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The use of many of South Africa’s medicinal plants has shown marked increase with over 27 million users in South Africa alone. Most plants are still being unsustainably wild-harvested, a major concern for biodiversity conservation. Commercial interest in certain more commonly-used species has increased, with potential to cultivate medicinal plants on a more sustainable basis. Focus has shifted from conventional use of synthetic fertilisers, pesticides and fungicides to more organic methods of plant propagation. Aqueous extract derived from earthworm composted food waste (vermitea) was used to study the germination and rooting success of selected species. Also survival and growth performance of selected plants grown in a medium amended with commercial NPK fertiliser was compared to those grown in the same medium amended with compost and to those grown in the same medium amended with compost with weekly applications of vermitea. No change in germination success was noted. Vermitea showed promising results on the rooting of cuttings. The application of NPK improved growth performance (biomass) significantly for all species tested. However, they had lower root:shoot ratios as well as lower survival rates compared to plants under the compost and compost/vermitea treatments. The improved survival of these plants highlights the potential of these organic treatments on the propagation of selected medicinal plants.

Multiple shoots were produced from nodal explants of thirty-day-old in vitro grown seedlings and from pretreated three, five- and nine-month-old greenhouse grown Acacia mearnsii plants, respectively. Explants were sterilized for 15 minutes using 0.1 % HgCl₂ for the three-month-old explants and 0.2 % for the five and nine-month-old explants. Nodal explants were induced to form multiple shoots when placed on Murashige and Skoog (MS) medium supplemented with 2.0 mg l ¯¹ benzyladenine (BA). Rooting of these shoots was achieved on MS medium supplemented with 1.0 mg l ¯¹ indole-3-butyric acid (IBA). Plantlets were acclimatized in transparent plastic containers under greenhouse conditions with a 90 % success rate. These plantlets were successfully acclimatised under greenhouse conditions and planted in the field together with plants regenerated by cuttings. In an attempt to overcome maturation effects and loss of juvenile characteristics, when using adult plant material in vitro, investigations were undertaken into the use of coppice material, as an alternative explant source. A. mearnsii trees from five ages (two, four, six, eight and ten-years old, respectively) were decapitated to a height of 1.5 m. After three weeks, coppice was noted on the stumps of trees from all ages. A linear response to coppice production was noted, with the greatest coppice production being on the two-year-old tree stumps and the least on the ten-year old tree stumps. Decontamination of the coppice was successful and multiple shoot production was obtained from coppice taken from all age groups on MS medium supplemented with 2.0 mg l ¯¹ BA. The effect of various sucrose concentrations were investigated. Greater shoot production occurred with increased sucrose concentrations (20 and 30 g l ¯¹). It was evident that rejuvenation of mature tissue could be achieved through the use of coppice material. A second approach to rejuvenate adult material and to overcome the deleterious effects of maturation, was in the use of apical meristems. Meristems were taken from 30-day-old in vitro grown plants, from coppice (rejuvenated tissue) and adult material of five various tree ages (two, four, six, eight and ten-years-old, respectively). Plant material were taken over two seasons (1997 to 1999) and the use of agar and liquid support media were tested under both light and dark conditions. The coppice and adult material was successfully decontaminated in both seasons. In the first season (1997/1998), shoot production was obtained from meristems of in vitro grown plants, coppice and adult material from all trees on MS medium alone or MS medium supplemented with 2.0 mg l ¯¹ BA. In the following season (1998/1999), the use of a solidified agar medium was superior to the use of a liquid culture. There appeared to be no significant difference (p<0.05) between the use of light or dark culture conditions. Various media were tested and maximum shooting occurred on half-strength MS medium and Woody Plant Medium (WPM). However, once multiple shoot primordia were initiated, shoot elongation posed a problem. It was for this reason that the size of the meristems excised from the coppice material was increased from 0.5 mm to 1.0 mm in the 1997/1998 season, to 1.0 to 2.0 mm in the 1998/1999 season. The use of gibberellic acid and 100 ml jars were also investigated to see if this might enhance shoot elongation. Sufficient plant material was not available for a thorough investigation. Environmental conditions under which the plant material (adult or coppice) was harvested was similar in both seasons, with respect to temperature, but differed in rainfall. Rainfall was high (105.1 mm) in 1997/1998 season and low (ranging from 59.8 to 71.45 mm) in the 1998/1999 season. Shoot production from meristems taken from coppice material in the 1998/1999 season was significantly greater (p>0.05) than that in the 1997/1998 season, whereas shooting from the adult plant material remained unchanged. The disadvantage with using coppice material is that its production on decapitated tree stumps is dependant on rainfall, which is unpredictable. The differences in results from coppice material could be attributed to the fact that the trees felled in the two seasons were not related to each other in any way. In both seasons meristems, tree age was not a limiting factor, for meristems from adult and from coppice material. Meristems from the ten-year-old trees were as productive as those taken from the two-year-old trees. In the 1997/1998 season the results from the meristems from the adult material was equal if not greater than those obtained from the coppice material. In the 1998/1999 season, there was no significant difference (p<0.05) in percentage shoot production between the meristems from the adult and coppice material throughout the age groups. This suggests that the use of rejuvenated tissue in the form of coppice is not essential. This re-emphasized the advantage of using meristems taken from adult plant material. This study provided suitable protocols for the micropropagation of both in vitro and ex vitro grown nodal explants of A. mearnsii. However, as the plant material obtained from the field matures so the ease of obtaining sterile material decreased, thus reducing the chances of in vitro micropropagation. For this reason suitable pretreatments and rejuvenation methods are necessary if explants from mature field tissue are to be introduced into culture and successfully micropropagated. This study has shown that through the use of nodal material (taken from coppice produced on adult tree stumps) and apical meristems taken from both coppice and mature plant material, adult material can be successfully decontaminated, introduced into culture and stimulated to produce shoots. Analysis of tannin production was conducted to see if there was any indication that the presence of tannins in the plant material effected in vitro culture of nodal explants. However, no trends were obtained suggesting any influence of tannins on in vitro performance. In future years after further optimisation, these techniques could be incorporated in an A. mearnsii clonal programme, with the advantage of possibly eliminating maturation effects, commonly noted in vegetative practices. This will allow for easy manipulation and amplification of superior quality adult material. / Thesis (Ph.D.)-University of Natal, Pietermaritzburg, 1999.

Cussonia species (commonly known as Cabbage trees) are indigenous to South Africa and are used in traditional medicine to treat an assortment of diseases. Due to their attractive growth form, they are assets in gardens. However, there are no developed methods for propagating these species. The use of three selected species, Cussonia paniculata (Eckl. & Zeyh.), C. spicata (Thunb.) and Schefflera umbellifera (Sond.) Baill, = C. umbellifera), in traditional medicine was validated. Rapid propagation protocols for C. paniculata and C. spicata were investigated and ultimately developed for the former species. Cussonia paniculata, C. spicata and C. umbellifera were screened for their medicinal properties, mainly focussing on anti-bacterial, anti-inflammatory and anti-malarial activities. In the anti-bacterial screening, C. spicata bark and root extracts showed activity against selected Gram-positive and Gram-negative bacterial strains at a concentration of 50 mg ml ¯¹ . The highest inhibition was observed with ethanol and ethyl acetate root extracts against Staphylococcus aureus. The other two species did not show anti-bacterial activity. Ethanol and ethyl acetate extracts of all species showed anti-inflammatory activity in the cyclooxygenase assay (COX-1) at a concentration of 8 μg ml ¯¹, These active extracts showed an inhibition percentage that was greater than 50 % against cyclooxygenase. In the anti-malarial screening , bark extracts were screened. C. umbellifera bark extracts exhibited the best inhibition against P. falciparum, a malaria-causing agent in humans. The percentage inhibition of these extracts was up to 100% at a concentration of 200 μg ml ¯¹ . While C. spicata is known to be used to treat malaria, the screening results showed much less activity (less than or equal to 35 %) as compared to C. umbellifera, which is preferably used to treat malaria. The results obtained from screening these three species validated their use in traditional medicine. This means that the people or traditional healers use these species for different treatments by possibly relying on past knowledge about the effects after administering the medicine. Fingerprinting using Thin Layer Chromatography (TLC) was used in an attempt to determine whether there are any chemical differences or similarities between the three species. There were similarities between the plant parts across the species as well as some differences. However, this method cannot be used as an unequivocal test to deduce that compounds that are present in a certain species and not in others are the ones responsible for bringing about a certain biological activity. That can only be achieved by a bioassay-guided isolation of possible compounds. A tissue culture protocol was developed to produce a large -number of plants of C. paniculata. Explants were derived from nodal explants of in vitro germinated seeds and cultured on Murashige and Skoog (MS) (1962) medium supplemented with 3% sucrose, 2.5 mg l ¯¹ BA and solidified with 3 g l ¯¹ Gelrite. These explants produced multiple shoots. The average number of shoots per explant ranged between 1 to 3.5. Multishoots were subcultured on to rooting media and roots were produced on MS with 0.75 mg l ¯¹ IBA and 1 mg l ¯¹ NAA. Callus from zygotic embryos also produced plantlets on MS supplemented with 1.5 mg l ¯¹ 2,4-D and 0.5 mg l ¯¹ BA. Hyperhydricity was encountered in this study. This problem was reversed successfully by transferring the shoots from medium solidified with 3 g l ¯¹ Gelrite to medium solidified with 8 g l ¯¹ agar. Plantlets were successfully acclimatized for planting ex vitro. The percentage of healthy plants after a 35-day acclimatization period was 63 %. C. spicata was not successfully micropropagated from shoot-tip explants. However, a protocol was developed for decontaminating shoot-tips from the mother plants. The plant material was successfully decontaminated with 0.01% HgCl₂ for 15 min. The decontamination percentage was up to 80 %. Browning of the explants was observed and it was successfully treated with soaking the explants in a 15 mg l ¯¹ ascorbic acid solution for 15 min. A high percentage of shoot-tip regeneration (80 %) was observed when they were cultured on MS medium supplemented with 2 mg l ¯¹ BA, 1 mg l ¯¹ IAA and 1 mg l ¯¹ GA₃. However, multishoots were not observed as in C. panicualata. Shoot elongation in vitro was similar to shoot elongation as it occurs in nature. The shoots elongated and a flush of palmitately arranged leaves were produced. Further research is required to investigate a commercially viable protocol for rapid propagation and conservation of the germplasm of Cussonia species. / Thesis (M.Sc.)-University of Natal, Pietermaritzburg, 2000.

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.

Graduation date: 1998

Research into the micropropagation of black wattle in South Africa is important for two reasons. Firstly micropropagation technology allows breeders to select and propagate mature tissue, which in turn allows them to better capture selected traits. Secondly, tissue culture may control the highly invasive nature of black wattle. If triploid black wattle can be developed, foresters will then have to rely on clonal propagation to supply material for their growing operations. This research was part of the Institute for Commercial Forestry’s Acacia mearnsii vegetative propagation programme. The main focus of this research was to overcome various problems associated with direct organogenesis of ex vitro material. The shoot apex region was used as the explant in all studies because this region is thought to harbour relatively few internal microbial contaminants and is of sufficient size to withstand stresses associated with micropropagation. The initial research was focussed on the screening of sterilants, searching for a viable alternative to mercuric chloride. Surface sterilisation is integral to any micropropagation technique. This process should do the least amount of plant damage, whilst reducing microbial contamination to an acceptable level. Explants were cultured on Murashige and Skoog (MS) medium supplemented with 2.0 mg L-1 BA and monitored for signs of contamination and shooting. Household bleach proved an excellent alternative to mercuric chloride because it did significantly less damage to the explants than mercuric chloride and is handled easily. There was no significant effect of sterilant exposure time on explant decontamination levels, whilst the shortest exposure time resulted in significantly higher levels of shoot development than the other two times tested. The results of this initial research was developed into a protocol and utilised in subsequent investigations. Due to a considerable variation in the success of the developed surface sterilisation protocol according to different times of the year, a further investigation into the effects of season and mother plant material on shoot apex culture of Acacia mearnsii was undertaken. The success of any tissue culture technique depends on a large array of ex vitro and in vitro variables. The objective of this research was to determine the ii effect of two ex vitro variables, season and mother plant, on shoot apex culture of Acacia mearnsii. Explants from individual mother plants were cultured on MS medium supplemented with 2.0 mg L-1 BA during four separate seasons and monitored for signs of contamination and shooting. Spring was found to be the best harvesting season because spring explants showed significantly higher decontaminated explant levels and shooting levels than explants harvested in the other three seasons. The effect of mother plant selection on the performance of Acacia mearnsii explants during shoot apex culture was also found to be significant, especially with regard to shooting levels. Finally factors influencing shoot elongation of A. mearnsii during shoot apex culture were investigated. In the past, induction of shoot elongation during micropropagation of A. mearnsii was attained through the addition of plant growth regulators and other supplements to the basal culture medium. However, some micropropagation methods in other species have utilised red light as a means of promoting shoot elongation. The objective of this study was to test the effects of an alternative basal medium, red light and differing concentrations of chemical additions to the culture medium on shoot elongation of Acacia mearnsii during shoot apex culture. Four independent experiments were undertaken comparing: shoot elongation on Woody Plant Medium (WPM) to the MS basal medium control; shoot elongation under a red cellophane box compared to control culture light conditions; shoot elongation on media supplemented with various concentrations of GA3 to the un-supplemented control and shoot elongation on media supplemented with combinations of BA and IBA compared to a control. Although no significant effects were observed, many trends were noted. The results indicated that there was no advantage to using WPM instead of MS medium when attempting to elongate shoots, rejuvenated through shoot apex culture of A. mearnsii, whilst the effect of GA3 showed a negative trend. The effects of red light and some BA and IBA combinations showed positive trends on the elongation of initiated shoots. This research successfully addressed some of the problems associated with micropropagation of A. mearnsii. Shoot apex culture shows promise and further research into this technique should be considered. A viable surface sterilant alternative to mercuric chloride was successfully identified. This alternative is not only iii safer to use but shows a large reduction in phytotoxic effects. The effects of season and mother plant on shoot apex culture was successfully investigated, resulting in a better understanding of mother plant influences on tissue culture as well as the identification of an optimum season for explant selection. Finally two possible shoot elongation promoters were identified for further research and a more affordable alternative to red light sources and screens was identified. / Thesis (M.Sc.)-University of KwaZulu-Natal, Pietermaritzburg, 2007.