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Cadmium (Cd) is a widely distributed pollutant that adversely affects plants' metabolism and productivity. Phytohormones play a vital role in the acclimation of plants to metal stress. On the other hand, phytohormones trigger systemic resistances, including systemic acquired resistance (SAR) and induced systemic resistance (ISR), in plants in response to biotic interactions. The present study aimed to investigate the possible induction of SAR and ISR pathways in relation to the hormonal alteration of barley seedlings in response to Cd stress. Barley seedlings were exposed to 1.5 mg g−1 Cd in the soil for three days. The nutrient content, oxidative status, phytohormones profile, and expression of genes involved in SAR and ISR pathways of barley seedlings were examined. Cd accumulation resulted in a reduction in the nutrient content of barley seedlings. The specific activity of superoxide dismutase and the hydrogen peroxide content significantly increased in response to Cd toxicity. Abscisic acid, jasmonic acid, and ethylene content increased under Cd exposure. Cd treatment resulted in the upregulation of NPR1, PR3, and PR13 genes in SAR pathways. The transcripts of PAL1 and LOX2.2 genes in the ISR pathway were also significantly increased in response to Cd treatment. These findings suggest that hormonal-activated systemic resistances are involved in the response of barley to Cd stress. [ABSTRACT FROM AUTHOR]

Background: One of the problems of diabetic patients is the formation of ulcers in the lower areas, especially the legs, and the treatment of these ulcers is very important. In wounds, grampositive and Gram-negative opportunistic and biofilm-forming bacteria accumulate, and these bacteria often become resistant to antibiotics over time. For this reason, researchers are looking for new alternatives to break the barrier of antibiotic resistance. Due to their unique properties and natural origin, antimicrobial peptides can be a suitable alternative to common antibiotics. Methods: In this research, several antimicrobial peptides of animal origin were investigated using docking and molecular dynamics techniques. Then, the antibacterial disk diffusion test was performed and the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) were measured. Results: According to the Insilico results, the melittin peptide, which is present in Apis mellifera bee venom, was selected as the best candidate for measuring the antibacterial effect on the Gram-positive bacterium Staphylococcus aureus and the Gram-negative bacterium Pseudomonas aeruginosa. Then, a wound dressing was prepared using Apis mellifera bee venom, and an antibacterial disk diffusion test and the minimum inhibitory concentration (MIC) and the minimum bactericidal concentrate (MBC) measurements were performed. The results of the invitro tests showed that compared with the antibiotic ciprofloxacin, the wound dressing containing melittin could effectively inhibit the target bacteria and create a non-growth halo with an acceptable diameter. Conclusion: According to the results of this research, wound dressing containing the antimicrobial peptide melittin can be a suitable option for treating diabetic foot ulcers. The inhibitory effect observed on Pseudomonas aeruginosa and Staphylococcus aureus bacteria can be generalized to all Gram-positive and Gram-negative bacteria present in diabetic foot ulcers. [ABSTRACT FROM AUTHOR]

Abiotic stresses limit the quantity and quality of rice grain production, which is considered a strategic crop in many countries. In this study, a meta-analysis of different microarray data at seedling stage was performed to investigate the effects of multiple abiotic stresses (drought, salinity, cold situation, high temperature, alkali condition, iron, aluminum, and heavy metal toxicity, nitrogen, phosphorus, and potassium deficiency) on rice. Comparative analysis between multiple abiotic stress groups and their control groups indicated 561 differentially expressed genes (DEGs), among which 422 and 139 genes were up-regulated and down-regulated, respectively. Gene Ontology analysis showed that the process of responding to stresses and stimuli was significantly enriched. In addition, pathways such as metabolic process and biosynthesis of secondary metabolites were identified by KEGG pathway analysis. Weighted correlation network analysis (WGCNA) uncovered 17 distinct co-expression modules. Six modules were significantly associated with genes involved in response to abiotic stresses. Finally, to validate the results of the meta-analysis, five genes, including TIFY9 (JAZ5), RAB16B, ADF3, Os01g0124650, and Os05g0142900 selected for qRT-PCR analysis. Expression patterns of selected genes confirmed the results of the meta-analysis. The outcome of this study could help introduce candidate genes that may be beneficial for use in genetic engineering programs to produce more tolerant crops or as markers for selection. [ABSTRACT FROM AUTHOR]

Giardiasis, which is caused by Giardia duodenalis, has clinical symptoms such as steatorrhea and can be very dangerous in children. In addition, some documents reported that this parasite is present inside the tissue of patients with cancer. In this study, we analyzed the gene expression profiles of some main genes important to apoptosis and anti-apoptosis in humans. Expression profile arrays of Genomic Spatial Event (GSE) 113666, GSE113667, and GSE113679 obtained from Gene Expression Omnibus were used for meta-analysis using R commands. Cytoscape and STRING databases used the protein–protein Interaction network. Then, the Kyoto Encyclopedia of Genes and Genomes and Gene Ontology analysis was performed. Similar genes in Homo sapiens were identified using Basic Local Alignment Search Tool analysis. The validation was performed on eight people using real-time Polymerase chain reaction. In addition to the candidate genes, the gene expression of some other genes, including Serine/Threonine Kinase 1 (AKT1), Cyclin Dependent Kinase Inhibitor 2A (CDKN2A), Kirsten Rat Sarcoma (KRAS), and Phosphatidylinositol-4,5-Bisphosphate 3-Kinase Catalytic Subunit Alpha (PIK3CA) were also examined. Analysis of the expression of serum amyloid A1 (SAA1), Regenerating Islet-Derived 3 Gamma (REG3G), and REG3A genes did not show any difference between the two groups of healthy and diseased people. Examining the mean expression of the four genes AKT1, CDKN2A, KRAS, and PIK3CA showed that three genes of AKT1, CDKN2A, and KRAS had increased expression in people with a history of giardiasis compared to healthy people. We showed that the gene expression pattern differs in apoptosis and anti-apoptosis signaling in people with a history of giardiasis. Giardia duodenalis seems to induce post-non-infectious symptoms with stimulation of human gene expression. [ABSTRACT FROM AUTHOR]

Nisin, derived from Lactococcus lactis, is a well-known natural food preservative. In the present study, the gene of nisin was transformed to carrot by Agrobacterium tumefaciens strain LBA4404 harboring the recombinant binary vector pBI121 containing neomycin phosphotransferase II (nptII) gene, peptide signal KDEL, and Kozak sequence. The integration of nisin and nptII transgenes into the plant genome was confirmed by polymerase chain reaction (PCR) and dot blot analysis. The gene expression was also performed by RT-PCR and Enzyme-Linked Immunosorbent Assay. The level of nisin expressed in one gram of transgenic plant ranged from 0.05 to 0.08 μg/ml. The stability of nisin varied in orange and peach juices depending on the temperature on the 70th day. The leaf protein extracted from the transgenic plant showed a significant preservative effect of nisin in peach and orange juice. A complete inhibition activity against Staphylococcus aureus and Escherichia coli in orange juice was observed within 24 h. After 24 h, log 1 and log 2 were obtained in a peach juice containing Staphylococcus aureus and Escherichia coli, respectively. Results of HPLC indicated that Chlorogenic and Chicoric acid compounds were increased in transgenic plants, but this increase was not significant. The study of determining the genetic stability of transgenic plants in comparison with non-transgenic plants showed high genetic stability between non-transgenic plants and transgenic plants. This study confirmed the significant inhibitory effect of nisin protein on gram-positive and gram-negative bacteria. [ABSTRACT FROM AUTHOR]

Ovarian cancer (OC) incidence and mortality rates continue to escalate globally. Early detection of OC is challenging due to extensive metastases and the ambiguity of biomarkers in advanced High-Grade Primary Tumors (HGPTs). In the present study, we conducted an in-depth in silico analysis in OC cell lines using the Gene Expression Omnibus (GEO) microarray dataset with 53 HGPT and 10 normal samples. Differentially-Expressed Genes (DEGs) were also identified by GEO2r. A variety of analyses, including gene set enrichment analysis (GSEA), ChIP enrichment analysis (ChEA), eXpression2Kinases (X2K) and Human Protein Atlas (HPA), elucidated signaling pathways, transcription factors (TFs), kinases, and proteome, respectively. Protein–Protein Interaction (PPI) networks were generated using STRING and Cytoscape, in which co-expression and hub genes were pinpointed by the cytoHubba plug-in. Validity of DEG analysis was achieved via Gene Expression Profiling Interactive Analysis (GEPIA). Of note, KIAA0101, RAD51AP1, FAM83D, CEP55, PRC1, CKS2, CDCA5, NUSAP1, ECT2, and TRIP13 were found as top 10 hub genes; SIN3A, VDR, TCF7L2, NFYA, and FOXM1 were detected as predominant TFs in HGPTs; CEP55, PRC1, CKS2, CDCA5, and NUSAP1 were identified as potential biomarkers from hub gene clustering. Further analysis indicated hsa-miR-215-5p, hsa-miR-193b-3p, and hsa-miR-192-5p as key miRNAs targeting HGPT genes. Collectively, our findings spotlighted HGPT-associated genes, TFs, miRNAs, and pathways as prospective biomarkers, offering new avenues for OC diagnostic and therapeutic approaches. [ABSTRACT FROM AUTHOR]

Today, the culture of hairy roots is a suitable tool for studies in plant biology, such as gene expression, gene function analyses, enzymes and metabolic pathways. Lythrum salicaria is a herbaceous perennial that has great potential as a medicinal plant and pollution purifier. To the best of our knowledge, the present study is the first to report on hairy root production of L. salicaria. In this research, hairy root cultures of L. salicaria were evaluated with two strains of Agrobacterium rhizogenes, A4 and ATCC. The hairy roots induced by A. rhizogenes were confirmed with PCR analysis using the rolC genes specific primers. The highest percentage of transformation was obtained in the excised shoots of L. salicaria in inoculation with A. rhizogenes strain A4 (65 ± 2.3%). The percentage of explants with transgenic roots was increased in solid MS medium containing 100 µM acetosyringone at a 10 min infection time and a 48 h co-cultivation period. The hairy roots obtained from the shoot explants were extremely fertile and had extensive lateral branching compared to normal roots. In conclusion, the aim of the present study was to develop an efficient protocol for hairy root induction in L. salicaria. The results of this study can be used in the hairy root cultures of L. salicaria for various applications. [ABSTRACT FROM AUTHOR]

Linum album is a well-known rich source of anticancer compounds, i.e., podophyllotoxin (PTOX) and other lignans. These compounds play an important role in the plant's defensive system. The RNA-Seq data of flax (L. usitatissimum) were analyzed under various biotic and abiotic stresses to comprehend better the importance of lignans in plant defense responses. Then, the association between the lignan contents and some related gene expressions was experimented with HPLC and qRT-PCR, respectively. Transcriptomic profiling showed a specific expression pattern in different organs, and just the commonly regulated gene EP3 was detected with a significant increase under all stresses. The in silico analysis of the PTOX biosynthesis pathway identified a list of genes, including laccase (LAC11), lactoperoxidase (POD), 4-coumarate-CoA ligase (4CL), and secoisolariciresinol dehydrogenase (SDH). These genes increased significantly under individual stresses. The HPLC analysis showed that the measured lignan contents generally increased under stress. In contrast, a quantitative expression of the genes involved in this pathway using qRT-PCR showed a different pattern that seems to contribute to regulating PTOX content in response to stress. Identified modifications of critical genes related to PTOX biosynthesis in response to multiple stresses can provide a baseline for improving PTOX content in L. album. [ABSTRACT FROM AUTHOR]

We conducted a comparative analysis of the effects of two key stress-tolerance genes, spinach betaine aldehyde dehydrogenase (BADH) and cyanobacterial flavodoxin (Fld), on enzymatic, non-enzymatic antioxidants, content of glycine betaine (GB) and proline osmo-protectants and relative water content (RWC) of ajowan (Trachyspermum ammi L.) transgenic lines (obtained previously by Agrobacterium-mediated transformation) under in vitro and greenhouse salinity and drought stress. The heterologous expressions of BADH and Fld genes significantly enhanced salinity and drought tolerance of ajowan transgenic lines in comparison with wild-type plants. BADH- and Fld-overexpressing ajowan plants (TaBADH and TaFld) showed higher activity of catalase, peroxidase and superoxide dismutase enzymatic antioxidants under in vitro salinity and drought conditions. Compared with wild-type plants, TaBADH and TaFld lines had higher accumulation of GB and proline osmo-protectants under in vitro salinity and drought stress. During the water-stress experiment in greenhouse conditions, TaBADH and TaFld ajowan plants showed significantly higher RWC than their non-transformed counterparts. Results of both in vitro and greenhouse assessments reported the superiority of BADH over Fld in enhancing salinity and drought stress tolerance in ajowan plants. Further experiments are required to accurately demonstrate the superiority of each of these two genes over the other. The co-delivery of BADH and Fld transgenes would be more effective than their individual heterologous expressions, as there two genes affect the stress response of plants through different mechanisms. Key Message: The heterologous expression of BADH and Fld transgenes was compared in ajowan medicinal plant. The antioxidant activities, glycine betaine and proline contents of transgenic lines were higher than wild types. BADH was superior to Fld in enhancing stress tolerance of ajowan. [ABSTRACT FROM AUTHOR]

Milk thistle is an oil and medicinal crop known as an alternative oil crop with a high level of unsaturated fatty acids, which makes it a favorable edible oil for use in food production. To evaluate the importance of Milk thistle lipids in drought tolerance, an experiment was performed in field conditions under three different water deficit levels (Field capacity (FC), 70% FC and 40% FC). After harvesting seeds of the plant, their oily and methanolic extracts were isolated, and subsequently, types and amounts of lipids were measured using GC–MS. Genes and enzymes engaged in biosynthesizing of these lipids were identified and their expression in Arabidopsis was investigated under similar conditions. The results showed that content of almost all measured lipids of milk thistle decreased under severe drought stress, but genes (belonged to Arabidopsis), which were involved in their biosynthetic pathway showed different expression patterns. Genes biosynthesizing lipids, which had significant amounts were selected and their gene and metabolic network were established. Two networks were correlated, and for each pathway, their lipids and respective biosynthesizing genes were grouped together. Four up-regulated genes including PXG3, LOX2, CYP710A1, PAL and 4 down-regulated genes including FATA2, CYP86A1, LACS3, PLA2-ALPHA were selected. The expression of these eight genes in milk thistle was similar to Arabidopsis under drought stress. Thus, PXG3, PAL, LOX2 and CYP86A1 genes that increased expression were selected for protein analysis. Due to the lack of protein structure of these genes in the milk thistle, modeling homology was performed for them. The results of molecular docking showed that the four proteins CYP86A1, LOX2, PAL and PXG3 bind to ligands HEM, 11O, ACT and LIG, respectively. HEM ligand was involved in production of secondary metabolites and dehydration tolerance, and HEM binding site remained conserved in various plants. CA ligands were involved in synthesis of cuticles and waxes. Overall, this study confirmed the importance of lipids in drought stress tolerance in milk thistle. [ABSTRACT FROM AUTHOR]

Excessive heavy metal (HM) levels in soil have become a source of concern due to their adverse effects on human health and the agriculture industry. Soil contamination by HMs leads to an accumulation of reactive oxygen species (ROSs) within the plant cell and disruption of photosynthesis-related proteins. The response of tobacco lines overexpressing flavodoxin (Fld) and betaine aldehyde dehydrogenase (BADH) to cadmium (Cd) toxicity was investigated in this study. PCR results demonstrated the expected amplicon length of each gene in the transgenic lines. Absolute qRT-PCR demonstrates a single copy of T-DNA integration into each transgenic line. Relative qRT-PCR confirmed overexpression of Fld and BADH in transgenic lines. The maximum quantum yield of photosystem II (Fv/Fm) was measured under Cd toxicity stress and revealed that transgenic lines had a higher Fv/Fm than wild-type (WT) plants. Accumulation of proline, glycine betaine (GB), and higher activity of antioxidant enzymes alongside lower levels of malondialdehyde (MDA) and hydrogen peroxide (H2O2) was indicative of a robust antioxidant system in transgenic plants. Therefore, performing a loop in reducing the ROS produced in the photosynthesis electron transport chain and stimulating the ROS scavenger enzyme activity improved the plant tolerance to Cd stress. [ABSTRACT FROM AUTHOR]

Background: Soil drought stress is a limiting factor of productivity in walnut (Juglans regia L). Ferredoxin (Fd) level decreases under adverse environmental stress. Functional replacement of decreased Fd by Fld (Flavodoxin) had been shown to have protective effect under abiotic stress condition. This study aimed to evaluate four transgenic lines (L3, L4, L13 and L17) along with non-transgenic line under three osmotic stresses levels (0, 10 and 12% PEG). Methods and results: This experiment carried out based on a completely randomized design with four replications. To confirm that the Fld gene is successfully integrated into the walnut genome, PCR and dot blot analysis were carried out. The transgenic lines of walnut expressing Fld displayed increased tolerance to osmotic stress at 10 and 12% PEG condition. Lines expressing Fld exhibited increasing tolerance to drought stress and maintained health of plants under osmotic conditions. Results of real time PCR showed that expression level of Fld gene in L4 was higher than the others. Among transgenic lines, L4 was more tolerant than other lines under osmotic stress. Conclusions: These findings indicate that expression of Fld gene can increase tolerance to osmotic stress in Persian walnut and is useful tool for walnut production in arid and semi-arid regions. [ABSTRACT FROM AUTHOR]

The development of artificial biocrust using cyanobacterium Phormidium tenue has been suggested as an effective strategy to prevent soil degradation. Here, a combination of in silico approaches with growth rate, photosynthetic pigment, morphology, and transcript analysis was used to identify specific genes and their protein products in response to 500 mM NaCl in P. tenue. The results show that 500 mM NaCl induces the expression of genes encoding glycerol-3-phosphate dehydrogenase (glpD) as a Flavoprotein, ribosomal protein S12 methylthiotransferase (rimO), and a hypothetical protein (sll0939). The constructed co-expression network revealed a group of abiotic stress-responsive genes. Using the Basic Local Alignment Search Tool (BLAST), the homologous proteins of rimO, glpD, and sll0939 were identified in the P. tenue genome. Encoded proteins of glpD, rimO, and DUF1622 genes, respectively, contain (DAO and DAO C), (UPF0004, Radical SAM and TRAM 2), and (DUF1622) domains. The predicted ligand included 22B and MG for DUF1622, FS5 for rimO, and FAD for glpD protein. There was no direct disruption in ligand-binding sites of these proteins by Na+, Cl−, or NaCl. The growth rate, photosynthetic pigment, and morphology of P. tenue were investigated, and the result showed an acceptable tolerance rate of this microorganism under salt stress. The quantitative real-time polymerase chain reaction (qRT-PCR) results revealed the up-regulation of glpD, rimO, and DUF1622 genes under salt stress. This is the first report on computational and experimental analyses of the glpD, rimO, and DUF1622 genes in P. tenue under salt stress to the best of our knowledge. [ABSTRACT FROM AUTHOR]

Shallow water tropical reefs are widely threatened by anthropogenic ocean warming which sometimes exceeds their thermal tolerance limit. The majority of reefs have been currently predicted to entirely disappear if CO2 emissions continue at the present rate. However, certain species are thermally resilient and can quickly adapt to anomalous thermal stress events by altering the expression of heat stress-related genes, known as gene expression plasticity. Here, we examined the expression of key genes (i.e., heat-shock proteins, oxidative stress, immune response, ion transport, apoptosis, anti-apoptosis) in the scleractinian submassive coral Porites harrisoni and cytochrome P450 gene of its algal symbionts collected from the northeastern Persian Gulf during warm (August) and cold (December) periods. The potential for recovery from severe heat stress in August was additionally examined through transplantation of partially bleached coral fragments from shallow (~ 2.5 m) to a deeper site (~ 9 m), close to colonies of the same species experiencing much less heat stress. To evaluate the acclimation capability, several healthy deep (~ 9 m) coral fragments were transplanted to the shallow (~ 2.5 m) site experiencing severe heat stress. A significant upregulation of heat-shock proteins in response to heat stress was found and identified as the primary defense mechanism against heat stress-induced bleaching. In conjunction with the apoptotic gene downregulation, the immune response gene in acclimation samples showed pronounced upregulation, indicating high compatibility of deep corals to thermal changes. In recovery samples, the expression of the studied genes was similar to the expression of samples that underwent much less heat stress, which implies they were fully recovered. The expression of the cytochrome P450 gene showed no significant changes in all experiments. Overall, the findings suggest that P. harrisoni in the Persian Gulf is a thermal-tolerant species compared with counterpart species elsewhere in the world and can be classified as a thermally resilient coral. Thus, transplanting P. harrisoni in warm regions, where native corals have been subjected to climate change-driven mortality, might maintain the coral community under enhanced global warming scenarios. [ABSTRACT FROM AUTHOR]

Background: Trachyspermum ammi is one of the key medicinal plant species with many beneficial properties. Thymol is the most important substance in the essential oil of this plant. Thymol is a natural monoterpene phenol with high anti-microbial, anti-bacterial, and anti-oxidant properties. Thymol in the latest research has a significant impact on slowing the progression of cancer cells in human. In this research, embryos were employed as convenient explants for the fast and effectual regeneration and transformation of T. ammi. To regenerate this plant, Murashige and Skoog (MS) and Gamborg's B5 (B5) media were supplemented with diverse concentrations of plant growth regulators, such as 6-benzyladenine (BA), 1-naphthaleneacetic acid (NAA), 2,4-dichlorophenoxyacetic acid (2,4-D), and kinetin (kin). Transgenic Trachyspermum ammi plants were also obtained using Agrobacterium-mediated transformation and zygotic embryos explants. Moreover, two Agrobacterium tumefaciens strains (EHA101 and LBA4404) harboring pBI121-TPS2 were utilized for genetic transformation to Trachyspermum ammi. Results: According to the obtained results, the highest plant-regeneration frequency was obtained with B5 medium supplemented with 0.5 mg/l BA and 1 mg/l NAA. The integrated gene was also approved using the PCR reaction and the Southern blot method. Results also showed that the EHA101 strain outperformed another strain in inoculation time (30 s) and co-cultivation period (1 day) (transformation efficiency 19.29%). Furthermore, HPLC method demonstrated that the transformed plants contained a higher thymol level than non-transformed plants. Conclusions: In this research, a fast protocol was introduced for the regeneration and transformation of Trachyspermum ammi, using zygotic embryo explants in 25–35 days. Our findings confirmed the increase in the thymol in the aerial part of Trachyspermum ammi. We further presented an efficacious technique for enhancing thymol content in Trachyspermum ammi using Agrobacterium-mediated plant transformation system that can be beneficial in genetic transformation and other plant biotechnology techniques. [ABSTRACT FROM AUTHOR]

Data-driven models in a combination of optimization algorithms could be beneficial methods for predicting and optimizing in vitro culture processes. This study was aimed at modeling and optimizing a new embryogenesis medium for chrysanthemum. Three individual data-driven models, including multi-layer perceptron (MLP), adaptive neuro-fuzzy inference system (ANFIS), and support vector regression (SVR), were developed for callogenesis rate (CR), embryogenesis rate (ER), and somatic embryo number (SEN). Consequently, the best obtained results were used in the fusion process by a bagging method. For medium reformulation, effects of eight ionic macronutrients on CR, ER, and SEN and effects of four vitamins on SEN were evaluated using data fusion (DF)–non-dominated sorting genetic algorithm-II (NSGA-II) and DF-genetic algorithm (GA), respectively. Results showed that DF models with the highest R2 had superb performance in comparison with all other individual models. According to DF-NSGAII, the highest ER and SEN can be obtained from the medium containing 14.27 mM NH4+, 38.92 mM NO3−, 22.79 mM K+, 5.08 mM Cl−, 3.34 mM Ca2+, 1.67 mM Mg2+, 2.17 mM SO42−, and 1.44 mM H2PO4−. Based on the DF-GA model, the maximum SEN can be obtained from a medium containing 0.61 μM thiamine, 5.93 μM nicotinic acid, 0.25 μM biotin, and 0.26 μM riboflavin. The efficiency of the established-optimized medium was experimentally compared to Murashige and Skoog medium (MS) for embryogenesis of five chrysanthemum cultivars, and results indicated the efficiency of optimized medium over MS medium. Key points • MLP, SVR, and ANFIS were fused by a bagging method to develop a data fusion model. • NSGA-II and GA were linked to the data fusion model for establishing and optimizing a new embryogenesis medium. • The new culture medium (HNT) had better efficiency than MS medium. [ABSTRACT FROM AUTHOR]

Tissue browning is a major problem in tissue culturing of woody plants, especially for Ficus religiosa which occurs by the accumulation and oxidation of phenolic compounds. This study aimed to determine the effect of different concentrations of sodium nitroprusside on the appearance of callus browning from leaf explants. The results indicate that callus browning was significantly reduced by supplementation of sodium nitroprusside to the MS medium and supplemented with 2.26 μM of 2,4-dichlorophenoxyacetic acid and 0.22 μM of 6-benzyl amino purine. The accumulation of hydrogen peroxide and phenolic compounds in the callus tissues decreased at the 50 μM concentration of sodium nitroprusside. Although catalase and peroxidase activities decreased at the 50 μM concentration, the activity of superoxide dismutase and polyphenol oxidases, as well as proline content, increased exponentially. Sodium nitroprusside could be useful for the formation of non-embryogenic callus with high levels of metabolic activity for the production and isolation of secondary metabolites. [ABSTRACT FROM AUTHOR]

The successful introduction of isopentenyl transferase (IPT) gene into perennial ryegrass, cultivars Numan and Grassland using Agrobacterium tumefaciens via three explants (callus, seed and meristem tip) under three individual experiment was evaluated. In the first experiment, the calli were inoculated with LBA4404 Agrobacterium strain under vacuum, heat and in combination of both at 42 °C for 5 min followed by vacuum treatment (390 mm Hg pressure) for 15 min. Sonication-assisted Agrobacterium-mediated transformation (SAAT) was applied for seed and meristem tip transformation of perennial ryegrass for the first time. Results showed positive effects of heat treatment on transformation efficiency during Agro-infection in both cultivars. However, heat shock treatment was more effective in ʻGrasslandʼ than ʻNumanʼ (14.2% vs 9.2%). In addition, high transformation efficiency of about 46.65% and 29.15% was observed using meristem tip explants of ʻGrasslandʼ and ʻNumanʼ based on IPT and RD29A positive PCR results, respectively. Seed transformation efficiency in ʻGrasslandʼ and ʻNumanʼ under SAAT method reached to 37.5% and 16.65%, respectively. Results of these experiments revealed that LBA4404 strain was more efficient than GV3101 in transformation of both perennial ryegrass cultivars. The DNA-blot analysis confirmed that a single T-DNA copy of the IPT gene was integrated into the genomic DNA of the positive transgenic T0 plants which obtained from callus and meristem tip explants of ʻGrasslandʼ after heat and SAAT treatment, respectively. Because monocots are not the host of Agrobacterium tumefaciens, this novel protocol can be used in further experiments on genetic transformation of perennial ryegrass cultivars. [ABSTRACT FROM AUTHOR]

Licorice is a well-known medicinal plant, containing various secondary metabolites of triterpenoid and phenolic families. The aim of this study is to evaluate the effect of salinity stress on the expression of key genes involved in the biosynthetic pathway of triterpenoids such as glycyrrhizin, betulinic acid, soyasaponins, and phytosterols in licorice root, as well as providing a phonemic platform to characterize antioxidant properties, glycyrrhizin, and total phenolic content. This study also includes measuring the gene expression level and glycyrrhizin content in leaves and roots of control plants. The studied genes included squalene synthase (SQS1 and SQS2), β-amyrin synthase (bAS), lupeol synthase (LUS), cycloartenol synthase (CAS), β-amyrin 11-oxidase (CYP88D6), and β-amyrin 24-hydroxylase (CYP93E6). Our results revealed that all of the mentioned genes were upregulated following the stress condition with different transcription rates. The highest increase (12-fold) was observed for the expression of the LUS gene, which is related to the betulinic acid pathway. Also, the highest content of glycyrrhizin was observed at 72 h post-treatment, which was consistent with the upregulated transcription levels of the glycyrrhizin pathway genes especially SQS1 and CYP88D6 at the same time. Correlation and stepwise regression analysis proved the key role of SQS1 gene in the biosynthetic pathway of glycyrrhizin. Antioxidant activity and phenolic content also were increased following stress condition. A comparison between the expression levels of SQS1 and other genes involved in the production of glycyrrhizin, phytosterols, and soyasaponins revealed a similar transcription trend, which shows the gene expression in the roots was significantly higher than the leaves. In contrast, SQS2 and LUS genes displayed a higher expression in leaf tissues. The genes related to betulinic acid biosynthetic pathway exhibited an expression rate different from other triterpenoid pathway genes, which could be observed in the leaves and roots of control plants and the roots of salt-treated plants. Furthermore, results showed that these two SQS genes have different expression rates due to different plant tissues (roots and leaves) and stress conditions. Importantly, in contrast to previous reports, we detected the glycyrrhizin in leaf tissues. This result may indicate the presence of a different genetic background in native Iranian licorice germplasm. [ABSTRACT FROM AUTHOR]

Abstract Triterpenoid saponins are a diverse group of bioactive compounds, which are used for possessing of many biomedical and pharmaceutical products. Generally, squalene synthase (SQS) is defined as an emerging and essential branch point enzyme far from the major pathway of isoprenoids biosynthetic and a latent adjusting point, which manages carbon flux into triterpenes biosynthesis and sterols. The present study deals with the detailed characterization of SQS by bioinformatics approaches to evaluate physicochemical properties, structural characteristics including secondary and 3D structure prediction and functional analysis from eight plants related to Fabaceae family and Arabidopsis thaliana. Bioinformatics analysis revealed that SQS proteins have two transmembrane regions in the C-terminal. The predicted motifs were used to design universal degenerate primers for PCR analysis and other molecular applications. Phylogenetic analysis showed conserved regions at different stretches with maximum homology in amino acid residues within all SQSs. The secondary structure prediction results showed that the amino acid sequence of all squalene synthases had α helix and random coil as the main components. The reliability of the received model was confirmed using the ProSA and RAMPAGE programs. Determining of active site by CASTp proposes the possibility of using this protein as probable medication target. The findings of the present study may be useful for further assessments on characterization and cloning of squalene synthase. [ABSTRACT FROM AUTHOR]

Glycyrrhiza glabra is one of the most important and well-known medicinal plants which produces various triterpene saponins such as glycyrrhizin. Beta-amyrin 11-oxidase (CYP88D6) plays a key role in engineering pathway of glycyrrhizin production and converts an intermediated beta-amyrin compound to glycyrrhizin. In this study, pBI121GUS-9:CYP88D6 construct was transferred to G. glabra using Agrobacterium rhizogene ATCC 15834. The quantitation of transgene was measured in putative transgenic hairy roots using qRT-PCR. The amount of glycyrrhizin production was measured by HPLC in transgenic hairy root lines. Gene expression analysis demonstrated that CYP88D6 was over-expressed only in one of transgenic hairy root lines and was reduced in two others. Beta-amyrin 24-hydroxylase (CYP93E6) was significantly expressed in one of the control hairy root lines. The amount of glycyrrhizin metabolite in over-expressed line was more than or similar to that of control hairy root lines. According to the obtained results, it would be recommended that multi-genes of glycyrrhizin biosynthetic pathway be transferred simultaneously to the hairy root in order to increase glycyrrhizin content. [ABSTRACT FROM AUTHOR]

A new protocol for in vitro regeneration through direct somatic embryogenesis for two muskmelon cultivars (Cucumis melo L., “Mashhadi” and “Eivanaki”) is reported. Somatic embryos were obtained culturing 4- and 8-day-old cotyledons, seeds, and hypocotyls on Murashige and Skoog medium supplemented with three different hormonal combinations never tested so far for melon (naphthoxyacetic acid (NOA) + thidiazuron (TDZ), NOA + 6-banzylaminopurine (BAP), and 2,4-dichlorophenoxyacetic acid (2,4-D) + N-(2-chloro-4-pyridyl)-N′-phenylurea (4-CPPU)). Results were compared with those obtained when explants were cultivated in the presence of 2,4-D + BAP, previously used on melon. Embryogenesis occurred more successfully in 4-day-old cotyledons and seeds than hypocotyls and 8-day-old cotyledons. The best result was achieved with NOA + BAP. Genotypes significantly affected embryogenesis. The number of embryos in “Eivanaki” was significantly higher than that in “Mashhadi.” Embryo proliferation when explants were maintained in jars (9.3%) was found to be higher compared to that in petri dishes. For the first time, genetic stability of regenerated melon plants was evaluated using inter-simple sequence repeat markers. Polymerase chain reaction (PCR) products demonstrated a total of 102 well-resolved bands, and regenerants were 93% similar compared to the mother plant. Somaclonal changes during embryogenesis were evaluated by flow cytometry, showing 91% of the same patterns in regenerated plants. The results suggest that the new hormone components are effective when applied for in vitro embryogenesis of muskmelon as they show a high frequency in regeneration and genetic homogeneity. [ABSTRACT FROM AUTHOR]

Trachyspermum ammi (L.) Sprague (Ajowan) is an endangered medicinal plant with useful pharmaceutical properties. Ex situ conservation of this medicinal plant needs the development of an in vitro regeneration protocol using somatic embryogenesis. In the present study, a high-precision image-processing approach was successfully applied to measure physical properties of embryogenic callus. Explant age and the concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D), kinetin (Kin), and sucrose were used as inputs, and an artificial intelligence technique was applied to predict physical properties of embryogenic callus, and the number of somatic embryos produced. Artificial neural network (ANN) models were tested to find the best combinations of input variables that affected output variables. The lower values of root mean square error, and mean absolute error, and the highest values of determination coefficient, were achieved when all four input variables were applied to predict the number of somatic embryos, the area of the callus, the perimeter of the callus, the Feret diameter of the callus, the roundness of the callus, and the true density of the callus in ANN models. The highest measured and predicted number of somatic embryos were achieved from the interaction of 15-d-old explants × 1.5 mg L−1 2,4-D × 0.5 mg L−1 Kin × 2.5% (w/v) sucrose. Based on sensitivity analysis, the 2,4-D concentration was the most important component in the culture medium that affected the number of somatic embryos and physical properties of the embryogenic callus tissue. [ABSTRACT FROM AUTHOR]

Tissue-specific expression of recombinant proteins in transgenic plants is of particular importance. Production of tissue-specific transplastomic plants is challenging due to lack of tissue-specific promoter in the plastome. The objective of the present study was to produce a transgenic platform enhancing tissue-specific transgene expression in the green organs of Nicotiana tabacum Cv. Jafarabadi. A bacteria/plant hybrid sigma factor sequence ( hsig) under control of the PEP Carboxylase promoter (PEPC) was designed and cloned in a plasmid construct without selectable marker gene and finally transformed into the nucleus genome by Agrobacterium. Transformation was confirmed by PCR, RT-PCR, southern blot and real-time PCR analysis. The resulted platform was re-transformed using a plastid construct pFNGi containing gfp (reporter gene) and neo genes (a kanamycin resistance selectable marker gene). Expression of GFP was assayed under Epifluorescence microscopy. The results showed that the constructed platform was functional, and the GFP was expressed. Finally, it can be concluded that application of the hybrid sigma factor ( hsig) under control of a tissue-specific promoter could be a suitable platform for tissue-specific expression of plastome integrated transgenes, and also it is proposed to use other more safer marker genes for plastid transformation instead of the antibiotic resistance gene. [ABSTRACT FROM AUTHOR]

Biohydrogen production through water-gas shift (WGS) reaction by a biocatalyst was conducted in batch fermentation. The isolated photosynthetic bacterium Rhodopseudomonas palustris PT was able to utilize carbon monoxide and simultaneously produce hydrogen. Light exposure was provided as an indispensable requirement for the first stage of bacterial growth, but throughout the hydrogen production stage, the energy requirement was met through the WGS reaction. At ambient pressure and temperature, the effect of various sodium acetate concentrations in presence of CO-rich syngas on cell growth, carbon monoxide consumption, and biohydrogen production was also investigated. Maximal efficiency of hydrogen production in response to carbon monoxide consumption was recorded at 86 % and the highest concentration of hydrogen at 33.5 mmol/l was achieved with sodium acetate concentration of 1.5 g/l. The obtained results proved that the local isolate; R. palustris PT, was able to utilize CO-rich syngas and generate biohydrogen via WGS reaction. [ABSTRACT FROM AUTHOR]

Compositional studies comparing transgenic with non-transgenic counterpart plants are almost universally required by governmental regulatory bodies. In the present study, two T transgenic cotton lines containing chitinase (Line 11/57) and Bt lines (Line 61) were compared with non-transgenic counterpart. To do this, biochemical characteristics of leaves and seeds, including amino acids, fatty acids, carbohydrates, anions, and cations contents of the studied lines were analyzed using GC/MS, high-performance liquid chromatography (HPLC), and ion chromatography (IC) analyzers, respectively. polymerase chain reaction (PCR) and Western blot analyses confirmed the presence and expression of Chi and Bt genes in the studied transgenic lines. Although, compositional analysis of leaves contents confirmed no significant differences between transgenic and non-transgenic counterpart lines, but it was shown that glucose content of chitinase lines, fructose content of transgenic lines (Bt and chitinase) and asparagine and glutamine of chitinase lines were significantly higher than the non-transgenic counterpart plants. Both the transgenic lines (Bt and chitinase) showed significant decrease in the amounts of sodium in comparison to the non-transgenic counterpart plants. The experiments on the seeds showed that histidine, isoleucine, leucine, and phenylalanine contents of all transgenic and non-transgenic lines were the same, whereas other amino acids were significantly increased in the transgenic lines. Surprisingly, it was observed that the concentrations of stearic acid, myristic acid, oleic acid, and linoleic acid in the chitinase line were significantly different than those of non-transgenic counterpart plants, but these components were the same in both Bt line and its non-transgenic counterpart. It seems that more changes observed in the seed contents than leaves is via this point that seeds are known as metabolites storage organs, so they show greater changes in the metabolites contents comparing to the leaves. [ABSTRACT FROM AUTHOR]

An efficient in vitro propagation is described for Punica granatum L. using shoot tip and nodal explants. The influence of two basal medium, WPM and MS, and different plant growth regulators was investigated on micropropagation of the Iranian pomegranate cultivars, 'Malas Saveh' and 'Yousef Khani'. For proliferation stage, media supplemented with different concentrations (2.3, 4.7, 9.2 and 18.4 μM) of kinetin along with 0.54 μM NAA was used. WPM proved to be more efficient medium compared to MS. The best concentrations of kinetin were 4.7 μM for 'Malas Saveh' and 9.2 μM for 'Yousef Khani', resulting in the highest number of shoots per explants, shoot length and leaf number. For both cultivars, half-strength WPM medium supplemented with 5.4 μM NAA was most effective for rooting of shoots. Rooted plantlets were successfully acclimatized and transferred into soil. The micropropagated plants were morphologically uniform and exhibited similar growth characteristics and vegetative morphology to the mother plants. [ABSTRACT FROM AUTHOR]

Developing a reliable technique to transform unicellular green algae, Chlorella vulgaris, could boost potentials of using microalgae feedstock in variety of applications such as biodiesel production. Volumetric lipid productivity (VLP) is a suitable variable for evaluating potential of algal species. In the present study, the highest VLP level was recorded for C. vulgaris (79.08 mg lday) followed by 3 other strains studied; C. emersonii, C. protothecoides, and C. salina by 54.41, 45 and 18.22 mg lday, respectively. Having considered the high productivity of C. vulgaris, it was selected for the preliminary transformation experiment through micro-particle bombardment. Plasmid pBI 121, bearing the reporter gene under the control of CaMV 35S promoter and the kanamycin marker gene, was used in cells bombardment. Primary selection was done on a medium supplemented by 50 mg l kanamycin. After several passages, the survived cells were PCR-tested to confirm the stability of transformation and then were found to exhibit β-glucuronidase (GUS) activity in comparison with the control cells. Southern hybridization of npt II probe with genomic DNA revealed stable integration of the cassette in three different positions in the genome. The whole process was successfully implemented as a pre-step to transform the algal cells by genes involved in lipid production pathway which will be carried out in our future studies. [ABSTRACT FROM AUTHOR]

To introduce genetic resistance against alfalfa weevil ( Hypera postica), leaves and petiole explants of three commercial alfalfa genotypes, including Km-27, Kk-14 and Syn-18 were transformed with Agrobacterium tumefaciens strains GV101, LBA4404 and AGL01. All the Agrobacterium strains used harbored the recombinant binary vector pBI121 containing a synthetic cry3a gene under the control of CaMV35S promoter as well as the nptII gene as selectable marker. Transformed explants were cultured on callus-induction medium, and the germinated somatic embryos were then transferred to the regeneration medium. The primary transformants were evaluated by PCR and Southern blot analysis. The results indicated successful integration of the target gene into the genomes of primary transgenic lines. Moreover, the expression of Cry3a protein in the transgenic plants was confirmed by ELISA method. Three transgenic lines, including TL6, TL8 and TL11 showed significantly higher levels of insect resistance against H. postica larvae (mortality rate of 73-90 % after infestation), in comparison with the control plants during the two-year bioassays. All transgenic plants were fertile and no irregular behavior in terms of growth and the morphological traits were observed. Transgenic plants developed during the course of this study are currently being grown in greenhouse and will be crossed with each other for seed production. [ABSTRACT FROM AUTHOR]

The aim of the current study was modeling and optimizing medium compositions for shoot proliferation of chrysanthemum, as a case study, through radial basis function- non-dominated sorting genetic algorithm-II (RBF-NSGAII). RBF as one of the artificial neural networks (ANNs) was used for modeling four outputs including proliferation rate (PR), shoot number (SN), shoot length (SL), and basal callus weight (BCW) based on four variables including 6-benzylaminopurine (BAP), indole-3-butyric acid (IBA), phloroglucinol (PG), and sucrose. Afterward, models were linked to the optimization algorithm. Also, sensitivity analysis was applied for evaluating the importance of each input. The R2 correlation values of 0.88, 0.91, 0.97, and 0.76 between observed and predicted data were obtained for PR, SN, SL, and BCW, respectively. According to RBF-NSGAII, optimal PR (98.85%), SN (13.32), SL (4.83 cm), and BCW (0.08 g) can be obtained from a medium containing 2.16 µM BAP, 0.14 µM IBA, 0.29 mM PG, and 87.63 mM sucrose. The results of sensitivity analysis indicated that PR, SN, and SL were more sensitive to BAP, followed by sucrose, PG, and IBA. Finally, the performance of predicted and optimized medium compositions were tested, and results showed that the difference between the validation data and RBF-NSGAII predicted and optimized data were negligible. Generally, RBF-NSGAII can be considered as an efficient computational strategy for modeling and optimizing in vitro organogenesis. [ABSTRACT FROM AUTHOR]

Triterpenic acids (TAs), a large group of natural compounds with diverse biological activity, are produced by several plant taxa. Betulinic, oleanolic, and ursolic acids are the most medicinally important TAs and are mainly found in plants of the mint family. Metabolic engineering is strongly dependent on identifying the key genes in biosynthetic pathways toward the products of interest. In this study, gene expression tracking was performed by transcriptome mining, co-expression network analysis, and tissue-specific metabolite-expression analysis in order to identify possible key genes involved in TAs biosynthetic pathways. To this end, taxa-specific degenerate primers of six important genes were designed using an effective method based on the MEME algorithm in a phylogenetically related group of sequences and successfully applied in three members of the Lamiaceae (Rosmarinus officinalis, Salvia officinalis, and Thymus persicus). Based on the results of in-depth data analysis, genes encoding squalene epoxidase and oxido squalene cyclases are proposed as targets for boosting triterpene production. The results emphasize the importance of identifying key genes in triterpene biosynthesis, which may facilitate genetic manipulation or overexpression of target genes. [ABSTRACT FROM AUTHOR]

This article is part of a PhD thesis of the first author, with work being carried out both at Azad University and AREEO listed as A. Abbasi's affiliations in the original version. [ABSTRACT FROM AUTHOR]