Box 1. Acquisition and maintenance of BY-2 cells

The tobacco BY-2 cell line can be used for non-commercial scientific studies without any restrictions and investigators can obtain samples from nearby users. The authors can supply the transgenic cell lines as well (please see our website: http://www.biol.s.u-tokyo.ac.jp/users/hasezawa/eng/mta.html). We usually maintain the original and transgenic BY-2 lines as liquid cultures on a rotary shaker set at 130 r.p.m., 27 °C, in the dark. Every 7 days, we transfer 1–1.5 ml of culture at the stationary phase to 95 ml of fresh medium. Investigators can transfer BY-2 cells by normal aseptic procedures using a sterilized graduated pipette with a large mouth. After transfer, the BY-2 cells begin proliferation within a day and undergo active cell division for 4–5 days, after which they reach the stationary phase. Stationary cultures can be recognized by their thick appearance. Ideally, the cell number should increase 100-fold in 7 days. ? TROUBLESHOOTING

CRITICAL Often we hear the difficulty in reproducing the high synchronization. From our experience, it largely depends on the starting material. Therefore, the maintenance of the culture condition is the most critical step to acquire high cell synchrony constantly.

BY-2 cells can also be maintained as calli on modified LS agar plates. When liquid cultures are initiated from calli, start with a small culture (about 10 ml in a 50-ml flask) and then gradually increase the volume. It will take several weeks until the growth rate becomes rapid enough to yield acceptable synchronization.