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Masters Degrees (Research Centre for Plant Growth and Development)

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    A comparative evaluation of the biological activities and phytochemical properties in Ehretia obtusifolia and Ehretia rigida.
    (2021) Mnikathi, Mzamo Mpendulo Ntethelelo.; Finnie, Jeffrey Franklin.; Van Staden, Johannes.
    Ehretia from the Boraginaceae family is predominantly found in parts of Asia and North America, with fewer species found in Africa, Europe, and Australia. The genus consists of more than 150 species, and species such as E. microphylla, E. accuminata, E. laevis have been reported on their medicinal prowess. Distribution of Ehretia in Southern Africa is found among a variety of habitats such as the lush forests of the Eastern Cape and Arid parts of Namibia. In South Africa, two species have been identified, namely E. rigida and E. obtusifolia and are used in traditional medicine. African and Asian countries traditional medicine is highly recommended because of its affordability. The aim of the study was to establish a baseline and compare different biological activities and phytochemical properties exhibited by the two South African species. In the study, phenolics, saponins, flavonoids, and tannins were detected in bark, roots, and leaves, of both species, but no detection of alkaloidsf. E. obtusifolia had a higher quantity of flavonoids than E. rigida. Both species exhibited high phenolic quantities in leaves with E. rigida having the highest quantity. Condensed tannins were found with a higher content in leaves than roots and bark for both species, with E. rigida containing higher quantities. E. rigida had the lower MIC’s compared to E. obtusifolia (0.195 mg/ml against M. luteus from ethyl-acetate root extracts). E. rigida had more samples with a MIC lower than 1 mg/ml than E. obtusifolia. The lowest MIC for leaves was 0.39 from ethyl-acetate extracts against S. aureus while methanol bark extracts also achieved 0.39 mg/ml against M. luteus. E. obtusifolia’s lowest MIC was 0.195 mg/ml from methanol leaf extracts against K. pneumoniae. Activity against C. albican was not as good as against the bacterial strains, as the lowest MIC was 0.78 mg/ml for both species. E. rigida and E. obtusifolia had dose-dependent antioxidant activity, with methanol and ethyl-acetate bark, leaf, and root extracts having the highest activities for both species. This study revealed that in comparison to literature, the activity achieved was similar or better when compared to the likes of E. laevis extracts. The α-glucosidase inhibitory activity reported in this study was dose-dependent. The relationship between antioxidant activity and antidiabetic activity is well documented and this study found that extracts with high antioxidant activity also had similarly high α-glucosidase inhibitory activity. It was E. rigida methanol extracts from bark and roots which exhibited the highest activities compared to E. obtusifolia. However, based on the dosedependent activity, E. obtusifolia is more potent because of the higher activity observed at the lowest concentrations. The study demonstrated that both species have good ethnopharmacological properties and were rich in phytochemicals, particularly phenolics and flavonoids. With E. rigida being the least studied of the two species with only one reported study, it was important to carry out this investigation as it has yielded further evidence that the genus Ehretia has multiple species with medicinal potential.
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    Propagation of Sceletium tortuosum (L.) N. E. Br.: a South African medicinal plant.
    (2019) Sreekissoon, Amrisha.; Van Staden, Johannes.; Finnie, Jeffrey Franklin.
    Abstract available in PDF.
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    Impacts of climate change on cowpea (Vigna unguiculata L. Walp) treated with biostimulants.
    (2020) Voko, Mxolisi Peter.; Van Staden, Johannes.; Finnie, Jeffrey Franklin.
    Vigna unguiculata L. Walp, commonly known as cowpea, is a warm-season herbaceous legume considered native in Africa and Asia. The crop is traditionally consumed as both a leafy vegetable and staple pulse. Although the growth behaviour and nutritional composition of V. unguiculata have been explored by the research community, information regarding the plant’s response to biostimulants under abiotic stresses remain limited. Therefore, studies of this nature are pertinent, especially in the presence of climate change which manifests into global warming, drought episodes and dissipating of natural resources. Hence, a better understanding of the effects of temperature and drought stress on V. unguiculata physiology, morphology, nutrition and phytochemistry are important to ensure high yields which is important for meeting the goals of global food security. Firstly, this study investigated the effects of seed priming with biostimulants [vermicompost leachate, VCL (1:20 v/v), commercial seaweed extract Kelpak®, KEL (0.6%) and smoke-water, SW (1:1000 v/v)] and distilled water (dH2O) on V. unguiculata germination and seedling parameters under constant day/night temperatures of 30/30, 35/35 and 40/40 °C in Conviron® plant chambers. In addition, hydroponic experiments were set-up to evaluate biostimulant efficacy on rooting. Secondly, post-germination effects of VCL 1:20 (v/v), KEL (0.6%) and SW 1:1000 (v/v) were evaluated under similar temperature conditions to ascertain the influence on morphological parameters 28 days after sowing (DAS). Thirdly, postgermination effects of VCL 1:20 (v/v), KEL (0.6%) and SW 1:1000 (v/v) were investigated under different watering regimes using greenhouse protocols to ascertain biostimulation influence on growth variables and flowering after 13 weeks. Finally, the effects of VCL 1:20 (v/v), KEL (0.6%) and SW 1:1000 (v/v) were tested on V. unguiculata’s photosynthetic pigments [chlorophyll a, b, (a + b) and carotenoids], carbohydrates, proteins and phytochemicals (total phenolics and flavonoids) grown for 11 weeks under different watering regimes in the greenhouse. Despite the biostimulants not differing significantly with the corresponding controls, KEL and SW induced marked germination at 30, 35 and 40 °C while VCL being potent under 30 and 40 °C. Seed priming (i.e. biostimulant and hydropriming) significantly improved shoot length and root length over non-priming, with biostimulant-priming being more effective under 40 °C by also inducing significantly higher leaf number, fresh weights and seedling areas compared to non-primed controls. At 30 °C, priming with the three biostimulants improved peduncle diameter, fresh weight and established a significant increase on root length and dry biomass over hydropriming. VCL was most effective at promoting shoot length, root elongation and dry weight under 30 °C while KEL was most effective in increasing seedling leaves, shoot length, root number, fresh and dry biomass of plants exposed to 35 °C. Although overall, biostimulant-non-priming was second best after biostimulantpriming, non-priming with biostimulants was able to promote key variables compared to both controls (i.e. non-primed and hydroprimed controls). Hydroponic results revealed that non-priming with VCL and SW increased root number by 3 and 4-fold, respectively, at 40 °C. SW also stands out at enhancing a significant increase in leaf number and seedling area whereas KEL was the most significant solution promoting shoot length and seedling area. At 30 °C, SW-non-priming promoted significant root elongation, improved fresh and dry weights while VCL was best at promoting root number and root length. KEL and SW also exhibited post-germination effects over the control at 40 °C by improving leaf number on a weekly basis. Fresh and dry weight were improved similarly with significant improvements at 30 °C by KEL and SW. Increasing watering regimes from once to thrice a week significantly increased the number of leaves, root length and flower number. The number of nodules, however, did not differ significantly. Restricting watering frequency to once a week significantly increased shoot length, root length and leaf area in SW-treated plants compared to the control. Shoot length and root length of KEL-treated plants were also increased similarly. Remarkably, VCL increased the number of nodules and shoot length by 4 and 3-fold, respectively. Relative leaf weekly growth in SW, VCL and KEL was higher by 1, 4 and 5 leave(s), respectively, after 11 weeks under high water deficits. This foliage increase remained high by more than 4 leaves in KEL and SW-treated plants watered twice and thrice a week. Accompanying increase in number of flowers was only established in SW water-stressed plants. However, raising watering frequency to twice a week increased flower number in SW, VCL and KEL-treated plants by 2, 4 and 7-fold, respectively, compared to the control. This floral increase was still comparatively high by 2, 4 and 2-fold, respectively, in plants watered thrice a week with biostimulants. VCL also induced a marked significant increase on root length, peduncle diameter and dry weights of plants watered thrice a week. Decreasing substrate water availability from thrice to once a week induced a general increase in leaf soluble proteins, total phenolics and flavonoids. This watering transition significantly enhanced root soluble carbohydrates and proteins while root phenolics and flavonoids markedly declined. VCL, KEL and SW promoted leaf carbohydrates coupled with significant increases in those of roots of SW plants compared to the corresponding controls. Remarkably, leaf soluble proteins of biostimulant plants significantly declined to within the ranges of the plants watered twice and thrice a week. Root proteins were significantly greater to those of leaves in high water-stressed plants and statistically the same to those of roots of plants watered twice and thrice a week. Total phenolics and flavonoids of foliage of the biostimulant plants were lowered and relatively the same in the different watering regimes. Root total phenolics were highly inhibited in less watered plants and gradually increased with an increase in watering regimes. Similar trends were established in flavonoids although they were greater than in the corresponding controls. Biostimulant photosynthetic pigments [i.e. chlorophyll a, b, (a + b) and carotenoid contents] did not differ significantly with those of the control in plants watered once a week. However, the three biostimulants were able to improve chlorophyll a and a + b. KEL and SW induced higher increase in chlorophyll b and carotenoid concentrations. The biostimulants increased chlorophyll a in 3-day-watered plants by more than 2-fold. These biostimulants also improved the chlorophyll a + b and carotenoid contents. Both increasing and decreasing trends in compatible solutes (i.e. soluble sugars and proteins), photosynthetic pigments and phytochemicals under water deficits indicated biostimulant-induced capacity in cowpea for osmotic adjustment, drought tolerance or adaptive mechanisms to water stress. These findings demonstrated the biological potential of VCL, KEL and SW to improve germination, seedling/plant growth and yield in legumes even under temperature stress and drought stress. Thus, establishing their stress amelioration properties to offset negative impacts of climate change on plants and yield.
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    A phylogeny-based comparative study of the phytochemical and pharmacological characteristics of Croton species occurring in KwaZulu-Natal.
    (2021) Mathe, Tanya.; Bytebier, Benny Leopold Germaine.; Finnie, Jeffrey Franklin.; Van Staden, Johannes.
    Ethnobotanical enquiries often lead to the discovery of phytocompounds with pharmacological activities. Against this background a comparative and quantitative evaluation of the phytochemical and antioxidant activity of extracts from six Croton species; C. gratissimus Burch., C. sylvaticus Hochst., C. menyhartii Pax, C. pseudopulchellus Pax, C. steenkampianus Gerstner and C. rivularis Müll.Arg., all collected from KwaZulu-Natal (KZN), South Africa, was conducted. The analysis included a comparison of the different plant organs to explore the possibility of using leaves rather than bark for medicinal purposes. The latter would result in less destructive harvesting and would contribute to sustainable use of these medicinal plant resources. Extraction of the different plants and their organs were done in water and, in different organics solvents, including methanol (MeOH), dichloromethane (DCM), and petroleum ether (PE). The extracts were screened for antibacterial and antifungal activities using the microdilution technique. All of the tested plant samples showed some notable antibacterial activity in one or two of their organs, except for C. rivularis, which was the only species in the list that had no record of medicinal use. The most potent antibacterial activity was exhibited by the dichloromethane (DCM) extracts of C. steenkampianus leaves and the petroleum ether (PE) extracts of C. pseudopulchellus stem bark, both sampled from the Durban Botanic Gardens, which yielded a minimum inhibition concentration (MIC) value of 0.04 mg/ml against Enterococcus faecalis (E. faecalis). The DCM stem bark and leaf extracts, as well as the PE twig extracts of C. pseudopulchellus, (Durban Botanic Gardens) also exhibited noteworthy activities against S. aureus (MIC value of 0.08 mg/ml). A broad spectrum of activity was observed in the DCM and PE twig extracts of C. sylvaticus collected from Umdoni Park, with a MIC ranging from 0.31-0.94 mg/ml. This activity was against E. faecalis, Staphylococcus aureus (S. aureus) and Klebsiella pneumoniae (K. pneunomiae). Noteworthy antifungal activity against Candida albicans was only observed in one extract, the MeOH leaf extract of C steenkampianus collected at Kosi Bay. The MIC of this extract was 0.6 mg/ml. Water extracts did not show any antimicrobial activity. The results of the pharmacological study suggested that the aerial plant organs, such as the leaves and the twigs, could replace bark as they exhibited significant antimicrobial activity when compared to the preferred bark. The study also revealed that the same species collected from different regions may not necessarily exhibit similar biological activities, as pharmacological activities are as a result of the phytochemicals present in the plant, which are triggered by environmental stimuli. The phenolic profiles of aqueous (50%) methanol extracts obtained from the plants were assessed using the Folin and Ciocalteu (Folin C), butanol-hydrochloric acid and aluminium chloride assays. Aqueous (50%) methanol extracts were also run on thin layer chromatography plates and the plates were later stained with the Dragendorff reagent to determine the possible presence of alkaloids. Antioxidant activity was determined with two different assays, β-carotene /Linoleic model system and 1,1-diphenyl-2-picryl hydrazyl (DPPH) radical scavenging assay. The overall EC50 values of the different Croton species displayed by the DPPH assay ranged from 1.76 to 5.35 μg/ml. The β-carotene /Linoleic model system displayed antioxidant activity that ranged between 48.66 to 81.97%. In the phytochemical study; the leaf extracts of C. pseudopulchellus from Mkuze exhibiting the highest phenolic content at 23.8±1.1 mg GAE/g Dry Weight. The highest condensed tannin content was from the leaf extracts of C. gratissimus from Southport at a concentration of 31.3± 0.1 mg CCE/g Dry Weight. The highest flavonoid content observed in the leaf extracts of C. gratissimus from Southport at a concentration of 31.2±0.7 mg CE/g Dry Weight. Higher phytochemical contents were also observed in the leaves and twigs. These phytochemicals are believed to be the reason for most of the notable antimicrobial activities exhibited by these plant organs. The mutagenic potential of the most biologically active Croton extracts was tested. An Ames with two Salmonella tester strains (TA98 and TA102) revealed that the species are not toxic as they did not produce His+ revertant colonies in Salmonella tester strains that were more than twice the number of His+ revertant produced by the positive control, Nitroquinoline-N-oxide (4-NQO). Standard DNA barcodes of the Croton species occurring in KZN were generated, since these are useful in plant identification and authentication. These were used to run a phylogenetic analysis in order to assess whether the phytochemical profile is clade-specific. The results showed that the quantity and quality of phytochemicals closely related species may vary. DNA barcoding may be a useful tool in medicinal plant identification, especially where fragments of the plants are traded and morphological identification is not possible. In this study, the biological activity of different parts of six Croton species occurring in KZN, was investigated with the aim of replacing bark as the source of local medicine with other plant parts (leave and twigs) that can be more sustainably harvested, as this will contribute to the conservation of these species.
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    Antidiabetic and phytochemical properties of four selected medicinal plants.
    (2019) Ratsoma, Manchela Francinah.; Van Staden, Johannes.; Finnie, Jeffrey Franklin.
    Abstract available in pdf.
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    Enhancing phenolic compound production in medicinal plants.
    (2017) Paine, Christine Susan.; Finnie, Jeffrey Franklin.; Van Staden, Johannes.
    Abstract available in PDF file.
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    Micropropagation of three Brachystelma species and investigations of their phytochemical content and antioxidant activity.
    (2017) Hlophe, Nqobile Prisca.; Finnie, Jeffrey Franklin.; Van Staden, Johannes.
    Local communities in most African countries possess a wealth of knowledge on the uses of plants in the environment. Documentation of this knowledge is not a common practice among these communities and therefore the knowledge is in the process of disappearing as the younger generation is more inclined towards the western lifestyle. The knowledge of African communities on the use of medicinal plants has provided and continues to provide leads towards therapeutic concepts which accelerate the pace of drug discovery. This makes the use of medicinal plants an important line of research to be pursued. The use of plants, particularly medicinal plants, as a resource is generally accompanied by the concern of over exploitation. Exponential population growth along with newly emerging and resistant diseases as well as habitat destruction due to human developmental activities are among the reasons for the decline of plant biodiversity. There is therefore an urgent need to develop effective means of conservation specific to Brachystelma species as a medicinal group, and also to document not only the uses but also the constituents and pharmacological activity of these plants. Brachystelma R. Br. ex Sims is a genus of geophytic plants used traditionally in some parts of the world including East Africa, southern Africa, West Africa, northern and western India. Apart from being used as a food source, they are used for the treatment of illnesses such as colds, chest pains, wounds, and as an appetite suppressant and for enhancing fertility. The aim of this study was to establish efficient micropropagation protocols for Brachystelma species, namely B. ngomense (Endangered - EN), B. pulchellum (Vulnerable - VU) and B. pygmaeum (Least Concern - LC), as a means of ensuring their survival, and to explore their phytochemical and pharmacological properties. A number of Brachystelma species have been listed as medicinal herbs. There is currently no documented approach towards the mass propagation of Brachystelma species and there are no available scientific studies validating the ethnomedicinal use of these plants. Traditional uses indicate that Brachystelma is commonly used and this is accompanied by the concern that wild populations, which are the only available ones, are under threat for various reasons. Development of optimal tissue culture protocols such as micropropagation and callus cultures may alleviate conservation concerns. In addition, these protocols would make available the possibility of utilization of the plant as a daily food and towards production of biological compounds with potential health benefits. The development of a micropropagation protocol for the three Brachystelma species made use of nodal explants (~10 mm in length). The effects of different concentrations of N6-benzyladenine (BA), 2-isopentenyladenine (iP) and meta-topolin riboside (mTR) supplemented into Murashige and Skoog (MS) (1962) media were tested over a period of 6 weeks. The specific concentrations used were 1.0, 5.0, 10 and 25 μM for each of the cytokinins without auxins. An increase in concentration of all the cytokinin treatments was found to typically result in significantly higher shoot proliferation. However, each species differed in response to each specific cytokinin, the optimal concentrations were 25 μM mTR, 25 μM iP and 25 μM BA for B. ngomense, B. pulchellum and B. pygmaeum respectively. The conclusion drawn from these results is that the application of BA, iP or mTR is beneficial for in vitro shoot proliferation from nodal explants of the three species. Regenerated shoots were rooted ex vitro after exposure to a 3 min pulse treatment using 100 mgl-1 indole-3-butyric acid (IBA). Shoots regenerated from a plain MS medium were typically found to have rooted prior to IBA treatment, however, these shoots as well as the ones derived from other treatments yielded poor rooting ex vitro. Survival of these shoots in the greenhouse was short-lived. As a result, acclimatization of all three species has been extremely limited thus the micropropagation protocol is not effective on a commercial scale. A number of assays were used to evaluate the phytochemical content of the three species of Brachystelma. Vacuum filtered methanolic extracts were used for the determination of phenolics and flavonoids. Absorbance readings obtained using a spectrophotometer were further converted to concentration of compound per gram of extract. The phenolic and flavonoid contents varied between the three species. Higher phenolic and flavonoid content was found in leaf extracts. The effect of plant growth regulators (PGRs) i.e. BA, iP and mTR on specific phenolic acids was also observed. The cytokinin treatments were found to have a stimulatory effect on some of the phenolic acids. Pharmacological properties against oxidative stress were tested using the 2,2-Diphenyl-1-picryl hydrazyl (DPPH) and Oxygen radical absorbance capacity (ORAC) assays. The potency of the antioxidant activity of the three species was compared to the standard antioxidant, ascorbic acid. The activity of the standard antioxidant was found to not be significantly different to the plant extracts. B. ngomense leaf extracts (13.5 μg/ml) were more potent in comparison to ascorbic acid (14.5 μg/ml). The effect of plant growth regulators (PGRs) i.e. BA, iP and mTR on antioxidant activity was also observed. In some instances, cytokinin-treated plant extracts showed an increased antioxidant capacity compared to those not treated.
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    Evaluation of plants used in African traditional medicine for asthma and related conditions.
    (2014) Motlhatlego, Katlego Ellena.; Van Staden, Johannes.; Finnie, Jeffrey Franklin.
    Traditional medicine is a form of discipline that has been applied within most South African societies with the objective of enhancing the physical and psychological health system in the country. Asthma is a complex inflammatory disease that involves the narrowing of the airways. The prevalence of asthma is increasing worldwide, and this chronic disease has been identified as a significant cause of morbidity and mortality. Asthma poses a major threat to health across the population of South Africa. The adverse effects of current treatments have encouraged the use of traditional medicine. The primary aim of the research study was to evaluate the efficacy of plants used in African traditional medicine against asthma and chest infections. This was achieved by screening Adansonia digitata, Ballota africana, Catha edulis, Datura stramonium, Pelargonium sidoides, Siphonochilus aethiopicus, Xerophyta retinervis and Zantedeschia aethiopica for their pharmacological properties against key bacteria; Staphylococcus aureus (ATCC 12600), Klebsiella pneumonia (ATCC 13883), Streptococcus pyogenes (ATCC 12344) and Haemophilus parainfluenzae (ATCC 7901) as well as the fungus Candida albicans (ATCC 10231) these microorganisms are known to cause chest infections. In the microdilution antibacterial assay, the crude extracts of the screened medicinal plants showed activity at minimal inhibitory concentrations (MICs) ranging from 0.098 to >12.5 mg/ml. In the disc-diffusion assay, only the ethanol extract of stems from Siphonochilus aethiopicus and water extract of leaves from Zantedeschia aethiopica showed zones of inhibition of 13.24 and 21.10 mm. All the other screened extracts showed no zones of inhibition, which may possibly indicate that plants were ineffective against Haemophilus parainfluenzae. One or more extracts from the tested plants were effective against one or both Gram-positive bacteria investigated in the study. There was no good antifungal activity shown in the study as the MIC and minimal fungicidal concentrations (MFCs) values were higher than 1 mg/ml. Genotoxicity of medicinal plant extracts that showed good antibacterial activity ≤ 0.5 mg/ml was evaluated using the Salmonella microsome assay without S9 metabolic activation. Two strains of Salmonella TA98 and TA102 were used in the Ames test. Most tested extracts were non-mutagenic in the Ames test except for the Siphonochilus aethiopicus roots which showed a dose dependent increase. The ethanolic crude extracts were screened in an immunological assay to determine the level of competitive binding to the receptors for the treatment of asthma and related conditions. Histamine is intimately associated with allergies. Datura stramonium flowers and fruits experienced remarkable histamine binding of approximately 97% at both concentrations (400 and 800 μg/ml). The immunological activity may be attributed to the various phytochemical constituents in the crude extracts. Ballota africana leaves and stems, Datura stramonium flowers and fruits, roots and stems as well as Zantedeschia aethiopica leaves showed excellent affinity with histamine ranging between 88 and 97% and these medicinal plants could potentially serve as a new effective antihistamine when compared to the currently available pharmaceuticals. Most of the medicinal plants tested may potentially provide remedies for asthma and related conditions such as eczema, rhinitis (hayfever), anaphylaxis, sinusitis, chronic obstructive pulmonary disease (COPD), emphysema, bronchiectasis and bronchitis.
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    Breeding systems of some cold tolerant eucalyptus species.
    (2002) Jones, Wayne Russell.; Van Staden, Johannes.
    Seasonal flowering times for Eucalyptus nitens, E. dunnii, E. smithii, E. macarthurii and E. grandis were evaluated in clonal grafted orchards located at the Shaw Research Centre (SRC) in KwaZulu-Natal, South Africa. The orchards are situated at 29° 29 'South, 30° 11 'East at 1100 m above sea level. The climate is cool (MAT 16.7° C) with a January mean monthly maximum of 25.8° C and July minimum of 4.4° C. An estimated mean annual rainfall of 998 mm and median annual rainfall of 899 mm has been reported (PALLETT and MITCHELL 1993). It is evident that the different species flower consistently from one year to the next during the same period with similar mean flowering peaks. Long reproductive sequences where identified for all species relative to E. grandis, particularly E. smithii and E. dunnii. Paclobutrazol was used to initiate flowering to facilitate the study of the breeding systems of the different species. When applied as a soil drench during early summer an increase in the flower bud production in E. nitens, E. smithii and E. grandis was achieved. The use of various cytochemical methods to test pollen viability, were shown to be mere indicators of potential viability and lack the reliability for adequate testing of stored pollen. From the range of in vitro, pollen viability studies the most successful media for all species tested was 30 % sucrose with 150 mg r¯¹ boric acid. Without boric acid in the media, the response after 24 h was significantly poorer (p<0.001). Significant differences (p<0.05) in the area of pollen grains were found between and within species. There was no significant difference between E. dunnii and E. macarthurii at the species level. Pollen of E. smithii, E. grandis and E. nitens were significantly smaller than that of both E. dunnii and E. macarthurii. From isolation experiments which limited potential pollinators it is apparent that a reduction of pollinators not only leads to poorer capsule survival but also poorer seed set. Following an initial survey of pollinators of E. grandis, very few insects were recorded relative to surveys conducted in the natural habitats with indications that an association does exist between the presence of active pollinators and temperature. The potential of flowers to set seed is clearly demonstrated by the difference between open pollinated flowers and controlled pollinated flowers following intraspecific crosses where differences in seed yield per capsule are very often more than double for species such as E. nitens and E. macarthurii. Similarly with interspecific crosses, higher seed yields are extracted from crosses between closely related species. An extensive survey of orchards clearly demonstrates that E. nitens has the lowest clean seed recovery (13.8 %) significantly less than that of E. smithii (18.0 %) and both E. macarthurii and E. dunnii at 26.1 % and 26.0 % respectively.
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    Factors influencing controlled pollination of Pinus patula.
    (2002) Nel, André.; Van Staden, Johannes.
    A study of factors contributing to successful controlled pollinations of Pinus patula Scheide et Deppe was undertaken. The pollen morphology of P. patula, P. oocarpa, P. greggii, P. elliottii, P. tecunumanii, P. caribaea and P. radiata was studied and the mean size of pollen grains was determined for these species. Clonal differences in pollen size within P. patula were also determined. The impact of pollen management practices on pollen viability was highlighted and a protocol for in vitro pollen viability testing of P. patula and other pine species was determined. A one percent agar solidified distilled water medium gave the best germination results after 72 hours incubation at 30 °C for a number of different Pinus species and P. patula clones. The addition of boric acid increase germination, although not significantly. The addition of sucrose to the pollen germination medium had a negative effect on pollen germination of P. patula, P. greggii and P. caribaea. Re-hydration of pollen for two hours prior to in vitro germination testing improved germination significantly. Incubation temperatures of above 38 °C were detrimental to germinating pollen grains. Stored pollen with low humidity (less than 10 %) of P. patula, P. greggii and P. caribaea could tolerate temperatures of up to 70 °C while still retaining some level of viability. The initiation and growth of the pollen tube was also studied and differences in pollen tube-lengths germinated at 30 °C for 72 hours were found between species studied. Flowering of different P. patula clones was monitored over seven seasons. Flowering periods varied in length between 4 and 14 days amongst five clones over the different seasons. The best cone-survival after controlled pollination was achieved with breathable micro-fibre material. Seed yields were also highest when breathable material was used for controlled pollination. The role of pollen viability in controlled pollination was also determined in pollination studies with low viability resulting in low cone survival and low seed yields. The temperature and relative humidity inside isolation bags were monitored and temperatures above 40 °C were reached inside bags constructed of nonbreathable material. These temperatures were lethal to pollen germinating in vitro. Relative humidity of between 80 and 100 % was maintained in non-breathable bagging material, constituting a risk of diseases causing cone-mortality. The application of fungicide before, during and after controlled pollination was ineffective in improving cone survival.
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    Micropropagation and acclimatization of Aloe polyphylla and Platycerium bifurcatum.
    (2001) Chukwujekwu, Jude Chinedu.; Van Staden, Johannes.; Fennell, Catherine W.
    Shoot cultures of Aloe polyphylla were initiated from young shoot explants of in vitro grown plants. The basal medium was MS medium (MURASHIGE and SKOOG, 1962), supplemented with 100 mgl ¯¹ myo-inositol, and 30 gl ¯¹ sucrose. Agar (0.8 %) was used as the gelling agent. Different cytokinins, singly or in combination with auxins (IBA and NAA), were tested for shoot proliferation activity. All the cytokinins tested (kinetin, zeatin, iP, and BA) gave a good shoot proliferation response. The optimal concentrations for shoot proliferation of each of the cytokinins tested were: zeatin (0.5 mgl ¯¹), kinetin (1.5 mgl ¯¹), iP (1.0 mg ¯¹) and BA (1.5 mgl ¯¹). In combination with auxins, the optimal combinations were kinetin/NAA (2.0/0.1 mgl ¯¹), kinetin/lBA (1.5/1.0 mgl ¯¹), zeatin/lBA (1.0/0.5 mgl ¯¹), zeatin/NAA (1.0/1.0 mgl ¯¹), BA/IBA (1.0/1.0 mgl ¯¹), BA/NAA (1.5/0.1 mgl ¯¹). Although it gave the highest number of shoots per explant, BA was responsible for hyperhydricity. Temperature and sucrose also influenced shoot proliferation. The optimal temperature was 25°C, while 30 gl ¯¹ was the optimal concentration of sucrose for shoot proliferation. Plants rooted well in plant growth regulator-free MS medium. Amongst the potting mixtures tested, soil: sand: vermiculite (1:1:1 v/v) was the best with 98 % plantlet survival. In the second part of this project, Platycerium bifurcatum cultures were established using leaf explants. The basal medium was MS medium (MURASHIGE and SKOOG, 1962), supplemented with 100 mgl ¯¹ myo-inositol and 30 g l ¯¹ sucrose. For bud initiation, 1.0 mgl ¯¹ BA was used, while 0.8 % agar was used as the gelling agent. Three different strengths of MS medium (full, half, and one-quarter strength) without plant growth regulators were tested for further bud growth and development. Half-strength MS proved to be the best for further bud growth and development. Rooting was best achieved in one-quarter strength MS medium without plant growth regulators. In vitro grown plantlets were successfully acclimatized using peat as the potting medium.
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    An investigation of the medicinal properties of Siphonochilus aethiopicus.
    (2002) Light, Marnie Elizabeth.; Van Staden, Johannes.; Jäger, Anna Katharina.
    Siphonochilus aethiopicus (Schweinf.) B.L. Burtt (Zingiberaceae), commonly known as wild ginger, is a highly sought after plant for use in traditional medicine in South Africa. Over-exploitation of this medicinal plant has resulted in regional extinction in the wild. As a result, there is great interest in the medicinal properties of S. aethiopicus, and as a plant for small scale cultivation to increase the supply for use in traditional medicine. Water, ethanol and ethyl acetate extracts were prepared from the leaves, rhizomes and roots of S. aethiopicus. These extracts were tested for in vitro anti-inflammatory activity in the cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2) assays, and in the microdilution antibacterial assay. The aqueous extracts showed no significant prostaglandin synthesis inhibition in the COX-1 and COX-2 assays. The ethanol and ethyl acetate extracts of the leaves showed the highest levels of activity at a concentration of 250 µg ml¯¹ per test solution, in both the COX-1 and COX-2 assays. The ethanol and ethyl acetate extracts of the rhizomes and roots also had moderate levels of activity in the COX-1 assay. These results provide some evidence for the rational use of S. aethiopicus in traditional medicine for anti-inflammatory purposes. In the microdilution antibacterial assay, no inhibitory activity against the test bacteria was detected with the aqueous extracts. The ethanol and ethyl acetate extracts tested showed greater antibacterial activity at minimal inhibitory concentrations ranging from 0.78 to 3.13 mg ml¯¹ against the gram-positive bacteria (Bacillus subtilis, Staphylococcus aureus) than the Gram-negative bacteria (Escherichia coli, Klebsiella pneumoniae). No distinct differences were observed between the ethanol and ethyl acetate extracts, or between the different plant parts. A serial extraction of S. aethiopicus rhizome material was conducted and the extracts were tested in the COX-1 assay and the microdilution assay as a preliminary investigation for a bulk extraction. The hexane and ethyl acetate extracts gave slightly higher COX-1 inhibition than the ethanol extract. No distinct differences were observed in the microdilution assay. A bulk ethyl acetate extract of S. aethiopicus rhizome material was prepared, yielding 6.3 g of a thin orange oil. Vacuum liquid chromatography (VLC) was used to fractionate ≈4 g of the extract. The VLC fractions were evaluated using thin layer chromatography (TLC) and a bioautographic assay, using S. aureus as a test organism. The fractions were also tested in the COX-1 assay. The bioautography revealed a number of compounds which exhibited antibacterial activity. Fraction C was purified further using preparative TLC, and 24.9 mg of a pure compound from R,0.54 (toluene:ethyl acetate 93:7) was isolated. The structure of the compound was elucidated from nuclear magnetic resonance (NMR) spectra, and mass spectroscopy of the compound was also recorded. The compound was identified as the sesquiterpenoid furanoeremophil-2-en-1-one, which is structurally identical to the recently reported compound 4aαH-3,5α,8aβ-trimethyl-4,4a,9-tetrahydro-naphtho[2,3-b]-furan-8-one. The compound showed only a very minimal bacteriostatic effect in the microdilution assay. S. aethiopicus plants were harvested before and after seasonal senescence. Ethanol extracts were prepared from fresh or dried material of the leaves, rhizomes and roots, and tested in the COX-1 assay and the microdilution assay TLC fingerprints of the various extracts were also prepared. No noteworthy changes in COX-1 inhibition, due to senescence, were observed with extracts prepared from fresh material, although there did appear to be a slight decrease in activity in the α-roots and an increase in the β-roots after senescence (fresh and dry). A decrease in the antibacterial activity of the leaves and an increase in the antibacterial activity of the α-roots was observed after senescence. These results suggest that the time of harvest may only have a minimal influence on the degree of anti-inflammatory and antibacterial activity.
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    Cytokinins and the germination of Tagetes minuta L.
    (2003) Gold, John David.; Van Staden, Johannes.; Stirk, Wendy Ann.
    Tagetes minuta L. is a weedy herb that has been a rich source of fragrant oils, used as in the perfume and flavour industry. T. minuta achenes germinate erratically under field conditions. However, at the optimal germination temperature of 25 °C, 100 % germination is attained within 48 h of imbibition. The achenes are thermoinhibited at 35 °C. The aims of this project were to assess the role of cytokinins (CKs) in normal germination at 25 °C, and to investigate the factors that regulate thermoinhibition at 35 °C. CKs were extracted from achenes germinating at 25 °C at 0, 24; 48; 96 and 144 h after imbibition. Two different purification techniques were used, namely Dowex cation exchange resin followed by paper chromatography, or high performance liquid chromatography (HPLC). CK-like activity was tested with the soybean callus bioassay. With both techniques, a peak in CK-like activity appeared 24 h after imbibition, which coincides with the period during which most of the achenes germinated. For quantitative analysis, HPLC\mass spectrometry (MS) techniques were used. The isoprenoid CKs were far more abundant in T. minuta achenes than the aromatic CKs. cis-Zeatin (cZ) and its derivatives were the most abundant CKs. In total, 19 CK compounds were detected, including 4 free bases and a number of corresponding conjugates. Benzyladenine (BA) was the only aromatic CK detected. There was no common time at which active free base maximal concentrations were detected, suggesting that different CKs may have specific roles in the germination process, and thus peak at different times. This in turn suggests that germination is not a single process, but rather a correlative process involving a number of events, with specific CKs having specific roles relating to these correlative events. There is sufficient evidence obtained from both the soybean callus bioassay and HPLC/MS analysis to suggest that CKs have an active role in T. minuta germination. A decline in free BA during germination without corresponding conjugation, suggests that BA is actively used in early germination processes, possibly in the stimulation of DNA synthesis. Secondly, there was a distinct dihydrozeatin (DHZ) peak obtained at 24 h. Roughly 75 % of the achenes germinate between 16 and 26 h, thus it is likely that DHZ has an active role during the germination of T. minuta. Although CKs are probably not involved in the breaking of dormancy per se, the distinct peak in CK-like activity obtained in the bioassays, 24 h after imbibition, suggests that CKs have an active role in the germination of T. minuta. With respect to the regulation of thermoinhibition, a number of exogenous treatments were applied, including hormones [gibberellins (GA₄₊₇), abscisic acid (ABA), ethylene and a number of CKs], adenosine triphosphate (ATP) and incubation in 100 % oxygen. ABA was extracted from thermoinhibited and germinating achenes to assess the role of ABA in thermoinhibition and germination. While exogenous 0.1 mg L¯¹ GA₄₊₇ application slightly improved normal germination at 25°C, no treatments were effective in alleviating thermoinhbibition in T. minuta achenes. Thermoinhibition in T. minuta achenes may be under hormonal regulation, as there is strong evidence for the role of ABA in the maintenance of dormancy and thermoinhbition. High ABA levels were found in dry control samples. Additionally, exogenous ABA application inhibited normal germination, and the commencement of germination was accompanied by a decrease in endogenous ABA levels. A number of experiments relating to the imposition of thermoinhibition were carried out. Thermoinhibition appears to be very rapidly imposed. Germination is rapidly inhibited following shifting to higher thermoinhibitory temperatures, even after prolonged exposure to optimal germination temperatures. Results suggest active de novo biosynthesis of ABA in thermoinhibited achenes. Active biosynthesis of ABA during thermoinhibition suggests that this phytohormone is essential in the maintenance of thermoinhibition of T. minuta achenes. It thus appears that ABA is synthesized in the achenes in response to elevated temperatures that are unfavourable for germination to proceed. Unfavourable environmental conditions result in an achene-mediated inhibition of germination, which appears to be initiated and maintained by elevated levels of endogenous ABA.
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    In vitro conservation of endangered Dierama species.
    (2004) Madubanya, Lebogang Angelo.; Fennell, Catherine W.; Makunga, Nokwanda P.; Van Staden, Johannes.
    No abstract available.
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    Micropropagation of Brunsvigia undulata F.M. Leight.
    (2009) Rice, Laura Jane.; Finnie, Jeffrey Franklin.; Van Staden, Johannes.
    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.