Morphological Analysis of Cell Growth Mutants in Physcomitrella - Plant Cell Morphogenesis: Methods and Protocols - page 198

3. Plate cells on cellophane-covered PRM-B plates using liquid
plating medium.
4. Four days after transformation, transfer plants to regular
growth medium (PPNH4) plates containing 15 μ g/ml

hygromycin.
5. Grow for 3 more days.

1. Using the fl uorescence channel that allows the visualization of
both the nuclear GFP and the chloroplast auto-fl uorescence,
identify actively silencing plants. Living plants can be easily
distinguished from dead plants by the level of chlorophyll fl u-
orescence; dead plants have very weak or no fl uorescence,
while live plants show intense red fl uorescence. At this stage,
there will be several dead plants that were not transformed and
were killed by the antibiotic. There will also be two popula-
tions of living plants, some containing nuclear GFP and some
without nuclear GFP ( see Note 11 ).
2. The presence of the internal silencing reporter allows one to
visually identify the plants that are actively silencing. Under
optimal conditions, it should be possible to fi nd 50-100 of
these plants per Petri dish of transformed plants.
3. At this point you can remove the lid of the Petri dish under
observation to improve the quality of the images. Try to mini-
mize the time that the dishes are open because the level of
moisture will rapidly diminish resulting in plant damage. It is
also important to avoid constant illumination of a single plant
with the blue light; this can result in “crumbling” of plants
with extensive exposure to the air.
4. We routinely capture images of individual plants with a color
digital camera using a fi lter that allows for both red and green
channels to be captured simultaneously; an example of the
types of images acquired is presented in Fig. 1 . Alternatively a
black and white camera may be used to capture images; under
this condition it is important to avoid multiple plants per
image; when this is unavoidable the plants that are actively
silencing should be identifi ed in a separate record for later
morphological analysis.
5. Sometimes two or three actively silencing plants may be pres-
ent in one fi eld; in those cases we recommend acquiring two
or three pictures of the same fi eld to simplify downstream
analysis ( see Note 12 ).

3.4 Image
Acquisition

3.5 Examples

Example images are provided to test the ImageJ macro and to
become familiar with the types of data sets generated by the mor-
phological analysis. The data set is composed of two separate TIF
stacks of images. They can be downloaded from http://users.wpi.
edu/~lvidali/MMB_moss/ . One data set has 30 images of the