Chloroplast Genetic Engineering

Chloroplasts are ideal hosts for the expression of transgenes. Once integrated via homologous recombination, these transgenes express large amounts of protein (up to 46% of total leaf protein) due to the high copy number of the chloroplast genome in each plant cell. Foreign proteins that are toxic when present in the cytosol, such as vaccine antigens, trehalose and xylanase, are non-toxic when sequestered within transgenic plastids. Because transgenes are maternally inherited in most crops, there is little danger of cross-polination with wild-type plants. By using chloroplast DNA sequences that flank transgenes, higher plants have efficiently and stably integrated transgenes imbuing important agronomic traits, including herbicide, insect and disease resistance, drought and salt tolerance, and phytoremediation. More recently, highly efficient, soybean, carrot and cotton plastid transformation have been accomplished via somatic embryogenesis using species-specific vectors. Chloroplast transgenic carrot plants withstand salt concentrations that only halophytes could tolerate. Previously an exclusively mitochondrial-encoded trait, cytoplasmic male sterility is now possible through β-ketothiolase expression via the chloroplast genome. This is a valuable tool towards transgene containment, in addition to the maternal inheritance of transgenes integrated into the chloroplast genome, in most crops. Crops such as tobacco have expressed transgenes for a variety of biopharmaceuticals, vaccines and biomaterials. Due to the high biomass of tobacco plants (~40 mtons/acre), large amounts of vaccines preventing anthrax, plague, tetanus and cholera, and pharmaceuticals like human somatotropin, serum albumin, interferons and insulin-like growth factor have been produced in transgenic chloroplasts. The chloroplast also contains machinery that allows for correct folding and disulfide bond formation, resulting in fully functional human blood proteins or vaccine antigens. Additionally, expression of the Rubisco small subunit gene (RbcS) via the chloroplast genome restored normal photosynthetic activity in a nuclear RbcS antisense line, a goal that has been elusive for decades. Multigene operons engineered into the chloroplast genome do not require processing of polycistrons to monocistrons for efficient translation. Secondary structures formed by intergenic spacer regions in bacterial operons are efficiently recognized by the chloroplast processing machinery; when such processing occurs, 3’ UTRs are not required for transcript stability. Extension of chloroplast genetic engineering technology to other useful crops will depend on the availability of the plastid genome sequences and the ability to regenerate transgenic events and advance them towards homoplasmy. In addition to biotechnology applications, plastid transformation system has been extensively used to study chloroplast biochemistry and molecular biology.