مجله بیوتکنولوژی کشاورزی
سال دوم شماره 1 (پیاپی 3، بهار 1389)

Cotton (Gossipium spp) is ranked first among the fibers producing plants in the world and is also considered as an important source of oil. Although significant progress has been made in traditional cotton breeding programs, but there are still limitations including limitedgene resources, lack of crosses, lack of efficient selection and high timeconsumption.Through recent advances in gene transformation to cotton, not only facilitated transformation of useful genes has become possible, but also promising results have been obtained in gene identification as well asfunction and regulation studies. Agrobacterium mediated transformation is one of the most used transformation methods in plants, including cotton. Several factors could affect transformation efficiency such as bacterial strains and concentrations, added phenolic regent in plant culture medium, plant species and genotypes, use of plant growth regulators, explants type, light and temperature during co cultivation, antibiotics, wounding the target tissue and suitable method for selection of transgenic cells. Hypocotyls, cotyledons and cell suspensions are used as explants in cotton transformation using Agrobacterium. These explants have some limitations such as low regeneration rate and genotype dependence. Therefore, regeneration could be obtained in only a limited number of species using these explants. On the other hand, establishment of a regeneration system using shoot apex meristem in cotton has increased its transformation efficiency. This article is trying to review the factors shown to be effective in gene transformation into cotton.

Thioredoxins (Trxs) are small heat-stable proteins that participate in dithiol-disulfideexchange reactions. In contrast to other organisms, plants contain six different Trx types: f, m, x, y, o and h. The h-type Trx consists of multiple forms that involved in differentprocesses such as cellular protection against oxidative, biotic and abiotic stresses. Twothioredoxin h-type genes, called VvTrxh4 and VvCxxS2, were isolated of grape (Vitisvinifera L. cv. Askari) berry tissue and were cloned into pUC19 plasmid vector. Reversetranscription was carried out using Polymerase Chain Reaction (RT-PCR), to analyzebiochemical, structural and phylogenetic characteristics. Nucleotide sequence analysisrevealed that the open reading frame of VvTrxh4 and VvCxxS2 genes are 345 bp and 381bp long, respectively and encode for proteins of 114 and 126 amino acid residues,respectively. Protein sequence analysis showed that VvTrxh4 gene contains a typicalcatalytic site WCGPC, whereas VvCxxS2 gene harbors the non-typical active site WCIPS.The calculated molecular mass and the predicted isoelectric point of the deducedpolypeptides for both VvTrxh4 and VvCxxS2 were 12.76 kDa and 5.22, and also 14.25kDa and 4.68, respectively. Structural analysis showed that deduced proteins contain aidentical 3D structure, whereas phylogenetic study of cloned genes with thioredoxin fromother plants revealed that these genes are belonging to different subgroups. In the present research project, VvTrxh4 gene belongs to class IA, while VvCxxS2 gene belongs to class IIIB from h-type thioredoxins.

Safflower, Carthamus tinctorius L., is a member of Asteraceae, cultivated mainly for itsseed and flower, which are used as edible oil and for medicinal applications. Safflower is aresistant plant to the environmental stresses; therefore scientists use this plant as a modelfor studying the mechanism of plant resistance to the environmental stresses. Antioxidantenzyme system is one of the defense mechanisms that plants use for various environmental stresses. In this study some biochemical properties of catalase, an antioxidant enzyme system, from safflower and it sensivity to azide and cyanide was investigated. Carthamus tinctorious L.cv.IL-111 was grown hydroponically in perlite for 40 days and catalase was extracted from their leaves using phosphate buffer 0.1 M, pH 7.2. Study of pH profile, temperature and different concentration of substrate and inhibitors (azide and cynide) on catalase activity in Safflower and non-denaturant polyacrylamide gel electrophoresis of crude extract, showed that at least two isoenzymes of catalase were present in safflower leaves with optimal pH at 6.5 and 8.5, respectively. Isoenzyme active at pH 8.5 was more resistant to temperature in comparison to isoenzyme active at pH 6.5. Effects of inhibitors (azide and cynide) on catalase activity showed that isoenzyme active at pH 8.5 was more sensitive to both inhibitors (azide: 4.6 fold, and cynide: 2.6 fold) in comparison to isoenzyme active at pH 6.5.

Streptokinase is the most utilized drug for thrombolytic therapy. However, due to its nonhuman origin and nonspecific activity in the absence of fibrin, its application may encounter side effects such as allergic reactions and hemorrhage respectively. It is suggested that some truncated forms of streptokinase may show better fibrin-specificity and decreased antigenicity while retaining most of the enzymatic activity. In the present study, different truncated forms of streptokinase gene,”Skc60-386 and skc143-386” together with the intact form of skc (skc1-414) were cloned in T5 and T7 based-E.coli expression vectors, “pQE30 and pET41a” respectively. While expression of intact streptokinase could equally be achieved in both systems, but pET41a was the only effective system to produce truncated forms. pET41a vector contains three tandem tags before multiple cloning sites which possibly prevent intrinsic problems encountered in heterologous recombinant protein expression in E.coli. Analyses of the purified proteins by SDS-PAGE and Western blotting evidenced for the true expression and the expected MWs. While caseinolysis method was not able to demonstrate exact difference between specific activity of SK143-386, SK60-386 and SK1-414 but truncated molecules showed high reduction of specific activity (83 to 91 percent) compared to the full-length streptokinase by more exact techniques such as colorimetric assay. This study provided a primary comparative study for expression of streptokinase and its truncated forms and analyses of their specific activity. Thus further analyses would determine their specificity to fibrin and antigenicity.

Bone morphogenetic protein 15 (BMP15) is member of the transforming growth factor-beta superfamily that has crucial roles in fecundity of goat and sheep. Previous investigations confirmed that the fecundity mutations of sheep presented in goat. Different mutations in the bone morphogenetic protein-15 (BMP-15) gene has increased ovulation rate and infertility in sheep. To illuminate polymorphisms in BMP15 gene, was characterized the coding region of BMP15. Two exons (1 and 2) encoded prepropeptide of 394 amino acids in BMP15. Blood sample collected randomly from 100 goats from Red Jabalbarez goat from 10 farms and genomic DNA was isolated using the DNA kit. Two primer pairs were used to amplify genomic DNAof Bmp15 gene, and the PCR products were separated on agarose gels. The PCR products were electrophoresed and visualized by Ethidim Bromide staining. The results showed that amplication products had good specificity, which could be directly analyzed. Single nucleotide polymorphism of FecXB and FecXG loci in BMP-15 gene were determined using PCR-RFLP technique. In this study, there was no evidence of mutation in FecXB and FecXG in these goats, all of which were monomorph for exon 2 of BMP-15 gene. Further investigation should be directed at other loci of BMP-15 gene or other genes, using larger sample sizes.

Production of transgenic wheat (Triticum Aestivum) was studied by chitinase and glucanase genes by Agrobacterium-mediated transformation and also pBI121 plasmid containing neomycin phospho transferasa selectable marker gene under control of Nos promoter. In addition, chitinase and glucanase genes were individually expressed under control of CaMV35S promoter. Immature embryo explants excised from seeds then were cocultivated with bacterial suspension containing the recombinant plasmid and they were placed to callus induction medium supplemented with 50 mg/l kanamycin. Embryonic calluses, were selected and they were transferred to regeneration medium with 25 mg/l kanamycin in order to producing shoots and roots. Maximum percent of transformation by Agrobacterium mediated transformation was 0.31% for transformed ARTA with C58 strain and then 0.074% for transformed ARTA with LBA4404. There was not any transgenic plant in other cultivars or strains and transformation percent was 0%. PCR and Dot blot analysis showed that the putative transgenic plants consist at least one copy of either, chitinase, glucanase and neomycin phospho transferase genes in their genome in comparison with control plants.

After cereals, oilseed crops are included as the second source of supplying calorie forhuman populations. In recent years, safflower was considered due to quality and highcontent of fatty acids. For optimization of tissue culture of safflower, an experiment wascarried out as a factorial arrangement in a completely randomized design with threereplications in 2008-2009. Callus induction and shoot regeneration percentage ofcotyledons and hypocotyl explants were measured on basal MS medium for two cultivars(Dincer and Sina) of safflower in different concentrations of NAA and BAP hormones.Analysis of interaction effects indicated that the cultivars were independent in response ofdifferent level of NAA. Mean comparison showed that the highest percentage of callusinduction occurred on MS medium containing 0.5 mg/l NAA and 0.5 mg/l BAP (94.33%)and 1 mg/l NAA and 1 mg/l BAP (97%). Furthermore, hypocotyl explant of Dincer has agood response to callus induction. The highest percentage of shoot regeneration also wasachieved on MS medium supplemented with 0.1 mg/l NAA and 2 mg/l BAP fromcotyledon explant of Dincer.

GF677 hybrid is one the suitable somaclone rootstock for almond and peach in worldwide, and its mass-propagation is demanded highly. For its in vitro micropropagation MS medium is used, in which it critically involves with the some problems such as low shooting efficiency, vitrification and rosetting growth. In order to overcome to these problems the effects of three concentrations of pectin, three types of media and three plant growth regulators (PGRs) at different concentration on the rate of adventitious shooting and quality using the axial buds and factorial experimental design under in vitro condition was investigated. The results indicated that the rate of adventitious shoot regeneration on TK and WPM media contain pectin is high and on MS contain pectin is low; but on contrary, the rate and quality of normal growth of shootlets was vice versa. The combination of 0.5mg/l ZR and 0.25 mg/l BAP on MS contain 0.5% pectin exhibited as the best treatment for the shooting rate and normal growth of shootlets, in which it seems that the BAP mostly responsible for shoot regeneration and normal leaves development and the ZR responsible for inter node growth and increasing shoot length. However, IBA has no significance effect on mentioned characters in comparison with other PGRs in this context.