The use of the phosphomannose-isomerase/mannose selection system to recover transgenic apple plants

Abstract

A selection system based on the phosphomannose-isomerase gene (pmi) as a selectable marker and mannose as the selective agent was evaluated for the transformation of apple (Malus domestica Borkh.). Mannose is an unusable carbon source for many plant species. After uptake, mannose is phosphorylated by endogenous hexokinases to mannose-6-phosphate. The accumulation of mannose-6-phosphate leads to a block in glycolysis by inhibition of phosphoglucose-isomerase, resulting in severe growth inhibition. The phosphomannose-isomerase is encoded by the manA gene from Escherichia coli and catalyzes the conversion of mannose-6-phosphate to fructose-6-phosphate, an intermediate of glycolysis. Transformed cells expressing the manA gene can therefore utilize mannose as a carbon and survive on media containing mannose. The manA gene along with a beta-glucuronidase (GUS) gene was transferred into apple cv. 'Holsteiner Cox' via Agrobacterium tumefaciens-mediated transformation. Leaf explants were selected on medium supplemented with different concentrations and combinations of mannose and sorbitol to establish an optimized mannose selection protocol. Transgenic lines were regenerated after an initial selection pressure of 1-2 g l(-1) mannose in combination with 30 g l(-1) sorbitol followed by a stepwise increase in the mannose concentration up to 10 g l(-1) and simultaneous decrease in the sorbitol concentration. Integration of transgenes in the apple genome of selected plants was confirmed by PCR and southern blot analysis. GUS histochemical and chlorophenol red (CPR) assays confirmed activity of both transgenes in regenerated plants. The pmi/mannose selection system is shown to be highly efficient for producing transgenic apple plants without using antibiotics or herbicides.

2-deoxyglucose selection system for Agrobacterium-mediated transformation of Solanum lycopersicon and Nicotiana tabacum plants

“…Lizixiang) (Sheng-Jun et al, 2004), while 200 mg/l kanamycin was used to select transformed tobacco leaf disc (Mentewab and Steward, 2005). Apple young leaves required 2.5, 5 and 10 g/l of mannose for their selection (Degenhardt et al, 2006) while for the transformation of mature embryoderived calli of Indica rice (Oryza sativa L.), 30 g/l of mannose was required (Penna et al, 2008).…”

Phosphomannose-isomerase as a selectable marker to recover transgenic orchid plants (Oncidium Gower Ramsey)

“…The pmi/Man system has been successfully used in several plant systems such as sugar beet (Joersbo et al 1998), cassava (Zhang and Puonti-Kaerlas 2000), maize (Negrotto et al 2000;Wright et al 2001), Arabidopsis (Todd and Tague 2001), wheat , tobacco and potato (Kunze et al 2001), pepper (Kim et al 2002), sweet orange (Boscariol et al 2003), pearl millet (O'Kennedy et al 2004), tomato (Sigareva et al 2004, papaya (Zhu et al 2005), bentgrass (Fu et al 2005), apple (Degenhardt et al 2006), onion (Aswath et al 2006), almond (Ramesh et al 2006), cucumber (He et al 2006), cabbage (Min et al 2007), sugarcane (Jain et al 2007), flax (Lamblin et al 2007), citrus (Ballester et al 2008), sorghum (Gurel et al 2009) and chickpea (Patil et al 2009). However, the pmi/Man system has not been tested previously in Oncidium orchid.…”

Recent Advances in Fruit Species Transformation

“…Sensitivity to the toxic effect of mannose-6-phosphate is different between species, and can be avoided by combining with other sugars such as sucrose, maltose and fructose (Joersbo et al, 1999). Diverse fruit trees were selected with this system, alone or in combination with sucrose, i.e., 12 g/L mannose and 5 g/L sucrose in orange (Ballester et al, 2008); 30 g/L mannose without any sugar more in papaya (Zhu et al, 2005); 2,5 g/L www.intechopen.com mannose and 5 g/L sucrose in almond (Ramesh et al, 2006) or 1-10 g/L mannose and 5-30 g/L in apple (Degenhardt et al, 2006). In Citrus sinensis, the best results were obtained when 13 g/L mannose as unique source of carbon was added into the selection media.…”