No description available.

Univ., Diss., 2010--Marburg.

Thèse de doctorat : Sciences agronomiques : INPL : 2008. / Titre provenant de l'écran-titre.

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.

Hairy roots are a syndrome of the plant pathogen Agrobacterium rhizogenes, which induces the aggressive growth of roots in the plants it infects. Hairy roots are shown to have increased production of secondary metabolites when compared to roots that are not transformed, especially when they are exposed to plant signaling hormones called elicitors. Two popular elicitors are Methyl Jasmonate (MeJA) and Salicylic Acid (SA), which are also potent plant signaling compounds involved in plant defense and immunity. Many studies have reported on the secondary metabolites of hairy roots and their production of metabolites after exposure to elicitors. However, there is a gap in current knowledge of how hairy roots and non-transformed roots of Helianthus annuus (the common sunflower) compare in their secondary metabolite profiles, which include the volatile terpenoids they produce. This experiment used solid-phase microextraction gas chromatography-mass spectrometry (SPME GC-MS) to compare the volatile terpenoid profiles of hairy roots and non-transformed roots of H. annuus after their exposure to 0.2 mM MeJA and MeSA, the methyl ester of SA. The experiment identified several differences in the production of volatile compounds across elicitor treatments and time points yet hairy roots largely maintained the properties of their native counterparts. This project provides information on the secondary metabolism and volatile terpenoid profiles of hairy roots and explores the biotechnological applications of such insights.

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

Universiẗat, Diss--Karlsruhe, 2002.

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).

Submitted by Reginaldo Soares de Freitas (reginaldo.freitas@ufv.br) on 2017-04-24T12:12:21Z No. of bitstreams: 1 texto completo.pdf: 1281900 bytes, checksum: 4472f78480864646e3d483fe0dffb30a (MD5) / Made available in DSpace on 2017-04-24T12:12:21Z (GMT). No. of bitstreams: 1 texto completo.pdf: 1281900 bytes, checksum: 4472f78480864646e3d483fe0dffb30a (MD5) Previous issue date: 1999-12-15 / Conselho Nacional de Desenvolvimento Científico e Tecnológico / Foram realizados dois trabalhos no laboratório de testes de progênie do Departamento de Fitotecnia da Universidade Federal de Viçosa, em Viçosa-MG. Um com a finalidade de desenvolver um protocolo de transformação de plantas de mandioca (Manihot esculenta Crantz) via Agrobacterium rhizogenes resistente à canamicina, e o outro para avaliar o efeito de fotoperíodo, filtros coloridos, nitrato de amônio, 2,4-D, picloram, ABA, AgNO 3 e STS na micropropagação de plântulas de mandioca. Na transformação da mandioca foram testados: a) meio seletivo com canamicina (0,0; 100; 200; 300; 400; 500; 600; 700; 800; 900; e 1.000 mg/L), b) influência da idade e luz nos discos foliares co-cultivados com a bactéria, c) efeito do ANA (0,0; 0,1; 1,0; 2,0; 3,0; 4,0; e 5,0 mg/L) no co- cultivo, d) efeito de concentrações de BAP (1,0; 10,0; 50; e 100,0 mg/L), e) efeito do GA 3 (0,05) + BAP (2,0 mg/L) e picloram (8,0 mg/L). Foi também realizada a análise isozimática do material. Avaliaram-se: peso da matéria seca, número de folhas e raízes, comprimento da parte aérea, presença de injúrias e coloração de folhas e caule. A partir de 100 mg/L de canamicina, as plântulas apresentaram fitotoxidez; folhas novas e no claro apresentam os melhores resultados; o melhor resultado com ANA foi obtido a 2,0 mg/L; calos transformados apresentaram maior peso de matéria seca com 10 mg/L de BAP e com picloram; na análise isozimática constatou-se diferença no padrão de bandas. Na micropropagação, avaliaram-se as mesmas características dos experimentos de transformação; nos experimentos com STS e AgNO 3 foram avaliados também o teor de clorofila, e nos de nitrato de amônio, fotoperíodo, filtros coloridos e ABA foi feita também uma análise mineral do material vegetal. Avaliou-se o efeito do nitrato de amônio (0; 1/8; 1/4; 1/2; 3/4; 1; e 2 da concentração original) com e sem BAP. Estudou-se o efeito dos fotoperíodos: 0; 8 e 16 h luz. Quanto ao efeito do STS e AgNO 3, as concentrações utilizadas foram: de STS (0, 20, 30 e 50 μM) e de AgNO 3 (0, 10, 30 e 50 μM). Foram avaliados os efeitos de 2,4-D (0; 4,4; e 8,8 mg/L) e picloram (0; 4,8; e 9,6 mg/L) no claro e no escuro. Verificou-se também o efeito de 2,4-D (0; 2,2; 4,4; 6,6; e 8,8 mg/L) e ABA (0,0; 1,0 e 10 mg/L). Para avaliar o efeito da qualidade de luz, as plântulas foram colocadas sob filtros de acrílico vermelho, amarelo e azul. Os resultados obtidos indicaram que, com ou sem o BAP, pode-se usar uma concentração de nitrato de amônio abaixo da normalmente utilizada; o 2,4-D teve efeito melhor na concentração de 4,4 mg/L no escuro; e o picloram não induziu nenhum tipo de mudança morfogênica. O ABA impediu o crescimento da mandioca. O filtro amarelo foi o mais indicado para um estudo na área de enraizamento. O fotoperíodo age diferente conforme, o cultivar. Tanto para o AgNO 3 como para o STS a resposta da mandioca não foi benéfica. / There were two researches done in the progenie test laboratory at the Plant Technology Department at the Federal University of Viçosa, in Viçosa-MG. One of them had as its aim to develop a manioc plant transformation protocol (Manihot esculenta Crantz) by means of Agrobacterium rhizogenes which is resistant to kanamicine, and the other was to evaluate the effect of the photoperiod, colour filters, ammonium nitrate; 2,4-D, picloram, ABA, AgNO 3 and STS in the small plant of cassava. The following were tested during the cassava transformation: a) selective means with kanamicine (0,0; 100; 200; 300; 400; 500; 600; 700; 800; 900; and 1.000 mg/L), b) the influence of age and light over the leaf disks which were cultivated in bacteria, c) the effect of ANA (0,0; 0,1; 1,0; 2,0; 3,0; 4,0; and 5,0 mg/L) in the co-cultivate, d) the effect of the BAP (1,0; 10,0; 50; and 100,0 mg/L) concentrations, e) the effect of the GA3 (0,05) + BAP (2,0 mg/L) and picloram (8,0 mg/L). There was also a isozimatic analysis of the material. The following were evaluated: dry matter weight, the number of leaves and roots, the length of the aerial part, the presence of damage and the coloring of the leaves and stem. With 100 mg/L of kanamicine, the small plants presented phitotoxidity; the new leaves and under light present better results; the best result with ANA was obtained with 2,0 mg/L; the calluses that were formed presented heavier weight, 10 mg/L of BAP, within the dry matter and with picloram; there was a difference with the band pattern in the isozimatic analysis. The same characteristics of the transformation experiments were evaluated; the chlorophyll content was evaluated in the experiments that had STS and AgNO 3, and there was also a mineral analysis of the vegetal material done for the photoperiod, colored filters and ABA. The effect of the ammonium nitrate (0; 1/8; 1/4; 1/2; 3/4; 1; and 2 from the original concentration) with and without BAP was evaluated. The effects of the photoperiods of light were studied : 0; 8 and 16 light hours. For the effect of the STS and the AgNO 3, the concentrations that were used were: STS (0, 20, 30 e 50 μM) and AgNO 3 (0, 10, 30 and 50 μM). The effects of the 2,4-D (0; 4,4; and 8,8 mg/L) and picloram (0; 4,8; and 9,6 mg/L) in the light and in the dark were evaluated. The effect of 2,4-D (0; 2,2; 4,4; 6,6; and 8,8 mg/L) and ABA (0,0; 1,0 and 10 mg/L) was verified. To evaluate the light quality effect, the small plants were put under red, yellow and blue acrylic filter. The results that were obtained indicated that, with or without BAP, a ammonium nitrate concentration can be used below the usual amount that is used; the 2,4-D had a better effect in the concentration of 4,4 mg/L in the dark; and the picloram did not induce any type of morphogenic change. The ABA inhibited the growth of the manioc. The yellow filer was more indicated for a study in the rooting area. The photoperiod works differently according the cultivar. Either for the AgNO 3 or for the STS the return from the cassava was not beneficial.

This project is focused on transformation of Silene vulgaris mediated by Agrobacterium tumefaciens and A. rhizogenes. S. vulgaris is a good model plant to study gynodioecy, an evolutionary step from bisexuality to dioecy. Gynodioecious plants form in some individuals bisexual flowers, while the others possess only female flowers. The aim of this research is do develop a technique to introduce foreign genes into this plant to study its developmental consequences. Using A. rhizogenes we successfuly prepared hairy root cultures, which unfortunately do not form shoot regenerants. We have prepared a protocol to induce plant regenerants from S. vulgaris leaf fragments. The first results do not confirm that A. tumefaciens infected plant regenerants harbor reporter transgenes. We used Nicotiana tabacum as a positive control.