Chloroplast Transformation: Current Results and Future Prospects

Genetic engineering of proteins is a powerful tool used in both basic and applied research. In vitro alteration of the primary structure of a gene and subsequent introduction of the mutated DNA into the genome of a living cell allows the directed manipulation of protein structure. Such an approach, often termed ‘reverse genetics,’ has been widely used to investigate the complex relationship between the structure of a protein and its function, and to explore the intricacies of biochemical and developmental pathways. An obvious prerequisite for genetic engineering is the ability to introduce DNA into a living cell in such a way that it is stably maintained and properly expressed in the appropriate genome of the host cell. The genome of a prokaryote is in the cell cytoplasm and generally consists of one or a few copies of a large DNA molecule. In contrast, photosynthetic eukaryotes contain three distinct genomes, each located within a subcellular organelle enveloped by one or more membranes and hence separated from the cytoplasm. The three plant cell genomes are those of the nucleus, the mitochondrion, and the plastid. The genome of the chloroplast, the plastid type found in photosynthetic cells, presents a complex genetic target because there are often hundreds of copies of the circular chloroplast DNA molecule per chloroplast, and often hundreds of chloroplasts per cell. Obtaining a plant cell in which every resident copy of a given chloroplast gene has been replaced by an engineered, mutant gene copy is an essential step in experiments involving DNA-mediated chloroplast transformation. An ideal model organism for such studies is provided by the unicellular green alga Chlamydomonas reinhardtii, which contains a single large chloroplast. This chapterpresents current results and future prospects for chloroplast transformation, both in Chlamydomonas and in plants of agronomic interest.