Plant Molecular Biology 52: 415–419, 2003. © 2003 Kluwer Academic Publishers. Printed in the Netherlands. 415 Stable wheat transformation obtained without selectable markers Hugo R. Permingeat +,∗ , Mar´ ıa L. Alvarez + , Gerardo D.L. Cervigni, Ricardo A. Ravizzini and Rub´ en H. Vallejos Centro de Estudios Fotosint´ eticos y Bioqu´ ımicos (CEFOBI) (CONICET-Fund.M. Lillo-Universidad Na- cional de Rosario), Suipacha 531, 2000 Rosario, Argentina ( ∗ author for correspondence; e-mail per- mingeath@arnet.com.ar); + these authors contributed equally to the work Received 11 June 2002; accepted in revised form 16 December 2002 Key words: cereal transformation, hygromycin, selective agents Abstract Transgenic wheat plants without the selectable marker gene were obtained either in the presence or in the ab- sence of selective pressure during the transformation protocol. When using hygromycin as selective agent in a co-transformation experiment involving a mixture of plasmids pGL2, containing the hpt gene, and pAI 1 Gus, containing the uidA gene, 3 out of 19 transgenic wheat plants had the uidA gene alone as shown by Southern blots. The gene was transmitted to the progeny following Mendelian rules. Segregation and loss of the selectable marker gene was also found in three out of six events from other experiments where high-molecular-weight glutenin genes were expressed or over-expressed. On the other hand, in 7 experiments where no selective pressure was applied and that involved 1016 bombarded explants, 23 transgenic wheat plants were obtained. The uidA gene was stably integrated as suggested by its transmission to the progeny. Introduction Genetic engineering of monocots is usually carried out by direct methods such as particle bombardment or protoplast transformation and involves the use of selective agents and marker genes to favor the multi- plication of the initially transformed cells (Christou, 1996; Hansen and Wright, 1999). Antibiotics or her- bicides are used as selective agents. However, both may have some undesirable effects. It has been argued that there might be a risk, although small, that the antibiotic-resistance gene could be transferred from plants to pathogenic bacteria (Puchta, 2000; Smalla et al., 2000). On the other hand, herbicide resis- tance may not be of interest in a given crop or the marker gene might be transferred to weeds (Puchta, 2000). Moreover, the presence of a marker gene in a plant prevents the use of the same marker gene for further transformation of that plant (Goldsbrough, 1992). Thus, several strategies have been suggested for removal the marker genes (for a review, see Hohn et al., 2001). Among them, the elimination of marker genes by recombinases or transposases such as the lox P-Cre (Odell et al., 1990), the Ac/Ds system (Golds- brough et al., 1993) or the use of a chimeric ipt gene inserted into the transposable element Ac (Ebinuma et al., 1997). Removal of the marker gene by co- transformation with Agrobacterium has also been re- ported (Daley et al., 1998). Avoiding the use of a se- lectable marker gene has also been discussed (Puchta, 2000; Hohn et al., 2001). Selection and screening strategies depend, among other factors, on the trans- formation efficiency (Birch, 1997). Thus, with high efficiency it may be possible and useful to do it without introducing a selectable marker gene and screening for the gene of interest. In this paper we report that it is possible to ob- tain transgenic wheat plants without the marker gene in co-transformation experiments using the gene-gun method with mild selective pressure by screening for the gene of interest, and that transgenic wheat can also be obtained without selection.