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Use of fluorescent reporter genes in olive (Olea europaea L.) transformation

Cerezo, Sergio, Palomo-Ríos, Elena, Ben Mariem, Sinda, Mercado, José A., Pliego-Alfaro, Fernando
Acta physiologiae plantarum 2019 v.41 no.4 pp. 49
Agrobacterium radiobacter, Olea europaea, beta-glucuronidase, callus, confocal laser scanning microscopy, fluorescence, fluorescent proteins, genetic transformation, genetically modified organisms, genotype, leaves, olives, plasmids, reporter genes, roots, somatic embryos
Fluorescent proteins (FPs) can be used for different purposes in plant transformation studies such as the evaluation and improvement of transformation parameters or the isolation of transgenic cells in the absence of selective agents. In this research, the applicability of green (GFP) and red (DsRed) fluorescent proteins in olive transformation has been investigated. Olive embryogenic callus was transformed with Agrobacterium tumefaciens AGL1 strain carrying pXK7FNF2 (harbouring the gfp gene), pXK7RNR2 (DsRed), or pXK7S*NF2 (gfp and β-glucuronidase) binary plasmids. After 3 months of selection in the presence of paromomycin, several resistant calli were recovered for each construct, obtaining transformation rates in the range of 2–8%. The expression of FPs was studied during the different stages of olive plant regeneration using epi-fluorescence and confocal laser scanning microscopy. GFP from pXK7SN*F2 plasmid could be easily detected in olive somatic embryos (SE) during proliferation whereas SE transformed with pXK7FNF2 showed weak GFP signal. After embryo conversion, plants transformed with both vectors were analysed, but GFP could be detected neither in leaves nor in roots. By contrast, DsRed was highly expressed in SE and could also be visualized in leaf and root tissues of regenerated plants using confocal laser microscopy and epi-fluorescence zoom microscope, respectively. In addition, pXK7RNR2 was used to transform a different olive embryogenic line, detecting DsRed expression in SE transformed from this genotype. These results show that FPs can be a useful tool in genetic transformation of olive embryogenic cells, being DsRed gene more useful than gfp for this purpose.