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

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.

Isolation of gametes from <i>Brachypodium distachyon</i>

“…The isolated putative egg cells showed a granularstructured cytoplasm and a developed nucleolus ( Figure 1E), being consistent with cellular characteristics of egg cells isolated from rice (Uchiumi et al 2006) and maize (Kranz et al 1991;Kranz and Lörz 1993). In addition, vacuoles, ranging in size from 3-10 µm, existed at the peripheral region of the cells ( Figure 1E), and these peripheral localization of vacuoles has been reported in isolated egg cells of rice (Uchiumi et al 2007), maize (Faure et al 1992) and wheat (Kovacs et al 1994). These cytological characteristics observed in the isolated cells suggest that the cells released from cut Brachypodium ovaries are egg cells.…”

“…rice (Kranz and Lörz, 1993;Uchiumi et al 2007). In the case of wheat, although methods of isolation of egg cells, zygotes, and early embryos have been established (Kovacs et al 1994;Kumlehn et al 1999;Sprunck et al 2005), a complete IVF system with wheat gametes has not yet been established.…”

“…Distribution of mitochondria in the putative Brachypodium egg cell was observed, since it has been reported that mitochondria and starch granules are abundantly localized in the granular-structured cytoplasm (Diboll 1968;Faure et al 1992Faure et al , 1993Uchiumi et al 2006Uchiumi et al , 2007. To stain mitochondria, the putative Brachypodium egg cells were transferred into 2 µl droplets of mannitol solution (370 mOsmol kg −1 H 2 O) containing 2 µg/ml MitoTracker Red CMXRos (Molecular Probes, USA) for 40 min at room temperature in darkness.…”

“…Kranz and Lörz (1993) first developed a complete IVF system 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 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), fertilizationinduced/suppressed gene expression (Okamoto et al 2005), epigenetic resetting in early embryos (Jahnke and Scholten 2009), have been successfully observed and investigated.…”

Karyogamy in rice zygotes: Actin filament-dependent migration of sperm nucleus, chromatin dynamics, andde novogene expression

“…24 However, how the male nucleus approaches and fuses with the female nucleus remains unclear, partly because of the zygote's location within the embryo sac, which is deeply embedded in the ovaries, making the nuclear dynamics in zygotes difficult to trace. We prepared zygotes by in vitro fusion of rice gametes 25 in which nuclei, nuclear membranes, and Figure 1. Schematic diagram of the progression of zygotic development (A) and karyogamy (B) in rice.…”