Iranian Journal of Biotechnology
Volume:9 Issue: 1, Winter 2011

Sclerotinia sclerotiorum is a phytopathogenic fungus which causes serious yield losses in canola. A pathogen inducible-promoter can facilitate the production of Sclerotinia-resistant transgenic canola plants. In this study, the “gain of function approach” was adopted for the construction of a pathogen-inducible promoter. The synthetic promoter technique was used, which involved the insertion of the dimerized form of the cis-acting element (F) upstream of the minimal CaMV 35S promoter, which drives the expression of the Beta-glucronidase (GUS) gene. The pGFF construct containing this synthetic promoter (SynP-FF) was used for stable transformation of the canola plant. Fluorometric GUS expression analysis indicated that the SynP-FF promoter is responsive to methyl jasmonate and S. sclerotiorum treatments. The results of histochemical GUS expression patterns showed strong reporter expression in leaf, flower and stem tissues of canola. Hence, the SynP-FF synthetic promoter, carrying fungal pathogen-inducible features, could be considered as a valuable tool for controlling the expression of transgenes to improve resistance against the same lifestyle pathogens

Nuclear simple sequence repeats (nSSRs), together with 16 different enzyme loci, were used to analyze genetic diversity and differentiation among beech (Fagus orientalis Lipsky) populations along two altitudinal gradients in Hyrcanian forests of Iran. Both enzymes and nSSR analyses revealed a high level of genetic diversity in natural populations of F. orientalis. The genetic diversity, estimated by expected heterozygosity, was 0.19 (by enzymes) and 0.65 (by nSSRs). Genetic variation across both markers did not reveal genetic structuring along altitudinal transects. There was less genetic variation among altitudinal gradients within transects compared to transect sites. Differentiation assays and analysis of molecular variance (AMOVA) indicated that there was a relatively low genetic differentiation among populations, and just 1% and 5% of the genetic variation occurred among populations by nSSR and enzyme data, respectively. Mantel tests showed that there was not a significant correlation between the genetic distances among populations and the distance of elevation. The results of the present study indicate that the relatively low genetic differentiation among F. orientalis populations at different elevations was not caused by ecological factors. These patterns suggest that higher rates of gene flow along altitudinal gradients within transects, than between transects; a process that could question altitudinal adaptation.

Salinity stress is one of the most widespread soil problems next to drought, in rice growing areas. Reducing Sodium (Na+), while maintaining Potassium (K+) uptake in rice are traits that would aid in salinity tolerance. Therefore, the identification of quantitative trait loci (QTLs) associated with those for Na+ and K+ uptake, will enable breeders to use marker-assisted selection to transfer QTLs into elite lines in rice improvement programs. In view of this, 62 advanced backcross-inbred lines (BILs), at the BC2F5 generation, derived from the cross of Tarome-Molaei (salt tolerant) and Tiqing (Salt sensitive), were used to identify the QTLs involved in salinity stress tolerance, using SSR markers. Advanced backcross inbred lines along with their parents were evaluated for six parameters viz. Sodium (Na+) and Potassium (K+) in roots and shoots and the Na+/K+ ratio, using the modified Yoshida’s nutrient solution at an electrical conductivity of 6 and 12 dS/m. A total of 114, out of 235 simple sequence repeats (SSRs) markers that showed polymorphism in the parents, were used to genotype the BILs. A linkage map was constructed with an average interval of 15.3 centiMorgan (cM) between the markers, spanning 1747.3 cM across all 12 rice chromosomes. Using the composite interval mapping (CIM) and a minimum logarithm of the odds (LOD) threshold of 3.0, a total of 14 QTLs were detected as follows; on chromosome 1 (5 QTLs), 3 (1QTL), 4 (3 QTLs), 5 (2 QTLs), 6 (1 QTL), and 8 (2 QTLs) for all six traits except, Sodium (Na+) in the shoot. The phenotypic variation explained by these QTLs ranged from 9 to 30% of the total variation. A QTL (QKr1.2) for K+ content in the root was identified with the highest LOD score (7.8), on chromosome 1. This QTL explicated 30% of the total variation and was identified as a major QTL conferring salt tolerance in rice.

Evening primrose (Oenothera biennis L.) is a wild flower with high and valuable oil content. High seed shattering, indeterminate inflorescence and heterogeneous germination limit the commercial cultivation of this plant. Besides agronomical research, breeding programs can also remedy the above mentioned problems. Since traditional breeding methods take a long time, using techniques such as tissue culture and somatic embryogenesis accelerate the breeding process. In the present study, callus formation was accomplished in MS (Murashik and Skoog) medium, but no embryogenesis was observed in the presence or absence of plant hormones like 2,4-D (2,4-dichlorophenoxy) acetic acid. In contrast, B5 (Gamborg) medium containing 2,4-D induced embryogenesis. Different parts of plants exhibited good callus production potency and the hypocotyl was found as the best plant explants. In B5 medium, various concentrations of 2,4-D (0,2.26 mM, 4.52 mM, and 9.04 mM) were applied as a complete randomized design experiment with 4 replications. For embryogenesis, hypocotyls (1cm long) were cultured in B5 liquid medium at the induction phase. After 3 weeks, induced organs were sub-cultured to realization phase and the number of embryos (different stages of embryogenesis) was counted 4 weeks later. The results indicated that variation in hormone concentrations caused significant differences with respect to somatic embryogenesis. Embryo development were not observed in hormone-free media. The highest numbers of globular, heart, torpedo, cotyledonary, and total embryo was recorded at 9.04 mM of 2, 4-D. Histological studies of embryos after the realization phase revealed large nuclei and abundance of starch grains indicating the presence of embryonic cells in evening primrose hypocotyls.

The biological phosphorus removal is a microbial process widely used for removing phosphorus from wastewater to avoid eutrophication of water bodies. The study was aimed to screen the efficient phosphate reducing isolates and used to remove phosphate from synthetic wastewater using batch scale process. The three most efficient phosphate reducers were isolated and screened from eutrophic lake water and forest soil samples. The total heterotrophic bacterial analysis of the samples showed the presence of about 38 phosphate reducers based on the minimum inhibitory concentration (MIC) test. Among them, Bacillus sp. RS-1, Pseudomonas sp. YLW-7 and Enterobacter sp KLW-2 were found to be efficient in phosphate reduction. Among the individual strains, Pseudomonas sp. YLW-7 was noticed to be 68% removal in MSM with glucose at neutral pH. The consortium with combination of Bacillus sp. RS-1, Pseudomonas sp. YLW-7 and Enterobacter sp. KLW-2 was effectively removed the phosphate in the synthetic medium when compared to individual strains. The phosphate removal was observed to be maximum of 92.5% in mineral salts medium (MSM) at pH 7and 5, and 63.4% in synthetic phosphate solution at neutral pH with lactose as a carbon source by the consortium after 72 h. Thus the microorganisms may use the contaminants as nutrients and as energy sources or it may be utilized by co-metabolism. Therefore, these bacterial isolates might be used in the remediation of phosphate contaminated environments.

The continuous production of polygalacturonases (PGases) that Exo-polygalacturonase (exo-PGase) and Endo-polygalacturonase (endo-PGase) are two members of this group by a strain of Aspergillus awamori in surface culture fermentation was investigated. Surface culture fermentation is usually done in batch mode. Wheat flour acted as a good substrate for the cultivation of the fungus and production of PGases in surface culture fermentation. Fermentation started in batch mode until mycelia completely occupied the medium following growth of the microorganism, after which it was turned to the continuous mode by the introduction of fresh feed. The process continued for 34 days, and the thickness of the microbial layer on the surface of the liquid medium became almost constant after approximately one week. The production of PGases, however, continued throughout the experiment, and maximum activities of 1.2 U/ml and 0.014 U/ml were obtained for exo-polygalacturonase (exo-PGase) and endo-polygalacturonase (endo-PGase), respectively. An increase in production was observed when a similar system was used with a line for medium recycling. Lowering the residence time to 12 h decreased the exo-PGase and endo-PGase activities. Reducing the residence time from 24 h to 12 h almost halved the concentrations of the enzymes at the outlet.

Information on the genetic structure of marine species is essential for stock improvement programs. In order to analyses the genetic diversity of the Hawksbill turtle (Eretmochelys imbricte) by the microsatellite genetic method, 64 samples were caught from the beaches located in Kish and Qeshm islands. Polymerase chain reactions (PCR) of genomic DNA extracted from the samples were carried out using 5 pairs of microsatellite primers. The results of this study indicated that all 5 pairs of primers were polymorphic. Average numbers of real allele and effective allele were 4.90 and 2.99, respectively. Average rate of observed heterozygosity was 0.570 and that for expected heterozygosity was 0.616. Study of the Hardy-Weinberg equilibrium was shown the entire locus had not equilibrium except Cm3 and Ei8 locus in Kish area. Fst (0.166) and Rst (0.634) calculated by the Analysis of Molecular Variance (AMOVA) test illustrated that there are separate populations of Hawksbill turtle in this part of the Persian Gulf (Kish and Qeshm islands). It seems that Kish’s turtles live under better conditions in contrast to their Qeshm counterparts. Diminution of genetic variation within examined population decreases its adaptation to environmental alterations. We identified two different E. imbricte populations from north of the Persian Gulf.

Despite the widespread prevalence of canine parvovirus disease (CPV) in Iranian dog population, molecular diagnosis of CPV variants, and investigation of the trends of its genetic changes is a new effort. In this study 50 samples from dogs suspicious of infection with clinical signs of diarrhea and vomiting, and 25 samples from dogs suspected of infection with general symptoms such as depression and anorexia were collected from dogs presented to the veterinary clinic of Shiraz University, Shiraz, Iran. Viral DNA was extracted from feces. Three specific pairs of primers, P2, Pab, and Pb, were used in a PCR assay for differential diagnosis of the virus type. Pab primer pairs detect the new type-strains, CPV-2a and 2b. The primer pairs P2 and Pb detect CPV types 2 and 2b, respectively. Our results showed that 44 individuals with clinical signs of diarrhea and vomiting were positive for CPV-2. 39 individuals (89%) were positive for CPV-2b and 5 individuals (11%) for CPV-2a. Therefore, the CPV-2b was identified as the predominant virus type. All dogs without symptoms of diarrhea and vomiting were CPV-negative. The relationship of breed, age and sex with PCR results was not significant (P>0.05). For the first time in the country, the causative agent of CPV-2 was identified, and presence of new antigenic variants, CPV-2a and CPV-2b was confirmed.

In plants, secondary metabolites and polysaccharides interfere with genomic isolation procedures and downstream reactions such as restriction enzyme analysis and gene amplification. The removal of such contaminants needs complicated and time-consuming protocols. In this study, a simple, rapid and efficient method for leaf DNA extraction was optimized. This method use small amount of plant material to reduce inhibitory agents (alkaloids, phenolic). The procedure involves homogenization of the plant leaf in extraction buffer, incubation at 60ºC, extraction by chloroform: iso-amyl alcohol and finally DNA precipitation by cold isopropanol. The results showed that the extracted DNA could be used directly for PCR.