Gene Expression and Genome Editing Systems by Direct Delivery of Macromolecules Into Rice Egg Cells and Zygotes

Abstract

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.

Keywords: PEG-Ca2+ transfection; CRISPR/Cas9; Electro-fusion; Gamete; Genome editing; In vitro fertilization; Rice; Zygote.