Polyacetylenes in hairy roots of a panax hybrid

From the hairy roots of an interspecific hybrid ginseng ( Panax ginseng x P. quinquefolium) known as Pgq, three polyacetylenes were isolated: panaxynol, panaxydol and 1,8-heptadecadiene-3,10-diol. These compounds isolated from the hairy roots were used for quantitative analysis to investigate the polyacetylene production of the hairy roots cultured in Gamborg B5 (B5) and 1/8 Murashige-Skoog (MS) liquid media. Maximum growth of the hairy roots was observed (ca. 5.4 g fresh weight/100 mL flask) at 8 weeks of culture in B5 medium. The highest total content of total polyacetylenes was 0.18 % of dry weight at week 8 when cultured in 1/8 MS medium. In addition, we compared the yields of polyacetylenes and ginsenosides in hairy roots cultured in B5 with those in 1/8 MS media and found the highest yields were obtained in the hairy roots cultured in B5 medium (1.24 mmol/flask polyacetylenes and 4.45 mmol/flask ginsenosides at week 8).     

In vitro plant regeneration from leaf-derived callus of Cimicifuga racemosa

Cimicifuga racemosa (L.) Nutt., also known as Black Cohosh, is among the top 10 selling medicinal herbs in the United States. The rhizomes have been used to relieve menopausal discomfort. This plant is wild crafted and conservationists have expressed concerns with the sustainability of C. racemosa. Excised tissues from young leaves of C. racemosa were cultured on Murashige and Skoog's medium (MS) supplemented with various concentrations of NAA and TDZ for production of callus. The optimum callus growth and maintenance was in 1.0 microM NAA plus 0.5 microM TDZ. Two-month-old calli were sub-cultured on different concentrations of cytokinins (BA, kinetin, 2ip, TDZ) or in combination with GA(3) for shoot induction. The rate of shoot induction and proliferation was higher in MS media supplemented with 2.0 or 4.0 microM of TDZ. Concentrations of TDZ greater than 4.0 microM suppressed shoot growth. Adding 3.5 microM of GA(3) into media containing BA increased shoot growth. The presence of GA(3) with kinetin or TDZ did not affect shoot production. For rooting, shoots were transferred to MS medium with activated charcoal supplemented with various auxins (IAA, IBA and NAA), roots were noticed 20 days after transference. Activated charcoal was an essential component for vigorous rooting formation. Our results suggest that conservation of C. racemosa is possible through in vitro multiplication of leaf-derived callus.     

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.     

Transformation of ipecac (Cephaelis ipecacuanha) with Agrobacterium rhizogenes

Transformed root cultures of ipecac (Cephaelis ipecacuanha A. Richard), one of the recalcitrant woody plant species for Agrobacterium-mediated transformation, were established by co-culturing of in vitro petiole segments with Agrobacterium rhizogenes ATCC 15 834. Southern blot analysis of the established roots revealed that only the TL-DNA was integrated into the plant genome without incorporation of the TR-DNA. The transformed roots grew slowly on phytohormone-free solid medium and adventitious shoots were regenerated after over 6 months of culture on HF, half-strength Murashige and Skoog (1/2 MS) medium in the dark. The individually separated transformed shoots developed into plantlets on phytohormone-free solid medium at 25 degrees C under 16 h/day light, and the plants demonstrated wider leaves, shorter internodes and vigorous root growth compared to non-transformed plants. Effects of basal media and auxins on the growth and the ipecac alkaloid production of the transformed roots were investigated either under light or in the dark. The roots cultured in the dark grew well in Gamborg B5 (B5) liquid medium containing 0.5 mg/L IBA and yielded 112 mg/L of cephaeline and 14 mg/L emetine after 8 weeks of culture.     

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.     

A study of the production of essential oils in chamomile hairy root cultures.

The active substances in chamomile (Matricaria recutita L.) belong to chemically different structural types. The largest group of medically important compounds forming the essential oils are primarily chamazulene, (-)-alpha-bisabolol, bisabololoxides, bisabolonoxide A, trans-beta-farnesene, alpha-farnesene, spathulenol and the cis/trans-en-in-dicycloethers. Flavonoids, coumarins, mucilages, mono- and oligosaccharides also have pharmacological effects. We studied the production of essential oils in genetically transformed cultures. Sterile juvenile chamomile plants were infected with A4-Y strains of Agrobacterium rhizogenes. They are known plant pathogens and are capable of inducing so-called hairy roots. The transfer DNA segment of the Ri-virulence plasmid of A. rhizogenes becomes integrated in the genome of the plant cells. The isolated hairy roots grow rapidly on hormone-free media. In order to obtain bacteria-free media, we cultured the transformed roots on Murashige-Skoog (MS) medium supplemented with carbenicillin (800 mg/l). To study the production of essential oils, the clones were propagated on liquid and solid MS and Gamborg (B5) media, respectively. According to gas chromatography, the composition of the essential oil of hairy root cultures on different media was found to be similar, but differing in proportion. The main component of the essential oil which was identified by gas chromatography and mass spectrometry was trans-beta-farnesene, as in the intact roots.     

掌叶大黄毛状根培养及培养物中蒽醌类成分分析

大黄是我国特产的世界著名的传统中药，历代本草均有记载，始载于《神农本草经》，“味苦寒，有毒。主下瘀血、血闭、寒热，破征瘕积聚、留饮宿食，荡涤肠胃，推陈致新，通利水谷，调中化食，安和五脏”。作者已于近期报道了天山大黄发根的诱导方法［１］掌叶大黄Ｒｈｅｕ...     

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.     

Quantitative determination of secoiridoid glucosides in in vitro propagated plants of Gentiana davidii var. formosana by high performance liquid chromatography

A simple protocol for in vitro mass propagation of Gentiana davidii var. formosana (Gentianaceae) has been developed. Multiple shoot development was achieved by culturing the stem node explants on Murashige and Skoog (MS) medium supplemented with 4.44 microM N6-benzyladenine (BA). The shoots were multiplied by subculturing on MS medium supplemented with 1.07-10.74 microM alpha-naphthaleneacetic acid (NAA) and 8.88 microM BA. Shoots were rooted on MS basal medium supplemented with various auxins. Shoots rooted on growth regulator-free medium were transferred to peat moss:vermiculite mixture and acclimatized in the growth chamber. The contents of gentiopicroside and swertiamarin, the two important secoiridoid glucosides, in different plant material were determined by high performance liquid chromatography (HPLC). The analysis revealed that the content of gentiopicroside and swertiamarin in the aerial and underground parts of G. davidii var. formosana was higher than the marketed crude drug (underground parts of G. scabra) and varied with the age of the plant.     

The inhibitory effects of ginsenosides on protein tyrosine kinase activated by hypoxia/reoxygenation in cultured human umbilical vein endothelial cells

27 individual ginsenosides and aglycones, together with five extracts from ginseng roots, ginseng leaves, American ginseng roots, American ginseng leaves and non-saponin fraction from roots of Panax ginseng, were tested for their effects on protein tyrosine kinase (PTK) activation induced by an in vitro hypoxia/reoxygenation (H/R) model in cultured human umbilical vein endothelial cells (HUVEC). The results indicated that ginsenoside-Rb1 (3), -Rd (7), -Ra1 (1) and -Ro (27) showed significant inhibitory effects on PTK activation induced by H/R. Dose-response experiments revealed that ginsenoside-Rb1 was the most active compound and it completely blocked PTK activation at a wide range of concentrations. Most protopanaxadiol-type ginsenosides and some protopanaxatriol-type saponins also showed significant effects on PTK activation. However, the crude extracts did not protect against H/R-induced PTK activation.     

Red American ginseng: ginsenoside constituents and antiproliferative activities of heat-processed Panax quinquefolius roots

Red Asian ginseng ( Panax ginseng C. A. Meyer, Araliaceae) is used in many Oriental countries. In this study, the saponin constituents and anticancer activities of steamed American ginseng ( Panax quinquefolius L.) roots were evaluated. The contents of 12 ginsenosides in the roots were determined using high performance liquid chromatography (HPLC). After the steaming treatment (100 - 120 degrees C for 1 h and 120 degrees C for 0.5 - 4 h), the quantity of 7 ginsenosides decreased and that of 5 others increased. The content of ginsenoside Rg3, a previously recognized anticancer compound, increased significantly when the root was steamed at 120 degrees C for 0.5 - 3 h. The antiproliferative effects of unsteamed and steamed (120 degrees C for 1 h and 2 h) American ginseng root extracts were assayed by the modified trichrome stain (MTS) method using three cancer cell lines (SW-480, HT-29, NSCLC). Heat-processing increased the antiproliferative effect of American ginseng significantly, and the activity of the extract from roots steamed for 2 h was greater than that of roots steamed for 1 h. Chemical constituents and antiproliferative activities of white and red Asian ginseng have also been evaluated. Five representative ginsenosides, Rb1, Rd, Re, Rg2 and Rg3, were studied. Ginsenoside Rg3 had the most potent effect. The antiproliferative activities of red American ginseng are augmented when ginsenoside Rg3 is increased.     

Shoot Tip Culture of Arnica montana for Micropropagation

Multiple shoots were regenerated from shoot tips of ARNICA MONTANA on MS and B5 media supplemented with BA (1 mg/l) and NAA (0.1 mg/l). Sections of 1-2 mm in length cultured from IN VITRO germinated seedlings regenerated 7.7 (mean) shoots on the MS medium, whereas sections cultured from greenhouse plants regenerated 9.0 (mean) shoots on the B5 medium within 6 weeks. Subsequent subcultures of shoots on the same media but without NAA resulted in similar or lower multiplication rates (1.6 to 3.1 in 3 weeks). Shoot development was promoted, whereas shoot initiation was simultaneously inhibited by the addition of activated charcoal to the media. Rooting was induced by culturing shoots from seedling as well as from greenhouse plant shoot tips on MS or B5 medium supplemented with NAA. The plantlets were transplanted into soil and grown successfully under greenhouse and field conditions.     

Impact of nutrient components on production of the phytoestrogens daidzein and genistein by hairy roots of <Emphasis Type="Italic">Psoralea corylifolia</Emphasis>

Transformed hairy roots of Psoralea corylifolia were established by infection with Agrobacterium rhizogenes LBA 9402. The aim of this work was to elucidate the effects of media constituents on production of the phytoestrogenic isoflavones daidzein and genistein. A. rhizogenes strain LBA 9402 harboring Ri plasmid was used to transform stem segments of in vitro seedlings. The resultant hairy roots were confirmed by polymerase chain reaction (PCR) and exhibited Ri T-DNA. Transformed hairy root clones were cultured in Murashige and Skoog’s (MS) medium altered with different concentrations of NH₄ ⁺ and NO₃ ⁻ and their growth and production of isoflavones were assessed. Biomass and productivity increased when MS medium was supplemented with NH₄ ⁺ and NO₃ ⁻ at a ratio of 20:10. Increased yield of daidzein was obtained when sucrose level in the culture medium increased, whereas decreased level of sucrose favored genistein production. The hairy roots produced the highest levels of daidzein (2.06% dry wt.) and genistein (0.37% dry wt.) in the presence of low concentrations of PO₄ ³⁻. Hairy roots secreted trace amounts of daidzein and genistein into the culture medium. The present results demonstrated that the productivity of daidzein was 2.2-fold more than that of untransformed roots.     

Genetic transformation of Salvia austriaca by Agrobacterium rhizogenes and diterpenoid isolation

Hairy roots of Salvia austriaca Jacq. transformed with Agrobacterium rhizogenes strain A4 were obtained and transgenic status of the roots was confirmed by polymerase chain reaction (PCR) using rolB and rolC specific primers. The root cultures growing in half-strength Gamborg (1/2 B5) liquid medium supplemented with sucrose (30 g L(-1)) under light conditions (photoperiod: 16 h light/8 h dark) were examined for their ability to produce diterpenoids. From n-hexane extract the abietane-type diterpenoids royleanone, 15-deoxyfuerstione and taxodione were isolated and identified. This is the first report on the genetic transformation of S. austriaca.     

Embryoidogenesis and plant regeneration from leaf tissue of Gloriosa superba

The induction, maturation and germination of embryoids from leaf tissue of Gloriosa superba L. were developed by exploiting solid and liquid culture. Nodular calli were obtained from SH medium supplemented with 2,4-D and 2iP. In solid culture, the nodular calli when transferred to 2,4-D along with glycerol gave the best response (68.4 %) in embryoid induction after 20 days. After two subcultures at 7-day intervals in a medium with thiamine instead of glycerol, the embryoids matured. When mature embryoids were transferred to BAP and IBA medium, they gave rise to plantlets with single shoots and roots. In liquid culture, the medium supplemented with NAA and L-glutamine with continuous agitation, the embryoidogenic calli produced embryoids (85 %) after 21 days. The mature embryoids began to turn green and produced shoots and elongated "radicles" after 35 days.     

人参愈伤组织培养的初步研究

本工作初步研究了人参愈伤组织培养的适宜培养基和培养条件、各种生长物质和补充物质对其生长及其人参皂甙含量的影响。此外,还对不同的无性繁殖系与不同年龄的人参愈伤组织的生长特性,进行了初步的观察。应用薄层层析-光电比色法测定证明:愈伤组织中人参皂甙的成分和含量和栽培的人参根相近;所获得的愈伤组织具有合成人参皂甙的能力。     

Cultures ofGinkgo biloba,effect of nutritional and hormonal factors on the growth of cultured cells derived fromGinkgo biloba

Calli and suspension cultures were obtained following inoculation of the explant from leaves ofGinkgo biloba L. on the supplemented MS basal medium. The obtained calli and suspension cultured cells were able to produce detectable amounts of ginkgolides which are known as natural specific PAF antagonists. The production of ginkgolides in the calli and suspension cultured cells were identified using GC/MS, GC and HPLC with authentic compounds. Since the production of ginkglides A and B in the calli and suspension cultured cells had been confirmed, effects of types and concentration of plant growth regulators, media and illumination on the induction and growth of the callus were studied. The concentrations of growth regulators for optimal callus induction were 1.0 to 2.0 mg/L for NAA and 0.1 mg/L for kinetin. The growth of the callus seemed to be more stimulated with the combination of NAA and kinetin than NAA and BA with illumination at all concentration ranges of 1.0 to 4.0 mg/L for NAA and 0.1 to 1.0 mg/L for kinetin or BA studied. Among 8 different media used, the induction rate of callus on Anderson, Eriksson, and Schenk and Hildebrant at 4 weeks after the innoculation was almost the same as that of MS. However, callus was rarely induced on Heller or White medium. Suspension cultures were easily initiated with 3 g of callus (fresh weight) derived from ginkgo leaves on supplemented MS medium. A typical growth curve of suspension cultured cells could be obtained by measuring the fresh weight of the suspension cultured cells at every 3 days. To improve the growth of suspension cultured cells of ginkgo, effects of concentrations of NAA, sucrose, phosphate ions and molar ratio of NH₄ ⁺ to NO₃ ^? ions in the culture medium were studied. The maximum growth of the cells was achieved when the culture medium contained 1.0 mg/L of NAA, 30 g/L sucrose, 1.75 mM phosphate ions and 1∶5 molar ratio of NH₄ ⁺ to NO₃ ^? ions.     

Production of Forskolin by Axenic Coleus forskohlii Roots Cultivated in Shake Flasks and 20-l Glass Jar Bioreactors*

Root cultures of COLEUS FORSKOHLII Briq. were initiated from primary callus or IBA-treated suspension cultures and maintained on Gamborg's B5 medium containing 1 mg/l IBA. Transformed root cultures were established by infecting surface-sterilized leaves with AGROBACTERIUM RHIZOGENES strain 15834. Transformation was confirmed by mannopine detection. These cultures displayed the typical characteristics of hairy root cultures, with the sole exceptions of slow growth in hormone-free medium and accelerated growth on medium containing phytohormones. All root cultures examined formed forskolin and its derivatives in amounts ranging from 500 to 1300 mg/kg dry weight, corresponding to about 4 to 5 mg/l. During cultivation roots could be cut into small pieces without affecting growth and forskolin production. Scale-ups of the cultivation procedure were performed in 20-l glass jars with a working volume of 10 to 13l. Forskolin production in bioreactors was better than in shake flasks. Levels of almost 14 mg/l could be reached after 21 d of cultivation. As in the shake flask experiments cutting the roots did not affect growth or productivity in a negative way.     

Ginsenosides-induced nitric oxide-mediated relaxation of the rabbit corpus cavernosum.

1. Ginsenosides, the active ingredients extracted from Panax ginseng, have been shown to promote nitric oxide (NO) release in bovine aortic endothelial cells. Since the endothelial cells and the perivascular nerves in penile corpus cavernosum contain NO synthase and an NO-like substance has been shown to be released from these cells which relaxes corpus cavernosum, the possibility that ginsenosides may relax corpus cavernosum by releasing endogenous NO was examined. 2. With an in vitro tissue superfusion technique, ginsenosides (250, 500 and 750 micrograms ml-1) relaxed corpus cavernosum, concentration-dependently. 3. Using an in vitro tissue bath technique, acetylcholine (ACh)-induced relaxations were increased in the presence of ginsenosides (250 micrograms ml-1). 4. Ginsenosides at 100 micrograms ml-1 significantly enhanced the tetrodotoxin (TTX)-sensitive relaxation of corpus cavernosum elicited by transmural nerve stimulation. 5. The ginsenosides-induced, ACh-induced and ginsenosides-enhanced transmural nerve stimulation-elicited relaxations were significantly attenuated by NG-nitro-L-arginine (100 microM) and oxyhaemoglobin (oxyHb; 5-10 microM), and were enhanced by superoxide dismutase (SOD; 50 u ml-1). 6. The relaxations and their attenuation by NG-nitro-L-arginine and TTX were associated with increase and decrease in tissue cyclic GMP levels, respectively. 7. It is concluded that ginsenosides may release NO from endothelial cells, and enhance NO release from endothelial cells elicited by other vasoactive substances and from perivascular nitrergic nerves in the corpus cavernosum. These endothelial and neurogenic effects of ginsenosides in inducing relaxation of the corpus cavernosum may account for the aphrodisiac effect of Panax ginseng.     

Protective effect of fermented Red ginseng on a transient focal ischemic rats

Red ginseng and fermented red ginseng were prepared, and their composition of ginsenosides and antiischemic effect were investigated. When ginseng was steamed at 98-100 degrees C for 4 h and dried for 5 h at 60 degrees C, and extracted with alcohol, its main components were ginsenoside Rg3> ginsenoside Rb1 > ginsenoside Rb2. When the ginseng was suspended in water and fermented for 5 days by previously cultured Bifidobacterium H-1 and freeze-dried (fermented red ginseng), its main components were compound K > ginsenoside Rg3 > or = ginsenoside Rh2. Orally administered red ginseng extract did not protect ischemia-reperfusion brain injury. However, fermented red ginseng significantly protected ischemica-reperfusion brain injury. These results suggest that ginsenoside Rh2 and compound K, which was found to be at a higher content in fermented red ginseng than red ginseng, may improve ischemic brain injury.