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In the present research, nicotine alkaloid production by Nicotiana tabacum (tobacco) hairy roots and tropane alkaloid production by Hyoscyamus niger hairy roots were investigated. The first objective of this research was to improve the oxygen mass transfer in hairy root cultures with microbubbles. Oxygen was shown as a critical nutrient for the growth of tobacco and H. niger hairy roots. In a 1-liter fermentor, microbubble dispersion improved the oxygen mass transfer, tobacco hairy root growth, and nicotine production in the medium. In a novel ground-joint column bioreactor, microbubbles enhanced the oxygen mass transfer and the growth of H. niger hairy roots. The second objective of this research was to enhance the release of alkaloids from the hairy roots into the culture medium. In a l-liter fermentor, nicotine concentration in medium was improved by adjusting the medium pH to 6. Unlike the nicotine alkaloid, hyoscyamine concentration in medium was not detectable at medium pH 6, whereas hyoscyamine in medium increased to 42 mg l-1 at medium pH 3. Similar to the hyoscyamine, scopolamine in medium increased from 0.1 to 11 mg l-1 when the medium pH was adjusted from 6 to 3. The release of alkaloids into culture medium provides opportunities to isolate a high-value alkaloid directly from the culture fluid, and reduces the cost of product recovery.

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.

Chemical and biological aspects of thiarubrine, a highly antifungal dithiacyclohexadiene polyine, were investigated. A tissue culture system for the production of thiarubrines was developed by culturing hairy roots of Chaenactis douglasii induced by Agrobacterium rhizogenes strain TR7. One culture line accumulated two times the levels of thiarubrines of nontransformed control root cultures, while maintaining rapid growth. The combination of fast growth and high thiarubrine accumulation could not be duplicated in controls by adding exogenous NAA to the culture medium. Hairy root cultures also produced less thiarubrine B relative to thiarubrine A compared to controls. Thiarubrine synthesis appears to be closely correlated with degree of tissue differentiation; it is suggested that it may be more practical to improve the growth rate of thiarubrine-producing root cultures by transformation rather than seek to induce synthesis in fast-growing suspension cultures. The biosynthetic relation between thiarubrines and the always co-occurring thiophenes was investigated by performing ³⁵S tracer experiments with C. douglasii hairy root cultures. It is possible that the thiophenes are not actively synthesized by the roots but rather are products of thiarubrine decomposition resulting from the extraction procedures and other manipulations of the cultures. The in vitro conversion of thiarubrine to thiophene can be induced by light, heat and other agents. No turnover of thiarubrines could be detected in the cultures in late logarithmic or stationary phases of the growth cycle. I Thiarubrines show strong light-independent antibacterial and antifungal activity. The mechanism of action of thiarubrine against E. coli and S. cerevisiae was investigated using comparative disk bioassays. A very similiar polyine from Rudbeckia hirta was as active as thiarubrine in the dark, indicating the central role of the disulfide ring in toxicity of the compounds. Visible light enhanced this activity suggesting that decomposition of the disulfide ring is important for its antibiotic effects. The photodegradation product, a thiophene, is phototoxic, probably via both type I and type II photosensitization mechanisms. The root culture extracts of Rudbeckia hirta yielded a new isomer of a known dithiacyclohexadiene polyine. MS and NMR analyses confirmed the cis configuration of this isomer. / Science, Faculty of / Botany, Department of / Graduate

Thesis (Ph. D.)--University of Wisconsin--Madison, 1935. / Typescript. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references (leaves 34-37).

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.

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.

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.

No description available.