Production of ginsenoside and polysaccharide by two-stage cultivation of Panax quinquefolium L. cells

Based on the results of carbon source consumption in cell suspension culture of Panax quinquefolium L., 30 g L⁻¹ sucrose was fed into a 5-L stirred tank bioreactor on day 16 of culture to enhance cell density and metabolite production. Using a fed-batch cultivation strategy, polysaccharide production was enhanced to 1.608 g L⁻¹, which was 1.96-fold greater than with batch cultivation. The maximum saponin yield (7.828 mg L⁻¹) was obtained on day 24 and was about 36% higher than the yields obtained using batch cultivation. In a two-stage culture process, a combined treatment with sucrose, lactoalbumin hydrolysate, and methyl jasmonate caused a significant increase in total saponin yield (31.52 mg L⁻¹) in cell cultures after 27 d. This value represents an increase of 4.03-fold compared with the total saponin yield in fed-batch cultivation. The two-stage culture mode provided the best method for the in vitro production of secondary metabolites from P. quinquefolium.     

Combination effect of lactoalbumin hydrolysate and methyl jasmonate on ginsenoside and polysaccharide production in <Emphasis Type="Italic">Panax quinquefolium</Emphasis> L. cells cultures

Lactoalbumin hydrolysate (LH) at 100 mg L⁻¹ with methyl jasmonate (MJ) at 2 mg L⁻¹ synergistically stimulated ginsenoside accumulation in Panax quinquefolium cells compared with 100 mg L⁻¹ LH. Combination elicitors led to higher ginsenoside productivity (45.93 mg L⁻¹) than single treatment of 100 mg L⁻¹ LH (31.37 mg L⁻¹). This present result will be helpful in providing a tool for enhancing the productivity of ginsenoside by Panax quinquefolium cell cultures on a commercial scale.     

Dynamics of ginsenoside biosynthesis in suspension culture of <Emphasis Type="Italic">Panax quinquefolium</Emphasis>

Biosynthesis of six saponins (ginsenosides) in suspension culture of P. quinquefolium Z₅ was investigated. Ginsenoside content in biomass reached the highest level, nearly 30 mg g⁻¹ d.w., between 25 and 30 days of the culture. Saponins were synthesized simultaneously with cell growth but their synthesis rate was not proportional to the growth rate. During the phase of rapid biomass multiplication, after which biomass reached 90% of its maximum yield, only half examined ginsenosides was produced. The second half of the final saponins yield was produced during the slow growth phase, in which only 10% of biomass was grown. During the intensive growth phase the productivity of six saponins examined per biomass (dry weight) unit was 3.4 μg mg⁻¹ d.w. day⁻¹, however, this parameter calculated for slow growth phase reached nearly 30 μg mg⁻¹ d.w. day⁻¹. There were differences in increase of the contents of six saponins determined in biomass, and it was the highest for saponins Re (20(S)-protopanaxatriol-6-[O-α-l-rhamnopyranosyl(1 → 2)-β-d-glucopyranoside]-20-O-β-d-glucopyranoside) and Rg₁ (20(S)-protopanaxatriol-6,20-di-O-β-d-glucoside).     

Inoculum size and auxin concentration influence the growth of adventitious roots and accumulation of ginsenosides in suspension cultures of ginseng ( Panax ginseng C.A. Meyer)

The effect of the root-inoculum size and axuin concentration on growth of adventitious roots and accumulation of ginsenosides were studied during suspension cultures of ginseng (Panax ginseng C.A. Meyer). Of the various concentrations of indole-3-butyric acid (IBA) and γ-naphthaleneacetic acid (NAA) used as supplementary growth regulators along with Murashige and Skoog medium, 25 μM IBA was found suitable for lateral root induction and growth, as well as accumulation of ginsenosides. Inoculum size of 5 g L⁻¹ was found suitable for optimal biomass (10.5 g L⁻¹ dry biomass) and ginsenosides (5.4 mg g⁻¹ DW) accumulation. Of the various length of root inocula tested (chopped to 1–3, 4–6, 7–10 mm and un-chopped), root inocula of 7–10 mm was found suitable for biomass and ginsenoside accumulation.     

Bioreactor application on adventitious root culture of <Emphasis Type="Italic">Astragalus membranaceus</Emphasis>

Astragalus membranaceus is one of the most widely used traditional medicinal herbs in China, but the time required to generate a useful product in the field production is long. The growth of adventitious root cultures was compared between cultures grown in solid, liquid, or a 5-L balloon-type bubble bioreactor. The maximum growth ratio (final dry weight/initial dry weight) was determined for adventitious roots grown in the bioreactor. Studies carried out to optimize biomass production of adventitious roots compared adventitious root growth from various inoculum root lengths, inoculum densities, and aeration volume in the bioreactors. The maximum growth ratio occurred in treatments with a 1.5-cm inoculum root length, with 30 g (fresh weight) of inoculum per bioreactor or with an aeration volume of 0.1 vvm (air volume/culture medium volume per min). The polysaccharide, saponin, and flavonoid content of roots from bioreactor-grown cultures were compared to roots from field-grown plants grown for 1 and 3 yr. Total polysaccharide content of adventitious roots in the bioreactor (30.0 mg g⁻¹ dry weight (DW)) was higher than the roots of 1-yr-old (13.8 mg g⁻¹ DW) and 3-yr-old (21.1 mg g⁻¹ DW) plants in the field. Total saponin (3.4 mg g⁻¹ DW) and flavonoid (6.4 mg g⁻¹ DW) contents were nearly identical to 3-yr-old roots and higher than that of 1-yr-old roots under field cultivation.     

Dynamic change of metabolites and nutrients in suspension cells of <Emphasis Type="Italic">Panax Quinquefolium</Emphasis> L. in bioreactor

In order to understand how the nutrient elements were taken up during the cell growth as well as the production of metabolites, it was quite necessary to identify the dynamic change of metabolites and nutrients in suspension cells of Panax quinquefolium in bioreactor. In this study, dynamic accumulation of biomass and ginsenosides Re, Rb1 and polysaccharide as well as major nutrients consumption in cell suspension culture of P. quinquefolium in a 5-L stirred tank bioreactor were investigated. The dry cell weight and the contents of ginsenosides Re, Rb1 and polysaccharide reached the maximum peak simultaneously on about 21 days and the results showed that cell growth and metabolites synthesis related to nutrients consumption. For this reason, we supposed that the contents of metabolites can be increased through added nutrient at the right moment. These results provided theory reference for two-stage or continuous perfusion culture in suspension cells of P. quinquefolium in bioreactor.     

Phenylpropanoid production in callus and cell suspension cultures of <Emphasis Type="Italic">Buddleja cordata</Emphasis> Kunth

Plant tissue cultures represent a potential source for producing secondary metabolites. In this work, Buddleja cordata tissue cultures were established in order to produce phenylpropanoids (verbascoside, linarin and hydroxycinnamic acids), as these metabolites are credited with therapeutic properties. Highest callus induction (76.4–84.3%) was obtained in five treatments containing 2,4-Dichlorophenoxyacetic acid (2,4-d: 0.45–9.05 μM) with Kinetin (KIN: 2.32, 4.65 μM), whereas highest root induction (79.6%) corresponded to the α-Naphthaleneacetic acid (9.05 μM) with KIN (2.32 μM) treatment. Verbascoside was the major phenylpropanoid produced in in vitro cultures (root, white and green callus) [66.24–86.26 mg g⁻¹ dry weight (DW)], while linarin and hydroxycinnamic acid production was low (0.95–3.01 mg g⁻¹ DW). Verbascoside and linarin production were improved in cell suspension culture (116 mg g⁻¹ DW and 8.12 mg g⁻¹ DW, respectively).     

Camptothecin accumulation in various organ cultures of <Emphasis Type="Italic">Camptotheca acuminata</Emphasis> Decne grown in different culture systems

Levels of camptothecin (CPT) and 10-hydroxycamptothecin (HCPT) were determined in different cultures of Camptotheca acuminata grown either in a Temporary Immersion System (TIS) or on solid medium. CPT was also detected in liquid culture medium. HPLC analysis showed significant differences in CPT contents in all tissues analysed and the highest CPT contents were found in shoots grown on solid medium and in TIS with a mean of 2.2 and 2.5 mg g⁻¹ DW, respectively. The highest content of CPT detected in seedlings was 1.96 mg g⁻¹ DW; while that of somatic embryos at cotyledonary stage and regenerated plants were 0.87 and 1.23 mg g⁻¹ DW, respectively. It was also shown that shoots cultured in TIS secreted substantial amount of CPT into the liquid medium. After 4 weeks in culture a mean of 6, 05 and 12, 6 μg g⁻¹ FW were determined at 4 and 8 immersion cycles daily (IC d⁻¹), respectively. This aspect opens new possibilities regarding the isolation of CTP using TIS culture systems.     

Production of high-density Chlorella culture grown in fermenters

The freshwater microalga Chlorella vulgaris was grown heterotrophically in fed-batch 50–600-L fermenters at 36°C, on aerated and mixed nutrient solution with urea as a nitrogen and glucose as a carbon and energy source. Cell density increased from the initial value 6.25 to 117.18 g DW L⁻¹ in 32 h in the fermenter 50 L at a mean growth rate 3.52 g DW L⁻¹ h⁻¹. The DW increase in the fermenter 200 L was from 7.25 to 94.82 g DW L⁻¹ in 26.5 h at a mean growth rate 3.37 g DW L⁻¹ h⁻¹. Mean specific growth rate μ was about 0.1 h⁻¹ in the both fermenters, if nutrients and oxygen were adequately supplied. The DW increase in the fermenter 600 L was from 0.8 to 81.6 g DW L⁻¹ in 66.5 h at a mean growth rate 1.22 g DW L⁻¹ h⁻¹ and μ = 0.07 h⁻¹. A limitation of the cell growth rate in 600 L fermenter caused by a low dissolved oxygen concentration above cell densities higher than 10 g DW L⁻¹) occurred. Specific growth rate decreased approximately linearly with increasing glucose concentration (25–80 g glucose L⁻¹) at the beginning of cultivation and decreased with the time of cultivation. The cell yield was 0.55–0.69 g DW (g glucose)⁻¹. The content of proteins, β-carotene, and chlorophylls in the cells steadily increased and starch content decreased, by keeping aerated and mixed culture another 12 h in fermenter after the cell growth was stopped due to glucose deficiency.     

Morphactin and 2iP markedly enhance accumulation of stilbenes in cell cultures of Cayratia trifolia (L.) Domin

The cell cultures of Cayratia trifolia (Vitaceae) a tropical lianas, were maintained in Murashige and Skoog’s medium containing 0.25 mg l⁻¹ naphthalene acetic acid, 0.2 mg l⁻¹ kinetin and 250 mg l⁻¹ casein hydrolysate. Cell suspension cultures of C. trifolia accumulate stilbenes (piceid, resveratrol, viniferin, ampelopsin) which on addition of 0.1–0.5 mg l⁻¹ morphactin in the medium containing naphthalene acetic acid and kinetin declined. Morphactin or 2 isopentenyl adenine alone at 0.1 mg l⁻¹ concentration enhanced stilbenes which on combination markedly enhanced the yield to ~5 mg l⁻¹ at 15th day.     

In vitro hormone-regulated growth and floral induction of <Emphasis Type="Italic">Cuscuta reflexa</Emphasis>: a parasitic angiosperm

The role of different growth regulators in callus induction, shoot regeneration, floral induction and chlorophyll content of the obligatory parasitic plant Cuscuta reflexa has been studied. Callus development was excellent from the nodal part of the shoot explants in modified Murashige and Skoog (MMS) media supplemented with 2 mg L⁻¹ benzyl adenine (MMS1c). Supplementation of 2 mg L⁻¹ naphthalene acetic acid (NAA) along with MMS1c (MMS2c) was responsible for estimable shoot induction and development in callus. 2,4-Dichloro acetic acid (2,4-D) played a crucial role in the floral induction of C. reflexa in vitro. MMS supplemented with 2 mg L⁻¹ NAA and 2 mg L⁻¹ 2,4-D (MMS3b) supported floral induction after shooting in vitro. MMS supplemented with 3 mg L⁻¹ 2,4-D (MMS4a) rapidly induced flower directly from the stem explants without showing any elongation of shoot. MMS1c along with MMS3b (MMS5a) showed callus proliferation followed by shoot elongation and floral induction. In vitro MMS5a grown plants show a sharp increase in the chlorophyll contents. Cytokinin treatment further increases the chlorophyll level of the plant.     

Manipulation of ginsenoside heterogeneity of <Emphasis Type="Italic">Panax notoginseng</Emphasis> cells in flask and bioreactor cultivations with addition of phenobarbital

Structure-similar ginsenosides have different or even totally opposite biological activities, and manipulation of ginsenoside heterogeneity is interesting and significant to biotechnological application. In this work, addition of 1 mM phenobarbital to cell cultures of Panax notoginseng at a relatively high inoculation size of 7.6 g dry cell weight (DW)/L enhanced the production of protopanaxatriol-type (Rg₁ + Re) ginsenosides in both shake flask and airlift bioreactor (ALR, 1 L working volume). The content of Rg₁ + Re in the ALR was increased from 42.5 ± 4.0 mg per gram DW in untreated cell cultures (control) to 56.4 ± 4.6 mg per gram DW with addition of 1.0 mM phenobarbital. The maximum productivity of Rg₁ + Re in the ALR reached 5.66 ± 0.38 mg L⁻¹ d⁻¹, which was almost 3.3-fold that of control. The maximum ratio of the detectable ginsenosides protopanaxatriol:protopanaxadiol (Rb₁) was 7.6, which was about twofold that of control. The response of protopanaxadiol 6-hydroxylase (P6H) activity to phenobarbital addition coincided with the above-mentioned change of ginsenoside heterogeneity (distribution). Phenobarbital addition is considered as a useful strategy for manipulating the ginsenoside heterogeneity in bioreactor with enhanced biosynthesis of protopanaxatriol by P. notoginseng cells.     

Two-stage culture for producing berberine by cell suspension and shoot cultures of <Emphasis Type="Italic">Berberis buxifolia</Emphasis> Lam

In vitro cultures of Berberis buxifolia were established using thidiazuron (4.5, 23 and 45 mM) or picloram (4 and 40 mM) as plant growth regulators for sustaining growth. For producing berberine, a two-stage culture was performed. In the first step, thidiazuron or picloram were used for biomass production followed by the production stage where benzylaminopurine (4.4 mM) was added as a plant growth regulator. Berberine yields (102 mg g⁻¹ DW) and in vitro shoot cultures (200 mg g⁻¹ DW) were significantly lower than those of whole plants in the field (416 mg g⁻¹ DW). The highest productivity (0.18 mg 1⁻¹ day⁻¹) was attained using picloram (either 4 on 40 mM) in the first stage for producing biomass.     

Zinc tolerance and accumulation in stable cell suspension cultures and in vitro regenerated plants of the emerging model plant Arabidopsis halleri (Brassicaceae)

Arabidopsis halleri is increasingly employed as a model plant for studying heavy metal hyperaccumulation. With the aim of providing valuable tools for studies on cellular physiology and molecular biology of metal tolerance and transport, this study reports the development of successful and highly efficient methods for the in vitro regeneration of A. halleri plants and production of stable cell suspension lines. Plants were regenerated from leaf explants of A. halleri via a three-step procedure: callus induction, somatic embryogenesis and shoot development. Efficiency of callus proliferation and regeneration depended on the initial callus induction media and was optimal in the presence of 1 mg L⁻¹ 2,4-dichlorophenoxyacetic acid, and 0.05 mg L⁻¹ benzylaminopurine. Subsequent shoot and root regeneration from callus initiated under these conditions reached levels of 100% efficiency. High friability of the callus supported the development of cell suspension cultures with minimal cellular aggregates. Characterization of regenerated plants and cell cultures determined that they maintained not only the zinc tolerance and requirement of the whole plant but also the ability to accumulate zinc; with plants accumulating up to 50.0 μmoles zinc g⁻¹ FW, and cell suspension cultures 30.9 μmoles zinc g⁻¹ DW. Together this work will provide the experimental basis for furthering our knowledge of A. halleri as a model heavy metal hyperaccumulating plant.     

Elicitor-induced accumulation of stilbenes in cell suspension cultures of Cayratia trifolia (L.) Domin

Cell cultures of Cayratia trifolia (Vitaceae), a tropical lianas, were maintained in Murashige and Skoog’s medium containing 0.25 mg l⁻¹ NAA, 0.2 mg l⁻¹ kinetin and casein hydrolysate 250 mg l⁻¹. Cell suspension cultures of C. trifolia accumulate stilbenes (piceid, resveratrol, viniferin, ampelopsin), which on elicitation by any of 500 μM salicylic acid, 100 μM methyl jasmonate, 500 μM ethrel and 500 mg l⁻¹ yeast extract, added on the 7th day, were enhanced by 3- to 6-fold (5–11 mg l⁻¹) by the 15th day.     

Effects of donor plants and plant growth regulators on naphthoquinone production in root cultures of <Emphasis Type="Italic">Impatiens balsamina</Emphasis>

The effects of type of explant (leaves and roots), donor plants, and plant growth regulators on naphthoquinone (NQ) production of Impatiens balsamina L. root cultures were evaluated. The root cultures were initiated in liquid Gamborg’s B5 medium supplemented with 0.1 mg l⁻¹ α-naphthaleneacetic acid (NAA), 0.1 mg l⁻¹ kinetin (Kn) and 1.0 mg l⁻¹ 6-benzyladenine (BA). The present investigation indicated that the root cultures established from the leaf explants produced higher total NQ content [1.01 ± 0.046 mg/g dry weight (DW)] than those established from the root explants (0.62 ± 0.023 mg/g DW). The leaf explants of four I. balsamina strains including white flower plant (IbW), pink flower plant (IbP), violet flower plant (IbV) and red flower plant (IbR) were used to establish the root cultures. Based on HPLC analysis, IbP strain produced the highest total NQ content (3.39 ± 0.072 mg/g DW), while IbR strain produced the lowest one (1.45 ± 0.055 mg/g DW). The root cultures established from the IbP explant were capable of producing higher content of total NQs (2.76 ± 0.093 mg/g DW) than those established from the other strains. The results suggest that the tissue cultures initiated from the high-yielding donor plants should be capable of producing higher content of secondary compounds than those initiated from low-yielding donor plants. In addition, plant growth regulator manipulation exhibited that a combination of 0.1 mg l⁻¹ NAA, 1.0 mg l⁻¹ Kn and 2.0 mg l⁻¹ BA is capable of increasing NQ production (2.97 ± 0.072 mg/g DW) in I. balsamina root cultures.     

Arabinogalactan proteins are involved in cell aggregation of cell suspension cultures of <Emphasis Type="Italic">Beta vulgaris</Emphasis> L.

Arabinogalactan proteins (AGPs) are glycoproteins present at cell surfaces. Although exact functions of AGPs remain elusive, they are implicated in plant growth and development. The aim of this study was to evaluate the role of AGPs in the process of cell aggregation of Beta vulgaris L. suspension cultures. It was observed that B. vulgaris suspension cultures accumulated AGPs in parallel form to its cell growth. The AGPs maximum content in the stationary phase was 0.330 mg g⁻¹ dry weight (DW) in the cell wall (CW) and 1.534 mg g⁻¹ DW in the culture medium (CM), generating cell aggregates >500 μm (93.21% DW). The addition of tunicamycin (TM) caused a reduction of AGPs content in CW and CM of 46 and 64%, respectively. These changes were associated with inhibition of growth and the reduction of the cell aggregates >500 μm (50.0% DW). When TM was removed from the CM, cell growth, aggregation, and AGPs content on CW and CM were recovered. Precipitation of AGPs with Yariv reagent generated a reduction of 61.14% of AGPs content in CW and a total inhibition of AGPs secretion in CM. This Yariv treatment generated a reduction in the cell aggregates >500 μm of 51.31% of DW. When the Yariv reagent was removed from the culture, cells did not recover their AGPs accumulation. In addition, cell cultures did not recover their ability to grow and aggregate. These results indicate that AGPs are molecules required in the cellular aggregation process of B. vulgaris L. suspension cultures.     

The effects of jasmonic acid and methyl jasmonate on rosmarinic acid production in <Emphasis Type="Italic">Mentha</Emphasis> × <Emphasis Type="Italic">piperita</Emphasis> cell suspension cultures

The effects of two elicitors: jasmonic acid and methyl jasmonate on cell growth as well as on rosmarinic acid accumulation in cell suspension cultures of Mentha × piperita were investigated. The highest rosmarinic acid accumulation 117.95 mg g⁻¹ DW (12% DW) was measured 24 h after addition of 100 μM methyl jasmonate. A similar concentration 110.12 mg g⁻¹ DW was detected 48 h after application of 200 μM jasmonic acid. Those values were nearly 1.5 times higher compared to the control sample, without elicitation. There was no substantial influence of elicitors on rosmarinic acid secretion into the culture media. Extracellular concentrations of rosmarinic acid were similar to the values from the control variants. It was documented that suspension cultures of M. piperita treated with elicitors showed a decrease in biomass accumulation when compared to the control.     

Improved monoterpene biotransformation with <Emphasis Type="Italic">Penicillium</Emphasis> sp. by use of a closed gas loop bioreactor

A closed gas loop bioprocess was developed to improve fungal biotransformation of monoterpenes. By circulating monoterpene-saturated process gas, the evaporative loss of the volatile precursor from the medium during the biotransformation was avoided. Penicillium solitum, isolated from kiwi, turned out to be highly tolerant towards monoterpenes and to convert α-pinene to a range of products including verbenone, a valuable aroma compound. The gas loop was mandatory to reproduce the production of 35 mg L⁻¹ verbenone obtained in shake flasks and also in the bioreactor. Penicillium digitatum DSM 62840 regioselectively converted (+)-limonene to the aroma compound α-terpineol, but shake flask cultures revealed a pronounced growth inhibition when initial concentrations exceeded 1.9 mM. In the bioreactor, toxic effects on P. digitatum during biotransformation were alleviated by starting a sequential feeding of non-toxic limonene portions after a preceding growth phase. Closing the precursor-saturated gas loop during the biotransformation allowed for an additional replenishment of limonene via the gas phase. The gas loop system led to a maximum α-terpineol concentration of 1,009 mg L⁻¹ and an average productivity of 8–9 mg L⁻¹ h⁻¹ which represents a doubling of the respective values previously reported. Furthermore, a molar conversion yield of up to 63% was achieved. M. Pescheck and M. A. Mirata have contributed equally to this work.     

Response of Chara globularis and Hydrodictyon reticulatum to lead pollution: their survival, bioaccumulation, and defense

The responses of the pioneer submerged macroalga (Chara globularis) and the rapidly spreading floating macroalga (Hydrodictyon reticulatum) to high levels of lead (40, 80, and 160 mg L⁻¹) at pH 7.14 were studied. Growth rate, Pb bioaccumulation, and physiological response of plants were measured after 5 and 15 days exposure. Both macroalgae efficiently postponed the deposition process of Pb from water column to soil. The Pb bioaccumulation in C. globularis was concentration- and time-dependent increase during the experiment and the maximum bioaccumulation activity was about 3,650 mg Pb kg⁻¹ DW in 160 mg L⁻¹ Pb at pH 7.14 after 15 days, whereas H. reticulatum showed saturable bioaccumulation in 5 days and the maximum was approximately 4,000 mg Pb kg⁻¹ DW; in addition, H. reticulatum exhibited higher tolerance to Pb pollution than C. globularis. The results also showed that the antioxidant defense systems of both tested macroalgae were overwhelmed under high Pb levels with superoxide radical and malondiadehyde levels increasing significantly. The antioxidant enzymes, superoxide dismutase, catalase, and guaiacol peroxidase activities were inhibited severely increasing Pb levels and exposure time. These results indicate that the pioneer species C. globularis would have difficulty growing in a habitat polluted by Pb >40 mg L⁻¹and the rapidly spreading H. reticulatum may not grow in an environment polluted by >80 mg L⁻¹ Pb. Because Pb levels in most water bodies are lower than 40 mg L⁻¹, both C. globularis and H. reticulatum can be considered for phytoremediation of Pb pollution.