Agrobacterium Mediated Transformation of Fld and GUS Genes into Canola for Salinity Stress

Salinity is one of the major abiotic stress which limits wide spread canola cultivation. One way to overcome this problem could be transfection, to produce tolerable species. Cotyledonary and hypocotyls explants obtained from 4 and 7 days old seedling of Elite and RJS003 varieties were utilized in this study. Genetic transformation was implemented through Agrobacterium tumefaciens LBA4404 containing PBI121 plasmid and Agrobacterium tumefaciens C58, LBA4404, AGL0 and EHA 101 strains which contain P6u- ubi- fvt1 construct. The T-DNA region of P6u- Ubi- Fvt1 plasmid included HPT (Hygromycin phosphotransferase) plant selectable marker and Fld (flavodoxin) gene. PBI121 plasmid had NptII (Neomycin phosphotransferase) plant Selectable marker and β-glucuronidase (GUS) reporter genes. Transfected explants were analyzed by PCR and histochemical assay for Fld and Gus genes, respectively. Our data indicated that the cotyledonary explants of both cultivars were incompetent to be infected with Fld gens. However, the transformation in Elite hypocotyls explants with Agrobacterium tumefaciens C58 and LBA 4404 strains were confirmed through PCR product and histochemical evaluation for Fld and GUS genes, respectively. Therefore, the result of this manuscript may to certain degree fulfill the endeavor appointed to this oilseed

Modeling and Optimizing in vitro Sterilization of Chrysanthemum via Multilayer Perceptron-Non-dominated Sorting Genetic Algorithm-II (MLP-NSGAII)

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

A review on the application of hairy roots in removing phenolic compounds from aqueous solutions

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

Regeneration of Trachyspermum ammi L. Sprague ecotypes via indirect somatic embryogenesis using hypocotyl and epicotyl explants

Trachyspermum ammi L. Sprague, commonly known as ajwain, carom or thymol seeds, is one of the oldest medicinal plants with numerous health benefits. The classical breeding process of medicinal plants is not only time consuming but also requires much efforts. Biotechnological techniques such as in vitro regeneration which increase the capacity of production of medicinal plants rely upon a plenty of components. Here, we studied regeneration of T. ammi by indirect somatic embryogenesis by employing MS and B5 media supplemented with a diverse concentration of plant growth regulators (PGRs), including 2,4-D, kinetin, NAA, IAA, and BAP with four explants (hypocotyl, epicotyl, root and node) and 3 ecotypes (Qom, Yazd, and Rafsanjan). The highest callus induction was obtained from hypocotyl explants on MS medium supplemented with 8.917 µm/L 2,4-D and 2.32 µm/L KIN and epicotyl explants on B5 medium supplemented with 1.074 µm/L NAA and 0.89 µm/L BAP. The highest indirect regeneration (83%) was observed in hypocotyl explants on MS medium supplemented with 0.93 µm/L kinetin. To the best of our knowledge, the present study (2017) is the first report on indirect somatic embryogenesis from hypocotyl and epicotyl of the Iranian Trachyspermum ammi ecotypes that can be beneficial for genetic transformation and other plant biotechnology techniques

Regeneration of Trachyspermum ammi L. Sprague ecotypes via indirect somatic embryogenesis using hypocotyl and epicotyl explants

263-269Trachyspermum ammi L. Sprague, commonly known as ajwain, carom or thymol seeds, is one of the oldest medicinal plants with numerous health benefits. The classical breeding process of medicinal plants is not only time consuming but also requires much efforts. Biotechnological techniques such as in vitro regeneration which increase the capacity of production of medicinal plants rely upon a plenty of components. Here, we studied regeneration of T. ammi by indirect somatic embryogenesis by employing MS and B5 media supplemented with a diverse concentration of plant growth regulators (PGRs), including 2,4-D, kinetin, NAA, IAA, and BAP with four explants (hypocotyl, epicotyl, root and node) and 3 ecotypes (Qom, Yazd, and Rafsanjan). The highest callus induction was obtained from hypocotyl explants on MS medium supplemented with 8.917 µm/L 2,4-D and 2.32 µm/L KIN and epicotyl explants on B5 medium supplemented with 1.074 µm/L NAA and 0.89 µm/L BAP. The highest indirect regeneration (83%) was observed in hypocotyl explants on MS medium supplemented with 0.93 µm/L kinetin. To the best of our knowledge, the present study (2017) is the first report on indirect somatic embryogenesis from hypocotyl and epicotyl of the Iranian Trachyspermum ammi ecotypes that can be beneficial for genetic transformation and other plant biotechnology techniques

In silico Coding Sequence Analysis of Walnut GAI and PIP2 Genes and Comparison with Different Plant Species

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

Somatic Embryogenesis Induction in Narcissus papyraceus cv. Shirazi

PTC&B Explants from bulb scales of Narcissus papyraceus cv. Shirazi cultured in MS and Nitsch media containing different concentrations of BAP, 2,4-D, GA3, Kn, IBA and NAA produced highest number of regenerated bulblets in Nitsch medium containing BAP (2.2 mg/l) and 1.1 mg/l 2,4-D. The highest number of direct somatic embryos were observed in MS containing 0.5 mg/l GA3, 1.6 mg/l BAP and 1.6 mg/l 2,4-D. Induced somatic embryos were multiplied by transferring them to hormone free MS

Forecasting and optimizing Agrobacterium-mediated genetic transformation via ensemble model- fruit fly optimization algorithm: A data mining approach using chrysanthemum databases.

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

Plant Epigenetics: Mechanisms and Applications

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

Effect of Iranian Bt cotton on life table of Bemisia tabaci (Hemiptera: Alyrodidae) and Cry 1Ab detection in the whitefly honeydew

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