Semi-continuous production of sanguinarine and dihydrosanguinarine by Papaver somniferum L. cell suspension cultures treated with fungal homogenate

Papaver somniferum L. (opium poppy) cells were elicited with a Botrytis sp. homogenate and cultured by a semi-continuous process. Elicitation induced synthesis of sanguinarine and dihydrosanguinarine. Significant release of both alkaloids into the culture medium occurred. Medium exchange at 2-day intervals enabled product recovery from spent medium and maintained culture viability. Culture growth was not inhibited by elicitor treatment necessitating sub-culture prior to re-elicitation. Re-elicited cultures displayed an increasing sensitivity (reduced growth rate, higher alkaloid yield) to the elicitor with each successive treatment and did not survive a fourth elicitation.Abbreviations DW dry weight - FW fresh weight - 2,4-D 2,4-dichlorophenoxyacetic acid - SGE sanguinarine - DSGE dihydrosanguinarine Publication 29143 from the National Research Council of Canada     

Statistical analysis of elicitation strategies for thiarubrine A production in hairy root cultures of Ambrosia artemisiifolia

Elicitation strategies were studied for yield enhancement of thiarubrine A, a secondary metabolite and a potential pharmaceutical, produced by hairy root cultures of Ambrosia artemisiifolia. Abiotic elicitation was performed using vanadyl sulfate solution and biotic elicitation using autoclaved cell wall filtrates of the fungi Protomyces gravidus, a pathogen of A. artemisiifolia and Botrytis cinereae. The factors considered were age of the culture, concentration of the elicitor used and the time period of exposure or contact. Statistical methods were used to determine the strength of the interaction between the various factors and their response on the yield of the secondary metabolite. The maximum increase in the yield relative to the control, 8-fold corresponding to 569 microg g(-1) of biomass, was observed when 16-day-old cultures were elicited with 50 mg l(-1) of vanadyl sulfate for a time period of 72 h. The maximum yield of 647 microg g(-1) was achieved when the cultures were exposed to 5 microM autoclaved cell wall filtrates of P. gravidus for a time period of 48 h. The yield increase was 3-fold in the case of elicitation with autoclaved cell wall filtrates of B. cinereae. The methodology used in this report can be extended to determine the optimum conditions of other elicitors.     

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.     

Mapping meristem respiration of Prunus persica (L.) Batsch seedlings: potential respiration of the meristems, O2 diffusional constraints and combined effects on root growth

Root system architecture partially results from meristem activities, which themselves depend on endogenous and environmental factors, such as O2 depletion. In this study, meristem respiration and growth was measured in the root systems of three Prunus persica (L.) Batsch seedlings. The spatial distribution of meristem respiration within the root system was described, and the relationship between the respiration rates and meristem radii was analysed, using a model of radial O2 diffusion and consumption within the root. Histological observations were also used to help interpret the results. Respiration rates were linearly correlated to the root growth rates (rho 2 = 0.9). Respiration reached values greater than 3.5 x 10(-13) mol O2 s-1 for active meristems. The taproot meristem consumed more O2 than the rest of the entire root system meristems. Similarly, the first order lateral meristems used more O2 than the second order ones. A near hyperbolic relationship between respiration rates and meristem radii was observed. This can be explained by a model of radial O2 diffusion and consumption within the root. Therefore, only one maximum potential respiration rate and one O2 diffusion coefficient was estimated for all the meristems.     

内生真菌Colletotrichum sp. B501的寡糖提取物对黄花蒿发根中青蒿素生物合成的诱导

在黄花蒿(Artemisia annua L.)发根液体培养中,黄花蒿内生炭疽菌(Colletotrichum sp. B501)细胞壁寡糖提取物可促进发根青蒿素的合成.经寡糖诱导子(20 mg/L)处理4 d后,发根青蒿素含量达1.15 mg/g, 比对照高出64.29%.诱导作用与诱导子浓度、作用时间相关.诱导处理1 d后,X射线能谱分析表明黄花蒿发根细胞中Ca2+积累量显著增高,电镜观察发现液泡内出现高电子致密物,具活性氧清除作用的过氧化物酶表现出高活性(6.5 unit*min-1*g-1 FW).诱导处理第三天,细胞核DNA呈梯度条带降解,部分细胞出现程序化死亡.内生菌细胞壁寡糖提取物引起的生理反应有利于细胞中青蒿素的生物合成.     

内生真菌Colletotrichum sp. B501的寡糖提取物对黄花蒿发根中青蒿素生物合成的诱导(英文)

在黄花蒿 (ArtemisiaannuaL .)发根液体培养中 ,黄花蒿内生炭疽菌 (Colletotrichumsp .B5 0 1)细胞壁寡糖提取物可促进发根青蒿素的合成。经寡糖诱导子 (2 0mg/L)处理 4d后 ,发根青蒿素含量达 1.15mg/g ,比对照高出6 4 .2 9%。诱导作用与诱导子浓度、作用时间相关。诱导处理 1d后 ,X射线能谱分析表明黄花蒿发根细胞中Ca2 积累量显著增高 ,电镜观察发现液泡内出现高电子致密物 ,具活性氧清除作用的过氧化物酶表现出高活性 (6 .5unit·min-1·g-1FW)。诱导处理第三天 ,细胞核DNA呈梯度条带降解 ,部分细胞出现程序化死亡。内生菌细胞壁寡糖提取物引起的生理反应有利于细胞中青蒿素的生物合成。     

Efficient production and recovery of diterpenoid tanshinones in Salvia miltiorrhiza hairy root cultures with in situ adsorption, elicitation and semi-continuous operation

In Salvia miltiorrhiza hairy root cultures, the desired secondary metabolites diterpenoid tanshinones are normally produced at low yields and stored within the roots. To enhance tanshinone production and the secondary product recovery, we employed three means, elicitation with a yeast elicitor (YE), in situ adsorption of tanshinones with a hydrophobic polymeric resin (X-5) and semi-continuous mode of operation. YE treatment stimulated the tanshinone biosynthesis, increasing the total tanshinone (TT) content of root by about two-fold, from 0.46 to 1.37 mg/g dry weight (dw) (TT content=total content of three major tanshinones, cryptotanshinone, tanshinone I and tanshinone IIA). The addition of X-5 resins to the culture only increased the tanshinone yield slightly, but recovered more than 80% of tanshinones from the roots. With the application of a semi-continuous culture process involving repeated medium renewal, elicitor addition and resin replacement, starting at the late exponential growth phase, the root biomass was increased to 30.5g dw/l (versus 8-10g dw/l in batch mode) and the volumetric tanshinone yield to 87.5mg/l (about 15-fold increase), with 76.5% adsorbed to the resin. The volumetric productivity of total tanshinone reached 1.46 mg/lday, more than 7.4 times that of the batch culture. The results demonstrate that the integration of multiple elicitation, in situ adsorption and semi-continuous operation can synergistically enhance tanshinone production in S. miltiorrhiza hairy root cultures.     

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.     

Improvement of artemisinin accumulation in hairy root cultures of Artemisia annua L by fungal elicitor

The effect of fungal elicitor, derived from mycelial extracts of Penicillium chysogenum 3446, on artemisinin production in hairy root cultures of Artemisia annua L was studied. Various concentrations of elicitor were added to the culture medium after 18 days. Time course experiments were carried out using a defined concentration of elicitor after 18 days. Various ages of hairy root cultures were elicited using a defined concentration of elicitor for 3 days. Artemisinin production in 21-day hairy root cultures treated with 0.3 mg total sugar/ml medium elicitor for 3 days reached to 549.1 mg/l.     

真菌诱导子对青蒿发根细胞生长和青蒿素积累的影响

３种真菌诱导子（大菌丽花轮枝孢（Ｖｅｒｔｉｃｉｌｌｉｕｍ ｄａｈｉａｅ Ｋｌｅｂ．）、葡枝根霉（Ｒｈｉｚｏｐｕｓ ｓｔｏｌｏｎｉｆｅｒ（Ｅｈｒｅｎｂ．ｅｘＦｒ．）Ｖｕｉｌｌ）和束状刺盘孢（Ｃｏｌｌｅｔｏｒｉｃｈｕｍ ｄｅｍａｔｉｕｍ（Ｐｅｒｓ．）Ｇｒｏｖｅ）处理青蒿（Ａｒｔｅｍｉｓｉａ ａｎｎｕａＬ．）的发根,均能促进发根中青蒿素的积累,其中以大丽花轮枝孢的诱导效果最好;对细胞生长均没有明显影响,     

Interpreting the role of phosphorus and growth rate in enhanced fungal induction of sesquiterpenes from Hyoscyamus muticus root cultures

The effect of thiamine limitation in combination with fungal elicitation on sesquiterpene (solavetivone) production was studied in Agrobacterium-transformed hairy-root cultures of Hyoscyamus muticus as a potential means of manipulating the growth rate independent of phosphorus availability. Limiting the initial supply of thiamine did not affect the growth of these cultures compared to growth at the control level of thiamine (0.01 g/l). There was also no enhancement in sesquiterpene production when thiamine supply was limited. Serial culturing in thiamine-free media suggests that these root cultures are not strictly auxotrophic for thiamine, in contrast to previously published results for untransformed root culture. The effect of phosphate limitation combined with elicitation on the production of solavetivone was examined at constant media volume to provide a constant elicitor concentration and to eliminate feedback-inhibition effects. Limiting the initial supply of phosphate to elicited cultures resulted in a twofold increase in solavetivone production as compared to the elicitation at control media phosphate levels (1.1mm). Because growth was attenuated, production per unit cell mass increased 11-fold compared to the control. The effect of phosphate limitation on solavetivone production at constant cell mass and elicitor per root mass was studied. Limiting the initial supply of phosphate to elicited cultures under these conditions did not result in enhanced production of solavetivone. The initially observed enhanced production of solavetivone at limiting initial phosphate concentrations is therefore due to factors other than the growth rate or phosphate involvement in secondary metabolism. Correspondence to: W. R. Curtis     

Some common signal transduction events are not necessary for the elicitor-induced accumulation of silymarin in cell cultures of Silybum marianum

A variety of pharmacological effectors of signal transduction pathways were used to investigate the elicitor-activated sequence of cellular responses by which yeast extract (YE) or methyljasmonate (MeJA) enhanced production of silymarin in cell cultures of Silybum marianum. As we recently showed that inhibition of external and internal calcium fluxes significantly increased flavonolignan production in S. marianum cultures, we examined whether calcium mediates signaling events leading to enhancement of silymarin production upon YE or MeJA elicitation. Pre-treatment of cultures with calcium chelators, calcium blockers or intracellular antagonists enhanced the elicitor effect of YE or MeJA. The increase of intracellular-free Ca(2+) level also promoted the elicitor effect, suggesting that an external source of calcium or alterations in internal calcium fluxes were not required for the elicitation to occur. Activation of phosphorylation/dephosphorylation cascades did not appear to mediate the elicitation mechanism; the increase in silymarin induced by elicitation was not suppressed by inhibitors of protein phosphatases or by protein kinase inhibitors. No H(2)O(2) generation was detected at any time after elicitation. Also, diphenyleneiodonium, a potent inhibitor of NAD(P)H-oxidase, did not block silymarin production in elicited cultures. From these results, we conclude that S. marianum cell cultures do not appear to employ conserved signaling components in the transduction of the elicitor signal to downstream responses such as silymarin production.     

Influence of fungal elicitors on production of ajmalicine by cell cultures of Catharanthus roseus

Suspension cultures of Catharanthus roseus (C. roseus) were elicited with fungal cell wall fragments of Aspergillus niger (A. niger), Fusarium moniliforme (F. moniliforme), and Trichoderma viride (T. viride). The effects of elicitor dosage, exposures time, and age of subculture on ajmalicine accumulation were studied. A higher concentration of elicitor extract responded positively to C. roseus suspension cultures. Ajmalicine accumulation increased by about 3-fold when cells were treated with A. niger, F.moniliforme, and T. viride. The maximum ajmalicine production (75 microg g(-1) dry weight (DW)) was observed in cells treated with T. viride. Cell cultures were elicited with 5% preparation of A. niger, F. moniliforme, and T. viride and exposed for 24, 48, 72, and 96 h. for elicitation. Suspension cultures elicited with T. viride for 48 h showed a 3-fold increase (87 microg g(-1) DW) in ajmalicine contents, whereas A. niger and F. moniliforme synthesized a 2-fold increase in alkaloid and yielded 52 and 56 microg g(-1) DW ajmalicine, respectively. C. roseus cells of different age (5,10, 15, 20, and 25 days old) were treated with a 5% elicitor of A. niger, F. moniliforme, and T. viride and investigated elicitors activity at different age of cell cultures. Maximum yield 166 microg g(-1) DW of ajmalicine was synthesized in 20 day old suspension cultures treated with T. viride. A longer period of incubation of cell cultures with elicitors adversely affected the ajmalicine synthesis.     

Intrinsic oxygen use kinetics of transformed plant root culture

Root meristem oxygen uptake, root tip extension rate, and specific growth rate are assessed as a function of dissolved oxygen level for three transformed root cultures. The influence of hydrodynamic boundary layer was considered for all measurements to permit correlation of oxygen-dependent kinetics with the concentration of oxygen at the surface of the root meristem. Oxygen uptake rate is shown to be saturated at ambient conditions, and a saturation level of approximately 300 micromole O2/(cm(3) tissue.hr) was observed for all three of these morphologically diverse root types. In nearly all cases, the observation of a minimum oxygen pressure, below which respiration, extension, or root growth would not occur, could be accounted for as a boundary layer mass transfer resistance. The critical oxygen pressure below which respiration declines is below saturated ambient oxygen conditions. In contrast, critical oxygen pressures for root tip extension were much higher; extension was nearly linear for the two thicker root types (Hyoscyamus muticus, henbain; Solanum tuberosum, potato) above ambient oxygen levels. The performance of the thinnest root, Brassica juncea (Indian mustard) was consistent with reduced internal limitations for oxygen transport. Extension rates did not correlate with biomass accumulation. The fastest growing henbain culture micro = 0.44 day(-)(1)) displayed the slowest extension rate (0.16 mm/hr), and the slowest growing mustard culture (micro = 0.22 day(-)(1)) had the fastest tip extension rate (0.3 mm/hr). This apparent paradox is explained in terms of root branching patterns, where the root branching ratio is shown to be dependent upon the oxygen-limited mersitem extension rate. The implications of these observations on the performance of root culture in bioreactors is discussed.     

Effect of elicitation on the accumulation of solasodine by immobilized cells of Solanum eleagnifolium Cav.

The effect of a fungal elicitor obtained from Alternaria sp. on growth and solasodine production by free and alginate-entrapped cells of Solanum eleagnifolium Cav. was studied. Fourteen-day-old cultures were elicited with 1% FW/V autoclaved homogenates. The solasodine production increased from 0.9 to 1.5 mg g^-1 DW (65%) in suspension cultures and from 0.75 to 1.4 mg g^-1 DW (about 95%) in entrapped cells. The maximum accumulation was obtained after 72 h of elicitation. In order to induce alkaloid release from cells (suspension and entrapped cells), permeabilization with 10% dimethylsulfoxide (DMSO) for 30 min was used. In both cases (free and entrapped cells), about 50–60% of intracellular solasodine was released into the medium. The reuse of elicited and permeabilized entrapped cells was also carried out for three production cycles.     

Effects of Fungal Elicitors on Cell Growth and Artemisinin Accumulation in Hairy Root Cultures of Artemisia annua

The artemisinin accumulation in the hairy root cultures of Artemisia annua L. was enhanced via a treatment of three fungal elicitors separately ( Verticillium dahliae Kleb., Rhizopus stolonifer (Ehrenb. ex Fr. ) Vuill and Colletotrichum dematium (Pers.) Grove). Among these three elicitors, V. dahliae had the highest inducing efficiency, but none of them manifests any noticeable effects on the cell growth of the hairy root cultures. The artemisinin content of the hairy root cultures treated with V. dahliae elicitor was 1.12 mg/g DW, which was 45% higher than the control (0.77 mg/g DW). The results showed that elicitation was dependent on the elicitor concentration, the incubation period and the physiological stage at which the hairy root cultures were treated. In addition, the authors found that for V. dahliae, the optimum concentration was 0.4 mg carbohydrate per millilitre medium, the strongest response of A. annua hairy root cultures to the elicitation was at the late exponential growth stage, and the highest artemisinin content of the hairy root cultures was on the 4th day post treatment.     

Pretreatment of Parsley (Petroselinum crispum L.) Suspension Cultures with Methyl Jasmonate Enhances Elicitation of Activated Oxygen Species

Suspension-cultured cells of parsley (Petroselinum crispum L.) were used to demonstrate an influence of jasmonic acid methyl ester (JAME) on the elicitation of activated oxygen species. Preincubation of the cell cultures for 1 d with JAME greatly enhanced the subsequent induction by an elicitor preparation from cell walls of Phytophtora megasperma f. sp. glycinea (Pmg elicitor) and by the polycation chitosan. Shorter preincubation times with JAME were less efficient, and the effect was saturated at about 5 [mu]M JAME. Treatment of the crude Pmg elicitor with trypsin abolished induction of activated oxygen species, an effect similar to that seen with elicitation of coumarin secretion. These results suggest that JAME conditioned the parsley suspension cells in a time-dependent manner to become more responsive to elicitation, reminiscent of developmental effects caused by JAME in whole plants. It is interesting that pretreatment of the parsley cultures with 2,6-dichloroisonicotinic and 5-chlorosalicylic acid only slightly enhanced the elicitation of activated oxygen species, whereas these substances greatly enhanced the elicitation of coumarin secretion. Therefore, these presumed inducers of systemic acquired resistance exhibit a specificity different from JAME.     

Elicitor-mediated induction of isochorismate synthase and accumulation of 2,3-dihydroxy benzoic acid in Catharanthus roseus cell suspension and shoot cultures

After elicitation, cell suspension cultures of Catharanthus roseus accumulate phenolic compounds. The major phenolic compound produced was isolated and identified as 2,3-dihydroxybenzoic acid (DHBA). The accumulation of this compound is a rapid response to the addition of elicitor; within 6 h after the addition of elicitor, DHBA concentration reached 6.3 mg/l cell suspension. DHBA was not detected in non-elicited cells. The formation of DHBA in elicited cells was correlated with the induction of the enzyme isochorismate synthase (ICS). Shoot cultures of C. roseus also presented a strong induction of ICS after elicitation. Due to its biological activity, DHBA could play a role in the defence mechanism of C. roseus.     

Polyacetylenes accumulation in <Emphasis Type="Italic">Ambrosia maritima</Emphasis> hairy root and cell cultures after elicitation with methyl jasmonate

Three lines of hairy root culture of Ambrosia maritima induced by Agrobacterium rhizogenes ATCC15834 were established. Thiarubrine A, thiarubrine A epoxide, thiarubrine A diol and their precursor pentayneene were produced by the hairy roots after elicitation with methyl jasmonate, the common signal molecule in the plant defense and development. Thiarubrine A diol was the main form detected in the medium. Maximum yield was achieved when the 13-day-old hairy root cultures were exposed to 40 M methyl jasmonate for 72 h. Callus and cell suspension cultures were established and maintained on Murashige and Skoog medium supplied with -naphthylacetic acid (NAA) and kinetin. When the cell suspension cultures were elicited with methyl jasmonate, pentayneene was the only polyacetylene produced. The yield of pentayneene in hairy root cultures was much higher (9.6 times) than that of cell suspension cultures.     

Phenylpropanoid defence responses in transgenic Lotus corniculatus: II. Modelling plant defence responses in transgenic root cultures using thiol and carbohydrate elicitors

Transformed root cultures of Lotus corniculatus L. cv. Leo weretreated with a range of thiol and carbohydrate elicitors. Boththiol reagents and fungal carbohydrate preparations resultedin an increase in the activity of phenylalanine ammonia lyase(PAL) in a concentration-dependent manner. One representativethiol elicitor, glutathione (GSH), and one fungal elicitor,derived from Rhynchosporium orthosporum autoclaved cell walls(Ro), were analysed in more detail. Both elicitors induced thetransient accumulation of vestitol, an isoflavan phytoalexin,in tissue and in culture medium. Treatment of Lotus root cultureswith the Ro elicitor resulted in a more rapid initial accumulationof this end product when compared with GSH, however, sativan(the 2–methoxy ester of vestitol) previously reportedto co-accumulate in Lotus leaves was only detected followingelicitation with high concentrations of GSH. Ro and GSH elicitorsalso induced the accumulation of a number of other phenylpropanoidcompounds putatively identified as chalcones. The addition ofthiol and carbohydrate elicitors to Lotus root cultures alsoresulted in characteristic changes in root morphology. Glutathione,in particular, resulted in the inhibition of root growth dueto differential damage of meristem cells. Key words: Lotus corniculatus, hairy roots, elicitors, phytoalexins.