Effect of growth hormone modifications on shikonin production fromLithospermum erythrorhizon cell cultures within situ extraction

Summary The effect of growth hormone modifications on shikonin production was studied with the cell cultures ofLithospermum erythrorhizon. The cells grown in SH–H or SHA medium were effective for shikonin production in M–9 medium and maximum shikonin concentrations reached 43 and 63 mg/L, respectively, within situ extraction. In the case of the cells grown in SHA medium, induction time required for shikonin production was very short and the maximum shikonin concentration was obtained within 6 days.     

Increased shikonin production by hairy roots of Lithospermum erythrorhizon in two phase bubble column reactor

Summary Plant hairy root cultures of Lithospermum erythrorhizon were carried out to produce shikonin derivatives by employing in situ extraction with n-hexadecane in a shake flask and a bubble column bioreactor. Over 95 % shikonin produced was recovered in the n-hexadecane layer. In flask cultures the maximum concentration of shikonin with n-hexadecane extraction was 3 times higher than that obtained without extraction. In the two phase bubble column reactor, 572.6 mg/L of shikonin and 15.6 g/L of dry cell mass were obtained after 54 days. Shikonin was produced at a constant level of 10.6 mg/L day during this period.     

The effects of CA2+ during the elicitation of shikonin derivatives inOnosma paniculatum cells

Summary A fungal elicitor extracted fromAspergillus oryzae (Ahlb.) Cobn mycelia promoted the production of shikonin derivatives inOnosma paniculatum Bur et Franch cell suspension cultures. Elicitor treatment also increased Ca²⁺ concentration in RM₉ medium, which could be measured earlier than the elicited increase of shikonin formation. Several reagents known to induce Ca²⁺-influx and increase the intracellular-free Ca²⁺ level, such as the addition of Ca (NO₃)₂·4H₂O, the Ca²⁺ ionophore A₂₃₁₈₇, and abscisic acid (ABA), appreciably suppressed the elicitor-promoted shikonin formation inOnosma cells. In contrast, the decrease of intracellular-free Ca²⁺ level by the specific Ca²⁺-chelator ethylene glycol bis (β-aminoethylether)-N,N,N′,N′-tetraacetic acid (EGTA) or the Ca²⁺—channel blocker, verapamil, enhanced the biosynthesis of shikonin even in the absence of elicitor. Treatment of cells with trifluoperazine (TFP) also stimulated shikonin formation inOnosma cell cultures. A rapid and transient drop of free Ca²⁺ level in one protoplast was directly determined after the addition of elicitor toOnosma cell cultures. The inhibitory effect on shikonin formation by ABA was largely on account of its ability to restore the intracellular Ca²⁺ level lowered by the elicitor. These results suggest that Ca²⁺ play a significant role in an early stage of the elicitation process ofOnosma cells. The rapid drop of cytoplasmic Ca²⁺ carries the elicitor signal and in turn regulates the biosynthesis of shikonin derivatives.     

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。     

Fed-batch hairy root cultures within situ separation

Fed-batch cultures ofL. erythrorhizon hairy root were carried out by controlling sucrose concentration and media conductivity in a shake flask and a modified stirred tank reactor. For the efficient product recovery from the culture,in situ adsorption by XAD-2 was also conducted. When sucrose was used as a carbon source, the highest shikonin production and hairy root growth were obtained. When glucose or fructose was used instead, the growth was severely inhibited. In addition, it was found that alternating feeding of sucrose could be used as an effective strategy for enhancing the productivity of shikonin derivatives., As the XAD-2 amount was increased up to 1.5 g/L, shikonin production was enhanced by removing shikonin produced and other products which might be inhibitory to cell growth. Most amount of shikonin produced was successfully recovered in XAD-2 (Over 99%). Using hairy root culture in a modified stirred tank reactor, the shikonin productivity and hairy root growth rate on the average were 9.34 mg/L day and 0.49 g DCW/L · day, respectively.     

Elicitation of camptothecin production in cell cultures ofCamptotheca acuminata

Campothecin production was increased with elicitors, methyl jasmonate, jasmonic acid, yeast extract elicitor, and ferulic acid in suspension cultures ofCamptotheca acuminata. jasmonic acid was found to be the most efficient elicitor. Camptothecin production increased 11 times by using the optimum dosing concentration of jasmonic acid which was 50 μM. The kinetics of camptothecin accumulation in response to the treatment with jasmonic acid showed that the camptothecin accumulation reached the maximum value at 4 days after jasmonic acid dosing and then a rapid decrease in camptothecin accumulation was observed.     

Fractionation and Biological Activity of Aspergillus oryzae Elicitor Promoting Biosynthesis of Shikonin Derivatives

The Aspergillus oryzae elicitor was extracted from mycelia. The concentrated crude preparation of which was treated through DEAE-Cellulose, Sepharose-4B. Bio-Gel p- 4 and 732 columns. Elicitor activity was associated with fraction F Ⅰ b2-H, which had no affinity for DEAE-Cellulose and 732 resin. Its molecular weight was 1200～2200 D and its carbohydrate content was 6.7% of that of the crude. The elicitor activity was 120 times higher than that of crude preparation. There were also fractions F Ⅱ of nucleic acids and F Ⅰ b2-Na of nucleotides, amino acids in crude elicitor preparation. They did not affect shikonin derivative formation at low concentration, but inhibited shikonin derivative formation at high concentration. Fraction FIa of polysaccharid nature in the crude preparation which strongly inhibited shikonin derivative formation was another kind of elicitor of a new metabolite yellow pigment.     

真菌诱导子与吸附树脂对新疆紫草毛状根中萘醌积累的影响

通过考察真菌诱导子与吸附树脂对新疆紫草毛状根中萘醌积累的影响,获得真菌诱导子与吸附树脂对萘醌类物质积累的最佳处理,为规模化生产提供依据.以新疆紫草毛状根为试验材料,将黑曲霉、米曲霉诱导子及其混合诱导子、大孔吸附树脂添加到M-9培养基中,采用分光光度法测定毛状根总萘醌含量.试验结果表明:在毛状根培养10d时以2.5∶50的比例添加混合诱导子,总萘醌含量是对照的2.28倍;在此结果基础上,在培养第0天添加大孔吸附树脂NKA-9,总萘醌含量最高是对照的3.71倍;黑曲霉诱导子与米曲霉诱导子有协同效应;在生物反应器中添加混合诱导子及大孔吸附树脂NKA-9,其总萘醌含量是对照的4.17倍.米曲霉诱导子、混合诱导子对毛状根增殖有促进作用;同时添加大孔吸附树脂NKA-9及混合诱导子能提高毛状根总萘醌含量.生物反应器培养毛状根为今后利用新疆紫草毛状根规模化生产总萘醌提供了理论参考.     

Enhanced shikonin production from Lithospermum erythrorhizon by in situ extraction and calcium alginate immobilization

Plant cell cultures of Lithospermum erythrorhizon were carried out to produce shikonin by in situ extraction and cell immobilization in calcium alginate bead in shake flask cultures. In situ product extraction and cell immobilization enhanced shikonin production and facilitated product recovery. In situ extraction by n-hexadecane and cell immobilization by calcium alginate gave higher specific shikonin productivities of 7.4 and 2.5 times, respectively, than those from the cultures of free cells without extraction. Simultaneous use of both techniques increased specific and volumetric productivities of shikonin 25- and 15-fold, respectively. In calcium alginate immobilized cell cultures, n-hexadecane addition at an early stage (before 15 days) was effective for shikonin production, and solvent addition after 15 days of the culture significantly reduced shikonin production. Higher numbers of plant cell immobilized bead inoculation did not increase shikonin production and sucrose consumption. Most of the produced shikonin was dissolved in the solvent layer.     

Induction of antimicrobial compounds in alfalfa callus by cell wall components fromFusarium oxysporum

Callus cultures ofMedicago sativa were treated with a preparation of elicitor from the mycelium ofFusarium oxysporum. The ability of the elicitors to induce the formation of antimicrobial substances was tested biologically. The preparation of the cell walls ofF. oxysporum induced the occurrence of substances with a strong antimicrobial effect. Thermal sterilization had no effect on the activity of the elicitor.     

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.     

Selection of Cell Lines with High Productivity of Shikonin Derivatives by Protoplast Culture of Lithospermum erythrorhizon Cells

We selected high-yield cell lines, using protoplast culture of Lithospermum erythrorhizon cells. Three cell lines having different shikonin productivities were used as parent cells for the selection, and cell lines with high productivity were obtained efficiently in every case. The best cell line had 6.45 g shikonin/g inoculum/23 days of production which was almost 1.5 times higher than that of the original cell line. The productivities of protoplast-derived cell lines were distributed widely and their average productivity was similar to the original one. The subculture of such a protoplast-derived cell line for eight months showed that its shikonin productivity was stabler than the original cell line.     

Differential induction of phenylalanine ammonia-lyase activity in date palm roots in response to inoculation with Fusarium oxysporum f. sp. albedinis and to elicitation with fungal wall elicitor

The inoculation of the roots of resistant (BSTN) and susceptible (JHL) cultivars of date palm seedlings byFusarium oxysporum f. sp.albedinis (Foa) induces an increase in activity of phenylalanine ammonia-lyase (E.C. 4. 3. 1. 5., PAL). The post-infectional response in the PAL activity in the resistant cultivar roots was faster and higher than that in the susceptible cultivar. However, the elicitation of the seedlings by the hyphal wall preparation (HWP) ofFoa induces an identical PAL response in the resistant and the susceptible cultivars. The elicitor activity of HWP was dose-dependent, the optimal concentration which induces a maximum PAL activity was 10 mg of mycelium per mL. The elicitor present in the HWP was thermostable since its elicitor activity was maintained after heat treatment (121 °C for 45 min). The treatment of the HWP with protease (Pronase E) does not have an effect on the HWP elicitor activity. However, the treatment of the HWP with sodium periodate inhibits its elicitor activity. This data suggests that the HWP elicitor is a carbohydrate compound. In addition, the HWP elicitor is non-specific since it induces identical responses of the PAL activity in two cultivars showing different behaviors to the pathogen. The absence of specificity of HWP elicitors and the differential response of the PAL activity to the infection byFoa and to the elicitation by the HWP are discussed. An explanation of the general interactions between plant and parasite is proposed.     

Improved Taxol production in suspension cultures of Taxus chinensis var. mairei by in situ extraction combined with precursor feeding and additional carbon source introduction in an airlift loop reactor

Taxus chinensis var. mairei was grown in two-liquid-phase culture in a 2.5 l airlift external loop reactor for production of Taxol. A mixture of oleic acid and dibutyl phthalate (1:1, v/v) in a volume ratio of 8:92 to the culture medium gave higher production of Taxol (up to 12 mg l^–1) with an aeration rate of 1 vvm. By combination of the in situ extraction, precursor feeding and additional sugar introduction, Taxol production (16.7 mg l^–1) was 5 times higher than that in the case without any additives (3.4 mg l^–1) and 1.4 times of that in the case with only thein situ extraction (12 mg l^–1). Taxol was extracted from the organic solvents with water and methanol (1:1:1.4, by vol.) and lyophilized giving a total yield of ca. 90%.     

Cell-Specific Production and Antimicrobial Activity of Naphthoquinones in Roots of Lithospermum erythrorhizon

Pigmented naphthoquinone derivatives of shikonin are produced at specific times and in specific cells of Lithospermum erythrorhizon roots. Normal pigment development is limited to root hairs and root border cells in hairy roots grown on “noninducing” medium, whereas induction of additional pigment production by abiotic (CuSO₄) or biotic (fungal elicitor) factors increases the amount of total pigment, changes the ratios of derivatives produced, and initiates production of pigment de novo in epidermal cells. When the biological activity of these compounds was tested against soil-borne bacteria and fungi, a wide range of sensitivity was recorded. Acetyl-shikonin and β-hydroxyisovaleryl-shikonin, the two most abundant derivatives in both Agrobacterium rhizogenes-transformed “hairy-root” cultures and greenhouse-grown plant roots, were the most biologically active of the seven compounds tested. Hyphae of the pathogenic fungi Rhizoctonia solani, Pythium aphanidermatum, and Nectria hematococca induced localized pigment production upon contact with the roots. Challenge by R. solani crude elicitor increased shikonin derivative production 30-fold. We have studied the regulation of this suite of related, differentially produced, differentially active compounds to understand their role(s) in plant defense at the cellular level in the rhizosphere.     

Cell death unlikely contributes to taxol production in fungal elicitor-induced cell suspension cultures of Taxus chinensis

Cell suspension cultures ofTaxus chinensis, with 20, 40 and 100 mg fungal elicitor l^–1 from Aspergillus niger, underwent rapid cell death after 24 h, which was about 2, 3.7 and 5-fold of that of the control. At the same time, Taxol production was increased, respectively, to about 5, 8 and 3-fold of that of the control. Inhibition of phenolics biosynthesis resulted in a 150% increase in cell death but a 54% decrease in Taxol production compared with 40 mg elicitor l^–1 alone. O₂-free N₂ inhibited cell death but had little effect on Taxol production as induced by 40 mg fungal elicitor l^–1.     

Thiophene accumulation in hairy roots ofTagetes patula in response to fungal elicitors

Summary Hairy roots ofTagetes patula were treated with fungal extracts from 3 different fungi. Treated roots were found to accumulate thiophenes at a higher level than untreated control cultures. The kinetics of thiophene formation varied with the fungus from which the elicitor was prepared, elicitor concentration and duration of exposure.     

Enhancement of growth and secondary metabolite biosynthesis: Effect of elicitors derived from plants and insects

Plant-derived natural products have been and will continue to be valuable sources. Elicitors have been employed to modify cell metabolism in order to enhance the productivity of useful metabolites in plant cell/tissue cultures. In this study, several elicitors were used to improve the productivity of useful metabolites and to reduce culture time for archiving high concentration inP. ginseng hairy root cultures. The addition of chitosan, chitosan oligosaccharide and alginate oligosaccharide to the culture ofP. ginseng hairy roots caused growth to be inhibited with the increase in elicitor concentration. The usage of the chitosan elicitor andd-glucosamine caused a slight decrease in hairy root growth, whereas total ginseng saponin accumulated slightly with the increase in elicitor concentration. When gel beads were added to the culture medium at the initial period, hairy root growth was enhanced. The maximum growth was 1.35 times higher than that of the control at 1% (w/v). Total ginseng saponin content decreased due to the addition of alginate beads. This would result in consistent diffusion of lower levels of calcium ions during the culture period that promotes biomass growth.     

Interaction of cotton tissue culture cells andVerticillium dahliae

Summary Elicitation of sesquiterpenoid aldehyde phytoalexins inGossypium arboreum cell suspension cultures was confirmed by thin layer chromatography, high performance reverse phase liquid chromatography, and an aniline-reaction assay after inoculation with heat-treated conidia ofVerticillium dahliae A 2.3X mean increase in total terpenoids was observed. Component phytoalexins varied, with either hemigossypol and gossypol being detected or the O-methylated terpenoids hemigossypol-6-methyl ether and related compounds. Long-termGossypium suspension cultures were mixoploid with an increase in chromosome number and mean DNA content. Addition ofV. dahliae elicitor(s) to the medium for embryo-proliferating callus ofG. hirsutum inhibited growth and embryo production with a linear correlation (r=−0.87;P<0.01) between the elicitor concentration and the number of embryos. Addition of¹⁴C-labeled NaOAc to suspension cells gave 30% incorporation, and from¹³C-NaOAc addition, labeled sesquiterpenoid aldehydes were recovered. The cotton-Verticillium system is another case of secondary metabolite elicitation in plant tissue culture and might be used for basic studies of hostpathogen interaction as well as for a selection tool to obtain resistance to an important disease.