Pearl millet transformation system using the positive selectable marker gene phosphomannose isomerase

Abstract

Fertile transgenic pearl millet plants expressing a phosphomannose isomerase (PMI) transgene under control of the maize ubiquitin constitutive promoter were obtained using the transformation system described here. Proliferating immature zygotic embryos were used as target tissue for bombardment using a particle inflow gun. Different culture and selection strategies were assessed in order to obtain an optimised mannose selection protocol. Stable integration of the manA gene into the genome of pearl millet was confirmed by PCR and Southern blot analysis. Stable integration of the manA transgene into the genome of pearl millet was demonstrated in T1 and T2 progeny of two independent transformation events with no more than four to ten copies of the transgene. Similar to results obtained from previous studies with maize and wheat, the manA gene was shown to be a superior selectable marker gene for improving transformation efficiencies when compared to antibiotic or herbicide selectable marker genes.

Methods to produce marker‐free transgenic plants

“…Only transformed cells are capable of utilizing mannose as a carbon source [66]. PMI has been used as a selectable marker for transformation of many plant species, such as sugar beet [67], maize [68,69], wheat [70], rice [71], pearl millet [72], and canola [66]. However, this system may not be as effective in plant species that contain endogenous PMI.…”

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

“…The mannose positive selection system favors the regeneration and growth of transgenic cells expressing the 6-phosphomannose isomerase (PMI) gene, manA, while the growth of non-transgenic cells is inhibited through carbohydrate starvation (positive selection). The utility of the pmi gene from Escherichia coli (Miles and Guest 1984) as a positive selectable marker in conjunction with mannose for recovery of transgenic plants (pmi/Man) has been demonstrated in a number of crops, including sugar beet (Joersbo et al 1998), cassava , maize (Wang et al 2000;Wright et al 2001), wheat , 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), onion (Aswath et al 2006), almond (Ramesh et al 2006), cucumber (He et al 2006), maize (Privalle 2002), rice (He et al 2004), barley , apple (Degenhardt 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). …”

In vitro transformation of pearl millet (Pennisetum glaucum (L). R. BR.): Selection of chlorsulfuron-resistant plants and long term expression of the gus gene under the control of the emu promoter

“…Stable calli have been selected and analysed by Lambé et al (1995) after introducing the neomycin phosphotransferase gene (NPTII). Transgenic plants resistant to phosphinothricin and hygromycin (Goldman et al, 2003;O'Kennedy et al, 2004;Ceasar and Ignacimuthu, 2009;Ramadevi et al, 2014), kanamycin (Plaza-Wüthrich and Tadele, 2012) and mildew (Latha et al, 2006) have been studied. However, to date, no stable P. glaucum transformed calli and plant resistant to the chlorsulfuron herbicide have been reported yet.…”