There is growing evidence that the achievement of biodiversity conservation objectives at the regional scale will have to take into account not only habitats within reserves, but also the contribution of semi-natural habitats in production landscapes. This requires knowledge of the conditions that maintain or enhance biodiversity in these suboptimal habitats. I focused on a system of forest patches and hedgerows in an agricultural landscape as habitats for dispersal-limited native forest herbs to (1) identify species-specific traits that appear to either limit or facilitate survival or colonization of forest herbs in hedgerows compared to forest patches, and (2) to evaluate the relative contribution of landscape and local variables in explaining patterns of forest herb species richness, abundance, and diversity in hedgerows. A fourth-corner analysis revealed that traits such as early spring flowering, ant dispersal or a lack of vegetative propagation can reduce the likelihood of native forest species to benefit from hedgerows. Conditional autoregressive models showed that landscape variables, specifically hedgerow age and neighboring forest area, largely account for species diversity in hedgerows.

Sclerocarya birrea (marula, Anacardiaceae) is a highly-valued indigenous tree in most parts of sub-Saharan Africa because of its medicinal and nutritional properties. The marula tree is adapted to the semi-arid conditions that characterise most parts of sub-Saharan Africa and renders them unsuitable for conventional crop agriculture. The unique nutritional properties of marula and its high tolerance to dry conditions provide opportunities for its development into a plantation crop. On the other hand, the demand for marula plant parts, mainly the bark and roots as medicinal remedies, poses a great threat to wild populations. In the long term, the growing demand of marula products in the food, pharmaceutical and cosmetic industries will not be sustainable from wild populations alone. Plant tissue culture technologies can be useful for in vitro manipulation and mass propagation of the plant in the process of domestication and conservation. The aims of the project were to determine the optimum conditions for seed germination, in vitro propagation and plant regeneration, and to evaluate the potential bioactivity of secondary metabolites from its renewable plant parts as an alternative option in the conservation of S. birrea. An ex vitro seed germination study indicated that after-ripening and cold stratification are critical factors. Cold stratification (5 °C) of marula nuts for 14 days improved germination (65%) as compared to non-stratified nuts (32%). Direct shoot organogenesis was achieved from leaf explants through the induction of nodular meristemoids on Murashige and Skoog (MS) (1962) medium and woody plant medium (WPM) supplemented with 6-benzyladenine (BA) in combination with naphthalene acetic acid (NAA), indole-3-butryric acid (IBA) and indole-3-acetic acid (IAA). Induction of nodular meristemoids from 86% of the leaf cultures was achieved on a MS medium with 4.0 ìM BA and 1.0 ìM NAA. High levels (78–100%) of induction were also achieved on WPM with different concentrations of BA (1.0–4.0 ìM) and IBA (1.0–4.0 ìM). The highest conversion of nodular meristemoids into shoots on MS initiation medium was only 22% for 4.0 ìM BA and 1.0 ìM NAA. This was improved to 62% when nodular clusters were cultured in MS liquid medium. Histological studies revealed high numbers of unipolar meristematic buds developing from globular nodules. These embryo-like structures have in the past been mistaken for true somatic embryos. The initiation of high numbers of nodular meristemoids per explant provides potential for automated large-scale clonal propagation in bioreactors, in vitro phytochemical production and the development of synthetic seed technology, similar to somatic embryogenesis. Plant regeneration through nodule culture has potential for application in mass micropropagation and plant breeding of S. birrea. Adventitious shoot and root induction are important phases in micropropagation. Plant growth regulators play an important role in these developmental processes, and the type and concentration used have major influences on the eventual organogenic pathway. Three auxins (IAA, IBA and NAA) and four aromatic cytokinins (6-benzyladenine, meta-topolin, meta-topolin riboside, and meta-methoxytopolin riboside) were evaluated for their potential to induce adventitious shoot and root formation in S. birrea shoots, hypocotyls and epicotyls. Among the evaluated cytokinins, the highest adventitious shoot induction (62%) was achieved on MS medium supplemented with meta-topolin (8.0 ìM). The lowest adventitious shoot induction (2.5%) was obtained on MS basal medium containing 2.0 ìM meta-methoxytopolin riboside. The highest adventitious shoot induction for hypocotyls was 55% on MS medium supplemented with 8.0 ìM meta-topolin. For the tested auxins, IBA induced adventitious rooting in 91% of shoots at a concentration of 4.0 ìM after 8 weeks in culture. However, the in vitro rooted plants only survived for two weeks when transferred ex vitro. A temperature of 25 °C and 16-h photoperiod were optimum for adventitious root induction. Stomatal density (number per mm2) on the abaxial leaf surfaces was higher for the 16-h photoperiod treatment (206.6 ± 15.28) compared to that for a 24-h photoperiod (134.6 ± 12.98). Normal mature stomata with kidney-shaped guard cells and an outer ledge over the stomatal pore were observed for in vitro plants growing under a 16-h photoperiod. Total phenolic content, proanthocyanidins, gallotannins, flavonoids, and antioxidant activities of S. birrea methanolic extracts were evaluated using in vitro bioassays. Methanolic extracts of the young stem bark and leaves contained high levels of these phytochemicals. Sclerocarya birrea young stem extracts contained the highest levels of total phenolics (14.15 ± 0.03 mg GAE g-1), flavonoids (1219.39 ± 16.62 ìg CE g-1) and gallotannins (246.12 ± 3.76 ìg GAE g-1). Sclerocarya birrea leaf extracts had the highest concentration of proanthocyanidins (1.25%). The EC50 values of the extracts in the DPPH free radical scavenging assay ranged from 5.028 to 6.921 ìg ml-1, compared to ascorbic acid (6.868 ìg ml-1). A dose-dependent linear curve was obtained for all extracts in the ferric-reducing power assay. All the extracts exhibited high antioxidant activity comparable to butylated hydroxytoluene based on the rate of â-carotene bleaching (89.6 to 93.9%). Sclerocarya birrea provides a source of secondary metabolites which have potent antioxidant properties and may be beneficial to the health of consumers. Sclerocarya birrea young stem and leaf ethanolic extracts exhibited high bioactivity (MIC < 1.0 mg ml-1) against both Gram-positive (Bacillus subtilis and Staphylococcus aureus) and Gram-negative (Escherichia coli and Klebsiella pneumoniae) bacteria. The highest activity (MIC = 0.098 mg ml-1 and total activity = 1609.1 ml g-1) was recorded for young stem extracts against B. subtilis. The highest activity (MIC = 1.56 mg ml-1 and MFC = 1.56 mg ml-1) in the antifungal assay against Candida albicans was observed for young stem ethanolic extracts. Sclerocarya birrea extracts had moderate acetylcholinesterase (AChE) inhibition activity. The dichloromethane (DCM) and methanol (MeOH) fractions exhibited dose-dependent acetylcholinesterase inhibitory activity. The highest AChE inhibitory activities were from leaves (DCM fraction, IC50 = 0.1053 mg ml-1) and young stems (MeOH fraction, IC50 = 0.478 mg ml-1). High inhibitory activity against cyclooxygenase (COX-1 and COX-2) enzymes was observed. All extracts and fractions showed high COX-1 enzyme inhibition (90.7-100%). Petroleum ether (PE) and dichloromethane fractions also exhibited high inhibition against COX-2 enzyme (77.7-92.6%). The pharmacological activities observed suggest that S. birrea renewable plant parts (leaves and young stems) provide a substantial source of medicinal secondary metabolites. Based on these results, plant part substitution can be a practical conservation strategy for this species. / Thesis (Ph.D.)-University of KwaZulu-Natal, Pietermaritzburg, 2009.

The crisis of newly emerging diseases and the resistance of many pathogens to currently used drugs, coupled with the adverse side-effects of many of these drugs have necessitated the continuous search for new drugs that are potent and efficacious with minimal or no adverse side-effects. The plant kingdom is known to contain many novel biologically active compounds, many of which could potentially have a higher medicinal value when compared to some of the current medications. Indeed, the use of plants in traditional medicine, especially in African communities, is gaining more importance due to their affordability and accessibility as well as their effectiveness. Exponential population growth rates in many developing countries has resulted in heavy exploitation of our plant resources for their medicinal values. In addition, plant habitat destruction arising from human developmental activities has contributed to the fragmentation or loss of many plant populations. Owing to these factors, many plant species with horticultural and/or medicinal potential have become either extinct or are threatened with extinction. These threatened species cut across different taxonomic categories including shrubs, trees and succulents. Without the application of effective conservation strategies, the medicinal and/or horticultural potential of such threatened species may be totally lost with time. The extinction of such species could lead to the loss of potential therapeutic compounds and/or genes capable of being exploited in the biosynthesis of new potent pharmaceutical compounds. / Thesis (Ph.D.)-University of KwaZulu-Natal, Pietermaritzburg, 2009.

Micropropagation of Aloe polyphylla, an endangered species with a high ornamental and medicinal value, is an important part of its conservation. However, the in vitro culture was hindered by the phenomenon of hyperhydricity. The research reported in this thesis was undertaken for two reasons. Firstly, to understand the role of various culture factors involved in the process of hyperhydricity in A. polyphylla and to identify the in vitro conditions, under which this disorder can be prevented. Secondly, we conducted an investigation into the underlying mechanisms of this phenomenon by probing if it was mediated through internal cytokinins. Ammonium (NH4 +) ions, applied cytokinins (CKs) and CK concentrations were tested in multifactorial combinations and significantly influenced the regeneration rate and occurrence of hyperhydricity. Shoots were grown on media with different NH4 + concentrations (10.3, 20.6 and 61.8 mM) and supplemented with BA, zeatin or TDZ at 0, 5 or 15 ìM. Elevating the levels of NH4 +, in the absence of CKs, could not induce hyperhydricity. Similarly, very low hyperhydricity was observed when CKs were added to media containing low NH4 + (10.3 mM). However, in the presence of higher NH4 + concentrations, CKs increased hyperhydricity in a concentrationdependant manner, suggesting that they were capable of inducing this syndrome only when other factors in the culture system were not optimised. High numbers of healthy looking shoots were produced on media with low NH4 + and low BA or zeatin (5 ìM). The use of TDZ resulted in the formation of buds, which did not develop into shoots. In view of the fact that NH4 + was supplied in the form of NH4NO3, it was difficult to determine if NH4 + or nitrate (NO3 -) ions were associated with the increase in hyperhydricity. We further examined the role of nitrogen (N) supplied as inorganic NH4 + or NO3 -, or organic glutamine. The omission of total N from the culture medium resulted in low multiplication and hindered shoot growth. Ammonium as the sole source of N depressed shoot regeneration and growth and escalated the frequency of hyperhydricity to ca. 50%. When NO3 - was used as the sole N source, shoots of fine quality were produced and hyperhydricity was completely eliminated. Overall, the MS N mix was superior to any single N source for multiplication and growth of shoots, suggesting a synergistic effect between NH4 + and NO3 - on shoot regeneration. Furthermore, not only the absolute amount of N, but also the relative amounts of NH4 + and NO3 - influenced the multiplication rate, frequency of hyperhydricity and shoot quality. The highest regeneration was obtained with NH4 + : NO3 - ratios (mM) of 20 : 40, 30 : 30 and 40 : 20. Decreasing the ratio of NH4 + : NO3 - lowered the occurrence of hyperhydricity. The potential of glutamine as the sole source of N was also demonstrated, since its application resulted in the production of good quality shoots and almost no hyperhydricity. Shoot explants grown in static liquid media became hyperhydric and lost their ability to regenerate. The type of gelling agent used to solidify the medium affected greatly hyperhydricity and shoot multiplication. Gelrite resulted in a significantly lower multiplication rate and four times higher hyperhydricity (64.7%) compared to when agar was used. Gelrite was further selected to test the hypothesis if hyperhydricity can be overcome by decreasing the relative matric potential of the media, and respectively the availability of water, as represented by increasing gelrite concentrations. Satisfactory reduction in hyperhydricity was achieved only at 16 g l-1 gelrite, however the regeneration also decreased. The nature of the gelling agent is therefore essential for the successful control of this phenomenon. It appears that a crucial prerequisite for the reduction of hyperhydricity in tissue cultures of A. polyphylla is the gaseous exchange between the in vitro atmosphere and the outside environment. In ventilated cultures, achieved by using a modified lid with a hole (d = 7 mm) covered with polyester or cotton mesh, hyperhydricity was completely eliminated, irrespectively of the type of gelling agent. Ventilation was further advantageous for the in vitro regenerants by increasing their leaf chlorophyll content as well as epicuticular wax deposition, the last one being indicative of the development of the water loss regulation mechanisms of explants. The increased culture ventilation, however, was negatively correlated with the regeneration rate and shoot growth. Endogenous CKs were measured in in vitro regenerants after an eight-week cycle to examine whether the hyperhydricity-inducing effect of exogenous CKs and gelling agents is associated with changes in the endogenous CK content. The content of endogenous CKs, determined by HPLC-mass spectrometry, in the shoots grown on CK-free media comprised isopentenyladenine-, trans-zeatin- and cis-zeatin-type CKs. The application of exogenous CKs resulted in an increase in the CK content of the shoots. Following application of zeatin, dihydrozeatin-type CKs were also detected in the newly-formed shoots. Application of BA to the media led to a transition from isoprenoid CKs to aromatic CKs in the shoots. Shoots grown on gelrite media contained higher levels of endogenous CKs compared to those on agar media. Total CK content of hyperhydric shoots was higher than that of normal shoots grown on the same medium. We suggest that the ability of exogenous CKs and gelrite to induce hyperhydricity in shoots of Aloe polyphylla is at least partially due to up-regulation of endogenous CK levels. However, hyperhydricity is a multifactor process in which different factors intervene. / Thesis (Ph.D.)-University of KwaZulu-Natal, Pietermaritzburg, 2009.

The rhizomes of Alepidea amatymbica and Alepidea natalensis are used for medicinal purposes. Because of the increase in demand for these plants the species is becoming scarce. As the seed biology of neither species is well defined, conditions as well as treatments required for optimum germination and vigour were studied. Seeds were exposed to various physical factors such as varying light and temperature conditions and cold stratification, sowing depth and seed storage. The effects of smoke-water, butenolide (3-methyl-2H-furo [2, 3-c] pyran-2-one) a novel smoke compound and chemical substances (gibberellins, kinetin and KNO3) were also tested in order to improve seed germination. Alepidea amatymbica and A. natalensis achieved the highest seed germination (72.5% and 80%, respectively) at 25 °C under a 16 h photoperiod with a mean germination time (MGT) of 18 and 12 days, respectively. Phytochrome studies showed that A. natalensis requires light for germination. Cold stratification (5 °C) for 14-28 days significantly improved the percentage germination of both species (> 90%) compared to non-stratified seeds (control) at 25 °C under a 16 h photoperiod. Sowing A. amatymbica and A. natalensis seeds at a depth of 0.5 cm resulted in higher percentage germination compared to 2.5 cm. The highest emergence rate for A. amatymbica was 40% at a sowing depth of 0.5 cm and the lowest emergence rate was 3% at 2.5 cm. Six months storage of A. natalensis seeds at room temperature (25 ± 2 °C) showed maximum germination (99%) with a MGT of 9 days. Smoke-water treatment of A. amatymbica seeds significantly enhanced germination from 72% to 91%. Smoke and butenolide at 10 °C and 25 °C promoted germination of A. natalensis seeds in a 16 h photoperiod. Smokewater application significantly improved both germination and seedling vigour of A. natalensis. GA3 (10-8 M) was the best treatment for achieving maximum percentage germination of A. natalensis seeds. Antibacterial (two Gram-positive bacteria: Bacillus subtilis, Staphylococcus aureus and two Gram-negative bacteria: Escherichia coli, Klebsiella pneumoniae), antifungal (Candida albicans), anti-inflammatory (COX-1 and -2) and genotoxicity tests (Ames test) were carried out on petroleum ether (PE), dichloromethane (DCM), 80% ethanol (EtOH) and water extracts of the two Alepidea species. Water extracts of A. natalensis rhizomes exhibited high activity (MIC values of 0.78 mg/ml) against the four bacterial strains. High activity was also observed in the PE and DCM leaf extracts of the same plant against the Gram-positive bacteria. The PE and DCM extracts of A. amatymbica rhizomes exhibited the best activity (MIC values of 0.39 mg/ml) against Bacillus subtilis. The rest of the extracts showed low activity (MIC values >1 mg/ml). All the extracts showed activity against Candida albicans, with A. natalensis leaf extracts exhibiting the highest antifungal activity with MIC values of 0.88, 0.20 and 0.78 mg/ml for PE, DCM and EtOH, respectively. EtOH extracts had inhibition less than 40% for both A. natalensis and A. amatymbica. All the PE extracts showed higher inhibitory activity for COX-2 than for COX-1. PE and DCM extracts had percentage inhibitions above 70% in both COX-1 and COX-2 assays. The Ames test for genotoxicity revealed that none of the plant extracts were genotoxic to the Salmonella TA98 tester strain. / Thesis (M.Sc.)-University of KwaZulu-Natal, Pietermaritzburg, 2009.

Traditional medicine in South Africa is part of the culture of the people and has been in existence for a long-time. Although animal components form part of the ingredients used, plant material constitutes the major component. South Africa is endowed with vast resources of medicinal and aromatic plants which have been employed for treatment against various diseases for decades. A large number of South Africans still depend on traditional medicine for their healthcare needs due to its affordability, accessibility and cultural importance. Helminth infections are among the variety of diseases treated by traditional healers. These infections are regarded as neglected tropical diseases (NTDs) due to their high prevalence among the economically disadvantaged living in rural areas in different regions of the world. / Thesis (M.Sc.)-University of KwaZulu-Natal, Pietermaritzburg, 2009.

Eucomis L’ Hér. is a genus of 10 species that fall within the Hyacinthaceae family. Eucomis zambesiaca Baker is a summer-blooming bulbous geophyte occurring from northern South Africa to Malawi. Eucomis species are used in southern African traditional medicine for the treatment of various ailments, in particular, pain and inflammation. As a result, the bulbs are heavily harvested for trade in South Africa’s traditional ‘muthi’ markets. Over-collection of Eucomis species has seriously depleted natural populations and now Eucomis plants are among the 15 scarcest medicinal species to be traded. Micropropagation is a useful technique for rapid clonal multiplication of plant material which could potentially yield useful secondary metabolites as well as alleviate the pressure on the wild plant populations. The in vitro induction of storage organs is especially beneficial as it can limit the loss of plants during acclimatization as bulblets are hardier than shoots or plantlets. The aim of this research was to determine optimal growth conditions for bulblet induction of Eucomis zambesiaca. The effect of environmental and physiological parameters on the initiation and growth of bulblets was investigated. These included the effect of temperature, photoperiod, various carbohydrates at different concentrations and combinations as well as various plant growth regulators. Maximum number of bulblets per explant was obtained at 20 °C, with an average of three bulbs p er leaf explant. The average bulblet mass was 57 mg, which was significantly higher than bulblets formed at other tested temperatures. An 8 h light regime was the optimum photoperiod. The highest mean number of bulblets (1.4 per leaf explant) developed under the 8 h photoperiod and the bulblets that formed were large in size. They had a mean bulb diameter of 3.4 mm and a mean bulb weight of 42 mg. Different carbohydrates such as fructose, sucrose and glucose were tested at concentrations of; 1, 3, 6, 9 and 12%. Fructose at a concentration of 3% was found to produce the best results. An average of 1.2 bulbs formed per explant. The mean bulb diameter was 3.4 mm and mean bulb weight was 56.6 mg. Plant growth regulators (GA3, IAA, IBA, NAA, BA, zeatin, iP and others) were tested at concentrations of 1, 2 and 5 mg/L. 1 mg/L IBA was found to be the optimum hormone treatment for bulblet induction. Bulblets were large, had good leaves and well established roots. Medium supplemented with 1 mg/L IBA produced bulblets that had an average bulb diameter of 4.36 mm and a mean bulblet weight of 79.1 mg. Bulblets grown in vitro were transferred to vermiculite and placed in a misthouse to acclimatize. After 2 months the plantlets were transferred to pots containing a sand:soil mixture of 1:1 and placed in a greenhouse. There was a 80 to 90% survival rate. / Thesis (M.Sc.)-University of KwaZulu-Natal, Pietermaritzburg, 2009.

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