Effects of immobilizing agents and procedures on viability of cultured celery (Apium graveolens) cells

Summary In an attempt to develop concurrent permeabilization/immobilization systems for the production of secondary plant metabolites, the effects of chitosan, alginate, carrageenan gel and carrageenan/chitosan copolymers as immobilizing agents and immobilization procedures on viability of culturedApium graveolens cells have been examined. Chitosan immobilization, ascorbic and succinic acid resulted in low viability of plant cells but use of carrageenan/chitosan copolymers enabled maintenance of viable cell lines providing the potential for concurrent immobilization/permeabilization of cells and elicitation of secondary metabolites by chitosan.     

Medium composition and methyl jasmonate influence the amount and spectrum of secondary metabolites in callus cultures of Zanthoxylum stenophyllum Hemsl.

Callus cultures of Zanthoxylum stenophyllum were initiated in vitro and the effect of growth regulators and elicitors was tested both upon callus growth and secondary metabolite production. On a medium containing naphthaleneacetic acid, kinetin, and 2,4-dichlorophenoxyacetic acid, a yellowish and friable callus was obtained from 90% of cotyledon explants. Callus growth and secondary metabolite accumulation was followed after sub-culturing the established callus culture on different media containing various hormonal combinations. Results indicate that medium containing naphthaleneacetic acid and a higher concentration of 2,4-dichlorophenoxyacetic acid gave the highest stimulation of growth. Addition of an organic nitrogen source also had a positive effect on growth. Rapid HPLC screening of methanol extractable secondary metabolites from calluses showed that phytohormones and nutrients were able to modify the chromatographic pattern of compounds. Calluses grown on the medium giving the highest stimulation of growth show a reduced accumulation of some secondary products, but not all. In response to elicitation by methyl jasmonate, metabolite production was different for the different classes of compounds, and hormonal composition of the culture medium influenced the response. Thus, results confirm the importance of the reciprocal interactions between hormones, nutrients, and elicitors when attempts are made to enhance secondary metabolite accumulation in in vitro cultures.     

Stimulation of ajmalicine production and excretion from Catharanthus roseus: effects of adsorption in situ,elicitors and alginate immobilization

Summary More efficient bioreactors for the production and recovery of secondary metabolites from plant cell cultures are needed. Three factors that have the potential to increase productivity are adsorption in situ, elicitors, and cell immobilization. The effects of these factors on ajmalicine production from Catharanthus roseus are reported in this paper. Elicitation using autoclaved cultures of the mold, Phytophthora cactorum, stimulates a 60% increase in ajmalicine production. The response time to elicitor addition was under 11 h. Adsorption of ajmalicine from the extracellular medium with the neutral resin, Amberlite XAD-7, greatly enhanced the release of ajmalicine (less than 10% extracellular to 40%) with a 40% increase in total productivity. Immobilization in Caalginate beads resulted in a significant increase in the accumulation of ajmalicine in the medium. The effects of elicitation, adsorption and immobilization were synergistic. For a 23-day culture period the amount of ajmalicine in the medium for cells subjected to all three treatments was 90 mg/L compared to 2 mg/L for suspension cultures cultured under otherwise identical conditions. These results suggest that immobilized cell bioreactors may be feasible for continuous production of products normally stored intracellularly in vacuoles in plant cells.     

Permeabilization of cultivated plant cells by electroporation for release of intracellularly stored secondary products

Summary Plant cell suspension cultures producing secondary metabolites have been permeabilized for product release by electroporation. The two cell cultures studied, i.e. Thalictrum rugosum and Chenopodium rubrum, require about 5 and 10 kV cm^–1, respectively, for complete permeabilization (release of all the intracellularly stored product). The number of electrical pulses and capacitance used had a relatively limited effect on product release while the viability of the cells was strongly influenced by the latter. Conditions for complete product release resulted in total loss of viability of the cells after treatment. The release of product from immobilized cells was also achieved by electroporation. Cells entrapped in alginate required less voltage for permeabilization than free or agarose entrapped cells.     

Elicitation: A biotechnological tool for enhanced production of secondary metabolites in hairy root cultures

Elicitation is a possible aid to overcome various difficulties associated with the large‐scale production of most commercially important bioactive secondary metabolites from wild and cultivated plants, undifferentiated or differentiated cultures. Secondary metabolite accumulation in vitro or their efflux in culture medium has been elicited in the undifferentiated or differentiated tissue cultures of several plant species by the application of a low concentration of biotic and abiotic elicitors in the last three decades. Hairy root cultures are preferred for the application of elicitation due to their genetic and biosynthetic stability, high growth rate in growth regulator‐free media, and production consistence in response to elicitor treatment. Elicitors act as signal, recognized by elicitor‐specific receptors on the plant cell membrane and stimulate defense responses during elicitation resulting in increased synthesis and accumulation of secondary metabolites. Optimization of various parameters, such as elicitor type, concentration, duration of exposure, and treatment schedule is essential for the effectiveness of the elicitation strategies. Combined application of different elicitors, integration of precursor feeding, or replenishment of medium or in situ product recovery from the roots/liquid medium with the elicitor treatment have showed improved accumulation of secondary metabolites due to their synergistic effect. This is a comprehensive review about the progress in the elicitation approach to hairy root cultures from 2010 to 2019 and the information provided is valuable and will be of interest for scientists working in this area of plant biotechnology.     

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.     

Yeast extract and methyl jasmonate-induced silymarin production in cell cultures of Silybum marianum (L.) Gaertn

The biosynthesis of the flavonolignan silymarin, a constitutive compound of the fruits of Silybum marianum with strong antihepatotoxic and hepatoprotective activities, is severely reduced in cell cultures of this species. It is well known that elicitation is one of the strategies employed to increase accumulation of secondary metabolites. Our study here reports on the effect of several compounds on the production of silymarin in S. marianum cultures. Yeast extract (YE), chitin and chitosan were compared with respect to their effects on silymarin accumulation in S. marianum suspensions and only yeast extract stimulated production. Jasmonic acid (JA) potentiated the yeast extract effect. One of the jasmonic acid derivatives, methyl jasmonate (MeJA), strongly promoted the accumulation of silymarin. Methyl jasmonate acted in a number of steps of the metabolic pathway of flavonolignans and its stimulating effect was totally dependent of "de novo" protein synthesis. Chalcone synthase (CHS) activity was enhanced by methyl jasmonate; however there did not appear to be a temporal relationship between silymarin accumulation and increase in enzyme activity. Also, this increase was not blocked by the protein synthesis inhibitor cycloheximide (CH). This study indicates that elicitor treatment promotes secondary metabolite production in S. marianum cultures and that jasmonic acid and its functional analogue plays a critical role in elicitation.     

Efficient production and recovery of diterpenoid tanshinones in Salvia miltiorrhiza hairy root cultures with in situ adsorption, elicitation and semi-continuous operation

In Salvia miltiorrhiza hairy root cultures, the desired secondary metabolites diterpenoid tanshinones are normally produced at low yields and stored within the roots. To enhance tanshinone production and the secondary product recovery, we employed three means, elicitation with a yeast elicitor (YE), in situ adsorption of tanshinones with a hydrophobic polymeric resin (X-5) and semi-continuous mode of operation. YE treatment stimulated the tanshinone biosynthesis, increasing the total tanshinone (TT) content of root by about two-fold, from 0.46 to 1.37 mg/g dry weight (dw) (TT content=total content of three major tanshinones, cryptotanshinone, tanshinone I and tanshinone IIA). The addition of X-5 resins to the culture only increased the tanshinone yield slightly, but recovered more than 80% of tanshinones from the roots. With the application of a semi-continuous culture process involving repeated medium renewal, elicitor addition and resin replacement, starting at the late exponential growth phase, the root biomass was increased to 30.5g dw/l (versus 8-10g dw/l in batch mode) and the volumetric tanshinone yield to 87.5mg/l (about 15-fold increase), with 76.5% adsorbed to the resin. The volumetric productivity of total tanshinone reached 1.46 mg/lday, more than 7.4 times that of the batch culture. The results demonstrate that the integration of multiple elicitation, in situ adsorption and semi-continuous operation can synergistically enhance tanshinone production in S. miltiorrhiza hairy root cultures.     

Production of 8-epi-tomentosin by plant cell culture ofXanthium strumarium

This study was conducted to establish a plant cell culture system for the production of medically important secondary metabolites fromXanthium strumarium. The effects of plant growth regulators including NAA, 2,4-D, kinetin, and ABA were examined in terms of callus induction, maintenance of callus and suspension cultures. It was shown that callus was induced upon treatment with NAA while embryo was induced after treatment with 2,4-D. Callus formation was further improved by treatment with ABA and NAA. The level of callusing increased by 17–29% for the seed case, cotyledon, leaf, and hypocotyl and by 96% in the case of the root. Suspension cell lines were established using calli produced from cotyledon, hypocotyl and root and cultured at 25°C under light conditions. The cells grew up to 15 g/L with NAA 2 ppm, BA 2 ppm, and ABA 1 ppm treatment. Supernatants of suspension cultures of cell lines derived from coyledon and hypocotyl produced some distinctive secondary metabolites, one of which was identified as 8-epi-tomentosin, which belongs to the xanthanolides. The amounts of 8-epi-tomentosin produced by the cotyledon-and hypocotylderived cell lines were 13.4 mg/L and 11.0 mg/L, respectively.     

Induction of anthraquinone biosynthesis in Rubia cordifolia cells by heterologous expression of a calcium-dependent protein kinase gene

Calcium-dependent protein kinases (CDPKs) play an important role in plant cell responses to stress and pathogenic attack. In this study, we investigated the effect of heterologous expression of the Arabidopsis CDPK gene, AtCPK1, on anthraquinone production in transgenic Rubia cordifolia cells. AtCPK1 variants (a constitutively active, Ca(2+) -independent form and a non-active form used as a negative control) were transferred to callus cells by agrobacterial transformation. Overexpression of the constitutively active, Ca(2+) -independent form in R. cordifolia cells caused a 10-fold increase in anthraquinone content compared with non-transformed control cells, while the non-active form of AtCPK1 had no effect on anthraquinone production. Real-time PCR measurements showed that the activation of anthraquinone biosynthesis in transgenic calli correlated with the activation of isochorismate synthase gene expression. The activator effect of AtCPK1 was stable during prolonged periods of transgenic cell cultivation (more than 3 years) and the transgenic cultures exhibited high growth. Our results provide the first evidence that a CDPK gene can be used for the engineering of secondary metabolism in plant cells.     

Enhanced production of plumbagin in immobilized cells of Plumbago rosea by elicitation and in situ adsorption

Cell cultures of Plumbago rosea were immobilized in calcium alginate and cultured in Murashige and Skoog's basal medium containing 10 mM CaCl(2) for the production of plumbagin, an important medicinal compound. Studies were carried to find out the impact of immobilization on the increased accumulation of this secondary metabolite. Immobilization in calcium alginate enhanced the production of plumbagin by three, two and one folds compared to that of control, un-crosslinked alginate and CaCl(2) treated cells respectively. Cell loading at a level of 20% to the polymer volume (Na-alginate) was optimal and maximum plumbagin was obtained. At higher cell loading (40-50%), lower plumbagin accumulation was noticed. Addition of 200 mg l(-1) chitosan as an elicitor to the immobilized cells resulted in eight and two folds higher accumulation of plumbagin over control and immobilized cells. Also, more than 70% of the plumbagin was released into the medium, which is highly desirable for easy recovery of the product. Sucrose utilization rate of the cells was higher when cells were subjected to in situ product removal using Amberlite XAD-7. This may indicate that the toxicity of plumbagin was reduced on cells when it was removed from the medium. Cells subjected to combined treatments of chitosan, immobilization and in situ extraction showed a synergistic effect and yielded 92.13 mg g(-1) DCW of plumbagin which is 21, 5.7, 2.5 times higher than control, immobilized, immobilized and elicited cells respectively.     

Biosynthesis of avenanthramides in suspension cultures of oat (<Emphasis Type="Italic">Avena sativa</Emphasis>)

Oats produce a group of secondary metabolites termed avenanthramides (avn). These compounds are biosynthesized through the action of the enzyme hydroxycinnamoyl CoA: hydroxyanthranilate N-hydroxycinnamoyl transferase (HHT) which catalyzes the condensation of one of several cinnamate CoA thioesters with the amine functionality of anthranilic acid, 4-hydroxy- or 5-hydroxy-anthranilic acid. In oat leaf tissue the biosynthesis of avenanthramides appears to result from elicitation by fungal infection. Here we demonstrate the biosynthesis of several avenanthramides in suspension cultures of oat apical meristem callus tissue. This phenomenon appears as a generalized pathogen response, evidenced by the production of PR-1 mRNA, in response to elicitation with chitin (poly-N-acetyl glucosamine). The suspension cultures also produce relatively large quantities of avnA and G in response to chitin elicitation. Under certain culture conditions avnB and C are also produced as well as three additional metabolites tentatively identified as avnH, O and R. These findings portend the utility of oat suspension culture as a tool for more detailed investigation of the mechanisms triggering their biosynthesis as well as the factors dictating the particular types of avenanthramides biosynthesized.     

Improvement of phenylethanoid glycosides biosynthesis in Cistanche deserticola cell suspension cultures by chitosan elicitor

Effect of chitosan elicitor on growth and phenylethanoid glycosides (PeGs) accumulation in Cistanche deserticola cell suspension cultures was investigated. PeGs accumulation was dramatically improved by addition of selected chitosan at optimal elicitation conditions. Furthermore, a strategy of repeated addition of the chitosan elicitor for enhancing PeGs accumulation was developed. The chitosan elicitor of 10 mg l(-1)-medium repeatedly added on days 15 and 17 improved PeGs accumulation further, and the final PeGs production in the treated cell cultures of C. deserticola reached 364.6 mg l(-1), which was 3.4-fold higher than that of the control without elicitation. The increase of PeGs accumulation in C. deserticola cell suspension cultures was related to the increase of phenylalanine ammonium lyase activity stimulated by the chitosan elicitor.     

Enhanced production and secretion of rutin and GABA in immobilized cells of mulberry tree (<Emphasis Type="Italic">Morus bombycis</Emphasis> K.)

Cell immobilization has been proposed as a useful technique for mass production and efficient purification of secondary metabolites. In this study, we compared the bio-productivity of ligand-free and Ca-alginate-immobilized mulberry cells for rutin and γ-amino butyric acid (GABA). In the leaves of Subong mulberry plants (M. bombycis K.) grown in a greenhouse, GABA accumulated as the leaves aged; a more than a 20-fold increase of GABA was observed in leaves undergoing senescence than in younger leaves. In contrast, more rutin was detected in mature leaves than in young leaves and those undergoing senescence. The production of total proteins in ligand-free leaf callus cells dramatically increased until 6 days after incubation in liquid suspension media (from 6.5 mg/g callus at day 0–14.5 mg/g callus), and by day 15 dropped to levels similar to those seen in the 0-day control. In contrast, immobilized cells showed a slight increase and then an insignificant decrease in protein content during the 15-day incubation period. Interestingly, immobilized mulberry cells more efficiently produced and secreted rutin and GABA into the suspension media than ligand-free cells. KN, a cytokinin, enhanced this production while 2,4-dichlorophenoxyacetic acid(2,4-D), an auxin, alleviated the effect of KN. As a result, incubation of the immobilized Subong cells in a full-strength Murashige and Skoog (MS) liquid medium containing 1 mg/l of 2,4-D and 0.1 mg/l KN, among the hormone combinations in the medium we tested, produced the highest amounts of rutin (8.2 μg/g callus cells) and GABA (305 μg/g callus cells) and secreted the largest amounts into the suspension media.     

Metabolism of 2,4-dichlorophenoxyacetic Acid in soybean root callus and differentiated soybean root cultures as a function of concentration and tissue age

The metabolism of [1-¹⁴C]2,4-dichlorophenoxyacetic acid (2,4-D) in soybean (Glycine max [L.] Merrill var. Amsoy) root callus and in differentiated soybean root cultures was investigated as a function of pesticide concentration and age of tissue. The chronological age of the tissue was found to be correlated with the mitotic index which reached a peak at 2 weeks and then declined. The metabolism of 2,4-D changed with age of the root callus tissue. The amount of free 2,4-D found in 3-week-old root callus tissue rapidly increased as the concentration of 2,4-D in the medium was increased from 10⁻⁶ to 10⁻⁵ molar, whereas the low level of aqueous (glycosides) and ether soluble metabolites (2,4-D amino acid conjugates) increased slowly. With 9-week-old root callus tissue, the amount of free 2,4-D remained at a relatively low, constant level (saturation level) as the concentration of 2,4-D in the medium increased. Under these conditions the aqueous metabolites increased only slightly but the ether fraction (2,4-D amino acid conjugates) rapidly increased. Thus, the older root callus tissue appeared to regulate the level of free 2,4-D at about 4 nanomoles per gram by converting any excess 2,4-D into amino acid conjugates.

In 3-week-old, differentiated root cultures the metabolism of 2,4-D closely paralleled the metabolism found in the older 9-week-old callus tissue. The saturation level of free 2,4-D found in this tissue was only about 1 to 2 nanomoles per gram.

     

Exudation: an expanding technique for continuous production and release of secondary metabolites from plant cell suspension and hairy root cultures

This review addresses methods of obtaining secondary metabolites from plant cell suspension and hairy root cultures and their exudates, particularly the physiological mechanisms of secondary metabolites release and trafficking. The efficiency for product recovery of metabolites can be increased by various methods, based on the principle of continuous product release into the cultivation medium. The most common methods for metabolite recovery are elicitation, influencing membrane permeability, and in situ product removal. The biosynthetic pathways can be influenced by cultivation conditions, transformation, or application of elicitors. The membrane permeability can be altered through the application of chemical or physical treatments. Product removal can be greatly increased through a two-phase system and the introduction of absorbents into the cultivation medium. In this review, we describe some improved approaches that have proven useful in these efforts.     

Cell line selection through gamma irradiation combined with multi-walled carbon nanotubes elicitation enhanced phenolic compounds accumulation in Salvia nemorosa cell culture

The current study focused on improving the production of phenolic acids in the Woodland Sage cell suspension culture (CSC) through attaining high-yielding cell lines and carboxyl functionalized multi-walled carbon nanotubes (MWCNT-COOH) elicitation. The leaf-derived callus was irradiated at different doses of gamma irradiation 10 to 100 Gy. The maximum content of rosmarinic acid (RA), salvianolic acid B (SAB), ferulic acid (FA), and cinnamic acid (CA) was recorded in callus cultures irradiated with 70 Gy, which was 18.53, 5.21, 1.9, and 7.59 mg/g DW, respectively. The CSC that established from 70 Gy γ-irradiated calli accumulated 1.7-fold RA more higher irradiated callus culture. The CSC elicited with various concentrations of MWCNT-COOH in range 25 to 100 mg/l significantly increased fresh weight (FW), dry weight (DW), and phenolic acid contents of cells. The highest FW with 268.47 g/l and DW with 22.17 g/l was obtained in 100 mg/l MWCNT-COOH treatment. The RA, SAB, CA and FA content of CSC treated with 100 mg/l MWCNT-COOH were 13-fold, 14.2-fold, 20-fold, and 3- fold higher than wild S. nemorosa plant at flowering stage, respectively. The antioxidant activity of cultures significantly enhanced with both gamma and MWCNT-COOH based on DPPH and FRAP assay. Our results showed that the combination of cell line selection and MWCNT-COOH elicitation significantly improved the production of secondary metabolites in Woodland Sage, which is useful for large-scale production of phenolic compounds.

     

Morphactin influences guggulsterone production in callus cultures of <Emphasis Type="Italic">Commiphora wightii</Emphasis>

Guggulsterone, a hypolipidemic natural agent, is produced in resin canals of the plant Commiphora wightii. In this study, the efficacy of different plant growth regulators was evaluated for optimizing its production. Morphactin was found to be effective in enhancing the accumulation of guggulsterones in callus cultures. Maximum callus growth was recorded on medium containing morphactin (0.1 mg l⁻¹) and 2iP (2.5 mg l⁻¹), whereas maximum guggulsterone production occurred when the calluses were cultured on medium containing 0.1 mg l⁻¹ morphactin and 1.0 mg l⁻¹ 2iP. Morphactin and 2iP interacted significantly to enhance the callus growth and guggulsterone production by about 8-folds in one-year-old cultures. However, the effect of morphactin on callus growth and guggulsterone production was not uniform over the levels of 2iP tested. Such an effect of morphactin has never been reported on the production of secondary metabolites.     

Comparison of secondary product accumulation in photoautotrophic,photomixotrophic and heterotrophic Nicotiana tabacum cell suspension cultures

Summary Photoautotrophic, photomixotrophic and heterotrophic Nicotiana tabacum cell suspension cultures were compared for the constitutive accumulation of secondary metabolites and the elicitor-induced formation of the phytoalexin capsidiol. Nicotine and chlorogenic acid were found in high amounts in the heterotrophic cultures and in moderate concentrations in photomixotrophic but not in photoautotrophic cells. Nicotinic acid-N-glucoside occured in all culture types; in photoautotrophic and photomixotrophic cells the formation of N-methylnicotinic acid (trigonelline) was also observed. Treatment with a fungal elicitor led to substantial accumulation of capsidiol in heterotrophic and photomixotrophic cells and in only low levels in photoautotrophic cultures. Elicitor-treated photomixotrophic cells showed a pronounced increase in cell wall-bound phenolics. The levels of nicotine, nicotinic acid-N-glucoside and trigonelline were not affected by elicitation.Abbreviations hcc heterotrophic cell culture - mcc photomixotrophic cell culture - pcc photoautotrophic cell culture - fr.wt. freshweight - nic-N-glc nicotinic acid-N-glucoside - PMG Phytophthora megasperma f. sp. glycínea - HPLC high performance liquid chromatography - GC gas chromatography - TLC thin layer chromatography - 2,4D 2,4-dichlorophenoxyacetic acid - Kin kinetin - BAP 6-benzylaminopurine - NAA -naphthylacetic acid     

Effect of Hormone Treatment on Growth Bud Formation and Free Amine and Hydroxycinnamoyl Putrescine Levels in Leaf Explant of Nicotiana tabacum Cultivated in Vitro

Foliar explants of Nicotiana tabacum cv Xanthi n.c. were cultured on four different media: a basal medium, basal medium plus benzyladenine, basal medium plus 2,4-dichlorophenoxyacetic acid (2,4-D), and the basal medium containing both hormones. No differentiation or cell division occurred in leaf explants cultured on the basal medium. Addition of benzyladenine caused the formation of buds on the explants, while 2,4-D caused callus formation and proliferation. Likewise, only callus was formed when explants were cultured on medium containing both hormones, but growth was significantly greater than that of callus grown on a medium containing 2,4-D alone. The levels of amines and hydroxycinnamoyl putrescines were determined in the four types of explants. In nongrowing explants, amines (except an aromatic amine, tyramine) and hydroxycinnamoyl putrescines were always at a low level and only small changes in their concentrations were observed. In callus cultures, amine (except an aromatic amine, phenethylamine) and hydroxycinnamoyl putrescine levels were higher than those found in bud cultures. In all the media, transitory accumulation of aromatic amines occurred after a few days of culture. Higher levels of hydroxycinnamoyl putrescines were attained in callus cultures with a slow growth rate (2,4-D alone) than in callus cultures with a fast growth rate (benzyladenine + 2,4-D). The formation of buds was accompanied by significant changes in putrescine and hydroxycinnamoyl putrescine levels. Increasing levels were found during the first 14 days in culture when cell multiplication was rapid, followed by a sharp decline after 20 days in culture as the rate of cell division decreased and differentiation took place. The relationship among amines, hydroxycinnamoyl putrescines, and cell division and bud formation is discussed.