Characteristics of growth and tropane alkaloid production in Hyoscyamus albus hairy roots transformed with Agrobacterium rhizogenes A4

Summary Hairy root culture of Hyoscyamus albus was established by transformation with Agrobacterium rhizogenes strain A4. The growth and production of five tropane alkaloids were investigated under various culture conditions. Among the four basal culture media tested, Woody Plant medium was the best for growth of the hairy roots, but a high amount of tropane alkaloids was obtained with Gamborg's B5 medium. Sucrose concentration in B5 medium had little effect on the growth, while 3% sucrose was suitable for the alkaloid production. Addition of KNO₃ to Woody Plant medium affected the growth, whereas the alkaloid content was not markedly improved. Supplement of some metal ions to B5 medium stimulated the alkaloid production. In particular, Cu²⁺ remarkably enhanced both the growth and the alkaloid yield. The hairy roots cultured under 16 h/day light survived for more than 32 days compared with those cultured in the dark.Abbreviations EDTA ethylenediaminetetraacetic acid - HPLC high performance liquid chromatography - MeOH methanol - MS medium Murashige and Skoog medium - WP medium McCown's Woody Plant medium - B5 medium Gamborg B5 medium - wt weight     

Effect of differentiation on the regulation of indole alkaloid production in Catharanthus roseus hairy roots

In vitro cultures of hairy root derived from Catharanthus roseus accumulate higher levels of indole alkaloids than cell suspension cultures. Hairy roots were interconverted to undifferentiated cells by manipulation of the culture medium. When the concentration of micronutrients in the culture medium was five times that of Phillips and Collins (1979) medium, cell suspensions formed from the hairy roots. The alkaloid content was five times lower in the cell suspensions than in the control, but upon regeneration of the roots the alkaloid content regained its original level. The formation of cell suspensions from hairy roots was also accompanied by a reduction in tryptophan decarboxylase and the strictosidine synthase activity to less than 5% and 30%, respectively. 3-Hydroxymethylglutaryl coenzyme A reductase activity was the same in the cell suspension and in the regenerated line. Received: 12 February 1998 / Revision received: 21 May 1998 / Accepted: 5 June 1998     

The effect of temperature on hairy root cultures of Catharanthus roseus: Growth,indole alkaloid accumulation and membrane lipid composition

Cultivation of Catharanthus roseus hairy root cultures at different temperatures was found to have an effect on growth rate and indole alkaloid content as well as lipid composition. When lowering the temperature, the roots responded by increasing the degree of unsaturation of cellular lipids, which was mainly due to an increased proportion of linolenic acid in the main lipid classes. The modifications in lipid composition were obviously necessary for the roots to retain the proper cell membrane fluidity at each temperature. Despite of changes in membrane lipids, no effect on the distribution of indole alkaloids between the roots and the medium could be detected. Instead, the level of alkaloid accumulation showed a clear increase with lowering temperature.Abbreviations PC phosphatidylcholine - PE phosphatidylethanolamine - PI phosphatidylinositol - PS phosphatidylserine - PG phosphatidylglycerol - CL cardiolipin - DGD digalactosyldiglyceride - MGD monogalactosyldiglyceride - NL neutral lipids - DU degree of fatty acid unsaturation     

Influence of feeding precursors on tropane alkaloid production during an abiotic stress in Datura innoxia transformed roots

The effects of feeding tropane alkaloid precursors in transformed root culture of Datura innoxia Mill. were studied during a stress treatment. The permeabilizing effect of Tween 20 on tropane alkaloid production by hairy root cultures was studied in flasks with different feeding of precursors (L-ornithine, L-arginine, L-phenylalanine, DL-β-phenyllactic acid, and tropinone). It has been shown that the addition of various precursors alone (0.5 m mol l ^-1) was ineffective in stimulating hyoscyamine production. In contrast, a short treatment with Tween 20, combined with L-phenylalanine feeding, amplified the level of hyoscyamine released into the medium compared with the Tween treatment alone. Thus, the total hyoscyamine content per flask was increased (+ 40%) compared with the control. When DL-β-phenyllactic acid (0.5 m mol l ^-1) was used, this last effect became more pronounced (+ 60%). These results show that permeabilization with Tween modulates tropane alkaloid accumulation by a release of alkaloids into the medium and a restoration of hyoscyamine root content. The simultaneous feeding of DL-β-phenyllactic acid and tropinone during the Tween treatment gave a similar effect to that obtained with DL-β-phenyllactic acid and Tween, suggesting that the synthesis of the tropate moiety determines the flux at the level of the esterification of tropine. This revised version was published online in June 2006 with corrections to the Cover Date.     

Effect of tween series surfactants on alkaloid production by submerged cultures of Claviceps species

Supplementation of Tween surfactants promoted alkaloid production by submerged cultured of Claviceps sp. strain SD-58. Tween 80 (0.5%) exhibited the maximum (2-fold) stimulatory effect when added to the medium at the initial stage of cultivation. The stimulation of alkaloid production by Tween 80 was found to be associated with the increase in cell mass, higher consumption of nutrients and enhanced excretion of alkaloids from the cells. The results are discussed in relation to the physiology of alkaloid production in Claviceps sp. strain SD-58.     

Effect of tweens on the production of ergot alkaloids byAspergillus fumigatus

Tween 80, which caused increased biomass formation, also produced the highest increase in the uptake rate of all components of the medium. The fatty acid components of the respective Tweens,i.e. palmitic acid (Tween 40), stearic acid (Tween 60), and oleic acid (Tween 80), have no effect either on alkaloid production or on substrate uptake. The fatty acid composition was different in the cell membrane of the culture supplemented with Tween 60 and facilitated the transport of metabolites into the cells.     

Up-Regulation of Licochalcone A Biosynthesis and Secretion by Tween 80 in Hairy Root Cultures of <Emphasis Type="Italic">Glycyrrhiza uralensis</Emphasis> Fisch

We evaluated the effect of Tween 80 as elicitor on licochalcone A from hairy root cultures of Glycyrrhiza uralensis Fisch. After a 15-days treatment with 2% Tween 80, hairy roots still grew well and produced higher levels of licochalcone A and total flavonoids than the control (without treatment). Licochalcone A content and total flavonoid content were 3.103 and 127.095 mg per flask (9- and 11-fold higher), respectively, compared with controls. Secretion of licochalcone A and total flavonoids into the culture medium was remarkably high, up to 98 and 94% of the total production, respectively. The enhanced flavonoid production was associated with elevated mRNA levels and enzyme activities of phenylalanine ammonia-lyase (PAL), 4-coumarate:coenzyme A ligase (4CL), and cinnamate-4-hydroxylase (C4H). These results clearly demonstrated that Tween 80 treatment permeabilized the roots to enhance secretion, but also acted as an efficient elicitor of licochalcone A and total flavonoid production in hairy roots of G. uralensis Fisch.     

Growth and tropane alkaloid production inAgrobacterium transformed roots and derived callus ofDatura

Small callus pieces excised from theAgrobacterium transformed root line D₂ ofDatura stramonium, were cultured onto solidified MS medium supplemented with a 1.0 μM kinetin and three different concentrations (0.1, 0.5 and 1.0 μM) of 2,4-dichlorophenoxyacetic acid (2,4-D), and were examined for their alkaloid productivity in relation to organization level and growth rate. Growth of transformed roots (in a MS liquid medium without plant growth regulators) was greater than that of transformed calli excised from them and cultured separately. The addition of 1.0 μM 2,4-D to the culture medium had a positive effect on callus biomass production, while it inhibited root formation by this tissue (the lower the 2,4-D concentration in the medium the greater the number of roots which emerged from the calli). Hyoscyamine production was also higher in the transformed roots than in the transformed calli, and in these tissues the production of hyoscyamine was positively correlated with organogenesis index (i.e. its ability for rooting). At the same time, the epoxidation of hyoscyamine to scopolamine only took place in the transformed calli. This occurred to a greater extent at the lower concentrations of 2,4-D in the culture medium. The mode through which the 2,4-D could control the alkaloid production of transformed callus is discussed.     

Bioreactor studies on hairy root cultures of Catharanthus roseus: Comparison of three bioreactor types

Summary Hairy roots of Catharanthus roseus were cultivated in three different types of bioreactors. The best growth and indole alkaloid production was achieved in an airsparged bioreactor with no other mixing. In the stirred bioreactor or in the bioreactor with medium circulation the roots did not grow, suggesting that hairy roots of C. roseus are more sensitive to stress than root cultures of many other plant species.     

Effects of growth conditions on the ion composition of Bifidobacterium bifidum subsp. pennsylvanicum

The cation content of Bifidobacterium bifidum subsp. pennsylvanicum was markedly influenced by the washing procedure of the cells, by the growth phase and the temperature, and by the composition of the culture medium. Optimal retention of cations was achieved by washing with 0.25 M MgCl₂ at 20 C. The intracellular Na⁺ concentration rose during growth in normal medium to a constant value in the stationary phase, the K⁺ concentration rose in the exponential phase, but fell in the stationary phase. Cells from 29-C cultures contained more Na⁺ and less K⁺ in the stationary phase than did cells from 37-C cultures, but the total cation content was the same at 29 and 37 C.Intracellular Na⁺ and K⁺ concentrations were dependent on the concentrations in the medium and on its osmolarity. The intracellular Na⁺/K⁺ ratio varied from 0.04 to 2.3. The concentrations of Na⁺, K⁺ and phosphate in the medium hardly affected growth. Mg²⁺-deficiency of the medium markedly decreased the concentration of Mg²⁺ within the cell; its concentration in the cell sap was greatly affected, but the amount of sedimentable, bound Mg²⁺ only slightly. The content of K⁺ within the cell decreased in Mg²⁺-deficient medium, but the concentration of Na⁺ did not. Omission of Tween 80 as well as its substitution by Tween 20 caused a decrease of intracellular K⁺. Cells from Tween 40 and Tween 60 cultures additionally contained markedly less Na⁺.The present investigations have been carried out with financial support from the Netherlands Organization for the Advancement of Pure Research (ZWO) through the Netherlands Foundation for Chemical Research (SON).     

Genetic variation in constitutive and inducible pyrrolizidine alkaloid levels inCynoglossum officinale L.

The constitutive pyrrolizidine alkaloid (PA) concentration of both shoots and roots differed significantly between 17 selfed families. The broad-sense heritability accounted for 33–43% of the variation in PA levels. Families also differed significantly in the amount and the direction of PA induction in both shoots and roots, 24 h after punching 15 holes in the leaves. We found a significantly negative relationship between the changes in PA content of the shoots and changes in PA content of the roots. The total PA content of the plants did not increase. We thus concluded that changes in PA distribution over the plant resulted from transport of PAs within the plant. The direction of transport differed between families: some transported PAs to the shoots, others to the roots. This makes it questionable whether PAs act as damage-induced defences. The effect of damage on the PA concentration is far less than the differences found between families in the constitutive PA concentration. This again strongly suggests that damage-induced defences inCynoglossum officinale do not play an important role. We argue that the general lack of attention that is given to genotype in induction experiments, has led to false conclusions.     

Heterologous expression of Vitreoscilla hemoglobin (VHb) and cultivation conditions affect the alkaloid profile of Hyoscyamus muticus hairy roots

Fast-growing hairy root cultures of Hyoscyamus muticus induced by Agrobacterium rhizogenes offer a potential production system for tropane alkaloids. Oxygen deficiency has been shown to limit growth and biomass accumulation of hairy roots, whereas little experimental data is available on the effect of oxygen on alkaloid production. We have investigated the effect of Vitreoscilla hemoglobin (VHb) expression and cultivation conditions on the complete alkaloid profile of H. muticus hairy roots in shake flasks and in a laboratory scale bioreactor. We optimized the growth medium composition and studied the effects of sucrose, ammonium, nitrate, and phosphate on growth and alkaloid production. Maximum biomass accumulation was achieved with the highest and maximum hyoscyamine content with the lowest sucrose concentration. The optimum nitrate concentration for growth was higher for the VHb line than the control. Neither VHb expression nor aeration improved the hyoscyamine content significantly, thus suggesting that hyoscyamine biosynthesis is not limited by oxygen availability. Interestingly, the effect of VHb expression on the alkaloid profile was slightly different from that of aeration. VHb expression did not affect the concentrations of cuscohygrine, which was increased by aeration. Therefore, the effect of VHb is probably not related only to its ability to increase the intracellular effective oxygen concentration.     

Effect of phosphate on ergot alkaloid synthesis inAspergillus fumigatus

High concentration of inorganic phosphate in the culture medium ofAspergillus fumigatus inhibited ergot alkaloid synthesis. Addition ofl-tryptophan but not mevalonate or 5-methyltryptophan to the above culture restored the alkaloid synthesis to the level found in normal cultures. The decrease in alkaloid synthesis in the fungus accompanies an increase in cell mass, cellular protein and sterol content. Aspartate aminotransferase and alanine aminotransferase activities were significantly increased in the high-phosphate culture. Part of the work was presented at the seminar on “Enzymatic Methods in Mycology” organised by the Czechoslovak Microbiological Society in Brno, Czechoslovakia, in June 1975.     

Bioreactor production of camptothecin by hairy root cultures of Ophiorrhiza pumila

A large-scale culture of hairy root of Ophiorrhiza pumila using a modified 3 l bioreactor was established. The hairy roots, incited by infection of Agrobacterium rhizogenes were grown in the bioreactor equipped with a stainless net. The final concentration of camptothecin was 0.0085% fresh wt of tissue, and the total production of camptothecin, an anti-neoplastic quinoline alkaloid, reached 22 mg over 8 weeks' culture in the reactor. Approx. 17% (3.6 mg) of the total camptothecin produced was excreted into the culture medium.     

Growth and Alkaloid Patterns of Roots of Tabernaemontana pachysiphon and Rauvolfia mombasiana as Influenced by Environmental Factors

The influence of environmental factors on the indole alkaloid content and biomass of the roots of Tabernaemontana pachysiphon and Rauvolfia mombasiana, two species of considerable local medicinal use in tropical East Africa, was investigated. Both species, belonging to the Apocynaceae, are frequent constituents of the residual tropical forests, prefering sites of different ecological conditions. Experimental plants, raised from seeds, were grown for 16 months in a temperature- and humidity-controlled greenhouse. Environmental factors at variance were water and nutrient supply, and light intensity. At sufficient water and nutrient supply, the more drought and nutrient shortage-tolerating heliophilous Rauvolfia mombasiana showed increased alkaloid accumulation, concurrently with reduced root biomass. Under the same conditions, the drought-sensitive and higher levels of nutrient-requiring ombrophilous Tabernaemontana pachysiphon produced more root biomass but accumulated less alkaloids in the roots. The results indicate that the accumulation of indole alkaloids in the roots, as well as biomass allocation to the roots, is influenced in an opposite manner by the nutrient and water supply to the heliophilous and the ombrophilous species.     

Some Accounts on Culture Conditions of Callus Tissues of Solanum laciniatum Ait. for Producing Solasodine

Production of solasodine in callus cultures of Solanum laciniatum Ait. was examined under several culture conditions. The steroidal alkaloid was produced more actively in rapidly proliferating callus tissues cultured on PN medium. The alkaloid concentration in the tissue was about 0.05% (dry weight basis) during the first 5 weeks’ culture. The highest accumulation of the alkaloid per culture was obtained with 2,4-d concentration in the medium at 1~2 ppm. It is noteworthy that the alkaloid production was not inhibited by such high concentration of 2,4-d as up to 10 ppm in the medium. Supplementation of kinetin slightly increased the alkaloid production.     

Effect of Tween 80 on the production of curdlan by Alcaligenes faecalis ATCC 31749

This study aims to investigate the effects of Tween 80 on curdlan production, cell growth, and glucosyltransferase activity. The addition of Tween 80 to the culture medium increased curdlan production. However, curdlan production did not increase further when excessive Tween 80 (>0.3% Tween 80) was added to the culture medium. The addition of Tween 80 to the culture medium did not affect cell growth. The glucosyltransferase activity involved in the curdlan synthesis increased with the increase of Tween 80 concentration. The glucosyltransferase activity did not increase further when excessive Tween 80 (>0.3% Tween 80) was added to the culture medium. Maximum curdlan was observed at day 5 and then levelled off. The biomass continued to increase until the end of the experimental period (6 d). Maximum glucosyltransferase activity was also observed at day 5 and decreased thereafter. The results indicate that the enhanced curdlan production by Tween 80 is highly correlated with glucosyltransferase activity.     

Product enhancement and recovery from transformed root cultures of Nicotiana glauca

Transformed roots of Nicotiana glauce synthesize the alkaloids nicotine and anabasine at levels reflecting the parent plants. Media composition, strength, and pH were evaluated with respect to biomass yield and productivity. Full-strength Gamborg's B5 medium proved the best for biomass yield while half-strength, or low-salt, medium enhanced alkaloid accumulation. A detailed investigation of media nitrate levels demonstrated how these may be manipulated to promote growth and intracellular or extracellular alkaloid levels. High nitrate concentrations were found to significantly enhance media alkaloid levels at the end of the growth phase. Media pH is also important, although transformed roots will grow in Gamborg's B5 medium between pH 3 and 9, root biomass is favored by an increase in medium alkalinity, while alkaloid release is encouraged by mildly acidic pH.Transformed roots release a proportion of their secondary metabolites into the growth medium. By continually removing root products, any feedback inhibition on enzymatic reactions is reduced, as are the toxic effects resulting from product accumulation. In this article we describe the use of Amberlite resins (XAD-2 and XAD-4) to enhance alkaloid levels (nicotine and anabasine) of hairy root cultures of Nicotiana glauca by a factor of 10 with no adverse effect on root growth. The performance of the Amberlite columns was subsequently investigated with respect to alkaloid adsorption and desorption, including an evaluation of the effects of pH and loading capacity. The resins also adsorb media constituents which are identified and quantified as part of this work. Resulting nutritional stresses are thought to be partly responsible for enhancing secondary metabolism at the expense of biomass yield. However, the net effects of using Amberlite resins as a means of product removal significantly increases the overall product yield and the extent to which products are released into the growth medium.     

Physiological Aspects of Biosynthesis of Lignin Peroxidases by Phanerochaete chrysosporium

Methods based on UV-visible diffuse reflectance spectroscopy were used to study the physiological aspects of lignin-peroxidase biosynthesis by Phanerochaete chrysosporium. Here we introduce the use of cytochrome aa₃ as an indicator of active fungal biomass and of its redox state to calculate the oxygen mass transport coefficient between the growth medium and the fungal cell interior. When lignin peroxidase biosynthesis was enhanced by the addition of Tween 80 or Tween 20 to the growth medium, a higher proportion of reduced cytochrome aa₃ and a higher oxygen diffusion barrier were observed compared with control cultures. In cultures supplemented with Tween 80 or Tween 20, a higher oxygen mass transport coefficient between the growth medium and the interior of the fungal cell was also found. The beginning of the lignin peroxidase activity in these cultures was found to coincide with a temporary cessation in the dry biomass increase and a reduction in the relative active-biomass concentration. During the lignin peroxidase activity, a decrease in the intracellular pH and an increase in the growth medium pH were determined in cultures supplemented with Tween 80.