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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. [ABSTRACT FROM AUTHOR]

Key message : Overexpression of both native and mutant forms of AtCPK1 in Rubia cordifolia cells increased anthraquinone production and transcript abundance of the RcIPPI, RcOSBL, RcOSBS , and RcICS genes to different extents. Abstract: Calcium-dependent protein kinases (CDPKs) are involved in various cell processes and are regulated by a calcium signal system. CDPKs also function in plant defense against stress factors such as pathogens, temperature, and salinity. In this study, we compared the effect of heterologous expression of two forms of the Arabidopsis AtCPK1 gene, native and constitutively active (Ca-independent), on anthraquinone production in transgenic Rubia cordifolia cells. Significant qualitative and quantitative differences were found in the content of anthraquinone derivatives in control and AtCPK1-transgenic calli. Expression of the AtCPK1 gene increased anthraquinone production by 3 and 12 times for native and constitutively active forms, respectively, compared with control cells. In addition, we identified and quantified the expression of genes encoding key enzymes of the anthraquinone biosynthesis pathway, including isochorismate synthase (ICS), o-succinylbenzoate synthase (OSBS), o-succinylbenzoate ligase (OSBL), and isopentenyl diphosphate isomerase (IPPi). In all AtCPK1-transgenic cell lines, expression of ICS, OSBS, OSBL, and IPPi increased considerably at 14-15 days of subculture and decreased at the end of cultivation (30 days). The results suggest that both native and constitutively active AtCPK1 forms induced anthraquinone accumulation at the logarithmic growth stage via enhancement of expression of genes involved in the metabolism of anthraquinones or their regulatory mechanisms. [ABSTRACT FROM AUTHOR]

Cell cultures of Arabidopsis thaliana ( A. t.) respond to changes in the gravitational field strength with fluctuations of the amount of cytosolic calcium (Ca). In parabolic flight experiments, where hyper- and μg phases follow each other, μg clearly increased Ca, while hyper-g caused a slight reduction. Since the latter observation had not been reported before, we studied this effect in more detail. Using a special centrifuge for heavy items (ZARM, Bremen, Germany), we determined the hyper-g-dependent intracellular Ca level with transgenic cell lines expressing the Ca sensor, cameleon. This sensor exhibits a shift in fluorescence from 480 to 530 nm in response to Ca binding. The data show a drop in the intracellular Ca concentration with a threshold gravity of around 3 g. This is above hypergravity levels achieved during parabolic flights (1.8 g). The use of mutants with different sub-cellular targets of cameleon expression (nucleus, tonoplast, plasma membrane) gave the same results, i.e. Ca is obviously exported from several intracellular compartments. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Somatic mutations of the nuclear and mitochondrial DNA and alterations in DNA methylation levels in mammals are well known to play important roles in ageing and various diseases, yet their specific contributions await further investigation. For plants, it has also been proposed that unrepaired DNA damage and DNA polymerase errors accumulate in plant cells and lead to increased somatic mutation rate and alterations in transcription, which eventually contribute to plant ageing. A number of studies also show that DNA methylation levels vary depending on the age of plant tissue and chronological age of a whole plant. Recent studies reveal that prolonged cultivation of plant cells in vitro induces single nucleotide substitutions and increases global DNA methylation level in a time-dependent fashion. Changes in DNA methylation are known to influence DNA repair and can lead to altered mutation rates, and, therefore, it is interesting to investigate both the genetic and epigenetic integrity in relationship to ageing in plants. This review will summarise and discuss the current studies investigating somatic DNA mutation and DNA methylation levels in relation to plant ageing and senescence. The analysis has shown that there still remains a lack of clarity concerning plant biological ageing and the role of the genetic and epigenetic instabilities in this process. [ABSTRACT FROM AUTHOR]

The metabolism of14C-clodinafop-propargyl (CfP) was examined in cell cultures of wheat (Triticum aestivumL. cv. ‘Heines Koga II’) and tobacco (Nicotiana tabacumL.). Besides the non-transgenic tobacco culture, cultures transformed separately with cDNA of human cytochrome P450-monooxygenases (P450s) CYP1A1, CYP1A2, CYP3A4, CYP2B6 and CYP2C19 were examined. Experiments with wheat were executed in the presence and absence of safener cloquintocet-mexyl (CqM). After 48 h of incubation, only about 10% of applied14C was found in media (both tobacco and wheat). Non-extractable residues of14C-CfP in wheat cells were 16.54% (without CqM) and 30.87% (with CqM). In all tobacco cultures, 82.41–92.46% of applied radioactivity was recovered in cell extracts. In contrast to wheat, non-extractable residues amounted only to 1.50–2.82%. As determined by radio-thin layer chromatography (TLC) and -high-performance liquid chromatography (HPLC), the parent CfP was not found in the cell extracts of wheat; in tobacco cell extracts, only traces of CfP were detected. After a hydrolysis of assumed carbohydrate conjugates of CfP derived polar14C-labeled compounds, TLC and HPLC analysis showed that in wheat, a more complex pattern of metabolites of CfP were observed as compared to all tobacco cultures. In hydrolysates resulting from wheat, the identity of three primary products was confirmed by means of GC-EI-MS: free acid clodinafop (Cf), hydroxy-Cf hydroxylated at the pyridinyl moiety, and 4-(5-chloro-3-fluoropyridin-2-yloxy)phenol. In hydrolysates derived from all tobacco cultures, main metabolite was Cf besides only traces of further unidentified products. Differences among the different tobacco cultures (non-transgenic, transgenic) did not emerge. According to kinetics of disappearance of primary metabolite Cf as well as formation of polar soluble products and non-extractable residues, metabolization of CfP proceeded at a noticeably higher rate in wheat cells treated with safener CqM than in cells without CqM treatment. Thus, these results indicated a stimulation of CfP's metabolism by CqM, although metabolic profiles observed in CqM treated and non-treated cells (after hydrolysis) were qualitatively similar. The findings obtained from all tobacco cultures suggested that with the exception of ester cleavage to Cf, CfP cannot be metabolized by tobacco itself or by the human P450s examined. [ABSTRACT FROM PUBLISHER]

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. [ABSTRACT FROM AUTHOR]

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.

Cell cultures of Mandevilla velutina have proved to be an interesting production system for biomass and secondary metabolites able to inhibit the hypotensive activity of bradykinin, a nonapeptide generated in plasma during tissue trauma. The crude ethyl acetate extract of cultured cells contains about 31- to 79-fold more potent anti-bradykinin compounds (e.g., velutinol A) than that obtained with equivalent extracts of tubers. Somaclonal variation may be an explanation for the wide range of inhibitor activity found in the cell cultures. The heterogeneity concerning morphology, differentiation, carbon dissimilation, and velutinol A production in M. velutina cell cultures is reported. Cell cultures showed an asynchronous growth and cells in distinct developmental stages. Meristematic cells were found as the major type, with several morphological variations. Cell aggregates consisting only of meristematic cells, differentiated cells containing specialized cell structures such as functional chloroplasts (cytodifferentiation) and cells with embryogenetic characteristics were observed. The time course for sucrose metabolism indicated cell populations with significant differences in growth and metabolic rates, with the highest biomass-producing cell line showing a cell cycle 60% shorter and a metabolic rate 33.6% higher than the control (F2 cell population). MALDI-TOF mass spectrometric analysis of velutinol A in selected cell lines demonstrated the existence of velutinol A producing and nonproducing somaclones. These results point to a high genetic heterogeneity in general and also in terms of secondary metabolite content.

The response of two Taxus cell systems to the action of cyclodextrin (CD) and coronatine (CORO), supplied to the culture medium either separately or together, was studied. Two-stage Taxus globosa and Taxus media cell cultures were established and the elicitors were added at the beginning of the second stage. Growth, taxane production, and the expression of known taxol biosynthetic genes, including the recently characterized CoA ligase gene, were studied. Although CORO reduced the growth capacity of both cell lines, CD apparently counteracted this negative effect. Taxane production was significantly enhanced by the simultaneous addition of CD and CORO to the medium. The total taxane production in the T. media cell line was more than double that of T. globosa , but in the latter more than 90% of the taxanes produced were excreted to the medium. Individual taxane patterns also differed: at the height of production, the main taxanes in T. globosa cultures were cephalomannine and 10-deacetyltaxol, and in T. media , taxol and baccatin III. The low transcript levels of taxane biosynthetic genes found in T. globosa cells mirrored the lower taxane production in these cultures, while a high expression was strongly correlated with a high taxane production in T. media . [ABSTRACT FROM AUTHOR]

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.

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-ß-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. [ABSTRACT FROM AUTHOR]

Global translational repression under abiotic stress influences translation of both endogenous and transgene mRNAs. Even in plant cell culture, hypoxia and nutrient deficient stress arise during the growth process. In this study, we first demonstrated the existence of global translational repression in Arabidopsis T87 cultured cells over a time course following inoculation. Next, we performed genome-wide analysis, which revealed that the translational states of endogenous mRNAs differed significantly between growth and stationary phase cells. This analysis showed that translation from most mRNAs was repressed upon stationary phase. Otherwise, a part of mRNA including alcohol dehydrogenase ( ADH ) gene was recalcitrant to the repression. Furthermore, by polysome analysis and followed quantitative reverse transcription PCR analysis of transformants having 5′untranslated regions (UTRs) of ADH or translationally repressed At3g47610 mRNA fused to reporter gene, we demonstrated that polysomal associations of reporter mRNAs were in accordance with those the mRNAs from which their 5′UTR derived, suggesting that the 5′UTR is an important determinant of the translational state of mRNAs in stationary phase cells. Finally, we demonstrated the effectiveness of 5′UTR of ADH mRNA in transformants derived from the BY-2 tobacco cell line. These results suggested that 5′UTR of ADH mRNA would be a useful element for efficient transgene expression upon stationary phase. [ABSTRACT FROM AUTHOR]

Highlights: [•] Dendrobium candidum protocorm suspension cultures in balloon type bubble bioreactors have been established. [•] Enhanced accumulation of biomass and bioactive compounds in bioreactor cultures was achieved. [•] The results of the current study are useful for the production of D. candidum bioactive compounds. [Copyright &y& Elsevier]

Plant cell and organ cultures have emerged as potential sources of secondary metabolites, which are used as pharmaceuticals, agrochemicals, flavors, fragrances, coloring agents, biopesticides, and food additives. In recent years, various strategies have been developed to assess biomass accumulation and synthesis of secondary compounds in cultures. Biomass accumulation and metabolite biosynthesis are two-stage events, and the parameters that control the growth and multiplication of cultured cells/organs and biomass accumulation are controlled in the first stage. Parameters that assist with the biosynthesis of metabolites are controlled in the second stage. The selection of high-producing cells or organ clones; optimization of medium parameters such as suitable medium, salt, sugar, nitrogen, phosphate, and plant growth regulator levels; and physical factors such as temperature, illumination, light quality, medium pH, agitation, aeration, and environmental gas (e.g., oxygen, carbon dioxide, and ethylene) are controlled in the first stage of the culture process. Elicitation, replenishment of nutrient and precursor feeding, permeabilization, and immobilization strategies assist with the accumulation of metabolites and can be applied in the second stage of the culture process. By following stage-specific strategies, it is possible to produce large amounts of biomass with an increase in the accumulation of secondary compounds. [ABSTRACT FROM AUTHOR]

Extracts of Salvia species are used in traditional medicine to treat various diseases. The economic importance of this genus has increased in recent years due to evidence that some of its secondary metabolites have valuable pharmaceutical and nutraceutical properties.The bioactivity of sage extracts is mainly due to their content of terpenes and polyphenols. The increasing demand for sage products combined with environmental, ecological and climatic limitations on the production of sage metabolites from field-grown plants have led to extensive investigations into biotechnological approaches for the production of Salvia phytochemicals. The purpose of this review is to evaluate recent progress in investigations of sage in vitro systems as tools for producing important terpenoids and polyphenols and in development of methods for manipulating regulatory processes to enhance secondary metabolite production in such systems. [ABSTRACT FROM AUTHOR]

Highlights: [•] Four-baffled flasks elicited mechanically the AQs synthesis in R. tinctorum cultures. [•] AQs obtained in the baffled flasks was similar to that obtained in the bioreactor. [•] Shear stress was similar in both culture systems, according to Nishikawa's method. [•] Darkness played an important role on AQs synthesis. [•] This down-scaled system is a valid instrument for the study of shear stress effects. [Copyright &y& Elsevier]

Plant cell suspension cultures can be used to make safe vaccines at a lower cost than conventional procedures. An inducible gene expression system provides an opportunity to optimize the conditions of vaccine production in a plant system. In this investigation, a dexamethasone-inducible Norwalk virus capsid protein (NVCP) gene expression system has been developed in cell suspension cultures for four different plant species: tobacco ( Nicotiana tabacum), rice ( Oryza sativa L.), cotton ( Gossypium hirsutum L.), and slash pine ( Pinus elliottii Engelm.) via Agrobacterium-mediated transformation. Resulting transgenic cell lines were confirmed by Southern blot analyses and NVCP gene expression was confirmed by Northern blot analysis. NVCP gene expression was observed in all 24 cell lines tested, but there were minor differences in transgene expression among the transgenic cell lines. The highest level of NVCP gene expression was observed 48 h after addition of the glucocorticoid hormone dexamethasone (10 mg/l), for all transgenic cell lines derived from four different plant species. This investigation demonstrated that expression of NVCP in different transgenic cell lines and in different species was tightly controlled by the inducer, and the inducible gene expression system could be useful in controlling expression of NVCP or similar proteins for production of vaccines in cultured plant cells. [ABSTRACT FROM AUTHOR]

Key message: VaCDPK3a is actively expressed in leaves, stems, inflorescences, and berries of Vitis amurensis and may act as a positive growth regulator, but is not involved in the regulation of resveratrol biosynthesis. Abstract: Calcium-dependent protein kinases (CDPKs) are known to play important roles in plant development and defense against biotic and abiotic stresses. It has previously been shown that CDPK3a is the predominant CDPK transcript in cell cultures of wild-growing grapevine Vitis amurensis Rupr., which is known to possess high resistance against environmental stresses and to produce resveratrol, a polyphenol with valuable pharmacological effects. In this study, we aimed to define the full cDNA sequence of VaCDPK3a and analyze its organ-specific expression, responses to plant hormones, temperature stress and exogenous NaCl, and the effects of VaCDPK3a overexpression on biomass accumulation and resveratrol content in V. amurensis calli. VaCDPK3a was actively expressed in all analyzed V. amurensis organs and tissues and was not transcriptionally regulated by salt and temperature stresses. The highest VaCDPK3a expression was detected in young leaves and the lowest in stems. A reduction in the VaCDPK3a expression correlated with a lower rate of biomass accumulation and higher resveratrol content in calli of V. amurensis under different growth conditions. Overexpression of the VaCDPK3a gene in the V. amurensis calli significantly increased cell growth for a short period of time but did not have an effect on resveratrol production. Further subculturing of the transformed calli resulted in cell death and a decrease in expression of the endogenous VaCDPK3a. The data suggest that while VaCDPK3a acts as a positive regulator of V. amurensis cell growth, it is not involved in the signaling pathway regulating resveratrol biosynthesis and resistance to salt and temperature stresses. [ABSTRACT FROM AUTHOR]

Context: Catharanthus roseus (L.) G. Don (Apocynaceae) is a medicinal plant that produces more than 130 alkaloids, with special attention given to the production of the anti-hypertensive monomeric indole alkaloids, serpentine and ajmalicine, and the antitumor dimeric alkaloids, vinblastine and vincristine. Objective: This study evaluated the cytotoxic activity of the indole alkaloid-enriched bioactive extract obtained from suspension cultured-cells of C. roseus elicited with methyl jasmonate (MJ) and cyclodextrins (CDs) in three cell lines: JURKAT E.6 human lymphocytic leukemia, THP-1 human monocytic leukemia and BL 1395 non-tumor human B-cell line. Materials and methods: An indole alkaloid-enriched bioactive extract was obtained from C. roseus cell cultures elicited with MJ and CDs. The indole alkaloids were identified using an HPLC-diode array system coupled to a time-of-flight mass spectrometer using electrospray ionization (ESI) source. The cytotoxic assays were made using the colorimetric assay 2, 3-bis (2-methoxy-4-nitro-5-sulfophenyl)- S-[(phenylamino)carbonyl]-2 tetrazolium hydroxide (XTT). Results: Four indole alkaloids were identified (catharanthine, ajmalicine, tabersonine and lochnericine) but only catharanthine and ajmalicine were quantified. The concentration of the indole alkaloid-enriched bioactive extract that inhibited cell growth by 50% was 211 and 210 ng/mL for the JURKAT E.6 and THP-1 cell lines, respectively. Discussion and conclusion: The results confirm that the powerful antitumor activity of this indole alkaloid-enriched bioactive extract is not due to the effect of a single compound but depends on the synergistic action of the four compounds identified. [ABSTRACT FROM AUTHOR]

The Arabidopsis thaliana bZIP60 ( AtbZIP60) transcription factor regulates stress signaling. However, its involvement in plant salt tolerance is not fully understood. In this investigation, cell suspension cultures of three different plant species: tobacco ( Nicotiana tabacum), rice ( Oryza sativa L.), and slash pine ( Pinus elliottii Engelm.) were transformed via Agrobacterium tumefaciens LBA4404 harboring pBI- AtbZIP60deltaC. Integration of AtbZIP60deltaC into the genome of tobacco, rice, or slash pine has been confirmed by polymerase chain reaction, Southern blotting, and Northern blotting analyses. Six transgenic cell lines from each of three species were used to analyze the salt tolerance conferred by the overexpression of AtbZIP60deltaC. Our results demonstrated that expression of AtbZIP60deltaC enhanced salt tolerance of transgenic cells in all transgenic lines and that improved salt tolerance was associated with increased activities of both ascorbate peroxidase and superoxide dismutase, and decreased lipid peroxidation (thiobarbituric acid reactive substances). This investigation has demonstrated that the expression of AtbZIP60deltaC gene in transgenic cell lines attenuated salt-induced oxidative damage by increasing the activities of antioxidant enzymes and the overexpression of AtbZIP60deltaC gene could be an alternative choice for engineering plant salt tolerance. [ABSTRACT FROM AUTHOR]

In the last two decades, the use of plants to produce recombinant proteins and particularly biopharmaceuticals has become an attractive alternative to established systems. This is due to advantages in scalability, economy and safety. In addition the expression of recombinant proteins in plants can also be achieved utilizing in vitro cell suspensions with all the advantages such systems confer, such as product consistency, production 'on demand' and the ability to perform the entire process according to good manufacturing practices. In this study we have produced the glycosylated human hormone Erythropoietin (EPO), in Medicago truncatula and Arabidopsis thaliana plants and also in cultured cell lines of tobacco, Medicago and Arabidopsis. We have also tested two different versions of the protein, one with a KDEL tag for targeted expression in the Endoplasmic Reticulum, and an untagged version expected to be secreted to the apoplast. The recombinant protein was detected in the plant leaf extracts and in the cultured cell lines. In the latter, the rEPO was detected in the cell extracts and in the spent culture medium. It was possible to recover the KDEL version of rEPO from crude cell extracts by nickel affinity chromatography, however the secreted form did not bind to the Ni- agarose beads which may indicate a possible internalization of the his-tag in the folded protein. Although the yield of rEPO obtained in cell suspensions was not as high as expected, the protein was successfully produced and secreted into the culture medium, reinforcing that plant cell suspension cultures are a promising system for production of human biopharmaceuticals. [ABSTRACT FROM AUTHOR]

Resveratrol is a polyphenol, present in grapes, peanuts, and other plant sources, with a wide range of valuable biological activities. We established a Vitis amurensis cell culture accumulating high levels of resveratrol by introducing the rolB gene of Agrobacterium rhizogenes in the V. amurensis genome, and studied the stability of resveratrol accumulation during 27 months of continuous subculturing. This study demonstrates a decline in the high level of resveratrol production by the rolB transgenic cell line during its long-term cultivation. Elicitation of the rolB transgenic calli with methyl jasmonate and salicylic acid, which are known to stimulate the production of plant secondary metabolites, resulted in a recovery of resveratrol accumulation in the rolB transgenic cell culture, while the empty vector-transformed culture with trace starting content of resveratrol exhibited low inducibility to the treatment. [ABSTRACT FROM AUTHOR]

Echinacea angustifolia cell suspension cultures are usually grown and maintained in the dark, but we also exposed cells to light for one culture cycle (14 days) and then compared the metabolomes of dark-grown and illuminated cells by liquid chromatography-mass spectrometry. Among 256 signals, we putatively identified 159 molecules corresponding to 56 different metabolites plus their fragments, adducts and isotopologs. The E. angustifolia metabolome consisted mainly of caffeic acid derivatives, comprising (a) caffeic acid conjugated with tartaric, quinic and hexaric acids; and (b) caffeic acid conjugated with hydroxytyrosol glycosides (e.g., echinacoside, verbascoside and related molecules). Many of these metabolites have not been previously described in E. angustifolia, which currently lacks detailed metabolic profiles. Exposure to light significantly increased the levels of certain caffeic acid derivatives (particularly caffeoylquinic acids and hydroxytyrosol derivatives lacking rhamnose residues) and reduced the level of hydroxytyrosol derivatives with rhamnose residues, revealing that light specifically inhibits the rhamnosylation of caffeoyl phenylethanoid glycosides. These results are significant because they suggest that the metabolic profile of cell cultures can be manipulated by controlling simple environmental variables such as illumination to modulate the levels of potentially therapeutic compounds. [ABSTRACT FROM AUTHOR]

Abstract: The moss Physcomitrella patens is a promising expression system for the production of recombinant human biopharmaceuticals. Highly standardized cultivation conditions are required to maintain the desired chloronemal level of cell differentiation during cultivation. Although the life cycle of P. patens is well known and described in literature only little is known about morphology development in closed photo bioreactors (PBR). In the current work we describe the relationship between light intensity and development of moss-morphology in a stirred draft tube PBR during cultivation: pellet formation occurred for high initial photon flux densities (PFD). The application of a low initial PFD followed by subsequent increase could prevent the moss culture from forming dense pellets and the filamentous morphology could be preserved. A high bio dry mass (BDM) of 3–4.5gL−1 could be achieved as well as high specific chlorophyll content of the filaments. Pellet formation is discussed on basis of particle size distributions and data obtained from image analysis. Adjusting the level of illumination is a non-invasive method to maintain the filamentous morphology of P. patens and thus homogeneity of the medium because cells do not need to be disrupted mechanically. [Copyright &y& Elsevier]

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 Ca2+-sensitive photoprotein widely used as a reliable Ca2+ 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 Ca2+ concentrations. Plant cells incubated for just 5 min with TAT–aequorin responded to different environmental stimuli with the expected Ca2+ 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 Ca2+ concentration values. These results indicate that TAT-mediated aequorin transduction is a promising alternative to traditional plant transformation methods to monitor intracellular Ca2+ dynamics rapidly and effectively in plant cells. [ABSTRACT FROM PUBLISHER]

Benzo-(1,2,3)-thiadiazole-7-carbothioic acid S-methyl ester (BTH), a particularly efficient inducer of systemic acquired resistance (SAR), was developed as an immunizing agent to sensitize various crop species against pathogen infections. Recent works highlighted its activating effect on different metabolic pathways, concerning both primary and secondary metabolites. In this study, we investigated the effect of BTH treatment on sterol levels and vitamin D metabolism in Solanum malacoxylon cultures. Calli of S. malacoxylon were incubated in Gamborg B5 liquid medium alone or added with 50 μM BTH for different times (one, two or three cycles of light). Histocytochemical investigations performed on our experimental system using 3,3′-diaminobenzidine (DAB) for hydrogen peroxide (HO) detection and phloroglucinol for lignin staining showed that BTH causes HO accumulation and lignin deposition in treated calli. Gas chromatographic analysis of principal cell membrane sterols (β-sitosterol, campesterol, stigmasterol) showed that BTH transiently increases their cellular levels. Callus cultures were found to contain also cholesterol, 7-dehydrocholesterol, the putative precursor of vitamin D, and the hydroxylated metabolites 25-hydroxyvitamin D [25(OH)D] and 1α,25-dihydroxyvitamin D [1α,25(OH)D]. BTH treatment enhanced 7-dehydrocholesterol while reduced cholesterol. HPLC analysis of sample extracts showed that BTH does not affect the cell content of vitamin D, though results of ELISA tests highlighted that this elicitor moderately enhances the levels of 25(OH)D and 1α,25(OH)D metabolites. In conclusion, BTH treatment not only causes cell wall strengthening, a typical plant defence response, as just described in other experimental models, but in the same time increases the cellular level of the main sterols and 7-dehydrocholesterol. [ABSTRACT FROM AUTHOR]

microwave-assisted extraction (MAE) method was used to recover the anticancer agent paclitaxel from plant cell cultures, and the extraction efficiency of the paclitaxel was determined using various organic solvents (acetone, chloroform, ethanol, methanol, and methylene chloride) and solvent concentrations. Methanol provided the highest recovery of paclitaxel (∼93%) and resulted in the most severe rupturing of the biomass surface during MAE. Most of the paclitaxel (>99%) was recovered using a methanol concentration of 90% (water content: 10%), suggesting that the addition of a small amount of water improves the efficiency of MAE. Furthermore, analysis of the surface of the biomass using an electron microscope revealed that the higher the recovery of paclitaxel, the more severe the damage to the biomass surface. A comparison of the extraction efficiency between MAE and conventional solvent extraction (CSE) showed that with CSE, only up to 54% of the paclitaxel could be recovered in one extraction whereas with MAE, most of the paclitaxel (>99%) in the biomass could be recovered in one extraction. [ABSTRACT FROM AUTHOR]

emisinin is a sesquiterpene antimalarial compound produced, though at low levels (0.1-1% dry weight), in Artemisia annua in which it accumulates in the glandular trichomes of the plant. Due to its antimalarial properties and short supply, efforts are being made to improve our understanding of artemisinin biosynthesis and its production. Native β-cyclodextrins, as well as the chemically modified heptakis(2,6-di- O-methyl)-β-cyclodextrin (DIMEB) and 2-hydroxypropyl-β-cyclodextrins, were added to the culture medium of A. annua suspension cultures, and their effects on artemisinin production were analysed. The effects of a joint cyclodextrin and methyl jasmonate treatment were also investigated. Fifty millimolar DIMEB, as well as a combination of 50 mM DIMEB and 100 μM methyl jasmonate, was highly effective in increasing the artemisinin levels in the culture medium. The observed artemisinin level (27 μmol g dry weight) was about 300-fold higher than that observed in untreated suspensions. The influence of β-cyclodextrins and methyl jasmonate on the expression of artemisinin biosynthetic genes was also investigated. [ABSTRACT FROM AUTHOR]

Abstract: A cell suspension culture was developed from calli of grape rootstock 41B in order to study the bioproduction of resveratrol. While 41B grape cultures produced no resveratrol, methyljasmonate (MeJA) elicitor treatment activated its production in a dose dependent manner. The concentration of 0.2mM MeJA was optimal for efficient production and high accumulation of resveratrol (150mg/L) in flask experiments. Microscopic analysis of cells monitored for viability showed that MeJA elicitor triggered expression of resveratrol fluorescence within the cells. These results led to scale-up of the culture in a 2L stirred bioreactor where a resveratrol production of 209mg/L being secreted into the liquid medium, corresponding to 90% of the total production. Liquid/liquid extraction of the culture medium and a solid/liquid extraction of the cells showed that other stilbenes were also produced. For the first time, trans-ɛ-viniferin, trans-δ-viniferin, and a trans-3-methylviniferin as well as trans-piceatannol were identified in a 2L bioreactor cell cultures of grapevine. Furthermore, a one step FPLC method was developed for the purification of resveratrol and ɛ-viniferin. [Copyright &y& Elsevier]

Abstract: 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. [ABSTRACT FROM AUTHOR]

Calcium-dependent protein kinases (CDPKs) are proposed to play an essential role in plant defense responses. In this study, we aimed to define the full sequence of a CDPK gene of Panax ginseng and analyze its expression in roots, leaves, and cell cultures of P. ginseng, one of the most valuable Chinese traditional medicinal herbs. We isolated the full-length cDNA of a P. ginseng CDPK gene, which was designated PgCDPK1a. PgCDPK1a shares high sequence identity at the amino acidic level with previously reported CDPK sequences for other plant species. We analyzed PgCDPK1a expression in the leaves of wild-growing P. ginseng plants, and in the roots and leaves of cultivated P. ginseng plants growing in an open experimental nursery at a natural ginseng habitat. PgCDPK1a was more actively expressed in the young leaves of cultivated P. ginseng plants than in that of wild-growing ones. Finally, we analyzed the expression of the gene in control GV and five rolC and rolB transgenic callus cultures of P. ginseng with different levels of fresh biomass accumulation, pathogen-related gene expression, and ginsenoside production. We observed a strong positive correlation between fresh biomass accumulation of P. ginseng cell cultures and expression of the PgCDPK1a gene. There was a less clear negative correlation between the expression of pathogen-related genes and the content of ginsenosides with the PgCDPK1a expression in cell cultures of P. ginseng. Perhaps, PgCDPK1a is involved in ginseng growth, as a positive regulator. [ABSTRACT FROM AUTHOR]

A dynamic model for plant cell metabolism was used as a basis for a rational analysis of plant production potential in in vitro cultures. The model was calibrated with data from 3-L bioreactor cultures. A dynamic sensitivity analysis framework was developed to analyse the response curves of secondary metabolite production to metabolic and medium perturbations. Simulation results suggest that a straightforward engineering of cell metabolism or medium composition might only have a limited effect on productivity. To circumvent the problem of the dynamic allocation of resources between growth and production pathways, the sensitivity analysis framework was used to assess the effect of stabilizing intracellular nutrient concentrations. Simulations showed that a stabilization of intracellular glucose and nitrogen reserves could lead to a 116% increase in the specific production of secondary metabolites compared with standard culture protocol. This culture strategy was implemented experimentally using a perfusion bioreactor. To stabilize intracellular concentrations, adaptive medium feeding was performed using model mass balances and estimations. This allowed for a completely automated culture, with controlled conditions and pre-defined decision making algorithm. The proposed culture strategy leads to a 73% increase in specific production and a 129% increase in total production, as compared with a standard batch culture protocol. The sensitivity analysis on a mathematical model of plant metabolism thus allowed producing new insights on the links between intracellular nutritional management and cell productivity. The experimental implementation was also a significant improvement on current plant bioprocess strategies. [ABSTRACT FROM AUTHOR]

Abstract: A review of over 15 years of research, development and commercialization of plant cell suspension culture as a bioproduction platform is presented. Plant cell suspension culture production of recombinant products offers a number of advantages over traditional microbial and/or mammalian host systems such as their intrinsic safety, cost-effective bioprocessing, and the capacity for protein post-translational modifications. Recently significant progress has been made in understanding the bottlenecks in recombinant protein expression using plant cells, including advances in plant genetic engineering for efficient transgene expression and minimizing proteolytic degradation or loss of functionality of the product in cell culture medium. In this review article, the aspects of bioreactor design engineering to enable plant cell growth and production of valuable recombinant proteins is discussed, including unique characteristics and requirements of suspended plant cells, properties of recombinant proteins in a heterologous plant expression environment, bioreactor types, design criteria, and optimization strategies that have been successfully used, and examples of industrial applications. [Copyright &y& Elsevier]

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 β-glucoside (60%) and β-gentiobioside (10%). 4-Fluorophenol was also glycosylated to its β-glucoside (32%) and β-gentiobioside (6%) by N. tabacum cells. On the other hand, N. tabacum glycosylated 3-fluorophenol to β-glucoside (17%). [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

A simple single step ion exchange chromatography method has been developed for purification of SA from plant cell culture extracts. The optimum washing and elution solvents are 25 mM sodium phosphate (pH 7.0-7.5) and 0.3 M HCl in 60% acetonitrile, respectively. Using the developed method the SA recoveries were found 69-80% calculated from the whole purification process. In particular, in the second 10 mL elution, 70 µg SA in 1 g resin yielded 65% SA recovery, which provided a well-detectable amount of SA with a relatively low level of impurities as analyzed by 400 MHz 1H-NMR. [ABSTRACT FROM AUTHOR]

Abstract: An approach for automated, continuous and rapid extraction of flavonoids from Saussurea medusa Maxim dried cell cultures has been developed in a new-designed dynamic microwave-assisted extraction system. The main factors affecting the extraction process namely power of microwave irradiation, liquid/solid ratio, flow rate of solvent and irradiation time were optimized. The yield of flavonoids reached 4.97% in 60min under the optimum microwave-assisted extraction conditions: 1200W of radiation power, 50:1 (v/w) of the liquid/solid ratio, and 50mls−1 of solvent flow rate. The dynamic microwave-assisted extraction showed obvious advantages in short duration and high efficiency to extract flavonoids without causing degradation of target components from the S. medusa dried cell cultures in comparison with the dynamic solvent extraction without microwave assistance. [Copyright &y& Elsevier]

Abstract: 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. [Copyright &y& Elsevier]

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. [ABSTRACT FROM AUTHOR]

The potential of strategies for increasing secondary metabolite accumulation from plant cell cultures may still be partially limited by unfavorable conditions for biosynthesis (i.e. feedback inhibition, unfavorable equilibrium state) or product metabolism. In this paper, the rate of product removal was altered and used as a tool for probing the impact of these limiting conditions on product accumulation, using the production of ajmalicine and serpentine from Catharanthus roseus suspensions as a model system. The method for altering the in situ product removal rate was simple; Amberlite® XAD-7HP resin was enclosed in either a porous or less porous material, and experiments confirmed that the product removal rate was higher with resin enclosed in the porous Miracloth versus the less porous nylon.When resin was added to suspensions, higher ajmalicine production and recovery were associated with Miracloth-enclosed resin which had higher product removal rates. This simple experiment illustrated that further improvements in production and recovery were possible, indicating that conditions unfavorable for biosynthesis and/or product metabolism existed in cultures with nylon-enclosed resin. These results also suggest the importance of optimizing the product removal rate for improving the overall production and recovery of secondary metabolites from plant cell cultures. [Copyright &y& Elsevier]

A dexamethasone-inducible transgene expression system has been developed in transgenic loblolly pine (Pinus taeda L.) cells. This system uses green fluorescent protein (gfp) gene as a reporter gene and is based on the chimaeric transcriptional activator GVG, which is originally developed by T. Aoyama and N.-H. Chua [Plant J. 11 (1997) 605] and improved by P.B.F. Ouwerkerk et al. [Planta 213 (2003) 370]. We have inserted a fragment of m-gfp5-ER into an optimized binary vector pINDEX3 and used this vector to produce transgenic loblolly pine cell lines via Agrobacterium-mediated transformation. Three transgenic cell lines were used to test the function of inducer. Though there are differences in transgene expression among transgenic cell lines, the highest level of green fluorescent protein expression was observed 48 h after addition of the glucocorticoid hormone dexamethasone and by the concentration of 5 mg/l for all three transgenic cell lines. Differential expressions of gfp at 24, 48, and 72 h after treatment of 0.5, 1, 5, and 10 mg/l dexamethasone, respectively, were confirmed by northern blot analysis and by quantitative fluorescence analyses of confocal images taken by a laser scanning microscope (LSM 510). This investigation has demonstrated that the gfp expressions in transgenic cell lines were tightly controlled by inducer and the inducible gene expression system could be useful in molecular physiology and functional identification of novel genes in cultured pine cells. [Copyright &y& Elsevier]

Aryltetralin lignans (podophyllotoxin type) are the main lignan constituents of species belonging to Linum section Syllinum (Linaceae). Linum persicum, a perennial plant native to Iran closely related to L. album, has not yet been studied. To evaluate the lignan profile, fresh plants of L. persicumwere collected and divided into different parts and analyzed by HPLC. The main aryltetralin lignans found in L. persicumplant parts, callus and cell cultures were podophyllotoxin (PTOX), 6-methoxypodophyllotoxin (MPTOX) and α- and β-peltatin. Furthermore, the systematic relationship between L. persicum and other Linum species are discussed in the light of morphological and phytochemical aspects. Abbreviations: MPTOX – 6-methoxypodophyllotoxin; PTOX – podophyllotoxin; DOP – deoxypodophyllotoxin. [ABSTRACT FROM AUTHOR]

S-Adenosyl-l-methionine:β-peltatin 6-O-methyltransferase was isolated and characterized from cell suspension cultures of Linum nodiflorum L. (Linaceae), a Linum species accumulating aryltetralin lignans such as 6-methoxypodophyllotoxin. The enzyme transfers a methyl group from S-adenosyl-l-methionine to the only free OH-group of β-peltatin in position 6 thus forming β-peltatin-A methylether. This reaction is a putative biosynthetic step in the biosynthesis of 6-methoxypodophyllotoxin from deoxypodophyllotoxin. The enzyme has a pH-optimum at pH 7.7 and a temperature optimum at 40 °C. The enzyme activity is strongly inhibited by MnSO4, FeCl3, FeSO4 and ZnSO4 as well as S-adenosyl-homocysteine. Mg2+ and EDTA did not influence the methylation of β-peltatin. Substrate saturation curves were obtained for S-adenosyl-methionine and β-peltatin and apparent Km-values of 15 μM and 40 μM, respectively, were determined for these substrates. Substrate inhibition was observed for β-peltatin. No other lignan substrate tested nor caffeic acid were accepted. The suspension cell line of Linum nodiflorum was characterized with respect to growth, medium alterations and lignan production as well as activity of SAM:β-peltatin 6-O-methyltransferase. Highest specific activities of β-peltatin 6-O-methyltransferase were determined on day 7 of the culture period corresponding to the highest levels of 6-methoxypodophyllotoxin on days 7 to 12. [Copyright &y& Elsevier]