Proceedings Volume 2010 (format .pdf) - SimpBTH

CO-TRANSFORMATION OF POTATO (SOLANUM TUBEROSUM L.)CV. DESIREE BY TWO SEPARATE T-DNAs CARRYINGORYZACYSTATIN GENESCINGEL ALEKSANDAR 1 , VINTERHALTER BRANKA 1 , VINTERHALTERDRAGAN 1 , MILOŠEVIĆ SNEŽANA 1 , SAVIĆ JELENA 1 , SMIGOCKI ANN 2 ,NINKOVIĆ SLAVICA 11 Institute for Biological Research “Siniša Stanković”, University of Belgrade, Bul Despota Stefana 142, 11060Belgrade, Republic of Serbia. E-mail: cingel@ibiss.bg.ac.rs2 USDA-ARS, Molecular Plant Pathology Laboratory, 10300 Baltimore Ave., Beltsville, MD 20705, USAKeywords: potato, Agrobacterium tumefaciens, co-transformation, oryzacystatin I,oryzacystatin IIABSTRACTIn an effort to develop plants with enhanced insect and pathogen resistance rice cystatin geneswere introduced into potato via Agrobacterium-mediated transformation. Plants of Solanumtuberosum L. cv. Desiree were co-transformed using the mixture method with two separateAgrobacterium tumefaciens EHA101 strains carrying oryzacystatin I (OCI) or oryzacystatin II (OCII)genes. PCR analysis indicated integration frequency of 38.0 ± 2.9% and 42.0 ± 5.8% for OCI andOCII genes, respectively, while co-transformation frequency of both rice cystatin genes was 20.0 ±2.9% of total number of analyzed transformants.1. INTRODUCTIONSterility and tetraploidy, together with a high degree of heterozygosity,significantly reduce the efficiency of conventional breeding as a methods forimproving potato genotypes. Agrobacterium-mediated gene transfer has appearedas promising technique to introduce heterologous genes into potato since it wasfirst reported by Ooms et al. (1986). A large number of agronomically useful geneshave already been transferred to potato. Most of these genes play role in aresistance to potato predators/pathogens (Chue et al., 2004; Naimov et al., 2001;Urwin et al., 2001; Lyapkova et al., 2001; Hefferon et al., 1997; Wierenga et al.,1996) and modification of important metabolic and physiological pathwayssuch as starch and sucrose synthesis (Edwards et al., 1999; Wolters et al., 1998).Co-transformation with multiple Agrobacterium strains carrying different T-DNA is mainly used to study the mechanism of T-DNA integration (De Neve etal., 1998; De Block et al., 1991), to facilitate selectable marker removal (Park etal., 2004; Vidal et al., 2003) or for multiple gene enhancements via geneticengineering (Li et al., 2009). This approach has not been widely used for potato,although it provides an effective method for rapidly stacking transgenes. Chang etal. (2002) reported a co-transformation efficiency of 24% for potato cv. RussetBurbank obtained using two plasmids cloned in separate A. tumefaciens LBA4404containing the same selectable marker. Using same approach Morris et al. (2006)25