Articles
TRUNCATED VERSIONS OF THE CITRUS TRISTEZA VIRUS (CTV) REPLICASE AND BASTA RESISTANCE GENES INCORPORATED IN TRANSGENIC TROYER CITRANGE
Article number
535_27
Pages
223 – 230
Language
Abstract
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 x 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.
Explants 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.
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 x 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.
Explants 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.
Authors
D. Piestun, O. Batuman, X. Che, R. Gofman, V. Filatov, S. Zypman, R. Gafny, M. Bar-Joseph
Keywords
Citrus spp., genetic engineering, pathogen derived sequences, Agrobacterium tumefaciens, virulence induction, vacuum infiltration, root-crown-stem
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