Time-lapse imaging establishes a roadmap for Brassica microspore embryogenesis

Abstract

Microspore embryogenesis describes a process whereby the haploid cell that usually develops into pollen is reprogrammed to become an embryo. For that, microspores are dissected from developing anthers and cultured in vitro. Abiotic stress, such as heat treatment, can trigger their development into haploid embryos. The chromosome number of the haploid embryos can then be doubled, either spontaneously or chemically, to produce diploid (‘doubled-haploid’) plants with two sets of chromosomes. This results in homozygous diploid plants, as each chromosome in the haploid state is replicated. Having homozygous plants (i.e. genetic stability) available early in a breeding program significantly enhances breeding efficiency (Hale et al., 2022). Microspore embryogenesis of oilseed rape (Brassica napus) has been studied since the 1980s, and the induction treatment is simple and short (Lichter, 1982).

Charlotte Siemons, first author of the highlighted publication and a PhD student in Kim Boutilier's group at Wageningen University & Research at the time of the study, was fascinated by this remarkable plasticity of plant cells. For Siemons, the ability of the microspore to switch cell fate from developing into mature pollen to forming an embryo provided an exciting opportunity to explore plant cell totipotency.

The application of heat stress to microspore cultures induces B. napus microspores to develop into four distinct types of embryogenic tissue (Li et al., 2014). Two are differentiated embryos, either with or without a suspensor, while the other two are either compact or loose embryogenic calli. Both embryo types show high viability in culture and can develop into seedlings, but embryogenic calli have a low viability and generally never develop into differentiated embryos (Corral-Martínez et al., 2020). Currently, these tissue types can only be identified after about 5 days in culture, making it impossible to deduce the cell division dynamics leading to the different developmental pathways. To address this, Siemons et al. used time-lapse imaging of B. napus microspores to monitor the development of embryogenic structures from the single- to few-cell stage, allowing them to trace the cell divisions that lead to the formation of the different embryo types (Siemons et al., 2025).

For the study, Boutilier's group teamed up with John van Noort's group at the University of Leiden to combine their expertise in in vitro biology with John's expertise in high-resolution live imaging. Previously, they had used time-lapse imaging with confocal microscopy, but it negatively affected embryo development, most likely due to photo-induced damage. Two-photon microscopy, however, resulted in less photodamage due to the reduced absorption in near-infrared light when relatively low light doses were used, which allowed for long-term time-lapse imaging.

To track the development from single to few-cell embryogenic structures, the authors used fluorescent reporter lines for LEAFY COTYLEDON1 (LEC1) (LEC1:LEC1-GFP) and an auxin response reporter line (DR5v2:ntdTomato). LEC1 expression has served as a marker for embryo identity across various in vitro embryo culture systems (Li et al., 2014). Therefore, it was used as a marker for early developmental events in microspore embryogenesis.

Both the LEC1 and DR5v2 reporters specifically identified the development of embryogenic structures at the single- to few-cell stage, though they exhibited distinct temporal and spatial expression patterns in the different types of embryogenic structures that formed afterward (Figure 1). Their expression transiently decreased in the few-celled suspensorless embryos, which could indicate a significant event in suspensorless embryo development. Before the pollen wall (exine) ruptured, all cells in suspensor-bearing embryos expressed LEC1 and DR5v2 reporters. After exine rupture, the future basal (suspensor) and apical (embryo proper) regions became distinguishable. LEC1 was expressed in the embryo proper and maintained in the suspensor cells, while DR5v2 expression was confined to the embryo proper. This pattern was in contrast with the sudden loss of DR5v2 and LEC1 reporter expression observed in embryogenic calli after exine rupture.

By tracing back the cell divisions leading to the formation of the different embryo types, the authors found that the orientation and symmetry of the first embryogenic cell division predicted the developmental fate and timing of exine rupture. Suspensorless embryos started with a symmetric division of either the microspore or the vegetative cell of bicellular pollen, followed by late exine rupture. These structures developed into globular embryos or later into calli (Figure 1). Suspensor-bearing embryos and embryogenic calli originated from an asymmetric division followed by early exine rupture. In the case of compact calli, the exine ruptured only partially, and in the case of loose calli, the exine ruptured completely. Suspensor embryos developed from asymmetric divisions into a larger apical embryo proper and a smaller basal suspensor, and both were initially surrounded by the pollen wall. Suspensor-bearing embryos could also develop later into calli.

The authors hypothesize that both the division plane and exine rupture are related to how committed the microspore is to develop into pollen at the time of the induction treatment. When pollen germinates, the pollen tube cell walls expand and the cell volume increases. If suspensor embryos and embryogenic calli retain some pollen characteristics, they might start pollen germination processes like cell wall softening and cell expansion, leading to earlier exine rupture than in those that become a suspensorless embryo.

The diverse developmental pathways that lead to the formation of embryogenic structures in B. napus microspore cultures are markedly different from the consistent cell division patterns seen in zygotic embryos. This contrast suggests that embryo development pathways are inherently flexible and influenced by their specific context.