f-g Limited (f) or extensive (g) loss of cell adhesion between two cells Both embryo proper and suspensor identity is established ab initio in embryogenic callus Our cell tracking data indicates that loose embryogenic callus forms suspensor embryos in culture, but it is not clear whether suspensor identity is already present in callus structures or whether it is acquired later. of an alternative asexual reproduction pathway or in vitro in response to inducer treatments (Vijverberg et al. 2019; Mndez-Hernndez 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). Haploid embryos develop in vitro from single cells, most commonly from the unicellular microspore or from the vegetative cell of bicellular pollen (Sunderland 1974; de F. Maraschin et al. 2008; Daghma et al. 2014). The predominately single cell origin of microspore embryos makes ME a tractable system to study embryo development in the absence of parental and filial tissues. is a well-studied model system for ME, in part due to Sacubitrilat the large number of responding genotypes that differ in the extent to which they are able to form haploid embryos (Bhowmik et al. 2011). In ME has been used to study various aspects Slit3 of (in vitro) embryo development, including totipotency (Joosen et al. 2007; Malik et al. 2007; Li et al. 2014), cell wall architecture (El-Tantawy et al. 2013; Sols et al. 2016; Corral-Martnez 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; Rodrguez-Sanz et al. 2015), and the role of the suspensor in patterning the embryo proper (Supena et al. 2008; Soriano et al. 2014). A heat stress treatment is used to induce ME in microspore culture. We show that embryogenic callus, despite its poor cell morphology and low growth potential, develops into suspensor-bearing embryos. Different pathways to suspensor-bearing embryo development from embryogenic callus could be defined based on the orientation of the first cell division, the extent of exine rupture, and the degree of cell adhesion. This newly discovered route to haploid embryo development highlights the high degree of developmental plasticity found in haploid embryo cultures. Materials and methods Plant material and culture The L. DH12075 genotype was used for haploid embryo culture. Plants were grown and cultured as in Li et al. (2014). The reporter line was previously described (Soriano et al. 2014; Li et al. 2014). Trichostatin?A (TSA) was dissolved in DMSO (Sigma) and applied as described below. Sample preparation for light microscopy Whole-mount samples for light microscopy were collected by centrifugation and fixed overnight at 4?C with 4% paraformaldehyde in PBS (pH 7.4) before being washed three times with PBS and stored at 4?C in 0.1% paraformaldehyde in PBS until use. Samples prepared for a separate transmission electron microscopy study were also used for light microscopy. Samples were centrifuged, fixed in Karnovsky solution (Karnovsky 1965), and then post-fixed with 2% OsO4. The buffer was replaced with one to two drops of warm liquid gelatin (15%) to immobilize and concentrate the samples. The samples were centrifuged (1?min at 8000?rpm), cooled on ice for gelatin solidification and then incubated overnight at 4?C with 20?l of 1% paraformaldehyde to harden the gelatin. Gelatin-embedded samples were cut in small pieces and kept in buffer until use. All of the above solutions were prepared in cacodylate buffer (Hayat 1981). Samples were dehydrated in a progressive ethanol series and embedded and polymerized in Embed-812 resin (Electron Microscopy Science). Sacubitrilat One micron-thick sections were made with a Leica UC6 ultramicrotome. At least three blocks were used for each treatment, and at least 30 to 50 embryogenic structures were analyzed per block. Light and confocal microscopy Pectins were stained in whole mounts with Ruthenium Red (500?mg/L; Merck Sigma-Aldrich) dissolved in PBS (Luft 1971). The whole mounts were stained for 10?min and then observed using a Nikon Eclipse E1000 microscope. At least 100 embryogenic structures were examined per treatment. Cell walls were stained by a 30?min incubation in Sacubitrilat 0.1% (v/v) SCRI Renaissance 2200 (Musielak et al. 2015) prior to observation. Staining of cytoplasmic membranes was performed directly in live samples using CellBrite? Orange (Biotium) (200??dilution; 20?min. incubation). Nuclei were stained with 1?g/ml DAPI as in Custers et al. (Custers et al. 1994). Cell viability was assessed using fluorescein diacetate (FDA) and propidium iodide (PI) (Merck Sigma-Aldrich) as in Ibidi Application Note 33. Fluorescence was observed with Leica SP5 confocal laser scanning microscope. SR2200 and DAPI.