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In angiosperms, many developmental events, including seed germination, seedling growth, vegetative growth, reproductive growth, flowering, double fertilization, and seed formation, occurs during life cycle. Among these developmental events in angiosperms, our research interests are the molecular and cellular mechanisms in fertilization, zygotic embryogenesis and seed formation, since these are critical reproductive processes for generating next generation.

[Lab members]
Professor: Takashi Okamoto
Assistant professor: Toshiko Furukawa
Ph.D. student: Yukinosuke Ohnishi, Hiromi Suzuki, Rahman MD Hassanur, Yoriko Watanabe
Master student: Yumiko Sukawa
Undergraduate student: Kokubu Iwao, Koiso Narumi, Nakahira Orika, Takahara Mirei, Iwami Momoka
Cooperative associate professor: Mitsunori Seo
Guest professor: Tomokazu Koshiba

[Updated on May 18, 2016]
Faculty
Prof Takashi Okamoto e-mail
Ast Prof Toshiko Furukawa e-mail
In vitro fertilization with rice gametes
In contrast to animals and lower plants, which use naturally free-living gametes, in angiosperms, the fertilization and subsequent events such as gamete fusion, embryogenesis and endosperm development occur in the embryo sac deeply embedded in ovular tissue. Difficulties associated with research directly addressing the biology of the female gamete, zygote and early embryo have impeded investigations into the molecular mechanisms of fertilization and embryogenesis. Therefore, such investigations have been conducted predominantly through mutant analyses using Arabidopsis models. However, for two decades, in vitro fertilization (IVF) has been utilized as a tool in angiosperm to observe and analyze fertilization and post-fertilization processes directly.
The rice IVF system was established to take advantage of the abundant resources stemming from rice research for investigations into the mechanisms of fertilization and early embryogenesis (Figure 1). Fusion of gametes can be performed using electrofusion (movie of gamete fusion) and the fusion product, a zygote, forms a cell wall and an additional nucleolus. The zygote divides into an asymmetric two-celled embryo and develops into an early globular embryo, as in planta. The embryo further develops into irregularly shaped cell masses and fertile plants can be regenerated from the cell masses. This rice IVF system is a powerful tool for studying the molecular mechanisms involved in the fertilization and early embryogenesis of angiosperms and for making new cultivars.
Image
Figure 1. In vitro fertilization system with rice gametes.
Egg cell activation and subsequent zygotic development
To understand how the egg cell is activated by the fusion with sperm cells, resulting fused egg cell (zygote) stars the genetic and cellular programs for embryonic development, we currently conducting the projects described below.

(1) Egg cell activation and cell cycle regulation
(2) Functional analyses of genes expressing in zygote with monoallelic manner
(3) Regulation system of nuclear fusion (karyogamy) in zygote
(4) Epigenetic regulation in zygote
(5) Establishment of cell polarity in zygote and subsequent asymmetric zygote division

Figrue 2 shows the kayogamy progression in rice zygote.
Image
Figure 2. Karyogamy in rice zygote.
Toward making new cultivars
Image
Figure 3. Embryogenic callus from rice-wheat hybrid zygote
By the artificial fusion of gametes from different and same plant species with a combination of as intended, hybrid or polyploidy zygotes have been prepared for making new cultivars. Figure 3 shows the hybrid embryogenic callus from rice and wheat gametes.
Recent Publications
  1. Toda, E., Ohnishi, Y., Okamoto, T. (2016) Development of polyspermic rice zygotes. Plant Physiol., 171: 206-214.
  2. Matsumura T. and Okamoto T. (2016) Isolation of gametes from Brachypodium distachyon. Plant Biotech. 33: 39-43.
  3. Ohnishi, Y., Okamoto, T. (2015) Karyogamy in rice zygotes: Actin filament-dependent migration of sperm nucleus, chromatin dynamics, and de novo gene expression. Plant Signal. Behav., DOI:10.4161/15592324.2014.989021
  4. Ohnishi, Y., Okamoto, T. (2015) Microscopic observation, three-dimensional reconstruction, and volume measurements of sperm nuclei. Bio-protcol, in press
  5. Ohnishi, Y., Hoshino, R., Okamoto, T. (2014) Dynamics of male and female chromatin during karyogamy in rice zygotes. Plant Physiol., 165: 1533-1543.
  6. Okamoto, T. (2014) Gene and protein expression profiles in rice gametes and zygotes: a cue for understanding the mechanisms in gametic and/or early zygotic development of angiosperms. In gSexual Reproduction in Animals and Plantsh, Eds, Sawada H., Inoue H., Iwano M., Springer, pp369-382.
  7. Abiko, M., Maeda, H., Tamura, K., Hara-Nishimura, I., Okamoto, T. (2013) Gene expression profiles in rice gametes and zygotes: Identification of gamete-enriched genes and up- or down-regulated genes in zygotes after fertilization. J. Exp. Bot. 64: 1927–1940.
  8. Abiko M., Furuta K., Yamauchi Y., Fujita C., Taoka M., Isobe T., Okamoto T. (2013) Identification of proteins enriched in rice egg or sperm cells by single-cell proteomics. PLOS ONE 8(7): e69578
  9. Okamoto T. (2011) In vitro fertilization with isolated rice gametes: production of zygotes and zygote and embryo culture. Methods Mol. Biol. 710: 17-27.
  10. Ohnishi T., Takanashi H., Mogi M., Takahashi H., Kikuchi H., Yano K., Okamoto T., Fujita M., Kurata N. and Tsutsumi N. (2011) Distinct gene expression profiles in egg and synergid cells of rice as revealed by cell type-specific microarrays. Plant Physiol. 155: 881-891.
  11. Okamoto T. (2010) Gamete fusion site on the egg cell and autonomous establishment of cell polarity in the zygote. Plant Signaling & Behavior, 5: 1464-1467
  12. Nakajima K., Uchiumi T. and Okamoto T. (2010) Positional relationship between the gamete fusion site and the first division plane in the rice zygote. J. Exp. Bot. 61: 3101-3105.
  13. Sato A., Toyooka K., and Okamoto T. (2010) Asymmetric cell division of rice zygotes located in embryo sac and produced by in vitro fertilization. Sex Plant Reprod. 23: 211–217.
  14. Takanashi, H., Ohnishi, T., Mogi, M., Okamoto, T., Arimura, S and Tsutsumi N. (2010) Studies of mitochondrial morphology and DNA amount in the rice egg cell. Curr. Genet. 56:33-41.
  15. Wang, S., Okamoto, T. (2009) Involvement of polypyrimidine tract-binding protein (PTB) related proteins in pollen germination in Arabidopsis. Plant Cell Physiol. 50: 179-190.
  16. Kranz, E., Hoshino, Y., Okamoto T. (2008) In vitro fertilization with isolated higher plant gametes. Methods Mol. Biol. 427: 51-69.
  17. Uchiumi, T., Uemura, I., Okamoto, T. (2007) Establishment of an in vitro fertilization system in rice (Oryza sativa L.). Planta 226:581-589.
  18. Uchiumi T., Komatsu S., Koshiba T. and Okamoto T. (2006) Isolation of gametes and central cells from Oryza sativa L. Sex Plant Reprod. 19: 37-45.
©2015 Department of Biological Sciences, Tokyo Metropolitan University