Metabolites In Cell Suspension Cultures Research Articles

Stimulation of Secondary Metabolites and γ-Terpinene Synthase by Silver Nanoparticles in Callus Cultures of Carum carvi.

Biotechnology and nanotechnology are important tools for understanding biochemical pathways. They can be used efficiently for stimulating and increasing the production of secondary metabolites in medicinal plants. The present study aimed to identify the γ-terpinene synthase gene (CcTPS2) as an effective contributor to the biosynthetic pathway of monoterpenes. The effects of silver nanoparticles (AgNPs; 50 and 100mg l- 1) and time (24 and 48h) were examined on secondary metabolites in cell suspension cultures of Carum carvi. This involved the identification, isolation, and sequencing of a partial sequence in the CcTPS2 gene of C. carvi. The genomic sequence of CcTPS2 comprised 292bp which were organized into two exons (110 and 82bp) and one intron (100bp), while the cDNA was 192bp. In the scale of nucleotides, the CcTPS2 gene showed 96% similarity with the TPS2 gene of Oliveria decumbens. We generated sequence data of the CcTPS2 gene for the first time in this species, thereby enabling further developments in understanding the molecular mechanisms responsible for terpene biosynthesis and other chemical derivatives in C. carvi. The results of GC/MS and GC/FID showed that AgNPs strongly affected the secondary metabolites in cell suspension cultures of C. carvi. According to the results, the AgNPs (50mg l- 1) increased p-cymene and carvone contents in comparison with the control. The exposure of plants to 100mg l- 1 AgNPs induced the production of thymol and carvacrol. The results of real-time PCR revealed that the exposure of plants to 100mg l- 1 AgNPs caused a significant upregulation of CcTPS2 expression for 24h. These cell suspension cultures were elicited by AgNPs, the application of which proved as an effective method to improve the production of secondary metabolites in vitro.

Effects of Nanoparticles on Activity of Lignan Biosynthesis Enzymes in Cell Suspension Culture of Linum usitatissimum L.

Flax (Linum usitatissimum L.), as an important commercial crops, is a rich source of fatty acids, protein, dietary fiber and lignans. Flax plant has an effective anticancer activity due to lignan contents. Nanoparticles have recently been used as efficient non-biologic elicitors to improve the biosynthesis of secondary metabolites. In this study, the effects of different concentration of ZnO (0, 30, 60, and 120 mg/L) and TiO2 (0, 50, 100, and 150 mg/L) nanoparticles at different time (0, 24, 48, and 72 h) were investigated on enzyme activities and production of secondary metabolites in cell suspension cultures of flax. The results indicated that the highest activity of phenylalanine ammonia lyase (PAL) was observed in 30 mg/L nano-ZnO treatment at 48 h, whereas the effect of nano-TiO2 on PAL enzyme activity was not statistically significant. According to the results, the highest activity of CAD (cinnamyl alcohol dehydrogenase) was observed in 60 mg/L concentration of nano-ZnO at different intervals. The use of 150 mg/L nano-TiO2 led to increased activity of CAD. The maximum content of total phenol was detected at 150 mg/L nano-TiO2. Different concentrations of nano-TiO2, caused to an increase in total lignan at all intervals. The highest amount of total phenol and lignans was observed in 30 and 60 mg/L ZnO. In the present study, we were observed different effects of nanoparticle on enzymes activity and secondary metabolite production in cell suspension cultures of flax plant, depending on concentration and type of nanoparticles.

Influence of fungal elicitor on bio production of Azadirachtin in cell cultures of Azadirachta indica

The present investigation involves the influence of elicitor feeding on bioproduction of important secondary metabolites in cell suspension cultures of Azadirachta indica. The content of azadirachtin in MS media supplemented with NAA (1 mg/l) + Kinetin (0.5 mg/l) and Dicamba (2 mg/l) + Kinetin (1 mg/l) hormonal combinations was found to be (0.42±0.04 mg/l) and (0.30 mg/l±0.07) respectively. Out of these two combinations, NAA (1 mg/l) + Kinetin (0.5 mg/l) combination was found optimum for bioproduction of azadirachtin. The addition of Fusarium solani significantly enhanced the amount of azadirachtin over control cultures. The cell extracts (CE) and culture filtrates (CF) of Fusarium solani were added in different concentrations (5% v/v and 10% v/v) to the cell cultures of A. indica separately, while maintaining suitable control. There is no significant change in the amount of azadirachtin between control and cell extract (CE) treated cultures. The addition of culture filtrate (5% v/v) of Fusarium solani showed significant improvement in bioproduction of azadirachtin (0.87±0.12 mg/l) over to control cultures (0.38±0.07 mg/l). It showed approximately 2-fold improvement in the bioproduction of azadirachtin. Present study constitute an important milestone in realizing commercial production of these metabolites.

Sucrose-enhanced biosynthesis of medicinally important antioxidant secondary metabolites in cell suspension cultures of Artemisia absinthium L.

Natural products are gaining tremendous importance in pharmaceutical industry and attention has been focused on the applications of in vitro technologies to enhance yield and productivity of such products. In this study, we investigated the accumulation of biomass and antioxidant secondary metabolites in response to different carbohydrate sources (sucrose, maltose, fructose and glucose) and sucrose concentrations (1, 3, 5, 7 and 9%). Moreover, the effects of 3% repeated sucrose feeding (day-12, -18 and -24) were also investigated. The results showed the superiority of disaccharides over monosaccharides for maximum biomass and secondary metabolites accumulation. Comparable profiles for maximum biomass were observed in response to sucrose and maltose and initial sucrose concentrations of 3 and 5%. Maximum total phenolic and total flavonoid contents were displayed by cultures treated with sucrose and maltose; however, initial sucrose concentrations of 5 and 7% were optimum for both classes of metabolites, respectively. Following 3% extra sucrose feeding, cultures fed on day-24 (late-log phase) showed higher biomass, total phenolic and total flavonoid contents as compared to control cultures. Highest antioxidant activity was exhibited by maltose-treated cultures. Moreover, sucrose-treated cultures displayed positive correlation of antioxidant activity with total phenolics and total flavonoids production. This work describes the stimulatory role of disaccharides and sucrose feeding strategy for higher accumulation of phenolics and flavonoids, which could be potentially scaled up to bioreactor level for the bulk production of these metabolites in suspension cultures of A. absinthium.

Enhancement of the productivity of tea (Camellia sinensis) secondary metabolites in cell suspension cultures using pathway inducers

Tea cell suspension culture is an alternative method for the synthesis of secondary metabolites. For the separation of cells, different concentrations of pectinase were used and a concentration of 0.5% was found to be the optimum concentration for the separation of cells (41.7%) in the culture medium than the other two concentrations (33.3 and 25.0%). The separated cells were cultured in liquid MS medium using different PGR combinations. The time taken for the cells to reach stationary phase, under different PGRs, ranged from 17 to 21 d. The maximum cell density was found in IAA and 2, 4-D medium at 21 d followed by 2, 4-D. Results revealed that the amount of secondary metabolites such as catechins were high with stationary phase when compared to other growth phases (lag and log phases). Different concentrations of shikimic acid (10, 20, and 30 mM) were added to the stationary phase of cell culture in the bioreactor and the secondary metabolite content was analyzed. Synthesis of polyphenols, catechins, caffeine, and other secondary components were high (33.87, 22.85, and 4.66%) with 20 mM shikimic acid treatment than the other two concentrations.

Accumulation of stress metabolites in cell suspension cultures of Hyoscyamus albus

Cell suspension cultures of Hyoscyamus albus treated with freeze-dried Phytophthora cinnamomi autoclaved mycelium of freeze-dried Candida albicans autoclaved cells accumulated mainly lubimin and 10- 1epilubimun, along with small amounts of rishitin and solavetivone. Cultures treated with cellulase did not accumulate any of the reported compounds. The pattern and the amount of sesquiterpenoids produced were dependent on cell growth stage, incubation time with the elicitor and elicitor concentration. Higher yields of lubimin and 10- epilubimin occurred in cultures inoculated two days after subculturing and incubated for 48 hr. Some elicitor-treated cultures and old untreated ones accumulated acetophenones: acetovanillone, acetosyringone and hydroxyacetosyringone.

Cell Suspension Cultures of Cassia didymobotrya: Optimization of Growth and Secondary Metabolite Production by Application of the Orthogonal Design Method

The optimization of both the cell growth and the accumulation of secondary metabolites in cell suspension cultures of Cassia didymobotrya Fres. was achieved for the first time by applying an orthogonal design method. The influence of the concentration of 2,4-D, kinetin, and sucrose as well as of the amount of the inoculum was studied.

High Field Phosphorus-31 Nuclear Magnetic Resonance Investigation of the Phosphate Metabolites in Cell Suspension Cultures of Nicotiana tabacum

31P NMR measurements have been made of both in vivo cell suspension cultures and cell extracts of Nicotiana tabacum. Comparison of the spectra with those of reference compounds and pH variation studies on the extracts has allowed signals attributable to inorganic phosphate, glucose-6-phosphate, sugar phosphates, phosphodiester, ATP, ADP and a nucleoside-diphosphoglucose to be identified. The in vivo study has allowed most of these signals to be monitored throughout the growth cycle and hence changes in the composition of the cytoplasm and vacuole have been followed. ATP and ADP are both present as the Mg2+-complex in the cytoplasm.

Attempts to demonstrate Incorporation of labelled Precursors into Aromatic Metabolites in Cell Suspension Cultures of Galium mollugo L.

Key Word Index Galium mollugo - Fracer Experiments, Cell Suspension Cultures, Anthraquinones, Anthraquinone Glycosides, Naphthols