Bifunctional selection–reporter systems for genetic transformation of citrus: mannose- and kanamycin-based systems

Abstract

Agrobacterium-mediated transformation of Carrizo citrange [Citrus sinensis (L.) Osbeck × Poncirus trifoliata (L.) Raf.] with a binary vector containing a novel bifunctional reporter-selection fusion gene comprising an inframe fusion between the manA gene and egfp gene is detailed. This system combined the phosphomannose isomerase positive selection system with the ability to monitor gene expression in a non-destructive manner using EGFP. Transgenic plants stably expressing the EGFP protein were regenerated following Agrobacterium-mediated transformation using a vector containing this fusion gene. We also obtained comparable transformation efficiencies when Carrizo explants were transformed using another Agrobacterium strain containing a binary vector with a bifunctional egfp-nptII fusion gene. Regenerating shoots were selected on medium containing 15 g L −1 mannose supplemented with 5 g L −1 sucrose for the manA-based selection or on medium containing 100 mg L −1 kanamycin for the nptII-based selection. Our results indicated that the mannose-based antibiotic-free selection combined with visual identification of transgenic shoots using EGFP allows for early elimination of escape non-transgenic shoots and can provide a viable alternative to antibiotic-based selection systems in the genetic transformation of citrus and other crops.

Successful recovery of transgenic cowpea (Vigna unguiculata) using the 6-phosphomannose isomerase gene as the selectable marker

“…Leaves of untransformed plants incubated in chlorophenol red medium did not show medium acidification, leaving the red color of the medium unchanged. The cholorophenol red assay has been shown to be valuable for rapid visual screening of PMI positive transgenic plants (Lucca et al 2001;Patil et al 2009;Dutt et al 2010). …”

Genetic Transformation in Citrus

“…Citrus cultivars vary in their response to in vitro organogenesis and genetic transformation. This results in the need for cultivar-specific optimization of in vitro protocols [20, 22]. …”

“…However, this method is not suitable for the transformation of any seedless cultivar. Also, special cultivars in the mandarin group remain robust to transform using this method [20, 22, 24]. …”

Transformação genética de laranja doce com o gene codificador de defensina de Citrus sinensis, sob controle dos promotores 35S (Cauliflower mosaic virus) ou AtSuc2 (Arabidopsis thaliana)

“…Com o desenvolvimento da cultura de tecidos e da engenharia genética, diferentes métodos de transformação genética de citros têm sido empregados, como a transformação de protoplasto, via polietilenoglicol (PEG) (KOBAYASHI; UCHIMYA, 1989;FLEMING et al, 2000;OMAR;GROSSER, 2008) ou eletroporação (FROMM; TAYLOR; WALBOT, 1985), a biobalística (YAO et al, 1996), além da transformação genética via Agrobacterium tumefaciens (HIDAKA et al, 1990;PEÑA et al, 1997;MENDES et al, 2002;MUNIZ et al, 2012, MYATA et al, 2012, que é hoje o método mais utilizado para a obtenção de plantas transgênicas de citros GROSSER, 2010;FEBRES et al, 2011;PEÑA et al, 1995;MACHADO et al, 2005). (PEÑA et al, 2005;RODRÍGUEZ, et al, 2008;.…”

“…(CARDOSO et al, 2010;BOSCARIOL, et al 2006, MUNIZ et al, 2012. Por outro lado, o uso de promotores tecido-específicos também tem sido relatado, pois, evita uma expressão desnecessária do gene e reduz a possibilidade de interferência no desenvolvimento da planta (DUTT et al, 2012;DUTT et al, 2014;PORTO et al, 2014). Estudos utilizando promotores tecido-específicos para expressão gênica no floema foram reportados em diversos trabalhos (ATÍLIO et al, 2013;MYIATA et al, 2012;TAVANO, 2013), visando a obtenção de plantas cítricas resistentes a Candidatus Liberibacter spp..…”