Transplastomic Plants Homoplasmic for Foreign Transgenes.

Scientists pay more and more attention to the research on plastid engineering for its following advantages foreign genes can be integrated site-specifically into the plastid genome (plastome) there are no position effects as experienced with random insertion of transgenes in nuclear transformation pollen-mediated dispersion of transgenes can be avoided because chloroplasts are maternally transmitted gene silencing does not occur in plastids and therefore transgene expression is stable in progeny of transplastomic plants and the high ploidy level of the plastome in leaf cells makes high levels of transgene expression feasible. At the same time, however, the highly polyploid plastome makes it difficult to get transplastomic plants homoplasmic for foreign transgenes. In this work, chloroplast transforming vector pTRCH205, which carries two psbA5'-nifH-psbA3'and Prrn-aadA-TpsbA cassettes flanked by plastid DNA sequence to target their insertion between psbA and trnK operons, was constructed. Plastid transformation of Nicotiana tabacum was carried out by the biolistic delivery of transforming plasmid pTRCH205 DNA into leaf cells. Integration of nifH and aadA by two homologous recombination events via the flanking ptDNA sequences, and selective amplification of the transplastomes on MS medium with high concentration of spectinomycin, yielded resistant cell lines. All the independent transplastomic lines were subjected to three additional rounds of regeneration and were subcultured for 6--10 times through stem sections on MS medium containing 500 mg/L spectinomycin, to obtain homoplasmic tissues. The results of PCR assay and Southern blot hybridization, probed with 0.9 kb BglII/SnaBI homologous fragment, indicated that foreign genes had been integrated into the plastomes of transgenic plants, which finally became homoplasmic for foreign transgenes.