We shall develop the tools for introducing genes into the plastid genome of higher plants using Nicotiana tabacum, a suitable model species. Lack of plastid transformation in higher plants has been due to problems of a large plastid genome copy number in a cell, lack of appropriate selective markers, and an effective way of DNA delivery. First, we shall transform wild-type plastids with plastid 16S rRNA genes that confer resistance to antibiotics. Introduction of DNA will be by bombardment with DNA-coated tungsten particles. Transformation will result in gene replacement. Research will be extended to study the requirements of inserting foreign DNA into the plastid genome. Oligonucleotide, and gene- size segments of DNA will be linked to the selective markers. Incorporation of oligonucleotides will be used to select suitable sites for gene insertion. Larger segments of DNA will include chimeric genes. Involvement of copy correction will be determined in the formation of stable transgenic plastid genomes. Information derived from proposed research will make feasible the study of plastid gene regulation and function in transgenic plants. This research will develop a plastid transformation protocol for higher plants. This is a very important tool for further genetic engineering of plants.
