According to generally accepted knowledge, two classes of cell organelles, i.e. plastids and mitochondria, are derived from initially independent prokaryotes that were taken up into a predecessor of present day eukaryotic cells by separate endosymbiotic events (Gray, 1991). As a consequence, these organelles contain their own DNA, DNA transcripts in the form of messenger RNA, ribosomes, and at least some of the necessary tRNAs that are required for decoding of genetic information (Marechal-Drouard et al., 1991).
While, shortly after endosymbiotic uptake, these organelles were genetically autonomous, since they contained all the elements necessary to drive prokaryotic life, this autonomy was reduced during evolution by transfer of genetic information to the cell's nucleus. Nevertheless, their genetic information is of sufficient complexity to make recent cell organelles an attractive target for gene technology. This is particularly the case with plastids, because these organelles still encode about 50% of the proteins required for their main function inside the plant cell, photosynthesis. Plastids also encode their ribosomal RNAs, the majority of their tRNAs and ribosomal proteins. In total, the number of genes in the plastome is in the range of 120 (Palmer, 1991). The vast majority of proteins that are found in plastids are, however, imported from the nuclear/cytosolic genetic compartment.
Plastids can be Genetically Transformed
With the development of general molecular cloning technologies, it became soon possible to genetically modify higher plants by transformation. The main emphasis in plant transformation was and still is on nuclear transformation, since the majority of genes, ca. 26,000 in the case of Arabidopsis thaliana, the complete sequence of which was recently published (The Arabidopsis Genome Initiative, 2000), is found in the cell's nucleus. Nuclear transformation was easier to achieve, since biological vectors such as Agrobacterium tumefaciens were available, which could be modified to efficiently enable nuclear transformation (Galvin, 1998). In addition, the nucleus is more directly accessible to foreign nucleic acids, while the organelles are surrounded by two envelope membranes that are, generally speaking, not permeable to macromolecules such as DNA.
A capability of transforming plastids is highly desirable since it could make use of the enormous gene dosage in these organelles—more than 10000 copies of the plastome may be present per cell—that bears the potential of extremely high expression levels of trangenes. In addition, plastid transformation is attractive because plastid-encoded traits are not pollen transmissible; hence, potential risks of inadvertent transgene escape to wild relatives of transgenic plants are largely reduced. Other potential advantages of plastid transformation include the feasibility of simultaneous expression of multiple genes as a polycistronic unit and the elimination of positional effects and gene silencing that may result following nuclear transformation.
Methods that allow stable transformation of plastids could indeed be developed for higher plants. To date, two different Methods are available, i.e. particle bombardment of tissues, in particular leaf tissues (Svab et al., 1990), and treatment of protoplasts with polyethylene glycol (PEG) in the presence of suitable transformation vectors (Koop et al., 1996). Both methods mediate the transfer of plasmid DNA across the two envelope membranes into the organelle's stroma.
One significant disadvantage of all multicellular plant transformation procedures used today is the occurrence of marker genes in the transgenic plants. These marker genes that are needed for the selection of transgenic plant cells from a vast background of untransformed cells code for antibiotic or herbicide resistance genes. Examples for plastid resistance genes are aadA conferring resistance to spectinomycin and streptomycin (Svab & Maliga, 1993), or nptII conferring resistance to kanamycin (Carrer et al., 1993). As these marker genes are stably integrated into the genome together with the genes of interest (GOI), they will stay in the homoplastomic transgenic plants although they are not required for GOI function. These remaining marker genes are a main issue of criticism of plant biotechnology as they could theoretically increase antibiotic resistance of pathogens or herbicide resistance of weeds. Construction of a selection system which does not result in a resistance gene in the transgenic plant is, therefore, highly desirable (Iamtham and Day, 2000).
Another problem in plastid transformation is the shortage of selectable marker genes available. The aadA gene is the only selectable marker gene that is used routinely (Heifetz, 2000), and the nptII gene is the only alternative that has been shown to function in higher plant plastid transformation (Carrer et al., 1993). Since neither the aadA nor the nptII gene can be used universally, the number of higher plant species that have been transformed in the plastome is still very low (Heifetz, 2000). Plastid transformation in higher plants cannot at present be exploited to its full potential.
USPTO Backgrounds
USPTO Backgrounds