Live Imaging of embryogenic structures in Brassica napus microspore embryo cultures highlights the developmental plasticity of induced totipotent cells

Abstract

Key message In vitro embryo development is highly plastic; embryo cell fate can be re-established in tissue culture through different pathways.

Cell Wall Composition and Structure Define the Developmental Fate of Embryogenic Microspores in Brassica napus

“…Finally, loose bicellular structures (LBS) (Figure 1C right; 0.16%) comprised 2-3 loosely connected cells surrounded by partially ruptured exine, but with a level of cell organization higher than LC and similar to EE. These structures strongly resembled the few-celled suspensors observed in DH4079 under milder heat stress treatment (Supena et al, 2008) or in DH12075 (Corral-Martinez et al, 2020). However, in the absence of cell fate tracking data, we refer to these as LBS.…”

“…This idea changed when two different types of structures, EE, suspensorless embryos (EE), and suspensor-bearing embryos (SUS) were found to coexist in DH4079 microspore cultures (Supena et al, 2008). This was further refined in B. napus DH12075 microspore cultures, where besides EE and SUS, embryogenic compact and loose calli (CC and LC) were also described (Corral-Martinez et al, 2020). Embryo identity and fate were unambiguously assigned to these multicellular structures using embryo fluorescent reporters and time-lapse imaging (Li et al, 2014;Soriano et al, 2014;Corral-Martinez et al, 2020).…”

“…In DH12075, it was previously shown that callus-like structures (LC and CC) have thicker cell walls than EE (Corral-Martinez et al, 2020). We therefore used SCRI Renaissance Cell 2200 staining to examine cell wall thickness in the DH4079 proliferating structures, paying special attention to the two cell wall layers observed previously in embryogenic microspores, the subintinal layer and the intine (Figure 2A).…”

“…There are several ways to induce microspore embryogenesis, all of which rely on the application of a stress treatment (Seguí-Simarro et al, 2021). Brassica napus is considered a model species to study this process, since most of the knowledge gained in the last decades comes from the use of this species to investigate molecular, cellular, physiological, and developmental aspects of microspore embryogenesis (Jouannic et al, 2001;Corral-Martínez et al, 2013;Soriano et al, 2013;Rivas-Sendra et al, 2017Corral-Martinez et al, 2020). In B. napus and many other species, microspore embryogenesis is triggered by the application of a heat stress treatment to isolated microspores.…”

“…Previous studies in B. napus showed the presence of exine-enclosed (EE), suspensorless structures, and suspensorbearing structures, both described as embryogenic based on the expression of embryo-reporter genes (Soriano et al, 2013(Soriano et al, , 2014, and two types of embryogenic callus-like structures, loose and compact (Telmer et al, 1995;Li et al, 2014;Soriano et al, 2014). Recently, a time-lapse imaging study defined the developmental fate of these structures (Corral-Martinez et al, 2020) and distinguished two main groups of microspore-derived structures according to their ability to form differentiated embryos. EE structures and suspensor-bearing embryos (SUS) are considered highly embryogenic because a high proportion of them develop into differentiated embryos, while only a low percentage become callus-like structures or die.…”