Seed-expressed fluorescent proteins as versatile tools for easy (co)transformation and high-throughput funtional genomics in Arabidopsis

A.R. Stuitje, E.C. Verbree, K.H. van der Linden, E.M. Mietkiewska, J.P.H. Nap, T.J.A. Kneppers

    Research output: Contribution to journalArticleAcademicpeer-review

    Abstract

    We demonstrate that fluorescent proteins can be used as visual selection markers for the transformation of Arabidopsis thaliana by the floral dip method. Seed-specific expression of green fluorescent protein (GFP) variants, as well as DsRed, permits the identification of mature transformed seeds in a large background of untransformed seeds by fluorescence microscopy. In planta visualization of transformed seeds in siliques shows that susceptibility to floral dip transformation is limited to a small, defined window in flower development. In the competent stage, the random transformation of up to 25% of the seeds within a single silique may occur. The use of fluorescent proteins with different spectral characteristics allows a rapid identification and genetic analysis of seeds that have received multiple genes-of-interest in co-transformation experiments. The data reveal that co-transformation does not occur at random, since the co-transformed genes are integrated at a single genetic locus in ¿ 70% of the cases. This genetic linkage of the co-transformed genes greatly simplifies metabolic pathway engineering by reverse genetics in Arabidopsis. Additional advantages of using visual selection instead of antibiotic resistance include a rapid identification of the effect of the T-DNA insertion or the transgene on seed development and/or germination. This technology, of tagging and identifying transformed seeds by fluorescence provides a novel high-throughput screening system with many potential applications in plant biotechnology.
    Original languageEnglish
    Pages (from-to)301-309
    JournalPlant Biotechnology Journal
    Volume1
    Issue number4
    DOIs
    Publication statusPublished - 2003

    Keywords

    • agrobacterium-mediated transformation
    • t-dna integration
    • vacuum infiltration
    • transgenic plants
    • brassica-napus
    • thaliana
    • recombination
    • tumefaciens
    • gene

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