Neomycin phosphotransferase gene (nptII) as a selectable marker for Brachypodium BD21

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Brachypodium distachyon BD21 is an embryogenic diploid accession and is the most commonly used material for genetic transformation. Unfortunately hygromycin selection can be unclear in this and other embryogenic Brachypodium accessions and the established protocol (Alves et al, 2009) uses GFP as an additional selection. Without GFP this protocol allows many escapes as selection is reduced from 40 to 30 to 20 mgl-1 hygromycin as calli grow and regenerate plants. This author found only calli that grow readily and regenerate on continuous selection of 40mgl-1 hygromycin to be definitely transformed. Experiments with the hygromycin gene under the CaMV promoter (pROB5,
Bilang et al, 1991) have yielded few such easily identifiable transformants and
hygromycin under the actin promoter (pAct1HPT, Cho et al, 1998) is preferred. This selection gene was compared with the bar gene (pUBA, Toki et al, 1992) and nptII gene (pBKS ubi-nptII, Paul Robson, IBERS) under the maize ubiquitin promoter and cobombarded with gus under an enhanced rubi3 rice ubiquitin promoter (pRESQ48, Sivamani and Qu, 2006). Calli bombarded with pUBA and selected on bialophos became friable and were not continued. In this experiment calli bombarded with pAct1HPT produced no obvious
transformants, although weak plants grew. These were not GUS positive however. Calli bombarded with pUbi-nptII and selected on 100-150 mgl-1 paromomycin were obviously transformed or not and plants were regenerated under selection. One plant constitutively expressed GUS and is the first grass to do so with the pRESQ48 construct, although the plant may be infertile. The nptII gene appears to be a useful and easily selectable gene for Brachypodium, and the enhanced rice ubiquitin promoter appears to be constitutive and to not co-suppress the maize ubiquitin promoter.
Original languageEnglish
Publication statusPublished - 2011
Event1st European Brachypodium Workshop - Versailles, France
Duration: 19 Oct 201121 Oct 2011


Conference1st European Brachypodium Workshop
Period19 Oct 201121 Oct 2011


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