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Establishment of an in vitro fertilization system in rice (Oryza sativa L.)

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Abstract

In vitro fertilization (IVF) systems using isolated male and female gametes have been utilized to dissect fertilization-induced events in angiosperms, such as egg activation, zygote development and early embryogenesis, as the female gametophytes of plants are deeply embedded within ovaries. In this study, a 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. Fusion of gametes was performed using a modified electrofusion method, and the fusion product, a zygote, formed cell wall and an additional nucleolus. The zygote divided into a two-celled embryo 15–24 h after fusion, and developed into a globular-like embryo consisting of an average of 15–16 cells by 48 h after fusion. Comparison of the developmental processes of zygotes produced by IVF with those of zygotes generated in planta suggested that zygotes produced by IVF develop and grow into early globular stage embryos in a highly similar manner to those in planta. Although the IVF-produced globular embryos did not develop into late globular-stage or differentiated embryos, but into irregularly shaped cell masses, fertile plants were regenerated from the cell masses and the seeds harvested from these plants germinated normally. The rice IVF system reported here will be a powerful tool for studying the molecular mechanisms involved in the early embryogenesis of angiosperms and for making new cultivars.

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Abbreviations

DAPI:

4′,6-Diamidino-2-phenylindole

IVF:

In-vitro fertilization

NAA:

1-Naphtylacetic acid

PEG:

Polyethyleneglycol

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Acknowledgments

This work was supported in part by Grants-in-Aid (Grant no. 17657075 to T.O.), and by the “The Initiative for Attractive Education in Graduate Schools” fund to the Department of Biological Sciences, Graduate School of Tokyo Metropolitan University from the Ministry of Education, Science, Sport, and Culture of Japan. We are grateful to the Riken Bio Resource Center (Tsukuba, Japan) for providing cultured rice cells (Line, Oc).

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  1. Department of Biological Sciences, Tokyo Metropolitan University, Minami-osawa 1-1, Hachioji, Tokyo, 192-0397, Japan

    Takao Uchiumi, Isao Uemura & Takashi Okamoto

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  1. Takao Uchiumi
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  2. Isao Uemura
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  3. Takashi Okamoto
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Correspondence to Takashi Okamoto.

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Uchiumi, T., Uemura, I. & Okamoto, T. Establishment of an in vitro fertilization system in rice (Oryza sativa L.). Planta 226, 581–589 (2007). https://doi.org/10.1007/s00425-007-0506-2

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