Micropropagation of three Brachystelma species and investigations of their phytochemical content and antioxidant activity.

Master of Science in Plant Growth. University of KwaZulu-Natal, Pietermaritzburg 2017.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