Cre-mediated marker gene removal for production of biosafe commercial oilseed rape

Abstract

The Cre/loxP-based self-excision represents a promising strategy for removal of hazardous transgene(s) from genomes of targeted crops prior to their introduction into environment. Here, we applied the Cre/loxP self-excision strategy in which the cre recombinase gene is driven by the embryo-specific CRUC promoter from Arabidopsis thaliana. Besides, the Cre/loxP cassette, the T-DNA also consisted of the β-glucuronidase gene, controlled by the double dCaMV 35S promoter and the selectable neomycin phosphotransferase gene; the latter was aimed to be removed from the transgenic genome. The Cre/loxP self-excision cassette was introduced into genomes of four commercial oilseed rape cultivars Haydn, Heros, Hunter and Topas (Brassica napus L.) via Agrobacterium tumefaciens. Transgenic T0 plants were regenerated from all cultivars. Their detailed molecular analyses revealed premature activation of the CRUC promoter that resulted in partial excision of the nptII gene. Progenies of four self-pollinated T0 lines were further analysed, and the data confirmed complete excision event in 5 out of 105 (4.8%) of T1 transgenic oilseed rape plants. The excision efficiency does not seem to depend on the target cultivar. However, the poor transformation efficiency of rapeseed and the limited specificity of the CRUC promoter are clearly the bottleneck of this approach, and the feasibility of (tissue-specific) self-excision of selectable marker gene from genomes of each commercial rapeseed variety adds to their perspective to cope the increasing negative impacts of climate changes.