morphogenesis of organelles offers a promising field of study which will contribute also to our knowledge of embryo physiology and development.

The work described in this thesis is in the main an attempt to throw additional light on a very controversial aspect of sieve tube structure, namely the condition of the functioning sieve plate pores. The avoidance of surge artifact has been attempted by employing two main approaches, the second quite novel. In the first very minute seedlings have been fixed whole. The ultrastructure of their sieve tubes has been investigated and compared with that of maturer plants of the same species. In the second, seedlings, some minute, have been subjected to neutron irradiation to the point of flaccidity before being conventionally fixed for electron microscopy. The results of all these investigations have confirmed the usual picture of pores occluded with P-protein. As a contribution to the understanding of the function of P-protein several species of primitive vessel-less angiosperms have also been investigated. These prove to have typical P-protein distributed as usual in the sieve tube lumens and pores. This material must therefore be understood in the context of transport in the woody plant since the herbaceous habit is recognised as secondary and P-protein only appears historically with the angiosperms.

This study examines the potential for increasing natural L-DOPA drug biosynthesis in Mucuna pruriens by silencing or “knocking down” expression of putative DOPA/tyrosine decarboxylase (Mp-ty/ddc) in situ. Mp-ty/ddc codes for DOPA/tyrosine decarboxylase (Mp-TY/DDC) which converts L-DOPA to dopamine in plants. The hypothesis of the work was that silencing the Mp-ty/ddc gene would result in accumulation of L-DOPA in the plant tissues. This work involved isolation and characterisation of 1.73 kb putative full-length ORF of Mp-ty/ddc. The gene showed 74% homology with TY/DDC protein alignments of other plants in the same taxa, although no enzyme activity was detected when the gene product was heterologously expressed. In addition, a protocol was developed for Agrobacterium mediated transformation of M. pruriens so as to be able to manipulate expression of the DOPA genes in situ. The cotyledonary nodal and hypocotyl tip explants regenerated shoots on M.S media supplemented with 50 μM BA, 0.5 μM NAA and 50 mg l-1 kanamycin selection also the nptII transgene was detected by PCR. The Agrobacteria strains GV3101 harbouring a pGREEN vector and carrying an Mp-ty/ddc antisense were used for the plant transformation experiments. Further work showed that the Mp-ty/ddc gene copy number was 1, the gene expression was highest in roots and stems, followed by seeds and was very low in leaves. On the other hand, L-DOPA-content in seeds was 17-fold higher relative to leaves and 15 fold relative to stems and roots.

A study was conducted to investigate the effects of varying levels of ozone (0, 1.5, 2.5, 3.5 or 5.0 ppm) for 96 h on 1. the in vitro and in vivo growth of Colletotrichum gloeosporioides, the causal organism of anthracnose; 2. the reactive oxygen species generation and spore mitochondria of C. gloeosporioides using transmission electron microscope, fluorescence microscope and laser scanning confocal microscope; 3. the production of defence-related enzymes in papaya; 4. microbiological analysis on ozone-treated and non-treated papaya; 5. the biochemical, physiological, gas exchange and sensory characteristics of papaya fruit during storage (25 ± 3 °C, 70 ± 5 %RH) for 14 days; 6. the changes in total phenols, total carotenoids and antioxidant activity; and 7. gene expression of ozone-fumigated papaya fruit. Data were analyzed using analysis of variance and differences among treatment means were separated by Duncan Multiple Range Test (DMRT). The results of antifungal studies showed that mycelial growth of C. gloeosporioides was reduced significantly (p < 0.05) at all concentrations compared to the control. The maximum inhibition in mycelium growth (41.2 %) was obtained at 5.0 ppm ozone. Similarly, conidial germination inhibition was 100 % for 5 ppm ozone. In vivo analysis revealed that 2.5 ppm ozone was the optimal concentration for controlling anthracnose disease incidence (72.5 %) and disease severity after 10 days of storage, showing that a moderate concentration of ozone is effective in the reduction of C. gloeosporioides in artificially inoculated papaya fruit without affecting the quality aspect of the fruit. The results of scanning electron microscopy (SEM) also confirmed that ozone fumigated fungus at levels above 3.5 ppm deformed and disintegrated spore and mycelia structure. Further to that, transmission electron microscopy (TEM) illustrated that the mitochondria of ozonized fungus was disintegrated and had ruptured membrane. In spores treated with 3.5 ppm ozone, mitochondrial cristae were distorted, whereas the mitochondria were almost completely degraded in spores treated with 5.0 ppm. Meanwhile, the results from microscopy studies using laser scanning confocal microscope and fluorescence microscope showed that ozone treatment caused production of reactive oxygen species (ROS) in mitochondria of C. gloeosporioides. With increased concentration of ozone, higher levels of ROS were induced in the spores. Besides its direct antifungal activity, the study strongly suggested that ozone induces a series of defense reactions through production of compounds such as total phenols, polyphenol oxidase (PPO), peroxidase (POD) and phenylalanine ammonia-lyase (PAL) in ozone-fumigated papaya. Likewise, content of ascorbic acid, β-carotene, lycopene and antioxidant activity of papaya increased as fruit ripened and was further enhanced by exposure to ozone for 96 hours from day 4 until day 8. Twenty-four hours of ozone treatment at the level of 0.5, 2, 3.5 and 5.8 ppm reduced the total mesophilic microorganism counts of fruit with initial values of 4.48 to 2.18 log cfug-1. In addition, no coliform bacteria were initiated after 24 hours at all levels of ozone exposure. In addition, 2.5 ppm ozone treated fruit showed maximum beneficial effects in reducing weight loss, maintaining firmness, reduced rate of respiration, delaying changes in peel colour and containing the highest soluble solids concentration (SSC) as compared to the control. The titratable acidity declined throughout the storage period with slower rate in ozone-fumigated fruits. Overall sensory assessment of quality after ripening showed fruit were significantly better in quality when fumigated with 2.5 ppm ozone which were assigned highest sensory score in terms of appearance, sweetness, pulp colour, texture, aroma and overall acceptability than the control. The discovery of the gene expression of papaya in defense response induced by ozone fumigation has further clarified the understanding on how specific gene involved in controlling its expression when the plant changes during stress or in any plant lifecycle event. Among those genes, some involved in ethylene biosynthesis, generation of reactive oxygen species and stress responses of plant defense were found (mitochondrion, chloroplast, heat shock proteins, polygalacturonase-inhibiting protein, hydroxyproline-rich glycoprotein, ethylene responsive factor and acyl-CoA oxidase). Thus, the findings from all the experiments carried out during this study showed that 2.5 ppm ozone reduced anthracnose incidence and extended the storage life for up to 12 days while maintaining acceptable quality of papaya fruit. Ozone exposure at 1.5 ppm resulted in poorer quality fruit as compared to 2.5 ppm ozone treated fruit. Higher concentration of ozone exposure at 3.5 ppm and 5 ppm ozone seems non-physiological and caused phytotoxic effect on the quality of papaya fruit. As a non-toxic, biodegradable product, eco-friendly and safe sanitizer, ozone has the potential to become a natural preservative for prolonging the shelf life and retaining quality of papaya by combating fungal disease, particularly fungus C. gloeosporioides, thus promoting the marketability of the crop and minimizing postharvest losses in the papaya industry.

There is a need to breed for root systems architectures (RSAs) that optimise soil resource acquisition. This requires high resolution and high-throughput quantification of RSA in as natural an environment as possible. Current imaging techniques are limited by cost, reproducibility, throughput and complexity. This thesis describes (1) the construction of a low cost, high-resolution, root phenotyping platform that requires no sophisticated equipment which is adaptable to most laboratory and glasshouse environments and (2) its application to quantify environmental and temporal variation in RSA between genotypes of Brassica rapa L. The high resolution root phenotyping system (HRP) that was constructed employed 24 scanners and could screen up to 72 individual plants at any time, with the possibility of capturing thousands of root images daily depending on the operational number of scanners and scanning periodicity. Plants were supplied with a complete nutrient solution through the wick of a germination paper. Images of RSA were acquired automatically, over extended periods, using multiple scanners controlled by customised software. The RSA data was used to validate a mechanistic model and mixed effects models were used to describe the sources of variation in traits contributing to RSA. Plants were also grown in rhizoboxes and under varying concentrations of P ([P]ext). Broad-sense heritability (H2), was highest (≥ 0.70) for shoot biomass, length of primary roots (PRs), number of lateral roots (LRs). Coefficients of variation in RSA traits within a genotype were large and ranged between 5 and 103%. It was found that between 4 and 48 replicates were needed to detect a significant difference (95% CI, 50% difference between trait means). Significant differences were found between genotypes in root traits with strong positive correlations among RSA traits and between biomass and RSA traits. Principal component analyses identified 5 significant axes of variation, accounting for approximately 86 and 78% of the variation in the genotypes on paper and soil substrates, respectively. Cluster analysis of the genotypes produced 5 discrete groups. Genotypes with more or less shoot biomass or with bigger or smaller RSA could be distinguished. A density-based 2D model reproduced experimental results accurately by simulating PR length and total length of LRs. Mixed-effects statistical models demonstrated that root traits show temporal variations of various types with significant effects of genotype. All genotypes followed a similar growth pattern with time, but differed in their maximum total root length (TRL), primary root length (PRL) and LR growth. A 3-parameter logistic model satisfactorily described TRL and PRL when genotypes were grown on both paper and soil substrates. On paper substrate, TRL required only a single, random-effect parameter (asymptote), describing maximum TRL. On soil substrate, TRL required two random-effects parameters, asymptote and inflection, describing maximum TRL and time at which ½ of maximum TRL occurs, respectively. Primary root length on both paper and soil substrates required only a single, random-effect parameter, describing maximum PRL. The growth rate of LRs of all ages followed a quadratic function and required only a single, random-effect parameter, describing maximum growth rate. There was variation in specific RSA traits and plasticity in response to [P]ext among genotypes. Length of the apical un-branched zone of the PR increased with increasing [P]ext. Total root length, total LR length and number of LRs was positively correlated with total plant tissue P concentration at low [P]ext but not at high [P]ext. Paper substrate was more suitable for screening seedling root traits but root phenotypes must be validated in situ in the field or in soil media because some differences were evident between data observed on paper and soil substrates. Scanner-based phenotyping of RSA provides economical means of studying the mechanisms underlying the plant-soil interactions and can be used to quantify environmental and temporal variation in traits contributing to RSA. The HRP system can be extended to screen the large populations required for breeding for efficient resource acquisition. The necessity for high replication and time-consuming image analysis could however limit throughput in the phenotyping system.

The present work was taken up by me at the suggestion of Professor Sir William Wright Smith and has been done under the guidance and supervision of himself and Dr. P.H.Davis. Taxonomic studies have undergone considerable changes during the last three quarters of a century since the publication of the great Flora Orientalis of Boissier (I867) and the Flora of British India of Hooker f. (1872). Many nomenclatorial changes have been made and the taxonomic status of several species has been clarified in addition to numerous taxa added to our knowledge. Both the above mentioned Floras, therefore, need a complete revision - a fact emphasized by several other taxonomists before Blatter, E. (1930); Chakravarty, H.L. (1948).

UV-B radiation is an integral component of natural sunlight reaching the Earth’s surface. Although being a potentially harmful and damaging agent, UV-B is a key environmental signal for plants initiating diverse responses that affect their metabolism, development and viability. The majority of these responses involve the differential regulation of gene expression and all require accurate perception of the effective light quality by a photoreceptor. The recent identification of UV RESISTANCE LOCUS8 (UVR8) as a UV B photoreceptor has been an important milestone in plant UV B research (Rizzini et al., 2011; Christie et al., 2012; Wu et al., 2012). The aim of this study was to investigate how the structure of the UVR8 protein determines its function in the UV-B response in Arabidopsis.

A detailed understanding of the mechanisms by which plants sense and respond to major environmental stress factors will significantly contribute towards the prospects of developing crops capable of yielding well over a wider geographical range, including marginalised lands. One of the important stress response mechanisms in eukaryotes is mediated through phosphorylation of the eIF2α-subunit (serine 51/56) by specific kinases, namely double stranded RNA activated protein kinase (PKR), General Control Non-repressible 2 protein kinase (GCN2), Pancreatic eIF2α kinase (PERK) and Heme-regulated inhibitor protein kinase (HRI). This mechanism is a highly conserved phenomenon in eukaryotes and occurs in response to various stress conditions. Unlike in yeast and mammals, the mechanism is however not well established in higher plants, although its components such as eIF2α and GCN2 kinase have been identified in plants. The objective of the study reported herein was therefore to elucidate this mechanism in Arabidopsis, a model plant species. Initially the presence of yeast GCN2 kinase (ScGCN2), human PKR (HsPKR), human HRI (HsHRI) and human PERK (HsPERK) kinase homologues in Arabidopsis and Viridiplantae (green plants and algae) was evaluated through homology and phylogenetic analysis using TAIR10 and NCBI protein sequences, respectively. Arabidopsis lacked homologues of HsPKR, HsHRI and HsPERK however the presence of ScGCN2 homologue, herein referred to as AtGCN2 (Arabidopsis GCN2 kinase), was confirmed. Further evaluation of translation control mechanism through phosphorylation of AteIF2α (Arabidopsis eukaryotic initiation factor 2 α-sub-unit) was conducted using Atgcn2-1 null mutant plants (plants expressing a copy of truncated non-functional AtGCN2 kinase). Unlike WT Col-0, the Atgcn2-1 seedlings failed to induce phosphorylation of AteIF2α after exposure to amino acid starvation (150 µM glyphosate), NaCl (50 and 100 mM), heat (37oC) and cold (4oC) acclimation. On the other hand no strong phenotype of Atgcn2-1 was observed under optimal growth conditions and NaCl stress, except seedlings had relatively shorter roots compared with WT Col-0 seedlings. Failure of Atgcn2-1 seedlings to induce phosphorylation of AteIF2α Ser 56, after exposure to various stress confirmed that Arabidopsis possesses only one GCN2 kinase, as is in the case of yeast, and unlike mammalian systems. Further characterisation was conducted by exposing WT Col-0, Atgcn2-1, jar-1 and NahG seedling to biotic stress; Cauliflower Mosaic Virus (CaMV) and Pseudomonas syringae DC3000 (P. syringae) and positive control treatment using 150 µM glyphosate. The jar-1 and NahG seedlings are mutants defective in jasmonate and salicylic pathways, respectively. Inoculation with CaMV and P. syringae failed to induce phosphorylation of AteIF2α, unlike glyphosate. These results suggested that activation of the AtGCN2 kinase may be independent of jasmonate and salicylic pathways. Due to lack of a strong Atgcn2-1 phenotype, two mutants expressing AtGCN2 under the control of a 35S promoter, namely p35S:AtGCN2 and p35S:GFP:AtGCN2 were generated for further characterisation and localisation of AtGCN2 kinase, respectively. For characterization experiments the p35S:AtGCN2 seedlings were subjected to salinity stress, osmotic stress and temperature shock. In localisation experiments, GFP activities were assessed in non-stressed 7-day old p35S:GFP:AtGCN2 seedlings. During characterisation, higher germination rates were generally obtained with p35S:AtGCN2 and Atgcn2-1 compared with WT Col-0 seeds on ½ MS media containing NaCl, KCl and mannitol. On media infused with PEG6000 however p35S:AtGCN2 had the lowest germination rates. There were also no strong p35S:AtGCN2 phenotypes observed, except for increased root growth compared with WT Col-0 and Atgcn2-1 seedlings. In contrast, Atgcn2-1 seedlings subjected to PEG6000 osmotic stress had the highest increase in root growth compared with both WT Col-0 and p35S:AtGCN2 seedlings. On the other hand localisation of the GFP:AtGCN2 fusion protein was observed in the root and shoot tip tissues of p35S:GFP:AtGCN2 seedlings. The results obtained with Atgcn2-1 and p35S:AtGCN2 seedlings suggested that mutation of Atgcn2 produced root phenotypes. There were no significant differences in the survival of all the three genotypes when seedlings were subjected to heat shock stress. In cold shock experiments however Atgcn2-1 survival was significantly (p<0.05) lower than that of WT Col-0 and p35S:AtGCN2 seedlings, thus suggesting that null mutation of Atgcn2 increased susceptibility of seedlings to cold shock. The homologues of the yeast General Control Non-repressible 4 (ScGCN4) and human Activating Transcriptional Factor 4 (HsATF4), that are activated, when yeast and mammalian eIF2α is phosphorylated, respectively are yet to be identified in plants. To identify putative Arabidopsis ScGCN4 and HsATF4 homologues both in vitro and in silico approaches were explored. In vitro translation experiments using Wheat Germ Lysate (WG) mimicking plant translation under stress (WGeIF2α-P) and non-stress (WGeIF2α) conditions were conducted. To mimic stress conditions mPKR kinase was added into the translation reaction and significantly inhibited protein synthesis compared to control treatment. However, due to technical difficulties it was not possible to identify all translated transcripts under stress conditions (WGeIF2α-P) thereby identifying potential Arabidopsis ScGCN4 and HsATF4 homologues. This prompted the use of in silico tools to identify putative Arabidopsis homologues of ScGCN4 and HsATF4 using the FivePrime Viewer programme (Webb, 2008). A total of 99 TAIR10 transcripts with 5′ upstream Open Reading Frames (uORFs) were identified and only two transcripts, AT4G31590.1 and AT1G58120.1, were identified as putative homologues of ScGCN4 and non for HsATF4. The AT4G31590.1 and AT1G58120.1 transcripts encode for proteins involved in cellulose synthase/ gylcosyl transferase and methyl transferase activities, respectively. Although these genes are involved in key plant growth and developmental activities, there is need to assess translation control of their main open reading frame (mORF) by uORFs through phosphorylation of AteIF2α. Overall the data presented in this study suggest that stress response translation regulation mechanism mediated by phosphorylation of eIF2α is present in Arabidopsis. Plants are known, however, to carry out unique biological processes such as photosynthesis and cellulose biosynthesis that other eukaryotes lack. It would therefore not be surprising for them to have translation regulation mechanisms like other eukaryotes but with unique differences.

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