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.     

Biosynthetic origin of 2-geranyl-1,4-naphthoquinone and its related anthraquinone in a Sesamum indicum hairy root culture

In order to clarify the biosynthetic origin of 2-geranyl-1,4-naphthoquinone and its biogenetically related anthraquinone, which are possible intermediates of anthrasesamones, [1-¹³C]glucose was administered to a hairy root culture of Sesamum indicum. The labeling patterns of these quinone derivatives indicated that the naphthoquinone ring and geranyl side-chain of geranylnaphthoquinone were respectively biosynthesized through the shikimate and methylerythritol phosphate pathways, and that these quinone derivatives have the same biosynthetic origin.     

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.     

Structures of Naphthoquinones Produced by the Fungus,Fusarium sp., and Their Biological Activity toward Pollen Germination

From an unidentified Fusarium sp., eight substances possessing a 1,4-naphthoquinone skeleton were isolated (F2~F9) and their structures were elucidated from the physical and chemical evidence. The effect of these compounds together with 1,4-naphthoquinone, 5-hydroxy-1,4-naphthoquinone and 5,8-dihydroxy-1,4-naphthoquinone on the pollen germination of Pinus thunbergii Pari was examined. 5,8-Dihydroxy-1,4-naphthoquinone showed marked inhibitory activity.     

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     

Transformation of <Emphasis Type="Italic">Saussurea medusa</Emphasis> for hairy roots and jaceosidin production

Axenically grown Saussurea medusa plantlets were inoculated with four Agrobacterium rhizogenes strains, and hairy root lines were established with A. rhizogenes strain R1601 in N6 medium. PCR and Southern hybridization confirmed integration of the T-DNA fragment of the Ri plasmid from A. rhizogenes into the genome of S. medusa hairy roots. In N6 medium, maximum biomass of the hairy root cultures was achieved [8 g (dry weight) per liter; growth ratio 35-fold] after 21 days of culture. The amount of jaceosidin extracted from the hairy root cultures was 46 mg/l (production ratio of 37-fold) after 27 days of culture. The maximum jaceosidin content obtained using N6 medium was higher than that obtained with Modified White, MS or B5 medium. In N6 medium, the tip segments were more efficient for hairy root growth and jaceosidin production than the middle and basal regions of the root.Abbreviations AS Acetosyringone - BA Benzyladenine - cef Cefotaxime sodium - DW Dry weight - FW Fresh weight - HPLC High-performance liquid chromatography - IAA Indole-3-acetic acid - km Kanamycin - NAA -Naphthaleneacetic acid - SDS Sodium dodecyl sulfate     

Statistical design of the effect of inoculum conditions on growth of hairy root cultures of Catharanthus roseus

Summary The factors in the inoculum conditions that influence the growth rate of hairy roots of Catharanthus roseus in liquid culture were investigated in a randomized two-level, three-factor statistical design. The three variables were the number of root tips, the length of root tips, and the initial volume of media. The experiments and analysis demonstrated the effect of inoculum conditions on hairy root growth, with the length of hairy root tips being the dominant variable without any clonal variability. The best growth rates (doubling times) were obtained with an inoculum of 5 hairy root tips, each 35–40 mm long in 50 mL media: 3.7 and 3.1 days for C. roseus hairy root clones LBE-6-1 and LBE-4-2, respectively.     

Traits of Panax ginseng hairy roots after cold storage and cryopreservation

Summary Panax ginseng hairy root cultures were established by infecting petiole segments with Agrobacterium rhizogenes strain 15834. Hairy root segments including root tips placed onto phytohormone-free 1/2 Murashige and Skoog solid medium and stored at 4 °C in the dark for 4 months, resumed elongation when the temperature was raised to 25 °C in the dark. For cryopreservation, a vitrification method was applied. Root tips precultured with 0.1 mg/l 2,4-D for 3 days and dehydrated with PVS2 solution for 8 minutes prior to immersion into liquid nitrogen had a survival rate of 60 % and could regenerate. The hairy roots regenerated from cryopreserved root tips grew well and showed the same ginsenoside productivity and patterns as those of the control hairy roots cultured continuously at 25 °C. The conservation of T-DNAs in the regenerated hairy roots was proved by PCR analysis.Abbreviations 1/2 MS a half strength Murashige and Skoog (1962) - B5 Gamborg B5 (Gamborg et al. 1968) - WP woody plant (Lloyd and McCown 1980) - RC root culture (Thomas and Davey 1982) - RCI root culture medium containing 100 mg/l myoinositol - HF phytohormone-free - IAA indole-3-acetic acid - IBA indole-3-butyric acid - 2,4-D 2,4-dichlorophenoxyacetic acid - TIBA 2,3,5-triiodobenzoic acid - PCR polymerase chain reaction - PVS2 plant vitrification solution 2 (Sakai et al., 1990) - FDA fluorecein diacetate     

Cytotoxicity of new polyfluorinated 1,4-naphtoquinones with diverse substituents in the quinone moiety

Fluorinated derivatives of 1,4-naphthoquinones are highly potent inhibitors of Cdc25A and Cdc25B phosphatases and growth of tumor cells. Eight new derivatives of polyfluoro-1,4-naphthoquinone were synthesized and their cytotoxicity in human myeloma, human mammary adenocarcinoma, mouse fibroblasts and primary mouse fibroblast cells as well as their mutagenic and antioxidant properties in a Salmonella tester strain were studied. The efficiency of suppressing the growth of two lines of tumor cells decreased in the order: 2-(2-hydroxy-ethylamino)-3,5,6,7,8-pentafluoro-1,4-naphthoquinone (1), 2,3-dimethoxy-5,6,7,8-tetrafluoro-1,4-naphthoquinone (2), 2-[2-hydroxyethyl(methyl)amino]-3,5,6,7,8-pentafluoro-1,4-naphthoquinone (3), 2-morpholino-3,5,6,7,8-pentafluoro-1,4-naphthoquinone (4), 2-[bis-(2-hydroxyethyl)amino]-3,5,6,7,8-pentafluoro-1,4-naphthoquinone (5), 2-[(2-hydroxy)ethylsulfanyl)]-5,6,7,8-tetrafluoro-1,4-naphthoquinone (6), 2-methoxy-3,5,6,7,8-pentafluoro-1,4-naphthoquinone (7), and 1,4-dioxo-3-(1-pyridinio)-1,4-dihydro-5,6,7,8-tetrafluoronaphthalene-2-olate (8). Taking into account these data together with the better cytotoxic effect against cancer cells as compared with normal mammalian cells, protecting of bacterial cells from spontaneous and H₂O₂-dependent mutagenesis, and lower general toxicity of the compounds towards different cells, one can propose that compounds 3-5 may be considered as useful potential inhibitors of growth of tumor cells.     

金铁锁毛状根构型对其生长的影响

毛状根的构型是影响其生长速度和生物量积累的重要因素,为了规模化培养金铁锁毛状根,进一步解决金铁锁资源短缺问题,该研究以金铁锁毛状根为材料,通过改变培养基类型、碳源及碳源浓度,观察和分析了毛状根的生长状态,找出影响毛状根构型的因素。结果表明:最适合金铁锁毛状根生长的培养基为B5+蔗糖30 g·L~(-1),金铁锁毛状根主根长而粗壮,一级、二级侧根生长量大,根系表面积较大,生长效果最佳。经液体悬浮培养验证,测定毛状根的生长量,与在固体培养基培养的毛状根生长状态基本一致。通过该项研究,优化了培养基中营养成分的配比,实现了金铁锁毛状根的快速生长和生物量的积累。     

Scopolamine release into media by Duboisia leichhardtii hairy root clones

Summary The hairy root clones of Duboisia leichhardtii were found to release scopolamine into the medium. Among media examined, a modified Heller's medium that had 37 mm nitrate and no ammonium was suitable for scopolamine release. Scopolamine in the medium was efficiently recovered by the use of an Amberlite XAD-2 column. A reactor system consisting of a 2-1 airlift reactor and a 25-ml column packed with Amberlite XAD-2 was constructed for production of scopolamine by the culture of the hairy root clone. The culture medium was passed through the column and the eluent from the column was back into the reactor continuously by using a low-pressure pump. When the hairy root clone DL47-1 was cultured in the reactor, 245 mg/l of scopolamine was released into the medium during 6 weeks and 97% scopolamine in the medium was recovered by the column. The scopolamine production was about five times higher in the column-combined reactor than in the reactor without the column. Scopolamine was recovered as the hydrobromide salt with more than 90% purity. Correspondence to: T. Muranaka     

A model for a bioconversion system with the promoter of the parAt gene,which confers a high level of expression of a transgene in hairy roots

The promoter of the protoplast auxin-regulated (parAt) gene of tobacco, which is expressed throughout the tissues of hairy roots, can be useful for developing a bioconversion system with hairy roots. The parAt gene is shown to be expressed in roots of seedlings and in those of mature tobacco plants. The 5-upstream region of parAt was fused to the coding sequence of the ß-d-glucuronidase (GUS) gene to generate the parAt-GUS fusion gene, which was introduced into the binary vector for Agrobacterium. Hairy roots that carried the fusion gene were obtained (parAt-GUS/hairy root) by infecting tobacco plants with A. rhizogenes carrying the fusion gene in the binary vector. Biochemical analysis with 4-methylumbelliferyl ß-d-glucuronide (MUG), a substrate for GUS, showed that the level of GUS activity was tenfold higher than that of hairy roots carrying the reporter GUS gene, which is linked to the cauliflower mosaic virus 35S RNA promoter (35S-GUS/hairy root). We also examined the rate of conversion of MUG to 4-methylumbel-liferone (MU) by hairy roots when MUG was added to the culture medium of the parAt-GUS/hairy roots. The hairy roots converted MUG to MU at more than ten times as high efficiency as the 35S-GUS/hairy roots. In addition to tobacco, the parAt-GUS gene was similarly expressed in hairy roots from Atropa and Arabidopsis. These results suggest that the promoter of the parAt gene is a useful tool for conversion of various metabolites by hairy root cultures. Correspondence to: Y. Machida     

Insecticidal activities of a Diospyros kaki root-isolated constituent and its derivatives against Nilaparvata lugens and Laodelphax striatellus

Diospyros kaki root-derived materials were examined for insecticidal properties against Nilaparvata lugens and Laodelphax striatellus. Based on the LD₅₀ values, the chloroform fraction of D. kaki extracts showed the most activity against N. lugens (3.78 μg/female) and L. striatellus (7.32 μg/female). The active constituent of the chloroform fraction was isolated by various chromatographic methods and was identified as 5-hydroxy-2-methyl-1,4-naphthoquinone by spectroscopic analyses. To establish the structure–activity relationships, the insecticidal effects of 5-hydroxy-2-methyl-1,4-naphthoquinone and its derivatives against N. lugens and L. striatellus were determined using micro-topical application bioassays. On the basis of LD₅₀ values, 5-hydroxy-1,4-naphthoquinone was the most effective against N. lugens (0.072 μg/female) and L. striatellus (0.183 μg/female). 2-Bromo-1,4-naphthoquinone, 2-hydroxy-1,4-naphthoquinone, and 5-hydroxy-2-methyl-1,4-naphthoquinone also had potent insecticidal activities against N. lugens and L. striatellus. In contrast, no insecticidal activity was observed with 2-methoxy-1,4-naphthoquinone or 2-methyl-1,4-naphthoquinone. These results indicate that the functional group (bromo- and hydroxyl-) at the C-2 position of the 1,4-naphthoquinone skeleton and the change in position of the hydroxyl group play important roles in insecticidal activity. Therefore, naturally occurring D. kaki root-derived 5-hydroxy-2-methyl-1,4-naphthoquinone and its derivatives may be suitable as insecticides.     

Hernandulcin in hairy root cultures of Lippia dulcis

The hairy root culture of Lippia dulcis Trev., Verbenaceae, was established by transformation with Agrobacterium rhizogenes A4. The transformed roots grew well in Murashige and Skoog medium containing 2% sucrose. The roots turned light green when they were cultured under 16 h/day light. The green hairy roots produced the sweet sesquiterpene hernandulcin (ca. 0.25 mg/g dry wt) together with 20 other mono- and sesquiterpenes, while no terpenes were detected in the nontransformed root cultures. The growth and hernandulcin production in the hairy root cultures were influenced by the addition of auxins to the medium. The addition of a low concentration of chitosan (0.2 – 10.0 mg / l) enhanced the production of hernandulcin 5-fold.Abbreviations Cht chitosan - IAA indole-3-acetic acid - NAA 1-naphthaleneacetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - MS Murashige and Skoog(1962)     

Eugenol from normal and transformed root cultures of Coluria geoides

Transformed root cultures of Coluria geoides Ledeb. were established with the use of Agrobacterium rhizogenes LBA 9402. Both normal and transformed root cultures were investigated for their growth and yield of eugenol. Normal roots were grown in B5 medium-supplemented with 0.2 mg l^-1 of kinetin and 0.2 mg l^-1 of 1-naphthaleneacetic acid (NAA). Hairy roots grew well in hormone-free B5 medium. Both hairy roots and normal roots produced glycosidic bound eugenol. as with the roots of intact plants, eugenol was the main component of the total essential oils obtained from hairy root and normal root cultures. The yield of eugenol from normal roots was 0.1–0.25% of the dry wt. and depended on the development stage of the culture. Yield of eugenol from hairy roots was 0.08–0.1% of the dry wt. NAA modified the hairy root morphology and influenced the yield of eugenol.Abbreviations NAA 1-naphthaleneacetic acid     

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     

Optimization of induction and culture conditions and tropane alkaloid production in hairy roots of <Emphasis Type="Italic">Anisodus acutangulus</Emphasis>

In this study, an efficient transformation system for the medicinal plant Anisodus acutangulus was successfully developed and optimized using Agrobacterium rhizogenes. Three bacterial strains, A4, R1601, and modified C58C1 and three explant types, leaf blade, petiole, and stem, were examined. The highest transformation efficiency of 94.44% was achieved using strain C58C1 with stem explants. Over 20 independent hairy root lines were successfully established with strain C58C1 using stem explants, all of which contained the ro/B and ro/C genes as confirmed by polymerase chain reaction (PCR). Out of four media compositions, the liquid 1/2 MS medium was found the most suitable for hairy root growth. The maximum biomass of one hairy root line increased up to 80 times in liquid 1/2 MS medium after a 30 day culture period. Different hairy root lines displayed a varied capacity for tropane alkaloid production and the best hairy root line (T4) from the C58C1-stem combination produced up to 10.21 mg/g (dw) of hyoscyamine, which was about 1.5-fold higher than in the wild type plants. To our knowledge, this is the first report to demonstrate the production of tropane alkaloids in hairy roots of A. acutangulus.     

Cryopreservation and post freeze molecular and biosynthetic stability in transformed roots of Beta vulgaris and Nicotiana rustica

Crypopreservation methods were firstly developed for root-tips from hairy root cultures of Beta vulgaris, established after transformation by Agrobacterium rhizogenes. The effects of culture age, pre-growth, cryoprotection, freezing rate and post-freeze culture conditions were determined. The resulting freezing protocol was then used to cryopreserve transformed root cultures of Nicotiana rustica. Both species were viable after freezing (ca. 80%), according to fluorescein diacetate vital staining. However, on average the regeneration of proliferating roots from surviving root-tips was low (<20%). Growth rates, secondary metabolite production and T-DNA structure of a number of hairy root lines were examined and found to be unchanged after cryopreservation.Abbreviations DMSO dimethyl sulphoxide - 2,4-D dichlorophenoxyacetic acid - FDA fluoroscein diacetate - IAA indole-3-acetic acid - NAA -napthalene acetic acid     

Modulation of artemisinin biosynthesis by elicitors,inhibitor, and precursor in hairy root cultures of Artemisia annua L.

Artemisinin is frequently used in the artemisinin-based combination therapy to cure drug-resistant malaria in Asian subcontinent and large swath of Africa. The hairy root system, using the Agrobacterium rhizogenes LBA 9402 strain to enhance the production of artemisinin in Artemisia annua L., is developed in our laboratory. The transgenic nature of hairy root lines and the copy number of transgene (rol B) were confirmed using polymerase chain reaction and Southern Blot analyses, respectively. The effect of different concentrations of methyl jasmonate (MeJA), fungal elicitors (Alternaria alternate, Curvularia limata, Fusarium solani, and Piriformospora indica), farnesyl pyrophosphate, and miconazole on artemisinin production in hairy root cultures were evaluated. Among all the factors used individually for their effect on artemisinin production in hairy root culture system, the maximum enhancement was achieved with P. indica (1.97 times). Increment of 2.44 times in artemisinin concentration by this system was, however, obtained by combined addition of MeJA and cell homogenate of P. indica in the culture medium. The effects of these factors on artemisinin production were positively correlated with regulatory genes of MVA, MEP, and artemisinin biosynthetic pathways, viz. hmgr, ads, cyp71av1, aldh1, dxs, dxr, and dbr2 in hairy root cultures of A. annua L.