Effect of sugar concentration on ginsenoside biosynthesis in hairy root cultures of Panax quinquefolium cultivated in shake flasks and nutrient sprinkle bioreactor

The study assessed the influence of sugar concentration (10, 20, 30, 50, 70, 100, 120 g l^?1) on growth and ginsenoside biosynthesis in Panax quinquefolium hairy roots cultivated in shake flasks and a nutrient sprinkle bioreactor. The highest growth rate was achieved in medium containing 3–5 % sucrose. More than 70 g l^?1 or less than 20 g l^?1 sugar content in the medium induces significant inhibition of root growth when cultivated in shake flasks. The saponin content was determined using HPLC. The maximum yield (above 9 mg g^?1 d.w.) of the sum of six examined ginsenosides (Rb₁, Rb₂, Rc, Rd, Re and Rg₁) in hairy roots cultivated in shake flasks was obtained with 30 g l^?1 sucrose in the medium. The sucrose concentration in the medium was found to correlate with saponin content in bioreactor-cultured specimens. A higher level of protopanaxadiol derivatives was found for lower (20 and 30 g l^?1) sucrose concentrations; higher sucrose concentrations (50 and 70 g l^?1) in the medium stimulated a higher level of Rg group saponins.     

Enhanced production of hairy root metabolites using microbubble generator

Previously, increased partitioning of the natural product nicotine from tobacco hairy roots into the culture media was achieved by altering the expression of the nicotine uptake permease gene. The present study demonstrated that further increases in nicotine yield in the media were attained by using surfactant-stabilized microbubbles. Compared to other non-ionic surfactants (Tween 20 and Tween 80) and the ionic surfactant SDS, Triton X-100 (TX100) both increased total nicotine production and exudation into the hairy root culture media. In comparison to surfactant-free medium, TX100 at 10, 25, and 50 mg l^?1 did not show strong inhibition of hairy root growth. At 4,000 rpm shear speed, microbubbles stabilized by 10, 25, and 50 mg l^?1 TX100 had k _L a of 22.3, 36.2, and 44.1 h^?1 in Gamborg’s B5 medium, respectively, in comparison to 16.4 h^?1 with conventional air sparging. In a 1-l bioreactor, microbubbles stabilized by TX100 were applied to hairy roots after the inoculated root tips were self-immobilized by branching. With microbubble dispersion, dissolved oxygen rapidly increased from 60 to 85 %, and hairy root growth rate increased. Nicotine accumulation in culture medium with microbubbles reached 146 mg l^?1 after 30 days cultivation. These results show that combining genetic modification with surfactant-stabilized microbubble dispersion can substantially increase levels of nicotine in the media of hairy root cultures.     

Source of nitrogen as a factor limiting saponin production by hairy root and suspension cultures of <Emphasis Type="Italic">Calendula officinalis</Emphasis> L.

Triterpenic saponins represented in Calendula officinalis L. by oleanolic acid (OA) glycosides are pentacyclic triterpene compounds with a wide range of biological and medicinal properties. This report demonstrates nitrogen source impact on growth, saponin accumulation, and secretion in hairy root and suspension cultures of marigold. Hairy roots preferred nitrate as a mineral source of nitrogen, but its impact on growth, OA glycosides accumulation, and secretion were line-dependent. The best productivity of OA glycosides was found in CC16 line (74.86 mg flask^?1) in ½ MS medium modified by 2.5× KNO₃ and ammonium elimination with 2.5 g l^?1 peptone. Organic nitrogen source at 27.5-g l^?1 impairs the growth rate of hairy roots. Its effect on saponin accumulation and secretion to the surrounding medium depended on line and media composition. Nitrate:ammonium ratio of 4:2 for CC16 resulted in 5.7-fold increment of saponin secretion comparing to the standard medium. Embryo roots, apical bud, and hypocotyls explants were crucial for induction of suspension culture synthesizing saponins; however, effect of mineral form of nitrogen in cultivating medium had to be considered. The highest OA glycosides level (171.97 μg g^?1 of dry weight) was recorded in the root derived culture with nitrate as a sole mineral form of nitrogen. Peptone from lactalbumin decidedly inhibited the saponin formation; however, it was essential for culture initiation, proliferation, and organ differentiation.     

The production and antiprotozoal activity of abietane diterpenes in Salvia austriaca hairy roots grown in shake flasks and bioreactor

The biomass and concentration of bioactive quinone methide-type diterpenes in hairy roots of Salvia austriaca were determined and compared with levels of these metabolites in roots of field-grown plants. The cultures were maintained in shake flasks and a nutrient sprinkle bioreactor. Diterpene production was more efficient in the shake flask root culture than the bioreactor one. Biomass and diterpene production within the shake flask culture was evaluated using Schenk and Hildebrandt (SH), Gamborg (B5), and woody plant medium (WPM), with both full- and half-strength macro and micronutrient concentrations (1/2 SH, 1/2 B5, and 1/2 WPM). Among the tested media, SH medium proved to be most effective for biomass and diterpene production. In this medium, the transformed roots accumulated the levels of taxodone (3.89?mg?g^?1 DW; equivalent to 63.3?mg?L^?1), taxodione (1.15?mg?g^?1 DW; equivalent to 17.4?mg?L^?1), 15-deoxy-fuerstione (2.15?mg?g^?1 DW; equivalent to 32.5?mg?L^?1), and 7-(2′-oxohexyl)-taxodione (0.076?mg?g^?1 DW; equivalent to 1.1?mg?L^?1). Three diterpenes were also detected in the roots of S. austriaca intact plants, but their concentrations were lower than those in hairy root culture. No 7-(2′-oxohexyl)-taxodione was found in the roots of field-grown plants. The hairy roots were able to maintain high metabolite levels even for 6 years of cultivation. Taxodone, taxodione, 15-deoxy-fuerstione, and 7-(2′-oxohexyl)-taxodione were tested for in vitro activity against Trypanosoma brucei rhodesiense, T. cruzi, and Plasmodium falciparum and their cytotoxicity was determined using L6 cells. Among these compounds, taxodione was the most active against T. brucei rhodesiense [IC₅₀?=?0.05?µM with high selectivity, selectivity index (SI)?=?38]. Taxodione was found to inhibit the growth of P. falciparum and T. cruzi by 50% at respective concentrations of 1.9 and 7.1?µM (SI values of 1.0 and 0.27). Other diterpenoids demonstrated weaker activity against tested parasites (IC₅₀ values ranging from 0.62 to 194.7?µM) and lower selectivity (SI value ranged from 0.4 to 5.0).     

Effect of elicitation and precursor feeding on accumulation of 20-hydroxyecdysone in <Emphasis Type="Italic">Achyranthes aspera</Emphasis> Linn. cell suspension cultures

20-Hydroxyecdysone is one of the most common ecdysteroids in plants with potential therapeutic applications. In this study, cell suspension cultures of Achyranthes aspera were raised in shake flasks to investigate the production of 20-hydroxyecdysone. The quantification and characterization of 20-hydroxyecdysone in the cultures were done by High performance liquid chromatography (HPLC) and Liquid Chromatography-quadrupole time-of- flight mass spectrometry (LC-Q-TOF) analyses. For raising the suspension, calli initiated from in vitro grown leaf explants were cultured in liquid Murashige and Skoog (MS) medium augmented with combinations of 2, 4-dichlorophenoxyacetic acid (1 mg L^?1) and α-naphthaleneacetic acid (1 mg L^?1). Maximum growth index of the cell suspension was 9.9, which was achieved during 20th day of culture (final phase of exponential growth). At this stage, the biomass accumulated was 1.09 ± 0.09 g dry weight (DW) and the 20-hydroxyecdysone concentration was 0.24 mg g^?1 DW. Eliciting the cultures with 0.6 mM Methyl jasmonate for 6 days; enhanced the production of 20-hydroxyecdysone production to 0.35 mg g^?1 DW. By augmenting the cultures with the precursors namely cholesterol (10 mg L^?1) and 7-dehydrocholesterol (10 mg L^?1), production of 20-hydroxyecdysone was boosted to 0.31 mg g^?1 DW and 0.28 mg g^?1 DW respectively.     

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.     

Biomass production of hairy roots of Artemisia annua and Arachis hypogaea in a scaled‐up mist bioreactor

Hairy roots have the potential to produce a variety of valuable small and large molecules. The mist reactor is a gas phase bioreactor that has shown promise for low‐cost culture of hairy roots. Using a newer, disposable culture bag, mist reactor performance was studied with two species, Artemisia annua L. and Arachis hypogaea (peanut), at scales from 1 to 20 L. Both species of hairy roots when grown at 1 L in the mist reactor showed growth rates that surpassed that in shake flasks. From the information gleaned at 1 L, Arachis was scaled further to 4 and then 20 L. Misting duty cycle, culture medium flow rate, and timing of when flow rate was increased were varied. In a mist reactor increasing the misting cycle or increasing the medium flow rate are the two alternatives for increased delivery of liquid nutrients to the root bed. Longer misting cycles beyond 2–3 min were generally deemed detrimental to growth. On the other hand, increasing the medium flow rate to the sonic nozzle especially during the exponential phase of root growth (weeks 2–3) was the most important factor for increasing growth rates and biomass yields in the 20 L reactors. A. hypogaea growth in 1 L reactors was µ = 0.173 day^?1 with biomass yield of 12.75 g DW L^?1. This exceeded that in shake flasks at µ = 0.166 day^?1 and 11.10 g DW L^?1. Best growth rate and biomass yield at 20 L was µ = 0.147 and 7.77 g DW L^?1, which was mainly achieved when medium flow rate delivery was increased. The mist deposition model was further evaluated using this newer reactor design and when the apparent thickness of roots (+hairs) was taken into account, the empirical data correlated with model predictions. Together these results establish the most important conditions to explore for future optimization of the mist bioreactor for culture of hairy roots. Biotechnol. Bioeng. 2010;107: 802–813. © 2010 Wiley Periodicals, Inc.     

Effect of biotic and abiotic elicitors on production of betulin and betulinic acid in the hairy root culture of Betula pendula Roth

Abstract

Betulin (B) and betulinic acid (BA) are two triterpenoids with a wide range of biological and medicinal activities in different organs of Betula pendula. This research aimed to increase the accumulation of B and BA in the hairy root culture of B. pendula by seven biotic and abiotic elicitors. Hairy root was induced in the stem’s inner bark of B. pendula using the C58C1 strain in the WPM (Woody Plant Medium). The effects of different concentrations of elicitors and different time of root harvest in hairy root culture of B. pendula showed that highest level of growth index (GI), B, and BA was acquired in treated hairy roots with chitosan (CTS), chlorocholine chloride (CCC) and chitosan nano-fiber (CTS NF). Highest GI of B. pendula hairy roots was 13 that was obtained in the roots treated with CTS 150?mg l^?1 on the 8th day. The highest content of BA was 1.3?mg g^?1 DW after treatment with 1?mg l^?1CCC on the 4th and 6th days and 200?mg l^?1CTS NF on the 10th day. The highest B content (0.94?mg g^?1DW) was obtained in the treated hairy root by 2?mg l^?1 CCC after 4 and 6?days.     

Effects of dynamic changes of nutrients on adventitious roots growth and periplocin accumulation in culture of Periploca sepium Bunge

In this study, the dynamics of biomass production, accumulation of periplocin, medium conditions and consumption of carbon, nitrogen and phosphate were investigated in adventitious roots culture of Periploca sepium in shake flasks over a period of 4 weeks. The biomass reached the maximum peak on day 24 (2.46 and 0.213 g of fresh and dry weight, respectively). Similarly, periplocin production got to a peak of 0.083 mg g^?1 on day 24, simultaneously. PH in medium had a decrease tendency at the beginning and then remained stable around 4.0, however, EC declined during the whole culture with the nutrients consumed. Sucrose was almost used up on the first 12 days which led to the increase in glucose and fructose. In case of nitrogen, consumption of ammonium is faster than nitrate at the beginning. Phosphate was almost consumed during the first 8 days. Based on the nutrients consumption, the adding time of nutrients (1/2 MS medium and 30 g L^?1 sucrose) was investigated and it obtained highest content of periplocin (0.106 mg g^?1) and yield (0.513 mg L^?1) on day 12.     

Establishment of Gymnema sylvestre hairy root cultures for the production of gymnemic acid

Gymnema sylvestre is an important medicinal plant that bears bioactive compound namely gymnemic acid. In the present study, G. sylvestre was transformed by Agrobacterium rhizogenes. Seedling explants namely roots, stems, hypocotyls, cotyledonary nodal segments, cotyledons and young leaves were inoculated with A. rhizogenes strain KCTC 2703. Transformed (hairy) roots were induced from cotyledons and leaf explants. Six transgenic clones of hairy roots were established and confirmed by polymerase chain reaction (PCR) and RT-PCR using rolC specific primers. Hairy roots cultured using MS liquid medium supplemented with 3 % sucrose showed highest accumulation of biomass (97.63 g l^?1 FM and 10.92 g l^?1 DM) at 25 days, whereas highest accumulation of gymnemic acid content (11.30 mg g^?1 DM) was observed at 20 days. Nearly 9.4-fold increment of biomass was evident in suspension cultures at 25 days of culture and hairy root biomass produced in suspension cultures possessed 4.7-fold higher gymnemic acid content when compared with the untransformed control roots. MS-based liquid medium was superior for the growth of hairy roots and production of gymnemic acid compared with other culture media evaluated (B5, NN and N6), with MS-based liquid medium supplemented with 3 % sucrose was optimal for secondary metabolite production. The current results showed great potentiality of hairy root cultures for the production of gymnemic acid.     

Embryogenesis,plant regeneration and cardiac glycoside determination in Digitalis ferruginea subsp. ferruginea L

The present study reports, for the first time, an efficient in vitro plant regeneration protocol for Digitalis ferruginea subsp. ferruginea L. (rusty foxglove). We have used different concentrations of gibberellic acid (GA₃) on Murashige and Skoog (MS) medium to assess the germination frequency of seeds. High frequency of germination was achieved on MS medium with 1.0 mg l^?1 GA₃. 6-Benzylaminopurine (BAP) combined with α-naphtaleneacetic acid (NAA) or 2, 4-dichlorophenoxy acetic acid (2, 4-D) in the induction MS medium induced both somatic embryogensis and shoot organogenesis. The highest percentage of callus growth (85 %) was obtained when hypocotyl explants were cultured on MS medium containing 0.5 mg l^?1 2, 4-D plus 1.0 mg l^?1 BAP. The maximum mean number of somatic embryos (7.3 ± 1.3 embryos) or shoots (12.0 ± 1.1 shoots) per callus was obtained when medium contained 0.25 mg l^?1 NAA plus 1.0 mg l^?1 BAP or 0.5 mg l^?1 NAA plus 2.0 mg l^?1 BAP. The regenerated shoots easily rooted on MS medium. Higher amounts of lanatoside C [13.2 ± 0.5 mg 100 g^?1 dry weight (dw)] and digoxin (2.93 ± 0.31 mg 100 g^?1 dw) accumulation were obtained when shoots were obtained by indirect regeneration. We also investigated derivatives of cardenolides, i.e., digitoxigenin (730 ± 180 mg 100 g^?1 dw), gitoxigenin (50 ± 20 mg 100 g^?1 dw) and digoxigenin (490 ± 170 mg 100 g^?1 dw) from natural samples.     

Hairy roots of Dracocephalum moldavica: rosmarinic acid content and antioxidant potential

Hairy roots of Dracocephalum moldavica L. were induced using Agrobacterium rhizogenes strain A4. Transformed roots were obtained from shoot explants with low transformation frequency of up to 3 %. The effects of different liquid media: Murashige and Skoog (MS), Gamborg et al. (B5) and Woody Plant (WP) with full- and half-strength (½MS, ½B5, ½WP), on biomass accumulation and rosmarinic acid (RA) content were investigated. The hairy roots were cultured in photoperiod (16 h light/8 h dark) and darkness. Biomass of D. moldavica hairy roots was the highest (7.23 g flask^?1 of fresh weight and 0.89 g flask^?1 of dry weight) in the cultures grown in WP medium under periodic light. Ultra performance liquid chromatography analysis revealed the highest RA content (78 mg g^?1 dry wt) in roots cultured in ½B5 medium under photoperiod conditions. It was about tenfold higher compared to roots of field-grown mother plants. Antioxidant activities and total phenolic contents of methanolic extracts of D. moldavica hairy roots cultured in ½B5 and WP media under photoperiod and darkness and roots of field grown plants were compared. All extracts were investigated using 1,1-diphenyl-2-picrylhydrazyl (DPPH) scavenging and phosphomolybdenum reduction assays. Total phenolic contents were estimated by the Folin–Ciocalteu method. The methanolic extract of D. moldavica hairy roots grown in ½B5 medium under photoperiod possessed the strongest effects on reducing Mo and DPPH radical scavenging. The activities were significantly higher (p ≤ 0.05) than those of methanolic extract of roots of intact plants grown in the field. The most active methanolic extract of hairy roots was characterized by the highest level of rosmarinic acid and total content of phenolic compounds.     

Methyl jasmonate effect on diterpenoid accumulation in Salvia sclarea hairy root culture in shake flasks and sprinkle bioreactor

Hairy root cultures of Salvia sclarea were grown in shake flasks and 10 L nutrient sprinkle bioreactor, running for 30 days and the effects of methyl jasmonate (MJ) on their growth and capacity to accumulate diterpenoids were measured. We found that MJ concentration and exposure time to the elicitor were factors that strongly affected the diterpenoid production. The highest diterpenoid accumulation (67.5 ± 7.1 mg g⁻¹ dry weight, calculated as a sum of ferruginol, salvipisone, aethiopinone and 1-oxoaethiopinone) without reduction of biomass, was achieved when the 23-day-old hairy roots in bioreactor culture were exposed to 125 μM MJ for 7 days. The roots produced 9 and 3.8 times as much aethiopinone (40 ± 5.9 mg g⁻¹ dry weight) and salvipisone (12.6 ± 0.4 mg g⁻¹ dry weight), respectively, as roots cultured in shake flasks. Our results imply that cultivation of S. sclarea hairy roots in sprinkle bioreactor after elicitation with MJ may be valuable to enhance production of the bioactive diterpenoids.     

<Emphasis Type="Italic">In vitro</Emphasis> propagation,micromorphological studies and <Emphasis Type="Italic">ex vitro</Emphasis> rooting of cannon ball tree (<Emphasis Type="Italic">Couroupita guianensis</Emphasis> aubl.): a multipurpose threatened species

In vitro propagation methods using seeds and nodal segments of a 21-year old Couroupita guianensis - a medicinally important but threatened tree have been developed. Hundred percent of the seeds germinated on half strength Murashige and Skoog (MS) medium with 2.0 mg l^?1 indole-3 butyric acid (IBA). Nodal segments were found most suitable for the establishment of cultures. About 90 % explants responded and 4.1 ± 0.23 shoots per node were induced after five weeks of inoculation on MS medium +4.0 mg l^?1 6-benzylaminopurine (BAP). Further shoot multiplication was achieved by repeated transfer of mother explants and subculturing of in vitro produced shoots on fresh medium. Maximum number (8.2 ± 0.17) of shoots were regenerated on MS medium with 1.0 mg l^?1 each of BAP and Kinetin (Kin) + 0.5 mg l^?1 α-naphthalene acetic acid (NAA) with additives (50 mg l^?1 of ascorbic acid and 25 mg l^?1 each of adenine sulphate, L-arginine and citric acid). The multiplied shoots rooted (4.3 ± 0.26 roots/shoot) on half strength MS medium with 2.5 mg l^?1 IBA. All the shoots were rooted ex vitro when pulse treated with 400 mg l^?1 of IBA for five min with an average of 7.3 ± 0.23 roots per shoot. Nearly 86 % of these plantlets were acclimatized within 7–8 weeks and successfully transferred in the field. Biologically significant developmental changes were observed during acclimation particularly in leaf micromorphology in terms of changes in stomata, veins and vein-islets, and trichomes. This study helps in understanding the response by the plants towards outer environmental conditions during acclimatization. This is the first report on micropropagation of C. guianensis, which could be used for the large-scale multiplication, restoration and conservation of germplasm of this threatened and medicinally important tree.     

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.     

Tryptophan decarboxylase plays an important role in ajmalicine biosynthesis in Rauvolfia verticillata

Tryptophan decarboxylase (TDC) converts tryptophan into tryptamine that is the indole moiety of ajmalicine. The full-length cDNA of Rauvolfia verticillata (RvTDC) was 1,772 bps that contained a 1,500-bp ORF encoding a 499-amino-acid polypeptide. Recombinant 55.5 kDa RvTDC converted tryptophan into tryptamine. The K _m of RvTDC for tryptophan was 2.89 mM, higher than those reported in other TIAs-producing plants. It demonstrated that RvTDC had lower affinity to tryptophan than other plant TDCs. The K _m of RvTDC was also much higher than that of strictosidine synthase and strictosidine glucosidase in Rauvolfia. This suggested that TDC might be the committed-step enzyme involved in ajmalicine biosynthesis in R. verticillata. The expression of RvTDC was slightly upregulated by MeJA; the five MEP pathway genes and SGD showed no positive response to MeJA; and STR was sharply downregulated by MeJA. MeJA-treated hairy roots produced higher level of ajmalicine (0.270 mg g^?1 DW) than the EtOH control (0.183 mg g^?1 DW). Highest RvTDC expression level was detected in hairy root, about respectively 11, 19, 65, and 109-fold higher than in bark, young leaf, old leaf, and root. Highest ajmalicine content was also found in hairy root (0.249 mg g^?1 DW) followed by in bark (0.161 mg g^?1 DW) and young leaf (0.130 mg g^?1 DW), and least in root (0.014 mg g^?1 DW). Generally, the expression level of RvTDC was positively consistent with the accumulation of ajmalicine. Therefore, it could be deduced that TDC might be the key enzyme involved in ajmalicine biosynthesis in Rauvolfia.     

Plant regeneration of the arsenic hyperaccumulator <Emphasis Type="Italic">Pteris vittata</Emphasis> L<Emphasis Type="Italic">.</Emphasis> from spores and identification of its tolerance and accumulation of arsenic and copper

An in vitro plant regeneration system was established from the spores of Pteris vittata and identification of its tolerance, and accumulation of gametophytes and callous, to arsenic (As) and copper (Cu) was investigated. The highest frequency (100%) of callus formation was achieved from gametophyte explants treated with 0.5 mg l^?1 6-benzylaminopurine (6-BA) + 0.5 mg l^?1 gibberellin acid (GA). Furthermore, sporophytes were differentiated from the callus tissue derived from gametophyte explants on MS medium supplemented with 0.5 mg l^?1 6-BA, 0.5–1.0 mg l^?1 GA and additional 300 mg l^?1 lactalbumin hydrolysate (LH) for 4 weeks. The optimum combination of ½ MS + 1.0 mg l^?1 GA + 0.5 mg l^?1 6-BA + 300 mg l^?1 LH promoted sporophyte formation on 75 ± 10% of the callus. Every callus derived from gametophyte explants could achieve 3–4 sporophytes. The in vitro growth of gametophyte and callus was accelerated in the medium containing Na₃AsO₄ lower than 0.5 mM, but this growth was inhibited with 2 mM Na₃AsO₄. And with the increase of Na₃AsO₄ in the culture medium from 0 to 2 mM, the As accumulation in gametophytes and callus increased and achieved a level of 763.3 and 315.4 mg kg^?1, respectively. Gametophytes and calluses transplanted to culture medium, supplemented with different concentrations of CuSO₄, are similar to those in Na₃AsO₄, and the Cu accumulation in gametophytes could achieve 7,940 mg kg^?1 when gametophytes were subcultured in medium containing 3 mM CuSO₄. These results suggested that the high efficiency propagation system could be a useful and rapid means to identify other heavy metal tolerance and accumulation. Further, the regeneration ability of callus made it possible for genetic transformation of this fern.     

Enhanced production of isoflavones by elicitation in hairy root cultures of Soybean

A combined treatment of sonication (2 min) and vacuum infiltration (2 min) stimulated isoflavones production of 75.26 mg g^?1 DW which was 15.11-fold higher than control hairy root line at optimal harvest time of 40 days. Addition of MeJ at 100 μM concentration with 72 h exposure time on 30 day-old hairy root culture further enhanced total isoflavones production of 53.16 mg g^?1 DW (10.67-fold) and SA at 200 μM concentration with 96 h exposure period enhanced the production of isoflavones (28.79 mg g^?1 DW; 5.78-fold). MeJ-treated hairy roots reduced biomass accumulation whereas sonication, vacuum infiltration and SA did not exhibit a negative effect on biomass growth.     

Modeling of growth and laccase production by Pycnoporus sanguineus

Production of extracellular laccase by the white-rot fungus Pycnoporus sanguineus was examined in batch submerged cultures in shake flasks, baffled shake flasks and a stirred tank bioreactor. The biomass growth in the various culture systems closely followed a logistic growth model. The production of laccase followed a Luedeking-Piret model. A modified Luedeking-Piret model incorporating logistic growth effectively described the consumption of glucose. Biomass productivity, enzyme productivity and substrate consumption were enhanced in baffled shake flasks relative to the cases for the conventional shake flasks. This was associated with improved oxygen transfer in the presence of the baffles. The best results were obtained in the stirred tank bioreactor. At 28 °C, pH 4.5, an agitation speed of 600 rpm and a dissolved oxygen concentration of ~25 % of air saturation, the laccase productivity in the bioreactor exceeded 19 U L^?1 days^?1, or 1.5-fold better than the best case for the baffled shake flask. The final concentration of the enzyme was about 325 U L^?1.     

Production of salidroside and polysaccharides in Rhodiola sachalinensis using airlift bioreactor systems

Rhodiola sachalinensis is widely used in traditional Chinese medicine, and salidroside and polysaccharides are its important bioactive compounds. This study used airlift bioreactor systems to produce mass bioactive compounds through callus culture. Several factors affecting callus biomass and bioactive compound accumulation were investigated. Callus growth was vigorous in a bioreactor system, and the growth ratio was 2.8-fold higher in bioreactor culture than in agitated-flask culture. Callus biomass and polysaccharide content were favorable at 0.1 air volume per culture volume per min (vvm), whereas favorable salidroside content was observed at a high air volume (0.2 vvm). The maximum yields of salidroside (7.90 mg l^?1) and polysaccharide (2.87 g l^?1) were obtained at 0.1 vvm. Inoculum density greatly affected callus biomass and bioactive compound accumulation, and the highest biomass and contents or yields of salidroside and polysaccharide were determined at a high inoculum density of 12.5 g l^?1. The level of hydrogen ion concentration (pH) at 5.8 improved callus biomass accumulation. Acidic medium (pH 4.8) stimulated salidroside synthesis but higher pH level (7.8) promoted polysaccharide accumulation. The highest yields of both bioactive compounds were obtained at pH 5.8. Methyl jasmonate (MeJA) participated in synthesis promotion of bioactive compounds, and the contents and yields of salidroside [4.75 mg g^?1 dry weight (DW), 58.43 mg l^?1] and polysaccharides (392.41 mg g^?1 DW, 4.79 g l^?1) were at maximum at 125 and 150 μmol of MeJA. Therefore, bioreactor systems can be used to produce R. sachalinensis bioactive compounds, and callus culture in a bioreactor can be as an alternative method for supplying materials for commercial drug production.