Excision of a selectable marker gene in transgenic banana using a Cre/ lox system controlled by an embryo specific promo
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Excision of a selectable marker gene in transgenic banana using a Cre/lox system controlled by an embryo specific promoter Borys Chong-Pe´rez • Maritza Reyes • Luis Rojas • Ba´rbara Ocan˜a • Adolfo Ramos Rafael G. Kosky • Geert Angenon
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Received: 27 November 2012 / Accepted: 8 April 2013 Ó Springer Science+Business Media Dordrecht 2013
Abstract Antibiotic and herbicide resistance genes have been used in transgene technology as powerful selection tools. Nonetheless, once transgenic events have been obtained their presence is no longer needed and can even be undesirable. In this work, we have developed a system to excise the selectable marker and the cre recombinase genes from transgenic banana cv. ‘Grande Naine’ (Musa AAA). To achieve this, the embryo specific REG-2 promoter was isolated from rice and its expression pattern in banana cell clumps, somatic embryos and regenerated plantlets was characterized by using a pREG2::uidA fusion construct. Subsequently, the REG-2 promoter was placed upstream of the cre gene, conferring Cre functionality in somatic embryos and recombination of lox sites resulting in excision of the selectable marker and cre genes. PCR analysis revealed that 41.7 % of the analysed transgenic plants were completely marker free, results that were thereafter confirmed by Southern blot hybridization. These results demonstrate the feasibility of using developmentally controlled promoters to mediate marker excision in banana. This system does not require any extra handling compared to the conventional transformation procedure and might be useful in other species regenerating through somatic embryogenesis.
B. Chong-Pe´rez M. Reyes L. Rojas B. Ocan˜a A. Ramos R. G. Kosky Instituto de Biotecnologı´a de Las Plantas, Universidad Central ‘‘Marta Abreu’’ de Las Villas, Carretera A Camajuanı´ Km 5.5, Santa Clara, Villa Clara, Cuba B. Chong-Pe´rez G. Angenon (&) Laboratory of Plant Genetics, Institute for Molecular Biology and Biotechnology, Vrije Universiteit Brussel, Pleinlaan 2, 1050 Brussels, Belgium e-mail: [email protected]
Keywords Agrobacterium Banana Cre-lox Marker gene excision REG-2 Transgenic plant
Introduction Plant genetic engineering is a technology that is critical for future food, feed, energy, and even pharmaceutical needs (Castle et al. 2008; Eckardt et al. 2009; Edgerton 2009; Farre´ et al. 2010; Varshney et al. 2011). It has a notable potential to engage important socioeconomic problems, especially in the developing world (Farre´ et al. 2010). In order to produce genetically engineered plants, firstly a recombinant DNA has to be introduced in the plant cell and secondly, cells that have integrated the DNA into the appropriate plant genome (nuclear or plastid) should be differentiated from the cells that do not contain the recombinant DNA. This distinction is possible by the use of selectable marker genes (SMGs), which in most cases are genes that encode proteins conferring resistance to antibiotics or herbicides (Miki and McHugh 2004). Thus, SMGs are cons
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