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combined with transient complementation to evaluate the effect of
point mutations or gene chimeras [
3
-
7
]. The morphological analy-
sis described here can also be used to evaluate the growth charac-
teristics of small moss plants derived from protoplasting and
regenerating viable mutants [
8
,
9
], which greatly simplifi es the
protocol and allows the analysis of a large number of plants with-
out the need for transient transformation.
This methodology takes advantage of the chlorophyll auto-
fl uorescence generated by the abundant chloroplasts present in
moss protonemal cells; this fl uorescence allows determining the
plant's shape. Independent images are acquired with a simple fl uo-
rescence stereo microscope from regenerating plants. The images
are analyzed by an ImageJ macro that provides morphological infor-
mation. Several hundred plants can be rapidly measured and aver-
aged, and effects can be evaluated by robust statistical analysis. The
described protocol can provide evidence for the participation of a
gene product in polarized cell growth, protonemal branching, or
cell division. In addition, transient complementation analysis allows
for the evaluation of fl uorescent protein fusion functionality and
detailed structure-function analysis using site-directed mutagenesis.
2
Materials
The protocol used here is optimized for polyethylene glycol
(PEG)-mediated transient transformation of moss protoplasts, a
simple and highly reproducible procedure ([
10
,
11
],
see
Note 1
).
A detailed description of the materials required for moss culture
and transformation has been recently compiled and can be easily
accessed (together with the relevant experimental protocols) from
our recent open-access publication in the Journal of Visual
Experiments [
10
]. This publication includes a detailed video to
help those that have no previous experience working with
Physcomitrella
, which can be accessed at
http://www.jove.com/
transformation-moss
(
see
also
Note 2
).
2.1 Moss Cultures
2.2 Vectors
and Cell Lines
The biological materials listed below are available upon request
from the authors or from the Bezanilla Laboratory (
http://beza-
1. pUGGi-RNAi silencing Gateway-based vector designed by
Bezanilla et al. [
12
]; via a simple LR reaction, this vector gen-
erates constructs that silence the gene(s) of interest plus the
internal nuclear reporter NLS-GFP-GUS.
2. pUGi-control RNAi plasmid to silence only the NLS-GFP-
GUS construct.
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