Abstract
Key messageIn vitro embryo development is highly plastic; embryo cell fate can be re-established in tissue culture through different pathways.In most angiosperms, embryo development from the single-celled zygote follows a defined pattern of cell divisions in which apical (embryo proper) and basal (root and suspensor) cell fates are established within the first cell divisions. By contrast, embryos that are induced in vitro in the absence of fertilization show a less regular initial cell division pattern yet develop into histodifferentiated embryos that can be converted into seedlings. We used the Brassica napus microspore embryogenesis system, in which the male gametophyte is reprogrammed in vitro to form haploid embryos, to identify the developmental fates of the different types of embryogenic structures found in culture. Using time-lapse imaging of LEAFY COTYLEDON1-expressing cells, we show that embryogenic cell clusters with very different morphologies are able to form haploid embryos. The timing of surrounding pollen wall (exine) rupture is a major determinant of cell fate in these clusters, with early exine rupture leading to the formation of suspensor-bearing embryos and late rupture to suspensorless embryos. In addition, we show that embryogenic callus, which develops into suspensor-bearing embryos, initially expresses transcripts associated with both basal- and apical-embryo cell fates, suggesting that these two cell fates are fixed later in development. This study reveals the inherent plasticity of in vitro embryo development and identifies new pathways by which embryo cell fate can be established.
Highlights
Plant embryogenesis begins with formation of a totipotent zygote that develops after fusion of the male and female gametes
DH12075 haploid embryo cultures are induced by heat stress (HS; 33 °C) from isolates with a high proportion of mid-uninucleate microspores (Supplemental Fig. 1)
Three types of embryogenic structures are observed at this time: (1) exineenclosed embryos; (2) suspensors or suspensors-bearing embryos, 3) and two types of embryogenic callus, termed compact and loose callus (Fig. 1a–h, t)
Summary
Plant embryogenesis begins with formation of a totipotent zygote that develops after fusion of the male and female gametes. The establishment of an apical-basal axis is a common theme in both monocot and dicot embryo development, the regularity of the cell division pattern and the timing and presence of key developmental processes often differ greatly between plant species (Kaplan and Cooke 1997; Radoeva and Weijers 2014; Zhao et al 2017). Many plant cells are able to form embryos in the absence of fertilization, either in vivo as part of an alternative asexual reproduction pathway or in vitro in response to inducer treatments (Vijverberg et al 2019; Méndez-Hernández et al 2019; Testillano 2019). Microspore embryogenesis (ME) is a form of in vitro totipotency in which cultured immature male haploid gametophytes (microspores and pollen) are induced to form embryos, usually in response to a stress treatment (Soriano et al 2013; Testillano 2019). In B. napus ME has been used to study various aspects of (in vitro) embryo development, including totipotency (Joosen et al 2007; Malik et al 2007; Li et al 2014), cell wall architecture (ElTantawy et al 2013; Solís et al 2016; Corral-Martínez et al 2019; Rivas-Sendra et al 2019), hormone signalling (Hays 2000; Dubas et al 2013, 2014; Soriano et al 2014; Robert et al 2015; Rodríguez-Sanz et al 2015), and the role of the suspensor in patterning the embryo proper (Supena et al 2008; Soriano et al 2014)
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