Evaluation of selection strategies alternative to nptII in genetic transformation of citrus

Abstract

The neomycin phosphotransferase (nptII) selection system has proved successful in citrus transformation; however, it may be recommendable to replace it given the pressure exerted against antibiotic-resistance selectable marker genes in transgenic plants. The present work investigates three different selection alternatives, comparing them to nptII selection in two citrus genotypes, Carrizo citrange and Pineapple sweet orange. The first method used the beta-glucuronidase (uidA) reporter marker gene for selection; the second attempted to generate marker-free plants by transforming explants with a multi-auto-transformation (MAT) vector, combining an inducible R/RS-specific recombination system with transgenic-shoot selection through expression of isopentenyl transferase (ipt) and indoleacetamide hydrolase/tryptophan monooxygenase (iaaM/H) marker genes; while the third exploited the phosphomannose isomerase (PMI)/mannose conditional positive selection system. Firstly, GUS screening of all regenerated shoots in kanamycin-free medium gave 4.3% transformation efficiency for both genotypes. Secondly, workable transformation efficiencies were also achieved with the MAT system, 7.2% for citrange and 6.7% for sweet orange. This system affords an additional advantage as it enables selectable marker genes to be used during the in vitro culture phase and later removed from the transgenic plants by inducible recombination and site-specific excision. Thirdly, the highest transformation rates were obtained with the PMI/mannose system, 30% for citrange and 13% for sweet orange, which indicates that this marker is also an excellent candidate for citrus transformation.

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

“…Complete excision of marker genes ranged from 5 to 12 months, however, only 41% of the regenerated transgenic shoots R-mediated recombination occurs correctly. In Citrus sp., it was also reported that anomalous excision of marker genes promoting failures in the expression of the reporter genes (Ballester et al, 2007(Ballester et al, , 2008.…”

“…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.…”

Citrus Transformation: Challenges and Prospects

“…No Agrobacterium was involved and there was no selective agent applied. Ballester et al (2008) compared the most common citrus transformation and selection system, using kanamycin selection and scorable GUS staining to three methods that did not utilize antibiotic selection, in 'Carrizo' citrange and 'Pineapple' sweet orange. The alternative methods included scoring for GUS staining without applying selection, transforming explants with a multi-autotransformation (MAT) vector, combining an inducible recombinase-specific recombination system (R/RS) with transgenic-shoot selection through expression of isopentenyl transferase (ipt) and indoleacetamide hydrolase/tryptophan monooxygenase (iaaM/H) marker genes, and selection with the PMI/mannose conditional positive selection system .…”