Thesis (PHD)--University of Montana, 2009. / Contents viewed on April 30, 2010. Title from author supplied metadata. Includes bibliographical references.

Plant cell and tissue cultivations are of growing interest for the production of structurally complex and expensive plant-derived products, especially in pharmaceutical production. Problems with up-scaling, low yields and high-priced process conditions result in an increased demand for models to provide comprehension, simulation, and optimization of production processes. In the last 25 years, many models have evolved in plant biotechnology; the majority of them are specialized models for a few selected products or nutritional conditions. In this article we review, delineate, and discuss the concepts and characteristics of the most commonly used models. Therefore, the authors focus on models for plant suspension and submerged hairy root cultures. The article includes a short overview of modeling and mathematics and integrated parameters, as well as the application scope for each model. The review is meant to help researchers better understand and utilize the numerous models published for plant cultures, and to select the most suitable model for their purposes.

Formation of segments during vertebrate embryogenesis is regulated by a biological clock. Models and experimental data indicate that the core of this clock consists of a cell- autonomous single cell oscillator. This oscillator likely involves a genetic feedback loop of transcriptional repressors belonging to the hairy gene family. In zebrafish, three her genes, her1, hes6 and her7, have been identified as core oscillator components. The main purpose of this project was to study the molecular mechanism of the hairy gene negative feedback oscillator in single cells. To determine whether a single cell oscillator is part of the zebrafish segmentation clock, a cell dissociation protocol was established to track the expression of Her1 ex vivo. Upon dissociation, Her1 expression continued to oscillate for up to three cycles. The period of oscillations was significantly slower than that of the segmentation clock, but appears to speed up in the presence of serum. To test whether the hairy gene interactions are sufficient to generate oscillations in single cells, a protocol was established that uses synthetic biology principles to design, construct and characterize hairy gene networks in yeast. First a library of network parts, containing hairy genes, promoters and Her binding sites was generated and subsequently assembled into simple devices to test their functionality in yeast. The three core oscillator components, Her1, Hes6 and Her7, were characterized and optimized for expression in yeast. In the SWITCH-OFF assay, the Her1 protein, modified with a MigED yeast repressor domain, was found to function as a transcriptional repressor in yeast, while Hes6 with the same modification can not. The dissociation of segmentation clock cells provides the first direct evidence that single cell oscillators exist in zebrafish. In this system, oscillator dynamics can be studied without the interactions of higher level clock components. In parallel, establishing a yeast chassis for hairy gene networks provides a novel technique to directly test predicted oscillator mechanisms by constructing them ’bottom up’.

In dieser Arbeit wird die Eignung eines Haarwurzelsystems (HR) aus transgenen Sonnenblumen-hairy roots zur biotechnologischen Produktion von α-Tocopherol untersucht. Es wurden hairy roots mit und ohne Transgene erzeugt. Transgene HR exprimieren Tocopherolbiosynthesegene aus Arabidopsis thaliana. Die HR wurden unterschiedlichen Stressoren unterworfen, um die α-Tocopherolproduktion binnen eines Zeitraums zu überprüfen. Stressoren waren verringerte Kohlenstoffquelle, Beleuchtung und der Zusatz von Zytokininen. Die Produktionsleistung des Systems ist schlussendlich ungenügend zur kostengünstigen Produktion von α-Tocopherol.

Formation of segments during vertebrate embryogenesis is regulated by a biological clock. Models and experimental data indicate that the core of this clock consists of a cell- autonomous single cell oscillator. This oscillator likely involves a genetic feedback loop of transcriptional repressors belonging to the hairy gene family. In zebrafish, three her genes, her1, hes6 and her7, have been identified as core oscillator components. The main purpose of this project was to study the molecular mechanism of the hairy gene negative feedback oscillator in single cells. To determine whether a single cell oscillator is part of the zebrafish segmentation clock, a cell dissociation protocol was established to track the expression of Her1 ex vivo. Upon dissociation, Her1 expression continued to oscillate for up to three cycles. The period of oscillations was significantly slower than that of the segmentation clock, but appears to speed up in the presence of serum. To test whether the hairy gene interactions are sufficient to generate oscillations in single cells, a protocol was established that uses synthetic biology principles to design, construct and characterize hairy gene networks in yeast. First a library of network parts, containing hairy genes, promoters and Her binding sites was generated and subsequently assembled into simple devices to test their functionality in yeast. The three core oscillator components, Her1, Hes6 and Her7, were characterized and optimized for expression in yeast. In the SWITCH-OFF assay, the Her1 protein, modified with a MigED yeast repressor domain, was found to function as a transcriptional repressor in yeast, while Hes6 with the same modification can not. The dissociation of segmentation clock cells provides the first direct evidence that single cell oscillators exist in zebrafish. In this system, oscillator dynamics can be studied without the interactions of higher level clock components. In parallel, establishing a yeast chassis for hairy gene networks provides a novel technique to directly test predicted oscillator mechanisms by constructing them ’bottom up’.

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.

Plant cell and tissue cultivations are of growing interest for the production of structurally complex and expensive plant-derived products, especially in pharmaceutical production. Problems with up-scaling, low yields and high-priced process conditions result in an increased demand for models to provide comprehension, simulation, and optimization of production processes. In the last 25 years, many models have evolved in plant biotechnology; the majority of them are specialized models for a few selected products or nutritional conditions. In this article we review, delineate, and discuss the concepts and characteristics of the most commonly used models. Therefore, the authors focus on models for plant suspension and submerged hairy root cultures. The article includes a short overview of modeling and mathematics and integrated parameters, as well as the application scope for each model. The review is meant to help researchers better understand and utilize the numerous models published for plant cultures, and to select the most suitable model for their purposes.

In dieser Arbeit wird die Eignung eines Haarwurzelsystems (HR) aus transgenen Sonnenblumen-hairy roots zur biotechnologischen Produktion von α-Tocopherol untersucht. Es wurden hairy roots mit und ohne Transgene erzeugt. Transgene HR exprimieren Tocopherolbiosynthesegene aus Arabidopsis thaliana. Die HR wurden unterschiedlichen Stressoren unterworfen, um die α-Tocopherolproduktion binnen eines Zeitraums zu überprüfen. Stressoren waren verringerte Kohlenstoffquelle, Beleuchtung und der Zusatz von Zytokininen. Die Produktionsleistung des Systems ist schlussendlich ungenügend zur kostengünstigen Produktion von α-Tocopherol.

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.

Arsenic is an element which belongs to metaloids. Contamination with arsenic is a problem all over the world. Basically it is a part of Earth's crust, but with anthropogenic activities it could overspread into soil, water and air in large scale a thus it could mean health hazard. Fytoremediation is kind of environment decontamination, which is quite effective and cheap as well. Publications about arsenic and its influence on plant metabolism are mostly focused on important crop plants like rice. Rice is mostly used for experiments and questions on anatomical and morphological changes are widely being solved by these experiments, but it has only insignificant relevance for fytoremediation. There are only few publications about arsenic influence on carbohydrate metabolism, thus little is known about this problem. That is why I have decided to study this topic more deeply and get more information about carbohydrate metabolic changes under influence of arsenic and partly also under influence of mercury, because information about influence of mercury are completely lacking. My experimental material includes tobacco plant, tobacco tissue cultures and horseradish hairy roots cultures. Accumulation of starch and soluble carbohydrate spectrum and content was determined by HPLC. Furthermore arsenic influence...