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.     

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.     

Influence of abiotic elicitation on production of dipyranocoumarins in suspension cultures of <Emphasis Type="Italic">Calophyllum inophyllum</Emphasis> L.

Effects of elicitation with heavy metals such as copper, cadmium, chromium (abiotic elicitation) and supplementation of CaCl₂ on production of dipyranocoumarins (inophyllums) in suspension cultures of leaf and stem callus of Calophyllum inophyllum were studied. The optimum timing for elicitor introduction was found to be the 10th day after initiating the suspension cultures. Cadmium as abiotic elicitor in suspension cultures of stem callus was found best to elicit maximum production of inophyllums A, C, and calophyllolide while cadmium in suspension cultures of leaf callus was found best for eliciting maximum production of inophyllums B and P. Inophyllum D was the only dipyranocoumarin whose highest production was achieved when 1.0 mM chromium was used as abiotic elicitor in suspension cultures of stem callus. Out of the three abiotic elicitors used, none could result biomass growth. Only incorporation of CaCl₂ in suspension cultures resulted biomass growth. A maximum of 35.26-fold biomass growth was achieved when suspension cultures of stem callus were incorporated with 2.0 mM CaCl₂. CaCl₂ was noted to have no positive influence on production of most of the dipyranocoumarins under study.     

<Emphasis Type="Italic">Fusarium oxysporum</Emphasis> cell elicitor enhances betalain content in the cell suspension culture of <Emphasis Type="Italic">Celosia cristata</Emphasis>

We started a cell suspension culture from magenta coloured calli of cockscomb to study the effect of biotic and abiotic elicitors on the biosynthesis of betalain pigments. The cultures were grown in a flask containing 30 ml MS media fortified with 13.5 μM 2,4-D and 0.44 μM BAP. These cultures were elicited during its log-phase of growth using fungal elicitors (prepared from mycelia of Fusarium oxysporum), yeast extract, copper sulphate and cobalt chloride. The elicitation reduced the cell count, cell viability and percent pigmented cell in the suspension culture. Similarly, it also resulted in reduced betalain content by all the elicitors except 0.125 × 10^?3% fungal elicitor. Rather, fungal elicitor at this concentration significantly enhanced the amaranthin, betanin, betalamic acid and betaxanthin content in the culture. Besides this, copper sulphate doubled the pigment contribution (ratio of particular pigment content to total pigment content) of betaxanthin at all the concentrations. Therefore, we conclude that fungal elicitor can further be investigated to enhance the content of betalain pigments in suspension culture at a larger scale.     

Phytoalexin Induction in French Bean : Intercellular Transmission of Elicitation in Cell Suspension Cultures and Hypocotyl Sections of Phaseolus vulgaris

Treatment of hypocotyl sections or cell suspension cultures of dwarf French bean (Phaseolus vulgaris L.) with an abiotic elicitor (denatured ribonuclease A) resulted in increased extractable activity of the enzyme l-phenylalanine ammonia-lyase. This induction could be transmitted from treated cells through a dialysis membrane to cells which were not in direct contact with the elicitor. In hypocotyl sections, induction of isoflavonoid phytoalexin accumulation was also transmitted across a dialysis membrane, although levels of insoluble, lignin-like phenolic material remained unchanged in elicitor-treated and control sections. In bean cell suspension cultures, the induction of phenylalanine ammonia-lyase in cells separated from ribonuclease-treated cells by a dialysis membrane was also accompanied by increases in the activities of chalcone synthase and chalcone isomerase, two enzymes previously implicated in the phytoalexin defense response. Such intercellular transmission of elicitation did not occur in experiments with cells treated with a biotic elicitor preparation heat-released from the cell walls of the bean pathogen Colletotrichum lindemuthianum. The results confirm and extend previous suggestions that a low molecular weight, diffusible factor of host plant origin is involved (in French bean) in the intercellular transmission of the elicitation response to abiotic elicitors.     

Elicitation of benzophenanthridine alkaloid synthesis in Eschscholtzia cell cultures

Quaternary benzophenanthridine alkaloids (sanguinarine, chelerythrine, chelirubine, chelilutine and macarpine) are specifically induced by cell wall components of Penicillium and Saccharomyces in a colorless strain of Eschscholtzia californica cell suspension cultures. Classical elicitors such as the Phytophthora megasperma elicitor are inactive. The alkaloid synthesis is, however, strongly induced by certain polypeptide antibiotics. Out of 190 tested plant species the yeast elicitor provoked benzophenanthridine synthesis in 13 cultures. One of the branch point enzymes, namely the berberine bridge enzyme, catalysing the formation of (S)-scoulerine from (S)-reticuline, is strongly stimulated during the elicitation process. These results clearly demonstrate the induction of the benzophenanthridine biosynthetic pathway by microbial elicitors.Abbreviations ACC 1-Aminocyclopropane-1-carbonic acid - EDTA Ethylenediaminetetraacetic acid - LS-medium Linsmaier and Skoog medium - Pmg Phytophthora megasperma     

Enhancement of anthocyanin production in callus cultures ofDaucus canota L. under the influence of fungal elicitors

Aqueous extracts of fungal biomass ofAspergillus niger, A. flavus, Penicillium notatum, Fusarium oxysporum and the filtrates of their culture media were analysed for elicitation capability to enhance anthocyanin production in callus cultures ofDaucus carota. The mycelial extract ofA. flavus at the 2.5% level gave maximum elicitation, which resulted in a two-fold increase in anthocyanin with maximal productivity of 23.7% on a dry weight basis. Whereas the media filtrates ofA. flavus induced a 1.25-fold increase in anthocyanin production with a yield of 20.6% on a dry weight basis,P. notatum andF. oxysporum were not as effective asA. flavus orA. niger. The contact time and the concentration required for maximal elicitation of anthocyanin differed with the type of elicitor used. Qualitative analysis of anthocyanins revealed the presence of cyanidin glycosides in control and elicited cultures.     

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.     

Amino Acids, Iron, and Growth Rate as Key Factors Influencing Production of the Pseudomonas Putida BTP1 Benzylamine Derivative Involved in Systemic Resistance Induction in Different Plants

The biological control bacterium Pseudomonas putida BTP1 exerts its protective effect mostly by inducing an enhanced state of resistance in the host plant against pathogen attack [induced systemic resistance (ISR)]. We previously reported that a specific compound derived from benzylamine may be involved in the elicitation of the ISR phenomenon by this Pseudomonas strain. In this article, we provide further information about the N,N-dimethyl-N-tetradecyl-N-benzylammonium structure of this determinant for ISR and show that the benzylamine moiety may be important for perception of the molecule by root cells of different plant species. We also investigated some regulatory aspects of elicitor production with the global aim to better understand how in situ expression of these ISR elicitors can be modulated by physiological and environmental factors. The biosynthesis is clearly related to secondary metabolism, and chemostat experiments showed that the molecule is more efficiently produced at low cell growth rate. Interestingly, the presence of free amino acids in the environment is necessary for optimal production, and a specific positive effect of phenylalanine was evidenced in pulsed continuous cultures. The influence of other abiotic factors, such as mineral content, oxygen concentration, or pH, on elicitor production is also reported and discussed with respect to the specific conditions that the producing strain undergoes in the rhizosphere environment.     

Preparation of mixed alginate elicitors with high activity for the efficient production of 5′-phosphodiesterase by Catharanthus roseus cells

When various autoclaved microbial cells suspensions (exogenous elicitors) were added to Catharanthus roseus cell cultures, its growth was inhibited but 5′-phosphodiesterase (PDase) production was stimulated. The greatest effect was with autoclaved Alteromonas macleodii: the dry cell concentration decreased from 13 to 10.9 mg/ml while PDase production increased from 0.022 to 0.235 U/ml. A combination of A. macleodii (as exogenous elicitor) and 0.1%(w/v) alginate oligomers (AO: acting as both endogenous elicitor and scavenger of active oxygen species) minimized the cell growth inhibition but enhanced PDase production (0.474 U/ml) about 20 times higher than the control (no addition). The method for the preparation of mixed alginate elicitors with high activities containing exogenous elicitor (autoclaved A. macleodii), endogenous elicitor (AO), and trans-4,5-dihydroxy-2-cyclopenten-1-one was developed. The mixed alginate elicitors significantly promoted PDase production (2.67 U/ml) by C. roseus, and the productivity was increased 120-fold compared to the control without cell growth inhibition.     

Polysaccharide elicitors enhance anthocyanin and phenolic acid accumulation in cell suspension cultures of <Emphasis Type="Italic">Vitis vinifera</Emphasis>

The effects of yeast extract and selected polysaccharide elicitors on secondary metabolite production, particularly of anthocyanin and phenolic acid, in cell suspension cultures of Vitis vinifera were investigated. All elicitors either maintained or promoted cell growth in culture. Overall, secondary metabolite production in V. vinifera cell suspension cultures responded differently to different elicitors. Chitosan, pectin, and alginate enhanced production of anthocyanin within 13 days of culture with levels of 2.5-, 2.5-, and 2.6-fold increase, respectively, over that of control. Chitosan, alginate, and gum arabic significantly promoted accumulation of phenolic acids, particularly 3-O-glucosyl-resveratrol, in V. vinifera cultures, as well as in the culture medium. Intracellular phenolic acid production was significantly enhanced by alginate and chitosan, with 1.7- and 1.5-fold levels, respectively, of that of control. Extracellular phenolic acid production was also significantly increased in the presence of chitosan and gum arabic, with levels of 3.3- and 1.7-fold higher, respectively, than those of control. In addition, DPPH (1,1-diphenyl-2-picrylhydrazyl) radical scavenging activity was enhanced in the presence of elicitors, and this was positively correlated with increased accumulation of anthocyanin in V. vinifera cell suspension cultures.     

米根霉诱导因子对紫草细胞培养中紫草宁色素分泌的影响

在紫草细胞培养中,加入采根霉粗提物可显著提高紫草宁色素产量,并可加快胞内色素分泌到培养液中的速率和数量。在细胞培养的第6天加入米根霉诱导因子时,其促进紫草宁色素分泌的作用最大,培养液中紫草宁色素含量是对照的2．24倍。此外,同时加入正十六烷和米根霉诱导因子对紫草宁色素的分泌具有协同作用。     

Cutin Monomers and Surface Wax Constituents Elicit H2O2 in Conditioned Cucumber Hypocotyl Segments and Enhance the Activity of Other H2O2 Elicitors

Hypocotyls from etiolated cucumber (Cucumis sativus L.) seedlings were gently abraded at their epidermal surface and cut segments were conditioned to develop competence for H₂O₂ elicitation. Alkaline hydrolysates of cutin from cucumber, tomato, and apple elicited H₂O₂ in such conditioned segments. The most active constituent of cucumber cutin was identified as dodecan-1-ol, a novel cutin monomer capable of forming hydrophobic terminal chains. Additionally, the cutin hydrolysates enhanced the activity of a fungal H₂O₂ elicitor, similar to cucumber surface wax, which contained newly identified alkan-1,3-diols. The specificity of elicitor and enhancement activity was further elaborated using some pure model compounds. Certain saturated hydroxy fatty acids were potent H₂O₂ elicitors as well as enhancers. Some unsaturated epoxy and hydroxy fatty acids were also excellent H₂O₂ elicitors but inhibited the fungal elicitor activity. Short-chain alkanols exhibited good elicitor and enhancer activity, whereas longer-chain alkan-1-ols were barely active. The enhancement effect was also observed for H₂O₂ elicitation by ergosterol and chitosan. The physiological significance of these observations might be that once the cuticle is degraded by fungal cutinase, the cutin monomers may act as H₂O₂ elicitors. Corrosion of cutin may also bring surface wax constituents in contact with protoplasts and enhance elicitation.     

Enhanced anthocyanins and resveratrol production in Vitis vinifera cell suspension culture by indanoyl-isoleucine, N-linolenoyl-L-glutamine and insect saliva

The effects of two synthetic elicitor indanoyl-isoleucine (In-Ile), N-linolenoyl-l-glutamine (Lin-Gln) and one biotic elicitor insect saliva (from Manduca sexta larvae) on plant cell cultures with respect to the induction of secondary metabolite production were investigated. Stimulated production of secondary metabolites, particularly anthocyanins in plant cells and phenolic acids in culture medium, was studied by using suspension culture of Vitis vinifera L. cv. Gamay Fréaux as a model system. In the treatments with In-Ile, the production of anthocyanins was enhanced 2.6-fold. In-Ile, Lin-Gln and saliva significantly elevated the accumulation of phenolic acids, particularly 3-O-glucosyl-resveratrol. The used elicitors did not suppress cell growth. Secondary metabolites were differently responsive to elicitation. 3-O-glucosyl-resveratrol was the predominant phenolic acid in V. vinifera cell culture, and its production was significantly stimulated by saliva, with 7.0-fold of the control level 24 h after treatment. The production of 4-(3,5-dihydroxy-phenyl)-phenol was significantly stimulated by In-Ile with 6.4-fold of the control level 24 h after treatment.     

Significance of protein elicitor isolated from Tuber melanosporum on the production of ganoderic acid and Ganoderma polysaccharides during the fermentation of Ganoderma lucidum

Under the elicitation of protein elicitor isolated from the culture mycelia of Tuber melanosporum, the biosynthesis of ganoderic acids (GA) was significantly stimulated during Ganoderma lucidum fermentation. Compared with our previous results that, GA content was inhibited by polysaccharide elicitor isolated from T. melanosporum, while improved by the elicitor of polysaccharide and protein, protein was identified to be the exact component inducing GA biosynthesis in this work. G. lucidum cell growth was significantly inhibited by elicitor of polysaccharide and protein, and polysaccharide elicitor did not inhibit the cell growth. In this work, the remarkable inhibition on the cell growth was considerably eliminated under the elicitation of protein elicitor isolated from T. melanosporum. These suggested maybe the interaction of polysaccharide and protein components existed in the inhibition on the cell growth of G. lucidum. Not only GA content but also total GA accumulation obtained the highest values after the elicitation of protein elicitor. The maximal GA production of 260.5 ± 5.6 mg/L was 31.2% higher than the control. Under the elicitation of protein elicitor, the production of extracellular polysaccharide (EPS) and the content of intracellular polysaccharide (IPS) were also enhanced; however, total IPS accumulation was lower. GA biosynthesis was also significantly affected by the addition time of protein elicitor, whose optimal value was the culture of day 4.     

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.     

Host-Pathogen Interactions : XXI. Extraction of a Heat-Labile Elicitor of Phytoalexin Accumulation from Frozen Soybean Stems

An extract of frozen and thawed soybean (Glycine max L. Merr. cv. Wayne) stems is active, in wounded soybean cotyledons, as a heat-labile elicitor of phytoalexins. The elicitor activity of the extract is destroyed by heating to 95°C for 10 minutes. The fraction that contains heat-labile elicitor activity releases heat-stable elicitor-active molecules from purified soybean cell walls. Heat-labile elicitor activity voids a Bio-Gel P-6 column and can be absorbed onto and eluted from a DEAE Sephadex ion exchange column. Using the cotyledon phytoalexin elicitor assay, maximum heatlabile elicitor activity was obtained when soybean stems were extracted with acetate buffer at pH 6.0. Addition of 1 millimolar CaCl₂ increased apparent heat-labile elicitor activity. The heat-labile elicitor stimulated maximum phytoalexin accumulation when applied to cotyledons immediately after the cotyledons were cut. Partially purified stem extracts lost heat-labile elicitor activity during storage for several days at 3°C. The possible role of a heat-labile elicitor in stimulation of phytoalexin accumulation by both abiotic and biotic elicitors is discussed.     

Effects of elicitation on the production of saponin in cell culture of Panax ginseng

This study was initiated to investigate the impacts of elicitor concentration and elicitor-adding time on the saponin synthesis and the cell growth of Panax ginseng cell suspensions. Both of the elicitors tested, yeast extract and methyl jasmonate, significantly improved saponin production. The highest additive level of the seven ginsenosides tested was 2.07% (dry weight basis), which was 28-fold higher than that in the control. The optimum time to add either elicitor was found to be on the day of inoculation. The addition of either elicitor did not show as significant an influence on cell growth as on saponin production. It was advisable to remove 2,4-dichlorophenoxyacetic acid (2,4-D) from the medium when methyl jasmonate was used as the elicitor as methyl jasmonate interacts antagonistically with 2,4-D. These results suggest that the addition of an elicitor to ginseng cell suspension cultures could stimulate saponin production.