Biology Reference
In-Depth Information
Fig. 4
Protoplasts from
Arabidopsis thaliana
cell culture under light microscope. (
a
) Seven-day-old cells and
(
b
) intact protoplasts produced by degradation of the cell wall using a buffer containing 0.4 % (w/v) cellulase,
0.05 % (w/v) pectolyase, 0.4 M mannitol. (
c
) Released cellular contents of disrupting protoplasts with fi ve
strokes of a Potter-Elvehjem homogenizer on ice. Scale bars: 20
μ
m
4. Note that the original recipe by [
12
] includes 1 % (w/v)
bovine serum albumin (BSA). However, this may interfere
with protein quantifi cation if not adequately removed by
washing. An alternative is to include BSA in this buffer and
wash with Chloroplast Buffer without BSA.
5. The ratio of plant cells fresh weight (FW) to enzyme buffer
solution for protoplast generation may require some adjust-
ment as it depends on the plant origin and its growth. The
optimal ratio can be determined by the amount of released
cellular contents during the homogenization step (Fig.
4
).
For our
Arabidopsis
cell cultures, the optimal ratio range is
in between 1:4 and 1:5 plant cells (FW) to enzyme buffer
solution. Ratios below this range are generally ineffective at
producing protoplasts.
6. For the successful production of protoplasts from cell cultures,
two critical factors are the use of a wide-base conical fl ask and
the speed of the orbital shaker during digestion. The depth of
Enzyme Buffer solution containing
Arabidopsis
cells in the
conical fl ask should be relatively shallow (i.e., no more than
~3 cm) and the shaker should be set at a speed in where cells
are maintained in suspension. Setting the shaker speed too
slow will allow cells to settle to the bottom of the fl ask during
incubation and likely result in ineffi cient enzymatic digestion
of cell walls. Conversely, setting the shaker speed too high may
cause protoplasts to rupture from higher energy collisions, by
either hitting each other or the fl ask.
7. After protoplasts are removed from the Enzyme Buffer and
washed free of cellulase and pectolyase, they should be homog-
enized as soon as possible.
