TRANSFORMING THE PLASTOME - GENETIC-MARKERS AND DNA DELIVERY SYSTEMS

Stable chloroplast transformants were first obtained following particl e bombardment of tobacco leaves, and later by PEG-mediated uptake of D NA by protoplasts. The transforming DNA in these studies was itself of plastid origin and carried double (streptomycin, spectinomycin) antib iotic resistance which was used to select transformants. Integration w as by homologous recombination, and both donor and recipient were Nico tiana species. Recent characterisation of plastid mutants of Solanum n igrum has allowed the extension of this gene replacement approach to i nclude 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 flank ed by sequences homologous to a region of the recipient plastome. Thus homologous recombination in the flanking sequences allows introductio n of a foreign gene. A large number of putative transformants can be g enerated 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 select ion step using plastome-encoded antibiotic resistance markers. The pla stome is an attractive target for the introduction of useful genes int o crop plants, as maternal inheritance acts as an insurance against un wanted spread of the foreign gene, and the large plastome copy number ensures immediate gene amplification and may influence levels of expre ssion. Specific characters encoded on the plastid DNA, including compo nents of photosynthesis and other aspects of metabolism, will also bec ome open to manipulation as a consequence of developments in plastid t ransformation.