Biosynthesis of anthraquinone derivatives in a Sesamum indicum hairy root culture

In order to investigate the intermediacy of 2-(4-methylpent-3-en-1-yl)anthraquinone (MPAQ), a possible intermediate for the biosynthesis of anthraquinone derivatives in sesame (Sesamum indicum), ²H-labeled MPAQ was administered to a hairy root culture of S. indicum. Efficient conversion of fed MPAQ to 2-[(Z)-4-methylpenta-1,3-dien-1-yl]anthraquinone ((Z)-MPDEAQ) was observed. Furthermore, administration experiment with ²H-labeled 2-geranyl-1,4-naphthohydroquinone, another possible intermediate, showed that it was converted to MPAQ and (Z)-MPDEAQ. The results clearly demonstrated that these substrates are the actual precursors for the production of (Z)-MPDEAQ. In contrast, neither MPAQ nor 2-geranyl-1,4-naphthohydroquinone was converted to anthrasesamone B and 2,3-epoxyanthrasesamone B, other anthraquinone derivatives in the hairy roots, suggesting that these substrates may not be the common precursors in the biosynthesis of anthraquinone derivatives.     

2-Geranyl-1,4-naphthoquinone, a possible intermediate of anthraquinones in a Sesamum indicum hairy root culture

2-Geranyl-1,4-naphthoquinone was isolated from the hairy root culture of Sesamum indicum. The structure was determined to be 2-[(E)-3,7-dimethylocta-2,6-dienyl]-1,4-naphthoquinone on the basis of spectroscopic evidence and chemical synthesis. The production of anthrasesamones A, B and C by the hairy root culture was also confirmed for the first time.     

Isolation and photoisomerization of a new anthraquinone from hairy root cultures of Sesamum indicum

An unstable anthraquinone was isolated from hairy root cultures of Sesamum indicum after preventing light throughout all experimental procedures. The structure of the (Z)-isomer of a previously isolated anthraquinone was determined to be 2-[(Z)-4-methylpenta-1,3-dien-1-yl]anthraquinone by spectroscopic methods. This compound was readily isomerized to the known (E)-isomer under light.     

Effect of biotic and abiotic elicitors on production of betulin and betulinic acid in the hairy root culture of Betula pendula Roth

Abstract

Betulin (B) and betulinic acid (BA) are two triterpenoids with a wide range of biological and medicinal activities in different organs of Betula pendula. This research aimed to increase the accumulation of B and BA in the hairy root culture of B. pendula by seven biotic and abiotic elicitors. Hairy root was induced in the stem’s inner bark of B. pendula using the C58C1 strain in the WPM (Woody Plant Medium). The effects of different concentrations of elicitors and different time of root harvest in hairy root culture of B. pendula showed that highest level of growth index (GI), B, and BA was acquired in treated hairy roots with chitosan (CTS), chlorocholine chloride (CCC) and chitosan nano-fiber (CTS NF). Highest GI of B. pendula hairy roots was 13 that was obtained in the roots treated with CTS 150?mg l^?1 on the 8th day. The highest content of BA was 1.3?mg g^?1 DW after treatment with 1?mg l^?1CCC on the 4th and 6th days and 200?mg l^?1CTS NF on the 10th day. The highest B content (0.94?mg g^?1DW) was obtained in the treated hairy root by 2?mg l^?1 CCC after 4 and 6?days.     

An unusual root tip formation in hairy root culture of Hyoscyamus muticus

Summary Hairy root cultures of Hyoscyamus muticus were established using Agrobacterium rhizogenes ATCC 15834. In one out of 8 clones established, an unusual root tip formation was observed after transfer of cultures from half-strength Murashige and Skoog (1962) to White's medium (1939). This phenomenon was associated with the production of a fine brownish cell suspension culture. Hairy root development resumed after transfer of the root tips from White to half-strength Murashige and Skoog medium. After plating the isolated brownish cells on hormone-free half-strength Murashige and Skoog or White solid medium, callus proliferation was observed, and then redifferentiation of hairy roots occurred. The polymerase chain reaction analysis of the H. muticus hairy root (clone Z2) revealed that only the tl region of the T-DNA was integrated. The growth and the production of five tropane alkaloids by this clone were examined.Abbreviations PCR Polymerase Chain Reaction - MS medium Murashige and Skoog Medium - 1/2 MS medium half-strength MS medium - WP medium Woody Plant medium - RC medium Root Culture medium - WH medium White medium - HPLC High Performance Liquid Chromatography - wt. weight     

Diterpenoid production in hairy root culture of Salvia sclarea L

Growth and diterpenoid accumulation (salvipisone, ferruginol, aethiopinone and 1-oxoaethiopinone) during the growth cycle of a Salvia sclarea hairy root culture are described. The roots transformed by Agrobacterium rhizogenes (LBA 9402) were cultured in half-strength B5 liquid medium supplemented with 30 g L(-1) sucrose under light (16 h/8 h light/dark). A culture period of 30 days was optimal for both biomass and diterpenoid production. The total content of four diterpenoids in the hairy roots [(27.3 +/- 0.6) mg g(-1) dry weight] was higher than that of roots of field-grown S. sclarea plants [(3.15 +/- 0.15) mg g(-1) dry weight]. In transformed roots, aethiopinone was the main diterpenoid, whereas the principal diterpenoid of natural roots was salvipisone.     

Micropropagation and hairy root culture of Ophiorrhiza alata Craib for camptothecin production

An efficient system was developed for the in vitro micropropagation and hairy root culture of Ophiorrhiza alata Craib for camptothecin (CPT) production. Shoot multiplication on leaf and node explants from germinated seeds of O. alata was successful on half-strength Murashige and Skoog medium supplemented with varying amounts of kinetin and α-naphthaleneacetic acid. Node explants grown in vitro were successfully infected by Agrobacterium rhizogenes TISTR 1450 for the establishment of hairy root culture. The amount of CPT in various parts of O. alata was analyzed by HPLC. The accumulation of CPT in transformed hairy roots was twice that in soil-grown plants (785 ± 52 and 388 ± 32 μg/g dry wt, respectively). In the presence of a polystyrene resin (Diaion HP-20) that absorbed CPT, the CPT content in the culture media increased sevenfold compared with controls (1,036 and 151 μg per 250 ml medium, respectively). These results enable the feasible production of CPT of O. alata by means of a cell culture strategy. These measures can help safeguard the plant from extinction.     

Effect of calcium chloride on heavy metal induced alteration in growth and nitrate assimilation of Sesamum indicum seedlings

In the early growth phase of Sesamum indicum cv. PB-1, the decrease in fresh and dry mass was higher with 1.0 mM Cd²⁺ than with the same level of Pb²⁺ and Cu²⁺. Recovery from the metal stress was considerable in the root fresh weight and almost completely in the root dry weight when 10.0 mM (1.9 EC), calcium chloride was supplied to the growing seedlings along with the metal salts in various combinations. Accumulation of Pb²⁺, Cd²⁺ and Cu²⁺ was differential to the metals and the plant parts when supplied without or with 10.0 mM calcium chloride. The order of endogenous metal accumulation was Cu²⁺Cd²⁺Pb²⁺ and roots accumulated more metal than the leaves in the absence, as well as in the presence, of calcium chloride. Calcium chloride could recover loss of in vivo NRA in roots caused by either of the metal combinations, whereas the salt could recover the loss in leaf NRA caused only by Pb²⁺Cd²⁺ (1.0 mM each). Response of root and leaf NRA was on the other hand, different when the enzyme was assayed directly using an in vitro assay method, and the salt accelerated the loss in enzyme activity drastically. The organic-N content of root and leaf was, however, increased significantly (p < 0.001) with calcium chloride alone and with the metals supplied in various combinations. Our data indicate that instead of a high endogenous accumulation of Cu²⁺, Cd²⁺ and Pb²⁺ in roots and leaves the metal toxicity is recovered to a great extent in the presence of 10.0 mM calcium chloride in the root environment regarding growth and nitrate reduction of the roots and leaves of young sesame seedlings.     

Continuous production of glycosides by a bioreactor using ginseng hairy root culture

Ginseng (Panax ginseng) hairy-root culture, established by transformation with the Ri plasmid of Agrobacterium rhizogenes, had a higher potential to biotransform (RS)-2-phenylpropionic acid (PPA) to (RS)-2-phenylpropionyl -d-glucopyranoside (1) (71% conversion ratio), (2RS)-2-O-(2-phenylpropionyl)-d-glucose (2) (8%), (2S)-2-phenylpropionyl 6-O--d-xylopyranosyl--d-glucopyranoside (3) (10%) and a myo-inositol ester of (R)-2-phenylpropionic acid (4) (5%). Moreover, the hairy root excreted about a half of the conversion products, 46.8%. The continuous glycosylation of PPA was carried out using a bioreactor with ginseng hairy root, and the continuous long-term reaction for 2 months was successfully made at a high conversion ratio, 30% or more on average.This work is Part 84 in the series Studies on Plant Tissue Culture. For Part 83, see Asaka et al. (1993) Correspondence to: T. Furuya     

Optimization of the sucrose and ion concentrations for saikosaponin production in hairy root culture ofBupleurum falcatum

Saikosaponin productivity was examined in aBupleurum falcatum L. BFHR2 hairy root culture in response to changes in the sucrose content (2≈8%), nitrogen content (0≈250 mM NH₄NO₃), phosphate content (0≈12 mM NaH₂PO₄), and the potassium content (0≈87.2 mM KCl) of the culture media. We found that the conditions for maximal saikosaponin production differed from those for optimal root growth. Highest saikosaponin yield was achieved for 8% sucrose, 62 mM NH₄NO₃, 1.2 mM NaH₂PO₄, and 0.5 mM KCl.     

Statistical medium optimization for enhanced azadirachtin production in hairy root culture of Azadirachta indica

Azadirachtin, a well-known biopesticide, is a secondary metabolite extracted from the seeds of Azadirachta indica. In the present study, azadirachtin was produced in hairy roots of A. indica, generated by Agrobacterium rhizogenes-mediated transformation of leaf explants. Liquid cultures of A. indica hairy roots were developed with a liquid-to-flask volume ratio of 0.15. The kinetics of growth and azadirachtin production were established in a basal plant growth medium containing MS medium major and minor salts, Gamborg’s medium vitamins, and 30 g l⁻¹ sucrose. The highest azadirachtin accumulation in the hairy roots (up to 3.3 mg g⁻¹) and azadirachtin production (∼44 mg l⁻¹) was obtained on Day 25 of the growth cycle, with a biomass production of 13.3 g l⁻¹ dry weight. To enhance the production of azadirachtin, a Plackett–Burman experimental design protocol was used to identify key medium nutrients and concentrations to support high root biomass production and azadirachtin accumulation in hairy roots. The optimal nutrients and concentrations were as follows: 40 g l⁻¹ sucrose, 0.19 g l⁻¹ potassium dihydrogen phosphate, 3.1 g l⁻¹ potassium nitrate, and 0.41 g l⁻¹ magnesium sulfate. Concentrations were determined by a central composite design protocol and verified in shake-flask cultivation. The optimized medium composition yielded a root biomass production of 14.2 g l⁻¹ and azadirachtin accumulation of 5.2 mg g⁻¹, which was equivalent to an overall azadirachtin production of 73.84 mg l⁻¹, 68% more than that obtained under non-optimized conditions.     

Continuous production of scopolamine by a culture of Duboisia leichhardtii hairy root clone in a bioreactor system

The scopolamine-releasing hairy root clone DL47-1 of Duboisia leichhardtii was cultured in an Amberlite XAD-2 column-combined bioreactor system for continuous production of scopolamine. The medium used was continuously exchanged during culture to maintain the electrical conductivity of the medium constant. After culturing the hairy roots in the system for 11 weeks, 0.5 g/l of scopolamine was obtained in the column. When the roots were cultures in the reactor system containing polyurethane foam or stainless-steel mesh to support the hairy roots, scopolamine recovery was increased. Thereafter, a two-stage culture, the first stage in the medium for hairy root growth and the second stage in the medium for scopolamine release, was carried out in this system by using a turbine-blade reactor with stainless-steel mesh as a support. Under these conditions, 1.3 g/l of scopolamine was recovered during 11 weeks of culture in the medium for scopolamine release. This bioreactor system seems applicable for the production of various plant metabolites by cultures of hairy roots. Correspondence to: T. Muranaka     

Sesquiterpene lactones in a hairy root culture of Cichorium intybus

A transformed root culture of Cichorium intybus L. (Asteraceae) was found to produce sesquiterpene lactones of guaiane and germacrane type. Lactucopicrin, 8-desoxylactucin and three sesquiterpene lactone glycosides: crepidiaside B, sonchuside A and ixerisoside D were isolated from the roots. The yield of 8-desoxylactucin reached 0.03 g l(-1) at the early stationary phase of the culture.     

Enhanced production of valerenic acid in hairy root culture of Valeriana officinalis by elicitation

Valerenic acid (VA) is a pharmacologically-active sesquiterpene found in valerian (Valeriana officinalis L., Valerianaceae) roots and rhizomes. The plant produces only small amounts of this metabolite naturally. So, induction of hairy roots as well as elicitation can be useful to increase its commercial production. In this study, Wild-type strain ‘A13’ of Agrobacterium rhizogenes was used to induce hairy roots in valerian. The influence of three different elicitors including Fusarium graminearum extract (FE), methyl jasmonate (MJ) and salicylic acid (SA) on VA production in the selected hairy root line ‘LeVa-C4’ was also investigated. The 23-day-old cultures were treated with different concentrations of the elicitors at exposure time of 3 and 7 days. FE (1%) and MJ (100 µM L^?1) highly promoted VA production at 7 days after elicitation, to a level of 12.31- and 6-fold higher than that of non-elicited controls, respectively, and FE did not exert any negative effects on biomass yield of hairy root. SA did not significantly increase the production of VA. This is the first time study to assess the elicitation of hairy root cultures to promote VA biosynthesis in valerian and the resulting experiments demonstrated that F. graminearum extract and MJ were indeed a potent inducer of VA biosynthesis.     

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.     

Effect of fungal elicitor on thiophene production in hairy root cultures of Tagetes patula

Summary The effect of fungal elicitor, derived from mycelial extracts of Fusarium conglutinans, on thiophene production in hairy-root cultures of Tagetes patula was studied. Various concentrations of elicitor were added to the culture media. Time-course experiments were carried out using a defined concentration of elicitor. Thiophene production increased with the addition of elicitor. The major thiophenes produced were 5-(4-aceoxy-1-butenyl)-2,2-bithiophene and 5-(buten-1-enyl)-2,2'bithiophene.On leave from Department of Biological Sciences, R. J. College, Bombay 86, India Offprint requests to: M. A. Hjortso     

Enhanced glucotropaeolin production in hairy root cultures of Tropaeolum majus L. by combining elicitation and precursor feeding

We have studied the effects of precursor amino acids (phenylalanine and cystein), elicitors (salicylic and acetylsalicylic acids, methyl jasmonate, β-aminobutyric acid, yeast extract), PAL-inhibitor (1-amino-2-phenylethylphosphonic acid) applied alone or in combination on glucotropaeolin production and myrosinase activity in hairy root cultures of Tropaeolum majus. Short, 24-h treatment, and subsequent transfer of hairy roots to the fresh medium enabled avoiding detrimental effect of studied stimulators on biomass growth. In control cultures the highest glucotropaeolin content, 58.1 ± 6.7 mg g⁻¹ DW, was detected on the 3rd day after transfer of the roots to the fresh medium but glucotropaeolin yield (mg 100 ml^–1 culture volume) had been increasing until the 9th day after transfer as a result of continuous biomass growth. Glucotropaeolin content and yield were 2-fold enhanced after treatment with precursor amino acids or PAL-inhibitor alone, but their combination additively led to 4-fold increase in glucotropaeolin production. Among the studied elicitors acetylsalicylic acid induced the highest, 3-fold increase in glucotropaeolin production, it also enhanced myrosinase activity, but to a smaller extend (by about 50%). Acetylsalicylic acid also potentiated induced by precursors, PAL-inhibitor, methyl jasmonate and yeast extract production of glucotropaeolin. The highest, 4.8-fold increase in glucotropaeolin production was found after combined acetylsalicylic acid and precursors treatment. Additive effect of acetylsalicylic acid-combined treatment on myrosinase activity was not detected. The obtained results indicate that amino acid precursors, phenylalanine and cystein, availability may be a limiting factor in the process of stimulation of glucotropaeolin production in T. majus hairy root cultures.