Thesis (Ph. D.)--Virginia Polytechnic Institute and State University, 1994. / Vita. Abstract. Includes bibliographical references (leaves 125-138). Also available via the Internet.

Freshly isolated protoplasts from pea leaves were used to investigate the responses of antioxidant enzymes to oxidative stress. Two cultivars, Progress (tolerant) and Nugget (sensitive), that have differing resistance with respect to oxidative stress at the whole plant level were used. Sulfite and the superoxide generating herbicide, paraquat, were used as the oxidants. Final sulfite concentrations during photosynthetic incubations ranged from 1.5 mM to 30.0 mM. During the polarographic estimation of photosynthesis, CO₂-dependent O₂ evolution did not decrease. At sulfite concentrations of 3.0 mM or less, light-dependent O₂ evolution increased and was probably due to a concomitant SO₂-dependent O₂ evolution. Photosynthesis determined as ¹⁴CO₂ fixation was not increased at these low concentrations of sulfite. Concentrations greater than 7 mM = sulfite inhibited photosynthetic ¹⁴CO₂ fixation. No difference in these responses was found between the two cultivars. At 0.1 µM paraquat, the relative resistance to oxidative stress was reversed compared to previous studies at the whole plant level. With the tolerant cultivar, activity of the plastid antioxidant enzyme, glutathione reductase, increased after a three-hour exposure. Changes in the steady state level of glutathione reductase protein, as judged by immunoblots, did not correlate with the observed changes in enzyme activity. No change in the de novo synthesis of glutathione reductase occurred over the same period as a consequence of paraquat application. A mechanism, unrelated to oxygen free radical scavenging, may contribute to the relative tolerance to low concentrations of paraquat. On the other hand, after an eight-hour exposure to 0.1 mM PQ in the presence of Gamborg’s basal salts, superoxide dismutase activity of Progress protoplasts was enhanced 288% above the preexposure levels while glutathione reductase activity decreased 70% and ascorbate peroxidase activity decreased 90%. The relationship of these changes to oxidative damage to the photosynthetic machinery remains to be assessed. / Ph. D.

An in vitro system of generating protoplasts from their callus cultures was established. The friable callus was more productive in terms of producing protoplasts than the green compact callus. The concentration of the various cell wall degrading enzymes had an effect on the viability of the protoplasts in the medium. The protoplast system developed from the experiments was stable and could be used for the transformation experiments of Albizia lebek and for other plant improvement practices.

Thesis (MTech (Agriculture))--Cape Peninsula University Of Technology, 2016. / Bambara groundnut (Vigna subterranea), a dicotyledonous plant is a legume which has a potential to contribute to food security and nutrition. Protoplasts are naked plant cells lacking cell walls. Viable protoplasts are potentially totipotent. Therefore, when given the correct stimuli, each protoplast is capable, theoretically, of regenerating a new wall and undergoing repeated mitotic division to produce daughter cells from which fertile plants may be regenerated through the tissue culture process. Protoplast systems are valuable and versatile cell based systems that are useful in observing cellular processes and activities. In this study, the isolation of protoplast from the leaves of Bambara groundnut plant was extensively optimised. The factors affecting protoplast isolation considered in this study were ages of plant material, mannitol concentration, combinations and concentrations of enzymes and duration of incubation. Effects of ages of Bambara groundnut plant (4, 6, 8, 10 weeks), molarities of mannitol (0.4 M, 0.5 M. 0.6 M and 0.7 M), concentration and combination of enzymes (1%, 2% and 4% cellulase, 0.5% and 1% macerozyme and, 0.5% and 1% pectinase) at different incubation duration (4, 18, 24, 42 hours) were investigated. Overall, it can be deduced from this study that the optimal protoplast yield (4.6 ± 0.14×105ml-1/gFW) and viability (86.5 ± 2.12%) were achieved by digesting the leaves of four week old Bambara groundnut plant with 2% cellulase and 0.5 % macerozyme with 0.5M mannitol for 18 hours. Freshly isolated protoplasts were then cultured at different densities of 1 × 104 - 2 ×106 protoplasts/ml using MS in three different culture (Liquid, agar and agarose bead) methods. First cell division was observed only in liquid medium. With several attempts, no division was achieved in the agar and agarose bead methods, division also did not progress in the liquid medium and hence, plant regeneration from Bambara groundnut protoplasts could not be achieved in this study. Consequently, a further study is underway to compare the proteomic profiles of freshly isolated protoplasts and cultured protoplasts in order to gain insights into the expression of proteins that could perhaps be contributing to the difficulty in regenerating Bambara groundnut plant through protoplast technology. The present study is novel because it is the first study to optimise the various factors that could affect protoplast isolation from the leaves of Bambara groundnut and thus developed an efficient protocol for protoplasts isolation from leaves of Bambara groundnut for cell manipulation studies.

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

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.

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.

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.