Medium salt strength induced changes in growth, physiology and secondary metabolite content in adventitious roots of Morinda citrifolia: the role of antioxidant enzymes and phenylalanine ammonia lyase

In an attempt to improve growth and secondary metabolite production, and to understand the possible mechanism involved in relation to the changes in physiology and activities of antioxidant enzymes, we cultured Morinda citrifolia adventitious roots in different strength (0.25, 0.50, 0.75, 1.0, 1.5 and 2.0) of Murashige and Skoog (MS) medium supplemented with 5 mg l⁻¹ indole butyric acid and 30 g l⁻¹ sucrose. Quarter-strength MS medium was proven suitable for the production of both root biomass and secondary metabolites [anthraquinone (AQ), phenolics and flavonoids]. With the increasing salt strength, root growth and AQ accumulation decreased significantly. Higher (1.5 and 2 MS) salt strength provoked osmotic stress resulted in more than twofold free proline accumulation than lower salt strength treated roots and induced free radical scavenging activity. Phenylalanine ammonia lyase activity showed a positive correlation in relation to salt strength that leads to an increase in phenol biosynthesis in expense of AQ formation. The elevated catalase (CAT), guaiacol peroxidase (G-POD) and superoxide dismutase activities and decreased ascorbate peroxidase (APX) activities were observed in roots treated with 2.0 MS. On the other hand, APX activity was strongly activated along with considerable increase in CAT activity at 0.25 MS treated culture. However, the joint functions of CAT, G-POD and APX at 0.25 MS treated cultures were efficient to eliminate the potential danger of hydrogen peroxide (H₂O₂) as evidenced from low H₂O₂ accumulation and low level of lipid peroxidation.     

Production of saponions and polysaccharide in the presence of lactoalbumin hydrolysate in <Emphasis Type="Italic">Panax quinquefolium</Emphasis> L. cells cultures

In this article, ginsenosides and polysaccharide contents in suspension cells and native roots of Panax quinquefolium L. were studied. In order to enhance the contents of ginsenosides and polysaccharide in P. quinquefolium suspension cells, we tested the effects of lactoalbumin hydrolysate on the growth of P. quinquefolium suspension cell, synthesis of ginsenosides and polysaccharide in flask and bioreactor. In flask culture, cells growth ratio was significantly enhanced by the addition of lower concentration of lactoalbumin hydrolysate. Addition of 100 mg L⁻¹ lactoalbumin hydrolysate significantly enhanced the contents of total saponins (5.44 mg g⁻¹ DW) and the contents were 3.89-fold over the control group. Addition of lactoalbumin hydrolysate significantly promoted the accumulation of polysaccharide, except 200 mg L⁻¹ lactoalbumin hydrolysate. The highest total saponins yield (36.72 mg L⁻¹ DW) and polysaccharide yield (0.83 g L⁻¹ DW) were obtained at 100 mg L⁻¹ lactoalbumin hydrolysate. In a 5-L stirred tank bioreactor, the highest contents of total saponins and TRb group ginsenosides were achieved on day 26, while the effect of lactoalbumin hydrolysate on the contents of TRg group ginsenosides were insignificant. This result suggests that lactoalbumin hydrolysate might have triggered the enzyme activities for the synthesis of TRb group ginsenosides. Overall, the highest total saponins yield (31.37 mg L⁻¹ DW) and polysaccharide yield (1.618 g L⁻¹ DW) were obtained on day 26 and day 24 respectively and the polysaccharide yield was 1.95-fold higher than the shake flask culture (0.83 g L⁻¹ DW). These results provided theoretical reference for two-stage culture in suspension cells of P. quinquefolium in bioreactor.     

Sucrose-induced osmotic stress affects biomass,metabolite, and antioxidant levels in root suspension cultures of <Emphasis Type="Italic">Hypericum perforatum</Emphasis> L.

In this study, we investigated the influence of initial sucrose concentration on the accumulation of biomass, phenols, flavonoids, chlorogenic acid, and hypericin in adventitious root cultures of Hypericum perforatum L. Cultures were initiated in shake flasks by using half-strength Murashige and Skoog (MS) medium, 1.0 mg l⁻¹ indolebutyric acid (IBA), 0.1 m g l⁻¹ kinetin, and different concentrations 0, 1, 3, 5, 7, or 9% in w/v) of sucrose and were maintained in darkness. The medium supplemented with 3% (w/v) sucrose resulted in the optimum biomass accumulation, but higher sucrose concentrations (5, 7, and 9%) inhibited biomass accumulation due to the relatively higher osmotic pressure. However, the amount of total phenols, flavonoids, chlorogenic acid, and total hypericin was increased with the roots grown in the medium supplemented with 5, 7, and 9% (w/v) sucrose. The antioxidant potential of methanolic extract [1,1-diphenyl-2-picrylhydrazyl (DPPH) and 2,2-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid; ABTS) radical scavenging activities] of H. perforatum adventitious roots was also assessed and correlated with the metabolite accumulation. Cultures maintained with higher initial sucrose concentration (5, 7, and 9% w/v) showed increased accumulation of phenols, flavonoids, chlorogenic acid, and total hypericin, and this might be due to the osmotic stress at elevated sucrose concentrations. To verify the effect of osmotic stress on lipid peroxidation, the levels of hydrogen peroxide (H₂O₂), malondialdehyde (MDA), and proline were determined in the adventitious roots and the results revealed a marked increase in the concentrations of these compounds. These results suggest that optimal adventitious root biomass could be achieved in the MS medium with 3% (w/v) sucrose and increased sucrose concentration resulted in osmotic stress and, in turn, induces the accumulation of secondary metabolites.     

Growth,secondary metabolite production and antioxidant enzyme response of <Emphasis Type="Italic">Morinda citrifolia</Emphasis> adventitious root as affected by auxin and cytokinin

Morinda citrifolia adventitious roots were cultured in shake flasks using Murashige and Skoog medium with different types and concentrations of auxin and cytokinin. Root (fresh weight and dry weight) accumulation was enhanced at 5 mg l⁻¹ indole butyric acid (IBA) and at 7 and 9 mg l⁻¹ naphthalene acetic acid (NAA). On the other hand, 9 mg l⁻¹ NAA decreased the anthraquinone, phenolic and flavonoid contents more severely than 9 mg l⁻¹ IBA. When adventitious roots were treated with kinetin (0.1, 0.3 and 0.5 mg l⁻¹) and thidiazuron (TDZ; 0.1, 0.3 and 0.5 mg l⁻¹) in combination with 5 mg l⁻¹ IBA, fresh weight and dry weight decreased but secondary metabolite content increased. The secondary metabolite content (including 1,1-diphenyl-2-picrylhydrazyl activity) increased more in TDZ-treated than in kinetin-treated roots. Antioxidative enzymes such as catalase (CAT) and guaiacol peroxidase (G-POD), which play important roles in plant defense, also increased. A strong decrease in ascorbate peroxidase activity resulted in a high accumulation of hydrogen peroxide. This indicates that adventitious roots can grow under stress conditions with induced CAT and G-POD activities and higher accumulations of secondary metabolites. These results suggest that 5 mg l⁻¹ IBA supplementation is useful for growth and secondary metabolite production in adventitious roots of M. citrifolia.     

Production of Cold-Adapted Amylase by Marine Bacterium <Emphasis Type="Italic">Wangia</Emphasis> sp. C52: Optimization,Modeling, and Partial Characterization

The aim of this work was to optimize the fermentation parameters in the shake-flask culture of marine bacterium Wangia sp. C52 to increase cold-adapted amylase production using two statistical experimental methods including Plackett–Burman design, which was applied to find the key ingredients for the best medium composition, and response surface methodology, which was used to determine the optimal concentrations of these components. The results showed starch, tryptone, and initial pH had significant effects on the cold-adapted amylase production. A central composite design was then employed to further optimize these three factors. The experimental results indicated that the optimized composition of medium was 6.38 g L⁻¹ starch, 33.84 g L⁻¹ tryptone, 3.00 g L⁻¹ yeast extract, 30 g L⁻¹ NaCl, 0.60 g L⁻¹ MgSO₄ and 0.56 g L⁻¹ CaCl₂. The optimized cultivation conditions for amylase production were pH 7.18, a temperature of 20°C, and a shaking speed of 180 rpm. Under the proposed optimized conditions, the amylase experimental yield (676.63 U mL⁻¹) closely matched the yield (685.60 U mL⁻¹) predicted by the statistical model. The optimization of the medium contributed to tenfold higher amylase production than that of the control in shake-flask experiments.     

Effects of plant growth regulators and sucrose on post harvest physiology,membrane stability and vase life of cut spikes of gladiolus

Effects of post-harvest application of two plant growth regulators viz., gibberellic acid (GA₃) and benzyl adenine (BA) with sucrose in the vase solution on cell membrane stability and vase life of gladiolus were investigated. The vase solution treatment combinations of GA₃ and BA with sucrose significantly increased the membrane stability index and enhanced the vase life as compared to the sucrose alone treatments or the controls. Vase solution treatment of GA₃ (50 mg l⁻¹), followed by BA (50 mg l⁻¹) with sucrose (50 g l⁻¹) significantly increased solution uptake, fresh weight and dry weight of cut spikes. The same treatments also enhanced the concentration of reducing and non-reducing sugars in gladioli petals 4 days after treatment (DAT). Cut spikes in vase solution enriched with 50 mg l⁻¹ GA₃ + 50 g l⁻¹ sucrose showed higher antioxidative enzyme activities of superoxide dismutase (SOD) and glutathione reductase (GR), lower lipoxygenase (LOX) activity and lipid peroxidation (measured as TBARS). Petal membrane stability index was also highest in cut spikes 6 DAT with 50 mg l⁻¹ GA₃ + 50 g l⁻¹ sucrose vase solution. Treatment of gladiolus cut spikes with 50 mg l⁻¹ GA₃ + 50 g l⁻¹ sucrose vase solution showed two fold increase in vase life and improved flower quality with a higher number of open flower per spike at any one time. These results suggest that post-harvest application of GA₃ (50 mg l⁻¹) with sucrose (50 g l⁻¹) maintains higher spike fresh and dry weight, improves anti-oxidative defence, stabilizes membrane integrity leading to a delay in petal cell death.     

Large-scale cultivation of adventitious roots of Echinacea purpurea in airlift bioreactors for the production of chichoric acid, chlorogenic acid and caftaric acid

Adventitious roots of Echinacea purpurea were cultured in airlift bioreactors (20 l, 500 l balloon-type, bubble bioreactors and 1,000 l drum-type bubble bioreactor) using Murashige and Skoog (MS) medium with 2 mg indole butyric acid l⁻¹ and 50 g sucrose l⁻¹ for the production of chichoric acid, chlorogenic acid and caftaric acid. In the 20 l bioreactor (containing 14 l MS medium) a maximum yield of 11 g dry biomass l⁻¹ was achieved after 60 days. However, the amount of total phenolics (57 mg g⁻¹ DW), flavonoids (34 mg g⁻¹ DW) and caffeic acid derivatives (38 mg g⁻¹ DW) were highest after 50 days. Based on these studies, pilot-scale cultures were established and 3.6 kg and 5.1 kg dry biomass were achieved in the 500 l and 1,000 l bioreactors, respectively. The accumulation of 5 mg chlorogenic acid g⁻¹ DW, 22 mg chichoric acid g⁻¹ DW and 4 mg caftaric acids g⁻¹ DW were achieved with adventitious roots grown in 1,000 l bioreactors.     

Effects of cytokinin on callus proliferation associated with physiological and biochemical changes in <Emphasis Type="Italic">Vitis vinifera</Emphasis> L.

The essential role of 6-benzylaminopurine (BA) in plant tissue culture has been widely known; however, physiological and biochemical mechanisms behind BA requirement have not been fully understood yet. BA may have an important role on callus growth by regulating antioxidant enzyme activities and acting as an effective free radical scavenger. To test this hypothesis, the impact of exogenous BA concentrations on antioxidative system in Vitis vinifera L. cv. ‘Bogazkere’ callus was investigated under in vitro conditions. Relative fresh weight growth (RFWG) of calli, total phenolics (TP) content, endogenous hydrogen peroxide (H₂O₂), malondialdehyde (MDA), proline concentrations, percentage of electrolyte leakage (EL), and some of the antioxidant enzyme activities; such as superoxide dismutase (SOD), and guaiacol peroxidase (POD), catalase (CAT), and ascorbate peroxidase (APX) were measured. Inhibitory effect of high concentrations of BA on antioxidant enzyme activities and RFWG was found. In the presence of BA at 0.1 mg L⁻¹, SOD, POD, and APX activities decreased, while CAT activity increased in comparison with the controls. Exogenous BA treatments higher than 0.1 mg L⁻¹ resulted in an increase in cellular TP, H₂O₂, MDA, proline contents, and percentage of EL, while RFWG of calli decreased. Based on the findings, it may be concluded that only 0.1 mg L⁻¹ BA concentration combined with NAA could play a direct role in reducing the level of free radicals and phenolic production associated with proliferation capacity of grape cells under in vitro conditions. Furthermore, cytokinin was effective in the antioxidative enzyme system, lipid peroxidation, and electrolyte leakage.     

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.     

Characteristics of cadmium accumulation and tolerance in <Emphasis Type="Italic">Rorippa globosa</Emphasis> (Turcz.) Thell., a species with some characteristics of cadmium hyperaccumulation

Characteristics of cadmium (Cd) accumulation and tolerance in Rorippa globosa (Turcz.) Thell., a species with some characteristics of cadmium hyperaccumulation were further investigated and compared with a closely related species, Rorippa islandica. The results showed that there was no phytotoxicity for R. globosa leaves or reduction in biomass when treated with 25 μg Cd g⁻¹, although the concentration of Cd accumulated in the leaves was up to 218.9 μg Cd g⁻¹ dry weight (DW). On the contrary, Cd toxicity was observed in R. islandica leaves by way of determining changes in fresh weight (FW), malondialdehyde (MDA) level and chlorophyll content while treated with 25 μg Cd g⁻¹ DW. R. globosa had stronger self-protection ability than R. islandica to adapt to oxidative stress caused by Cd. Application of Cd significantly increased the activity of superoxide dismutase (SOD) in leaves, the activity of peroxidase (POD) in roots, and the activity of catalase (CAT) in leaves and roots of R. globosa. By contrast, in R. islandica, the activity of antioxidant enzymes was inhibited or unchanged by various Cd treatments. However, R. globosa leaves had higher activity of antioxidant enzymes such as SOD and POD than that of R. islandica. The antioxidative defense systems in R. globosa might play an important role in Cd tolerance. The Cd treatments significantly induced the synthesis of phytochelatins (PCs) in the two species. Leaf PCs and Cd accumulation by R. globosa were much greater than those by R. islandica, but root PCs and Cd accumulation by R. islandica were much greater than those by R. globosa, suggesting that PCs in leaves may be a biomarker of Cd hyperaccumulation, and the synthesis of PCs may be related to an increase in the uptake of Cd ions into the cytoplasm, not the primary mechanism for Cd tolerance.     

Effects of macroelements and nitrogen source on biomass accumulation and withanolide-A production from cell suspension cultures of <Emphasis Type="Italic">Withania somnifera</Emphasis> (L.) Dunal

Withania somnifera is an important medicinal plant that contains withanolides and withaferins, both bioactive compounds. We have tested the effects of macroelements and nitrogen source in W. somnifera cell suspension cultures with the aim of optimizing the production of biomass and withanolide A. The effects of the macroelements NH₄NO_3, KNO_3, CaCl_2, MgSO₄ and KH₂PO₄ at concentrations of 0.0, 0.5, 1.0, 1.5 and 2.0× strength and of the nitrogen source [NH₄ ⁺/NO₃ ⁻ (mM/mM) ratio of: 0.00/18.80, 7.19/18.80, 14.38/18.80, 21.57/18.80, 28.75/18.80, 14.38/0.00, 14.38/9.40, 14.38/18.80, 14.38/28.20, and 14.38/37.60 (mM)] in Murashige and Skoog medium were tested for biomass and withanolide A production. The highest accumulation of biomass [147.81 g l⁻¹ fresh weight (FW) and 14.02 g l⁻¹ (dry weight (DW)] was recorded in the medium containing a 0.5× concentration of NH₄NO₃, and the highest production of withanolide A content was recorded in the medium with 2.0× KNO₃ (4.36 mg g⁻¹ DW). The NH₄ ⁺/NO₃ ⁻ ratio also influenced cell growth and withanolide A production, with both parameters being larger when the NO₃ ⁻ concentration was higher than that of NH₄ ⁺. Maximum biomass growth (110.45 g l⁻¹ FW and 9.29 g l⁻¹ DW) was achieved at an NH₄ ⁺/NO₃ ⁻ ratio of 7.19/18.80, while withanolide A production was greatest (3.96 mg g⁻¹ DW) when the NH₄ ⁺/NO₃ ⁻ ratio was 14.38/37.60 mM.     

Production of withanolide-A from adventitious root cultures of Withania somnifera

Withanolides are biologically active secondary metabolites present in roots and leaves of Withania somnifera. In the present study, we have induced adventitious roots from leaf explants of W. somnifera for the production of withanolide-A, which is having pharmacological activities. Adventitious roots were induced directly from leaf segments of W. somnifera on half strength Murashige and Skoog (MS) semisolid medium (0.8% agar) with 0.5 mg l⁻¹ indole-3-butyric acid (IBA) and 30 g l⁻¹ sucrose. Adventitious roots cultured in flasks using half strength MS liquid medium with 0.5 mg l⁻¹ IBA and 30 g l⁻¹ showed higher accumulation of biomass (108.48 g l⁻¹FW and 10.76 g l⁻¹ DW) and withanolide-A content (8.8 ± 0.20 mg g⁻¹ DW) within five weeks. Nearly 11-fold increment of fresh biomass was evident in suspension cultures and adventitious root biomass produced in suspension cultures possessed 21-fold higher withanolide-A content when compared with the leaves of natural plants. An inoculum size of 10 g l⁻¹ FW favoured the biomass accumulation and withanolide-A production in the tested range of 2.5, 5.0, 10.0 and 20.0 g l⁻¹ FW. Among different media tested [Murashige and Skoog (MS), Gamborg’s (B5), Nitsch and Nitsch (NN) and Chu’s (N6)], MS medium favoured both biomass accumulation and withanolide-A production. Half strength MS medium favoured the biomass accumulation and withanolide-A production among the different strength MS medium tested (0.25, 0.5, 0.75, 1.0, 1.5 and 2.0). The current results showed great potentiality of adventitious roots cultures for the production of withanolide-A.     

Somatic embryogenesis and plant regeneration in oil palm using the thin cell layer technique

An efficient procedure has been developed for inducing somatic embryogenesis and regeneration of plants from tissue cultures of oil palm (Elaeis guineensis Jacq.). Thin transverse sections (thin cell layer explants) of different position in the shoot apex and leaf sheath of oil palm were cultivated in Murashige and Skoog (MS) (Physiol Plant 15:473–497, 1962) medium supplemented with 0–450 μM picloram and 2,4-D with 3.0% sucrose, 500 mg L⁻¹ glutamine, and 0.3 g L⁻¹ activated charcoal and gelled with 2.5 g L⁻¹ Phytagel. Embryogenic calluses were evaluated 12 wk after inoculation. Picloram (450 μM) was effective in inducing embryogenic calluses in 41.5% of the basal explants. Embryogenic calluses were maintained on a maturation medium composed of basal media, plus 0.6 μM NAA and 12.30 μM 2iP, 0.3 g L⁻¹ activated charcoal, and 500 mg L⁻¹ glutamine, with subcultures at 4-wk intervals. Somatic embryos were converted to plants on MS medium with macro- and micronutrients at half-strength, 2% sucrose, and 1.0 g L⁻¹ activated charcoal and gelled with 2.5 g L⁻¹ Phytagel.     

Adventitious root suspension cultures of <Emphasis Type="BoldItalic">Hypericum perforatum</Emphasis>: effect of nitrogen source on production of biomass and secondary metabolites

The present study investigated the effect of nitrogen source (NH₄⁺; NO₃⁻) at different concentrations on the accumulation of biomass and secondary metabolites in adventitious root cultures of Hypericum perforatum L. Cultures were initiated in shake flasks by using half-strength Murashige and Skoog (MS) medium with B5 vitamins, 1.0 mg l⁻¹ indole-3-butyric acid, 0.1 mg l⁻¹ kinetin, 3% (w/v) sucrose, and different ratios of ammonium and nitrate (0:30, 5:25, 10:20, 15:15, 20:10, 25:5, and 30:0 mM, using NH₄Cl and KNO₃). The cultures were maintained in darkness. The medium supplemented with 5:25 (mM) NH₄⁺/NO₃⁻ resulted in the optimum accumulation of biomass and total phenols and flavonoids. The antioxidant potential of a methanolic extract, measured as the 1, 1-diphenyl-2-picrylhydrazyl and 2, 2-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid) radical scavenging activities, of H. perforatum adventitious roots showed that antioxidant activity was high from root extracts that were grown on higher concentrations of NO₃⁻ nitrogen (15, 20, and 25 mM). Further, assessment of hydrogen peroxide (H₂O₂) and malondialdehyde content of the root extracts revealed that cultures supplemented with higher levels of NO₃⁻ nitrogen (15–30 mM) were under oxidative stress, which boosted the levels of secondary metabolites in the adventitious roots. These results suggest that optimal adventitious root biomass could be achieved with the supplementation of cultures with 5:25 ratios of MS nitrogen sources.     

Cultivation of <Emphasis Type="Italic">Chlorella emersonii</Emphasis> with flue gas derived from a cement plant

The present study reviews the options of cultivating the green alga, Chlorella emersonii, under photoautotrophic conditions with flue gas derived from a cement plant. It was conducted in the Lafarge Perlmooser plant in Retznei, Austria, where stone coal and various surrogate fuels such as used tyres, plastics and meat-and-bone meal are incinerated for heating limestone. During 30 days of cultivation, flue gas had no visible adverse effects compared to the controls grown with pure CO₂. The semi-continuous cultivation with media recycling was performed in 5.5-L pH-stat photobioreactors. The essay using CO₂ from flue gas yielded a total of 2.00 g L⁻¹ microalgal dry mass and a CO₂ fixation of 3.25 g L⁻¹. In the control, a total of 2.06 g L⁻¹ dry mass was produced and 3.38 g L⁻¹ CO₂ was fixed. Mean growth rates were between 0.10 day⁻¹ (control) and 0.13 day⁻¹ (flue gas). No accumulation of flue gas residues was detected in the culture medium. At the end of the experiment, however, the concentration of lead was three times higher in algal biomass compared to the control, indicating that cultures aerated with this type of flue gas should not be used as food supplements or animal feed.     

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.     

Soluble sugar,starch and phenolic status during rooting of easy- and difficult-to-root magnolia cultivars

The aim of the study was to determine the effect of indole-3-butyric acid (IBA) and exogenous sucrose concentrations on in vitro rooting, soluble sugar, starch and phenolic production, and ex vitro survival of four magnolia cultivars. There was a significant linear increase in rooting of most magnolia genotypes with an increase in IBA concentration in the medium from 1 to 6 mg L^?1. A further increase of IBA concentration to 10 mg L^?1 decreased (‘Elizabeth’, ‘Burgundy’) or had no effect on rooting frequency (‘Spectrum’). The effect of IBA on rooting of magnolia shoots was modified by sucrose supply. The three out of four magnolia cultivars showed the highest rooting efficiency in the presence of 6 mg L^?1 IBA and 30 g L^?1 of sucrose. Generally, decreasing and increasing the sucrose supply from 30 g L^?1 significantly lowered the rooting frequency. In ‘Yellow Bird’, sucrose at 40 g L^?1 totally blocked root formation. It has been found that the poor rooting ability of ‘Yellow Bird’ coincided with a low soluble sugar content, and high production of starch and phenolics in the shoot bases during the whole rooting period as compared to easy-to-root cultivars. After 5 weeks of the growth on IBA medium, rooted and unrooted shoots were transferred to ex vitro conditions. Both types of shoots showed a high survival and rooting rate (85.4–100%), but they differed in their growth activity and quality. Sucrose concentration in the rooting medium had a post-effect on ex vitro root formation and survival of magnolia plantlets. Ex vitro establishment (13.3%) of recalcitrant ‘Yellow Bird’ was obtained only when the shoots were taken from rooting medium containing the lowest level of sucrose (20 g L^?1).

     

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).     

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.