Transgenic rice for animal feed with high tryptophan content generated by a selectable marker- and vector backbone-free technology

Abstract

We reported previously that the genetically modified rice lines expressing OASA1D, a feedback-insensitive anthranilate synthase (AS) α-subunit gene of rice, showed remarkable increase of free tryptophan (Trp) in calli. In the present study, we investigated transformation of lKusahonami” (Oryza sativa L.), a rice variety for animal feed with this gene. In order to avoid the co-integration of vector backbone sequences and taking into account the fact that OASA1D gene exert resistance to 5-methyltryptophan (5MT), gene cassette vector consisting of a promoter, OASA1D cDNA and terminator was constructed and introduced directly to rice calli by use of potassium titanate whisker-supersonic method. Transgenicity of regenerated plants and the copy number of the transgene in the genome were analyzed by Southern blot. Fertile transgenic plants carrying low copy number of transgene and producing high level of free Trp in leaves and seeds were obtained. Genetic stability of the transgene has been demonstrated. This cultivar is a common variety for animal feed, these transgenic plants may be useful to establish an actual variety that is nutritionally improved.