Prospecting the utility of a PMI/mannose selection system for the recovery of transgenic sugarcane (Saccharum spp. hybrid) plants

Abstract

For the first time, the phosphomannose isomerase (PMI, EC 5.3.1.8)/mannose-based "positive" selection system has been used to obtain genetically engineered sugarcane (Saccharum spp. hybrid var. CP72-2086) plants. Transgenic lines of sugarcane were obtained following biolistic transformation of embryogenic callus with an untranslatable sugarcane mosaic virus (SCMV) strain E coat protein (CP) gene and the Escherichia coli PMI gene manA, as the selectable marker gene. Postbombardment, transgenic callus was selectively proliferated on modified MS medium containing 13.6 microM 2,4-D, 20 g l(-1) sucrose and 3 g l(-1) mannose. Plant regeneration was obtained on MS basal medium with 2.5 microM TDZ under similar selection conditions, and the regenerants rooted on MS basal medium with 19.7 microM IBA, 20 g l(-1) sucrose, and 1.5 g l(-1) mannose. An increase in mannose concentration from permissive (1.5 g l(-1)) to selective (3 g l(-1)) conditions after 3 weeks improved the overall transformation efficiency by reducing the number of selection escapes. Thirty-four vigorously growing putative transgenic plants were successfully transplanted into the greenhouse. PCR and Southern blot analyses showed that 19 plants were manA-positive and 15 plants were CP-positive, while 13 independent transgenics contained both transgenes. Expression of manA in the transgenic plants was evaluated using a chlorophenol red assay and enzymatic analysis.

Genetic Engineering of Saccharum

“…This selection system is also called positive selection since cells expressing PMI acquire a growth advantage due to their ability to utilize mannose-6-phosphate, which otherwise is toxic to plants when mannose is supplied as a carbon source (Brumbley et al 2008 ;Darbani et al 2007 ;Privalle et al 2000 ) . In sugarcane, Jain et al ( 2007 ) were the fi rst to show the successful application of the PMI/mannose selection system, using 3 g/l of mannose in the presence of sucrose during selection and regeneration, and 1.5-3 g/1 of mannose for rooting.…”

Biolistic Transformation of Citrullus vulgaris Schrad (Watermelon)

“…This were significantly reduced. This may be due to decrease target distance is similar to the target distance used for in velocity of the microcarriers as a result of increasing sugarcane and hardwood tree (Paulownia elongata) flight distance which consequently reduced penetration (Jain et al, 2007;Castellanos-Hernandez et al, 2009). In force and followed by fewer cells receiving the hop (Humulus lupulus L.) and garden balsam (Impatiens incoming DNA.…”

“…Some of the GUS spots dispersed near the garden balsam (Ikea et al, 2003;Ramesh and Gupta, 2005; meristematic region. Comparatively, the number of GUS Sreeramanan et al, 2005;Jain et al, 2007;Sailaja et al, spots at 9 cm target distance was only at 92% and many 2008; Taha et al, 2009). However, in barley, mothbean and of the GUS spots spread out of the meristem region.…”

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.…”