Thèses sur le sujet « Hairi root cultures »

2013 - 2014
Hyperpigmentation is the process by which an excess of melanin is produced by the skin. Typically, hyperpigmentation occurs as a result of stress, damage or prolonged inflammation of the skin. The most common cause is sun damage, though hyperpigmentation is often a consequence of inflammation following acne, eczema, psoriasis, dermatitis etc. Hyperpigmentation may also occur in the skin due to hormonal changes in the body typically associated with pregnancy or the taking of oral contraception. Beside this medical aspects, the global skin depigmenting product market has been forecast to reach a value of $19.8 billion by 2018, driven by the growing desire for light-coloured skin among both men and women primarily from the Asian, African and Middle East regions. Although products do exist that can actually bleach the skin, these products contain dangerous or toxic ingredients (such as hydroquinone and mercury) and are banned in most countries. Blocking or reducing the accumulation of melanin in the skin can be obtained either by switching off one or more components of the pathway that go from the receptor activation to the enzymatic inhibition of melanin formation catalyzed by the tyrosinase. For this purpose, several antimelanogenic reagents have been developed and discovered nowadays. However, only a few of these inhibitors have been introduced and used due to their problems in cytotoxicity (affecting the cell growth and survival), selectivity, solubility and stability. The present project was aimed at identifying new total plant extracts exerting beneficial effects in skin care, with special emphasis on the development of novel plant-derived actives with hypopigmenting effects. Experimental activities were carried out in collaboration with Arterra Bioscience S.r.l, in the frame of the programme “Dottorato di Ricerca in Azienda”, funded by European Commission and Regione Campania (POR Campania FSE 2007-2013). Arterra is an Italian research-based Biotech company mostly involved in developing new plant-derived extracts to be used as active ingredients with cosmetic application. Hairy root cultures of three different plant species (Cichorium intybus, Brassica rapa subsp. pekinensis and Helianthus annuus) were generated. Hairy roots of Brassica rapa subsp. pekinensis were selected for further studies on the base of a preliminary screening for anti-oxidant activity of a total crude ethanol extract and a sugar/peptides mixture derived from cell walls, coupled to an active growth. Crude ethanol extract and a sugar/peptides mixture derived from cell wall of Brassica rapa subsp pekinensis hairy roots were tested in murine melanoma cells (B16-F1) and human epidermal melanocytes isolated from lightly pigmented adult skin (HEMa-LP), by using a panel of in vitro and in vivo biological assays to assess their role in modulating melanogenesis. Both extracts at different concentrations demonstrated to inhibit the cellular tyrosinase, a key enzyme in melanin production, and to reduce melanin content in murine melanoma cells. In addition, the sugar/peptides mixture of Brassica rapa susp. pekinensis hairy roots significantly inhibited the levels of cyclic adenosine monophosphate (cAMP), an important second messenger within melanogenesis signalling pathway. Furthermore, the same extract significantly decreased the expression of microphtalmia-associated transcription factor (MITF) and its promoter activity of about 30%, analyzed by in vitro reporter (luc+)-assay. Altogether these data indicates that the sugar/peptides mixture isolated from cell wall of Brassica rapa subsp. pekinensis hairy roots might exert its inhibitory effect on melanogenesis through the downregulation of MITF transcription. Furthermore, Brassica rapa subsp. pekinensis ethanol extract was able to enhance the expression levels of important genes encoding for proteins involved into extracellular matrix (ECM) assembly. Finally, a competitive industrial production hairy-root based platform was developed by Brassica rapa subsp. pekinensis hairy root biomass scaling-up and improved extraction procedures. Overall, these results, under pending patent application, will contribute to introduce product and process innovations at Arterra Bioscience s.r.l, for the identification of new and safer plant-derived melanogenesis inhibitors. In general, the developed industrial production platform will be also extended to the screening of actives from other plant species and to the release of novel plant-derived products in different segments of the cosmetic market. [edited by author]
XIII n.s.

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.

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.

Thesis (MSc)--Stellenbosch University, 2012.
ENGLISH ABSTRACT: Bacterial and fungal pathogens have developed numerous defence mechanisms against antimicrobial chemical agents, and resistance to old and new produced drugs are on the rise. Discovery of natural products derived from plants with diverse chemical structures and novel mechanisms of action to treat these notorious pathogens is a priority. Biotechnology (discussed in Chapter 1) has much to offer as a pharmacological tool and in the general study of medicinal plants. The Genus Salvia (Lamiaceae) has gathered much interest as these plants manufacture a diverse range of secondary metabolites including flavonoids, tannins and terpenoids. Of particular interest are the terpenoids which are largely implicated in the efficacy of Salvia plants as traditional medicines contributing to their pharmacological actions (discussed in Chapter 2). Due to the importance of these plants as herbal remedies, in this study, biotechnological techniques such as tissue culture and Agrobacterium-mediated transformation were applied on Salvia runcinata L.f., a South African medicinal plant, in an attempt to enhance the metabolomic profile and its bioactivity. Like so many other sages, S. runcinata has been used in folk medicine to treat a variety of ailments. Application of biotechnology was viewed as an important value adding platform for this species, assisting with its commercialisation for the cosmeceutical and pharmaceutical industries. Therefore the study had three foci: (1) to determine the seed germination behaviour and optimal conditions for micropropagation; (2) to develop a protocol that would be efficient whilst being simple for genetic transformation; and lastly, (3) to conduct phytochemical studies on in vitro generated S. runcinata transgenic hairy root and in vitro organ cultures by comparing these to glasshouse plants as potential therapeutic sources of natural compounds used in the treatment of infections in plants and humans. Data generated is thus summarised in three research chapters and Chapter 3 describes the formulated procedures assisting with in vitro seed germination and micropropagation of S. runcinata. The efficacy of smoke and scarification treatments for germination improvement was initially tested coupled to the evaluation of different hormonal combinations and different explant types which would aid with inducing adventitious shoot formation in vitro. The most effective germination treatment proved to be a 3 min exposure of seeds to 25% (w/v) H2SO4 combined with a concentration of 10-5 M smoke solution, resulting to more than 80% germination. Shoot proliferation was significantly higher using nodal explants with the addition of 4.43 μM BA. The protocol established in this part of the study is viable for large scale commercial production of S. runcinata as it would yield 1296 to 46656 viable plants in 4 to 6 months from one nodal explant. Micropropagation was applied also as a pre-emptive measure to ease pressure on the wild plants as the demand for S. runcinata is anticipated to increase due to its growing economic value as it is one of two South African sages with epi-α-bisabolol that is sought after by the pharmaceutical and cosmeceutical industries. This makes the protocol developed in this part of the study suitable for ex situ conservation of S. runcinata plantlets. Evaluations on the transgene transfer capacities of two different agropine strains (A4T and LBA 9402) of Agrobacterium rhizogenes to induce hairy root cultures of S. runcinata explants on nodal and leaf explants were conducted (reported in Chapter 4). Hairy roots formed 3 to 4 weeks after inoculation of the explants and these agropine strains showed different abilities for genetic transformation with the LBA 9402 strain producing significantly more roots on each explant compared to the A4T strain (P=0.0075). However, none of the LBA 9402 derived clones and only 2 clones generated through A4T transformation survived subculturing. The polymerase chain reaction (PCR) and reverse transcriptase-polymerase chain reaction (RT-PCR) confirmed the presence and transcription (respectively) of rol A, rol B, rol C and ags genes which are mobilised from the transfer-DNA (T-DNA) fragment of the root-inducing (Ri) plasmid of A. rhizogenes to the plant genome during transformation. The two A4T clones, termed here A4T3 and A4T5, were stably transformed, Southern blot analysis using rol A as a probe further validated the integration of one copy of the rol A gene. Transformed hairy roots, untransformed roots from tissue cultured plants, tissue culture-derived plants and glasshouse-grown plants were profiled for secondary metabolites by thin layer chromatography (TLC) and gas chromatography-mass spectrometry (GC-MS) in Chapter 5. In this part of the study, it is clear that the use of tissue culture as a propagation system did not negatively affect the volatile compound profile of S. runcinata and plants had a similar essential oil content to that reported by Kamatou et al. (2008), leading to a conclusion that in vitro plants maintained their biochemical integrity even under an alternative micro-controlled environment. Similarly to others, Ri-transformation was explored as an avenue to alter secondary metabolism creating inter-clonal variation. Transformed clones were distinguishable, displaying more of some primary metabolites including sucrose, galactose, sorbose and fructose than the leaf extracts. With the current GC-MS methods used, this clear distinction was not obvious at the secondary metabolite level. In general, solvent extracts (acetone and methanol:dichloromethane (MetOH: DCM) (1:1 v/v) exhibited good to moderate antibacterial activity with the minimum inhibitory concentration (MIC) values ranging from 0.39 to 0.78 mg ml-1. However, in vitro plant cultures were the most potent against two Gram-negative bacterial strains: Escherichia coli (ATCC 11775) and Klebsiella pneumoniae (ATCC 13883), and two Gram-positive bacterial strains: Bacillus subtilis (ATCC 6051) and Staphylococcus aureus (ATCC 12600). The hairy root extracts did not show any activity against fungi, Fusarium subglutinans (MRC 0115) and Fusarium proliferatum (MRC 6908). Micropropagation therefore proves to be an interesting avenue for commercial production of S. runcinata, supplying plants with an improved pharmacological activity. Hence the biotechnological approach applied here is a viable strategy for the production of medicinal bioactives from S. runcinata.
AFRIKAANSE OPSOMMING: Bakterieë en fungi patogene het baie verskeie meganismes ontwikkel teen antimikrobiese chemiese agente, en weerstand teen ou en nuwe chemise stowwe is besig om te vergroot. Daarom is dit belangrik om natuurlike plantaardige produkte met diverse chemiese strukture en unieke werkings meganismes te ontdek waarmee hierdie berugte patogene beveg kan word. Biotegnologie (wat in Hoofstuk 1 bespreek word) kan gebruik word as 'n farmakologiese hulpmiddel in die algemene studie van plante. Die Klas (Genus) Salvia (Lamiaceae) het al baie aandag getrek aangesien hierdie plante 'n wye reeks sekondêre metaboliete vervaardig wat flavonoïede, tanniene en terpenoïede insluit. Veral van belang is die terpenoïde wat betrokke is by die doeltreffendheid van die Salvia plante as tradisionele medisyne, aangesien dit bydra tot hulle farmalogiese aksie (wat in Hoofstuk 2 bespreek word). Aangesien hierdie plante sulke belangrike kruie is, word daar in hierdie studie, biotegnologiese tegnieke soos die kweek van weefsel en Agrobacterium-bemiddelde transformasie op Salvia runcinata L.f. toegepas om die metabologiese profiel en die bioaktiwiteit daarvan te verbeter. Soos baie van die salies is S. runcinata tradisioneel dikwels gebruik om allerhande siektetoestande te behandel. Die toepassing van biotegnologie word beskou as 'n belangrike manier om waarde by te voeg sodat hierdie plant kommersieei deur die kosmetiese en farmakeutiese bedrywe gebruik kan word. Daarom is daar op drie dinge gefokus: (1) die ontkiemings gedrag van saad en die optimale toestande vir mikrovoortplanting (2) die ontwikkeling van protokol wat eenvoudig maar doeltreffend is vir genetiese transformasie, en die (3) fito-chemise studies op in vitro genereerde S. runcinata transgeniese harige wortels en in vitro orgaan kwekings deur om hulle te vergelyk met kweekhuis plante as potentiële terapeutiese bronne van natuurlike samestellings vir die behandeling van infeksies in beide plante en mense. Die data wat gegenereer is, is opgesom in drie hoofstukke, en in Hoofstuk 3 word die prosedures wat gebruik word in die in vitro saad ontkieming en die mikro voortplanting van S. runcinata, bespreek. Die doeltreffendheid van rook en skarifikasie behandeling vir die verbetering van ontkieming is eers getoets en gekoppel aan die evaluering van verskillende hormoonkombinasies en verskillende eksplant tipes wat lei tot die formasie van uitloopsels in vitro. Daar is gevind dat die effektiefste behandeling vir ontkieming, 'n 3-minuut blootstelling van saad aan 25% (w/v) H2SO4 gekombineer met 'n konsentrasie 10-5 M rook oplossing is. Dit het gelei tot meer as 80% ontkieming. Daar was baie meer uitloopsels toe nodale eksplante gebruik is met die byvoeging van 4.43 μM BA. Die proktokol wat hier gevestig is, kan op groot skaal gebruik word vir die kommersiële produksie van S. runcinata, want 1296 tot 46656 lewensvatbare plante kan binne 4 ot 6 maande van een nodale eksplant gemaak word. Mikro voortplanting is toegepas as 'n voorkomende maatreel om die druk op die natuur te verminder omdat daar verwag word dat die vraag na S. runcinata sal toeneem na gelang die groeiende ekonomiese waarde daarvan toeneem. Dit is een van twee Suid-Afrikaanse salies met epi-α-bisabolol wat deur die farmakeutiese en die kosmetiese bedrywe gebruik word. Dit beteken dat die protokol wat hier ontwikkel is, geskik is vir die ex situ bewaring van S. runcinata plante. Die transgeen oordrag van twee verskillende agropien tipes (A4T and LBA 9402) van Agrobacterium rhizogenes is geevalueer (en in Hoofstuk 4 beskryf). Harige wortels het 3 tot 4 weke na die inenting van die eksplante gevorm en hierdie agropien tipes het verskillende vermoëns vir genetiese transformasie getoon, met die LBA 9402 tipe wat baie meer wortels op elke eksplant voorgebring het in vergelyking met die A4T tipe (P=0.03116). Geen van die LBA 9402-afgeleide klone en slegs 2 klone wat deur A4T transformasie genereer is, het oorleef. The polimerase ketting reaksie (PCR) en die teenoorgestelde trenskriptasie-polimerase (RT-PCR) ketting reaksie het die teenwoordigheid en transkipsie (onderskeidelik) van rol A, rol B en rol C en ags gene, wat oorgedra word deur die oordrag DNA (T-DNA) fragment van die wortel induserende (Ri) plasmied van A. rhizogenes na die plant genoom tydens transformasie, bevorder. A4T klone, hier A4T3 and A4T5 genoem, is stabiel transformeer. Southern blot ontleding het met die gebruik van rol A, die integrasie van een kopie van die rol A geen, bevestig. In Hoofstuk 5 is transformeerde harige wortels, ongetransformeerde wortels van weefsel gekweekte plante, weefsel gekweekte plante, en kweekhuis plante deur dun-laag chromatografie (TLC) en gas-chromatografie-massa spektrometrie (GC-MS) geprofiel vir sekondêre metaboliete. In hierdie deel van die studie is dit duidelik dat die gebruik van weefsel kwekery as 'n voortplantsisteem nie 'n negatiewe effek gehad het op die vlugtige samestelling profiel van S. runcinata nie en dat plante 'n sootgelyke essentiële olie inhoud het as wat deur Kamatou et al. (2008) bevind is. Dit lei tot die gevolgtrekking dat in vitro plante hulle biochemiese integriteit behou selfs onder alternatiewe mikro-beheerde omgewings. Ri-transformasie is ondersoek as 'n manier om sekondêre metabolisme te verander om interkloon variasie te skep. Getransformeerde klone kon uitgeken word, aangesien dit meer primêre metaboliete soos sukrose, galaktose en fruktose insluit as die blaar ekstrakte. Hierdie verskil was nie met die huidige GC-MS metodes so duidelik sigbaar op die sekondêre metabolitiese vlak nie. Oor die algemeen toon ekstraksie met asetoon en methanol dichlorometaan (MetOH: DCM) (1:1 v/v) goeie tot gemiddelde antibakteriese aktiwiteit met die minimum remmende konsentrasie (MIC) waardes van 0.39 tot 0.78 mg ml-1. Die in vitro plant kulture het egter sterker weerstand gebied teen twee Gram-negatiewe bakteriese tipes: Escherichia coli (ATCC 11775) en Klebsiella pneumoniae (ATCC 13883), en teen twee Gram-positiewe bakteriese tipes: Bacillus subtilis (ATCC 6051) en Staphylococcus aureus (ATCC 12600). Die harige wortel ekstrakte het geen aktiwiteit teen die swamme, Fusarium subglutinans (MRC 0115) en Fusarium proliferatum (MRC 6908) getoon nie. Mikro-voortplanting is dus 'n interessante manier om S. runcinata kommersieel te produseer aangeien die plante verbeterde farmalogiese aktiwiteit toon. Die biotegnologiese benadering wat hier toegepas word, is 'n praktiese strategie vir die produksie van geneesmiddels van S. runcinata.

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.

Catharanthus roseus (l. . ) G. Don synthétise des alcaloïdes indoles monoterpéniques dimères à activité anti-néoplastique, la vinblastine et la vincristine, utilisés en chimiothérapie cancéreuse. Ces alcaloïdes, faiblement accumulés in planta, sont obtenus par couplage in vitro de deux monomères précurseurs, la vindoline et la catharanthine. Les cultures in vitro de hairy roots ou "chevelus de racines", génétiquement et biochimiquement stable, peuvent produire des alcaloïdes à des niveaux égaux ou supérieurs à ceux trouvés chez la plante entière. Une méthode puissante et rapide de transformation génétique de feuilles par Agrobacterium rhizogenes, non destructive pour la plante, à partir de différentes espèces Catharanthus, nous a permis de générer des clones de hairy roots et de les sélectionner pour leur production en catharantine. Parallèlement, des paramètres de cultures susceptibles d'augmenter la biosynthèse de catharanthine ainsi que sa sécrétion dans le milieu ont été recherchés
Catharanthus roseus (L. ) G. Don biosynthesises terpenoid indole alkaloids whose dimeric form, vinblastine and vincristine, posses anti-neoplastic activity conducing to their using in anti-cancer chemotherapies. Currently such compounds, founded at low levels from whole plant, were obtained by semi synthesis from monomeric precursors, vindoline and cathranthine. In vitro cultures of hairy roots or "chevelus de racines", cause of genetical and biochemical stability, could produce alkaloids to equal or more important level to these found in the whole plant. Establishment of an efficient, fast and non destructive for the plant method to genetic transformation of leaves with Agrobactrium rhizogenes have permitted to generate hairy root clones from different Catharanthus species and to select these clones for their catharanthine production. Parallel, we have searched for culture parameters able to strongly increasing catharanthine biosynthesis as well as this secretion in medium

Salvia sensu lato includes more than about 9000 species and is considered one of the largest taxa of the Lamiaceae (Will et al., 2015). For a long time, Salvia species were known for a wide variety of medicinal used in folk medicine for the relief of pain, protecting the body against oxidative stress, free radical damages, angiogenesis, inflammation, bacterial and virus infection, etc… (Hamidpour et al., 2014). In vitro cultures have been considered as an alternative agricultural processes for producing secondary metabolites (Y. Kim et al., 2002). The research activity of this PhD project was aimed to the application of in vitro biotechnology techniques applied to aromatic plants for the production of bioactive compounds. The selected plants are species of Salvia showing an important antibacterial activity. The investigation study was carried out on S. corrugata and S. tingitana. Salvia corrugata Vahl. is an ornamental plant that grown easily along the Mediterranean coast. The aerial part is rich in terpenoid compounds like the diterpene quinones fruticuline A and demethylfruticuline A that present high antibacterial activity. Protocols of tissue culture and genetic transformation were set up with the aim to obtain in vitro shoot and hairy root biomass to grown in controlled condition for metabolite extraction. Two strains of Agrobacterium rhizogenes (wild type ATCC 15834 and hypervirulent LBA9402) were tested for their ability to induce hairy root on wounded leaves. The best response (75 %) was achieved by infection with ATCC 15834 about thirty days after the infection onto the hormone-free MS basal solid medium. Two hairy-root lines from ATCC 15834 treatment and one from LBA 9402 treatment were established. Transformation of selected clones was confirmed by polymerase chain reaction analysis of bacterial rolC and virC1 genes. The growth evaluation in TIS RITA® bioreactor put in evidence the best cultural conditions for the biomass production; the clone SCO-HR-FA8 had the best increase on Murashige and Skoog (1962) and ½ Woody plant medium (Lloyd et al., 1980) salt compositions in comparison to other tested media. 30 mg/L was the best sucrose concentration that guaranteed the highest biomass production. The growth curve of HR lie SCO-HR-FA8 was elaborated and then, three classes of elicitors and their combination were tested: a heavy metal ions (Ag+), the yeast extract and plant response-signaling compound (methyl jasmonate MJ). Among them, Ag+ and yeast extract (YE) at high concentration were most effective to stimulate the biomass production. The scale up of the biomass was performed using the bioreactor RITA®. The methanolic extracts of the biomass (16.8 g) was fractionated by Si gel MPLC to obtain 16 fractions. The methanolic extract and the semi-purified fractions were tested against several multidrug resistant clinical strains of various bacterial species: Staphylococci and Enterococci and E. coli. The total extract was poorly effective while the semi purified fractions displayed variable potency with MIC values ranging from 8 to >128 μg/mL and from 4 to >128 μg/mL respectively against the Staphylococci and Enterococci strains considered. Our results suggest that the application of biotechnology approach on S. corrugata, it is possible to induce hairy roots from leaf, optimize the condition to increase the production of biomass, scale-up using TIS bioreactor and produce secondary metabolites with antibacterial activity. The study carried out on S. tingitana aimed to develop an in vitro culture protocol to produce different plant tissues and investigate the antibacterial activity of the extract. The callus formation from sterilized leaves was evaluated on MS medium added with different PGRs in presence of ascorbic acid in light or in dark conditions. The result showed the importance of the presence 2,4-D and darkness for callus development. The high development percentage (94.4 %) was achieved by combining KIN and 2,4-D at the concentration of 0.5:0.5 or 1:1 mg/L respectively. The optimization of the medium for the callus growth was determined evaluating the type and concentration of cytokinin and auxin in dark. The suitable condition for callus proliferation was MS medium supplemented with 2,4-D 4.52 μM, KIN 2,32 μM and 10 mg /L of ascorbic acid. The elicitation with MJ or light intensity was investigated. The methanolic extract and fractions obtained by MPLC were inactive against Staphylococci species and E. coli. The fractions 11 and 12 displayed an antibacterial activity only against E. faecalis and E. faecium with MIC values ranging from 32 to 64μg/mL. In S. tingitana, a successful protocol for callus production was established; this in vitro culture system can be used as good method to obtain medicinally-useful secondary compounds from S. tingitana.

Plants are used to produce many secondary metabolites that are too difficult, expensive or impossible to make by chemical synthesis. Conventional cultivation of plants is of course subject to vagaries of weather, pests and availability of land; hence, the interest in highly controlled culture of plant cells and hairy roots in bioreactors as methods of producing various products. This project focussed on production of blue and red colors of Lavandula augustifolia and Beta vulgaris, respectively. Callus and suspension cell culture were successfully produced from L. augustifolia after extensive trials, but hairy roots could not be generated from this species. In contrast, a successful protocol was developed for consistently producing hairy roots from B. vulgaris, but calli could not be generated from this species. Effects of medium composition on growth of L. augustifolia calli and freely suspended cells and production of the blue pigment by the latter, were investigated. Optimal production of callus occurred in full-strength Murashige and Skoog (MS) medium supplemented with 2 mg/l of indole-3-acetic acid (IAA) and 1 mg/l of kinetin. Stable suspension cultures could be produced and maintained in full-strength MS medium supplemented with 1 mg/l each of IAA and kinetin. In suspension culture in full-strength MS medium, the following hormone combinations were tested: (1) 1 mg/l each of indole-3-acetic acid (IAA) and kinetin; (2) 2 mg/l of IAA and 1 mg/l of kinetin; (3) 2 mg/l of IAA and 1 mg/l of benzyl amino purine (BAP); and (4) 2 mg/l each of IAA and BAP. Combination (3) maximized cell growth, but the highest cell-specific production of the blue pigment was seen in combination (2), although pigment production occurred at all hormone combinations. The medium formulation that gave the best production of the pigment in shake flasks was scaled up to a 2 L aerated stirred tank bioreactor, but both the biomass and pigment productivities were reduced in the bioreactor apparently due to the high shear stress generated by the Rushton turbine impeller. Compared to suspension cultures of L. augustifolia, the hairy root cultures of B. vulgaris grew extremely rapidly. Hairy roots also produced large amounts of the red pigments. Growth of hairy roots was influenced by the composition of the medium. Although the full strength MS medium better promoted biomass growth compared to the half-strength MS medium, the final concentration of the biomass and the pigment were nearly the same in both media. Attempts were made to enhance production by using various hormones (i.e. naphthalene acetic acid, BAP, IAA added individually at a concentration of 0.5 mg/l), but none of the hormones proved useful. BAP adversely affected the growth of hairy roots. In summary, production of pigments by suspension culture of L. augustifolia and hairy root culture of B. vulgaris, is technically possible, but requires substantial further optimization for enhancing productivity than has been possible in this project. iii

Secondary metabolism in plants produces numerous compounds with wide-ranging activities, including the antineoplastic compound taxol and related taxanes. The biotechnological production of taxol has so far been based on empirical studies. The aim of the present work has been to study how the factors that enhance taxane production affect the metabolic profiles and gene expression in productive cells. As a consequence of this work, new potential candidates have been obtained for unknown taxane biosynthetic genes, some bottle-neck steps of taxane biosynthesis (in vitro and in silico) have been identified and a master regulator, not only for taxane biosynthesis, but also for other secondary metabolism routes, has been characterized. Coronatine, a powerful and less harmful elicitor than methyl jasmonate, has been successfully assayed and found to increase taxane production. In the different studies of this work, the expression level of genes that participate in taxol biosynthesis has been determined, clarifying their involvement in the production of this anti-cancer agent.
El metabolismo secundario de las plantas produce numerosos compuestos con un amplio rango de actividades, entre los que se encuentra el compuesto antineoplásico taxol y los taxanos relacionados, la producción biotecnológica del cual se basa en estudios empíricos. El objetivo de este trabajo ha sido estudiar como los factores que incrementan la producción de taxanos afectan los perfiles metabólicos y la expresión génica en los cultivos. De esta manera se han identificado nuevos genes candidatos que codifican para los genes desconocidos de la biosíntesis, algunos pasos limitantes de ésta y se ha caracterizado un regulador relacionado con el metabolismo secundario. Se ha ensayado la coronatina, un elicitor más eficiente para mejorar la producción de taxanos y menos dañino que el jasmonato de metilo. En los diferentes ensayos de este trabajo han sido determinados los niveles de expresión de genes que participan en la biosíntesis de taxol, ayudando a comprender su papel en la producción de este anticancerígeno.

This dissertation examines how Philippine (or Filipino) authors emphasise the need for articulating or “re/membering” cultural identity. The researcher mainly draws from the theory of Caribbean critic, Stuart Hall, who views cultural identity as an articulation which allows “the fragmented, decentred human agent” to be considered as one who is both “subject-ed” by power but/and one who is capable of acting against those powers (Grossberg 1996 [1986]: 157, emphasis mine). Applied to the Philippine context, this writer argues that, instead of viewing an apparent fragmented Filipino identity as a hindrance to “defining” cultural identity, she views the “damaged” (Fallows 1987) Filipino history as a the material itself which allows articulation of identity. Instead of reducing the cultural identity of a people to what-they-could-have-been-had-history-not-intervened, she puts forward a vision of identity which attempts to transfigure these “damages” through the efforts of coming-to-terms with history. While this point of view has already been shared by other critics (such as Feria 1991 or Dalisay 1998:145), the author’s contribution lies in presenting re/membering to describe a specific type of articulation which neither permits one to deny wounds of the past nor stagnate in them. Moreover, re/membering allows one to understand continuous re-articulations of “new” identities (due to current migration), while putting an “arbitrary closure” (Hall) to simplistic re-articulations which may only further the “lines of tendential forces” (such as black or brown skin bias) or hegemonic practices.

Written as such (with a slash),“re/membering” encapsulates the following three-fold meaning: (1) a “re-membering”, to indicate “a putting together of the dismembered past to make sense of the trauma of the present” (Bhabha 1994:63); as (2) a “re-membering” or a re-integration into a group and; as (3) “remembering” which implies possessing “memory or … set [ting] off in search of a memory” (Ricoeur 2004:4). As a morphological unit, “re/membering” designates, the ways in which Filipino authors try to articulate cultural identity through the routes of colonisation, migration and dictatorship.

The authors studied in this thesis include: Carlos Bulosan, Bienvenido Santos, N.V.M. Gonzalez, Nick Joaquin, Frank Sionil José, Ninotchka Rosca, Jessica Hagedorn, and Merlinda Bobis. Sixty-years separate Bulosan’s America is in the Heart (1943) from Hagedorn’s Dream Jungle (2003). Analysis of these works reveals how articulation is both difficult and hopeful. On the one hand, authors criticize the lack of efforts and seriousness towards articulation of cultural identity as re/membering (coming to terms with the past, fostering belonging and cultivating memory). Not only is re/membering challenged by double-consciousness (Du Bois 1994), dismemberment and forgetting, moreover, its necessity is likewise hard to recognize because of pain, trauma, phenomena of splitting, escapist attitudes and preferences for a “comfortable captivity”.

On the other hand, re/membering can also be described as hopeful by the way authors themselves make use of literature to articulate identity through research, dialogue, time, reconciliation and re-creation. Although painstaking and difficult, re/membering is important and necessary because what is at stake is an articulated Philippine cultural identity. However, who would be prepared to make the effort?

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Cette thèse démontre que, pour les auteurs philippins, l’articulation ou « re/membering » l'identité culturelle, est nécessaire. Le chercheur s'appuie principalement sur la théorie de Stuart Hall, qui perçoit l'identité culturelle comme une articulation qui permet de considérer l’homme assujetti capable aussi d'agir contre des pouvoirs (cf. Grossberg 1996 [1986]: 157). Appliquée au contexte philippin, cet auteur soutient que, au lieu de la visualisation d'une identité fragmentée apparente comme un obstacle à une « définition » de l'identité culturelle, elle regarde l’histoire philippine «abîmée» (Fallows 1987) comme le matériel même qui permet l'articulation d’identité. Au lieu de réduire l'identité culturelle d'un peuple à ce qu’ ils auraint pû être avant les interventions de l’histoire, elle met en avant une vision de l'identité qui cherche à transfigurer ces "dommages" par un travail d’acceptation avec l'histoire.

Bien que ce point de vue a déjà été partagé par d'autres critiques (tels que Feria 1991 ou Dalisay 1998:145), la contribution de l'auteur réside dans la présentation de « re/membering » pour décrire un type d'articulation sans refouler les plaies du passé, mais sans stagner en elles non plus. De plus, « re/membering » permet de comprendre de futures articulations de « nouvelles » identités culturelles (en raison de la migration en cours), tout en mettant une «fermeture arbitraire» (Hall) aux ré-articulations simplistes qui ne font que promouvoir des “lines of tendential forces” (Hall) (tels que des préjugés sur la couleur brune ou noire de peau) ou des pratiques hégémoniques.

Rédigé en tant que telle (avec /), « re/membering » comporte une triple signification: (1) une «re-membering », pour indiquer une mise ensemble d’un passé fragmenté pour donner un sens au traumatisme du présent (cf. Bhabha, 1994:63); (2) une «re-membering» ou une ré-intégration dans un groupe et finalement, comme (3)"remembering", qui suppose la possession de mémoire ou une recherche d'une mémoire »(Ricoeur 2004:4). Comme unité morphologique, « re/membering » désigne la manière dont les auteurs philippins tentent d'articuler l'identité culturelle à travers les routes de la colonisation, les migrations et la dictature.

Les auteurs inclus dans cette thèse sont: Carlos Bulosan, Bienvenido Santos, NVM Gonzalez, Nick Joaquin, Frank Sionil José, Ninotchka Rosca, Jessica Hagedorn, et Merlinda Bobis. Soixante ans séparent America is in the Heart (1943) du Bulosan et le Dream Jungle (2003) du Hagedorn. L'analyse de ces œuvres révèle la façon dont l'articulation est à la fois difficile et pleine d'espoir. D'une part, les auteurs critiquent le manque d'efforts envers l'articulation en tant que « re/membering » (confrontation avec le passé, reconnaissance de l'appartenance et cultivation de la mémoire). Non seulement est « re/membering » heurté par le double conscience (Du Bois 1994), le démembrement et l'oubli, en outre, sa nécessité est également difficile à reconnaître en raison de la douleur, les traumatismes, les phénomènes de scission, les attitudes et les préférences d'évasion pour une captivité "confortable" .

En même temps, « re/membering » peut également être décrit comme plein d'espoir par la façon dont les auteurs eux-mêmes utilisent la littérature pour articuler l'identité à travers la recherche, le dialogue, la durée, la réconciliation et la re-création. Bien que laborieux et difficile, « re/membering » est important et nécessaire car ce qui est en jeu, c'est une identité culturelle articulée des Philippines. Mais qui serait prêt à l'effort?

Doctorat en Langues et lettres
info:eu-repo/semantics/nonPublished

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.

碩士
中國醫藥大學
中國藥學暨中藥資源學系碩士班
101
Rehmannia glutinosa Libosch. (Scrophulariaceae), known as Di Huang, is widely used in Chinese medicine with hypoglycemic property. Its mainly biological active compounds are glycosides (catalpol, rehmanniosides A-D, verbascoside and stachyose). The activities of these compounds include anti-tumoral, anti-inflammatory, cancer-chemopreventive and antioxidant effects. In this study, in order to enhance the verbascoside content and biomass from hairy root culture of R. glutinosa, the effects of light and elicitors, including salicylic acid (SA), chitosan (CH), jasmonic acid (JA) and methyl jasmonate (MJ), have been investigated . The results show that the highest verbascoside content and production in R. glutinosa hairy root culture was on the 28th day during the culture. The highest content (4.08 mg/g) and production (61.64 mg/L) were achieved under light condition, and compared with the control group (dark culture) were 3.00 times and 3.13 times, respectively. It means that light is the role to stimulate verbascoside biosynthesis in R. glutinosa hairy root cultures. In addition, the R. glutinosa hairy roots were incubated in 10 L stirred tank bioreactors under light and dark conditions. The biomass for each condition were achieved 10.30 times and 5.15 times compared with the initial embedded weight, respectively. Moreover, The added methyl jasmonate (MJ, 20 mg/mL), salicylic acid (SA, 40 mg/mL) and chitinase (CH, 40 mg/mL) in the hairy root cultures of R. glutinosa were able to produce the highest verbascoside content (4.54, 6.85 and 2.15 mg/g ) and production ( 47.62, 47.27 and 27.71 mg/L ) than control one. Furthermore, different exposure time of methyl jasmonate (MJ, 20mg/mL) on hairy root cultures of R. glutinosa was examined. . The highest production of verbascoside (0.10 mg/L ) was achieved on the 14th day and reached to 1.65 times with the control group. In the meanwhile, the methanol extract of hairy root of R. glutinosa showed the radical scavenging activity on the 1,1-diphenyl-2-picrylhydrazyl (DPPH) bioassay. The radical scavenging activity of DPPH from the light cultured hairy root of R. glutinosa was higher than the dark cultured one with 1.45 times. This result may be caused by the production of verbascoside in the hairy root cultures of R. glutinosa. In addition, the methanol extract of hairy root of R. glutinosa showed the activity of DCs as well. The methanol extract of hairy root cultures of R. glutinosa with the treatment of MJ, SA and CH had activating effects on DCs. Contrary to activation, that the methanol extract from light condition and JA treatment had inhibitory effect on DCs. In conclusion, the light and elicitors can enhance the verbascoside content on the hairy root culture of R. glutinosa. Meanwhile, its methanol extract also had antioxidant activity and immunomodulatory effects, respectively.

碩士
國立臺灣大學
化學工程學研究所
91
The transformed hairy roots of the plants generally grow fast and are genetically stable. This is advantageous in productivity of the secondary metabolites. In this work the transformed Stizolobium hassjoo was cultivated in shake flask for producing L-DOPA. The correlations between “sugar consumption rate” and “conductivity decline rate“ with increase of biomass were established with which the on-line measurement of biomass can be realized. During the culture, conductivity, DO and pH were on-line monitored continuously. Sugar consumption rate was also measured. It was found that the change of conductivity and sugar consumption rate were inversely proportional to the hairy root mass. This kind of on-line measurement facilitated the indirect measurement of hairy root in a reactor. The growth behavior and productivity of L-DOPA using 3L Mist Trickling Reactor (MTR) were investigated. The drawback of browning of hairy roots in MTR during the later stage of cultivation was remedied by employing the intermittent feeding strategy. The optimum time intervals of on/off times for feeding mist medium were investigated. It was found that the 1hr on/1hr off for medium fed to the MTR resulted 25% increase in L-DOPA content. The productivity of total L-DOPA increased 11% compared to the control run. The supplementation of nitrogen source at the time the sugar consumption rate was slow down enhanced the root growth and sugar consumption rate remarkably.

碩士
國立嘉義大學
森林暨自然資源學系研究所
98
In this study, we inoculated paulownia (Paulownia fortunei Hemsl.) to produce hairy roots for aceteoside (As) production. Hairy root lines 7-6, 23-1 and 55-1 were first cultured for high-yield screening. Among them, 55-1 incubated on MS liquid medium (50 mL/explant) for 35 days achieved the highest yield (13.12±2.36 mg/explant). Subsequent examination using 55-1 as explants demonstrated that both MS and 1/2 MS media provided equal supporting for a better aceteoside production (p＜0.05). Medium supplemented with 6% (w/v) sucrose also achieved the best yield, and aceteoside content was reduced at lower sucrose concentration. Ethanol (EtOH) is a common solvent for preparation of various elicitors. This present study demonstrated that at high EtOH concentration (100 mL/L) the production of As from hairy root culture was reduced. The elicitor methyl jasmonate (MeJA) dissolved in a proper amount of ethanol and supplemented in the medium (with final concentration 0~50 μM ) did not promote As production during a 24 hr pulse treatment. A better combination of pulse duration and elicitor concentration is necessary to be further investigated.

Catharanthus roseus, a tropical plant, produces the valuable anti-cancer compounds, vincristine and vinblastine in extremely low amounts. My research objectives were to examine the response of plant secondary metabolism to various metabolic perturbations, and indicate limitations in the reaction network. In hairy roots, tabersonine is an important intermediate in the synthesis of vindoline, a monomer in the formation of the anti-cancer compounds that are generally absent from cell and hairy root cultures. To understand how much metabolic flux is directed toward the tabersonine branchpoint, transient profiles of lochnericine and horhammericine in relation to tabersonine in both dark and light-adapted cultures were quantified. The results demonstrated that the accumulation of lochnericine was growth related, similar to tabersonine, and that light repressed the formation of all three alkaloids. Using enzyme inhibitors, the involvement of separate P-450 mononoxygenase dependent enzymes in the biosynthesis of horhammericine and lochnericine was demonstrated. Furthermore, horhammericine and lochnericine were observed to be turned over. A search for rate limiting regions of the pathway was accomplished through precursor feeding studies. By identifying precursors, which after feeding, significantly enhance the production of alkaloids, the specific precursor branch that is limiting flux to alkaloids can be elucidated. Precursors fed from the terpenoid portion of the pathway at 21 days in the culture cycle were found to enhance the specific yield of tabersonine. This result suggests that during the early stationary phase period (21--24 days) flux limitations may occur upstream of geraniol. On the other hand, the rate-limiting pathway could not be identified during the late growth phase (17--21 days). In part, this was due to the fact that tryptophan served as a precursor of indole acetic acid (IAA), a plant growth regulator. Therefore, changes in indole alkaloid accumulation and root growth due to tryptophan feeding were similar to those induced by exogenously added IAA. Since feeding tryptamine or terpenoid precursors did not significantly enhance indole alkaloid accumulation the rate-limitation may be downstream of loganin. The metabolic flux distribution between separate alkaloid branches was quantified by monitoring the transient profiles of multiple alkaloids. The flux towards the Iboga alkaloids decreased while the flux to the Aspidosperma alkaloids increased between 12 and 26 days. In contrast, the flux distribution between the Corynanthe branch and the sum of the Iboga and Aspidosperma branches remained unchanged during this period. Despite significant changes in extracellular nutrient levels during this period, the total flux to the alkaloids remained constant between 12 and 26 days. Through the precursor feeding and biogenetic flux analysis studies, flux limitations to alkaloids likely exist in the terpenoid pathway leading to secologanin. With the tools developed to quantify metabolic flux through a simple model, metabolic flux in metabolically engineered plant cell and tissue cultures can be routinely analyzed.

博士
國立臺灣大學
微生物與生化學研究所
93
Gynostemma pentaphyllum (Thunb.) Makino, which belongs to the Cucurbitaceae family, is a perennial, deciduous, creeping herb. Phytochemical studies of G. pentaphyllum have identified roughly 90 dammarane-type saponin glycosides known as gypenosides which closely resembles the ginsenosides found in Panax ginseng. Hairy root cultures of G. pentaphyllum were established by infecting leaf discs with Agrobacterium rhizogenes ATCC 15834. Seven transformed root clones were established and examined for their growth rate and gypenoside productivity in various basal media. Clone GP-01 was selected from 7 hairy root clones based on its gypenosides productivity and stability. Kinetic studies revealed the dry biomass of the hairy roots in MS medium was 7.3 g L-1 and the gypenoside content was 3.8 % DW in a 49 days’ culture. The pattern of gypenoside accumulation was “growth–associated” with a peak value of 280 mg L-1. No gypenosides were released into the medium during the cultures. Elicitor can be used to improve the production of secondary metabolites in hairy root cultures because of its ability to induced phytoalexins in plant. To investigate the effects of different elicitors on gypenoside production in hairy root cultures of G. pentaphyllum. The results showed that elicitation was dependent on the elicitor type, concentration, the incubation period and the physiological stage at which the hairy root cultures were treated. Among these three elicitors, 200 μM MJ had the highest inducing efficiency at the late exponential growth stage. The gypenoside content of the hairy root cultures treated with MJ elicitor was 4.5% DW, which was 21% higher than the control (3.7% DW). For the enhancement of gypenoside production by G. pentaphyllum hairy roots, the effects of medium constituents on hairy root cultures were investigated in flasks. Based on these results, a step-wise medium shift strategy was developed in which GM (growth medium) was used for cultivation for the first 38 days and PM (production medium) for the remainder of the cultivation period. The gypenoside production obtained by the strategy was 586 mg L-1, which was 126% higher than those in the single medium (GM). Two areas that have been identified as key factors for large-scale implementation are: the development of suitable inoculation techniques and strategies to provide uniform root distribution within bioreactors. In this study, inoculation of large-scale hairy root culture reactors can be carried out by briefly homogenizing bulk root tissue, followed by aseptic transfer as a slurry to the reactor. Uniform root distribution can be achieved in bioreactors by entrapment of the growing root inoculum onto stainless steel mesh in gas-phase reactors operation. Hairy root cultures of G. pentaphyllum were cultivated in three different culture systems: a bubble column, a modified stirred tank (without impeller) and a stirred tank (with impeller). The growth rate and gypenoside contents of hairy root cultures in the bubble column and the modified stirred tank were higher than that in the stirred tank. The hairy root distribution in the bubble column and the modified stirred tank were also better than in the stirred tank. The roots grown in modified stirred tank reactor showed the best performance in terms of dry weight (7.1 g L-1) and gypenoside content (4.7 g L-1), the latter of which was better than that of shake-flask data. In this study high levels of gypenosides were obtained from hairy roots despite distribution results showing relatively low gypenoside levels in native roots of G. pentaphyllum. This study demonstrated that transformed roots can synthesize and store significant quantities of secondary metabolites. Although the hairy roots under these conditions produced approximately 10% to 30% less gypenosides than commercial sources of G. pentaphyllum, the growing time was much shorter when compared to field-grown plants. Therefore, in this study, we demonstrate the induction of hairy roots and the establishment of the culture followed by the infection with A. rhizogenes to grow more rapidly and to produce the gypenosides more efficiently than the ordinary field-grown cultivars. With hairy root cultures, product quality and quantity are easy to control because natural variances in seasonal climates and geographical environments are excluded and culture conditions and process variables are easily optimized. The hairy root cultures have been considered as a potential alternative for production of gypenosides.

碩士
中州科技大學
保健食品系
103
Red beetroot (Beta vulgaris) contents rich nutrients, bioactive compounds with antioxidative activity and function in human benefits. Hairy-root cultures, were infected via Agrobacterium rhizogenes, with the high production capability and genetic stability have been used on production of secondary metabolites in plants. In this study we investigated various leaves grown in greenhouse or outdoor for their total phenols, chlorophyll and betalains (betacyanins, BC and betaxanthins, BX) contents along with antioxidative ability. Hairy roots derived from various leaves were cultured and their betalains contents were analyzed. The results showed that the total phenols, chlorophyll and betalains amounts in green young leaves were significantly (p < 0.05) higher than green old leaves. BC concentration is slight higher than that of BX in of beetroot leaves. Chlorophyll showed the positive correlation (Person factor= 0.994, p < 0.001) with betalains contents. A dose-dependent kinetics in DPPH scavenging ability and in reducing power was found on beetroot leaves extracts. Red hairy-root has higher batalains than that of the yellow hairy root. It was found that inoculation amount and sucrose concentration would contribute to the betalains synthesis on hairy root culture with higher BX contents than that of BC.

C. roseus is the source of the important anticancer indole alkaloids, vincristine and vinblastine. Hairy root cultures are an attractive alternative for the production of plant derived secondary metabolites. C. roseus hairy root cultures were established in our laboratory by A. rhizogenes mediated transformation of C. roseus seedlings prior to the beginning of this project. The objectives of this project were twofold: (i) Alkaloid analysis of C. roseus hairy root cultures, (ii) Scaleup of hairy root cultures from shake flasks to bioreactors. An extraction method was adapted for the rapid quantification of C. roseus hairy root extracts, and was used for quantifying alkaloid yields from five different root clones. Optimum analytical wavelengths were chosen for quantification after analyzing chromatograms at five different wavelengths using PDA detection. Extracts were analyzed using different analytical techniques--GC/MS, MS, HPLC-PDA and TLC--and several alkaloids were identified. The presence of ajmalicine, akuammine, apparicine, catharanthine, hoerhammericine, lochnericine, serpentine and tabersonine was confirmed. The presence of vindoline--an important precursor of vinblastine and vincristine--could not be confirmed although GC/MS indicated the presence of vindoline fragment ions. A novel method based on non-linear regression analysis was developed for online measurement of oxygen uptake rates. Thus, mass transfer rates could be adjusted in bioreactor cultures to provide sufficient oxygen. Three different bioreactor configurations--two recirculation reactors (60 mL, 1L) and a 500 mL spinner flask--were designed for scaleup studies. Growth, nutrient utilization and alkaloid productivities were measured. This is the first study where several biochemical parameters have been measured in multiple bioreactor configurations and found to be comparable--without exception--to shake flask cultures. Cultures were cultivated in different gas phase environments--'air' and '2.5% CO$\sb2$'--in each bioreactor configuration. This is the first study of the effect of CO$\sb2$ on hairy roots cultivated in liquid culture. A beneficial effect ($>$98% confidence) of CO$\sb2$ was observed on biomass accumulation. Possible metabolic importance of CO$\sb2$ on heterotrophic plant cultures is discussed.

碩士
國立中興大學
農藝學系所
99
Using plant tissue culture system has advantages on producing better quality and higher content plant secondary metabolites. Optimization factors for biomass and platycodin D production for Platycodon grandiflorus hairy root culture was conducted in this study. Hairy root cultured in a flask containing 20 mL medium was shaked on a gyratory shaker in darkness. Time course experiment showed the highest dry weight of 14.97 g/L at the eighth week culturing and the highest platycodin D content of 6.16 mg/g at the ninth culturing week for a total 16-week-culture period. Inoculation experiment showed that inoculation density and culture period had interaction on dry weight production. In other words, culture period could be reduced when inoculation density was increased. Medium formulas of MS, B5 and WPM were compared for P. grandiflorus hairy root culture, and the B5 medium showed the best results among tested media. Sucrose concentrations from 29.2 mM to 262.9 mM for hairy root culture were investigated. Significant enhancement was found using 204 mM sucrose on dry weight, platycodin D content and its production with 16.96 g/L, 7.76 mg/g and 134.01 mg/L, respectively. In order to increase platycodin D content, additional elicitors into the culture medium during culture period was tested. Result showed 3 days after adding 1.25 mg/L ABA had the highest platycodin D content of 19.95 mg/g, and with a total platycodin D production reached to 281.83 mg/L. In addition, platycodin D content of 9.20 mg/g with a total 111.86 mg/L platycodin D production was obtained by adding elicitor of 5 mg/L jasmonic acid. Response surface methodology support a clearly recognition in effects and interactions of input variables such as medium components. Result for the screening experiment showed that sucrose, potassium nitrate, ammonium sulfate, sodium dihydrogenate phosphate and pH of the medium had significant influences on hairy root biomass production. Factors of sucrose, potassium nitrate and pH of the medium had significant influences on platycodin D content. Factors of sucrose, potassium nitrate, sodium dihydrogen phosphate and pH of medium had significant influences on platycodin D production. Optimum concentration range for sucrose, potassium nitrate and sodium dihydrogenate phosphate on biomass production of P. grandiflorus hairy root culture was obtained through using central composite design in this study. Currently, the highest hairy root dry weight 23.06 g/L, a 1.54-fold production comparing with original B5 medium, was obtained using response surface methodology however optimum valve for platycodin D production needs to be continued.

碩士
國立臺灣大學
微生物與生化學研究所
92
Culture of Rheum palmatum L. hairy roots were established after transformation with Agrobacterium rhizogenese A4, and the transformation was confirmed by PCR for the presence of a T-DNA sequence in their genomes. Quantitative determination of anthraquinones were performed by colorimetry, thin layer chromatography (TLC) and high performance liquid chromatography (HPLC). Among these anthraquinones produced by Rheum palmatum L., emodin and rhein were considered more important. In the studies, rhein is taken as the reference standard. Root growth and production of rhein were investigated in various basal media. It was found that B5 medium was superior to MS medium in terms of both root growth and rhein production. The results indicate that Rheum palmatum L. hairy root can be cultivated in the B5 medium enriched with 2 % sucrose, 20.25 mM KNO3, 6.75 mM NH4NO3 and 0.75 mM NaH2PO4. The rhein production is 10.06 mg/L in a 42 days` culture. The productivity is 1.38 folds as that in normal flask culture. A scale up culture of Rheum palmatum L. hairy root from 125 mL flask to 1 L bubble column reactor was conducted. The rhein production is 17.245 mg/L in a 49 days` culture. The productivity is 2.37 folds as that in flask culture.

碩士
臺灣大學
微生物與生化學研究所
95
Achyeanthes bidentata Blume, a perennial herbaceous plant, contains the oleanane-type saponins as one of the main pharmacologically active compounds. Hairy roots of A. bidentata were induced by the infection of Agrobacterium rhizogenes. The main constituents of basal medium significantly affected the growth of hairy roots, and the saponin content was also increased by higher concentration of sucrose. The addition of elicictors to the hairy root cultures is an effective method for inducing the biosynthesis of plant secondary metabolites by increasing the accumulation of phytoalexins in plant cells. In this study, using different types of elicitors to investigate the effect of the saponin content in A. bidentata hairy roots and found that the most effective one was jasmonic acid. Three days after the addition of jasmonic acid to the hairy root cultures by final concentration of 250 μM, the saponin content was 2.8-fold increase compared with the controls. To analyze the cytotoxicity of the hairy root methanolic extracts, using two human carcinoma cell lines, HepG2 and WiDr cells. As the result, treating with the hairy root extracts, significantly inhibiting the proliferation of WiDr cells. Furthermore, the morphological changes in HepG2 and WiDr cells were observed when exposed to the hairy root extracts, including membrane shrinking, and nuclear fragmentation. The further isolation and purification of hairy root extracts may be needed for the future work to find new sources of anti-cancer drugs.

碩士
朝陽科技大學
生化科技研究所碩士班
98
Stephania tetrandra S. MOORE is an important Chinese medicinal herb and it has been traditionally used as anti-allergic, anti-inflammatory, anti-cancer and against coronary diseases. Tetrandrine, a bis-benzylisoquinoline alkaloid has been identified as an active ingredient in S. tetrandrae and it is mainly exists in the root. Extraction of these metabolites from plants grown in the natural habitat is tedious and several factors can alter their yield. In vitro hairy root culture can be the probable solution to extract this alkaloid. The present study describes the consistent production of tetrandrine, achieved by Agrobacterium mediated transformation using A. rhizogenes strains ATCC 15834 and ATCC 43057. WPM was the most suitable media for in vitro hairy root culture. The integration of foreign gene into the genome was confirmed by PCR using rolB and rolC gene specific primers in different transgenic lines. There was variation in the growth rate and alkaloid content within different transgenic hairy root lines. Two transgenic lines HR10-1 and HR11-1 were selected and their HPLC analysis showed that the content of tetrandrine was 8.57 mg g-1 and 11.66 mg g-1, respectively. The tetrandrine contents were higher than that in the marketed crude drug (4.81 mg g-1). Furthermore, the HR10-1 transgenic line produced maximum biomass (6.5 g L-1) and tetrandrine (68.69 mg L-1) after 5 weeks cultivation. Affect three additives total biomass Response Surface Methodology (RSM). For media optimization were used to the higher biomass by alterity three different component of WPM media ie (NH4NO3, Ca(NO3)2 and sucrose). According to the central composite design experiment and mathematic calculation, the optimal concentration of NH4NO3, Ca(NO3)2 and sucrose was 564.34 mg L-1, 624.14 mg L-1, 41.24 g L-1, respectively, and the best hairy root biomass was 8.09 g L-1.Our results indicated that transgenic lines of S. tetrandra hairy root culture exhibited potential for mass production of tetrandrine. Key words: Stephania tetrandra S. MOORE, Hairy root, Tetrandrine, RSM, Bioreactor

碩士
朝陽科技大學
生化科技研究所碩士班
99
Anisomeles indica O. Kuntze belongs Labiatae, the major active ingredients mainly exist in the whole plant, including ovatodiolide, acteoside and apigenin. In the previous studies, the acteoside shown increase the coefficient of sexual organs significantly and improved pathology changes of testes. Acteoside inhibits human promyelocytic HL-60 leukemia cell proliferation and significantly inhibited the cytotoxicity induced by aflatoxin B1 (AFB1) and used anti-oxidant, anti-inflammatory. In this study, the hairy-root cultures were established by infecting sterile leave segments of Anisomeles indica with Agrobacterium rhizogenes strains ATCC15834 and ATCC43057. The integration of foreign gene into the genome was confirmed by PCR using rolB and rolC gene specific primers in different transgenic lines. The transformed hairy roots are characterized by high growth rate and genetic stability. After 28 days cultivation on soild-agar plate, the hairy root line (HR10-11) which exhibited high biomass (1.25g dw/L) and acteoside content (10.94 mg/g) was obtained. The acteoside content of hairy root was higher than that in different parts of plant (stem: 1.23 mg/g；adventitious roots:3.09 mg/g ). The hairy roots were cultured on 3 different media such as 1/2MS, B5 and WPM to obtain the suitable culture medium. The result shown the WPM was the most suitable media for in vitro hairy root culture. A time-course experiment was carried out in Erlenmeyer flask with WPM medium, in the exponential phase (fifth week) of growth, the highest acteoside content (11.6 mg/g) was obtained, furthermore, in the stationary phase (seventh week) of growth, the maxium biomass (7.5 g dw/L) and acteoside production (83.6 mg/L) were obtained. For increasing the biomass, acteoside accumulation and production of hairy root, the nutritional composition of WPM, such as sucrose, ammonium nitrate, calcium nitrate, copper sulfate and potassium phosphate and elicitors (aba meja sa and yeast elicitor) were conducted to evaluated. The results shown the maximum biomass of 12.02 g dw/L was obtained with 50 g/L sucrose, it was 2 fold compared to the control. Moreover, a 1.66 fold increases in acteoside content (18.72 mg/g) was observed with 5 mg/L MeJA in the WPM medium. Meanwhile, the maximum acteoside production was obtained with supplementing 800 mg/L ammonium nitrate.

The regulation of primary metabolism and induction of secondary metabolism in Catharanthus roseus hairy root cultures were studied using noninvasive in vivo NMR spectroscopy. A micro-perfusion system was first developed and characterized for the in vivo NMR metabolic studies which include both short-term bioassays and long-term micro-reactor experiments. In vivo $\sp{31}$P NMR experiments indicated that hairy roots stored P$\sb{i}$ in the vacuoles. Cytoplasmic and vacuolar pH of C. roseus hairy roots were regulated at 7.40 $\pm$ 0.08 and 5.45 $\pm$ 0.12 respectively with initial medium pH at 5.7, 6.5 or 7.3. A more acidic vacuolar pH and abnormal root morphology were observed at external pH of 4.2. Utilization of inorganic phosphates was unaffected by this morphological variation. Significantly higher specific yields of vindoline and catharanthine were found in the cultures initiated at pH 4.2. In vivo $\sp{13}$C NMR data indicated active pentose phosphate pathway in the metabolism of C. roseus hairy root cultures. Transient profiles of glucan resonances has led to a dynamic model for glucan synthesis. Under hypoxic conditions, C. roseus hairy roots accumulated ethanol, malate and $\gamma$-aminobutyric acid, and the cytoplasmic pH dropped. Glycerol was synthesized uniquely in C. roseus hairy roots perfused with medium containing glucose. Variations in shear force or in gas composition are possible factors affecting this glycerol formation. In response to the chemical elicitor A. niger pectinase, both the cytoplasmic and vacuolar pH of C. roseus hairy roots decreased immediately, and phosphate uptake by the hairy roots was hampered. These initial responses were followed by a shift in carbon fluxes as indicated by the transient increases in alanine and succinate, and decreases in glutamine and pyruvate. Glucan synthesis, malate and ethanol accumulation were significantly enhanced by the elicitor treatment. The Ca$\sp{+2}$ ion is an important factor in the glucan synthesis. Alkaloid analysis showed enhanced ajmalicine and catharanthine production by the A. niger pectinase-treated C. roseus hairy root cultures.

Plant in vitro cultures can be considered as an important and efficient tool for many purposes, including controlled production of valuable natural products, as well as useful experimental systems for studying regulatory mechanisms of plant development and metabolism. Two lines of Calendula officinalis in vitro hairy root culture, obtained as a result of transformation with the wild type Agrobacterium rhizogenes strain ATCC 15834, were subjected to elicitation by different biotic and abiotic factors in the context of possible stimulation of biosynthesis, accumulation and secretion of triterpenoids. Selected biotic (jasmonic acid, salicylic acid, chitosan) and abiotic (heavy metals, ultrasound, UV radiation) elicitors were applied in different concentration, intensities and duration of exposure. Some of applied elicitors are supposed to mimic the stress factors or conditions occurring in natural environment and triggering a complex network of reactions leading to specific metabolic changes. The obtained results showed the diverse efficiency of various factors in stimulation of triterpenoid biosynthesis/accumulation and saponin secretion in Calendula officinalis hairy root cultures. Among all tested elicitors, jasmonic acid was the most effective, increasing both the accumulation of oleanolic acid saponins in the hairy root tissue (20-fold) and particularly effectively the secretion of these compounds to the medium (113-fold). In turn, despite to 12-fold increase of saponin secretion, the advantage of the use of heavy metals as elicitors is debatable according to the harmful influence on the hairy root growth and the visible changes in their morphology and branching. The relationship between primary and secondary metabolism (represented respectively by sterols and pentacyclic triterpenes), modifications of compositional profile and fluctuations in triterpenoid content as a response or adaptation to stress were monitored in performed experiments. The symptoms of the competition between biosynthetic pathways of sterols and pentacyclic triterpenoids were the most visible in response of Calendula hairy roots to stimulation with biotic elicitors: jasmonic acid, salicylic acid and chitosan, whereas less evident in reactions to abiotic stressors, particularly UV radiation that caused the increase of the content of all investigated groups of triterpenoids. Heavy metals seem to disturb deeply the triterpenoid biosynthesis pathway since their influence includes modifications concerning not only the switch between sterols and pentacyclic compounds, but also the significant changes in the sterol profile. The performed study shows that Calendula officinalis in vitro hairy root culture can serve as a model system to investigate the regulation of the production and secretion of oleanolic acid saponins as well as for investigation of metabolic changes in plant response to stress imitated by chosen elicitors.
Roślinne kultury in vitro mogą być wykorzystywane do wielu celów, między innymi do kontrolowanej produkcji wartościowych metabolitów, a także jako modele doświadczalne do badania mechanizmów regulacyjnych procesów wzrostu i rozwoju oraz metabolizmu roślin. Dwie linie korzeni włośnikowatych nagietka Calendula officinalis, otrzymanych w wyniku transformacji przy pomocy Agrobacterium rhizogenes ATCC 15834 (szczep dziki), poddano elicytacji różnymi czynnikami biotycznymi i abiotycznymi w celu stymulacji procesów biosyntezy, nagromadzania i sekrecji triterpenoidów. Wybrane elicytory biotyczne (kwas jasmonowy, salicylowy i chitozan) oraz abiotyczne (jony metali ciężkich, ultradźwięki, promieniowanie UV) stosowano w różnych stężeniach, natężeniu i czasie działania. Niektóre ze stosowanych elicytorów miały imitować warunki stresu występujące w naturalnym środowisku i uruchomić reakcje prowadzące do specyficznych zmian metabolicznych. Otrzymane wyniki wykazały różną skuteczność badanych czynników w stymulacji biosyntezy i akumulacji triterpenoidów w kulturach korzeni włośnikowatych oraz wydzielania przez nie saponin. Kwas jasmonowy okazał się najbardziej efektywnym elicytorem, stymulującym zarówno nagromadzanie saponin kwasu oleanolowego w tkankach korzeni włośnikowatych (12 razy), jak i wydzielanie tych związków do pożywki (113 razy). Pomimo 12-krotnego zwiększenia sekrecji saponin, jony metali ciężkich wydają się nie być dobrymi elicytorami, gdyż jednocześnie powodują zahamowanie wzrostu korzeni i widoczne zmiany w ich morfologii i sposobie rozgałęziania. Podczas przeprowadzanych eksperymentów obserwowano także zależności między pierwotnym i wtórnym metabolizmem (reprezentowanym odpowiednio przez sterole i triterpenoidy pentacykliczne) oraz modyfikacje składu i zawartości triterpenoidów towarzyszące odpowiedzi na stres. Działanie elicytorów biotycznych (kwasu jasmonowego, salicylowego, chitozanu) wywołuje współzawodnictwo szlaków biosyntezy steroli i triterpenoidów pentacyklicznych, które nie jest tak wyraźnie widoczne przy działaniu czynników abiotycznych, zwłaszcza po elicytacji promieniowaniem UV, gdy nastąpił wzrost zawartości wszystkich badanych grup triterpenoidów. Jony metali ciężkich wydają się silnie zaburzać szlak biosyntezy triterpenoidów, gdyż powodują modyfikacje dotyczące nie tylko współzawodnictwa między sterolami i triterpenoidami pentacyklicznymi, ale także zmiany w profilu steroli. Przeprowadzone badania wykazały, że kultury korzeni włośnikowatych nagietka mogą służyć jako model do badań regulacji biosyntezy i wydzielania saponin kwasu oleanolowego oraz zmian metabolicznych zachodzących w odpowiedzi na czynniki stresowe.

Plant metabolic engineering is a developing field still in need of improved tools and an increased understanding of relevant pathways. This thesis addresses both those needs by testing a new tool for improved metabolic engineering studies and utilizing that tool for the exploration of monoterpenoid indole alkaloid biosynthetic pathways. Using GFP as a model protein in Catharanthus roseus hairy roots, we report that the glucocorticoid inducible promoter system is active and has a tightly controlled, reversible, and dosage-dependent response to dexamethasone. Furthermore, it provides an improved negative control useful for the study of genes affecting alkaloid synthesis. The most exhaustive transgenic studies reported here focus on the indole pathway. Expressing a feedback-resistant Arabidopsis anthranilate synthase alpha subunit results in dramatic increases in tryptophan and tryptamine yields. On induction, tryptophan increases from undetectable levels to 2.5 mg/g DW, while tryptamine increases from 25 mug/g DW to 267 mug/g DW. Additionally, a transient improvement in lochnericine yield indicates a possible increase in alkaloid flux countered by tight regulation of alkaloid levels. In lines transgenic for inducible tryptophan decarboxylase, serpentine specific yields increased by as much as 129% on induction. The reported studies on the indole pathway demonstrate successful methods to improve indole flux and show that increased indole flux can lead to improvements in certain alkaloids. Precursors from the complementing terpenoid pathway are also required for alkaloid synthesis. A feeding study utilizing an intermediate and a specific inhibitor of the nonmevalonate pathway validates the importance of this pathway for improved alkaloid yields and points to the potential success of upstream metabolic engineering efforts. In preliminary results, improved yields of certain alkaloids are reported for hairy root lines transgenic for 1-deoxy-D-xylulose-5-phosphate synthase and geraniol 10-hydroxylase, while an ORCA3 study highlights some potential problems with the use of transcriptional activators. In the last study, we focus on the engineering of a valuable alkaloid pathway and report a transgenic hairy root line overexpressing the full coding sequence of tabersonine 16-hydroxylase. On induction, the line produces 16-methoxytabersonine. Overall, a valuable new tool is introduced and subsequently used to manipulate the indole, terpenoid, and alkaloid pathways.

碩士
朝陽科技大學
應用化學系生化科技碩博士班
102
Salvia miltiorrhiza Bunge, a perennial plant belongs to Lamiaceae Family. Its roots contain tanshinones and phenolic acids. In the recent years, roots of S. miltiorrhiza have been used for the treatment of cardiovascular diseases. In the present study, hairy roots were induced in leaves of S. miltiorrhiza by infecting with Agrobacterium rhizogenes. In a liquid culture system, these hairy roots were cultured for the production of Salvianolic acid B (SAB). Three basal salt media (MS, B5, and WPM) in half (½x) and full (1x) strengths were evaluated for hairy root growth and SAB accumulation. Among these three basal salt media, B5 was selected because here nitrogen source in form of NO3- and NH4+ are separate and can be controlled to investigate their influence on production of hairy root biomass and SAB. Thus, different concentrations of six medium components viz. sucrose, KNO3, (NH4)2SO4, CuSO4, CoCl2 and NaH2PO4 were investigated for hairy roots biomass and SAB content. At 4 weeks of culture, results showed that ½ strength of MS basal medium gave the least hairy root biomass and was not suitable for the hairy roots culture of S. miltiorrhiza. In case of WPM (1x and ½x), production of the maximum root biomass (6.36 g dw/L) and SAB content (33.6 mg/g) was obtained after 3 weeks of culture. Thereafter, there was a decreasing trend in SAB content in the hairy roots. In the B5 medium (1x and ½x), the maximum production of root biomass (7.87 g dw/L) was obtained at 8 weeks of culture. Thereafter, there was a decreasing trend in the SAB content. It was found that there was no significant difference in biomass when hairy roots were cultured in the B5 basal medium containing different concentrations of (NH4)2SO4 from 0 to 2.0 mM for 4 weeks. However, with the increasing concentrations of (NH4)2SO4, there was a decreasing trend in the SAB content. The SAB content was 3.1-fold higher in the medium without (NH4)2SO4 compared to the medium with 1.0 mM (NH4)2SO4 indicating the influence of (NH4)2SO4 on SAB production. Results showed that growth of hairy roots varied with concentration of CuSO4 (0-75 µg/L) in the medium. B5 medium containing 50 µg/L CuSO4 produced 1.5-fold higher root biomass compared to medium with 25 µg/L CuSO4 demonstrating its influence on root biomass of S. miltiorrhiza. Thus, the present study demonstrates that concentrations of NH4+ and Cu+ in the culture medium effect hairy root production and its SAB content. To verify the effect of NH4+ ions on growth of hairy roots, a further experiment was carried out. The hairy roots were grown in the B5 medium without (NH4)2SO4 for 3 weeks, before adding different concentrations of (NH4)2SO4 in the medium. Results showed that production of biomass was directly proportional to concentration of (NH4)2SO4 (2 mM > 1 mM > 0 mM > CK) at the 4th week of evaluation. However, further culture of hairy roots beyond 4 weeks retarded the biomass production. In conclusion, this study demonstrates that the type of basal medium, concentrations of Cu+ ions and (NH4)2SO4 in the culture medium significantly influence the biomass and SAB production in hairy root culture of S. miltiorrhiza, thus, needing culture medium optimization for the optimum results.

碩士
朝陽科技大學
應用化學系生化科技碩博士班
103
Gentiana scabra Bunge, commonly known as ‘Longdan’ is a perennial plant belonging to Gentianaceae family. In traditional Chinese medicine, roots of ‘Londan’ are used to prevent indigestion, anorexia and gastric infections. Due to an extensive medicinal use, wild G. scabra plants are in huge demand resulting in its over exploitation and decline in wild populations. Hence, it is necessary to find alternative method for production of active compounds without destruction of wild plants. The aim of the present study is to optimize hairy root culture system in order to improve production of root biomass and pharmaceutically important compounds of G. scabra. Hairy roots obtained from leaf explants of G. scabra infected with Agrobacterium rhizogenes were used as starting material. The rapidly growing hairy roots of five transgenic lines HR-32, HR-35, HR-36, HR-39, and HR-49 were cultured in semi-solid Woody Plant Medium (WPM) for four weeks. Of these five, line HR-32 yielded the highest root biomass (2.74 g dw/petri-dish), hence, it was selected for further experiments. Hairy roots of HR-32 were cultured in liquid medium of five different basal media like Murashige and Skoog (MS), WPM, B5, 1/2B5, and N6. After four weeks of culture, the highest root biomass (4.56 g dw/L) was obtained in B5 liquid medium. Also, it produced maximum Gentiopicroside (29.0 mg dw/g) and Swertiamain (2.2 mg/g dw), therefore, all further experiments were carried out with B5 medium alone. In order to determine whether liquid or semi-solid medium is better for production of root biomass and bioactive compounds, experiments were performed with liquid and semi-solid media or alternate subculture on solid to liquid or solid to solid medium. Also, certain medium components play an important role in production of root biomass and active compounds. Therefore, further experiments were carried out to optimize components like sucrose (10 – 70 g/L), Potassium nitrate (6.2, 12.4, 24.7 and 37.1 mM), ammonium sulfate (05, 1, 1.5 and 2 mM) and copper sulfate (12.5, 25, 50 and 75 μg/L) in the B5 medium. Root biomass in both semi-solid medium and liquid cultures was recorded. Production of Gentiopicroside and Swertiamarin in semi-solid and liquid cultures was estimated by High Performance Liquid Chromatography (HPLC) analysis. Among different sucrose concentrations in the medium, a supplement of 50g/L sucrose resulted in the highest biomass (5.49 g dw/L) production. While, sucrose at 70g/L supported maximum Gentiopicroside (29.2 mg/g) and Swertiamarin (1.6 mg/g) production. Potassium nitrate at 24.7mM in the medium supported the highest biomass of 8.51 g dw/L with Gentiopicroside and Swertiamarin contents as 49.2 mg/g and 3.1 mg/g, respectively. The maximum root biomass (6.68g d.w/L) among different concentrations of Ammonium sulfate was recorded at 2mM. The contents of Gentiopicroside and Swertiamarin were 56.6 mg/g and 2.9 mg/g, respectively. There was a marginal difference in biomass production at different levels of Copper sulfate, however, Copper sulfate at 75 μg/L in the medium supported highest contents of Gentiopicroside (40.0 mg/g) and Swertiamarin (2.1 mg/g). The results obtained in present study indicate that the concentration of carbon source (sucrose) and nitrogen level in the medium affect the hairy root growth and production of Swertiamarin and Gentiopicroside in cultures of G. scabra. Also, present study affirms that there is a possibility of producing the bioactive compounds through hairy root cultures without sacrificing the wild plants of G. scabra Bunge.

碩士
中國醫藥大學
中國藥學暨中藥資源學系碩士班
102
This study is focus on the growth and secondary metabolite productionof hairy rootsculture of Salvia miltiorrhiza using the stimulation from ultrasound (US). The hairy roots of S. miltiorrhiza was exposed to low-energiedUS for short durations(5、10、15 min), and thedetection of rosemary acid (RA) and salvianolic acid B (SAB) cumulativecontent in S. miltiorrhiza hairy roots was carried out.The results showThat the US exposure was able to significantly stimulated the productionof the secondary metabolites on hairy roots, increased the yieldofrosemary acid salvianolic acid B by 40% and 30%, respectively. The plant cells will generate the oxidative burstand increase oftheir secondary metabolitesproductionwhen plants receivethe stimulationfrom shock waves. Therefore, in order to understand the relation between the stimulationshock waves and harvest timeof hairy root cultures , the comparison of accumulation of secondary metabolites of S. miltiorrhiza hairy roots at different harvest time under a fixed ultrasound frequency and10 minutesprocessing duration was also carried out. The resultindicates the yields of rosemary acid and salvianolic acid B were increased by 160% and 80% in S. miltiorrhizahairy roots when they were exposed with 10 minutesUS stimulationand cultured for 12 hoursafterward. . These results indicatethat the stimulation using ultrasound on S. miltiorrhizahairy root is a positive treatment for their secondary metabolites production and able to be applied for further studies. The specific aim of this study is to establish the tetraploidhairy roots culture of S. miltiorrhiza using transformation of Agrobacterium rhizogenes LBA 1334 and treatment of Colchicine. During the solid medium cultures, the productive levels of rosemarinic acid (RA), salvianolic acid B (SAB) and tanshinone IIA (TSIIA) from tetraploid hairy roots were 1.5 times, 2 times and 2.2 times higher than the diploid ones, respectively.In addition, the contents of RA and TSIIA from tetraploid hairy roots were 6.2 times and 4 times higher than the original plant root.It means that the production of secondary metabolitesin polyploid hairy rootsof S.miltiorrhizais able to be considered asa potential method.

"Hairy" roots hold the potential for economically feasible biotechnological routes to the controlled biosynthesis of complex, plant-derived, 'natural' molecules. A novel transgenic root system of the tropical plant Catharanthus roseus was established and analyzed for the synthesis of indole alkaloids, including the valuable anti-cancer drugs vinblastine and vincristine. A structured approach to developing the biosynthetic potential of hairy roots is presented: transformation, screening, selection, optimization of culture protocols, and product enhancement. Five hairy root clones with unoptimized doubling times of 3-4 days and vindoline output of 0.005-0.07% dry weight, were screened from 150 transformants. Hairy root morphology likely under the control of rol genes transferred from the Agrobacterium plasmid, was identified as a key determinant of fitness in liquid culture and a target for transgenic design for large-scale bioreactor environments. The hairy root inoculum was optimized and standardized to facilitate the assessment of culture performance under diverse environmental treatments and in process scale-up. The length of the root tip has a dominant effect on growth, uninfluenced by clonal variability. The optimum inoculum is comprised of 5 root tips, each 35-40 mm long, in 50 mL media. Long-term dose-response and transient studies examined heterotrophic and photoheterotrophic carbon regimes. These studies are unique in the metabolic adaptation of cultures, and examined the putatively antagonistic kinetics of nutrient utilisation and secondary metabolite accumulation. The activities of the cathenamine and bisindole alkaloid pathways responded, respectively to high and moderate sucrose concentrations. The cultures were nitrogen limited with 2-4% sucrose in B5/2 salt. Organic acids were excreted in the presence of excess sugars. The ordered assimilation of macronutrients--ammonium, phosphate and nitrate--corroborated by changes of extracellular pH, have important implications on fed-batch strategies. Tabersonine accumulation was growth-associated, while serpentine accumulated in a non-growth manner. Ajmalicine, catharanthine, vindoline, and compounds tentatively identified as vinblastine and vincristine, accumulated optimally in the late-exponential or early-stationary phase. Photoheterotrophic conditions incremented peak biomass by 60-300%, doubling times by 60%, and vindoline levels by an order of magnitude, likely due to the anapleurotic activity of PEPCase and the light induction of nitrate reductase and vindoline synthesis.

Trabalho Final de Mestrado Integrado, Ciências Farmacêuticas, Universidade de Lisboa, Faculdade de Farmácia, 2013
Os compostos anti-cancerígenos têm tido uma grande importância devido ao número crescente de indivíduos que vive diariamente com o diagnóstico de cancro. Uma vez que a cura ainda não foi alcançada, os tratamentos disponíveis para esta doença recaem na quimioterapia, radioterapia e cirurgia, sendo que a quimioterapia baseia-se principalmente em produtos naturais ou derivados. O objectivo desta monografia residiu em consolidar os compostos anti-cancerígenos obtidos através de culturas vegetais mais usados em quimioterapia e os métodos usados na sua obtenção de modo a concluir quais as limitações dos mesmos e quais as perspectivas futuras que facilitariam a obtenção e a rentabilização dos processos de produção destes compostos. Uma vez que a obtenção destes compostos através de culturas de células vegetais é um processo moroso e no qual são obtidos rendimentos baixos, o uso de bioreactores para o cultivo em larga-escala é uma boa alternativa para melhorar os sistemas de obtenção de metabolitos secundários. Uma possibilidade explorada nesta monografia são os avanços no design de bioreactores com tecnologias baseadas em sistemas “hairy root”, mantendo o seu potencial biosintético e ao mesmo tempo promovendo uma produção em larga escala.
Anticancer compounds have had a great importance due to the increasing number of daily living individuals diagnosed with cancer. Because the cure has not been achieved, available treatments for this disease relapse in chemotherapy, radiotherapy and surgery being chemotherapy mainly based on natural products or derivatives. The purpose of this monography resided in consolidating the most used plant-derived anticancer agents and the methods used to obtain them, in order to take conclusions about their limitations and which are the future prospects that would facilitate the production and enhancement of the yield of these anticancer compounds. Since these compounds obtained by plant cell cultures is a time-consuming process, with low yields , the use of bioreactors for large-scale cultivation is a good alternative to improve systems to obtain these compounds. One possibility explored in this monography are the advances in bioreactors design based in hairy-root technologies, maintaining biosynthetic potential while promoting a large-scale production.

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...