Charles University in Prague, Faculty of Pharmacy in Hradec Králové Department of Pharmacognosy Candidate: Hana Novotná Supervisor: PharmDr. Marie Kašparová, Ph.D. Title of diploma thesis: Secondary metabolites of plant tissue culture of Trifolium pratense L. Explant cultures are perspective sources of secondary metabolites. Nevertheless production of flavonoids and isoflavonoids by the suspension culture of Trifolium pratense L. is not high. Elicitation is one of the methods used to enhance the biosynthesis of secondary metabolites. Elicitation induces physiological changes, stimulates defensive or stress-induced reactions in plants and subsequently triggers the synthesis of secondary metabolites. The objective of this study was to observe the influence of two elicitors - abscisic acid and ascorbic acid - on the production of flavonoids and isoflavonoids by the Trifolium pratense L. suspension culture (Sprint variety). The culture was cultivated in Gamborg medium to which 2 mg.l-1 of 2,4- dichlorophenoxyacetic acid and 2 mg.l-1 of 6-benzylaminopurine were added, at the temperature of 25 řC and 16 hours light / 8 hours dark period. The best elicitation effect of abscisic acid on the production of flavonoids and isoflavonoids was observed after a 6-hour application of the highest 500 µmol.l-1...

Call number: LD2668 .T4 1986 R35 / Master of Science / Horticulture, Forestry, and Recreation Resources

No description available.

A mist reactor was used to study plant growth and development under various environmental conditions towards the production of healthy plantlets ready for soil transplant in one step from inoculation. In addition, a 3D type of cultivation via surface attachment of explants to vertically hanging strips inside the mist reactor was also investigated to maximize productivity with minimal footprint. Using carrot as the model species, pre-embryogenic cell suspensions were successfully spray-inoculated onto hanging poly-L-lysine (PLL)-coated nylon mesh to which they then attached and remained for several weeks while they developed into rooted plantlets. To study single step micropropagation from shoot explants to fully acclimatized plantlets, Artemisia annua was used as the model species. Nodal cuttings of A. annua were inoculated onto PLL-coated mesh strips by briefly immersing the strips in the suspension of nodal cuttings. Investigation of medium, phytohormones, CO2, ventilation level and humidity ensued resulting in selection of a preferred final process that reduced physiological aberrations like hyperhydricity and was time efficient. The nodal cuttings that attached to the strips were first misted with half strength shooting medium for 7 days to develop new shoots. Then the new shoots were misted with the rooting medium supplemented with NAA for 12 days to develop roots. Rooted plantlets were acclimatized in the same rooting medium for 9 days to acquire fully functional stomata prior to planting into soil. Taken together this study suggested that fully developed plantlets ready for planting into soil could be obtained in a single step in a bioreactor from embryogenic cells or from nodal explants.

Xanthomonas albilineans est l'agent causal de l'échaudure des feuilles, une des principales maladies bactériennes de la canne à sucre dont l'impact peut être très important lorsque des variétés sensibles sont infectées au champ. Les mécanismes qui régissent les interactions entre cet agent pathogène et la canne à sucre sont encore très peu connus. Les objectifs de ce travail étaient (i) d'identifier des déterminants moléculaires impliqués dans la survie épiphyte de X. albilineans et (ii) de préciser la localisation de la bactérie dans les tissus de la canne à sucre. Parmi les facteurs étudiés, les polysaccharides de surface et une protéine de la membrane externe (XaOmpA1) de X. albilineans s'avèrent indispensables pour la survie épiphyte de cet agent pathogène. En revanche, la molécule signal diffusible DSF et les métabolites secondaires codés par les gènes NRPS (« Non Ribosomal Peptide Synthetases ») ne sont pas requis pour l'installation de la bactérie en surface des feuilles, au moins en l'absence d'autres microorganismes compétiteurs. Toutefois, la colonisation optimale de la phyllosphère de la canne à sucre nécessite la présence d'un système DSF/RpfGC intact. Dans la deuxième partie de ce travail, nous avons vérifié la localisation in planta de X. albilineans par microscopie confocale, immunocytochimie, et microscopie électronique à transmission. Les observations microscopiques réalisées ont permis de montrer que X. albilineans n'est pas limitée au xylème de la canne à sucre, comme on le considérait jusqu'à présent. Bien au contraire, cette bactérie est capable de quitter le système vasculaire de sa plante hôte et de pénétrer dans d'autres types cellulaires, notamment les cellules du parenchyme non vasculaire. Il s'agit là, à notre connaissance, d'un nouveau mécanisme de colonisation d'une plante par une bactérie phytopathogène qui reste à décrypter. / Xanthomonas albilineans is the causal agent of leaf scald, a lethal disease of sugarcane that can significantly impact infected susceptible varieties in the field. The mechanisms that govern the interactions between this bacterial pathogen and its host plant are not well known. The objectives of this study were (i) to identify molecular factors involved in epiphytic survival of X. albilineans and (ii) to verify the localization of X. albilineans in sugarcane tissues. Among the studied factors, surface polysaccharides and an outer-membrane protein (XaOmpA1) of X. albilineans were crucial for epiphytic survival of this pathogen. Secondary metabolites synthesized by non-ribosomal peptide synthetases and the diffusible signal factor DSF were not critical for survival of X. albilineans on the sugarcane leaf surface, at least in absence of competing microorganisms. However, an intact DSF/RpfGC system was necessary for optimal colonization of the phyllosphere. In the second part of this study, we verified in planta localization of X. albilineans by confocal microscopy, immunochemistry and transmission electron microscopy. Microscopic observations allowed us to show that X. albilineans is not a xylem limited bacterium as it was believed until now. This pathogen is able to invade numerous cellular types including vascular and non-vascular parenchyma cells. To our knowledge, this is a novel invasion strategy of a plant pathogenic bacterium that has not previously been described, and that remains to be deciphered.

A regeneração de plantas, por organogênese ou embriogênese somática, a partir do cultivo de células e tecidos vegetais in vitro é a base para a utilização da biotecnologia no melhoramento. Realizaram-se estudos com cinco cultivares de laranja doce (Citrus sinensis L. Osbeck), Pêra, Natal, Lima Verde, Hamlin e Westin. Este trabalho objetivou a avaliação da eficiência de isolamento de protoplastos das cultivares de laranja doce; o estudo da eficiência de plaqueamento em função de cinco densidades de protoplastos e diferentes meios de cultura e avaliação da embriogênese somática em função da composição dos meios de cultura e concentração da fonte de carboidrato. As soluções enzimáticas testadas para o isolamento de protoplastos foram: 1. Grosser e Chandler (1987), composta de 1% de celulase Onozuka RS (Yakult), 1% macerase R- 10 (Yakult Honsha) e 0,2% de pectoliase Y-23 (Seishin); 2. Grosser e Chandler (1987) modificado, composta de 1% de celulase Onozuka RS (Yakult), 1% macerase R-10 (Yakult Honsha); 3. Solução enzimática composta de 4% de celulase Onozuka R-10 (Yakult), 1% macerase R-10. O plaqueamento dos protoplastos foi realizado em cinco densidades, 2 x 104, 5 x 104; 105; 2x 105 e 3 x 105 protoplastos . mL-1,nos meios de cultura EME 0,7M, BH3 0,7M e BH3 + EME 0,7M em ausência de luz, a 25 ± 1 ºC. A solução enzimática 2 proporcionou um maior rendimento no isolamento de protoplastos das cultivares Hamlin, Natal e Pera e solução enzimática 1 foi melhor para a cultivar Westin. A eficiência final de plaqueamento avaliada aos 90 dias foi superior nas densidades de de 3 x 105 e 2 x 105 protoplastos. mL-1 para as cultivares Hamlin, Natal e Lima Verde, e na densidade de 2 x 105 e 105 protoplastos. mL-1 para a cultivar Westin. A indução da embriogênese somática ocorreu em meio de cultura MT modificado com 500 mg.L-1 de extrato de malte, acrescido de sacarose, galactose, glicose, sorbitol, lactose e maltose, nas concentrações de 18, 37, 75, 110 e 150 mM à temperatura de 27 °C. A formação de embriões somáticos variou com o genótipo, sendo a cultivar Lima Verde e Westin apresentaram menor número de embriões somáticos. As melhores fontes de carboidratos foram a maltose, seguida pela lactose nas concentrações de 37 e 75 mM para a cultivar Pêra, 37 mM para a cultivar Natal e 37, 75 e 110 mM para a cultivar Hamlin. / Plant regeneration, by organogenesis or somatic embryogenesis from cell cultures and in vitro plant tissue culture is the basis for the use of biotechnology in plant breeding. Studies were conducted with five cultivars of sweet orange (Citrus sinensis L. Osbeck), Pêra, Natal, Lima Verde, Hamlin and Westin. This work aimed to evaluate the isolation efficiency of protoplasts, to evaluate platting efficiency of protoplasts based on five densities of cells and different culture media and to evaluate somatic embryogenesis based on culture medium composition and concentration. The enzymatic solutions tested were: 1. Grosser and Chandler (1987): 1% de cellulase Onozuka RS (Yakult), 1% macerase R-10 (Yakult Honsha) and 0,2% de pectoliase Y-23 (Seishin); 2. Grosser and Chandler (1987) modified: 1% de cellulase Onozuka RS (Yakult) and 1% macerase R-10 (Yakult Honsha); 3. Enzimatic solution containing 4% cellulase Onozuka R-10 (Yakult) and 1% macerase R-10. Protoplasts were cultured at densities of 2 x 104; 5 x 104; 105; 2 x 105 e 3 x 105 protoplasts.mL-1 in EME 0,7M, BH3 0,7M and BH3 + EME 0,7M, in the dark, at 25 ± 1 ºC. The enzymatic solution 2 provided higher yield for the cultivars Hamlin, Natal and Pêra, and enzymatic solution 1 resulted in better protoplast isolation for cultivar Westin. Final platting efficiency, evaluated 90 days after culture, was higher at the densities of 3 x 105 e 2 x 105 protoplasts.mL-1 for Hamlin, Natal and Lima Verde, and at the density of 2 x 105 e 105 protoplasts.mL-1 for Westin. Somatic embryogenesis stimulation occurred in cultured medium MT (MURASHIGE AND TUCKER, 1969) modified with 500 mg. L-1 of malt extract, supplemented with sucrose, galactose, glucose, maltose, lactose and sorbitol at concentrations of 18, 37, 75, 110 and 150 mM, at 27 ± 1 ºC. Somatic embryos produced varied with the genotype, the smaller number of somatic embryos was observed in cultivars Lima Verde and Westin. The best source of carbohydrate were maltose, followed by lactose at concentrations of 37 and 75 mM for cultivar Pêra, 37 mM for cultivar Natal, and 37, 75 and 110 mM for cultivar Hamlin.

Imagens multiespectrais são utilizadas em diferentes aplicações, que vão desde sensoriamento remoto a processos médicos. No caso de imagens multiespectrais oriundas de microscopia confocal de varredura à laser (Confocal Laser Scanning Microscopy-CLSM), a extração da informação se inicia pela conversão das assinaturas espectrais, em uma imagem RGB. Esta imagem é a referência para a seleção da região de interesse, da qual se obtém a assinatura espectral média, originada do arquivo multiespectral (LSM). Mesmo utilizando um padrão muito bem estabelecido de conversão, alguns pontos devem ser considerados: i) o processo de conversão reduz a informação, a uma ordem de 10-145%; ii) a cor é uma experiência sensorial, subjetiva e pessoal, interferindo na seleção da região de interesse e; iii) a assinatura é obtida pela média espectral, da região de interesse, selecionada manualmente.Assim, esta tese de doutorado propõem um método de mapeamento e visualização das informações de imagens multiespectrais, combinando um algoritmo de agrupamento não supervisionado(kmeans) e um algoritmo que define uma paleta de cores coerentes com a informação espectral das regiões mapeadas. Aplicou-se o método em três casos de estudos de tecidos vegetais: i) no pré-tratamento de paredes celulares da cana-de-açúcar; ii) na plasticidade foliar do Jacaranda caroba e; iii) no uso de assinaturas espectrais na classificação de plantas do Cerrado. Os resultados demonstraram que o método é bastante robusto, permitindo de forma inovadora a: visualização, análise e comparação de imagens multiespectrais qualitativa e quantitativamente, e que seu uso é viável em qualquer área de pesquisa que utilize imagens multiespectrais. / Multispectral images are used in different applications, ranging from remote sensing images to medical images. In the case of multispectral images derived from confocal laser scanning microscopy (CLSM), the extraction of information begins with the conversion of spectral signatures in an RGB image. This is the reference for selecting the region of interest, from which it gets the average spectral signature, originated from multispectral file (LSM). Even using a very well established pattern of conversion, some points should be considered: i) the conversion process reduces the information on the order of 10-145%; ii) the color is a sensory experience, subjective and personal, interfering in the selection of the interest region and; the signature is obtained by the spectral average, from interest region which is selected manually. Thus, this doctoral thesis proposes a method of mapping and visualization of multispectral imaging information, combining an unsupervised clustering algorithm (kmeans) and an algorithm that defines a consistent color palette with the spectral information of mapped regions. The proposed method was applied in three cases plant tissue studies: i) in the pre-treating the cell walls of sugarcane; ii) in the leaf plasticity of Jacaranda caroba; iii) in the use of spectral signatures in the Cerrado plant classification. The results showed that the proposed method is quite robust. It presents innovation to the visualization and analysis of multispectral images and makes possible a qualitative and quantitative comparison of a group of multispectral images. Besides that, its use is feasible in any area of research, which are using multispectral images.

A regeneração de plantas, por organogênese ou embriogênese somática, a partir do cultivo de células e tecidos vegetais in vitro é a base para a utilização da biotecnologia no melhoramento. Realizaram-se estudos com cinco cultivares de laranja doce (Citrus sinensis L. Osbeck), Pêra, Natal, Lima Verde, Hamlin e Westin. Este trabalho objetivou a avaliação da eficiência de isolamento de protoplastos das cultivares de laranja doce; o estudo da eficiência de plaqueamento em função de cinco densidades de protoplastos e diferentes meios de cultura e avaliação da embriogênese somática em função da composição dos meios de cultura e concentração da fonte de carboidrato. As soluções enzimáticas testadas para o isolamento de protoplastos foram: 1. Grosser e Chandler (1987), composta de 1% de celulase Onozuka RS (Yakult), 1% macerase R- 10 (Yakult Honsha) e 0,2% de pectoliase Y-23 (Seishin); 2. Grosser e Chandler (1987) modificado, composta de 1% de celulase Onozuka RS (Yakult), 1% macerase R-10 (Yakult Honsha); 3. Solução enzimática composta de 4% de celulase Onozuka R-10 (Yakult), 1% macerase R-10. O plaqueamento dos protoplastos foi realizado em cinco densidades, 2 x 104, 5 x 104; 105; 2x 105 e 3 x 105 protoplastos . mL-1,nos meios de cultura EME 0,7M, BH3 0,7M e BH3 + EME 0,7M em ausência de luz, a 25 ± 1 ºC. A solução enzimática 2 proporcionou um maior rendimento no isolamento de protoplastos das cultivares Hamlin, Natal e Pera e solução enzimática 1 foi melhor para a cultivar Westin. A eficiência final de plaqueamento avaliada aos 90 dias foi superior nas densidades de de 3 x 105 e 2 x 105 protoplastos. mL-1 para as cultivares Hamlin, Natal e Lima Verde, e na densidade de 2 x 105 e 105 protoplastos. mL-1 para a cultivar Westin. A indução da embriogênese somática ocorreu em meio de cultura MT modificado com 500 mg.L-1 de extrato de malte, acrescido de sacarose, galactose, glicose, sorbitol, lactose e maltose, nas concentrações de 18, 37, 75, 110 e 150 mM à temperatura de 27 °C. A formação de embriões somáticos variou com o genótipo, sendo a cultivar Lima Verde e Westin apresentaram menor número de embriões somáticos. As melhores fontes de carboidratos foram a maltose, seguida pela lactose nas concentrações de 37 e 75 mM para a cultivar Pêra, 37 mM para a cultivar Natal e 37, 75 e 110 mM para a cultivar Hamlin. / Plant regeneration, by organogenesis or somatic embryogenesis from cell cultures and in vitro plant tissue culture is the basis for the use of biotechnology in plant breeding. Studies were conducted with five cultivars of sweet orange (Citrus sinensis L. Osbeck), Pêra, Natal, Lima Verde, Hamlin and Westin. This work aimed to evaluate the isolation efficiency of protoplasts, to evaluate platting efficiency of protoplasts based on five densities of cells and different culture media and to evaluate somatic embryogenesis based on culture medium composition and concentration. The enzymatic solutions tested were: 1. Grosser and Chandler (1987): 1% de cellulase Onozuka RS (Yakult), 1% macerase R-10 (Yakult Honsha) and 0,2% de pectoliase Y-23 (Seishin); 2. Grosser and Chandler (1987) modified: 1% de cellulase Onozuka RS (Yakult) and 1% macerase R-10 (Yakult Honsha); 3. Enzimatic solution containing 4% cellulase Onozuka R-10 (Yakult) and 1% macerase R-10. Protoplasts were cultured at densities of 2 x 104; 5 x 104; 105; 2 x 105 e 3 x 105 protoplasts.mL-1 in EME 0,7M, BH3 0,7M and BH3 + EME 0,7M, in the dark, at 25 ± 1 ºC. The enzymatic solution 2 provided higher yield for the cultivars Hamlin, Natal and Pêra, and enzymatic solution 1 resulted in better protoplast isolation for cultivar Westin. Final platting efficiency, evaluated 90 days after culture, was higher at the densities of 3 x 105 e 2 x 105 protoplasts.mL-1 for Hamlin, Natal and Lima Verde, and at the density of 2 x 105 e 105 protoplasts.mL-1 for Westin. Somatic embryogenesis stimulation occurred in cultured medium MT (MURASHIGE AND TUCKER, 1969) modified with 500 mg. L-1 of malt extract, supplemented with sucrose, galactose, glucose, maltose, lactose and sorbitol at concentrations of 18, 37, 75, 110 and 150 mM, at 27 ± 1 ºC. Somatic embryos produced varied with the genotype, the smaller number of somatic embryos was observed in cultivars Lima Verde and Westin. The best source of carbohydrate were maltose, followed by lactose at concentrations of 37 and 75 mM for cultivar Pêra, 37 mM for cultivar Natal, and 37, 75 and 110 mM for cultivar Hamlin.

Thesis (M. Sc. (Agricultural Science)) -- University of Limpopo, 2001 / Refer to document

This work was aimed to investigate the basic viral infection protocols mainly focusing on Nicotiana benthamiana hairy root cultures and wild-type tobacco mosaic virus (TMV). The application of transgenic virus containing the gene for green fluorescent protein (GFP) for foreign protein production in plant tissue cultures and whole plants was also studied. The effect on viral accumulation of the form of plant tissue culture used, such as hairy roots, shooty teratomas and suspended cells, was investigated. Viral infection was shown to have no effect on culture growth and morphology. Hairy root cultures are a superior host for viral propagation and production in vitro. The maximum specific rate of viral accumulation occurred mainly during the root growth phase. The average maximum virus concentration in the hairy roots was 0.82 ?? 0.14 mg g-1 dry weight and virus protein represented a maximum of approximately 6% of total soluble protein in the root biomass. Proportional scale-up of TMVinfected hairy roots in shake flasks and bioreactors can be achieved without changing the average virus concentration accumulated in the hairy roots. The level of viral accumulation was much lower in N. benthamiana hairy roots infected with transgenic virus containing GFP (TMVGFPC3) compared with TMV and low levels or no GFP was detected. Viral accumulation and GFP production in whole plants was studied using different generations of transgenic TMV-GFPC3 virus. Hybrid viruses with the foreign gene GFPC3 deleted may have been formed in successive TMV-GFPC3 generations, resulting in the loss of GFP production and enhanced viral infectivity. In vitro generated RNA transcript and first generation TMV-GFPC3 were found to be more suitable for infection than the second generation TMV-GFPC3. However, the accumulation of GFP and virus concentration did not occur at the same ratio. Provided a more genetically stable transgenic viral vector is used for infection, transient viral infection of hairy roots can be a potential alternative system for foreign protein production than plants grown in the field as the containment or safety issues can be addressed.