Production and secretion of resveratrol in hairy root cultures of peanut

Resveratrol and its derivatives are natural stilbenes associated with many health benefits that include those conferred by their antioxidant and anticancer properties. While stilbenes can be recovered as an extract from a selected number of plants, these products are not suitable for many applications in the food/pharmaceutical sectors due to high levels of impurities as well as the overall low concentration of resveratrol and its derivatives in the extract. To deliver a highly defined and enriched resveratrol product, hairy root cultures of peanut (Arachis hypogaea) were established and tested as a bioproduction system for resveratrol and associated derivatives. Analyses by HPTLC and GC-MS of ethyl acetate extracts showed that a single 24 h sodium acetate elicitation resulted in a 60-fold induction and secretion of trans-resveratrol into the medium of peanut hairy root cultures. trans-Resveratrol accumulated to levels of 98 microg/mg of the dried extract from the medium representing 99% of the total resveratrol produced. Other stilbenes, including trans-pterostilbene, were also detected in the medium. Our results demonstrate the capacity of hairy root cultures as an effective bioprocessing system for valued nutraceuticals like resveratrol and resveratrol derivatives. In being able to effectively induce and recover high levels of resveratrol and associated derivatives from the media fraction, hairy roots may offer a scalable and continuous product recovery platform for naturally-derived, high quality, enriched nutraceuticals.     

Selection of reliable reference genes for qPCR studies on chondroprotective action

Background  

Chondroprotective agents (CPA) such as glucosamine, curcumin and diacerein represent potential remedies for the management of osteoarthritis and several studies have been performed on their effects in-vitro and in-vivo. For the investigation of chondroprotective action on chondrocyte gene expression, quantitative real-time RT-PCR is the method of choice. However, validation of applied normalization strategies represents a crucial and sometimes neglected step in the analysis process. Therefore, the present study aimed to determine the expression stability of common reference genes (ACTB, Beta actin; GAPDH, Glyceraldehyde-3-phosphate; B2M, Beta-2-microglobulin; HPRT1, Hypoxanthine phosphoribosyl-transferase I; SDHA, Succinate dehydrogenase complex, subunit A; YWHAZ, Tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, zeta polypeptide) under the influence of glucosamine, curcumin and diacerein in the IL-1β-stimulated C-28/I2 chondrocyte model, using the geNorm software tool.     

Hernandulcin in hairy root cultures of Lippia dulcis

The hairy root culture of Lippia dulcis Trev., Verbenaceae, was established by transformation with Agrobacterium rhizogenes A4. The transformed roots grew well in Murashige and Skoog medium containing 2% sucrose. The roots turned light green when they were cultured under 16 h/day light. The green hairy roots produced the sweet sesquiterpene hernandulcin (ca. 0.25 mg/g dry wt) together with 20 other mono- and sesquiterpenes, while no terpenes were detected in the nontransformed root cultures. The growth and hernandulcin production in the hairy root cultures were influenced by the addition of auxins to the medium. The addition of a low concentration of chitosan (0.2 – 10.0 mg / l) enhanced the production of hernandulcin 5-fold.Abbreviations Cht chitosan - IAA indole-3-acetic acid - NAA 1-naphthaleneacetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - MS Murashige and Skoog(1962)     

Secondary metabolism of hairy root cultures in bioreactors

Summary In vitro cultures are being considered as an alternative to agricultural processes for producing valuable secondary metabolites. Most efforts that use differentiated cultures instead of cell suspension cultures have focused on transformed (hairy) roots. Bioreactors used to culture hairy roots can be roughly divided into three types: liquid-phase, gas-phase, or hybrid reactors that are a combination of both. The growth and productivity of hairy root cultures are reviewed with an emphasis on successful bioreactors and important culture considerations. The latter include strain selection, production of product in relation to growth phase, media composition, the gas regime, use of elicitors, the role of light, and apparent product loss. Together with genetic engineering and process optimization, proper reactor design plays a key role in the development of successful large scale production of secondary metabolites from plant cultures.     

Selection of reference genes for expression studies with fish myogenic cell cultures

Background  

Relatively few studies have used cell culture systems to investigate gene expression and the regulation of myogenesis in fish. To produce robust data from quantitative real-time PCR mRNA levels need to be normalised using internal reference genes which have stable expression across all experimental samples. We have investigated the expression of eight candidate genes to identify suitable reference genes for use in primary myogenic cell cultures from Atlantic salmon (Salmo salar L.). The software analysis packages geNorm, Normfinder and Best keeper were used to rank genes according to their stability across 42 samples during the course of myogenic differentiation.     

Flavonoids from Glycyrrhiza pallidiflora hairy root cultures

Three flavonoids named licoagrosides D, E and F together with four known flavonoids, medicarpin 3-O-glucoside, calycosin 7-O-glucoside, formononetin 7-O-(6"-malonylglucoside) and 2'-hydroxyformononetin 7-O-glucoside were isolated from Glycyrrhiza pallidiflora hairy root cultures. Their structures were determined on the basis of spectroscopic evidence. Licoagrosides E and F are the first examples of a 6a-hydroxypterocarpan glycoside and an alpha-O-glycosidic alpha-hydroxydihydrochalcone, respectively.     

Flavonoids from Glycyrrhiza pallidiflora hairy root cultures

Glycyrrhiza pallidiflora hairy roots were induced from axenic young plants by direct infection with Agrobacterium rhizogenes. The chemical constituents were then investigated after mass culture. The isoflavone, licoagroisoflavone and the coumestan, licoagroside C, were isolated along with seven known flavonoids. Their structures were determined on the basis of spectroscopic evidence.     

Fed-batch hairy root cultures within situ separation

Fed-batch cultures ofL. erythrorhizon hairy root were carried out by controlling sucrose concentration and media conductivity in a shake flask and a modified stirred tank reactor. For the efficient product recovery from the culture,in situ adsorption by XAD-2 was also conducted. When sucrose was used as a carbon source, the highest shikonin production and hairy root growth were obtained. When glucose or fructose was used instead, the growth was severely inhibited. In addition, it was found that alternating feeding of sucrose could be used as an effective strategy for enhancing the productivity of shikonin derivatives., As the XAD-2 amount was increased up to 1.5 g/L, shikonin production was enhanced by removing shikonin produced and other products which might be inhibitory to cell growth. Most amount of shikonin produced was successfully recovered in XAD-2 (Over 99%). Using hairy root culture in a modified stirred tank reactor, the shikonin productivity and hairy root growth rate on the average were 9.34 mg/L day and 0.49 g DCW/L · day, respectively.     

Genista tinctoria hairy root cultures for selective production of isoliquiritigenin

Hairy root cultures were established after inoculation of Genista tinctoria in vitro shoots with Agrobacterium rhizogenes, strain ATCC 15834. In transformed roots of G. tinctoria grown in Schenk-Hildebrandt medium without growth regulators the biosynthesis of isoflavones, derivatives of genistein and daidzein, and flavones, derivatives of luteolin and apigenin, characteristic for the intact plant, was completely inhibited. The only compound synthesized in G. tinctoria hairy roots was isoliquiritigenin (2.3 g/100 g DW), a daidzein precursor absent in the intact plant. This compound was stored entirely within cells and it was not until abscisic acid was added (37.8 microM supplement on day 42) that approx. 80% of it was released into the experimental medium. The paper discusses the effect of abscisic acid on the growth of G. tinctoria hairy root cultures, the biosynthesis of isoliquiritigenin and the way it is stored. A prototype basket-bubble bioreactor was designed and built to upgrade the scale of the G. tinctoria hairy root cultures. With immobilized roots and a new aeration system, large amounts of biomass were obtained (FWmax 914.5 g l(-1)) which produced high contents of isoliquiritigenin (2.9 g/100 g DW). The abscisic acid-induced release of the metabolite from the tissue into the growth medium greatly facilitated subsequent extraction and purification of isoliquiritigenin.     

Establishment of hairy root cultures of Psoralea species

Eight Psoralea species (Leguminosae) were inoculated with Agrobacterium rhizogenes, strains 8196 and 9402. Hairy roots were only induced by strain 9402. Attention was focussed on Psoralea lachnostachys. Transformed roots grew very rapidly in Gamborg B5 liquid medium with a doubling time of the culture of 38 hours. Whatever the culture conditions, the two furanocoumarins usually found in roots of Psoralea plants, psoralen and angelicin, were not detected in cultured transformed and non transformed roots even when some chitosan was added to the medium. However, 669 g.g^–1 dry matter of psoralen and 215 g.g^–1 dry matter of angelicin were found in roots from soil grown plants. A possible translocation of these compounds from the aerial parts to the roots is suggested.Abbreviations B5 medium Gamborg's medium (Flow laboratories's formulation) - NAA Naphthaleneacetic acid     

Anti-Prelog reduction of ketones by hairy root cultures

Raphanus sativus hairy roots were used in the anti-Prelog stereoselective reduction of a series of prochiral alkylaryl-ketones. Most of the bioreactions proceeded with high yields and excellent enantioselectivities. This novel biocatalyst is an easy handle system that allows the employment of the immense potential of plant enzymes in preparative asymmetric chemistry.     

Production of flavonoids by Psoralea hairy root cultures

Eighteen transformed root cultures from 7 Psoralea plant species (Leguminosae) were established with the objective of producing daidzein and related flavonoids. All the 18 hairy root lines grew fast and had the same capacities for biomass production. Each of them produced daidzein as an intracellular secondary metabolite. The Lach5 hairy root line, obtained from P. lachnostachys, was a high producing line for daidzein and was further studied for biomass and flavonoid production. This root line showed exponential growth. Chitosan was used for elicitation purposes as well as for its permeabilizing effect. Little elicitation effect could be demonstrated and the metabolite release in the medium was weak (about 1%) and limited to the first 29 h after chitosan addition. Daidzein was demonstrated to be more concentrated in young parts (apexes) whereas coumestrol content was higher in older parts (brown tissues). Compared to callus cultures from the same plant species, hairy roots displayed comparable concentrations. However, high-producing lines were more frequently found with hairy roots (4 out of 18) than with callus cultures (4 out of 217) This revised version was published online in June 2006 with corrections to the Cover Date.     

Alkaloid production by hairy root cultures in Atropa belladonna

Hairy roots were induced by inoculation of stems of sterile plants of Atropa belladonna with Agrobacterium rhizogenes. The axenic culture of the hairy roots isolated from the stems proliferated 60 fold as based on the initial fresh weight after one month of culture. The presence of atropine and scopolamine in hairy roots were examined by TLC and HPLC. Their amounts were analyzed by GLC. The results show that the amount of the two alkaloids in the axenic cultures was the same as or even higher than those of normal plants grown in the field.     

Development of Linum flavum hairy root cultures for production of coniferin

Linum flavum hairy roots were initiated from leaf discs using Agrobacterium rhizogenes strains LBA9402 and TR105 though two other strains, 15834 and A4, were relatively ineffective for induction. Significant variation in coniferin accumulation was observed between hairy root lines originating from different L. flavum seedlings and/or A. rhizogenes strains. Coniferin reached 58 mg g^–1 dry wt by culturing the roots in Linsmaier and Skoog (LS) medium with 2,4-dichlorophenoxyacetic acid and naphthaleneacetic acid as growth regulators.     

Flavonoid constituents from Glycyrrhiza glabra hairy root cultures

An unusual biflavonoid named licoagrodin was isolated from the hairy root cultures of Glycyrrhiza glabra (Leguminosae) along with three prenylated retrochalcones, licoagrochalcones B, C, D, a prenylated aurone, licoagroaurone and four known prenylated flavonoids, licochalcone C, kanzonol Y, glyinflanin B and glycyrdione A. From the glycosidic fraction, a isoflavone glycoside, licoagroside A, and a maltol glycoside, licoagroside B were isolated together with four known isoflavone glycosides, two flavone C-glycosides, and three other glycosides. Their structures were elucidated on the basis of spectroscopic evidence.     

Genetic stability of hairy root cultures of Datura stramonium

The karyotypic status of three root lines, transformed with Agrobacterium rhizogenes, versus a non-transformed root line of Datura stramonium, as well as grown plants (control), was investigated. In the transformed cultures, the karyotype as well as the number of chromosomes (2n = 24) remained the same as in the control plants. In the normal root line there were clear differences at a morphological level despite showing the same chromosomic number (2n = 24) at a high frequency. The existence of aneuploids (2n = 2n ± x) and tetraploids (2n = 4x = 48) was also observed in the latter. The frequency of chromosomic alterations was very low in all the cultures. Cytogenetic analysis of the transformed root cultures of D. stramonium reported here shows a great chromosomic stability, which is not the case with normal root cultures. This revised version was published online in June 2006 with corrections to the Cover Date.