KANAMYCIN RESISTANCE AS A SELECTABLE MARKER FOR PLASTID TRANSFORMATION IN TOBACCO

We report on a novel chimeric gene that confers kanamycin resistance o n tobacco plastids. The kan gene from the bacterial transposon Tn5, en coding neomycin phosphotransferase (NPTII), was placed under control o f plastid expression signals and cloned between rbcL and ORF512 plasti d gene sequences to target the insertion of the chimeric gene into the plastid genome. Transforming plasmid pTNH32 DNA was introduced into t obacco leaves by the biolistic procedure, and plastid transformants we re selected by their resistance to 50 mug/ml of kanamycin monosulfate. The regenerated plants uniformly transmitted the transplastome to the maternal progeny. Resistant clones resulting from incorporation of th e chimeric gene into the nuclear genome were also obtained. However, m ost of these could be eliminated by screening for resistance to high l evels of kanamycin (500 mug/ml). Incorporation of kan into the plastid genome led to its amplification to a high copy number, about 10000 pe r leaf cell, and accumulation of NPTII to about 1% of total cellular p rotein.