Expression of Genes from Paternal Alleles in Rice Zygotes and Involvement of OsASGR-BBML1 in Initiation of Zygotic Development.

Upon fertilization in angiosperms, one sperm cell fuses with the egg cell to produce a zygote, and, via karyogamy, the parental genetic information is combined to form the diploid zygotic genome. Recently, analyses with parentally imbalanced rice zygotes indicated that parental genomes are utilized synergistically in zygotes with different functions, and that genes transcribed from the paternal or maternal allele might play important roles in zygotic development. Herein, we first conducted single nucleotide polymorphism-based mRNA-sequencing using intersubspecific rice zygotes. Twenty-three genes, with paternal allele-specific expression in zygotes, were identified, and, surprisingly, their allele dependencies in the globular-like embryo tended to be biallelic. This suggests that the paternal-dependent expression of these genes is temporary, occurring during the early stages of zygote development. Of the 23 genes, we focused on Oryza sativa Apospory-specific Genome Region (ASGR)-BABY-BOOM LIKE (BBML) 1 (OsASGR-BBML1), presumed to encode an AP2-transcription factor, due to its reported role in zygotic development. Interestingly, ectopic expression of OsASGR-BBML1 in egg cells induced nuclear and cell divisions, indicating that exogenously expressed OsASGR-BBML1 converts the proliferation status of the egg cell from quiescent to active. In addition, the suppression of the function of OsASGR-BBML1 and its homologs in zygotes resulted in the developmental arrest, suggesting that OsASGR-BBML1 possesses an important role in initiating zygotic development. Monoallelic or preferential gene expression from the paternal genome in the zygote might be a safety mechanism allowing egg cells to suppress the gene expression cascade toward early embryogenesis that is normally triggered by fusion with a sperm cell.

In Vitro Production of Zygotes by Electrofusion of Rice Gametes.

In angiosperms, fertilization and embryogenesis occur in the embryo sac, which is deeply embedded in ovular tissue. In vitro fertilization (IVF) systems using isolated gametes have been utilized to dissect postfertilization events in angiosperms, such as egg activation, zygotic development, and early embryogenesis. In addition, using IVF systems, interspecific zygotes and polyploid zygotes have been artificially produced, and their developmental profiles/mechanisms have been analyzed. Taken together, the IVF system can be considered a powerful technique for investigating the fertilization-induced developmental sequences in zygotes and generating new cultivars with desirable characteristics. Here, we describe the procedures for the isolation of rice gametes, electrofusion of gametes, and the culture of the produced zygotes and embryo.