Submitted in complete fulfillment for the Degree of Master of Technology: Biotechnology, Durban University of Technology, 2012. / Many secondary metabolites that have been extracted from medicinal plants have been used as source of clinical drugs. However, the concentration of the active metabolites in plants is generally low. An attractive alternative for producing these important secondary metabolites is via plant tissue culture technology. More particularly, the genetic transformation of a plant tissue by Agrobaterium rhizogenes has been employed for producing high yields of secondary metabolites. In a previous study, three structurally similar anthraquinones: 9,10-Anthracenedione, 1-Hydroxy-4-methylanthraquinone and 5,8-Dimethoxy-2,3,10,10a-tetrahydro-1H,4aH-phenanthrene-4,9-dione, and one steroid; Androst-5-ene-3, 17, 19-triol were isolated from the root extracts of C. triloba. The anthraquinones have shown to exhibit the anticancer mechanism which involves the inhibition of the activity of the human topoisomerase II enzyme that transforms supercoiled DNA to linear DNA. However, these anthraquinones were found in very low concentrations. Therefore, in this study we used plant cell and tissue culture systems (cell suspension, shoot and hairy root cultures) of C. triloba to increase the production of anthraquinones. Since the establishment of C. triloba in vitro plant systems required a source sterile explants, a protocol that involved the use of NaCIO was optimized for the sterilization and subsequent germination of C. triloba seeds which were micro-propagated into shoot cultures. These cultures provided a source explants for the induction of callus and hairy root cultures. The biomass of these plant cell and tissue cultures were subsequently bulked up for the extraction for anthraquinones and the yields were compared followed by fractionation and identification of the major compounds. The bioactivity of the fractions was evaluated by testing their cytotoxicity on cancer cells and anti-topoisomerase activity. The sterilization protocol that provided sterile seeds was found to be a solution of 30% NaCIO at an exposure time of 10 minutes. From the sterilized seeds shoot cultures were established on MS medium. The leaf explants of the shoot cultures were then used to induce callus cultures which subsequently were transferred to liquid medium whereby the total biomass of suspension cultures increased from 4 g to 134.18 g (wet weight). Also hairy roots cultures were established from stem explants with a low cell density inoculum of A. rhizogenes at a transformation efficiency of 73%. The growth of these hairy roots was slow in hormone free medium. This was overcomed with the use NAA and IAA which increased the xvii biomass from 1.03 g in the control culture (without hormone) to 23.91 g and 46.13 g respectively. An evaluation of the anthraquinones in the field root and hairy root, cell suspension and shoot culture extracts was carried out by using their Thin Layer Chromatography profiles and the High Performance Liquid Chromatography profiles as well as the standards, 9,10-Anthracenedione and 1-Hydroxy-4-methylanthaquinone. TLC analysis showed that the RF values of the fractions CT01 and CT02 matched the RF values of anthraquinones standards while HPLC analysis revealed that hairy root cultures supplemented with IAA (125.03 μg.mg-1) or NAA (98.25 μg. mg-1) produced a higher concentration of anthraquinones than the control culture (without hormone) (13.33 μg.mg-1), the field roots (33.51 μg. mg-1) and the shoot (3.23 μg.mg-1) and cell suspension cultures (13.17 μg.mg-1). Due to co-elution of the compounds in HPLC analysis, six fractions were isolated by Preparative Thin Layer Chromatography from the hairy root extract (obtained from the culture supplemented with NAA) and were coded as CT01, CT02, CT03, CT04, CT05 and CT06. The compounds in these fractions were identified by Electron Ionization-Liquid chromatography-Mass Spectroscopy and it was found that the hairy roots produced one acridone derivative; 5-Methoxy-2-nitro-10H-acridin-9-one, one naphthoquinone derivative; 2H-Naphto[2,3-b]pyran-5,10-dione,3,4-dihydro-2,2-dimethyl- and seven anthracenedione derivatives. These were: i) 5,8-Dimethoxy-2,3,10,10a-tetrahydro-1H,4aH-phenanthrene-4,9-dione, ii) 9,10-Anthracenedione, 2-methyl-, iii) 1-Hydroxy-4-methylanthraquinone, iv) 9,10-Anthracenedione, 2-ethyl-, v) 1,5-Diaminoanthraquinone, vi) Phenanthrene, 3,6-dimethoxy-9-methyl-, vii) 9,10-Anthracenedione, 1,4-dimethyl-. Fractions CT01 (5,8-Dimethoxy-2,3,10,10a-tetrahydro-1H,4aH-phenanthrene-4,9-dione, 9,10-Anthracenedione, 2-methyl- and 1-Hydroxy-4-methylanthraquinone) and CT02 (9,10- Anthracenedione, 2-ethyl-) were cytotoxic to the DU-145 cancer cell line at concentrations of 125 μg.mg-1 to 1000 μg.mg-1. These fractions also showed anti-topoisomerase activity as they inhibited the conversion of supercoiled DNA into linear DNA. In conclusion this is the first study that describes the transformation of C. triloba by A. rhizogenes mediated transformation and compares the production of anthraquinones in C. triloba hairy roots to the field roots, shoot and cell suspension cultures. This study has xviii indicated that hairy root cultures is a high-yielding production system for anthraquinones (5,8-Dimethoxy-2,3,10,10a-tetrahydro-1H,4aH-phenanthrene-4,9-dione, 1-Hydroxy-4-methylanthraquinone, 9,10-Anthracenedione, 2-methyl- and 9,10- Anthracenedione, 2-ethyl-) which could have the potential to be used in cancer therapy. In addition the discovery of C. triloba hairy roots having the biosynthetic capacity to synthesize five valuable anthraquinone derivatives that are not found the field roots has also been revealed.

No description available.

Fundação Instituto de Pesquisas Econômicas / The herbicide glyphosate is commonly used to control weeds in the soybean crop, with some species tolerant to it. The weed species Ipomoea triloba (bindweed) is tolerant to the usual doses of the herbicide glyphosate, so there is a need to increase your dose to control effectively. It is also used to spray adjuvants and additives to improve the performance of control with this herbicide. In view of this, four experiments were conducted, three in the field, and one in the greenhouse. The first three were to evaluate the control of I. triloba with formulations of glyphosate associated with the additives and processing of water, where the Experiment I was set to low relative humidity (RH) (below 40%) and Experiments II and III to RH ideal (above 60%). The fourth experiment aimed to evaluate the control of I. triloba with different doses of glyphosate Trop associated with different adjuvants and additives for water. The results showed that the addition of the herbicide glyphosate and adjuvants reduces the pH of the solution. The herbicide used in treatments I and II should be applied in higher doses to have efficient control of the species. There was improvement in the efficiency of control with the addition of adjuvants to the herbicide, the recommended dose of the product. The application of adjuvants associated with glyphosate use has not improved the efficiency of control in the experiment III, the lowest dose used. With the experiment of dose-response curve only the addition of additives urea and A-20 showed glyphosate results in control. Considering the dry matter, the addition of adjuvants and aditives, except for Impact Plus, provided an improvement in the performance of control. / O herbicida glifosato é usualmente utilizado no controle de plantas daninhas na cultura da soja, existindo algumas espécies tolerantes a ele. A espécie daninha Ipomoea triloba (corriola) é tolerante às doses usuais do herbicida glifosato, portanto há a necessidade de aumentar a sua dose para o controle eficaz. Tem-se também, utilizado adjuvantes e aditivos de calda para melhorar o desempenho de controle com este herbicida. Em vista disto, foram então realizados quatro experimentos, três em campo, e um em casa de vegetação. Os três primeiros tinham como objetivo avaliar o controle de I. triloba com formulações de glifosato associado a aditivos e adjuvantes de calda, onde o Experimento I foi programado para a aplicação em condições de baixa umidade relativa do ar (UR) (abaixo de 40%) e os Experimentos II e III para UR ideal (acima de 60%). O quarto experimento visava avaliar o controle de I. triloba com diferentes doses de glifosato Trop associada a diferentes adjuvantes e aditivos de calda. Os resultados mostraram que a adição do herbicida glifosato e de adjuvantes reduz o pH da calda. O herbicida utilizado nos tratamentos I e II deve ser aplicado em doses maiores para que ocorra controle eficiente da espécie. Não ocorreu melhora na eficiência de controle com a adição de adjuvantes ao herbicida, na dose recomendada do produto. A aplicação de adjuvantes associado ao glifosato utilizado não melhorou a eficiência de controle no experimento III, na menor dose usada. E para o experimento de curva de dose-resposta concluiu-se que somente a adição dos aditivos uréia e A-20 ao glifosato demonstraram resultados no controle. Quanto à massa de matéria seca, a adição dos adjuvantes e aditivos, com exceção do Impact Plus, proporcionou uma melhora no desempenho de controle do glifosato sobre I. triloba.

Pawpaw (Asimina triloba (L.) Dunal) has significant potential as a new fruit crop. During ripening, loss of firmness is extremely rapid, and this trait may be the biggest obstacle to the development of a broader market as handling without injury is difficult. Cold storage of pawpaw seems limited to 4 weeks at 4 C. A study of several cultivars with commercial appeal showed that ripening traits such as ethylene production, respiration and loss of firmness were similar in all genotypes, and that no cultivar showed superior responses to cold storage. Cold storage for longer than 4 weeks caused the development of cold injury symptoms such as black discoloration, rapid loss of firmness, impaired respiration, tissue acidification, decrease in antioxidant content, decrease in volatile ester production and development of off-flavor volatile compounds. Overall cold storage injury symptoms observed in pawpaw may be due to oxidative damage linked to the failure of the two major antioxidant systems that could protect against such damage: phenolics and the ascorbateglutamate system. With the aim of enhancing pawpaw low temperature tolerance and prolonging cold storage length, different techniques such as hot air exposure and hot water dips of fruit prior to beginning cold storage, and intermittent warming periods during cold storage, were evaluated. Despite positive results with these techniques for other commodities, all the strategies failed to appreciably alter fruit ripening, loss of firmness or maintain fruit quality during and/or after cold storage.

The North American Pawpaw [Asimina triloba (L.) Dunal] shows great potential as a new fruit crop. Kentucky State University in Frankfort, Ky. is the site for the USDA National Clonal Germplasm Repository (NCGR) for Asimina species. Both the fruit and the trees themselves are of high value to growers and nursery producers. Pawpaw cultivars are currently propagated by grafting or budding onto seedling rootstock; no method currently exists to clonally propagate pawpaw on its own roots. Three methods of layering were attempted in this study to clonally propagate pawpaw: trench layering, pot layering, and mound layering. Both trench layering and pot layering experiments showed the importance of both juvenility and auxin application in adventitious rooting of pawpaw. Although rooting of more mature pawpaw shoots in these experiments did not exceed 30%, these propagation methods were more successful then previous attempts at rooting more mature pawpaw stems. Mound layering was less successful, but an easierto-root genotype of pawpaw in the KSU-USDA NCGR for Asimina spp. was discovered that may show promise for future propagation studies. Diversity in reproductive characteristics of pawpaw was also assessed in this study. Accessions in the KSU-USDA repository orchard collected from six different geographic regions were selected and trunk cross sectional area, total number of flowers, length of flowering, flowering peak, fruit set, total number of clusters, total number of fruit, number of fruit per cluster, average fruit weight, yield by weight, yield efficiency, length of harvest, harvest peak, and growing degree days required for ripening were evaluated. Significant differences were found among the regions in most of the characteristics evaluated. Correlations were also found between several vegetative and reproductive characteristics. This indicates that a significant level of reproductive diversity exists within KSUs repository collection, and between pawpaws collected from different regions of the native range.

Submitted in complete fulfillment for the Degree of Master of Technology: Biotechnology, Durban University of Technology, 2012. / Many secondary metabolites that have been extracted from medicinal plants have been used as source of clinical drugs. However, the concentration of the active metabolites in plants is generally low. An attractive alternative for producing these important secondary metabolites is via plant tissue culture technology. More particularly, the genetic transformation of a plant tissue by Agrobaterium rhizogenes has been employed for producing high yields of secondary metabolites. In a previous study, three structurally similar anthraquinones: 9,10-Anthracenedione, 1-Hydroxy-4-methylanthraquinone and 5,8-Dimethoxy-2,3,10,10a-tetrahydro-1H,4aH-phenanthrene-4,9-dione, and one steroid; Androst-5-ene-3, 17, 19-triol were isolated from the root extracts of C. triloba. The anthraquinones have shown to exhibit the anticancer mechanism which involves the inhibition of the activity of the human topoisomerase II enzyme that transforms supercoiled DNA to linear DNA. However, these anthraquinones were found in very low concentrations. Therefore, in this study we used plant cell and tissue culture systems (cell suspension, shoot and hairy root cultures) of C. triloba to increase the production of anthraquinones. Since the establishment of C. triloba in vitro plant systems required a source sterile explants, a protocol that involved the use of NaCIO was optimized for the sterilization and subsequent germination of C. triloba seeds which were micro-propagated into shoot cultures. These cultures provided a source explants for the induction of callus and hairy root cultures. The biomass of these plant cell and tissue cultures were subsequently bulked up for the extraction for anthraquinones and the yields were compared followed by fractionation and identification of the major compounds. The bioactivity of the fractions was evaluated by testing their cytotoxicity on cancer cells and anti-topoisomerase activity. The sterilization protocol that provided sterile seeds was found to be a solution of 30% NaCIO at an exposure time of 10 minutes. From the sterilized seeds shoot cultures were established on MS medium. The leaf explants of the shoot cultures were then used to induce callus cultures which subsequently were transferred to liquid medium whereby the total biomass of suspension cultures increased from 4 g to 134.18 g (wet weight). Also hairy roots cultures were established from stem explants with a low cell density inoculum of A. rhizogenes at a transformation efficiency of 73%. The growth of these hairy roots was slow in hormone free medium. This was overcomed with the use NAA and IAA which increased the xvii biomass from 1.03 g in the control culture (without hormone) to 23.91 g and 46.13 g respectively. An evaluation of the anthraquinones in the field root and hairy root, cell suspension and shoot culture extracts was carried out by using their Thin Layer Chromatography profiles and the High Performance Liquid Chromatography profiles as well as the standards, 9,10-Anthracenedione and 1-Hydroxy-4-methylanthaquinone. TLC analysis showed that the RF values of the fractions CT01 and CT02 matched the RF values of anthraquinones standards while HPLC analysis revealed that hairy root cultures supplemented with IAA (125.03 μg.mg-1) or NAA (98.25 μg. mg-1) produced a higher concentration of anthraquinones than the control culture (without hormone) (13.33 μg.mg-1), the field roots (33.51 μg. mg-1) and the shoot (3.23 μg.mg-1) and cell suspension cultures (13.17 μg.mg-1). Due to co-elution of the compounds in HPLC analysis, six fractions were isolated by Preparative Thin Layer Chromatography from the hairy root extract (obtained from the culture supplemented with NAA) and were coded as CT01, CT02, CT03, CT04, CT05 and CT06. The compounds in these fractions were identified by Electron Ionization-Liquid chromatography-Mass Spectroscopy and it was found that the hairy roots produced one acridone derivative; 5-Methoxy-2-nitro-10H-acridin-9-one, one naphthoquinone derivative; 2H-Naphto[2,3-b]pyran-5,10-dione,3,4-dihydro-2,2-dimethyl- and seven anthracenedione derivatives. These were: i) 5,8-Dimethoxy-2,3,10,10a-tetrahydro-1H,4aH-phenanthrene-4,9-dione, ii) 9,10-Anthracenedione, 2-methyl-, iii) 1-Hydroxy-4-methylanthraquinone, iv) 9,10-Anthracenedione, 2-ethyl-, v) 1,5-Diaminoanthraquinone, vi) Phenanthrene, 3,6-dimethoxy-9-methyl-, vii) 9,10-Anthracenedione, 1,4-dimethyl-. Fractions CT01 (5,8-Dimethoxy-2,3,10,10a-tetrahydro-1H,4aH-phenanthrene-4,9-dione, 9,10-Anthracenedione, 2-methyl- and 1-Hydroxy-4-methylanthraquinone) and CT02 (9,10- Anthracenedione, 2-ethyl-) were cytotoxic to the DU-145 cancer cell line at concentrations of 125 μg.mg-1 to 1000 μg.mg-1. These fractions also showed anti-topoisomerase activity as they inhibited the conversion of supercoiled DNA into linear DNA. In conclusion this is the first study that describes the transformation of C. triloba by A. rhizogenes mediated transformation and compares the production of anthraquinones in C. triloba hairy roots to the field roots, shoot and cell suspension cultures. This study has xviii indicated that hairy root cultures is a high-yielding production system for anthraquinones (5,8-Dimethoxy-2,3,10,10a-tetrahydro-1H,4aH-phenanthrene-4,9-dione, 1-Hydroxy-4-methylanthraquinone, 9,10-Anthracenedione, 2-methyl- and 9,10- Anthracenedione, 2-ethyl-) which could have the potential to be used in cancer therapy. In addition the discovery of C. triloba hairy roots having the biosynthetic capacity to synthesize five valuable anthraquinone derivatives that are not found the field roots has also been revealed. / National Research Foundation.

Orientador: Ana Carolina Conti e Silva / Banca: Karina de Lemos Sampaio / Banca: Natália Soares Janzantti / Resumo: A demanda por alimentos e ingredientes funcionais tem aumentado, em particular pelos prebióticos. A incorporação de inulina e oligofrutose às bebidas de frutas, como o néctar, torna-se uma alternativa para agregar valor nutritivo aos produtos. Dentre as frutas, o mamão representa uma excelente opção devido ao fácil cultivo, alta produtividade, baixo custo, alto valor nutritivo e particularmente à escassez do néctar dessa fruta no mercado brasileiro. O trabalho objetivou desenvolver néctares de mamão com diferentes concentrações de açúcar, oligofrutose e inulina. Foram elaborados néctares, sem frutanos, com 6, 8, 10 e 12% de açúcar e determinado o ideal de doçura em açúcar. A partir da formulação considerada ideal em doçura, foi aplicada a metodologia de modelagem de misturas para avaliar os efeitos da interação entre açúcar, oligofrutose e inulina na aceitação sensorial e características químicas dos néctares de mamão (variáveis dependentes). As formulações foram avaliadas sensorialmente quanto à aceitação (aparência, aroma, viscosidade, sabor, doçura e avaliação global) e intenção de compra, empregando-se escala hedônica de nove pontos e escala categórica de cinco pontos, respectivamente. Os resultados da aceitação sensorial também foram avaliados por meio da análise de Cluster e Análise de Componentes Principais para construção dos mapas de preferência internos. As formulações foram caracterizadas quimicamente quanto ao teor sólidos solúveis e totais, acidez total titulável, açúcares totais, cinzas e pH. A formulação mais aceita para avaliação global foi monitorada durante quinze dias quanto ao pH, acidez total titulável, bactérias mesófilas, psicrotróficas, bolores e leveduras, coliformes totais e termotolerantes. A formulação com 12% de açúcar foi considerada como ideal em doçura por 36% dos julgadores. A interação entre os ingredientes açúcar, oligofrutose e ... / Abstract: The demand for food and functional ingredients has increased, in particular by the prebiotics. The incorporation of inulin and oligofructose in fruit drinks, as nectars, is an alternative to increase nutritional value to products. Among fruits, the papaya is an excellent choice because of its easy cultivation, high productivity, low cost, high nutritional value and, particularly, the shortage of this fruit nectar in the Brazilian market. The study aimed to develop papaya nectars with different concentrations of sugar, oligofructose and inulin. Nectars were prepared with 6, 8, 10 and 12% sugar (without fructan) and the ideal sweetness of sugar was determined. From the formulation considered ideal sweetness, the mixtures modeling methodology was applied to assess the effects of the interaction between sugar, oligofructose and inulin on the acceptance sensory and chemical characteristics of papaya nectars (dependent variables). The sensory acceptance regarding to appearance, aroma, viscosity, flavor, sweetness and overall evaluation and purchase intent was evaluated, using a nine point hedonic scale and categorical scale of five points, respectively. The results of sensory acceptance were also assessed by cluster analysis and principal component analysis to construct internal preference maps. The formulations were characterized regarding to soluble solids and total, titratable acidity, total sugars, ash and pH. The best formulation for overall assessment was monitored for two weeks for pH, titratable acidity, mesophilic, psychrotrophic, yeasts and molds, total and fecal coliforms. The formulation with 12% sugar was considered ideal for sweetness by 36% of the panelists. The interaction between the ingredients sugar, oligofructose and inulin influenced the acceptance for the taste and the sweetness of the nectar, and the formulation with 6% sugar and 6% inulin was the most acceptable for these attributes. The preference for attributes of this ... / Mestre

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Made available in DSpace on 2014-06-11T19:28:32Z (GMT). No. of bitstreams: 0 Previous issue date: 2011-03-06Bitstream added on 2014-06-13T19:16:34Z : No. of bitstreams: 1 ferreiraneto_me_me_jabo.pdf: 1445654 bytes, checksum: b179a42d4eeb46cdbbec60b82a6761b5 (MD5) / Monsanto / A adoção da tecnologia Roundup-Ready® de controle de plantas daninhas na cultura da soja foi bastante rápida e extensa expondo-a a uma grande diversidade de situações de composição especifica de comunidades infestantes, características edáficas e climáticas e de práticas agrícolas. Em alguns anos, passaram a serem identificadas plantas daninhas com maiores dificuldades de controle pelo glyphosate e que passaram a ser selecionadas e suas importâncias aumentadas nos agroecossistemas. Com o objetivo de estudar o controle de plantas daninhas em soja geneticamente modificada para tolerância ao glyphosate dois ensaios foram conduzidos um a campo e outro em condições de casa de vegetação. O ensaio de campo foi conduzido no Pontal do Paranapanema e foram avaliados os controles de plantas daninhas e seletividade à cultura da soja do glyphosate aplicado isolado e em combinação com diclosulam, cloransulam-methyl, flumioxazin e s-metolachlor. Foram dois grupos de tratamentos: um com aplicação única e outro com aplicação seqüencial de glyphosate. No campo, o experimento obedeceu ao delineamento em blocos ao acaso. Os resultados mostraram que aplicações seqüenciais proporcionaram melhor controle de Commelina benghalensis, mas não incrementaram o de Ipomoea triloba. Os melhores níveis de produtividade foram obtidos nas parcelas tratadas com a combinação de flumioxazin ou s-metolachlor com glyphosate, com ou sem aplicação seqüencial. Um segundo grupo de ensaios foi conduzido em vasos com o objetivo de avaliar a atividade biológica de diferentes doses e formulações do glyphosate em Macroptilium lathyroides, Euphorbia heterophylla e Senna obtusifolia em estádio de desenvolvimento em que regularmente há dificuldade de controle nas condições de campo. Todos os ensaios obedeceram ao delineamento... / The adoption of the Roundup-Ready® technology for weed control in soybeans was very quick and extensive. With the geographic expansion, the Roundup-Ready® soybeans met a big diversity of situations of weed composition, spil and climate features, and agronomic practices. After some years, certain weeds were identified as more tolerant to glyphosate and were selected in Roundup-Ready® soybean growing areas. So, two assays were carried out aiming to evaluate the weed control in Roundup-Ready® soybean. The first experiment was conducted under field conditions and the use of glyphosate alone or mixed with diclosulam, cloransulammethyl, flumioxazin, and s-metolachlor were evaluated under two situations: single spraying or followed of a complementary spraying of glyphosate, 14 days after the first one. The complementary application of glyphosate increased the Commelina benghalensis control, but did not change Ipomoea triloba control. The better productivity levels were achieved using the treatments with flumioxazin or s-metolachlor + glyphosate, with or without the complementary applicatrin. The second group of assays was carried under pot conditions out aiming to evaluate the biological activity of different doses and formulations of glyphosate in the control of Macroptilium lathyroides, Euphorbia heterophylla and Senna obtusifolia at growth stage in which some control difficulties were reported, under field conditions. A greenhouse essay was carried out under for each weed in the completely randomized experimental design with four replications. The treatments were arranged in a 3 x 4 factorial design considering three commercial formulations (Roundup Original®, Roundup-Ready® e Roundup Ultra®) and four doses (2,32; 1,68; 1,15 e 0,57 kg.ha-1) of glyphosate. Additionally, a control plot without chemical control was considered. The results showed that the three weeds had differential... (Complete abstract click electronic access below)