Biogenic zinc oxide nanoparticles-enhanced biosynthesis of lignans and neolignans in cell suspension cultures of Linum usitatissimum L.

Zinc oxide nanoparticles (NPs) have emerged as a novel elicitor for enhanced biosynthesis of secondary metabolites in in vitro plant cell cultures. The current study was aimed to explore elicitation abilities of ZnO-NPs for enhanced accumulation of lignans and neolignans in cell cultures of Linum usitatissimum. We optimized concentration of zinc oxide NPs before carrying out a full-fledged experiment. Subsequently, an optimum dose of 100 mg/l was introduced into the culture medium on day 0, days 0 and 15, and finally days 0 and 25. We observed that repeated elicitation stimulated various parameters and physiological responses in Linum usitatissimum cell cultures than one-time elicitation. Repeated elicitation of cell cultures on day 0 and 15 resulted in highest fresh weight (412.16 g/l) and lignans production (secoisolariciresinol diglucoside 284.12 mg/l: lariciresinol diglucoside 86.97 mg/l). Contrarily, repeated elicitation on day 0 and 25 resulted in highest DW (13.53 g/l), total phenolic production (537.44 mg/l), total flavonoid production (123.83 mg/l) and neolignans production (dehydrodiconiferyl alcohol glucoside 493.28 mg/l: guaiacylglycerol-ß-coniferyl alcohol ether glucoside 307.69 mg/l). Enhancement in plant growth and secondary metabolites accumulation was several fold higher than controls. Furthermore, a linear relationship existed between total phenolic and flavonoid contents which in turn was correlated with higher antioxidant activities.

Salicylic acid-enhanced biosynthesis of pharmacologically important lignans and neo lignans in cell suspension culture of Linum ussitatsimum L.

Linum usitatsimum L. (flax) is a perennial herb with magnitude of medicinal and commercial applications. In the present study, we investigated the effects of salicylic acid (SA) on biosynthesis of lignans (secoisolariciresinol diglucoside (SDG) and lariciresinol diglucoside (LDG)) and neolignans (dehydrodiconiferyl alcohol glucoside (DCG) and guaiacylglycerol-ß-coniferyl alcohol ether glucoside (GGCG)) in cell cultures of flax. Moderate concentration of SA (50 µM) enhanced biomass accumulation (10.98 g/L dry weight (DW)), total phenolic content (37.81 mg/g DW), and antioxidant potential (87.23%) to two-fold than their respective controls after 72 h of exposure. However, higher levels of total flavonoid content (5.32 mg/g DW) were noted after 48 h of exposure to 50 µM of SA. HPLC analyses revealed that 50 µM SA, significantly enhanced biosynthesis of SDG (7.95 mg/g DW), LDG (7.52 mg/g DW), DCG (54.90 mg/g DW), and GGCG (16.78 mg/g DW), which was almost 2.7, 1.8, 3.88, and 3.98 fold higher than their respective controls after 72 h of exposure time, respectively. These results indicated that moderate concentrations of SA had significant effects on biosynthesis and productivity of lignans and neolignans in cell culture of L. usitatissimum.

LED-enhanced biosynthesis of biologically active ingredients in callus cultures of Ocimum basilicum.

Ocimum basilicum is a medicinal plant with multiple health benefits including cardiovascular, cancer and diabetics. In the present study, the influences of light emitting diodes (LEDs) were investigated on the accumulation of biologically active ingredients in callus cultures of Ocimum basilicum. Among the various tested treatments optimum levels of Total phenolic content (TPC) was noted in callus culture grown under blue lights as compared to control, while maximum accumulation of Total flavonoid content (TFC) was noted in callus culture grown under red light as compared to control. HPLC analyses showed that highest concentrations of Rosmarinic acid (96.0 mg/g DW) and Eugenol (0.273 mg/g DW) were accumulated in blue light which was 2.46 and 2.25 times greater than control (39.0 mg/g DW, 0.171 mg/g DW), respectively. Chicoric acid (81.40 mg/g DW) optimum accumulation was noted in callus grown under the continuous white light, which was almost 4.52 times greater than control. Anthocyanins content were also analyzed, the highest amount of Peonidin (0.127 mg/g DW) and cyanidin (0.1216 mg/g DW) were found in callus culture grown under red light. These findings suggest that application of LED's is a promising strategy for enhancing production of biologically active ingredients in callus cultures Ocimum basilicum.

In vitro cultures of Linum usitatissimum L.: Synergistic effects of mineral nutrients and photoperiod regimes on growth and biosynthesis of lignans and neolignans.

The multipurpose plant species Linum usitatissimum famous for producing linen fibre and containing valuable pharmacologically active polyphenols, has rarely been tested for it's in vitro biosynthesis potential of lignans and neolignans. The current study aims at the synergistic effects of mineral nutrients variation and different photoperiod treatments on growth kinetics and biomass accumulation in in vitro cultures of Linum usitatissimum. Both nutrient quality and quantity affected growth patterns, as cultures established on Gamborg B5 medium had comparatively long exponential phase compared to Murashige and Skoog medium, while growth was slow but steady until last phases of the culture on Schenk and Hildebrandt medium. Similarly, we observed that boron deficiency and nitrogen limitation in culture medium (Gamborg B5 medium) enhanced callus biomass (fresh weight 413 g/l and dry weight 20.7 g/l), phenolics production (667.60 mg/l), and lignan content (secoisolariciresinol diglucoside 6.33 and lariciresinol diglucoside 5.22 mg/g dry weight respectively) at 16/8 h light and dark-week 4, while that of neolignans (dehydrodiconiferyl alcohol glucoside 44.42 and guaiacylglycerol-ß-coniferyl alcohol ether glucoside 9.26 mg/g dry weight, respectively) in continuous dark after 4th week of culture. Conversely, maximum flavonoids production occurred at both Murashige and Skoog, Schenk and Hildebrandt media (both media types contain comparatively higher boron and nitrogen content) in the presence of continuous light. Generally, continuous dark had no significant role in any growth associated parameter. This study opens new dimension for optimizing growing conditions and evaluating underlying mechanisms in biosynthesis of lignans and neolignans in in vitro cultures of Linum usitatissimum.

Synergistic effects of drought stress and photoperiods on phenology and secondary metabolism of Silybum marianum.

Silybum marianum is an important medicinal plant of the family Asteraceae, well known for its set of bioactive isomeric mixture of secondary metabolites "silymarin", primarily acting as a hepato-protective agent. Abiotic stress augments plant secondary metabolism in different plant tissues to withstand harsh environmental fluctuations. In the current study, our aim was to induce drought stress in vitro on S. marianum under the influence of different photoperiod treatments to study the effects, with respect to variations in secondary metabolic profile and plant growth and development. S. marianum was extremely vulnerable to different levels of mannitol-induced drought stress. Water deficiency inhibited root induction completely and retarded plant growth was observed; however, phytochemical analysis revealed enhanced accumulation of total phenolic content (TPC), total flavonoid content (TFC), and total protein content along with several antioxidative enzymes. Secondary metabolic content was positively regulated with increasing degree of drought stress. A dependent correlation of seed germination frequency at mild drought stress and antioxidative activities was established with 2 weeks dark + 2 weeks 16/8 h photoperiod treatment, respectively, whereas a positive correlation existed for TPC and TFC when 4 weeks 16/8 h photoperiod treatment was applied. The effects of drought stress are discussed in relation to phenology, seed germination frequency, biomass build up, antioxidative potential, and secondary metabolites accumulation.