JOURNAL OF AGRICULTURAL BIOTECHNOLOGY
Year:2020 | Volume:11 | Issue:4 |Papers Count:12

Objective Amaryllis is one of the ornamental bulbous plants, whose reproduction occurs slowly in natural conditions. Therefore, using tissue culture technique can be a suitable way to increase the multiplication rate of this ornamental plant. Material and methods In this study, a factorial experiment was conducted in a completely randomized design with 5 replications. The effect of different concentrations of BA and Kin (0, 0. 5, 1 and 2 mg/L) in combination with 0. 1 mg/L NAA on regeneration of different twin scale type of amaryllis was evaluated. Explants were divided as twin scales and classified in 4 groups, so that the outermost twin scales was grouped in class 1 and the innermost twin scales was grouped in class 4. Results The results showed that the diameter of produced bulblet was influenced by the position of the twin scales in the mother bulb. The twin scale group one, which was made from the outer layers of the bulb, produced thicker bulblets, while the twin scales group four showed the minimum average of this trait. However, the explants cultured in a culture medium containing 0. 5 mg/l BA in combination with 0. 1 mg/l NAA had the highest bulblet diameter. Conclusion Generally, the application of twin scales group 1 and MS medium containing 0. 5 mg/l BA in combination with 0. 1 mg/l NAA is recommended for regeneration and proliferation of Amaryllis twin scale explants.

Objective Identifying the genetic diversity of native goat breeds of Iran can play an important role in the management and conservation of these breeds. The purpose of the current study was genetic and phylogenetic investigation of mitochondrial genome cytochrome b region in Lori, Pakistani, Zaboli and Adeni goats. Materials and Methods Blood samples were collected from 16 goats of each breed 4 blood samples. After extracting DNA, fragment 684 bp of cytochrome b region genome mitochondrial amplified by primers were designed and fragment amplified after purification was sequenced. Using the DNAsp software was determined in six different haplotypes. Results The result of comparative analysis between goat breeds indicates that nucleotide diversity Pakistani and Zaboli breeds 0. 35461 and 0. 11199 respectively. Tajima D was obtaining 1. 06945 that was significant. This confirmed the evolution by natural selection. Results from the analysis of molecular variance indicated that genetic variation was 80% of within and 20% was between populations that indicates high gene flow, migration among the studied populations or little sample size. Conclusions Based on a phylogenetic tree analysis, Lori and adni goat form a group with Capra hircus and Pakistani and Zabli goats form a separate group. This indicates the goat breeds have a common origin area.

Objective The aim of the present study was to investigate genetic diversity in six Iranian horse populations including Ardabil native horses, Ardabil clubs horses and Arab, Drehshoori, Kordi and Qarabakh breeds. Material and Methods To perform the present study, 100 horses were sampled. After extraction and quality assessment of DNA, polymerase chain reaction was performed for eight microsatellite markers. Genetic diversity and statistical analysis were performed using GenAlEx 6. 5 software. The phylogenetic tree was constructed using microsatellite marker information indicating genetic distances of analyzed horse breeds. Also, STRUCTURE software was used to analysis of genetic structure of Iranian horse populations. Results The obtained results showed that the number of observed alleles in Ardabil native horses was 22, while this value for Dareshoori breed was 7. 5. The observed heterozygosity was ranged from 0. 865 for Kordi to 1 for Qarabakh breed. The average of observed alleles was calculated 13. 729. Investigation phylogenetic tree showed that Ardabil native horses and Ardabil clubs horses have low genetic distance. Conclusion The investigation of genetic diversity of Iranian horses using eight microsatellite markers indicated that there is significant genetic variation in Iranian horses in the Northwest of Iran. Part of this variation is due to use of uncontrolled mating among breeds.

Objective According to economic value of cumin (Cuminum cyminum) medicinal plant, it is essential to do its genetic and phytochemical assessment to conserve genetic sources and achieve better quality of essential oil in breeding programs. This study purpose is to investigate genetic and phytochemical diversity of different cumin ecotypes in Yazd province. Materials and methods To study genetic diversity using 10 ISSR markers, cumin plants DNA extraction of four habitats, was done using CTAB method. The quantity and quality of DNA were determined using spectrophotometer and electrophoresis. Clear Bands resulting from electrophoresis of PCR products on 1. 5% agarose gel were scored. To investigate phytochemical diversity using GC-MS method, essential oil from cumin seeds was extracted via water steam distillation method. Results The results showed that ISSR-14 and ISSR-13 primers had higher amount of PIC (0. 39 and 0. 38) and MI (13. 3 and 12. 48) and number of bands (35 and 32). Cluster analysis using Jacquard's coefficient and UPGMA method, grouped four populations into 8 groups. PCA analysis showed three components explained 57. 17% of total variance. The results of this analysis correspond with cluster analysis and match with the geographical regions. Analysis of molecular variance showed genetic variation within and among populations 93% and 7% of total variation, respectively. On the basis of GC-MS results, Propanal, Benzenmethanol, 1-phenyl-1-butanol, γ-terpinene, β-Pinene and P-cymene compounds were identified as the main compounds. Cluster analysis of samples on the basis of phytochemical parameters put Taft, Mehriz and Ardakan habitats in one group and Bahabad habitat in another group so that it doesn’ t correspond with geographical distance. Conclusions Based the results, it can be concluded that there is high potential of diversity among cumin ecotypes. ISSR application in assessing genetic diversity of the ecotypes was successful. Grouping on the basis of phytochemical compounds didn’ t correspond with genetic grouping.

Objective The aim of this study was to amplify and cloning complete, as well as the primary part of the α-toxin gene from Clostridium perfringens with the size of 1100 and 750 bps, in E. coli using the expression vector pET28a. Materials and Methods Genomic DNA was extracted using Kiagen kit. Forward and reverse primers were designed in the lab, as the HindIII and XhoI enzymes rows were added in the initial regions of the oligonucleotides according to the cloning region of the pET28a plasmid. The genes of interests were amplified by PCR and cloned in the pET28a vector using the heat-shock method. Extraction of plasmid DNA was done using alkaline lysis method. Results The results showed the PCR products, clear and unique bands for the complete, as well as the initial portion of the desired genetic components in accordance with the expected sizes. In addition, by using single and double enzyme digestion experiments with HindIII and XhoI enzymes, cloning of alpha toxin genes in host bacteria and production of recombinant bacteria was confirmed. Conclusions Due to the successful cloning of alpha toxin genes in expression host bacteria done by this work, it can be said that primers designed in this study are highly specific for the α-toxin gene amplification. These primers can be also used as markers for the identification and classification of Clostridium perfringens bacteria. Also, by development construction of genetically engineered recombinant vaccines, it is possible to control and cope with important pathogens such as Clostridium perfringens bacteria.

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

Objective Lactic acid bacteria (LAB) synthesize valuable industrial and pharmaceutical important biopolymers, such as Alternan, by expressing glycosyltransferase enzymes by utilizing extracellular sucrose. In this study, due to the importance of such versatile biopolymers in the industry and medicine, a gene encoding an Altranansucrase (Asr) was introduced to sugar beet plants. Materials and methods A gene encoding the Asr gene was isolated from Leuconostoc mesenteroides bacterium and introduced to sugar beet plants by using Agrobacterium-mediated transformation. Molecular techniques were used to analyse transgenic plants and sugar content of sugar beet lines. Results Out of 131 transformed explants, only three transgenic plants were produced, showing a transformation efficiency of 2. 3%. The Asr gene integration in transgenic plants genome and expression were confirmed by specific PCR and semi-quantitative RT-PCR analysis, respectively. Sugar analysis of sugar beet transgenic plants showed that control plants with a 19. 6% brix value (Sucrose) had more sucrose than transgenic plants with an average brix value of 14. 4%. Brix in transgenic plants was lower than that of control plants. The amount of sugar (sucrose) in the transgenic asr-expressing plants reduced by 36. 1% relative to the untransformed control plants. Conclusions The results of this study showed that Altrenansucrase can convert substantial amount of sugar beet sucrose into Alternan biopolymer, producing 36. 6 mg/g FW alternan biopolymer with pharmaceutical and industrial applications.

Objective Herbal essential oils are added to the poultry diet for a variety of reasons, such as improving performance, production and increasing the level of immunity. This study was conducted to investigate the effects of fennel and savory essential oils and their mixtures on growth performance, carcass characteristics, antioxidant parameters and hepatic interleukin-6 gene expression in broilers. Materials and methods The study was conducted in a completely randomized design with a 3 × 3 factorial arrangement with 9 treatments and 4 replications, each replication containing 15 birds. (540 Ross 308 broiler chickens). The experimental diets included the levels of 0 g/kg, 0. 15 g/kg and 0. 25 g/kg of fennel and savory essential oils and their mixture. Feed intake, weight gain and feed conversion ratio were calculated at starter (1-10 days), grower (11-24 days) and finisher (25-42 days). Each repeated three birds were bled at day 42. Interleukin-6 gene expression was performed on day 42. The ileum contents were sampled at 42 days of age to determine the microbial population. Results The results showed that in the finisher (25-42), the experimental groups that received the essential oil had a lower conversion ratio than the control treatment. Among the experimental groups, the mixture of 0. 25 fennel + 0. 25 savory essential oils (g/kg feed) had the best production index (p<0. 05). The essential oils used reduced the amount of malondialdehyde produced in the broiler drumstick (p<0. 05). Herbal compounds increased the lactobacillus and reduced the coliforms and E. coli in the contents of the ileum. All savory supplementated groups except 0. 25 (g/kg) + fennel 0. 15 (g/kg) increased expression of hepatic interleukin-6 gene. Conclusions Diets containing 0. 25 (g/kg) fennel + 0. 25 (g/kg) savory essential oils had the highest effect on improvment of the performance and expression of the broiler liver gene.

Objective The main goals of this study were investigation of genetic diversity in 103 Aegilops accessions and comparison of the efficiency of start codon targeted (SCoT) and CAAT box-derived polymorphism (CBDP) markers. Materials and methods In this study, the genetic diversity in 103 Aegilops accessions belonging to seven species including seven samples of Ae. caudata, 14 samples of Ae. crassa, 19 samples of Ae. cylindrica, 11 samples of Ae. neglecta, 20 samples of Ae. tauschii, 15 samples of Ae. triuncialis and 17 samples of Ae. umbellulata was evaluated using 15 SCoT and 15 CBDP primers. Results In total, 15 SCoT and 15 CBDP primers amplified 164 and 141 polymorphic bands, respectively. SCoT primers showed the highest values for all of the informativeness parameters than CBDP primers. However, both molecular markers indicated the same PIC values. The results of analysis of molecular variance (AMOVA) revealed that the highest proportion of genetic variance referred to within species. Among all species, Ae. cylindrica had the highest values of genetic parameters. Although cluster analysis based on each marker system classified all accessions into two main groups, the grouping pattern obtained from CBDP data indicated a clear phylogenetic relationship among Aegilops species compared to SCoT data. Besides, the results of clustering were confirmed by principal coordinates analysis (PCoA) analysis. Conclusion On the whole, both molecular markers revealed good capability in depicting of polymorphism among tested accessions. However, CBDP markers provided a vivid grouping pattern for evaluated samples. Hence, the use of this technique individually or in combination with other molecular markers is recommended for phylogenetic assessments.

Objective Potato virus X (PVX) is a member of the genus Potexvirus in the family Alfaflexiviridae. This virus is one of the most common and widespread viruses infecting potato worldwide. Due to the wide distribution and economic damage of the virus in Iran, identification and detection of the virus is necessary using non-expensive methods. Production of polyclonal antibody through molecular approaches can be a useful method for detection of virus in the nature. Materials and methods In this research, an infected potato sample was collected from Zarand (Kerman province) and its PVX infection was confirmed by ELISA test. This sample was inoculated on test plant and the coat protein (CP) gene of this isolate was amplified in RT-PCR test with specific primers comprising BglII and NcoI restriction enzymes. Amplified product was cloned into expression prokaryotic vector (pQE60 plasmid). followed by transformation of the E. coli strain M15 competent cells. Results Subsequent to induction of CP expression, total protein was extracted and run onto 12. 5% SDS-PAGE. An approximate 24 kDa band was observed on the gel corresponded to the PVX coat protein. Furthermore, expression of the PVX CP was confirmed by dot blot technique. Conclusions In this study, a recombinant antigen suitable for the detection of potato X virus was prepared which was well identified by antiserum produced by injection of complete virus particles into rabbits in dot blot analysis. The production of multiclonal antibodies by recombinant viral antigen will be an important step in accelerating and facilitating the serological identification process of plants infected with this virus.

Objective Identification of associated markers with important agricultural traits is one of the most important methods for accelerating the transfer of desirable traits to other genotypes and their tracking. The aim of this research was to study the genetic diversity and identification of molecular markers related to drought tolerance traits in foxtail millet [Setaria italica (L. ) P. Beauv. ] genotypes using the AFLP molecular marker. Materials and methods In this study, 21 genotypes of foxtail millet were studied in three growing seasons (2013, 2014 & 2016). Drought tolerance indices and correlation between them with grain yield and biological yield were calculated. In order to identify molecular markers associated with drought tolerance traits, association analysis was performed by using the Mixed Linear Model (MLM) and 12 primer combinations of AFLP. Also, marker indices and principal components analysis (PCA) were performed using GenAlEx 6. 5 software Results The primer combinations used in this study generated a total of 443 scorable bands, of which 316 (71%) were polymorphic. The mean of polymorphism information content (PIC) was 0. 24, the mean of Shannon index was 0. 38, and the mean of Marker index was 6. 22. Also, M-CTG/E-ACC, M-CAA/E-ACC and M-CTA/E-AAC were the most efficient combinations in investigating the diversity of genotypes studied. Based on the results of PCA, the first and second components justify 61. 13 percent of the changes. The results of this study showed that HARM, GMP, and MP indices were the best indices for differentiation of drought tolerant samples, and M-CAA/E-AAC-14 and MCTG/ E-ACC-283 markers according to Grain yield and M-CTT/E-ACC-264 markers according to biological yield, were related to these indicators. Conclusions According to the results of this study, the HARM, GMP and MP indices could be considered as the best indicators for differentiation of drought tolerant foxtail millet samples. Also, molecular markers associated with these indices can be used to evaluate the drought tolerance of other genotypes in future breeding programs.

Objective Calpastatin has been found in skeletal muscle tissue of animals and could prevent unrestrained cellular growth by suppressing calpain activity. The involvement of calpains in apoptosis has been a subject of debate, although their involvement is limited to certain cell types and to specific stimuli. Various studies have shown that this gene is involved in growth performance and meat quality, therefore the aim of this study was to investigate Calpastatin gene expression in different tissues of Raini cashmere goat using Real Time PCR. Materials and Methods Totally 21 tissue samples including muscle, adipose, kidney, spleen, liver, lung and heart tissues were taken from 3 Raini cashmere goats. RNA was extracted and cDNA was synthesized. Real Time PCR was performed using SYBR Green method to study relative gene expression. GAPDH gene was used as housekeeping gene. Pfaffl method was used to analyze achieved data. Results For calpastatin gene 89bp fragment and for GAPDH 101bp fragment were observed in all studied tissues. Real Time PCR results of this study showed that Calpastatin gene is expressed in all studied (muscle, adipose, kidney, spleen, liver, lung and heart) tissues and the highest level of expression was observed in heart, spleen and liver tissues and the lowest level was seen in adipose tissue. Conclusions Results showed that Calpastatin are widespread in different tissues of goat. This study would also lay a foundation for further Calpastatin research in Raini cashmere goat. It is suggested that this study be conducted with greater number of livestock, different sexes, different ages and different physiological stages in different breeds of goats in order to reach a comprehensive conclusion.