A Novel Strategy for Promoting Homoplasmic Plastid Transformant Production Using the Barnase-barstar System

Plastid transformants form biofactories that are able to produce extra proteins in plastids when they are in a homoplasmic state. To date, plastid transformation has been reported in about twenty plant species; however, the production of homoplasmic plastid transformants is not always successful or easy. Heteroplasmic plants that contain wild-type plastids produce fewer target proteins and do not always successfully transfer transgenes to progeny. In order to promote the generation of homoplasmic plants, we developed a novel system using barnase-barster to eliminate wild-type plastids from heteroplasmic cells systematically. In this system, a chemically inducible cytotoxic under a plastid transit signal was introduced into nuclear DNA and , which inhibits barnase, was integrated into plastid DNA with the primary selection markers aminoglycoside 3'-adenylyltransferase () and green fluorescence protein () gene. As expected, the expression of the plastid was lethal to cells as seen in leaf segments, but expression in plastids rescued them. We then investigated the regeneration frequency of homoplasmic shoots from heteroplasmic leaf segments with or without expression. The regeneration frequency of homoplasmic-like shoots expressing system was higher than that of shoots not expressing this. We expect that the application of this novel strategy for transformation of plastids will be supportive to generate homoplasmic plastid transformants in other plant species.