Use of fused gfp and gus reporters for the recovery of transformed Medicago truncatula somatic embryos without selective pressure

Abstract

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 b-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.

Agrobacterium-mediated genetic transformation of yam (Dioscorea rotundata): an important tool for functional study of genes and crop improvement

“…Our results demonstrate that both gusA and gfp can be used as reporter genes for developing transgenic yam. The use of the gusA gene as a marker for transformation is effective and is widely applied in many species, including monocots such as rice ( Wakasa et al, 2012 ) and orchard grass ( Lee et al, 2006 ), and dicots such as common bean ( Mukeshimana et al, 2013 ) and alfalfa ( Duque et al, 2007 ). The presence of an intron in the gusA gene guards against false positives that may result from expression of the gene in A. tumefaciens ( http://www.cambia.org ).…”

“…GFP fluorescence visualization is also a useful tool for selecting transgenic yam plants. It has been widely used in the transformation of many plant species such as pepper ( Jung et al, 2011 ), Petunia hybrida ( Muβmann et al, 2011 ), and alfalfa ( Duque et al, 2007 ). The fluorescent marker GFP is a highly versatile reporter gene, because the gfp gene expression can be monitored any time in living cells under a fluorescence microscope in a non-destructive manner ( Chalfie et al, 1994 ).…”

Enhanced triterpene saponin biosynthesis and root nodulation in transgenic barrel medic (Medicago truncatula Gaertn.) expressing a novel β‐amyrin synthase (AsOXA1) gene

“…These triterpenes are probably derived from β‐amyrin, the initial product of the cyclization of 2,3‐oxidosqualene, mediated by a specific OSC, namely β‐amyrin synthase (Figure 1) (Dixon and Sumner, 2003). Medicago truncatula can also be genetically transformed and regenerated (Chabaud et al ., 2003; Araújo et al ., 2004; Zhou et al ., 2004; Duque et al ., 2007), and offers an opportunity for the manipulation of triterpenoid saponin content in transgenic plants by ectopic expression, over‐expression or gene silencing.…”

Gene expression and metabolite profiling of gibberellin biosynthesis during induction of somatic embryogenesis in Medicago truncatula Gaertn

“…Explanation of gibberellin metabolism during this phase could be based on developing a highly repetitive system, particularly with respect to light dependence of gibberellin biosynthesis [ 74 ]. Previous experiments established that Medicago truncatula SE may be induced similarly in light or in darkness [ 71 , 75 , 76 , 77 ]. This study involved two Medicago truncatula lines with remarkably different phenotypes: the non-embryogenic (M9) and the embryogenic (M9-10a), the later one comes from somaclonal variation of M9 line.…”