Transcriptomic data reveals the dynamics of terpenoids biosynthetic pathway of fenugreek.

Medicinal plants are rich sources for treating various diseases due their bioactive secondary metabolites. Fenugreek (Trigonella foenum-graecum) is one of the medicinal plants traditionally used in human nutrition and medicine which contains an active substance, called diosgenin, with anticancer properties. Biosynthesis of this important anticancer compound in fenugreek can be enhanced using eliciting agents which involves in manipulation of metabolite and biochemical pathways stimulating defense responses. Methyl jasmonate elicitor was used to increase diosgenin biosynthesis in fenugreek plants. However, the molecular mechanism and gene expression profiles underlying diosgening accumulation remain unexplored. In the current study we performed an extensive analysis of publicly available RNA-sequencing datasets to elucidate the biosynthesis and expression profile of fenugreek plants treated with methyl jasmonate. For this purpose, seven read datasets of methyl jasmonate treated plants were obtained that were covering several post-treatment time points (6-120 h). Transcriptomics analysis revealed upregulation of several key genes involved in diosgenein biosynthetic pathway including Squalene synthase (SQS) as the first committed step in diosgenin biosynthesis as well as Squalene Epoxidase (SEP) and Cycloartenol Synthase (CAS) upon methyl jasmonate application. Bioinformatics analysis, including gene ontology enrichment and pathway analysis, further supported the involvement of these genes in diosgenin biosynthesis. The bioinformatics analysis led to a comprehensive validation, with expression profiling across three different fenugreek populations treated with the same methyl jasmonate application. Initially, key genes like SQS, SEP, and CAS showed upregulation, followed by later upregulation of Δ24, suggesting dynamic pathway regulation. Real-time PCR confirmed consistent upregulation of SQS and SEP, peaking at 72 h. Additionally, candidate genes Δ24 and SMT1 highlighted roles in directing metabolic flux towards diosgenin biosynthesis. This integrated approach validates the bioinformatics findings and elucidates fenugreek's molecular response to methyl jasmonate elicitation, offering insights for enhancing diosgenin yield. The assembled transcripts and gene expression profiles are deposited in the Zenodo open repository at https://doi.org/10.5281/zenodo.8155183 .

In vitro and in silico analyses reveal the interaction between LysM receptor-like kinase3 of Solanum tuberosum and the carbohydrate elicitor Riclin octaose.

As a carbohydrate elicitor, Riclin octaose (Rioc) activates the pattern-triggered immunity of Solanum tuberosum L., while how the plant perceives Rioc is unknown. Here, a pattern recognition receptor StLYK3 (LysM receptor-like kinase3) whose transcription level was significantly up-regulated after Rioc elicitation was investigated in vitro and in silico. The nucleotide that encoded the ectodomain of StLYK3 (StLYK3-ECD) was heterologously expressed in the Pichia pastoris strain GS115. The purified StLYK3-ECD had the molecular weight of 25.08 kDa and pI of 5.69. Afterwards interaction between StLYK3-ECD and Rioc was analyzed by isothermal titration calorimetry. The molar ratio of ligand to receptor, dissociation constant, and enthalpy were 1.28 ± 0.04, 26.7 ± 3.1 µM, and -45.0 ± 1.8 kJ mol-1 , respectively. Besides, molecular dynamics results indicated that StLYK3-ECD contained three carbohydrate-binding motifs and the first two motifs probably contributed to the interaction with Rioc via hydrogen bond and van de Waals' forces. Amino acids containing hydroxyl, amidic, and sulfhydryl groups took the main portion in the docking site. Moreover, replacing the 92nd threonyl (T) of StLYK3-ECD with valyl (V) resulted in the alteration of the preferred docking site. The dissociation constant drastically increased to 841.6 ± 232.4 µM. In conclusion, StLYK3 was a potential receptor of Rioc.

Methyl jasmonate conferred Arsenic tolerance in Thymus kotschyanus by DNA hypomethylation, stimulating terpenoid metabolism, and upregulating two cytochrome P450 monooxygenases.

Arsenic (As) is a highly cytotoxic element impairing normal cellular functions, and its bioremediation has become one of the environmental concerns. This study explored the molecular and physiological responses of thyme (Thymus kotschyanus) seedlings to incorporating As (0 and 10 mgl-1) and methyl jasmonate (MJ; 0 and 10 µM) into the culture medium. The MJ treatment reinforced root system and mitigated the As cytotoxicity risk. MJ contributed to hypomethylation, a potential adaptation mechanism for conferring the As tolerance. Two cytochrome P450 monooxygenases, including CYP71D178 and CYP71D180 genes, were upregulated in response to As and MJ. The MJ treatment contributed to up-regulation in the γ-terpinene synthase (TPS) gene, a marker gene in the terpenoid metabolism. The As presence reduced photosynthetic pigments (chlorophylls and carotenoids), while the MJ utilization alleviated the As toxicity. The MJ supplementation increased proline accumulation and soluble phenols. The application of MJ declined the toxicity sign of As on the concentration of proteins. The activities of peroxidase, catalase, and phenylalanine ammonia-lyase (PAL) enzymes displayed an upward trend in response to As and MJ treatments. Taken collective, MJ can confer the As tolerance by triggering DNA hypomethylation, regulating CYPs, and stimulating primary and secondary metabolism, especially terpenoid.

Multifunctionality of AsCFEM6 and AsCFEM12 effectors from the potato early blight pathogen Alternaria solani.

Plant pathogens secrete fungal-specific common in several fungal extracellular membrane (CFEM) effectors to manipulate host immunity and contribute to their virulence. Little is known about effectors and their functions in Alternaria solani, the necrotrophic fungal pathogen causing potato early blight. To identify candidate CFEM effector genes, we mined A. solani genome databases. This led to the identification of 12 genes encoding CFEM proteins (termed AsCFEM1-AsCFEM12) and 6 of them were confirmed to be putative secreted effectors. In planta expression revealed that AsCFEM6 and AsCFEM12 have elicitor function that triggers plant defense response including cell death in different botanical families. Targeted gene disruption of AsCFEM6 and AsCFEM12 resulted in a change in spore development, significant reduction of virulence on potato and eggplant susceptible cultivars, increased resistance to fungicide stress, variation in iron acquisition and utilization, and the involvement in 1,8-dihydroxynaphthalene (DHN) melanin biosynthesis pathway. Using maximum likelihood method, we found that positive selection likely caused the polymorphism within AsCFEM6 and AsCFEM12 homologs in different Alternaria spp. Site-directed mutagenesis analysis indicated that positive selection sites within their CFEM domains are required for cell death induction in Nicotiana benthamiana and are critical for response to abiotic stress in yeast. These results demonstrate that AsCFEM effectors possess additional functions beyond their roles in host plant immune response and pathogen virulence.

Biological and Chemical Characterization of Musa paradisiaca Leachate.

There is a growing demand for molecules of natural origin for biocontrol and biostimulation, given the current trend away from synthetic chemical products. Leachates extracted from plantain stems were obtained after biodegradation of the plant material. To characterize the leachate, quantitative determinations of nitrogen, carbon, phosphorus, and cations (K+, Ca2+, Mg2+, Na+), Q2/4, Q2/6, and Q4/6 absorbance ratios, and metabolomic analysis were carried out. The potential role of plantain leachates as fungicide, elicitor of plant defense, and/or plant biostimulant was evaluated by agar well diffusion method, phenotypic, molecular, and imaging approaches. The plant extracts induced a slight inhibition of fungal growth of an aggressive strain of Colletotrichum gloeosporioides, which causes anthracnose. Organic compounds such as cinnamic, ellagic, quinic, and fulvic acids and indole alkaloid such as ellipticine, along with some minerals such as potassium, calcium, and phosphorus, may be responsible for the inhibition of fungal growth. In addition, jasmonic, benzoic, and salicylic acids, which are known to play a role in plant defense and as biostimulants in tomato, were detected in leachate extract. Indeed, foliar application of banana leachate induced overexpression of LOXD, PPOD, and Worky70-80 genes, which are involved in phenylpropanoid metabolism, jasmonic acid biosynthesis, and salicylic acid metabolism, respectively. Leachate also activated root growth in tomato seedlings. However, the main impact of the leachate was observed on mature plants, where it caused a reduction in leaf area and fresh weight, the remodeling of stem cell wall glycopolymers, and an increase in the expression of proline dehydrogenase.

In situ self-induced electrical stimulation to plants: Modulates morphogenesis, photosynthesis and gene expression in Vigna radiata and Cicer arietinum.

Specific stimuli to plants influence intracellular and intercellular communications, activation of ion channels, gene expression, growth and development. The functional role of self-induced in situ electrical stimuli at the rhizosphere of the plant by placing electrode assembly in a defined circuit mode was studied on the growth and development of Vigna radiata and Cicer arietinum plants. Experiments were designed with three-circuit mode configurational variations (CC-P, OC-P and SC-P) and compared with the relative performance of control system (non-potential). The plants cultivated under the in situ electrical stimuli (low-current) showed a marked influence on growth and photosynthetic performance of the plants. CC-P operation showed improved vegetative growth, characterized by increased roots, shoots and biomass along with accelerated plant growth from seed germination to vegetation, flowering and pod formation leading towards earlier and more robust flowering compared to control system. Plants also showed higher aquaporin gene expression levels in CC-P operation. The control operation showed 10 days additional maturation time compared to CC-P operation. The strategy can be beneficially applied to augment the bioremediation capacity of complex pollutants with reference to phytoremediation or constructed wetland systems where the plant and its roots are the main enabler apart from agriculture applications specific to nursery-raised or transplanted plants.

Exogenous Allantoin Confers Rapeseed (Brassica campestris) Tolerance to Simulated Drought by Improving Antioxidant Metabolism and Physiology.

Allantoin is an emerging plant metabolite, but its role in conferring drought-induced oxidative stress is still elusive. Therefore, an experiment was devised to explore the role of allantoin (0.5 and 1.0 mM; foliar spray) in rapeseed (Brassica campestris cv. BARI Sarisha-17) under drought. Seedlings at fifteen days of age were subjected to drought, maintaining soil moisture levels at 50% and 25% field capacities, while well-irrigated plants served as the control group. Drought-stressed plants exhibited increased levels of lipid peroxidation and hydrogen peroxide, electrolyte leakage, and impaired glyoxalase systems. Thus, the growth, biomass, and yield attributes of rapeseed were significantly impaired under drought. However, the allantoin-supplemented plants showed a notable increase in their contents of ascorbate and glutathione and decreased dehydroascorbate and glutathione disulfide contents under drought. Moreover, the activity of antioxidant enzymes such as ascorbate peroxidase, dehydroascorbate reductase, glutathione reductase, glutathione peroxidase, and catalase were accelerated with the allantoin spray and the glyoxalase system was also enhanced under drought. Moreover, the improvement in water balance with reduction in proline and potassium ion contents was also observed when allantoin was applied to the plants. Overall, the beneficial effects of allantoin supplementation resulted in the improved plant growth, biomass, and yield of rapeseed under drought conditions. These findings suggest that allantoin acts as an efficient metabolite in mitigating the oxidative stress caused by reactive oxygen species by enhancing antioxidant defense mechanisms and the glyoxalase system.

Endophytic fungus Colletotrichum sp. AP12 promotes growth physiology and andrographolide biosynthesis in Andrographis paniculata (Burm. f.) Nees.

Endophytic fungi can promote host plant growth, enhance antioxidant defense enzyme activity, and induce the biosynthesis and accumulation of secondarymetabolites. Therefore, using endophytic fungi to improve the quality and yield of medicinal plants or important crops is an effective means of regulation. Colletotrichum sp. AP12 has been reported to produce andrographolide compounds (ADCs). This study aimed to investigate the effects of AP12 and its elicitors on the growth, defense enzyme activity, accumulation, and transcription levels of key genes in Andrographis paniculata (Burm. f.) Nees (A. paniculata). Using fermentation method to prepare AP12 into the inactivated fermentation solution (IFS), fermentation solution (FS), inactivated mycelium solution (IMS), and mycelium solution (MS), and the results showed that all four fungal elicitor components (ECs) could promote A. paniculata growth, enhance antioxidant defense enzymes, and increase ADC content and yield, especially the IMS group that had the highest leaf area, whole plant dry weight, superoxide dismutase (SOD), catalase (CAT) enzyme activities, total lactone contents, and yields, which were 2.37-, 1.60-, 2.20-, 3.27-, 1.59-, and 2.65-fold of the control, respectively. The 14-deoxyandrographolide (NAD) in the host irrigated with MS was 3.35-fold that of the control. In addition, AP12-infected A. paniculata sterile seedlings could significantly increase ADC content and expression levels of key enzyme genes, especially on day 12, when the total lactone content of the host reached 88.881± 5.793 mg/g DW, while on day 6, CPS gene expression level reached 10.79-fold that of the control, in turn promoting the biosynthesis and accumulation of andrographolide. In conclusion, the endophytic fungus AP12 is beneficial to the growth and secondary metabolism of A. paniculata, which is helpful for the cultivation and application of the biological bacterial fertilizer in A. paniculata, providing a theoretical and research basis for the use of endophytic fungi as a microbial resource to improve the quality and yield of medicinal plants.

Enhanced podophyllotoxin production of endophyte Fusarium proliferatum TQN5T by host extract and phenylalanine.

Fermentation technology using endophytes is considered a potential alternative approach for producing pharmaceutical compounds like podophyllotoxin (PTOX). In this study, fungus TQN5T (VCCM 44284) was selected from endophytic fungi isolated from Dysosma versipellis in Vietnam for PTOX production through TLC. The presence of PTOX in TQN5T was further confirmed by HPLC. Molecular identification indicated TQN5T as Fusarium proliferatum with 99.43% identity. This result was asserted by morphological characteristics such as white cottony, filamentous colony, layer and branched mycelium, and clear hyphae septa. Cytotoxic assay indicated both biomass extract and culture filtrate of TQN5T presented strong cytotoxicity on LU-1 and HepG2 with IC50 of 0.11, 0.20, 0.041, and 0071, respectively, implying anti-cancer compounds were accumulated in the mycelium and secreted into the medium. Further, the production of PTOX in TQN5T was investigated in the fermentation condition supplemented with 10 µg/ml of host plant extract or phenylalanine as elicitors. The results revealed a significantly higher amount of PTOX in the PDB + PE and PDB + PA at all studied time points in comparison with PDB (control). Especially, after 168 h of culture, PTOX content in the PDB with plant extract reached the peak with 314 µg/g DW which is 10% higher than the best yield of PTOX in previous studies, denoting F. proliferatum TQN5T as a promising PTOX producer. This is the first study on enhancing the PTOX production in endophytic fungi by supplementing phenylalanine-a precursor for PTOX biosynthesis in plants into fermented media, suggesting a common PTOX biosynthetic pathway between host plant and endophytes. KEY POINTS: • Fusarium proliferatum TQN5T was proven for PTOX production. • Both mycelia extract and spent broth extract of Fusarium proliferatum TQN5T presented strong cytotoxicity on cancer cell lines LU-1 and HepG2. • The supplementation of 10 µg/ml host plant extract and phenylalanine into fermentation media of F. proliferatum TQN5T improved the yield of PTOX.

Improving Flavonoid Accumulation of Bioreactor-Cultured Adventitious Roots in Oplopanax elatus Using Yeast Extract.

Oplopanax elatus is an endangered medicinal plant, and adventitious root (AR) culture is an effective way to obtain its raw materials. Yeast extract (YE) is a lower-price elicitor and can efficiently promote metabolite synthesis. In this study, the bioreactor-cultured O. elatus ARs were treated with YE in a suspension culture system to investigate the elicitation effect of YE on flavonoid accumulation, serving for further industrial production. Among YE concentrations (25-250 mg/L), 100 mg/L YE was the most suitable for increasing the flavonoid accumulation. The ARs with various ages (35-, 40-, and 45-day-old) responded differently to YE stimulation, where the highest flavonoid accumulation was found when 35-day-old ARs were treated with 100 mg/L YE. After YE treatment, the flavonoid content increased, peaked at 4 days, and then decreased. By comparison, the flavonoid content and antioxidant activities in the YE group were obviously higher than those in the control. Subsequently, the flavonoids of ARs were extracted by flash extraction, where the optimized extraction process was: 63% ethanol, 69 s of extraction time, and a 57 mL/g liquid-material ratio. The findings provide a reference for the further industrial production of flavonoid-enriched O. elatus ARs, and the cultured ARs have potential application for the future production of products.

Arbuscular mycorrhizal fungi trigger danger-associated peptide signaling and inhibit carbon-phosphorus exchange with nonhost plants.

In soil, arbuscular mycorrhizal fungi (AMF) meet the roots of both host and presumed nonhost plants, but the interactional mechanisms of AMF with and functional relevance for nonhost plants is little known. Here we show AMF can colonize an individually grown nonhost plant, Arabidopsis thaliana, and suppress the growth of Arabidopsis and two nonhost Brassica crops. This inhibitory effect increased with increasing AMF inoculum density, and was independent of AMF species or nutrient availability. 13 C isotope labeling and physiological analyses revealed no significant carbon-phosphorus exchange between Arabidopsis and AMF, indicating a lack of nutritional function in this interaction. AMF colonization activated the danger-associated peptide Pep-PEPR signaling pathway, and caused clear defense responses in Arabidopsis. The impairment of Pep-PEPR signaling in nonhost plants greatly compromised AMF-triggered defensive responses and photosynthesis suppression, leading to higher colonization rates and reduced growth suppression upon AMF inoculation. Pretreatment with Pep peptide decreased AMF colonization, and largely substituted for AMF-induced growth suppression in nonhosts, confirming that the Pep-PEPR pathway is a key participant in resistance to AMF colonization and in mediating growth suppression of nonhost plants. This work greatly increases our knowledge about the functional relevance of AMF and their mechanisms of interactions with nonhost plants.

Therapeutic compounds from medicinal plant endophytes: molecular and metabolic adaptations.

During the last few decades, endophytes have attracted increased attention due to their ability to produce a plethora of bioactive secondary metabolites. These compounds not only help the endophytes to outcompete other plant-associated microbes or pathogens through quorum sensing, but also enable them to surmount the plant immune system. However, only a very few studies have described the interlink between various biochemical and molecular factors of host-microbe interactions involved in the production of these pharmacological metabolites. The peculiar mechanisms by which endophytes modulate plant physiology and metabolism through elicitors, as well as how they use transitional compounds of primary and secondary metabolism as nutrients and precursors for the synthesis of new compounds or enhancing existing metabolites, are still less understood. This study thus attempts to address the aspects of synthesis of such metabolites used in therapeutics by the endophytes in the light of their ecological significance, adaptation, and intercommunity interactions. Our study explores how endophytes adapt to the specific host environment, especially in medicinal plants that produce metabolites with pharmacological potential and simultaneously modulate host gene expression for the biosynthesis of these metabolites. We also discuss the differential interactions of fungal and bacterial endophytes with their hosts.

An endophytic fungus Schizophyllum commune isolated from Panax ginseng enhances hairy roots growth and ginsenoside biosynthesis.

Using endophytic fungal elicitors to increase the accumulation of valuable secondary metabolites in plant tissue culture is an effective biotechnology strategy. In this study, a collection of 56 strains of endophytic fungi were isolated from different organs of cultivated Panax ginseng, of which seven strains can be symbiotically co-cultured with the hairy roots of P. ginseng. Further experiments observed that strain 3R-2, identified as endophytic fungus Schizophyllum commune, can not only infect hairy roots but also promote the accumulation of specific ginsenosides. This was further verified because S. commune colonization significantly affected the overall metabolic profile of ginseng hairy roots. By comparing the effects of S. commune mycelia and its mycelia extract (EM) on ginsenoside production in P. ginseng hairy roots, the EM was confirmed to be a relatively better stimulus elicitor. Additionally, the introduction of EM elicitor can significantly enhance the expressions of key enzyme genes of pgHMGR, pgSS, pgSE, and pgSD involved in the biosynthetic pathway of ginsenosides, which was deemed the most relevant factor for promoting ginsenosides production during the elicitation period. In conclusion, this study is the first to show that the EM of endophytic fungus S. commune can be considered as an effective endophytic fungal elicitor for increasing the biosynthesis of ginsenosides in hairy root cultures of P. ginseng.

Optimizing elicitation strategy of salicylic acid for flavonoid and phenolic production of fed-batch cultured Oplopanax elatus adventitious roots.

Oplopanax elatus is a valuable medicinal plant, but its plant resource is lacking. Adventitious root (AR) culture of O. elatus is an effective way for the production of plant materials. Salicylic acid (SA) exerts enhancement effect on metabolite synthesis in some plant cell/organ culture systems. To clarify the elicitation effect of SA on fed-batch cultured O. elatus ARs, this study investigated the effects of SA concentration, and elicitation time and duration. Results showed that flavonoid and phenolic contents, and antioxidant enzyme activity obviously increased when the fed-batch cultured ARs were treated with 100 µM SA for 4 days starting on day 35. Under this elicitation condition, total flavonoid and phenolic contents reached 387 rutin mg/g DW and 128 gallic acid mg/g DW, respectively, which were significantly (p < 0.05) higher than those in the SA-untreated control. In addition, DPPH scavenging and ABTS+ scavenging rates, and Fe2+ chelating rate also greatly increased after SA treatment, and their EC50 values were 0.0117, 0.61, and 3.34 mg/L, respectively, indicating the high antioxidant activity. The findings of the present study revealed that SA could be used as an elicitor to improve the flavonoid and phenolic production in fed-batch O. elatus AR culture.

Investigation of the combined effects of cadmium chloride, silver nitrate, lead nitrate, methyl jasmonate, and salicylic acid on morphometric and biochemical characteristics of St. John's wort.

Hypericum perforatum L., is a sprawling, leafy herb that grows in open, disturbed areas, known as St. John's wort, has a variety of secondary metabolites that can be used for medicinal and therapeutic purposes. Heavy metals have become the most dangerous pollutants in the environment. The effect of cadmium chloride, lead nitrate, silver nitrate, methyl jasmonate, and salicylic acid was studied on several morphometric and biochemical features of St. John's wort simultaneously using the Taguchi statistical method. The results showed cadmium chloride and lead nitrate reduced the morphometric and biochemical properties of St. John's wort while salicylic acid compensated for the adverse effects of heavy metals. Simultaneously, use of salicylic acid and silver nitrate with cadmium chloride and lead nitrate reduced the toxic effects of these metals on morphometric properties. Methyl jasmonate improved growth characteristics at low levels and inhibited at higher levels. Also, according to the results, salicylic acid could reduce the effects of heavy metals on the biochemical properties, while silver nitrate acts like heavy metals, especially at higher levels. Salicylic acid reduced the harmful effects of these heavy metals and at all levels was able to create a better induction effect on St. John's wort. These elicitors mainly changed the adverse effects of heavy metals by strengthening the pathways of the antioxidant system in St. John's wort. The research assumptions were validated, which suggests that the Taguchi method could be considered in an optimum culture of medicinal plants under different treatments such as heavy metals and elicitors.

Supplementation with Ascophyllum nodosum extracts mitigates arsenic toxicity by modulating reactive oxygen species metabolism and reducing oxidative stress in rice.

Ascophyllum nodosum extract (ANE) is considered as an effective source of biostimulants that have the potential of ameliorating the negative impact of different abiotic stresses in plants. Considering the growth-promoting ability and other regulatory roles of ANE, the present investigation was executed to evaluate the role of ANE in conferring arsenic (As) tolerance in rice (Oryza sativa L. cv. BRRI dhan89). Rice seedlings (35-d-old) were exposed to two doses of sodium arsenate (As1 - 50 mg As kg-1 soil; As2 - 100 mg As kg-1 soil) at 25 days after transplanting through irrigation, whereas only water was applied to the control. Foliar application of 0.1% ANE was also supplemented under control as well as As-stressed conditions at 7 days intervals for 5 times. Arsenic-induced oxidative stress was evident through a sharp increase in lipid peroxidation, hydrogen peroxide, methylglyoxal, and electrolyte leakage in the As-treated plants. As a consequence, plant growth and biomass, leaf relative water content, as well as yield attributes were reduced noticeably. On the other hand, ANE supplemented plants accumulated enhanced levels of ascorbate and glutathione, their redox balance, and the activities of antioxidant and glyoxalase enzymes viz. ascorbate peroxidase, monodehydroascorbate reductase, dehydroascorbate reductase, glutathione reductase, catalase, glutathione peroxidase, and activities of glyoxalase I and glyoxalase II, respectively. Furthermore, relative water content, plant growth, yield attributes and yield were increased in the As-treated rice plants with ANE supplementation. The results reflected that foliar spray with ANE alleviated As-induced oxidative stress in rice plants by modulating the antioxidative defense and glyoxalase system.

Biosynthesized Ag nanoparticles on urea-based periodic mesoporous organosilica enhance galegine content in Galega.

The biological approach for synthesizing nanoparticles (NPs) using plant extracts is an efficient alternative to conventional physicochemical methods. Galegine, isolated from Galega (Galega officinalis L.), has anti-diabetic properties. In the present study, silver nanoparticles (AgNPs) loaded onto urea-based periodic mesoporous organosilica (AgNPs/Ur-PMO) were bio-synthesized using G. officinalis leaf extract. The synthesized NPs were characterized and confirmed via analysis methods. Different concentrations of biosynthesized AgNPs/Ur-PMO nanoparticles (0, 1, 5, 10, and 20 mg L-1) were used as elicitors in cell suspension culture (CSC) of G. officinalis. The callus cells from hypocotyl explants were treated at their logarithmic growth phase (8th d) and were collected at time intervals of 24, 72, 120, and 168 h. The viability and growth of cells were reduced (by 17% and 35%, respectively) at higher concentrations and longer treatments of AgNPs/Ur-PMO; however, the contents of hydrogen peroxide (H2O2) and malondialdehyde (MDA) were increased (1.23 and 3.01 fold, respectively in comparison with the control average). The highest total phenolic (2.43 mg g-1 dry weight) and flavonoid (2.22 mg g-1 dry weight) contents were obtained 168 h after treatment with 10 mg L-1 AgNPs/Ur-PMO. An increasing tendency in the antioxidant enzyme activities was also observed in all the elicitor concentrations. Treatment with AgNPs/Ur-PMO (in particular 5 mg L-1 for 120 h) significantly enhanced the galegine content (up to 17.42 mg g-1) about 1.80 fold compared with the control. The results suggest that AgNPs/Ur-PMO can be used as an effective elicitor for enhancing galegine production in the CSC of G. officinalis. KEY POINTS: • The green biosynthesis of AgNPs/Ur-PMO was done using G. officinalis leaf extract • Its toxicity as an elicitor increased with increasing concentration and treatment time • AgNPs/Ur-PMO significantly increased the antioxidant capacity and galegine content.

In vitro elicitation and detection of apigenin, catalpol and gallic acid in hairy root culture of Plantago major L. and assessment of cytotoxicity and anti-bacterial activity of its methanolic extract.

The aim of this study was to establish the hairy root (HR) culture of Plantago major to evaluate the accumulation of apigenin, catalpol and gallic acid after elicitation and investigate the biological activity of its methanolic extraction. The highest transformation frequency was obtained by Agrobacterium rhizogenes strain A4, 0.5 mg/L 6-Benzylaminopurine in pre-cultivation medium, 150 µM acetosyringone in co-cultivation medium (1/2 MS), and immersion method for inoculation of leaf explants. The production of apigenin, catalpol and gallic acid compounds were significantly affected by treatment of 1.18 mM AgNO3 at 24 h which yielded 4.30, 8.24 and 2.89-fold increase, respectively. The assessment of anti-bacterial activity showed that the methanolic extracts of the HRs elicited with 1.18 mM AgNO3 were significantly active against Proteus vulgaris (PTCC 1182) (MIC = 25 mg/mL and MBC = 25 mg/mL). Furthermore, the MTT assay revealed that the methanolic extracts of the HRs were cytotoxic on the SW-480 cell (IC50=337.56 ± 1.82 µg/mL).

Biostimulation can prime elicitor induced resistance of grapevine leaves to downy mildew.

Using plant defense elicitors to protect crops against diseases is an attractive strategy to reduce chemical pesticide use. However, development of elicitors remains limited because of variable effectiveness in the field. In contrast to fungicides that directly target pathogens, elicitors activate plant immunity, which depends on plant physiological status. Other products, the biostimulants, can improve certain functions of plants. In this study, the objective was to determine whether a biostimulant via effects on grapevine physiology could increase effectiveness of a defense elicitor. A new methodology was developed to study biostimulant activity under controlled conditions using in vitro plantlets. Both biostimulant and defense elicitor used in the study were plant extracts. When added to the culture medium, the biostimulant accelerated the beginning of plantlet growth and affected the shoot and root development. It also modified metabolomes and phytohormone contents of leaves, stems, and roots. When applied on shoots, the defense elicitor changed metabolite and phytohormone contents, but effects were different depending on whether plantlets were biostimulated or controls. Defense responses and protection against Plasmopara viticola (downy mildew agent) were induced only for plantlets previously treated with the biostimulant, Therefore, the biostimulant may act by priming the defense elicitor action. In this study, a new method to screen biostimulants active on grapevine vegetative growth was used to demonstrate that a biostimulant can optimize the efficiency of a plant defense elicitor.

Biotechnology and In Vitro Culture as an Alternative System for Secondary Metabolite Production.

Medicinal plants are rich sources of bioactive compounds widely used as medicaments, food additives, perfumes, and agrochemicals. These secondary compounds are produced under stress conditions to carry out physiological tasks in plants. Secondary metabolites have a complex chemical structure with pharmacological properties. The widespread use of these metabolites in a lot of industrial sectors has raised the need to increase the production of secondary metabolites. Biotechnological methods of cell culture allow the conservation of plants, as well as the improvement of metabolite biosynthesis and the possibility to modify the synthesis pathways. The objective of this review is to outline the applications of different in vitro culture systems with previously reported relevant examples for the optimal production of plant-derived secondary metabolites.