The effect of proteolytic enzymes on the transformation and transfection of Streptococcus lactis LM2301 protoplasts was examined in an attempt to eliminate the variability observed. By using both chymotrypsin and mutanolysin to form protoplasts followed by spread plating, consistent frequencies of 104 to 105 transformants per μg of pGB301 DNA, and 105 transfectants per μg of c2 bacteriophage DNA where achieved. Optimum transformation and transfection frequencies were obtained when 16 h cultures were treated simultaneously with 25 U/ml mutanolysin and 1.25 U/ml chymotrypsin for 15 min. Trypsin and pronase in conjunction with mutanolysin also increased transformation frequencies higher than when mutanolysin was used alone, but pronase was not as effective as chymotrypsin or trypsin. These results may explain the variability in transformation of mutanolysin-treated cells of S. lactis since commercial sources of mutanolysin contain varying amounts of proteolytic enzyme activity. Transformation of Streptococcus cremoris CS224 at low frequency (5 transformants per μg of pGB301 DNA) was achieved. Plasmid pGB301 was able to replicate and express antibiotic resistance in the resultant transformant (designated S. cremoris SW301). The presence of pGB301 in S. cremoris SW301 was confirmed by agarose gel electrophoresis.

Thesis (Ph. D.)--Institute of Paper Chemistry, 1980. / Includes bibliographical references (p. 58-59).

Toxic elements are present in polluted water from mines, industrial outlets, storm water etc. Wetland plants take up toxic elements and increase the pH of the medium. In this thesis was investigated how the shoots of submerged plants and roots of emergent plants affected the pH of the surrounding water in the presence of free toxic ions. The aim was to clarify the mechanisms by which these plants change the surrounding water pH in the presence of toxic ions. The influence of Elodea canadensis shoots on the pH of the surrounding water was studied in the presence of cadmium (Cd) at low initial pH (4-5). The involvement of photosynthetic activity in the pH changes was investigated in the presence and absence of Cd. The cytosolic, vacuolar and apoplasmic pH changes as well as cytosolic Cd changes in E. canadensis were monitored. The influence of Eriophorum angustifolium roots on the pH of the surrounding water was investigated in the presence of a combination of Cd, copper, lead, zinc and arsenic at low initial pH (3.5). Eriophorum angustifolium root exudates were analyzed for organic acids. Elodea canadensis shoots increased the pH of the surrounding water, an effect more pronounced with increasing Cd levels and/or increasing plant biomass and increased plant Cd uptake. The pH increase in the presence of free Cd ions was not due to photosynthesis or proton uptake across the plasmalemma or tonoplast. Cadmium was initially sequestered in the apoplasm of E. canadensis and caused its acidosis. Eriophorum angustifolium roots increased the surrounding water pH and this effect was enhanced in the presence of arsenic and metals. This pH increase was found to depend partly on the release of oxalic acid, formic acid and succinic acid by the plants. In conclusion, E. canadensis shoots and E. angustifolium roots were found to increase the low initial pH of the surrounding water. The pH modulation by these species was enhanced by low levels of free toxic ions in the surrounding water. / At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 2: Submitted. Paper 3: Submitted. Paper 4: Manuscript.

Thesis (Ph. D.) -- Lincoln University, 2007. / Also available via the World Wide Web.

Initially, protoplasts were isolated to detect various parameters using flow cytometric analysis. The most efficient ratio of cells to enzyme solution, for digestion of cell walls, needed to be established. To detect whether the time of incubation with the enzyme solution influenced the state or viability of the protoplasts, they were observed periodically under the light microscope during digestion at different concentrations of enzyme solution. After 2 h digestion with light swirling every 20 min, the protoplasts were still intact (Figure 1), and viable as detected with Trypan blue staining (results not shown). Increasing the digestion period led to a decrease in cell membrane integrity. The size of the protoplasts varied between 60 mm and 90 mm. Figure 1 shows the difference between cells before digestion with an enzyme solution and protoplasts after digestion. Size determination of protoplasts was important since the flow tip of the flow cytometer is limited to 100 mm and if the protoplasts exceeded this size, could lead to blockages in the flow tip of the flow cytometer, with ineffective readings and a time consuming clean-up process. / Dr. Marianne J. Cronje

O objetivo deste trabalho foi desenvolver uma metodologia para identificação de regiões promotoras funcionais a partir de segmentos de um genoma qualquer. O genoma da cana-de-açúcar foi escolhido para o desenvolvimento desta estratégia na qual envolve a obtenção de fragmentos de DNA os quais foram clonados em vetores de expressão. A triagem destes fragmentos foi realizada através de biobalística e resultou no isolamento de quatro clones. Um ensaio de transformação permanente em arroz com três clones gerou 12 plantas. Foi detectada expressão do marcador GUS em calos, folhas e raízes, comprovando sua funcionalidade. Desta maneira, o presente trabalho permitiu estabelecer uma metodologia de recuperação de sequências regulatórias funcionais com ampla possibilidade de serem explorados biotecnologicamente. / The aim of this work is develop a strategy to identify functional promoter regions from any genome. The modern sugarcane genome was chosen as a model for the development of this strategy that involves the generation of fragments of DNA and cloning them into expression vectors. These fragments were then screened by a transient expression assay using biolistic particle delivery resulting in the isolation of four clones. Three clones were permanently transformed in rice, and 12 plants were obtained. GUS expression was detected in the callus, leaves and roots of the rice plants thus confirming the functionality of sequences in these clones. The present work has established a strategy to identify and extract functional regulatory sequences containing functional regulatory regions which show great potential of being useful in both the biotechnology field and in the field of basic science.

Please open electronic version for Abstract. / Thesis (M.Sc.)-University of Natal, Pietermaritzburg, 1996.

M.Sc. (Biochemistry) / Lipopolysaccharide (LPS) is a complex lipoglycan that is found in the outer membrane of Gram-negative bacteria and is composed of three regions namely the fatty acid Lipid A, a core region of short oligosaccharide chains and an O-antigen region of polysaccharides. When LPS is recognized as a microbe-/pathogen-associated molecular pattern (M/PAMP), it not only induces an innate immune response in plants but also stimulates the development of defence responses such as the immediate release of reactive oxygen species/intermediates (ROS/I), pathogen-related (PR) gene expression and activation of the hypersensitive response (HR), resulting in stronger subsequent pathogen interactions. The identification and characterisation of the elusive LPS receptor/receptor complex in plants is thus of importance, since understanding the mechanism of perception and specific signal transduction pathways will clarify, and lead to the advancement of, basal resistance in plants in order to decrease crop plant losses due to pathogen attack. In mammals, LPS binds to a LPS binding protein (LBP) to form a LPS-LBP complex which is translocated to myeloid differentiation 2 (MD2) with the presence/absence of its co-receptor, a glycosylphosphatidylinositol (GPI)-linked protein, CD14. The interaction occurs on the host membrane and triggers an inflammatory defence response through the signalling cascade activated by the interaction with Toll-like receptor 4 (TLR4). A similar LPS-receptor interaction is, however, unknown in plants. To address the LPS perception mechanism in plants, biological binding studies with regard to concentration, incubation time and temperature, affinity, specificity and saturation were conducted in Arabidopsis thaliana protoplasts using LPS labeled with Alexa 488 hydrazide. Quantum dots (Qdots), which allow non-covalent hydrophobic labeling of LPS, were further also employed in similar Arabidopsis protoplast binding studies. These studies were conducted by fluorescence determination through the use of a BD FACS Aria flow cytometer. Although Alexa-labeling does not affect the biological activity in mammalian studies, the same cannot necessarily be said for plant systems, and hence Qdots were included to address this question. The conjugation of Qdots to LPS was confirmed by transmission electron microscopy (TEM) and results illustrated higher fluorescence values as compared to Alexa-LPS fluorescence analysis. Furthermore, inhibition of the perception process is also reported using Wortmannin and Brefeldin A as suitable endo- and exocytosis inhibitors. Affinity, specificity and saturability as well as the role of endo- and exocytosis inhibition in LPS binding to protoplasts was ultimately demonstrated by both fluorophores, with the use of Qdots as a label proving to be a more sensitive strategy in comparison to the conventional Alexa 488 hydrazide label.

A hibridação somática via fusão de protoplastos é um método de combinação genética que agrega integralmente os dois genomas genitores, assim como suas respectivas organelas citoplasmáticas, sendo uma ferramenta alternativa aos métodos convencionais de melhoramento. O objetivo deste trabalho foi a obtenção de híbridos somáticos interespecíficos de laranja doce (Citrus sinensis L. Osbeck) \'Pêra\' e \'Westin\' com a tangerina \'Fremont\' (C. clementina hort. ex Tan. x C. reticulata Blanco), \'Thomas\' (C. reticulata Blanco) e \'Nules\' (C. clementina hort. ex Tan), e o tangelo \'Nova\' (C. reticulata Blanco x C. paradisi Macf.) visando à produção de genitores tetraplóides com características agronômicas desejáveis. Como fontes de protoplastos foram utilizados calos embriogênicos de laranja e folhas jovens de tangerina e tangelo, coletadas de plantas cultivadas em casa-de-vegetação. Para o isolamento dos protoplastos, as células de ambas as fontes foram imersas em solução enzimática e incubadas no escuro por 14 horas, sob agitação. Após o isolamento, os protoplastos isolados de calos foram fundidos quimicamente (polietilenoglicol - PEG) com protoplastos não embriogênicos, isolados de mesofilo foliar. Em seguida, os protoplastos foram plaqueados nos meios de cultura líquido BH3, EME e BH3 + EME, e incubados em ausência de luz, sob temperatura de 27 ºC. Após 20 dias de incubação, foram iniciados subcultivos sequenciais para nutrição e redução do potencial osmótico do meio de cultura. Os microcalos desenvolvidos foram transferidos para meio de cultura EME + maltose (13 g.L-1) para a indução da embriogênese somática. Os embriões desenvolvidos foram transferidos para meio de cultura EME + sacarose (25 g.L-1) e em seguida para o meio de cultura 1500 (1,5 g.L-1 de extrato de malte) e finalmente para o meio B+ para desenvolvimento e germinação. As plantas regeneradas foram individualizadas, enraizadas ou microenxertadas e aclimatizadas em casa-devegetação. A confirmação da hibridação somática foi feita por análise morfológica, determinação da ploidia, por citometria de fluxo e análise do DNA com marcadores moleculares do tipo SSR e RAPD. Foram obtidos híbridos somáticos de três combinações (Pêra + Fremont, Pêra + Nules e Pêra + Nova) os quais serão avaliados para utilização diretamente como copa ou como genitor tetraplóide em programas de melhoramento de citros. / Somatic hybridization by protoplasts fusion is a method of genetic combination of two parental genomes, and their cytoplasmic organelles. It is an alternative tool to conventional breeding methods. The objective of this work was to obtain interspecific somatic hybrids of \'Pera\' and \'Westin\' sweet oranges (Citrus sinensis L. Osbeck) with \'Fremont\' (C. clementina hort. ex Tan. x C. reticulata Blanco), \'Thomas\' (C. reticulata Blanco) and \'Nules\' (C. clementina hort. ex Tan) mandarins and Nova tangelo (C. reticulata Blanco x C. paradisi Macf.) to produce tetraploid parents with desirable agronomic characteristics. The sources of protoplasts were sweet orange embryogenic callus and mandarin and tangelo young leaves, collected from plants cultivated in screenhouses. For protoplasts isolation, cells from both sources were immersed in enzyme solution and incubated in the dark for 14 hours (40 rpm). After isolation, the protoplasts isolated from callus were fused chemically (polyethylene glycol - PEG) with non embryogenic protoplasts, isolated from leaf mesophyll. The protoplasts were cultivated in BH3, BH3 + EME and EME liquid culture media and incubated in the dark, under temperature of 27 ºC. After 20 days of incubation, subcultures were initiated in order to reduce the osmotic potential of culture medium. The microcalus developed were transferred to EME medium supplemented with maltose (13 g.L-1) to induce somatic embryogenesis. The developed embryos were transferred into EME + sucrose (25 g.L- 1), culture medium 1500 (1.5 g.L-1 of malt extract) or B+ culture medium for development and germination. The regenerated plants were individually rooted or micrografted, and further acclimatized in screenhouse. Somatic hybridization was confirmed by analysis of leaf morphology, ploidy determination by flow cytometry and molecular analysis by SSR and RAPD markers. Somatic hybrids were obtained of the three combinations (Pera + Fremont, Pêra + Nules and Pêra + Nova) which will be evaluated for direct are as scion or as a tetraploid parent in citrus breeding programs.

O objetivo deste trabalho foi aplicar a técnica de fusão química de protoplastos para desenvolver híbridos somáticos interespecíficos entre tangerinas (Citrus reticulata) e toranjas (Citrus grandis), visando a obtenção de porta-enxertos semelhantes à laranja azeda Citrus aurantium). Como fonte de protoplastos foram utilizadas suspensões celulares embriogênicas de tangelo \'Page\' (C. reticulata x C. paradisi) e tangor \'Murcote\' (C. reticulata x C. sinensis) e folhas jovens de seedlings de toranjas \'Lau Tau\' e \'Ogami\' (Citrus grandis). Após a fusão os protoplastos foram cultivados sob ausência de luz, até a formação de microcolônias, que foram então cultivadas em meio de cultura EME em dupla-fase, suplementado com 13,33 g/L de maltose para a indução da embriogênese. Os embriões globulares formados foram transferidos para meio EME com 25 g/L de sacarose e quando em estádio cotiledonar foram transferidos para meio de cultura suplementado com 1,5 g/L de extrato de malte. As brotações obtidas foram enxertadas in vitro sobre laranja \'Hamlin\' e \'Valência\'. As plantas obtidas foram levadas para casa de vegetação e cultivadas em substrato comercial. As 17 plantas regeneradas de tangelo \'Page\' + toranja \'Lau Tau\' apresentaram conformação fenotípica adversa aos genitores, com folhas de tamanho reduzido, ápice arredondado, coloração verde escura, mesófilo enrugado e ausência de pecíolo alado. A análise de citometria de fluxo confirmou o caráter diplóide das plantas regeneradas. Marcadores moleculares RAPD apresentaram padrão de bandas similar entre a planta regenerada e o genitor tangelo \'Page\'. O protocolo utilizado para isolamento, fusão e cultura de protoplastos, bem como para a regeneração e aclimatização de plantas permitiram a obtenção 17 plantas da combinação entre tangelo \'Page\' e toranja \'Lau Tau\' com conformação fenotípica diferente dos genitores, duas plantas da combinação entre tangor \'Murcote\' e toranja \'Ogami\' e uma planta da combinação entre tangor \'Murcote\' e toranja \'Lau Tau\'. / The aim of this work was to apply the technique of chemical fusion of protoplasts, in order to develop interspecific somatic hybrids between mandarins (Citrus reticulata) and pummelos (Citrus grandis), in order to produce similar to sour orange (Citrus aurantium). The sources for protoplasts were embryogenic suspension cultures of \'Page\' tangelo (C. reticulata x C. paradisi) and \'Murcott\' tangor (C. reticulata x C. sinensis) and young leaves from seedlings of \'Lau Tau\' and \'Ogami\' pummelos (Citrus grandis). After the fusion, the protoplasts were cultivated in the absence of light, until the formation of microcolonies, and were then cultivated in double-phase EME medium, supplemented with 13.33 g/L of maltose for embryogenesis induction. The globular embryos thus formed were transferred to EME medium with 25 g/L of sucrose and, when in cotyledonal stage, were transferred to a culture medium supplemented with 1.5 g/L of malt extract. The shoots obtained were grafted in vitro onto \'Hamlin\' and \'Valencia\' sweet oranges. The regenerated plants were cultivated in a greenhouse, over commercial substrate. In this process, 17 plants were obtained. These plants presented phenotypic conformation different from the genitors, with leaves with reduced size, round apex, dark green coloration, rough leaf blade and absence of developed petiole. The analysis by flow cytometry confirmed the diploid character of the regenerated plants. RAPD molecular markers presented a similar band pattern between the regenerated specimen and the genitor \'Page\' tangelo. The protocol used for isolation, hybridization and cultivation of protoplasts, as well as for the regeneration and acclimatization of the plants allowed the obtainment of 17 plants from the combination of \'Page\' tangelo + \'Lau Tau\' pummelo, with phenotypic conformation different from the genitors, two plants from the combination of \'Murcott\' tangor + \'Ogami\' pummelo and one plant from the combination of Murcote\' tangor + \'Lau Tau\' pummelo.