Time-Lapse Imaging of Developing Meristems Using Confocal Laser Scanning Microscope - Plant Cell Morphogenesis: Methods and Protocols - page 115

However, even when using 11-12 days old seedlings for time-
lapse imaging (at 12 h interval), we occasionally observe the tran-
sition at the fourth or fi fth time point. During the fl oral transition,
the shape of meristem is changing, i.e., the meristem is more
bulgy (and instead of leaf primordia, fl oral meristems are formed).
5. Immersion of the meristem can be done in any water solution,
including tap water, although sterile water must be preferred
when doing time-lapse imaging over several days, to prevent
contaminations. While this may represent a hypotonic stress, in
our hands, we found little impact on signals in Arabidopsis mer-
istem. This is likely to depend on the species, as tomato meri-
stems seem to have a higher isotonic point than Arabidopsis
meristems ( see , e.g., ref. [ 27 ]).
6. We fi nd that the extent of FM4-64 staining can be unpredict-
able. Depending on the biological question, using membrane-
bound fl uorescent reporters (e.g., p35S::GFP-LTI6b line) can
be preferable, notably when focusing on the epidermal layer of
the tissue.
7. Many physiological processes are regulated by light; thus, light
conditions in a growth chamber, in which in vitro culture is con-
ducted, are very important. For example, to follow morphogen-
esis at the tomato meristem, we use a constant light for in vitro
culture, as organogenesis can be controlled by light. In particu-
lar, light can change the distribution of auxin at the meristem and
PIN1 localization at plasma membranes and affect cytokinin sig-
naling and expression of key regulatory genes [ 11 ].

Acknowledgments

This work was supported by a bilateral grant from INRA, France,
and Ministry of Science and Higher Education, Poland, and by a
grant from Agence Nationale de la Recherche ANR-10-
BLAN-1516 “Mechastem.” We thank Marion Louveaux for help-
ful comments on this manuscript.

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