7.3@ Cell cycle inhibitors
The moss Physcomitrella patens has an amazing ability for regeneration, whose leaf cells readily change to protonemal apical cells with excision signals without any exogenous phytohormones, resulting in regeneration of new whole plants. In particular, given that a part of a leaf is excised from a gametophore, leaf cells facing to the dissected plane start to behave similarly to a protonemal apical cell and readily reenter cell cycle in a few days. To investigate early events during these processes in excised leaves, we use chemical inhibitors for cell cycle studies, roscovitine and aphidicolin.
1. Inoculate protonemata tissues blended by a polytron homogenizer, glass beads or motor and pestle.
2. Cultivate 3 to 4 weeks on BCDAT solid medium (until growth of gametophores with approximately 10 leaves or more) at 25˚C under continuous light.
3. Collect gametophores, of which rhizoids and protonema tissues should be removed, within 1 hour with forceps.
4. Put the collected gametophores in 50 ml tube containing 10 ml of BCDAT liquid medium or sterilized water.
5. Blend with a polytron homogenizer or glass beads to cut leaves apart from the gametophores. (By the treatment, the number of leaf cells facing to the excised planes in a leaf is also increased.)
6. Wash with BCDAT liquid medium twice.
7. Suspend in 5~10 ml BCDAT liquid medium.
8. A part of suspension (1~2 ml) is inoculated in a 35 mm dish or a well of 24-well plate.
9. Add roscovitine (final conc. 100 µM) or aphidicolin (final conc. 30 µM) and incubate 25˚C for 48 hours at under continuous white light conditions.
10. Harvest the leaves.
E Roscovitine inhibits the CDK activity, resulting in the G1 arrest of the P.patens leaf cells after treatment with a polytron homogenizer.
E Aphidicolin inhibits DNA polymerase a and d, resulting in the G1/S phase arrest. At 48 hours or more after the treatment, you may see leaves with leaf cells changed to protonemal apical cells, despite of the cell cycle arrest by aphidicolin.
Planchais S, Glab N, Inzé D, Bergounioux C. FEBS Lett. (2000) 476: 78-83.