Construction of plant transformation vectors carrying beet necrotic yellow vein virus coat protein gene (i) - transformation vectors

Coat protein gene of beet necrotic yellow vein virus (BNYVV) was isolated from inoculated sugar beet roots and leaves of Chenopodium quinoa and Tetragonia expansa, by RT-PCR and imuno capture RT-PCR. Specific primers were made to complement coat protein gene and untranslated leader sequence, so that two fragments were obtained: long (731 bp), which contained coat protein gene and leader sequence, and short (587 bp), with coat protein gene. Fragments were cloned in two plant transformation vectors: pCAMBIA 3301M and pCAMBIA 1304M, which were modified by removing multicloning site and NcoI restriction site at the 5' end of the reporter genes. Vector pC3301M had bar gene which confers resistance against the herbicide gluphosinate ammonium as selectable marker, and pC1304M had gene for resistance to antibiotic hygromycin. Three constructs were made from each vector: CPL, containing coat protein gene with leader sequence; CPS with gene for coat protein, and CPSas with coat protein gene in antisense orientation. All constructs were transfered to Agrobacterium tumefaciens strain LBA4404

Construction of plant transformation vectors carrying beet necrotic yellow vein virus coat protein gene (ii)- plant transformation

Fragments containing the coat protein gene of beet necrotic yellow vein virus were cloned in two plant transformation vectors: pCAMBIA3301M with the bar gene as selectable marker, and pCAMBIA1304M, with resistance to hygromycin. Three constructs were made of each vector: CPL, containing coat protein gene with leader sequence; CPS with coat protein gene, and CPSas with coat protein gene in antisense orientation. Vectors pC3301MCPL, pC3301MCPS. and pC3301MCPSas were used in Agrobacterium—mediated transformation of Nicotiana tabacum (tobacco), Nicotiana excelsior and Nicotiana benthamiana. Regenerants that developed roots on selective media were tested for the presence of CP fragments and the bar gene, but most regenerants were nontransformed (50-83% escapes). After all rooted plants had been selfed, and T1 seed germinated on selective media, only plants descending from one N. excelsior regenerant transformed with pC3301MCPS were positive for presence of bar gene and CPS fragment. Tobacco and Nicotiana benthamiana were transformed with constructs pC1304MCPS and pC1304MCPSas. Transformation efficiency was much higher and approximately 50% of regenerants that rooted on media with 20 mg l−1 hygromycin were positive for the presence of CP fragments. All T1 plants were positive for presence of CP fragments