Establishment of hairy root cultures ofDatura stramonium. Characterization and stability of tropane alkaloid production during long periods of subculturing

The aim of this paper was the screening of the variability of growth patterns, biomass and tropane alkaloid production of 500 hairy root lines ofDatura stramonium. Data on the long term stability in alkaloid production of these lines for more than 5 years are also provided. In an effort to obtain high alkaloid-producing root clones, it is demonstrated that systematic selection is necessary. Comparisons are made, mainly concerning alkaloid production and its stability, with normal root cultures initiated from the same mother plants when necessary. Hairy root cultures were found to have a hyoscyamine and scopolamine bioproductivity of 2 orders of magnitude higher than mother plants.     

Tropane alkaloids production in transgenic Hyoscyamus niger hairy root cultures over-expressing Putrescine N-methyltransferase is methyl jasmonate-dependent

The cDNA from Nicotiana tabacum encoding Putrescine N-methyltransferase (PMT), which catalyzes the first committed step in the biosynthesis of tropane alkaloids, has been introduced into the genome of a scopolamine-producing Hyoscyamus niger mediated by the disarmed Agrobacterium tumefaciens strain C58C1, which also carries Agrobacterium rhizogenes Ri plasmid pRiA4, and expressed under the control of the CaMV 35S promoter. Hairy root lines transformed with pmt presented fivefold higher PMT activity than the control, and the methylputrescine (MPUT) levels of the resulting engineered hairy roots increased four to fivefold compared to the control and wild-type roots, but there was no significant increase in tropane alkaloids. However, after methyl jasmonate (MeJA) treatment, a considerable increase of PMTase and endogenous H6Hase as well as an increase in scopolamine content was found either in the transgenic hairy roots or the control. The results indicate that hairy root lines over-expressing pmt have a high capacity to synthesize MPUT, whereas their ability to convert hyoscyamine into scopolamine is very limited. Exposure to MeJA strongly stimulated both polyamine and tropane biosynthesis pathways and elicitation led to more or less enhanced production simultaneously.     

Influence of hairy root ecotypes on production of tropane alkaloids in Brugmansia candida

Hyoscyamine and scopolamine are tropane alkaloids widely applied in medicine. Differences in alkaloid production and growth kinetics have been observed in Argentinian and Colombian ecotypes of Brugmansia candida hairy roots. The aim of this work was to analyze the production of key intermediates in tropane alkaloid synthesis in both ecotypes to determine differences in the biosynthetic pathway. Additionally, rolC gene expression was analyzed to determine its correlation with hairy root growth. The results showed a higher accumulation of polyamines in Colombian hairy roots, suggesting that there may be a rate-limiting enzyme in the last steps of hyoscyamine biosynthesis. Additionally, rolC gene expression was correlated with an improvement in hairy root growth, which supports the function of rol genes as growth modulators and suggests that metabolic engineering approaches involving rolC manipulation may be useful for the development of more efficient B. candida hairy root cultures for biotechnological applications.     

Production of tropane alkaloids in diploid and tetraploid plants and in vitro hairy root cultures of Egyptian henbane (Hyoscyamus muticus L.)

In this study, the effects of ploidy level and culture medium were studied on the production of tropane alkaloids. We have successfully produced stable tetraploid hairy root lines of Hyoscyamus muticus and their ploidy stability was confirmed 30?months after transformation. Tetraploidy affected the growth rate and alkaloid accumulation in plants and transformed root cultures of Egyptian henbane. Although tetraploid plants could produce 200% higher scopolamine than their diploid counterparts, this result was not observed for corresponding induced hairy root cultures. Culture conditions did not only play an important role for biomass production, but also significantly affected tropane alkaloid accumulation in hairy root cultures. In spite of its lower biomass production, tetraploid clone could produce more scopolamine than the diploid counterpart under similar growth conditions. The highest yields of scopolamine (13.87?mg?l^?1) and hyoscyamine (107.7?mg 1^?1) were obtained when diploid clones were grown on medium consisting of either Murashige and Skoog with 60?g/l sucrose or Gamborg??s B5 with 40?g/l sucrose, respectively. Although the hyoscyamine is the main alkaloid in the H. muticus plants, manipulation of ploidy level and culture conditions successfully changed the scopolamine/hyoscyamine ratio towards scopolamine. The fact that hyoscyamine is converted to scopolamine is very important due to the higher market value of scopolamine.     

Tropane alkaloid production by Agrobacterium rhizogenes transformed hairy root cultures of Atropa baetica Willk. (Solanaceae)

Atropa baetica hairy root cultures were induced after infecting stem segments with Agrobacterium rhizogenes strain ATCC 15834. Accumulation of the tropane alkaloids atropine and scopolamine by hairy roots cultured in half- and full-strength Murashige and Skoog (MS) medium was high, although this was not growth associated. These alkaloids were also released into both liquid media. Higher tropane alkaloids present both in hairy roots and liquid medium occurred in half MS medium, showing a clear relationship between slow growth of cultures and higher product accumulation. The pH of both nutrient media varied as culture progressed, and seemed to be associated with the release of scopolamine. GC-MS analyses showed the presence of a new compound, namely tigloylpseudotropine; moreover, 3α-isobutyryloxytropane, formerly found only in plant leaf tissue, was also identified in the hairy roots. Received: 18 August 1997 / Revision received: 30 November 1997 / Accepted: 20 January 1998     

Enhanced production of hyoscyamine and scopolamine from genetically transformed root culture of <Emphasis Type="Italic">Hyoscyamus reticulatus</Emphasis> L. elicited by iron oxide nanoparticles

The medicinal plant Hyoscyamus reticulatus L. is a rich source of hyoscyamine and scopolamine, the tropane alkaloids. The use of hairy root cultures has focused significant attention on production of important metabolites such as stable tropane alkaloid production. Elicitation is an effective approach to induce secondary metabolite biosynthetic pathways. Hairy roots were derived from cotyledon explants inoculated with Agrobacterium rhizogenes and elicited by iron oxide nanoparticles (FeNPs) at different concentrations (0, 450, 900, 1800, and 3600 mg L^?1) for different exposure times (24, 48, and 72 h). The highest hairy root fresh and dry weights were found in the medium supplemented with 900 mg L^?1 FeNPs. Antioxidant enzyme activity was significantly increased in induced hairy roots compared to non-transgenic roots. The highest hyoscyamine and scopolamine production (about fivefold increase over the control) was achieved with 900 and 450 mg L^?1 FeNPs at 24 and 48 h of exposure time, respectively. This is the first report of the effect of FeNP elicitor on hairy root cultures of a medicinal plant. We suggest that FeNPs could be an effective elicitor in hairy root cultures in order to increase tropane alkaloid production.     

Enhanced production of tropane alkaloids in <Emphasis Type="Italic">Scopolia parviflora</Emphasis> by introducing the <Emphasis Type="Italic">PMT</Emphasis> (putrescine <Emphasis Type="Italic">N</Emphasis>-methyltransferase) gene

Summary In wild-type Scopolia parvilfora (Solanaceae) tissues, only the roots express the enzyme putrescine N-methyltransferase (PMT; EC 2.1.1.53), which is the first specific precursor of the tropane alkaloids. Moreover, the tropanane alkaloid levels were the highest in the root (0.9 mg g⁻¹ on a dry weight basis), followed by the stem and then the leaves. We metabolically engineered S. parviflora by introducing the tobacco pmt gene into its genome by a binary vector system that employs disarmed Agrobacterium rhizogenes. The kanamycin-resistant hairy root lines were shown to bear the pmt gene and to overexpress its mRNA and protein product by at least two-fold, as determined by polymerase chain reaction (PCR) and Northern and Western blottings, respectively. The transgenic lines also showed higher PMT activity and were morphologically aberrant in terms of slower growth and the production of lateral roots. The overexpression of pmt markedly elevated the scopolamine and hyoscyamine levels in the transgenic lines that showed the highest pmt mRNA and PMT protein levels. Thus, overexpression of the upstream regulator of the tropane alkaloid pathway enhanced the biosynthesis of the final product. These observations may be useful in establishing root culture systems that generate large yields of tropane alkaloids. These authors contributed equally to this paper (co-first authors).     

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.     

Effects of acetylsalicylic acid and UV-B on gene expression and tropane alkaloid biosynthesis in hairy root cultures of Anisodus luridus

Anisodus luridus hairy root cultures were established to test biological effects of acetylsalicylic acid (ASA) and ultraviolet ray-B (UV-B) on gene expression, tropane alkaloid (TA) biosynthesis and efflux. The TAs-pathway gene expression was ASA dosage dependant. The expression of PMT, TRI and CYP80F1 showed no significant difference in hairy root cultures in treatment of 0.01 and 0.1 mM ASA, compared with those without ASA treatment; while 0.01 or 0.1 mM ASA slightly upregulated H6H expression. All the four genes including PMT, TRI, CYP80F1 and H6H had a dramatic increase in 1 mM ASA-treated hairy root cultures compared with control. The expressing levels of all the four genes were much significantly higher in 1 mM ASA-treated hairy root cultures than those in 0.01 and 0.1 mM ASA-treated ones. As expected, hairy root cultures treated with 1 mM ASA had the highest capacity of TAs biosynthesis, in which the content of scopolamine and hyoscyamine reached respectively 57.2 and 14.7 μg g^?1 DW. Surprisingly, it was found that 1 mM ASA dramatically induced the efflux of scopolamine. In the liquid medium with 1 mM ASA, the content of scopolamine was 153.4 μg flask^?1, about 6.2 folds compared with that of control. At the same time, hyoscyamine was detected at trace levels in liquid medium. In the UV-B stressed hairy root cultures, all the four genes had a very strong increase of gene expression that led to more accumulation of scopolamine and lower accumulation of hyoscyamine. Only trace amounts of hyoscyamine and scopolamine were detected in the liquid medium when hairy root cultures were stressed under UV-B, and this suggested that UV-B did not affect TAs efflux.     

Effects of gibberellin GA7 on kinetics of growth and tropane alkaloid accumulation in hairy roots of Brugmansia candida

Summary Brugmansia candida, an indigenous South American plant, produces the tropane alkaloids scopolamine and hyoscyamine, which are widely employed in medicine as anticholinergic agents. In this research, hairy roots of Brugmansia candida, obtained through infection with Agrobacterium rhizogenes LBA 9402, were employed to produce these tropane alkaloids in vitro. The effects of different concentrations of GA₇ on kinetics of growth and alkaloid accumulation on two different hairy root clones of B. candida were analyzed, and the influence of GA₇ on the number of new branches and rates of elongation was also studied. On clone 7A, GA₇ at concentrations of 10⁻⁴, 10⁻¹, and 1 mg/l increased the exponential growth rate. Levels of 10⁻¹ and 10⁻⁴ mg/l GA₇ elevated the scopolamine/hyoscyamine (S/H) ratios in the early phases of growth, but the sum of scopolamine plus hyoscyamine per flask (S + H) decreased during that period. When 1 mg/l GA₇ was used, the highest S/H ratios were observed in late exponential/early stationary phases, but the highest S + H totals were obtained in mid-exponential phase. GA₇ at levels of 10⁻¹ and, especially, 1 mg/l exerted a positive effect on formation, emergence, and rate of elongation of lateral roots (clone 7A). On clone 7B, levels of 10⁻¹ and 1 mg/l GA₇ did not alter significantly the exponential growth rate. GA₇ in concentrations of 10⁻¹ mg/l induced increases in both S/H ratio and S + H totals in late phases of growth.     

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     

Effect of pmt gene overexpression on tropane alkaloid production in transformed root cultures of Datura metel and Hyoscyamus muticus

In order to increase the production of the pharmaceuticals hyoscyamine and scopolamine in hairy root cultures, a binary vector system was developed to introduce the T-DNA of the Ri plasmid together with the tobacco pmt gene under the control of CaMV 35S promoter, into the genome of Datura metel and Hyoscyamus muticus. This gene codes for putrescine:SAM N-methyltransferase (PMT; EC. 2.1.1.53), which catalyses the first committed step in the tropane alkaloid pathway. Hairy root cultures overexpressing the pmt gene aged faster and accumulated higher amounts of tropane alkaloids than control hairy roots. Both hyoscyamine and scopolamine production were improved in hairy root cultures of D. metel, whereas in H. muticus only hyoscyamine contents were increased by pmt gene overexpression. These roots have a high capacity to synthesize hyoscyamine, but their ability to convert it into scopolamine is very limited. The results indicate that the same biosynthetic pathway in two related plant species can be differently regulated, and overexpression of a given gene does not necessarily lead to a similar accumulation pattern of secondary metabolites.     

Indole alkaloid production by transformed and non-transformed root cultures ofCatharanthus roseus

Summary Ten transformed and two non-transformed root lines ofCatharanthus roseus were established. A systematic study of the growth kinetics and alkaloid content was performed over a culture cycle and showed significant differences between transformed and non-transformed cultures. Mean doubling times for transformed and normal root lines were 2.8 and 19.5 days, respectively. Alkaloid content in hairy roots was from two- to threefold higher than in the non-transformed tissues. The established transformed root lines produced a wide variety of indole alkaloids as can be observed from their complex thin layer chromatography patterns. A large quantity of serpentine was determined in two of the transformed root cultures. Alkaloid content, both quantitatively and qualitatively, has been stable in the hairy root cultures for more than 2 yr of subculturing.     

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     

Establishment of Physalis minima hairy roots culture for the production of physalins

This report describes the technique used to induce the hairy roots in Physalis minima (Linn.). Different types of explants obtained from in vitro germinated seedlings were aseptically co-cultivated with A. rhizogenesstrain LBA9402 in different media. Root growth and production of physalins were investigated in various basal media grown under dark and light conditions, and compared to that of normal root cultures. Transformed hairy root cultures grew rapidly and reach stationary phase after 15 days on a B5 medium. HPLC analysis of extracts of hairy root cultures showed that the maximum content of physalin B and F was 1.82 and 4.15 mg g^–1 DW, respectively, when grown under dark conditions. Normal root cultures produced higher physalin B (1.60–1.62 mg g^–1 DW) and F (3.30–3.75 mg g^–1 DW) under the same culture conditions. Physalin F synthesis in light-grown root cultures was reduced significantly.     

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     

Rose-scented geranium (<Emphasis Type="Italic">Pelargonium</Emphasis> sp.) generated by <Emphasis Type="Italic">Agrobacterium rhizogenes</Emphasis> mediated Ri-insertion for improved essential oil quality

Transgenic plants of rose-scented geranium (Pelargonium graveolens cv. Hemanti) have been produced from Agrobacterium rhizogenes (strains A₄ and LBA9402) mediated hairy root cultures. Amongst the explants tested, leaves were most responsive followed by the petioles and internodal segments, respectively. The A₄ strain performed better for all the three explants both in terms of frequency of response and time requirement for hairy root induction. Transgenic shoots could be obtained by spontaneous regeneration without intervening callus phase amongst 16% and 12% root lines of A₄ and LBA 9402 origin, respectively, or they were induced in 29% and 22% hairy root lines of A₄ and LBA9402 origin, respectively, with different hormonal supplementation. These transgenic plants showed 30% survival as against 90% of their control under the confined environment of glasshouse. The transgenic plants were of similar morphotype having increased branching, higher number of leaves with increased dentations, short and round stature, highly branched root system and absence of leaf wrinkling. These transgenic plants showed opine positive results even after 5 months of their transfer to the glasshouse. The essential oil compositions of 81% of these transgenics were qualitatively similar to that of the wild type parent. However, two transgenic plants (LZ-3 and 14TG) showed increase in concentrations of geraniol and geranyl esters signifying improved oil quality with respect to the citronellol:geraniol ratio. These two oils having better olfactory value represent an improvement over that of the wild type parent from the commercial point of view.     

Effects of Oxygen on Nicotine and Tropane Alkaloid Production in Cultured Roots Duboisia myoporoides

The effects of oxygen on nicotine and tropane alkaloid production in root cultures of Duboisia myoporoides were investigated. Duboisia roots cultured in air produced both nicotine and tropane alkaloids equally. However, when roots were cultured in pure oxygen, the metabolic flux to tropane alkaloids increased, and that to nicotine alkaloids decreased. Intermediate product analysis by GC-MS showed an increase in tropine, but decreases in acetyl derivatives of tropane alkaloids and tropine esters with low-class fatty acids. Furthermore, hyoscyamine 6β-hydroxylase (H6H, EC 1.14.11.11, the key enzyme in the pathway from hyosyamine to scopolamine) also increased. These results suggest that pure oxygen contributes to scopolamine production not only by activating the biosynthetic steps for scopolamine, but also by inactivating the biosynthetic steps for nicotine and other tropine derivatives.     

Recovery of phenotypically normal transgenic plants of Brassica oleracea upon Agrobacterium rhizogenes-mediated co-transformation and selection of transformed hairy roots by GUS assay

In this paper we describe the production of transgenic broccoli and cauliflower with normal phenotype using an Agrobacterium rhizogenes-mediated transformation system with efficient selection for transgenic hairy-roots. Hypocotyls were inoculated with Agrobacterium strain A4T harbouring the bacterial plasmid pRiA4 and a binary vector pMaspro::GUS whose T-DNA region carried the gus reporter gene. pRiA4 transfers T_L sequences carrying the rol genes that induce hairy root formation. Transgenic hairy-root production was increased in a difficult-to-transform cultivar by inclusion of 2,4-D in the medium used to resuspend the Agrobacterium prior to inoculation. Transgenic hairy roots could be selected from inoculated explants by screening root sections for GUS activity; this method eliminated the use of antibiotic resistance marker genes for selection. Transgenic hairy roots were produced from two cauliflower and four broccoli culivars. Shoots were regenerated from transgenic hairy root cultures of all four cultivars tested and successfully acclimatized to glasshouse conditions, although some plants had higher than diploid ploidy levels. Southern analysis confirmed the transgenic nature of these plants. T₀ plants from seven transgenic lines were crossed or selfed to produce viable seed. Genetic analysis of T₁ progeny confirmed the transmission of traits and revealed both independent and co-segregation of Ri T_L-DNA and vector T-DNA. GUS-positive phenotypically normal progeny free of T_L-DNA were identified in three transgenic lines out of the six tested representing all the cultivars regenerated including both cauliflower and broccoli.     

Rapid in vitro adventitious shoot propagation of <Emphasis Type="Italic">Scopolia parviflora</Emphasis> through rhizome cultures for enhanced production of tropane alkaloids

A rapid micropropagation system for Scopolia parviflora Nakai (Solanaceae), a rare medicinal plant native to Korea, was established using rhizome cultures. Shoots that originated from adventitious shoots of the rhizome were multiplied when the rhizomes were cultured on half-strength B5 liquid medium supplemented with various growth regulators. Optimum shoot multiplication was observed in half-strength B5 medium containing 3% (w/v) sucrose and 5.77 M gibberellic acid (GA₃). Each rhizome gave rise to an average of 12 shoots. Shoot elongation and root induction from multiple shoots occurred on growth regulator-free half-strength B5 solid medium. Healthy plantlets were transferred to a peat moss:vermiculite mixture for acclimatization, which was successful. The concentrations of tropane alkaloids, hyoscyamine and scopolamine were determined in different tissues of native growing plants, in vitro-propagated plants and acclimatized plants by high-performance liquid chromatography. The analysis revealed that the levels of hyoscyamine and scopolamine were higher in in vitro-propagated plants than in the native growing plants. When the rhizome was cut into segments and transferred to optimal culture conditions for multiple shoot propagation, only 12 weeks were required to produce a mature plant. We conclude that in vitro propagation techniques through rhizome cultures provide an efficient and rapid method for shoot propagation of S. parviflora.Abbreviations BA Benzyladenine - 2,4-D 2,4-Dichlorophenoxyacetic acid - GA₃ Gibberellic acid - HPLC High-performance liquid chromatography - IBA Indole-3-butyric acid - NAA -Naphthaleneacetic acid