In this thesis I synthesized different aspects related to diversity, distribution, uses and conservation of medicinal plants in Nepal and also have attempted to recommend guidelines for sustainability of two highly used alpine plant species. The over-harvesting or human induced activities are not the only problem for biodiversity but recently invasion of alien species has also emerged as serious problem in Nepal. I thus also attempted to analyze the effect of invasive species on community composition in the last paper. The first two papers deal with diversity, distribution, uses and harvesting. Paper I showed that medicinal plants in Nepal have unimodal relationship with elevation and the maximum total species richness is at 1000 m. Paper II which deals with the uses of medicinal plants in the Humla region, west Nepal showed that there are 161 medicinal plant species belonging to 61 families and 106 genera used for treating 72 human and 7 veterinary ailments. Medicinal plants in Humla were mostly collected in wild. This induces a serious threat to diversity of the medicinal plants and it is therefore necessary to develop proper management guidelines for their harvesting in wild and/or their domestication. Rheum australe, an endemic plant to west Himalayan region, is widely used plant in traditional...

Thesis (MTech (Horticulture))--Cape Peninsula University of Technology, 2018. / Deteriorating living and environmental conditions have contributed to the increasing prevalence of diseases in plants and animals. In humans, accumulation of abnormally high levels of free radicals in the tissues has been implicated in many non-communicable diseases, such as diabetes, cancer, arthritis, ischemia, gastritis, obesity and asthma. Worldwide, there is recognition of need to improve plant and animal health. Tulbaghia violacea (Alliaceae) is a medicinal plant that is extensively harvested by traditional healers in the wild for its medicinal uses and if this practice continues, it may result in an unsolicited decline of the species in situ. Therefore, there is a need for cultivation of this species. Plant cultivation in a controlled environment for conservation purposes as well as the enhancement of yield and quality is gaining favour among farmers and consumers. The main aim of this study was to investigate the effects of altering the growing conditions by applying environmental stresses on the plant growth, antifungal and antioxidant activities of T. violacea, with the view of enhancing the future cultivation of this species for pharmaceutical companies, traditional healers and the horticulture industry. This study was divided into two parts, and the first part, which was further sub-divided into two separate preliminary experiments, is presented in chapter three. Simultaneous assessments of the effects of i) varied pH levels (pH 4, pH 6, pH 8) and ii) light intensity on plant growth, antioxidant-content and -capacity of extracts of T. violacea were carried out. The second part of the thesis consisted of a more detailed assessment of the above-mentioned independent variables and interactions thereof on plant growth, and antifungal activity of extracts of T. violacea. Results obtained from the first part of the study, showed that plants exposed to pH 6 showed a marked increase in plant height (from 25-37 cm) after 2 months of treatment although, generally, the variations of the different growth parameters among the pH treatments were not significant (p > 0.05). Antioxidant-contents and -capacity were not significantly different (p > 0.05) when pH treatments were compared. However, a high polyphenol content value (of 3 mg/g) occurred in leaves of plants exposed to pH 8. Overall, comparatively, there was no significant difference (p > 0.05) in antioxidant-content and -capacity when pH treatments. In the light experiment, decreasing light intensity led to the elongation of plant height. A higher mean shoot length of 34.6 cm was obtained under low light compared to normal light (26.5 cm) two months post-treatment. The results obtained in this study indicated that light had a significant affect (p < 0.05) on the vegetative growth of this species. In contrast, normal light intensity yielded higher antioxidant-content and -capacity. The polyphenol and flavanol content were fluctuating between the averages of 5.8 mg/g to 8.5 mg/g. Overall, there was a significant difference (p < 0.05) in the antioxidant-content and -capacity when low and normal light intensity treatments compared. In conclusion, both normal light intensity and at pH 8 induced better antioxidant results. In the second part of the study, chapter four, one-month old T. violacea plantlets were grown under two light intensities (low light and normal light) in a greenhouse and concurrently exposed to varying pH levels: pH 4, pH 6 and pH 8. Plants exposed to normal light received natural sunlight through the roof of the greenhouse, while low light intensity (40% reduction) was achieved using shade nets. Plants were drip irrigated with Nutrifeed fertilizer. Plant growth parameters such as height and fresh and dry weights were determined. Leaf samples were analysed for macro-and micro-nutrients contents. Antifungal tests were carried out on the plant extracts from the various treatments in an antifungal bioassay (minimum inhibitory concentration [MIC]). The experimental data collected were analysed using one and two-way analyses of variance (ANOVA), and Tukey HSD was used to separate the means at p < 0.05 level of significance. Varied effects of different pH levels (4, 6 and 8) and light intensities (low and normal) on plant height, and fresh and dry weights were recorded in the current study. A significant interactive (df, 2; F = 0.001; p < 0.001) effect between pH and light on fresh weight was observed. The results revealed that there was a significant difference (df, 2, 57; F = 12.63; p < 0.001) in dry weights with plants under normal light intensity and pH 4 treatment (8.285 ± 0.802 g) producing the highest dry weight. There was a significant interaction (df, 2; F = 6.4; p < 0.001) between pH and light intensity on plant dry weight. Extracts from plants grown under normal light intensity showed stronger antifungal activity at pH level 4, and MIC values ranged from 0.18 ± 0 to 0.375 ± 0.04 mg/ml at 6h and 1.5 ± 0 to 0.97 ± 0.18 mg/ml at 18h. In conclusion, this study demonstrated the interactive effects of pH and light intensity on the growth of T. violacea. These findings also confirmed that it is possible to enhance the cultivation of T. violacea under greenhouse conditions. Chapter 5 focused on the interactive effects of pH and watering regime on plant growth, nutrient uptake and antifungal activity of T. violacea plant extracts, grown hydroponically. The results showed that there were significant differences (p < 0.05) on plant growth parameters amongst the different watering regimes under normal light intensity. Broadly, two trends occurred in the results: firstly, more macro-nutrients were taken up by plants in the higher frequency watering intervals as opposed to higher tissue micronutrient nutrient values for plants grown under the lower light intensity conditions. The levels of N, P, K, Mg nutrient uptake differed significantly in plants (p < 0.001) among watering interval periods. On the other hand, plants simultaneously exposed to extended watering intervals of 21-day and low light intensity showed more bioactivity of the crude extracts against F. oxysporum in the MIC bioassay. Based on the current results, a combination of shorter watering interval and normal light intensity favoured plant growth and development, while plants grown under low light intensity with longer watering interval showed good bioactivity. Broadly, these results demonstrated that varying pH, light intensity, and watering regime can influence plant growth, secondary metabolite contents and antifungal activity of crude extracts of T. violacea. These findings will contribute to the current body of knowledge around cultivation of indigenous medicinal plants. The study will further benefit the conservation of medicinal plant initiatives, increased income of small-scale farmers and potentially promote indigenous knowledge by increasing the availability of South African medicinal plants.

Dierama is a genus of plants with a potential to be developed as ornamental plants. It falls under the Iridaceae family and comprises of 44 species. Dierama erectum Hilliard, an attractive species with horticultural potential is mainly found in rough wet grasslands. Its corms are used for enemas and treating stomach ailments in southern African traditional medicine. Due to its habitat transformation by afforestation and the exploitation of its underground parts (corms) in traditional medicine, this plant is among the most vulnerable and rare species within its genus. Seed parasitism by Urodon lilli also hampers its conventional propagation. The increase in demand for ornamental and medicinal plants increases pressure on wild plant populations. Micropropagation is a useful tool for clonal propagation of plants as it does not only help in alleviating pressure on wild plants but an effective micropropagation protocol could also provide a foundation for plant genetic transformation, which could result in the development and introduction of new ornamental varieties into commercial markets. This research was aimed at developing a micropropagation protocol for D. erectum to ensure readily available source material for medicinal and horticultural use as well as serving as an alternative for its conservation. Seed decontamination and germination were successful when 0.2% HgCl2 or 2.5% NaOCl + 1% Benlate® were used. However, for safety reasons, 2.5% NaOCl + 1% Benlate® was used in all subsequent experiments. The shoot regenerative capacity of leaf, hypocotyl and root explants obtained from in vitro germinated seedlings was evaluated by culturing them individually on MS medium supplemented with various concentrations of BA. Only hypocotyl explants produced adventitious shoots. Since no shoots or callus was produced from leaf and root explants, hypocotyl explants were used in the development of a micropropagation protocol. Different types and concentrations of cytokinins (BA, mT, KIN and Z) with or without NAA were evaluated for their effect on adventitious shoot production. Maximum shoot number per explant (4.20 ±0.51) was obtained in MS medium supplemented with 1.0 ìM Z after 8 weeks. This was followed by a combination of KIN (2.0 ìM) and NAA (0.5 ìM) resulting in a production of 3.67 ± 0.81 shoots per explant. For BA treatments, the highest shoot multiplication (3.20 ± 0.22 shoots per explant) was achieved when 2.0 ìM was combined with 1.0 ìM NAA. mT gave maximum shoot production (3.09 ± 0.99 shoots per explant) when 2.0 ìM mT was combined with 2.0 ìM NAA. The effects of photoperiod and light intensity were investigated for the purpose of optimizing shoot multiplication. An average of 12.73 ± 1.03 shoots per explant were obtained after 8 weeks from shoots grown in 16 h light at a 100 ìmol m-2 s-1 light intensity. The 24 h light treatments and a light intensity lower than 100 ìmol m-2 s-1 negatively affected growth and regeneration of D. erectum. These results highlighted the need for evaluating environmental conditions when developing micropropagation protocols. Corm induction experiments were done with the intention of facilitating acclimatization of D. erectum ex vitro. Various concentrations of ancymidol, activated charcoal and sucrose did not promote in vitro corm formation, thus auxins (IAA, IBA and NAA) were tested for their efficiency in rooting. Plants rooted successfully after 8 weeks on MS medium supplemented with 1.0 ìM IBA, yielded the longest roots (4.63 ± 0.70 cm) and an average root number of 2.73 ± 0.40. All NAA treatments resulted in stunted roots. Plants grown in vitro were potted in trays containing a 1:1 ratio of soil: vermiculite and placed in the mist house for 2 weeks. They were then transferred to the greenhouse for further acclimatization. After 2 months, plants had formed corms. The largest corms (0.45 ± 0.026 cm in diameter) were found in plants pre-treated with 0.5 ìM IBA. Maximum plant survival percentage (73%) was also associated with this treatment. A successful micropropagation system for Dierama erectum was therefore developed. The utilisation of this protocol can yield about 15137 plants from one explant in a year. This will expand our existing knowledge about micropropagation of plants in the genus Dierama and will be useful in the conservation of this species. / Thesis (M.Sc.)-University of KwaZulu-Natal, Pietermaritzburg, 2009.

Thesis (MScConsEcol(Conservation Ecology and Entomology)--University of Stellenbosch, 2009.

Medicinal bulbs form part of the diversified flora in South Africa. The plants are used extensively in South African traditional medicine in the treatment of various ailments. Due to the ever-increasing demand and the unrestricted collection of medicinal plants from the wild, many of these slow growing bulbous plant species are driven into over-exploitation and extinction. The main parts collected for use are the underground bulbs, leading to the destructive harvesting of the whole plant. This form of plant harvesting poses threats to the long term sustainability of these plant resources from their natural habitats. Sustainable harvesting of these plants should be within the limits of their capacity for self-renewal. However, this seldom occurs with the often inconsiderate medicinal plant gatherers. Conservation of these plants is therefore necessary. A strategy that would take into consideration the sustainable harvesting and perhaps simultaneously provide similar medicinal benefits, would be the substitution of bulbs with leaves of the same plant. This study was aimed at evaluating the seasonal pharmacological and phytochemical properties in bulbs/corms and leaves of medicinal bulbs with a view of promoting the substitution of bulbs with leaves in traditional medicinal use. Four medicinal bulbous plants, Tulbaghia violacea, Hypoxis hemerocallidea, Drimia robusta and Merwilla plumbea were evaluated for the pharmacological and phytochemical properties in their bulbs/corms and leaves in spring, summer, autumn and winter seasons, with a view of promoting the use of leaves as a conservation strategy. Dried plant materials were sequentially extracted with petroleum ether (PE), dichloromethane (DCM), 80% ethanol (EtOH) and water in each season. The extracts were tested for activities against Gram-positive (Bacillus subtilis and Staphylococcus aureus), Gram-negative (Escherichia coli and Klebsiella pneumoniae) bacteria and the fungus Candida albicans using the in vitro microdilution assays to obtain minimum inhibitory concentrations (MIC) and minimum fungicidal concentrations (MFC). The four plant species were also evaluated for their ability to inhibit cyclooxygenase (COX-1 and COX-2) enzymes. Spectrophotometric methods were used to evaluate saponin and phenolic contents of samples from the four plant species in each season. Antibacterial activity was fairly comparable between bulbs/corms and leaves of H. hemerocallidea, T. violacea, and M. plumbea, with at least one extract showing some good activity (MIC < 1 mg/ml) in most of the seasons. Bulb extracts of D. robusta did not show good antibacterial activity while the leaf extracts showed good activity (0.78 mg/ml) against B. subtilis in spring, summer, and autumn and S. aureus (0.78 mg/ml) in autumn. The best antibacterial activity was recorded in winter, with MIC values as low as 0.195 mg/ml from the DCM bulb extracts of T. violacea against K. pneumoniae and S. aureus and PE corm extracts of H. hemerocallidea (0.195 mg/ml) against B. subtilis. Good antibacterial activity from water extracts were only recorded from corm extracts of H. hemerocallidea in summer, autumn and winter, H. hemerocallidea leaf extracts in autumn and winter, and M. plumbea bulb extracts in autumn. The leaf extracts of all the screened plant species demonstrated good fungicidal activity in autumn, with H. hemerocallidea corm water extracts recording an MFC value as low as 0.39 mg/ml. The leaf extracts of H. hemerocallidea (water), D. robusta (DCM) and M. plumbea (DCM) had good MFC values of 0.78 mg/ml each, in spring. The DCM leaf extracts of T. violacea also showed good fungicidal activity (0.78 mg/ml) in summer, while corm water extracts of H. hemerocallidea had an MFC value of 0.39 mg/ml in winter. There were no fungicidal activities recorded from all the bulb extracts in all the seasons. All the PE and DCM extracts in all the tested plant samples recorded between moderate (40-70%) and high (> 70%) COX-1 and COX-2 inhibition levels across all seasons. The EtOH corm extracts of H. hemerocallidea also demonstrated moderate to high inhibitory activity against COX-1 enzyme across all seasons. Bulb and leaf extracts of T. violacea showed selective inhibitory activity for COX-2 enzyme in all the seasons. The highest COX inhibitory levels were recorded in COX-2 from the PE leaf (spring) and bulb (autumn) extracts of T. violacea, with both recording 100% inhibitory activity. Phytochemical analysis revealed higher total phenolic compounds in bulbs/corms and leaves of all the analysed plant species, to be either higher in spring or winter. Plant material collected in autumn had the least levels of total phenolics. An almost similar trend to that of total phenolics was observed for flavonoids, gallotannins and condensed tannins in most plant samples, with higher levels either in spring or winter. Total saponins were consistently higher in winter than in the other seasons in all the screened plant species. There were in some cases, relationships between the peaks in the levels of some phytochemical compounds and the observed levels of bioactivity in different assays. The results obtained from this study demonstrate that the leaves of the screened plant species may substitute or complement bulbs in the treatment of certain ailments in traditional medicine. Thus, plant part substitution can be sustainably utilised in the conservation of these plant species while retaining the same medicinal benefits. / Thesis (M.Sc.)-University of KwaZulu-Natal, Pietermaritzburg, 2010.

Many South African medicinal plants face the threat of over-collection for use in traditional medicines. Many bulbous plants suffer as the whole plant is removed from the wild so that the bulb may be used for medicine. Micropropagation is a technique which can be used as an alternative to conventional propagation methods. Micropropagation produces many plantlets in a relatively short period of time. Different plant parts of Brunsvigia undulata F.M. Leight, a rare South African species of medicinal value, were used in an attempt to produce in vitro plantlets using micropropagation techniques. Although leaf and floral explants were successfully formed from seedling explants and twin-scales. Seeds germinated quickly in culture. Seedlings which grew from seeds were cut into sections and used to initiate bulblets. Seedling explants formed bulblets, shoots and callus best when the explants included a meristematic region. Callus from seedling explants formed shoot clusters readily when placed on hormone-free MURASHIGE and SKOOG (1962) (MS) medium. Shoots from shoot clusters formed bulblets and rooted on medium supplemented with IBA. The greatest rooting response was achieved by bulblets on 1 mgl-1 IBA. The callus which was left after shoot clusters were separated was placed back onto hormone-free MS medium. Callus explants continued to form shoot clusters. Twin-scales, cut from large parent bulbs, were cultured on 25 hormone treatments. Bulblets formed on twin-scales even in the absence of plant growth hormones. Bulblets formed by twin-scales were used to determine the effects of both medium constituents and environmental factors on bulblet multiplication. Bulblet multiplication was greatest when bulblets were split in half and cultured as half-bulblets. Optimal multiplication was achieved on hormone-free MS, with 4% sucrose, kept at high temperatures in the dark. Bulblets were successfully initiated and multiplied from both seedlings and twin-scales. Bulblets which were produced via both protocols were acclimatized relatively easily. Both explant types could be used to mass propagate Brunsvigia undulata. / Thesis (M.Sc.)-University of KwaZulu-Natal, Pietermaritzburg, 2009.

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.