نشریه مهندسی ژنتیک و ایمنی زیستی
سال دوازدهم شماره 1 (پیاپی 23، بهار و تابستان 1402)

Tomato big bud is one of the most important diseases of tomato caused by phytoplasma in Iran and all over the world. In infected tomato plants, leaves turn to purple and buds are large, swollen and green that fail to develop normally and do not set fruit. For detection of the causal pathogen, we collected some samples from leaves and swollen buds of infected tomato plants as well as from leaves of some weeds in tomato fields such as Orobanche purpurea, Reseda sp, Amaranthus sp. Xanthium strumarium. Total DNA from all collected samples was extracted. To survey the pathogen in the body of some insects that are known as vectors, samples of two species that appeared more frequently in the tomato fields (leafhoppers and whiteflies) were collected and the total DNA was extracted. Molecular detection of the big bud phytoplasma in all samples was carried out by running PCR using two specific primers (P1/P7). The results showed that in infected samples of the tomato plants, a DNA fragment of 1800 bp in size was multiplied while in the control plant no DNA fragment was observed when the PCR products were visualized in the gel electrophoresis. The same size specific DNA fragment was detected only in PCR reaction coming from leafhoppers and samples of the Orbanche plant parasite. This research showed that the pathogen can be transmitted by leafhoppers and Orbanche while it cannot be transmitted by seeds and whiteflies. This molecular approach could be an effective and fast method for detection and survey of phytoplasma and other fastidious plant pathogens.

With the discovery and development of CRISPR, genome engineering and gene therapy have been revolutionized. Genome editing research is expanding globally, and this technology is increasingly used in agriculture and medicine. One of the advantages of this technology is that it can perform targeted gene editing in a short time, unlike traditional breeding methods. Despite the many advantages that CRISPR has for treating diseases and improving human health, there are also biosafety concerns associated with the development and use of this technology, such as the presence of off-targets, concerns about the appropriate vectors used in this technology, the different transfer methods and gene drive. The rules for using products from this technology in some countries are the same as the existing regulatory rules for transgenic products. Some other countries do not consider these products as transgenic products and consider them exempt from these regulatory rules, just like the products resulting from traditional breeding. This review has explained the safety considerations that must be considered when designing sgRNA when working in the laboratory, and after the creation of CRISPR products. Finally, the global market of Crisper and the rules for the use of CRISPR products have been discussed in some countries.

CRISPR systems or clustered regularly interspaced short palindromic repeats and related proteins are natural microbial immune systems, which have been used as a tool for gene editing. To produce therapeutic effects, CRISPR components must be delivered directly to the target cells. There are different methods of delivery. In this study, some delivery systems through the cell membrane have been reviewed, including viral and non-viral delivery systems. The viral delivery system is the use of viruses such as adeno-associated virus vectors but there are concerns in its clinical use. The non-viral delivery system consists of various methods, including physical delivery and chemical delivery. In the physical delivery; the dose, duration, and specificity of the transfer are controlled more precisely, which includes the methods of electroporation, hydrodynamic injection, and microinjection. In the electroporation method, irreversible changes may occur in the physiology of the membrane, which threatens the cell's viability. In hydrodynamic injection, the volume required to start the injection is large and it is not a suitable method for human applications. Microinjection has high efficiency and precisely controlled dose, but it is very impractical for high-capacity cases. Although physical approaches are typically very successful in the laboratory, they generally do not scale well and are therefore less applicable for delivery to live conditions or high-throughput applications in the laboratory. In chemical delivery methods, instead of inducing a change in the target cell, the transferable cargo itself can be changed. There are two main forms of chemical delivery, including the method of encapsulation of the transferable substance and the method of modification of the transferable substance. Encapsulation can protect the cargo from enzymatic degradation or immune responses and enhance the specificity of delivery. Modification does not protect against degradation by proteases, nor are they capable of specific cell targeting. Therefore, successful use needs to be combined with encapsulation or other delivery methods. Considering that each delivery method has advantages and disadvantages, it is necessary to choose the most effective delivery method so that the CRISPR system is efficient for gene editing.

In order to identify the endophytic fungi of Azarshahr red onion variety, sampling was conducted from the main cultivation areas of this plant in East Azarbaijan province. A total of 360 fungal isolates were obtained from different parts of onion (root, base, scale, flowering stem and leaf). The isolates were grouped based on their phenotypic characteristics on culture media and comparing the DNA fingerprinting patterns using the ISSR molecular markers. In order to accurate identification of the endophytic fungi, the morphological characteristics were combined with the phylogeny inferred from analysis of ITS-rDNA genomic region sequence. Examined endophyte isolates were belonged into eight genera and 12 species namely Alternaria atrum, A. tenuissima, Aspergillus niger, A. kevei, Fusarium equiseti, F. oxysporum, F. proliferatum, F. solani, Mortierella alpina, Setophoma terrestris, Stachybotrys chartarum and Trichoderma longibrachiatum. The results of the inhibitory effect experiments of the selected endophyte Trichoderma longibrachiatum strain ASh268 on the growth of several pathogenic fungal isolates including Rhizoctonia solani, Fusarium oxysporum, Fusarium solani and Setophoma terrestris showed that this strain has a good ability to produce cellulase enzyme in carboxymethyl cellulose medium and significantly reduces the growth of all the above pathogens on the culture media. To our knowledge, this is the first report of Aspergillus kevei for the funga of Iran. Also, this research is the first study on the isolation and identification of endophytic fungi of red onion verity in its major cultivation areas in Iran.

It is possible to treat Coronavirus disease with perilla (Perilla ferutescens (L.) Britton), a medicinal plant. The current study's objective is to examine how Agrobacterium rhizogenes induces hairy roots in Perilla to produce secondary metabolites. Three replications of a completely randomized design were used for this factorial experiment. Explant with four levels hypocotyl, cotyledon, leaf, and internode as well as bacterial strain with two levels were among the factors examined (ATCC-15834 and A4). The findings showed that the interaction effect of the factors under investigation was significant for hairy root features such as day to induction, percentage of induction, and fresh and dry weight after two months. The treatment combination of cotyledon explant infected with ATCC18534 produced the highest percentage of hairy root induction, fresh weight, and dry weight compared to other treatments. By utilizing a PCR reaction to track a portion of the rolB gene, it was possible to determine that the hairy roots were transgenic because the corresponding band was not amplified in the normal or non- transformed roots. In general, the findings of the present study indicated that the bacterial strain and explant had an impact on the induction of hairy roots in Perilla, and these findings are a necessary prerequisite for hairy root culture studies to produce secondary metabolites.

Transgenic plants are important tools that can complement integrated pest management programs. One of the concerns about transgenic plants is their effects on non-target organisms such as predators and parasitoids. This effect can occur directly (lethal effects) and indirectly. Natural enemies may be affected by feeding on herbivores that feed on leaves, stems or flowers of transgenic plants. In this research, the effect of transgenic varieties of broad bean (G-faba-560, G-faba-5, G-faba-20 and G-faba-21) and also, a native variety (Qaramalek) on the biological parameters of black bean aphid, Aphis fabae Scolopi and its parasitoid, Lysiphlebus fabarum Staey was investigated in the laboratory. Results showed that mean of nymphs produced by female aphids on all transgenic cultivars was significantly reduced compared to Qaramalek cultivar, which indicated that these varieties are unfavorable for black bean aphid. It was also found that the native variety (Qaramalek) was more sensitive to A. fabae than the transgenic varieties, and the pest population decreased in transgenic varieties. On the other hand, developmental time, adult longevity, parasitism rate and emergency rate have significant differences in broad beans varieties. Because aphids fed on transgenic varieties were small in size, parasitoid did not prefer them for parasitization. As a result, in transgenic varieties, the L. fabarum was affected by population reduction and lack of A. fabae. Also, in this research, it was found that transgenic varieties of broad bean are able to reduce the population of A. fabae.

Endophytes are a large and various group of microorganisms that live in the plant tissues without any appreant disease symptoms. Endophytes play an important role in protecting the plants against the biotic and abiotic stresses. The objectives of this study were to isolate and identify the endophytic fungi of hazelnut (Corylus avellana) tree and evaluate the possible effects of the hazelnut different tissues on their communities. Healthy samples (fruit including nut, shell and involucre, leaf, stem and root) were collected from hazelnut tree of Fandoghlo forest in Ardabil, Iran. After surface sterilization, the endophytic fungi were isolated using standard culture methods and identified based on morphological characteristics. Representatives of each taxon were identified by sequencing of ITS1-5.8S-ITS2 rDNA region. Tissue colonization frequency, species isolation rate and diversity indices were calculated. In total, 79 endophytic fungal isolates were isolated from fruits (29.9%), leaves (9.1%), stems (54.5%) and roots (9.1%). The isolates belonged to the taxa of Clonostachys sp., Briansuttonomyces sp., Chaetomium sp., Colletotrichum sp., Fusarium sp., Curvularia sp., Epicoccum sp. and Biscogniauxia sp. Accordingly, 15.2% isolates (12 isolates) were not identified due to lack of sporulation. The stem and leaf of the hazelnut tree showed the highest and the lowest colonization frequency, respectively. The indices of diversity of leaf and fruit endophytes were higher than those of roots and stems. The results of thin layer chromatography analysis and polymerase chain reaction (with DBAT gene primer) showed that three isolates are probably able to produce paclitaxel.

The widely used silver nanoparticles made them readily accessible to the bacteria. The interactions between silver nanoparticles and proteins are still not fully understood. In this research, the effect of silver nanoparticles on glycerol dehydrogenase which is located in the respiratory chain of the Gluconobacter was investigated. Response Surface Methodology and a central composite design were used to evaluate the activity of enzyme and the interactions of pH and temperatures. Enzyme activity was evaluated by colorimetric method. The results showed that increasing the concentration of silver nanoparticles up to 50 mg/L decreased the activity of glycerol dehydrogenase enzyme. The inhibitory effect of nanoparticles decreased beyond this concentration. The glycerol dehydrogenase activity increased 35ºC and pH 7, but the activity decreased thereafter. The parameters of temperature, pH and nanoparticles concentration influenced the activity of glycerol dehydrogenase enzyme independently. It could be concluded that silver nanoparticles decrease the activity of glycerol dehydrogenase, which can ultimately decrease the activity of the respiratory chain and subsequently energy production in Gluconobacter. The results of this research can be used in the targeted control of glycerol dehydrogenase activity to produce dihydroxyacetone.

Apple chlorotic leaf spot virus (ACLSV) is one of the most important viruses infecting fruit trees. Preparation antibodies, ELISA kit and, biosensors are one of the applicable strategies to rapid and efficient screen a number of viral samples. To aim, the recent study was conducted to raise recombinant polyclonal antibodies to rapid detection of Apple chlorotic leaf spot virus by expressing coat protein gene in the prokaryotic system. First, the pTG19-ACLSV-CP was transformed to E. coli DH5α, the replicated plasmids were extracted and double digested was optimized by restriction enzymes, then the released fragment (Coat protein gene) was cloned into pET28 as an expression vector. Next, the expression construct (pET28a-ACLSV-CP) was transformed into E. coli strain BL21 (DE3) to express the coat protein gene. After optimization of expression the transformed cells, a protein band an approximate size of 27 kDa (22 kDa for coat protein and about 5 kDa for histidine tags) was observed at four hours after induction. The recombinant protein was purified by native methods using Ni-NTA Agarose column, then purified proteins were injected into New Zealand female rabbits in four steps as antigen. IgGs were purified from serums raised from immunized rabbits using an IgG purification kit. The efficiency of purified IgGs was approved that a 1: 1000 concentration of recombinant anti-ACLSV-CP antibodies are efficient to detect the desired antigens. The results showed the raised antibodies have enough specificity to detect Apple chlorotic leaf spot virus in gardens and seedlings.

Regarding the importance of biological control of plant pathogens, the present research was aimed to find antagonists with inhibitory effects on the causal agent of Cantaloupe Fusarium wilt, Fusarium oxysporum f. sp. melonis (FOM). At first, the inhibitory effect of four isolates of Trichoderma (including Trichoderma harzianum Tr3, T. harzianum 2, T. longibrachiatum and T. asperellum Tr14) and 52 bacteria (isolated from rhizosphere of Sophora alopecuroides( against FOM was investigated in laboratory conditions. Based on the results, three isoaltes of Trichoderma and two bacterial isolates (B1 and B2) were selected and along with Bacillus subtilis MCC0067 were assayed in green house experiments. The antagonists were applied on seed and in the soil and their potential for control of Cantaloupe Fusarium wilt and plant growth promotion were evaluated. In dual culture assay, the growth inhibition percentage of Trichoderma isoaltes varied from 62 to 75% and of bacterial isoaltes varied from 46 to 60%. In greenhouse conditions, the antagonist isolates were not able to prevent the incidence of Fusarium wilt disease on cantaloupe; but they delayed the onset of the disease and reduced disease progression. In 40 days after planting, the disease severity in T. harzianum Tr3 and T. longibrachiatum treatments was significantly lower than the others. The antagonists had no significant effect on increasing root length but they triggered an increase in root volume. Their effect on other indices was different. In general, B2 and the isolates of T. harzianum Tr3 and T. longibrachiatum treatmens were the most effective treatments in increasing growth indices and in control of disease, respectively.

Salt stress is one of the important environmental factors that adversely affects plant growth and yield. Identification of key components involved in the stress response networks is the important task in development of tolerant varieties through genetic engineering and molecular breeding programs. Here, we have performed comprehensive bioinformatics analysis to identify conserved genes and mechanisms involving in salt stress response across monocots, wheat and barley and dicot Arabidopsis. The microarray data were retrieved from GEO dataset at NCBI, which were already generated through experiments on the leaves of mentioned species under various stress conditions. Arabidopsis orthologues of salt responsive genes from wheat and barley were recognized by performing BLASTx compared with Arabidopsis protein database. We identified 208, 1336, and 4527 salt responsive genes from barley, wheat, and Arabidopsis, respectively, where 25 genes were common and considered as conserved salt responsive genes among species. The conserved genes are involved in stress signaling, ion homeostasis of ions, reactive oxygen species homeostasis, and energy, osmoprotectant mechanisms, and cell wall lignification pathways by exposing to salt stress in the three species. The theoretical gene co-expression network during signaling pathway was also presented in the salt stress condition.

Offshoot planting is the most common way of propagating date palms. Due to the limitation of the number of offshoots produced by each palm tree and the problems with their transferring and planting, the tissue culture technique is recommended for palm propagation. Tissue culture plantlets are completely similar to the mother stock. But in some cases (like as date palm cv. Barhee) this method shows somaclonal variation, such as abnormalities in pollination and fertility. Gene expression analysis can determine the molecular mechanism of somaclonal variation. Therefore, in this research, we investigated the expression of some genes affecting date palm pollination and fertilization, including Ubiquitin (UBQ), Metallothionein (MT), Phosphofructokinase (PFK), and Polyadenylate binding (PABP), in the Barhee cultivar at three stages of the inflorescence spathe growth (15, 25, and 35 cm long) with three replicates. The transcription amplification results showed that there was a significant difference in the mutual effects of spathe growth stages and propagation methods on gene expression. The expression levels of PFK and PABP were 1.6 and 1.3 times respectively, in tissue culture propagated palms comparing to offshoot propagated palms only at the initial stage of spot growth. However, the expression level of UBQ and MT genes was lower in tissue culture palms than in offshoot palms in all stages of spathe growth. We also investigated the protein network interacting with MT and found a relationship and interaction of this protein with proteins effective in male sterility and seed development.