Piestun, D., S. Tolkowsky Laboratory, Department of Virology, Agricultural Research Organization, P.O. Box 6, 50250 Bet Dagan, Israel
Batuman, O., S. Tolkowsky Laboratory, Department of Virology, Agricultural Research Organization, P.O. Box 6, 50250 Bet Dagan, Israel
Che, X., S. Tolkowsky Laboratory, Department of Virology, Agricultural Research Organization, P.O. Box 6, 50250 Bet Dagan, Israel
Gofman, R., S. Tolkowsky Laboratory, Department of Virology, Agricultural Research Organization, P.O. Box 6, 50250 Bet Dagan, Israel
Filatov, V., S. Tolkowsky Laboratory, Department of Virology, Agricultural Research Organization, P.O. Box 6, 50250 Bet Dagan, Israel
Zypman, S., S. Tolkowsky Laboratory, Department of Virology, Agricultural Research Organization, P.O. Box 6, 50250 Bet Dagan, Israel
Gafny, R., S. Tolkowsky Laboratory, Department of Virology, Agricultural Research Organization, P.O. Box 6, 50250 Bet Dagan, Israel
Bar-Joseph, M., S. Tolkowsky Laboratory, Department of Virology, Agricultural Research Organization, P.O. Box 6, 50250 Bet Dagan, Israel

Bar gene coding for resistance to the Basta (Hoechst) herbicide and cDNAs of citrus tristeza virus (CTV), utilized as pathogen-derived sequences (PDS), were incorporated into transgenic citrus plants. Two truncated constructs of ORF1b, the putative coding region of the RNA-dependent RNA polymerase (RdRp) (Δ1RdRp and Δ2RdRp of 1380 and 1413 bp respectively) were integrated into Troyer citrange (C. sinensis × P. trifoliata) plants. Binary vector pBINPlus, carrying cDNAs of Δ1RdRp along with the uidA (GUS) reporter gene, and Δ2RdRp driven by the CaMV-35S promoter and a termination signal NOS proved successful. Bar gene and uidA (GUS) were incorporated with the aid of a unique construct, based on pGA482 binary vector. Expiants of 2-3 cm, consisting of parts of the root, the crown, and a 0.5-cm long stem segment cleared of axillary buds and cotyledons, were vacuum-infiltrated with cells of Agrobacterium tumefaciens (strain EHA105) that had undergone a comprehensive virulence induction (phosphate starvation, glucose nutrition, pH 5.5, 100 μM acetosyringone, at 25°C), using a recalcitrant plant induction medium. Selection was achieved with 300 μg/ml kanamycin sulphate and resistant shoot tips were excised and top-grafted on in vitro-grown seedlings. The selected plantlets were re-grafted onto potted sour orange rootstocks. Southern blot and nucleic acid hybridization with ΔRdRp and nptII probes showed integration of several copies of the CTV-Δreplicase into the genomes of the transgenic Troyer plants. Specific GUS staining and PCR confirmed the integration of the bar-uidA constructs in whole transgenic plants.