Tagetes minuta L. is a weedy herb that has been a rich source of fragrant oils, used as in the perfume and flavour industry. T. minuta achenes germinate erratically under field conditions. However, at the optimal germination temperature of 25 °C, 100 % germination is attained within 48 h of imbibition. The achenes are thermoinhibited at 35 °C. The aims of this project were to assess the role of cytokinins (CKs) in normal germination at 25 °C, and to investigate the factors that regulate thermoinhibition at 35 °C. CKs were extracted from achenes germinating at 25 °C at 0, 24; 48; 96 and 144 h after imbibition. Two different purification techniques were used, namely Dowex cation exchange resin followed by paper chromatography, or high performance liquid chromatography (HPLC). CK-like activity was tested with the soybean callus bioassay. With both techniques, a peak in CK-like activity appeared 24 h after imbibition, which coincides with the period during which most of the achenes germinated. For quantitative analysis, HPLC\mass spectrometry (MS) techniques were used. The isoprenoid CKs were far more abundant in T. minuta achenes than the aromatic CKs. cis-Zeatin (cZ) and its derivatives were the most abundant CKs. In total, 19 CK compounds were detected, including 4 free bases and a number of corresponding conjugates. Benzyladenine (BA) was the only aromatic CK detected. There was no common time at which active free base maximal concentrations were detected, suggesting that different CKs may have specific roles in the germination process, and thus peak at different times. This in turn suggests that germination is not a single process, but rather a correlative process involving a number of events, with specific CKs having specific roles relating to these correlative events. There is sufficient evidence obtained from both the soybean callus bioassay and HPLC/MS analysis to suggest that CKs have an active role in T. minuta germination. A decline in free BA during germination without corresponding conjugation, suggests that BA is actively used in early germination processes, possibly in the stimulation of DNA synthesis. Secondly, there was a distinct dihydrozeatin (DHZ) peak obtained at 24 h. Roughly 75 % of the achenes germinate between 16 and 26 h, thus it is likely that DHZ has an active role during the germination of T. minuta. Although CKs are probably not involved in the breaking of dormancy per se, the distinct peak in CK-like activity obtained in the bioassays, 24 h after imbibition, suggests that CKs have an active role in the germination of T. minuta. With respect to the regulation of thermoinhibition, a number of exogenous treatments were applied, including hormones [gibberellins (GA₄₊₇), abscisic acid (ABA), ethylene and a number of CKs], adenosine triphosphate (ATP) and incubation in 100 % oxygen. ABA was extracted from thermoinhibited and germinating achenes to assess the role of ABA in thermoinhibition and germination. While exogenous 0.1 mg L¯¹ GA₄₊₇ application slightly improved normal germination at 25°C, no treatments were effective in alleviating thermoinhbibition in T. minuta achenes. Thermoinhibition in T. minuta achenes may be under hormonal regulation, as there is strong evidence for the role of ABA in the maintenance of dormancy and thermoinhbition. High ABA levels were found in dry control samples. Additionally, exogenous ABA application inhibited normal germination, and the commencement of germination was accompanied by a decrease in endogenous ABA levels. A number of experiments relating to the imposition of thermoinhibition were carried out. Thermoinhibition appears to be very rapidly imposed. Germination is rapidly inhibited following shifting to higher thermoinhibitory temperatures, even after prolonged exposure to optimal germination temperatures. Results suggest active de novo biosynthesis of ABA in thermoinhibited achenes. Active biosynthesis of ABA during thermoinhibition suggests that this phytohormone is essential in the maintenance of thermoinhibition of T. minuta achenes. It thus appears that ABA is synthesized in the achenes in response to elevated temperatures that are unfavourable for germination to proceed. Unfavourable environmental conditions result in an achene-mediated inhibition of germination, which appears to be initiated and maintained by elevated levels of endogenous ABA. / Thesis (M.Sc.)-University of Natal, Pietermaritzburg, 2003.

A review of the literature indicated that the purine cytokinin 6-(benzylamino)purine (SA) may be converted to a wide range of metabolites. Although the functional significance of these metabolites remains obscure, cytokinin physiologists have essentially classed them as either active or inactive. Inactivation of cytokinins is considered to proceed via catabolic oxidation (side-chain cleavage), or N-conjugation with glucose or alanine moieties. The literature survey was hampered by the confusing array of synonyms which have been coined for cytokinin metabolites. Accordingly, a working system of (semi-systematic) abbreviations was devised which accommodated all groups and classes of purine cytokinins. Prior to commencing metabolic interactive studies, it was necessary to resolve the contentious issue associated with the successful extraction of cytokinin nucleotides. Five-week-old soybean callus was fed [8[-14]C]BA and subsequently extracted using four widely used cytokin in extraction techniques. Techniques compared were a modified Bieleski method, 80% ethanol with tissue homogenisation, 80% ethanol without homogenisation, and boiling ethanol. All four procedures produced similar results, showing that all metabolites of SA, including the nucleotide, were adequately extracted. It was concluded that the extraction of nucleotides with Bieleski solvents did not warrant the inconvenience. Auxins have been shown to interact with cytokinins in the regulation of many physiological processes, although little is known of the mechanisms of interaction which proceed at the metabolic level. Previous investigators have shown that auxin promoted cytokinin degradation through catabolic oxidation, Shoot-apex and seed derived cell suspensions of Dianthus zevheri subsp. natalensis were incubated with [8[-14] C]BA for between 30 minutes and 48 hours in the presence of both low (2 mg l-1) and high (4 mg 1¯¹) levels of exogenously supplied 2,4-dichlorophenoxyacetic acid (2,4-D), In both systems, the auxin 2,4-D was shown to promote SA inactivation through 7-glucosylation (N-conjugation). This observation represents the first report of auxin-promoted cytokinin N-conjugate formation. The auxin effect on metabolism was transient in the case of shoot-apex, but not in seed-derived systems over a 48 hour period. Formation of the 7-glucoside of SA was dose-dependent in apex-derived cultures. Further studies were undertaken with indole-3-acetic acid (lAA) and α-naphthaleneacetic acid (NAA). It was found that auxin-promoted 7-glucosylation of SA was only minimally effected by these two auxins. In comparable studies with soybean suspension cultures (Glycine max cv. Acme), 2,4-D-promoted catabolic oxidation was observed between 18 and 48 hours, following application of phytohormones. The main catabolite was tentatively identified as adenosine-5'-monophosphate (AMP), based on chromatographic characteristics. Carrot (Daucus carota) cell suspensions similarly supplied with 2,4-D and SA maintained a large active cytokinin pool. Neither substantial oxidative nor Nconjugative processes were observed. Instead, there was a transient effect by 2,4- D on the relative formation of the riboside and the 7- and 9-glucosides of SA. The effect of auxin on the metabolism of SA thus varied with the species and system investigated. Generally, auxin promoted (rather than inhibited), the formation of inactivated metabolites and catabolites of SA, possibly by the induction of relevant enzyme systems. Transient auxin effects on the metabolism of SA are discussed in relation to the role of the auxin/cytokinin balance in the induction of developmental processes. / Thesis (Ph.D.)-University of Natal, Pietermaritzburg, 1993.

Solasodine, a steroidal alkaloid, is used by the pharmaceutical industry in certain parts of the world, as a raw material in the synthesis of steroid drugs. The compound is contained in many members of the genus Solanum, including S. mauritianum Scop., a common weed in South Africa. The levels of solasodine in three culture systems of S. mauritianum under various cultural conditions were examined. A high performance liquid chromatographic (HPLC) method was developed for the detection of solasodine. In order that low-cost, fixed wavelength ultra-violet detectors could be used, which would make the technique more widely applicable, a derivatization step, namely benzoylation, was included in the sample preparation. An extraction and purification protocol was then established, that complemented the HPLC technique and allowed successful detection of solasodine levels in a whole range of different sample types, including callus, suspension cultured cells, roots, stems and leaves. The three culture systems examined were callus, suspension and hairy root cultures. The callus system was used to establish which cultural parameters affected solasodine content in vitro to the greatest extent. A control culture was grown on a MURASHIGE and SKOOG (1962) medium (excluding glycine) supplemented with 3 % sucrose, 0.1 g I ¯¹ myo-inositol and lacking hormones. This culture contained an average of 9.2 μg g ¯¹ DW of solasodine. Many factors, including alteration of the carbon : nitrogen ratio and substitution of Gelrite for agar as the gelling agent, had no significant effect on the solasodine content of the callus or its growth. Greatly increased solasodine productivity of the callus was recorded when glucose was substituted for sucrose, the medium strength was reduced by half, or certain combinations of the hormones benzyladenine and naphthaleneacetic acid were added to the medium. The maximum levels of solasodine recorded in these cultures, on a per gram dry weight basis, equalled those of the vegetative parts of an intact S. mauritianum plant, but were approximately three times lower than those of the green berries. Suspension cultures could not be grown on the same medium as the callus cultures. Substitution of the vitamin complement of MURASHIGE and SKOOG (1962) with the so-called RT vitamin complement of KHANNA and STAB A (1968), resulted in successful growth and maintenance of S. mauritianum suspension cultures. The auxin, 2,4-dichlorophenoxyacetic acid (1 mg I ¯¹) was included in the medium. None of the suspension cultures grown on this medium, or slight modifications thereof, contained any trace of solasodine. This system could therefore not be used for the synthesis of solasodine in vitro. Hairy root cultures were initiated by inoculation of an excised hypocotyl of an in vitro-grown seedling of S. mauritianum with a 48 hour culture of Agrobacterium rhizogenes LBA 9402. Transformation frequency was extremely low. The transformed roots could be excised and grown successfully on a phytohormone-free medium, either in the solid or liquid form. Solasodine was extracted from hairy roots grown in a full-strength liquid MURASHIGE and SKOOG (1962) medium (excluding glycine) supplemented with 3 % sucrose and 0.1 g I ¯¹ myo-inositol, a half-strength such medium and a full-strength medium with 3 % glucose substituting for 3 % sucrose. In the latter medium, growth was very poor, whereas in the other two media, growth was very rapid . Both solasodine content (126 μg g¯¹DW) and root growth were greatest in the full-strength medium supplemented with 3 % sucrose. This level of solasodine was greater than that found in any of the callus cultures or vegetative parts of the plant and approached that of the green berries of S. mauritianum. Overall, of the culture types of S. mauritianum tested, the hairy root culture system appears to be most favourable for the in vitro production of solasodine. / Thesis (Ph.D)-University of Natal, Pietermaritzburg, 1993.

Using the Scanning Electron Microscope (SEM), fruit surface and pericarp anatomy were investigated in the African species of Bolboschoenits (Ascherson) Palla [B. maritimus (L.) Palla; B. glaucus (Lam.) S.G. Smith; B. nobilis (Ridley) Goetghebeur & D.A. Simpson; B. grandispicus (Steud.) K. Lewejohann & W. Lobin] and extended worldwide to cover most known species. There was consideration of type specimens wherever possible. Two main patterns of pericarp construction were revealed, with modifications. Using fruit samples from field populations, a surveillance of embryos was carried out to gain some information on percentages of perfect embryo development and variability in embryo outline (as seen in optical, median sagittal section), within and between selected species. Inflorescence structure within African species was studied and illustrated photographically and diagrammatically. The collective information obtained was directed towards better understanding of species limits within the genus and towards gaining evidence of the significance of natural hybridisation as a cause of morphological variability within some taxa. Formal taxonomy including a key to the identification of the African species is provided. Profuse illustrations of pericarp structure of world species are given, as are colour photographs of the African species, excepting B. grandispicus. A tentative pattern for subgeneric division of Bolboschoenus based primarily on pericarp morphology, is suggested supplemented by a world map illustrating the presently known distribution of the suggested groupings. / Thesis (Ph.D.)-University of Natal, Pietermaritzburg, 1998.

This study extends an earlier M.Sc. research project on the narrow-leaved species of the genus Asclepias L. (30 species) to cover the entire subtribe Asclepiadinae sensu K. Schum. in southern Africa- (182 species in 23 genera). Two genera (Eustegia R. Br. and Pentarrhinum E. Mey.) are revised and then removed from this tribe. The remaining 177 species and 21 genera form the focus of this thesis, whose principal objectives are to reevaluate the taxonomic and evolutionary significance of various macro, micro and chemical characters and then use them to produce a classification that, more closely, reflects the overall similarity and phylogeny of the taxa involved. Species and genera are recircumscribed based on the wealth of data that has come to light since the subtribe was last revised by N.E. Brown (1907-1908) some 90 years ago. This process was supplemented by extensive field work, observations on pollination and reproductive biology, ecology, biogeography, conservation and ethnobotany. The majority of this thesis consists of a compilation of 17 papers, 12 of these published and most, but not all, of the remainder in preparation for press. Two of these papers form the bulk of the taxonomy. The first deals with what was the genus Asclepias in southern Africa. The genus is now believed to be confined to the Americas. The • Botswana, Lesotho, Namibia, South Africa and Swaziland. southern African species have diverse origins and are partitioned into 7 genera, one of them (Gomphocarpus R. Br.) resurrected, two of them (Aidomene Stopp and Aspidonepsis Nicholas & Goyder) expanded and four of them (Paulforstera, Sigridia, Bruynsia and Pachyacris) described as new. Gomphocarpus is divided into two subgenera and Aidomene into four subgenera. Three new species are also described. The second paper investigates the bulk of most of the remaining genera. Kanahia R. Br., Cordylogyne E. Mey. and Fanninia Harv. remain as is. Xysmalobium R. Br., previously a genus of 19 species in southern Africa, is reduced to three species in two subgenera. Trichocodon is segregated off from Pachycarpus E. Mey. as a new genus. While two species, previously placed in Xysmalobium, are added to Pachycarpus, but placed in the new subgenus Parapodiopsis. Parapodium E. Mey. is reduced from three to two species and Periglossum Decne. is reduced from five to three species, one of them newly described. Woodia Schltr. and Stenostelma Schltr. are both considerably expanded, mainly with species previously housed in Xysmalobium), and the former divided into two subgenera. The third paper briefly looks at the Schizoglossum E. Mey., Miraglossum Kupicha and Aspidoglossum E. Mey. Some changes are suggested but, as further work is needed, none are formalised. As a corollary to the taxonomy, secondary metabolite profiles of 38 species and 17 genera were done using Thin layer Chromatography. The results sometimes confirmed morphological patterns and sometimes were at odds with them. A trend from simple profiles to more complex profiles seems to echo the suspected phylogeny of the genera within this sub tribe. Some species and genera have greater chemical diversity than others and secondary metabolites are shown to vary considerably in different parts of a single plant. As a supplement to the above work or because they are cited elsewhere in the dissertation, published papers dealing with floral structure, the asclepiadaceous work of Rudolf Schlechter, as well as miscellaneous works in the tribe Stapelieae, are also given. / Thesis (Ph.D.)-University of Durban-Westville, 1999.

This thesis examines the application of the heat pulse velocity technique (HPV) to plantation-grown Eucalyptus grandis in the Eastern Transvaal, South Africa. The work addresses the application of the technique per se and is ultimately focused on improving the prediction of the hydrological impact of afforestation, to assist in the equitable management of South Africa's limited water resources. The verification of the HPV technique on E. grandis against the cut-tree method showed that the technique accurately reflected the water uptake in four three-year-old trees and a sixteen-year-old tree. It was found that accurate measurement of wound size and probe separation was essential for accurate water use estimates. The optimal probe allocation strategy for accurate measurements of transpiration in individual trees and stands of trees was examined. Stratifying the depths of implanted probes resulted in greater precision and repeatability in the HPV-derived estimates of sap flow in E. grandis. Given a limitation in the number of probes available to estimate stand transpiration, the results showed that sampling many individuals with a low sampling intensity (few probes per tree), rather than sampling few individuals intensively, improved the estimate of stand transpiration. An examination of the influence of tree age and season on transpiration rates showed that the transpiration rate per unit leaf area of E. grandis declined with age. Also, transpiration rates were higher in summer than under equivalent conditions of evaporative demand in winter. A seasonal change in the response of transpiration to VPD was implicated as the primary cause of this shift. A number of models were derived to predict transpiration from E. grandis. The variables vapour pressure deficit (VPO) and photosynthetically active radiation (PAR) were found to account for a large proportion of the observed variation in transpiration from the age sequence of trees studied. The models developed are applicable to trees of varying age, but are valid only for conditions where minimal soil water stress is experienced. The derived models were tested against two sets of independent data. This confirmed that a simple linear multiple regression adequately describes the relationship between transpiration and the two driving meteorological variables, PAR and VPO, in E. grandis. The application of a selection of the developed models on a sample data set from Sabie showed that transpiration from a three-year-old stand of E. grandis in summer may be more than double that for a sixteen-year-old stand under the same conditions. Simulated results also showed that transpiration in summer was about 25 to 50% higher than that from the same stand during winter conditions. Simulated transpiration rates from the young E. grandis stands were high, suggesting that further validation of the estimated rates is required before the models are applied. It is concluded that the HPV method is an ideal technique to estimate water use in E. grandis trees. The models developed represent a major advancement on previous models used to predict the hydrological impact of afforestation on mountain catchments. / Thesis-(Ph.D.)-University of Natal, Durban, 1994.

The cancer bush (Lessertia frutescens L.) is an important leguminous perennial native to southern Africa and has been used for centuries in traditional medicine by the continent’s diverse cultural groups. Like many other legumes, the seeds of this species exhibit dormancy. Moreover, woody plants are typically difficult to propagate in in vitro culture systems. But in vitro shoot cultures are valuable in providing an alternative means of deriving desired secondary metabolites or phytocompounds, under controlled conditions. This study describes novel protocols for breaking seed dormancy, rapid and efficient in vitro propagation, bioreactor culture, and comprehensive phytochemical data following screening and analysis of in vitro and field extracts of L. frutescens. Experiments using physical, mechanical and chemical pre-sowing treatments were conducted to determine the germination response of this species. The results indicated that seeds of L. frutescens exhibited exogenous dormancy due to the inhibitory effect of the hard coat on germination. Seed dormancy was released by mechanical scarification in which 100 % germination was achieved. In vitro propagation studies using single node explants in Murashige and Skoog (MS) medium supplemented with combinations of different concentrations of benzyladenine and naphthaleneacetic acid revealed a maximum number of 10 shoots per explant in solid medium, and 12.9 shoots per explant in liquid medium inside a temporary immersion bioreactor. Indirect shoot organogenesis and plant regeneration using rachis and stem segments was achieved with the highest percentage of explants forming shoots (88.8 %) from rachis explants cultured onto MS medium supplemented with thidiazuron. Direct shoot organogenesis from hypocotyl and cotyledon segments was also achieved in L. frutescens. The highest shoot regeneration using hypocotyls (83 %) was obtained in MS medium supplemented with kinetin whilst the highest shoot regeneration using cotyledons (46 %) was obtained in MS medium supplemented with kinetin in combination with benzyladenine. Successful rooting (up to 80 %) and acclimatization (up to 90 % survival rate) was attained. Spectrophotometric and gravimetric methods indicated that saponins were the most abundant, followed by phenolics, flavonoids and then alkaloids in in vitro leaf extracts then in field leaf extracts and seed extracts, respectively. After qualitative analysis these extracts were also found to contain tannins, phlobatannins and cardiac glycosides of medicinal interest. By using gas and liquid chromatography the presence of the medicinally important L-canavanine, gamma amino-butyric acid and D-pinitol was verified in in vitro leaf, field leaf and seed extracts. In vitro leaves had higher quantities of all compounds, except for D-pinitol. Phytocompound analysis of shoots derived from several of the cytokinin-enhanced media showed that these organs contained higher quantities of L-canavanine compared to the control. This study, therefore, highlights the potential techno-economic production of medicinal phytocompounds from in vitro leaves of L. frutescens following large scale production using the protocols described in this study. / Thesis (Ph.D.)-University of KwaZulu-Natal, Westville, 2011.

Six different lots of soya beans (Glycine max (L.) Merr.) were examined. Seed hydroperoxide levels were highly correlated with viability, but not with moisture contents. It was proposed that moisture contents may exert a similar antioxidant effect at intermediate levels as has been observed in dry foods. Seeds treated with ferrous sulphate were significantly (S% level) invigorated. Furthermore, this treatment was observed to give rise to a reduction in the peroxide value of soya bean axes over the first hour of imbibition, an increase in 2,3,S-triphenyl-tetrazolium chloride reduction and protein synthesis, and a decline in electrolyte leakage. It was proposed that this was due to the antioxidant activity of the ferrous iron, leading to an attenuation of free-radical induced autoxidation. Ferrous sulphate treated seeds produced more aldehydes than untreated seeds. This result suggested that aldehydes may not be responsible for declining seed vigour. Hexanal, pentanal and butanal production from heated dry seeds was significantly correlated with seed germination, CVG and hydroperoxide levels. The thermal breakdown of the hydroperoxides was postulated to be the source of these compounds. A GC technique was developed using model systems of oxidized methyl oleate, linoleate, linolenate and soya bean bulk oil. The analysis of seed lipid oxidation products revealed marked differences in the proportions of the products compared to bulk and monolayer oxidation. The selective production of the 13-hydroperoxide implicated enzymatic or metalloprotein involvement. The implications of the results of this study with regard to the present theories of seed ageing were discussed. / Thesis (Ph.D.)-University of Natal, Durban, 1990.

In South Africa, large quantities of Scilla natalensis are harvested from wild populations and sold as traditional medicine, which is reducing the density, distribution and genetic diversity of wild populations. The enforcement of existing legislation, however, has proved ineffective with plants being traded locally and internationally. It has therefore, been suggested that ex situ conservation through cultivation may alleviate pressures on natural resources. Conventional propagation of these plants, however, is usually fairly slow. In vitro propagation provides a rapid means of propagating selected chemotypes or cultivars, serving both conservation and commercial interests. In the first part of the study, continuous culture systems were established for the three forms of Scilla natalensis, S. natalensis sensu stricto (Form A), S. natalensis syn. S. kraussii (Form B) and S. natalensis syn. S. dracomontana (Form C). The efficiency of the systems was strongly influenced by genetic factors, viz the form and epigenetic factors, viz the explant type, carbohydrate source, plant growth regulators and gelling agents. The form, Form A, Form B or Form C respectively, influenced shoot initiation with the larger forms generally producing more shoots than the smaller forms (Form A > Form B > Form C). The data confirmed that the three forms are significantly different in terms of their physiological response to carbohydrates, plant growth regulators and gelling agents in vitro. Since the effect of form could not be compensated for by the addition of either carbohydrates, plant growth regulators or gelling agents, this may provide some support for the reinstatement of these forms as three species, Scilla natalensis Planch., S. kraussii Bak. and S. dracomontana Hilliard & Burtt. The explant type, that is bulb or leaf explants respectively, significantly influenced shoot initiation. Leaf explants generally produced more shoots than bulb explants. The carbohydrate source significantly influenced shoot initiation. The explants generally produced more shoots when cultured on media containing glucose or sucrose than on media containing fructose, lactose, maltose and particularly mannitol. The combination of cytokinins and auxins significantly influenced shoot initiation. Shoot initiation was higher for combinations of kinetin: IAA than for combinations of kinetin: NAA or TDZ: NAA. Optimal shoot initiation for Form A, Form B and Form C occurred on media containing 1 to 2 mg I-1 kinetin and 1 to 2 mg I-1 IAA. The gelling agent also influenced shoot initiation with media solidified with Gelrite® producing more shoots than media solidified with Oxoid or Unilab agar. Shoots were then rooted on media containing IAA, IBA or NAA and the plantlets were successfully acclimatised. These continuous culture systems can be used to produce large quantities of plantlets, which may alleviate pressures on natural resources and provide an alternative source of high quality plants for the burgeoning medicinal plant market. In the second part of the study, the effect of carbohydrate source and concentration on growth and development of shoots of S. natalensis syn. sensu stricto (Form A) were determined. This has applications for the acclimatisation and germplasm storage of bulbous plants. The carbohydrate source and concentration significantly influenced the growth and development of shoots. In the absence of carbohydrates, the shoots were short with spindly leaves and short roots. When media were supplemented with high concentrations of fructose, the shoots were long with broad leaves, small bulbs, and few short to medium length roots at low concentrations. At higher fructose concentrations, however, the shoots were robust and short with narrow, sometimes deformed leaves, large bulbs, and few stunted, brown roots. When sucrose was substituted for fructose, the shoots were robust and long with narrow and often red-pigmented leaves, large bulbs, and many long, thick roots. When AC was used in combination with sucrose, however, the shoots were robust and short with few, and occasionally red-pigmented leaves, small to medium bulbs, and few, severely stunted roots. Optimal shoot growth and development in terms of shoot weight (FW) and quality occurred on media containing glucose or sucrose (40 to 60 g I-1). The carbohydrate source and concentration also significantly influenced the physical properties of media particularly pH, electrical conductivity (EC) and gel-strength. The pH decreased slightly with increasing glucose concentration but decreased significantly with increasing fructose concentration when fructose was used alone or in combination with glucose. The pH also decreased significantly with increasing sucrose concentrations when sucrose was used in combination with Sigma AC. The EC decreased significantly with increasing fructose concentration when fructose was used alone but remained fairly constant irrespective of glucose concentration when glucose was used alone or in combination with fructose. The EC also remained fairly constant irrespective of the sucrose concentration but decreased with increasing sucrose concentration when used in combination with AC. The gel-strength remained fairly constant irrespective of glucose. The gel-strength decreased with increasing fructose concentration when used alone or in combination with glucose. The gel-strength of media increased with increasing sucrose concentration although the addition of Sigma AC significantly decreased the gel-strength of media, which decreased with increasing sucrose concentration. The brand and concentration of AC also influenced gel-strength. The matrix plot suggested that the effect of carbohydrate source and concentration on the growth of shoots may be largely due to the indirect effects of these physical properties such as hydrolysis of carbohydrates, the spectrum and quantity of the breakdown products and the availability of nutrients, plant growth regulators and water rather than the direct effects of pH, EC and gel-strength per se. / Thesis (Ph.D.)-University of Natal, Pietermaritzburg, 1999.

Clonal propagation of Eucalyptus spp. and its hybrids allows for competitiveness in the commercial forestry industry through the propagation and preservation of superior/elite genotypes. Vegetative propagation through rooted cuttings is the industry‟s standard and the choice of clones selected for plantations are determined by their rooting ability. However, as many potentially valuable genotypes are recalcitrant to adventitious rooting, micropropagation is the only effective means of propagating them. Micropropagation results in high plantlet yields, achieved primarily through the empirical use of the key plant growth regulators (PGRs) cytokinins and auxins, for shoot and root production, respectively. Their selection for use in vitro is driven by their effects on percent rooting rather than root quality. Little is known regarding the quality of the roots of the plantlets ex vitro, but there is some evidence that they are different from those of seedlings and cuttings. It was therefore hypothesized that the properties of exogenous PGRs and their interaction with other exogenous and endogenous PGRs, influenced root development and subsequent root quality. This was tested in vitro using a good-rooting E. grandis (TAG31) and two poor-rooting E. grandis x nitens hybrid clones (GN155 and NH58). In the former, the auxins supplied during the pre-rooting culture stages (multiplication and elongation) were sufficient for 100% rooting in an auxin-free rooting medium. Different combinations of PGRs in the two pre-rooting stages, followed by rooting without auxins, revealed a direct relationship between the stability of the supplied auxin and the rooting ability of TAG31. Gas chromatographymass spectrometry (GC-MS) analyses indicated that endogenous shoot levels of indole- 3-acetic acid (IAA) influenced graviperception. Also, low IAA content was associated with atypical starch grain accumulation or its absence from root tips (53.1 nmol IAA gˉ¹ DW compared with 325.7 nmol IAA g-¹ DW in gravisensing roots). The specific roles of the natural auxins IAA and IBA on root morphogenesis were then investigated using 2,3,5-triiodobenzoic acid (TIBA; inhibits IAA transport), ρ-chlorophenoxyisobutyric acid (PCIB; inhibits auxin signal transduction), and the auxin antagonist kinetin in the rooting medium, following root induction. After 3 weeks, the mean root diameter was significantly reduced from 552.8μm (control) to 129.2μm (with PCIB) and 278.6μm (with kinetin). TIBA increased root diameter to 833.4μm, decreased Δ root length, increased root vasculature and resulted in agravitropism. Hence, whereas rooting could be induced by IBA, IAA was necessary for the maintenance of vascular integrity and graviperception. This critical role of IAA in root development is of importance as IBA, owing to its higher stability, has been traditionally relied upon for root induction in the majority of micropropagation protocols. The potential of incorporating IAA into the media formulations of in vitro protocols for poor-rooters that do not respond well to IAA was then investigated, using GN155 and NH58. While PCIB in the rooting medium of GN155 completely inhibited rooting, the addition of dihydroxyacetophenone (DHAP), an inhibitor of auxin conjugation, to the rooting medium, did not significantly increase % rooting in the presence of 0.1 mg 1ˉ¹ IBA (i.e. 50% rooting with 2mM DHAP and IBA, compared with 45% with IBA alone). The results suggested that the inability of some eucalypts to induce roots easily in vitro was not due to a deficiency in auxin signal transduction or to auxin conjugation. Instead, rooting was inhibited by an accumulation of kinetin within shoots during the pre-rooting culture stages. The endogenous levels of PGRs in shoots of GN155 and NH58 showed a strong relationship (R² = 0.943) between the shoot kinetin:auxin and shoot rootability. Substituting kinetin with the relatively less stable natural cytokinin trans-zeatin in the elongation stage resulted in a significant increase in % rooting in both clones, from 19% to 45% (GN155) and from 31% to 52% (NH58), with 0.1 mg 1ˉ¹ IAA in the rooting medium. However, omitting all cytokinins from the elongation medium, resulted in over 95% and 75% rooting of shoots of GN155 and NH58, respectively, with 0.1 mg 1ˉ¹ IAA. These results suggest that IAA is a requirement for root development and cannot be substituted by its analogues in certain root developmental events. Hence, IAA should be the preferred auxin for eucalypt micropropagation. As fundamental research, the approach taken in this study circumvents the empirical method used in improving micropropagation protocols. The importance of the properties and the interactions between endogenous and exogenous PGRs in regulating root morphogenesis, and the practical implications of these findings is emphasised. / Thesis (Ph.D.)-University of KwaZulu-Natal, Westville, 2011.