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Asiatic citrus canker is a devastating disease resulting in drastic economic losses in citriculture worldwide. Amongst three different types of the disease, i.e. A, A* and A^w, the A* type is genetically less known. In order to comprehend the behavior of the Asiatic citrus canker A*-type strain (Xanthomonas citri subsp. citri) in the vicinity of the host cells, a targeted semi-quantitative transcript analysis approach via RT-PCR was carried out. A subset of sixteen genes, as representative of different steps involved in phytopathogencity, was analyzed on the culture medium (as uninduced) and compared with the subset isolated from the infected Mexican lime (Citrus auarntifolia L.) plants (as induced). The results showed that certain genes were up-regulated in induced condition, suggesting a putative role in bacteria-host interaction. Furthermore, the transcripts in induced condition could be classified into constitutive, early- and late-responsive genes, demonstrating their functional relevance during the host-pathogen interaction.    

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Background: Adhesion and biofilm formation are two important steps in Candida pathogenesis. The aim of the current study was to investigate the presence of bcr1 gene in Candida albicans (C. albicans) isolates from women with vaginal candidiasis and its impact on biofilm formation. Methods: We used 50 clinical isolates which confirmed C. albicans by PCR-RFLP. Then total RNA was extracted from C. albicans isolates by glass bead and lysis buffer, and cDNA was synthesized using reverse transcriptase enzyme. RT-PCR (Reverse Transcriptase PCR) was used to evaluate the expression of bcr1 gene. Biofilm formation was evaluated in 96-well microplate and then tetrazolium reduction was assayed. All data were analyzed using t-test by SPSS software. Results: Fifty clinical isolates out of 150 were confirmed as C. albicans by using PCR-RFLP method. All the isolates were resistant to fluconazole, 47/50(94%) isolates had bcr1 gene by using PCR, and 45(95.7%) out of 47 isolates, showed BCR1 expression by the RT-PCR. Isolates which harbored bcr1 gene was succeed to form a dense biofilm on microplate. Comparison of the results of the tetrazolium reduction assay on the two isolates that had BCR1expression and two isolates that had no BCR1 expression showed significant differences (p=0.014). Conclusion: According to our result, all of the isolates that had bcr1 gene expression according to RT-PCR, were also resistant to fluconazole in disk diffusion test and additionally, their adherence was higher compared to the control group. These results indicate that there is a positive relation between expression of bcr1 gene and biofilm formation.

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Aims: Hepatitis B Virus (HBV) has infected more than million hundreds of people worldwide. Hence, a high rate of morbidity and mortality caused by liver-related diseases is due to HBV infection. However, a strong and effective treatment should be based on an accurate and correct diagnostic method. Hence, the present research provided a multidimensional study comparing and analyzing patients’ molecular and serological tests results.
Materials & Methods: In this research, the HBV DNA molecular tests results were studied by examining patients’ gender, age, and HBsAg strip results.
Findings: Among the female patients (29 persons) studied in this research, 55.1% were positive for HBV DNA and HBsAg strip tests, and 17.3% were negative for both tests. Also, among the male patients  (44 persons), 65.9% were positive, and 6.8% were negative for both tests.
Conclusion: The present study results shed light on the correlation between the HBV DNA and HBsAg tests. Also, the significance of HBV DNA tests was highlighted for particular diagnostic purposes and for the differentiation and interpretation of the pathophysiological conditions of patients with hepatitis B.

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Background: Calf scours (diarrhea) in unweaned calves play a major role in economic losses of animal farming industry worldwide. The present study was conducted to investigate and interpret the presence of BRV, BVDV, and Escherichia coli K99 by molecular and serological approaches simultaneously.
Materials & Methods: A total of 73 E. coli-negative diarrheic fecal samples were collected from one-week to less than one-month-old calves of Holstein dairy cattle herds of some provinces of Iran during autumn and winter. The samples were directed to antigen detection by ELISA (Enzyme Linked ImmunoSorbent Assay), RNA extraction by semi-manual approach, and cDNA synthesis for PCR amplification.
Findings: Out of 73 calves’ diarrheic  fecal samples, 28 (38.3%) and 1 (1.36%) were positive for BRV and BVDR by ELISA, respectively. However, 31 (42.4%) samples were positive for BRV and non for BVDV by RT-PCR. The Kappa coefficient showed significant differences in BRV and BVDR detection between ELISA and RT-PCR methods. The distribution of the BRV-positive samples among bovine diarrheic calves was 80, 52.6, and 50% in Eslamshahr, Qazvin, and Hamedan, respectively.
Conclusion: ELISA and RT-PCR indicated high prevalence rate of BRV in autumn and winter, respectively. The present study results showed that positive cases detected by RT-PCR were more than those detected by ELISA. Further studies are needed to achieve a comprehensive preventive and therapeutic strategy to address  diarrhea bovine pathogens.

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Abstract:
Background: Negative sence RNA genome of Influenza A virus contains 8 segments coding for 12-14 proteins depending on strains. Genetically modified virus is caused world wide spread of a new Influenza in the human population. Developing a rapid and accurate diagnostic method to identify new species is necessary. The aim of this study was rapid detection of new species of Influenza A subtypes using specific RT-PCR based on hemagglutinin gene.
Methods: In this study 30 Nasopharynx samples of patient cultured in embryonated eggs. Then RNA was extracted, cDNA prepared and PCR was performed using specific primers designed from hemagglutinin gene. PCR products purified and sequenced.
Findings: PCR products sequences compared with Influenza A sequences obtained from the Gene Bank database. All positive isolates most closely related to the influenza reference strain. This Result showed that the specific RT-PCR used was able to amplified and detect Influenza A subtypes from clinical specimen.
Conclusion: The results of this study confirmed that PCR based on hemagglutinin gene with sequencing is a sensitive and accurate method for rapid detection of influenza A new subtypes directly from clinical specimen which is useful in preparation and production of vaccine.

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Zucchini yellow mosaic virus (ZYMV) is a potyvirus with a worldwide distribution. This virus causes serious economic losses in Iran in many cucurbits. During 2002-2003, sam-ples were collected from squash fields in Tehran Province. Five isolates (Z1, Z2, Z3, Z4 and Z5) were inoculated on 27 species of Cucurbitaceae, Chenopodiaceae, Amarantha-ceae,Solanacae, Leguminosae and Ranunculaceae. Chenopodium quinoa and C. amaranti-color showed chlorotic local lesions. Gomphrena globosa developed necrotic local lesions. Systemic symptoms were produced in the members of Cucurbitaceae and Ranunculus sardous. Z2, Z4 and Z5 caused mosaic symptoms on Phaseolus vulgaris cv. Red Kidney and P. vulgaris cv. Khorram but Z1 and Z3 caused chlorotic local lesions.Virus was puri-fied from Cucurbita pepo. Virus particles in immunoelectron microscopy were filamentous flexuous. The molecular weights of coat protein using SDS-PAGE and western blotting were estimated at 32 kDa. Reverse transcription polymerase chain reaction (RT-PCR) was performed using one primer pairs designed by Desbiez et al. An approximately 458 bp fragment was amplified with a specific primer.

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The two-spotted spider mite, Tetranychus urticae (Acari: Tetranychidae) is one of the most important pests of field crops, orchard trees and ornamentals around the world. The short life cycle, high reproductive potential, accompanied by frequent acaricide applications have caused resistance development to wide range of acaricides. In this study the susceptibility of two populations, collected from Karaj and Mahllat, was investigated against fenpyroximate. The bioassay test was carried out by using the leaf-dip method. The results showed that the LC₅₀ values for Karaj and Mahallat population were 2.1 and 92 (mg/ml), respectively. The resistance ratio was 43.8. The enzyme assay results revealed that the activity ratios of esterase in Mahallat to Karaj populations were 2.5 and 1.2 when α-NA and β-NA were used as a substrate, respectively. The activity of cytochrome P₄₅₀ in Mahallat population was 1.37 times higher than the Karaj population. There was no significant difference in glutathion S-transferase activity between the two populations. The gene expression (qRT-PCR) results showed that the expression level of CYP392A11 in Mahallat population was 3.52 times higher than Karaj population. These results suggested that esterase and cytochrome P₄₅₀ monoxygenase are probably involved in resistance of T. urticae to fenpyroximate. 

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The purpose of this study was to determine the distribution of Tobacco streak virus (TSV) in the vegetable fields of the Markazi, Qom, Lorestan and Hamadan provinces. This study was performed in 2017, and a total of 475 samples of parsley plants were collected. Using the specific antibody of the virus, the TSV infection of these samples was investigated by the immunosorbent assay through the double antibody sandwich ELISA (DAS-ELISA) method. The results of this study indicated that the TSV infection of parsley samples in Qom, Markazi, Hamedan and Lorestan provinces were 14, 18.8, 15.4 and 20.1%, respectively. Also, the phylogenetic analysis of nucleotide and amino acid sequences of the coat protein of these isolates showed that Iranian and Indian isolates could be clustered along with each other. The phylogenetic tree obtained based on nucleotide and amino acid sequences of the coat protein gene, showed that the isolates were divided into two and three clusters, respectively. Iranian isolates were clustered along with global TSV isolates and other Ilarviruses formed a separate cluster. This is the first report of TSV genetic diversity in Iran, and also the first report of TSV infection in the vegetable fields of Qom, Markazi, Hamedan and Lorestan provinces.

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Objectives: Tissue homeostasis is the result of strict regulatory mechanisms, which control self-renewal, differentiation, prevention of premature senescence and apoptosis of stem cells. Bmi-1, a Polycomb group repressor protein, represses genes that induce cellular senescence and cell death, and can contribute to cancer when improperly expressed. Material and methods: Bladder tumoral and nontumoral samples were collected from Labbafi-Nejad hospital. RNA was extracted from each sample, reverse transcribed and amplified by RT-PCR technique, using specific primers for Bmi-1 and β2-microglobolin, as an internal control. The production and distribution of Bmi-1 protein was also examined by western blotting and immunohistochemistry technique. Results: To clarify the role of Bmi-1 in bladder tumors, we examined the expression of Bmi-1 in tumoral and nontumoral samples. RT-PCR generated a 683 bp product, corresponding to the expected size of the Bmi-1 amplified region. The identity of the amplified fragment was then confirmed by direct DNA sequencing. The mean of expression of the Bmi-1 detected in tumoral tissues was significantly higher than the non-tumoral tissues and there is also a significant correlation between the mean of gene expression with stage of malignancy (p < 0.05). The expression of Bmi-1 at protein level was further confirmed by western blotting and immunohistochemistry. Conclusion: The tumor suppressor locus Cdkn2a (Ink4a/Arf locus) codes for two proteins, p16ink4a and p14arf. Ink4a and Arf are playing important roles in the retinoblastoma (pRB) and p53 pathways, respectively. Bmi-1 is a potent repressor of both pathways and hence elucidating its role in tumorigenisis is very important. Here, for the first time we are reporting the expression of Bmi-1 and its correlation with malignancy in bladder tumors.

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Objective: Inhibition of apoptosis may favor the onset and progression of cancer. Survivin is an inhibitor of apoptosis that has been considered as a potential marker for diagnostic and/or prognostic of bladder cancer. The survivin protein regulates both cell division and cell death and is overexpressed in the vast majority of human cancers. In this study, the expression pattern and potential prognostic value of survivin was assessed in Formalin-Fixed Paraffin-Embedded (FFPE) samples of bladder tumor. Materials and Methods:FFPE samples, from patients with a well-known five-year survival record, were assessed by semi-quantitative RT-PCR technique. 51 samples from 30 patients were analyzed on the basis of Survivin expression. Tissue distribution and subcellular localization of survivin protein in tumor tissues was also examined by immunohistochemistry (IHC). Results: The expression of survivin was detected in 66.6% of the samples, with an increase of expression in higher grades of tumor. Furthermore, survivin was overexpressed in 2nd and 3rd recurrences of the same patients. Also, with the increased malignancy and accordingly increased expression of surviving, the overall 5-year survival rate of patients was significantly declined (P=0.036). IHC results also localized a nuclear localization for Survivin protein in tumor tissues. Conclusion: In conclusion, we were able to detect the expression of survivin in FFPE samples of bladder tissues, at the level of mRNA and protein and find a correlation between the level of Survivin expression and the degree of malignancy of the tumors. Our findings introduce Survivin as a suitable prognostic marker for predicting the bladder tumors.

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Objective: Hepatitis C virus is the major cause of viral hepatitis and its diagnosis in suspected specimens is of great importance. The risk of transfusion- transmitted virus infection is primarily the result of failure in serological screening tests to detect recently infected donors in the pre-seroconversion window period of infection. Therefore, sensitive and accurate diagnosis of HCV prior to antibody production to reduce window period is necessary. Materials and Methods: In the present study, a sensitive and specific RT-Nested PCR method for detection of a conserved HCV 5'UTR sequence was developed. Two pairs of primers for amplification of the target sequence in two rounds of PCR were selected. The developed RT-Nested PCR assay was performed on HCV-antibody confirmed positive samples as well as negative controls and standard samples. In order to compare the results, One Step RT-PCR kit was used in this study. Results: 25 HCV-positive plasma samples whose positivity were confirmed by ELISA and Western Blot tests, also as well as 10 fold dilutions of a high viral load plasma sample obtained from a HCV-positive patient as standard samples and 25 negative control plasmas from healthy blood donors were collected and tested by this assay. In all of positive samples a 175bp band was observed on agarose gel electrophoresis, but no band could be detected in negative control plasma. Results from developed RT-PCR assay and One Step RT-PCR kit showed a good correlation. Conclusion: According to the results of this study, the developed RT-Nested PCR assay has a good sensitivity and specificity for diagnosis of HCV infection. It has the advantage of viral genome detection prior to seroconversion and can be used to detect HCV infection during window period

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Objective: In this study, a SYBR Green real-time RT-PCR assay for quantification of HIV-1 viral RNA was developed. Materials and Methods: This assay was performed based on amplification of the pol region of HIV-1 and product analysis by an ABI 7500 system. We quantified HIV-1 viral load in 26 seropositive patients by this system and the data were subsequently compared with results obtained with a reference technique represented by COBAS AMPLICOR HIV-1 Monitor test. Results: The results demonstrated that this technique could detecte up to 500 HIV-1 RNA copies/ml of plasma. The linearity of this approach was conserved over a wide range of HIV-1 copy numbers (5×102-5×109). Since no positive signal was observed in seronegative volunteers, the specificity of the test was calculated as 100%. Comparison of the results with those obtained by the reference quantification method, revealed a significant correlation between the results (R2= 0.95). Conclusion: On the basis of the most recent recorded cases for HIV-1 infection and AIDS in Iran, the prevalence of this disease is rising rapidly and the situation has been called to be alarming by national health representatives. Determination of HIV-1 viral load in plasma has been considered as the most effective single prediction tool of clinical outcome. Indeed, the development and stabilization of HIV-1 RNA assays have given physicians a unique tool for monitoring HIV-1 patients treated with antiviral drugs. In this study, we have developed a SYBR-Green Real Time RT-PCR assay for quantitative analysis of HIV-1 in infected patients. Since a synthetic RNA standard was used in this assay, the upper limit of detection was detected to be higher than the standard test (5×10 9 versus 7.5×10 5). This can be important in patients with acute high viral load infections. Reproducibility was assessed by Intra assay and Inter assay analysis. Coefficient of variations Ct, in reproducibility tests for Intra assay and Inter assay variability were less than

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Beet virus Q (BVQ) is a soilborne pomovirus (family Virgaviridae) associated with rhizomania syndrome in sugar beet. In the present study, BVQ was investigated in sugar beet farms of 12 provinces in Iran by RT-PCR. Thirty-five out of 214 root samples resulted in a positive reaction to BVQ (16.3%). Moreover, 501-bp- long fragments of the coat protein gene of 11 Iranian isolates were purified, cloned, and sequenced. Phylogenetic analysis using the 501 bp fragment of 17 BVQ isolates (11 from this study and six retrieved from GenBank) showed that all isolates clustered in two main groups. Iranian isolates belonged to group I alongside isolates from France (AJ810289) and Germany (AJ223597). Iranian isolates shared 98.80–100% nucleotide sequence identity (98.19–100% amino acid identity) and 97.21–99.60% nucleotide sequence identity (96.99–99.40% amino acid identity) with the corresponding sequence of six other BVQ isolates available in the GenBank. Iranian isolates displayed the highest nucleotide and amino acid sequence identities of 98.80-99.60% and 98.19-99.40%, respectively, with the French isolate FP71 (AJ810289). To our knowledge, this is the first molecular characterization of BVQ in Iran. This information can be used in plant breeding to obtain virus-resistant plants.

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Objective: Despite sensitive antibody based blood donor screening, infection can be transmitted during window period. Therefore sensetive methods bosed on nucleic acid tests (NATs) have been considered. The aim of this project was to design a more sensitive method for detection of PCR products and diagnosis of HIV-1/HCV co-infection accurately. Materials and Methods: After designing specific primers and probes, the Multiplex RT-PCR method was optimized and the PCR products were labeled with Digoxigenin. The PCR product was denatured under alkaline condition and was hybridized with the specific probe that had a biotin at 5' end, and then was added to streptavidin coated wells. After washing an antibody against DIG, conjugated with alkaline phosphates enzyme was used, following second washing, the substrate (ABTS) solution was added to each reaction well. Development of green color shows the positive where as no color shows negative results. Results: 35 samples were tested with the developed method including 27 positive samples, 8 confirmed negative and 4 standard panels. False negative or positive reactions were not observed. Conclusion: This method had acceptable sensitivity and specificity for detecting HCV and HIV-1 infections during window period, also the method can be quantified which can be used for the flow-up and treatment of patients. In addition to the very high sensetivity of the test, it is cost effective and takes less time to performe.

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Soybean mosaic virus (SMV) which belongs to the virus family Potyviridae, causes a disease in soybean that is present in soybean-growing areas of the world, and is widely distributed in northern Iran. Detection of SMV is very important for disease management. In the present study several serological and molecular (nucleic acid- based) methods of rapid virus detection were compared. Serological studies including DAS- ELISA, DAC-ELISA, TPIA and DIBA were optimized and compared to identify the virus by using a polyclonal antibody. Among the serological methods, TPIA and DIBA are simple and TPIA is rapidly and easily applicable in the field. However, TPIA was found to be preferable. TPIA is time-saving, not requiring conventional sap extraction and also nitrocellulose membranes used for printing can be used in the field and stored for a long time or transported to other laboratory to be processed. RT-PCR and Immunocapture RT-PCR (IC-RT-PCR) were performed as molecular methods for detecting SMV using a pair of primers designed to amplify a fragment in the coding region of the SMV coat protein. To extract total RNA for RT-PCR, two methods including RNAWIZ and phenol-chloroform were used. A part of the coat protein genome of SMV was converted to cDNA using a reverse transcription (RT) reaction. For IC-RT-PCR method, virus partial purification was carried out by solid-phase (0.2 ml microfuge tube) adsorbed polyclonal antibody, and then the RT reaction was carried out in the tube. In both methods cDNAs were amplified by PCR. Both methods amplified the expected fragment in virus-infected plants. Whereas RT-PCR requires total RNA extraction, ICRT- PCR do not have total RNA extraction problems. Our findings suggest that TPIA and IC- RT- PCR can be routinely used for SMV detection, with high efficiency.

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The incidence of grapevine virus A (GVA) is reported from almost all of the major grapevine growing regions in Iran. Grapevine is vegetatively propagated by rooting of cuttings or grafting. In such plants, viral diseases are transmitted from stock plants to the progeny. Therefore, the control of grapevine viruses can be achieved primarily through production of healthy stock plants. In the present research, cryotherapy and electrotherapy were employed for elimination of GVA from naturally infected vine (Vitis vinifera L. cv Black) and their efficiency was compared. In cryotherapy, 59% of the shoot tips survived and regenerated into whole plants, of which 42% were free of GVA detected by RT-PCR. In the electrotherapy, the effects of electric current value and treatment duration were investigated on plant survival and virus elimination. The best results were obtained by using 30 milliamps (mA) for 15 minutes. With this treatment, survival and virus-free frequencies were about 62% and 40%, respectively. This is the first report of electrotherapy of grapevine shoot tips as a potential tool for GVA elimination. The results showed that cryotherapy was a more efficient and convenient protocol than electrotherapy for elimination of GVA from infected grapevine.

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Alfalfa crops were surveyed for the incidence of alfalfa mosaic virus (AMV), cucumber mosaic virus (CMV), peanut stunt virus (PSV), bean leaf roll virus (BLRV), bean yellow mosaic virus (BYMV) and bean common mosaic virus (BCMV) in the major growing areas in the southeast and central regions of Iran. Samples were collected between May 2009 and March 2011 and analyzed for viral infection initially by enzyme-linked immunosorbent assay (ELISA) followed by RT-PCR using capsid protein gene specific primers. In total, 634 symptomatic leaf samples were collected in four southeastern and central provinces of Iran representing 20 regions. Our results revealed a high incidence of AMV over a wide geographical area. AMV and BLRV were identified in most regions, whereas BYMV was found only in Yazd Province. PSV was detected in three regions, but not in Sistan- Balouchestan and Hormozgan Provinces. The highest incidence of viral infection amongst the surveyed provinces was recorded in Kerman (66.8%), followed by Yazd (39%), Sistan and Balouchestan (20.8 %), and Hormozgan (4.5%). AMV, BLRV, PSV and BYMV were present in 23.3%, 12%, 0.70% and 0.28% of the samples, respectively. CMV and BCMV were not detected in any surveyed region. Multiple virus infections were recorded in 42 samples. This is the first report on the detected occurrence of BLRV, PSV and BYMV in alfalfa in the southeast and central regions of Iran.

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Objective: Common variable immunodeficiency (CVID) is one of the most frequent cases of primary immunodeficiency. It is likely that this heterogeneous disease is caused by several distinct genetic disorders. The activation-induced cytidine deaminase (AID) enzyme is involved in class switching, somatic hypermutation (SHM) and processes associated with gene conversion in the germinal center. In order to clarify the possible role of AID in the pathogenesis of CVID, we have studied the AID gene expression in CVID patients. Methods: Peripheral blood mononuclear cells (PBMC) from 21 patients and healthy controls were isolated. The isolated cells were stimulated by CD40L and IL-4 to induce AID gene expression. After five days, total RNA from the stimulated cells was extracted and AID gene expression was investigated by RT-PCR. Results: RT-PCR results showed that after stimulation by CD-40L and IL-4, the AID gene was expressed in all of the samples. The control samples were also positive for AID gene mRNA expression. Conclusion: In this investigation we studied the expression of AID gene in CVID patients' B lymphocytes for the first time. Regards to our results which showed that all patients normally expressed the AID gene mRNA and considering that one of the main problems in a number of CVID patients is disorders in phenomena related to the germinal center and complete differentiation of B lymphocytes, it can be concluded that possible defects in other molecules involved in class switching is responsible for this disease. Understanding the various genetic defects responsible for this heterogeneous disease could lead to its division into more homogenous subtypes with distinct therapeutic strategies, so further investigations is recommended.

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In this study, accumulation of H₂O₂, malondialdehyde (MDA) (as cold-induced oxidative stress indicators), the transcript levels of dehydrin and beta-glucosidase genes (involved in metabolic responses) was evaluated in chickpea cv. Jam, using qRT-PCR during control, cold acclimation (CA), cold stress (CS), recovery, and freezing phases. Results showed the existence of wide range of genetic capacity in the cultivar to increase cold tolerance when environmental conditions change. Significant increase in H₂O₂ and MDA content during CA phase indicated that seedlings perceived cold signaling that resulted in remarkable increase in the transcript levels of dehydrin and beta-glucosidase genes as part of defense responses of plants. Balancing the expression of these genes and oxidative stress indicators showed the interplay between two major defense and injury pathways. During freezing phase, the higher transcript levels of these genes in acclimated plants compared to non-acclimated plants showed a more active role for plant cells. An incapability of defense machine in non-acclimated plants was a limiting factor determining the low potential of chickpea plants to freezing phase. It was suggested that adjustment and metabolic alterations like dehydrin and beta-glucosidase genes, especially after CA phase and, thereby, decrease in oxidative stress indicators, could be a reason for relative cold tolerance in chickpea.