Effects of exogenous methyl jasmonate in elicited anthocyanin-producing cell cultures of ohelo (Vaccinium phalae)

Summary Elicitation of anthocyanin-producing cells of ohelo (Vaccinium pahalae) by both biotic (purified β-glucan and chitosan) and abiotic [sodium ferric ethylenediamine di-(o-hydroxyphenylacetate) FeEDDHA, and CuSO₄] elicitors resulted in significant enhancement of anthocyanin accumulation. Anthocyanin production increased up to 1.8 and 1.5-fold over the control in the presence of abiotic elicitors (90 μM FeEDDHA and 20 μM CuSO₄, respectively), and increased 1.9 and 1.6-fold in the presence of biotic elicitors (10 mg L⁻¹ β-glucan and 100 mg L⁻¹ chitosan). Maximum anthocyanin production with the two most effective elicitors was achieved when cultures were treated on Day 3 (β-glucan) or Day 0 (FeEDDHA) after the initiation of fresh cell cultures. A concentration-dependent response was exhibited by cultures treated with exogenous methyl jasmonate (MJ). The addition of 0.5 μM MJ alone provoked a 2–3-fold increase in anthocyanin production over that of the control; however, no additive effect on anthocyanin production was observed in any treatments which combined MJ and β-glucan or FeEDDHA. Conditioning of the cells with a preculture in either MJ, β-glucan, or FeEDDHA similarly did not enhance anthocyanin production. Inoculation of cultures elicited by MJ or β-glucan with ibuprofen, a reported inhibitor of jasmonate biosynthesis, dramatically stimulated, rather than inhibited, anthocyanin production, resulting in levels of accumulation beyond any of the tested elicitor combinations. Hypotheses for the observed influence of ibuprofen in this system are discussed.     

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.     

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.     

茉莉酸甲酯与水杨酸对肉苁蓉悬浮细胞中苯乙醇甙合成的影响

向肉苁蓉悬浮细胞培养系中添加茉莉酸甲酯(MJ)和水杨酸(SA) ,分别考察了这两种诱导子的添加浓度及添加时间对肉苁蓉悬浮细胞系中苯乙醇甙含量的影响。研究结果表明:MJ和SA能够促进肉苁蓉悬浮细胞系中苯乙醇甙(PeG)和松果菊甙(Echinacoside)的合成,但两者的适用的浓度范围和最佳添加时间存在差异。与未经诱导子处理的细胞培养结果相比,MJ在对数生长初期(培养14d) ,添加浓度为5 μmol L条件下,可使肉苁蓉悬浮细胞系中PeG含量提高2 5 9倍,Echin含量提高3 82倍;而SA在对数生长后期(培养2 8d) ,添加浓度为5 0 μmol L条件下,可使PeG含量提高2 71倍,Echin含量提高3 16倍。     

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.     

Improvement of biosynthesis and accumulation of bioactive compounds by elicitation in adventitious root cultures of Polygonum multiflorum

Applied Microbiology and Biotechnology - We examined the effects of abiotic (methyl jasmonate [MeJA] and salicylic acid [SA]) and biotic (yeast extract and chitosan) elicitors for improvement of...     

Elicitor enhanced production of protoberberine alkaloids from in vitro cell suspension cultures of <Emphasis Type="Italic">Tinospora cordifolia</Emphasis> (Willd.) Miers ex Hook. F. &amp; Thoms

The present research investigates the effect of Piriformospora indica, an endophytic fungus, on production of protoberberine alkaloids in in vitro cell suspension cultures of Tinospora cordifolia. Although T. cordifolia produces a number of protoberberine alkaloids, the simultaneous production of jatrorrhizine and palmatine in cell suspension cultures of T. cordifolia was observed for the first time with the use of P. indica as biotic elicitor. The cells in suspension cultures were elicitated with P. indica on 14th day of culture initiation and the production of the alkaloids on 16th day was monitored. The autoclaved as well as filter sterilized cultures of P. indica were used in addition to the use of fungal cell extract. The elicitor effect of P. indica was analyzed and compared with other abiotic elicitor (methyl jasmonate) and biotic elicitors (chitin and chitosan). The culture filtrate of P. indica in the filter sterilized (5.0% v/v) form gave better response with enhanced 4.2-fold production of jatrorrhizine (10.72 mg/g DW) and 4.0-fold production of palmatine (4.39 mg/g DW). The production of these compounds was at par with that achieved in methyl jasmonate (at 250 µM) treated cell suspension cultures.     

前体饲喂、诱导子和光照联合使用对葡萄细胞培养合成花青素的影响

苯丙氨酸前体饲喂分别和环糊精、葡聚糖、茉莉酸甲酯、黑曲霉和直喙镰孢菌提取液五种诱导子联合作用,其中以与茉莉酸甲酯的联合作用对葡萄细胞培养生产花青素的影响最大,可使单位鲜细胞花青素含量提高2.7倍,花青素产量提高3.4倍,实验证明两者在培养后第4天加入效果最好。在30μmol/L苯丙氨酸、218μmol/L茉莉酸甲酯和3000~4000lx光照条件下,不同花青素产量的细胞株都能显著提高花青素产量,但低产株VV06比高产株VV05具有更大的产率提高潜力。该条件下VV05和VV06花青素产量分别达到2975和4090CV/L,是对照组的2.5倍和5.2倍。     

Betacyanin biosynthesis in the isolated hypocotyls bei Acetabularia mediterranea

Summary Isolated hypocotyls synthesize betacyanin after light exposure in Amaranthus caudatus L. Pigment synthesizing capacity is reduced in the hypocotyls with increased incubation of seedlings in dark after 24h. External feeding of precursors of betacyanin L-tyrosine and DOPA enhances pigment synthesis in the isolated hypocotyls to equal that of intact hypocotyls. Cotyledons are probably the source of precursors while both cotyledons and hypocotyls are the sites of betacyanin synthesis. Betacyanin synthesizing capacity is progressively lost from the base of the hypocotyl and precursors could not induce pigment synthesis in these regions.     

The influence of different biotic and abiotic elicitors on the production and profile of tropane alkaloids in hairy root cultures of Brugmansia candida

Hairy root cultures of Brugmansia candida produce the tropane alkaloids scopolamine and hyoscyamine. In an attempt to increase productivity, several biotic and abiotic elicitors were tested. Salicylic acid increased significantly the release of both alkaloids (2- to 12-fold) and it also acted positively on specific production without altering the production profile. AgNO(3) increased significantly scopolamine release (3-fold) and both alkaloid's accumulation (5- to 8-fold) in the roots, thus favoring the production of scopolamine (up to 2-fold). The inhibiting effects of AgNO(3) and salicylic acid on ethylene could be partly responsible for these responses. Yeast extract incremented the intracellular content of both alkaloids (ca. 3-fold), but particularly increased the release of scopolamine (7-fold). CaCl(2) had little effect on accumulation or release of either alkaloid. CdCl(2) acted positively on the release of both alkaloids (3- to 24-fold), but was highly detrimental to growth. Hairy roots of B. candida are therefore susceptible to elicitation by biotic and abiotic elicitors, with variations in the kinetics of induction and the extent of release of each metabolite, thereby also exerting different effects on the alkaloid profile.     

Improved cardenolide production in Calotropis gigantea hairy roots using mechanical wounding and elicitation

A hairy root culture system of Calotropis gigantea was established and effects of mechanical wounding (MW) and elicitors [methyl jasmonate (MJ), yeast extract (YE) and chitosan (CS)] on cardenolide production were investigated. All treatments stimulated the production of cardenolide in hairy root cultures of C. gigantea. CS was the most effective elicitor, followed by MJ. YE and MW also improved cardenolide yield in individual treatments. The highest cardenolide yield (1,050 ± 55 mg/l) was obtained after adding 50 mg CS/l for 20 days, which was 2.7-fold higher than the control.     

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     

Effects of sucrose on betacyanin accumulation and growth in suspension cultures of Phytolacca americana

The effects of sucrose on betacyanin accumulation and growth in suspension cultures of Phytolacca americana L. were investigated. Maximal betacyanin accumulation was observed at 88 m M sucrose on cell number basis and at 175 m M sucrose on fresh weight basis. This is because cell size decreased as the initial sucrose concentration was increased. Supplementary studies using mannitol indicated that sucrose itself caused increased cell number and that cell size was affected by both sucrose concentration and water potential. Betacyanin accumulation per cell and per fresh weight at a constant concentration of sucrose (88 m M ) decreased with decreasing water potential. When sucrose concentration increased at a constant water potential (–0.7 MPa), betacyanin accumulation per fresh weight increased up to 88 m M and remained at constant level at higher concentrations, while betacyanin accumulation per cell decreased remarkably, due to a dramatic increase in cell number.     

Synergistic effects of sequential treatment with methyl jasmonate, salicylic acid and yeast extract on benzophenanthridine alkaloid accumulation and protein expression in Eschscholtzia californica suspension cultures

To develop an optimal bioprocess for secondary metabolite production and explain the bioprocess at the molecular level, we examine the synergistic effects of sequential treatment with methyl jasmonate (MJ), salicylic acid (SA) and yeast extract (YE) on benzophenanthridine alkaloid accumulation and protein expression in Eschscholtzia californica suspension cultures. Serial treatment of MJ, SA and YE at 24 h intervals enhanced the accumulation of dihydrosanguinarine (2.5 times) and sanguinarine (5.5 times). This sequential treatment using different signal elicitors was more effective than single elicitor or simultaneous treatment of the elicitors; it induced benzophenanthridine alkaloid accumulation to 917.7 ± 42.0 mg/L. Also, (S)-methylcoclaurine-3′-hydroxylase (CYP80B1) and 3′-hydroxy-(S)-N-methylcoclaurine-4′-O-methyltransferase (4′OMT) expressions among enzymes in sanguinarine biosynthetic pathway explained the synergistic effects by sequential treatment of the elicitors. The sequential treatment strategy using elicitors related to different signal transduction pathways can be used to design better processes to increase accumulation of secondary metabolites in plant cell culture. Analysis of protein expression provides the detailed information about metabolite accumulation through the correlated results.     

Oxidative enzymes activity during abiotic and biotic stresses in <Emphasis Type="Italic">Zea mays</Emphasis> leaves and roots exposed to Cu,methyl jasmonate and <Emphasis Type="Italic">Trigonotylus caelestialium</Emphasis>

The activities of antioxidative enzymes, i.e. superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX), and guaiacol peroxidase (GPX), in the leaves and roots of Zea mays L. plants exposed to abiotic (methyl jasmonate, MJ, or/and copper, Cu) and biotic (Trigonotylus caelestialium) factors were examined. The contribution of MJ as a signal molecule in the defense mechanism against abiotic and biotic stresses was studied. All plants were cultivated hydroponically and divided into three groups: not treated by abiotic factors (control), treated by MJ only (MJ) and by MJ and Cu (MJ + Cu) and in each group half of the plants were exposed to T. caelestialium attack. The enzymatic activities of SOD, CAT, APX, and GPX in the leaves were higher in the insect-treated than non-insect-treated control plants, but lower in both MJ + Cu- or MJ- and insect-treated plants. In the roots, the enzyme activities were elevated in all insect-treated plants with the highest rise in MJ + Cu, in comparison with the MJ-treated plants. The results showed that MJ and MJ + Cu were efficient in reducing the activity of the antioxidative enzymes in the leaves under the insect influence by elevating enzyme activity in the roots.     

Biotic elicitor enhanced production of psoralen in suspension cultures of Psoralea corylifolia L.

Cell cultures of Psoralea corylifolia L. were established from the leaf disk derived callus. The effect of different biotic elicitors prepared from the fungal extract (Aspergillus niger and Penicillium notatum), yeast extract and chitosan with different concentrations was studied. The increased synthesis of psoralen in 16-day old cell cultures under 16 h of light and 8 h of dark period was studied. Elicitation of psoralen in A. niger elicitor treated cells was found 9-fold higher over control cells. Treating the cells with P. notatum, yeast extract and chitosan elicitors lead to four to seven-fold higher psoralen accumulation over control cells. The extract of A. niger at 1.0% v/v increased the significant accumulation of psoralen (9850 μg/g DCW) in the cultured cells. Our study clearly shows that all the elicitors had the potential to increase the accumulation of psoralen but the A. niger elicitor at 1.0% v/v induced maximum accumulation.     

Enhancement of centelloside production from cultured plants of Centella asiatica by combination of thidiazuron and methyl jasmonate

In order to produce centellosides from whole plant cultures of Centella asiatica (L.) Urban, we evaluated the synergistic effects of thidiazuron (TDZ) and methyl jasmonate (MJ) on whole plant growth and centelloside production. After 4 weeks of treatment with 0.025 mg/L of TDZ coupled with 0.1 mM MJ, the production of madecassoside and asiaticoside from whole plant cultures was estimated to be 2.40- and 2.44-fold, respectively, above that of MJ elicitation alone. When whole plants were treated with a growth regulator and an elicitor, the growth of whole plants, as compared to the controls, did not differ. Additionally, total phytosyterol content in the leaves of whole plants co-treated with MJ and TDZ was 1.08-fold greater than those of MJ alone. These results demonstrate that combined treatments not only stimulate the accumulation of centellosides in the leaves but also inhibit the reduction of phytosterol levels caused by MJ elicitation.     

Effect of plant growth regulators and elicitors on rhinacanthin accumulation in hairy root cultures of Rhinacanthus nasutus (L.) Kurz

Several biologically active secondary metabolites like anthraquinones, sterols, triterpenes, flavonoids and naphthoquinones are present in Rhinacanthus nasutus. Naphthoquinones are important group of compounds generally known as rhinacanthin (RC) consists of 15 derivatives named RC A–D and G–Q of which RC-C, RC-D and RC-N have various medicinal properties. The individual role of two auxins i.e. indole-3-butyric acid (IBA) and α naphthalene acetic acid (NAA) and two elicitors i.e. methyl jasmonate (MJ) and salicylic acid (SA) in Murashige and Skoog medium on hairy root growth and RC (RC-C, RC-D and RC-N) accumulation was investigated in the present study. Time course study revealed that IBA and NAA at 2.5 μM showed maximum fresh weight (FW) and dry weight (DW) 4 weeks after culture. However, RC production was maximum after 6 weeks of culture on both media. A concentration-dependent response was observed when various concentrations of MJ (2.0, 5.0, 10 and 15 μM) and SA (10, 50, 100 and 150 μM) were supplemented in the medium. On MJ and SA media the FW and DW decreased as the concentration of elicitors increased. However, this decrease was more severe in MJ treated cultures. All the MJ and SA treated cultures showed significantly higher amount of RC-C, RC-D and RC-N in hairy roots harvested 7 days after elicitation as compared to control. Of the two elicitors, MJ was more efficient in inducing RC accumulation than SA. The highest RC content (6.3 mg/g DW RC-C; 1.1 mg/g DW RC-D and 0.61 mg/g DW RC-N) was observed after treatment with 10 μM MJ which was about 1.7-, 2.5- and 3.5-fold higher RC-C, RC-D and RC-N respectively than the control.     

Improved gymnemic acid production in the suspension cultures of Gymnema sylvestre through biotic elicitation

Enhancement of secondary metabolite accumulation in cultured plant cells through biotic and abiotic elicitation has been recognised as an important biotechnological strategy. Gymnema sylvestre is a rich source of triterpenoid saponins—gymnemic acids used mainly in the treatment of diabetes I and II. The cell suspension cultures initiated from the leaves and stalks of in vitro-grown plantlets have shown to accumulate large amounts of gymnemic acid. The cell-free extracts of Aspergillus niger, Saccharomyces cerevisiae, Agrobacterium rhizogenes, Bacillus subtilis and Escherichia coli were employed as sources of biotic elicitors to study the effect on secondary metabolite production. All the elicitors have shown a positive response in terms of gymnemic acid, with the highest response induced by A. niger [98.65 ± 0.93 mg/gram dry cell weight (gDCW)], 11.2-fold, and the lowest by E. coli (33.25 ± 1.38 mg/gDCW), 3.8-fold, in comparison to the untreated cultures (8.79 ± 0.82 mg/gDCW). The suspension cultures of G. sylvestre can serve as a continuous source of gymnemic acids throughout the year, irrespective of the climatic and geographical barriers.