A transplastomic plant is a genetically modified plant in which genes are inactivated, modified or new foreign genes are inserted into the DNA of plastids like the chloroplast instead of nuclear DNA.
Currently, the majority of transplastomic plants are a result of chloroplast manipulation due to poor expression in other plastids. However, the technique has been successfully applied to the chromoplasts of tomatoes.
Chloroplasts in plants are thought to have originated from an engulfing event of a photosynthetic bacteria (cyanobacterial ancestor) by a eukaryote. There are many advantages to chloroplast DNA manipulation because of its bacterial origin. For example, the ability to introduce multiple genes (operons) in a single step instead of many steps and the simultaneous expression of many genes with its bacterial gene expression system. Other advantages include the ability to obtain organic products like proteins at a high concentration and the fact that production of these products will not be affected by epigenetic regulation.
The reason for product synthesis at high concentrations is because a single plant cell can potentially carry up to 100 chloroplasts. If all these plastids are transformed, all of them can express the introduced foreign genes. This is may be advantageous compared to transformation of the nucleus, because the nucleus typically contains only one or two copies of the gene.
The advantages provided by chloroplast DNA manipulation has seen growing interest into this field of research and development, particularly in agricultural and pharmaceutical applications. However, there are some limitations in chloroplast DNA manipulation, such as the inability to manipulate cereal crop DNA material and poor expression of foreign DNA in non- green plastids as mentioned before. In addition, the lack of post- translational modification capability like glycosylation in plastids may make some human- related protein expression difficult. Nevertheless, much progress has been made into plant transplastomics, for example, the production of edible vaccines for Tetanus by using a transplastomic tobacco plant.
Transformation and selection procedure
Gene construct
The first requirement for transplastomic plant generation is to have a suitable gene construct that can be introduced into a plastid like a chloroplast in the form of an E. coli plasmid vector. There are several key features of a suitable gene cassette including but not limited to (1) selectable marker (2) flanking sequences (3) gene of interest (4) promoter sequences (5) 5' UTR (6) 3' UTR (7) intercistronic elements. The selectable marker typically tends to be an antibiotic resistant gene, which would give the plant cell the ability to tolerate being grown on antibiotic containing agar plates. Flanking sequences are crucial for introduction of the gene construct at precise predetermined points of the plastid genome through homologous recombination. The gene of interests introduced have many different applications and can range from pest resistance genes to vaccine antigen production. Intercistronic elements (IEE) are important for facilitating high levels of gene expression if multiple genes are introduced in the form of an operon. Finally, the 5' UTR and 3' UTR enhances ribosomal binding and increases transcript stability respectively.