Chloroplast Vector Systems for Biotechnology Applications

Abstract

Chloroplasts are ideal hosts for expression of trans- genes. Transgene integration into the chloroplast genome occurs via homologous recombination of flanking sequences used in chloroplast vectors. Iden- tification of spacer regions to integrate transgenes and endogenous regulatory sequences that support opti- mal expression is the first step in construction of chloroplast vectors. Thirty-five sequenced crop chlo- roplast genomes provide this essential information. Various steps involved in the design and construction of chloroplast vectors, DNA delivery, and multiple rounds of selection are described. Several crop species have stably integrated transgenes conferring agro- nomic traits, including herbicide, insect, and disease resistance, drought and salt tolerance, and phytore- mediation. Several crop chloroplast genomes have been transformed via organogenesis (cauliflower (Brassica oleracea), cabbage (Brassica capitata), lettuce (Lactuca sativa), oilseed rape (Brassica napus), petunia (Petunia hybrida), poplar (Populus spp.), potato (Sola- num tuberosum), tobacco (Nicotiana tabacum), and to- mato (Solanum lycopersicum)) or embryogenesis (carrot (Daucus carota), cotton (Gossypium hirsutum), rice (Oryza sativa), and soybean (Glycine max)), and maternal inher- itance of transgenes has been observed. Chloroplast- derived biopharmaceutical proteins, including insulin, interferons (IFNs), and somatotropin (ST), have been evaluated by in vitro studies. Human INFa2b trans- plastomic plants have been evaluated in field studies. Chloroplast-derived vaccine antigens against bacterial (cholera, tetanus, anthrax, plague, and Lyme disease), viral (canine parvovirus (CPV) and rotavirus), and protozoan (amoeba) pathogens have been evaluated by immune responses, neutralizing antibodies, and pathogen or toxin challenge in animals. Chloroplasts have been used as bioreactors for production of bio- polymers, amino acids, and industrial enzymes. Oral delivery of plant cells expressing proinsulin (Pins) in chloroplasts offered protection against development of insulitis in diabetic mice; such delivery eliminates expensive fermentation, purification, low temperature storage, and transportation. Chloroplast vector sys- tems used in these biotechnology applications are described. ADVANTAGES OF PLASTID TRANSFORMATION