Ajmalicine production in methyl jasmonate-induced Catharanthus roseus cell cultures depends on Ca2+ level

Cytosolic Ca²⁺ and jasmonate mediate signals that induce defense responses in plants. In this study, the interaction between Ca²⁺ and methyl jasmonate (MJ) in modulating defense responses was investigated by monitoring ajmalicine production in Catharanthus roseus suspension cultures. C. roseus suspensions were treated with nine combinations of CaCl₂ (3, 23, and 43 mM) and MJ (0, 10, and 100 μM) on day 6 of growth. Increased Ca²⁺ influx through the addition of extracellular CaCl₂ suppressed ajmalicine production in MJ-induced cultures. The highest ajmalicine production (4.75 mg/l) was observed when cells were treated with a low level of calcium (3 mM) combined with a high level of MJ (100 μM). In the presence of 3 mM CaCl₂ in the medium, the addition of Ca²⁺ chelator EGTA (1, 2.5, and 5 mM) or Ca²⁺ channel blocker verapamil (1, 10, and 50 μM) to MJ-induced (100 μM) cultures on day 6 also inhibited ajmalicine production at higher levels of the Ca²⁺ inhibitors. Hence, ajmalicine production in MJ-induced C. roseus cultures depended on the intracellular Ca²⁺ concentration and a low extracellular Ca²⁺ concentration (3 mM) enhanced MJ-induced ajmalicine production.     

Methyl jasmonate increases the production of valepotriates by transformed root cultures of Valerianella locusta

Transformed roots of V. locusta (Valerianaceae) were obtained through transformation with Agrobacterium rhizogenes strains A4 and ATCC 15834. Six known valepotriates, including diavaltrate, acevaltrate, didrovaltrate, IVHD-valtrate, isovaltrate, and valtrate were the major components detected. An LC/PDA method was used in the quantitation of these compounds in the transformed root extracts. The treatment of transformed roots with biotic (methyl jasmonate, salicylic acid, yeast extract) and abiotic elicitors (CuSO₄, HgCl₂, CaCl₂) was used as a strategy to improve the production of valepotriates. Methyl jasmonate appeared to be the best elicitor for valepotriate production, yielding up to a 7-fold increase in total valepotriate content, while HgCl₂ had the most deteriorating effect on the production of valepotriates. Salicylic acid-, CuSO₄- and CaCl₂-treated roots showed significant increases in the production at a short duration of exposure; the production decreased as the time of elicitation increased. The highest total valepotriate content achieved in this study was 139 mg g^–1 DW (13.9%) from transformed roots treated for 10 days with 100 M methyl jasmonate. This amount was >50- and 12-fold higher than the values reported from the cultivated plants and callus culture, respectively, and was comparable to the amount reported from the high valepotriate-producing species Valeriana thalictroides Graebn. The production of diavaltrate, acevaltrate, didrovaltrate, and isovaltrate were significantly higher, while the production of IVHD-valtrate was lower and that of valtrate was similar to that of the control. The IVAL/VAL production ratio was affected by the treatment with methyl jasmonate but not by other elicitors. The use of transformed root cultures in combination with the treatment with biotic and abiotic elicitors offer a new route for high valepotriate production.     

Elicitation of rosmarinic acid by <Emphasis Type="Italic">Lavandula vera</Emphasis> MM cell suspension culture with abiotic elicitors

The growth of Lavandula vera MM plant cell suspension culture and rosmarinic acid biosynthesis under elicitation with benzothiadiazole and methyl jasmonate were investigated. Upon elicitation with 50 μM methyl jasmonate, the production of rosmarinic acid was enhanced 2.4-fold (3348 mg/l) compared to the non-elicited cells. The influence of benzothiadiazole on rosmarinic acid biosynthesis was weaker and 12 h after its addition the achieved yields were 20–30% higher compared to the control variant at this time. The influence of both elicitors on rosmarinic acid secretion into the culture medium was also discussed.     

Enhanced glucotropaeolin production in hairy root cultures of Tropaeolum majus L. by combining elicitation and precursor feeding

We have studied the effects of precursor amino acids (phenylalanine and cystein), elicitors (salicylic and acetylsalicylic acids, methyl jasmonate, β-aminobutyric acid, yeast extract), PAL-inhibitor (1-amino-2-phenylethylphosphonic acid) applied alone or in combination on glucotropaeolin production and myrosinase activity in hairy root cultures of Tropaeolum majus. Short, 24-h treatment, and subsequent transfer of hairy roots to the fresh medium enabled avoiding detrimental effect of studied stimulators on biomass growth. In control cultures the highest glucotropaeolin content, 58.1 ± 6.7 mg g⁻¹ DW, was detected on the 3rd day after transfer of the roots to the fresh medium but glucotropaeolin yield (mg 100 ml^–1 culture volume) had been increasing until the 9th day after transfer as a result of continuous biomass growth. Glucotropaeolin content and yield were 2-fold enhanced after treatment with precursor amino acids or PAL-inhibitor alone, but their combination additively led to 4-fold increase in glucotropaeolin production. Among the studied elicitors acetylsalicylic acid induced the highest, 3-fold increase in glucotropaeolin production, it also enhanced myrosinase activity, but to a smaller extend (by about 50%). Acetylsalicylic acid also potentiated induced by precursors, PAL-inhibitor, methyl jasmonate and yeast extract production of glucotropaeolin. The highest, 4.8-fold increase in glucotropaeolin production was found after combined acetylsalicylic acid and precursors treatment. Additive effect of acetylsalicylic acid-combined treatment on myrosinase activity was not detected. The obtained results indicate that amino acid precursors, phenylalanine and cystein, availability may be a limiting factor in the process of stimulation of glucotropaeolin production in T. majus hairy root cultures.     

A combination of elicitation and precursor feeding leads to increased anthocyanin synthesis in cell suspension cultures of <Emphasis Type="Italic">Vitis vinifera</Emphasis>

Both elicitation and precursor feeding are effective strategies for improving secondary metabolite production in plant cell suspension cultures. In this study, cell suspension cultures of Vitis vinifera subjected to methyl jasmonate treatment resulted in a significant increase in levels of anthocyanin production. Moreover, a combination of 5 mg/L phenylalanine and 50 mg/L methyl jasmonate promoted the highest level of anthocyanin biosynthesis, resulting in 4.6- and 3.4-fold increases in anthocyanin content and yield, respectively, over the control. The optimum period for elicitation of anthocyanin synthesis was 4 days following incubation in the presence of elicitors, at the beginning of the exponential growth phase. V. vinifera cell lines of different anthocyanin-producing capabilities responded differently to elicitation and precursor feeding. Anthocyanin production of a low-producing cell line, VV06, could be enhanced with addition of elicitors and precursor feeding. Methyl jasmonate was the only elicitor that increased anthocyanin production of the high-producing cell line VV05, but contributed to moderate enhancement of anthocyanin production compared with VV06. For cell line VV06, synergistic effects were observed for all treatment combinations of methyl jasmonate along with other elicitors and precursors. In addition, 6.1- and 4.6-fold increases in anthocyanin content and yield, respectively, were obtained in the presence of 5 mg/L phenylalanine, 50 mg/L methyl jasmonate, and 1 mg/L dextran. However, none of these treatment combinations exhibited synergistic effects in cell line VV05.     

Precursor limitations in methyl jasmonate-induced Catharanthus roseus cell cultures

Jasmonates enhance the expression of various genes involved in terpenoid indole alkaloid (TIA) biosynthesis in Catharanthus roseus. We applied precursor feeding to our C. roseus suspensions to determine how methyl jasmonate (MJ) alters the precursor availability for TIA biosynthesis. C. roseus suspensions were induced with MJ (100 μM) on day 6 and fed loganin (0.30 mM), tryptamine (0.15 mM), loganin plus tryptamine, or geraniol (0.1–1.0 mM) on day 7. While MJ increased ajmalicine production by 3-fold, induced cultures were still limited by terpenoid precursors. However, both induced and non-induced cultures became tryptamine-limited with excess loganin. Geraniol feeding also increased ajmalicine production in non-induced cultures. But MJ appeared to increase geraniol availability in induced cultures, due presumably to the increased expression of Dxs with MJ addition.     

Production of steroidal alkaloids by hairy roots of Solanum aviculare and the effect of gibberellic acid

Cultures of Solanum aviculare hairy roots were established after transformation with Agrobacterium rhizogenes A4. High levels of steroidal alkaloids measured as solasodine equivalents were produced in shake-flasks and bioreactor, even though relatively low concentrations are found in roots in vivo. In shake flasks the maximum alkaloid yield was 32 mg g^-1 dry weight; in a 3-1 air-driven bioreactor the yield was 29 mg g^-1. These yields represent a 5-fold increase over previous reports for in vitro production, and are comparable with levels found in the aerial parts of intact S. aviculare plants. Production of steroidal alkaloids was growth-associated. High sugar levels at stationary phase and insensitivity to increased levels of medium components suggest that root cultures were limited by oxygen mass-transfer. In Petri-dish culture with and without exogenous gibberellic acid, root length and number of root tips increased exponentially; growth proceeded with a constant length per root tip of about 35 mm. Addition of gibberellic acid enhanced growth but reduced the specific steroidal-alkaloid level. Taking into account both growth and alkaloid yield, accumulation of steroidal alkaloids was improved by about 40% at gibberellic-acid concentrations of 10 and 100 g l^-1.     

10-Hydroxycamptothecin produced by a new endophytic <Emphasis Type="Italic">Xylaria</Emphasis> sp., M20, from <Emphasis Type="Italic">Camptotheca acuminata</Emphasis>

A new 10-hydroxycamptothecin (HCPT)-producing fungus was isolated from Camptotheca acuminata. The strain was classified as a Xylaria sp. based on the internal transcribed spacer and 18S rDNA gene analysis. All elicitors tested, except methyl jasmonate, increased HCPT production in submerged culture. The maximum yield was 5.4 mg HCPT/l⁻¹, when salicylic acid was added at 0.1 mM to the culture medium     

Production of ajmalicine and ajmaline in hairy root cultures of Rauvolfia micrantha Hook f., a rare and endemic medicinal plant

Hairy roots of Rauvolfia micrantha were induced from hypocotyl explants of 2–3 weeks old aseptic seedlings using Agrobacterium rhizogenes ATCC 15834. Hairy roots grown in half-strength Murashige & Skoog (MS) medium with 0.2 mg indole 3-butyric acid l^–1 and 0.1 mg -naphthaleneacetic acid l^–1 produced more ajmaline (0.01 mg g^–1 dry wt) and ajmalicine (0.006 mg g^–1 dry wt) than roots grown in auxin-free medium. Ajmaline (0.003 mg g^–1 dry wt) and ajmalicine (0.0007 mg g^–1 dry wt) were also produced in normal root cultures. This is the first report of production of ajmaline and ajmalicine in hairy root cultures of Rauvolfia micrantha.     

Baicalin production in transformed hairy root clones ofScutellaria baicalensis

A transformed hairy root clone ofScutellaria baicalensis was established following infection withAgrobacterium rhizogenes ATCC15834. Three root clones ofS baicalensis were selected by growth habit and baicalin content. The most active strain-the SR-03 clone-was examined for its growth and baicalin content under various culture conditions. The root growth and baicalin content were maximized in a Schenk and Hildebrandt medium supplemented with 4 and 6% sucrose, respectively. The accumulation of baicalin in transformed hairy roots was enhanced through exposure to various elicitors. Elicitation was attained by the addition of methyl jasmonate, salicylic acid, and various concentrations of fungal cell wasll elicitors to the medium. The accumulation of baicalin in the elicited cultures ranged from 10.5 to 18.3 mg/g dry weight of the roots, which was 1.5-to 3-fold the amount attained in controls.     

Elicitor-stimulation of Monoterpene Indole Alkaloid Formation in Suspension Cultures of Catharanthus roseus

Upon treatment of 5 cell lines of Catharanthus roseus with homogenates of various fungi, as well as with chemically defined phytoalexin elicitors, all except one (non-alkaloid producing #916) responded with browing and accumulation of tryptamine within 6 – 24 h. Cells of line #615 responded with not only accumulating tryptamine, but also N-acetyl tryptamine, strictosidine lactam, ajmalicine, tabersonine, lochnericine, and catharanthine. Based on amounts of alkaloids accumulated, cells of line #615 performed best when treated with homogenates of Alternaria zinnae, Pythium apbanidermatum, Verticillium dabliae, and Rhodotorula rubs. A Pythium homogenate concentration of 5 % and a Rhodotorula homogenate concentration of 0.5 % effected maximum alkaloid yields, and, thus, were used in subsequent studies. These revealed a temporary increase of the level of alkaloids in cells and in their medium after 12 – 24 h of treatment. Ten-day-old subcultures responded better than younger and older ones. The elicitor stimulated accumulation of alkaloids and alkaloid composition did not depend on the use of 1-B5 or alkaloid production medium. A 5 l cell suspension of #615 grown in a 7.5 l bioreactor and treated with 5 % Pythium homogenate for 18 h was found to contain strictosidine lactam, ajmalicine, and catharanthine in concentrations of 27, 10, and 13 μg/g DW respectively, the medium contained 42 % of total ajmalicine.     

Impact of hypoxia on the growth and alkaloid accumulation in <Emphasis Type="Italic">Catharanthus roseus</Emphasis> cell suspension

The Madagascar periwinkle (Catharanthus roseus) produces numerous indole alkaloids, several of which have an important pharmaceutical uses such as ajmalicine, vinblastine and vincristine. The relationship between hypoxia and ajmalicine production in a cell suspension culture of C. roseus were investigated during the cycle of cell culture, in correlation with the effects on growth. The results show that the lack of oxygenation in C20D cells provokes a very strong inhibition in accumulation of the alkaloids and of other possible substances. Moreover, the addition of loganin, a metabolic intermediate of the biosynthetic pathway, in the culture medium of cells subjected to hypoxia restored the alkaloid production. Also, the results showed that the addition of benzyladenine (BA) to the culture medium increased the ajmalicine production and that the inhibitory effect of hypoxia was almost absent in these conditions. Therefore, it could be suggested that BA can without doubt decrease the effects of the hypoxia and increase the ajmalicine production in periwinkle cell suspensions.     

Enhanced catharanthine production in catharanthus roseus cell cultures by combined elicitor treatment in shake flasks and bioreactors

Chemical and fungal elicitors were added to Catharanthus roseus cell suspension cultures so as to improve the production of indole alkaloids. A synergistic effect on alkaloid accumulation was observed in C. roseus cell cultures when treated with some combined elicitors of fungal preparations and chemicals. Among them, the combination of tetramethyl amminium bromide and Aspergillum niger mycelial homogenate gave the highest ajmalicine yield (63 mg l(-1)) and an improved catharanthine accumulation (17 mg l(-1)). The combined elicitors of malate and sodium alginate resulted in the highest catharanthine yield (26 mg l(-1)) and a high ajmalicine accumulation (41 mg l(-1)) in the cell cultures. Based on the synergistic effect of malate and sodium alginate, a process with enhanced catharanthine production in Catharanthus roseus cell cultures was developed in shake flasks and a bioreactor. After 10 days of culture, 25 mg l(-1), 32 mg l(-1) and 22 mg l(-1) catharanthine yield were obtained in 500-ml flasks, 1000-ml flasks and in a 20-l airlift bioreactor, respectively. Upon malate-alginate combining treatments, peroxidase, catalase and superoxide dismutase activities decreased in elicited cells but phenylalanine ammonia lyase and lipoxygenase activities increased dramatically. That suggests a typical defense responses took place in the combined elicitors-treated cell cultures. Furthermore, the combined elicitors also caused a significant increase of malondialdehyde level in cell cultures, which suggests a serious lipid peroxidation occurred in the elicited cell cultures. Comparison of these results suggests that malate and alginate combining treatment also stimulates defense responses, such as lipid peroxidation, in all C. roseus culture processes and this may mediate the indole alkaloid production via jasmonate pathway.     

The effects of jasmonic acid and methyl jasmonate on rosmarinic acid production in <Emphasis Type="Italic">Mentha</Emphasis> × <Emphasis Type="Italic">piperita</Emphasis> cell suspension cultures

The effects of two elicitors: jasmonic acid and methyl jasmonate on cell growth as well as on rosmarinic acid accumulation in cell suspension cultures of Mentha × piperita were investigated. The highest rosmarinic acid accumulation 117.95 mg g⁻¹ DW (12% DW) was measured 24 h after addition of 100 μM methyl jasmonate. A similar concentration 110.12 mg g⁻¹ DW was detected 48 h after application of 200 μM jasmonic acid. Those values were nearly 1.5 times higher compared to the control sample, without elicitation. There was no substantial influence of elicitors on rosmarinic acid secretion into the culture media. Extracellular concentrations of rosmarinic acid were similar to the values from the control variants. It was documented that suspension cultures of M. piperita treated with elicitors showed a decrease in biomass accumulation when compared to the control.     

Elicitation of different Panax ginseng transformed root phenotypes for an improved ginsenoside production

We tested the effect of the presence in the culture medium of chitosan, vanadyl sulfate or methyl jasmonate on growth and ginsenoside production of three stable hairy root lines of Panax ginseng C.A. Meyer showing different morphological phenotypes C-M, HR-M and T-M. The response depended upon line phenotype, specificity of the elicitor and the stage of growth at which the lines were treated. The highest ginsenoside yield was found when methyl jasmonate was added during the progressive deceleration growth phase (on day 25 of culture). In this case, the ginsenoside content reached at the end of the culture (day 28) by root lines C-M, HR-M and T-M was, respectively, 2, 1.8 and 4 times higher than the highest content achieved, also at the end of the culture, by the corresponding untreated root lines. Under the same conditions, the ginsenoside content in the presence of vanadyl sulfate also increased considerably, while with chitosan it clearly decreased. The ginsenoside pattern in response to the presence of the elicitors is also considered.     

Differential Secretion and Accumulation of Terpene Indole Alkaloids in Hairy Roots of <Emphasis Type="Italic">Catharanthus roseus</Emphasis> Treated with Methyl Jasmonate

The induction of several secondary metabolites in plants is one of the most commonly observed effects after the external addition of methyl jasmonate (MeJA). After the elicitation of Catharanthus roseus hairy roots with different concentrations of MeJA, changes in the accumulation of alkaloids such as ajmalicine, serpentine, ajmaline and catharanthine were observed. In addition to the increased accumulation of alkaloids in the tissues, the root exudation of phytochemicals increased compared to that of the non-treated control hairy roots. Moreover, MeJA induced differential secretion of several C. roseus hairy root metabolites.     

Enhanced accumulation of decursin and decursinol angelate in root cultures and intact roots of <Emphasis Type="Italic">Angelica gigas</Emphasis> Nakai following elicitation

Angelica gigas root cultures were elicited with various elicitors, including yeast extract, chitin, methyl jasmonate, salicylic acid, and copper, with the aim of increasing the production of decursin and decursinol angelate. The treatment of A. gigas root cultures with a combination of yeast extract (2 g l⁻¹) and copper ion (0.5 mM) at the late exponential growth phase increased decursinol angelate accumulation up to 6.86 mg l⁻¹. The best elicitor preparation selected through in vitro experiments was also applied to roots of A. gigas whole plants grown in the field in order to investigate the potential of elicitation as a novel cultivation practice for producing medicinal herbs of improved quality. Biweekly treatments with the elicitor at 70 mg g l⁻¹ FW roots for 10 weeks before the annual harvest resulted in an increment in both plant yields and specific productivity of decursins by 1.5- and 1.7-fold, respectively. This result implies that in vitro screening of elicitors with the ultimate aim of in planta elicitation of whole plants could be effective in terms of time and expense. The elicitation technique reported here demonstrates it potential as a strategy for improving growth and active compounds productivity of medicinal plants through short-term and pre-harvest treatment of the elicitor preparation.     

Improved isoflavonoid production in Pueraria candollei hairy root cultures using elicitation

The effect of abiotic and biotic elicitors (methyl jasmonate, chitosan, salicylic acid, Agrobacterium, and yeast extract) at various concentrations on total isoflavonoid accumulation was studied in the hairy root cultures of Pueraria candollei. All elicitors stimulated isoflavonoid production. Yeast extract (0.5 mg/ml) was the most efficient giving total isoflavonoids at 60 ± 1 mg/g dry wt, which was 4.5-fold higher than control hairy roots on day 3 of elicitation.     

Improvement of indole alkaloid production in Catharanthus roseus cell cultures by osmotic shock

Catharanthine production in Catharanthus roseussuspension cell cultures was increased by about 4-fold to 28 mg l^–1, 23 mg l^–1and 24 mg l^–1by adding sodium alginate, mannitol or polyvinyl pyrrolidone, respectively. Sodium alginate and polyvinyl pyrrolidone also enhanced ajmalicine production to 28 mg l^–1and 31 mg l^–1, respectively. Up to 55–70% of the total alkaloids were released into the medium. These treatments could stimulate higher alkaloid production in C. roseuscell cultures than NaCl and KCl stresses. The possible mechanisms for these treatment effects are discussed.     

Effects of different elicitors on yield of tropane alkaloids in hairy roots of Anisodus acutangulus

The four tropane alkaloids have played a pivotal role in controlling diseases such as the toxic and septic shock, the organophosphorus poison and the acute lung injury. Here, the elicitation effect of different elicitors on the production of tropane alkaloids and the molecular mechanism of enzyme genes in the pathway was firstly demonstrated in hairy roots of Anisodus acutangulus. The results showed ethanol, methyl jasmonate and Ag⁺ could improve the accumulation of tropane alkaloids up to 1.51, 1.13 and 1.08 times after 24 h treatment, respectively (P < 0.05), whereas salicylic acid decreased the average content of tropane alkaloids. Furthermore, expression profile analysis results revealed that up-regulation of hyoscyamine-6b-hydroxylase (AaH6H) and little regulation of tropinone reducase II (AaTR2) elicited by ethanol, increased expression of putrescine N-methyltransferase I (AaPMT1) elicited by Ag⁺, elevated expression of tropinone reducase I (AaTR1) elicited by methyl jasmonate, respectively, resulted in tropane alkaloids improvement. Our results showed that hairy root culture of A. acutangulus in combination with elicitors was a promising way for production of tropane alkaloids in the future.