Tobacco is an important crop in South Africa. The genetic basis of tobacco breeding is very narrow and cultivars are closely related. The production of new tobacco hybrids with novel characteristics through classical breeding techniques is difficult. Genetic engineering could assist plant breeders to introduce new herbicide, disease and pest resistance traits into existing proven cultivars. Plant genetic engineering has not previously been applied to the improvement of South African commercial tobacco cultivars. Agrobacterium-mediated leaf disc transformation was used to create transgenic tobacco plants from South African commercial tobacco cultivars TL33, J6 and 20/19. The cultivar samsun was also used to create transgenic plants. The Agrobacterium tumefaciens helper strain C58C1 (pGV2260) containing the binary vector pJIT119 was used to carry out the transformation. As well as the leaf disc transformation method, other methods of obtaining transgenic tobacco plants were explored. These methods included the use of Agrobacterium-mediated transformation of tobacco cell cultures and direct DNA-mediated transformation of tobacco protoplasts. The vector pJIT119 encodes the uidA gene for the β-glucuronidase (GUS) enzyme, the nptl/ gene for neomycin phosphotransferase (NPTII) and the sul I gene for the dihydropteroate enzyme conferring asulam resistance. The presence and expression of these three foreign genes uidA , npt/l and sul I from pJIT119 in transgenic tobacco plants was confirmed by a variety of experimental approaches, including the culture of transgenic plants on medium containing kanamycin or asulam, the GUS histochemical assay, the neomycin phosphotransferase assay, DNA dot-blot analysis, in situ hybridization, computerized image analysis, polymerase chain reaction and progeny analysis. A detailed analysis of individual transgenic plants is necessary in order to select those plants which express the foreign genes maximally. Only these plants would be given to plant breeders for field trial assessment. A high level of foreign gene inactivation was observed in transgenic tobacco plants obtained from the Agrobacterium-mediated leaf disc transformation method. Approximately 20% of the original transgenic plants were discarded as "escapes" as they contained a defective npt/l gene. The remaining kanamycin resistant plants, however, had inactive copies of either the sul I or the uidA gene, or both. The use of in situ hybridization and the polymerase chain reaction (PCR) helped to explain the foreign gene inactivation. The lack of foreign gene expression in individual transgenic plants was not due to the physical loss of entire foreign genes, DNA methylation or the position effect. The lack of expression was due to possible T-DNA rearrangements or deletions which disabled certain genes carried on the T-DNA. Transcription and translation of these foreign genes occurred, but the final uidA and sul I gene products (β-glucuronidase and dihydropteroate synthase, respectively) were possibly defective and did not confer GUS activity or asulam resistance on the transgenic plants The tissue specific activity of the uidA gene under the control of the cauliflower mosaic virus (CaMV) 358 promoter was studied. In the vegetative structures of transgenic tobacco plants, the uidA gene activity was located within the cells surrounding the vascular traces and within the glandular hairs. The effects of stress on 358 promoter activity was also investigated. Chemical and nutrient stess in vitro did not have a significant effect to decrease uidA gene expression under 358 promoter control. Foreign gene expression (uidA) under CaMV 358 promoter control may be enhanced by in vitro stress. Oxygen stress (anaerobic culture under waterlogged conditions) induced uidA expression in areas of the plant which usually did not show usual tissue specific patterns of uidA expression. The stage of differentiation in tissue culture when compared to the mature hardened off transgenic plant, also had an effect on the amount of uidA gene expression. Mature hardened off plants expressed less GUS activity than immature in vitro plants. The tissue specific pattern of foreign gene expression under CaMV 358 promoter direction was conserved in the reproductive structures of transgenic tobacco plants. In floral organs, the pattern of uidA gene expression was essentially the same as that found in vegetative tissues. In all floral organs examined, uidA expression was found associated with the vascular system and within the glandular hairs. The uidA gene with a CaMV 358 promoter was not expressed in pollen. Because of the ease of transformation of tobacco, it is possible that genes for pharmaceutically valuable proteins and peptides could be expressed in tobacco, for agricultural scale fine chemical production("pharming"). This could be of economic advantage for the survival of tobacco as a commercial agricultural crop in the future when tobacco smoking is no longer popular. / Thesis (Ph.D.)-University of Natal, Pietermaritzburg, 1994.

Soil erosion and vegetation destruction have reached serious proportions in disturbed areas of the montane grasslands of the Natal Drakensberg. Little is known of the inherent ability of indigenous grasses to produce viable seed and establish seedlings for rehabilitation programmes. This study examined the effects of fire and small-scale disturbance on the seedling dynamics of the dominant grass, Themeda triandra Forssk. Annual seed production of T. triandra increased with length of burning rotation, ranging from 21 seeds m⁻² in the annual winter burn to 485 seeds m⁻² in the five-year burn. High predation of seeds (70-98%) and low viability (37% in 15 month-old seeds) contributed to the poor representation of T. triandra in the seed bank when compared to the aboveground vegetation. Seedling densities and survival were monitored by mapping quadrats at six-weekly intervals from November 1986 to September 1989. Highest establishment of T. triandra was recorded in the biennial spring burn treatment (< 184 seedlings m⁻²). Microclimatic studies of the seedling environment demonstrated that increases in photosynthetically active radiation following burning, and high soil moisture associated with accumulation of litter, favour seedling establishment. The main constraint to seedling establishment was high mortality in winter which resulted in a 1% survival of seedlings of T. triandra during the three-year study. This indicates that in the fire-climax grasslands of the study, seedlings contribute little to the population structure. The most significant factor contributing to low survivorship is intraspecific competition between seedlings. Spacing of seedlings in rehabilitation programmes is therefore a critical factor in optimizing seedling establishment. Analysis of the fate of seeds of T. triandra between their production and establishment, showed that the main constraints for seedling establishment were predation, low viability and poor germination. A population model, based on transitions between seeds, seedlings and mature tufts, predicts that a biennial spring burn is the optimal treatment for maximising both seedling establishment and vegetative growth. / Thesis (Ph.D.)-University of Natal, Pietermaritzburg, 1994.

Acid phosphatases (apase) are important hydrolytic enzymes that function in the acquisition, production; transport and recycling of inorganic phosphate (Pi), thus making a significant contribution towards nutrients dynamic of many ecological niches. The aim of this study was to characterize the apase enzyme found in the lichen Cladonia portentosa at the molecular level. The initial experiment entailed cloning the apase gene by PCR using degenerate primers designed from close relatives of C. portentosa from the Ascomycete family. The isolation of apase gene from Cladonia portentosa using PCR was not successful. Attempts were then made to purify the secreted apase and to determine its biochemical and molecular properties and to allow comparison with already characterized secreted phosphatases from other fungal sources existing in the NCBI database. It was anticipated that the partial sequence of the purified enzymes would provide a corresponding apase gene. The acid phosphatase enzyme was partial purified to 45 fold by a gel filtration with a yield of 18%. It gave a single, broad glycoprotein band on native PAGE and SDS-PAGE corresponding in size to 250 and 148 kDa, respectively. Under reducing conditions, the purified enzyme migrated as two bands of 116 and 32 kDa, indicating the heterodimer nature of this enzyme. Only one distinct band, (pI 6.4) was observed after electrofocusing. The optimum temperature for the enzyme was 65 °C where an optimal pH was detected at 2.5. The enzyme was inhibited by known acid phosphatase inhibitors (fluoride, molybdate, orthovanadate and tartrate) and the metals (Cu²⁺ and Zn²⁺). The purified enzyme demonstrated broad substrates selectivity and had a KM of 31.2±0.25 μM for phytic acid. Peptide analysis by Mass Spectrometry (MS) MALDI-TOF indicated the presence of two apase proteins. The amino sequences of purified apase/s from Cladonia portentosa were FLAETNPAPFGH, AVGLGYVEELLAR and AQGLGYVQEVLAR. Comparing the amino acids of the sequenced protein with that of already known proteins confirmed the enzyme to be a secreted histidine acid phosphatase, resembling other acid phosphatases and phytase from several filamentous fungi with respect to amino acid composition. To investigate the effect of phosphorus on C. portentosa apase, the mycelium was grown under different concentrations of Pi [0.05, 1.0, 3.0, 10 and 100 mM (KH₂PO₄)]. The aim was to localize the apase enzyme and to screen for the occurrence of the gene coding for the acid phosphatase enzyme. A treatment of 3.0 mM Pi induced high levels of apase compared to all other treatments. In addition, cultures of C. portentosa were grown in axenic cultures to study the effect of pH and Pi versus menadione on the production of acid phosphatase and mycelia growth. A culture media of pH 4.8 and 6.0 resulted in higher apase secretion than when compared with pH 2.5 medium. The presence of 2.0 μM menadione marginally increased levels of the apase compared to the control treatment. Apase was further localized cytochemically using fluorescent substrate-enzyme-labelled fluorescence (ELF-97) which forms a fluorescent crystalline precipitate at the site of phosphate activity. Fluorescent microscope revealed that the enzyme was present in all treatments, irrespective of Pi concentration, however, the fluorescence signals were intense in low Pi concentrations (0.05 and 1.0 and 3.0 mM Pi). Ultrastructure localization using live mycelium under confocal microscopy using Vector blue III substrate revealed that the enzyme was localized in the cytoplasm, cell membrane, vacuole and small organelles, presumed to be endosomes. Co-staining with FM4-64, confirmed the punctuate structure to be secretory vesicles or a vacuolar network. To investigate the effect of P starvation on C. portentosa at a molecular level, the effect of Pi on the gene expression profile was examined. The generation of a cDNA library from axenic grown mycelium treated with P provided a foundation for the identification and characterization of genes expressed in the P treated mycelium through expressed sequence tags (ESTs). Several genes were identified whose transcriptional profiles have been significantly changed by phosphorus treatment and menadione. They include genes required for signal transduction and vesicular transport, cell biosynthesis and protein metabolism and stress response. In conclusion, this study constitutes the first step towards understanding the molecular mechanism governing acid phosphatase in C. portentosa. / Thesis (Ph.D.)-University of KwaZulu-Natal, Pietermaritzburg, 2011.

In this study adaptations of bryophytes and lichens to desiccation stress were examined. The aim was to test whether desiccation tolerance in the selected species is constitutive or if desiccation tolerance could be induced by various hardening treatments. In addition, some putative tolerance mechanisms were investigated, including the accumulation of sugars, increase in ROS scavenging systems and other mechanisms e.g. energy dissipating processes. To determine if hardening treatments prior to desiccation stress increased desiccation tolerance, mosses and lichens were partially desiccated or treated with ABA. The effect of hardening treatments on the physiology of the moss Atrichum androgynum and lichens Peltigera polydactyla, Ramalina celastri and Telochistes capensis during a desiccation-rehydration cycle was investigated. Photosynthesis, respiration and chlorophyll fluorescence measurements were used as rapid tools to determine the metabolic activities in these lichens and moss species. In A. androgynum partial desiccation following slow drying at 52% RH increased the rate of recovery of net photosynthesis. Net photosynthesis recovered almost completely following slow drying in the material that was partially dehydrated and/or treated with ABA. This suggests that partial dehydration hardens the moss, and that ABA can fully substitute for partial dehydration. In R celastri and P. polydactyla both partial dehydration and ABA treatments displayed some improvement in desiccation tolerance depending on the duration and severity of stress. The reduction in the re-saturation respiration burst in P. polydactyla, although not quite significant, strongly suggests that hardening increases mycobiont tolerance. However, it is more difficult to establish whether the hardening treatments improve photobiont performance. In the moss A. androgynum ABA treatment increased the rate of recovery of photosynthesis and PSII activity, and also doubled non-photochemical quenching (NPQ). Increased NPQ activity will reduce ROS formation, and may explain in part how ABA hardens the moss to desiccation. In ABA treated, but not untreated mosses, desiccation significantly increased the concentration of soluble sugars in A. androgynum. Sugar accumulation may promote vitrification of the cytoplasm and protect membranes during desiccation. Starch concentrations in freshly collected A. androgynum and R. celastri were only c. 40 and 80 mg g ¯¹ dry mass respectively, and slightly rose during desiccation, but were only slightly affected by ABA pretreatment. ABA did not reduce chlorophyll breakdown during desiccation. In P. polydactyla ABA pretreatment had little effect on any of these parameters. Changes in the activities of the free radical scavenging enzymes ascorbate peroxidase, catalase and superoxide dismutase were measured during wetting and drying cycles in the moss A. androgynum and in the lichens P. polydactyla, R. celastri and T capensis. These species normally grow in the understorey of the Afromontane forest, moist, xeric, and extremely xeric miicrohabitats respectively. In A. androgyum, enzyme activity was measured shortly after collection, after 3 d storage following hardening by partial dehydration and/or 1 h treatment with ABA or distilled water and during desiccation and rehydration. In A. androgynum enzyme activities of CAT and SOD in untreated material were always higher than in the hardened treatments, while both partial dehydration and ABA treatments tended to reduce both CAT and the induction of SOD activity, although these effects were not significant between the treatments. This suggests that ABA may not be involved in the induction of free radical scavenging enzymes and probably these enzymes are not important in desiccation tolerance of A. androgynum. In lichens, the enzyme activity was measured shortly after collection, after hydration for 48 hat 100% RH, after desiccation for 14 d and 28 d, and during the first 30 min of hydration with liquid water. Enzyme activities tended to rise or stay the same following rehydration in all the species tested. After desiccation for 14 d, enzyme activities decreased, and then decreased further to very low values after 4 weeks desiccation. In all species, including T capensis from an extremely xeric habitat, the activities of all enzymes remained at very low values during the 30 min following rehydration, and were therefore unavailable to remove any reactive oxygen species accumulating in lichen tissues as a result of desiccation. Results suggests that the enzymic antioxidants are more likely to be involved in removing reactive oxygen species produced during the normal metabolic processes of lichens than having a role in desiccation tolerance. The Afromontane understorey moss Atrichum androgynum displayed an oxidative burst of H₂O₂ during rehydration following desiccation. Maximum rates of H₂O₂ production occur during the first 15 min of rehydration. While the production of H₂O₂ increases with increasing desiccation times, the moss produced significant amounts of H₂O₂ during rehydration after desiccation for times that did not inhibit photosynthesis or cause K⁺ leakage. A. androgynum may produce more H₂O₂ during desiccation than rehydration, because desiccation artificially induced using polyethylene glycol strongly stimulates production. Experiments involving inhibitors and exogenously supplied reductants indicate that peroxidases are responsible for the synthesis of H₂O₂. Factors that influence the rate of H₂O₂ production during rehydration include light and the hormone ABA. Patterns of H₂O₂ production are discussed in terms of their possible role as a defence against pathogenic fungi and bacteria. / Thesis (Ph.D.)-University of Natal, Pietermaritzburg, 2002.

Plants have evolved numerous strategies for the adaptation to drought. Although many investigations reported on the potential value of proline accumulation during environmental stress, it is still unknown whether or not a constitutive higher level of proline accumulation enhances plant tolerance. Thus, it was investigated if underproduction and overproduction of proline will influence the susceptibility to drought stress in soybean plants. This was made possible with the transformation of soybean plants with an L-Δ¹-pyrroline-5-carboxylate reductase (P5CR) gene. First, an Agrobacterium-mediated vacuum infiltration transformation system, using partially germinating Carnia 2233 soybean seed, was established through the assessment of several conditions that can affect transformation efficiency with the use of β-glucuronidase reporter genes. Transformation was confirmed with PCR and Southern blot analysis and results indicated that stable transgenic soybean plants were obtained within one generation with a transformation rate of± 30%. This technique was used in the transformation of Carnia 2233 soybean seed with the P5CR gene in the antisense orientation under the control of an inducible heat shock gene promoter (IHSP). It was confirmed that the P5CR-IHSP gene construct was integrated into the soybean cells and was conserved over three generations. Physiological screening of the antisense P5CR transgenic plants in the greenhouse proved that, with activation of the promoter, an under-expression of the P5CR gene and subsequent inhibition of the accumulation of proline were experienced during drought and osmotic stress. The decline of the viability of the transgenics with prolonged drought stress, as monitored with a woodenbox screening test, is an indication that proline is needed for survival of soybean plants under drought stress conditions. The transgenic plants demonstrated a sensitive reaction in contrast to the control plants that displayed a tolerant reaction to osmotic stress in a TTC assay. The underexpression of the P5CR gene resulted in a decline protein synthesis due to proline shortage as was observed with the evaluation of the efficiency of protein synthesis. All these results suggest that a decrease in the proline level due to the antisense P5CR gene, yielded plants that are more osmotic and drought stress sensitive. Subsequently, the soybean cultivar Ibis was successfully transformed with the P5CR-IHSP construct in the sense and antisense directions in order to test the reproducibility of the transformation process and to assessed the link between the biochemical traits involved in the drought stress mechanism. Three different experiments were conducted: a mild heat and drought stress on "To" transgenic plants exploring changes in chlorophyll fluorescence transients, a mild heat stress on "T1" transgenic plants comparing proline accumulation and chlorophyll fluorescence transients and a severe drought and heat stress on the "T1" transgenic plants comparing proline accumulation NADP⁺synthesis and chlorophyll fluorescence transients. Chlorophyll fluorescence transients were successfully used as a screening method for transgenic soybean plants during this study. The sense transgenics responded to the mild stresses with a significant decrease in their electron transport, trapping and absorption compared to the antisense plants that displayed significant increases in electron transport and trapping. During the severe stress, the antisense transgenics experienced total photoinhibition indicated by the enormous loss of electron transport but the sense plants had the ability to overcome the stress as is revealed in the increase in the electron transport. It was demonstrated that although proline accumulation yielded no significant differences during the mild heat stress, the sense plants accumulated substantially more proline than the control and antisense plants during the severe heat and drought stress. It was demonstrated that proline plays an important role in the plant's response to a drought stress as well as in the recovery phase after drought, as the sense plants also had the ability to reduce the accumulated proline during the recovery period in contrast to the antisense transgenics that experienced protein degradation. The transgenics responded to a period of heat and drought stress with a reduction in NADP⁺ levels in the antisense plants and increasing levels in the sense plants. The sense plants were able to fully recover after the stress period, thus adaptation to drought may depend on different mechanisms, including the capacity to maintain high levels of proline and to regenerate them through the "reduction" of NADP⁺. It was possible to alter the drought tolerance of Ibis by transformation with antisense and sense P5CR gene constructs, which resulted in respectively more sensitive and more tolerant Ibis plants. It can be concluded that over-expression of P5CR during a drought stress resulted in higher proline levels, better photosynthetic efficiency, higher NADP⁺ production and thus a more drought tolerant plant. This study gave additional proof that a constitutively higher level of proline accumulation enhances drought tolerance in soybean. / Thesis (Ph.D.)-University of Natal, Pietermaritzburg, 2000.

It is believed that the process-based model 3-PG (Physiological Principles Predicting Growth; Landsberg and Waring, 1997) can potentially play a useful role within South African forestry, both as an operational and a strategic tool. Strategic applications may include the prediction of potential productivity on a site-by-site basis; broadscale productivity estimates based on remote sensing and the spatial application of 3-PG; identification of production constraints; and estimation of carbon fluxes to help address sustainability issues. Operationally, 3-PG could complement empiricallybased models or be used in conjunction with them as a hybridised product. The challenges of this study were therefore to see whether it is possible to adapt 3-PG to predict the growth and yield of E. grandis under South African conditions, test that model predictions are consistent with observed growth data and are biologically reasonable, and to assess the practicality of using 3-PG as either a strategic or operational tool. The main emphasis of this study was to understand the internal logic of 3-PG and how physiological processes are represented, and to develop methods to objectively parameterise and initialise the model. Thereafter a detailed model validation study was performed, ending off with selected potential applications of 3-PG within the South African context. The sensitivity of predicted stand volume (SV) and leaf area index (LAI) to the values of the species-specific parameters in 3-PG was examined. These analyses enabled the development of three distinct parameter sensitivity classes: insensitive parameters (i.e. those that can be varied widely without affecting the outputs studied), sensitive parameters (i.e. those whose value strongly affects the outputs, and non-linear parameters (i.e. those for which the outputs depend in a non-linear manner on the parameter value). Minimum data requirements for the parameterisation and initialisation of 3-PG are considered in detail. Conventional methods used for the parameterisation of models, specifically 3-PG, are reflected upon. An automated parameter estimation technique was examined and used for the estimation of selected parameters. Species-specific parameters were categorised according to data source estimation and sensitivity classes. Parameters describing allometric and age-dependent relationships were assigned values using observed data from biomass harvests. Critical parameters that could not be directly assigned using observed data were the ratio of foliage to stem allocation (i.e. P2 and p2o), allocation of net primary production (NPP) to roots (TJRX and T]Rn), optimum temperature for growth (7^,) and maximum canopy quantum efficiency (acx)- These were estimated using Parameter ESTimation, by fitting model output to corresponding observed growth data. As well as species-specific parameter values, mandatory inputs required by 3-PG include weather data, site-specific factors such as site fertility (FR) and physical properties of the soils, and stand initialisation data. Objective techniques to determine these site-specific factors and the initial values for the biomass pools were proposed. Most of these data are readily available for sites where experimental trials exist, or where monitoring networks are in place. However, this is the exception rather than the rule, so alternative data and information sources are required. These, together with the need for accurate weather inputs (especially monthly rainfall) and physical properties (especially soil texture, maximum available soil water and FR) of the sites being modelled were explored. 3-PG was validated using four simple tests by comparing predicted versus observed SV. Results showed that 3-PG predictions are relatively consistent with observed stand data. Analyses performed using time-series data showed model predictions accurately tracked observed growth in response to erratic and fluctuating weather conditions. Results from the initial model validation showed production on high and low productivity sites was under- and over-predicted, respectively. Further results presented here show a similar, but less marked trend (i.e. over- and under-predictions are not as extreme), and individual biases are less than those from predictions made using another locally developed parameter set. The application of 3-PG showed that the model is able to make estimates of tree growth that are consistent with those used within the forestry site classification. This showed the considerable potential 3-PG has for strategic planning by the forest industry (i.e. projected wood supplies etc) and in research planning (refining existing site classifications). The model could be useful in predicting growth in various areas where E. grandis is not grown, assisting in future decision making. 3-PG was able to identify growth constraints on a site-by-site basis and distinguish among them, and was able to identify environmental and site limitations to plantation growth, and how they vary in space and time. These results together with predictions of site productivity demonstrate the potential for 3-PG to be used to improve existing forest site classifications. The model comparison study between empirically-based models and 3-PG showed that although the empirical models made accurate predictions of volume under static climatic conditions, under fluctuating weather conditions empirical estimates of volume were less accurate than those made with 3-PG. 3-PG can therefore be used operationally with minimum input data to predict growth using enumeration data. This is useful in saving time and cutting costs. The use of process-based models (PBMs) in general, and 3-PG in particular, needs to be "championed'' to the South African forest industry. This is necessary for two reasons. Firstly, the model and the manner with which it describes physiological processes of growth need to be explained in layman's terms. This will demonstrate how and why a process-based model can work better in a fluctuating environment than empirically based models. Secondly the comparison between 3-PG and the local empirical models needs to be presented as an example of how 3-PG can be applied on an operational basis. It is accepted that much convincing is still required. / Thesis (Ph.D.)-University of KwaZulu-Natal, Durban, 2005.

Three questions are considered in the context of the possible effects of seedassociated mycoflora, typified by Fusarium moniliforme, during hydrated storage of recalcitrant seeds of the tropical species, Avicennia marina. These are: 1) whether fungal infection reduces storage lifespan; 2) whether seeds become more susceptible to fungal attack during storage and whether they posses defence mechanisms that might suppress fungal proliferation in hydrated storage (production of antifungal compounds and 13-1,3-glucanase (EC 3.2.1.39) and chitinase (EC 3.2.1.14)] and 3) whether it is possible to discriminate ultrastructurally between inherent deteriorative changes and those that are fungally-induced. 1) The data indicate unequivocally that if fungal activity is curtailed, then the hydrated storage lifespan of A. marina seeds can be considerably extended. 2) When inoculated immediately with F. moniliforme, newly harvested seeds were extremely susceptible to the adverse effects of the fungus, while seeds that had been wet-stored for 4 days showed a considerably heightened resilience to the effects of the fungus prior to inoculation. The enhanced resilience, although declining, persisted in seeds stored hydrated for up to 10 days prior to inoculation, being lost after 12 days. This finding was supported by significant increase in 13-1,3-glucanase and chitinase and in antifungal compound production during 10 days of wet storage. After 14 days of wetstorage, seeds become more susceptible to the effects of fungusthanthose in the newly harvested condition. 3) The resilience of seeds that had been stored in the short-term was associated with ultrastructural changes indicative of enhanced metabolic activity associated with the onset of germination (e.g. increase in vacuolation, well-developed mitochondria and endomembrane system [ER and Golgi bodies]). However, with sustained stress associated with wet-storage IV conditions, the seeds became increasingly badly affected by the fungus, showing some ultrastructural fungally-induced abnormalities (e.g. nuclear lobing, presence of lipid bodies and prevalence of Golgi bodies that had many associated vesicles) and a decrease in 13-1,3-glucanase and chitinase activity. It is suggested that the decreased susceptibility of A. marina seeds during short-term storage relies on the ability to create an antifungal environment prior to infection (through synthesis and accumulation of pre-formed and induced antifungal compounds and antifungal enzymes), which would also be an effective strategy during germination in the natural environment. / Thesis (Ph.D.)-University of KwaZulu-Natal, Durban, 2004

Pinus patula Schiede ex Schlect. & Cham. is the most widely planted softwood species for both pulpwood and saw timber in the South African forestry industry. High mortality of this species, often in excess of 20%, following planting is currently of major concern and has the potential to limit future deployment for commercial timber. Water stress is often reported to be a cause of mortality during regeneration in commercial forestry plantations yet, prior to 2007, there was no published research on the water relations of P. patula during regeneration in South Africa. This, together with questions raised by the industry as to the role of using water in the planting operation, initiated the series of studies conducted for this thesis. Water planting (application of water into the planting hole at the time of planting) of P. patula seedlings has been used commercially to reduce post-planting water stress and buffer against potentially extreme weather conditions immediately after planting. However, the primary role of the water, as well as its success in increasing survival following planting, has never been critically assessed. Since the use of water in the planting operation is expensive, it was essential that the benefits to using water were quantified, in terms of survival and growth, and justified, in terms of any monetary investment. In addition, there was a lack of local studies investigating the physiological characteristics of P. patula seedlings, particularly their tolerance to low soil water availability. To understand the role of water during the regeneration of P. patula in terms of plantation management and seedling physiology, a variety of research methodologies were used that included: applied field trials, multivariate methods (a retrospective investigation), pot trials and the development of a simple financial model. Four field trials were implemented to test the response in P. patula survival to water applied at planting. Two trials each were situated in the KwaZulu-Natal (KZN) Midlands and Mpumalanga Escarpment. The first trial at each site was planted in spring (October) and the second in summer (February). Watering treatments consisted of different quantities of water used in the planting operation and included 0.5 litres, 2 litres, 4 litres and no water (dry plant). Only at the spring planted trial in the KZN Midlands was survival of the dry planted seedlings significantly lower than that of the seedlings planted with water, at 90 days after planting. This may have been due to low rainfall during the week before and two weeks after planting, or the small size of the seedlings used in the trial. Application of 0.5 litres of water to the planting pit was sufficient to increase survival to a level equivalent to that where 2 or 4 litres of water was used, yet only increased soil moisture in the area immediately surrounding the seedling. This suggested that the role of the water applied during planting was increased root to soil contact. Overall, these four trials indicated that planting with water had the potential to increase survival only when soil water availability was low and rainfall sporadic. There was no effect of water applied at planting on early tree growth. While the results of the four field trials provided an indication of the effect of planting with water on subsequent survival of P. patula seedlings, there was concern that the results of the four trials may not be a true reflection of a dynamic situation. Survival in response to water applied at planting may vary from year to year and across forestry regions due to the unpredictable nature of rainfall and high air temperatures during the planting season, as well as the wide range of forestry sites across which P. patula seedlings are planted. To improve our understanding, a database of 58 trials was compiled where water and dry planting had been carried out. In this way it was possible to investigate whether the results from the four field trials were reflected in a range of previously conducted field trials implemented across time and space. The trials incorporated into the dataset were all planted to P. patula between 1990 and 2005 in the summer rainfall region of southern Africa. Data related to the climate, local weather, physiography and site management at each trial were also included. Summary statistics, linear correlation and multiple regression were used to determine if site-associated variables were related to an increase in survival in the water relative to the dry planted treatments. The analyses indicated that for all 58 trials, survival was lowest during the summer months, regardless of planting treatment. Planting with water was most likely to increase survival when used during spring, autumn and winter planting, although (as with the four applied field trials) there was no overall significant relationship between water planting and survival. Based on these results it was anticipated that an understanding of the water stress physiology of P. patula seedlings was required to explain the observed trends from a more fundamental perspective; if planting with water did not always increase survival, why not? Three pot trials were conducted to increase the understanding of the water relations of P. patula seedlings. These trials were also used to provide benchmark physiological data related to stressed (water) and unstressed seedlings. The first pot trial highlighted the importance of root plug moisture at the time of planting for increasing subsequent survival. The subsequent two pot trials were aimed at investigating the interaction between planting stock quality (as determined by measures of size) and soil water availability and the effect on survival, growth and physiology of P. patula seedlings. These results indicated that P. patula seedlings were not as sensitive to high air and soil temperatures (above 30°C) and low soil water availability (below -1.5 MPa) as previously thought. The seedlings were able to tolerate low soil water availability for several weeks and, following rewatering, were able to recover from moderate and severe water stress (a shoot water potential of below -1.5 MPa). This data supported the results from the four applied field trials and retrospective study of 58 trials, where the application of water to the seedlings at planting did not substantially increase survival. In the pot trials, stomatal conductance started to decrease when shoot water potential approached -0.8 to -0.9 MPa. Stomatal closure occurred at a shoot water potential between -1.2 MPa to -1.5 MPa. Mortality due to water stress occurred only in response to extended periods of low soil water and was associated with a shoot water potential of below -3.0 MPa. There was variability between seedlings in their potential for survival and growth. Inherently bigger seedlings had a greater capacity for new root growth following planting. New root growth, as well as a greater mass of new roots, was associated with higher shoot water potentials and higher rates of transpiration under conditions of low soil water availability. This indicated that seedling quality, as determined by size, may play a role in sensitivity to water stress. The field trials, retrospective study and pot trials indicated that the practice of planting with water was not always critical to the survival of P. patula seedlings. A simple financial model was developed to estimate whether planting with water represented a cost that could be used as a decision criterion, given certain growth parameters and management scenarios. The data projected by the model were also compared to actual research data for water versus dry planting (and the inclusion of an insecticide in the water). While these comparisons were specific to the parameters included in the model for this study, as well as the results of the research trials used in the benchmarking exercises, the model indicated that; 1) costs for planting with water were likely to be recovered only when no blanking (replacing of dead trees) was carried out, with capital invested at a low return rate (3%), 2) including an insecticide in the water increased the likelihood of cost recovery, and 3) site quality had an impact on the increase in survival required to recover planting method costs, with a greater percentage increase in survival required on lower quality sites. Lower quality sites often have a lower mean annual precipitation (associated with higher rainfall variability), or shallow soils (associated with lower soil water availability) and therefore are also likely to be sites where foresters may want to use water to reduce (drought related) mortality. The impact of site quality is thus also an important factor to include in any decisions regarding planting methods (i.e. using water) and their costs. Further investigations should be aimed at examining; 1) the interaction of root plug size (as determined by container type) and soil water availability on growth and physiology of P. patula seedlings, 2) the methods of grading seedlings within a population to select those that have a high potential for survival and growth, and 3) the effects of soil water availability on the physiology, survival and growth of P. patula cuttings, as well as other pine species and hybrids grown in South Africa, such as P. elliottii, P. elliottii x P. caribaea and P. patula x P. tecunumanii. It is likely that the proportion of forestry regions planted to these hybrids will increase in the future. / Thesis (Ph.D.)-University of KwaZulu-Natal, 2008.

The C4 plant, sugarcane (Saccharum spp. hybrids), accumulates sucrose to high concentrations and, as a result, has been the focus of extensive research into the biochemistry and physiology of sucrose accumulation. Despite this, the relationship between source leaf photosynthetic rates and sucrose accumulation in the culm has not been well documented. The observations that photosynthetic activity declines during culm maturation in commercial cultivars and that high-sucrose accumulating ancestral genoptypes photosynthesize at rates two-thirds of those of low-sucrose ancestral Saccharum species indicate that source-sink communication may play a pivotal role in determining sucrose yield. The relationship between source and sink tissues in sugarcane was investigated using a supply-demand paradigm, an approach novel in the study of the crop. The demand for photosynthate from the primary culm growth sink was shown to be closely linked to photosynthetic rates, sucrose export and the eventual physiological decline of source leaves. Results from initial field experiments revealed that leaf assimilation rates were negatively correlated with leaf hexose concentrations, but not those of sucrose. Further manipulation of leaf sugar status, through sugar-feeding and cold-girdling techniques, demonstrated the regulatory role of leaf sugar concentrations on photosynthetic activity, thus revealing sucrose, and particularly hexose, as key signal molecules in the modulation of the amount of photosynthate available for export to the sink. Gene expression profiling, by means of array technologies, indicated that changes in leaf sugar status and photosynthetic rates result in concurrent modifications in the expression of several genes involved in fundamental metabolic pathways, including photosynthesis, carbohydrate metabolism, stress response and sugar-signaling. Notable amongst these, was the identification of a potential trehalose 6-phosphate (T6P) sugar-signaling mechanism, thus implicating the trehalose pathway as a central regulatory system in the communication of sink carbon requirements to the source leaf. This study demonstrated that maturation of the culm results in a decreased demand for sucrose, which invokes a sugar-mediated feedback signal to decrease leaf photosynthetic supply processes. However, sugarcane leaves appear to retain the capacity to increase the supply of assimilate to culm tissues under conditions of increased assimilate demand. Uncoupling of the signaling pathways that mediate negative feedback between source and sink tissues may result in improved leaf assimilation rates and, consequently, lead to increased sugarcane sucrose yields. / Thesis (Ph.D.) - University of KwaZulu-Natal, Westville, 2007.

The response of three Eucalyptus spp. clones (GC550, GU210 and TAG14) to water availability was assessed in terms of growth, plant water status, leaf gas exchange, whole plant hydraulic characteristics (both root and shoot), stem xylem vulnerability. Furthermore, to experimentally assess the influence of hydraulic conductance on leaf physiology and plant growth, specimens of two of the clones were subjected to long-term root chilling. Prior to harvesting data were collected on the diurnal variation in leaf water potential (ΨL), transpiration rate (E), stomatal conductance (gs) and net CO2 assimilation rate (A). Main stem xylem vulnerability was assessed using ultrasonic acoustic emissions (UAE). Vulnerability of the main stem was assessed as the leaf water potential corresponding to the maximum rate of acoustic emissions (ΨL, EPHmax), and as the critical water potential triggering cavitation events, calculated as the mean of the water potentials of data points lying between 5 and 10% of the total accumulated emissions (ΨCAV,cUAE,%). Hydraulic conductance was measured on roots and shoots using the high-pressure flow meter (HPFM). Root data were expressed per unit root dry mass (Kr/trdw) and per unit leaf areas (Kr/LA), shoot data expressed per unit shoot dry mass (Ks/tsdw) and per unit leaf area (Ks/LA), and whole plant conductance was expressed per unit leaf area (KP/LA). Soil-to-leaf hydraulic conductance was also assessed as the inverse of the slope of the relationship between leaf water potential and transpiration rate (the evaporative flux, EF, method). A field study was undertaken on three month old TAGl4 and GU210 plants. Diurnal values of leaf water potential ΨL, E and gs were consistently higher in TAG14 than GU210, but A did not differ among the clones. Main stem xylem vulnerability (ΨCAV, cUAE,%) was higher in TAG14 than GU210. In both clones midday ΨL fell below ΨcAv,cUAE,%, suggesting lack of stomatal control of xylem cavitation. Kr/LA was higher in TAGl4 than GU210, whereas, Ks/LA and Ks/tsdw was higher in GU210 than TAGI4. A greater proportion of hydraulic resistances resided in the roots, particularly in GU210. Kp/LA was higher in TAGl4 than GU210 clone, although the significance was marginal (P=0.089). However, all the physiological measurements, were consistent with the concept of higher hydraulic conductances in TAGl4 leading to lower leaf level water stress. Above ground biomass was higher in TAG14 than GU210, in agreement with this concept, although this clone was more vulnerable than GU210. Material grown for 14 months in 25 l pots clones showed no differences in ΨSoil between the high and low watering supply, indicating that even the 'high' supply was inadequate to prevent water stress. In accordance with this, diurnal values of ΨL, gs, E and A did not differ significantly between treatments and clones. Early stomatal closure was apparent, maintaining ΨL constant during the middle of the day. Stem xylem vulnerability, assessed as both ΨL,EPHrnax and ΨCAV,cUAE,% showed that the main stem of GC550 was more vulnerable than other two clones, and that low watered plants were more resistant to xylem cavitation than those receiving high water. Midday ΨL fell below the vulnerability values assessed by both measures across treatments and clones, suggesting lack of stomatal control preventing stem xylem cavitation. There was no relationship between stem xylem cavitation and the shoot hydraulic conductances. Root pressures did not differ between either treatment or clones. Kr/LA was marginally higher in high watered plants, and Ks/LA and Ks/tsdw were higher in low watered plants, possibly by adjustment of leaf hydraulic architecture, and there were no clonal differences. Kr/LA was much lower than Ks/LA. Kp/LA did not differ between the watering treatment, but there was a clonal effect. Growth in dry mass was higher in high watered than low watered plants, but there were no differences among clones. As KP/LA was not affected by watering treatment there was no relationship between KP/LA and growth in total biomass. In plants grown for 21 months in 85 l pots low water treatment decreased midday ΨL, gs, E and A relative to high watered plants. Interclonal differences occurred at midday. Stem xylem vulnerability assessed as ΨCAV,cUAE,% and as ΨL,EPHrnax show similar trends as in the 14 months saplings, clonal differences being significant in ΨL, EPHmax. There was a 1:1 relationship between minimum leaf water potential and ΨL, EPHmax, suggesting that the water potential developed was limited by stem vulnerability. This implies stomatal control to reduce transpiration rates to prevent extensive cavitation occurring. These plants did not develop positive root pressures, indicating that recovery from xylem cavitations occurred through some other process. Kr/LA was higher in high watered plants than those receiving low water, and clonal differences were observed in Kr/trdw. There was no treatment effect in KS/LA and KS/tsdw, but a clonal effect was apparent. KP/LA was significantly different between treatment, and was reduced by low water in two clones, and increased by this in TAGI4. Reduced water availability reduced biomass production, with a greater effect on roots than shoots, such that low watering reduced root:shoot ratios. There was a weak but significant relationship between whole plant hydraulic conductance and maximum stomatal conductance, and between plant conductance and total biomass produced; these data are consistent with the concept of some hydraulic limitation to growth. Root chilling (achieved through chilling the soil) of two of the clones was used to experimentally manipulate hydraulic conductance to test the hydraulic limitation hypothesis. Short-term root chilling decreased both Kr/LA and KP/LA in both clones, but had marginal effects on leaf gas exchange. With long-term chilling the decrease in Kr/LA was observed only in GU210, with TAGl4 showing some adjustment to the treatment. As the roots constitute the major hydraulic resistance, KP/LA largely reflected those of the roots. Long-term root chilling significantly affected leaf physiological characteristics, despite the lack of effect on hydraulic conductance in TAGI4. Long term chilling decreased the whole plant dry mass, but the effect was smaller in TAGI4, and this clone also showed morphological adjustment, in that root growth was less adversely affected than shoot growth. The data from GU210 support the hydraulic limitation hypothesis; because of the morphological and physiological adjustment to long-term root chilling in TAGI4, the data are unsuitable to directly assess the hypothesis. / Thesis (Ph.D.)-University of Natal, Durban, 2002.