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Heat shock induced excision of selectable marker genes in transgenic banana by the Cre-lox site-specific recombination system

Chong-Pérez, Borys, Kosky, Rafael G., Reyes, Maritza, Rojas, Luis, Ocaña, Bárbara, Tejeda, Marisol, Pérez, Blanca, Angenon, Geert
Journal of biotechnology 2012 v.159 no.4 pp. 265-273
Arabidopsis, Musa, Southern blotting, bananas, biotechnology, crops, excision, genes, genetic markers, heat shock response, polymerase chain reaction, site-specific recombination, soybeans, transfer DNA, transgenic plants
Selectable marker genes are indispensable for efficient production of transgenic events, but are no longer needed after the selection process and may cause public concern and technological problems. Although several gene excision systems exist, few have been optimized for vegetatively propagated crops. Using a Cre-loxP auto-excision strategy, we obtained transgenic banana plants cv. Grande Naine (Musa AAA) devoid of the marker gene used for selection. We used T-DNA vectors with the cre recombinase gene under control of a heat shock promoter and selectable marker gene cassettes placed between two loxP sites in direct orientation, and a gene of interest inserted outside of the loxP sites. Heat shock promoters pGmHSP17.6-L and pHSP18.2, from soybean and Arabidopsis respectively, were tested. A transient heat shock treatment of primary transgenic embryos was sufficient for inducing cre and excising cre and the marker genes. Excision efficiency, as determined by PCR and Southern hybridization was 59.7 and 40.0% for the GmHSP17.6-L and HSP18.2 promoters, respectively. Spontaneous excision was not observed in 50 plants derived from untreated transgenic embryos. To our knowledge this is the first report describing an efficient marker gene removal system for banana. The method described is simple and might be generally applicable for the production of marker-free transgenic plants of many crop species.