Cultivation of Rice Protoplasts and Their Transformation Mediated by Agrobacterium Spheroplasts

Improvement of the cultivation of rice (Oryza saliva L.) protoplasts isolated from suspension cultures led to their division at a frequency of 5 to 10%. Rapidly growing colonies were obtained on a hormone-free medium when Agrobacterium tumefaciens spheroplasts were introduced into the protoplasts by polyethylene glycol treatment. Opines corresponding to the strains of A. tumefaciens used for the spheroplast treatments were detected in some of these colonies at a frequency of about 10−4. Using radioactive precursors, [14C]-α-ketoglutaric acid and [3H]-arginine, activities of nopaline synthase, a marker enzyme of nopaline-type crown gall, were also detected in some of these clones. These results show that the rice cells were transformed by Ti plasmid introduced by the spheroplast method.