EPSPS gene encode 5-enolpyruvylshikimate 3-phosphate synthase - EPSPS enzyme catalysis for 5enolpyruvylshikimate 3-phosphate (EPSP) synthesis process. EPSPS is a key enzyme in way of aromatic amino acids biosynthetic as tyrosine, phenylalanine and tryptophan. The herbicide glyphosate is a large herbicide range to inactivate EPSPS enzyme. It will interact to crops no biosynthesis essential amino acids and death. EPSPS encode gene is isolated from Agrobacterium CP4, has high resistance character. In this paper, the authors constructed a binary vector earring plant optimal EPSPS. This gene was transferred into N. tabacum K326 cultivar. Transgenic plants were confirmed by PCR and glyphosate treatment. The results show that in the total plants have 20 transgenic cultivars, which were confirmed by PCR. After that, the transgenic plants were assayed to detect different glyphosate resistance: E1, E12, E16 cultivars are good resistance
E4, E7, E11, E18 cultivars are medium resistance and the remaining cultivars are bad resistance. There are the significant results to provide material sources for glyphosate resistant breeding and assay in the lab and fields conditions.