Search

Modern plant biotechnology often faces the problem of obtaining a stable and powerful vector for gene overexpression. It is known that introns carry different regulatory elements whose effects on transgene expression have been poorly studied. To study the effect of an intron on transgene expression, the stilbene synthase 11 (VaSTS11) gene of grapevine Vitis amurensis Rupr. was selected and overexpressed in grapevine callus cell cultures and several plant generations of Arabidopsis thaliana as two forms, intronless VaSTS11c and intron-containing VaSTS11d. The STS genes play an important role in the biosynthesis of stilbenes, valuable plant secondary metabolites. VaSTS11d contained two exons and one intron, while VaSTS11c contained only two exons, which corresponded to the mature transcript. It has been shown that the intron-containing VaSTS11d was better expressed in several generations of transgenic A. thaliana than VaSTS11c and also exhibited a lower level of cytosine methylation. As a result, the content of stilbenes in the VaSTS11d-transgenic plants was much higher than in the VaSTS11c-transgenic plants. Similarly, the best efficiency in increasing the content of stilbenes was also observed in grapevine cell cultures overexpressing the intron-containing VaSTS11d transcript. Thus, the results indicate that an intron sequence with regulatory elements can have a strong positive effect on both transgene expression level and its biological functions in plants and plant cell cultures.

Stilbenes are plant defense compounds known to rapidly accumulate in grapevine and some other plant species in response to microbial infection and several abiotic stresses. Stilbenes have attracted considerable attention due to valuable biological effects with multi-spectrum therapeutic application. However, there is a lack of information on natural signaling pathways and transcription factors regulating stilbene biosynthesis. It has been previously shown that MYB R2R3 transcription factor genes VaMyb40 and VaMyb60 were up-regulated in cell cultures of wild-growing grapevine Vitis amurensis Rupr. in response to UV irradiation. In this study, the effects of VaMyb40 or VaMyb60 overexpression in cell cultures of V. amurensis on their capability to produce stilbenes were investigated. Overexpression of the VaMyb60 gene led to a considerable increase in the content of stilbenes in three independently transformed transgenic lines in 5.9–13.9 times, while overexpression of the VaMyb40 gene also increased the content of stilbenes, although to a lesser extent (in 3.4–4.0 times) in comparison with stilbene levels in the empty vector-transformed calli. Stilbene content and stilbene production in the VaMyb60-transgenic calli reached 18.8 mg/g of dry weight (DW) and 150.8 mg/L, respectively. Using HPLC analysis, we detected eight individual stilbenes: t-resveratrol diglucoside, t-piceid, t-resveratrol, ε-viniferin, δ-viniferin, cis-resveratrol, cis-piceid, t-piceatannol. T-resveratrol prevailed over other stilbenoid compounds (53.1–89.5% of all stilbenes) in the VaMyb-overexpressing cell cultures. Moreover, the VaMyb40- and VaMyb60-transformed calli were capable of producing anthocyanins up to 0.035 mg/g DW, while the control calli did not produce anthocyanins. These findings show that the VaMyb40 and VaMyb60 genes positively regulate the stilbene biosynthesis as strong positive transcription regulators and can be used in biotechnological applications for stilbene production or high-quality viticulture and winemaking.

Key message Sustainability and safety aspects of plant cell cultures as food are presented. Applicability of dairy side streams as carbon source and use of natural growth enhancers in cultivation are shown. Abstract Biotechnologically produced cellular products are currently emerging to replace and add into the portfolio of agriculturally derived commodities. Plant cell cultures used for food could supplement current food production. However, still many aspects need to be resolved before this new food concept can enter the market. Issues related to sustainability and safety for human consumption are relevant for both consumers and regulators. In this study, two plant cell cultures, deriving from arctic bramble (Rubus arcticus) and birch (Betula pendula), were cultivated using lactose-rich dairy side streams as alternative carbon sources to replace sucrose. Biomasses were comparable to those of original plant cell culture media when up to 83% and 75% of the original sucrose was replaced by these side streams for arctic bramble and birch cell cultures, respectively. Furthermore, nutritional composition or sensory properties were not compromised. Synthetic plant growth regulators were replaced by natural components, such as coconut water and IAA for several subculture cycles. Finally, it was shown that only trace amounts of free growth regulators are present in the cells at the harvesting point and assessment by freshwater crustaceans assay indicated that toxicity of the cells was not exceeding that of traditionally consumed bilberry fruit.

Stilbenes are plant phenolics known to rapidly accumulate in grapevine and other plants in response to injury or pathogen attack and to exhibit a great variety of healing beneficial effects. It has previously been shown that several calmodulin-like protein (CML) genes were highly up-regulated in cell cultures of wild-growing grapevine Vitis amurensis Rupr. in response to stilbene-modulating conditions, such as stress hormones, UV-C, and stilbene precursors. Both CML functions and stilbene biosynthesis regulation are still poorly understood. In this study, we investigated the effect of overexpression of five VaCML genes on stilbene and biomass accumulation in the transformed cell cultures of V. amurensis. We obtained 16 transgenic cell lines transformed with the VaCML52, VaCML65, VaCML86, VaCML93, and VaCML95 genes (3–4 independent lines per gene) under the control of the double CaMV 35S promoter. HPLC-MS analysis showed that overexpression of the VaCML65 led to a considerable and consistent increase in the content of stilbenes of 3.8–23.7 times in all transformed lines in comparison with the control calli, while biomass accumulation was not affected. Transformation of the V. amurensis cells with other analyzed VaCML genes did not lead to a consistent and considerable effect on stilbene biosynthesis in the cell lines. The results indicate that the VaCML65 gene is implicated in the signaling pathway regulating stilbene biosynthesis as a strong positive regulator and can be useful in viticulture and winemaking for obtaining grape cultivars with a high content of stilbenes and stress resistance.

Stilbenes are plant phenolics known to rapidly accumulate in grapevine and other plants in response to injury or pathogen attack and to exhibit a great variety of healing beneficial effects. It has previously been shown that several calmodulin-like protein (CML) genes were highly up-regulated in cell cultures of wild-growing grapevine Vitis amurensis Rupr. in response to stilbene-modulating conditions, such as stress hormones, UV-C, and stilbene precursors. Both CML functions and stilbene biosynthesis regulation are still poorly understood. In this study, we investigated the effect of overexpression of five VaCML genes on stilbene and biomass accumulation in the transformed cell cultures of V. amurensis. We obtained 16 transgenic cell lines transformed with the VaCML52, VaCML65, VaCML86, VaCML93, and VaCML95 genes (3–4 independent lines per gene) under the control of the double CaMV 35S promoter. HPLC-MS analysis showed that overexpression of the VaCML65 led to a considerable and consistent increase in the content of stilbenes of 3.8–23.7 times in all transformed lines in comparison with the control calli, while biomass accumulation was not affected. Transformation of the V. amurensis cells with other analyzed VaCML genes did not lead to a consistent and considerable effect on stilbene biosynthesis in the cell lines. The results indicate that the VaCML65 gene is implicated in the signaling pathway regulating stilbene biosynthesis as a strong positive regulator and can be useful in viticulture and winemaking for obtaining grape cultivars with a high content of stilbenes and stress resistance.

International audience; The growing knowledge in genetics, epigenomics, epitranscriptomics, and the 3D - or even 4D - genome structure provides an increasing number of detection targets that can be used to identify species or genetic lines, whether modified or not. Biotic and abiotic stresses also induce numerous unintentional genetic, epigenetic, and epitranscriptomic modifications. Those changes are transmissible and can be ordered in regions and classified. The detection target is characterised by the mutagenesis technique used. For instance, the detection of transgenic GMO or SDN3 modification of New Breeding Techniques (NBTs) will target their insertion's junction fragments into the genome. Each insertion induces epigenetic, and probably epitranscriptomic, changes which can also be targeted. In addition, one group of markers is linked to the trait(s) introduced or modified by the breeder whose sequence could be used in quantification and "screening". The other target will be a subset of the elements of a matrix approach (as described in the previous chapter). General selection markers, such as those used for plant breeding, together with mutagenesis techniques specific markers, could differentiate genetically modified organisms (GMOs) of any origin. They can be used to quantify and certify, through a global approach to the organism, that the trait modification is artefactual and not "natural." The growing mastery of single-cell sequencing techniques should soon make it possible to differentiate the modifications due, for example, to each step of a Crispr-Cas transformation of cells in culture. This chapter will focus mainly on detection targets based on nucleic acids, DNA, RNA, modified or unmodified, for their routine use in private and enforcement detection laboratories to comply with food labelling and European traceability rules. The chapter use the wording “hidden” GMOs and “new” GMOs as defined by the French NGOs and farmers’ union at the origin of the 2018 European Court of Justice ruling.

Thevetia peruviana is a medicinal plant that has valuable secondary terpenoid-type metabolites and phenolic compounds. Some flavonoid compounds of pharmaceutical interest stand out in the latter group. The concentration of these bioactive compounds in natural conditions is limited by environmental; therefore, it has been considered necessary to make in vitro plant cell suspension cultures that admit the use of elicitors to increase the content of active principles. Accordingly, in this study, for the optimization of flavonoid production in cell suspension culture of T. peruviana, different parameters related to elicitation with methyl jasmonate (MeJa), and salicylic acid (SA) were evaluated, at stirred flask scale. Firstly, 3 μM MeJa and 300 μM SA were added separately in cell cultures of T. peruviana, to assess their potential effects. Secondly, several experimental conditions were evaluated, for optimization purpose. In the first part, MeJa and SA increased the total flavonoid content, in 1.07 and 1.3 times, respectively, compared to the control culture; in the second part, total flavonoid content produced in MeJa mediated cell suspension cultures were 4.14 mg QE/g DW (milligrams of quercetin equivalent per gram of dry biomass) with: concentration 0.3 μM, addition time day 5 and harvest time 90 h. On the other hand, total flavonoid content produced in SA mediated cell suspension cultures were 3.75 mg QE/g DW with: concentration 100 μM, addition time day 0 and harvest time 96 h. Elicitation of cell suspension cultures of T. peruviana with MeJa and SA under their ideal parameter values increased flavonoid content.

Cultivated cardoon (Cynara cardunculus var. altilis L.) is a promising candidate species for the development of plant cell cultures suitable for large-scale biomass production and recovery of nutraceuticals. We set up a protocol for Agrobacterium tumefaciens-mediated transformation, which can be used for the improvement of cardoon cell cultures in a frame of biorefinery. As high lignin content determines lower saccharification yields for the biomass, we opted for a biotechnological approach, with the purpose of reducing lignin content, we generated transgenic lines overexpressing the Arabidopsis thaliana MYB4 transcription factor, a known repressor of lignin/flavonoid biosynthesis. Here, we report a comprehensive characterization, including metabolic and transcriptomic analyses of AtMYB4 overexpression cardoon lines, in comparison to wild type, underlining favorable traits for their use in biorefinery. Among these, the improved accessibility of the lignocellulosic biomass to degrading enzymes due to depletion of lignin content, the unexpected increased growth rates, and the valuable nutraceutical profiles, in particular for hydroxycinnamic/caffeoylquinic and fatty acids profiles.

The biomass of plant cell culture can serve as an important source of enviromentaly friendly herbal medicine. Moreover, it is considered a food source of the controlled high quality enriched with microelements, vitamins and other biologically active substances. The use of plant in vitro culture enables the strict control of the cultivation conditions in order to optimize the way of accumulation of the valuable substances, to select the plant lines, characterized by the increased productivity of the medicinal substances, and to standardize the conditions for growing crops in order to increase the yield of the target substances. Basing on the published data of the high level of polyfructans in the seeds of fenugreek, the aim of our work was to initiate the callus, suspension cultures and "hairy" root culture, to measure the polyfructan content in the obtained cultures and evaluate the possibility of usage of the plant in vitro culture for biotechnological production of polyfructans. In order to initiate callus and suspension culture the 14-day hypocotyl fenugreek explants of were cultivated on the nutrient medium with 2 mg/l 2,4-dichlorophenoacetic acid. Agrobacterium rhizogenes-mediated transformation of the hypocotyl fenugreek explants was carried out according to the commonly accepted method. To confirm the transgenic nature of the obtained "hairy root" culture, the PCR analysis was performed. The polyfructan content in the extracts of the samples of the initiated callus, suspension cultures and "hairy" root culture was mesuared by the method of McRarry and Slatery, which is based on Selevanov reaction. The highest polyfructan level (up to 0.9% FW) was shown for the obtained Ri-root culture, which corresponds the fructan rate in fenugreek seeds. At the same time, the "hairy root" culture is characterized by fast unlimited growth rate, so that it seems much more promising source for their production. The lower level of fructans was demonstrated for the suspension culture and callus culture, possibly due to the low cell differentiation activity in these cultures., Важливим джерелом отримання екологічно чистої сировини лікарських рослинних препаратів може бути біомаса культивованих клітин. Вона є також джерелом харчової сировини контрольованої високої якості, за необхідності збагаченої мікроелементами, вітамінами, іншими біологічно активними речовинами. Використання рослинної культури in vitro дозволяє за строго контрольованих умов визначити особливості накопичення цінних речовин, відібрати лінії рослин, що характеризуються підвищеною продуктивністю цінних лікарських речовин, а також стандартизувати умови вирощування культур з метою підвищення рівня виходу цільових речовин. Зважаючи дані щодо високого рівня вмісту поліфруктанів у насінні гуньби сінної, метою нашої роботи було ініціювати утворення калусної, суспензійної культури та культури «бородатих» коренів, виміряти вміст поліфруктанів у отриманих культурах та оцінити можливість використання рослинної культури in vitro для біотехнологічного виробництва поліфруктанів. З метою ініціації калусної та суспензійної культури 14-денні гіпокотильні експланти гуньби сінної культивували у живильному середовищі з додаванням 2 мг/л 2,4-дихлорфенооцтової кислоти. Agrobacterium rhizogenes-опосередковану трансформацію гіпокотильних експлантів гуньби проводили за загально прийнятою методикою. Для підтвердження трансгенної природи отриманої культури «бородатих» коренів проводили ПЛР аналіз. Вміст поліфруктанів у екстрактах ініційованих калусних, суспензійних культур та зразків культури «бородатих» коренів визначали за методом Мак-Рері та Слатері, який базується на реакції Селеванова. Виявлено, що найбільше поліфруктанів – до 0,9% сирої ваги – міститься у культурі Ri-коренів, що відповідає даним вмісту фруктанів у насінні. В той же час культура «бородатих» коренів характеризується швидким необмеженим ростом та є значно рентабельнішою у виробництві. Менше фруктанів містилося у суспензійній культурі та калюсній культурі, можливо, через низьку диференціацію клітин у даних культурах.

Metabolite profiling of plant cell cultures under stress is an important approach towards the understanding of the biology of stress responses in plants by identifying by-products of the stress metabolism, either stress signal transduction molecules or molecules that are involved in adaptive responses. To model in a comprehensive way such complex biological phenomena, it is important to analyse the time-dependent behaviour of the cell metabolism in response to external stimuli. A single point measurement indeed gives only a snapshot of the cellular state in which all the dynamical information is not captured. In this respect, high resolution time-course experiments are expected to provide more information in the phase of data analysis than a series of measurements collected only at a few time-points. The main objective of this thesis is to study the metabolic response of grapevine cell cultures subjected to an external stimulus, designed to mimic the interaction of the plant with environment factors. The PhD thesis firstly describes the optimization of the sample preparation method, extraction protocol and targeted metabolomics analysis of compounds isolated from the grape cells. This optimized metabolomics protocol was then applied to study the long-term dynamic response of elicited grapevine (Vitis vinifera cv. Gamay) cell suspension cultures treated by methyl jasmonate or sodium nitroprusside. Generalized additive models were applied to study the time dependent profile of secondary metabolites in grapevine cell suspension cultures. The results suggested that this class of non-parametric statistical models in combination with metabolomics could be an effective tool to explore the complete time-dependent changes of secondary metabolites and their responses to external stimuli in plant cells and foundation for further integrative omics studies., Inhaltsstoffanalysen (metabolite profiling) in pflanzlichen Zellkulturen, die sich unter Stress befinden, tragen nicht unerheblich zum Verständnis der biologischen Mechanismen der Stressantworten der Pflanzen durch die Identifizierung von Produkten des Stressstoffwechsels, der Signaltransduktion oder von Molekülen, die an adaptiven Reaktionen beteiligt sind, bei. Darüber hinaus kann die zielgerichtete (targeted) und nicht zielgerichtete (non-targeted) Inhaltsstoffanalyse heutzutage eine komplexe Sicht auf die Reaktion des pflanzlichen Stoffwechsels auf einen bestimmten Stimulus liefern. Um solche komplexen biologischen Phänomene umfassend zu modellieren, ist es wichtig das zeitabhängige Verhalten des Zellstoffwechsels als Reaktion auf äußere Reize zu analysieren. Eine Einzelpunktmessung liefert tatsächlich nur eine Momentaufnahme des zellulären Zustands, in dem nicht alle dynamischen Informationen erfasst werden. In dieser Hinsicht wird erwartet, dass die Inhaltsstoffanalysen von hochaufgelösten Zeitprofilen in der Phase der Datenanalyse weit mehr Informationen liefert als eine Reihe von Messungen, die nur zu wenigen Zeitpunkten gesammelt wurden. Das Hauptziel dieser Arbeit ist die Untersuchung der metabolischen Reaktion von Weinzellkulturen nach Gabe eines externen Stimulus, der die Interaktion mit Umweltfaktoren nachahmt. Die vorliegende Doktorarbeit beschreibt zunächst die Optimierung der Probenvorbereitungsmethode, des Extraktionsprotokolls und der gezielten Inhaltsstoffanalyse der aus den Traubenzellen isolierten Verbindungen. Dieses optimierte Protokoll wurde dann angewendet, um die langfristige dynamische Antwort von aus Weinreben (Vitis vinifera cv. Gamay) gewonnenen Zellsuspensionskulturen zu untersuchen. Das biologische System wurde unter der Behandlung von Methyljasmonat oder Natriumnitroprussid untersucht. Generalisierte additive Modelle wurden angewendet, um das zeitabhängige Profil von Sekundärmetaboliten in den Suspensionskulturen zu untersuchen. Die Ergebnisse deuten darauf hin, dass der Ansatz, generalisierter additiver Modelle und Inhaltsstoffanalysen anzuwenden, eine effektive Methode sein könnte, um die vollständigen zeitabhängigen Veränderungen von Sekundärmetaboliten und deren Reaktionen auf externe Stimuli in Pflanzenzellen zu untersuchen und so die Grundlagen für weitere integrative Omics-Studien zu legen.

Cannabis sativa L. is one of the most-studied species for its phytochemistry due to the abundance of secondary metabolites, including cannabinoids, terpenes and phenolic compounds. In the last decade, fiber-type hemp varieties have received interest for the production of many specialized secondary metabolites derived from the phenylpropanoid pathway. The interest in these molecules is due to their antioxidant activity. Since secondary metabolite synthesis occurs at a very low level in plants, the aim of this study was to develop a strategy to increase the production of such compounds and to elucidate the biochemical pathways involved. Therefore, cell suspensions of industrial hemp (C. sativa L. var. Futura) were produced, and an advantageous elicitation strategy (methyl jasmonate, MeJA) in combination with precursor feeding (tyrosine, Tyr) was developed. The activity and expression of phenylalanine ammonia-lyase (PAL) and tyrosine aminotransferase (TAT) increased upon treatment. Through 1H-NMR analyses, some aromatic compounds were identified, including, for the first time, 4-hydroxyphenylpyruvate (4-HPP) in addition to tyrosol. The 4-day MeJA+Tyr elicited samples showed a 51% increase in the in vitro assay (2,2-diphenyl-1-picrylhydrazyl, DPPH) radical scavenging activity relative to the control and a 80% increase in the cellular antioxidant activity estimated on an ex vivo model of human erythrocytes. Our results outline the active metabolic pathways and the antioxidant properties of hemp cell extracts under the effect of specific elicitors.

Many medicinal plant species are currently threatened in their natural habitats because of the growing demand for phytochemicals worldwide. A sustainable alternative for the production of bioactive plant compounds are plant biofactories based on cell cultures and organs. In addition, plant extracts from biofactories have significant advantages over those obtained from plants, since they are free of contamination by microorganisms, herbicides and pesticides, and they provide more stable levels of active ingredients. In this context, we report the establishment of Satureja khuzistanica cell cultures able to produce high amounts of rosmarinic acid (RA). The production of this phytopharmaceutical was increased when the cultures were elicited with coronatine and scaled up to a benchtop bioreactor. S. khuzistanica extracts enriched in RA were found to reduce the viability of cancer cell lines, increasing the sub-G0/G1 cell population and the activity of caspase-8 in MCF-7 cells, which suggest that S. khuzistanica extracts can induce apoptosis of MCF-7 cells through activation of the extrinsic pathway. In addition, our findings indicate that other compounds in S. khuzistanica extracts may act synergistically to potentiate the anticancer activity of RA.

All parts of Neem (Azadirachta indica A. Juss) show a broad spectrum efficacy against insect pests including insecticidal, anti-feedant or insect repellent activities. Several studies have shown that plant cell cultures can produce azadirachtins. We induced more than 40 novel Neem cell lines in modified Murashige and Skoog (MS) media containing different concentrations of auxins and cytokinins. To enhance the Azadirachtin production from 1 mg/l, it was necessary to optimize the media composition separately for biomass and secondary metabolite production. In light of this complex challenge we used our novel fully automated high-throughput microbioreactor system that allows us a fast and controlled batch and fedbatch screening in 48-well microtiter plates. There is increasing evidence that plants like Azadirachta indica contain endophytes which are able to colonize internal plant tissue without causing visible disease symptoms. The estimated high species diversity of endophytes suggests a rich and almost untapped source of new secondary metabolites. We isolated more than 340 endophytes from various plant tissues and tested if they were able to produce Azadirachtin-A. Here, we present data on isolation of endophytes and induction of callus as well as first results of our microbioreactor system.

The successful cultivation of plant cell and tissue cultures for the production of valuable chemical components requires the selection of an appropriate bioreactor. Selection criteria are determined based on a number of factors that are intrinsic to particular plant cell or tissue cultures and are influenced by the process objectives. Due to the specific properties of plant cell and tissue cultures, bioreactor systems may differ significantly from those used for microorganism or animal cell cultures. Furthermore, the differences from one plant culture to another can be immense; it is obvious that the optimal bioreactor system for a plant suspension cell culture is different to one for a plant tissue culture in many ways. General considerations are presented, and based on these key points, selection criteria are used to establish a “bioreactor chooser” tool. The particular details of the most relevant bioreactor types for plant cell and tissue cultures are listed and described. To produce valuable products, the process also needs to be scaled up to an economically justifiable size, which is usually done either by scaling up the size of the bioreactor itself or by bioreactor parallelization. Therefore, the most significant influencing factors are also discussed.

The growing demand for t-resveratrol for industrial uses has generated considerable interest in its production. Heterologous resveratrol production in plant cell suspensions, apart from requiring the introduction of only one or two genes, has the advantage of high biomass yield and a short cultivation time, and thus could be an option for large-scale production. Silybum marianum is the source of the flavonolignan silymarin. Phenylpropanoid synthesis in cultures of this species can be activated by elicitation with methyl jasmonate and methylated β-cyclodextrins, with products of the pathway (coniferyl alcohol and some isomers of the silymarin complex) being released into the medium. Given that stilbene synthase shares the same key precursors involved in flavonoid and /or monolignol biosynthesis, we explored the potential of metabolically engineered S. marianum cultures for t-resveratrol production. Cell suspensions were stably transformed with Vitis vinifera stilbene synthase 3 and the expression of the transgene led to extracellular t-resveratrol accumulation at the level of milligrams per litre under elicitation. Resveratrol synthesis occurred at the expense of coniferyl alcohol. Production of silymarin was less affected in the transgenic cultures, since the flavonoid pathway is limiting for its synthesis, due to the preferred supply of precursors for the monolignol branch. The fact that the expressed STS gene took excessively produced precursors of non-bioactive compounds (coniferyl alcohol), while keeping the metabolic flow for target secondary compounds (i.e. silymarin) unaltered, opens a way to extend the applications of plant cell cultures for the simultaneous production of both constitutive and foreign valuable metabolites. This work has been supported by a grant from the Spanish Ministry of Science and Innovation (BIO2014-51861-R). Diego Hidalgo is a predoctoral fellow of Mexican CONACYT.

Plant cell cultures as valuable tools for the production of specific metabolites can be greatly improved by the application of elicitors including cyclodextrins (CDs) for enhancing the yields of the desired plant compounds. Here the effects of 2,6-dimethyl-β-cyclodextrins (DIMEB) on the production of carotenoids and quinones from Artemisia annua L. cell suspension cultures were investigated. The addition of 50 mM DIMEB induced an early increase of intracellular carotenoid and quinone contents, which could be observed to a higher extent for lutein (10-fold), Q9 (3-fold) and Q10 (2.5-fold). Real Time PCR analysis revealed that the expression of 1-deoxy-d-xylulose-5-phosphate reductoisomerase (DXR) gene in DIMEB treated cell cultures after three days was 2.5-fold higher than in untreated samples, thus suggesting that the DIMEB induced increase of carotenoids and quinones could be due to the induction of the plastidial isoprenoid biosynthetic route. In addition, the DIMEB treatment induced an enhanced release of carotenoids and quinones into the culture medium of A. annua cell suspension cultures possibly due to the ability of CDs to form inclusion complexes with hydrophobic molecules.

The last decades witnessed a great demand of natural remedies. As a result, medicinal plants have been increasingly cultivated on a commercial scale, but the yield, the productive quality and the safety have not always been satisfactory. Plant cell cultures provide useful alternatives for the production of active ingredients for biomedical and cosmetic uses, since they represent standardized, contaminant-free and biosustainable systems, which allow the production of desired compounds on an industrial scale. Moreover, thanks to their totipotency, plant cells grown as liquid suspension cultures can be used as “biofactories” for the production of commercially interesting secondary metabolites, which are in many cases synthesized in low amounts in plant tissues and differentially distributed in the plant organs, such as roots, leaves, flowers or fruits. Although it is very widespread in the pharmaceutical industry, plant cell culture technology is not yet very common in the cosmetic field. The aim of the present review is to focus on the successful research accomplishments in the development of plant cell cultures for the production of active ingredients for cosmetic applications.

Cannabis sativa L. is one of the most-studied species for its phytochemistry due to the abundance of secondary metabolites, including cannabinoids, terpenes and phenolic compounds. In the last decade, fiber-type hemp varieties have received interest for the production of many specialized secondary metabolites derived from the phenylpropanoid pathway. The interest in these molecules is due to their antioxidant activity. Since secondary metabolite synthesis occurs at a very low level in plants, the aim of this study was to develop a strategy to increase the production of such compounds and to elucidate the biochemical pathways involved. Therefore, cell suspensions of industrial hemp (C. sativa L. var. Futura) were produced, and an advantageous elicitation strategy (methyl jasmonate, MeJA) in combination with precursor feeding (tyrosine, Tyr) was developed. The activity and expression of phenylalanine ammonia-lyase (PAL) and tyrosine aminotransferase (TAT) increased upon treatment. Through 1H-NMR analyses, some aromatic compounds were identified, including, for the first time, 4-hydroxyphenylpyruvate (4-HPP) in addition to tyrosol. The 4-day MeJA+Tyr elicited samples showed a 51% increase in the in vitro assay (2,2-diphenyl-1-picrylhydrazyl, DPPH) radical scavenging activity relative to the control and a 80% increase in the cellular antioxidant activity estimated on an ex vivo model of human erythrocytes. Our results outline the active metabolic pathways and the antioxidant properties of hemp cell extracts under the effect of specific elicitors.

The comparison between the structured and unstructured models was performed in order to determine the mathematical approach that best fit the biological synthesis of anthocyanins from plant cell cultures. A structured model was used for the production of secondary metabolites using cell cultures of strawberry (Fragaria ananassa) taking into account the cell viability of pigmented and non-pigmented cells. Moreover, growth and anthocyanin production of cell suspensions of grape (Vitis vinifera) were evaluated by an unstructured model. Both models adequately described the growth of plant cells and substrate uptake. However, anthocyanin production is intrinsically related to cell growth, especially degradation thereof; therefore the structured model was selected to describe the production of this secondary metabolite. La comparación entre los modelos estructurados y no estructurados se realizó con el fin de determinar la aproximación matemática que mejor se ajustara a la síntesis biológica de antocianinas a partir de cultivos de células vegetales. Se utilizó un modelo estructurado para producir metabolitos secundarios empleando cultivos celulares de fresa (Fragaria ananassa), teniendo en cuenta la viabilidad celular de las células pigmentadas y no pigmentadas. Por otra parte, el crecimiento y la producción de antocianinas de las suspensiones celulares de uva (Vitis vinífera) se evaluaron por medio de un modelo no estructurado. Ambos modelos describieron adecuadamente el crecimiento de las células vegetales y la captación de sustrato. Sin embargo, la producción de antocianinas está relacionada intrínsecamente con el crecimiento celular, sobre todo su degradación; por lo tanto, se seleccionó el modelo estructurado para describir la producción de este metabolito secundario.

The growing interest in rosmarinic acid (RA), an ester of caffeic acid and 3,4‐dihydroxyphenyl lactic acid, is due to its biological activities, which include cognitive‐enhancing effects, slowing the development of Alzheimer's disease, cancer chemoprotection, and anti‐inflammatory activity. Inspired by the challenge of meeting the growing demand for this plant secondary metabolite, we developed a biotechnological platform based on cell suspension cultures of Satureja khuzistanica. The high amounts of RA produced by this system accumulated mainly inside the cells. To further improve production, two elicitors, 100 μM methyl jasmonate (MeJA) and 40 mM cyclodextrin (CD), were tested, separately and together. MeJA increased RA productivity more than 3‐fold, the elicited cultures achieving an RA production of 3.9 g L−1 without affecting biomass productivity. CD did not have a clear effect on RA production, and under the combined treatment of MeJA + CD only a small amount of RA was released to the medium. When the cell culture was transferred from a shake flask to a wave‐mixed bioreactor, a maximum RA production of 3.1 g L−1 and biomass productivity of 18.7 g L−1 d−1 was achieved under MeJA elicitation, demonstrating the suitability of S. khuzistanica cell suspensions for the biotechnological production of this bioactive plant secondary metabolite.

Plant cell cultures are valuable tools for the production of bioactive compounds. Recently, many efforts have been made in order to develop and optimize strategies for increasing the yields of the desired plant metabolites by eliciting their biosynthesis or improving the efficiency of product recovery. We established Artemisia annua L. (Asteraceae) cell suspension cultures to investigate the biosynthesis of the antimalarial artemisinin (AN) [1] and other healthful phytochemicals such as carotenoids and quinones. The use of culture supplementation with cyclic oligomers ?-cyclodextrins was successful to significantly increase intracellular and extracellular levels of AN, lutein, Q9 and Q10 [2, 3]. In order to investigate the mechanism underlying these effects, other oligosaccharides having a linear structure were explored as putative elicitors in A. annua cell suspension cultures. For this purpose, oligogalacturonides (OGA) were obtained by hydrolysis of polygalacturonic acid with pectolyase from Aspergillus japonicus. Various OGA fractions were used for culture medium supplementation at different concentrations. The results showed that the 4-5 OGA fraction induced significant increases of AN and the intermediate dihydroartemisinic acid. These results suggest that the ability of ?-CDs to elicit the production of bioactive compounds in A. annua cell cultures could be due to their chemical similarity to pectic oligosaccharides often occurring in plants after fungal infection.

High temperatures are a significant stress factor for plants. In fact, many biochemical reactions involved in growth and development are sensitive to temperature. In particular, heat stress (HS) represents a severe issue for plant productivity and strategies to obtain high yields under this condition are important goals in agriculture. While selenium (Se) is a nutrient for humans and animals, its role as a plant micronutrient is still questioned. Se can prevent several abiotic stresses (drought, heat, UV, salinity, heavy metals), but the action mechanisms are poorly understood. Se seems to regulate reactive oxygen species (ROS) and to inhibit heavy metals transport. In addition, it has been demonstrated that Se is essential for a correct integrity of cell membranes and chloroplasts, especially the photosynthetic apparatus. Previous results showed that in tobacco (Nicotiana tabacum cv. Bright-Yellow 2) cultures HS (5 min at 50 &deg, C) induced cell death with apoptotic features, accompanied by oxidative stress and changes in the levels of stress-related proteins. In this work we investigated the effect of Se on the responses induced by HS. The obtained results show that Se markedly reduces the effects of HS on cell vitality, cytoplasmic shrinkage, superoxide anion production, membrane lipids peroxidation, activity of caspase-3-like proteases, and the levels of some stress-related proteins (Hsp90, BiP, 14-3-3s, cytochrome c).

Fluoroaromatic compounds are used as agrochemicals and released into environment as pollutants. Glycosylation of 2-, 3-, and 4-fluorophenols using plant cell cultures of Nicotiana tabacum was investigated to elucidate their potential to metabolize these compounds. Cultured N. tabacum cells converted 2-fluorophenol into its beta-glucoside (60%) and beta-gentiobioside (10%). 4-Fluorophenol was also glycosylated to its beta-glucoside (32%) and beta-gentiobioside (6%) by N. tabacum cells. On the other hand, N. tabacum glycosylated 3-fluorophenol to beta-glucoside (17%).

Calcium has been largely hypothesized to be involved in the arbuscular mycorrhizal (AM) signal transduction. A transient elevation in cytosolic Ca2+ concentration is considered one of the earliest responses detected in plant cells challenged with different stimuli. In this paper the detection of Ca2+ transients was selected as a tool to analyse whether plant cells perceive diffusible molecules released by the AM fungus Glomus intraradices. An aequorin-transformed line of soybean cells was used as experimental system. When cells were treated with the medium in which Gl. intraradices spores germinated, a rapid and significant increase in cytosolic Ca2+ level was induced and dissipated within 30 min. The medium did not induce either intracellular accumulation of reactive oxygen species or cell death. A detectable Ca2+ elevation was triggered also by the medium of fungal extraradical mycelium, associated to mycorrhizal hairy roots of carrot. These results indicate that Gl. intraradices releases diffusible molecules that are sensed by host plant cells through a Ca2+-mediated signalling during different steps of its life cycle. Key words: aequorin method, arbuscular mycorrhizal symbiosis, calcium signalling, Glomus intraradices, plant cell cultures.

Plant cell cultures represent a high valuable source for the production of bioactive secondary metabolites which can be used in food industry, medicine and cosmetic industry. In our study, we focused on obtaining phenolic acids from plant cell cultures. We compared cell cultures obtained from nine plant species of two families with a high potential for the production of phenolic substances. The most productive cell cultures for phenolic acids were Artemisia frigida and Silybum marianum. Isochlorogenic acid and chlorogenic acid were the most abundant phenolic acids identified in the analyzed cell cultures. Cell culture of Nicotiana tabacum contained only one hydroxycinnamic acid derivative in low amounts. Altogether S. marianum cell culture was verified as the most suitable for production of phenolic acids.Keywords: Plant cell cultures, secondary metabolite, phenolic acids, chlorogenic acid

Over the past few years, the increasing demand for natural products in the food industry has encouraged remarkable efforts towards the development of biotechnological processes for the production of flavour compounds. The present paper reviews the recent major achievements reported in this field, with a special emphasis on the potential lying in plant cell, microbial cultures and enzyme technology for the production of a wide range of flavours. The use of solid-state fermentation as a means to improve economical feasibility of these processes is also considered.

Plant cell culture is traditionally viewed as a unique artificially created biological system represented a heterogenous population of dedifferentiated cells. This system undergoes a continuous process of autoselection based on the intensity and stability of cell proliferation. Obviously, secondary metabolism must be differ in cell culture compared to the plant per ser, because in cell culture metabolites are synthesized and compartmentalized within a single proliferating heterotrophic cell with sparse or underdeveloped vacuoles and plastids. The specifics of formation and regulation of secondary metabolism biosynthesis in plant cells in vitro based on literature survey and our research results has been discussed. We investigated secondary metabolites formation in plant cell cultures of Panax spp., (ginsenosides); Dioscorea deltoidea (steroid glycosides); Ajuga reptans, Serratula coronata, Rhaponticum carthamoides (ecdisteroids); Polyscias spp., (triterpene glycosides), Taxus spp. (taxoids), Stevia rebaudiana (diterpene steviol-glycosides), Stephania glabra (alkaloids). They are some regular trends of secondary metabolites synthesis in the plant cell culture: It can be noted the stable synthesis of the compound promoting cell proliferation. Indeed, cell cultures of Dioscorea deltoidea were demonstrated to accumulate only furostanol glycosides, which promoted cell division. Furostanol glycoside content of Dioscorea strain DM-0.5 was up to 6 - 12% by dry biomass. Panax ginseng and P. japonicus plant cell cultures synthesize as minimum seven triterpene glycosides (ginsenosides), the productivity of these compounds was up to 6.0 - 8.0% on dry biomass. By contrast, the detectable synthesis of diterpene steviol-glycosides in cultivated cells of Stevia rebaudiana initiated in the mixotrophic cultures during chloroplast formation only. Despite these differences, or mainly due to them, plant cell cultures have become an attractive source of phytochemicals in alternative to collecting wild plants. It provides a guideline to bioreactorbased production of isoprenoids using undifferentiated plant cell cultures.

Cell suspension cultures of Linum album were established, which were able to synthesize and accumulate lignans. Podophyllotoxin and 5-methoxypodophyllotoxin were the main products and were present as glycosides, together with small amounts of deoxypodophyllotoxin, 5′-demethoxy-5-methoxypodophyllotoxin, lariciresinol, pinoresinol, matairesinol, α- and β-peltatin, as well as the monolignol glucoside, coniferin. In dark and light grown cultures, maximal product yields of lignans of up to 0.2% and 0.5% of the dry weight, respectively, were achieved, mainly consisting of podophyllotoxin.

In the present work the production of the human monoclonal Anti-HIV Antibody 2G12 in Nicotiana tabacum L. cv. BY-2 cell cultures has been optimised. Two independent cell lines have been used. The antibody produced from the cell line GFD was secreted into the apoplast, whereas the antibody produced from the cell line GFERD was localized in the endoplasmic reticulum. The main topics studied in this work were the analysis and characterisation of the cell lines, the development of a production medium by design of experiments, the modulation of the antibody production with mathematic modells and the process development and optimisation. In the first part the method for the selection of stable transgenic cell lines was investigated in more detail. The results showed that the the reporter protein DsRed can be used initially for identifying promising cell lines, however, a subsequent screening under production conditions should be done. The analysis of the cell line GFD#5 showed that this line was heterogeneous, i.e. oligoclonal, and that monoclonalization would be important to further increase the production yields. During the work, the cell line showed a drop in productivity, probably resulting from a stress response. The reduction of expression was epigenetic due to promoter methylation because productivity has been restored after cultivating exponentially growing cells in the presence of aza-cytidine. A new production medium called MSNP for Nicotiana tabacum L. cv. BY-2 cell cultures have been developed. The use of this medium led to an increase of the antibody yields in shaking flask as well as in bioreactor experiments: Cell line GFD#5, secreted mAb 2G12• 20-x higher concentration compared to the standard MS-medium • 2-x higher concentration compared to the already optimised MSN-Medium• No impact on the product quality: high activity, homogeneous N glycan-patternCell line GFERD#17, ER-retarded mAb 2G12• 2,6-x higher volumentic product yield compared to the standard MS-Medium• No impact on the product quality: high activity, homogeneous N glycan-patternFurthermore the use of design of experiments enabled the generation of mathematic models for predicting the extracellular 2G12-concentration and the ceell biomass. Process analytical technologies (PAT) have successfully been implemented within this work for Nicotiana tabacum L. cv. BY 2 suspension cell cultures. Dielectric spectroscopy was established as an inline biomass measurement and linear correlations between the conventional offline parameters such as the packed cell volume, the fresh weight and the dry weight and the corresponding conversation factor have been determined.The use of a fed-batch strategy with a mixed feed of sucrose and potassium nitrate enabled the successful uncoupling of the growth and production phase.

The application of cyclodextrins as precursor solubilizers in biotechnological processes, in which plant cells are involved, is new. In this paper the possibilities for cyclodextrin facilitated bioconversions by freely suspended and/or immobilized plant cells or plant enzymes are demonstrated. After complexation with beta-cyclodextrin, the phenolic steroid 17 beta-estradiol could be ortho-hydroxylated into a catechol, mainly 4-hydroxyestradiol, by a phenoloxidase from in vitro grown cells of Mucuna pruriens. By complexation with beta-cyclodextrin the solubility of the steroid increased from almost insoluble to 660 mu M. In addition, by complexation with beta-cyclodextrin, a solution of 3 mM coniferyl alcohol could be fed to cell cultures of Podophyllum hexandrum in order to enhance the accumulation of podophyllotoxin. Finally, the glucosylation of podophyllotoxin by cell cultures derived from Linum flavum was investigated. Four cyclodextrins: beta-cyclodextrin, gamma-cyclodextrin, hydroxypropyl-beta-cyclodextrin and dimethyl-beta-cyclodextrin were used to improve the solubility of podophyllotoxin. Dimethyl-beta-cyclodextrin met our needs the best and the solubility of podophyllotoxin could be enhanced from 0.15 to 1.92 mM. Podophyllotoxin-beta-D-glucoside was formed at a rate of 0.51 mmol l(-1) suspension per day by the L. flavum cells growing in the presence of 1.35 mM podophyllotoxin, complexed with dimethyl-beta-cyclodextrin.

Potato cDNA microarray slides (TIGR 1 K) were used to compare changes in gene expression of two potato cell populations subjected to shock or gradual exposure to PEG-mediated water stress. A total of 180 transcripts were found to be statistically up- or down-regulated in PEG-shocked or gradually stressed cells, compared to untreated control cells. Up-regulated genes in shocked cells were prevalently involved in carbohydrate metabolism, cellular communication, and signal transduction. In cells stressed gradually, the most represented induced genes were those involved in signal transduction, in response to environmental signals and in the regulation of transcription and translation machineries, including the heat shock protein 90-2, ribosomal and RNA-binding proteins. The expression of selected up-regulated genes was also tested in potato plants subjected to slow soil-drying conditions. Interestingly, the rgga gene, encoding an RNA-binding protein, was strongly induced in both leaves and roots of water-stressed potato plants, supporting its putative function in adaptive mechanisms to water stress. Pointing to differences in gene expression between shock-like and adaptive response, our findings might contribute to the controversial debate on the biological function of stress-responsive genes in cell damage repair or in restoring homeostasis and elaborating longer term responses for adaptation.

Cell-penetrating peptides are short cationic peptides with the property of translocating across the plasma membrane and transferring macromolecules otherwise unable to permeate cell membranes. We investigated the potential ability of the protein transduction domain derived from amino acids 47-57 of the human immunodeficiency virus type 1 (HIV-1) TAT (transactivator of transcription) protein to be used as a nanocarrier for the delivery of aequorin, a Ca(2+)-sensitive photoprotein widely used as a reliable Ca(2+) reporter in cell populations. The TAT peptide, either covalently linked to apoaequorin or ionically bound to plasmids encoding differentially targeted aequorin, was supplied to plant suspension-cultured cells. The TAT-aequorin fusion protein was found to be rapidly and effectively translocated into plant cells. The chimeric molecule was internalized in fully active biological form and at levels suitable to monitor intracellular Ca(2+) concentrations. Plant cells incubated for just 5 min with TAT-aequorin responded to different environmental stimuli with the expected Ca(2+) signatures. On the other hand, TAT-mediated plasmid internalization did not provide the necessary level of transformation efficiency to allow calibration of luminescence signals into Ca(2+) concentration values. These results indicate that TAT-mediated aequorin transduction is a promising alternative to traditional plant transformation methods to monitor intracellular Ca(2+) dynamics rapidly and effectively in plant cells.

The present paper reports the study of the optimal conditions for grape (Vitis vinifera cv. Barbera) cell suspensions in batch and fed-batch bioreactor cultures, in order to specifically improve the production of mono-glucosylated stilbenes, which are resveratrol derivatives. These compounds are physiologically as active as free resveratrol in cardio- and chemoprotection, but are more stable and bioavailable after ingestion through diet. In fed-batch conditions the production of mono-glucosides was considerably increased together with that of free resveratrol. For the first time, an elicitor (chitosan) was tested in a bioreactor system, demonstrating its efficacy in inducing the production of stilbenes. The bioreactor culture conditions also allowed the accumulation of other polyphenols, such as catechins. The vast majority of the produced compounds was released into the culture media, which represents a relevant advantage for the recovery of specific molecules or of polyphenol-enriched mixtures. These results represent a further step toward the employment of grape cells in fed-batch cultures, as a promising alternative to whole plant extraction for the industrial production of plant polyphenols, considering the necessity for developing sustainable processes.

Tocopherols, collectively known as vitamin E, are lipophilic antioxidants, essential dietary components for mammals and exclusively synthesized by photosynthetic organisms. Of the four forms (alpha, beta, gamma and delta), alpha-tocopherol is the major vitamin E form present in green plant tissues, and has the highest vitamin E activity. Synthetic alpha-tocopherol, being a racemic mixture of eight different stereoisomers, always results less effective than the natural form (R,R,R) alpha-tocopherol. This raises interest in obtaining this molecule from natural sources, such as plant cell cultures. Plant cell and tissue cultures are able to produce and accumulate valuable metabolites that can be used as food additives, nutraceuticals and pharmaceuticals. Sunflower cell cultures, growing under heterotrophic conditions, were exploited to establish a suitable in vitro production system of natural alpha-tocopherol. Optimization of culture conditions, precursor feeding and elicitor application were used to improve the tocopherol yields of these cultures. Furthermore, these cell cultures were useful to investigate the relationship between alpha-tocopherol biosynthesis and photomixotrophic culture conditions, revealing the possibility to enhance tocopherol production by favouring sunflower cell photosynthetic properties. The modulation of alpha-tocopherol levels in plant cell cultures can provide useful hints for a regulatory impact on tocopherol metabolism.

The nearly unlimited enzymatic potential of cultured plant cells can basically be employed for bioconversion purposes. Plant enzymes are able to catalyze regio- and stereospecific reactions and can therefore be applied to the production of compounds of pharmaceutical interest. Naturally occurring as well as related synthetic compounds may be used as precursors. A review of the current status of such bioconversions is given. It includes the performance of bioconversions by freely suspended and immobilized plant cells or enzyme preparations. In addition, the kinetic aspects of immobilized plant cells are discussed. Special attention is paid to the bioconversion of poorly or water insoluble precursors. Finally, a model scheme for the development of a commercially available drug, produced by bioconversion, and perspectives are discussed.

Hairy roots of Tagetes patula have been grown in aqueous two-phase systems. After selecting suitable polymers from single-phase experiments (in which salt phases were unable to support growth in the desired concentrations) several two-phase systems were tested for their influence on cell growth and thiophene production. Cell growth occurred in all aqueous two-phase systems, but the highest growth rate was achieved in normal medium. There was no difference in thiophene production between medium and aqueous two-phase systems. The partition of thiophenes favoured slightly the more hydrophobic top phase in most cases, while the cells were confined to the bottom phase. One aqueous two-phase system (15% polyethyleneglycol 10,000 and 15% Reppal PES 200) was tested in a stirred tank reactor with normal medium as a control. The growth rate in medium was higher than in the aqueous two-phase system, while the thiophene production per unit cell weight was in the same range for both systems. The excretion of thiophene...

Twenty-four callus, and eleven cell suspension, cultures were established from Turkish medicinal plants, and crude extracts prepared from them tested against microorganisms to assess their antimicrobial activities in vitro. Of the extracts tested, those belonging to the cell cultures of five of the plant species showed antibacterial activity against mainly three bacteria and a yeast. No activity was observed against herpes simplex viruses, HSV-I and II, but an extract from Hypericum capitatum showed a slight anti-retroviral activity against HIV-I.

Agrobacterium-mediated transient assays for the analysis of gene function are used as alternatives to genetic complementation and stable plant transformation. Although such assays are routinely performed in several plant species, they have not yet been successfully applied to grapevines. We explored genetic background diversity of grapevine cultivars and performed agroinfiltration into in vitro cultured plants. By combining different genotypes and physiological conditions, we developed a protocol for efficient transient transformations of selected grapevine cultivars. Among the four cultivars analyzed, Sugraone and Aleatico exhibited high levels of transient transformation. Transient expression occurred in the majority of cells within the infiltrated tissue several days after agroinfiltration and, in a few cases, it later spread to a larger portion of the leaf. Three laboratory strains of Agrobacterium tumefaciens with different virulence levels were used for agroinfiltration assays on grapevine plants. This method promises to be a powerful tool to perform subcellular localization analyses. Grapevine leaf tissues were transformed with fluorescent markers targeted to cytoplasm (free GFP and mRFP1), endoplasmatic reticulum (GFP::HDEL), chloroplast (GAPA1::YFP) and mitochondria (²::GFP). Confocal microscope analyses demonstrated that these subcellular compartments could be easily visualized in grapevine leaf cells. In addition, from leaves of the Sugraone cultivar agroinfiltrated with endoplasmic reticulum-targeted GFP-construct, stable transformed cells were obtained that show the opportunity to convert a transiently transformed leaf tissue into a stably transformed cell line. Electronic supplementary material The online version of this article (doi:10.1007/s00299-008-0510-4) contains supplementary material, which is available to authorized users.

Within the present thesis transgenic BY-2 cultures should be tested regarding their potential as industrial expression system for therapeutic proteins. For this purpose, BY-2 cultures expressing recombinant HSA under control of the CaMV 35SS promoter were developed. A leader peptide ensured the secretion of the protein to the apoplast. Main focus of the described studies are such parameters, which are of essential importance for the industry, like productivity, genetic stability of the organism as well as the production of a functional protein and the economy of the production process. In general the reproducibility of developed and optimised industrial production processes are ensured by cell stocks of the relevant organisms. Such cell banks guarantee especially the genetic stability of initially used organisms. Traditionally periodic subculturing in shake flasks performs the storing of plant cell cultures. Thus the cells are weekly supplied with fresh medium to maintain their vitality. During this kind of maintenance changes regarding expression and productivity of recombinant proteins could be detected. Hence the development of alternative storing opportunities is of crucial relevance for the industry. Within these studies a protocol for the long-time storage of transgenic BY-2 cultures was developed. In the process development media optimisation is a helpful tool to increase the productivity of the used organisms. Especially for plant cell cultures commercialised media are overaged and not defined for single cell lines. This indicates the enormous room of improvement, which could also be shown within these studies: by variation of the concentration of only one media component the extracellular HSA amount could be increased 17-fold. All results generated during shake flasks trials could be transferred to a larger scale performed in stirred tank bioreactors. The combination of two different enhancing substances did not yield in a summation of the effects during shake flask trials. In stirred tank bioreactors the combination of two enhancing substances increased the productivity by a factor of 20. Even if these results correspond to data, which were generated in other studies performed at the IME (data not shown), a generalization about the production of recombinant proteins in plant cell cultures is not allowed yet. The development and optimisation of a fermentation strategy for the effective production of recombinant proteins in plant cell cultures was an important part of this thesis. Different fermentation strategies, their variations as well as different process parameters should be investigated. All process strategies of industrial interest like the conventional batch fermentation as well as the continuous fermentation were applicable. An outstanding result was the continuous fermentation: during cultivation time of 50 days an extensively constant amount of HSA could be daily harvested. This thesis was supported by the RWTH Aachen Postgraduate Sponsorship for Young Scientists.

A 23-kDa antifungal thaumatin-like protein was isolated and purified from Cassia didymobotrya (Fres.) cell cultures for the first time. The protein was secreted in the culture medium, but it could be also isolated after elution of whole cells with a 0.5 M CaCl2 solution. Treatment of the cells with laminarin oligosaccharides or salicylic acid, but not with NaCl, resulted in enhancement of expression of the protein. A rapid purification protocol was used based on cationic exchange chromatography. The protein, with a highly basic character (pI 10), has an exact molecular mass of 23034 Da, as determined by MALDI-ToF mass spectrometry analysis. N-terminal sequencing of the intact polypeptide and the sequencing of two internal tryptic peptides indicated significant identity with other thaumatin-like proteins (TLP). The protein exerted antifungal activity towards some Candida species showing EC50 values comparable to those of other antifungal TLPs. The collected data lead to classify this TLP as a new PR-5 protein.

Posljednjih su godina sve veći zahtjevi za prirodnim proizvodima prehrambene industrije ohrabrili nevjerojatne pokušaje razvoja biotehnoloških procesa u proizvodnji aromatskih spojeva. Ovaj revijalni prikaz donosi nova dostignuća iz tog područja, osobito ističući mogućnosti primjene biljnih stanica i mikrobnih kultura te enzimske tehnologije u proizvodnji velikog broja aromatskih sastojaka hrane. Razmatra se i poboljšanje ekonomske isplativosti proizvodnje uzgojem mikroorganizama na čvrstoj podlozi., Over the past few years, the increasing demand for natural products in the food industry has encouraged remarkable efforts towards the development of biotechnological processes for the production of flavour compounds. The present paper reviews the recent major achievements reported in this field, with a special emphasis on the potential lying in plant cell, microbial cultures and enzyme technology for the production of a wide range of flavours. The use of solid-state fermentation as a means to improve economical feasibility of these processes is also considered.

Os estudos da biotransformação têm-se mostrado um método eficiente para reações que envolvam a obtenção de compostos com interesse comercial. Dentre estes produtos utilizados podemos destacar monoterpenos por constituírem uma variedade de compostos com grande aplicação tanto no âmbito farmacêutico, como em perfumes, cosméticos e alimentos. As suspensões de células vegetais e os fungos filamentosos têm sido largamente usados em reações de biotransformação destes metabólitos secundários como substratos exógenos. A habilidade das culturas celulares e fungos em transformar tais compostos vêm sendo dirigidos principalmente para a obtenção de derivados oxigenados de maior valor agregado, principalmente visando à produção de flavorizantes, aromatizantes e fragrâncias. As suspensões de células vegetais apresentam sistemas enzimáticos mais complexos, podendo levar a produtos específicos, enquanto os microrganismos, por serem mais simples e de crescimento mais rápido, geralmente promovem mais rapidamente a metabolização dos substratos.Neste contexto, o presente trabalho descreve a investigação do potencial de bioconversão dos monoterpenos (R)-(+)-α-pineno, (S)-(-)-α-pineno e (S)-(-)-β- pineno, bem como do óleo de terebentina, para avaliação de suas potencialidades frente a reações de biotransformação mediadas pelos microrganismos Cunninghamella echinulata ATCC 9245, Mortierella isabellina NRRL 1757 e Cunninghamella elegans ATCC 36112 e por suspensões de células vegetais de Catharanthus roseus. Todos os sistemas estudados mostraram habilidade na bioconversão dos substratos testados. Biotransformation has been an efficient method to obtain important commercial compounds such as monoterpenes, that have a range of applications in the pharmaceutical, perfumery, cosmetics and food industries. Plant cell cultures and filamentous fungi suspensions have been widely used in biotransformation reactions of monoterpenes as exogenous substrates. The ability of the cells and fungi cultures to transform these compounds has being mainly used for the synthesis of oxygenated derivatives aiming at the production of flavours and fragrances. The plant cell cultures suspensions have complex enzymatic systems with the production of specific compounds, where as filamentous fungi, which are structurally simpler and fast growing, can promote quicker transformations reactions. In this context, the present work describes the evaluation of bioconversion of the monoterpenes (R)-(+)-α-pinene, (S)-(-)-α-pinene, (S)-(-)-β-pinene, as well as turpentine oil using the filamentous fungi suspensions of Cunninghamella echinulata ATCC 9245, Cunninghamella elegans ATCC 36112 and Mortierella isabellina NRRL 1757, aswer as Catharanthus roseus plant cell cultures. All evaluated systems were capable of bioconverting the studied substrates. The main product obtained from pinenes and turpentine oil was α-terpineol.

Shoot, callus and hairy root cultures of Linum strictum ssp. strictum L. were initiated. The lignan 6-methoxypodophyllotoxin was detected and quantified., Uzgojena je kultura stanica mladica, kalusa i čupavog korijenja biljke Linum strictum ssp. strictum L. U njima je određena količina lignana 6-metoksipodofilotoksina.

[no abstract]

Plant enzymes are able to catalyze regio- and stereospecific reactions. Freely suspended and immobilized plant cells as well as enzyme preparations can therefore be applied for the production of pharmaceuticals by bioconversion, as such or in combination with chemical syntheses. This review paper deals with bioconversions of added precursors from natural or synthetic origin by several biocatalytic systems.

In this study, laboratory scale biotechnical production processes were developed for different types of plant cell cultures: embryogenic cell culture, undifferentiated cell suspension culture and hairy root culture. The production of somatic embryos, secondary metabolites and biomass were optimized. The growth as well as the production characteristics and the nutrient uptake of the different plant cell systems were determined. The suitabilities of different bioreactor configurations for the various culture types were tested in laboratory scale. In the optimization of growth and production, statistical experimental designs were used in addition to the conventional method of parallel testing of different media were used. The embryo production medium for an embryogenic cell line of birch (Betula pendula Roth) was optimized using a 22-factorial design. Factorial design was also tested for the optimization of alkaloid production medium for a cell suspension of Catharanthus roseus (L.) G. Don, but the conventional method of parallel testing of different media proved to be more successful. Factorial experimental design was used to determine the effects of low phosphate and sugar concentrations on strawberry (Fragaria x ananassa Duch.) hairy roots. In this way a slowly growing root system was obtained for infection studies with the native Finnish arbuscular mycorrhizal fungus, Glomus fistulosum V128. The embryogenic cell line of birch was cultivated in four different types of bioreactors: a sparged blade-stirred bioreactor, a diffusion-aerated bioreactor with a marine impeller (ChemCell, Chemap), an air-lift and a bubble column bioreactor. Embryo development was monitored during the cultivations by computer vision. The highest embryo production was achieved in the bubble column bioreactor. However, this was only 20% of the production in control shake flasks. In the mechanically stirred bioreactors production was even lower, indicating shear damage of the embryos. The suspension culture of Catharanthus roseus L. was cultivated in three different types of bioreactors: a standard aerated and stirred bioreactor (control bioreactor), a recirculation bioreactor in which part of the air is recirculated with a peristaltic pump, and a bubble column bioreactor. The recirculation bioreactor was used to mimic the gaseous regime of a shake flask. When cultured in a turbine stirred tank reactor aerated through a sparger and in the bubble column the cells did not grow well and the production of ajmalicine was low. When cultivated in the recirculation bioreactor the biomass concentration and production of ajmalicine were similar to the level observed in shake flasks. Hairy roots of C. roseus were cultivated in three different types of bioreactors: an air-sparged bioreactor with no stirring, an air-sparged bioreactor in which the medium is slowly circulated through the vessel with a peristaltic pump and a standard air-sparged and stirred bioreactor. The best biomass yield and root morphology were obtained in the air-sparged bioreactor with no stirring. The indole alkaloid contents of the roots cultivated in the bioreactor with stirring and in the bioreactor with medium circulation were significantly lower than that of the roots in the air-sparged bioreactor. Hairy roots of strawberry (Fragaria x ananassa Duch.) were cultivated in an air-sparged bioreactor with no stirring. The biomass yield was good and the roots were long and branching. The roots produced were used as a host for an endophyte, arbuscular mycorrhizal fungus, Glomus fistulosum V128.