Somatiska embryogenes är en mikroförökningsmetod som med hjälp av tillväxtreglerande medel får en växtdel att bilda skott och rötter i ett speciellt odlingssubstrat sterila förhållanden. Kunskapen vad gäller förutsättningar för groning och plantetablering efter detta laboratoriestudium är dock begränsad. I denna studie undersökte hur två kloner av SE-groddar av gran etablerade sig i olika ljusförhållanden avseende våglängd och intensitet i fyra växtmiljöer. Etableringen bedömdes utifrån plantornas överlevnad och höjdtillväxt. Vid sista inventeringstillfället var 62 SE-plantor vid liv och 18 plantor var döda. Ingen signifikant skillnad kunde påvisas i överlevnad mellan de olika växtmiljöerna, inte heller mellan klonerna i respektive miljö eller oberoende av miljö. Signifikant skillnad i höjdtillväxten mellan klonerna kunde påvisas inom alla växtmiljöer, men inte mellan växtmiljöerna oberoende av klon. Det fanns en tendens att ljusförhållanden med en ljusintensitet på 100 μmol och rött ljus inom våglängderna 610-760 nm gav bäst etablering för SE-groddarna. Studien visade också att det var skillnad i etablering mellan de två kloner som användes.

The increasing demand for flavors in the world market requires new strategies for obtaining these compounds which can be produced via fermentation processes using low cost substrates such as agroindustrial residues. This turns out to be a viable process and the aroma compounds generated are classified as natural. Large volumes of waste in the form of bagasse, husks, kernels or seeds including from the processing of fruit pulp are generated and these can be used as substrates in these fermentations. Thus the main purpose of this work was to use agro-industrial residues for the production of volatile aroma compounds by fermentation processes. In this regard, we investigated the potential of the production of volatile compounds by submerged fermentation using guava, passion fruit and umbu processing waste as substrates using endophytic microorganisms isolated from guava, passion fruit and umbu as well as by using the pure cultures (Aspergillus niger, Kluyveromyces marxianus and Kluyveromyces lactis) to produce volatile compounds characteristic of fruity or floral aroma. Initially the fruit pulp and wastes from the processing of guava, passion fruit and umbu were chemically characterized (moisture, ash, fiber, proteins, lipids, sugars, titratable acidity, Aw, pH and soluble solids). Fermentation with different microorganisms in synthetic medium was performed and the fermented products analyzed for aroma note. From the results of this sensory analysis, the guava residue and the yeast Kluyveromyces lactis were selected since these constituents in the medium produced fruity and floral aromas. The volatile compounds produced were captured by two headspace techniques (SPME and Purge & Trap) and analyzed in a system of gas chromatography coupled to mass spectrometry. Sixty-two microorganisms were isolated and out of these six were selected. The fermented products were analyzed for pH, soluble solids and reducing sugars as well as to establish the kinetics of the fermentation process at every 24 hours for five days. The main aroma descriptors in the fermented products were fruity, honey, fruity green, plum, guava, banana and alcohol type notes. Forty-three volatile compounds were detected in the fermented product obtained by using the Guava residue in the medium. Among the compounds identified were: Ò {caryophyllene, 2-phenylethyl acetate, × n- humulene, bisabolene, humulene, 3-methyl butanol, bergamoteno, × n-cadinene, caryophyllene oxide and 2-phenylethanol. The production of volatile compounds was quite rapid and in most cases, this production occurred on the first day of fermentation and had achieved its maximum concentration between 24-48h of fermentation. According to the response surface method analysis, the conditions of the manufacturing process were optimized. Increased production of 2- phenylethyl acetate was achieved at concentrations varying from 25 and 35g/L of yeast extract, 40 to 60 g/L glucose. Ò-caryophyllene was obtained at concentrations varying between 120 and 140g/L of guava waste, and an agitation performed between 160 and 180 rpm. The production of 2-phenylethanol was obtained at agitation varying from 228rpm and 5g/L glucose. From these optimized conditions, scale-up of fermentation was performed in a bioreactor wherein 6 times larger concentration of 2-phenylethanol and Ò-caryophyllene compounds, and 1.5 times higher concentration of 2-phenylethyl acetate were obtained. This study reveals that guava residue can be used to produce aroma compounds of great importance for the flavor and fragrance industry, the compounds being 2-phenylethyl acetate, 2-phenylethanol and Ò -caryophyllene. / A crescente demanda de aromas no mercado mundial exige novas estrategias de obtencao desses compostos. A obtencao via processos fermentativos utilizando substratos de baixo custo e uma alternativa economicamente viavel e o aroma gerado e considerado como natural. Grandes volumes de residuos na forma de bagacos, cascas, carocos ou sementes sao gerados pelo processamento de frutas, e estes podem ser utilizados como substratos nesses processos fermentativos. Diante desse contexto, esse trabalho teve como principal finalidade utilizar residuos agroindustriais para a producao de compostos volateis de aroma atraves da fermentacao submersa. Neste sentido, foi verificada a potencialidade da producao de compostos volateis atraves da fermentacao submersa utilizando residuos de goiaba, maracuja e umbu como substratos utilizando microrganismos endofitos da goiaba, maracuja e umbu de cultura puras (Aspergillus niger, Kluyveromyces marxianus e Kluyveromyces lactis) para produzir compostos volateis de aroma frutal ou floral. Inicialmente a polpa e os residuos de goiaba, maracuja e umbu foram caracterizados quimicamente (umidade, cinzas, fibras, proteinas, lipideos, acucares, acidez titulavel, Aw, pH e solidos soluveis). Foram realizadas fermentacoes com os diferentes microrganismos em meio sintetico, onde os produtos fermentados foram analisados sensorialmente. A partir dos resultados obtidos da analise sensorial, o residuo de goiaba e a levedura Kluyveromyces lactis foram selecionados uma vez que estes apresentaram a melhor capacidade de producao de aromas. Os compostos volateis produzidos foram capturados por duas tecnicas de headspace (MEFS e Purge & Trap) e analisados por cromatografia gasosa acoplada a espectrometria de massas. Foram isolados 62 microrganismos, dentre estes seis foram selecionados e os produtos fermentados foram analisados por cromatografia gasosa como tambem as caracteristicas de pH, solidos soluveis e acucar redutor para estabelecer a cinetica do processo fermentativo a cada 24h durante cinco dias. As principais descricoes de aroma para o fermentado de residuo de goiaba foram frutal, mel, fruta verde, ameixa, goiabada, banana, fermento e alcool. Foram detectados 43 compostos volateis no produto fermentado obtido pelo uso do residuo de goiaba. Dentre os compostos identificados destacam-se: Ò-cariofileno, acetato de 2-feniletil, ×-humuleno, bisaboleno, humuleno, 3-metil butanol, bergamoteno, ×-cadineno, oxido de cariofileno e 2-feniletanol. A producao dos compostos volateis foi bastante rapida, e na maioria dos casos, essa producao aconteceu a partir do primeiro dia de fermentacao e teve o seu maximo entre 24-48h de fermentacao. Segundo a analise da superficie de resposta, o processo de producao foi otimizado, onde a maior producao do acetato de 2-feniletil foi obtida em concentracoes entre 25 a 35g/L de extrato de levedura, entre 40 a 60 g/L de glicose; do Ò-cariofileno foi obtido em concentracoes entre 120 a 140g/L de residuo de goiaba, e agitacao entre 160 e 180 rpm enquanto a producao do 2-feniletanol foi obtida na agitacao entre 228rpm e 5g/L de glicose. A partir dessas condicoes otimizadas foi possivel realizar um escalonamento da producao desses compostos no biorreator e foi possivel obter uma producao 6 vezes maior para os compostos 2-feniletanol e Ò-cariofileno, e 1,5 vezes para o acetato de 2-feniletil. Desta forma, neste trabalho verifica-se que o residuo de goiaba e leveduras isoladas de frutas podem ser utilizados para produzir compostos de aroma de grande importancia para a industria de aromas e fragrancias, como o acetato de 2-feniletanol, o 2-feniletanol e o Ò-cariofileno.

Infectious diseases remain a significant cause of human deaths, as approximately 15 million deaths were attributed to infectious diseases in 2010 (Dye, 2014). One such disease is rabies, which causes around 59,000 human deaths worldwide annually according to some estimates (Kessels et al., 2017). However, 95% of human deaths attributed to rabies occur in Asia and Africa (Singh et al., 2017). Rabies is preventable, yet it is still a major concern in developing, low-income countries that lack access to the medical care necessary to combat it (Hampson et al., 2015). Alternative techniques for low-cost vaccine production have the potential to resolve this issue. This research investigates the use of recombinant DNA techniques and plant biotechnology to produce a more cost-effective vaccine for rabies. Gene sequences from the rabies glycoprotein were inserted at the end of the coat protein portion of the Tobacco Mosaic Virus (TMV) genome. Plants were then infected with this recombinant virus, with hopes that TMV particles would assemble with proteins produced from the inserted glycoprotein sequence fused to the TMV coat protein. Results thus far suggest some of the sequences could be producing recombinant TMV particles, although issues involving successful extraction and reversion to wild type are still a challenge. Additionally, other research suggests that this is an effective method for vaccine development in general and for rabies.

The demand for plant and other production is increasing with the growth of human population when the territory where plants can be grown is continually decreasing. The present agricultural production of food, industrial materials and drugs of plant origin is not enough for people, as for the effort to achieve higher environmental comfort, especially in developed countries. We are trying to find a simpler, cleaner and cheaper ways of crop production. The genetic modification is one of the modern ways how to achieve this goal. Students get a lot of information about this topic from various sources, but mainly just from the internet and television. It is recommended that students should be able to compare information received from the media with school knowledge as for avoiding opinion pressure and manipulations.

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Die Pflanzenbiotechnologie ermöglicht die nachhaltige Gewinnung pflanzlicher Wertstoffe mittels innovativer biotechnologischer Methoden. Bisher mangelt es auf diesem Gebiet jedoch an Grundlagenwissen und aussagekräftigen Studien, z. B. zur Anwendung biotechnologischer Standardverfahren beim Respirationsmonitoring. Im Rahmen der vorliegenden Arbeit werden grundlegende Untersuchungen zur Erzeugung (Induktion) pflanzlicher in-vitro-Kulturen und zu geeigneten Kultivierungssystemen sowie Prozessüberwachungsstrategien vorgestellt und diskutiert. Als Modellsystem dient die Einjährige Sonnenblume Helianthus annuus. Die Induktion pflanzlicher Zellkulturen (Kallus und Suspensionen) mit photomixotrophem Stoffwechsel wurde unter unterschiedlichen Bedingungen untersucht und geeignete Induktionsparameter ermittelt. Sowohl pflanzliche Gewebekulturen (Hairy roots) als auch die erzeugten photomixotrophen und heterotrophe Suspensionen konnten in verschiedenen Reaktorsystemen erfolgreich kultiviert und die Produktbildung nachgewiesen werden. Protokolle zu Induktion sowie Erhaltung von Zell- und Gewebekulturen von H. annuus wurden etabliert. Ein modernes Prozessüberwachungssystem für Schüttelkolben, das RAMOS® (Respiration Activity Monitoring System®) wurde erstmals umfassend für Untersuchungen des Wachstumsverhaltens und zum Screening pflanzlicher Zell- und Gewebekulturen eingesetzt. Dabei wurde die Problematik der Verdunstung (Evaporation) aus den Kulturgefäßen als signifikant bei den langen Kultivierungen von pflanzlichen in-vitro-Kulturen diagnostiziert und ein Modell zur Korrektur der Atmungstransferraten entwickelt. Erstmalig in der Pflanzenbiotechnologie kam das RAMOS® für Studien mit Zell- und Gewebekulturen von H. annuus im Speziellen sowie für Untersuchungen von Hairy roots im Allgemeinen zum Einsatz. Mit Hilfe der vorliegenden Arbeit werden relevante Kriterien zur Anwendung des innovativen Messsystems RAMOS® im Rahmen pflanzenbiotechnologischer Untersuchungen vorgestellt. Es wird ein Überblick über geeignete Kultivierungssysteme und zu publizierten Modellierungsstrategien für Applikationen in der Pflanzenbiotechnologie gegeben. Ein Literaturüberblick zu publizierten Modellierungsstrategien mit pflanzenbiotechnologischem Bezug vervollständigt die Arbeit. / Plant biotechnology enables a sustainable production of valuable plant resources using innovative biotechnological methods. However, a comprehensive knowledge base as well as significant studies, e. g. concerning the application of biotechnological standard procedures of respiration monitoring, are missing so far. In this work, basic investigations regarding the induction of plant in vitro cultures and appropriate cultivation systems as well as process monitoring strategies will be introduced and discussed. The annual sunflower Helianthus annuus serves as biological model system. The induction of plant cell cultures (callus and suspension) with photomixotroph metabolism was investigated at different conditions and appropriate induction parameter were determined. Both, plant tissues (Hairy roots) and induced photomixotroph as well as heterotrophic suspensions were cultivated successfully in various reactor systems. The production of desired metabolites was proven. Protocols concerning induction respectively maintenance of cell and tissue cultures of H. annuus have been established. For extensive investigations of growth behavior and for screening of plant cell and tissue cultures, a modern process monitoring tool for shake flasks, the RAMOS® (Respiration Activity Monitoring System®), was used for the first time. Thereby, the problem of evaporation off the culture vessels was identified as significant for time-intensive cultivations of plant in vitro cultures. A model for the correction of respiration transfer rates has been developed. For the first time in plant biotechnology, the RAMOS® has been applied for studies with H. annuus in special, and for studying the growth and respiration behavior of Hairy roots in general. With the help of the present work, relevant criteria concerning the application of the innovative measuring system RAMOS® for plant biotechnological investigations will be given. Furthermore, a survey over appropriate cultivation systems and published modelling strategies in plant biotechnology are introduced. A literature survey concerning model strategies regarding plant biotechnology completes this work.

A biotecnologia está revolucionando a atividade humana nas mais diversas áreas. No setor florestal, as novas tecnologias como transgenia, genômica, proteômica e bioinformática, estão sendo rapidamente incorporadas. A associação dessas tecnologias aos programas de melhoramento convencional de árvores pode acelerar o processo de obtenção de árvores com características específicas da madeira para a indústria de papel e celulose. UDP-glicose pirofosforilase (UGPase, EC 2.7.7.9) é uma enzima chave na produção de UDP-glicose, intermediário na interconversão de carboidratos envolvidos em várias funções metabólicas, como síntese de sacarose e de celulose. No presente trabalho, foi clonado o cDNA que codifica a UGPase. Este cDNA foi sintetizado por meio de RT-PCR utilizando-se mRNA extraído de tubérculo de batata (Solanum tuberosum L.). Os primers específicos para a ORF do gene da UGPase foram definidos com base em seqüências conservadas entre genes ortólogos, já disponíveis em bancos de dados. O cDNA foi clonado no vetor pENTR-D TOPO e inserido, por meio de transformação química, em células competentes de Escherichia coli. O DNA plasmidial foi purificado e após seu sequenciamento, a presença de um inserto de 1402 pb foi confirmada. O alinhamento da seqüência deduzida de aminoácidos com outras UGPases revelou alta homologia a UGPases de batata e de outras espécies vegetais. Na seqüência do cDNA clonado foram identificados sítios de N-glicosilação, resíduos lisina e motivos conservados que possivelmente estão relacionados tanto com a capacidade de ligação ao substrato, como com a atividade catalítica da enzima. Também foi realizada uma análise de Southern blot para confirmar a presença deste gene no DNA genômico de batata e de eucalipto (Eucalyptus grandis). Posteriormente, o cDNA clonado será utilizado na avaliação do impacto de sua superexpressão na biossíntese de celulose e hemiceluloses e no desenvolvimento de plantas de fumo (Nicotiana tabacum) e de eucalipto. / Biotechnology is revolutionazing human activity in several fields. In forestry, new technologies like transgenies, genomics, proteomics and bioinformatics have been quickly consolidated. Assossiating these technologies with conventional forestry breeding will accelerate the process of obtaining new trees with specific wood properties for the paper and pulp industry. UDP-glucose pyrophosphorylase (UGPase, EC 2.7.7.9) is a key enzyme producing UDP-glucose, an important intermediate in carbohydrate interconversion involved in various metabolic processes like sucrose and cellulose synthesis. In this work, it was cloned the cDNA that encodes UGPase. This cDNA was synthetized through RT-PCR using RNA extracted from potato (Solanum tuberosum L.) tuber. The UGPase primers were designed based on the conserved sequences of hortologous genes, already available in public data banks. The cDNA was cloned into pENTR-D TOPO vector and introduced into Escherichia coli competent cells, by chemically transforming. The plasmid DNA was purified and the insert PCR amplified. The presence of 1402 bp insert was confirmed after sequencing. The alignment of the derived amino acids sequence with other UGPases revealed high homology to UGPases from potato and other plant species. Along the cDNA sequence N-glicolisation sites, Lysyl residues and conserved domain were identified, which probably are involved in substrate binding capacity, as well as catalytic ativity of the enzyme. Southern blot analysis was carried to confirm the presence of this gene in potato and eucalypt (Eucalyptus grandis) genomic DNA. The cloned cDNA will be used to evaluate the impact of overexpression on celullose and hemicelluloses biosynthesis and in the tobacco (Nicotiana tabacum) and eucalypt plant development.

<p>The aim of this work was to develop a production process for the enzyme xyloglucan <i>endo</i>-transglycosylase from <i>Populus tremula x tremuloides</i> (<i>Ptt</i>XET16-34). The natural transglycosylating activity of this enzyme has previously been employed in a XET-Technology. This chemo enzymatic method is useful for biomimetic modification of cellulose surfaces and holds great potential for industrial applications. Thus, it requires that the XET-enzyme can be produced in larger scale.</p><p>This work also shows how the wildtype <i>Ptt</i>XET16-34 was modified into a glycosynthase. By mutation of the catalytic nucleophile into an alanine, glycine or serine residue, enzymes capable of synthesising defined xyloglucan fragments were obtained. These defined compounds are very valuable for further detailed studies of xyloglucan active-enzymes, but are also useful in molecular studies of the structurally important xyloglucan-cellulose interaction.</p><p>A heterologous production system for <i>Ptt</i>XET16-34 was previously developed in the methylotrophic yeast Pichia pastoris. A methanol-limited fed-batch process was also previously established, but the yield of active XET was low due to proteolysis problems and low productivity. Therefore, two alternative fed-batch techniques were investigated for the production of <i>Ptt</i>XET16-34: a temperature-limited fed-batch (TLFB) and an oxygen-limited high-pressure fed-batch (OLHPFB).</p><p>For the initial recovery of XET after the fermentation process, two different downstream processes were investigated: expanded bed adsorption (EBA) and cross-flow filtration (CFF).</p>

Thesis (M.Tech.: Food Technology)-Dept. of Biotechnology, Durban University of Technology, 2007 xv, 122 leaves / Several herbs are well known for their nutritional applications. The reported study tested some herbs for their efficacy as antioxidants. The herbs and combinations thereof were analyzed for antioxidant activity using two comparative methods: the beta-carotene bleaching method and through measurement by the Rancimat apparatus. The efficacy of the antioxidants was also tested in a cosmetic base cream. The concentration of the natural antioxidant vitamins, such as vitamin C, vitamin E and beta-carotene was determined by analytical methods. The methods used for analysis provided adequate results for interpretation. All herbs exhibited antioxidant activity, at comparable levels. The two methods used for analysis showed variable results, as previous researchers have shown. In terms of antioxidant potency, the herbs were ranked as follows: fenugreek > coriander > oregano > sage (beta-carotene bleaching method), oregano > sage > coriander > fenugreek (Rancimat method). Oregano and sage were good synergists, when combinations of herbs were used. Fenugreek, unlike the other herbs under investigation, is a prooxidant when used at higher concentrations. A direct relationship was observed between the concentration of herbs and antioxidant potency for sage, oregano and coriander. Fenugreek which is prooxidant at higher concentrations, should be used as an antioxidant independently rather than a synergist. This was the most important finding in the reported study. Similar antioxidant activities of the herbs were observed in the base cosmetic formulation. The reported study has provided an adequate base for further quantitative research into the innovative topic of antioxidants.

Diss. (sammanfattning) Uppsala : Sveriges lantbruksuniv. / Härtill 4 uppsatser.