12.5 Regeneration of protonemata from excised leaves

Yoshikatsu Sato


Cell fate change of a matured cell to a pluripotent stem cell is a well-known feat of plants. To understand the reprogramming it is useful to analyze simple systems. Because of the simplicity of its body structures, the leaf of P. patens is one of the most tractable systems for the study of plant regeneration.



Procedure is very simple. Gametophore leaves are cut off from the gametophore and plated on the BCDAT medium. Usually, cells faced to the cut end are regenerated and exhibit the protonemal growth within 48 h. However, the followings are important for the reproducible results. If the timing of regeneration is delayed or the position of regenerating cell is varied, please check them.


Key points

1. Use young leaves

Gametophores are grown for 4-5 weeks on the BCDAT medium. Young leaves of gametophores up to the 3rd leaf from the top should be used for the regeneration assay.  The older leaves will need the long time for regeneration.

2. Keep from drying of leaves

Drying leaves will result in the delay of regeneration and ectopic regeneration. Gametophores are pulled out and plated on the BCDAT medium covered with a sheet of cellophane quickly. The surface of the cellophane should be wet. Add a drop of sterilized water if the surface is dry.

3. Keep from damaging of leaf cells after excision

Regeneration progresses at the cells where they located at the face of cut plane. However, drying of a leaf and wounding a leaf except the excised region will exhibit regeneration ectopically. To prevent this, transfer a leaf to BCDAT medium at a time with tweezers. Put a leaf on the tip of tweezers (Do not grasp a leaf).