Effects of Age and Growth Regulators on Growth and Alkaloid Production in Cinchona ledgeriana Leaf-Shoot Organ Cultures

CINCHONA LEDGERIANA Moens et Trimen leaf-shoot organ cultures established and maintained on Murashige and Skoog's medium containing benzyladenine (BA 5 mg/l) were used to study the effects of age and growth regulators on alkaloid production. The total and individual alkaloid content of the leaf-shoot organ cultures increased with age and resembled closely that of the 1-year-old plant which favored quinine production. The 32-week-old tissue cultures contained the same amount of alkaloid as that of the 1-year-old plant (350 mg%). Quinine production was favored by the presence of benzyladenine (5 mg/l), gibberellic acid (5 mg/l) and/or shoots. Quinidine production was high in the presence of indole-3-acetic acid (5 mg/l), the absence of benzyladenine, and/or the presence of roots. High concentrations of abscisic acid and mefluidide inhibited growth and alkaloid production.     

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.     

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.     

Production of triterpenoids in liquid-cultivated hairy roots of Galphimia glauca

The production of three triterpenoids from Galphimia glauca hairy root cultures, the sedative principle galphimine E (2), the recently described glaucacetalin A (3), and maslinic acid (6), was quantified by HPLC in the biomass and the culture medium. Batch cultures of the hairy root line VYT, obtained through infecting cotyledons with Agrobacterium rhizogenes ATCC 15 834, were grown for 41 days in shake flasks containing B5 medium without phytohormones. A maximum biomass of 11 g/L DW was obtained on day 33, while the doubling time was 6 days. Throughout the growth cycle fresh and dry weights as well as triterpene production were registered. Glaucacetalin A (3), excreted into the culture media, reached a maximum amount of 2.14 mg/L after 21 days while galphimine E (2) and maslinic acid (6) were recovered from the root biomasses reaching maximum concentrations of 0.11 and 0.43 mg/g, respectively, on day 39.     

Growth and production of camptothecin by cell suspension cultures of Nothapodytes foetida

Callus cultures were initiated from stem parts of Nothapodytes foetida on Murashige and Skoog's medium supplemented with different growth regulators. Suspension cultures were established and the cell biomass was higher in the presence of NAA in comparison with 2,4-D. Culture medium supplemented with NAA (10.74 microM) and BA (2.22 microM) attained 31.3 g/l DW during 20 days of cultivation in shake flasks. In the presence of NAA, maximum concentrations of camptothecin (0.035 mg/ml) and 9-methoxycamptothecin (0.026 mg/ml) were found in the medium. Alkaloid production was reduced in presence of 2,4-D in the culture medium. Cells contained trace amount of alkaloids. Alkaloids were detected and identified by means of TLC and HPLC.     

Somatic embryogenesis and rhizogenesis of tissue cultures of two genotypes of Papaver somniferum: relationships to alkaloids production

PAPAVER SOMNIFERUM L. tissue cultures, issued from various explants (cotyledons, hypocotyls, roots) derived from plantlets belonging to two genotypes, were established on LS solid medium containing growth regulators (NAA, Kin) in various combinations. Hypocotyls and roots were found to be interesting explants to obtain cellular development. Many roots developed on calli growing on a medium containing NAA (1 mg/l) + Kin (0.1 mg/l) for the PS genotype while somatic proembryos redifferentiated on calli issued from PS 1639 genotype. The same growth substance combination was the most favourable for the production of morphinan alkaloids and papaverine: up to 10 x 10 (-3)% DW in roots redifferentiated from PS calluses.     

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。     

Production of amarogentin in root cultures of Swertia chirata

Conventional and Agrobacterium rhizogenes-transformed root cultures were studied with respect to growth and amarogentin content following cultivation in various growth media. The fastest growth rate was observed using Nitsch medium. The best amarogentin content was obtained after cultivation in root culture (RC) medium for which the slowest growth rate was noticed. Addition of sucrose at 6% and 9% (w/v), respectively, also resulted in better growth rates and increased total but unaltered relative amarogentin content compared to 3% (w/v) sucrose. No change in amarogentin content was observed upon addition of elicitors, putative precursors of amarogentin biosynthesis, and plant growth hormones with the exception of salicylic acid and chitosan: at 100 mM salicylic acid a reduction and at 25 mg/L chitosan an increase of amarogentin were observed at significant levels. The cultivation of S. chirata roots in a 2-L stirred-tank bioreactor was successful only with a stainless-steel mesh fitted inside the culture vessel for immobilization of the roots. A 15-fold enhancement of amarogentin content in the medium was achieved by a root permeabilisation treatment using Tween 20 at 1.3% (v/v) final concentration in the bioreactor.     

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.     

Withanolide production by root cultures of Withania somnifera transformed with Agrobacterium rhizogenes

Transformed root cultures of Withania somnifera Dunal (Solanaceae) were obtained by infecting shoots cultured in vitro with Agrobacterium rhizogenes LBA 9402. They grew axenically in the absence of exogenous plant growth regulators in Murashige and Skoog's medium containing 3% (w/v) sucrose. The root cultures synthesized several withanolides of which withanolide D was isolated and identified. Transformed root (clone HR (1)) showed a growth index of 20.07 and a withanolide D yield of 0.30 mg g(-1) DW. The productivity of withanolide D in transformed roots (0.181 mg 1(-1) d(-1)) was higher than in untransformed root cultures (0.026 mg 1(-1) d(-1)).     

Production of Taxol and its Analogues from Cell Cultures of Taxus wallichiana

Callus cultures of Taxus wallichiana young leaves were established on Gamborg’s (B5) medium supplemented with dicamba (2 mg/l), benzyladenine (0.2 mg/l) and glutamine (292 mg/l). The cell cultures were initiated and maintained on B5 medium supplemented with NAA (1mg/l) and benzyladenine (0.02 mg/l). 0.25 B5 medium supplemented with 2,4-D (2 mg/l) and zeatin (0.5 mg/l) was found to be suitable as production medium. On HPLC analysis using photodiode array detector, the extracts from cell cultures showed the presence of taxanes, viz., taxol, deacetyl baccatin III (DAB) and baccatin III (BA). Precursors and growth retardants significantly improved the production of taxol, DAB and BA. Phenylalanine (1 mM), sodium benzoate (0.2mM), hippuric acid (1 mM) and leucine (1 mM) addition enhanced the accumulation of taxol (6, 4.5, 25 and 2 times, respectively), DAB (7, 9.5, 7.5 and 3 times, respectively) and BA (36, 16, 57 and 2 times, respectively) in cultures grown in production medium. Ancymidol (50µM) and 2-chloroethyl phosphonic acid (50µM) addition has shown significant increase in bioproduction of taxol (3 and 11 times, respectively), DAB (3 and 1.5 times, respectively) and BA (16 and 9 times, respectively). However, chlorocholine chloride (1mM) improved the production of DAB (2.5 times) and taxol (1.7 times) only.     

In Vitro.–Enhanced Production of Podophyllotoxin in Phytohormonal-Induced and Regenerated Roots of Podophyllum hexandrum.

ABSTRACT

Sixty-day-old in vitro–induced roots and roots regenerated from totipotent calli of Podophyllum hexandrum Royle produced an enhanced quantity of podophyllotoxin. Induced root cultures originated from aseptically germinated seedlings, and regenerated roots were established on both Gamborg's B5 and Murashige and Skoog media. The podophyllotoxin content was compared with normal plant root/rhizomes by HPLC. The highest values, both in growth/proliferation and in podophyllotoxin production, were obtained using Gamborg's B5 medium.     

Comparative study of the effects of various culture conditions on cell growth and Gagaminine synthesis in suspension culture of Cynanchum wilfordii (MAXIM.) HEMSLEY

Gagaminine, a steroidal alkaloid isolated from the roots of Cynanchum wilfordii, exhibited potent inhibitory effects on aldehyde oxidase activity and lipid peroxidation. To determine whether it would be possible to mass produce this active component, which would be useful for animal tests, we tried to synthesize it using in vitro cell culture methods with various growth conditions. In a previous study it was found that calli were easily induced from the stem of this medicinal plant and cultivated effectively on MS medium containing 2,4-dichlorophenoxyacetic acid (2,4-D) 2 mg/l. In this work we attempted to determine the effects of various culture conditions on cell growth and gagaminine synthesis in suspension culture. Gagaminine production was increased markedly when cell growth proceeded to the death phase. Cell growth was more effective with 5% (v/v) sucrose, in the light (at 38 microE/m(2) x s), on medium containing 2,4-D 2 mg/l, with 2.5 g/10 ml medium as the initial cell concentration. The concentration of gagaminine was optimal with 3% sucrose, in darkness on medium 2,4-D 1 mg/l, with 2.5 g/10 ml medium as an initial cell concentration. However, the highest growth rate was 0.18 d(-1), when the gagaminine concentration was seven- and three-fold (at 140 mu/ml) that of the plant stem and 10 ml of medium respectively, on the 50 ml of medium in suspension culture.     

Clonal propagation of Isoplexis canariensis

Isoplexis is a plant genus closely related to Digitalis. Members of this genus contain cardenolides considered more "primitive" than those present in Digitalis. Isoplexis plants, tissue cultures, and isolated cardenolides may thus be used to elucidate the biosynthesis of cardenolides in the Scrophulariaceae. Therefore, a method was developed to cultivate and propagate Isoplexis canariensis (L.) Lindl. ex. G. Don in vitro. Seeds were germinated in liquid modified MS medium and shoot cultures were established and propagated in liquid modified MS medium containing 0.1 mg/l IAA and 1 mg/l BAP. Shoot cultures were also established from excised axillary buds and propagated on solid culture medium containing 0.1 mg/l IAA and 1 mg/l BAP. Shoots of either origin were rooted in medium containing 1 to 5 mg/l IAA and 0.5 to 4 mg/l IBA. Rooted plantlets were cultivated for 2 to 3 weeks in hormone-free modified MS medium and then transferred to the greenhouse, where they developed into healthy plants.     

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.     

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.     

In vitro plantlet regeneration in Panax sikkimensis

A three-step procedure for complete plantlet regeneration via somatic embryogenesis has been developed in Panax sikkimensis. Somatic embryos (SE) were induced in root callus upon lowering the level of 2,4-D from 1.0 mg/l to 0.25 mg/l in the callusing medium. Maturation of SE occurred on a half-strength MS medium with 0.5 mg/l each of BAP and GA3. An exposure for 15 days of cotyledonary and heart-shaped SE to 1.0 mg/l IBA in liquid shake 1/2 MS medium significantly improved the rate of embryo-to-plantlet conversion and plantlet quality. The procedure has now allowed the retention of high regeneration potential of the root callus for over three years.     

不同诱导子对黄芩不定根生长及黄芩苷累积的影响

目的研究蔗糖、茉莉酸甲酯和水杨酸3种诱导子对黄芩不定根生长及黄芩苷累积的影响。方法向黄芩不定根悬浮培养液中添加不同种类及浓度的诱导子,观察不定根生长情况并测量不定根的生物量及黄芩苷的含量。结果培养基中蔗糖浓度为50 g·L^-1时可使不定根生物量达到对照组（30 g·L^-1）的1.62倍,黄芩苷的含量达到对照组的1.71倍为20.81 mg·g^-1。加入44.860 mg·L^-1茉莉酸甲酯可使黄芩苷的含量为对照组的3.22倍,达到最高39.14 mg·g^-1,但却大大影响黄芩不定根的产量。低浓度水杨酸对不定根生长及黄芩苷累积均有利。当其浓度为4 mg·L^-1时,不定根的生物量及黄芩苷含量均达到最大。结论在黄芩不定根悬浮培养中加入适量的蔗糖、茉莉酸甲酯及水杨酸,对不定根的生长及黄芩苷的合成有一定的促进作用。     

Improved production of caffeic acid derivatives in suspension cultures ofEchinacea purpurea by medium replenishment strategy

The aim of this study was to produce caffeic acid derivatives from adventitious root cultures of Echinacea purpurea, which are of high pharmaceutical value. The effects of both media optimization and replenishment strategies were adopted to achieve improved production of E. purpurea adventitious roots and caffeic acid derivatives. Of the different media strengths (0.25 MS, 0.5 MS, 0.75 MS and 1 MS) tested for the culturing of adventitious roots in 5 L bioreactors, 0.5 MS medium was found to be most suitable for the growth of adventitious roots. The optima accumulation of biomass (73.6 g L(-1) FW and 10.03 g L(-1) DW), phenolics (61.14 mg g(-1) DW) and flavonoids (38.30 mg g(-1) DW) were achieved in this medium. Furthermore, fed batch cultivations (media replenishment with 0.25 MS, 0.5 MS, 0.75 MS and 1 MS at the end of 2nd and 3rd weeks) to further enhance the production of adventitious root biomass and metabolites were also attempted. High adventitious root biomasses (83.1 g L(-1) FW and 15.30 g L(-1) DW) were achieved with feeding of the 0.5 MS medium at the end of 2nd week. This led to slight decreases in the total production of phenolics and flavonoids; however, this feeding was responsible for increases in the accumulation of caftaric acid (5.76 mg g(-1) DW) and cichoric acid (26.12 mg g(-1) DW).     

The effect of creosote on the growth of an axenic culture of Myriophyllum spicatum L

Due to the importance of submersed, rooted macrophytes to the aquatic ecosystem and the use of creosote impregnated structures adjacent to or within water bodies, a study was conducted using an axenic culture of Myriophyllum spicatum to determine the effect of creosote on this aquatic macrophyte. Four plants were cloned and exposed to nominal creosote concentrations ranging from 0.16 to 200 mg/l for 14 days. A variety of response parameters were assessed, including shoot and root length, number of roots and nodes, and dry weight biomass, as well as visual observations on plant colouring and morphology. Regression and ANOVA analyses were conducted to determine EC50s and significant differences. Biphasic responses were observed for shoot length, node production and biomass, with shoot length showing statistically significant stimulation (hormesis) at creosote concentrations below 13.3 mg/l. EC50 values of 55.1 (CI 40-60) mg/l, 33.4 (CI 26-48) mg/l and 86 (CI 70-120) mg/l were determined for shoot length, dry weight and node production, respectively. Root number was significantly higher at 3.6 mg/l and root length was significantly reduced at 4.5 mg/l creosote, within the concentration range that stimulated shoot growth. Visual changes, including an increase in pink colouration and changes in the location of root initiation, were also observed in the same creosote concentration range that affected root length and numbers. Therefore, it appears that changes in root growth and location of root initiation may be the most sensitive endpoints for creosote effects on Myriophyllum.