Use of fused gfp and gus reporters for the recovery of transformed Medicago truncatula somatic embryos without selective pressure
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We developed an alternative methodology for in vitro selection of transgenic Medicago truncatula cv. Jemalong plants using a bifunctional construct in which the coding sequences for the green fluorescent protein (GFP) and the β-glucuronidase protein (GUS) are fused. An Agrobacterium-mediated transformation protocol was used followed by regeneration via somatic embryogenesis in the dark, to avoid the synthesis and the consequent autofluorescence of chlorophyll. This method is a clear advantage over antibiotic and herbicide selection in which survival of non-transformed tissue is commonly reported, with the reassurance that all the somatic embryos selected as GFP positive are transformed. This was subsequently corroborated by the detection of GUS activity in leaves, stems and roots of the regenerated plants. Without antibiotic selection, and performing the embryo induction in the dark, it was possible to attest the advantage of using GFP as an in vivo detectable reporter for early embryo selection. The fusion with the GUS coding sequence provided additional evidence for the transformation of the previously selected embryos.
KeywordsAgrobacterium-mediated transformation Antibiotic-free Dark embryo induction
Green fluorescent protein
Embryo induction medium
Embryo proliferation medium
Embryo conversion medium
S. Duque (BD/1164/2000), S. Araújo (BD/5225/2001) and D. Santos (BPD/21968/2005), are supported by Fundação para a Ciência e Tecnologia, Portugal. The authors wish to thank Dr. H. Spaink (Institute of Molecular Plant Sciences, Leiden) for providing the plasmid pMP2482. We also wish to thank to Dr. A. M. Vieira (Instituto Gulbenkian da Ciência, Oeiras) for helping with the software of image acquisition.
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