Tropane Alkaloids in Transformed Roots of Datura quercifolia

Hairy root cultures of DATURA QUERCIFOLIA were established following infection with AGROBACTERIUM RHIZOGENES strain LBA 9402. Eight tropane alkaloids were identified in the hairy roots, hyoscyamine being the major constituent. The growth and the hyoscyamine content of transformed roots were investigated under various conditions. Gamborg B5, Murashige and Skoog, and Woody Plant media were tested. Gamborg B5 medium was the best for growth as well as for hyoscyamine accumulation. The influence of sucrose concentration was examined and a 5% concentration was found to be the most appropriate for growth and for alkaloid production. After 35 days of incubation in this medium, the hyoscyamine content of the roots was 1.24% based on dry weight. The influence of gibberellic acid and of Amberlite XAD-4 resin on hyoscyamine production was tested.     

Tropane alkaloid production in transformed root cultures of brugmansia Candida

Transformed root cultures of BRUGMANSIA CANDIDA were established by infection with AGROBACTERIUM RHIZOGENES LBA 9402. Several clones with different growth index and tropane alkaloid pattern and content were obtained and two were examined in depth. The alkaloid content and pattern changed during the time course. At 21 days of culture clone 1 revealed an alkaloid spectrum dominated by 3alpha-acetoxytropane (about 50% of the total alkaloid) and hyoscyamine (about 25%), with a ratio of hyoscyamine to scopolamine of 11.2. In clone 40 this ratio was 1.5 and the content of 3alpha-acetoxytropane was low (2%). The maximum concentrations of hyoscyamine were obtained at 3 weeks of culture, and were 700 and 500 microg/g FW in clone 1 and 40, respectively.     

Expression of Vitreoscilla hemoglobin enhances growth of Hyoscyamus muticus hairy root cultures

The Vitreoscilla hemoglobin gene (vhb) was introduced into Hyoscyamus muticus with the aim of investigating its effect on growth and alkaloid production of Agrobacterium rhizogenes-induced hairy root cultures. We were able to generate several VHb-expressing hairy root lines with different integration patterns. Substantial somaclonal variation was observed in growth and hyoscyamine production amongst both VHb-expressing lines and controls. Despite this variation, the growth properties of single lines remained stable over time. Expression of VHb was found to improve growth of H. muticus hairy roots in shake-flask cultures. The dry weights of the root cultures expressing Vitreoscilla hemoglobin were on average 18 % higher than those of the controls. VHb expression also increased the volumetric hyoscyamine production, mainly due to the improved growth properties. However, this difference was not statistically significant due to the wide somaclonal variation and fluctuations over time in both VHb and control hairy root lines.     

Variation in the Tropane Alkaloid Content of Hyoscyamus muticus Plants and Cell Culture Clones

Systematic studies were carried out on two different strains (Gatersleben and Cairo) of HYOSCYAMUS MUTICUS L. (Solananaceae) in order to analyse the variation in the contents of the two main tropa-alkaloids in individual plants and protoplast-derived cell culture clones. The hyoscyamine content was determined by radioimmunoassay, the scopolamine content by radio- and enzymeimmunoassay and the total tropane alkaloid content by quinuclidinyl benzilate assay. The development stage of the plant is important for alkaloid production. Clear maximal foliar scopolamine and hyoscyamine contents were reached at the onset of flowering and during the full blooming stage, respectively. In the roots the changes in the production of these alkaloids were not considerable. Hyoscyamine is a major alkaloid in the plants as well as in the cell culture clones. In a few exceptions the scopolamine content was greater than that of hyoscyamine, but this phenomenon was not inherited by the cultured clones derived from these plants. High and low tropane alkaloid production was inherited by the selfed F (1), generation plants. Great variability in these alkaloids was observed among individual plants or clones in the same plant or clone population. There was a significant difference between the Cairo strain and the Gatersleben strain as regards their ability to produce tropane alkaloids. Haploid plants of both strains contained more hyoscyamine and scopolamine than the diploid ones. Hyoscyamine and scopolamine were the main alkaloids in the clones and in the plants. Via QNB-assay an interesting clone was found which contains remarkable amounts of unknown tropane-group alkaloids.     

Decreased scopolamine yield in field-grown Duboisia plants regenerated from hairy roots

Hairy root cultures were obtained from hybrid clones of Duboisia myoporoides x D. leichhardtii following transformation by Agrobacterium rhizogenes strain A4. Shoots spontaneously regenerating from the hairy root cultures were rooted and transferred to soil. The plants displayed typical morphological alterations known as hairy root syndrome to varying degrees. PCR analysis confirmed that all transformed plants contained the rolA, rolB and rolC genes, irrespective of the degree of morphological alterations. A field test of the transformed regenerated plants revealed that those plants displaying the strongest hairy root syndrome symptoms had the highest content of the tropane alkaloid scopolamine. However, the overall scopolamine and hyoscyamine yield of all transformed plants was clearly reduced compared to untransformed control plants. These results demonstrate that the A. rhizogenes-transformed plants tested in this study do not provide a viable alternative to agricultural farming of hybrid clones of D. myoporoides x D. leichhardtii obtained by conventional breeding.     

Plant Nutritional Elements and Tropane Alkaloid Production in the Roots of Henbane (Hyoscyamus niger)

The effect of calcium on the morphology, growth, tropane alkaloid production (hyoscyamine and scopolamine) and plant nutritional element (calcium, magnesium and potassium) content of roots and root cultures of Hyoscyamus niger L. was examined in this study. It was observed that the tropane alkaloid productivity of root cultures was significantly higher than that of the corresponding field cultures. Murashige-Skoog (MS) medium was found to be optimal for the production of tropane alkaloids of the root cultures of H. niger. Variation in the calcium content of the medium had no substantial effect on the morphology, growth or content of magnesium, potassium and sodium of the root cultures of H. niger. However, increased calcium content in MS medium increased the production of the scopolamine and decreased the production of alkaloid hyoscyamine in the root cultures of H. niger. Collectively, our results are helpful in the optimization of medium composition for the production of highly valuable tropane alkaloid scopolamine using root cultures of H. niger.     

Production of Hyoscyamine by 'Hairy Root' Cultures of Datura stramonium

'Hairy root' cultures of DATURA STRAMONIUM were established following infection of aseptic leaves with AGROBACTERIUM RHIZOGENES. Transformation was confirmed by Southern blotting using [ (32)P]-labelled fragments of the T-DNA as probes. The transformed cultures grew in the absence of added phytohormones and cell mass increased 55-fold during 28 days incubation. Hyoscyamine was a major component of the alkaloid fraction and accounted for at least 0.3% of the dry matter, comparable to pot-grown plants from which the cultures were initiated. Production of hyoscyamine followed growth during the first 15 days of incubation, but continued to increase during the early part of stationary phase. The alkaloid was retained almost entirely in the root tissue. The effects of medium composition and pH on growth and hyoscyamine production are reported.     

透明颤菌血红蛋白对黄芪甲苷生物合成的调控作用

为了研究透明颤菌血红蛋白(Vitreoscilla hemoglobin,VHb)对黄芪甲苷生物合成的调控作用,本文运用农杆菌介导法,将透明颤菌血红蛋白基因(Vitreoscilla hemoglobin gene,vgb)导入黄芪毛状根中,经PCR特征性引物扩增得到54个转基因株系。Southern blot分析,证明vgb基因已经整合到转基因黄芪毛状根基因组中。RT-PCR分析结果表明,vgb基因在转录水平获得表达。转基因黄芪毛状根培养15天,其生长量和增长倍数皆比非转基因黄芪毛状根高;黄芪甲苷含量测定结果显示,转基因黄芪毛状根黄芪甲苷含量是非转基因黄芪毛状根的5～6倍,是山西产黄芪药材的10～12倍。研究结果表明,vgb基因的表达促进了转基因毛状根的生长,提高了黄芪甲苷含量。     

Solanum chrysotrichum hairy root cultures: characterization, scale-up and production of five antifungal saponins for human use

The production of five antifungal saponins (SC-2 - SC-6) recently extracted from Solanum chrysotrichum leaves was performed using hairy root cultures grown in 250-mL flasks and in 2-L modified draught-tube internal-loop airlift reactors (2-L MR). In both scales, first order growth kinetics were observed, reaching specific growth rates of 0.08 and 0.12 d(-1), respectively. Root density at the end of the culture period was the same for the flasks and for the 2-L MR. In flasks the production of the most active saponin SC-2 was growth-associated, with a maximum yield of 0.04 % dry weight, while in the 2-L MR an SC-2 yield of 0.7 % dry weight was reached, representing a value six times greater than that observed for plant leaves. SC-3 was obtained from root biomasses grown in flasks, while SC-4 was recovered from biomasses at both levels under investigation. SC-5 and SC-6 were only detected in the culture medium of roots grown in 2-L MR. Solanum chrysotrichum hairy root cultures and their scale-up in reactors are feasible strategies for the production of these antifungal compounds for human use.     

Alkaloid Production in Diploid and Autotetraploid Plants of Datura stramonium

A comparative investigation of alkaloid production and accumulation in the roots and leaves of diploid (2n = 24), and C 4 generation of induced autotetraploid (4n = 48) Datura stramonium L. plants was performed. Fifteen alkaloids have been determined in the roots and two in the leaves, at a level of 1% or more of the crude alkaloid fractions in both ploidy levels. Two 3-tigloyloxy-6-isovaleryloxy-7- hydroxytropane isomers were detected for the first time in genus Datura. In the conditions of the prolonged photoperiod, hyoscyamine was the main alkaloid in the roots whereas scopolamine was the main alkaloid in the leaves of both ploid forms. In comparison to diploids, the roots and leaves of tetraploids had a higher alkaloid content and scopolamine/hyoscyamine ratio.     

Tropane alkaloid production and shoot regeneration in hairy and adventitious root cultures of Duboisia myoporoides-D. leichhardtii hybrid

Co-culture conditions for Duboisia myoporoides-D. leichhardtii hybrid hairy root induction were investigated using leaf explants and Agrobacterium rhizogenes ATCC 15834. The bacteria density and duration of co-culture greatly affected the induction rate; the highest rate of 50% was obtained when the leaf explants were co-cultured for 2 d with 10(6) bacteria. One hairy root clone that showed the fastest root growth was selected and used for comparison study with adventitious roots cultured with 0.5 mg/l indole-3-acetic acid (IAA). The hairy roots cultured in Murashige and Skoog (MS) liquid medium grew well and yielded much more tropane alkaloids (35 mg/l scopolamine and 17 mg/l hyoscyamine) than adventitious roots cultured in 0.5 mg/l IAA after 6 weeks of culture at 25 degrees C in the dark. The hairy and adventitious roots (2.5 cm) grown in liquid media were divided into 5 parts (each 0.5 cm) along the root axis. Distribution of scopolamine and IAA was then determined by enzyme-linked immunosorbent assay (ELISA). Inverse relationship between contents of scopolamine and IAA was observed in the hairy roots; increase of scopolamine and decrease of IAA were proportional to the distance from the root meristem. In contrast, the contents of scopolamine and IAA were relatively constant in the adventitious roots. In shoot regeneration experiments, the hairy and adventitious root segments (1 cm) were placed onto 1/2 MS solid medium containing various concentrations of IAA and BA cultured at 25 degrees C under 16 h light. In adventitious roots, the shoots regenerated on media containing 6-benzyladenine (BA) (0.5 to 5 mg/l), and 100% regeneration was observed in medium with 0.1 mg/l IAA and 2 mg/l BA. On the other hand, shoot regeneration was only observed in 33% of hairy roots cultured on medium containing 5 mg/l BA.     

Influence of Agrobacterium rhizogenes Strains on Biomass and Alkaloid Productivity in Hairy Root Lines of Hyoscyamus muticus and H. albus *

Using leaf explants of IN VITRO grown HYOSCYAMUS ALBUS and H. MUTICUS plantlets, hairy roots were induced following inoculation with AGROBACTERIUM RHIZOGENES strains A (4) and LBA-9402. The transformed roots, appearing after 14 - 17 days incubation on hormone-free MS medium containing 1 g/L cephalexin, were excised and maintained in the same medium. Ten randomly selected hairy root lines from each bacterial treatment of the two plant systems were compared for growth and alkaloid production in half-strength, hormone-free MS medium on 25 (th) day of culture. A. RHIZOGENES strain - A (4) induced hairy root lines of both H. ALBUS and H. MUTICUS were comparatively faster growing than those induced by strain LBA-9402. In contrast to earlier reports, some of the hairy root lines of H. ALBUS induced by A. RHIZOGENES strain A (4) were as fast growing as the hairy root lines of H. MUTICUS. The atropine yields of A (4) induced lines of H. ALBUS were significantly higher (3.5 fold) than the LBA-9402 induced lines. No such relationship between the bacterial strain and alkaloid productivity could, however, be obtained in case of hairy root lines of H. MUTICUS.     

Factors Affecting the Growth and Hyoscyamine Production during Batch Culture of Transformed Roots of Datura stramonium

The growth and hyoscyamine production of transformed roots of DATURA STRAMONIUM (cell line DS 1) were investigated in batch culture experiments at three different temperatures and in media of varying total ion composition and sucrose level. Growth rate was not greatly affected by the level of Gamborg's B5 salts or the sucrose concentration. Growth rates of roots in a range of media were similar at 25 degrees C (doubling time T (d) 1.2-1.6 days) and at 30 degrees C (T (d) 1.2-1.7 days) but roots grew slower at 20 degrees C (T (d) 1.9-3.5 days). The total root yield on a fresh weight basis was higher in full strength B5 media compared with half strength B5 media at all sucrose levels tested. However, the dry weight content of the roots increased significantly (4-20%) as the sucrose level in the medium was raised (2-10%) at each of the three temperatures. At 20 degrees C, the hyoscyamine content of the roots was higher than that at 25 degrees C or 30 degrees C at all sucrose levels tested and was unaffected by increasing sucrose levels. However, increasing the level of sucrose (1-5%) in either half or full strength B5 media at 25 degrees C or 30 degrees C increased the hyoscyamine content of the roots by up to eight fold. At 20 degrees C, the total yield of hyoscyamine per flask or per unit of sucrose supplied was also higher than at 25 degrees C or 30 degrees C with sucrose levels up to 5%. The highest rates of hyoscyamine production by cell line DS 1 were obtained with full strength B5 medium with 5% sucrose at either 20 degrees C or 25 degrees C. Under these conditions, rates of hyoscyamine formation of up to 7.4 mg/l/d were observed under batch culture conditions. The amount of hyoscyamine released into the culture medium was much greater at 30 degrees C (up to 14% of the total) than at either 20 degrees C or 25 degrees C (up to 4% of the total).     

Growth and ginsenoside production in hairy root cultures of Panax ginseng using a novel bioreactor

We tested the effect of three variables: the bioreactor system (Wave or Spray reactor), medium exchange and culture period, on the capacity of a selected hairy root line of Panax ginseng to produce ginsenosides. Among the reactors, the Wave bioreactor appeared to be the most efficient in promoting hairy root line growth. Periodic exchanges of the medium and a longer culture period increased the growth rate of cultured hairy root line and, consequently, its capacity to produce ginsenosides. Under established optimum conditions (medium exchange every 14 days over a culture period of 56 days using the Wave bioreactor), the initial root fresh weight was enhanced more than 28-fold, giving a root biomass of 284.9 g L(-1) and a ginsenoside content of 145.6 mg L(-1). It is noteworthy that this ginsenoside production exceeded by almost 3-fold that obtained during the shake flask culture of our hairy root line, although it often happens that the scale-up from shake flask to a bioreactor culture results in reduced productivities. To our knowledge this is the first time that a Wave bioreactor has been used for hairy root culture.     

Permeabilization of Datura innoxia hairy roots for release of stored tropane alkaloids

The effects of tween 20 as permeabilizing agent on tropane alkaloids from Datura innoxia Mill, hairy roots have been studied. For various tween 20 concentrations both hyoscyamine and scopolamine accumulated in the culture medium. Plant material viability could be preserved after a 24 hours-2% tween 20 concentration treatment. The time-course study of alkaloid release showed that the maximum of excretion occurred after a 20 hour contact with tween 20. At that time, a concentration of hyoscyamine superior to 25 mg/l was detected in the medium.     

黄花蒿发根的液体培养及青蒿素合成的动态特征研究

目的:探讨黄花蒿发根生长及青蒿素生物合成的动态特征,建立高效、稳定的黄花蒿发根液体培养体系。方法:测定不同培养基以及MS培养基中不同营养元素对黄花蒿发根生物量和青蒿素含量的影响。结果:筛选出优化的黄花蒿发根液体培养基,获得拟合的黄花蒿发根生长和青蒿素合成的Logistic方程。结论:在优化的MS培养基中黄花蒿发根生长迅速且能稳定合成青蒿素,为工业化大规模生产青蒿素提供了可能。     

Datura metel: in vitro production of tropane alkaloids

Hairy root lines of Datura metel were established following infection of aseptic stem segments with Agrobacterium rhizogenes strain A4 and cultured in hormone-free B5 solid medium. The growth and production of hyoscyamine and scopolamine (mg/g dry wt.) of these root cultures was encouraged by using B5 liquid medium with half-strength salts. In these culture conditions, the capacity of the highest productive root line 25 to excrete scopolamine into the culture medium rose from 8.7% to 70% when the permeabilizing agent Tween 20 was added for 24 h to the medium, after 2 and 4 weeks of culture. Using an airlift bioreactor (41) with modifications in order to increase root anchorage, the Tween 20 treatment encouraged both growth and alkaloid productivity of cultured root line 25. After 4 weeks their biomass yield was 2.3 mg/l/day and 0.84 mg/l/day of scopolamine was produced (70% extracellular). The scopolamine released into the culture medium was separated with an Amberlite XAD-2 column located in the media exit.     

甘草毛状根中甘草总黄酮和甘草酸的检测和分析

目的检测甘草毛状根中总黄酮和甘草酸的含量。方法采用紫外分光光度法检测甘草总黄酮含量,采用HPLC检测甘草酸含量。结果甘草毛状根中的总黄酮含量在不同根系中有很大差异,最高可达0.803%,接近生药根;大多数根系高于正常组培根;总黄酮含量随着培养时间的延长呈增加趋势,至9周时增加了68.5%。不同甘草毛状根系中的甘草酸含量差异显著,部分根系未检出甘草酸,能检出的根系最高干重含量为0.69mg·g^-1,远低于生药根（干重含量32.13mg·g^-1）,且含量不受培养时间影响。结论甘草毛状根在目前情况下不适于作为甘草酸的获取资源,但作为甘草总黄酮获取的新资源,是值得进一步研究的。     

Auxins affected ginsenoside production and growth of hairy roots in Panax hybrid

Hairy roots of interspecific hybrid ginseng (Panax ginseng x P. quinquefolium), induced by Agrobacterium rhizogenes ATCC 15834, grew well in B5 liquid media supplemented with 2.5 microM auxins (3-indole butyric acid (IBA), 1-naphtaleneacetic acid (NAA) and 3-indoleacetic acid (IAA)). The hairy roots cultured in B5 liquid medium supplemented with 2.5 microM IBA showed best growth (6.39 g fresh weight per a flask, at week 8). The highest content of the total ginsenosides was 1.63% as dry weight at week 8 when cultured with 2.5 microM NAA. The different auxins affected the numbers and lateral branching roots. Especially, 2.5 microM IBA promoted the lateral root formation (43.7+/-4.0 roots, at week 8), and 2.5 microM NAA promoted the lateral root growth (45.3+/-5.6 mm, at week 8). The growth and ginsenosides production of 8-week old hairy roots cultured in B5 liquid media supplemented with IBA and NAA combinations were also investigated. Hairy roots produced higher amounts of ginsenosides in B5 liquid media supplemented with 0.5-1.0 microM IBA and NAA combinations than that cultured in B5 liquid media supplemented with only IBA and NAA. The highest yield of ginsenoside was obtained when cultured with 0.5 microM IBA and 1.0 microM IBA combination (6.38 mg per a flask, at week 8).     

Quantitative determination of secoiridoid and gamma-pyrone compounds in Gentiana lutea cultured in vitro

The production of secondary metabolites was studied in shoots, roots, and hairy roots of Gentiana lutea obtained in vitro. In shoots, both secoiridoid and gamma-pyrone compounds were detected in amounts similar to those found in aerial parts of plants collected from nature. The most abundant secoiridoid was gentiopicrin while mangiferin was the main compound among the gamma-pyrones. The adventitious roots obtained in vitro showed a poor biosynthetic capacity. Upon infection with Agrobacterium rhizogenes, nine hairy root clones were established which differed in the amount of secondary metabolites.