مجله فنآوری زیستی در کشاورزی
سال شانزدهم شماره 1 (تابستان 1396)

Papaver somniferum remains the sole commercial source for narcotic and variety of semisynthetic drugs. Thebaine 6-O-demethylase (T6ODM) and codeine O-demethylase (CODM) genes are related the dioxygenase family that occur at the end of biosynthesis of these alkaloids in this plant. For silencing of CODM and T6ODM genes in Papaver somniferum, a TRV2-based construct containing a conserved sequence of DIOX from the coding region of T6ODM and CODM was designed for simultaneously silencing of two mentioned genes. Emerging first leaves of 2 to 3-week-old seedling were infiltrated with Agrobacterium tumefaciens harboring silencing construct, or the empty vector (TRV2) as a control. Analysis of gene expression at the transcript level by real-time quantitative PCR (polymerase chain reaction) technique showed 86 and 87 percent reduction in gene expression of CODM and T6ODM compared with control plants, respectively. This study demonstrated the efficiency of this method for knockdown the transcript levels of specific BIA biosynthetic genes.

The herbicide metribuzin is a triazine that remains in soil for long periods. It contaminates groundwater pollution and causes biological toxicity that causes concern worldwide. Microbial degradation plays a major role in removing triazine. This study was done to identify and isolate local bacteria for efficient degradation of metribuzin in the soils of Zanjan province. For this purpose, sampling of corn, apple and tomato fields from depths of 0 to 20 cm in spring was done. After preparation of soil samples and culture of them in the culture medium, the bacteria were purified. The synthetic growth of bacteria showed that ZT12, ZT15 and ZT19 isolates were from 21 isolates of bacteria with the increase of herbicide, the highest growth was observed at 500 mg/l, and the most significant isolate of ZT15 was detected by optical density of, 0.8629. It was, also determined that higher concentrations of 500 mg/L reduced growth of ZT15 isolate. Results of biochemical experiments and sequence investigation of bacterial isolates by interior primer indicate that bacterial strain ZT12, ZT15 and ZT19 are a strain of Enterobacter cancerogenus, Klebsiella sp. and Staphylococcus sp., respectively. Results of tests to determine presence or absence of metribuzin degrading gene or plasmid genomes of three bacteria isolates showed that the gene had been isolated on the plasmid. Cultivation of herbicide-degrading bacterial strains for release in a polluted environment is considered as appropriate for bioremediation.

The production of doubled haploid cucurbit plants can accelerate the breeding programs of these species. In this research, anther culture of Cucurbita pepo var. styriaca was studied to produce callus and haploid embryos. Cytological studies showed that 15 mm male buds containing 9-10 mm anthers in size and microspores at mid to late-uninucleate were desirable for anther culture experiments. Three experiments were conducted as factorial based on completely Randomized Design (CRD) with three replications. In first experiment, the effect of anther orientation on culture medium as well as type and concentration of the plant growth regulators of NAA (0, 0.5 and 1 mgl-1) and 2,4-D (0, 1 and 2 mgl-1) on callogenesis percentage of pumpkin anther cultures were studied. Results showed that the culturing of anthers from convex side in a culture medium supplemented with 2 mgl-1 2,4-D and 1 mgl-1 NAA resulted in the highest percentage of callogenesis. In second experiment, the interaction effects of medium type (B5 and E20) and combination treatments of 2,4-D (0, 2.5 and 5 mgl-1) and BAP (0, 0.5 and 1 mgl-1) on callogenesis percentage were studied. E20 culture medium supplemented with 2.5 mgl-1 2,4-D and 1 mgl-1 BAP produced the highest percentage of callogenesis compared to other treatments. In third experiment, the effect of pumpkin and wheat ovary conditioned medium on callogenesis percentage from pumpkin anthers were studied in two E20 culture media (Solid and liquid). In this experiment, co-culturing of pumpkin anthers with wheat ovaries in solid culture medium resulted in the highest percentage of callogenesis compared to other treatments.

Plant lipid transfer proteins, also known as plant LTPs, are a group of highly-conserved proteins of about 8-10kDa found in higher plant tissues. As its name implies, lipid transfer proteins are responsible for the shuttling of phospholipids and other fatty acid groups between cell membranes. Here we study the expression pattern of Lipid transfer protein gene from wheat (Triticum aestivum) responding to Septoria tritici Blotch (STB) by semi quantitative reverse transcriptase-polymerase chain reaction (RT-PCR). This method is used to qualitatively detect gene expression in different cells. level of LTP2 gene expression was measured over time, from 0h to 6 days after inoculation in Wangshuibai as a resistant wheat cultivar and Falat as a sensitive one. The results showed that inoculation of wheat by M. graminicola (asexual stage: Septoria tritici) cause different level of LTP2 gene expression in both resistant and susceptible cultivars over time. The pattern of gene expression indicated that LTP2 gene is an early expression gene in resistant cultivar and induced at 24h after inoculation and in sensitive cultivare the high level expression have been seen at 6 days after inoculation. Thus, according this results can be concluded that these genes, along the main genes, increase and maintain resistance to Septoria tritici blotch in wheat. In this study we cloned LTP2 genes from both cultivars and after sequencing the blast protein was studied. Blast results confirmed that these proteins contain eight cysteines that are conserved in all LTP peptides.

This research was conducted at two different experiments to optimization of in vitro propagation Alstroemeria. Experiment 1: effect of BAP (0, 0.5, 1, 1.5 and 2 mg/l-1) and IBA (0, 0.1 and 0.2 mg/l-1) on apical buds (0.8-1 cm) grown from young’s shoots tips of Alstroemeria c.v. Fujie and experiment 2: effect of 2,4-D (0, 1, 2, 4, 6 and 8 mg/l-1), picloram (0, 1, 2, 4, 6 and 8 mg/l-1) and BAP (0 and 0.5 mg/l-1) were studied on embryonic callus induction from first seedling leaves in A. ligtu hybrid. The MS medium was used in both experiments. At experiment 1: the highest shoots number was obtained in 1 mg.l-1 BAP.1 mg.l-1 IBA (4.27). The results showed that increasing of BAP concentration caused decreasing of shoot and internode length due to apical dominant decreasing via IBA effect. The highest root number (3.25) was obtained in 0.2 mg.l-1 IBA concentration without BAP and the highest rhizome number was recorded at 1 mg/l-1 BAP without IBA (3.5). At experiment 2: maximum of compact embryonic callus (29.13%) was obtained with 2 mg/l-1 picloram and 0.5mg/l-1 BAP, followed by 2 mg/l-1 2,4-D and 0.5 mg/l-1 BAP. To callus proliferation, both types of callus were maintained on MS with 4 mg/l-1 picloram.

Chitin and chitosan are two very important biopolymer products that have so many usages in the high cost industries. Chitin Converts into chitosan via de-acetylation of chitin. It occurs by alkaline melting method in the absence of oxygen. Chemical structure change, severe environmental pollution and De-polymerization are of the major problems in producing high quality chitosan. In this study for conversion of chitin into chitosan fungus Aspergillus niger strains (ATHUM-10864), the generator of de-acetylases enzymes were used instead of chemicals. Chitosan quality was determined via elemental analysis infrared spectroscopy, X-ray tomography, molecular weight determination and estimation of crystallinity percent, color and molecular structure.The results showed 80±5% efficiency in the conversion of chitin into chitosan or de-acetylation degree of chitin. The gained chitosan contained of 44.4% carbon, 8.9% nitrogen, 2.7% hydrogen and 39.5% oxygen. The physical characteristics were as 94.5% Crystallinity and pale brown color. The chemical structure of per unit of chitosan was obtained as C6H12NO4. The results showed that replacing biological methods instead of chemicals was possible to access well quality products. It also eliminates the use of chemical materials such as concentrate sodium hydroxide that is damaging the environment.

Considering the importance of identification and maintaining of genetic diversity in native animals, four sheep breeds existing in Iran (Pakistani (25), Kermani (102), Iran Black (44) and Lory-Bakhtiyari (25)) were evaluated in terms of genetic diversity, using four microsatellite markers McMA2, HSC, OarHH35 and BM6444. Genomic DNA was extracted from 196 blood samples by optimized salting-out DNA extraction procedure and PCR was performed using four primer pairs. The results showed that all loci are polymorph. Hardy-Weinberg equilibrium using chi-square test showed that some of the various components of loci - population were in Hardy-Weinberg disequilibrium (P

Members of Fusarium species are common Fungi that can metabolize of petroleum hydrocarbons in entire the world. These fungi are resistant in oil contaminated sites.Therefore, the objectives in this study were to isolate and identify of Fusarium spp. from oil-contaminated soils in oil refinery in Kermanshah province based on the morphological and molecular characteristics. Crude oil contaminated soil samples were collected from crude oil contaminated soils in oil refinery in Kermanshah province. Fusarium spp. was isolated using peptone pentachloronitrobenzene agar (PPA) plates supplemented with streptomycin and neomycin. In this research 30 Fusarium strains were isolated. Based on morphological characters, all isolates belonged to F. falciforme, F. keratoplasticum, and F. petroliphilum which belonged to Fusarium solani species complex. Based on the sequence data from ITS rDNA analysis, Fusarium isolates were divided into three major groups. Although the data obtained from morphological and molecular studies sufficiently supported each other, the resulting phylogenetic trees based on ITS rDNA dataset clearly distinguished closely related species and distinctly separated all morphological taxa. All the Fusarium spp. related from oil-contaminated soils was reported for the first time in Iran. Existing of fungi particularly Fusarium species in crude oil soils shows the probability of degradation and consumption of oil contaminated by these fungi.

Most popular binary vectors used in plant transformation via Agrobacterium have limeted common restriction recognition sites due to large vector size which has caused difficulties in cloning of target genes. To overcome this problem, new plant expression vectors including pBI1213 with three additional recognition sites downstream of gus gene and pBI121GUS-9 with nine recognition sites between CaMV 35S and Nos terminator (by using two primers designed upstream and downstream of Nos terminator in a PCR reaction) were constructed and identified. To analyze the efficiency of new vectors both in cloning and transformation steps, gus reporter gene was cloned and the new recombinant constructs were transferred to model plant Nicotiana tabbacum L. cv. Samsun via Agrobacterium tumefaciens (LBA4404). DNA was extracted from putative transgenic seedlings, which were regenerated on selective media and analyzed by PCR method. GUS assay confirmed the expression of the transgene in leaves of transgenic seedlings. After verification of the pBI1213 and pBI121GUS-9 in transformation and expression of the reporter gene, these vectors can be used for transformation of strategic plants with commercial transgenes.

Flowering branches formation in bulb production time were reduced the quality and quantity of product. Thus, study how this phenomenon for identifying bolting resistant genotypes is important. To evaluate diversity of some center and south Iranian onion genotypes in respect to flower production, 16 accessions of Iranian onions seeds (6 genotype received from Iranian Gene bank and 10 local accessions were collected from different regions of the center and south of the country) as well as two foreign cultivars were planted in the pots and in stage of eight leaves, were treated during 45 and 70 day. From 12 SSR primers, only 10 primers can produced right bands. The average polymorphic Information Content (PIC), observed and expected heterozygosity were 0.38, 0.49 and 0.59, respectively. Cluster analysis based on molecular data using Jaccard's similarity coefficient and UPGMA method, classified the evaluated genotypes in six groups. The results of binary logistic regression showed that some of SSR markers are linked to morphological and physiological characteristics.