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Electro-fusion of Gametes and Subsequent Culture of Zygotes in Rice   

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Original research article

A brief version of this protocol appeared in:
Plant Physiology
Mar 2016

Abstract

Electro-fusion system with isolated gametes has been utilized to dissect fertilization-induced events in angiosperms, such as egg activation, zygote development and early embryogenesis, since the female gametophytes of plants are deeply embedded within ovaries. In this protocol, procedures for isolation of rice gametes, electro-fusion of gametes, and culture of the produced zygotes are described.

Keywords: Egg cell, Embryogenesis, Fertilization, in vitro fertilization, Sperm cell, Rice, Zygote

Background

Fertilization and subsequent events in angiosperms, such as embryogenesis and endosperm development, occur in the embryo sac deeply embedded in ovular tissue (Nawaschin, 1898; Guignard, 1899; Russell, 1992; Raghavan, 2003). Therefore, isolated gametes have been used for in vitro fertilization (IVF) system to observe and analyze fertilization and postfertilization processes (reviewed in Wang et al., 2006). The IVF system used for angiosperms includes a combination of three basic micro-techniques: (i) the isolation and selection of male and female gametes; (ii) the fusion of pairs of gametes and (iii) single cell culture (Kranz, 1999). Procedures for isolating viable gametes have been established in a wide range of plant species, including monocotyledonous and dicotyledonous plants (reviewed in Kranz, 1999 and in Okamoto, 2011). The isolated gametes can be fused electrically (Kranz et al., 1991; Uchiumi et al., 2006 and 2007) or chemically using calcium (Faure et al., 1994; Kranz and Lörz, 1994; Khalequzzaman and Haq, 2005), polyethyleneglycol (Sun et al., 1995; Tian and Russell, 1997) or bovine serum albumin (Peng et al., 2005), as the gametes are generally protoplasts. Although gametes can be fused using these different procedures, only zygotes produced by electro-fusion are only known to divide and develop into embryo-like structures and plantlets. A complete IVF system was developed by Kranz and Lörz (1993) using maize gametes and electrical fusion, and, to take advantage of the abundant resources stemming from rice research, a rice IVF system was also established by Uchiumi et al. (2007). By the use of these electro-fusion based IVF systems, post-fertilization events, such as karyogamy (Faure et al., 1993; Ohnishi et al., 2014), egg activation and zygotic development (Kranz et al., 1995; Nakajima et al., 2010; Sato et al., 2010), paternal chromatin decondensation in zygote nucleus (Scholten et al., 2002), the microtubular architecture in egg cells and zygotes (Hoshino et al., 2004), fertilization-induced/suppressed gene expression (Okamoto et al., 2005), epigenetic resetting in early embryos (Jahnke and Scholten, 2009), have been successfully observed and investigated. Moreover, polyspermic triploid zygotes were produced by the modification of the rice IVF system and the triploid zygotes were grown into triploid mature plants (Toda et al., 2016). The rice IVF system described here might become an important technique for generating new cultivars with desirable characters as well as for investigating post fertilization events.

Copyright: © 2016 The Authors; exclusive licensee Bio-protocol LLC.
How to cite:  Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Toda, E., Ohnishi, Y. and Okamoto, T. (2016). Electro-fusion of Gametes and Subsequent Culture of Zygotes in Rice. Bio-protocol 6(24): e2074. DOI: 10.21769/BioProtoc.2074.
  2. Toda, E., Ohnishi, Y. and Okamoto, T. (2016). Development of polyspermic rice zygotes. Plant Physiol 171(1): 206-214.
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