An episomal vector system for plastid transformation in higher plants

We developed a new plastid transformation vector system using the putative replication origin of a minicircular chromosome from the marine dinoflagellate Heterocapsa triquetra. Transplastomic tobacco plants generated with this vector properly expressed the green fluorescent protein (GFP) gene without incorporating it into the plastid genome. To construct the episomal vector, a 610-bp DNA fragment containing the putative replication origin was fused to a dicistronic expression cassette encoding the aminoglycoside 3′-adenyltransferase ( aadA ) and gfp genes under control of the plastid rrn promoter. The vector was delivered to plastids of tobacco leaf explants by biolistic bombardment. After 8 weeks of bombardment, episomal transformant shoots were generated from leaf explants cultured on selection media containing 500 mg/L spectinomycin. Fluorescence microscopy and northern blot analysis demonstrated GFP expression in episomal transformant plants. PCR, Southern blot analysis, recovery of episomes, and sequencing analysis showed the vector to be maintained as self - replicating extrachromosomal circular DNA molecules for at least 6 months. Using a single construct for all plants, our episomal vector system may offer an advantage over the conventional plastid vector systems, which require species-specific constructs.