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Background and Objectives: Silybum marianum as a member of the Asteraceae family, commonly acknowledged as a weed. Nevertheless, silymarin as one of the major secondary metabolites in this plant proved to have possitive effects on liver, heart and cancer diseases. The aim of this study was to induce hairy roots using elicitors towards elevate silymarin production in milk tistle plants. Materials and Methods: The objective of this study was to induce hairy roots in milk thistle using salicylic acid and phenylalanine as elicitors to enhance silymarin production. Current research was conducted as two distinct experiments at University of Zanjan. The first trail was performed in a completely randomized design with nine replications to evaluate the production of hairy using three strains of Agrobacterium rhizogenesis including A7, R1000 and ATCC. Morphological traits, root number and root length were measured. The second experiment was conducted as a completely randomized design with three replicates to evaluate the impact of salicylic acid (0, 50 and 100 μM) and phenylalanine (0, 100 and 200 μM) on hairy roots induction, and total phenol, flavonoids, antioxidants and silymarin production. Results: Larger and higher number of hairy roots were induced by ATCC strain. The results showed that all physiological and biochemical attributes were significant (P>0.01). The highest content of total phenols was recorded in 200 μM phenylalanine while hairy roots induced by ATCC. The highest silymarin was induced following the treatment of 200 μM phenylalanine in roots were induced by R1000 and ATCC. Conclusion: Due to high potential of Agrobacterium rhizogenesis to induce hairy roots in diverse range of host plants, it considers as a robust approach for production of secondary metabolites. On the other hand, elicitors alongside hairy roots improved the biochemical and physiological traits of marigold. The amount of silymarin in hairy roots transfected with R1000 and ATCC alongside 200 μM phenylalanine was significantly higher. [ABSTRACT FROM AUTHOR]

Background & Objective: The aim of this experiment was to study the effect of inoculation with growth promoting bacteria and mycorrhiza-like fungi (Piriformospora indica) under water deficit stress on some morphological traits and essential oil yield of Moldavian balm (Dracocephalum moldavica L.). Materials & Methods: a factorial experiment was conducted in University of Tabriz, Iran based on randomized complete block design with three replications in 2017. The first factor was water deficit based on field capacity (FC) at four levels including 80-100 % FC, 70-85%FC, 55-70% FC, 40-55% FC and the second factor was inoculation of Moldavian balm with different microorganisms including Enterobacter, Pseudomonas, Agrobacterium, Piriformospora indica and control (non-inoculation). Results: The results showed that the effect of water deficit stress and inoculation treatments on all morphological traits were significant. With increasing water deficit stress, all growth traits including plant height, branch number, main branch length, number of cycle of flowers per main branches, essential oil content and yield were significantly reduced. The highest amount of dry flowering head yield (2.41g/pot) was obtained with Enterobacter sp. However, the highest dry mater yield (59 g/pot), essential oil percentage (0.32%) and essential oil yield (0.19 g/pot) were obtained under Agrobacterium treatment. Conclusion: In general, inoculations with Piriformospora indica and Agrobacterium, were the best treatments in terms of dry mater yield and essential oil yield under moderate drought stress conditions and can be recommended as treatments to reduce the application of chemical fertilizers in sustainable production of Moldavian balm.

Objective Tissue plasminogen activator is one of the most important drugs in the treatment of heart disease. This drug is produced in the expression system as a recombinant protein that has high production costs. Transient expression system is very suitable for protein expression because of its high expression, high speed, low cost and no spatial effect. Post-transcriptional silencing has been shown to affect expression levels. Therefore, the aim of this study was to investigate the effect of simultaneous expression of P19 silencing suppressor gene on transient expression of recombinant tissue plasminogen activator (rtPA) at transcriptional and protein levels in Nicotiana benthamiana. Material and methods To serve this purpose, the expression proportion of injected Agrobacterium tumefaciens containing a binary vector pCAMBIA1304-rtPA with agrobacterium containing pCAMBIA1304-P19 have been studied comparison with the expression level from Agrobacterium containing only the binary vector pCAMBIA1304-rtPA. Leaf samples were prepared on 4, 7, and 10 day post-inoculation with Agrobacterium. Transcription and then protein levels were calculated using the Real Time PCR and ELISA tests. Results The results of Real Time PCR test showed that rtPA transcript increased in the presence of P19. Also, 4 days after plant inoculation, the highest transcript levels were obtained from p19 and rtPA genes. ELISA results showed that the expression of rtPA protein in the presence of P19 was 89 and 84 µg.g-1 leaf weight at the 7 and 10 day after inoculation, respectively. This expression was 12 and 15% higher than of when agrobacterium-containing pCAMBIA1304-rtPA vector alone was used, respectively. Conclusion The results showed that the use of transient expression method could be a suitable method for rtPA protein production.

One of the main strategies to improve plant tolerance is the expression of stress-induced genes, which play a significant role in the ionic balance of plants. SOS3 is one of the important components of SOS-regulated ionic homeostasis pathway. Therefore, the expression of this gene could be an important step towards producing salt-resistant plants. In this work, we have transformed tomato (Solanum lycopersicum) by Agrobacterium (GV3101 and LBA4404) containing plasmids with SOS3 genes. The maximum regeneration rate was determined in cotyledons of CH genotype. The simultaneous use of cotyledons and hypocotyls in the culture medium had the best outcome. In addition, the best time was found to be one day after inoculation. Also, the best transgenic variety was detected for Agrobacterium GV3101, which can be attributed to the interaction between the genus Agrobacterium and the tomato variety. Transgenic plants were transferred to a culture medium containing sequestrene, which caused the acceleration of the seedling growth in particular. The presence of the SOS3 in the transgenic plants was verified by PCR and RT-PCR methods.

Objective Begonias are popular ornamental plants in the world so introducing of various forms of this plant is very important. Nowadays plant modification through biotechnology and genetic engineering for production of new genotypes with varied forms is highlighted in this genus. So optimization of regeneration in in vitro culture condition and gene transferring is in priority to provide a suitable condition for gene transferring in this plant specially those responsible in flower color. Materials and methods In the first step, thin cell layer (TCL) explants from petioles of B. soli-mutata were assayed in MS medium containing Kin or TDZ (0.2, 1 and 2 mg/l) in combination with NAA (0 and 0.2 mg/l). For co-culturing of petiole TCL explants with Agrobacterium tumefacians strain LBA4404 containing GUS and NPTII genes MS medium supplemented with 1 mg/l Kin and 0.2 mg/l NAA was used. Transformed plantlets were confirmed by PCR and Gus assay test. Results Based on the regeneration results, Kin was better than TDZ, also adding NAA to the medium has positive effect on both of cytokinins. In transformation process, out of 2000 explants which infected with Agrobacterium, after 4 months of culture only 17 plantlets were adopted. Of these 17 plantlets, only 7 plantlets were confirmed by Gus assay test and PCR as transgenic plants. Conclusions The result showed in this species transformation percentage is low and optimization of transformation condition is important.

Background and Aim: A variety of bacteria like Staphylococcus aureus utilize quorum sensing to perform their important activities such as biofilm formation and production of virulence factors. Interfering with the bacterial QS will disable the bacteria to perform above-mentioned vital activities. The principal aim of the study was to evaluate the effects of five plant extracts against bacterial Quorum sensing of Staphylococcus aureus. Materials and Methods: Thirteen strains of Staphylococcus aureus were isolated from patients with dental implant infection and identified. The plant species were collected from vicinity gardens of Fars Province and extracted using 96% ethanol. The anti-QS and antimicrobial susceptibility methods were then carried out to evaluate their bactericidal and QS properties with the use of Agrobacterium tumefaciens NTL/PZLR4. Furthermore, the biofilm production of the isolates was evaluated by microtiter plate (MTP) assay. Ethical Considerations: This study with research ethics codes 01-16-1-1959 and 73/118248 has been approved by research ethics committee at Islamic Azad University, Jahrom and research sciences branch, respectively. Findings: The results of the study disclosed that the extract of raspberry blossom possesses significantly (p21 mm). The anti-QS activity was proved by creating clear halo sides of the wells formed by Agrobacterium tumefaciens NTL/PZLR4. Moreover, the extracts of tarragon, wheat flower, flixweed and basil showed antimicrobial properties. Conclusion: According to the anti-biofilm and anti-QS properties of raspberry blossom extract against the isolates of Staphylococcus aureus, it could be considered as a mouthwash against dental bacterial infection with the identification of active compounds in the raspberry.

Introduction: The white button mushroom does not produce remarkable yield in the third flash. Nutritional deficiency and the inability of this mushroom to efficient use of compost are mentioned as its reasons. Basically, compost includes two major food components, lignocellulose and microbial biomass. But this microbial biomass provides just 10% of button mushroom food needs. According to research studies, differentenzymes in both white button mushroom and oyster mushroom are responsible for decomposition of lignin compounds in compost media, from begin of mycelium grows to the end of fruiting. Lacasse, manganese peroxidase, lignin peroxidase, glyoxal oxidase enzymes contribute to degradation of lignin compounds in degradation mushroom has proven by researchers however itis dependent on mushroom types. Manganese peroxidase enzyme (EC. 1.11.1.13) is an extracellular parser lignin enzyme that has a central peroxidase core. Manganese peroxidase enzyme oxidizesMn2+ to Mn3+ and then Mn3+ oxidizes phenolic structure to fonoxile radical. Produced Mn3+ is very active and makes complex by chelating organic acids that is produced by mushrooms such as oxalate or malate. Mn3+ ions become stable by helping of these chelates and it can penetrate through materials such as wood. On the other hand, in recent years, plant biotechnology provides new solutions for old problems such as use of microorganisms, particularly using bacteria for gene transfer and improvement of superlatives. For a sample of this method, Agrobacterium-mediated transformation system can be noted. In addition, the use of suitable promoters for heterologous genes expression in suitable hosts is an important strategy in functional biotechnology that has been raised in edible mushroom genetic engineering. The lack of efficient and sufficient use of compost, low power of white button mushroom in competition with other rivals, lack of yield per area unit due to production costs, pests and diseases, low flexibility and adaptability with environmental conditions changes are some of the problems that the mushroom reformers are faced. Unlike the great efforts made by researchers, conventional breeding techniques to produce the A. bisporus mushroom only have been led to produce a few new races. Therefore, todays some problems associated with traditional methods of breeding of edible mushrooms, including the need to provide races that have desired characteristics, the traditional method performance tests and low chances of success in the transfer of important agronomic characteristics such as functionality and disease resistance. So, they almost have been replaced with new biotechnology methods. Anexample of this method is to manipulateproperties transformation for the particular purpose. Modification of both expression or type of lignin degrading enzyme are possible solutions to deal with this problem, but these are not applicable or are difficult to be done with traditional breeding programs. In recent years, gene transformation mediated with Agrobacterium routinely is used for gene transformation to mushrooms and is proposed as a method for removing limitations of white button mushroom breeding. Materials and Methods: In this research, the oyster mushroom strain Florida was used as the source of manganese peroxidase (mnp) gene and white button mushroom strain 737 gill and cap tissue were used as transformation host. Agrobacterium strain LBA4404 harbors p133H88-FM plasmid thatcontainsmnp gene of oyster mushroom and also hph gene under control of gpdII promoter of the button white mushroom strain IM008 was used as a transformer. Selection medium containing 30 mg/ml Hygromycin B and was used for selecting transformed explants. To confirm transformation, PCR with specific primers of mnp and hph genes was performed on genomic DNA of selected colonies. Results and Discussion: Results showed the gill tissue explants, with transformation rate 5%, have a better response to applied transformation method than cap tissue explants, with transformation rate zero percent. As expected, polymerase chain reaction with specific primers ofhph and mnp genes amplified 1049 and 1086 bp fragments and verified the transformation of mycelium's grown on selection medium. It seems that Bacterial strain and also used plasmid were one of the responses for observed low rate transformation which is in accordance with leach and co-workers study. Finally, we could propose that cap tissue is more suitable for further gene transformation of this mushroombecause of high transformation rate of cap tissue.

The parasympatholytic tropane alkaloids, Atropine and Scopolamine, accumulated in Atropa belladonna, are of great interest for the pharmaceutical industry.In this research, Atropa belladonna transformed hairy roots were produced by Agrobacterium rhizogenes strain A4 mediated transformation. Effects of different concentrations of methyl jasmonate (0, 150 and 300 Micro molar) were investigated on production of Atropine and Scopolamine in 24 hours treatment. Then the contents of two tropane alkaloids, Atropine and Scopolamine, were assayed by HPLC method. The tropane alkaloids contents were shown to be significantly increased in transformed hairy roots. Results showed Atropine and Scopolamine content in transformed hairy roots reached respectively from 0.12 to 1.715 and 0.0195 to 0.43 of mg in 100 mg of dry weight. Methyl jasmonate dramatically enhanced both tropane alkaloids, Atropine and Scopolamine. Tropane alkaloids content increased in 300 Mµ concentration of methyl jasmonat in comparison with 150 Mµ concentration and control treatment. In conclusion, it is suggested that, hairy root can be used as a replacement of plants in further studies and for tropane alkaloids production in economical and commercial scales.

Background and Aim: A variety of bacteria like Staphylococcus aureus utilize quorum sensing to perform their important activities such as biofilm formation and production of virulence factors. Interfering with the bacterial QS will disable the bacteria to perform above-mentioned vital activities. The principal aim of the study was to evaluate the effects of five plant extracts against bacterial Quorum sensing of Staphylococcus aureus. Materials and Methods: Thirteen strains of Staphylococcus aureus were isolated from patients with dental implant infection and identified. The plant species were collected from vicinity gardens of Fars Province and extracted using 96% ethanol. The anti-QS and antimicrobial susceptibility methods were then carried out to evaluate their bactericidal and QS properties with the use of Agrobacterium tumefaciens NTL/PZLR4. Furthermore, the biofilm production of the isolates was evaluated by microtiter plate (MTP) assay. Ethical Considerations: This study with research ethics codes 01-16-1-1959 and 73/118248 has been approved by research ethics committee at Islamic Azad University, Jahrom and research sciences branch, respectively. Findings: The results of the study disclosed that the extract of raspberry blossom possesses significantly (p<0.05) anti-QS property (>21 mm). The anti-QS activity was proved by creating clear halo sides of the wells formed by Agrobacterium tumefaciens NTL/PZLR4. Moreover, the extracts of tarragon, wheat flower, flixweed and basil showed antimicrobial properties. Conclusion: According to the anti-biofilm and anti-QS properties of raspberry blossom extract against the isolates of Staphylococcus aureus, it could be considered as a mouthwash against dental bacterial infection with the identification of active compounds in the raspberry. [ABSTRACT FROM AUTHOR]

Introduction: Mortierella alpina is one of the most important fungi in food industry because of having ability of synthesizing unsaturated fatty acids, particularly Arashidonic Acid. This is a precursor of Eicosanoidregulate-lipoprotein metabolism which is involved in blood rheology, platelet activation and leukocyte-function, and the functional characteristics of the cell membrane. Materials and methods: In this study genetic transformation of M. alpina CBS754.68 fungus was evaluated via Agrobacterium tumefaciens and Agrobacterium rhizogenes. Agrobacteriums containing pBI121 vector were used for transformation of three days of old mycelia. Three days old hyphae were exposed to the bacteria with three level of time (one, two and three hours) in the present of acetosyringone. Mitotic stability of the third generation of transgenic (T2) was confirmed by GUS assay and amplification of CaMV 35S promoter by polymerase chain reaction. Results: The highest percentage of transformation and mitotic stability were obtained by using A. tumefaciens and A. rhizogenese, respectively. Discussion and conclusion: The results showed that to obtain more efficient and more stable transformation, the fundamental factor is the use of suitable species of Agrobacterium. It is the first report for transformation of autothroph strain of M. alpine via Agrobacterium.

Introduction: A variety of oxygen-transport and -binding proteins exist in organisms including bacteria, protozoans, and fungi all have hemoglobin-like proteins. In addition to dealing with transport and sensing of oxygen, they may also deal with NO2, CO2, sulfide compounds, and even O2 scavenging in environments. Also they detoxified chlorinated materials like P450 enzymes and peroxidases and use as a detector of nitrate and hydrogen peroxide. Pore-forming bacterial globins are interested for filtration. Materials and methods: Although there are data for bacterial toxin as a filter, here we used Agrobacterial hem to induce nano pore in the heme structure using point mutation. Results: Investigations showed that three amino acids leucine 76, alanine 83 and histidine 80 are important for pore formation in Agrobacterium hemoglobin. A point mutation on leucine 76 to glycine, histidine 80 to asparagine and alanine 83 to lysine step by step led to create the nano pore 0.7- 0.8 nm in the globin. Discussion and conclusion: These mutations in bacterial hemoglobin increase the stability when mutation is with it’s at pH7. This mutation decreases the aliphatic index however increase the stability index.

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

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

The application of recombinant DNA leads to production of a large number of therapeutic proteins and peptides. Lactoferricin, a strong cationic peptide N-terminal region of lactoferrin, is produced by a protein-digesting enzyme pepsin. So, this project was done to produce lactoferricin of camel in hairy roots of tobacco and investigate its antibacterial effect. For this reason camel lactoferricin peptide gene sequence was extracted from gene bank and after optimization of codon usage synthesized artificially. The mentioned sequence was subcloned into pBI121expression vector. The recombinant pBI121 vector was transferred to Agrobacterium rhizogenes bacterium and then was used to produce hairy roots. Hairy roots was evaluated and proved by specific primers of gene which coded lactoferricin peptide. Recombinant peptide of lactoferricin was extracted from hairy roots and evaluated against plant pathogenic bacteria including Pseudomonas syringae, Pseudomonas fluorescens, Pseudomonas syringae pv. syringae, Erwinia amylovora, Xanthomonas citri, Pectobacterium carotovorum by disk diffusion method. The results showed that this peptide was expressed in hairy roots and has significant antibacterial effect. [ABSTRACT FROM AUTHOR]

In this study, signal peptide sequences of Lillum longiflorum anther-specific protein LIM14 and ATCPrecA, a protein with the origin of A. thaliana that had the characteristics of chloroplast signal sequence were independently fused to the N terminus of the green fluorescent protein (GFP) and ß-glucoronidase (GUS) genes and subsequently transferred to potato (Solanum tuberosum cv. Kardal) as an important starch storage plant by Agrobacterium-mediated transformation. Fluorescence microscopy and GUS staining showed that the recombinant LIM14-GFP fusion protein was targeted to the amyloplast and chloroplast of transgenic potato plants. Quantitative assay of GUS showed that the level of expression in the enriched amyloplast fragment of signal peptide included plants is higher than the GUS control transformed plants. ATCPrecA is a protein with the origin of Arabidopsis thaliana that had the characteristics of chloroplast signal sequence in N-terminus. To study the function of the transit peptide of ATCPrecA, full length cDNA was fused to the N-terminus of the GFP to make recombinant protein. ATCPrecA-GFP was transferred to potato by Agrobacterium-mediated transformation and transiently expressed in onion (Allium cepa L.) epidermal cells via microprojectile bombardment. According to the results of this research, we concluded that the transit sequence of both LIM14 and ATCPrecA were functional and were capable of directing recombinant proteins into plastids. [ABSTRACT FROM AUTHOR]

In this study, signal peptide sequences of Lillum longiflorum anther-specific protein LIM14 and ATCPrecA, a protein with the origin of A. thaliana that had the characteristics of chloroplast signal sequence were independently fused to the N terminus of the green fluorescent protein (GFP) and β-glucoronidase (GUS) genes and subsequently transferred to potato (Solanum tuberosum cv. Kardal) as an important starch storage plant by Agrobacterium-mediated transformation. Fluorescence microscopy and GUS staining showed that the recombinant LIM14-GFP fusion protein was targeted to the amyloplast and chloroplast of transgenic potato plants. Quantitative assay of GUS showed that the level of expression in the enriched amyloplast fragment of signal peptide included plants is higher than the GUS control transformed plants. ATCPrecA is a protein with the origin of Arabidopsis thaliana that had the characteristics of chloroplast signal sequence in N-terminus. To study the function of the transit peptide of ATCPrecA, full length cDNA was fused to the N-terminus of the GFP to make recombinant protein. ATCPrecA-GFP was transferred to potato by Agrobacterium-mediated transformation and transiently expressed in onion (Allium cepa L.) epidermal cells via microprojectile bombardment. According to the results of this research, we concluded that the transit sequence of both LIM14 and ATCPrecA were functional and were capable of directing recombinant proteins into plastids. [ABSTRACT FROM AUTHOR]