This thesis presents an investigation into the transcriptional regulation of TaxolTM biosynthsis in Taxus cuspidata cell suspension cultures. The potent anticancer drug TaxolTM has been shown to be successful in the treatment of breast, lung and ovarian cancer and the acquired immunodeficiency syndrome (AIDS) related Kaposi’s sarcoma. Produced by all species of yew, TaxolTM belongs to the class of taxane diterpenoids and is of huge pharmaceutical importance. The plant material utilised in this thesis is a cell suspension culture initiated from isolated procambium cells of T. cuspidata. The latter is a meristematic tissue giving rise to the conductive tissue of plants. This un-differentiated cell suspension culture exhibits an increased and stable production of TaxolTM in response to the plant hormone elicitor methyljasmonate, limited cell aggregation and fast growth when compared to a cell suspension culture initiated from differentiated cells (somatic) of T. cuspidata. In order to assess the stem cell characteristics of the employed procambium cell suspension culture, the transcriptome of T. cuspidata was sequenced utilising Roche/ 454 and Illumina/ Solexa NlaIII tag sequencing technoloxiv gies. Statistical analysis uncovered differential expression profiles of 563 genes present within the procambium cell derived transcriptome by comparison with the somatic cell derived transcriptome. Gene ontology analysis of the latter identified that genes associated with response to stress and defence response were upregulated in the differentially expressed portion within the procambium cell suspension culture. This is consistent with the characteristics of animal stem cells which exhibit robust defence strategies to environmental stress. Furthermore PHLOEM INTERCALATED WITH XYLEM (PXY ) and TRACHEARY ELEMENT DIFFERENTIATION 2 (TED2), which are essential for ordered procambium cell division and differentiation into trachaery elements respectively in A. thaliana and Z. elegans, are up-regulated in the T. cuspidata procambium cell suspension culture. Further T. cuspidata homologues of the jasmonate signalling components JASMONATE ZINC FINGER LIKE ZIM DOMAIN 2 (JAZ2) and JAZ3 were identified among up-regulated transcripts in response to jasmonate treatment in both the procambium and the somatic cell line. Blast analysis identified 211 transcription factors within the APETELA 2 (AP2), BASIC-HELIX-LOOPHELIX (bHLH), WRKY, MYB and BASIC-LEUCIN-ZIPPER (bZIP) families. Further characterisation established 21 transcription factors which are significantly up-regulated in response to jasmonate treatment and show a higher expression level in procambium cells. These provide promising targets for further functional characterisation to elucidate their involvement within TaxolTM biosynthesis. In order to investigate transcriptional regulation of the TaxolTM structural genes, a 513 bp fragment corresponding to the TAXADIENE SYNTHASE (TASY ) promoter was cloned by genome walking. In-silico analysis of the TASY and 3’-N-DEBENZOYLTAXOL N-BENZOYLTRANSFERASE (DBTNBT) promoter resulted in the identification of methyljasmonate and pathogen-responsive elements which may significantly contribute to jasmonate mediated accumulation of TaxolTM. Analysis of a chimeric promoter construct driving the reporter gene β-GLUCURONIDASE (GUS) in N. benthamiana confirmed jasmonate-responsiveness of the TASY promoter. Finally, comparison of the expression level of genes coding for potentially rate-limiting enzymes within the TaxolTM pathway established a significantly increased expression of BACCATIN II PHENYLPROPANOYLTRANSFERASE (BAPT) in response to jasmonate treatment within the procambium cell suspension culture. Furthermore transcripts of TASY, PHENYLALANINE AMINOMUTASE (PAM) and DBTNBT show an overall higher expression and prolonged transcript accumulation in procambium compared to somatic cells. In this thesis jasmonate-signalling components, jasmonate-responsive transcription factors and differential gene expression profiles of TaxolTM structural genes were identified which, may contribute to an increased TaxolTM production in the utilised procambium cell suspension culture. Furthermore the T. cuspidata procambium cell suspension culture was found to have an increased level of stress- and defence-response reflected by differential gene expression profiles and content of phenolic compounds and TaxolTM.

Paclitaxel is an antitumor diterpene from Taxus spp. that binds tubulin, stabilizes microtubules and induces apoptosis in dividing human cells. It was originally isolated from the bark of Taxus brevifolia and approved for clinic uses by the FDA in 1992. Because of its excellent activity in treatment of various cancers, a significant supply shortage has been created by the enormous demand for this natural product. Thus, researchers have been focusing on the development of effective ways to increase the production of paclitaxel and related bioactive molecules. This shortage was initially solved by over-harvesting of T. brevifolia bark; however, it is not an environment-friendly, effective and sustainable way to supply paclitaxel. A semisynthetic route was then developed to convert the more readily available and renewable 10-deacetylbacatin III into paclitaxel. As an alternative, plant cell cultures have been employed to commercially produce paclitaxel and it is a more environment-friendly and sustainable route to end the supply crisis. However, problems associated with plant cell culturing at an industrial scale, such as cell aggregation and variability in yield, significantly affect paclitaxel production. Therefore, a discovery of a better-performing Taxus cell line might be a solution to overcome these culturing-associated problems. A cambial meristematic cell (CMC) line of Taxus cuspidata has been isolated, cultured and demonstrated to be a cost-effective and environmentally friendly platform for the sustainable production of paclitaxel (Lee et al. 2010). Compared to dedifferentiated cell (DDC) lines, CMC lines are undifferentiated cells and proved to have stem cell-like properties. When cultured at an industrial scale, this cell line contains much smaller cell aggregates with many cells appearing as singletons, the biomass of which is still increasing after 22-month culturing, and has much greater paclitaxel production after elicitation (Lee et al. 2010). In my project, we aimed to identify the transcription factors (TFs) that regulate the expression of paclitaxel biosynthesis genes. We performed Illumina Solexa sequencing on cDNA libraries derived from methyl jasmonate (MeJA)-elicitated CMCs to digitally profile gene expression. Analysis of differentially expressed gene (DEG) abundance led to the discovery of 19 putative TFs and bioinformatic analysis further showed that these 19 TFs belong to 5 different TF families. Further, the DNA binding motifs associated with these TFs can be found in the promoters of the two early, taxadiene synthase (TASY) and taxadiene 5α hydroxylase (T5αH), and three late, 10-deacetylbaccatin III-10-O-acetyltransferase (DBAT), phenylpropanoyltransferase (PAM) and 3’-N-debenzoyl-2-deoxytaxol-Nbenzoyltransferase (DBTNBT), paclitaxel biosynthesis pathway genes. Then, yeast one-hybrid analysis, gel shifting assays and plant transient expression assays (TEA) were employed to assay TFs that interact with these promoters. Although Y1H screening did not show any convincing TF-promoter interactions, the attempted plant transient expression assay in the leaves of Nicotiana benthamiana might be a more suitable system to screen the positive regulators. Finally, the elucidation of a TF regulatory network that controls paclitaxel biosynthesis will guide the rational engineering of CMCs to ultimately increase yields of this important pharmaceutical.

Numerous drugs in the current pharmacopoeia originate from plant sources. Plant cell culture represents an alternative source for producing high-value secondary metabolites including paclitaxel. Paclitaxel is mainly derived from the plant genus Taxus and has been widely used in cancer chemotherapy. However, plant cell culture is often not commercially viable because of difficulties associated with culturing dedifferentiated plant cells (DDCs) on an industrial scale. Therefore, we isolated and cultured innately undifferentiated cambial meristematic cells (CMCs) from Taxus cuspidata, which possess superior growth properties relative to DDCs. These CMCs have been demonstrated to be a cost effective platform for the sustainable production of paclitaxel. Using 454 sequencing, we determined the transcriptome of T. cuspidata CMCs. Utilizing this transcriptome as a reference, we then employed Solexa digital gene expression profiling to identify transcriptional regulators that were induced by methyl jasmonate, an activator of paclitaxel biosynthesis. This lead to the discovery of 19 putative transcription factors (TFs) belonged to 5 TF families which were further confirmed by associated molecular methods. We aimed to identify which of these 19 regulatory proteins drive the expression of 5 paclitaxel biosynthetic genes by employing yeast one-hybrid analysis and electrophoretic mobility shift assays. Further, the cis-regulatory elements associated with these TFs were identified in the promoter regions of the two early, taxadiene synthase (TASY) and taxadiene 5α hydroxylase (T5αH), and three late, 10-deacetylbaccatin III-10-O-acetyltransferese (DBAT), phenylalanine aminomutase (PAM) and 3'-N-debenoyl-2-N-benzoyltransferase (DBTNBT), paclitaxel biosynthetic genes to facilitate the TF-DNA binding studies. Finally, understanding the TF regulatory network underlying paclitaxel biosynthesis can guide the engineering of CMCs to elevate the production of this key pharmaceutical.

Thema of the thesis is to analyse actual state and predict future application of english yew (Taxus baccata L.) in forest sites at chosen parts of PLA Moravský kras. Structure of parent stand and natural regeneration is analyzed on locations of interest Habrůvecká tůň, Lažánky, Sloup and Macocha. There was measured height, dbh, height of the crown, projection of the crown and space situations were drawned for mature individuals. In case of natural regeneration every individual was measured for height, distance from assumed parent tree, age and damage by game. At locations of interest were analysed 82 mature individuals. Height ranges from 1,7 to 13,4 m and dbh ranges from 4,1 to 33,7 cm. Natural regeneration containing 260 individuals was found near 35 mature yews and 58% was younger than 5 years. Damage by browsing was observed at 51 individuals, respectively 19,6 % of population. Mostly, trees older than 5 years are damaged. Damage is often repetitive. Only two individuals from natural regeneration are higher than 1m. From research is apparent negative influence of game on natural regeneration. Future of yew population in PLA Moravský Kras is conditioned to elimination of negative influence of game, which can be achieved by lowering numbers of game or suitable protection of trees.

Human society has benefited greatly from plant secondary metabolites, often utilizing a variety of compounds as dyes, food additives, and drugs. In particular, pharmaceutical development has benefited greatly from plant secondary metabolites. One example of this utility is paclitaxel, a highly substituted diterpene approved in the treatment of breast cancer, ovarian cancer, non-small cell lung cancer, and the AIDSrelated Kaposi’s sarcoma. Demand of paclitaxel is likely to increase, due to the current examination of paclitaxel in numerous clinical trials against a variety of other cancers. Taxus cell culture represents a production source of paclitaxel to meet future demand. However, paclitaxel production through Taxus cell culture is often variable and low. Targeted metabolic engineering of Taxus to produce superior paclitaxelaccumulating lines is a viable strategy to address variable and low yields. To facilitate the production of genetically engineered Taxus cell lines, stable transformation is required to examine the long-term effect of gene expression in vitro. Additionally, suitable transient transformation systems are necessary to characterize novel Taxus genes related to paclitaxel accumulation. A transient particle bombardment-mediated transformation protocol was developed to introduce transgenes into Taxus cells in vitro. Additionally, agroinfiltration in Nicotiana benthamiana was examined as a system to express genes related to paclitaxel biosynthesis and lead to the accumulation of the first dedicated taxane, taxa- 4(5), 11(12)-diene. In regard to stable transformation, an Agrobacterium-mediated transformation protocol was developed, though this method requires further optimization for reliability and increased transformation efficiency. These transformation technologies will aid in the creation of elite paclitaxel-accumulating Taxus cell lines.

<p> Recently, the discovery that taxol (2), a highly modified diterpenoid available from Taxus species, possesses anticancer activity has attracted considerable interest. The synthesis of [1-3H]-geranylgeranyl pyrophosphate(1b), a labelled form of the precursor to the diterpenoids(1a), and its incubation with a cell-free enzyme preparation from Taxus x media densiformis, a species of yew tree, were investigated.</p> <p> The investigation into the biosynthesis of taxol(2) was to be accomplished by monitoring the enzymatic transformations of (1b). The synthesis of 1b was accomplished via a convergent method using geraniol, a ten carbon compound, as the starting material. Essentially two molecules of geraniol were chemically manipulated and condensed to give geranyl geraniol which was oxidized to the corresponding aldehyde and then reduced using sodium borotritide. The radioactive alcohol was subsequently converted to the pyrophosphate (1b) using standard methodology. Cell-free extracts of Taxus x media densiformis were prepared and incubated with [1-3H]-GGPP (1b), the intermediates were extracted, and then analyzed by scintillation counting and radioactive gas chromatography. Identification of these labelled intermediates was attempted in order to provide information about the biosynthesis of 2. (Diagram included in thesis)</p> / Thesis / Master of Science (MSc)

Die Gemeine Eibe (Taxus baccata L.) und die Stechpalme (Ilex aquifolium L.) gehören in Deutschland zu den seltenen Baumarten und sind nach Bundesartenschutzverordnung unter besonderen Schutz gestellt. Die Schutz- und Erhaltungsbemühungen erstrecken sich meist auf die Freistellung von Individuen und Verjüngung, um durch einen höheren Strahlungseinfall deren Wachstum zu fördern. Untersuchungen an Individuen in unterschiedlicher Belichtungssituation zeigen aber, dass das Wachstumsverhalten von Taxus baccata und Ilex aquifolium bei vermehrtem Strahlungsgenuss nicht den Strategien der meisten heimischen Baumarten folgt, die ihr Höhenwachstum in diesem Falle steigern. Das bedeutet, dass die durchgeführten Maßnahmen zur Förderung und Erhaltung nicht oder nur zu einem geringen Teil dem angestrebten Zweck dienen. Mögliche Ursachen für das abweichende Wachstumsverhalten können in den hydraulischen Eigenschaften der Baumarten liegen. Anhaltspunkt ist die Wasserversorgung der Nadeln bzw. Blätter, die vom Wasserhaushalt, speziell von der Xylemleitfähigkeit, beeinflusst wird. Ziel der Untersuchungen ist es, die hydraulischen Eigenschaften an Zweigen der Baumarten Gemeine Eibe und Gemeine Stechpalme auf anatomischer und physiologischer Ebene zu analysieren und nachfolgend übliche waldbauliche Förderungs- und Erhaltungsmaßnahmen unter dem Gesichtspunkt ihrer Eignung zu bewerten. Zur Durchführung der ökophysiologischen Untersuchungen wurden auf zwei sich in der Sonneneinstrahlung unterscheidenden Standorten (Standort „schattig“ und Standort „sonnig“) im Forstbotanischen Garten Tharandt an je vier bis fünf Individuen der Arten Stechpalme, Eibe und Rot-Buche das Minimum-Wasserpotential der Blätter gemessen und der Gaswechsel in physiologischen Lichtkurven und im Tagesgang unter Umgebungsbedingungen aufgezeichnet. Die Rot-Buche dient dabei als Referenzbaumart. Im Labor wurden an Zweigproben aller drei Baumarten Messungen der hydraulischen Leitfähigkeit durchgeführt, die Xylemanatomie anhand von Querschnitten erfasst, die Blattflächen analysiert sowie an Eibe die Stomatadichte aufgenommen. Die Ergebnisse zeigen, dass die untersuchten Eiben eine große Umweltstabilität in der Ausprägung ihrer hydraulischen Eigenschaften aufweisen. Unter sonnigen Standortbedingungen sind sie größerem Stress ausgesetzt, was sich z. B. im Auftreten von Photoinhibition und einer stärkeren Verminderung der Mittags-Blattwasserpotentiale bei ansteigenden Wasserdampfdefizit der Luft ausdrückt. Es konnten keine anatomischen Anpassungen an sonnige oder schattige Wuchsbedingungen gefunden werden. Die untersuchten Stechpalmen sind dagegen sowohl physiologisch als auch unter anatomischen Gesichtspunkten gut an ihren jeweiligen Standort angepasst. Es lassen sich z. B. Anpassungen einer erhöhten Leitfähigkeit unter sonnigen Wuchsbedingungen erkennen. Im Vergleich von Individuen der Art Stechpalme am Standort Tharandt, der östlichen Verbreitungsgrenze der Art, mit Individuen aus Schleswig-Holstein, aus dem ökologischen Optimum der Art, zeigt sich kein Unterschied in der Richtung der physiologischen Antworten unter verschiedenen Belichtungssituationen. Teilweise sind die Reaktionen an Stechpalmen aus Tharandt deutlicher zu erkennen. Die Referenzbaumart Rot-Buche weist in zahlreichen untersuchten Größen im Vergleich zu den beiden immergrünen Baumarten entgegengesetzte physiologische und anatomische Anpassungen auf die unterschiedlichen Strahlungsangebote an den Standorten auf. Es wird gezeigt, dass alle drei untersuchten Arten unterschiedliche hydraulische und ökologische Strategien verfolgen. Die Eibe weist eine sehr breite ökologische Amplitude auf und ist perfekt an schattige Wuchsbedingungen im Waldinnenklima angepasst. Daher ist es nicht förderlich, durch starke Eingriffe im Oberstand das Lichtregime und damit auch die kleinklimatischen Verhältnisse ihres Standortes plötzlich stark zu verändern. Im Gegensatz dazu ist für die Art Stechpalme eine Förderung der Wuchsform durch eine vorsichtige und langsame Auflichtung des Oberstandes im Rahmen von regulären Durchforstungseingriffen durchaus möglich und erfolgversprechend.:Inhalt 1 Abkürzungsverzeichnis 4 1 Einleitung 6 1.1 Forschungsziele 7 1.2 Hypothesen und Fragestellungen 8 2 Stand des Wissens 10 2.1 Verbreitung, ökologische Strategie und waldbauliche Behandlung der Baumarten 10 2.1.1 Reaktion der Arten auf den Standortfaktor Sonneneinstrahlung 12 2.1.2 Waldbauliche Behandlung 14 2.2 Untersuchung hydraulischer und physiologischer Eigenschaften und Anpassungen dieser an unterschiedliche Standort-bedingungen 17 3 Material und Methoden 20 3.1 Aufbau der Untersuchungen 20 3.1.1 Untersuchungsgebiet Forstbotanischer Garten Tharandt 20 3.1.1.1 Hemisphärische Fotografie 20 3.1.1.2 Standortcharakteristik 22 3.1.2 Versuchsdesign 23 3.1.3 Untersuchungsgebiet Fröruper Holz 24 3.1.4 Vergleich des Standortfaktors Licht der Untersuchungsgebiete Forstbotanischer Garten Tharandt und Fröruper Holz/Schleswig-Holstein 26 3.2 Bestimmung des volumetrischen Bodenwassergehaltes 27 3.3 Untersuchung der axialen hydraulischen Leitfähigkeit 27 3.4 Xylemanatomische Untersuchungen 29 3.5 Analyse der Blattflächen 31 3.6 Erfassung der Stomatadichte an Eibe 31 3.7 Gaswechseluntersuchung 32 3.7.1 Messungen von Tagesgängen der Photosynthese 35 3.8 Untersuchung des Minimum-Blattwasserpotentials 36 3.9 Statistische Auswertung 37 4 Ergebnisse und Diskussion 40 4.1 Volumetrischer Bodenwassergehalt 40 4.1.1 Einordnung und Bedeutung des volumetrischen Wassergehaltes der Standorte 40 4.2 Ergebnisse für die Baumart Eibe 44 4.2.1 Hydraulische Leitfähigkeit 44 4.2.2 Xylemanatomie der Zweigquerschnitte 46 4.2.3 Blattflächen 47 4.2.4 Stomatadichte 49 4.2.5 Gaswechsel 50 4.2.5.1 Netto-Photosyntheserate 50 4.2.5.2 Transpiration 52 4.2.5.3 stomatäre Leitfähigkeit gS 53 4.2.5.4 Wassernutzungseffizienz WUE 54 4.2.5.5 intrinsische Wassernutzungseffizienz WUEintr 55 4.2.6 Tagesgang der Photosynthese 56 4.2.7 Minimum-Blatt-Wasserpotentiale und Einfluss von Umweltparametern 64 4.2.8 Zusammenfassung Ergebnisse Eibe 67 4.3 Diskussion der Ergebnisse der Baumart Eibe 68 4.4 Ergebnisse für Stechpalme 76 4.4.1 Hydraulische Leitfähigkeit von Stechpalmen im Forstbotanischen Garten Tharandt 76 4.4.2 Hydraulische Leitfähigkeit von Stechpalme im Fröruper Holz/Schleswig-Holstein und Vergleich mit Stechpalme im Forstbotanischen Garten Tharandt 78 4.4.3 Xylemanatomie der Zweigquerschnitte von Stechpalmen im Forstbotanischen Garten Tharandt 82 4.4.4 Xylemanatomie der Zweigquerschnitte von Stechpalme im Fröruper Holz/Schleswig-Holstein und Vergleich mit Stechpalme im Forstbotanischen Garten Tharandt 84 4.4.5 Blattflächen von Ilex im Forstbotanischen Garten Tharandt 87 4.4.6 Blattflächen von Stechpalme im Fröruper Holz und Vergleich mit Stechpalme im Forstbotanischen Garten Tharandt 88 4.4.7 Gaswechsel 92 4.4.7.1 Netto-Photosyntheserate 92 4.4.7.2 Transpiration 93 4.4.7.3 stomatäre Leitfähigkeit gS 93 4.4.7.4 Wassernutzungseffizienz WUE 94 4.4.7.5 Intrinsische Wassernutzungseffizienz WUEintr 95 4.4.8 Tagesgang der Photosynthese 96 4.4.9 Minimum-Blattwasserpotentiale und Einfluss von Umweltparametern 102 4.4.10 Zusammenfassung Ergebnisse Stechpalme 105 4.5 Diskussion der Ergebnisse der Baumart Stechpalme 106 4.6 Ergebnisse für die Referenzbaumart Rot-Buche 110 4.6.1 Hydraulische Leitfähigkeit 110 4.6.2 Anatomie der Zweigquerschnitte 111 4.6.3 Blattflächen 113 4.6.4 Gaswechsel 114 4.6.4.1 Netto-Photosyntheserate 114 4.6.4.2 Transpiration 115 4.6.4.3 stomatäre Leitfähigkeit gS 116 4.6.4.4 Wassernutzungseffizienz WUE 117 4.6.4.5 intrinsische Wassernutzungseffizienz WUEintr 118 4.6.5 Minimum-Blattwasserpotentiale und Einfluss von Umweltfaktoren 118 4.6.6 Zusammenfassung Ergebnisse Rot-Buche 120 4.7 Diskussion der Ergebnisse der Referenzbaumart Rot-Buche 122 5 Vergleichende Darstellung der Ergebnisse der Baumarten 126 5.1 Hydraulische Leitfähigkeit 126 5.2 Anatomie der Zweigquerschnitte 128 5.3 Blattflächen 129 5.4 Gaswechsel 131 5.4.1 Netto-Photosyntheserate 131 5.4.2 Transpiration 131 5.4.3 stomatäre Leitfähigkeit gS 132 5.4.4 Wassernutzungseffizienz WUE 133 5.4.5 intrinsische Wassernutzungseffizienz WUEintr 134 5.5 Minimum-Blattwasserpotentiale 135 6 Zusammenführung, weiterführende Diskussion und Bedeutung der hydraulischen Eigenschaften 138 7 Bewertung waldbaulicher Handlungsmöglichkeiten 147 8 Ausblick 155 9 Zusammenfassung 157 10 Literatur 159 Abbildungsverzeichnis 171 Tabellenverzeichnis 176 Danksagung 178 Anhang I

No description available.

Taxol (generic name paclitaxel), a plant‐derived antineoplastic agent, was originally isolated from the bark of the Pacific yew, Taxus brevifolia. Obtaining taxol from this source requires destruction of trees. It has been used alone or in combination with other chemotherapeutic agents for the treatment of breast, ovarian as well as many other types of cancer, including non‐small cell lung carcinoma, prostate, head and neck cancer, and lymphoma, as well as AIDSrelated Kaposi’s sarcoma. The mode of action of taxol against a number of human cancer cells is by preventing the depolymerization of tubulin during cell division. This molecule increases microtubule stability in the cell and induces apoptosis. From yew trees, the yield of taxol is usually between 0.004 to 0.1% of the dry weight. The commercial isolation of 1 Kg of taxol requires about 6 to 7 tons of T. brevifolia bark obtained from 2000‐3000 well‐grown trees. The limited supply of the drug has prompted efforts to find alternative sources of taxol. Alternative methods for taxol production, such as chemical synthesis, tissue and cell cultures of the Taxus species are expensive and give low yields. A fermentation process involving any microorganism would be the most desirable means to lower the cost and increase availability. The first report on the isolation of taxol‐producing fungi from Taxus brevifolia appeared in 1993 (Stierle, et al., 1993). Several taxol‐producing fungi have been identified since, such as Taxomyces andreanae, Taxodium disticum, Tubercularia sp., Pestalotiopsis microspora, Alternaria sp., Fusarium maire and Periconia sp (Li, et al., 1996, Strobel, et al., 1996a, Strobel, et al., 1996b, Li, et al., 1998b, Ji, et al., 2006, Xu, et al., 2006). This thesis investigates the isolation of an endophytic fungus, isolated from the stem cuttings of Taxus celebica, which produces taxol and related taxanes. We observed morphological and cultural characteristics and analyzed the sequences of rDNA ITS from the strain. The isolated fungus grew on potato carrot agar (PCA) medium at 25 °C and the colonies were white to off‐white, floccose, with irregular margins. The reverse side of the culture was cream in color. The morphology was examined microscopically following staining with cotton blue in lactophenol. Cultures produced macroconidia on slender, 85 μm long phialides. The macroconidia were 25‐40 X 3.75 μm. Cultures also produced round or oval microconidia. Analysis of the ITS and D1/D2 26S rDNA sequence revealed 99 % identity with Fusarium solani voucher NJM 0271. Based on its morphological, cultural characteristics and 26S rDNA sequence, the fungus was identified as F. solani. This fungus is different from the previously reported endophytic taxol‐producing species of Fusarium. Taxol and baccatin III, produced by this fungus, were identified by chromatographic and spectroscopic comparison with standard compounds. The amount of taxol produced by F. solani in potato dextrose liquid medium is low (1.6 μg l‐1) (Chakravarthi, et al., 2008). We further investigated different growth media and various factors of cultivation to select the medium and conditions that maximize production of taxol and other taxanes by this fungus. F. solani was grown in five well‐defined culture media under stationary and shake conditions separately for various time intervals and the amounts of taxol, baccatin III and other taxanes produced were estimated by competitive immunoassay. The modified flask basal medium (MFBM) was shown to yield the highest production of taxol (128 μg l‐1) which is 80 times more than when grown in potato dextrose liquid medium, baccatin III (136 μg l‐1) and total taxanes (350 μg l‐1) under shake conditions. From our results the highest taxol production of F. solani was achieved when cultured in MFBM. The production in MFBM was 80 times higher than that cultured in the potato dextrose liquid medium. In conclusion, it was shown that the culture medium plays a major role in taxol and other taxanes production and fungal growth. MFBM is the best medium, among the media studied, to produce taxol and other taxanes. The higher concentrations of NH4NO3, MgSO4, KH2PO4 and FeCl3 in the FBM medium seem important for production of taxol and other taxanes. These results can be considered as starting‐point for the research directed to improve taxol and baccatin III production by F. solani via different approaches including fermentations, strain improvement and genetic engineering techniques. Finally, in order to get more insights into the mode of action of this fungal taxol and baccatin III (for the first time), their apoptotic activity on different cancer cell lines was determined. We elucidated the biochemical pathways leading to apoptotic cell death after fungal taxol‐ and baccatin III‐ treatment in different cancer cell lines. Experiments are done on various cancer cell lines namely JR4 Jurkat (T‐cell leukemia), J16 Bcl‐2 Jurkat T cells, HepG2 (hepatoma), caspase‐8‐deficient Jurkat T cells, HeLa (human cervical carcinoma), Ovcar3 (human ovarian carcinoma) and T47D (human breast carcinoma) cells. We were able to demonstrate that both fungal taxol and baccatin III can induce apoptosis in all the cell lines tested, by flow cytometric analysis. Hallmarks of apoptosis following the signaling pathway to far more upstream‐located events were investigated using biochemical and cell biological methods. It has shown that during fungal taxol‐ and baccatin III‐induced apoptosis, DNA is degraded resulting in a increased number of hypodiploid cells reaching up to 65‐70% after 48 h. Disruption of mitochondrial membrane potential was examined by flow cytometric analysis using mitochondrial membrane potential sensitive dye JC‐1 and JR4‐Jurkat cells were shown to undergo significant loss of mitochondrial membrane potential loss of mitochondrial membrane potential reaching up to 70% in 6 nM fungal taxol and 65 % in 3.5 μM baccatin III after 36 h. These results were similar to those observed with standard taxol and baccatin III. We further investigated the role of caspases in fungal taxol‐ and baccatin III‐induced apoptosis, caspase‐8‐deficient Jurkat cells, Bcl‐2‐over‐expressed J16‐Jurkat cells and caspase inhibitors were used. Results derived from caspase‐8‐deficient Jurkat cells show that caspase‐8 is not involved in fungal taxol‐ and baccatin IIIinduced apoptosis of Jurkat cells. Using the pan‐caspase inhibitor (Z‐VAD‐FMK), caspase‐9 inhibitor (Z‐LEHD‐FMK), caspase‐3‐inhibitor (Z‐DEVD‐FMK), caspase‐2‐ inhibitor (Z‐VDVAD‐FMK) and caspase 10‐inhibitor (Z‐AEVD‐FMK), it was shown that caspase‐10 is involved in fungal taxol‐ and baccatin III‐ induced apoptosis in JR4‐Jurkat cells. It was also shown that inhibitors of caspases‐9, ‐2 or ‐3 partially inhibited fungal taxol‐ and baccatin III‐ induced apoptosis, whereas the caspase‐ 10 inhibitor totally abrogated this process. With the use of a fluorescence microscope, several morphological features characteristic of apoptosis such as condensed chromatin and apoptotic bodies were identified in fungal taxol‐ and baccatin III‐treated JR4‐Jurkat and HeLa cells. DNA fragmentations were shown by agarose gel electrophoresis method. Our work showed that treatment of JR4‐ Jurkat and HepG2 cells with fungal taxol and baccatin III induces apoptosis as shown by DNA ladder formation. Herein it was demonstrated that fungal taxol and baccatin III have a similar mechanism of action, but the efficacy of fungal taxol to induce apoptosis is higher. In summary, fungal baccatin III is found to be effective in inducing apoptosis similar to taxol but at higher concentration and both fungal taxol and baccatin III induce apoptosis via caspase‐10 and mitochondrial pathway in Jurkat cells. In conclusion, the present study describes isolation of a taxol‐producing endophyte F. solani IISc.CJB‐1. The growth requirements of this fungus for production of taxol, baccatin III and other taxanes were studied. The apoptotic activity of taxol and baccatin III (for the first time) was observed. In addition, our results show that the culture medium plays a major role in taxol and other taxanes production and fungal growth. Among the media studied, modified flask basal medium (MFBM) is the best to produce taxol and other taxanes. It is evident from this data that this fungal strain can be promising candidate for large‐scale production of taxol and related taxanes.

Plant derived natural products represent some of the most effective anti-cancer and anti-infectious disease pharmaceuticals available today. However, uncertainty regarding the feasibility of commercial supply due to the limited availability of many plants in nature has resulted in a dramatic reduction in the use of natural products as leads in modern drug discovery. Plant cell suspension culture, consisting of dedifferentiated plant cells grown in vitro and amenable to large scale industrial biotechnology processes, is a production alternative which promises renewable and economical supply of these important drugs. The widespread application of this technology is limited by low product yields, slow growth rates, challenges in scale-up, and above all, variability in these properties, which is poorly understood. Plant cells grow as aggregates in suspension cultures ranging from two to thousands of cells (less than 100 micron to well over 2 mm). Aggregates have long been identified as an important feature of plant cell culture systems, as they create microenvironments for individual cells with respect to nutrient limitations, cell-cell signaling, and applied shear in the in vitro environment. Despite its purported significance, a rigorous engineering analysis of aggregation has remained elusive. In this thesis, aggregation was characterized, analyzed, and modeled in Taxus suspension cultures, which produce the anti-cancer drug paclitaxel. A technique was developed to reliably and routinely measure aggregate size using a Coulter counter. The analysis of aggregate size as a process variable was then used to evaluate the effect of aggregation on process performance, and the analysis of single cells isolated from different sized aggregates was used to understand the effect of aggregation on cellular metabolism and heterogeneity. Process characterization studies indicated that aggregate size changed over a batch cycle as well as from batch to batch, so a population balance equation model was developed to describe and predict these changes in the aggregate size distribution. This multi-scale engineering approach towards understanding plant cell aggregation serves as an important step in the development of rational strategies aimed at controlling the process variability which has heretofore limited the application of plant cell culture technology.