Due to the increasing prevalence and severity of Al phytoxicity in certain regions of the South African sugar industry, a research programme has been initiated at SASEX to elucidate the molecular mechanisms by which sugarcane detects and responds to the metal. As part of this larger investigation, the current study aimed to assess the response of a reportedly Al tolerant cultivar, Saccharum spp. hybrid cv. N12, to phytotoxic levels of Al. Hydroponically-grown plants of this commercial genotype were used in Al inhibition studies, the results of which indicated that exposure of plants to 250µM Al for 24 hours resulted in maximum reduction of root elongation. Under these conditions, root growth was inhibited by approximately 36%, compared with only 4% for the 50µM Al treatment. Subsequently, this exposure regime was used to gather the terminal 5 to 10mm of root tips, the site of the primary Al lesion, of challenged and control, unchallenged plants for molecular analysis. Total RNA was extracted from the Al challenged and control root tips, from which mRNA was subsequently isolated, reverse transcribed and converted to double-stranded cDNA. The two populations of cDNA were reciprocally subtracted from each other and used to construct subtractive cDNA libraries in Lambda ZAP®II phages. Randomly selected clones, 576 representatives from each of the libraries, were screened using membrane-based array technology. Results indicated that only 33% (190) of the Al-treatment specific library cDNAs were found to be more highly expressed under conditions of Al stress than under control conditions. Of these potentially Al response-related cDNAs, 25 were sequenced and submitted to sequence databases for the assignment of putative identities. No genic sequences known to be directly associated with the Al stress response were identified, however, several were found to be related to pathogenesis or general stress pathways. Although further Northern hybridisation work is required to validate these results, they suggest that the induction of general stress response pathways may be involved in the aluminium stress response of this sugarcane cultivar. Such Al stress-related sequences could have applications in marker-assisted breeding programmes and as candidate genes for the genetic engineering of tolerant genotypes. / Thesis (M.Sc.)-University of Natal, Durban, 2002.

Seeds remain the most convenient and successful way for storing the genetic diversity of plant species and for producing new plants routinely for agriculture and horticulture. The importance of seed storage and the ability to predict seed longevity must therefore not be underestimated. To be successful, storage conditions must maintain seed vigour and viability and ensure that normal seedlings are subsequently established under field conditions. Seed quality is best retained when deteriorative events are minimised, which is achieved by storage of low moisture-content seeds under cool to cold, or even sub-zero, temperatures. Such conditions are employed for 'orthodox' seeds, which are desiccation tolerant and able to survive at sub-zero temperatures in the dehydrated state for extended periods. It is seeds referred to as 'recalcitrant' that cannot be dehydrated and often not stored at low temperatures because they are desiccation sensitive and may not tolerate chilling. According to almost anecdotal records chilling temperatures for such seeds are those below 15°C down to 0°C, depending on the species. The limited storage lifespan of recalcitrant seeds presents a problem even for short-term storage, and as most research on chilling sensitivity has been conducted on vegetative tissue, relatively little data exist for seeds, especially recalcitrant types. The purpose of this study was to gain an understanding of the chilling response of recalcitrant seeds, as reduced temperature could have the potential to extend, rather than curtail, storage lifespan, depending on the species. Selected physiological, biochemical and ultrastructural responses of recalcitrant seeds of Avicennia marina and Ekebergia capensis were characterised. Seeds of the two species were stored at 25, 16 and 6°C. Germination, water content (determined gravimetrically), respiration (measured as CO2 production) and leachate conductivity (tissue electrolyte leakage over time) were assessed at regular intervals. Chilling response at the subcellular level was examined using transmission electron microscopy (TEM). Changes in sugar metabolism and activities of the antioxidant enzymes superoxide dismutase (SOD), catalase (CAT) and glutathione reductase (GR) were assessed for A. marina seeds, which were severely affected by the chilling temperature of 6°C, losing viability after 1 week. In contrast, the seeds of E. capensis retained viability after 12 weeks of storage at 6°C, indicating the marked difference in chilling response between seeds of the two recalcitrant species, despite their common tropical provenance. However, when E. capensis seeds were stored at 3°C viability decreased significantly after 8 weeks, thus indicating how critically temperature must be controlled if such conditions are to be profitably employed. Ultrastructural studies revealed that in both E. capensis and A. marina seeds vacuole formation was initiated more rapidly at lower temperatures than at higher temperatures, indicating that this was a response specific to the chilling stress imposed. Once again, 'lower temperatures' differed relative to the species concerned. In the E. capensis seeds, nucleolar morphology was affected and the extent of chromatin patches in the nuclei increased as the storage temperature was reduced. Other ultrastructural findings could not be linked specifically to the chilling stress imposed on the E. capensis and A. marina seeds. Activity of the antioxidant enzymes SOD and GR was detected in the A. marina seeds. No measurable CAT activity was detected. Glutathione reductase activity increased in response to chilling stress, the rate of the increase depending upon the severity of the chilling stress imposed. Other than when the A. marina seeds were placed directly at 6°C, there were no notable increases in SOD activity. Interestingly, SOD and GR activity was not the same in the axes as in the cotyledons. Superoxide dismutase activity was found to be higher in the axes and GR activity higher in the cotyledons. It would have been beneficial to determine the extent of antioxidant enzyme activity in the E. capensis seeds as well if this had been possible. Generally, chilling of recalcitrant seeds seems to evoke a response similar to that of dehydration below a critical water content. This could lead to the conclusion that recalcitrant seeds do not possess the genetic ability to cope with dehydration or chilling stress, if it were not for the existence of recalcitrant seed species that are more chilling tolerant. / Thesis (M.Sc.)-University of Natal, Durban, 2002.

Under storage conditions of ambient temperature and relative humidity in South Africa, seed-associated mycoflora proliferates. Fusarium moniliforme is ubiquitous in newly-harvested maize, persisting for variable periods in storage, while Aspergillus flavus may represent the final group of species in the succession of aspergilli after grain storage under high temperature and/or high humidity. Many strains of these fungi produce toxigenic secondary metabolites (mycotoxins) under local storage conditions. Since pathogenic fungi may be present within the tissues of stored seeds, these contaminants will not be eradicated by external fungicide treatment, therefore a possible alternative is biological control. The aim of the present investigation was to ascertain whether certain strains and/or species of Trichoderma have potential as biocontrol agents against the seed-associated pathogenic fungi, Aspergillus flavus and Fusarium moniliforme. A study of the fungal growth in dual cultures revealed that from nine isolates of Trichoderma spp. (T harzianum and T viride), four had a noticeable inhibitory effect on the growth of the pathogenic fungi. Scanning electron microscopical investigation of fungal interaction demonstrated no obvious hyphal penetration by - Trichoderma spp. In addition, significant alteration of Fusarium hyphae, with pronounced collapse and loss of turgor, and production of aberrant conidial heads and microheads by A. flavus were observed. Evidence derived from some biochemical studies revealed that antibiosis (by production of extracellular enzymes, volatile compounds and possible antibiotics) is probably the mechanism involved in the antagonistic effect of the four aggressive Trichoderma spp. The in vitro studies demonstrated that the use of Trichoderma spp. as biocontrol agents against A. flavus and F. moniliforme appears promising. / Thesis (M.Sc.)-University of Natal, 1995.

The consequences of toxigenic fungi associated with stored seed have stimulated these investigations aimed at developing treatments to minimise this mycoflora, without significantly reducing seed quality or viability. The effects of immersion in water at 55, 57 and 60 QC for durations of 5 to 60 min were assessed for maize (Zea mays L.) seed in terms of fungal status, water uptake, electrolyte leakage, germination and seedling establishment. These assessments were conducted immediately after treatment, after re-dehydration for 2 days in an ambient air stream, and following a 1 month storage period under either cold (4 QC) or ambient (25 QC) conditions (33% and 91% RH, respectively). In all cases, the results are compared with those of control seeds and seeds pre-imbibed for 4 h at ambient temperature. The level of internal contamination, represented almost entirely by Fusarium moniliforme Sheldon, declined significantly when assessed immediately after treatment, the efficacy of which increased with increasing temperature and duration of treatment. Seeds immersed in water at 55 QC for a duration of 15 min exhibited an 85% reduction in infection levels, when compared with those of the control, while those treated at 57 and 60 QC (same duration) were uninfected. Immersing seeds in hot water, however, resulted in a lag in germination rate and drop in germination totality, the degree of which was enhanced by increasing duration and temperature of treatment, suggesting the status of the manipulation to be an accelerated ageing treatment. The electrolyte leakage studies indicated that the reduced germination performance of these seeds was not due to plasmalemma disorganisation. These deleterious effects, however, were counter-balanced as seeds treated at 55, 57 and 60 QC for durations up to 60, 30 and 10 min, respectively, produced plants of superior quality than those of the control, which is ascribed to the reduction of systemically transmitted pathogens. The efficacy of the hot water treatment in reducing the levels of seed infection and improving seedling quality was enhanced by subsequent re-dehydration. The reduction in seed-associated mycoflora was maintained following storage for 1 month at both 4 QC (33% RH) and 25 QC (91% RH). However, both seed and seedling quality were adversely affected following storage even under cold, dry conditions, which may be a consequence of the pre-treatment history of the seeds, which had been cold-stored for two years prior to the experiments. Applied as a pre-sowing treatment, therefore, hot water treatment shows promise for producing a crop of superior quality, less prone to fusarial pathogenesis. This treatment may be of particular importance to Third-World subsistence communities. / Thesis (M.Sc.)-University of Natal, 1995.

In South Africa, Eucalyptus breeding programmes are aimed at the selection of fastgrowing varieties, with appropriate wood characteristics and/or resistance to pests and diseases. However, the slow growth rate, long generation time and heterozygosity of trees make this a difficult task. Such problems may be overcome by the adoption of a biotechnological approach for plant propagation and modification. Towards this end, the aims of this investigation were to establish protocols for the micropropagation of Eucalyptus grandis and for the Agrobacterium-mediated transformation and subsequent plant regeneration of this important species. The usefulness of transformed cells and/or tissues is dependent upon the availability of methods for their regeneration into plants. Consequently, methods for plant regeneration via indirect organogenesis from leaf discs and cell suspension cultures were investigated. Organogenic calli were produced from leaf explants on MS medium with 16 mg.1-1 &l:em•calcltrate, 20 g.I-I sucrose, 1mg.I-I NAA and 0.05 mg.1--<1 BA. Shoots were induced on MS medium containing 1 mg.1-1 ZEA and 0.2 mg.1-1 IAA, and subsequently rooted on medium containing MS nutrients supplemented with 1 mg.1- 1 IAA. Cell suspension cultures were established but not regenerated via indirect organogenesis. Additionally, various media were investigated in order to obtain somatic embryos from cell suspension cultures. The MS media supplemented with 30 g.1-1 sucrose, 12 mg.1- 1 ABA and/or 40 g.1-1 PEG were found to be most suitable, resulting in the production of embryoids; germination results are not available at this stage. In order to establish methods for the transformation of both leaf discs and cell suspension cultures of Eucalyptus, a triparental mating was performed between Escherichia coli pnT119 (donor), A. tumefaciens LBA4404 (recipient), and E. coli HBI0l::pRK2013 (helper), resulting in the transconjugant LBA4404 (pnT119); insertion of the pJIT119 plasmid was demonstrated using agarose gel electrophoresis. The transconjugant CS8C1 (pMP90) (pJIT119) was also used. Protocols for the transformation of both leaf discs and cell suspension cultures were established, and resulted in the production of putatively transformed calli which were GUS positive and with stood selection on kanamycin (50 Ilg.mr1) and/or sulfadiazine (50 Ilg.mr1). Also, Southern blotting analysis indicated that the gene transfer process was successful. Due to difficulties in the regeneration of plants from transformed calli transgenic plants were not obtained. Future research strategies and applications of the developed protocols to Eucalyptus breeding programmes are discussed. / Thesis (M.Sc.)-University of Natal, 1994.

The herbal medicine trade is thriving in KwaZulu Natal with an ever-increasing number of people harvesting and trading in indigenous plants, especially those species with medicinal and/or magical properties. The number of plants harvested has increased whereas the size of the plants collected has decreased, resulting in low recruitment into wild populations. As a result of these two factors, species diversity has decreased. To this end, the aim of these investigations was to establish micropropagation protocols for the selected species i.e. Bowiea volubilis, Haworthia_ limifolia and Cryptocarya latifolia. In addition, hardening-off protocols were also developed. The bulbous plant, Bowiea volubilis, was propagated via organogenesis using the inflorescence stem. Bulblet formation occurred directly without an intervening callus phase. Bulblets were produced on explants on Linsmaier and Skoog (1965) (LS) medium containing 30 g.r' sucrose and either I mg.r' BAP and I mg.r' 2,4-D or 1 mg.r' BAP and 1 mg.r' NAA. Shoots and roots were induced upon transfer to the basal medium devoid of plant growth regulators. Regenerated plantlets were successfully hardened-off. Haworthia limifolia, a succulent, was propagated via direct somatic embryogenesis using leaf material. Embryo formation was induced on a modified Murashige and Skoog (1962) (MS) medium containing 20 g.r' sucrose and 1 - 5 mg.r' 2,4-D. secondary embryogenesis occurred when the explants were transferred to the basal medium supplemented with activated charcoal and devoid of growth hormones. Healthy plantlets, produced from secondary embryos, were transferred to pots and acclimatised to greenhouse conditions. A large proportion of the plantlets regenerated were vitrified and as a result, this problem was addressed by changing the medium composition or culture environment. Silica gel, when placed in the culture vessel, was the best treatment for reversal of the vitrified condition. The establishment of leaf and nodal segment cultures of Cryptocarya latifolia required extensive investigation of sterilants to reduce fungal contamination. Several fungicides were tested and a successful sterilisation protocol was established. A number of media were tested for the induction of dormant axillary buds and multiplication of shoots. The best medium for both bud induction and proliferation was MS medium containing 30 g.r1 sucrose and 1 mg.r1 BAP and 0.01 mg.r1 NAA. Callus cultures were established on MS medium containing 30 g.r1 sucrose and 3 mg.rl 2,4-D. These calli, however, were non-embryogenic. Application of the established protocols and future research strategies are discussed. / Thesis (M.Sc.)-University of Natal, 1995.

Due to over-exploitation of commonly-used medicinal plants, mainly from KwaZuluNatal, because of ever-increasing human population growth, many of the useful medicinal plants are becoming depleted in their natural habitats. Some species like Warburgia salutaris, which is currently declared very rare in the KwaZulu-Natal province, appear to be on the verge of extinction. In order to counteract this overexploitation, this study sought to provide information that could help resource users to grow these threatened species through ex situ conservation methods. A short list of heavily utilised medicinal tree specles was selected from the approximately 700 tree species indigenous to KwaZulu/Natal. The criteria considered for short listing were; life form, species scarcity, past population status and part used. A total of 23 species were short listed, but a subset of 12 species was selected based on the availability of fruits and seeds. The aim of short-listing was to work on a manageable number of commonly utilised medicinal tree species. The seed physiology and growth of these species were studied. With the exception of Erythrophleum lasianthum and Curtisia dentata, all of them had a moisture content of 2': 20 % (on a dry mass basis), which is indicative of a recalcitrant behaviour. However, it could not be concluded that these seeds were truly recalcitrant because desiccation sensitivity was not directly assessed. Using the triphenyl tetrazolium chloride (TTC) viability test, most of the seeds of the 12 species seemed to be of good quality. Results of the TTC test for seed viability were similar to results obtained v using direct germination for most species. Results of flotation test for seed viability were different from the results obtained using direct germination for most spcies. The pre-treatment which achieved the highest germination percentage in almost all the seed types was cracking the outer coverings. Cracking pre-treatment appeared to be efficient in enhancing the removal of some substances which might inhibit germination of seeds. Hot water and acid pre-treatments frequently reduced germination. Growth of young seedlings was assessed in terms of stem diameter, height, and leaf area under sun and shade. Seedling growth in terms of stem diameter and height of most species did not show any significant difference. One of the few species which showed statistically significant differences in stem diameter growth was Ekebergia capensis. It was found that 3 out of lO of the species showed statistically significant differences in height growth. Two of the statistically significant differences in height occured on seedlings in the sun while one had statistically significant difference in the 40% shadecloth while 7 did not. Significant differences in leaf area occured on 7 out of lO species. Of these, 4 species had higher growth in the shade than in the sun while 3 had higher growth in the sun than in the shade. Generally, it appears that young developing seedlings establish themselves well under shade environment; this could be because most of the species used in this study are forest species. / Thesis (M.Sc.)-University of Natal, 1996.

Glutathione reductase (GR) and superoxide dismutase (SOD) enzymes are thought to play an important role in the plant chloroplast antioxidant system. Tobacco plants transformed with E. coli glutathione reductase and superoxide dismutase genes were used to investigate the role of these gene products (enzymes) in the chloroplast antioxidant system. These plants were T1318 (transformants with increased levels of cytoplasmic glutathione reductase activity) and GOR1OT (transformants with increased levels of cytoplasmic' glutathione reductase activity and chloroplastic superoxide dismutase). In addition, 10~M methyl violegen (paraquat), was used to perturb the system experimentally under high light, low light and in darkness. During these experiments GRA (glutathione reductase activity) was assayed and the results expressed as mg-1protein, mg-1 chlorophyll and g-1 tissue, using different types of transgenic plants. T131 B-cytosolic GOR transformants had a higher GRA under high light intensity. Under low light intensity T131B had a small increase in GRA compared to controls (T131 Bs in 1mM CaS04). Also leaf discs in the dark showed similar GRA as did controls. The three treatments had no effect on the GRA of untransformed plants. GOR1OT (cytoplamic GOR and chloroplastic SOD transformants) had a slight increase in GRA under high light intensity and in darkness. At low light intensity GOR10T showed similar results to controls. The results indicate the overall absolute increase in GRA in transgenic plants after methyl violegen treatment. The higher activity than that of nontransgenic controls indicate that bacterial GRA must have also increased following exposure to methyl violegen. / Thesis (M.Sc.)-University of Natal, 1996.

The aim of this study was to test the hypothesis that ryegrass and clover, when grown under optimal conditions in mixed stands, interact in response to available light energy. with other resources non-limiting, pasture yield is determined by the efficiency with which solar radiation is intercepted by canopies and converted into dry matter. The clover-ryegrass interaction under varying light regimes, as experienced in the canopies of these pastures, was studied by investigating the growth and production of ryegrass and clover in relation to light harvesting abilities and photosynthetic utilization of intercepted light. Pasture canopy structure and growth were studied under a four-weekly clipping treatment. The interception of photon flux density (PFD) in the pasture canopy was monitored diurnally and seasonally in mixed and mono cultures. Light use efficiency (C02 fixed/ unit absorbed PFD) as well as photochemical efficiency of photosystem II (Fv/Fm) were studied by monitoring CO2 assimilation rates and chlorophyll fluorescence respectively. The results obtained from this study indicated that interaction did occur between ryegrass and clover, cultivated in mixed pastures. The mixture was capable of more efficient light interception than the mono cultures, which resulted in higher productivity. Light interception abilities, as manifested in canopy architecture, and not physiological utilization of light energy, were found to govern the interaction between the two pasture components. / Thesis (M.Sc.)-University of Natal, Durban, 1993.

The geology, topography, soils and climate of the Namib Desert Park south of 23 S. Lat. are described briefly. The vegetation was investigated using the techniques of the Zurich - Montpellier school of phytosociology. Floristic and other site data were collected from 472 sites in the standard Braun - Blanquet manner and the techniques are described in some detail. These data are presented by means of phytosociological tables and detailed descriptions of each community. A total of 34 noda, communities and sub-communities, were delimited in the study area and the floristic and ecological relationships of these are discussed. Only a few of the communities can be assigned to syntaxa from other parts of the world or southern Africa because most of the Namib Desert communities seemed too different to be compared to associations from North Africa, the Middle East, South America or Australia on more than a superficial, structural basis. Very few data from other arid or semi-arid areas in southern Africa have received formal phytosociological treatment and there is therefore insufficient material to draw meaningful comparisons. The "foam structure" described by Volk & Geyger (1970) was found to be widely distributed in soils of the plains of the Namib Desert, and it was shown to have a profound influence on water penetration. The effects on vegetation development are discussed. The study generated a number of questions about the vegetation and ecosystems of the Namib Desert and some recommendations are made concerning future synecological and autecological studies. A list of species and synonyms of the names of all higher plants recorded in the study area are given in Appendix I. It is concluded that the Braun - Blanquet method is efficient in terms of time required to collect data which can be used for a number of purposes, but that a classification of vegetation ' should be followed up by autecological and detailed synecological studies of species (particularly those which characterize communities) and individual communities to determine the controlling factors more precisely and to enable more accurate predictions concerning the effects of management programmes to be made. / Thesis (M.Sc.)-University of Natal, Pietermaritzburg, 1976.