Establishment of an In Vitro Fertilization System in Wheat (Triticum aestivum L.) Tety Maryenti 1 , Norio Kato 1,2,3 , Masako Ichikawa 3 and Takashi Okamoto 1,2, * 1 Department of Biological Sciences, Tokyo Metropolitan University, Minami-osawa 1-1, Hachioji, Tokyo, 192-0397 Japan 2 Plant Breeding Innovation Laboratory, RIKEN Cluster for Science, Tsurumi, Yokohama, 230-0045 Japan 3 Plant Innovation Center, Japan Tobacco Inc., Higashihara 700, Iwata, Shizuoka, 438-0802 Japan *Corresponding author: Email, okamoto-takashi@tmu.ac.jp; Fax, +81-42-677-2559. (Received November 7, 2018; Accepted December 21, 2018) In vitro fertilization (IVF) systems using isolated gametes have been utilized to dissect post-fertilization events in angiosperms, since the female gametophytes of plants are deeply embedded within ovaries. In addition, IVF systems have been used to produce hybrid and polyploid zygotes. Complete IVF systems have been established in maize and rice, two of three major crop species providing the majority of human energy intake. Among those crop species, gametes of wheat have not been used to establish a complete IVF system successfully. In this study, a wheat IVF system was developed to introduce the advantages of this technology to wheat research. Fusion of gametes was performed via a mod- ified electrofusion method, and the fusion product, a zygote, formed a cell wall and two nucleoli. The first division of zygotes was observed 19–27 h after fusion, and the resulting two-celled embryo developed into 10–20-celled globular- like embryos and multicellular club-shaped embryos by 3 and 7–10 d after fusion, respectively. Such zygotic division profiles were mostly consistent with those in the embryo sac, suggesting that the division profile of IVF-produced early embryos reflects that of early embryos in planta. Although the IVF-produced club-shaped embryos did not develop into differentiated embryos but into compact em- bryonic calli, fertile plants could be regenerated from the embryonic calli, and the seeds harvested from those plants grew normally into seedlings. The IVF system described here might become an important technique for generating new genotypes of wheat and/or new hybrids as well as for inves- tigating fertilization-induced events in wheat. Keywords: Egg cell  Embryogenesis  In vitro fertilization  Sperm cell  Wheat  Zygote. Abbreviations: DAPI, 4 0 ,6-diamidino-2-phenylindole; IVF, in vitro fertilization; PEG, polyethylene glycol. Introduction Fertilization is the key event in the life cycle of higher organisms. In angiosperms, the female gametophyte, which is also referred to as the embryo sac or megagametophyte, develops in the ovule, and fertilization and subsequent events such as embryo- genesis and endosperm development also occur in the embryo sac, which is deeply embedded in ovular tissue (Russell 1992, Raghavan 2003). Difficulties in directly researching the biology of the embedded female gamete, zygote and early embryo have impeded investigations into the molecular mechanisms of fer- tilization and embryogenesis. Therefore, such studies have been conducted predominantly through analyses of Arabidopsis mu- tants or transformants coupled with live-imaging (reviewed in Berger 2011, Hamamura et al. 2012, Hamamura et al. 2014, Maruyama et al. 2015). Alternatively, direct analyses using iso- lated gametes or zygotes are possible because procedures for isolating viable gametes have been established, and in vitro fertilization (IVF) systems using isolated gametes can be used to observe and analyze fertilization and post-fertilization pro- cesses directly (reviewed in Wang et al. 2006). The IVF system used for angiosperms involves a combin- ation of three basic microtechniques: (i) the isolation and se- lection of male and female gametes, (ii) the fusion of pairs of gametes and (iii) single-cell culture (Kranz 1999). For the isola- tion of viable gametes, procedures have been established in a wide range of plant species, including both monocotyledonous and dicotyledonous plants (reviewed in Kranz 1999 and in Okamoto 2011). Fusion of isolated gametes can be performed electrically (Kranz et al. 1991, Uchiumi et al. 2006) or chemically via calcium (Faure et al. 1994, Kranz and Lo ¨rz 1994, Khalequzzaman and Haq 2005), polyethylene glycol (PEG) (Sun et al. 1995, Tian and Russell 1997) or bovine serum albu- min (Peng et al. 2005), since plasma membranes of isolated gametes are partly or largely exposed, and plasma membranes between the isolated gametes can be attached each other. Among these four kinds of fusion procedures, only zygotes produced by electrofusion are known to divide and develop into embryo-like structures and plantlets. A complete IVF system that involves maize gametes and electrical fusion was developed by Kranz and Lo ¨rz (1993), and to take advantage of the abundant resources stemming from rice research, a rice IVF system was also established by Uchiumi et al. (2007). These electrofusion-based IVF systems have been successfully utilized to observe and investigate post-fertilization events such as kar- yogamy (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 nuclei (Scholten et al. 2002), microtubular architecture in egg cells and zygotes (Hoshino et al. 2004), fertilization- Plant Cell Physiol. 60(4): 835–843 (2019) doi:10.1093/pcp/pcy250, Advance Access publication on 3 January 2019, available online at https://academic.oup.com/pcp ! The Author(s) 2019. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com Regular Paper Downloaded from https://academic.oup.com/pcp/article/60/4/835/5272502 by guest on 09 November 2022