Search

Abstract Introduction Developing somatic embryogenesis is one of the main steps in successful in vitro propagation and gene transformation in the carrot. However, somatic embryogenesis is influenced by different intrinsic (genetics, genotype, and explant) and extrinsic (e.g., plant growth regulators (PGRs), medium composition, and gelling agent) factors which cause challenges in developing the somatic embryogenesis protocol. Therefore, optimizing somatic embryogenesis is a tedious, time-consuming, and costly process. Novel data mining approaches through a hybrid of artificial neural networks (ANNs) and optimization algorithms can facilitate modeling and optimizing in vitro culture processes and thereby reduce large experimental treatments and combinations. Carrot is a model plant in genetic engineering works and recombinant drugs, and therefore it is an important plant in research works. Also, in this research, for the first time, embryogenesis in carrot (Daucus carota L.) using Genetic algorithm (GA) and data mining technology has been reviewed and analyzed. Materials and methods In the current study, data mining approach through multilayer perceptron (MLP) and radial basis function (RBF) as two well-known ANNs were employed to model and predict embryogenic callus production in carrot based on eight input variables including carrot cultivars, agar, magnesium sulfate (MgSO4), calcium dichloride (CaCl2), manganese (II) sulfate (MnSO4), 2,4-dichlorophenoxyacetic acid (2,4-D), 6-benzylaminopurine (BAP), and kinetin (KIN). To confirm the reliability and accuracy of the developed model, the result obtained from RBF-GA model were tested in the laboratory. Results The results showed that RBF had better prediction efficiency than MLP. Then, the developed model was linked to a genetic algorithm (GA) to optimize the system. To confirm the reliability and accuracy of the developed model, the result of RBF-GA was experimentally tested in the lab as a validation experiment. The result showed that there was no significant difference between the predicted optimized result and the experimental result. Conclutions Generally, the results of this study suggest that data mining through RBF-GA can be considered as a robust approach, besides experimental methods, to model and optimize in vitro culture systems. According to the RBF-GA result, the highest somatic embryogenesis rate (62.5%) can be obtained from Nantes improved cultivar cultured on medium containing 195.23 mg/l MgSO4, 330.07 mg/l CaCl2, 18.3 mg/l MnSO4, 0.46 mg/l 2,4- D, 0.03 mg/l BAP, and 0.88 mg/l KIN. These results were also confirmed in the laboratory

Abstract Fusarium head blight (FHB) is a devastating fungal disease affecting different cereals, particularly wheat, and poses a serious threat to global wheat production. Chitinases and β-glucanases are two important proteins involved in lysing fungal cell walls by targeting essential macromolecular components, including chitin and β-glucan micro fibrils. In our experiment, a transgenic wheat (Triticum aestivum) was generated by introducing chitinase and glucanase genes using Biolistic technique and Recombinant pBI121 plasmid (pBI-ChiGlu (-)). This plasmid contained chitinase and glucanase genes as well as nptII gene as a selectable marker. The expression of chitinase and glucanase was individually controlled by CaMV35S promoter and Nos terminator. Immature embryo explants from five Iranian cultivars (Arta, Moghan, Sisun, Gascogen and A-Line) were excised from seeds and cultured on callus induction medium to generate embryonic calluses. Embryogenic calluses with light cream color and brittle texture were selected and bombarded using gold nanoparticles coated with the recombinant pBI-ChiGlu plasmid. Bombarded calluses initially were transferred to selective callus induction medium, and later, they were transfferd to selective regeneration medium. The selective agent was kanamycin at a concentration of 25 mg/l in both media. Among five studied cultivars, A-Line showed the highest transformation percentage (4.8%), followed by the Sisun, Gascogen and Arta in descending order. PCR and Southern blot analysis confirmed the integration of genes into the genome of wheat cultivars. Furthermore, in an in-vitro assay, the growth of Fusarium graminearum was significantly inhibited by using 200 μg of leaf protein extract from transgenic plants. According to our results, the transgenic plants (T1) showed the resistance against Fusarium when were compared to the non-transgenic plants. All transgenic plants showed normal fertility and no abnormal response was observed in their growth and development.

Abstract 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 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 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 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.

Background and Objective: The presence of toxic compounds, including phenol, due to industrial development, poses a threat to the environment. Utilizing hairy roots has emerged as a potential method to remove these toxins. This review aims to explore the efficacy of hairy roots in absorbing phenol pollutants and the influencing parameters. Materials and Methods: This study was conducted using a descriptive-review method based on existing literature gathered from databases such as Science Direct, PubMed, and Google Scholar. The focus of the study was on the purification of phenol using hairy roots. Keywords such as Phytoremediation, Hairy root, Phenol, and Transgenic roots were used for data collection. Results: Results show successful phenol removal by hairy roots, potentially attributed to abundant production of peroxidase enzymes. Various factors, such as hydrogen peroxide (H2O2), incubation time, pH, plant species, and pollutant concentration, impact phenol removal efficiency. Notably, plants like Brassica napus, rich in peroxidase enzymes, exhibit high efficiency in removing phenol pollution up to 500 mg/L, with H2O2 and within a pH range of 4-9. Conclusion: In conclusion, hair roots possess significant adsorption capacity for phenol. However, phenol concentration, contact time, pH, and temperature influence their performance. Therefore, further research is required to explore optimal conditions for phenol removal.

Abstract 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 Background Nitrogen is very important for crop yield and quality. Crop producers face the challenge of reducing the use of mineral nitrogen while maintaining food security and other ecosystem services. The first step towards understanding the metabolic responses that could be used to improve nitrogen use efficiency is to identify the genes that are up- or downregulated under treatment with different forms and rates of nitrogen. We conducted a transcriptome analysis of barley (Hordeum vulgare L.) cv. Anni grown in a field experiment in 2019. The objective was to compare the effects of organic (cattle manure) and mineral nitrogen (NH4NO3; 0, 40, 80 kg N ha−1) fertilizers on gene activity at anthesis (BBCH60) and to associate the genes that were differentially expressed between treatment groups with metabolic pathways and biological functions. Results The highest number of differentially expressed genes (8071) was found for the treatment with the highest mineral nitrogen rate. This number was 2.6 times higher than that for the group treated with a low nitrogen rate. The lowest number (500) was for the manure treatment group. Upregulated pathways in the mineral fertilizer treatment groups included biosynthesis of amino acids and ribosomal pathways. Downregulated pathways included starch and sucrose metabolism when mineral nitrogen was supplied at lower rates and carotenoid biosynthesis and phosphatidylinositol signaling at higher mineral nitrogen rates. The organic treatment group had the highest number of downregulated genes, with phenylpropanoid biosynthesis being the most significantly enriched pathway for these genes. Genes involved in starch and sucrose metabolism and plant-pathogen interaction pathways were enriched in the organic treatment group compared with the control treatment group receiving no nitrogen input. Conclusion These findings indicate stronger responses of genes to mineral fertilizers, probably because the slow and gradual decomposition of organic fertilizers means that less nitrogen is provided. These data contribute to our understanding of the genetic regulation of barley growth under field conditions. Identification of pathways affected by different nitrogen rates and forms under field conditions could help in the development of more sustainable cropping practices and guide breeders to create varieties with low nitrogen input requirements.

Abstract Background Callus induction is the first step in optimizing plant regeneration. Fit embryogenesis and shooting rely on callus induction. In addition, using artificial intelligence models in combination with an algorithm can be helpful in the optimization of in vitro culture. The present study aimed to evaluate the percentage and speed of callus induction optimization in carrot with a Multilayer Perceptron-Single point discrete genetic algorithm (GA). Materials and methods In this study, the outputs included callus induction percentage and speed, while inputs were different types and concentrations of plant growth regulator (0. 5, 0.2 mg/l 2,4-D, 0.3, 0.2, 0.5 mg/l BAP, 1, 0.2 mg/l Kin, and 2 mg/l NAA), different explants (shoot, root, leaf, and nodal), a different concentration compound of MS medium (1 × MS, 4× MS, and 8× MS) and time of sampling. The data were obtained in the laboratory, and multilayer perceptron (MLP) and radial basis function (RBF), two well-known ANNs, were employed to model. Then, GA was used for optimization, and sensitivity analysis was performed to indicate the inputs’ importance. Results The results showed that MLP had better prediction efficiency than RBF. Based on the results, R2 in training and testing data was 95 and 95% for the percentage of callus induction, while it was 94 and 95% for the speed of callus induction, respectively. In addition, a concentration compound of MS had high sensitivity, while times of sampling had low sensitivity. Based on the MLP-Single point discrete GA, the best results were obtained for shoot explants, 1× MS media, and 0.5 mg/l 2, 4-D + 0.5 mg/l BAP. Further, a non-significant difference was observed between the test result and predicted MLP. Conclusions Generally, MLP-Single point discrete GA is considered a potent tool for predicting treatment and fit model results used in plant tissue culture and selecting the best medium for callus induction.

Abstract 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.

The important feature of petunia in tissue culture is its unpredictable and genotype-dependent callogenesis, posing challenges for efficient regeneration and biotechnology applications. To address this issue, machine learning (ML) can be considered a powerful tool to analyze callogenesis data, extract key parameters, and predict optimal conditions for petunia callogenesis, facilitating more controlled and productive tissue culture processes. The study aimed to develop a predictive model for callogenesis in petunia using ML algorithms and to optimize the concentrations of phytohormones to enhance callus formation rate (CFR) and callus fresh weight (CFW). The inputs for the model were BAP, KIN, IBA, and NAA, while the outputs were CFR and CFW. Three ML algorithms, namely MLP, RBF, and GRNN, were compared, and the results revealed that GRNN (R2≥83) outperformed MLP and RBF in terms of accuracy. Furthermore, a sensitivity analysis was conducted to determine the relative importance of the four phytohormones. IBA exhibited the highest importance, followed by NAA, BAP, and KIN. Leveraging the superior performance of the GRNN model, a genetic algorithm (GA) was integrated to optimize the concentration of phytohormones for maximizing CFR and CFW. The genetic algorithm identified an optimized combination of phytohormones consisting of 1.31 mg/L BAP, 1.02 mg/L KIN, 1.44 mg/L NAA, and 1.70 mg/L IBA, resulting in 95.83% CFR. To validate the reliability of the predicted results, optimized combinations of phytohormones were tested in a laboratory experiment. The results of the validation experiment indicated no significant difference between the experimental and optimized results obtained through the GA. This study presents a novel approach combining ML, sensitivity analysis, and GA for modeling and predicting callogenesis in petunia. The findings offer valuable insights into the optimization of phytohormone concentrations, facilitating improved callus formation and potential applications in plant tissue culture and genetic engineering.

The process of optimizing in vitro seed sterilization and germination is a complicated task since this process is influenced by interactions of many factors (e.g., genotype, disinfectants, pH of the media, temperature, light, immersion time). This study investigated the role of various types and concentrations of disinfectants (i.e., NaOCl, Ca(ClO)2, HgCl2, H2O2, NWCN-Fe, MWCNT) as well as immersion time in successful in vitro seed sterilization and germination of petunia. Also, the utility of three artificial neural networks (ANNs) (e.g., multilayer perceptron (MLP), radial basis function (RBF), and generalized regression neural network (GRNN)) as modeling tools were evaluated to analyze the effect of disinfectants and immersion time on in vitro seed sterilization and germination. Moreover, non‑dominated sorting genetic algorithm‑II (NSGA‑II) was employed for optimizing the selected prediction model. The GRNN algorithm displayed superior predictive accuracy in comparison to MLP and RBF models. Also, the results showed that NSGA‑II can be considered as a reliable multi-objective optimization algorithm for finding the optimal level of disinfectants and immersion time to simultaneously minimize contamination rate and maximize germination percentage. Generally, GRNN-NSGA-II as an up-to-date and reliable computational tool can be applied in future plant in vitro culture studies.

Introduction: Nowadays, the widespread use of antimicrobial peptides as a natural preservative due to the side effects of synthetic preservatives (cancers and liver damages in medicines and foods) has received much attention. Materials and methods: In the present study, five antibacterial peptides including Nisin A of Lactococcus lactis, melitin of Apis mellifera, copsin from Coprinopsis cinerea, Terpene of Erythrolobus australicus, and thionin from Arabidopsis thaliana were studied using bioinformatics analysis. Results: The results showed that these peptides (peptides studied in eukaryotics and prokaryotics) had cytoplasmic targeting and the studied peptides were not protected in different organisms. There was a variation in the number of α helixes and β sheets among the studied peptides. The results of the phylogenetic tree by Mega5 showed that besides Apis mellifera, four other species were located in the same cluster. The domains were different in the five studied species, but all domains had antibacterial properties. These peptides showed a wide range of physicochemical properties. The substitution template, substitution model, and D-Tajima sequence of the studied peptides showed that Apis mellifera was isolated from other species during evolution. Three-dimensional (3-D) modeling of peptides by the homology modeling method and Swiss Model database showed that the three-dimensional structure of terpene and thionin had high quality. Using peptidecutter and allermatch websites, it was found that nisin A, melitin, copsine, terpene, and thionin were not allergenic. To determine the minimum lethal concentration and minimum inhibitory concentration, the disk diffusion method was used. The highest inhibition of nisin of Lactococcus Lactis was obtained in Staphylococcus aureus and Escherichia coli by disk diffusion. Blank discs were 19, 23 mm, 5, and 1 mm, respectively. Discussion and conclusion: The results showed that nisin can be used as a natural preservative to delay food spoilage against gram-positive bacteria.

Magnaporthe grisea is one of the most destructive pathogen that encounters a challenge to rice production around the worldwide. The unique properties of ZnO nanoparticles (NPs), have high attractiveness as nanofungicide. In the present study, the response of fungi to ZnO NPs was evaluated using RNA sequencing (RNA-seq). Two different aligners (STAR and Hisat2) were used for aligning the clean reads, and the DEseq2 package was used to identify the differentially expressed genes (DEGs). In total, 1,438 and 761 fungal genes were significantly up- and down-regulated in response to ZnO NPs, respectively. The DEGs were subjected to functional enrichment analysis to identify significantly enriched biological pathways. Functional enrichment analysis revealed that “cell membrane components,” “ion (calcium) transmembrane transporter activity,” “steroid biosynthesis pathway” and “catalytic activity” were the contributed terms to fungal response mechanisms. The genes involved in aflatoxin efflux pumps and ribosome maturation were among the genes showing significant up- and down-regulation after ZnO NPs application. To confirm the obtained RNA-seq results, the expression of six randomly selected genes were evaluated using q-RT-PCR. Overall, the RNA-seq results suggest that ZnO NPs primarily act on the fungal cell membrane, but accumulation of ROS inside the cell induces oxidative stress, the fungal catalytic system is disrupted, resulting into the inhibition of ROS scavenging and eventually, to the death of fungal cells. Our findings provide novel insights into the effect of ZnO NPs as a promising nanofungicide for effective control of rice blast disease.

Abstract 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 Background Optimizing the somatic embryogenesis protocol can be considered as the first and foremost step in successful gene transformation studies. However, it is usually difficult to achieve an optimized embryogenesis protocol due to the cost and time-consuming as well as the complexity of this process. Therefore, it is necessary to use a novel computational approach, such as machine learning algorithms for this aim. In the present study, two machine learning algorithms, including Multilayer Perceptron (MLP) as an artificial neural network (ANN) and support vector regression (SVR), were employed to model somatic embryogenesis of chrysanthemum, as a case study, and compare their prediction accuracy. Results The results showed that SVR (R2 > 0.92) had better performance accuracy than MLP (R2 > 0.82). Moreover, the Non-dominated Sorting Genetic Algorithm-II (NSGA-II) was also applied for the optimization of the somatic embryogenesis and the results showed that the highest embryogenesis rate (99.09%) and the maximum number of somatic embryos per explant (56.24) can be obtained from a medium containing 9.10 μM 2,4-dichlorophenoxyacetic acid (2,4-D), 4.70 μM kinetin (KIN), and 18.73 μM sodium nitroprusside (SNP). According to our results, SVR-NSGA-II was able to optimize the chrysanthemum’s somatic embryogenesis accurately. Conclusions SVR-NSGA-II can be employed as a reliable and applicable computational methodology in future plant tissue culture studies.

Podophyllotoxin (PTOX), one of the most important natural medicinal compounds, has anticancer properties. Its effective medicinal derivatives, such as etoposide and teniposide, have been approved by the Food and Drug Administration (FDA) for cancer treatment. This compound is found as a specialized metabolite in the Linum album Kotschy ex Boiss., belonging to the Linaceae family. PTOX is the major aryltetralin lignan resulting from the shikimic acid/phenylpropanoid pathway, and it accumulates in the shoots and roots of L. album. PTOX plays a necessary role in plant defense systems, protecting against abiotic as well as biotic stresses and helping their adaptation to adverse environmental conditions. Therefore, the content of specialized metabolites increases under stress. In this study, the content of PTOX under stressful conditions (potassium deficiency stress at two time points: 12 and 48 h) was examined using High Performance Liquid Chromatography (HPLC) in a completely randomized design with three replications. The results of HPLC showed that the content of PTOX first decreased after 12 h, while after 48 h of treatment, compared with the control plants, it showed a significant increase, with a value of 135.8 in the shoots. In the roots, the results were consistent with the results of the aerial parts, and the amount increased significantly after 48 h. In general, the results show that L. album, as a suitable natural source for PTOX, has great potential to generate large-scale products for commercial and pharmaceutical purposes.

Plant epigenetic has become one of the key research topics not only as the subject of basic research, but also as a new source of useful traits for plant breeding. Epigenetic regulation is necessary for the production of differentiated cells throughout plant development, as well as maintaining the stability and integrity of the gene expression profiles. Although epigenetic processes are essential for natural growth, they can become misdirected led to abnormal phenotypes and diseases. Epigenetics is the study of heritable phenotype changes that do not involve alterations in the DNA sequence. The microstructure (not code) of DNA itself or the associated chromatin proteins may be modified, causing activation or silencing. This mechanism enables differentiated cells in a multicellular organism to express only the genes which are necessary for their own activity. In this review, our goal is to introduce epigenetics and its different applications in plants, especially in production of transgenic plants, plants tolerate to biotic and abiotic stresses and understanding the mechanisms of gene silencing. Also, in this review, we have referred to the role of transposons in epigenetic, epigenetic engineering methods, epigenetic fingerprinting and ultimately methods for epigenetic data analysis and related databases.

Objective Homospermidine synthase (HSS) is the first enzyme in the specific pathway of pyrrolizidiene alkaloid (PA). HSS catalyzes the NAD1-dependent transfer of an aminobutyl group of spermidine to putrescine. Putrescine, spermidine and spermine are three polyamines which are naturally positively charged compounds found in virtually all living cells. These compounds bind to DNA and have been implicated in a number of crucial processes such as cell division, differentiation and membrane function. Very few studies on expression of homospermidine synthase gene in Senecio vulgaris, particularly in natural conditions have been reported. The objective of current research was to study homospermidine synthase gene expression in different tissues of S. vulgaris by real-time PCR. Material and methods The total RNA was extracted from roots, shoots (stems and leaves) and flowers, and was converted to cDNA. Specific primers were designed for HSS1 gene and also for the Pol II which was applied as a reference housekeeping gene in the experiment and expression level of HSS1 was assessed using real-time PCR. Results Melting curves of two genes showed the specificity of PCR amplification. Evaluation of normalized expression level of HSS1 gene showed that the gene was differentially expressed in different tissues of S. vulgaris. Maximum relative expression (>59-fold change relative to Pol II reference gene) was observed in roots where the homospermidine synthase is activated and homospermidine is produced. Interestingly, the gene is also expressed in flowers but with a lower extent (4-fold change relative to housekeeping Pol II gene). As expected the gene showed non-significant relative expression in shoots where the homospermidine is transported. Conclusions Expression of HSS1 gene in S. vulgaris is tissue-specific and is higher in the roots relative to other tissues.

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. Keywords: Bioinformatics analysis, Fabaceae, Squalene synthase, Triterpenoid biosynthesis

Background: Leishmaniasis is caused by the protozoan Leishmania spp. In some loci from Iran, some isolates have ITS1 like the one in Crithidia spp. Therefore, in this study, we analyzed molecular characters of the mentioned isolates. Methods: DNA was extracted from all clinical isolates harboring ITS1 like the one in Crithidia spp. Amplification was performed using the specific primer pair of 13A/13B and followed by sequencing. The sequences were assessed using BLASTn, multiple alignment, T-coffee, and Vector NTI. Phylogenic tree was used for grouping and analyzing the isolates. Findings: Sequencing analyzing showed low heterogeneity among the mentioned isolates, but high heterogeneity between the mentioned isolates and the ones in genbank, therefore the mentioned isolates were grouped separately. Moreover, although ITS1 from the mentioned isolates were homolog to the ones in Crithodia spp., but 13A/13B loci on mitochondria showed homology with Leishmania. Conclusion: As Crithidia is a monoxenous, and its natural host is flies, finding its genes in the agents of cutaneous leishmaniasis may show these parasites may meet each other in sandfly. Till now, no sexual reproduction has been reported in Leishmania spp., therefore it is likely recombinant.

Abiotic stresses, mainly salinity and drought, are the most important environmental threats that constrain worldwide food security by hampering plant growth and productivity. Plants cope with the adverse effects of these stresses by implementing a series of morpho-physio-biochemical adaptation mechanisms. Accumulating effective osmo-protectants, such as proline and glycine betaine (GB), is one of the important plant stress tolerance strategies. These osmolytes can trigger plant stress tolerance mechanisms, which include stress signal transduction, activating resistance genes, increasing levels of enzymatic and non-enzymatic antioxidants, protecting cell osmotic pressure, enhancing cell membrane integrity, as well as protecting their photosynthetic apparatus, especially the photosystem II (PSII) complex. Genetic engineering, as one of the most important plant biotechnology methods, helps to expedite the development of stress-tolerant plants by introducing the key tolerance genes involved in the biosynthetic pathways of osmolytes into plants. Betaine aldehyde dehydrogenase (BADH) is one of the important genes involved in the biosynthetic pathway of GB, and its introduction has led to an increased tolerance to a variety of abiotic stresses in different plant species. Replacing down-regulated ferredoxin at the acceptor side of photosystem I (PSI) with its isofunctional counterpart electron carrier (flavodoxin) is another applicable strategy to strengthen the photosynthetic apparatus of plants under stressful conditions. Heterologous expression of microbially-sourced flavodoxin (Fld) in higher plants compensates for the deficiency of ferredoxin expression and enhances their stress tolerance. BADH and Fld are multifunctional transgenes that increase the stress tolerance of different plant species and maintain their production under stressful situations by protecting and enhancing their photosynthetic apparatus. In addition to increasing stress tolerance, both BADH and Fld genes can improve the productivity, symbiotic performance, and longevity of plants. Because of the multigenic and complex nature of abiotic stresses, the concomitant delivery of BADH and Fld transgenes can lead to more satisfying results in desired plants, as these two genes enhance plant stress tolerance through different mechanisms, and their cumulative effect can be much more beneficial than their individual ones. The importance of BADH and Fld genes in enhancing plant productivity under stress conditions has been discussed in detail in the present review.

Background Leishmaniasis is a prevalent tropical disease caused by more than 20 Leishmania species (Protozoa, Kinetoplastida and Trypanosomatidae). Among different clinical forms of the disease, cutaneous leishmaniasis is the most common form, with an annual 0.6–1 million new cases reported worldwide. This disease’s standard treatment is pentavalent antimonial (SbV) that have been used successfully since the first half of the 20th century as a first-line drug. However, treatment failure is an increasing problem that is persistently reported from endemic areas. It is important to define and standardize tests for drug resistance in cutaneous leishmaniasis. SbV must be reduced to its trivalent active form (SbIII). This reduction occurs within the host macrophage, and the resultant SbIIIenters amastigotes via the aquaglyceroporin1 (AQP1) membrane carrier. Overexpression of AQP1 results in hypersensitivity of the parasites to SbIII, but resistant phenotypes accompany reduced expression, inactivation mutations, or deletion of AQP1. Hence, in this study, a phylogenetic analysis using barcode gene COXII and kDNA minicircle and expression analysis of AQP1 were performed in treatment failure isolates to assess the isolates’ molecular characteristics and to verify possible association with drug response. Methods Samples in this study were collected from patients with cutaneous leishmaniasis referred to the Diagnosis Laboratory Center in Isfahan Province, Iran, from October 2017 to December 2019. Among them, five isolates (code numbers 1–5) were categorized as treatment failures. The PCR amplification of barcode gene COXII and kDNA minicircle were done and subsequently analyzed using MEGA (10.0.5) to perform phylogenetics analysis of Treatment failures (TF) and Treatment response (TR) samples. Relative quantification of the AQP1 gene expression of TF and TR samples was assessed by real-time PCR. Results All samples were classified as L. major. No amplification failure was observed in the cases of barcode gene COXII and kDNA minicircle amplification. Having excluded the sequences with complete homology using maximum parsimony with the Bootstrap 500 method, four major groups were detected to perform phylogenetic analysis using COXII. The phylogenetic analysis using the barcode target of minicircle showed that all five treatment failure isolates were grouped in a separate sub-clade. Conclusions We concluded that the barcode gene COXII and the minicircle kDNA were suitable for identification, differentiation and phylogenetic analysis in treatment failure clinical isolates of Leishmania major. Also, AQP1 gene expression analyses showed that treatment failure isolates had less expression than TR isolates. The isolate with TF and overexpression of the AQP1 gene of other molecular mechanisms such as overexpression of ATP-binding cassette may be involved in the TR, such as overexpression of ATP-binding cassette which requires further research.

Transcriptome resources can facilitate to increase yield and quality of walnuts. Finding the best transcriptome assembly has not been the subject of walnuts research as yet. This research generated 240,179,782 reads from 11 walnut leaves according to cDNA libraries. The reads provided a complete de novo transcriptome assembly. Fifteen different transcriptome assemblies were constructed from five different well-known assemblers used in scientific literature with different k-mer lengths (Bridger, BinPacker, SOAPdenovo-Trans, Trinity and SPAdes) as well as two merging approaches (EvidentialGene and Transfuse). Based on the four quality metrics of assembly, the results indicated an efficiency in the process of merging the assemblies after being generated by de novo assemblers. Finally, EvidentialGene was recognized as the best assembler for the de novo assembly of the leaf transcriptome in walnut. Among a total number of 183,191 transcripts which were generated by EvidentialGene, there were 109,413 transcripts capable of protein potential (59.72%) and 104,926 were recognized as ORFs (57.27%). In addition, 79,185 transcripts were predicted to exist with at least one hit to the Pfam database. A number of 3,931 transcription factors were identified by BLAST searching against PlnTFDB. Furthermore, 6,591 of the predicted peptide sequences contained signaling peptides, while 92,704 contained transmembrane domains. Comparison of the assembled transcripts with transcripts of the walnut and published genome assembly for the 'Chandler' cultivar using the BLAST algorithm led to identify a total number of 27,304 and 19,178 homologue transcripts, respectively. De novo transcriptomes in walnut leaves can be developed for the future studies in functional genomics and genetic studies of walnuts.

Optimizing the gene transformation factors can be considered as the first and foremost step in successful genetic engineering and genome editing studies. However, it is usually difficult to achieve an optimized gene transformation protocol due to the cost and time-consuming as well as the complexity of this process. Therefore, it is necessary to use a novel computational approach such as machine learning models for analyzing gene transformation data. In the current study, three individual machine learning models including Multi-Layer Perceptron (MLP), Adaptive Neuro-Fuzzy Inference System (ANFIS), and Radial Basis Function (RBF) were developed for forecasting Agrobacterium-mediated gene transformation in chrysanthemum based on eleven input variables including Agrobacterium strain, optical density (OD), co-culture period (CCP), and different antibiotics including kanamycin (K), vancomycin (VA), cefotaxime (CF), hygromycin (H), carbenicillin (CA), geneticin (G), ticarcillin (TI), and paromomycin (P). Consequently, best-obtained results were used in the fusion process by bagging method. Results showed that ensemble model with the highest R2 (0.83) had superb performance in comparison with all other individual models (MLP:063, RBF:0.69, and ANFIS: 0.74) in the validation set. Also, ensemble model was linked to Fruit fly optimization algorithm (FOA) for optimizing gene transformation, and the results showed that the maximum gene transformation efficiency (37.54%) can be achieved from EHA105 strain with 0.9 OD600, for 3.8 days CCP, 46.43 mg/l P, 9.54 mg/l K, 18.62 mg/l H, and 4.79 mg/l G as selection antibiotics and 109.74 μg/ml VA, 287.63 μg/ml CF, 334.07 μg/ml CA and 87.36 μg/ml TI as antibiotics in the selection medium. Moreover, sensitivity analysis demonstrated that input variables have a different degree of importance in gene transformation system in the order of Agrobacterium strain > CCP > K > CF > VA > P > OD > CA > H > TI > G. Generally, the developed hybrid model in this study (ensemble model-FOA) can be employed as an accurate and reliable approach in future genetic engineering and genome editing studies.

Introduction: Dwarfism is one of the important traits in breeding of crops and horticulture plants. A dwarfing rootstock will produce trees with 15-50% of standard trees size. In modern intensive fruit tree orchards, dwarfing rootstocks are commonly used to reduce trees size, enabling high-density planting and easy management, thus achieving higher yield. Trees on dwarfing rootstocks can also exhibit other economically important traits, such as precocious flowering, increased yield and increased disease resistance. Dwarf rootstocks have been extensively studied and released in stone and pome fruits, because of presence of genetic materials and the simplicity of budding methods. Control of tree size using genetically dwarf rootstocks for achievement to higher density and mechanized orchard systems is now very important for walnut production in the world especially in Iran. Many different genes can be involved in appear of this. Mutations in GAI and PIP2 genes cause dwarf trait by two different mechanisms in some plant species. In this case, we study in silico analysis of GAI and PIP2 genes consist of conserved sequences and domains, exon and intron number, function of their proteins, targeting, secondary and tertiary structure, and post translational modification. Materials and methods: The GAI and PIP2 mRNA and protein sequences (FASTA format) belonging to 17 monocotyledon and dicotyledon were downloaded from NCBI (http://www.ncbi.nlm.nih.gov) accessed, on September 2014. Several online web services and software were used for analysis of GAI and PIP2 mRNA and Proteins in plants. Comparative and bioinformatics analyses of PIP2 and GAI proteins were performed online at two websites NCBI (http://www.ncbi.nih.gov) and EXPASY (http://expasy.org/tools). Molecular Evolutionary Genetics Analysis (MEGA; version 4) program and CLUSTAL-W with default parameters were used for multiple alignments of sequences. The phylogenetic analysis of GAI and PIP2 protein was done with MEGA from aligned sequences. The motifs of protein sequences were found using the program of T-COFEE at website (http://www.ebi.ac.uk/Tools/msa/tcoffee/). The Neighbor-Joining (NJ) method was used to designing the phylogenetic tree. The predicted exons and introns in mRNA sequences were done by http://genes.mit.edu/GENSCAN.html website. The secondary structure of proteins was predicted by PSIORED online on http://bioinf.cs.ucl.ac.uk/psipred/. Prediction of 3D model of protein was performed using the 3D alignment of protein structure by BLASTp and PDB database as source. Also, targeting prediction of proteins was done online by TargetP at (http://www.cbs.dtu.dk/services/TargetP/) website. Results and discussion: In phylogenetic investigation among 17 different species, Walnut species evolutionary stand in dicotyledonous and woody plants by both of GAI and PIP2 genes and protein sequence clustering. By multiple alignments and investigation in conserved sequence of these genes in plant revealed that despite differences in cDNA length, there were very similarities in conserved region, secondary and tertiary structure. Protein analysis in the GAI gene family showed that the following domains including DELLA, TVHYNP, VHIID, RKVATYFGEALARR, AVNSVFELH, RVER, and SAW were conserved in this proteins. In secondary structure of protein, β-sheets and α-helixes specified by PSIPRED software for both of GAI and PIP2 proteins. GAI protein had 9 β-sheets and 15 α-helixes in its structure, also PIP2 protein had2 β-sheet (at 180-188 and 248-253) and 8 α-helixes. In comparison of 3D structure, walnut PIP2 protein was very similar to chain A of PIP2 protein of spinach (Spinacia oleracea) and GAI protein of walnut was similar to B-subunit of Arabidopsis GAI protein with 48% similarity. The length of GAI protein was varied from 636 aa in Malus baccata var. xiaojinensis to 336 aa in Physcomitrella patens among species. In walnut, the length of GAI and PIP2 protein was 613 aa and 287 aa, respectively. PIP2 protein length was similar in different species among 257 aa in Triticum aestivum to 290 aa in Zea mays. By exon-intron and targeting analysis of sequence, it was found that GAI gene target was in nuclear and had just one exon without intron, and PIP2 gene in walnut had 4 exons and 3 introns with cell membrane targeting. In results, Tcoffee analysis revealed that PIP2 gene was very conserved across the evolution between plant species in compared with GAI gene. Conclusion: Our results provide new insights into the evolutionary relationships of GAI and PIP2 proteins. The results of the sequence alignment showed that GAI and PIP2 in walnut and other species have high homology with each other. After this analysis, we can have a good perspective about molecular situation of walnut GAI and PIP2 genes. Result of this study can be used for make relationship between growth, flowering, and water uptake characteristics of these plants and their protein sequences. Also this research gives good information for us if we want to clone these genes from Iranian genotypes.

This investigation was carried out in Albroz research station complex (Karaj, Iran) during 2014 summer. Mount Atlas mustic (Pistacia atlantica Desf.) seedlings were treated by drought (moderate, sever and without drought stress) and shading (low, moderate and without shade) factors in a split plot experiment based on a randomized complete block design. After one month, physiological characteristics of leaves including RWC, soluble sugars, proline and pigments content, catalase enzyme activity and ion leakage were studied. Drought stress caused severe decrease in RWC and a, b and total chlorophyll contents. In contrast, shade by ameliorating drought stress significantly increased these parameters. Drought induced tolerance mechanisms in saplings and increased proline and carotenoid content while shade decreased these parameters significantly as well as a to b chlorophyll ratio. Interaction of drought and shade was significant for a chlorophyll and catalase activity, suggested that, drought treatments showed different responses to shade. Overall, results revealed positive effect of shade on drought induced saplings.

A hybrid artificial intelligence model and optimization algorithm could be a powerful approach for modeling and optimizing plant tissue culture procedures. The aim of this study was introducing an Adaptive Neuro-Fuzzy Inference System- Non-dominated Sorting Genetic Algorithm-II (ANFIS-NSGAII) as a powerful computational methodology for somatic embryogenesis of chrysanthemum, as a case study. ANFIS was used for modeling three outputs including callogenesis frequency (CF), embryogenesis frequency (EF), and the number of somatic embryo (NSE) based on different variables including 2,4-dichlorophenoxyacetic acid (2,4-D), 6-benzylaminopurine (BAP), sucrose, glucose, fructose, and light quality. Subsequently, models were linked to NSGAII for optimizing the process, and the importance of each input was evaluated by sensitivity analysis. Results showed that all of the R2 of training and testing sets were over 92%, indicating the efficiency and accuracy of ANFIS on the modeling of the embryogenesis. Also, according to ANFIS-NSGAII, optimal EF (99.1%), and NSE (13.1) can be obtained from a medium containing 1.53 mg/L 2,4-D, 1.67 mg/L BAP, 13.74 g/L sucrose, 57.20 g/L glucose, and 0.39 g/L fructose under red light. The results of the sensitivity analysis showed that embryogenesis was more sensitive to 2,4-D, and less sensitive to fructose. Generally, the hybrid ANFIS-NSGAII can be recognized as a powerful computational tool for modeling and optimizing in plant tissue culture.

In vitro sterilization is a primary step of plant tissue culture which the ultimate results of in vitro culture are directly depended on the efficiency of the sterilization. Artificial intelligence models in a combination of optimization algorithms could be beneficial computational approaches for modeling and optimizing in vitro culture. The aim of this study was modeling and optimizing in vitro sterilization of chrysanthemum, as a case study, through Multilayer Perceptron- Non-dominated Sorting Genetic Algorithm-II (MLP-NSGAII). MLP was used for modeling two outputs including contamination frequency (CF), and explant viability (EV) based on seven variables including HgCl2, Ca(ClO)2, Nano-silver, H2O2, NaOCl, AgNO3, and immersion times. Subsequently, models were linked to NSGAII for optimizing the process, and the importance of each input was evaluated by sensitivity analysis. Results showed all of the R2 of training and testing data were over 94%. According to MLP-NSGAII, optimal CF (0%), and EV (99.98%) can be obtained from 1.62% NaOCl at 13.96 min immersion time. The results of sensitivity analysis showed that CF and EV were more sensitive to immersion time and less sensitive to AgNO3. Subsequently, the performance of predicted and optimized sterilants × immersion times combination were tested, and results indicated that the differences between the MLP predicted and validation data were negligible. Generally, MLP-NSGAII as a powerful methodology may pave the way for establishing new computational strategies in plant tissue culture.

Transgenic cotton expressing the Cry 1Ab protein of Bacillus thuringiensis developing against Helocoverpa armigera may be affect on secondary pest such as Bemisia tabaci. In this study effects of Bt cotton on demographic parameters of B. tabaci were assessed and the data analyzed using the age specific, two-sex life table parameters. Results showed that getting to the adulthood stage, was faster on non-Bt cotton in comparison with Bt cotton. Also the fecundity was higher on non-Bt cotton than that on Bt cotton. Some of the population parameters (r, R0 and T) of B. tabaci were affected by the Bt cotton significantly. The intrinsic rate of increase (r) on Bt and non-Bt cotton was 0.07 day-1 and 0.1 day-1 , respectively. The net reproductive rate (R0) was 20.68 and 15.04 offspring/individual on Bt and non-Bt cotton, respectively. Mean generation time (T) in non-Bt cotton was 27.22 and 34.62 days in Bt cotton. The results indicated that the life history of B. tabaci in the laboratory condition was influenced by host plant quality and Bt cotton was not a suitable host for B. tabaci. The western immunoblot method showed that the Cry protein detection in honeydew was positive which indicated that the Cry protein was ingested. Results revealed that the transgenic cotton could adversely affect the secondary pest and the natural enemies which feed on such pests as a host or their honeydew as a food source should be considered.

One concern about using transgenic plants is the genetic variation that occurred from theirs tissue culture and regeneration. Molecular markers are an important element for efficient and effective determination of genetic variation. The present work was carried out to assess the genetic uniformity of transgenic cottons (Bt and chitinase lines), using RAPD, ISSR molecular markers and SDS-PAGE analysis. Similarity matrix for RAPD marker based on Nei and Li’s coefficient revealed that pairwise value between Bt and chitinase cottons and their conventional counterpart plant was 93% and 95% respectively. Also, similarity matrix for ISSR marker revealed that pair wise value between Bt and chitinase lines and their conventional counterpart was 94% and 96% respectively. Pairwise similarity coefficient among two transgenic plants and their conventional counterpart ranged from 0.94 to 0.99 SDS-protein patterns exhibited four major bands, molecular weight ranged from 10 KD to 35 KD. Similarity matrix for protein patterns revealed that pairwise value between Bt and chitinase lines and their conventional counterpart was 100%.All protein bands of three cultivars are the same and no polymorphic bands was detected. Pairwise similarity coefficient among two transgenic plants and their conventional counterpart ranged from 0.94 to 0.99. These lines could be used for more in-dept.evaluation and biosafety studies on their way to commercialization

To meet the growing demand of world’s current population for food, devising specific strategies are required. Production of genetically modified crops has been considered as a suitable strategy so that the number of countries producing GM crops rose from 29 countries in 2012 to 29 in 2013 with 175.2 million hectare cultivation area. Among the developed GM crops, Bt crops with more than 24 million ha cultivation area have been accepted greatly. Although they offer too many advantages, concerns regarding biosafety issues exist. However, no report against the of undesirable effects of GM crops exists, risk assessment of certain concerns relating to biosafety of Bt crops is necessary. Some of these concerns include risk to non-target organisms, persistence of Bt residue in Soil, acquisition of weediness potential, monoculture and eventual loss of biodiversity, unintended effects of gene transfer on plant metabolism and gene flow from Bt transgenic crops. The present reviews emphasis the considered concerns in relation to Bt transgenic crops, their assessment and management that can be used by policy maker or experts.

Background: Neisseria meningitidis, a life-threatening human pathogen with the potential to cause large epidemics, can be isolated from the nasopharynx of 5–15% of adults. The aim of the current study was to evaluate biophysical and biochemical properties and immunological aspects of chimeric acyl-carrier protein-macrophage infectivity potentiator protein-type IV pilus biogenesis protein antigen (ACP-MIP-PilQ) from N. meningitidis serogroup B strain. Methods: Biochemical properties and multiple alignments were predicted by appropriate web servers. Secondary molecular structures were predicted based on Chou and Fasman, Garnier-Osguthorpe-Robson, and Neural Network methods. Tertiary modeling elucidated conformational properties of the chimeric protein. Proteasome cleavage and transporter associated with antigen processing (TAP) binding sites, and T- and B-cell antigenic epitopes, were predicted using bioinformatic web servers. Results: Based on our in silico and immunoinformatics analyses, the ACP-MIP-PilQ protein (AMP) can induce high-level cross-strain bactericidal activity. In addition, several immune proteasomal cleavage sites were detected. The 22 epitopes associated with MHC class I and class II (DR) alleles were confirmed in the AMP. Thirty linear B-cell epitopes as antigenic regions were predicted from the full-length protein. Conclusion: All predicted properties of the AMP indicate it could be a good candidate for further immunological in vitro and in vivo studies.

Background: Sigma factors are proteins that regulate transcription in bacteria. Sigma factors can be activated in response to different environmental conditions. The rpoS (RNA polymerase, sigma S) gene encodes sigma-38 (σ38, or RpoS), a 37.8 kDa protein in Pseudomonas aeruginosa (P. aeruginosa) strains. RpoS is a central regulator of the general stress response and operates in both retroactive and proactive manners; not only does it allow the cell to survive environmental challenges; it also prepares the cell for subsequent stresses (cross-protection). Methods: The significance of RpoS for stress resistance and protein expression in stationary-phase P. aeruginosa cells was assessed. The goal of the current study was to characterize RpoS of P. aeruginosa PAO1 using bioinformatics tools. Results: The results showed that RpoS is an unstable protein that belongs to the sigma-70 factor family. Secondary structure analysis predicted that random coil is the predominant structure followed by extended alpha helix. The three-dimensional (3D) structure was modeled using SWISS-MODEL Workspace. Conclusion: Determination of sequence, function, structure, and predicted epitopes of RpoS is important for modeling of inhibitors that will help in the design of new drugs to combat multi-drug-resistant (MDR) strains. Such information may aid in the development of new diagnostic tools, drugs, and vaccines for treatment in endemic regions.

Amorpha-4,11-diene synthase (ADS) is a key enzyme in biochemical pathway of the antimalarial agent artemisinin. An Agrobacterium-mediated transformation was carried out to express a synthetic ADS gene in green microalga Chlamydomonas reinhardtii strain 125C with bacterial strains GV3101 and LBA4404. The foreign gene was optimized based on codon usage bias of the microalga. Integration of the ADS in nuclear genome of C. reinhardtii was confirmed by polymerase chain reaction assay. The transgenic colonies cultured on selective medium turned yellow after three days and gradually died. Transformation procedure, growth habit of the transgenic microalgae together with probable causes of transformants loss is discussed. The present study is the first investigation for production of ADS enzyme in a microalgal system.

Advanced generations of biofuels basically revolve around non-agricultural energy crops. Among those, microalgae owing to its unique characteristics i.e. natural tolerance to waste and saline water, sustainable biomass production and high lipid content (LC), is regarded by many as the ultimate choice for the production of various biofuels such as biodiesel. In the present study, manipulation of carbon flux into fatty acid biosynthesis pathway in Dunaliella salina was achieved using pGH plasmid harboring AccD and ME genes to enhance lipid content and to improve produced biodiesel quality. The stability of transformation was confirmed by PCR after several passages. Southern hybridization of AccD probe with genomic DNA revealed stable integration of the cassette in the specific positions in the chloroplast genome with no read through transcription by indigenous promoters. Comparison of the LC and fatty acid profile of the transformed algal cell line and the control revealed the over-expression of the ME/AccD genes in the transformants leading to 12% increase in total LC and significant improvements in biodiesel properties especially by increasing algal oil oxidation stability. The whole process successfully implemented herein for transforming algal cells by genes involved in lipid production pathway could be helpful for large scale biodiesel production from microalgae.

Fusarium wilt caused by Fusarium oxysporum f.sp. Lycopersici is one of the major obstacles to the production of tomato which causes huge losses in tomato products worldwide. In order to increase the tolerance to this disease, a triple structure containing PR1, chitinase and glucanase genes controlled by 35S promoter was transferred to tomato. Eight days after planting on pre-culture medium, explants were inoculated by Agrobacterium tumefaciens strain LBA4404 containing the aforementioned plasmid. When the regenerated shoots grew to 2-3 cm, they were cut and transferred to rooting medium.The plantlets were then transferred to pots filled with a soil mixture of peat moss and perlite for further acclimatization. The putative transgenic plant lines were analyzed by multiplex PCR and the transcription of the transgenes was confirmed by RT-PCR method using the specific primers. The estimated value for the frequency of the simultaneous transfer of chitinase, glucanase and PR1 genes to tomato was 2.7%. Protein extracts of transgenic plants expressing chitinase, glucanase and PR1 genes inhibited in vitro hyphal growth of F. oxysporum f.sp. lycopersici. Compared with non-transgenic control plants, despite some alterations in chlorophyll content no other morphological changes were observed in transgenic plants. The total content of chlorophyll “a” and “b” in transgenic plants were 31.8 and 36.2 % higher than that of control plants, respectively, which may be attributed to metabolic changes due to simultaneous expression of three transgenes.

Several studies have been investigated the effects of Yeast extract (YE) on morphological and biochemical properties of various plant species. In this research, isoflavone synthase (IFS) gene expression profile, flavonoid and anthocyanin contents and also activity of some antioxidant enzymes have been assessed in 9-day soybean (Glycine max) seedlings which were treated with various concentrations of YE for 8 and 16 hours. Soybean as an annual plant is a member of the Leguminoseae family. Its seeds are containing of isoflavonoid compound (such as genestin and dedzein) which produce through IFS catalyzes from flavonone precursor. Results showed flavonoid and anthocyanin contents, IFS gene expression and also protein content significantly increased in elicited seedlings in compared to that of the control, but more significantly at 16-hour treatment. On the other hand, activity of antioxidant enzymes including superoxid dismutase, catalase and peroxidase was also elevated in treated seedlings rather than of the control that was more remarkable at 8-hour treatment. Based on the results, it can be concluded that treated soybean seedlings with YE lead to induce an oxidative stress. In this condition, while at 8-hour elicitation time the enzymatic antioxidant systems is more active, higher activity of the non-enzymatic antioxidant system (phenyl propanoid pathway) was seen for 16 hours treatment. Therefore, it seems that YE-treated for 16 hours is optimal for inducing of gene expression and also biosynthesis of active ingredient in this plant.

Salinity imposes significant stresses in various living organisms including microalgae. High extracellular concentration of Na+ directly influences ionic balance inside the cell and subsequently the cellular activities. In the present study, the effect of such stress on growth and intracellular ions concentration (IIC) of Dunaliella salina and Chlorella Spp. was investigated. IIC was analyzed using Ion chromatography technique. D. salina showed the highest degree of resistance to increase in salinity as little changes occurred both in IIC and in growth parameters. D. salina could maintain the balance of K+ inside the cell and eject the excess Na+ even at NaCl concentrations above 1M. Moreover, D. salina accumulated β-carotene in order to protect its photosynthetic apparatus. Among Chlorella species, C. vulgaris showed signs of adaptation to high content of salinity, though it is a fresh water species by nature. Moreover, the response shown by C. vulgaris to rise in salinity was even stronger than that of C. salina, which is presumably a salt-water resistant species. In fact, C. vulgaris could maintain intracellular K+ better than C. salina in response to increasing salinity, and as a result, it could survive at NaCl concentrations as high as 0.75 M. Marine strains such as D. salina well cope with the fluctuations in salinity through the existing adaptation mechanisms i.e. maintaining the K+/N+ balance inside the cell, K+ accumulation and Na+ ejection, accumulation of photosynthetic pigments like β-carotene.

The cotton wilt disease caused by the fungus Verticillium dahliae reduces total yield and quality in cotton worldwide. Since traditional breeding has achieved only low levels of wilt resistance, we used a transgenic approach to produce wilt tolerant cotton. We produced fertile transgenic T2 cotton lines by Agrobacterium-mediated transfer of an endochitinase gene from Phaseolus. Transgene integration and expression were confirmed by PCR amplification, Southern blot and Western blot analyses. Seedlings of homozygous T2 plants with high levels of chitinase expression were evaluated for their tolerance to Verticillium dahliae by infecting them with a spore suspension in a greenhouse. The transgenic plants demonstrated higher degrees of tolerance to the fungus than non-transgenic plants, as measured by foliar disease symptoms, vascular discoloration and plant height. Transgenic plants were also taller than the control ones and are currently being grown in the greenhouse and will be crossed with Iranian cotton breeding lines in a future study.

Fungal diseases are among the most important biotic stresses causing considerable losses in agricultural products. Improving crop plants with broad resistance to fungal diseases is one of the main challenges in agriculture. Classical plant breeding is too timely and has many other limitations, hence using biotechnology for this purpose is inevitable. Expression of a complex of Pathogen Related proteins induces polygenic resistance in plants. This technique leads to a broad and stable resistance to fungal pathogens. Hence, in this study we isolated resistance genes and constructed treble plasmid vectors with multiple genes involving three important families of disease-related proteins namely PR1, PR2, and PR3. For this purpose we isolated two genes of PR1 family with special primer design from tobacco genome, first. After analysis the accuracy of gene isolation using 35S promoter and Nos terminator, they were cloned in binary vector pBI121. Then, we cloned two important antifungal genes including PR2 (glucanase) and PR3 (chitinase) under the control of regulatory independent regions in T-DNA of the above mentioned vector with parallel and antiparallel orientations. These treble vectors have good antibacterial potential too and can be used for resistance to bacteria and other biotic and abiotic stresses. They can be introduced into plants using particle gun or through Agrobacterium mediated transformation.

In an attempt to speed up the process of screening of transgenic cotton (G. hirsutum L.) plants, a visual and rapid loop-mediated isothermal amplification (LAMP) assay was adopted. Genomic DNA was extracted from fresh leaf tissues of T2 transgenic cotton containing chitinase (chi) and cry1A(b) genes. Detection of genes of interest was performed by polymerase chain reaction (PCR), LAMP and real-time PCR methods. In LAMP assay the amplification was performed after 30 min at 65°C when loop primers were involved in the reaction. The involvement of loop primers decreased the time needed for amplification. By testing serial tenfold dilutions (10-1 to 10-8) of the genes of interest, the detection sensitivity of LAMP was found to be 100-fold higher than that of PCR. The rapid DNA extraction method and LAMP assay can be performed within 30 min and the derived LAMP products can be directly observed as visually detectable based on turbidity in the reaction tube. The accuracy of LAMP method in the screening of transgenes was confirmed by PCR and real-time PCR. The developed method was efficient, rapid and sensitive in the screening of cotton transgenic plants. This method can be applied to any other crops.

Cotton (Gossipium spp ) is ranked first among the fibers producing plants in the world and is also considered as an important source of oil. Although significant progress has been made in traditional cotton breeding programs, but there are still limitations including limited gene resources, lack of crosses, lack of efficient selection and high timeconsumption. Through recent advances in gene transformation to cotton, not only facilitated transformation of useful genes has become possible, but also promising results have been obtained in gene identification as well asfunction and regulation studies. Agrobacterium mediated transformation is one of the most used transformation methods in plants, including cotton. Several factors could affect transformation efficiency such as bacterial strains and concentrations, added phenolic regent in plant culture medium, plant species and genotypes, use of plant growth regulators, explants type, light and temperature during co-cultivation, antibiotics, wounding the target tissue and suitable method for selection of transgenic cells. Hypocotyls, cotyledons and cell suspensions are used as explants in cotton transformation using Agrobacterium. These explants have some limitations such as low regeneration rate and genotype dependence. Therefore, regeneration could be obtained in only a limited number of species using these explants. On the other hand, establishment of a regeneration system using shoot apex meristem in cotton has increased its transformation efficiency. This article is trying to review the factors shown to be effective in gene transformation into cotton

Production of transgenic wheat (Triticum Aestivum) was studied by chitinase and glucanase genes by Agrobacterium-mediated transformation and also pBI121 plasmid containing neomycin phospho transferasa selectable marker gene under control of Nos promoter. In addition, chitinase and glucanase genes were individually expressed under control of CaMV35S promoter. Immature embryo explants excised from seeds then were co-cultivated with bacterial suspension containing the recombinant plasmid and they were placed to callus induction medium supplemented with 50 mg/l kanamycin. Embryonic calluses, were selected and they were transferred to regeneration medium with 25 mg/l kanamycin in order to producing shoots and roots. Maximum percent of transformation by Agrobacterium-mediated transformation was 0.31% for transformed ARTA with C58 strain and then 0.074% for transformed ARTA with LBA4404 . There was not any transgenic plant in other cultivars or strains and transformation percent was 0%. PCR and Dot blot analysis showed that the putative transgenic plants consist at least one copy of either, chitinase, glucanase and neomycin phospho transferase genes in their genome in comparison with control plants.

A study on the impacts of soil humidity and artificial shading on growth, biomass production and its allocation to different organs of wild pistachio (Pistacia atlantica Desf.)saplings was carried out in Alborz research station complex (Karaj, Iran). One year seedlings were treated by soil humidity (20, 50 and 100 percent of field capacity) and shading (100, 70 and 50 percent of full sunlight) factors in a split plot experiment based on a randomized complete block design from June to December 2014. Results showed significant impact of oil humidity on height, collar diameter, fresh and dry biomass of leaves and stem as well as on total fresh biomass. Shade significantly decreased height, root and total dry biomass and root to shoot fresh biomass ratio. Moreover, wild pistachio saplings with different soil humidity treatments show different responses to shade. In particular, shade significantly decreases height, fresh leaves biomass and R/S fresh biomass ratio for well-irrigated saplings and increases these parameters for saplings under drought stress. Response trends of different parameters under humidity × shade treatments showed that shade can reduce undesirable effects of drought stress on wild pistachio saplings.