Biological, chemical and mathematical sciences

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Browsing Biological, chemical and mathematical sciences by Subject "Agricultural biotechnology—Research"

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    Production of transgenic eggplant (Solanum Melongena L.) resistant to herbicide Bromoxynil
    (Universiti Teknologi Malaysia, 2013) Taghipour, Farhad
    Eggplant (Solanum melongena L.) is an economically important vegetable crop grown in various tropical and temperate parts of the world. Bromoxynil is a nitrile herbicide which inhibits electron transfer in photosystem II during photosynthesis and consequently led to leaf necrosis in eggplant. Synthetic bxn gene was ligated into pGS21a expression vector and introduced into E.coli strain BL21(DE3)pLySs for in vitro gene expression test and the recombinant protein was evaluated by SDS-PAGE. The synthetic bxn gene fragment was also cloned into pCAMBIA 1301 for plant genetic transformation. The cloning was carried out using standard method and the ligated bxn gene was checked by double digestion and sequencing analysis. The recombinant plasmid was designated as pCAMBIA(bxn+). pCAMBIA(bxn+) was then introduced into Agrobacterium tumefaciens strain LBA4404 and used for genetic transformation of tobacco control plant and target eggplant. The integration of bxn gene in tobacco genomic DNA was proved by PCR and Southern blotting followed by bio-assay test. Therefore, the genomic DNA of transformed tobacco was used as a positive control in molecular analyses of transgenic eggplant production. In vitro tissue culture of eggplant was established as the prerequisite for genetic transformation using cotyledon and leaf explants. Cotyledon explants was selected with efficiency of 86.67±6.67 % and mean of 4.3±0.33 in shoot regeneration than cultured leaf explants for genetic transformation. Shoot regeneration medium number 10 (SRM10) supplied with 0.5 mg/L NAA, 0.1 mg/L BAP and 2 mg/L TDZ in combination with MS medium was selected and used for regeneration of transformed cotyledon explants. The genetic transformation of 100 cotyledon explants of eggplant was carried out with 9% efficiency. The integration of synthetic bxn gene in eggplant genome was checked by PCR and Southern blotting. The bio-assay test showed plant resistance up to 50 mg/200ml ratio of [bromoxynil:water (1:4)]. This phenomenon suggests the eggplant plantlets successfully transformed with synthetic bxn gene