A binary vector for Agrobacterium mediated plant transformation with new glyphosate tolerant gene as a selectable marker.

Efficient and reproducible selection of transgenic plant cells is an essential component of a successful transformation and regeneration. Usually antibiotic resistant genes are used as selectable markers. In view of biosafety considerations, attempts have been made to substitute such genes with ones that are less harmful, such as herbicide resistance. Glyphosate is a non-selective, broad-spectrum herbicide that inhibits 5-enolpyruvylshikimate- 3-phosphate synthase, a key enzyme in the aromatic amino acid biosynthetic pathway in microorganisms and plants. In this project, a new binary vector (pSA101) hauling a glyphosate tolerant construct as a selectable marker for Agrobacterium-mediated transformation of plant cells was constructed. Precise attachment of different fragments of the synthetic construct was carried out by the SOEing PCR. This construct contains the first intron of Hsp70 from maize, a chloroplastic transient peptide of Rubico from Brassica napus and a bacterial glyphosate tolerant mutated EPSPS gene. It was expressed under the control of the CaMV 35S promoter and nos terminator. Transgenic tobacco plants with increased tolerance to glyphosate were directly selected on medium containing glyphosate. Molecular analyses confirmed the correctness of gene integration and expression. The results of transformation, regeneration and selection of transgenic tobacco plants with this new vector showed that is a usable and safer plasmid than those carrying the antibiotic resistance gene as a selectable marker.