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  • Title: Co-transformation of grapevine somatic embryos to produce transgenic plants free of marker genes.
    Author: Dutt M, Li ZT, Dhekney SA, Gray DJ.
    Journal: Methods Mol Biol; 2012; 847():201-13. PubMed ID: 22351010.
    Abstract:
    A cotransformation system using somatic embryos was developed to produce grapevines free of selectable marker genes. This was achieved by transforming Vitis vinifera L. "Thompson Seedless" somatic embryos with a mixture of two Agrobacterium strains. The first strain contained a binary plasmid with an egfp gene of interest between the T-DNA borders. The second strain harbored the neomycin phosphotransferase (nptII) gene for positive selection and the cytosine deaminase (codA) gene for negative selection, linked together by a bidirectional dual promoter complex. Our technique included a short positive selection phase of cotransformed somatic embryos on liquid medium containing 100 mg/L kanamycin before subjecting cultures to prolonged negative selection on medium containing 250 mg/L 5-fluorocytosine.
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