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     

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     

Tropane alkaloid production in cultured cells of Duboisia leichhardtii

Calluses were derived from explants of aerial parts of a Duboisia leichhardtii F. Muell. plant. After 3 months of culture we detected small amounts of hyoscyamine and scopolamine in the unorganized calluses, but the amounts decreased until they were undetectable with successive subculture. When shoots were produced from the unorganized calluses, the ability to convert hyoscyamine added into media to scopolamine appeared; when roots were initiated from the shoot-organizing calluses, hyoscyamine, scoporamine, and nicotine were synthesized. Cultured adventitious roots produced these alkaloids and converted exogenous hyoscyamine to scopolamine.Abbreviations NAA 1-naphthaleneacetic acid - BA benzyladenine - IBA indole-3-butyric acid     

Alkaloid production in Duboisia hybrid hairy root cultures overexpressing the pmt gene

Putrescine:SAM N-methyltransferase (PMT) catalyses the N-methylation of the diamine putrescine to form N-methylputrescine, the first specific precursor of both tropane and pyridine-type alkaloids, which are present together in the roots of Duboisia plants. The pmt gene of Nicotiana tabacum was placed under the regulation of the CaMV 35S promoter and introduced into the genome of a scopolamine-rich Duboisia hybrid by a binary vector system using the disarmed Agrobacterium tumefaciens strain C58C1 carrying the rooting plasmid pRiA4. The presence of the foreign gene in kanamycin-resistant hairy roots and its overexpression were confirmed by polymerase chain reaction and Northern blot analysis respectively. The N-methylputrescine levels of the resulting engineered hairy roots increased (2-4-fold) compared to wild type roots, but there was no significant increase in either tropane or pyridine-type alkaloids.     

Enhancement of scopolamine production in Hyoscyamus muticus L. hairy root cultures by genetic engineering

 In order to test the possibility of enhancing the production of pharmaceutically valuable scopolamine in transgenic cultures, the 35S-h6h transgene that codes for the enzyme hyoscyamine-6β-hydroxylase (EC 1.14.11.11) was introduced into Hyoscyamus muticus L. strain Cairo (Egyptian henbane). This plant was chosen for its capability to produce very high amounts of tropane alkaloids (up to 6% of the dry weight in the leaves of mature plant). To our knowledge, this is the first time such a large population of transgenic cultures has been studied at the morphological, chemical and genetic levels. A great variation was observed in the tropane alkaloid production among the 43 positive transformants. The best clone (KB7) produced 17 mg/l scopolamine, which is over 100 times more than the control clones. However, conversion of hyoscyamine to scopolamine was still incomplete. The expression of h6h was found to be proportional to the scopolamine production, and was the main reason behind the variation in the scopolamine/hyoscyamine ratio in the hairy-root clones. These results indicate that H. muticus strain Cairo has a potential for even more enhanced scopolamine production with more efficient gene-expression systems. Received: 24 December 1998 / Accepted: 13 January 1999     

Feasible production of camptothecin by hairy root culture of Ophiorrhiza pumila

Camptothecin derivatives are used clinically as anti-tumor alkaloids. Camptothecin and its related compounds are at present obtained by extraction from intact plants, but transformed plant cell cultures may be an alternative source of production. We have established a hairy root culture of Ophiorriza pumila (Rubiaceae) transformed by Agrobacterium rhizogenes strain 15834. This hairy root culture grew well, increasing by 16-fold during 5 weeks in liquid culture, and it produced camptothecin as a main alkaloid up to 0.1% per dry weight of the cells. Interestingly, not only the hairy root cells contained camptothecin, but the culture medium also accumulated substantial amounts. Camptothecin content in the medium was increased by the presence of a polystyrene resin (Diaion HP-20) that absorbed camptothecin. Camptothecin was easily recovered from the resin. Our method is the most feasible and commercially applicable way to produce camptothecin by in vitro cell culture.     

Lobeline production by hairy root culture of Lobelia inflata L.

Hairy roots were obtained following inoculation of the stems of Lobelia inflata L. with Agrobacterium rhizogenes strain ATCC 15834. These hairy roots contained agropine and mannopine. In addition, lobeline was detected by HPLC and confirmed by mass spectrometry. Various media were tested for the growth of hairy roots as well as for the content of lobeline in hairy roots. The growth rate of hairy roots cultured in Nitsch and Nitsch's medium was approximately one third of those cultured in other media. The lobeline content of hairy roots (18–54 g/g dry weight) cultured in these media was the same order of magnitude compared with that of roots of L. inflata (24 g/g dry weight) cultivated in pots. The hairy roots cultured in Nitsch and Nitsch's medium were morphologically different from those cultured in other media.Abbreviations MS medium Murashige and Skoog's medium - 1/2 MS medium one-half strength of the standard Murashige and Skoog's medium - B5 medium Gamborg's B5 medium - NN medium Nitsch and Nitsch's medium - FW fresh weight - DW dry weight     

Enhanced production of artemisinin by Artemisia annua L hairy root cultures in a modified inner-loop airlift bioreactor

Hairy root cultures of Artemisia annua L were cultured in a modified inner-loop airlift bioreactor for achieving maximum artemisinin production. The effects of initial pH, air flow rate, cycle of light irradiation and temperature on growth and artmisinin production in Artemisia annua L hairy root cultures were investigated. Under the optimum conditions, the maximum production of artemisinin reached to 577.5?mg/l after 20 days.     

Enhanced production of scopolamine by bacterial elicitors in adventitious hairy root cultures of Scopolia parviflora

In an attempt to increase productivity, the effect of elicitation on tropane alkaloids (TA) biosynthesis was studied in adventitious hairy root cultures of Scopolia parviflora. Two Gram-positive strains and one Gram-negative strain of bacteria were used as biotic elicitors. The raw bacterial elicitors affected the tropane alkaloid profile by increasing the scopolamine concentration, while the autoclaved bacterial elicitors produced similar effects on the control. The conversion ratio of hyoscyamine to scopolamine was increased following elicitation using raw bacterial elicitors. The bacterial elicitor inhibited the expression of H6H (hyoscyamine 6β-hydoxylase) whereas the expression of PMT (putrescine N-methyltransferase) was raised by elicitation. These results have important implications for the large-scale production of tropane alkaloids.     

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.     

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.     

Lateral root bridging as a strategy to enhance L-DOPA production in Stizolobium hassjoo hairy root cultures by using a mesh hindrance mist trickling bioreactor

Stizolobium hassjoo hairy roots exhibited a lateral root bridging behavior, enabling not only root dry weight but enhancement of intracellular L-DOPA content. When a single root tip was exerted a proper hindrance, the primary root growth was inhibited while lateral roots were profusely induced. The hindrance-induced lateral roots from individual primary root could bridge together under appropriate inoculation densities, leading to high density hairy root cultures producing secondary metabolites. In the present paper, a novel bioreactor was proposed based on a strategy of lateral root bridging by utilizing mesh as a hindrance, called "mesh hindrance mist trickling bioreactor (MHMTB)". Significant improvements of dry weight and L-DOPA production by using MHMTB were 1.8 and 2.2-folds, respectively, higher than those in the control run without the mesh hindrance within the root bed.     

Secoiridoid glycosides production by Centaurium maritimum (L.) Fritch hairy root cultures in temporary immersion bioreactor

Secoiridoid glycosides are naturally occurring phytochemicals of great importance for the food and pharmaceutical industry because of their various biological activities. Certain Gentiana and Centaurium species, which are recognized as the most important sources of these compounds, have become critically endangered due to overexploitation. In this study we describe a laboratory-scale approach for further implementation in large-scale production of secoiridoid glycosides, using a hairy root culture system of Centaurium maritimum L. Fritch, an underutilized and phytochemically unexplored species. Hairy roots were induced by Agrobacterium rhizogenes strain A40M70GUS and grown in Erlenmeyer flasks, as well as in RITA^® temporary immersion bioreactors (TIBs). About 2–4 times higher biomass production rate and up to 8 times higher total secoiridoid glycosides production rate were achieved in RITA^® bioreactors. Among the selected hairy root lines, line HR3 cultured in RITA^® TIBs proved to be the most efficient considering both biomass and secoiridoid glycosides production rate.     

Continuous biodiesel production using in situ glycerol separation by membrane bioreactor system

Biodiesel is one of the most promising renewable fuel sources. Candida antarctica lipase B (CalB) has been used for biodiesel production because of its high activity and stability. However, CalB can only be utilized in industrial biodiesel production if the enzyme deactivation by methanol and the negative effects of glycerol can be mitigated. Methanol inhibition can be avoided by utilizing a stepwise addition of methanol, but there is no suitable method to reduce the glycerol effect. This study aims to use a membrane bioreactor system to remove glycerol during biodiesel production. In addition, methanol inhibition can be reduced by continuously feeding methanol through the membrane system. This continuous membrane bioreactor system can be used for efficient biodiesel production.     

Tropane alkaloid production by shoot culture of Duboisia myoporoides R. Br

This work demonstrates the presence of hyoscyamine and scopolamine at different stages of shoot regeneration from non-organogenic and organogenic calli. The 11-week-old non-organogenic calli contained 0.41+/-0.03 and 0.23+/-0.02 microg g(-1) dry wt hyoscyamine and scopolamine respectively. However, no root meristem was found in the calli. The alkaloids were absent in 2-week-old organogenic calli. The shoot-buds induced on the non-organogenic and organogenic calli did not contain these alkaloids. Hyoscyamine and scopolamine contents of the 6-week-old non-rooted shoots regenerated from non-organogenic calli were 7.8+/-0.1 and 6.5+/-0.4 microg g(-1) dry wt respectively and those in the 9-week-old non-rooted shoot regenerated from organogenic calli were 38.5+/-0.4 and 3.6+/-0.1 microg g(-1) dry wt respectively. Hyoscyamine and scopolamine contents of the 4-week-old roots regenerated from non-organogenic and organogenic calli were higher than those in the non-rooted shoots. Since the presence of hyoscyamine and scopolamine in the non-rooted shoot depends on the stage of differentiation, manipulation of culture environment may improve hyoscyamine and scopolamine contents of the non-rooted shoots.     

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.     

Development of a bioreactor for high density culture of hairy roots

Summary The effects of agitation and aeration on the growth of carrot hairy roots were investigated. When hydrodynamic stress index was above 0.001 cm/s, the growth rate of hairy roots decreased sharply. When volumetric O₂ transfer coefficient was high, the specific growth rate was also high. However, the specific growth rate approached the maximum value when the volumetric O₂ transfer coefficient was over 4 h^–1 . It is therefore necessary to maintain low hydrodynamic stress and high volumetric oxygen transfer for high density culture of hairy roots. By considering hydrodynamic stress and oxygen transfer, a novel bioreactor type was suggested for hairy roots cultivation.This research was supported in part by the Genetic Engineering Research Fund Korean Ministry of Education     

Production of scopolamine by normal root culture of Hyoscyamus niger

Summary Normal root of Hyoscyamus niger was cultivated in various nutrient conditions to optimize root growth and scopolamine production. Nitrate or phosphate concentration had little effect on root growth, while the low levels of nitrate or phosphate enhanced specific scopolamine content by 44% and 39%, respectively. Glucose or fructose was not adequate for the root culture as a carbon source, while the root growth was markedly enhanced at high sucrose concentration (up to 9%). The growth rate (0.59g dw./L.d.) and scopolamine productivity (1.98 mg/L.d.) was increased greater in roots with 9% initial sucrose concentration as compared to 3% sucrose (0.28 g dw./L.d., 0.98 mg/L.d.)     

Alkaloid production by hairy root cultures in Atropa belladonna

Hairy roots were induced by inoculation of stems of sterile plants of Atropa belladonna with Agrobacterium rhizogenes. The axenic culture of the hairy roots isolated from the stems proliferated 60 fold as based on the initial fresh weight after one month of culture. The presence of atropine and scopolamine in hairy roots were examined by TLC and HPLC. Their amounts were analyzed by GLC. The results show that the amount of the two alkaloids in the axenic cultures was the same as or even higher than those of normal plants grown in the field.     

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.