No description available.

No description available.

no abstract provided by author / Master of Science

Tip growth is the primary form of growth in hyphal organisms and some plant cells. Tip growth in hyphae is highly dependent on F-actin, which acts to regulate and support growth. One of the models suggested for tip growth, the amebae model of tip growth, suggests that F-actin may also be the primary source of protrusive force for tip growth in some conditions, and that proteins with a similar function to animal integrins would be present an involved in tip growth (Heath and Steinberg 1999). In this thesis we examine the role of F-actin in the growth of the oomycete Phytophthora cinnamomi and the effects on growth of the F-actin disrupting compound Latrunculin B. We demonstrate that F-actin plays a critical role in the tip growth of Phytophthora cinnamomi with it's disruption causing rapid cessation in directional growth, followed by significant subapical swelling. Further more we examine Phytophthora cinnamomi for the presence of an B4 integrin like protein that has been previously reported in the oomycete Achlya bisexualis (Chitcholtan & Garrill 2005) and show that the B4 integrin like protein is not present in Phytophthora cinnamomi. These experiments help further our understanding of tip growth in Phytophthora cinnamomi an economically important plant pathogen.

Genetic diversity of cultivated species and their wild relatives, as well as of wild species encompasses plant genetic resources or germplasm, the ex situ preservation of which embodies a critical aspect of biological conservation. While seed storage affords an efficient ex situ conservation method, recalcitrant seeds are intolerant of desiccation and cannot be stored conventionally in seed banks. Seeds of the three indigenous tree species investigated in this study, viz. Trichilia emetica, T. dregeana and Protorhus longifolia are recalcitrant, with the species considered to be endangered. Cryopreservation, which involves storage at ultra-low temperatures of selected tissue(s) from which plants are subsequently able to be generated, is currently the only method available for long-term ex situ conservation of recalcitrant-seeded species and affords significant potential for the future. Many protocols that have been applied for the cryopreservation of the germplasm of recalcitrant zygotic embryonic axes excised from seeds of tropical/sub-tropical species have resulted in survival post-cryo which has been recorded only as root development or callus formation, with shoot formation seldom occurring. Successful cryostorage of genetic resources cannot be achieved until post-cryopreservation recovery facilitates normal seedling development, i.e. the formation of both a fully functional root and a shoot. Cryopreservation requires the utilisation of the smallest explant possible (greatest surface area to volume ratio), the most suitable for recalcitrant seeds in general being the zygotic embryonic axis. Based on preliminary studies it was demonstrated that shoot production by axes is inhibited in association with a burst of reactive oxygen species (ROS), produced in response to wounding upon excision of the axis from the cotyledons, when these are attached close to the shoot apical meristem. It was postulated that a combination of the oxidative burst at the site of excision coupled with inadequate antioxidant machinery within the recalcitrant axis tissue, precludes shoot production. It was further considered highly probable that each subsequent stressful manipulation throughout the cryopreservation process would be accompanied by a surge of uncontrolled oxidative activity within the tissue, in response to the stress. Therefore, the primary aim of the study was to investigate the underlying causes of failure of shoot production after procedures associated with cryopreservation and to focus on ways to ameliorate the consequences of unbalanced oxidative metabolism. Additionally, studies were carried out to optimise each step of the cryopreservation procedure, viz. cryoprotection, dehydration, rehydration and cooling, and subsequent recovery, in conjunction with assessment of oxidative responses, ultimately to achieve successful cryopreservation of the embryonic axes of these species. The experimental work conducted to achieve this aim assessed changes in various biomarkers of injury, those selected for this study being three ROS, viz. superoxide, the hydroxyl radical and hydrogen peroxide, after axes were exposed to various pre-treatments, cryopreservation and recovery. Concomitantly, the elicited responses of endogenous antioxidant systems accompanying these steps were assessed. Changes in the levels of ROS and antioxidant activity were determined using various biochemical assays, and these parametres, together with assessment of shoot development, were investigated after each step of the cryopreservation process. The effect of stress on oxidative metabolism was tested after exposure to pre-treatments with and without the provision of various antioxidants, viz. DMSO, ascorbic acid and cathodic water, so as to determine the efficacy of selected ROS scavengers and, in general, to develop the best protocol for cryopreservation of embryonic axes of the three species. Significant results, in terms of shoot development and regulated ROS generation, were obtained after three major processes of the cryopreservation procedure. The production of roots and shoots by excised axes of T. emetica, T. dregeana and P. longifolia after excision (75%, 80% and 75%, respectively), and by 40% of excised axes of T. dregeana after each of the two further stages, cryoprotection and desiccation, were major achievements towards cryopreservation of the recalcitrant germplasm. The modulation of ROS by ascorbic acid and cathodic protection significantly improved survival of axes of both Trichilia species. In its entirety, the present study made significant advancements towards cryopreservation of recalcitrant germplasm and also towards understanding oxidative events associated with cryogenic processing and exposure to cryogenic conditions. This study concludes that unregulated metabolism is one of the underlying causes of failure of recalcitrant germplasm represented by zygotic axes, to survive cryopreservation. The application of antioxidants and cathodic protection during cryopreservation facilitated survival that has been previously unattainable. The outcomes of this study provide an informative platform for further optimising cryopreservation procedures for the germplasm of the species investigated, and extending the work to other recalcitrant-seeded species, especially those of tropical/sub-tropical provenances. / Thesis (M.Sc.)-University of KwaZulu-Natal, Westville, 2012.

Philosophiae Doctor - PhD / Plants are continuously exposed to varying conditions in their environment, to which they have to adapt by manipulating various cellular processes. Environmental (abiotic) and pathogen (biotic) stress are challenges against which plants have to defend themselves. Many plant responses to stress stimuli are a result of cellular processes that can be divided into three sequential steps; namely signal perception, signal transduction m1d execution of a response. Stress signal perception is, in most of these cases, facilitated by cell surface or intracellular receptors that act to recognize molecules presented to the cell. In several cases, hormones are synthesized in response to stress signals and in turn these hormones are perceived by cellular receptors that trigger signal transduction cascades. Propagation of signal transduction cascades is a complex process that results from activation of various signaling molecules within the cell. Second messengers like calcium (Ca2+) and guanosine 3', 5'-cyclic monophosphate (cGMP) play a vital role in mediating many signal transduction processes. The result of these signal transduction cascades is, in most instances, expression of genes that contribute to the plant's ability to cope with the challenges presented to it. Plant natriuretic peptides (PNPs) are novel plant hormones that regulate water and salt homeostasis via cGMP-dependent signaling pathways that involve deployment of Ca2+. The aim of this study is to partially characterize a PNP and a guanylyl cyclase, both from Arabidopsis thaliana. Guanylyl cyclases synthesize cGMP from the hydrolysis of guanosine 5' -triphosphate (GTP) in the cell. The study also aims to investigate the effect of drought and salinity on cGMP levels in plants, using sorbitol to mimic the osmolarity/dehydration effect of drought and NaCl as a source of salinity stress and thus link NaCl and sorbitol responses to both AtPNP-A and cGMP up-regulation.

Thesis (Ph. D.)--University of Missouri-Columbia, 1999. / Typescript. Vita. Includes bibliographical references (leaves 99-102). Also available on the Internet.

Thèse--Paris.

No description available.

Achieving long-term storage of germplasm is critical for the conservation of plant biodiversity. Seed storage practices require that degradative reactions causing ageing be limited. By reducing the water content, cytoplasmic viscosity is increased to levels that minimise deteriorative reactions. Reducing the storage temperature additionally increases the storage lifespan by further reducing the rate at which such deleterious processes occur. Two broad categories of seeds can be distinguished based on their storage behaviour. Orthodox seeds are desiccation-tolerant; generally shed in the dry state and are metabolically quiescent. Such seeds are usually stored at low water contents (e.g. 5%), and their high cytoplasmic viscosity prevents freezing damage during cooling to subzero temperatures. On the other hand, desiccation-sensitive (recalcitrant) seeds do not undergo a maturation-drying phase, they are metabolically active at shedding, and sensitive to extreme or prolonged drying. Accordingly, recalcitrant seeds cannot be stored under conventional conditions because they do not survive drying to low water contents and are damaged by sub-zero temperatures, even when dried to the lowest water content tolerated. Therefore, procedures that facilitate harmless drying and cooling to low temperatures are required to achieve long-term storage of recalcitrant germplasm. Recalcitrant seeds that are dried rapidly can attain relatively lower water contents without injury. However, these seeds are usually large and this limits the drying rates that can be achieved even under favourable conditions. Isolating embryonic axes from the rest of the seed facilitates faster drying, and a consequent reduction in the water content at which damage occurs. In axes of many species, the level of drying attained before lethal desiccation damage occurs is sufficient to limit freeZing damage during cryogenic exposure and facilitate survival in vitro. However, many others are damaged when dried to water contents that preclude freezing, and also are killed if cooled to sub-zero temperatures at higher water contents. In such instances, the window of permissible water contents leading to survival may be small or nonexistent. A basic premise explored in this thesis is that by restricting the growth of intracellular ice crystals using increasingly rapid cooling rates, the range of permissible water contents can be widened, facilitating survival of axes at higher water contents. An overview of the problems associated with the long-term storage of recalcitrant germplasm, and the rationale behind such rapid cooling approach are presented in Chapter 1 of the present thesis. Subsequent chapters report investigations on the effects of variables required to dry and cryopreserve embryonic axes with minimum damage, in keeping with this approach. Collectively, those studies aimed at establishing a robust cryopreservation procedure for the conservation of recalcitrant germplasm with broad applicability across species. The approach presently adopted entailed manipulating the water content of excised axes using rapid drying to discrete water content ranges, and also using different methods to cool axes to cryogenic temperatures at various rates. The calorimetric properties of water in axes were investigated for Camellia sinensis (L.) O. Kuntze using differential scanning calorimetry (DSC). For all species, the effect of any drying or cooling treatment tested was determined by assessing the survival of axes in vitro, which provided the most reliable indicator of cellular damage. Additionally, the effects of different treatments upon the structural and functional integrity of axes were assessed using light and electron microscopy as well as measurement of electrolyte leakage. The studies undertaken are presented in a similar sequence to that in which they took place during the course of the experimental phase of this work. These are summarised below. Partial drying plays a pivotal role in the approach developed, and microscopy has contributed towards increasing present understanding of desiccation damage. Microscopy was used to determine the effects of drying rate upon the ultrastructure of recalcitrant axes. It was necessary to find reliable protocols to prepare specimens for light and electron microscopy that did not alter the architecture of the cells in the dry state. Freeze-substitution and conventional aqueous fixation were compared in Chapter 2 using variously dried material from three species. The results obtained revealed that an unacceptably high extent of artefactual rehydration occurs during aqueous fixation, and highlight the need for anhydrous processing of dehydrated samples. Significantly, that study also revealed that many cellular events commonly associated with desiccation damage (e.g. withdrawal, tearing and/or vesiculation of the plasmalemma) are not seen in freeze-substituted preparations, and are likely artefacts of aqueous fixation. Freeze-substitution was used subsequently (Chapter 3) to assess the effects of slow drying (2 - 3 days) or rapid drying (min) upon the survival of embryonic axes of jackfruit (Artocarpus heterophyllus Lamk.) Results confirmed the beneficial effects of rapid drying, and also provided insights into ultrastructural changes and probable causes underlying cellular damage that occur during a drying/rehydration cycle. Efforts subsequently focused on determining the effect of cooling rate upon survival of recalcitrant axes at various water contents. The study on embryonic axes of recalcitrant camellia sinensis (tea; Chapter 4) tested the hypothesis that rapid cooling facilitates survival of axes at higher water content by restricting the growth of ice crystals to within harmless dimensions. The presence of sharp peaks in DSC melting thermograms was indicative of decreased survival in vitro. These peaks were attributed to the melting of ice crystals sufficiently large to be detected by DSC as well as to cause lethal damage to axes. Increasing the cooling rate from 10°C min-1 to that attained by forcibly plunging naked axes into sub-cooled nitrogen increased the upper limit of water content facilitating survival in vitro from c. 0.4 to 1.1 - 1.6 g H20 g-1 (dry mass [dmb]). Subsequent studies tested whether a similar trend occurred in other recalcitrant species cooled under similar conditions. In order to investigate further the relationship between water content, cooling rate and survival it was necessary to achieve cooling rates reproducibly, and to quantify these reliably. The plunging device required to achieve rapid cooling, and instruments required to measure the cooling rates attained, are described in Chapter 5. That study investigated the effects of cryogen type, depth of plunge and plunging velocity on the cooling rates measured by thermocouples either bare or within tissues of similar size and water content as encountered in cryopreservation experiments. This plunger was used in subsequent studies to achieve the fastest cooling conditions tested. Favourable cooling conditions were selected, and the efficacy of this procedure to cryopreserve relatively large axes was tested (Chapter 6) using embryonic axes of horse chestnut (Aesculus hippocastanum L.) Axes at water contents above c. 0.75 g g-1 could not be cooled faster than c. 60°C S-1, but cooling rates of axes below this water content increased markedly with isopentane, and to a lesser extent with subcooled nitrogen. Axes were killed when cooled at water contents above 1.0 g g-1 but survived fully (albeit abnormally) when cooled in isopentane between 1.0 and 0.75 g g-1. Complete survival and increasingly normal development was attained at water contents below 0.75 g g-1, especially if isopentane was used. The study on horse chestnut axes emphasised that water content and cooling rate are co-dependent during non-equilibrium cooling. Accordingly, that study could not determine whether survival at lower water contents increased because of the corresponding increase in cooling rates measured, or because of the higher cytoplasmic viscosity that resulted from drying. That uncertainty was addressed by the study discussed in Chapter 7, using axes of the trifoliate orange (Poncirus trifoliata [L.] RAF.) That study investigated the effect of cytoplasmic viscosity upon survival of axes cooled and warmed at different rates. Survival and normal development was high at lower water contents, and seemingly independent of cooling rate at about 0.26 g g-1. At higher water contents the range of cooling rates facilitating survival became narrower and displaced towards higher cooling rates. This study revealed two conspicuous inconsistencies that questioned the beneficial effect of rapid cooling. Firstly, the fastest cooling rates did not necessarily facilitate the highest survival. Secondly, survival of fully hydrated axes was higher when cooled under conditions that encouraged - rather than restricted - the growth of intracellular ice crystals. These inconsistencies were explored further using embryonic axes of silver maple (Acer saccharinum L.). Freeze-fracture replicas and freeze-substitution techniques provided valuable insights into the way in which ice crystals were distributed in cells cooled using different methods at rates ranging between 3.3 and 97°C S-1. Extensive intracellular freezing was common to all treatments. Unexpectedly, fully hydrated axes not only survived cryogenic exposure, but many axes developed normally when cooled using the relatively slower methods (77 and 3.3°C S-1) if warming was rapid. The most conspicuous ultrastructural difference between plunge cooling and the relatively slower methods was the exclusion of ice from many intracellular compartments in the latter. It is possible that even the fastest warming cannot prevent serious cellular damage if ice crystals form within such 'critical' compartments. It is proposed that the intracellular location of ice is a stronger determinant of survival that the size attained by ice crystals. The study of A. saccharinum also investigated the recovery of axes cooled fully hydrated either rapidly (97°C S-1) or slowly (3.3°C S-1). This facet of the study showed that cell lysis became apparent immediately after warming only where damage was most extensive. In other cells damage became apparent only after 2.5 to 6 h had elapsed, thus cautioning against inferring survival from the ultrastructural appearance of cells immediately after warming. Microscopy enabled cell repair as well as the pattern of growth of cryopreserved tissues to be appraised at the cellular, tissue and organ levels. Similar studies are required to understand further the nature of freezing damage, and how those events affect cell function. The salient trends observed in previous chapters are brought together in Chapter 9. / Thesis (Ph.D.)-University of Natal, Durban, 2002.