Morphological Analysis of Cell Growth Mutants in Physcomitrella - Plant Cell Morphogenesis: Methods and Protocols - page 203

5. When possible we use exon sequences that can be amplifi ed
directly from genomic DNA, but on occasion it may be neces-
sary to use cDNA when two or more exons form part of the
construct.
6. Genomic sequence information and the most up-to-date gene
model annotations can be retrieved from http://www.cosmoss.
org . In addition, Phytozome ( http://www.phytozome.net )
provides useful comparative analysis that can help refi ne the
automatic models generated by the cosmoss.org site.

7. Sometimes 15 μ g is suffi cient for a transformation. We fi nd

g gives consistent results across several different
DNA preparations.
8. This medium is a modifi cation of the original protocols involv-
ing top agar for plating. The medium reduces the survival rate
of the protoplasts, but they regenerate and grow faster. The
other advantage is that the 1-week old plants can be recovered
for immunocytochemistry or quantitative PCR.
9. These plates can be prepared in advance; we usually prepare
enough for 1-2 months and keep them at 4 ° C. It is important
to avoid contamination of these plates since they sometimes
form condensation.
10. The concentration of complementing plasmid required can vary
considerably. Highly abundant proteins such as ADF and pro-
fi lin complement well at high concentrations of plasmid [ 3 , 5 ].
Formin, which is present at low concentrations, requires much
less plasmid for complementation [ 7 ] and, at high concentra-
tions, is toxic. In contrast, myosin XI, which is also expressed at
low concentrations, can tolerate high levels of plasmid [ 6 ].

that using 30 μ

11. The precise reason for the presence of these two populations is
not completely clear and it varies from construct to construct.
In general, constructs with a very weak or no phenotype tend
to have fewer of the no-silencing plants; for example, the
GUS-RNAi control shows very few plants with nuclear GFP,
while constructs with severe growth phenotypes accumulate
more plants with nuclear GFP that do not show the growth
phenotype.
12. The ImageJ macro requires that images from plants are
extracted or “shaved” to isolate them from debris present in
the background or from other plants; these isolated images are
transferred to a new image stack that automatically matches
the same number of frames as the original stack.
13. When processing an RGB stack, the macro assumes that the
red color channel, channel one, contains the chloroplast auto-
fl uorescence information. If this is not the case, the informa-
tion must be put in the fi rst RGB channel. This can be done
by splitting the RGB stack and rearranging it such that the
proper information is in channel one.