Autonomous development and regeneration of isolated rice egg cells <i>in vitro</i> in a fertilization-independent manner

Cell cycle and division are suppressed in rice egg cells to avoid precocious development before fertilization. Although cell cycle arrest in egg cells is released by particular environmental stimuli, how quiescence of egg cells is converted to the proliferative state remains open. Upon cold treatment, egg cells isolated from japonica (Nipponbare; NB) and indica (Kasalath; KS) subspecies of rice (Oryza sativa) autonomously divided and developed. Egg-derived plants were analysed for developmental profile, ploidy levels, single nucleotide polymorphism (SNP)-based genome sequencing, and transcriptome. Cold-treated haploid egg cells regenerated into mature plants, showing varied ploidy levels. Nuclear DNA quantification showed that genome duplication occurred during early parthenogenetic development. Owing to SNPs between NB and KS, inter-subspecific hybrid plants (NB-KS hybrids) were created via electrofusion. Egg cells from the NB-KS hybrid developed parthenogenetically into polyploid plants. Diploid and tetraploid plants originating from an NB-KS egg displayed the same homozygous SNP patterns throughout the genome, indicating that these plants were doubled and quadrupled haploids. Cold-treated eggs exhibited up-regulation of parthenogenesis-related genes. Cold treatment induces parthenogenesis of eggs isolated from crop species, offering cues for elucidating parthenogenetic mechanism at single-cell resolution and benefits for plant breeding technology as progenies possessing only maternal genes are produced by cold treatment and subsequent culture of isolated egg cells.