Anthraquinones in cell suspension cultures of Morinda citrifolia

From cell suspension cultures of Morinda citrifolia five known anthraquinones, rubiadin, lucidin, morindone, lucidin-3-β-primeveroside and morindone-6-β-primeveroside, and seven new anthraquinones were isolated. Six of the seven new quinones were characterized as 2-methyl-3,5,6-trihydroxyanthraquinone, 3-hydroxymorindone, 5,6-dihydroxylucidin, 2-methyl-3,5,6-trihydroxyanthraquinone-6-β-primeveroside, 3-hydroxymorindone-6-β-primeveroside and 5,6-dihydroxylucidin-3-β-primeveroside, respectively.     

Production of adventitious root biomass and secondary metabolites of Hypericum perforatum L. in a balloon type airlift reactor

The effects of inoculum density, aeration volume and culture period on accumulation of biomass and secondary metabolites in adventitious roots of Hypericum perforatum in balloon type airlift bioreactors (3 l capacity) were investigated. The greatest increment of biomass as well as metabolite content occurred at an inoculum density of 3 g l⁻¹ and an aeration volume of 0.1 vvm. After 6 weeks of culture, an approximately 50-fold increase in biomass was recorded containing 60.11 mg g⁻¹ dry weight (DW) of phenolics, 42.7 mg g⁻¹ DW of flavonoids and 0.80 mg g⁻¹ DW of chlorogenic acid. Liquid chromatography–mass spectroscopy/mass spectroscopy demonstrated that the presence of quercetin and hyperoside in adventitious roots at a level of 1.33 and 14.01 μg g⁻¹ DW, respectively after 6 weeks of culture. The results suggest scale-up of adventitious root culture of H. perforatum for the production of chlorogenic acid, quercetin and hyperoside is feasible.     

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

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

海巴戟(Morinda citrifolia L.)组织培养研究

以海巴戟（Morinda citrifolia L．）种子为试材,在不剥除种皮的情况下,在MS无激素培养基上播种1年内未见发芽,在剥除种皮的情况下,在MS无激素培养基上发芽率最高,50d内可达75％。海巴戟子叶和下胚轴均能单独由细胞分裂素BA0．7～2．0mg／L诱导不定芽发生,不定芽可直接从外植体发生,也可从愈伤组织发生,添加生长素NAA0．05～0．1mg／L则完全抑制不定芽发生,同时强烈促进愈伤组织生长和不定根发生。带芽茎段在BA1．5mg／L配合低浓度生长素时均能通过腋芽萌发和不定芽发生而增殖。芽梢在NAA0．1mg／L、IBA0．1mg／L或IAA0．1mg／L均有根群发生,但NAA0．1mg／L诱导生根时切口愈伤组织较多,部分不定根由愈伤组织发生。而IAA0．1mg／L诱导生根时根群欠发达,以IBA0．1mg／L最佳。     

Improvement of biosynthesis and accumulation of bioactive compounds by elicitation in adventitious root cultures of Polygonum multiflorum

Applied Microbiology and Biotechnology - We examined the effects of abiotic (methyl jasmonate [MeJA] and salicylic acid [SA]) and biotic (yeast extract and chitosan) elicitors for improvement of...     

Application of an airlift bioreactor system for the production of adventitious root biomass and caffeic acid derivatives of Echinacea purpurea

In this study, we evaluated the feasibility of using mass cultivation of the adventitious roots of Echinacea purpurea in balloon type bubble (air-lift) bioreactors to produce caffeic acid derivatives, which have pharmaceutical and therapeutic values. An approximately 10 fold increase in biomass and secondary compounds was observed after 4 weeks of culture in balloon type bubble bioreactors (5 L capacity containing 4 L of half strength MS medium). In addition, a linear relationship was observed between the concentration of biomass and the sucrose and ion consumption rate. Furthermore, the concentration of biomass in the bioreactor culture was found to increase as the conductivity decreased. An inoculum density of 7 g/L FW and an aeration rate of 0.1 vvm were found to be suitable for inducing the accumulation of biomass and secondary metabolites. Of the three caffeic acid derivatives evaluated (caftaric acid, chlorogenic acid, and cichoric acid), the concentration of cichoric acid was the highest (26.64 mg/g DW).     

Production of biomass and useful compounds from adventitious roots of high-value added medicinal plants using bioreactor

The increasing global demand for biomass of medicinal plant resources reflects the issues and crisis created by diminishing renewable resources and increasing consumer populations. Moreover, diverse usage of plants and reduced land for cultivation in the world accelerated the deficiency of plant resources. In addition, the preparation of safety of plant based medicine whips up demand for biomass of valuable medicinal plants. As one of alternative approach to upswing the productivity of plant-based pharmaceutical compounds, automation of adventitious root culture system in air-lift bioreactor was adopted to produce cosmic amount of root biomass along with enriched diverse bioactive molecules. In this review, various physiological, engineering parameters, and selection of proper cultivation strategy (fed-batch, two-stage etc.) affecting the biomass production and secondary metabolite accumulation have been discussed. In addition, advances in adventitious root cultures including factors for process scale-up as well as recent research aimed at maximizing automation of the bioreactor production processes are also highlighted. Examples of the scale-up of cultures of adventitious roots of Morinda citrifolia, Echinacea purpurea and angustifolia, Hypericum perforatum and Panax ginseng by applying 20L to 10,000L bioreactors in our lab were demonstrated with a view of commercial application.     

Effects of cytokinin on adventitious root formation in callus cultures ofVigna unguiculata (L.) walp

Summary Yellowish compact callus, induced from cowpea hypocotyls on Murashige and Skoog(MS) medium (1962) containing 0.2 mg/l(0.93 μM) kinetin and 0.4 mg/l (1.81 μM) 2,4-dichlorophenoxyacetic acid (2,4-D), was subcultured on MS medium containing cytokinin alone, auxin alone, or auxins plus cytokinins in order to determine the effect of cytokinins on root organogenesis in callus cultures. The callus actively proliferated on the same medium but did not show any organogenic activity macroscopically as well as microscopically. On medium with N⁶-benzyladenine (BA) and 1-naphthaleneacetic acid (NAA), the yellowish compact callus first changed to pale green compact callus and then many green spots appeared on its surface under light culture. But the yellowsih compact callus remained yellowish and white spots appeared on its surface in dark culture. These spots gradually became white nodular structures. Adventitious root formation from the nodular structures occurred not only on the same medium, but also on medium with either auxin or cytokinin but not both. Yellowish compact callus on medium with auxin alone was transformed to yellowish friable callus, which did not develop adventitious roots. The yellowish friable callus could gain rhizogenic activity only after morphological modification to pale green compact callus on medium with auxin plus cytokinin. The modified callus did not form adventitious roots on medium with auxins but only with cytokinins. Therefore, it is suggested that cytokinins have stimulating effects on root formation from callus that previously did not show rhizogenic activity on medium with auxins alone. In addition, the rhizogenic potential of cowpea callus was discriminated from that of leaf explants, which formed adventitious roots directly on medium with auxin alone.     

Scale-up of adventitious root cultures of Echinacea angustifolia in a pilot-scale bioreactor for the production of biomass and caffeic acid derivatives

In an attempt to scale-up of adventitious root cultures of Echinacea angustifolia for the production of biomass and caffeic acid derivatives, i.e. echinacoside, chlorogenic acid, cichoric acid, caftaric acid, and cynarin, the effects of Murashige and Skoog (MS) medium dilutions, and initial sucrose concentrations were investigated in a 5-L airlift bioreactor. In addition, the kinetics of adventitious root growth and accumulation of secondary metabolites were also studied. The greatest root dry weight (6.50 g L^?l) and accumulation of total phenolics [22.06 mg g^?1 DW (dry weight)], total flavonoids (5.77 mg g^?1 DW) and total caffeic acid derivatives (10.63 mg g^?1 DW) were obtained at quarter-strength MS medium. Of the various gradients of sucrose tested, 5 % sucrose supplementation was regarded as an optimal concentration for enhancing productivity of biomass and bioactive compounds. Neither higher salt strength (3/4–2 MS) nor sucrose concentrations (7 and 9 %) showed promotive effect on root growth and metabolite production. The kinetic studies revealed that 4 weeks of culture period is the optimal time to achieve highest productivity of metabolites. Based on these results, a large-scale (20 L) and a pilot-scale (500 L) adventitious root culture system was established. In the pilot-scale bioreactor, adventitious roots were elicitor-treated with 100 μM methyl jasmonate (MJ) on day 28. After 1 week of elicitation, 1.75 kg dry root biomass was harvested containing 60.41 mg g^?1 DW of total phenolics, 16.45 mg g^?1 DW of total flavonoids, and 33.44 mg g^?1 DW of total caffeic acid derivatives. Among the caffeic acid derivatives, the accumulation of echinacoside (the major bioactive compound) in MJ-treated adventitious roots grown in the 500-L bioreactor was the highest (12.3 mg g^?1 DW), which is approximately threefold more than the non-MJ-treated roots cultured in 5- and 20-L bioreactors.     

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

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

Production of hairy root cultures of lettuce (Lactuca sativa L.)

Hairy root cultures of lettuce (Lactuca sativa L.) were obtained by inoculation of cotyledonary leaves of in vitro lettuce seedlings (cvs. Nansen and Ljubljanska ledenka) with Agrobacterium rhizogenes A4M70GUS. Approximately in 96.7% cvs. Nansen and in 91.2% Ljubljanska ledenka inoculated explants produced hairy root when they were incubated on Murashige and Skoog (MS) half-strength medium without plant growth regulators. A total of 54% of all hairy root cultures expressed GUS activity. Every hairy root represented an independent transformation event. Line Ljubljanska ledenka 18 showed the highest biomass (5.5 times the biomass of control root). A PCR analysis of the genomic DNA confirmed the presence of marker and target genes in 15 hairy roots examined.     

Effect of explant type and genotype on the accumulation of bioactive compounds in adventitious root cultures of Polygonum multiflorum

Plant Cell, Tissue and Organ Culture (PCTOC) - The correlation between growth and accumulation of bioactive compounds was investigated in six genotypes of Polygonum multiflorum, grown in vitro,...     

Spectral lights trigger biomass accumulation and production of antioxidant secondary metabolites in adventitious root cultures of Stevia rebaudiana (Bert.)

Stevia rebaudiana (S. rebaudiana) is the most important therapeutic plant species and has been accepted as such worldwide. It has a tendency to accumulate steviol glycosides, which are 300 times sweeter than marketable sugar. Recently, diabetic patients commonly use this plant as a sugar substitute for sweet taste. In the present study, the effects of different spectral lights were investigated on biomass accumulation and production of secondary metabolites in adventitious root cultures of S. rebaudiana. For callus development, leaf explants were excised from seed-derived plantlets and inoculated on a Murashige and Skoog (MS) medium containing the combination of 2,4-dichlorophenoxy acetic acid (2, 4-D, 2.0 mg/l) and 6-benzyladenine (BA, 2.0 mg/l), while 0.5 mg/l naphthalene acetic acid (NAA) was used for adventitious root culture. Adventitious root cultures were exposed to different spectral lights (blue, green, violet, red and yellow) for a 30-day period. White light was used as control. The growth kinetics was studied for 30 days with 3-day intervals. In this study, the violet light showed the maximum accumulation of fresh biomass (2.495 g/flask) as compared to control (1.63 g/flask), while red light showed growth inhibition (1.025 g/flask) as compared to control. The blue light enhanced the highest accumulation of phenolic content (TPC; 6.56 mg GAE/g DW), total phenolic production (TPP; 101 mg/flask) as compared to control (5.44 mg GAE/g DW; 82.2 mg GAE/g DW), and exhibited a strong correlation with dry biomass. Blue light also improved the accumulation of total flavonoid content (TFC; 4.33 mg RE/g DW) and total flavonoid production (TFP; 65 mg/flask) as compared to control. The violet light showed the highest DPPH inhibition (79.72%), while the lowest antioxidant activity was observed for control roots (73.81%). Hence, we concluded that the application of spectral lights is an auspicious strategy for the enhancement of the required antioxidant secondary metabolites in adventitious root cultures of S. rebaudiana and of other medicinal plants.     

Production of nematocidal compounds by hairy root cultures of Tagetes patula L.

Summary Marigold (Tagetes patula L.) hairy roots induced by infection with Agrobacterium rhizogenes produced -terthienyl when grown in darkness, and an n-hexane extract of the roots showed nematocidal activity. Depending on the hairy root line used, the level of -terthienyl varied from 15 to 1268 g per g dry weight, a level that corresponded to 0.15 to 12.7-fold that in intact roots. Analysis by HPLC indicated that the nematocidal activity was due predominantly to -terthienyl. However, it is suggested that nematocidal compounds other than -terthienyl are present in hairy roots cultured in the dark for long periods or in the light.     

Focussed beam reflectance measurement (FBRM) monitoring of particle size and morphology in suspension cultures of Morinda citrifolia and Centaurea calcitrapa

Laser light scattering technology, as applied in the Lasentec focussed beam reflectance measurement (FBRM) system, was used to characterise two morphologically dissimilar plant cell suspension cultures, Morinda citrifolia and Centaurea calcitrapa. Shake-flask suspensions were analysed in terms of biomass concentration and aggregate size/shape over the course of typical batch growth cycles. For the heavily aggregated C. calcitrapa, biomass levels [from 10-160 g fresh weight (fw) l(-1))] were linearly correlated with FBRM counts. For M. citrifolia, which grows in unbranched chains of 2-10 elongated cells, linear correlation of biomass concentration with FBRM counts was applicable in the range 0-100 g fw l(-1); at higher levels (100-300 g fw l(-1)), biomass was non-linearly correlated with FBRM counts and length-weighted average FBRM chord length. For both cell systems, particle morphology (size/shape) was quantified using semi-automated digital image analysis. The average aggregate equivalent diameter (C. calcitrapa) and average chain length (M. citrifolia), determined using image analysis, closely tracked the FBRM average chord length. The data clearly demonstrate the potential for applying the FBRM technique for rapid characterisation of plant cell suspension cultures.     

Bacoside production by suspension cultures of Bacopa monnieri (L.) Pennell

Cell suspension cultures of Bacopa monnieri (L.) Pennell, grown in modified MS medium, grew some 5–6 fold over 40 days. Selected cell lines produced the important saponin, bacoside A, up to 1 g/100 g dry wt after this time.     

Synergistic effect of methyl jasmonate and cyclodextrins on anthraquinone accumulation in cell suspension cultures of Morinda citrifolia and Rubia tinctorum

Plant in vitro culture is a platform for producing secondary metabolites that combines safety, quality and low environmental impact. Besides, it is possible to increase the accumulation of these compounds by different strategies, such as elicitation. In this work, we analyzed the effects of the combination of methyl jasmonate (MeJ) and two cyclodextrins (CDs) on the production of anthraquinones (AQs) in cell cultures of Rubiaceae (Morinda citrifolia and Rubia tinctorum). These secondary metabolites have been traditionally used as dyes and have interesting therapeutic applications. The experiments were designed according to a full factorial design of two factors (MeJ and a CD) in two levels (0 and 0.1 mM for MeJ, and 0 and 20 mM of the CD). MeJ and (2-hydroxypropyl)-β-cyclodextrin (HPCD) synergistically increased intracellular AQ content in suspension cultures of R. tinctorum, and, to a lesser extent, in suspension cultures of M. citrifolia. Combination of MeJ with another CD, 2-methyl-β-cyclodextrin, led to a more intense and later increase in AQ content in cell cultures of R. tinctorum when compared to MeJ–HPCD treatment. However, the combination of CD and MeJ failed to induce a drastic AQ release to the culture media. Nevertheless, our results show that combination of strategies (using a CD and MeJ) was successful to increase secondary metabolite accumulation in suspension cultures. To our knowledge, this is the first report of synergistic effect of MeJ and CD on AQ accumulation in plant in vitro cultures.

     

Production of tropane alkaloids by small-scale bubble column bioreactor cultures of Scopolia parviflora adventitious roots

The mass production of tropane alkaloids from adventitious root cultures of Scopolia parviflora, in small-scale bubble column bioreactor (BCB) was attempted. Adventitious roots of S. parviflora produced relatively enhanced levels of scopolamine and hyoscyamine in bioreactor compared to flask type cultures, and rapidly produced root clumps, with continuously increasing biomass throughout the culture period. The production of scopolamine and hyoscyamine in the top and bottom regions of root clumps were higher than in the core region. The adventitious root cultures of S. parviflora in the BCB required a relatively high level of aeration. The optimized conditions for the bioreactor culture growth and alkaloid production were found to be 3g of inoculum, on a fresh weight basis, a 15-day culture period and 0.4vvm of airflow. The elicitation by Staphylococus aureus increased the specific compound of scopolamine, while the production of hyoscyamine was slightly inhibited in BCB cultures.