TY - JOUR
T1 - In vitro ovule cultivation for live-cell imaging of zygote polarization and embryo patterning in Arabidopsis thaliana
AU - Kurihara, Daisuke
AU - Kimata, Yusuke
AU - Higashiyama, Tetsuya
AU - Ueda, Minako
N1 - Funding Information:
Microscopy in this work was conducted at the Institute of Transformative Bio-Molecules (WPI-ITbM) of Nagoya University and supported by the Japan Advanced Plant Science Network. This work was supported by grants from the Japan Science and Technology Agency (ERATO project to T.H. and M.U.) and from the Japan Society for the Promotion of Science: a Grant-in-Aid for Scientific Research on Innovative Areas (Nos. JP24113514, JP26113710, JP15H05962, and JP15H05955 for M.U., and Nos. JP16H06465, JP16H06464 and JP16K21727 for T.H), a Grant-in-Aid for Young Scientists (B, Nos. JP24770045 and JP26840093 for M.U.), and a Grant-in-Aid for challenging Exploratory Research (No. JP16K14753 for M.U.).
Publisher Copyright:
© 2017 Journal of Visualized Experiments.
PY - 2017/9/11
Y1 - 2017/9/11
N2 - In most flowering plants, the zygote and embryo are hidden deep in the mother tissue, and thus it has long been a mystery of how they develop dynamically; for example, how the zygote polarizes to establish the body axis and how the embryo specifies various cell fates during organ formation. This manuscript describes an in vitro ovule culture method to perform live-cell imaging of developing zygotes and embryos of Arabidopsis thaliana. The optimized cultivation medium allows zygotes or early embryos to grow into fertile plants. By combining it with a poly(dimethylsiloxane) (PDMS) micropillar array device, the ovule is held in the liquid medium in the same position. This fixation is crucial to observe the same ovule under a microscope for several days from the zygotic division to the late embryo stage. The resulting live-cell imaging can be used to monitor the real-time dynamics of zygote polarization, such as nuclear migration and cytoskeleton rearrangement, and also the cell division timing and cell fate specification during embryo patterning. Furthermore, this ovule cultivation system can be combined with inhibitor treatments to analyze the effects of various factors on embryo development, and with optical manipulations such as laser disruption to examine the role of cell-cell communication.
AB - In most flowering plants, the zygote and embryo are hidden deep in the mother tissue, and thus it has long been a mystery of how they develop dynamically; for example, how the zygote polarizes to establish the body axis and how the embryo specifies various cell fates during organ formation. This manuscript describes an in vitro ovule culture method to perform live-cell imaging of developing zygotes and embryos of Arabidopsis thaliana. The optimized cultivation medium allows zygotes or early embryos to grow into fertile plants. By combining it with a poly(dimethylsiloxane) (PDMS) micropillar array device, the ovule is held in the liquid medium in the same position. This fixation is crucial to observe the same ovule under a microscope for several days from the zygotic division to the late embryo stage. The resulting live-cell imaging can be used to monitor the real-time dynamics of zygote polarization, such as nuclear migration and cytoskeleton rearrangement, and also the cell division timing and cell fate specification during embryo patterning. Furthermore, this ovule cultivation system can be combined with inhibitor treatments to analyze the effects of various factors on embryo development, and with optical manipulations such as laser disruption to examine the role of cell-cell communication.
KW - Arabidopsis thaliana
KW - Developmental biology
KW - Embryo
KW - Issue 127
KW - Live-cell imaging
KW - Plant biology
KW - Zygote
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U2 - 10.3791/55975
DO - 10.3791/55975
M3 - Article
C2 - 28930998
AN - SCOPUS:85030981081
VL - 2017
JO - Journal of Visualized Experiments
JF - Journal of Visualized Experiments
SN - 1940-087X
IS - 127
M1 - e55975
ER -