Master of Technology: Horticulture in the Faculty of Applied Sciences at the Cape Peninsula University of Technology, 2011 / A study was conducted to investigate the effects of: i) various media compositions and wounding treatments on the in vitro germination, growth and regeneration of Strelitzia reginae plantlets derived from zygotic embryos, ii) antioxidants, plant growth regulator (PGR) concentrations and plant tissue wounding treatments on phenolic compound production. One experiment consisted of 8 medium types including different combinations of Murashige and Skoog (MS) medium strength, activated charcoal and vitamin supplements. Twelve replicates were used for each treatment. In another experiment, germinated plantlets were subjected to 2 wounding treatments; (unwounded explants (control) and explants longitudinally sectioned through the apical meristem). The explants were transferred to ten different regeneration media consiting of different concentrations and combinations of auxin and cytokinin supplements and antioxidants. Ten replicates were used for each treatment. Results indicated the positive role of activated charcoal (AC) in reducing oxidative browning of embryo explants. The highest germination rate of embryos was observed in media containing AC without vitamin supplementation. Germination significantly decreased with the addition of vitamins. With regard to effects of various media compositions and wounding treatments on in vitro growth and regeneration of Strelitzia, significant results were achieved with 1-naphthalene acetic acid (NAA) and 6-benzylaminopurine (BAP) concentrations on explant discoloration and callus formation. The antioxidant treatments, activated charcoal (AC) and ascorbic acid (AA) significantly affected explant discoloration, the induction of callus and the length of roots developed. Wounding treatments affected plant height, increased explant height and callus formation. Interactions between higher NAA and BAP concentrations together with wounding resulted in the most effective treatment in reducing explant discoloration at the media contact point. Furthermore, results showed the various NAA and BAP concentrations to significantly affect phenolic exudation. The media containing the highest PGR concentration resulted in the highest phenol content. AC significantly reduced the total phenol content of media by 53%, compared with AA. Phenolic exudation was significantly increased as a result of explant wounding. Various interactions between the NAA and BAP concentrations, antioxidants and wounding affected phenolic exudation and the total phenol content of media. This study provides insight into the contributing factors and methods of overcoming the major problem of phenolic oxidation and promoting the in vitro growth and regeneration of Strelitzia.

Thesis (Ph. D. (Plant Production)) -- University of Limpopo, 2015 / Wild watermelon (Cucumis africanus LF.) and wild cucumber (Cucumis myriocarpus Naude.) are known for their ethnomedicine, ethnopesticide, ethnonematicide and nutritional properties, along with nematode resistance. The two Cucumis species were successfully used as inter-generic seedling rootstocks for watermelon (Citrullus lanatus Thunb.) cultivars, where nematode-resistant genotypes are not available. Also, the two Cucumis species are hardy and resilient to inland South Africa conditions, where temperatures are predicted to increase by 6°C in the year 2030. Seeds in the Cucurbitaceae Family contain high concentration of cucurbitacins, which induce auto-allelopathy that inherently inhibits plant growth and germination. Poor germination and non-uniform stands as a result of seed dormancy are a major challenge in sexual propagation of wild Cucumis species for various potential industries. Generally, true-to-type, uniform and disease-free plants in plant production are asexually-generated through in vitro propagation techniques. This study was therefore, initiated to address seed dormancy and related challenges of sexual propagation in the two wild Cucumis species by determining whether: (1) seed dormancy in C. africanus and C. myiocarpus would be ameliorated to allow for in vitro sexual propagation to establish pathogen-free parent stock, (2) the testa in C. africanus and C. myiocarpus seeds would possess structures, which interfere with imbibition and movement of water to the endosperm, (3) all organs of C. africanus and C. myriocarpus would be suitable for in vitro propagation, (4) suitable potting medium for in vitro propagated plantlets of C. africanus and C. myriocarpus would be available for acclimatisation of plantlets and (5) in vitro-produced xxviii plantlets from nematode-resistant C. africanus and C. myriocarpus would retain their resistance to Meloidogyne incognita race 2 under greenhouse conditions. In vitro and ex vitro experiments were conducted to achieve the stated objectives, with treatments in the laboratory and the greenhouse being arranged in completely randomised and randomised complete block designs, respectively. Validity was primarily ensured through the use of factorial trials, while the reliability of data was ensured by using appropriate levels of statistical significance. Leaching alone in C. africanus improved germination, while in C. myriocarpus this treatment had no effect on germination. The optimum leaching time in leached-control seeds of C. africanus was achieved at 7.1 h, with a 25-day mean germination time (MGT) and 52% optimum germination percentage (GP). In the two Cucumis species, the combined effect of leaching seeds in running tapwater and physical scarification of seeds at the chalaza region escalated germination in both Cucumis species, suggesting that both chemical and physical seed dormancies were involved. In C. africanus, cucurbitacin B (C32H48O8) was deposited exogenously to the testa, whereas in C. myriocarpus cucurbitacin A [cucumin (C27H4009) and leptodermin (C27H3808)], was deposited endogenously to the testa. The optimum leaching time in leached-scarified (LS) seeds of C. africanus was achieved at 5.7 h, with at least 40-day MGT and 89% optimum GP. In contrast, in C. myriocarpus LS seeds had the optimum leaching time of 6.3 h, with at least 41 days MGT and 93% optimum GP. Field emission SEM confirmed that there were two “water-gaps”, one at the micropylar region (hilum end) and the other at chalaza region (abaxial end) of seeds in both Cucumis species. Five distinct testa layers in seeds of C. myriocarpus were observed, namely, (i) epidermis, (ii) hypodermis, (iii) sclerenchyma, (iv) aerenchyma xxix and (v) chlorenchyma. In contrast, C. africanus seeds did not have the hypodermis between the micropylar and chalaza regions, but was present around both regions, which may provide some explanation of sporadic germination in non-leached and non-scarified seeds in this Cucumis species. The most suitable plant propagules for in vitro mass propagation of the two Cucumis species were nodal and apical buds. The optimum PGRs for shoot regeneration using both propagules in C. africanus and C. myriocarpus were at 0.80 and 0.35 μM 6-benzyladeninepurine (BAP), respectively. In contrast, the largest number of roots was regenerated at 0.31 and 0.44 μM indole-3-butyric acid (IBA) for C. africanus and C. myriocarpus, respectively. In vitro-produced plantlets were successfully acclimatised to ex vitro conditions, with sand + compost potting medium being the most suitable growing medium for weaning both Cucumis species. The in vitro-produced plantlets retained their resistance to M. incognita race 2. In conclusion, seeds of C. africanus and C. myriocarpus are structurally and chemically different, with strong evidence of chemical and physical dormancies. Structurally, C. myriocarpus seeds consist of five layers, four lignified and one non-lignified, whereas those of C. africanus have four layers, three lignified and one non-lignified. Evidence suggested that in C. africanus seeds, allelochemicals were primarily deposited outside the testa, whereas in C. myriocarpus they were deposited within the testa. The identified seed dormancies could successfully be ameliorated through combining leaching and scarification in both Cucumis species. The developed in vitro propagation protocols accord the two Cucumis species the potential for use as future crops in the context of climate-smart agriculture and research. / Flemish Interuniversity Council (VLIR)

Thesis ( PhD. (Biotechnology )) --University of Limpopo, 2005 / The marula tree (Sclerocarya birrea subsp. caffra), an indigenous, multipurpose, drought tolerant tree of Africa harbors great economic potential. Acceptance of marula-derived products internationally will directly increase the demand for marula resource. Rapid multiplication of marula trees of superior quality forms the basis of sustainable export growth. In vitro propagation and genetic improvement offer the opportunity for accelerated multiplication of selected tree material as well as to dramatically increase production, quality and efficiencies. The objectives of the study were therefore to develop a protocol for in vitro multiplication of marula and to determine the feasibility of Agrobacterium-mediated transformation of the marula tree. Nodal sections with axillary bud (s) were cultured on Murashige and Skoog (MS) medium supplemented with 4.8μM BA and 2.4μM KN and 0.1% polyvinylpyrrolidone (PVP) to obtain on average 2.5 microshoots per responding explant. The proliferated microshoots were elongated on MS medium supplemented with 1.2μM BA and 1.0μM KN. Elongated microshoots were rooted in MS medium at half salts strength supplemented with 10μM IBA and 0.3% activated charcoal (AC). On average 82% of the shoots rooted. Survival of acclimatized plantlets was 90%. RAPD analysis confirmed intraclonal genetic stability between parent plants and their clones within the limits of the technique.Nodal sections cocultivated with Agrobacterium tumefaciens for 3 days on MS multiplication medium supplemented with 100μM acetosyringone resulted on average in transient expression of 52.5% of the explants with 1.6 blue stained zones per explant. Cocultivated explants on MS selection medium containing 300mgl-1 kanamycin resulted in 1.5% chimeric putative transgenic shoots. This is the first report on the micropropagation and genetic transformation of marula, Sclerocarya birrea subsp caffra. / South Africa’s National Research Foundation Institutional Research Development Program (NRF-IRDP)

Thesis submitted in fulfilment of the requirements for the degree Master of Technology: Horticultural Sciences in the Faculty of Applied Sciences at the CAPE PENINSULA UNIVERSITY OF TECHNOLOGY Supervisor: Dr L Kambizi Co-supervisor: Dr NP Makunga Cape Town December 2013 / Agathosma betulina (Berg.) Pillans, previously known as Barosma betulina, is a member of the Rutaceae family, and indigenous to the fynbos botanical biome of the Western Cape of South Africa. It is commonly known as buchu. Extracts as well as powdered leaves have traditionally been used for the treatment of various ailments. The increase in the international demand for A. betulina for health as well as food and beverage benefits, have raised concerns over exploitation of wild populations and the lack of horticultural information necessitates this study to evaluate the propagation of this economical important species. The main objective of this study was to establish a simple and highly productive micropropagation protocol for A. betulina through experimenting with nodal explants. Testing of the effect of various treatments (physical scarification, chemical scarification, GA, stratification, smoke and combinations thereof) on the in vitro germination of A. betulina seeds was done to elucidate the factors which control seed germination. The study revealed that the physical scarification and smoke-induced germination had a significant effect on germination percentages. In terms of germination rate, the radical generally started to appear after approximately 10 days in the physical scarification with smoke treatment. Initial decontamination methods with the exposure of various concentrations of NaOCl gave fatal results, however 1.5% NaOCl had more phenolic reactions rather than fungal or bacterial contamination. Interestingly, contamination rates of explants were influenced by the stage of maturity of the explant material. This plant material was used to test different strengths of regeneration media, to ensure that the explants receive ample nutrients. Results made exhibited that ½ MS was the best strength for growing A. betulina nodal explants. Compared comparison between in vitro derived explants and ex vitro collected explants showed that the ex vitro derived explants had significant results, but the explants lost vigour soon after the initial exponential growth leading to the explants dying off. Furthermore, ex vitro decontaminated plant material was not economically viable to continue with. Seedlings derived from germinated seeds appeared to be the preferred method of propagation as this spent the least time in culture and produced a stable plant with an established root system, which is essential during the hardening off process after in vitro growth. When exposing nodal explants to phytohormone 2,4-D it responds best to dosages 0.5mg Lˉ¹ and 1mg Lˉ¹. Phytohormone BA was very effective in producing soft friable callus. The best results were shown when 0.5mg Lˉ¹ BA was applied to ½ MS media. For both shoot length and multiple shoot production, a combination of phytohormones BA-NAA (1: 0.5mgLˉ¹) had the most significant results. Interestingly, a higher phytohormone concentration of NAA is necessary to develop multiple adventitious roots. The effect of 3mg Lˉ¹ was significant in that it resulted in multiple adventitious roots, but fewer calli was observed in this treatment. Micropropagation becomes valuable as little attention between subcultures is needed; making it less labour intensive compared to conventional nursery propagation systems where weeding watering and spraying of plants are labour intensive. In the traditional world of medicine, more so in Southern Africa, extracts are prepared by adding boiling water to the plant material; however commercial ethanol is used as an extractant. Establishment of the essential oil quality of the in vitro cultures post exposure to various treatments was done. Analysis of essential oils from A. betulina resulted in the identification of twenty one compounds. The results showed qualitative as well as quantitative differences amongst the samples used in the study. The highest relative concentration of limonene was observed in the callus of nodal explants after it was exposed to 0.5mg lˉ¹ NAA. No pulegone was found in this treatment making it ideal for limonene production. This suggests that liquid culture with the same treatment may produce more calli making it ideal for the production of limonene.