Th.--Méd.--Montpellier, 1910-1911. / Montpellier, 1910-1911. N ° 126.

Thesis (MScAgric (Agronomy)--University of Stellenbosch, 2007. / An increase in the demand of buchu (Agathosma betulina) oil has lead to an increase in the commercial cultivation of buchu in fields and also in hydroponic systems. A nutrient solution for hydroponically grown buchu is still required to ensure optimal growth and yield. ASNAPP (Agribusiness in Sustainable Natural African Plant Products) South Africa has done some trials to achieve optimal EC and pH in the nutrient solution. Phosphate concentrations in the nutrient solution might play a significant role due to reports by a variety of researchers on the sensitivity of Protea plants to phosphate. Buchu and Proteas are both part of the Fynbos biome and are found in regions with similar soil (sandy soils with a low pH and mineral contents) and climatic conditions. Two separate experiments were conducted to determine the effect of increasing phosphate concentrations (ranging from 0.00 to 1.40 me L-1) in the nutrient solution on buchu growth. The first experiment was done in a plastic covered structure with a pad and fan and the objective of this trial was to determine the effect of increasing phosphate concentrations in the nutrient solution on the general growth, biomass production, oil composition, mortality rate and chemical composition of the buchu plants. The second experiment was done in a glasshouse with mechanical temperature control and the aim of this trial was to determine the response of buchu to increasing concentrations of P at two different root temperatures. A chemical analysis of the plants was done and the general growth, yield and root mass were recorded to determine the response of buchu plants to the phosphate and temperature treatments. In the greenhouse experiment an optimum growth and yield response of buchu plants was found at a phosphate concentration of 0.7 me L-1 in the nutrient solution. Phosphate concentrations lower or higher than 0.7 me L-1 lead to a decrease in growth and yield. An increase in the phosphate concentration in the nutrient solution lead to a general increase in N, P, K, Ca, Mg and B content in the buchu plants and a decrease in Fe content. The mortality rate of the buchu plants increased with an increase in the phosphate concentration from 0.0 to 1.4 me L-1 in the nutrient solution. The phosphate concentration in the nutrient solution only made a significant difference on one major component of the buchu oil which was Ψ-Diosphenol, but no general trend with Ψ-Diosphenol content and P concentration could be found and the significant difference in Ψ-Diosphenol observed in this trial may only have been due to genetic variation between the plants. The effect of the different root temperatures in the glasshouse experiment was very clear. The buchu plants grown at the high root temperature (20°C) produced a higher yield and better overall growth than the plants grown at lower (10°C) temperatures. The buchu plants grown at 20°C had a significantly higher N, K, Na and B content than plants grown at 10°C. Buchu plants grown at 10°C showed no significant response in terms of growth and yield to the phosphate concentration in the nutrient solution, but plants grown at 20°C exhibited growth and yield peaks at phosphate concentrations of 0.35 and 1.4 me L-1. The peak observed in the plants growth at high phosphate concentrations is unexplainable and can possibly be ascribed to the limitation of the plants per experimental unit and/or amount of replications. The increase in P concentration in the nutrient solution caused a general increase in N, P and K content in the buchu plants. A significant interaction between the phosphate concentration and root temperature was observed for the P, Mn en Zn contents of the plants which meant that the buchu plants respond differently towards phosphate concentrations at different root temperatures.

Thesis (MScAgric (Agronomy)--University of Stellenbosch, 2007. / The Cape Floristic Region is a highly distinctive phytogeographical unit which is recognized as a floral Kingdom on its own. Buchu (Agathosma betulina) plants fall under this important Kingdom. Buchu is one of the traditional medicinal plants originating in the Western Cape province of South Africa and the essential oil derived from the leaves is exported in large volumes. Due to high demand, under supply, restrictions of wild harvesting and high prices for Buchu essential oil, growers have started to introduce and commercialize this species as a crop. This commercialization of Buchu necessitated agronomic research to optimize production techniques. The objective of this study was to determine the optimum pH range for the cultivation of high yielding Buchu with acceptable essential oil quality under controlled conditions and compare this with the conditions in the natural habitat. Plant, soil and climatic data were gathered from eleven sites in the natural habitat of Buchu (A. betulina) in the Cederberg Mountains. At all sites most rainfall occurred from May to September, while high temperatures were recorded in summer. Soil analyses indicated low levels of nutrients and low soil pH, ranging from 3.7 to 5.3 at all the sites studied. Low levels of nutrients were also obtained from foliar analysis collected from plants at each of the different sites. Chemical analyses of the essential oil indicated that the plants were from a high quality diosphenol chemotype. In the greenhouse experiment, five different pH levels (pH 33.99, 4-4.99, 5-5.99, 6-6.99 and 7-7.99) were evaluated to determine the effect on growth, yield and quality of A. betulina. Complete nutrient solutions were used to irrigate the plants grown in pots filled with a sand and coco peat mixture. Although the plants subjected to the pH treatment of 4-4.99 tended to have the highest growth rate and yield, this did not differ significantly (P=0.05) from plants subjected to pH values between 3 and 6.99. In contrast, the pH 7-7.99 treatment lead to reduced growth and lower vegetative yields. Levels of nutrients obtained from the leaf mineral analysis differed significantly with different pH treatments. High pH levels resulted in high nitrogen, phosphorus, sodium, manganese and boron contents, but lower contents of copper. Nitrogen, phosphorus, calcium and zinc were higher than those recorded for plants from their natural habitat, but still within the norm reported for most plants. Levels of manganese, sodium, magnesium and copper were found to be more or less similar to the values obtained in plants from the natural habitat. No significant differences were found in essential oil quality in response to the pH treatments. However, high pulegone levels (10.8 to 13.2 %) were obtained from all the treatments in the greenhouse experiment. The high levels of this essential oil constituent could have a negative effect on the marketability of the oil and this aspect may need some attention in future studies.

Thesis (MSc (Plant Biotechnology))--University of Stellenbosch, 2006. / The development of a reliable and reproducible method for the genetic characterisation and identification of the commercially important Agathosma species was investigated. Previous research attempts aimed at developing a reliable and reproducible method of identifying these Agathosma species failed, mostly because these studies were based on phenotypic traits and these methods were therefore influenced by environmental factors. In this study amplified fragment length polymorphisms (AFLPs) were successfully used to quantify the genetic variation between the Agathosma species and as a result three distinct groups could be identified. The data obtained were elaborated with the Dice genetic similarity coefficient, and analysed using different clustering methods and Principle Coordinate Analysis (PCoA). Cluster analysis of the genotypes revealed an overall genetic similarity between the populations of between 0.85 and 0.99. The AFLP-based dendrogram divided the populations into three major groups: (1) the A. serratifolia and A. crenulata populations, (2) the putative hybrid, A. betulina X A crenulata populations, and (3) the A. betulina populations, confirming that this technique can be used to identify species. The question of hybridisation was also clarified by the results of the PCoA, confirming that the putative hybrid is not genetically intermediately spread between the A. crenulata and A. betulina populations, and that it is genetically very similar to A. betulina. The putative hybrid can therefore rather be viewed as a genetically distinct ecological variant of A. betulina. As the AFLP technique cannot be directly applied in large-scale, routine investigations due to its high cost and complicated technology, the development of polymerase chain reaction (PCR)-based molecular markers, able to accurately identify the species, was undertaken. Due to the superior quality of A. betulina oil, the development of such markers is especially critical for this species. Several species-specific AFLP markers were identified, converted to sequence characterised amplified regions (SCARs) and ultimately single nucleotide polymorphisms (SNPs) were characterised. The developed SCARs were unable to distinguish between the species. The conversion of AFLP fragments to SCARs is problematic due to multiple fragments being amplified with the AFLP fragment of interest. The diagnostic feature of the SNP-based markers was not sensitive enough, since this technique could not distinguish between the A. betulina and A. crenulata and/or the putative hybrid populations. The SNPs that were characterised were found not to be species-specific; they were only specific to the particular clone. Although a quick and robust marker specific for A. betulina has not yet been developed, this study sets the stage for future genetic studies on Agathosma species. Such a marker, or set of markers, would be an invaluable contribution to a blooming buchu oil industry.

Copies no. 3007841664 and 3007841665 are photocopies of the original. / Thesis (MScAgric)-- University of Stellenbosch, 2003. / ENGLISH ABSTRACT: Buchu (Agathosma betulina and A. crenulata) are grown commercially as an aromatic crop and are endemic to the Western Cape of South Africa. Poor rooting of cuttings have limited the development of superior clones. Under standard mist bed conditions terminal, sub-terminal or basal stem cuttings were taken from March to August. When not treated with an auxin, rooting percentages of between 20 and 25 were obtained. Rooting percentages increased to between 40 and 45 after treatment with 500-1000 ppm indolebutyric acid (lBA). Substituting lBA with naphthaleneacetic acid (NAA) did not improve rooting. There was a tendency for cuttings with fewer than four leaf pairs to give lower rooting percentages. Plants of Agathosma betulina x A. crenulata, grown in Paarl, and A. betulina, grown in Piketberg, were used as source plants for making cuttings. Paarl plants were shaded with 80 percent shade and Piketberg plants with 60 or 80 percent shade respectively from February to October 2002. Plants in full sun served as a control. Plants were pruned back initially in February and then two months before samples were taken in March, June, August and October at both locations. New shoots were used as cuttings. Terminal cuttings for rooting and for carbohydrate analyses were collected on four different dates (March, June, August and October). Cuttings were treated with 500 ppm indolebuteric acid (lBA) and placed in misting beds with bottom heating (18-25°C) for a period of three months. Shading reduced rooting of cuttings from the Paarl plants. However, it did not significantly increase rooting of cuttings taken from Piketberg plants. Rooting percentage was the highest in August (43%) for cuttings from sun grown plants in Paarl. No consistent relationship between, respectively, dry mass or carbohydrate content of cuttings and rooting could be established. Terminal current years' growth, taken from Agathosma crenulata x A. betulina (hybrid) softwood cuttings, collected in January 2002, were extracted with methanol and fractioned by thin layer chromatography (Silica gel) in isopropanol: acetic acid: water (4: 1:1 v/v). The chromatographs were divided in ten fractions and were bio-assayed for a rooting co-factor with the mung bean rooting test. Extracts from buchu cuttings showed significant activity at the Rf values of co-factor 3. Co-factors 1,2 and 4 do not seem to be present in significant quantities. However, co-factors with Rf values different from previous reported values were present in significant quantities. No inhibition was found in buchu. In fact, all Rf values stimulated rooting. / AFRIKAANSE OPSOMMING: Boegoe (Agathosma betulina x A. crenulata) word kommersieël verbon as 'n aromatiese gewas en is endemies tot die Wes-Kaap. Die ontwikelling van superieure klonale materiaal word beperk deur swakbeworteling. Terminale, sub-terminale en basale steggies is gesnyonder standaard misbed toestande van Maart tot Augustus. Beworteling was tussen 20 en 25 persent as geen ouksien gebruik word nie. As indolebottersuur (IBS) gebruik word tussen 500-1000 dpm, verhoog die bewortelingspersentasie tot tussen 40 en 45 persent. Die gebruik van naftaleen asynsuur (NAS) in plaas van IBS het nie beworteling verbeter nie. Daar was a tendens dat steggies wat minder as vier blaarpare gehad het 'n verlaging in bewortelingspersentasies gehad het. Plante van Paarl, A. betulina x A. crenulata, en Piketberg, A. betulina, is gebruik as plantmateriaal vir steggies. Plante in die Paarl was onder 80 persent skadu geplaas en plante in Piketberg onder 60 en 80 persent skadu van Februarie tot Oktober 2002. Plante in vol son was as 'n kontrole gebruik. Plante was eers in Februarie teruggesny en dan weer twee mande voor monsters geneem is. Die monsters is in Maart, Junie, Augustus en Oktober geneem in beide liggings. Terminale steggies is vier keer ingesamel (Maart, Junie, Augustus en Oktober) vir beworteling en koolhidraat analises. Die steggies is met 500 dpm IBS behandel. Daarna is die steggies vir drie maande in die misbed geplaas met bodem-verhitting (18- 25°C). Dit is gevind dat die gebruik van skadu die beworteling in Paarl verminder het alhoewel die beworteling in Piketberg nie beduidend beinvloed is nie. Die hoogste bewortelingspresentasies is waargeneem in Augustus (43%) in Paarl van plante wat in vol son was. Geen verband tussen onderskeidelik die droe massa of koolhidraat inhoud en beworteling kon gevind word nie. Terminale steggies van dieselfte jaar se groei van Agathosma betulina x A. crenulata (hibried) is in Januarie 2002 ingesamel. Die materiaal is geëkstraheer en gefraksioneer deur dunlaag kromatografie in isopropanol: asynsuur: water (4: 1:1 v/v). Die kromograaf is in 10 fraksies verdeel. Die fraksies was bioassaieer VIr beworteling ko-faktore met die mungboontjie bewortelingstoets. Die ekstrakte van boegoe het beduidende aktiwiteit by die Rf waardes van ko-faktor 3 getoon. Ko-faktore 1, 2 en 4 is nie in groot genoeg hoeveelhede waargeneem nie. Ko-faktore, wat nie voorheen gevind is nie, is waargeneem in beduidende hoeveelhede. Geen inhibitors is in boegoe gevind nie en al die getoetste ko-faktore het beworteling gestimuleer.

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.