The Hyrcanian forests have a remarkable variety of moss species which research on their taxonomy is of great importance. Since Forsstroemia remotifolia, Homalia besseri and Pseudoleskeella catenulata are exclusive and native mosses species of Hyrcanian forests, so in the current study, fourteen populations from three provinces in the north of Iran including Golestan, Mazandaran and Guilan were collected at the same altitudes in autumn 2017. In order to reveal the relationships among these species and populations, a cluster analysis based on numerical taxonomy and zymogram patterns of peroxidases and superoxide dismutase with Euclidean distances was performed. Numerical taxonomy analysis showed plant length, marginal laminal cell length and middle laminal cell length are appropriate traits to distinguish the species of F. remotifolia, H. besseri and P. catenulata from each other as well as their populations. The zymogram analysis showed genetic variability among species and also within populations of F. remotifolia, H. besseri and P. catenulata. Accordingly, the isozyme banding pattern of peroxidases showed a total of 6, 7 and 5 bands for F. remotifolia, H. besseri and P. catenulata, respectively. However, 4 isozyme bands were detected for superoxide dismutase for all three species. Furthermore, the morphological analyses in some populations was not matched with the isoenzyme banding pattern of enzymes in the current study. In conclusion, the biosystematics studies (morphometry and zymogram patterns of peroxidase and superoxide dismutase) indicate the close relationship between F. remotifolia and P. catenulata.
Algae are an enormous biological group, forming 50% of photosynthetic organisms. In addition to having chlorophyll for the absorption of light photons, algae are rich in red, orange, and yellow carotenoids, which mainly protect cells against harmful radiation and free radicals. Moreover, these organisms have phycobiliproteins (red and blue pigments), which are involved in capturing and passing light energy to chlorophylls during photosynthesis and have a wide range of antioxidant properties. Algae also play a key role in substituting artificial colorants with natural colorants due to the adverse side-effects of chemical colorants, especially since natural colors are commonly used by individuals and various industries. Recently, algal pigments have been widely used in medical, nutraceutical, cosmeceutical, and pharmaceutical industries owing to their antioxidant, antidiabetic, anti-obesity, anti-inflammatory, antiaging, antimalarial, and neuroprotective properties. The growing demand for algal bioproducts highlights the importance of evaluating the trends influential factors in their production. The current review study provided an introduction to algal pigment classification, distribution, function, application, and biological production. In addition, we have discussed crucial biochemical pathways, enzymes, and gene/biotechnological modifications, such as transformation and expression regulation, which noticeably affect the metabolism of their sink and source.
Chloride is considered as the most important micronutrient in tobacco production. But excessive amounts of chloride accumulation in leaves of tobacco has many adverse effects on the tobacco quality, such as burning capacity. Identification of quantitative trait loci (QTL) involved in chloride accumulation would be beneficial for the improvement of tobacco quality. The objective of this study was to identify genomic regions associated with chloride accumulation by using a mapping population consists of 225 F2:3 families derived from hybridization between ‘Basma Seres 31’ and ‘SPT 406’ lines. Linkage map was constructed with 23 microsatellite (SSR) and 29 inter simple sequence repeat (ISSR) polymorphic markers which covered 570.8 cM of the tobacco genome. Thirty-four of these polymorphic markers were mapped to 7 linkage groups. Distance between two adjacent markers was 17.3 cM. Composite interval mapping (CIM) was used to identify QTLs controlling chloride accumulation. One QTL for chloride accumulation was identified on linkage group 3. The percentage of phenotypic variance (R2) explained by this QTL was 12.7%. A significant association was not found between ISSR markers and chloride accumulation. The outcome of present effort can be a basis for marker aided selection (MAS) in tobacco breeding programs.
Buxus hyrcana is one of the endangered and evergreen species of the Hyrcanian forests in Iran. The genetic diversity assessment is an essential step towards the conservation of this species. High-quality DNA is required for molecular markers analysis; therefore we compared different DNA extraction methods on leaf samples of B. hyrcana. The quantity and quality of the extracted DNAs were evaluated by spectrophotometry and gel electrophoresis. Also, ISSR (Inter simple sequence repeats) markers were applied on the extracted DNAs to compare their quality for PCR amplification. Results showed that quantity, quality, and PCR efficiency and reproducibility were different for DNA extracted using different methods. The quality of the DNA at the absorbance A260/A280 ratio ranged from 1.02 to 1.97. The highest concentration of DNA measured by spectrophotometry belonged to the Cota-Sanchez extraction protocol (695.3 ng/l) and the lowest value was obtained with Edward4 method (204.7 ng/l). The modified Onate method (Onate2) was extracted the highest DNA concentration by comparison of brightness against the DNA ladder. Among the different extraction methods, the good quality and quantity were obtained in extracted DNA for Doyle and Doyle, Cota-Sanchez and modified Onate protocols; the latter method (Onate2) created both good quality and quantity of extracted DNA and operated effectively in terms of cost and time. Onate2 had the best amplification results with ISSR primers.
Garlic rust is one of the most important diseases of garlic worldwide, which hardly can be controlled by applying fungicides while the weather condition goes on the favor of the disease progress. The NBS-profiling approach is one of the effective methods for separating the replicated parts of resistance gene analogues (RGA). In this study, 12 primers (NBS-LRR) were used on 16 Iranian garlic clones. Out of 499 scored marker sites in the range of 100 to 800 bp for NBS, from which 477 sites were multi-faceted (95.59 percent). The highest number of marker sites was for the primer combination NBS1-AluI and the lowest was for the primer combination NBS7-RsaI. The highest polymorphism occurred with combination NBS2-AluI and NBS1-AluI with 70 alleles and the lowest polymorphic composition occurred in NBS7- RsaI combination.The results of cluster analysis using UPGMA divided the clones into eight separate groups. This study showed that there is a significant diversity in the homologous of resistance genes in the Iranian garlic clones, which can be exploited in plant breeding programs. In addition, the results indicated that the NBS profiling technique could be used as a method for investigating the diversity of resistance genes in various plant species, including garlic. The use of NBS-profiling technique to study the diversity of resistance genes in garlic clones for the first time in the world was evaluated in this study.
Silver nanoparticles are being extensively used in a broad range of applications in our daily routine life. In the present study, it was investigated if citric acid (CA) and hydrogen sulfide (H2S) can mitigate adverse effects of silver nanoparticles (AgNPs) in green bean plants. Green bean seedlings were applied with AgNPs either through soil drenching or foliar spray and were then treated with different concentrations of citric acid and NaHS, as H2S donor. Results indicated that AgNPs induced several stresses in green bean plants. Concomitant foliar and soil appliaction of nanoparticles caused adverse effects on photosynthetic pigments and reduced carotenoid and protein contents, while increasing H2O2 content and superoxide dismutase (SOD), and ascorbate peroxidase (APX) activities. It was revealed that citric acid and H2S application significantly alleviated adverse effects of AgNPs. In the plants challenged with AgNPs, the highest rates of catalase (CAT), glutathione S-transferase (GSTs), and malondialdehyde (MDA) activities were recorded, while these parameters were reduced when plants were also treated with H2S. Application of 1.5 g/L of citric acid caused sharp decreases in CAT, GST and MDA activities. Among the treatments, the highest levels of APX, SOD, and anthocyanin were observed in the plants treated with AgNPs trough both foliar and soil drench method without citric acid and H2S treatment. The findings of the present study would increase our knowledge of the interaction of plants with heavy metals and would be useful for designing sophisticated methods for reducing the damages in the stressed plants.
Sclerotinia stem rot disease caused by Sclerotinia sclerotiorum is one of the most important diseases of sunflower. Telomeres are nucleoprotein structures at the ends of chromosomes that are essential for maintaining the integrity of the genome. The aim of this study was to determine the alteration of telomerase enzyme gene (tert) expression under fungal infection stress. The expression of tert gene in both susceptible (SDR19) and resistant (LC1064-C) genotypes of sunflower was evaluated by qRT-PCR after infection with A37 isolate of S. sclerotiorum. The results showed significant and drastic decreased levels of tert expression in both susceptible and resistant genotypes of sunflower, immediately after fungal infection. This depletion followed by mild variation when infection continued for more hours, which was more constant in resistant line, compared with the susceptible one. In conclusion, the expression of tertgene in sunflower is downregulated in response to Sclerotinia rot disease.
One of the most effective biotechnological techniques for producing plant metabolites is the hairy roots (HRs) culture system. HRs are genetically and biologically stable and able to produce secondary metabolites in a short time. Ficus carica L. is one of the most important plant sources of valuable medicinal compounds, especially polyphenolic compounds. The aim of this study was to investigate the growth and morphological features of HRs, methyl jasmonate (MeJA) elicitation and plant regeneration potential of HRs induced by different strains of Agrobacterium rhizogenes on Ficus carica cv. Siah. Four bacterial strains (A4, A7, A13 and ATCC 15834) were used for HR induction in leaf and shoot samples. The MS medium containing 2 mg/l of 2,4-D in combination with 1 mg/l of TDZ or BAP was used to induce callus from HRs, and then the callus regeneration ability was evaluated in MS medium containing TDZ and NAA. Depending on explant type and bacterial strain, the roots were induced directly or indirectly (via callus formation) from the wound sites, and exhibited different morphology. The HRs showed high levels of phenolic compounds. A 4-day elicitation with MeJA, in dependence on the concentration, enhanced the phenolic capacity and antioxidant capacity of HRs. The calli obtained from HRs showed root (70-80%) and bud (23.33%) regeneration potential. The current study described that the HR culture systems, in addition to providing the possibility of plant regeneration from transgenic roots, could be a promising in vitro technique for high production of secondary metabolites through elicitation.
In the present experiment, the in vitro callus production from tea leaf explants was induced on three different basal media. Then, the efficiency of tea callus proliferation was enhanced through application of thiourea to the proliferation medium. Furthermore, the caffeine and Epigallocatechin Gallate content of the callus and leaf tissues were estimated through HPLC method. The calli mass volume in WPM (woody plant medium) supplemented with thiourea (either 0.1 or 1.0 mM) was significantly higher than other basal media. The callus tissue collected from media enriched with thiourea had significantly more caffeine content but the level of Epigallocatechin Gallate was not statistically affected by thiourea treatments. The amount of these two compounds was also estimated in young and old leaves of the tea mother plants and it was found that the callus tissue had little amount of caffeine and Epigallocatechin Gallate as compared to maternal tissues. It is concluded that, in species with low rate callus proliferation, addition of thiourea to the medium may be followed as an effective and low cost option for callus proliferation improvement.
Low temperature is a major abiotic stress which can significantly affect the grape production. microRNAs play an important role in the control of plant development and response to adverse environmental conditions. Although miRNAs and their targets have been identified in several Vitis species, their participation during cold accumulation remains largely unknown. One such microRNA is miR395, which is conserved and regulates sulfate assimilation and distribution in plants. In this study, the possible role of miR395 in cold stress response was investigated. Identification of target genes, gene ontology and biological system analysis were performed to identify the major networks in which this miRNA is involved. Finally, the effects of gradual chilling and also a shock chilling on the expression of miR395 were investigated. In total, five target genes were identified, which all of them are targeted by miR395s a to m, whereas of the five target genes, only one is identified by miR395n as a target. Three of these genes, including ATP sulfurylase, sat-1 and, LAST3-like are involved in the control of sulfur metabolism and transport. Pathway analysis showed that miR395 was involved in response to cold stress in grape through cellular response to sulfate and phosphate deficiency. Based on RT-PCR results, contrary expression patterns of miR395 under gradual (up-regulated) and shock chilling stress (down-regulated) were observed. The changes of sulfate assimilation process would influence the formation of sulfur-containing antioxidant compounds. These results provide an insight into the regulatory roles of miR395 in response to low-temperature stress in V. viniferae.
Regarding high potential of green plants for development of recombinant vaccines, this research was conducted to evaluate expression of a novel recombinant vaccines against Foot and Mouth Disease (FMDV) in tobacco plant. For this purpose, a synthetic gene encoding 129-169 amino acids of foot and mouth disease virus capsid protein VP1 was transferred to tobacco plant via Agrobacterium-mediated genetic transformation. Considering codon usage optimized for tobacco, ribosome binding site and endoplasmic reticulum signal peptide were included in the synthetic gene to enhance expression level. Expression of the synthetic gene in tobacco seedlings was analyzed at transcription and translation levels and production of recombinant protein was quantified. Moreover, an in vivo immunization assay was carried out to verify immunogenicity of the expressed peptide in model animals. Results showed that expression of the recombinant protein in two lines of transgenic plants was a high as 0.65% and 0.72% of total soluble protein. The recombinant protein was able to induce immunogenic response when parenterally administered in rabbit.
Grain and cooking qualities in rice are measured by several physical and chemical traits, each of them controlled by several genes. Molecular markers have become fast and reliable tools for screening genotypes for grain quality. However, As different populations may carry different genes, the efficacy of previously developed markers in new populations should be tested. In order to assess the effectiveness of molecular markers in predicting grain quality in Iranian rice genotypes, a total of 38 genotypes from three different backgrounds were fingerprinted by 9 grain quality specific molecular markers and 10 laboratory traits. A total of 31 alleles were detected with an average of 3.1 alleles per locus and the polymorphic information content values ranged from 0.245 to 0.74. Cluster analysis based on molecular markers divided the rice genotypes into three major clusters and effectively differentiated between various genotypes. However, the dendrogram based on the common set of qualitative traits didn't succeed in discriminating between original groups. There were significant associations between molecular markers and quality traits except for milling factor. However, these associations weren't necessarility specific to their supposed traits. Therefore, association of markers developed in other rice populations, specially those developed in Japonioca populations should be tested prior to application in Iranian rice marker assisted breeding programs.
Hypericum perforatum is a medicinal plant which Hypericin, Hyperforin and phenolic compounds are its active secondary metabolites. Hairy root induction by Agrobacterium rhizogenes in this plant is difficult and has low efficiency. In the present study two inoculation methods, immersion in bacterial suspension and direct injection of A. rhizogenes has been compared. For this purpose, the best conditions for H. perforatum hairy root induction including A. rhizogenes strains (A4, LBA9402, NCPPB2656), plant explants (Stem, Apical bud, leaves), co-cultivation media (MS, ½MS, B5, and ½B5) and Acetosyringone (AS) concentration (0 and 100 µM) were specified and used for comparative analysis. It was found that strain A4, Stem explants, ½MS co-cultivation medium without AS constitute the best conditions for hairy root induction of H. perforatum. Transgenic nature of the potential hairy roots was confirmed using PCR and specific rolB and rolC genes primers. The results showed that the efficiency of applying direct injection method is four times higher than immersion in bacterial suspension in H. perforatum hairy root induction. In general, the results indicate that direct injection can be the method of choice to successful hairy root induction in H. perforatum.
Agrobacterium-mediated gene transfer method is one of the used methods for genetic transformation in the plant regeneration program. Transformation efficiency can be optimized depending on the strain of bacteria, the genotype of plant and conditions of growth. In this study, the gfp gene was transferred into sugar beet, tobacco, and soybean by Agrobacterium rhizogenes strain AR15834. The effects of bacterial concentrations, antibiotic concentrations and the types of explants and genotypes on the gene transfer efficiency and transgenic hairy roots production were investigated. The explants were inoculated with the bacteria at the adjusted concentrations and two days after the transformation, the explants were transferred to a solid MS medium containing different concentrations of kanamycin antibiotic. According to the results and the examined factors, the optimal conditions to achieving of the maximum production of transgenic hairy roots included bacterial concentration with OD600 = 0.2, cotyledon explant, 50 mg/L kanamycin concentration and Djakel genotype for soybean; bacterial concentration with OD600 = 0.2, leaf with petiole explant and SBSI004 genotype for sugar beet, and bacterial concentration with OD600nm = 0.8 and 100 mg/L kanamycin concentration for tobacco.
High-molecular weight (HMW (glutenin subunits are encoded by the Glu-1 loci (Glu-A1, Glu-B1 and Glu-D1 (on the long arms of chromosomes 1A, 1B and 1D. In the present study, we constructed genetic linkage map of Glu-D1and then investigated the allelic variation of HMW glutenin subunits at Glu-A1 and Glu-D1 gene loci in 30 Iranian genotypes using Functional markers. Glu-D1 was located at 50.8 cM on chromosome 1D and it was tightly linked to wPt-3743 marker (>1 cM). QTL analysis using composite interval mapping detected one significant QTL for grain yield (GY) on chromosome 1D. This QTL (QYld.abrii-1D) was located between wPt-3743 and Glu-D1 gene locus. Allelic variation of HMW glutenin subunits showed the most frequent alleles were the null allele at Glu-A1and Dx2+Dy12 alleles at Glu-D1loci. The frequency of Null alleles or 1 and 2* were 40% and 60% respectively. Only 9 genotypes included allelic combination of Dx5+ Dy10 and the rest of genotypes had Dx2+Dy12 in the Glu-D1 locus. According to the Nei's genetic diversity index, alleles at Glu-A1 locus have more dispersion in genotypes compared to Glu-D1 locus. The cluster analysis of data based on the Simple Matching coefficient and UPGMC methods, classified the genotypes into four groups. Six genotypes including: Bezostaya, Tajan, Navid, Karaj1, Neyshabour, and Golestan had Ax2* and Dx5+ Dy10 subunits at Glu-A1 and Glu-D1 gene loci. Identification of genotypes with suitable allelic combinations can be used in breeding programs, especially in hybridization.
To improve Agrobacterium-mediated transformation of tobacco, factors influencing gene delivery, including genotype of the plant, bacterial strain, and Agrobacterium transformation procedure, were tested via direct somatic embryogenesis. Leaf tissue of three different tobacco genotypes (Nicotiana tabacum L. cvs. Samsun, and Xanthi, and N. benthamiana) were used as explant. Leaf explants were transformed using three Agrobacterium tumefaciens strains (EHA105, GV3101, and LBA4404) harboring the binary vector pCAMBIA1304 using three different types of transformation methods as named Agro-inoculation, Agro-infection and Agro-injection. Selection of hygromycin resistant shoots was conducted on MS medium containing 3.0 mgL-1 BAP and 0.2 mgL-1 IAA, 250 mgL-1 cefotaxime and 30 mgL-1 hygromycin. Hygromycin resistant shoots were then rooted on MS medium supplemented with 250 mgL-1 cefotaxime and 15 mgL-1 hygromycin. The results indicated that A. tumefaciens strain LBA4404 was more effective in gene delivery than EHA105 and GV3101 and Agro-infection method proved to be significantly better than two other methods. The highest transformation rate was obtained with the Agrobacterium strain LBA4404 and Agro-infection method with approximately 72.80%, 84.57%, and 93.33% for N. benthamiana, Samsun and Xanthi, respectively. Histochemical GUS assay confirmed the expression of gusA gene in putatively transformed plantlets. PCR and RT-PCR analysis using gene-specific primers confirmed the integration of the gusA and hpt genes and the expression of the gusA and hpt genes, respectively. Furthermore, Southern blot analysis confirmed stable integration of the gusA gene in selected T0 transformants.
Precise investigation of genetic diversity by means of novel molecular tools has made it possible to identify the superior genotypes among various male and female pistachio populations. Cytogenetic studies have shed light on the possible presence of distinct sex chromosomes in male and female genotypes. In this study, 22 start codon targeted (SCoT) primers were used to investigate the genetic diversity of 22 male genotypes and 22 female cultivars of pistachio. A total of 434 loci were produced that 339 loci were polymorphism. The average value of polymorphic information content (PIC), marker index (MI), and resolving power (Rp), ranged from minimum 10, 0.5, and 1, to maximum 31, 11.40, and 17.86% subsequently. The genetic similarity between genotypes, were calculated using Jaccard's coefficient, ranged from 35 to 66%. The cluster analysis divided pistachio genotypes into six groups, and could efficiently differentiate the male and female genotypes. Analysis of molecular variance (AMOVA) classified the total diversity into intra- and inter- population diversities with a high genetic variation (92%) within populations. This study reveals that SCoT marker is a useful and valuable molecular tool to separate male and female pistachios and to determine the genetic diversity among the populations.
In order to identify yield and yield component QTLs under control and salt-stress conditions, a population of 254 recombinant inbred lines (RILs), derived from a cross between two bread wheat cultivars, (Roshan / Sabalan), was assessed. Parents and their 254 recombinant inbred lines (RILs) were evaluated in an alpha-lattice design with two replications in two control and saline environments of Yazd in 2011-2012 cropping season. Yield and yield-related traits were evaluated at harvest time. The genotyping was carried out using SSR and DArT markers. A, B and D genomes were covered by 411.8, 620.4 and 67.5 cM, respectively. Also, a total of 48 QTLs were detected on 11 chromosomes for grain yield, biological yield, harvest index, thousand-kernel weight, grain number per spike, spike weight and spikelet number per spike. Roshan (salt tolerance) alleles were associated with an increase yield under saline conditions. SSR markers including gwm146, gwm577, gwm249 (on chromosomes 2A and 7B) were tightly associated with different QTLs. The major effect QTLs were located on chromosomes 1A and 7B for grain yield, harvest index and spike weight, which were explained 10.2%, 12.98% and 29 % of the total phenotypic variance, respectively. These QTLs and markers could be suitable for marker-assisted selection and gene stacking techniques. Moreover, co-located QTLs were detected on chromosome 2B for evaluated traits.
The present research aims to study the association and allelic diversity of linked microsatellite markers to grain quality QTLs of 84 exotic rice genotypes. To this end, 9 microsatellite markers (RM540, RM539, RM587, RM527, RM216, RM467, RM3188, RM246, RM5461) were used in which a total of 61 alleles were identified with a mean of 6 alleles per locus. The polymorphism information content (PIC) varied from 0.542 (RM540) to 0.812 (RM3188) for SSR markers. Cluster analysis was performed using UPGMA method and genotypes were divided into five groups. Furthermore, based on regression analysis, for rice grain quality properties in flooding conditions as long as drought stresses, 10 alleles were identified. Of these, four alleles with gelatinization temperature, an allele with protein content under flooding conditions, and three alleles with protein content and three alleles with gelatinization temperature were related under drought stress. It should be noted that the RM216-C and RM5461-D alleles were commonly identified in several traits. The presence of common markers for traits is probably due to the consistency of chromosomal locus controlling these traits or pleiotropy. The results of this study may imply that the important identified alleles for example RM216-A for gelatinization temperature (R2=30.1 %) can be used in rice quality improvement programs.
Salinity is one of the most important limitation factors in development of agricultural products. Cotton has a relative tolerance to salinity; however, salinity reduces its growth during germination and seedling stages. In this research, split-factorial design of time based on randomized complete block design with 3 replications was used. The real-time PCR results for, root, stem, and leaves of 14-day cotton seedlings of tolerant (Sepid) and sensitive (Thermus14) cotton cultivars with salinity levels from 0 to 16 ds.m-1 were analyzed at three time points, namely 0, 7 and 14 days after salinity stress. Selected genes for Real Time PCR reaction in current study were selected using Cytoscape 3.3.0 software. Results showed that the selected genes GhERF2, GhMPK2, GhCIPK6, GbRLK, GhNHX1, GhGST, GhTPS1 and Gh14-3-3 have positively responded to salinity stress and their expression in the root was higher than in stem and leaf. Moreover, the expression of tolerant genotype (Sepid) was higher than the sensitive cultivar (Thermus 14) one, however, a slight increase in sensitive genotypes was observed in a number of genes (GhERF2 and GhGST) 14 days after starting the stress treatment.
