Plant regeneration from protoplasts of a commercial Asian long-grain javanica rice

A reproducible plant regeneration system has been developed for protoplasts from embryogenic cell suspension cultures of the commercial Asian long-gra in javanica rice, Oryza sativa cv. Azucena. Protoplasts were isolated routi nely from cell suspensions with yields of 5.5-12.0 x 10(6) g(-1) fresh weig ht. A membrane filter nurse-culture method was adopted and was essential to support sustained mitotic division of protoplast-derived cells, leading to cell colony formation. The protoplast plating efficiency was higher when s uspension cells of Lolium multiflorum, rather than those of the japonica ri ce O. sativa L. cv. Taipei 309, were employed as nurse cells. A two-step sh oot regeneration procedure, in which protoplast-derived calli were cultured initially on medium semi-solidified with 1% (w/v) agarose followed by cult ure on medium containing 0.4% (w/v) agarose, induced plant regeneration fro m protoplast-derived calli. Fifteen percent of protoplast-derived tissues r egenerated shoots; tissues not subjected to this treatment failed to develo p shoots.