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     

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     

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     

Genetic transformation of <Emphasis Type="Italic">Harpagophytum procumbens</Emphasis> by <Emphasis Type="Italic">Agrobacterium rhizogenes</Emphasis>: iridoid and phenylethanoid glycoside accumulation in hairy root cultures

A genetic transformation method using Agrobacterium rhizogenes was developed for Harpagophytum procumbens. The influence of three factors on hairy root formation was tested: bacterial strains (A4 and ATCC 15834), various types of explants and acetosyringone (AS) (200 μM). The highest frequency of transformation (over 50% of explants forming roots at the infected sites after 6 weeks of culture on Lloyd and McCown (WP) medium) was achieved using a combination of nodal stem explants and A. rhizogenes strain A4. The addition of 200 μM AS to root induction medium was found to enhance hairy root induction but its effect varied depending on bacterial strain and explant type. Three of the most vigorously growing hairy root clones of H. procumbens were chosen and analyzed for accumulation of iridoid and phenylethanoid glycosides. The transgenic nature of these root clones was confirmed by PCR amplification; they were positive for rolB and rolC genes. Harpagoside, verbascoside and isoverbascoside were identified by HPLC and LC–ESI-MS as the major compounds from all analyzed hairy root clones. The Hp-3 root clone showed the higher harpagoside content (0.32 mg g⁻¹ dry wt.) compared with other analyzed transformed and non-tuberized untransformed roots of H. procumbens. However, the level of the compound in the hairy root clone was lower than that detected in a sample of commercially available root tubers of H. procumbens. The Hp-3 root clone also produced high amounts of verbascoside and isoverbascoside (8.12 mg g⁻¹ dry wt. and 9.97 mg g⁻¹ dry wt., respectively) comparable to those found in root tubers.     

Development of a potent <Emphasis Type="Italic">in vitro</Emphasis> source of <Emphasis Type="Italic">Phylanthus amarus</Emphasis> roots with pronounced activity against surface antigen of the hepatitis B virus

Summary In vitro culture of hairy roots of Phyllanthus amarus induced by Agrobacterium rhizogenes was established. Their growth and ability for in vitro inactivation of hepatitis B virus surface antigen was studied and compared with adventitious roots grown in vitro. The selected hairy root clone HR-1 was capable of growing at a very fast rate, and an approximately 900-fold increase in weight of root biomass was achieved after 4 wk of culture in hormone-free quarter-strength liquid Murashige and Skoog medium with continuous agitation. Non-transformed roots cultured in the presence of 1.0 mg l⁻¹ (5.71 μM) indole-3-acetic acid increased by 330-fold. The immuno-inactive property of roots was maximal in the crude extract. The hairy roots were shown to possess 85% inhibition (in contrast to 15% in the control) in binding of hepatitis B surface antigen (HBsAg) to its antibody (anti-HBs) after 24 h of incubation with HbsAg-positive sera in vitro at 37°C. Out of three fractions selected on the basis of molecular weight components of the extract, the Fraction III containing comparatively lower molecular weight substances (≤3500) yielded the highest activity. The extract from non-transformed roots was found to possess similar efficiency (87% inhibition). The levels of activity in both types of in vitro-raised roots were higher than those of naturally occurring roots and leafy shoots. The ability of P. amarus hairy root cultures to yield high biomass with the anti-viral property at high levels may provide an alternative source of raw material for more detailed study in the field of pharmaceutical research.     

High forskolin production in hairy roots of Coleus forskohlii

Hairy roots of Coleus forskohlii were induced by infection with the Agrobacterium rhizogenes MAFF 03-01724 strain. Growth and forskolin production of two hairy root clones cultured in various liquid media were examined. Hairy root clone B9 grew well in woody plant liquid medium and showed a high forskolin yield (ca. 1.3 mg/ 100 ml flask) after 5 weeks of culture. The time course of growth and forskolin production of the clone B9 cultured in woody plant liquid medium was also examined. Rapid growth started at week 2 and continued until week 5. The highest forskolin yield (ca. 1.6 mg/100 ml flask) was obtained at week 5. Productivity was much higher than that previously reported. Received: 19 June 1997 / Revision received: 6 October 1997 / Accepted: 18 October 1997     

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     

Isolation and identification of littorine from hairy roots of Atropa belladonna

A hairy root clone (M8) of Atropa belladonna, producing high levels of tropane alkaloids, was established by transformation with Agrobacterium rhizogenes (MAFF 03-01724). Littorine, an intermediate of tropane alkaloids, was detected by high-performance liquid chromatography and gas chromatography-mass spectrometry in the alkaloid fraction of the hairy roots and identified by nuclear magnetic resonance analysis. Littorine was also detected in the non-transformed root culture of A. belladonna. Received: 18 March 1998 / Revision received: 15 June 1998 / Accepted: 3 August 1998     

Production of Tropane Alkaloids by Hairy Root Cultures of Scopolia japonica

Hairy root clones of Scopolia japonica were established by selection of adventitious roots formed on the root segments inoculated with Agrobacterium rhizogenes strain 15834. Twenty-nine isolated hairy root clones displayed various phenotypes characterized by growth rate, opine production and tropane alkaloid production. Of these, two highly alkaloid productive clones SI and S22 were examined for their growth rate and alkaloid productivity under various cultural conditions. When the most scopolamine-productive clone SI was cultured for 4 weeks at 25°C in the dark, the weight of the root tissue was increased by 40 times and the content of scopolamine reached a level of 0.5% on a dry weight basis in each optimum medium. On culture of the most hyoscyamine-productive clone S22 under the same conditions as with S1, the weight was increased by 102 times and the content of hyoscyamine was 1.3% on a dry weight basis in each optimum medium.     

Variation of alkaloid productivity among several clones of hairy roots and regenerated plants ofAtropa belladonna transformed withAgrobacterium rhizogenes 15834

Hairy root cultures ofAtropabelladonna were established by transformation withAgrobacterium rhizogenes 15834. Five clones of them were employed to study the production of hyoscyamine, the main constituent of the plant, together with other tropane alkaloids. The growth and alkaloid production of each clone were differently affected by basal liquid culture media tested. The transgenic plants regenerated from each clone of the hairy roots had different phenotypes and diverse alkaloid productivity both in the cultured condition and in productivitiy both in the cultured condition and in hydroponics.Abbreviations ANOVA analysis of variance - B5 medium Gamborg B5 medium - BA N⁶-benzyladenine - B.S. Balanced Solution - dw dry weight - EC electric conductivity - fw fresh weight - GC/MS gas chromatography-mass spectrometry - HPLC high performance liquid chromatography - MS medium Murashige and Skoog medium - NAA naphthalene-l-acetic acid - PCR polymerase chain reaction - SDS sodium dodecyl sulfate - TMS trimethylsilyl - WP medium Woody Plant medium     

Production of ribosome-inactivating protein from hairy root cultures of Luffa cylindrica (L.) Roem.

Transformed root lines of Luffa cylindrica (L.) Roem. (Cucurbitaceae) were established by inoculation of in vitro grown plantlets with wild type Agrobacterium rhizogenes strain 1855. Cloned lines of hairy roots were tested for the presence of ribosome-inactivating proteins; crude extracts inhibited protein synthesis in a reaction mixture based on rabbit reticulocyte lysate. Inhibitory activity increased during culture period, reaching a maximum value in the stationary phase. No activity could be detected in the culture medium, nor in extracts from callus and/or suspension cultures. A ribosome-inactivating protein having specific activity of 62,100 U mg protein^–1 and a molecular mass of 26–28,000 Da was purified to homogeneity. The protein showed N-glycosidase activity on rat liver ribosomes. The results demonstrate that hairy root cultures can be successfully utilized for the in vitro production of ribosome-inactivating proteins.Abbreviations BAP benzylaminopurine - 2,4D 2,4dichlorophenoxyacetic acid - HPLC high pressure liquid chromatography - MS Murashige and Skoog - NAA naphthaleneacetic acid - NCPPB National Collection of Phytopathogenic Bacteria - Ri root-inducing - RIP ribosome-inactivating protein - UV ultra-violet - YMB yeast mannitol broth     

Agrobacterium rhizogenes-mediated transformation of Picrorhiza kurroa Royle ex Benth.: establishment and selection of superior hairy root clone

A protocol for induction and establishment of Agrobacterium rhizogenes-mediated hairy root cultures of Picrorhiza kurroa was developed through optimization of the explant type and the most suitable bacterial strain. The infection of leaf explants with the LBA9402 strain resulted in the emergence of hairy roots at 66.7% relative transformation frequency. Nine independent, opine and TL-positive hairy root clones were studied for their growth and specific glycoside (i.e., kutkoside and picroside I) productivities at different growth phases. Biosynthetic potentials for the commercially desirable active constituents have been expressed by all the tested hairy root clones, although distinct inter-clonal variations could be noted in terms of their quantity. The yield potentials of the 14-P clone, both in terms of biomass as well as individual glycoside contents (i.e., kutkoside and picroside I), superseded that of all other hairy root clones along with the non-transformed, in vitro-grown control roots of P. kurroa. The present communication reports the first successful establishment, maintenance, growth and selection of superior hairy root clone of Picrorhiza kurroa with desired phyto-molecule production potential, which can serve as an effective substitute to its roots and thereby prevent the indiscriminate up-rooting and exploitation of this commercially important, endangered medicinal plant species. CIMAP Publication No.: 2007-28J     

Production and release of pigments by culture of transformed hairy root of red beet

Adventitious roots were induced from red beet (Beta vulgaris L. cv. Detroit dark red) by infecting the plant with a soil bacterium, Agrobacterium rhizogenes. Based on analysis of opines which are uniquely produced in transformed hairy roots, the established clone was proved to be a transformed hairy root. In a shake culture of the beet hairy root clone with a liquid medium, it was found that significant amounts of pigments, mainly betanin and vulgaxanthin-I, were released into the medium by the cessation of culture shaking (temporary limitation of oxygen supply). The hairy root cells were capable of propagation even after the cells were subjected to shaking cessation. Repeated-batch culture of the beet hairy root was performed with the cell growth phases for 9 or 10 d and with pigment leakage phases during shaking cessation for 2 d, and more than 20% of the total intracellular pigments were recovered from the culture broth at a culture time of 35 d. The released pigments were confirmed to be substantially identical to those extracted from the hairy root and original plant cells of red beet.     

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。     

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 interchangeable system of hairy root and cell suspension cultures ofCatharanthus roseus for indole alkaloid production

Hairy roots ofCatharanthus roseus obtained by co-cultivation of hypocotyl segments withAgrobacterium rhizogenes, and cultured in SH (Schenk and Hildebrandt) basal medium, formed two types of calli when subcultured in SH medium with 1 mg/1 -naphthaleneacetic acid and 0.1 mg/l kinetin. One of them, a compact callus, when re-subcultured in SH basal medium gave rise to hairy roots again. A rhizogenic cell suspension culture was established from this type of callus. When cultured in SH medium with growth regulators, the rhizogenic callus produced catharanthine at a level of 41% of the level in the initial hairy roots. Upon transfer to SH basal medium, regenerated hairy roots produced this alkaloid at the original level of 1.5 mg/g dry wt. Using this cell/hairy root interchange system a new management system for hairy root culture in bioreactors has been devised and examined involving production of biomass in the form of a cell suspension in medium supplemented with growth regulators, and catharanthine production by hairy roots regenerated from these cells in medium without growth regulators.Abbreviations NAA -naphthaleneacetic acid - SH Schenk and Hildebrandt - SHNK SH medium + 1 mg 1^–1 NAA + 0.1 mg 1^–1 kinetin     

Production of peroxidase with horseradish hairy root cells in a two step culture system

Horseradish hairy root cells transformed by a soil bacterium Agrobacterium rhizogenes had peroxidase (POD) activity comparable to that of the original plant root tubers. To enhance POD production by the hairy root culture, various additives to the medium were tested including casein hydrolysates and plant extracts. Polypepton addition had a significant effect on the growth and POD production; at low concentrations (below 1 g/l) the growth was stimulated, while at high concentrations (3–10 g/l), POD activity in the cells was enhanced in spite of a low growth rate. Therefore, the hairy roots were at first cultured in the medium with 1 g/l Polypepton, and then 5 g/l Polypepton was added to enhance intracellular POD activity. POD activity in this two step culture system was 5.4 times higher than that in conventional culture in Polypepton-free medium.     

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.     

Virulence of different Agrobacterium strains on hairy root formation of Hyoscyamus muticus

Hyoscyamus muticus accession was evaluated for its response to inoculation with Agrobacterium rhizogenes strains LBA9402, A4, 15834, and Agrobacterium tumefaciens strain C58 CI pRT GUS104. The Agrobacterium strain used for the transformation has a significant influence on the phenotype of the clone as well as on the growth rate and hyoscyamine production of these root culture clones. The most virulent strains were C58 CI pRT GUS104 and LBA9402. More roots were obtained on LSO medium than on B50 medium. Acetosyringone addition and the time from wounding affected root formation. The alkaloid content was highest in clones C58 and A4 (90mg·l^-1). There are great differences between individual hairy root clones, and hence they are not as uniform as has often been speculated. The Agrobacterium strain used for the transformation has a great influence in this respect.