Synchronized tobacco protoplasts are efficiently transformed by DNA

A system for the synchronization of tobacco protoplasts was developed using aphidicolin, a mycotoxin which inhibits alpha-like DNA polymerase. Synchronized SR1 tobacco protoplasts were transformed with plasmid-DNA, derived from pLGV neo 11, at different stages of the cell cycle and the frequencies of kanamycin-resistant calli were measured. Compared to unsynchronized protoplasts synchronized cells show a clear increase in transformability provided that the transformation was performed at S- or M-phase. After completion of the M-phase trans-formation efficiencies dropped to the level of unsychronized cells. The efficiency of transformation for synchronized protoplasts was up to 3% of the surviving cells. This is approximately two orders of magnitude higher than the transformation efficiencies for unsynchronized protoplasts.