TRANSFORMING THE PLASTOME - GENETIC-MARKERS AND DNA DELIVERY SYSTEMS

Stable chloroplast transformants were first obtained following particle bombardment of tobacco leaves, and later by PEG-mediated uptake of DNA by protoplasts. The transforming DNA in these studies was itself of plastid origin and carried double (streptomycin, spectinomycin) antibiotic resistance which was used to select transformants. Integration was by homologous recombination, and both donor and recipient were Nicotiana species. Recent characterisation of plastid mutants of Solanum nigrum has allowed the extension of this gene replacement approach to include Nicotiana:Solanum combinations. The introduction of functional heterologous genes into the plastome is an alternative approach based on the use of constructs in which a bacterial resistance gene is flanked by sequences homologous to a region of the recipient plastome. Thus homologous recombination in the flanking sequences allows introduction of a foreign gene. A large number of putative transformants can be generated by the method, but this apparent attraction is partly offset by the need for repeated cycles of re-selection to obtain homoplasmic plants. In contrast, homoplasmy can be accomplished in a single selection step using plastome-encoded antibiotic resistance markers. The plastome is an attractive target for the introduction of useful genes into crop plants, as maternal inheritance acts as an insurance against unwanted spread of the foreign gene, and the large plastome copy number ensures immediate gene amplification and may influence levels of expression. Specific characters encoded on the plastid DNA, including components of photosynthesis and other aspects of metabolism, will also become open to manipulation as a consequence of developments in plastid transformation.