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Intl. Res. J. Appl. Basic. Sci. Vol., 3 (3), 642-649, 2012secondary metabolites in compared with suspension culture and even native cells. Due to receive a pieceof bacterial plasmid such a rolB gene Plant has these features.When the bacterium infects the plant, the T-DNA between the TR and TL regions of the Ri-plasmid in thebacterium is transferred and integrated into the nuclear genome of the host plant. Virulence genes thatform the vir region of the Ri plasmid, and chv genes found on bacterial chromosomes mediate transfer ofT-DNA(Kayser & Quax 2007). Recalcitrant plant species for transformation can be transformed byinducing the vir genes of the bacteria by signal molecules or it can be achieved in vitro by co-cultivatingAgrobacterium with wounded tissues or in media that contains signal molecules The transformationprocess produces a valuable by-product, hairy root, which will form at or near the site of infection(Chilton et al.1982).Hairy roots grow rapidly , show plagiotropic growth, and are highly branched on phytohormone-freemedium. These fast growing hairy roots have high biosynthetic capacity, in particular root-inherentmetabolites as found in original roots in vivo and they also can be used as a continuous source for theproduction of valuable secondary metabolites. In contrast with the instability and variability ofunorganized plant cell suspensions, transformed roots are able to regenerate whole viable plants andmaintain their genetic stability during further subculturing and plant regeneration. Many researchers havemade assiduous attempts to produce desired metabolites derived from the hairy roots (Kittipongpatana etal. 1998).To succeed in establishing a hairy root culture system for a certain plant species, several essentialconditions should be taken in consideration. Several factors including the type and age of explants,concentration and different strains of Agrobacterium rhizogenes are effective in production and increaseof transgenic hairy roots of the plants.In this research, we report the production of transformed hairy roots in portulaca oleracea byAgrobacterium rhizogenes in In vitro condition and examined Factors affecting the increasing inductionof transgenic hairy roots of P.oleracea and to increase noradrenaline production in transgenic hairy rootsof this plant than normal roots.Material and methodsPlant materialportulaca oleracea seeds were taken from garden plants of Hamedan in October 89 and transported tothe laboratory under Laminar flow condition ,sterilized by 2% sodium hypochloride and rinced withsterile distilled water. Then in a sterile Petri dishes containing liquid 1/2 MS medium cultured.bacterial preparationfor preparation of Suspension cultures of the Agrobacterium rhizogenes, which were obtained from theBiotechnology Research Center, Karaj, Iran, A colony of bacteria Agrobacterium rhizogenes grown onsolid LB medium was removed and were cultured In 10 ml of liquid LB medium containing 50 mg/l ofantibiotic rifampin under sterile conditions. For the next steps LB liquid medium contaminated withbacteria Was transferred to a growth chamber with 28 ° C and were shaked on the Shaker 110 rpm speed(RPM) for 24 h in dark conditions, till Bacteria to grow and be active. After this period, Concentration ofbacterial suspension was determined by using a spectrophotometer modle Lambda 45UV/Visibel inOD 600 (Optical Density). For transformation, OD was set at a wavelength of 600 nm between 0/2 to 0/8 ,And then used for infection of plant explants.Induction and production of hairy RootThree types of tissue (leaf, stem, root) from 14 to 56 day old seedlings germinated were used as anexplants for infecting. Explants were sectioned for inoculation with bacteria and then were placed in LBsuspensions containing active bacteria for 20 minutes. After the completion of infection period forbacteria cocultivation, explants were transferred to Petri dishes containing 1/2MS medium withouthormones, antibiotics and were put in a darkness growth chamber with temperature 25 ° C. At the sametime, the number of explants as a witness (Without putting the bacteria in suspension) were put in 1/2MS. After 48 hours cocultivation, to remove the bacteria, explants were transferred to 1/2 MS mediumcontaining the antibiotic cefotaxime.
Intl. Res. J. Appl. Basic. Sci. Vol., 3 (3), 642-649, 2012Factors examined in transgenic optimizationIn order to increase transgenic in P. oleracea some factors studied in a completely randomized design.Each of the treatments evaluated in triplicate and for each repeated was considered 10 explants. To reviewinformation obtained, Excel 2007 software and software SAS 9.1 were used for diagramming andanalysis of variance test treatments, respectively.Factors examined included:1- Type of explants: different explants of leaves, stems and roots were used.2- Age of explants: Seedlings of various ages 14, 28 and 56 days were studied.3- Different strains of A.rhizogenes : AR15834A4C318 9435 to determine the best strain were usedfor plant contamination.4- Concentration of bacteria: Suspension of bacteria at concentrations of 2/0, 4/0 6/0 and 8/0 was used toinfect.Molecular confirmation of transgenic rootsAfter the development of hairy roots, for molecular confirmation of transgenic roots, after several stagesof the subcultured in antibiotic-containing medium and ensure complete removal of bacteria, DNAextraction was performed by the method of Cai et al (Cai et al.1997) and PCR technique was used withspecific primers rol B gene in the T-DNA into the plant genome.Extraction and determination of noradrenaline from the transgenic rootsAfter the establishment of hairy roots, four lines were selected from the roots that have better growth andthey were cuted in to 3*3 pieces and then were placed in the 250 ml Erlenmeyer flask containing 50 ml of1/2 MS liquid medium. Natural root ,which were produced from control explants and their growth wasmuch lower compared with hairy roots, were in flasks as above. All of the Erlenmeyer flask were sealedwith parafilm and were put at 25 ° C with a growth period of 16 hours light and 8 hours dark on anelectric shaker with 110 rpm for 4 weeks. After 4 weeks, the method of Chen and colleagues was used forextraction of noradrenaline (Chen et al.2003). Also the HPLC system ,(Kanyvr,germany) with c18column(25 cm × 4.6 mm)and uv detector, was used for quantitative determination of noradrenaline. Themobile phase consisted of 0/02 molar solution of KH2 PO4 (95 percent), acetonitrile (5%), pH 3andwavelength was 280 nm.Three replicates for each treatment were considered and tests conducted in a completelyrandomizeddesign.Results and discussionInduction of transgenic rootsInitial results from sterilized seeds and cultured in 1/2 MS liquid medium showed germination seeds andseedling production after 3 days, so these seedling were used for the production of explants. The resultsof variable age of seedlings show that the 14-day seedlings more than seedlings 28 and 56 days reacted toAgrobacterium rhizogenes. The frequency of transgenic in 14-day seedlings with 62% was at the highestlevel that showed significant difference with the transgenic seedling 28 and 56 days at 5% level( fig 2-a).Results in the fig 2- b show that ,the tissue of the leaves, stems and roots were used as explants, levels intransgenic leaf explants with petioles was higher than other explants so at 5% level showed significantdifferences with other explants.
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