Iranian Journal of Biotechnology
Volume:8 Issue: 2, Spring 2010

Potato (Solanum tuberosum L.) an agro-economically important food crop in the world, is sensitive to many fungal pathogens including Rhizoctonia solani (AG-3), the causal agent of stem and root rot diseases. Chitinase and glucanase are cell wall degrading enzymes which have been shown to have high antifungal activity against a wide range of phytopathogenic fungi. In the present study, plasmid pBIKE3 harboring a double-gene cassette containing the chitinase (chit42) and β-1,3-glucanase (bgn13.1) genes was constructed. In this construct, the chit42 gene is located between the CaMV 35S promoter and nos terminator derived from pBI121, while the bgn13.1 gene is downstream of a modified CaMV 35S promoter, followed by the nos terminator both of which were derived from the pRTL plasmid. Micro-tubers of potato plants (the Savalan cultivar) were transformed with the pBIKE3 construct via the Agrobacterium delivery system. Integration of these two genes into the potato genome and their expression at the transcriptional level was confirmed by polymerase chain reaction (PCR) and reverse transcription-PCR (RT-PCR). The radial diffusion assay showed that the heterologous expressed chitinase and glucanase enzymes demonstrated antifungal activity on R. solani (AG-3).

A reliable method is described for rapid proliferation of axillary shoots from nodal segment of mature field-grown aromatic and medicinal plant Vitex negundo through in vitro culture. Of different concentrations of N6-benzyladenine (BA) evaluated as supplements to Murahige and Skoog (MS) medium, BA at 1 mg/l was effective in enhancing bud break and shoot regeneration. Response of explants was also influenced by explanting seasons. Early bud break and enhancement of shoot regeneration could be achieved by supplementing phloroglucinol (PG) at 100 mg/l and silver nitrate (20 mg/l) in addition to BA (1 mg/l). These compounds appear to have synergistic effects on enhancement of high frequency multiple shoot regeneration. The number of shoots obtained in the presence of AgNO3 and PG was significantly higher (15.12±0.33) than other media tested. The efficacy of culture establishment with high percentage of responding explants is depending on seasonal factors in addition to culture conditions. Among the different concentrations of IBA (0.1, 0.5 and 1.0 mg/l) tested, 0.5 mg/l IBA was more effective for inducing roots within two weeks of culture and 85% of the microshoots produced long healthy root system. Ex vitro rooting of micropropagated shoots was achieved by treating the basal end of the microshoots in IBA solution (4000 ppm) followed by planting in plastic pots filled with a mixture of soil, sand and vermicompost (6:2:1). Rooted microshoots were successfully established in the field with 80 percent survival rate after three months.

Fusarium wilt caused by Fusarium oxysporum Schltdl. f.sp. phaseoli (Fop), is one of the important diseases of the common bean in Iran and many of the bean growing countries. Incidence of the disease has been reported in tropical and semi-tropical regions of the world. This study was carried out to characterize genetic diversity of F. oxysporum isolates from the common bean by vegetative compatibility test and random amplified polymorphic DNA (RAPD) analysis, as well as determining the frequency of mating type alleles (idiomorphs) in F. oxysporum population. In the present study, a total of 82 Fusarium sp. isolates collected from the major common bean growing areas of Tehran province were purified and identified. Among the isolates, 20 (24.4%), which were identified as F. oxysporum, were selected for the study. Nitrate non-utilizing (Nit) mutants were used to identify vegetative compatibility groups (VCGs). Ten VCGs were identified based on heterokaryon formation, 6 of which were single isolate VCGs. In order to study genetic diversity by RAPD, 6 random primers were used. Amplicons were analyzed and a dendrogram was created. The 20 isolates were grouped into 10 fingerprint groups. The results of VCGs and RAPD analyses suggest high genetic diversity among the 20 F. oxysporum isolates obtained from the bean samples. To determine the frequency of mating type alleles (MAT1-1 and MAT1-2) in F. oxysporum isolates, PCR was carried out using two pairs of specific primers. Among the tested isolates, two isolates were identified as MAT1-1 and 15 isolates as MAT1-2. There may be a direct relationship between the existence of both mating types idiomorphs and the large genetic diversity among the isolates.

Several species of Magnetotactic bacteria have been discovered recently. These bacteria synthesize intracellular magnetic nanoparticles in specific sizes and shapes and arrange them in chains. These particles called magnetosomes and can be used for drug-delivery, cell-targeting and hyperthermia. Magnetotactic bacteria navigate along the magnetic field; this process is known as ‘magnetotaxis’ which may be very useful in robotic technology. In this research, two species of magnetotactic bacteria were isolated; freshwater specimens were collected from Karkheh River and marine specimens were collected from the Caspian Sea. After enrichment, two species of the Magnetotactic bacteria were isolated using a specific solid media culturing. The response of the two isolates to the magnetic field was observed by an optical microscope. SEM photos showed that the freshwater and marine bacteria are rod shaped. TEM images showed chains of magnetosomes in the bacterial cells. Also magnetic behavior of the magnetosomes was investigated by alternating-gradient-force magnetometer, indicating that the magnetosomes have superparamagnetic-to-single-domain properties.Key words: Magnetotactic bacteria; Isolation; Characterization

Bacillus sp. GUF8, isolated from acidic soil samples of a tea farm was identified as Bacillus cereus, based on 16S rDNA sequencing and standard bacterial identification methods. Following optimization of enzyme production, the resulting α-amylase was purified by acetone precipitation and ion exchange chromatography. Consequently, thermostability and kinetic parameters of the purified enzyme were determined. The temperature profile of the enzyme indicated a very broad temperature range (from 10 to 70°C) with 50°C representing the optimum temperature for enzyme activity, which is different from those of the known Bacillus α-amylases. This enzyme was optimally active at pH 6.0 and retained 75 and 50% of its maximal activity at pH 8.0 and 9.0, respectively. It was also strongly inhibited by Zn2+ and partially inhibited by Ni2+ and ethylenediaminetetraacetic acid (EDTA). The a-amylase enzyme was found to hydrolyze starch forming various maltooligosaccharides, such as maltose (G2) and maltopentaose (G5) as major products.

Fusarium head blight (FHB) caused by Fusarium graminearum is a serious disease of wheat (Triticum aestivum L.), through which grain quality losses are induced by fungal trichotecene mycotoxins such as deoxynivalenol (DON). A class of plasma membrane localized ABC transporter proteins related to the yeast PDR5 (pleiotropic drug resistance5) efflux pump seems to be responsible for partial resistance against trichothecenes in wheat. In order to develop and map a PDR5-specific marker linked to Fusarium head blight resistance in wheat, F3 and F5 generations obtained from a cross between ‘Wangshuibai’ and ‘Seri82’ were used. The analysis of nucleotide sequences of OSPDR5 revealed a high homology to the wheat EST BT009500. Among ten primer pairs developed from this PDR5-like EST, one was polymorphic between the parental lines. This PCR-based marker associated with FHB resistance in a ‘Wangshuibai’ derived mapping population. In this study, Composite Interval Mapping (CIM) analysis detected a QTL in the map interval of Xm12p17_2-Xpdr5 (consisting of the developed PDR5-like gene locus) on chromosome 6BS. This QTL accounted for up to 18% of AUDPC variation. Real-time quantitative analysis showed that wheat PDR5-like gene expression was up-regulated during the post-inoculation period of 96 hours in the spike. Our results are in agreement with the hypothesis that the PDR5-like gene may be considered as a FHB resistance gene candidate in wheat.

The possibility of occurrence of some rearrangements inside and or around coxII and atp6 genes and their relationship with male sterility in rice lines having wild abortive (WA) cytoplasm was studied. Two sterile (IR58025A and IR62829A) and two maintainer lines (IR58025B and IR62829B) were used. Radioactive Southern blotting was employed to carry out the experiments. The hybridization of coxII gene to the HindIII digests of total DNA from the maintainers and sterile lines was the same, indicating that this gene was not possibly related to male sterility. However, when the atp6 gene was used as a probe and the same lines and procedures were employed, the probe hybridised to different fragments in maintainer lines compared to the sterile lines and their hybridization pattern was totally different. These findings could be interpreted as the occurrence of some rearrangements inside and or around atp6 gene and its probable involvement in the male sterility of WA rice.

This study was conducted to optimize a highly efficient mRNA transfection into dendritic cells (DC) derived from esophageal squamous cell carcinoma (ESCC) patients. Applying an electroporation technique, in vitro synthesized Green Fluorescent Protein (GFP) mRNA was transfected as an indicator into the DCs derived from a healthy donor. Flow cytometry revealed 84.9% transfection efficiency for DCs transfected with GFP mRNA. Optimized condition (500V/300 ms) yielded 79.8% efficiency in transfecting GFP mRNA into DCs from ESCC patient. Applying this efficient method, tumoral mRNA was transfected into DCs. T cells were then primed with tumor RNA/DCs and cytotoxicity assay revealed significantly higher lyses of tumor/DC vs. Mock DC; approving the optimized method for further establishment of the preclinical phase of DC-based immunotherapy for ESCC.Keywords: Immuno-gene therapy; Esophageal squamous cell carcinoma (ESCC); Electroporation; mRNA transfection; Dendritic cell; GFP