Components of the South African indigenous flora are disappearing at an alarming rate, due to pressures on land use. The flora is protected by proclamation of reserves and conservation legislation, however these measures can never be wholly successful. For these reasons, methods for propagting Clivia miniata, Gloriosa superba and Sandersonia aurantiaca using in vitro techniques were investigated. The highly sought after Clivia miniata var citrina can be successfully cultured using fruit and floral explants. Use of these explants may limit the number of plants produced in culture due to the seasonal nature of flowering. Gloriosa superba and Sandersonia aurantiaca can be propagated using corm explants, with subsequent in vitro stimulation of cormlet formation. To establish a successful tissue culture procedure an integrated approach to all aspects of the culture is necessary. Sterilization techniques should be empirical and specific for each species and explant. The most critical factor in establishing a culture technique is the choice of a suitable explant. Without a suitable explant the success of the culture procedure may be severely limited. Nutritional and environmental variation may modify the explant response in culture, but initial culture response can be directly related to the origin of the explant, particularly, size, time of the year, age and physiological status. Since the discovery of colchicine in Gloriosa by CLEWER, GREEN and TUTIN (1915) a number of researchers have put forward the idea that Gloriosa would serve as a source of colchicine. The present trend in biochemical production is via artificial synthesis, however many desirable compounds still have to be extracted from plant material for biochemical production. The utilization of plant cells that are cultured in vitro provides a viable alternative to the problems involved in the production of chemical compounds. Levels of colchicine in Gloriosa and Sandersonia are very similar, in the range of ± 0,9%. From evidence presented by BELLET and GAIGNAULT (1985), levels of colchicine in the two study species is much higher than the recorded level (0,62%) of Colchicum. This higher level of the alkaloid makes these two plants a viable source for commercial colchicine production. Levels of colchicine recovered from in vitro grown roots and callus was 10 - 20 times lower than that found in -in -viv-o tissue. Levels of colchicine extracted from plantlets grown in vitro was the same as that normally recorded for parent tissue. Higher levels of colchicine in malformed roots adds to the evidence that differentiation increases colchicine production in Gloriosa tissue in vitro. It has been shown that Gloriosa and Sandersonia tissue can synthesize colchicine in vitro. The extent to which the cells synthetic capacity can be enhanced has yet to be determined. However, research into speedier and more wide ranging methods for metabolite production in culture is receiving attention throughout the world. / Thesis (Ph.D.)-University of Natal, Pietermaritzburg, 1988.

South Georgia is renowned for the abundance of Antarctic krill (Euphausia superba) and a range of krill predators. Variability in krill availability at a range of scales, and the consequences of this for predator-prey interactions, mean that quantifying the spatially explicit demand for krill by those predators is essential to understanding the mechanisms underlying ecosystem changes in the region. In this thesis demand within a distinct study box to the northwest of the island has been assessed. The thesis has three sections; (1) the number of predators; (2) the distribution of predators; and (3) the demand for krill by those predators. (1) Predator densities with confidence intervals were determined from appropriately designed shipboard transect survey; counts of Antarctic fur seals (Arctocephalus gazella), macaroni penguins (Eudyptes chrysolophus), gentoo penguins (Pygoscelis papua), and Antarctic prions (Pachyptila desolata) were adjusted for sea state, distance from observer and dive behaviour. Providing the first at-sea predator density estimates for the region. (2) A comparison of the distribution of female Antarctic fur seals engaged in pup-rearing (using satellite telemetry) and the whole population that were not restricted to a single part of the population (from shipboard transect survey) was undertaken. Using two general additive models based on the relationship between seal distribution (one derived from transect and the other from telemetry) and the physical environment indicated that the spatial distribution of lactating females is representative of the general population. (3) Using the derived predator density, the local krill demand estimate was 2581 tonnes krill per day, a consumption rate of 0.45% per day of the concurrently estimated krill biomass (using shipboard acoustics). Antarctic fur seals accounted for 75% of this demand. This level of demand was less than the increase in biomass resulting from krill growth. However, based on the length-specific demand, determined from concurrent predator diet samples demand exceeded growth for krill >48mm.

Proteins are a highly diversified group of molecules, andfor their study, advanced analytical tools are required. Inparticular, a need for high-throughput techniques has emergedin order to enable the characterization of large sets ofproteins. In this thesis, improved techniques for proteinseparations as well as new tools for the mass spectrometricanalysis of proteins are described. In the work, presented in the first part of the thesis, arefined extract containing proteases from Antarctic krill (Euphausia superba) was separated and characterized bymeans of capillary electrophoresis (CE) and mass spectrometry(MS). Tailored CE separations of the krill extract revealed thepresence of approximately 50 components. In addition, adetailed CE and MS analysis of fractions, containing individualkrill proteases has been carried out. Trypsin-like proteasesfrom krill exhibited a 12-fold and a 60-fold higher digestionefficiency at 37 °C and 2 °C respectively compared todigests performed with bovine trypsin. Furthermore, thecleavage specificity of the trypsin-like proteases wasstudied. In the last part of the thesis, novel concepts forchip-based nanoelectrospray (nanoESI) and matrix-assisted laserdesorption/ionization (MALDI) mass spectrometry are described.First, a micromachined silicon chip with a two-dimensionalmatrix of out-ofplane nanoESI needles for high-throughputanalysis was fabricated. A two-fold improvement insignal-to-noise reproducibility was obtained. Second, achip-based target for MALDI was developed, which featured pairsof elevated 50×50 µm anchors in close proximity. Theanchors were individually addressable with sample solution. Theminiaturized sample preparations at close distance to eachother allowed a simultaneous ionization of a physicallyseparated sample and standard by one single laser pulse. Thisresulted in a twofold reduction of relative mass errors.Moreover, ion suppression of the analyte was significantlyreduced. The effective utilization of the sample resulted in adetection limit of ca 200 zeptomole of angiotensin I. Key words:Proteins, peptides, proteases, Antarctickrill,Euphausia superba, capillary electrophoresis,fluorosurfactants, mass spectrometry, nanoelectrospray, ESI,MALDI, chip, high-throughput, reproducibility, sensitivity andmass accuracy.

<p>Proteins are a highly diversified group of molecules, andfor their study, advanced analytical tools are required. Inparticular, a need for high-throughput techniques has emergedin order to enable the characterization of large sets ofproteins. In this thesis, improved techniques for proteinseparations as well as new tools for the mass spectrometricanalysis of proteins are described.</p><p>In the work, presented in the first part of the thesis, arefined extract containing proteases from Antarctic krill (<i>Euphausia superba</i>) was separated and characterized bymeans of capillary electrophoresis (CE) and mass spectrometry(MS). Tailored CE separations of the krill extract revealed thepresence of approximately 50 components. In addition, adetailed CE and MS analysis of fractions, containing individualkrill proteases has been carried out. Trypsin-like proteasesfrom krill exhibited a 12-fold and a 60-fold higher digestionefficiency at 37 °C and 2 °C respectively compared todigests performed with bovine trypsin. Furthermore, thecleavage specificity of the trypsin-like proteases wasstudied.</p><p>In the last part of the thesis, novel concepts forchip-based nanoelectrospray (nanoESI) and matrix-assisted laserdesorption/ionization (MALDI) mass spectrometry are described.First, a micromachined silicon chip with a two-dimensionalmatrix of out-ofplane nanoESI needles for high-throughputanalysis was fabricated. A two-fold improvement insignal-to-noise reproducibility was obtained. Second, achip-based target for MALDI was developed, which featured pairsof elevated 50×50 µm anchors in close proximity. Theanchors were individually addressable with sample solution. Theminiaturized sample preparations at close distance to eachother allowed a simultaneous ionization of a physicallyseparated sample and standard by one single laser pulse. Thisresulted in a twofold reduction of relative mass errors.Moreover, ion suppression of the analyte was significantlyreduced. The effective utilization of the sample resulted in adetection limit of ca 200 zeptomole of angiotensin I.</p><p><b>Key words:</b>Proteins, peptides, proteases, Antarctickrill,<i>Euphausia superba</i>, capillary electrophoresis,fluorosurfactants, mass spectrometry, nanoelectrospray, ESI,MALDI, chip, high-throughput, reproducibility, sensitivity andmass accuracy.</p>

Na subfamília Bromelioideae o chamado Complexo Nidularioide apresenta gêneros intimamente relacionados que apresentam historicamente problemática circunscrição genérica. A busca por caracteres morfológicos, em especial os caracteres reprodutivos tem auxiliado a compreensão de processos evolutivos em diferentes grupos. Esse trabalho buscou descrever a estrutura anatômica do ovário e do rudimento seminal nas espécies do Complexo Nidularioide: Canistropsis billbergioides, Canistrum aurantiacum, Edmundoa lindenii, Neoregelia johannis, Nidularium innocentii e Wittrockia superba. Ovários dessas espécies em diferentes estádios do desenvolvimento foram coletados e fixados em glutaraldeído 1% e formaldeído 4%, lavados em tampão fosfato de sódio 0,1 M, pH 7,2, desidratados em série etílica (10- 100%), e incluídos em hidroxietilmetacrilato. Cortes de 4 μm de espessura foram montados em lâmina e corados com azul de toluídina O. Em todas as espécies analisadas foi observada a formação de aerênquima tipo esquizógeno no mesofilo ovariano. As espécies C. billbergioides, E. lindenii var. rosea, N. johannis, N. innocentii e Wittrockia superba apresentaram grande número de caracteres compartilhados, como a presença de três camadas de células no tegumento externo, epiderme nucelar e estrato parietal compostos por uma ou duas camadas de células. A espécie E. lindenii var. rosea apresentou tricomas epidérmicos na superfície do ovário, sendo esses tricomas descritos como uma nova morfologia de tricomas para a família Bromeliaceae. Considerando os caracteres observados, a espécie C. aurantiacum apresentou menor número de caracteres compartilhados com as demais espécies do Complexo Nidularioide. / In Bromelioideae subfamily, the so-called Nidularioid Complex presents intimately related genera, bearing historical generic circumscription issues. The search of morphological features, concerning mainly reproductive characters, has helped to enlighten evolution processes between different groups within the family. This work aimed to describe the ovary and ovule anatomical structure of the Nidularioid Complex species: Canistropsis billbergioides, Canistrum aurantiacum, Edmundoa lindenii var. rosea, Neoregelia johannis, Nidularium innocentii and Wittrockia superba. Ovaries in different development stages were collected and fixed in a 1% glutaraldehyde and 4% formaldehyde solution, washed in a sodium phosphate 0.1 M and 7.2 pH solution, and subsequently dehydrated in a crescent ethylic series, ranging from 10% to 100%; lately, the specimens were included in hydroxyethylmethacrylate. 4 μm cuts were mounted in glass blades, and stained with toluidine blue O. In all analyzed species a schizogenous type aerenchyma could be observed within the ovarian mesophyll. C. billbergioides, E. lindenii, N. johannis, N. innocentii and Wittrockia superba presented a great amount of common characters, such as a three layer cell in the outer tegument, nucellar epidermis and parietal tissue composed of two or more cell layers. E. lindenii var. rosea presented epidermic trichomes in the ovary surface, being these trichome described as a new kind of trichome morphology for the Bromeliaceae family. C. aurantiacum presented a minor quantity of common characters with the other analyzed species of the Nidularioid Complex.

Na subfamília Bromelioideae o chamado Complexo Nidularioide apresenta gêneros intimamente relacionados que apresentam historicamente problemática circunscrição genérica. A busca por caracteres morfológicos, em especial os caracteres reprodutivos tem auxiliado a compreensão de processos evolutivos em diferentes grupos. Esse trabalho buscou descrever a estrutura anatômica do ovário e do rudimento seminal nas espécies do Complexo Nidularioide: Canistropsis billbergioides, Canistrum aurantiacum, Edmundoa lindenii, Neoregelia johannis, Nidularium innocentii e Wittrockia superba. Ovários dessas espécies em diferentes estádios do desenvolvimento foram coletados e fixados em glutaraldeído 1% e formaldeído 4%, lavados em tampão fosfato de sódio 0,1 M, pH 7,2, desidratados em série etílica (10- 100%), e incluídos em hidroxietilmetacrilato. Cortes de 4 μm de espessura foram montados em lâmina e corados com azul de toluídina O. Em todas as espécies analisadas foi observada a formação de aerênquima tipo esquizógeno no mesofilo ovariano. As espécies C. billbergioides, E. lindenii var. rosea, N. johannis, N. innocentii e Wittrockia superba apresentaram grande número de caracteres compartilhados, como a presença de três camadas de células no tegumento externo, epiderme nucelar e estrato parietal compostos por uma ou duas camadas de células. A espécie E. lindenii var. rosea apresentou tricomas epidérmicos na superfície do ovário, sendo esses tricomas descritos como uma nova morfologia de tricomas para a família Bromeliaceae. Considerando os caracteres observados, a espécie C. aurantiacum apresentou menor número de caracteres compartilhados com as demais espécies do Complexo Nidularioide. / In Bromelioideae subfamily, the so-called Nidularioid Complex presents intimately related genera, bearing historical generic circumscription issues. The search of morphological features, concerning mainly reproductive characters, has helped to enlighten evolution processes between different groups within the family. This work aimed to describe the ovary and ovule anatomical structure of the Nidularioid Complex species: Canistropsis billbergioides, Canistrum aurantiacum, Edmundoa lindenii var. rosea, Neoregelia johannis, Nidularium innocentii and Wittrockia superba. Ovaries in different development stages were collected and fixed in a 1% glutaraldehyde and 4% formaldehyde solution, washed in a sodium phosphate 0.1 M and 7.2 pH solution, and subsequently dehydrated in a crescent ethylic series, ranging from 10% to 100%; lately, the specimens were included in hydroxyethylmethacrylate. 4 μm cuts were mounted in glass blades, and stained with toluidine blue O. In all analyzed species a schizogenous type aerenchyma could be observed within the ovarian mesophyll. C. billbergioides, E. lindenii, N. johannis, N. innocentii and Wittrockia superba presented a great amount of common characters, such as a three layer cell in the outer tegument, nucellar epidermis and parietal tissue composed of two or more cell layers. E. lindenii var. rosea presented epidermic trichomes in the ovary surface, being these trichome described as a new kind of trichome morphology for the Bromeliaceae family. C. aurantiacum presented a minor quantity of common characters with the other analyzed species of the Nidularioid Complex.

Na subfamília Bromelioideae o chamado Complexo Nidularioide apresenta gêneros intimamente relacionados que apresentam historicamente problemática circunscrição genérica. A busca por caracteres morfológicos, em especial os caracteres reprodutivos tem auxiliado a compreensão de processos evolutivos em diferentes grupos. Esse trabalho buscou descrever a estrutura anatômica do ovário e do rudimento seminal nas espécies do Complexo Nidularioide: Canistropsis billbergioides, Canistrum aurantiacum, Edmundoa lindenii, Neoregelia johannis, Nidularium innocentii e Wittrockia superba. Ovários dessas espécies em diferentes estádios do desenvolvimento foram coletados e fixados em glutaraldeído 1% e formaldeído 4%, lavados em tampão fosfato de sódio 0,1 M, pH 7,2, desidratados em série etílica (10- 100%), e incluídos em hidroxietilmetacrilato. Cortes de 4 μm de espessura foram montados em lâmina e corados com azul de toluídina O. Em todas as espécies analisadas foi observada a formação de aerênquima tipo esquizógeno no mesofilo ovariano. As espécies C. billbergioides, E. lindenii var. rosea, N. johannis, N. innocentii e Wittrockia superba apresentaram grande número de caracteres compartilhados, como a presença de três camadas de células no tegumento externo, epiderme nucelar e estrato parietal compostos por uma ou duas camadas de células. A espécie E. lindenii var. rosea apresentou tricomas epidérmicos na superfície do ovário, sendo esses tricomas descritos como uma nova morfologia de tricomas para a família Bromeliaceae. Considerando os caracteres observados, a espécie C. aurantiacum apresentou menor número de caracteres compartilhados com as demais espécies do Complexo Nidularioide. / In Bromelioideae subfamily, the so-called Nidularioid Complex presents intimately related genera, bearing historical generic circumscription issues. The search of morphological features, concerning mainly reproductive characters, has helped to enlighten evolution processes between different groups within the family. This work aimed to describe the ovary and ovule anatomical structure of the Nidularioid Complex species: Canistropsis billbergioides, Canistrum aurantiacum, Edmundoa lindenii var. rosea, Neoregelia johannis, Nidularium innocentii and Wittrockia superba. Ovaries in different development stages were collected and fixed in a 1% glutaraldehyde and 4% formaldehyde solution, washed in a sodium phosphate 0.1 M and 7.2 pH solution, and subsequently dehydrated in a crescent ethylic series, ranging from 10% to 100%; lately, the specimens were included in hydroxyethylmethacrylate. 4 μm cuts were mounted in glass blades, and stained with toluidine blue O. In all analyzed species a schizogenous type aerenchyma could be observed within the ovarian mesophyll. C. billbergioides, E. lindenii, N. johannis, N. innocentii and Wittrockia superba presented a great amount of common characters, such as a three layer cell in the outer tegument, nucellar epidermis and parietal tissue composed of two or more cell layers. E. lindenii var. rosea presented epidermic trichomes in the ovary surface, being these trichome described as a new kind of trichome morphology for the Bromeliaceae family. C. aurantiacum presented a minor quantity of common characters with the other analyzed species of the Nidularioid Complex.