12.5
Regeneration of protonemata from excised leaves
Yoshikatsu Sato
Introduction
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
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).