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.
Keywords
Agrobacterium-mediated transformation - Antibiotic-free - Dark embryo induction