Prevalence of crimean Congo hemorrhagic fever in Khyber Pakhtunkhwa, Pakistan.

BACKGROUND: CCHFV is well recognized as a major public health threat and its prevalence and epidemiological distribution in Pakistan and specifically in KP province is not well documented. METHODS: We used a gold-standard PCR-based diagnostic assay for confirmation of CCHFV among suspected patients. A total of 150 patients were enrolled from June 2022 to September 2022 and their blood samples were collected for PCR confirmation. RESULTS: The overall positivity rate for CCHFV was 26.67 %, with the virus mostly prevalent in the middle-aged group (21-40 years). In the July of 2022, a significant spike in the prevalence of CCHFV was observed in provincial capital Peshawar with the highest burden (31.57 %). CONCLUSION: Our findings indicate the necessity of strengthening CCHFV monitoring programs and intensifying efforts to identify hotspot regions for effective surveillance and control of CCHFV. The months before the Eid-ul-Adha are crucial in the context of CCHFV control.

Antibacterial and Antibiofilm Activity of Ficus carica-Mediated Calcium Oxide (CaONPs) Phyto-Nanoparticles.

The significance of nanomaterials in biomedicines served as the inspiration for the design of this study. In this particular investigation, we carried out the biosynthesis of calcium oxide nanoparticles (CaONPs) by employing a green-chemistry strategy and making use of an extract of Ficus carica (an edible fruit) as a capping and reducing agent. There is a dire need for new antimicrobial agents due to the alarming rise in antibiotic resistance. Nanoparticles' diverse antibacterial properties suggest that they might be standard alternatives to antimicrobial drugs in the future. We describe herein the use of a Ficus carica extract as a capping and reducing agent in the phyto-mediated synthesis of CaONPs for the evaluation of their antimicrobial properties. The phyto-mediated synthesis of NPs is considered a reliable approach due to its high yield, stability, non-toxicity, cost-effectiveness and eco-friendliness. The CaONPs were physiochemically characterized by UV-visible spectroscopy, energy-dispersive X-ray (EDX), scanning-electron microscopy (SEM), X-ray diffraction (XRD), and Fourier-transform infrared spectroscopy (FTIR). The biological synthesis of the calcium oxide nanoparticles revealed a characteristic surface plasmon resonance peak (SPR) at 360 nm in UV-Vis spectroscopy, which clearly revealed the successful reduction of the Ca2+ ions to Ca0 nanoparticles. The characteristic FTIR peak seen at 767 cm-1 corresponded to Ca-O bond stretching and, thus, confirmed the biosynthesis of the CaONPs, while the scanning-electron micrographs revealed near-CaO aggregates with an average diameter of 84.87 ± 2.0 nm. The antibacterial and anti-biofilm analysis of the CaONPs showed inhibition of bacteria in the following order: P. aeruginosa (28 ± 1.0) > S. aureus (23 ± 0.3) > K. pneumoniae (18 ± 0.9) > P. vulgaris (13 ± 1.6) > E. coli (11 ± 0.5) mm. The CaONPs were shown to considerably inhibit biofilm formation, providing strong evidence for their major antibacterial activity. It is concluded that this straightforward environmentally friendly method is capable of synthesizing stable and effective CaONPs. The therapeutic value of CaONPs is indicated by their potential as a antibacterial and antibiofilm agents in future medications.

The multipotent thidiazuron: A mechanistic overview of its roles in callogenesis and other plant cultures in vitro.

Thidiazuron (TDZ) is an active substituted phenyl urea compound that has found a significant role as a plant growth regulator. The most exciting aspect of its function is that it can mimic auxins and cytokinin but is chemically different from these two. Many theories have been put forward, and experiments performed to understand the mode of action of TDZ in callogenesis. One suggested mechanism presents that it works by inhibiting the cytokinin degrading enzymes that compete with cytokinin for an active site on the enzyme. An example is the TDZ-induced suppressed expression of gibberellic acid (GA) biosynthesis genes encoding GA3 and GA20 oxidases. This is entailed with a slightly increased expression of GA catabolism genes encoding GA20 oxidase. Similarly, one of the recommendations is that TDZ induces the expression of specific genes and transcription regulatory sequences that are either responsible directly for callus formation or in turn induce other auxins or cytokinin for callogenesis. There is no concise review available that discusses the details of TDZ-induced callus, specifically and other in vitro cultures in general. This review is an attempt to explore all these pathways and mechanisms involved in callogenesis in plants stimulated by TDZ.

Iron-doped zinc oxide nanoparticles-triggered elicitation of important phenolic compounds in cell cultures of Fagonia indica.

The callus cultures of Fagonia indica could prove as factories for the production of important phytochemicals when triggered through different types of stress. In this study, we initiated callus cultures from healthy stem explants in the presence of iron-doped zinc oxide nanoparticles (Fe-ZnO-NPs). We performed experiments with the callus cultures of F. indica to determine the impact of Fe-ZnO-NPs in concentrations (15.62-250 µg/mL) on biomass accumulation, production of important phenolic and flavonoids, and antioxidative potential. Our results showed that maximum callus biomass [Fresh weight (FW) = 13.6 g and Dry weight (DW) = 0.58 ± 0.01] was produced on day 40 when the media was supplemented with 250 µg/mL Fe-ZnO-NPs. Similarly, maximum total phenolic content (268.36 µg GAE/g of DW) was observed in 40 days old callus added with 125 µg/mL Fe-ZnO-NPs. Maximum total flavonoid content (78.56 µg QE/g of DW) was recorded in 20 days old callus grown in 62.5 µg/mL Fe-ZnO-NPs containing media. Maximum total antioxidant capacity (390.74 µg AAE/g of DW) was recorded in 40 days old callus with 125 µg/mL Fe-ZnO-NPs treated cultures, respectively. Similarly, the highest free radical scavenging activity (93.02%) was observed in callus derived from media having 15.62 µg/mL Fe-ZnO-NPs. The antioxidant potential was observed to have positive correlation with TPC (r = 0.44). HPLC analysis showed that Fe-ZnO-NPs produced compounds (e.g., Epigallocatechin gallate) that were either absent or in lesser quantities in the control group. These results showed that Fe-ZnO-NPs elicitors could increase the biomass and activate secondary metabolism in F. indica cells. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s11240-021-02123-1.

Interactive effects of zinc oxide nano particles and different light regimes on growth and silymarin biosynthesis in callus cultures of Silybum marianum L.

Silybum marianum L. commonly known as milk thistle is a medicinally potent plant with a multitude of pharmacological applications. The present investigations demonstrated the effects of Zinc Oxide nanoparticles (ZnO NPs) on callus growth and biosynthesis of silymarin in milk thistle under various light conditions. The callus cultures developed on Murashige and Skoog (MS) basal media containing ZnO NPs (0.15 mg/L), under the dark condition maintained for two weeks, followed by transference into normal light produced the maximum callus fresh weight (2294 mg/L FW). Further, the metabolite profiling revealed that ZnO NPs significantly augmented the production of silymarin and upregulated the antioxidant system in the callus cultures. Maximum TPC (total phenolic content: 37 ± 0.20 mg/g DW), TFC (total flavonoid content: 8.9 ± 0.023), DPPH antioxidant activity (91.5 ± 1.75%), Superoxide dismutase activity (SOD: 4.1 ± 0.045 nM/min/mg FW) and the highest silymarin content (14.6 ± 0.023 mg/g DW) were recorded in the callus cultures developed on MS media supplemented with solitary ZnO NPs (0.15 mg/L). While the callus culture evolved in presence of only PGRs (2,4 D and BA: 2 mg/L, each) accumulated the lesser fresh weight (562 mg/L FW). A higher concentration of ZnO NPs (0.15 mg/L) enhanced the secondary metabolite accumulation and silymarin content in the callus of Silybum marianum. This is the first standardized protocol to be applied on the industrial level for the production of silymarin.

Genetic Engineering Approaches for Enhanced Insect Pest Resistance in Sugarcane.

Sugarcane (Saccharum officinarum), a sugar crop commonly grown for sugar production all over the world, is susceptible to several insect pests attack in addition to bacterial, fungal and viral infections leading to substantial reductions in its yield. The complex genetic makeup and lack of resistant genes in genome of sugarcane have made the conventional breeding a difficult and challenging task for breeders. Using pesticides for control of the attacking insects can harm beneficial insects, human and other animals and the environment as well. As alternative and effective strategy for control of insect pests, genetic engineering has been applied for overexpression of cry proteins, vegetative insecticidal proteins (vip), lectins and proteinase inhibitors (PI). In addition, the latest biotechnological tools such as host-induced gene silencing (HIGS) and CRISPR/Cas9 can be employed for sustainable control of insect pests in sugarcane. In this review overexpression of the cry, vip, lectins and PI genes in transgenic sugarcane and their disease resistance potential is described.

Plant in vitro Culture Technologies; A Promise Into Factories of Secondary Metabolites Against COVID-19.

The current pandemic has caused chaos throughout the world. While there are few vaccines available now, there is the need for better treatment alternatives in line with preventive measures against COVID-19. Along with synthetic chemical compounds, phytochemicals cannot be overlooked as candidates for drugs against severe respiratory coronavirus 2 (SARS-CoV-2). The important role of secondary metabolites or phytochemical compounds against coronaviruses has been confirmed by studies that reported the anti-coronavirus role of glycyrrhizin from the roots of Glycyrrhiza glabra. The study demonstrated that glycyrrhizin is a very promising phytochemical against SARS-CoV, which caused an outbreak in 2002-2003. Similarly, many phytochemical compounds (apigenin, betulonic acid, reserpine, emodin, etc.) were isolated from different plants such as Isatis indigotica, Lindera aggregate, and Artemisia annua and were employed against SARS-CoV. However, owing to the geographical and seasonal variation, the quality of standard medicinal compounds isolated from plants varies. Furthermore, many of the important medicinal plants are either threatened or on the verge of endangerment because of overharvesting for medicinal purposes. Therefore, plant biotechnology provides a better alternative in the form of in vitro culture technology, including plant cell cultures, adventitious roots cultures, and organ and tissue cultures. In vitro cultures can serve as factories of secondary metabolites/phytochemicals that can be produced in bulk and of uniform quality in the fight against COVID-19, once tested. Similarly, environmental and molecular manipulation of these in vitro cultures could provide engineered drug candidates for testing against COVID-19. The in vitro culture-based phytochemicals have an additional benefit of consistency in terms of yield as well as quality. Nonetheless, as the traditional plant-based compounds might prove toxic in some cases, engineered production of promising phytochemicals can bypass this barrier. Our article focuses on reviewing the potential of the different in vitro plant cultures to produce medicinally important secondary metabolites that could ultimately be helpful in the fight against COVID-19.

Therapeutic potential of medicinal plants against COVID-19: The role of antiviral medicinal metabolites.

There are numerous trials underway to find treatment for the COVID-19 through testing vaccines as well as existing drugs. Apart from the many synthetic chemical compounds, plant-based compounds could provide an array of \suitable candidates for testing against the virus. Studies have confirmed the role of many plants against respiratory viruses when employed either as crude extracts or their active ingredients in pure form. The purpose of this review article is to highlight the importance of phytomedicine against COVID-19. The main aim is to review the mechanistic aspects of most important phytochemical compounds that have showed potential against coronaviruses. Glycyrrhizin from the roots of Glycyrrhiza glabra has shown promising potential against the previously epidemic coronavirus, SARS-CoV. Other important plants such as Artemisia annua, Isatis indigotica, Lindera aggregate, Pelargonium sidoides, and Glychirrhiza spp. have been employed against SARS-CoV. Active ingredients (e.g. emodin, reserpine, aescin, myricetin, scutellarin, apigenin, luteolin, and betulonic acid) have shown promising results against the coronaviruses. Phytochemicals have demonstrated activity against the coronaviruses through mechanisms such as viral entry inhibition, inhibition of replication enzymes and virus release blockage. However, compared to synthetic drugs, phytomedicine are mechanistically less understood and should be properly evaluated before application. Nonetheless, phytochemicals reduce the tedious job of drug discovery and provide a less time-consuming alternative for drug testing. Therefore, along with other drugs currently tested against COVID-19, plant-based drugs should be included for speedy development of COVID-19 treatment.

Micropropagation and Production of Health Promoting Lignans in Linum usitatissimum.

Linum usitatissimum commonly known as flax or linseed is an important medicinal plant, produces medicinally potent lignans, used in the treatment of several human diseases. Lignans limited production in the natural plants does not meet the increasing market demand. This study was conducted to establish an easy and rapid method for the in vitro micropropagation and production of potent lignans and antioxidant secondary metabolites in linseed. The results indicated that hypocotyl explants under the effects of thidiazuron (TDZ: 0.5 mg/L) + kinetin (Kn: 0.5 mg/L) in the basal growth media, resulted in the optimal shoot organogenesis parameters (shoot induction frequency: 86.87%, number of shoots: 6.3 ± 0.36 and shoots length: 6.5 ± 0.54 cm), in 4 weeks. Further, TDZ supplementation in the culture media efficiently activated the antioxidant system in the in vitro raised shoots, wherein maximum production of total phenolic content, TPC (34.33 ± 0.20 mg of GAE/g DW); total flavonoid content, TFC (8.99 ± 0.02 mg of QE/g DW); DPPH free radical scavenging activity (92.7 ± 1.32%); phenylalanine ammonia-lyase activity, PAL (8.99 ± 0.02 U/g FW); and superoxide dismutase expression, SOD (3.62 ± 0.01 nM/min/mg FW) were observed in the shoot cultures raised in presence of TDZ: 0.5 mg/L + Kn: 0.5 mg/L. Nonetheless, considerable levels of pharmacologically active lignans such as secoisolariciresinol (SECO: 23.13-37.10 mg/g DW), secoisolariciresinol diglucoside (SDG: 3.32-3.86 mg/g DW) and anhydrosecoisolariciresinol diglucoside (ANHSECO: 5.15-7.94 mg/g DW) were accumulated in the regenerated shoots. This protocol can be scaled up for the commercial production of linseed to meet the market demands for lignans.

Improved Production of Industrially Important Essential Oils Through Elicitation in the Adventitious Roots of Artemisia amygdalina.

The limited production of bioactive essential oils in natural plants does not meet the increasing worldwide market demand. Plant cell culture technology can be used for the higher production of industrially important essential oils. In the present study, a suitable method for production of essential oils was developed through establishment and elicitation of adventitious roots (AR) in a medicinally important plant Artemisia amygdalina D. The results indicated that leaf explants cultured on solid Murashige and Skoog (MS) media supplemented with 1.0 mg/L α- naphthalene acetic acid (NAA) and 4% sucrose instigated the higher AR induction frequency (90 ± 4.25) and maximum AR biomass (fresh biomass: 17.7 g/L). Furthermore, in the AR when transiently elicited with different elicitors for different time periods, methyl jasmonate (Me-J: 0.5 mg/L) resulted in the higher production of total phenolic content (TPC: 3.6 mg), total flavonoid content (TFC: 2.3 mg) and phenylalanine ammonia-lyase (PAL: 4.8 U/g×FW) activity, respectively. Nonetheless, considerable levels of the major bioactive compounds such as α-thujene (6.8%), α-pinene (8.3%), 1,8-cineole (16.2%), camphor (8.4%) and verbenole (10.2%) were recorded in the Me-J treated AR. Thus, a feasible protocol for production of essential oils through AR in A. amygdalina was established, which can be exploited for commercial production of the industrially important terpenes.

Plant-based gold nanoparticles; a comprehensive review of the decade-long research on synthesis, mechanistic aspects and diverse applications.

The worldwide focus on research in the field of green nanotechnology has resulted in the environmentally and biologically safe applications of a diversity of nanomaterials. Nanotechnology, in general, implies the production of nanoparticles having different but regular shapes, sizes, and properties. A lot of studies have been conducted on the synthesis of metal nanoparticles through biological, chemical, and physical methods. Owing to its safety, both environmental and in vivo, as well as the ease of synthesis, biogenic routes especially the plant-based synthesis of metal nanoparticles has been preferred as the best strategy. Among the metal nanoparticles, gold nanoparticles are recognized as the most potent, biocompatible and environment-friendly. A decade of research work has attempted the production of gold nanoparticles mediated by different parts of various plants. Further, these nanoparticles have been engineered through modification in the sizes and shapes for attaining enhanced activity and optimal performance in many different applications including biomedical, antimicrobial, diagnostics and environmental applications. This article reviews the fabrication strategies for gold nanoparticles via plant-based routes and highlights the diversity of the applications of these materials in bio-nanotechnology. The review article also highlights the recent developments in the synthesis and optical properties of gold nanoparticles.

Natural compounds from plants controlling leishmanial growth via DNA damage and inhibiting trypanothione reductase and trypanothione synthetase: an in vitro and in silico approach.

In the present study, four different natural compounds including quercetin, gallic acid, rutin, and lupeol were studied for their anti-leishmanial potentials with anticipated mechanism of action through in vitro and in silico approaches. Results showed that rutin was exceedingly active (IC50; 91.2 µg/ml) against the promastigote form of Leishmania tropica compared to quercetin (IC50; 182.3 µg/ml), gallic acid (IC50; 198.00 µg/ml) and lupeol (IC50; 200.77 µg/ml). Similarly, rutin was highly active against the amastigote form as well, followed by quercetin, gallic acid and lupeol with IC50 values of 101.3 µg/ml, 137.4 µg/ml, 277.2 µg/ml, and 298.9 µg/ml, respectively. These compounds were found to be nontoxic to human blood erythrocytes even at the highest concentration (1000 µg/ml) tested. Rutin and lupeol showed promising DNA degradation/fragmentation activity against the DNA of treated promastigotes which increased with the increase in concentration of the compounds. The in silico investigation revealed that these ligands have high affinity with the important catalytic residues of trypanothione reductase (Try-R) where, rutin showed the lowest docking score (i.e., - 6.191) followed by lupeol (- 5.799), gallic acid and quercetin. In case of ligands' interaction with trypanothione synthetase (Try-S), rutin again showed highest interaction with docking score of - 6.601 followed by quercetin (- 4.996), lupeol and gallic acid. The ADMET prediction of these compounds showed that all the parameters were within the acceptable range as defined for human use while molecular dynamics simulation supported the good interaction of quercetin and rutin against both enzymes. These findings suggest that the studied compounds may control leishmanial growth via DNA damage and inhibiting Try-R and Try-S, the two unique but critical enzymes for leishmania growth.

Comprehensive investigations on anti-leishmanial potentials of Euphorbia wallichii root extract and its effects on membrane permeability and apoptosis.

Clinically available synthetic chemotherapeutics to treat the vector-borne protozoan infection, leishmaniasis, are associated with serious complications such as toxicity and emergence of resistance. Natural products from plants consist of interesting biomolecules that may interfere with DNA or membrane integrity of the parasite and can possibly minimise the associated side effects. In the present study, various fractions of Euphorbia wallichii (EW) root extracts including n-hexane (EWNX), ethyl acetate (EWEA), chloroform (EWCH) and aqueous (EWAQ), were evaluated for their antileishmanial potential against Leishmania tropica followed by investigation of the possible mechanism of action via reactive oxygen species (ROS) quantification, membrane permeability (via sytox green dye) and apoptotic assay (via AO/EB method) using fluorescent microscopy. Two of the fractions i.e. EWEA and EWAQ inhibited the growth of promastigotes (IC50 7.8 and 10.2 µg/mL, respectively) and amastigotes (IC50 9.9 and 13.3 µg/mL, respectively) forms almost at similar concentrations as found for the standard antileishmanial drugs, tartar emetic (TA) and Glucantime (IC50 9.4 and 21.5 µg/mL, respectively). Both the active fractions remained non-toxic towards human blood erythrocytes and were able to cause membrane permeability and apoptotic induction (using Triton X-100 as a positive control) leading to death of Leishmania parasites. However, both the fractions could not triger significant and persistent ROS generation, compared to hydrogen peroxide used as a positive control. Antilesihmanial activity of the two active fractions might be attributed to the presence of high quantity of tannins and saponins.

Biosynthesis of anti-leishmanial natural products in callus cultures of Artemisia scoparia.

Clinically, available synthetic chemotherapeutics in the treatment for leishmaniasis are associated with serious complications, such as toxicity and emergence of resistance. Natural products from plants can provide better remedies against the Leishmania parasite and can possibly minimize the associated side effects. In this study, various extracts of the callus cultures of Artimisia scoparia established in response to different plant growth regulators (PGRs) were evaluated for their anti-leishmanial effects against Leishmania tropica promastigotes, followed by an investigation of the possible mechanism of action through reactive apoptosis assay using fluorescent microscopy. Amongst the different callus extracts, higher anti-leishmanial activity (IC50:19.13 µg/mL) was observed in the callus raised in-vitro in the presence of 6-Benzylaminopurine (BA) plus 2,4-Dichlorophenoxyacetic Acid (2,4-D) at the concentration of 1.5 mg/L, each. Further, the results of apoptosis assay showed a large number of early-stage apoptotic (EA) and late-stage apoptotic (LA) cells in the Leishmania under the effect of callus extract grown in-vitro at BA plus 2,4-D. For the determination of the potent natural products in the callus extracts responsible for the anti-leishmanial activity, extracts were subjected to Gas chromatography-mass spectrometry (GC-MS) for the metabolite analysis. Nonetheless, higher levels of the metabolites, such as nerolidol (22%), pelletierine (18%), aspidin (15%) and ascaridole (11%) were detected in the callus grown in vitro at BA plus 2,4-D (1.5 mg/L, each). This protocol determines a novel method of production of anti-leishmanial natural products through callus cultures of A. scoparia, a medicinal plant.

Production of biomass and medicinal metabolites through adventitious roots in Ajuga bracteosa under different spectral lights.

Ajuga bracteosa an important medicinal herb, is getting endangered worldwide due to destructive harvesting by pharmaceutical industries in its different habitats. It is in dire need for protection and demands conservation and sustainable utilization. In the present study, effects of α-naphthalene acetic acid (NAA) under different spectral lights were estimated on the growth, secondary metabolism and biosynthesis of phenolic acids in adventitious roots (AR) cultures of A. bracteosa. Among the different spectral lights, highest AR induction frequency (88%) and formation of biomass (72 g/L FW and 22 g/L DW) were recorded in explants incubated in the presence of 1.5 mg/L NAA under yellow light. Maximum production of poly phenols (TPC;44.2 mg) and flavonoids (TFC;2.51 mg) were recorded in the AR cultures grown in the presence of blue light. Further, highest total protein content of (401.6 µg) was detected in the AR in response to normal white light. Blue spectral light induced maximum superoxide dismutase (SOD; 2.5 nM) and peroxidase activity (POD;0.85 nM) respectively, in AR cultures. Compared with other monochromatic lights, red light significantly enhanced the antioxidant potential of the AR cultures. Analysis through High performance liquid chromatography (HPLC-DAD) revealed significant variations in the levels of important phenolic acids such as gallic acid, catechin, rutin, caffeic acid, myricetin and apigenin in the AR samples treated with the lights of different spectra.

Silver nanoparticles elicited in vitro callus cultures for accumulation of biomass and secondary metabolites in Caralluma tuberculata.

Elicited plant in vitro cultures are gaining more interest worldwide for their potential in the uniform production of industrially important secondary metabolites. In the present study, different ratios of silver nanoparticles (AgNPs) and plant growth regulators (PGRs) were supplemented to in vitro cultures for the sustainable production of biomass and antioxidant secondary metabolites through callus cultures of Caralluma tuberculata. Results indicated that various concentrations of AgNPs significantly affected the callus proliferation and substantially increased the callus biomass, when combined with PGRs in the MS (Murashige and Skoog) media. The highest fresh (0.78 g/l) and dry (0.051 g/l) biomass accumulation of callus was observed in the cultures raised in vitro at 60 µg/l AgNPs in combination with 0.5 mg/l 2,4-D plus 3.0 mg/l BA. Phytochemical analysis of the callus cultures showed higher production of phenolics (TPC:3.0 mg), flavonoids (TFC:1.8 mg), phenylalanine ammonialyase activity (PAL: 5.8 U/mg) and antioxidant activity (90%), respectively, in the callus cultures established on MS media in the presence of 90 ug/l AgNPs. Moreover, enhanced activities of antioxidant enzymes such as superoxide dismutase (SOD: 4.8 U/mg), peroxidase (POD: 3.3 U/mg), catalase (CAT: 2.5 U/mg) and ascorbate peroxidase (APX: 1.9 U/mg) were detected at higher level (90 ug/l) of AgNPs tested alone for callus proliferation in the MS media. It may be concluded that the AgNPs can be effectively utilized for the enhancement of bioactive antioxidants in the callus cultures of C. tuberculata, a highly medicinal and threatened plant. This protocol can be scaled up for the industrial production of plant biomass and pharmacologically potent metabolites in C. tuberculata.

Feasible production of biomass and natural antioxidants through callus cultures in response to varying light intensities in olive (Olea europaea. L) cult. Arbosana.

Light is the most important physical factor in growth and development of plants. Light intensity is directly proportional to the growth and accumulation of natural antioxidants during in vitro cultures of various medicinal plants. The present research study was designed to determine the effect of different light intensities i.e. normal light (2000-2500 lx), diffused light (500-1000 lx) and complete dark (0 lx) on callus growth dynamics and production of natural antioxidants in olive cult. Arbosana. Highest callus induction frequency (50%) was observed in the stem explants pre-treated with silver nanoparticles suspension (AgNPs: 50 ppm) and cultured on MS media supplemented with combination of 6-Benzylaminopurine (BAP: 2 mg/l), Gibberellic acid (GA3: 1.5 mg/l) plus Naphthalene acetic acid (NAA: 0.5 mg/l). Maximum callus biomass (FW = 1414 mg/l) was recorded when the cultured explants were incubated initially for seven days in complete darkness, followed by transference to diffused light for one week and then finally placed under normal light in total fifty six days culture period. Moreover, phytochemical analysis of the callus cultures showed significantly higher activities of antioxidant enzymes i.e. SOD, POD, CAT and APx (2.45, 2.96, 2.57 and 1.67 U/mg. protein) in the callus cultures grown under dark condition as compared with other light treatments. For non-enzymatic antioxidant potential, maximum activity of TPC, TFC, PAL and DPPH (2.42 mg GAE/g, 1.50 mg QAE/g, 3.95 U/mg and 75%) were recorded in the calli raised in vitro under diffused light. This is the first report on the production of natural antioxidants in response to different light intensities in callus cultures of Olea europaea. Future studies should focus on large scale production of callus cultures in order to yield maximum biomass from this high valued plant.

The interplay between light, plant growth regulators and elicitors on growth and secondary metabolism in cell cultures of Fagonia indica.

Manipulation in the light regimes combined with the effects of plant growth regulators (PGRs) and elicitors through plant cell culture technology is a promising strategy for enhancing the yield of medicinally important secondary metabolites. In this study, the effects of interplay between PGRs, elicitors and light regimes on cell cultures of F. indica have been investigated. The results showed that callus cultures resulted in maximum biomass formation (13.2 g/L) when incubated on solid MS (Murashige and Skoog) medium containing 1.0 mg/L BA under continuous light for 4 weeks. Among the other PGRs, compared with the auxins such as 2,4-D, and IBA, TDZ resulted in higher biomass accumulation (12.1 g/L). Elicitors (Me-J and PAA) resulted in a lower growth response, when compared with cytokinins and a higher response than auxins under all the light regimes on solid MS media. However, in liquid media, no significant increase in biomass was observed in response to the combined effects of PGRs and photoperiod regimes. Further, the highest phenolic content (TPC = 6.8 mg) and flavonoid content (TFC = 5.2 mg) were detected in the dark-grown cell cultures raised in vitro at 0.5 mg/L Me-J. The highest antioxidant activity (88%) was recorded in the dark-grown cell cultures harvested from LOG phase of the growth cycle supplemented with 0.5 mg/L Me-J. Furthermore, BA resulted in considerable flavonoids production (TFC = 4.7 mg) in the cell cultures grown under continuous light. However, overall dark treatment and elicitation with Me-J resulted in the optimal metabolic response in terms of secondary metabolites accumulation in cell suspension cultures of F. indica.

Impacts of hormonal elicitors and photoperiod regimes on elicitation of bioactive secondary volatiles in cell cultures of Ajuga bracteosa.

Light is an important physical factor necessary for the growth, morphogenesis and production of bioactive compounds in plants. In this study, effects of different photoperiod regimes and hormonal elicitors were investigated on the accumulation of biomass, antioxidant potential and biosynthesis of secondary volatiles in the cell cultures of Ajuga bracteosa. Maximum accumulation of biomass (13.2 g/L) was recorded in cell cultures established at 1.0 mg/L benzylaminopurine (BA) in exposure to continuous dark. Biochemical assays showed that in the presence of 0.5 methyl jasmonate (Me-J), cell cultures grown under continuous dark had the higher activities of superoxide dismutase (SOD: 4.5 U/mg), peroxidase (POD: 3.1 U/mg), total phenolic content (TPC: 8.1 mg GAE/g of DW) and total flavonoid content (TFC: 5.2 mg QE/g of DW) respectively. Nonetheless, the free radical scavenging activity (FRSA) was found correlated with the phenyl ammonia lyase (PAL) activity in the dark grown cell cultures. Analysis through gas chromatography-mass spectrometry (GC-MS) showed, biosynthesis of 29 compounds in the in vitro raised cell cultures. The major identified compounds consisted of monoterpene hydrocarbons such as ß-pinene (2.1-9.5%), ß-ocimene (1.4-8.3%), 1-terpinene-4-ol (5.8-9.6%), caryophyllene (1.3-6.2%), ß-farnesene (0.82-7.8), oxygenated monoterpenes including myrtenal (2.2-8.4%), citronellyl acetate (2.1-7.3%) and sesquiterpenes such as caryophyllene oxide (1.5-5.5) and ß-elemene (2.2-8.8%). This protocol has the potential for commercial production of important secondary volatiles.

Impacts of methyl jasmonate and phenyl acetic acid on biomass accumulation and antioxidant potential in adventitious roots of Ajuga bracteosa Wall ex Benth., a high valued endangered medicinal plant.

Ajuga bracteosa is a medicinally important plant globally used in the folk medicine against many serious ailments. In the present study, effects of two significant elicitors, methyl jasmonate (Me-J) and phenyl acetic acid (PAA) were studied on growth parameters, secondary metabolites production, and antioxidant potential in adventitious root suspension cultures of A. bracteosa. The results showed a substantial increase in biomass accumulation, exhibiting longer log phases of cultures growth in response to elicitor treatments, in comparison to control. Maximum dry biomass formation (8.88 DW g/L) was recorded on 32nd day in log phase of culture when  0.6 mg/L Me-J was applied; however, PAA at 1.2 mg/L produced maximum biomass (8.24 DW g/L) on day 40 of culture.  Furthermore, we observed the elicitors-induced enhancement in phenolic content (total phenolic content), flavonoid content (total flavonoid content) and antioxidant activity (free radical scavenging activity) in root suspension cultures of A. bracteosa. Application of 0.6 mg/L and 1.2 mg/L of Me-J, root cultures accumulated higher TPC levels (3.6 mg GAE/g DW) and (3.7 mg GAE/g DW) in the log phase and stationary phase, respectively, while 2.5 mg/L Me-J produced lower levels (1.4 mg GAE/g DW) in stationary phase of growth stages. Moreover, TFC and FRSA values were found in correspondence to TPC values in the respective growth phases at the similar elicitor treatment. Thus, a feasible protocol for establishment of adventitious roots in A. bracteosa was developed and enhancement in biomass and metabolite content in adventitious root was promoted through elicitation.