Seed-expressed fluorescent proteins as versatile tools for easy (co)transformation and high-throughput functional genomics in Arabidopsis.
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 approximately 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.
Stuitje
Antoine R
AR
Department of Genetics, Institute for Molecular Celbiology, Vrije Universiteit, de Boelelaan 1087, 1081 HV, Amsterdam, The Netherlands. stuitje@bio.vu.nl
Verbree
Elizabeth C
EC
van der Linden
Karin H
KH
Mietkiewska
Elzbieta M
EM
Nap
Jan-Peter
JP
Kneppers
Tarcies J A
TJ