A simple technology for plastid transformation with fragmented DNA

Plastid engineering has several unique advantages such as high expression of transgenes due to high polyploidy of plastid genomes and environmental biosafety because of maternal inheritance of transgenes, and has become a promising tool for molecular farming, metabolic engineering, and genetic improvement. However, there are no standard vectors available for plastid transformation. Moreover, the construction of plastid transformation vectors containing long operons or genes encoding proteins that are toxic to Escherichia coli was tedious or difficult. Here, we developed a simple plastid transformation technology without the need for in vitro vector construction by using multiple linear DNA fragments which share homologous sequences (HSs) at their ends. The strategy is based on homologous recombination between HSs of DNA fragments via endogenous recombination machinery in plastids, which subsequently are integrated into the plastid genome. We found that HSs of 200 bp or longer were sufficient for mediating the integration into the plastid genome with at least similar efficiency to that of plasmid DNA-based plastid transformation. Furthermore, we successfully used this method to introduce a phage lysin-encoding gene and a long operon into a tobacco plastid genome. The establishment of this technology simplifies the plastid transformation procedure and provides a novel solution for expressing proteins, which are either toxic to the cloning host or large operons in plastids, without need of vector cloning.