A parthenogenesis gene of apomict origin elicits embryo formation from unfertilized eggs in a sexual plant

Abstract

Apomixis is a naturally occurring mode of asexual reproduction in flowering plants that results in seed formation without the involvement of meiosis or fertilization of the egg. Seeds formed on an apomictic plant contain offspring genetically identical to the maternal plant. Apomixis has significant potential for preserving hybrid vigor from one generation to the next in highly productive crop plant genotypes. Apomictic Pennisetum/Cenchrus species, members of the Poaceae (grass) family, reproduce by apospory. Apospory is characterized by apomeiosis, the formation of unreduced embryo sacs derived from nucellar cells of the ovary and, by parthenogenesis, the development of the unreduced egg into an embryo without fertilization. In Pennisetum squamulatum (L.) R.Br., apospory segregates as a single dominant locus, the apospory-specific genomic region (ASGR). In this study, we demonstrate that the PsASGR-BABY BOOM-like (PsASGR-BBML) gene is expressed in egg cells before fertilization and can induce parthenogenesis and the production of haploid offspring in transgenic sexual pearl millet. A reduction of PsASGR-BBML expression in apomictic F1 RNAi transgenic plants results in fewer visible parthenogenetic embryos and a reduction of embryo cell number compared with controls. Our results endorse a key role for PsASGR-BBML in parthenogenesis and a newly discovered role for a member of the BBM-like clade of APETALA 2 transcription factors. Induction of parthenogenesis by PsASGR-BBML will be valuable for installing parthenogenesis to synthesize apomixis in crops and will have further application for haploid induction to rapidly obtain homozygous lines for breeding.

Keywords: AP2 transcription factor; BABY BOOM; Pennisetum; apomixis; parthenogenesis.

Fig. 1.

PsASGR-BBML expression in sexual embryo sacs. Ovaries from three sexual offspring derived from a T₀ PsASGR-BBMLpromoter-GUS line are shown (A–D). The embryo sacs with antipodals have been outlined. Picture Insets are magnified regions of the egg/synergid/central cell region. The Upper Inset in C is the next focal plane of the ovary to show the central cell. Two asterisks indicate the polar nuclei within the central cell. Arrows indicate synergids. GUS expression is detected in the egg cell of unfertilized sexual embryo sacs on the day of anthesis. GUS signal is not detected in the central cell or antipodal cells of the mature sexual embryo sac. GUS staining is detected in cells of the developing embryo (EM) 3 d after fertilization but not in developing endosperm (D). No other staining in ovary tissue is identified. (Scale bar, 50 µm.)

Fig. 2.

Parthenogenetic embryo development in ovaries of sexual tetraploid pearl millet containing the gPsASGR-BBML transgene. Images are from unpollinated ovaries collected and fixed 2 d after anthesis, cleared with methyl salicylate, and visualized using a Zeiss LSM 710 Confocal Microscope. Picture Insets are magnified regions of the egg or embryo/synergid/polar nuclei region. Arrows indicate the polar nuclei within the central cell. (A) A control ovary with a structurally mature embryo sac without fertilization derived from an untransformed tissue culture line. No embryo development is seen. Parthenogenesis in unfertilized ovaries is clearly seen in sexual transgenic line g3f carrying the gPsASGR-BBML transgene based on the appearance of an embryo-like structure at the micropylar end of the embryo sac, polar nuclei, and antipodal cells (B and C). (D) Offspring from line g52 #308 carrying the gPsASGR-BBML transgene and also showing parthenogenesis. Antipodal cells have been cropped from the picture. (Scale bar, 50 µm.)

Fig. 3.

Flow cytometry analysis to determine genome size of T₀ plants and offspring. Examples of genome size analysis using a BD-Accuri flow cytometer of T₀ plants and g3f offspring (T₁) are shown. (A) Peak analysis of sorghum and T₀ line g11a leaf tissue. (B) Peak analysis of sorghum and g3f offspring 108. (C) Peak analysis of sorghum and g3f offspring 101. (D) Peak analysis of g3f offspring 105 and 107. S2 and S4 designate sorghum 2n/2x/2c and 2n/2x/4c peaks, respectively. H2 and H4 designate T₁ diploid/dihaploid offspring (C and D) with 2n/2x/2c and 2n/2x/4c peaks, respectively. T2 and T4 designate tetraploid T₀ pearl millet (A) or tetraploid T₁ offspring (B and D) with 2n/4x/2c and 2n/4x/4c peaks, respectively.