Thesis (MSc (Botany and Zoology))--Stellenbosch University, 2011. / ENGLISH ABSTRACT: See full text for abstract / AFRIKAANSE OPSOMMING: sien volteks vir opsomming

Soyabean meal (SBM), the major protein source in feeds in sub-Saharan Africa, is in short supply. The shortage is a major constraint to intensified animal production to meet increased demand hence the dire need to search for alternatives. Kigelia africana, Mumisops zeyheri, Terminalia sericea and Ximenia caffra are indigenous fruit bearing trees (IFBTs) whose seeds’ potential as alternative protein sources in feeds were evaluated. The evaluation consisted of an initial physico-chemical characterization of the seeds followed by determining in vitro the safety of seed oils on cell lines. Based on the physico-chemical and in vitro evaluation, the most suitable seed was selected, defatted and its meal used as a dietary substitute to SBM in the in vivo trials using adult and weanling male Sprague Dawley rats. The T. sericea seed yield was not viable. Chemically K. africana and X. caffra seed demonstrated potential as protein sources in feeds. M. zeyheri seed demonstrated potential as an energy source. The IFBTs seeds oil yield surpassed that of some traditional oilseed crops. Oleic and linoleic acid were the major fatty acids contained in the oils. In vitro, K. africana, M. zeyheri and X. caffra seed oils suppressed Caco-2 and HEK-293 cell proliferation without causing cell death. X. caffra seed, deemed the most suitable, was defatted and its seed meal used in the in vivo trials. In mature rats, dietary substitution of SBM with the defatted X. caffra seed meal did not affect (P > 0.05) dry matter intake, apparent digestibility of nutrients and nitrogen absorption and retention. In weanling rats, the defatted X. caffra seed meal had no effect on termination (body mass at the end of the feeding trial) and empty carcass mass and linear growth of the rats. Metabolic substrate storage, fasting blood glucose concentration and the general health profile of the growing rats were not altered by dietary X. caffra seed meal. The defatted X. caffra seed meal increased the mass of the stomach and small intestine (P = 0.0071; P = 0.0001) of rats on the test diet where a 100% dietary crude protein (CP) from SBM was substituted by CP from the defatted X. caffra seed meal. Defatted X. caffra seed meal could substitute SBM in rat and possibly monogastrics feeds without compromising digestibility, nitrogen balance, growth and general health.

Thesis (MTech (Horticulture)--Cape Peninsula University of Technology, 2017. / A study was conducted to investigate the effects of various media composition and wounding treating on the in vitro propagation of Argyroderma subalbum and A. testiculare explants derived from mature plants, antioxidants and plant growth regulators (PGR) concentrations. One experiment consisted of 3 medium types including Murashige and Skoog (MS) medium strength, vitamin supplement. Fifteen replicates were used for each treatment. The shoots were then sub-cultured to ten replicate regenerated medium consisting of varying levels and combination of indole-3-acetic acid (IAA) and 10 μM 6-Benzyladenine (BA) supplements. In another experiment consisted of varying levels of auxins with MS medium strength, activated charcoal (AC) and vitamin supplements ten replicates were used for each treatment. Results indicated the positive role of cytokinins types’ 6-Benzyladenine (BA), 2-isopentyladenine (2iP) and Kinetin in inducing callus formation from wounded explants. The highest rate of friable callus formation of wounded explants was observed in media containing vitamin supplementation with BA at 10 μM. Callus formation significantly increased with the addition of vitamins at 10 μM on BA, 2iP and kinetin. With regards to the effects of various media composition and wounding explants on in vitro growth and regeneration of A. subalbum and A. testiculare, significant results were achieved with BA, 2iP and kinetin concentrations on explants discoloration and callus formation. The antioxidant treatment, AC did not reduce explants discoloration, but the induction of the callus was developed furthermore, results showed that IAA with BA concentrations without addition of AC there was significantly difference on both species but A. subalbum dominated with browning intensity (Chapter 3). Only sub-culturing of the explants succeeded in preventing explants discoloration and subsequently increased the number of shoots. The interaction between Indole-3-acetic acid (IAA) concentrations combined with BA resulted in the most effective technique in reducing explants discoloration at the media contact point. This study provides an insight into the contributing factor and methods of overcoming the major problem of phenolic oxidation and promoting the in vitro growth and regeneration of A. subalbum and A. testiculare.

Disa comprises 163 species, 131 of which occur in South Africa (SA). The genus is distributed across winter- and summer-rainfall areas, but few species transverse both climatic regions. Species are therefore regarded as winter-rainfall or summer-rainfall endemics - yet release their seeds in autumn, irrespective of provenance. Disa contributes 40 % of threatened Orchidaceae in SA, with half of the local species requiring conservation initiatives. In vitro seed germination is a potential conservation tool for producing large numbers of genetically diverse plants in relatively short periods. However, only 11 winter-rainfall Disa species are easily germinated ex situ. Studies were therefore undertaken on summer-rainfall taxa, which are ungerminated in vitro, in an effort to define their germination parameters. This thesis describes mechanisms that control germination in Disa and establishes practical propagation methods for seed culture. Two seed types occur in Disa; i) comparatively large, pale and pyriform seeds in members of the D. uniflora sub-c1ade, which populate streamside habitats under conditions of winter-rainfall maxima, and ii) smaller, variously brown and fusiform seeds in the remainder of the genus. Seed morphometrics distinguished seed types, although embryo dimensions were similar. Testa continuity, which is disrupted in the large seeds, also supported separation. Typically, small seeds are ungerminated in vitro, whilst large seeds germinated readily. Increased seed size did not necessarily impart increased germ inability, as several germinable, small-seeded species exist - being winter-rainfall species Attempts to establish in vitro germinability revealed that increased water availability and charcoal supplementation promoted germination in intractable species. The control of germination was therefore proposed as a trade-off between water availability and the presence of phyto-inhibitors - two features typical of seeds exhibiting water-impermeable dormancy. Three germinability categories were recognized; i) easily germinable species, ii) poorly germinable species through media manipulation, and iii) ungerminated species. Germination of immature seed in the absence of media modification was comparable to mature seed germination under modified conditions, providing evidence of the role of an impermeable seed testa in regulating germination. Testa impermeability in mature, small-seeded species was demonstrated using aqueous EVANS' blue dye and was linked to i) testa integrity and ii) increased levels of leachable phenolics (LPC) - which are hydrophobic and phytotoxic. In addition, this research revealed an impervious and elaborate embryo carapace in small seeds. Large-seeded species were highly permeable at dehiscence, with perforated testae and negligible LPC. Germinability was ultimately defined by a significant regression with LPC. Phenolic deposition increased exponentially with increasing seed maturity and reflected decreased permeability and the development of testa colouration. The testa precludes the use of viability stains such as nc and FDA, unless rendered permeable through scarification. This was achieved using NaOCI. Viability and germinability percentages did not correlate well for the small-seeded Disa species, indicating that i) the methods used to break dormancy are inadequate, ii) additional factors may be acting in concert with the testa to regulate germination and iii) that the determination of mature Disa seed viability is ineffective. As an alternative, the germination potential of immature seed was estimated as the ratio between the proportion of embryos stained with TTC and the proportion of seeds permeable to EVANS' blue. Attempts to relieve water-impermeable dormancy in Disa resulted in the formulation of a dual-phase protocol - with the specific aim of increasing water availability to the embryo. Dual-phase cultures comprised a solid, charcoal-rich medium overlaid with a reduced strength, liquid medium fraction of the same type. The solid fraction negated the influence of leached phenols and allowed protocorms to establish polarity, whilst the fluid fraction increased water availability. The dual-phase protocol allowed germination of nine summer-rainfall Disa species, usually in percentages that approximated their estimated germination potential. For the remaining species, germination is controlled by more complex factors. Large seeds are atypical in containing starch, the hydrolysis of which facilitated their rapid, autonomous germination. Small-seeded Disa species stored lipids and proteins and germinable species accumulated starch post-germination. The embryo protoplasts of all species contained appreciable amounts of soluble sugars, irrespective of germinability. However, decreased sucrose and increased fructose correlated significantly with decreased seed germinability. This study provides evidence of the nutritional value of mycotrophy, with endophytes liberating soluble carbohydrate and non-carbohydrate compounds upon lysis. However, few species were germinated symbiotically, suggesting that endophytes isolated from adult roots do not necessarily support germination in the same species. Similar endophytic fungi occur in Australian and Holarctic orchids. / Thesis (Ph.D.)-University of Natal, Pietermaritzburg, 2003.

No abstract available. / Thesis (Ph.D.)-University of Natal, Pietermaritzburg, 2003.

In South Africa, Eucalyptus breeding programmes are aimed at the selection of fastgrowing varieties, with appropriate wood characteristics and/or resistance to pests and diseases. However, the slow growth rate, long generation time and heterozygosity of trees make this a difficult task. Such problems may be overcome by the adoption of a biotechnological approach for plant propagation and modification. Towards this end, the aims of this investigation were to establish protocols for the micropropagation of Eucalyptus grandis and for the Agrobacterium-mediated transformation and subsequent plant regeneration of this important species. The usefulness of transformed cells and/or tissues is dependent upon the availability of methods for their regeneration into plants. Consequently, methods for plant regeneration via indirect organogenesis from leaf discs and cell suspension cultures were investigated. Organogenic calli were produced from leaf explants on MS medium with 16 mg.1-1 &l:em•calcltrate, 20 g.I-I sucrose, 1mg.I-I NAA and 0.05 mg.1--<1 BA. Shoots were induced on MS medium containing 1 mg.1-1 ZEA and 0.2 mg.1-1 IAA, and subsequently rooted on medium containing MS nutrients supplemented with 1 mg.1- 1 IAA. Cell suspension cultures were established but not regenerated via indirect organogenesis. Additionally, various media were investigated in order to obtain somatic embryos from cell suspension cultures. The MS media supplemented with 30 g.1-1 sucrose, 12 mg.1- 1 ABA and/or 40 g.1-1 PEG were found to be most suitable, resulting in the production of embryoids; germination results are not available at this stage. In order to establish methods for the transformation of both leaf discs and cell suspension cultures of Eucalyptus, a triparental mating was performed between Escherichia coli pnT119 (donor), A. tumefaciens LBA4404 (recipient), and E. coli HBI0l::pRK2013 (helper), resulting in the transconjugant LBA4404 (pnT119); insertion of the pJIT119 plasmid was demonstrated using agarose gel electrophoresis. The transconjugant CS8C1 (pMP90) (pJIT119) was also used. Protocols for the transformation of both leaf discs and cell suspension cultures were established, and resulted in the production of putatively transformed calli which were GUS positive and with stood selection on kanamycin (50 Ilg.mr1) and/or sulfadiazine (50 Ilg.mr1). Also, Southern blotting analysis indicated that the gene transfer process was successful. Due to difficulties in the regeneration of plants from transformed calli transgenic plants were not obtained. Future research strategies and applications of the developed protocols to Eucalyptus breeding programmes are discussed. / Thesis (M.Sc.)-University of Natal, 1994.

The herbal medicine trade is thriving in KwaZulu Natal with an ever-increasing number of people harvesting and trading in indigenous plants, especially those species with medicinal and/or magical properties. The number of plants harvested has increased whereas the size of the plants collected has decreased, resulting in low recruitment into wild populations. As a result of these two factors, species diversity has decreased. To this end, the aim of these investigations was to establish micropropagation protocols for the selected species i.e. Bowiea volubilis, Haworthia_ limifolia and Cryptocarya latifolia. In addition, hardening-off protocols were also developed. The bulbous plant, Bowiea volubilis, was propagated via organogenesis using the inflorescence stem. Bulblet formation occurred directly without an intervening callus phase. Bulblets were produced on explants on Linsmaier and Skoog (1965) (LS) medium containing 30 g.r' sucrose and either I mg.r' BAP and I mg.r' 2,4-D or 1 mg.r' BAP and 1 mg.r' NAA. Shoots and roots were induced upon transfer to the basal medium devoid of plant growth regulators. Regenerated plantlets were successfully hardened-off. Haworthia limifolia, a succulent, was propagated via direct somatic embryogenesis using leaf material. Embryo formation was induced on a modified Murashige and Skoog (1962) (MS) medium containing 20 g.r' sucrose and 1 - 5 mg.r' 2,4-D. secondary embryogenesis occurred when the explants were transferred to the basal medium supplemented with activated charcoal and devoid of growth hormones. Healthy plantlets, produced from secondary embryos, were transferred to pots and acclimatised to greenhouse conditions. A large proportion of the plantlets regenerated were vitrified and as a result, this problem was addressed by changing the medium composition or culture environment. Silica gel, when placed in the culture vessel, was the best treatment for reversal of the vitrified condition. The establishment of leaf and nodal segment cultures of Cryptocarya latifolia required extensive investigation of sterilants to reduce fungal contamination. Several fungicides were tested and a successful sterilisation protocol was established. A number of media were tested for the induction of dormant axillary buds and multiplication of shoots. The best medium for both bud induction and proliferation was MS medium containing 30 g.r1 sucrose and 1 mg.r1 BAP and 0.01 mg.r1 NAA. Callus cultures were established on MS medium containing 30 g.r1 sucrose and 3 mg.rl 2,4-D. These calli, however, were non-embryogenic. Application of the established protocols and future research strategies are discussed. / Thesis (M.Sc.)-University of Natal, 1995.

Due to over-exploitation of commonly-used medicinal plants, mainly from KwaZuluNatal, because of ever-increasing human population growth, many of the useful medicinal plants are becoming depleted in their natural habitats. Some species like Warburgia salutaris, which is currently declared very rare in the KwaZulu-Natal province, appear to be on the verge of extinction. In order to counteract this overexploitation, this study sought to provide information that could help resource users to grow these threatened species through ex situ conservation methods. A short list of heavily utilised medicinal tree specles was selected from the approximately 700 tree species indigenous to KwaZulu/Natal. The criteria considered for short listing were; life form, species scarcity, past population status and part used. A total of 23 species were short listed, but a subset of 12 species was selected based on the availability of fruits and seeds. The aim of short-listing was to work on a manageable number of commonly utilised medicinal tree species. The seed physiology and growth of these species were studied. With the exception of Erythrophleum lasianthum and Curtisia dentata, all of them had a moisture content of 2': 20 % (on a dry mass basis), which is indicative of a recalcitrant behaviour. However, it could not be concluded that these seeds were truly recalcitrant because desiccation sensitivity was not directly assessed. Using the triphenyl tetrazolium chloride (TTC) viability test, most of the seeds of the 12 species seemed to be of good quality. Results of the TTC test for seed viability were similar to results obtained v using direct germination for most species. Results of flotation test for seed viability were different from the results obtained using direct germination for most spcies. The pre-treatment which achieved the highest germination percentage in almost all the seed types was cracking the outer coverings. Cracking pre-treatment appeared to be efficient in enhancing the removal of some substances which might inhibit germination of seeds. Hot water and acid pre-treatments frequently reduced germination. Growth of young seedlings was assessed in terms of stem diameter, height, and leaf area under sun and shade. Seedling growth in terms of stem diameter and height of most species did not show any significant difference. One of the few species which showed statistically significant differences in stem diameter growth was Ekebergia capensis. It was found that 3 out of lO of the species showed statistically significant differences in height growth. Two of the statistically significant differences in height occured on seedlings in the sun while one had statistically significant difference in the 40% shadecloth while 7 did not. Significant differences in leaf area occured on 7 out of lO species. Of these, 4 species had higher growth in the shade than in the sun while 3 had higher growth in the sun than in the shade. Generally, it appears that young developing seedlings establish themselves well under shade environment; this could be because most of the species used in this study are forest species. / Thesis (M.Sc.)-University of Natal, 1996.

Micropropagation methods are often used to supplement existing clonal programmes for Eucalyptus species. However, genotypic differences among clones require the implementation of clone-specific protocols, an expensive and labour-intensive exercise. Hence, this study aimed at determining high-yielding hybrid-specific rather than clone-specific, micropropagation protocols for E. grandis x nitens (GN), E. grandis x nitens (NH), and E. grandis x urophylla (GU). Different conditions for surface sterilisation, bud-break (3 protocols, 2 media), multiplication (4 media), elongation (2 protocols) and rooting (4 media) were tested. A single successful surface sterilisation approach was possible for all clones of the tested hybrids (0.0-11.8% contamination, 0.0-22.9% necrosis). It involved rinsing nodal explants in a fungicide mixture (lg/l Benlate, 1g/1 boric acid, 0.5ml/1 Bravo, Tween 20) for 15 minutes followed by calcium hypochlorite (10g/l with Tween 20) for three minutes. Results at each culture stage were dependent on genotypes, and results indicated here represent ranges in values among the clones of each hybrid. The highest bud-break values for GN clones (87-90%) and NH clones (17-75%) were achieved on a medium containing MS, 0.1mg/1 biotin, 0.1mg/l calcium pantothenate, 0.04mg/1 NAA, 0.11mg/l BAP and 0.05mg/1 kinetin. In GU clones, bud-break values on this medium (84-97%) were not significantly different to those achieved directly on a multiplication medium (80-91%) (MS, 0.1 mg/l biotin, 0.1 mg/l calcium pantothenate, 0.2mg/l BAP, 0.01mg/1 NAA). Shoot multiplication yields for GN clones (4-13 shoots/bud) and GU clones (2-6 shoots/bud) were achieved on a medium consisting of MS, 0.1mg/1 biotin, 0.1 mg/l calcium pantothenate, 0.2mg/1 BAP and 0.01 mg/l NAA. As genotypic effects were highly significant among NH clones, a single multiplication medium for all clones of this hybrid could not be determined. The best method of elongation for clones of all three hybrids involved culturing shoots on MS, 0.1 mg/l calcium pantothenate, 0.1mg/1 biotin, 0.35mg/1 NAA, 0.1mg/l kinetin and 0.1mg/1 IBA, under photoperiod conditions, rather than total darkness, for 6 weeks. This resulted in 82.3-86.6% elongation and shoot lengths increasing by 22.9-35.2 mm for GN clones, 80.2-82.3 % elongation and an increase in length of 24.7-32.2 mm for NH clones and 70.8-78.1 % elongation, and shoot elongation of 21.6-29.3 mm for GU clones from passage 1-2. For all the above stages, media contained 20/25 g/l sucrose and 3.5g/l Gelrite, and cultures were maintained at 25°C ± 2°C day/ 21°C night with a 16 h light/ 8 h dark photoperiod (PPFD 66µmol/m2/s). In terms of rooting, cultures on different media were initially subjected to a 72 hour period of total darkness at room temperature, then a 16 h light/8 h dark photoperiod (PPFD 37µmol/m2 /s) at 24°C day/ 21°C night for 7 days. This was followed by a 16 h light/ 8 h dark photoperiod (PPFD 66µmol/m2/s) at 25°C ± 2°C day/ 21°C night for 21 days. Tested clones of the three hybrids were all rooted successfully (56-93% rooting in GN clones, 36-76% rooting in NH clones and 46-96% rooting in GU clones) on a medium containing ¼ MS, 0.1 mg/l biotin, 0.1 mg/l calcium pantothenate, 0.1mg/l IBA, 0.22g/1 CaCI2 .2H20, 0.185g/l MgS04.7H2O, 15g/l sucrose and 3.5g/1 Gelrite. Predicted yields from the established protocol are also presented (168-667 plants of E. grandis x nitens (GN), 35- 854 plants of E. grandis x nitens (NH) and 54-349 plants of E. grandis x urophylla from 100 initial nodal explants, depending on the clone). Hence, the established protocols can be used successfully for some of the clones, but the implementation of specific media and methods to obtain high yields may still be necessary for certain clones. / Thesis (M.Sc.)-University of Natal, Durban, 2001.

For the South African forestry industry, the patula pine (Pinus patula) is the most commercially important softwood species. A pine clonal programme has yet to be fully implemented in this country and at present much effort is being made to establish clonal plantings of selected trees. In order to accomplish this, it is essential that satisfactory commercially viable propagation technologies be developed for this species. This study examined the possibilities and constraints of three different in vitro systems for mass propagation of rare and important P. patula material. Seed germination and sterilisation techniques were developed for adventitious bud and somatic embryogenesis experimentation. Adventitious buds were initiated from excised 'mature P. patula embryos cultured on LM medium containing 5 mg 1-1 BA. Although, between 50 and 60% of the embryo explants produced adventitious buds, only 3-5 buds per explant actually developed further to form distinct shoots. The adventitious shoots elongated slowly (±8 mm in 2 months) on LM medium, containing 10 g 1-1 activated charcoal. Axillary buds were induced on 10 week-old juvenile shoots, after the development of an effective surface sterilisation procedure, using 0.02% HgCL2. The effect of removing the apex and trimming the needles on bud induction was significant. Dwarf shoots elongated at a rate of 25 mm in 5 weeks. Rooting studies conducted on juvenile P. patula shoots indicated that the most effective treatment was wounding the shoot base and placing the shoot in composted bark growing medium, under a greenhouse mist regime. Rooting percentages were low (50%). Included in this study is the first successful production of somatic pro-embryos from mature Pinus patula embryos. Calli were produced on LM induction medium containing 2 mg 1-1 2,4-D. Cultures were first placed in the dark for 4 weeks and then transferred to a 16 h photoperiod for a further 2 weeks, after which Stage 1 embryogenic cells were observed. When calli were placed on LM maturation medium, containing 12 mg 1-1 ABA, for a further 6-8 weeks, pro-embryo structures (maximum of 7 pro-embryos per callus) were detected embedded In the callus mass. Hence, investigations into the development of protocols for the micropropagation of Pinus patula, were undertaken. Two major constraints for applying in vitro techniques to the commercial production of pine were identified: the poor yield of shoots and pro-embryos and the length of time taken for plantlets to be produced. This study, however, provides some fundamental knowledge and background work required by tree breeders who wish to implement biotechnological techniques in the selection and improvement of P. patula genotypes. / Thesis (M.Sc.)-University of Natal, Durban, 1993.