Agricultural Biotechnology
Year:2016 | Volume:14 | Issue:2|Papers Count:12

Cell suspension culture is a suspension of rapidly growing and dividing cells which are cultured in liquid medium. The dividing capability of the cells in a suspension culture is much higher than that of calli. This potential has therefore been exploited for different purposes such as developing cell lines, producing secondary metabolites, investigating cell cycle phases, increasing gene transformation efficiency and studying biotic and abiotic stresses. In this study, callus was produced from nine oriental tobacco (Nicotiana tabacum) cultivars in MS medium containing NAA 1 mg.l-1 and Kinetin 1 mg.l-1. Then, callus growth rate and quality were examined in different media. The best growth rate and quality were achieved for the cultivars of OR-209, OR-205 and PDB 328 using LS medium treated by hormones 3 mg.l-1 IAA, 3 mg.l-1 NAA and 0.1 mg.l-1 Kinetin. The effects of different culture media and hormone compositions were then assessed on cell suspension. The best growth was acquired for OR-209 cultivar in liquid LS medium having the same hormone composition. Consequently, the growth rate of the developed cell suspension was compared with that of the TBY-2 cell line, revealing the high similarity. This result indicates the high potential of oriental tobacco cultivars as promising progenitors for the production of cell lines and their exploitation for research and applied purposes.

Several reports were published in regard to the comparison of the efficiency of different molecular markers in genetic diversity studies for estimating genetic parameters. Most researches have been conducted out in different plant genomes and in different breeding programs, but there isn`t any report on comparing the efficiency of SSR and RAPD markers in marker-assisted selection (MAS) programs in rice genome. In this line, a research was conducted out to compare the two marker systems for estimating some genetic parameters in advanced backcross lines in rice. One hundred SSR primer pairs and 20 RAPD primers were used for fingerprinting of the studied genotypes. Polymorphism of RAPD markers was higher than SSR markers, while polymorphism information content (PIC) of SSR markers was more than 2 fold relative to RAPD markers. A weak correlation was observed between the two systems for estimating genetic similarity of progenies to recurrent parent. Also, results showed that increasing the number of RAPD markers didn`t cause any increase in estimation precision. The results of this research suggested the use of SSR markers in selection programs.

Bacterial wilt disease caused by Ralstonia solanacearum is an important disease on potato in Hamedan province. During spring and summer 2011 infected plants were collected from potato fields to investigate the genetic diversity among R. solanacearum strains. A total of 52 strains were isolated on NA medium containing triphenyl tetrazolium chloride (TTC) from tubers and stems and their phenotypic characteristics revealed that they were belong to biovar 2A. PCR using ps96h and ps96i primers produced 148 bp bands in tested and standard strains. Genetic diversity of 26 selected strains were analyzed by PCR fingerprinting using, ERIC and BOX primers. The length of produced fragments ranged from 200bp to 1200bp in BOX primer and 200bp to 1500bp in rep primer. Tested bacterial strains were grouped based on the obtained molecular DATA from PCR using UPGMA method and Jaccard’s coefficient. Results showed five distinguished clusters. R. solanacearum strains were genetically divers although no direct connection was observed between geographic locations and PCR groups. The results of this study indicated that rep-PCR technique with two ERIC and BOX primers is capable to show genetic polymorphism among different populations of R. solanacearum.

Tomato yellow leaf curl disease (TYLCD) is one of the most important diseases of tomato (Solanum lycopersicum L.) plants in tropical, subtropical regions and greenhouses in temperate regions of the world. To produce an antibody against the viral agent of the disease for use in a diagnostic kits, the TYLCV cloned viral movement protein gene was expressed in an expression vector (pET26). The resulting recombinant protein was then injected into a rabbit. The prepared antiserum was used in western blot and ELISA analyses in order to assess its efficiency in virus detection. A 13KD protein band related to viral movement protein was detected in infected plants by using Western blot analysis at 1: 1500 antiserum dilutions, while in ELISA tests at 1: 1000 dilutions of antiserum, denaturation of the protein could increase the differences between the absorbance values of the infected plants and non-infected plants. However, these differences were not statistically significant. The results showed that the prepared antiserum against Tomato yellow leaf curl virus- movement protein was suitable for western blot analyses, but to be used in ELISA test the antibodies need to be extraction and purification of antibodies.

WRKY gene family encodes a large group of transcription factors which are involved in the regulation of biotic and abiotic stress responsive genes. To identify WRKY family members in wheat, multiple searches were done in the related databases. Rice WRKY conserved sequences were used as the templates for tblastn searches in the nr, EST, HTGS datasets for finding new members in wheat. EST and cDNA sequences were translated by the DNAstar software and NCBI ORF finder. Phylogenetic analyses were done using MEGA4 and BioEdit softwares. Expression pattern of this gene family studied by analysis of microarray data from the PLEXdb, GENEVESTIGATOR databases. According to the results, 94 members were found in the wheat WRKY gene family which 76 members of them had complete conserved domain. Wheat WRKY transcription factors such as rice WRKY TFs were divided into three groups based on the WRKY conserved domain.7 members of this family with differential expression at drought stress condition in sensitive and tolerant varieties were regarded as candidate genes for drought tolerance.

Myostatin gene belongs to the family of transforming growth factors (TGF-b), which in growth and development and maturation and specifically is expressed in adult skeletal muscle development. In this study protein amino acid sequence of Myostatin in various species, including cow, sheep, goats, horses, pigs, chickens, turkeys, ducks, goose, Columba, rats, and humans was investigated using by Bioinformatics tools. In alignment of the final section of Myostatin protein (active form of Myostatin protein that is expressed as a dimer), high degree of conservation was observed, so that the changes fluctuation in this section than the other parts of protein sequence, the least amount possible. Secondary structures such as alpha helices, beta sheets and three-dimensional structure of Myostatin proteins using specialized sites Proteus and Phyre2 was assessed. Amino acids leucine, lysine, isoleucine and glutamic acid in cow, sheep and goat showed the same frequency as compared with poultry, such as chicken, lots more. However, the frequency of the amino acid sequence of the protein myostatin in humans and pigs was moderate. Study of phylogenetic tree showed that difference between amino acid sequences among mentioned Myostatin species lead exposure in a group of birds was isolated from mammals. Investigation of 3-D structure showed that this gene is highly conserved across species.

Retrotransposons are current component of plants genomes. Their frequency, activity and even distribution thorough plant genomes make them useful as molecular markers. IRAP (Inter-retrotransposon amplified polymorphism) and REMAP (Retrotransposon-microsatellite amplified polymorphism) markers were used to study the activity, distribution and insertional polymophism of some LTR retrotransposons in Linum austriacum. The results showed that LTR retrotransposons used, are trsnspositionally active in L. austriacum genome and they are inserted near each other and in proximity of microsatellite regions as well. Out of 58 primers tested, 20 primers (7 IRAP and 13 REMAP) produced discernible and scorable banding patterns. In total, 199 loci were amplified by using 20 primers, of them 129 were polymorphic. The maximum and minimum number of polymorphic loci was amplified by primers LTR1868 and LTR1854-A13, respectively. The minimum and maximum Nei genetic distances based on IRAP, REMAP and IRAP+REMAP markers was observed between Urmia-Silvana and Khoy and Urmia-Sero and Urmia-Silvana populations, respectively. Mantel test between IRAP and REMAP cophenetic matrices evidenced no significant correlation. IRAP+REMAP-based cluster analysis using Minimum evolution method and Number of differences coefficients assigned individuals to four groups. The results make conclusion that IRAP and REMAP markers developed based on Linum active retrotransposons could be used as new tools in Linum breeding programs.

Pythium apanidermatum is the causal agent of cucumber damping-off disease. Regarding to development of sustainable agriculture, use of bio-fertilizers will be essential for plant disease management. In this study, the effect of nitroxin bio-fertilizer on resistance of cucumber seedlings (ES-2862 cultivar) to damping off disease was investigated. After treatment of infected seedlings with nitroxin, disease severity was measured in comparison with control. The leaves of seedling were used to total RNA extraction and gene expression analysis using Real Time PCR. The results of this assay showed that nitroxin bio-fertilizer reduced the disease severity. Also, expression of Chitinase and Cupi4 genes at 24 and 48 h after inoculation were increased. The the expression of Cupi4 were changed more than that of Chitinase gene. The results of this research indicate that application of nitroxin could increase resistance of sensitive cultivars against damping-off disease.

Medicinalchavil plant (Ferulago angulata B.) is an important plant belonging to the Apiaceae family. In order to determination of the best explants and hormonal combination for callus induction, this experiment carried out in Yasouj University usingtypes of explants including root, shoot, leaf (separated from seedling obtained from embryo culture) and fragmented embryo and hormone combinations of NAA with BAP each of at 5 levels (0, 0.5, 1, 1.5 and 2 mg/l) and 2, 4-D at 5 levels (0, 1, 2, 3 and 4 mg/l) with BAP at 5 levels (0, 0.5, 1, 1.5 and 2 mg/l) at 1/4 MS medium culture. These treatments arranged as factorial experiment using randomized completely design with three replications. Results of Analysis of Variances showed that main effects, two way interactions and explant´NAA´BAP interaction were significant (p£0.01) for callus induction percentage, callus fresh weight, callus dry weight, callus length, callus diameter and callus volum. In other experiment also, these effects were significant forcallus induction percentage, callus fresh weight, callus dry weight, callus length, callus diameter and callus volum. The result of experiment indicated that root explant at the all concentration of NAA had positive effect on callus induction. In addition root explant at 1 mg/l 2, 4-D and 1/5 mg/l BAP was the best treatment for callus induction. In general, because of good responses of chavil plant to callus induction, these results could be used in breeding programs of chavil plant.

Prunus necrotic ringspot virus (PNRSV) is one of the most devastating viruses that can affect stone fruit production. By considering negative impact of PNRSV on production of almond, objective of the present study was to develop a consistent and reliable method to detect PNRSV for studying the effect of it on pollen germination in the most important almond cultivars grown commercially in Saman region in Chahar Mahal va Bakhtiari province. One hundred almond trees of Mamaii, Rabii and Sefid cultivars were selected randomly during March, June and September of 2008 to 2009 and some parts of them as samples were checked by using ELISA and RT-PCR for the presence of PNRSV. Then healthy and infected pollen of Sefid cultivar were germinatad in vitro and compared pollen germination. Studying on the effect of PNRSV on germination of pollen grain, showed reduction of the germination capacity (34%) of infected pollen grain. In addition, Comparing ELISA test, RT-PCR was found more sensitive in detecting PNRSV. Detection of PNRSV by ELISA was most sensitive when plant material was collected in March, therefore sampling time was an important factor for detection of PNRSV.

Aspergillus niger is one of the microorganisms utilized to produce single cell protein. This filamentous fungus can produce a wide range of enzymes and may be cultivated by lot of substrates. Aspergillus niger is able to produce high amount of extracellular protein, therefore it is possible to enrich the substrate and also extract the protein with a solvent. Its growth efficiency is higher on solid substrate because it can grow naturally on pectin substrate. Saffron petal is now one of the agricultural wastes while it could be used as substrate for solid state fermentation. In this research A. niger was cultivated on dried saffron petals in a 15 day period and its protein content was extracted with two solvents and assayed by Biuret and Modified Lowry methods. Highest amount of protein assayed by Biuret was 98.53 mg per gram of dry substrate when extracted with distilled water and reached up to 167 mg/g dry substrate when using a mixture of NaOH and Sodium carbonate-bicarbonat buffer.

Tobacco is one of the industrial plants which plays important role in economy and income of producer contries. Potato Virus Y (PVY) is one of Viral diseases causing the most damage on tobacco plants and reduces the quality and quantity of tobacco products. Severity of damage depends on different factors such as tobacco variety and time of infection. Potato Virus Y (PVY) is belonging to the Potyvirus genus that infecting Solanaceae plants, such as potato, tobacco and tomato. The best way to reduce damage this virus, is breeding resistant or tolerance cultivars to this virus. DNA markers Linked with resistance genes and have high polymorphism Like SSR markers can be helpful in breeding programs on resistant cultivars. This study was performed to evaluation of tobacco relative resistance to Potato Virus Y using Microsatellite markers (SSR). The implementation of this research, the VAM variety (resistant to PVY) was crossed with susceptible cultivar K326. F1 seeds were planted and F2 seeds were obtained in 2011. F2 seeds were transplanted in 2011 in Tirtash tobacco research farm. These plants were inoculated with pure isolates PVY strain O. Leaf samples were prepared based on field symptoms from 10 tolerant and 10 susceptible plants. All the samples tested with ELISA and resistant and susceptible genotypes were identified. Genomic DNA of resistant susceptible genotypes to PVY, were extracted separately to Dellaporta method. Then, 100 SSR markers were tested on two parental DNA. Results showed that among 100 pairs of primers used in this study, 28 primer pairs were able to show different model between the resistant and susceptible parents. In the next stage of the 28 markers were tested on sensitive and resistant bulk to identifying marker genes associated with resistance. It was found that three SSR markers PT30002, PT20165, PT20357 have the highest polymorphism and can be considered as genetic markers for screening resistance to PVY in tobacco.