Phenolic Compounds from Wild Plant and In Vitro Cultures of Ageratina pichichensis and Evaluation of Their Antioxidant Activity.

Ageratina pichichensis, is commonly used in traditional Mexican medicine. In vitro cultures were established from wild plant (WP) seeds, obtaining in vitro plant (IP), callus culture (CC), and cell suspension culture (CSC) with the objective to determine total phenol content (TPC) and flavonoids (TFC), as well as their antioxidant activity by DPPH, ABTS and TBARS assays, added to the compound's identification and quantification by HPLC, from methanol extracts obtained by sonication. CC showed significantly higher TPC and TFC than WP and IP, while CSC produced 2.0-2.7 times more TFC than WP, and IP produced only 14.16% TPC and 38.8% TFC compared with WP. There were identified compounds such as epicatechin (EPI), caffeic acid (CfA), and p-coumaric acid (pCA) in in vitro cultures that were not found in WP. The quantitative analysis shows gallic acid (GA) as the least abundant compound in samples, whereas CSC produced significantly more EPI and CfA than CC. Despite these results, in vitro cultures show lower antioxidant activity than WP, for DPPH and TBARS WP > CSC > CC > IP and ABTS WP > CSC = CC > IP. Overall, A. pichichensis WP and in vitro cultures produce phenolic compounds with antioxidant activity, especially CC and CSC, which are shown to be a biotechnological alternative for obtaining bioactive compounds.

Enhanced specialized metabolite, trichome density, and biosynthetic gene expression in Stevia rebaudiana (Bertoni) Bertoni plants inoculated with endophytic bacteria Enterobacter hormaechei.

Stevia rebaudiana (Bertoni) Bertoni is a plant of economic interest in the food and pharmaceutical industries due its steviol glycosides (SG), which are rich in metabolites that are 300 times sweeter than sucrose. In addition, S. rebaudiana plants contain phenolic compounds and flavonoids with antioxidant activity. Endophytic bacteria promote the growth and development and modulate the metabolism of the host plant. However, little is known regarding the role of endophytic bacteria in the growth; synthesis of SG, flavonoids and phenolic compounds; and the relationship between trichome development and specialized metabolites in S. rebaudiana, which was the subject of this study. The 12 bacteria tested did not increase the growth of S. rebaudiana plants; however, the content of SG increased with inoculation with the bacteria Enterobacter hormaechei H2A3 and E. hormaechei H5A2. The SG content in leaves paralleled an increase in the density of glandular, short, and large trichome. The image analysis of S. rebaudiana leaves showed the presence of SG, phenolic compounds, and flavonoids principally in glandular and short trichomes. The increase in the transcript levels of the KO, KAH, UGT74G1, and UGT76G1 genes was related to the SG concentration in plants of S. rebaudiana inoculated with E. hormaechei H2A3 and E. hormaechei H5A2. In conclusion, inoculation with the stimulating endophytes E. hormaechei H2A3 and E. hormaechei H5A2 increased SG synthesis, flavonoid content and flavonoid accumulation in the trichomes of S. rebaudiana plants.

Production of indole-3-acetic acid by Bacillus circulans E9 in a low-cost medium in a bioreactor.

Bacillus circulans E9 (now known as Niallia circulans) promotes plant growth-producing indole-3-acetic acid (IAA), showing potential for use as a biofertilizer. In this work, the use of a low-cost medium containing industrial substrates, soybean, pea flour, Solulys, Pharmamedia, yeast extract, and sodium chloride (NaCl), was evaluated as a substitute for microbiological Luria Broth (LB) medium for the growth of B. circulans E9 and the production of IAA. In Erlenmeyer flasks with pea fluor medium (PYM), the maximum production of IAA was 7.81 ± 0.16 µg mL-1, while in microbiological LB medium, it was 3.73 ± 0.15 µg mL-1. In addition, an oxygen transfer rate (OTR) of 1.04 kg O2 m-3 d-1 allowed the highest bacterial growth (19.3 ± 2.18 × 1010 CFU mL-1) and IAA production (10.7 µg mL-1). Consequently, the OTR value from the flask experiments was used to define the conditions for the operation of a 1 L stirred tank bioreactor. The growth and IAA production of B. circulans cultured in a bioreactor with PYM medium were higher (8 and 1.6 times, respectively) than those of bacteria cultured in Erlenmeyer flasks. IAA produced in a bioreactor by B. circulans was shown to induce the root system in Arabidopsis thaliana, similar to synthetic IAA. The results of this study demonstrate that PYM medium may be able to be used for the mass production of B. circulans E9 in bioreactors, increasing both bacterial growth and IAA production. This low-cost medium has the potential to be employed to grow other IAA-producing bacterial species.

Engineering Considerations to Produce Bioactive Compounds from Plant Cell Suspension Culture in Bioreactors.

The large-scale production of plant-derived secondary metabolites (PDSM) in bioreactors to meet the increasing demand for bioactive compounds for the treatment and prevention of degenerative diseases is nowadays considered an engineering challenge due to the large number of operational factors that need to be considered during their design and scale-up. The plant cell suspension culture (CSC) has presented numerous benefits over other technologies, such as the conventional whole-plant extraction, not only for avoiding the overexploitation of plant species, but also for achieving better yields and having excellent scaling-up attributes. The selection of the bioreactor configuration depends on intrinsic cell culture properties and engineering considerations related to the effect of operating conditions on thermodynamics, kinetics, and transport phenomena, which together are essential for accomplishing the large-scale production of PDSM. To this end, this review, firstly, provides a comprehensive appraisement of PDSM, essentially those with demonstrated importance and utilization in pharmaceutical industries. Then, special attention is given to PDSM obtained out of CSC. Finally, engineering aspects related to the bioreactor configuration for CSC stating the effect of the operating conditions on kinetics and transport phenomena and, hence, on the cell viability and production of PDSM are presented accordingly. The engineering analysis of the reviewed bioreactor configurations for CSC will pave the way for future research focused on their scaling up, to produce high value-added PDSM.

Photosynthetic performance and stevioside concentration are improved by the arbuscular mycorrhizal symbiosis in Stevia rebaudiana under different phosphate concentrations.

In plants, phosphorus (P) uptake occurs via arbuscular mycorrhizal (AM) symbiosis and through plant roots. The phosphate concentration is known to affect colonization by AM fungi, and the effect depends on the plant species. Stevia rebaudiana plants are valuable sources of sweetener compounds called steviol glycosides (SGs), and the principal components of SGs are stevioside and rebaudioside A. However, a detailed analysis describing the effect of the phosphate concentration on the colonization of AM fungi in the roots and the relationship of these factors to the accumulation of SGs and photochemical performance has not been performed; such an analysis was the aim of this study. The results indicated that low phosphate concentrations (20 and 200 µM KH2PO4) induced a high percentage of colonization by Rhizophagus irregularis in the roots of S. rebaudiana, while high phosphate concentrations (500 and 1,000 µM KH2PO4) reduced colonization. The morphology of the colonization structure is a typical Arum-type mycorrhiza, and a mycorrhiza-specific phosphate transporter was identified. Colonization with low phosphate concentrations improved plant growth, chlorophyll and carotenoid concentration, and photochemical performance. The transcription of the genes that encode kaurene oxidase and glucosyltransferase (UGT74G1) was upregulated in colonized plants at 200 µM KH2PO4, which was consistent with the observed patterns of stevioside accumulation. In contrast, at 200 µM KH2PO4, the transcription of UGT76G1 and the accumulation of rebaudioside A were higher in noncolonized plants than in colonized plants. These results indicate that a low phosphate concentration improves mycorrhizal colonization and modulates the stevioside and rebaudioside A concentration by regulating the transcription of the genes that encode kaurene oxidase and glucosyltransferases, which are involved in stevioside and rebaudioside A synthesis in S. rebaudiana.

Production of anti-inflammatory compounds in Sphaeralcea angustifolia cell suspension cultivated in stirred tank bioreactor.

Sphaeralcea angustifolia is a plant used for the treatment of inflammatory processes. Scopoletin, tomentin, and sphaeralcic acid were identified as the compounds with anti-inflammatory and immunomodulatory effects. Successful establishment of the cell culture in Erlenmeyer flasks has been reported previously. The aim of this study was to evaluate the ability of cells in suspension from S. angustifolia grown in a stirred tank bioreactor and demonstrate their capacity to produce bioactive compounds. Cells in suspension grown at 200 rpm reached a maximal cell biomass in dry weight at 19.11 g/L and produced 3.47 mg/g of sphaeralcic acid. The mixture of scopoletin and tomentin was only detected at the beginning of the culture (12.13 µg/g). Considering that the profile of dissolved oxygen during the cultures was lesser than 15%, it is possible that the low growth at 100 rpm could be due to oxygen limitations or to cell sedimentation. At 400 rpm, a negative effect on cell viability could be caused by the increase in the hydrodynamic stress, including the impeller tip, average shear rate, and Reynolds number. The sphaeralcic acid content in the cell suspension of S. angustifolia obtained in the bioreactor was two orders of magnitude greater than that reported for the culture grown in Erlenmeyer flasks.

Cell Culture of Bursera linanoe in a Stirred Tank Bioreactor for Production of Linalool and Linalyl Acetate.

Bursera linanoe cell suspension cultures were initiated from callus grown in Murashige and Skoog medium supplemented with naphthalene acetic acid (3.0 mg L⁻¹) and 6-benzylaminopurine (0.5 mg L⁻¹). In flasks, B. linanoe cell cultures grew over a 9 day period, reaching a maximum biomass of 11.16 g DW L⁻¹. Throughout the growth phase, cell viability was constant at 60 - 70%. In contrast, B. linanoe cells growing in a bioreactor achieved a maximum biomass of 22.26 g DW L⁻¹ (after 7 days), and cell viability was constant at 75 - 85%. Production of linalool and linalyl acetate in the bioreactor (3.02 and 2.40 mg g⁻¹ DW, respectively) was significantly greater than that achieved from cells in flask cultures (1.05 and 0.97 mg g⁻¹ DW, respectively). B. linanoe cell suspension culture has potential as an alternative method for the production of essential oils.

Monoterpenoid oxindole alkaloid production by Uncaria tomentosa (Willd) D.C. cell suspension cultures in a stirred tank bioreactor.

Cell growth, monoterpenoid oxindole alkaloid (MOA) production, and morphological properties of Uncaria tomentosa cell suspension cultures in a 2-L stirred tank bioreactor were investigated. U. tomentosa (cell line green Uth-3) was able to grow in a stirred tank at an impeller tip speed of 95 cm/s (agitation speed of 400 rpm), showing a maximum biomass yield of 11.9 +/- 0.6 g DW/L and a specific growth rate of 0.102 d(-1). U. tomentosa cells growing in a stirred tank achieved maximum volumetric and specific MOA concentration (467.7 +/- 40.0 microg/L, 44.6 +/- 5.2 microg/g DW) at 16 days of culture. MOA chemical profile of cell suspension cultures growing in a stirred tank resembled that of the plant. Depending on culture time, from the total MOA produced, 37-100% was found in the medium in the bioreactor culture. MOA concentration achieved in a stirred tank was up to 10-fold higher than that obtained in Erlenmeyer flasks (agitated at 110 rpm). In a stirred tank, average area of the single cells of U. tomentosa increased up to 4-fold, and elliptical form factor increased from 1.40 to 2.55, indicating enlargement of U. tomentosa single cells. This work presents the first report of U. tomentosa green cell suspension cultures that grow and produce MOA in a stirred tank bioreactor.

Influence of auxins and sucrose in monoterpenoid oxindole alkaloid production by Uncaria tomentosa cell suspension cultures.

Growth and alkaloid production in Uncaria tomentosa cell suspension cultures were studied in Murashige and Skoog medium supplemented with 10 microM 2,4-dichlorophenoxyacetic acid, 10 microM kinetin, and 58 mM sucrose for maintenance and with 10 microM indole-3-acetic acid, 10 microM kinetin, and 58 mM sucrose for production. A U. tomentosa pale Uth-3 cell line, cultured in the production medium, showed a reduced lag phase and a specific growth rate (mu) of 0.27 day(-1), while cells growing in the maintenance medium showed mu = 0.20 day(-1). U. tomentosa cells growing in the production medium produced monoterpenoid oxindole alkaloids (MOA) in amounts of 10.2 +/- 1.6 microg g(-1) dry weight (DW). The chemical profile of MOA produced by in vitro cell cultures was similar to that found in the plant. After 10 subcultures, maximum MOA production decreased to 2.0 +/- 0.7 microg g(-1) DW, while tryptamine alkaloids (TA) were produced with a maximum of 6.2 +/- 0.4 microg g(-1) DW. The increase of initial sucrose concentration up to 145 mM in the production medium enhanced the cell biomass by 3.2-fold (from 10.2 +/- 0.1 to 32.8 +/- 1.1 g DW L(-1)), reduced mu from 0.27 to 0.23 day(-1), and provoked a substantial accumulation of TA (23.1 +/- 4.7 microg g(-1) DW). A high sucrose concentration stimulated MOA production in the maintenance medium (2.7 +/- 0.5 microg g(-1) DW), even in the presence of 2,4-dichlorophenoxyacetic acid.

Production of dihydroxylated betalains and dopamine in cell suspension cultures of Celosia argentea var. plumosa.

Betalains are plant pigments of hydrophilic nature with demonstrated chemopreventive potential in cancer cell lines and animal models. Among the betalains, those containing an aromatic moiety with two free hydroxyl groups possess the strongest antioxidant and free radical scavenging activities. The betaxanthins dopaxanthin and miraxanthin V and the betacyanins betanidin and decarboxy-betanidin are the only natural betalains with catecholic substructures. These four pigments have been produced in cell cultures established from hypocotyls of the plant Celosia argentea. Two stable and differentially colored cell lines, yellow and red, were maintained on Murashige and Skoog medium supplemented with the plant growth regulators 6-benzylaminopurine (6.66 µM) and 2,4-dichlorophenoxyacetic acid (6.79 µM). Derived suspension cultures showed increased production of dihydroxylated betalains in the cells and secreted to the medium with a maximum reached after 8 days of culture. In addition, precursor molecules betalamic acid and dopamine, with content up to 42.08 mg/g dry weight, were also obtained. The joint presence of the bioactive betalains together with the production of dopamine and betalamic acid show the ability of cell cultures of C. argentea to become a stable source of valuable phytochemicals.

Hydrodynamic stress induces monoterpenoid oxindole alkaloid accumulation by Uncaria tomentosa (Willd) D. C. cell suspension cultures via oxidative burst.

Uncaria tomentosa cell suspension cultures were grown in a 2-L stirred tank bioreactor operating at a shear rate gamma(.)(avg)=86 s(-1). The cultures showed an early monophasic oxidative burst measured as H2O2 production (2.15 micromol H2O2 g(-1) dw). This response was followed by a transient production of monoterpenoid oxindole alkaloids (178 +/- 40 microg L(-1) at 24 h). At the stationary phase (144 h), the increase of the shear rate gamma(.)(avg) up to 150 s(-1) and/or oxygen tension up to 85% generated H2O2, restoring oxindole alkaloid production. U. tomentosa cells cultured in Erlenmeyer flasks also exhibited the monophasic oxidative burst but the H2O2 production was 16-fold lower and the alkaloids were not detected. These cells exposed to H2O2 generated in situ produced oxindole alkaloids reaching a maximum of 234 +/- 40 microg L(-1). A positive correlation was observed between the oxindole alkaloid production and the endogenous H2O2 level. On the other hand, addition of 1 microM diphenyleneiodonium (NAD(P)H oxidase inhibitor) or 10 microM sodium azide (peroxidases inhibitor) reduced both H2O2 production and oxindole alkaloids build up, suggesting that these enzymes might play a role in the oxidative burst induced by the hydrodynamic stress.

Las proteínas arabinogalactanos en cultivos de células vegetales / Arabinogalactan proteins in plant cell cultures / As proteínas arabinogalactanos em cultivos de células vegetais

Las proteínas arabinogalactanos (AGPs) son macromoléculas que se encuentran prácticamente en todos los órganos de las plantas, siendo asociadas con varios aspectos del crecimiento y desarrollo vegetal. Estas moléculas se caracterizan bioquímicamente por contener carbohidratos y proteínas en relación 9:1. El carbohidrato está compuesto principalmente por arabinogalactanos tipo II; mientras que la parte proteica está organizada en dominios que definen a las AGPs como clásicas o no clásicas. Las primeras se caracterizan además por presentar una secuencia C-terminal que predice la incorporación de un grupo glicosilfosfatidilinositol (GFI), que permite su unión a la membrana plasmática. En cultivos de células vegetales se reportan varias especies que liberan AGPs al medio de cultivo. Se presenta una revisión de las características bioquímicas de las AGPs liberadas al medio y de las propuestas sobre los mecanismos bioquímicos y celulares por los cuales las AGPs participan en la diferenciación y crecimiento de las células vegetales. Los cultivos de células liberan al medio de cultivo AGPs clásicas y no clásicas, y se propone que podrían provenir de la membrana plasmática o la pared celular. Las AGPs intervienen en el control del crecimiento celular, además de estar relacionadas con la embriogénesis somática y la organogénesis, procesos de diferenciación celular importantes en los sistemas de micropropagación de plantas. El mecanismo bioquímico por el cual las AGPs participan en el crecimiento celular y la diferenciación implica que éstas, o los productos de su degradación, quizás actúen como moléculas de señalización.

The arabinogalactan proteins (AGPs) are macromolecules found in practically all plant organs, being associated with several aspects of the plant growth and development. These molecules contain carbohydrates and proteins in a 9:1 relation. The carbohydrate moiety is composed mainly of type II arabinogalactans, whereas the protein has particular amino acid domains that allow classifying the AGPs into two groups, classical and non-classical. In addition, the former are characterized by a C-terminal tail that predicts the incorporation of a glycosylphosphatidylinositol group (GPI) that allows the attachment of the AGPs to the plasma membrane. Plant cell cultures of several species release AGPs into the culture medium. The biochemical characteristics of the AGPs released into the medium, and the proposed biochemical and cellular mechanisms by which AGPs participate in plant cell differentiation and growth are reviewed. The plant cells release classical as well as non-classical AGPs into the culture medium. The origin of these AGPs could likely be the plasma membrane or the cell wall. They are involved in the control of cellular growth and differentiation processes, aspects that have fundamental importance in the induction of somatic embryogenesis and organogenesis, key steps in plant micropropagation programs. The biochemical mechanism by which the AGPs participate in cell growth and differentiation implies that the AGPs or their degradation products participate like signal molecules.

As proteínas arabinogalactanos (AGPs) são macromoléculas que se encontram praticamente em todos os órgãos das plantas, sendo associadas com vários aspectos do crescimento e desenvolvimento vegetal. Estas moléculas se caracterizam bioquímicamente por conter carboidratos e proteínas em relação 9:1. O carboidrato está composto principalmente por arabinogalactanos tipo II; enquanto que a parte protéica está organizada em domínios que definem as AGPs como clássicas ou não clássicas. As primeiras se caracterizam, além disso, por apresentar uma sequência C-terminal que prediz a incorporação de um grupo glicosilfosfatidilinositol (GFI), que permite sua união à membrana plasmática. Em cultivos de células vegetais se relatam várias espécies que liberam AGPs ao meio de cultivo. Apresenta-se uma revisão das características bioquímicas das AGPs liberadas ao meio e das propostas sobre os mecanismos bioquímicos e celulares pelos quais as AGPs participam na diferenciação e crescimento das células vegetais. Os cultivos de células liberam ao meio de cultivo AGPs clássicas e não clássicas, e se propõe que poderiam provir da membrana plasmática ou da parede celular. As AGPs intervêm no controle do crescimento celular, além de estar relacionadas com a embriogênese somática e a organogênese, processos de diferenciação celular importantes nos sistemas de micropropagação de plantas. O mecanismo bioquímico pelo qual as AGPs participam no crescimento celular e a diferenciação, implica que estas, ou os produtos de sua degradação, talvez atuem como moléculas de sinalização.

La agregación celular en la producción de metabolitos secundarios en cultivos vegetales in vitro / Celular aggregation in secondary metabolite production in in vitro plant cell cultures

El cultivo de células vegetales es una alternativa biotecnológica para la producción de metabolitos secundarios. Sin embargo, la productividad de los sistemas in vitro es menor a la obtenida de plantas. En esta revisión se ilustra la diferenciación y compartamentalización celular como eventos necesarios para la síntesis de metabolitos secundarios en las plantas. Se discute la inducción de la agregación celular en los cultivos in vitro como una de las estrategias para favorecer la acumulación de estos compuestos químicos. Este efecto positivo podría ser explicado como consecuencia de la formación de estructuras morfogénicas y/o por una condición de estrés por limitaciones de oxígeno al interior de los agregados. Finalmente, se muestra que la combinación de la agregación con otras estrategias tales como la selección de líneas celulares, la elicitación y la adición de precursores constituye una alternativa para desarrollar bioprocesos a partir de células vegetales in vitro para la producción de compuestos químicos de alto valor agregado.