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Introduction. Pests, diseases and weeds (biotic stresses) are significant limiting factors for crop yield and production. However, the limitations associated with conventional breeding methods necessitated the development of alternative methods for improving new varieties with higher resistance to biotic stresses. Molecular techniques have developed applicable methods for genetic transformation of a wide range of plants. Genetic engineering approach has been demonstrated to provide enormous options for the selection of the resistance genes from different sources to introduce them into plants to provide resistance against different biotic stresses. Literature. In this review, we focus on strategies to achieve the above mentioned objectives including expression of insecticidal, antifungal, antibacterial, antiviral resistance and herbicide detoxification for herbicide resistance. Conclusion. Regardless of the concerns about commercialization of products from genetically modified (GM) crops resistant to biotic stresses, it is observed that the cultivation area of these crops is growing fast each year. Considering this trend, it is expected that production and commercialization of GM crops resistant to biotic stresses will continue to increase but will also extend to production of crops resistant to abiotic stresses (e.g. drought, salinity, etc.) in a near future.

Fagus orientalis is one of the most important broadleaf species in Hyrcanian forests. The phenotypic and genetic adaptation potential of this species to climate change is very important. Quantifying and evaluating genetic diversity between and within oriental beech populations makes it possible to best preserve the diversity of populations. This study was conducted on mature trees of four different populations (regions) of this species in two drought index gradients from West to East and lowland to highland in Hyrcanian forests (Shafarood forest of Gilan, Khairudkenar forest of lowland and highland of Mazandaran and Shast Kalateh forest of Golestan). Genetic diversity was studied in native populations using seven microsatellite markers and one biochemical marker. The results showed high diversity in all populations, especially in the population of Shast Kalateh, Golestan. The results of molecular analysis of variance with SSR microsatellite marker showed that 77% of the total diversity was within populations and 23% was among the populations. The results of this study showed that the presence of more genetic diversity within the beech populations of Hyrcanian forests indicates their strong genetic basis for adaptation to environmental conditions and may facilitate shortterm adaptation to climate changes. On the other hand, the presence of higher genetic diversity in Shast Kalateh than in other regions indicates stronger evolutionary forces for adapting trees to environmental conditions. Additionally, higher gene flow among allogamous populations of oriental beech has led to low genetic differentiation between populations in the drought index gradient of Hyrcanian forests. [ABSTRACT FROM AUTHOR]

Cotton (Gossypium hirsutum) is one of the most important fiber crops with a raw material value of $5.5 billion per year worldwide. Cotton breeders with the help of conventional breeding programs have improved the cotton germplasm for resistance to pests and pathogens. However, there has been little success in producing cotton lines resistant to biotic stresses, due to limited cotton germplasm with adequate resistance to biotic stresses, especially pests and diseases. In this study, cry1Ab (resistance to cotton bollworm) and chitinase (resistance to cotton Verticillium wilt) genes were transferred to Iranian commercial cotton varieties (Varamin, Khordad, Sahel, and Bakhtegan), by crossing transgenic lines containing cry1Ab and chi transgenes with Iranian commercial varieties. To recover genetic background of commercial varieties, the obtained progenies were backcrossed with own commercial parent. The progenies of backcrosses were assessed by western blot and PCR analysis. Insect and fungus in vitro bioassay showed resistance against cotton bollworm and Verticillium dahliae in plants containing the stacked transgenes. According to the results, cry1Ab and chi genes were successfully transferred and stacked in Iranian commercial cotton varieties. Also, plants with two transgenes and plants with one transgene were protruded to BC1 and BC2 generations, respectively. [ABSTRACT FROM AUTHOR]

Somatic embryos of Persian walnut were transformed with two strains of Agrobacterium tumefaciens i.e. LBA4404 and C58, and two plasmids, namely, pBI121 with nptII and gus genes for improving the transformation protocol, and p6u-ubi-FVTI plasmid containing the hpt and fld genes. The transformation frequency was 10%. PCR and RT-PCR analysis proved the presence and expression of the genes. The transgenic and non-transgenic somatic embryos of Persian walnut were exposed to four salinity levels (0, 50, 100, and 200 mM NaCl) and four osmotic stress (0, 1.5, 5, and 10% PEG) levels. After 20 days, the number of survived, secondary and cotyledonary somatic embryos, as well as fresh and dry weights of embryos were evaluated. In addition, the transgenic and non-transgenic regenerated plantlets with 3 leaves and 2.5 cm length were subjected to 200 mM NaCl. In both experiments, the main effects of fld-transformation and stress treatments on evaluated parameters were significant. Transgenic somatic embryos showed no significant differences at 0 and 200 mM NaCl and 0 and 1.5% PEG. Significant differences of transgenic vs. non-transgenic somatic embryos were observed at 50 and 100 mM NaCl and 5 and 10% PEG. Non-transgenic plantlets on medium containing 200 mM NaCl showed complete necrosis and died after 10 days, while transgenic lines continued growth until 45 days. Our results clearly showed that expression of fld gene increased stress tolerance in fld transformant lines of walnut, and also revealed that expression of this specific cyanobacterial protein may provide a powerful tool to improve tolerance to environmental stresses. [ABSTRACT FROM AUTHOR]

Transgenic Bt cotton developed against lepidopteran pests may not be compatible with parasitoid of secondary pests such as Bemisia tabaci which attack many plants such as cotton. In this study, the effects of Bt cotton on the demographic parameters of Encarsia formosa, parasitoid of B. tabaci were assessed. The data were analysed using the age specific, two-sex life table parameters. The results indicated that pre-adult developmental time, the total preoviposition period (TPOP) and the adult preoviposition period (APOP) in the Bt cotton were significantly longer than in the non-Bt cotton. Also, fecundity and body size in both lines were significantly different. The fecundity was 23.64±0.73 and 43.75±0.89 eggs/females in the Bt and non-Bt cotton, respectively. All the population parameters were affected by the Bt cotton. The intrinsic rate of increase (r) was 0.15 day-1 in the non-Bt cotton but it was 0.10 day-1 in the Bt cotton. The finite rate of increase (λ) was 1.11 day-1 in the non-Bt cotton whilst it was 1.08 in the Bt cotton. The net reproductive rate (R0) in the non-Bt cotton was 36.75 but in the Bt cotton these parameters showed 19.62 offspring/individual. Also, the mean generation time (T ) in the non-Bt and Bt cotton was 22.69 and 27.79 days, respectively. The results illustrated, that although transgenic crops are effective tools for management of the target pests, they can adversely affect, either directly or indirectly, the natural enemies dependent on these plants.

Background: Adjuvants use several mechanisms to boost immunogenicity and to modulate immune response. The strength of adsorption of antigen by adjuvants can be a determinant factor for significant improvement of immunopotentiation., Methods: We expressed recombinant RBD-FC in PichiaPink Strain 4 and examined the vaccination of mice by vaccine formulation with different adjuvants (sodium alginate and aluminum hydroxide, alone and together)., Results: Sodium alginate significantly increased the immunogenicity and stability of RBD-FC antigen, so RBD-FC formulated with combined alginate and alum (AlSa) and sodium alginate alone showed higher antibody titer and stability. Immunogenicity of RBD-FC:AlSa was determined by serological assays including direct enzyme-linked immunosorbent assay (ELISA) and surrogate virus neutralization test (sVNT). High levels of IgGs and neutralizing antibodies were measured in serum of mice immunized with the RBD-FC:AlSa formulation. On the other hand, cytokines IL-10 and INF-γ were severely accumulated in response to RBD-FC:AlSa, and after 10 days, their accumulation was significantly declined, whereas IL-4 showed the highest and the lowest accumulation in response to alum and alginate, respectively., Conclusions: Our data may suggest that combination of alum and sodium alginate has a better compatibility with RBD-FC in vaccine formulation., (© 2024 The Author(s). Influenza and Other Respiratory Viruses published by John Wiley & Sons Ltd.)

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., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

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 (MgSO 4 ), calcium dichloride (CaCl 2 ), manganese (II) sulfate (MnSO 4 ), 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 MgSO 4 , 330.07 mg/l CaCl 2 , 18.3 mg/l MnSO 4 , 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., (© 2024. The Author(s).)

Cyanobacteria have developed acclimation strategies to adapt to harsh environments, making them a model organism. Understanding the molecular mechanisms of tolerance to abiotic stresses can help elucidate how cells change their gene expression patterns in response to stress. Recent advances in sequencing techniques and bioinformatics analysis methods have led to the discovery of many genes involved in stress response in organisms. The Synechocystis sp. PCC 6803 is a suitable microorganism for studying transcriptome response under environmental stress. Therefore, for the first time, we employed two effective feature selection techniques namely and support vector machine recursive feature elimination (SVM-RFE) and LASSO (Least Absolute Shrinkage Selector Operator) to pinpoint the crucial genes responsive to environmental stresses in Synechocystis sp. PCC 6803. We applied these algorithms of machine learning to analyze the transcriptomic data of Synechocystis sp. PCC 6803 under distinct conditions, encompassing light, salt and iron stress conditions. Seven candidate genes namely sll1862, slr0650, sll0760, slr0091, ssl3044, slr1285, and slr1687 were selected by both LASSO and SVM-RFE algorithms. RNA-seq analysis was performed to validate the efficiency of our feature selection approach in selecting the most important genes. The RNA-seq analysis revealed significantly high expression for five genes namely sll1862, slr1687, ssl3044, slr1285, and slr0650 under ion stress condition. Among these five genes, ssl3044 and slr0650 could be introduced as new potential candidate genes for further confirmatory genetic studies, to determine their roles in their response to abiotic stresses., (© 2024. The Author(s) under exclusive licence to Sociedade Brasileira de Microbiologia.)

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 (T 1 ) 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., (© 2024. The Author(s).)

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., (© 2024. The Author(s).)

In this study, a data-driven approach employed by utilizing the product called JRC-Global surface water mapping layers V1.4 on the Google Earth Engine (GEE) to map and monitor the effects of climate change on surface water resources. Key climatic variables affecting water bodies, including air temperature (AT), actual evapotranspiration (ETa), and total precipitation, were analyzed from 2000 to 2021 using the temperature-vegetation index (TVX) and Moderate Resolution Imaging Spectroradiometer (MODIS) products. The findings demonstrate a clear association between global warming and the shrinking of surface water resources in the LUB. According to the results, an increase in AT corresponded to a decrease in water surface area, highlighting the significant influence of AT and ETa on controlling the water surface in the LUB (partial rho of - 0.65 and - 0.68, respectively). Conversely, no significant relationship was found with precipitation and water surface area (partial rho of + 0.25). Notably, the results of the study indicate that over the past four decades, approximately 40% of the water bodies in the LUB remained permanent. This suggests a loss of around 30% of the permanent water resources, which have transitioned into seasonal water bodies, accounting for nearly 13% of the total. This research provides a comprehensive framework for monitoring surface water resource variations and assessing the impact of climate change on water resources. It aids in the development of sustainable water management strategies and plans, supporting the preservation and effective use of water resources., (© 2024. The Author(s).)

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., (© 2023. The Author(s).)

Bladder cancer (BC) is the 10th most common malignancy in worldwide, with substantial mortality and morbidity if not treated effectively. According to various research, inflammatory circumstances majorly impact the microenvironment of bladder cancer, and the chronic presence of cytokines and chemokines promotes tumor progression. In this investigation, we explored the impact of cell-free culture supernatant ofEscherichia colistrain 536 on inflammatory cytokines and chemokines in bladder cancer model microarray data (GSE162251). Then we examined in silico outcomes on human bladder cancer cell line 5637 to verify and extrapolate findings. This investigation revealed for the first time that this compound has potent suppressor effects on interleukin 1 beta (IL-1β), C-C motif chemokine ligand 2 (CCL2), and C-X3-C motif chemokine ligand 1 (CX3CL1) gene expression as well as increased NAD(P)H quinone dehydrogenase 1 (NQO1), as an anti-oxidant agent, gene expression in 4, 8, and 24 h. Moreover, we confirmed that c-MYC, a member of the MYC proto-oncogene family, gene expression reduced in 5637 cells in 4 h and then followed up its expression in 8 and 24 h. In addition, our investigation demonstrated that the supernatant raised the BCL2-Associated X Protein/B-cell lymphoma 2 (BAX/BCL2) ratio, and subsequent flow cytometry analysis demonstrated that the supernatant induction apoptosis and necrosis. In conclusion, our findings demonstrate that this compound is a potential candidate for the suppression of bladder cancer progression., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

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., (© 2023. The Author(s).)

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., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Rezaei et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

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., (© 2023. Springer Nature Limited.)

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., (© 2023. The Author(s).)

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., (© 2023. The Author(s).)

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., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Rezaei et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

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, R 2 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., (© 2022. The Author(s).)

Background: Soybean is an important oilseed crop that its development and production are affected by environmental stresses (such as saline-alkaline and water deficit)., Objectives: This experiment was performed with the aim of identifying candidate genes in saline-alkaline stress and water-deficit stress conditions using transcriptome analysis and to investigate the expression of these genes under water deficit stress conditions using RTqPCR., Materials and Methods: In this experiment, soybean transcriptome data under saline-alkaline and water-deficit stress were downloaded from the NCBI website, and then the co-expression modules were determined for them and the gene network was plotted for each module, and finally, the hub genes were identified. To compare the expression of genes in saline-alkaline and water deficit conditions, soybean plants were subjected to water deficit stress and their gene expression was determined using RTqPCR., Results: The filtered (Log FC above +2 and below -2) genes of soybean were grouped under saline-alkaline stress in 15 modules and under water-deficit stress in 2 different modules. Within each module, the interaction of genes was identified using the gene network, then three genes of ann11 , cyp450 and zfp selected as hub genes. These hub genes are highly co-expression with other network genes, which not only display differential expression but also differential co-expression. The results of RT-PCR indicated that cyp450 gene expression was not significantly different from the control, while ann11 gene expression significantly increased under water deficit stress, but zfp gene expression decreased significantly under water deficit stress., Conclusions: We identified three genes, ann11 , cyp450 and zfp , as hub genes. According to our results, ann11 gene had a significant increase in expression under water deficit stress, which can indicate the importance of this gene under drought conditions. Therefore, according to the results of this experiment as well as other researchers, we introduce this gene as a key gene in water deficit tolerance and recommend its use in genetic engineering to increase the tolerance of other plants., (Copyright: © 2021 The Author(s); Published by Iranian Journal of Biotechnology.)

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., (© 2022. The Author(s).)

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 (H 2 O 2 ) 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., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH Austria, part of Springer Nature.)

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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Ghamari, Ahmadikhah, Tohidfar and Bakhtiarizadeh.)

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., (© 2021. The Author(s), under exclusive licence to Springer Nature B.V.)

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., Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-021-03050-w., (© King Abdulaziz City for Science and Technology 2021.)

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.

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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Niazian, Sadat-Noori, Tohidfar, Mortazavian and Sabbatini.)

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 (Sb V ) 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. Sb V must be reduced to its trivalent active form (Sb III ). This reduction occurs within the host macrophage, and the resultant Sb III enters amastigotes via the aquaglyceroporin1 (AQP1) membrane carrier. Overexpression of AQP1 results in hypersensitivity of the parasites to Sb III , but resistant phenotypes accompany reduced expression, inactivation mutations, or deletion of AQP1. Hence, in this study, a phylogenetic analysis using barcode gene COX II 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 COX II 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 COX II. 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 COX II 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., Competing Interests: Yuriy Orlov and Gilda Eslami are Academic Editors for PeerJ., (© 2021 Eslami et al.)

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 R 2 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 NH 4 + , 38.92 mM NO 3 - , 22.79 mM K + , 5.08 mM Cl - , 3.34 mM Ca 2+ , 1.67 mM Mg 2+ , 2.17 mM SO 4 2- , and 1.44 mM H 2 PO 4 - . 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.

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., Competing Interests: The authors have declared that no competing interests exist.

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 (R 2  > 0.92) had better performance accuracy than MLP (R 2  > 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., Competing Interests: Competing interestsThe authors declare that they have no competing interests., (© The Author(s) 2020.)

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., Competing Interests: We have no conflicts of interest to disclose.

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 R 2 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.

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.

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 R 2 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.

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.

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 'Grassland' than 'Numan' (14.2% vs 9.2%). In addition, high transformation efficiency of about 46.65% and 29.15% was observed using meristem tip explants of 'Grassland' and 'Numan' based on IPT and RD29A positive PCR results, respectively. Seed transformation efficiency in 'Grassland' and 'Numan' 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 'Grassland' 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.

Background: In consideration for the increasing widespread use of genetically modified (GM) crops, one of the important issues for assessment is the effect of GM crops on soil microbial communities., Objectives: In this study, T 2 chitinase-transgenic cotton (line #57) and its non-transgenic line were investigated for bacterial and fungal dynamics during its development stages., Material and Methods: The assessments were performed by viable plate count and polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) assays., Results: Viable plate count analysis showed an increase in community structures and the number of culturable bacteria in rhizosphere of both transgenic and non-transgenic cultivars as compared to bulk soil. PCR-DGGE confirmed results of viable plate count assays of the changes in bacterial and fungal communities for all cotton development stages in rhizosphere and bulk zones. No significant differences in number of functional bacteria were observed between rhizosphere soil of chitinase transgenic and non-chitinase transgenic cotton at one particular stage., Conclusions: The results indicated that T 2 chitinase-transgenic cotton (line #57) might have no adverse effects on community structures and total number of culturable bacteria and fungi in the rhizosphere.

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 HgCl 2 , Ca(ClO) 2 , Nano-silver, H 2 O 2 , NaOCl, AgNO 3 , 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 R 2 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 AgNO 3 . 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.

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.

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, pBI121 GUS-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.

Background: Chrysanthemum; also commonly known as mums or chrysanths, is one of the most important ornamental crops worldwide. Introducing desirable traits into this valuable plant by the conventional breeding has so far been faced with some restrictions due to the limited gene pool and cross-incompatibility. Therefore, breeders have decided to exploit Agrobacterium -mediated transformation methods in order to satisfy the growing market demands. However, more efficient in vitro regeneration protocols are required for this approach., Objectives: The objective of this research was to develop an efficient protocol for an in vitro plant regeneration by the examining the effects of various combinations and concentrations of the plant growth regulators (PGRs) and different explants types., Materials and Methods: The leaf and petiole explants of the Chrysanthemum morifolium cv. 'Resomee Splendid' were collected from the in vitro grown plantlets. Murashige and Skooge (MS) medium was supplemented with different concentrations and combinations of benzylaminopurine (BAP), 1-naphthaleneacetic acid (NAA) and thidiazuron (TDZ). Thereafter, the effects of these hormonal treatments were investigated on shoot initiation percentage, the average number of shoots per explants, callogenesis, and the type of organogenesis in regard to both types of the explants., Results: Shoots were directly formed from leaf explants on the media that only contained BAP without callus formation. Amongst the other hormonal treatments, a combination of 4.5 mg.L -1 BAP plus 1 mg.L -1 NAA resulted in the direct organogenesis from the leaf explants, which was superior to the other combinations and concentrations. In regard to the petiole explants, direct shoot formation occurred in all the media except for the ones which were fortified with TDZ. In this case, considering the shoot initiation percentage and the mean shoot number per explants, the best results were achieved in the medium supplemented with 1.5 mg.L -1 BAP and 1 mg.L -1 NAA. Results showed that interaction of either BAP or TDZ with NAA was necessary for the callus induction., Conclusions: Significant differences in shoot initiation percentage and the average number of shoots per explants were observed both in leaves and petioles grown on different media. Moreover, the callogenesis rates, as well as organogenesis types, showed some differences among the studied explants when compared on the same media.

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.

This is the first report evaluating the relationship between the chemical compositions of cumin seeds (based on the analysis of the content of catalase, ascorbate peroxidase, proline, protein, terpenic compounds, alcohol/phenols, aldehydes, and epoxides) and the induction efficiency of somatic embryogenesis in two Iranian superior cumin landraces (Golestan and North Khorasan). Cotyledons isolated from Golestan landrace seeds cultivated on MS medium supplemented with 0.1 mg/L kinetin proved to be the best primary explant for the induction of somatic embryogenesis as well as the regeneration of the whole plantlet. Results indicated that different developmental stages of somatic embryos were simultaneously observed on a callus with embryogenic potential. The high content of catalase, ascorbate peroxidase, proline, and terpenic hydrocarbons and low content of alcoholic and phenolic compositions had a stimulatory effect on somatic embryogenesis. Band patterns of RAPD markers in regenerated plants were different from those of the mother plants. This may be related to somaclonal variations or pollination system of cumin. Generally, measurement of chemical compositions can be used as a marker for evaluating the occurrence of somatic embryogenesis in cumin. Also, somaclonal variations of regenerated plants can be applied by the plant breeders in breeding programs.

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.