Basic studies on the chloroplast transformation using photoautotrophic cultured cells

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Chloroplasts have an important role in photosynthesis, nitrogen metabolism and amino acid biosynthesis and many chloroplast proteins are encoded by the chloroplast genome. However, these properties make the isolation of chloroplast mutants of higher plants difficult ; the mutations are often lethal and the high copy number of the chloroplast genome makes the selection of mutants inefficient. Recently, we have shown that a photomixotrophic cell line of tobacco (Nicotiand tabacum cv Samsun. NN) was very useful to select a cell line resistant to the herbicide atrazine, as a model system for the isolation of chloroplast mutants. Based on this success, the basic studies were carried out to establish the method to transform chloroplast genome.1. A vector, in which kanamycin resistant gene (NPTII) was cloned downstream the promoter of psbA gene, was constructed to transform chloroplast genome. 2. Another vector, in which psbA gene was placed downstream the promoter and transit peptide of wheat Rubisco small subunit, was constructed to convert chloroplast psbA gene into a nuclear gene. 3. The conditions for the isolation and culture of protoplasts from photomixotrophic cells were established for the direct gene transfer of photomixotrophic cells. Further experiments to transform photomixotrophic cells is in progress. 4. Atrazine-resistant protoplasts and mesophyll protoplasts were fused asymmetrically to regenerate plants with mutant chloroplasts. By this method, putable atrazine-resistant shoots were obtained. 5. To understand the resistance mechanism of mutant cells and the function of D-1 protein, the projected secondary structures of the mutant D1 proteins were analyzed. This result indicates that the cross-resistantce of threonine264 D1 protein to triazine and urea herbicides is mainly due to a conformational change of the binding site for the herbicides.