Polyethylene glycol calcium (PEG-Ca²⁺)-mediated transfection allows rapid and efficient examination to analyze diverse cellular functions of genes of interest. In plant cells, macromolecules, such as DNA, RNA and protein, are delivered into protoplasts derived from somatic tissues or calli via PEG-Ca²⁺ transfection. To broaden and develop the scope of investigations using plant gametes and zygotes, a procedure for direct delivery of macromolecules into these cells has recently been established using PEG-Ca²⁺ transfection. This PEG-Ca²⁺-mediated delivery into rice egg cells/zygotes consists of four microtechniques, (i) isolation of gametes, (ii) production of zygotes by electrofusion of gametes, (iii) PEG-Ca²⁺-mediated delivery of macromolecules into isolated egg cells or produced zygotes, and (iv) culture and subsequent analyses of the transfected egg cells/zygotes. Because the full protocol for microtechniques (i) and (ii) have already been reported in Toda et al., 2016, microtechniques (iii) and (iv) are mainly described in this protocol.

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.

Karyogamy, a migration of the sperm nucleus toward the egg nucleus and their subsequent nuclear fusion, is an important biological event for initiating zygote formation toward early embryogenesis in angiosperms. However, how the male nucleus approaches and fuses with the female nucleus still remains unclear. Recently, time-lapse measurement of nuclear volume during karyogamic events revealed that the sperm nucleus enlarges during contact with the egg nucleus via possible one-directional migration of egg chromatin into sperm nucleus (Ohnishi et al., 2014). Here, we describe the protocol for microscopic observation, three-dimensional reconstruction, and volume measurements of sperm nuclei in rice zygotes/fused gametes, which are produced by an in vitro fertilization system (Uchiumi et al., 2006; Uchiumi et al., 2007). The present protocol will be applied for monitoring nuclear dynamics in cells during cell division, differentiation, de-differentiation and polarity formation as well as karyogamy progression.