Morphactin and 2iP markedly enhance accumulation of stilbenes in cell cultures of Cayratia trifolia (L.) Domin

The cell cultures of Cayratia trifolia (Vitaceae) a tropical lianas, were maintained in Murashige and Skoog’s medium containing 0.25 mg l⁻¹ naphthalene acetic acid, 0.2 mg l⁻¹ kinetin and 250 mg l⁻¹ casein hydrolysate. Cell suspension cultures of C. trifolia accumulate stilbenes (piceid, resveratrol, viniferin, ampelopsin) which on addition of 0.1–0.5 mg l⁻¹ morphactin in the medium containing naphthalene acetic acid and kinetin declined. Morphactin or 2 isopentenyl adenine alone at 0.1 mg l⁻¹ concentration enhanced stilbenes which on combination markedly enhanced the yield to ~5 mg l⁻¹ at 15th day.     

Elicitor-induced formation of free and cell-wall-bound stilbenes in cell-suspension cultures of Scots pine (Pinus sylvestris L.)

Treatment of Pinus sylvestris L. cell-suspension cultures with an elicitor preparation from the pine needle pathogen Lophodermium seditiosum, resulted in a severalhundredto thousandfold accumulation of the stilbenes pinosylvin and pinosylvin 3-O-methyl ether in methanolic cell extracts. There was a simultaneous induction of the biosynthetic enzymes phenylalanine ammonia-lyase (E.C. 4.3.1.5.) and stilbene synthase (pinosylvin-forming, E.C. 2.3.1.146). For the first time, an incorporation of stilbenes into the cell wall fraction as well as stilbene excretion into the extracellular space was demonstrated in addition to intracellular accumulation.Abbreviations PAL phenylalanine ammonia-lyase - PS pinosylvin - PSM pinosylvin 3-O-methyl ether - STS stilbene synthase (pinosylvin-forming) This study has been supported by the Bayerisches Staatsministerium für Landesentwicklung und Umweltfragen, Fonds der chemischen Industrie, and EUROSILVA. The authors wish to thank their colleagues L. Gößl for maintaining the Scots pine cell-suspension culture, and Dr. G. Bahnweg for supplying the Lophodermium mycelia.     

Differential accumulation of xanthones in methyl-jasmonate- and yeast-extract-treated cell cultures of Centaurium erythraea and Centaurium littorale

Cell-suspension cultures of Centaurium erythraea and Centaurium littorale (Gentianaceae) respond to methyl jasmonate and yeast extract with a differential accumulation of xanthones. Methyl jasmonate induced the formation of 1-hydroxy-3,5,6,7-tetramethoxyxanthone, the amount of which increased in both cell cultures around 10 h after addition. A substantial increase in the activity of phenylalanine ammonia-lyase (PAL) was not observed. When challenged with yeast extract the cell cultures accumulated l,5-dihydroxy-3-methoxyxanthone. This appeared rapidly after addition of yeast extract in C. erythraea but its amount in C. littorale increased only after a lag phase of 25 h. While PAL activity in C. erythraea was strongly suppressed a fourfold increase in its activity was found in C. littorale. Both elicited xanthones accumulated intracellularly. A scheme for xanthone biosynthesis in the two cell cultures is proposed.     

Influence of methyl jasmonate on podophyllotoxin and 6-methoxypodophyllotoxin accumulation in Linum album cell suspension cultures

The accumulation of podophyllotoxin (PTOX) and 6-methoxypodophyllotoxin (6MPTOX) was enhanced about twofold in the suspension culture of Linum album line 2-5 aH following the addition of methyl jasmonate (MeJas) to the cultivation medium, reaching 7.69±1.45 mg/g dry weight and 1.11±0.09 mg/g dry weight, respectively. There was no increase in 6MPTOX accumulation following the addition of MeJas to suspension cells of L. album line X4SF, whereas PTOX accumulation was enhanced about tenfold to 0.49±0.10 mg/g dry weight. Phenylalanine ammonia-lyase activity increased immediately after the addition of MeJas to a cell suspension culture of line X4SF, reaching a maximum between 4 h and 1 day after elicitation, while cinnamyl alcohol dehydrogenase activity and the lignin content of the cells were not affected.     

Methyl jasmonate-induced rosmarinic acid biosynthesis in Lithospermum erythrorhizon cell suspension cultures

Summary A dramatic increase in rosmarinic acid (RA) content in cultured cells of Lithospermum erythrorhizon was observed after their exposure to methyl jasmonate (MJ). Preceding the induced RA accumulation, phenylalanine ammonia-lyase (PAL) and 4-hydroxyphenylpyruvate reductase (HPR) activities increased rapidly and transiently, whereas tyrosine aminotransferase (TAT) activity showed only a slight increase. The elicitation activity of MJ was much higher than that of yeast extract (YE) in terms of the induction of PAL and HPR activities, RA accumulation and incorporation of both ¹⁴C-phenylalanine and ¹⁴C-tyrosine into RA. However, the response of the cultured cells to MJ-treatment was slower than that to YE-treatment.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - LS Linsmaier and Skoog - HPR 4-hydroxyphenylpyruvate reductase - PAL phenylalanine ammonia-lyase - TAT tyrosine aminotransferase - MJ methyl jasmonate - YE yeast extract     

Ethrel treatment enhanced isoflavonoids accumulation in cell suspension cultures of Pueraria tuberosa, a woody legume

The cell cultures of Pueraria tuberosa, a perennial leguminous lianas, were maintained in modified MS medium (KNO₃ 475 mg l⁻¹, thiamine 1 mg l⁻¹, biotin 1 mg l⁻¹, calcium pantothenate 1 mg l⁻¹) containing 0.1 mg l⁻¹ 2,4,5-trichloroacetic acid and 0.1 mg l⁻¹ kinetin. Isoflavonoids (puerarin, genistin, daidzein, genistein) accumulation in cell suspension cultures was increased by 14-fold to ~12 mg l⁻¹ after 48 h of adding 100 μM ethrel. Ethrel inhibitors (silver nitrate and silver thiosulfate) completely inhibited this effect in the presence of ethrel and isoflavonoids were not detected in the spent medium. The increase was dose dependent and can be explored to trigger high yield of isoflavonoids production.     

Metabolism of 2,4-dichlorophenoxyacetic acid in cell suspension cultures of soybean (Glycine max L.) and wheat (Triticum aestivum L.)

Cell suspension cultures of soybean (Glycine max L.) and wheat (Triticum aestivum L.) incorporated 2,4-dichlorophenoxyacetic acid (2,4-D) into a metabolite fraction which was insoluble in ethanol, water, and hot sodium dodecylsulphate. Further treatment with hot dimethylformamide solubilized a material which by the following criteria appeared to consist of 2,4-D derivatives covalently bound to lignin: i) co-chromatography of radioactivity and of UV-absorbing material upon gel permeation chromatography; ii) spectral similarity with authentic lignins (IR- and UV-spectra, phloroglucinol reaction), 2,4-D appeared to be incorporated as the intact molecule, as shown by comparison of ring- and sidechain-labeled 2,4-D and by detection of monohydroxylated and intact 2,4-D as the major radioactive products of acid hydrolysis. The same compounds were released from the metabolite material which could not be solubilized in dimethylformamide. The incorporation of xenobiotics or their metabolites into lignin, followed by deposition in the cell wall, is suggested as a general pathway for local excretion and detoxification by plant cells.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - 4-OH-2,5-D 4-hydroxy-2,5-dichlorophenoxyacetic acid - SDS sodium dodecylsulphate - DMF dimethylformamide     

Enhanced silymarin accumulation is related to calcium deprivation in cell suspension cultures of Silybum marianum (L.) Gaertn

Elimination of calcium ions from the medium of cell cultures of Silybum marianum (L.) Gaertn increased flavonolignan production. Silymarin accumulation was not altered by treatment of cultures with the calcium ionophore A23187. The specific Ca2+ chelator, EGTA, enhanced the silymarin content in cells by 200%, and its secretion by 3-4 times. The inorganic ion La3+, as well as the calcium channel inhibitor verapamil, also stimulated production. Several reagents known to block intracellular calcium movement, such as ruthenium red, thapsigargin and TMB-8 appreciably increased silymarin accumulation. These results suggest that inhibition of external and internal calcium fluxes plays a significant role in flavonolignan metabolism of S. marianum cell cultures.     

Stimulation of asiaticoside accumulation in the whole plant cultures of <Emphasis Type="Italic">Centella asiatica</Emphasis> (L.) Urban by elicitors

The effects of a number of different elicitors on asiaticoside production in whole plant cultures of Centella asiatica were studied, including yeast extract, CdCl₂, CuCl₂ and methyl jasmonate (MJ). Only MJ and yeast extract stimulated asiaticoside production—1.53 and 1.41-fold, respectively. Maximum asiaticoside production was achieved following treatment with 0.1 mM MJ (116.8 mg/l). The highest asiaticoside production (342.72 mg/l) was obtained after 36 days of elicitation in cultures treated with 0.1 mM MJ and 0.025 mg/l 1-phenyl-3-(1,2,3-thidiazol-5-yl)urea (TDZ). Interestingly, MJ not only stimulated the production of asiaticoside but also had an important role in the senescence of C. asiatica. Although asiaticoside content did not change when TDZ was added to medium containing an elicitor, TDZ did increase shoot growth of C. asiatica. We discuss the interactive roles of MJ and TDZ in secondary metabolic production and biomass in whole plants of C. asiatica     

Isolation of ESTs from cacao (<Emphasis Type="Italic">Theobroma cacao</Emphasis> L.) leaves treated with inducers of the defense response

Pathogenic diseases represent a major constraint to the growth and yield of cacao (Theobroma cacao L.). Ongoing research on model plant systems has revealed that defense responses are activated via signaling pathways mediated by endogenous signaling molecules such as salicylic acid, jasmonic acid and ethylene. Activation of plant defenses is associated with changes in the expression of large numbers of genes. To gain a better understanding of defense responses in cacao, we have employed suppressive subtractive hybridization (SSH) cDNA libraries, macroarray hybridization analysis, high throughput DNA sequencing and bioinformatics to identify cacao genes induced by these signaling molecules. Additionally, we investigated gene activation by a phytotoxic elicitor-like protein, Nep1. We have identified a unigene set of 1,256 members, including 330 members representing genes induced during the defense response.Electronic Supplementary Material Electronic supplementary material is available in the online version of this article at Sequences presented here are deposited with GenBank under accession numbers CF972636–CF974749     

First evidence of trans-resveratrol production in cell suspension cultures of cotton (Gossypium hirsutum L.)

For the first time, trans-resveratrol, a stilbene, has been identified in cotton cell suspensions. Cell suspensions of Coker 312, a cultivar which produces embryogenic structures, acccumulate trans-resveratrol contrary to those of cultivar R405-2000, which do not. This stilbene may be a good phenolic marker for induction of somatic embryogenesis in cotton.     

Furoquinoline alkaloids of Ertela (Monnieria) trifolia (L.) Kuntze from the Suriname rainforest

7-(2'-Hydroxy-3'-chloroprenyloxy)-4,8-dimethoxyfuroquinoline (1) and 6-(2'-hydroxy-3'-chloroprenyloxy)-4,7-dimethoxyfuroquinoline (2), together with ten known compounds, have been isolated from the aerial parts of Ertela (Monnieria) trifolia (L.) Kuntze. All the isolates were tested for antiproliferative activity against the A2780 human ovarian cancer cell line.     

High stilbenes accumulation in root cultures of <Emphasis Type="Italic">Cayratia trifolia</Emphasis> (L.) Domin grown in shake flasks

The Root cultures of Cayratia trifolia (Vitaceae) a tropical lianas, were maintained in liquid Murashige and Skoog’s medium containing 0.5 mg l⁻¹ NAA, 0.1 mg l⁻¹ kinetin with 3% sucrose. These root cultures when grown with 6% sucrose accumulated stilbenes (piceid, resveratrol, viniferin, ampelopsin) in high amounts, which on elicitation by 500 mg l⁻¹ yeast extract, 50 μM salicylic acid (SA), 50 μM methyl jasmonate (MeJa), 500 μM ethrel added at 25th day, increased up to ninefolds (7.1 mg l⁻¹). Addition of alar or phenylalanine along with the elicitors further enhanced the stilbenes content. In the present study, stilbenes accumulation up to 12 folds (9.2 mg l⁻¹) was obtained with SA and alar. The SA was the most effective in increasing the stilbenes contents while less than control values were recorded in the cells treated with MeJa. The roots could be grown up to 2 l flasks. The present work demonstrates that presence of precursor and sucrose during elicitation at an appropriate time combined with growth retardation significantly increased the production of stilbenes in C. trifolia cell cultures.     

Enhanced benzophenanthridine alkaloid production and protein expression with combined elicitor in <Emphasis Type="Italic">Eschscholtzia californica</Emphasis> suspension cultures

Production of the benzophenanthridine alkaloids in Eschscholtzia californica suspension cell cultures was optimized by adding 0.5 mg methyl jasmonate (MJ) and 0.02 mg salicylic acid (SA)/g FCW after 7 days cultivation. Sanguinarine reached 24 mg/g DCW by such treatment; 10 times higher than in control cell cultures. MJ and SA induced expression of berberine bridge enzyme and 3′-hydroxy-(S)-N-methylcoclaurine-4′-O-methyltransferase, respectively. MJ plus SA induced over-expression of both enzymes.     

Yew (Taxus x media Rehd.) cell suspension cultures as a source of taxanes

Natural resources of paclitaxel, an effective anticancer compound, were threatened with extinction soon after the discovery of this valuable substance. Cell suspension cultures derived from different Taxus species have rapidly become an alternative source of paclitaxel and other taxanes. In this paper we provide some insight into cell growth characteristics in cell suspension culture of Taxus x media cv. Hicksii, with emphasis on the effects of jasmonic acid (JA) on taxane production in cell lines with different initial taxane content. Additionally cell growth characteristics of two cell lines was followed during cultivation of cell suspension culture of Taxus x media cv. Hicksii. Packed cell volume (PCV) was shown to be a reliable and efficient alternative for measuring cell growth instead of fresh and dry weight. The initial total taxane content was screened in a number of cell lines, followed by observing the effect of JA on cell mass and total taxane production of selected lines. We showed a great variability in initial taxane content in different cell lines, which decreased during cell suspension maintenance. JA was shown to inhibit cell growth and increase total taxane production (14 to 106 fold).     

Pisatin metabolism in pea (Pisum sativum L.) cell suspension cultures

Cell suspension cultures were established from germinating pea (Pisum sativum L.) seeds. This cell culture, which accumulated pisatin, consisted mostly of single cells containing a few cell aggregates. The cells responded to treatment with a yeast glucan preparation with transient accumulation of pisatin in both cells and culture media. Addition of pisatin to cell cultures resulted in increased synthesis of pisatin. Phenylalanine ammonia-lyase, chalcone synthase and isoflavone reductase activities were present in untreated cells. Upon treatment with an elicitor preparation the activities of the first two enzymes showed a rapid, transient increase up to 20 hours after treatment. Isoflavone reductase showed a major and minor peak at 16 and 36 h, respectively, after elicitor treatment. The time course of the enzyme activity and pisatin accumulation is consistent with an elicitor-mediated response.Abbreviations CHS chalcone synthase - 2,4-D 2,4-dichlorophenoxyacetic acid - IBA indole-3-butyric acid - IFR isoflavone reductase - 2iP 6-(dimethylallylamino)-purine - MS Murashige & Skoog basal salt medium - PAL phenylalanine ammonia-lyase - PMSF phenylmethylsulfonyl fluoride - POPOP 1,4-bis-2-(4-methyl-5-phenyloxazolyl)-benzene - PPO 2,5-diphenyloxazole     

Calcium restriction induces cardenolide accumulation in cell suspension cultures of Digitalis thapsi L.

Summary The removal of calcium ions from Murashige and Skoog culture medium induced a marked increase in the accumulation of cardenolides in cell suspension cultures of Digitalis thapsi. Cell viability was not affected although growth was slightly reduced. Strontium ions could substitute for calcium in inhibiting cardenolide production, this effect of calcium being reversed by the addition of LaCl₃ or ethyleneglycol-bis-(-aminoethyl ether)-N,N-tetraacetic acid. The results suggest that calcium, apart from its general effects on growth, may play a role in the regulation of cardenolide metabolism in a concentration dependent manner.Abbreviations BA 6-benzylaminopurine - 2 4-D 2,4-dichlorophenoxyacetic acid - EGTA ethyleneglycol-bis-(-aminoethyl ether)-N,N-tetraacetic acid - FW fresh weight - MS Murashige and Skoog (1962)     

Bacoside production by suspension cultures of Bacopa monnieri (L.) Pennell

Cell suspension cultures of Bacopa monnieri (L.) Pennell, grown in modified MS medium, grew some 5–6 fold over 40 days. Selected cell lines produced the important saponin, bacoside A, up to 1 g/100 g dry wt after this time.     

Differential accumulation of monolignol-derived compounds in elicited flax (Linum usitatissimum) cell suspension cultures

Lignin and lignans share monolignols as common precursors and are both potentially involved in plant defence against pathogens. In this study, we investigated the effects of fungal elicitors on lignin and lignan metabolism in flax (Linum usitatissimum) cell suspensions. Cell suspension cultures of flax were treated with elicitor preparations made from mycelium extracts of Botrytis cinerea, Phoma exigua and Fusarium oxysporum F ssp lini. Elicitors induced a rapid stimulation of the monolignol pathway, as confirmed by the increase in PAL (phenylalanine ammonia-lyase, EC 4.1.3.5), CCR (cinnamoyl-CoA reductase EC 1.2.1.44) and CAD (cinnamyl alcohol dehydrogenase EC 1.1.1.195) gene expression and PAL activity. At the same time, CCR activity only increased significantly in F. oxysporum-treated cells 24 h post elicitation. On the other hand, CAD activity measured for coniferyl alcohol formation was transiently decreased but a substrate-specific activation of CAD activity was observed in F. oxysporum-treated cells when using sinapyl alcohol as substrate. The accumulation of monolignol-derived products varied according to the elicitor used. B. cinerea or P. exigua-elicited cell cultures were characterised by a reinforcement of the cell wall by a deposit of 8-O-4′-linked non-condensed lignin structures and phenolic monomers, while at the same time no stimulation of 8-8′-linked lignan or 8-5′-linked phenylcoumaran lignan accumulation was observed. Additionally, elicitation of cell cultures with F. oxysporum extracts even triggered a strong incorporation of monolignols in the non condensed labile ether-linked lignin fraction concomitantly with a decrease in lignan and phenylcoumaran lignan accumulation. Several hypotheses are proposed to explain the putative role of these compounds in the defence response of flax cells against pathogens. Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users. C. Hano and M. Addi contributed equally to this work.     

Metabolism of DDT and Kelthane in cell suspension cultures of parsley (Petroselinum hortense,Hoffm.) and soybean (Glycine max L.)

Cell suspension cultures of parsley and soybean were incubated for 44 to 48 h with¹⁴C-labeled DDT or Kelthane; autoclaved cultures were used as controls. Most of the radioactivity became associated with the cells, and metabolites were isolated by a sequential extraction procedure. The metabolites amounted to 0.6 to 2.2% of the applied pesticide. Relatively non-polar metabolites were identified as DDE in the case of DDT, and remained unidentified in the case of Kelthane. Polar metabolites were also isolated and are as yet unidentified. They were chromatographically different from the known and less polar metabolites of DDT and Kelthane reported from animal and insect studies. [DDT-1,1,1-Trichloro-2,2-bis-(4-chlorophenyl)-ethane; Kelthane=(1,1-bis-(4-chlorophenyl)-2,2,2-trichloro-ethanol; DDE=1,1-Dichloro-2,2-bis-(4-chlorophenyl)-ethylene.]Abbreviations DDT 1,1,1-Trichloro-2,2-bis-(4-chlorophenyl)-ethane - Kelthane (1,1-bis-(4-chlorophenyl)-2,2,2-trichloro-ethanol - DDE 1,1-Dichloro-2,2-bis-(4-chlorophenyl)-ethylene - DDA 2,2-bis-(4-chlorophenyl)-acetic acid - DDOH 2,2-bis-(4-chlorophenyl)-ethanol - DDD 1,1-Dichloro-2,2-bis-(4-chlorophenyl)-ethane - DBP 4,4-Dichloro-benzophenone - DDMU 1-Chloro-2,2-bis-(4-chlorophenyl)-ethylene - DDM Bis-(4-chlorophenyl)-methane - FW-152 1,1-Bis-(4-chlorophenyl)-2,2-dichloro-ethanol - SDS sodium dodecylsulphate