مجله بیوتکنولوژی کشاورزی
سال چهاردهم شماره 3 (پیاپی 48، پاییز 1401)

One of the most important methods for increasing the production of secondary metabolites is the use of elicitors in plant cell culture. A biopolymer made from D-glucosamine units, chitosan can be found in the cell walls of fungi and in the exoskeletons of arthropods. Plants respond to chitosan treatment by generating defense responses, increasing antioxidant enzyme activity, accumulating phenolic compounds, and releasing flavonoids. In the current study, chitosan was used as an elicitor to induce the production of phenolic acids in Lactuca undulata cells suspended in liquid medium.

First, cell suspension culture was prepared from 45 days old callus derived from leaf explants of Lactuca undulata on ½ MS medium supplemented with 0.1 and 1 mgL-1 2,4-D and Kin. The effect of different concentrations of chitosan (0, 50, 100 and 200 mgL-1) on cell suspension was evaluated during 24, 48 and 72 hours. After harvesting samples, the percentage of cell viability, total phenols and flavonoids, chicoric acid, chlorogenic acid and caffeic acid contents, as well as phenylalanine-ammonia lyase (PAL) activity and lipid peroxidation were measured.

In the present study, we found that the concentration and duration of chitosan treatment affect the production of phenolic compounds (including chicoric acid) in Lactuca undulata cell suspension culture. After 24 hours of treatment with 50 mgL-1 chitosan, chicoric acid levels had increased 2.8-fold compared to the control. After 24 and 48 hours of treatment with 200 mgL-1 chitosan, the highest levels of chlorogenic and caffeic acids were observed. Furthermore, the present study found the chitosan treatment resulted in an increase in the amount of phenol and total flavonoids as well as an increase in PAL enzyme activity. Chitosan induces lipid peroxidation and reduces cell viability in high concentrations, indicating a negative effect.

The present study found that low concentrations of chitosan could induce chicoric acid production in Lactuca undulata cell suspension cultures, which can be utilized in the pharmaceutical industry as a new method in the production of chicoric acid and its derivatives.

the first step in understanding a species genome is to study the number and shape of its chromosomes. determining the relationship between species of a genus, traits such as chromosome morphology, absolute chromosome size, diversity in coloring, centromere  position, chromosome base number and number of satellites must be considered. The aim of this study was to investigate cytogenetic diversity and to  determine the relationship between native garlic ecotypes of Iran (Shahroud, Bojnurd, Mashhad, Birjand, Talish) with foreign specimens (originated from Turkmenistan, Azerbaijan, Tajikistan) and to prepare genome analysis based on chromosomal information.

From the mitotic cells in the metaphase stage, which were prepared from the terminal meristem of the root and stained with acetoorcein, five suitable metaphase cells were selected and the length of short and long arms and the total length of chromosomes were measured using Karyotype Analysis Software (ver.1.5). including calculated. Data were analyzed by JMP8 statistical software in  an unbalanced completely randomized design. Mean comparisons were performed by Duncantest . To classify ecotypes, based on all chromosomal parameters, cluster analysis was performed using Ward method.

The results showed that the basic number of chromosoms in Turkmenistan and Bojnurd ecotypes was x=7, 2n=2x=14 and in other ecotypes x=8, 2n=2x=16. short arm length, long arm length, total chromosome length was significantly different (P≤0.01) between  ecotypes. Cluster analysis divided the ecotypes in two groups. Minimum Euclidean distance observed between Azerbaijan and Talish ecotypes, the smallest chromosome belonged to Mashhad and the largest chromosomes belonged to Shahroud. The most symmetric karyotype was Azerbaijan and the most asymmetric karyotype was Shahroud ecotype.

The results showed that the differences in the number of chromosomes could be explained by Robertsonian translocations. It seems that the ecotypes with 2n=2x=14 chromosomes had more antiquity, and the ecotypes with 16 chromosomes originated from them. Considering that Bojnourd ecotype with 14 chromosomes this region could be possibly in troduced as the oldest origin or nuclear center of variation for garlic in Iran. Chromosomes also differ in the size and location of the centromere, which is due to chromosomal breakdown and the formation of a new structure in their reconnections. This study revealed which ecotypes are distant among the studied ecotypes, and also showed to produce possible future hybrids, which direction will be more successful.

The objectives of this research were study of genetic diversity in Iranian and foreign pistachio cultivars using CBDP markers and the efficiency of these markers in differentiation and grouping of cultivars.

In this research, genetic diversity of 73 cultivars (63 Iranian and 10 foreign cultivars) was evaluated using 25 CBPD primers.

The results showed that, the bands polymorphism for Iranian and foreign cultivars was 77.56% and 67.80%, respectively. The PIC mean for CBPD markers was 0.76 and all CBDP primers had a high ability to separate pistachio cultivars. The MI mean for CBDP primers was 2.65 and CAAT2 primer showed the highest MI (8.52) value. Analysis of molecular variance determined 4% inter-group diversity and 96% intra-group diversity for studied genotypes. Gst value 0.04 verified low inter-group diversity as well. Nm value 11.03 showed the gene flow between foreign and Iranian pistachio cultivars. Iranian cultivars had higher values than foreign cultivars in terms of both Nei and Shannon gene diversity indices as well. Cluster analysis using Jaccard matrix and UPGMA method was performed and the results showed, the average of similarity coefficient between cultivars was 0.68 and the pistachio cultivars were divided in three main groups, in which the main groups were subdivided into several subgroups. The principal coordinate analysis also confirmed the result of the cluster analysis.

In conclusion, the use of CBDP markers is suitable for studying the genetic diversity of pistachio cultivars. Also, the efficiency and differentiation power of CBDP primers was confirmed due to the high mean of PIC and MI.

Potato (Solanum tuberosum) is an important food and economic crop in the world. However, potato shows susceptiblity to drought stress. Thus, the investigation of molecular mechanism of drought stress tolerance is essential considered. GF14/14-3-3 protein is one of the conserved dimeric proteins that regulate several cellular processes, ranging from metabolism to transport, growth, development, and stress response. However, only few reports are available regarding the effect of 14-3-3 genes in response to stress in potato.

In this study, twelve 14-3-3 genes were detected in the potato genome using bioinformatics methods.  Further, motif analysis, gene structure, phylogenetic analysis, TFBS and synteny analysis were performed on the 14-3-3 genes. In addition, expression analysis of two genes (StGF14i and StGF14h) in four tissues (root, stem, leaf, and tuber) and their expression under drought stress in greenhouse was investigated.  

Based on phylogenetic relationships, the StGF14 family members were categorized into two classes. Analysis of transcription factor binding sites (TFBS) in the promoter region of 14-3-3 genes revealed that the highest and the lowest number of TFBs were MYB and CSD, respectively, which found in promoter of 14-3-3 genes.  Moreover, different frequencies of TFSB in 14-3-3 genes could indicate that these genes control different developmental stages and are involved in complex regulatory mechanisms. Furthermore, genome evolution of S. tuberosum using orthologists and paralogues identification was studied. The number of exons in 14-3-3 genes was from four to seven and most of these genes in the same subfamily them had the same exon–intron patterns. The expression of two genes in leaves and tuber under drought stress as well as the gene expression of both genes in root, stem, leaf, and tuber tissues under drought were examined. Based on the expression analysis of two genes StGF14i and StGF14h in tissues and survey of drought stress, StGF14i gene has a maximum expression in four tissues and also, highest expression in tubers under drought stress. Our results revealed that two orthologous gene pairs between S. tuberosum and A. thaliana as well as eight paralogous genes among potato genomes were identified.

The expression patterns of StGF14i and StGF14h genes in different tissues and in response to drought stress that two genes had the conserved and necessary roles in potato growth and development potato. It is hope that the results of this study will be useful for further investigation of functional role and molecular mechanisms of 14-3-3 genes in response to drought stresses.

Recently, in East Azerbaijan province, while marketing of top sheep carrying fertility genes became suspected, because a number of farmers complained about the claim of the seller due to the low performance of such ewes on the farm. With this research motivation, the aim of the present study in the first step is to combine PCR-RFLP and subsequent PCR-Sequencing methods for genetic refinement of the FecB gene locus associated with litter size in the Booroola-Afshari sheep breed.

In this study, overall 62 ewes and rams of Booroola-Afshari sheep and 30 ewes of Ghezel- Romanov hybrids were obtained from different sheep farms around Tabriz. Then, two- separate phase were designed, after amplification of the 190 bp region of the BMPR15 gene by PCR, two molecular PCR- RFLP methods and direct sequencing were performed according to routine laboratory instructions. Genotype results were recorded and subsequently, using different software such as POPGENE, FinchTV and MAFFT, the genotypic, allelic, and alignment frequencies of the amplified sequences were performed to confirm the mutation, respectively.

The results of the present report demonstrated that the source of discrepancy between the identified genotype and the actual genotype can be due to the error of reading the genotype or the seller's fraud and false claim. Also, the combination and both outputs of both molecular techniques, PCR- RFLP and direct sequencing, can minimize the technical error caused by the genotype. In addition, high reproducibility observations confirmed the presence of 8/06% fraud in the FecB gene locus genotype.

The combination of two molecular methods, PCR- RFLP and PCR-sequencing direct sequencing to detect FecB mutations, has shown acceptable efficiencies, and the establishment of government-affiliated genotyping centers for fraud detection can assist regulators in genotyping sheep with fertility genes and moreover may reduce the number of frauds.

The unique characteristics of plant protoplasts have made them a powerful tool for researchers to study the genetic modification of plant cells. The most important prerequisite for the use of protoplasts is the ability to extract them easily and in large quantities, and to cultivate and regenerate them for cell colony formation and plant production. The most common method of extracting protoplasts from plant tissues is the enzymatic method. Concentration and composition of enzymes and isolation and culture steps are key factors in protoplast extraction. The aim of this study was to investigate the effect of these factors on protoplasts of in vitro potato (Solanum tuberosum L.) and to develop an efficient and suitable method for their extraction, transfection, callus formation and regeneration.

For protoplast isolation from commercial and common Agria cultivar of potato, different treatments of Cellulase and Macerozyme enzymes concentration, incubation time in enzyme solution and isolation steps on leaf tissue, stem and aerial part of in vitro plantlets were investigated. The effects of different hormone concentration on culture and regeneration of protoplasts were studied. After protoplast isolation, the efficiency of transfection by PEG 4000 using pBin61-GFP plasmid was evaluated.

The best results were obtained from leaf explants and the highest number of protoplasts (2×106 protoplasts per ml) were extracted from leaf mesophilic tissues after 16 hours' incubation in enzyme solution (1% Cellulase and 0.2% Macerozyme). The transfection efficiency of protoplasts by PEG with 40% concentration and duration of 30 minutes was 50%. The MS medium containing 5 mg/l NAA and 0.1 mg/l BAP was selected as the best culture medium for callus induction. In this hormonal treatment, 100% of callus induction from protoplasts was obtained after about one month. The best medium for plant regeneration and shooting from callus was the combination of 2 mg/l Zeatin with 0.1 mg/l GA3 and 0.01 mg/l NAA with 86% efficiency.

Based on the results, the described protocol for protoplast extraction and gene transfer in this study provides necessary steps for genetic engineering, transformation and gene expression in the potato plant.

The Hop stunt viroid has the widest host range among viroids, and is prevalent worldwide. This research aims to detect and characterize HSVd variants in the vineyards of Kerman province and some other parts of Iran and analyze the variants' phylogenetic status.

Seventy-four leaf samples were collected from grapevines in Kerman, East Azerbaijan (Tabriz), and Fars (Beyza) provinces during the summers of 2018–2020. The samples were processed and subjected to RT-PCR to detect HSVd using specific primers. Then, complete genomes of seven HSVd variants were sequenced and blasted into GenBank. In the MEGA 7.0 program, the neighbor-joining statistical method was used to construct a phylogenetic tree to consider relationships between the detected HSVd variants and those obtainable in GenBank. Furthermore, the homology of the grapevine-related variants was analyzed by SDT v1.2 software. Finally, eight plant species and varieties were experimentally inoculated by two HSVd variants to study symptom expression and determine host range.

Throughout the world, HSVd variants have been grouped into five phylogenetic groups: Citrus, Hop, Plum, Plum-Hop/cit3, and Plum-Citrus. Accordingly, the identified seven HSVd variants in this study and several previously reported variants from grapevines in Iran and Germany were classified under the main group, Hop. The HSVd variants from grapevine had genome lengths ranging from 297 to 300 nucleotides, with 93.3–98.7% nucleotide sequence similarity. Three of the seven HSVd isolates identified in Kerman province vineyards and four isolates previously reported from grapevines in Iran and Germany formed a subgroup within the Hop cluster. In addition, the Hop group included four Iranian pistachio HSVd isolates from the Kerman province and some Tunisian pistachio HSVd isolates with Iranian provenance.

Given that HSVd infection of grapevines has been documented in Iran prior to pistachio infection, pistachios were most likely infected via HSVd inocula transferred from affected grapevines. Therefore, based on the molecular studies and available evidence, grapevines can be assumed to be the source of infection for pistachio trees.

Medicinal plants have long been recognized as a rich source of essential oils and have been cultivated for a variety of uses since ancient times. White savory (Satureja mutica fisch & CA mey) is one of the medicinal plants belonging to the mint family with high content of carvacrol in its essential oil. A large body of literature has suggested that abiotic stresses, especially drought stress, could considerably boost the production of essential oils in plants. The aim of this study was to evaluate the effect of drought stress on the expression of genes affecting the biosynthesis of the main component of white savory essential oil carvacrol.

For this goal, a number of 20 pot-planted seedlings of white savory at the 6-8 leaf stage were subjected in a greenhouse to two conditions of control and drought stress (10 pots for each condition) with irrigation regimes of 100% and 30% of field capacity, respectively. In order to ensure the implementation of stress on plants and to determine the appropriate time for sampling, leaf proline and relative water content of plants were measured.

The results of both proline and relative water content tests revealed a significant statistical difference between the control and stress conditions in white savory at 30% of field capacity. The KEGG (Kyoto Encyclopedia of Genes and Genomes) database was used to obtain information on carvacrol biosynthesis pathway. The three key genes geranyl diphosphate synthase (gpps), terpene synthase (tps), and limonene hydroxylase (lh) were chosen for gene expression analysis. Due to the lack of genomic information on white savory, the sequence information of the genes in the closest genera within the family mint was used to design primers. The result of melting curve analysis from the real-time quantitative PCR (qPCR) assay confirmed the accuracy of the primer design. The raw qPCR data normalized to the housekeeping gene Actin, then differential expression analysis was preformed using the delta delta Ct method. qPCR result showed that expression of the genes gpps, tps, and lh were increased in stressed plants proximately 20, 18, 50 times, compared to those in control, respectively. 

The conclusion that must be drawn from the data available is that water stress leads to the process of plant response to stress and changes the expression of genes involved in the biosynthesis pathway of the active substance carvacrol.

Galega (Galega officinalis L.) from the leguminous family is used to treat diabetes. Hairy root culture is a valuable system for the production of secondary metabolites on a large scale. The use of elicitors in plant tissue and cell culture is one of the most efficient biotechnological tools for inducing biosynthesis and accumulation of secondary metabolites. This study aimed to investigate the effect of iron, silver, and silicon nanoparticles on growth and galegine content in hairy roots of G. officinalis.

In this study, induction of hairy roots in leaf, cotyledon, and hypocotyl explants of G. officinalis was performed using the A4 strain of Rhizobium rhizogenes, and the effect of different concentrations of silver, silicon, and iron oxide nano-elicitors on growth and galegine, phenol, and flavonoids content of hairy roots was investigated.

The highest rate of root induction was obtained in leaf explants with an average of 5 roots per explant. The highest root growth rate belonged to the treatments with 50 and 100 mg l-1 silicon nanoparticles for 72 h. Application of all levels of silver, iron, and silicon elicitors for 36 h resulted in a significant increase in the total phenol content of hairy roots. Thus that the highest amount of total phenol was obtained in 36 h treatment of roots with a concentration of 100 mg l-1 of silicon nanoparticles without significant difference with 10 and 20 mg l-1 silver nanoparticles. Also, the highest amount of total flavonoids was obtained at 200 mg l-1 iron nanoparticles for 36 h; and the highest content of galegine was obtained in the treatment of roots with 10 and 20 mg l-1 silver, and 100 mg l-1 silicon nanoparticles for 36 h.

The appropriate type and concentration of elicitors is an effective factor in the response of hairy roots to nano-elicitors. The highest yield of galegine (17.55 mg) was obtained in the treatment of roots with 50 mg l-1 of silicon nanoparticles for 72 h. This can be attributed to both better growth of hairy roots and high galegine content at this treatment.

One of the goals of the poultry industry is to select animals that increased growth rate and also muscle mass while maintaining meat quality. The present study was conducted to evaluate performance, some meat quality characteristics and genes expression associated with growth (IGF-I, IGF-II, TGF-β1, Myostatin) in three strains of Arian, Ross and Cobb.

To do this research 150 one day old (male and female) broiler chicks of Arian, Ross and Cobb strains were categorized in a completely randomized design with three treatments and five replicates and 10 observations per replicate. The function, some physical characteristics of meat and the relative expression of their genes were investigated. Strains types had significant effect on feed conversion ratio (FCR).

In the whole of rearing period, the lowest FCR was for Ross strain and the highest was for Cobb strain. TPA test of breast showed that hardness, cohesiveness and chewiness in Arian, Ross and Cobb strains were significantly different. Maximum hardness and chewiness were for Arian and Cobb strains and maximum cohesiveness was for Ross strain. There were no significant differences between strains for adhesiveness, springiness, gumminess. TPA test of thigh showed that, hardness, chewiness and gumminess were not significantly different, but cohesiveness, adhesiveness and springiness were significantly different between strains. Maximum cohesiveness and springiness were observed in Arian strain and maximum adhesiveness was for Ross strain. Breast and thigh pressure test of three strains showed that hardness and deformation of hardness were not significantly different. IGF-I and IGF-II relative genes expression of breast meat had no significant difference between strains. TGF-β1 and myostatin relative genes expression were significantly different and the Cobb strain had the highest relative gene expression of TGF-β1. Also IGF-I and IGF-II relative genes expression had no significant difference in liver.

Given the individual advantages of each strain in some traits, more research is needed with specialized diets for each strain to be able to comment conclusively on the superiority of their traits.

Oilseeds are the second-largest source of calories for human societies after cereals. Sesamum indicum has been considered recently due to its oil quality and content of 43-46% unsaturated fatty acids. To investigate the possibility of transferring the recombinant aroA-CYP81Q1 gene through Agrobacterium tumefaciens to the Sesamum indicum plant (reported for the first time in the country), after optimizing tissue culture and selecting the best hormonal combination, a factorial experiment was performed. 

In this experiment, the recombinant aroA-CYP81Q1 gene was synthesized and transformed into an Agrobacterium strain (LB4404). Gene cloning was confirmed by PCR and then confirmed by sequencing. The efficacy and frequency of transgenic Sesamum indicum were evaluated in the selected medium containing 50 mg/l kanamycin. Finally, to confirm the transgenicity of the regenerated plants, PCR analyzes were performed with specific primers on selected plants. Also, the expression of the sesamin-producing gene (CYP81Q1) in control groups and transgenic groups was measured at a significant level of P <0.01.   

Statistical analysis showed that the percentage of regeneration of transgenic seedlings using Agrobacterium (LB4404) in the selected medium containing kanamycin was 33%. Also, PCR analysis of transgenic plants showed a prevalence of transgenics of 33%. In addition, amplification of the bp fragment indicated the accuracy of cloning in transgenic plants. The results indicate the successful transfer of this gene to the sesame plant to increase its industrial properties. The results of the analysis of variance showed that there was a significant difference in the expression of sesamin-producing genes between transgenic cultivar and control groups (P <0.01). 

Since pBI121-based expression vectors are more efficient than other expression vectors and are widely used in the transfer of recombinant genes to plants, the recombinant vector constructed in this study indicates successful gene transfer. Sesame to sesame plant to increase its industrial properties.

It has been shown that mutation in DNAH1 (Dynein Axonemal Heavy Chain 1) gene is associated with MMAF (multiple morphological anomalies of the flagella) and PCD (primary ciliary dyskinesia) phenotypes. MMAF has been widely studied in male reproductive traits and is often associated with abnormal sperm, which seriously affects male reproductive traits and even leads to infertility. Indeed, as a MMAF-dependent gene, many studies have identified DNAH1 gene as a key and essential player in male and female gonadal development and have investigated the clinical applications of DNAH1 gene. PCD is often associated with ciliary motility disorder, which is also an autosomal recessive genetic disease. Animals with severe PCD show infertility. The aim of this study was to study the expression pattern of DNAH1 gene in the testicular tissue of Raini Cashmere goat using Real Time PCR.

Sampling was done from the testicular tissue of three heads (3 replicates from each head) of Raini Cashmere goat in the slaughterhouse. The isolated samples were placed in 1.5 mL microtubes. Total RNA was extracted from testicular tissue. Agarose gel electrophoresis and UV spectrophotometry were used to evaluate the quality and quantity of extracted RNA. Then cDNA was made and Real Time PCR was performed. Also, GAPDH housekeeping gene was used as a control. Melting curves were analyzed and data obtained from Real Time PCR were performed. 

The results of Real Time PCR curves and observing the results of electrophoresis of PCR products on 2% agarose gel showed that DNAH1 gene is expressed in testicular tissue. For the DNAH1 gene, a band of 140 bp was observed and for the GAPDH gene, a band of 143 bp was observed, and the accuracy of the amplification test was confirmed.

According to the results of the present study and the results of other researchers, it can be concluded that DNAH1 gene plays an important role in fertility. In the present study, it was found that DNAH1 gene is expressed in testicular tissue. Therefore, DNAH1 gene is most likely essential for male fertility, and the results of this research have provided the way for future studies to describe the role of DNAH1 gene as a candidate gene for better fertility and normal physiology in domestic animals, especially goats.