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. 2020 Jul 10;33(3):143–158. doi: 10.1007/s00497-020-00391-z

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© The Author(s) 2020

Open AccessThis article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 1 — Embryogenic structures found in B. napus microspore cultures. a–d Schematic representation of the different types of embryogenic structures found in 5-day-old cultures. Dotted line, exine; ep, embryo proper; s, suspensor. e–h CLSM images of LEC1:LEC1-GFP expressing (green) structures counter-stained with the SCRI Renaissance 2200 cell wall stain (white). LEC1:LEC1-GFP expression marks embryogenic cells. e compact exine-enclosed embryo; f suspensor-bearing embryo. g compact callus. h loose callus. Labels: sus, suspensor; ep, embryo proper. i–l CLSM images of Cell Brite-stained (green) membranes of i, an exine-enclosed embryo; j. a suspensor-bearing embryo; k compact embryogenic callus. l loose embryogenic callus Labels: sus, suspensor; ep, embryo proper. m–r FESEM images of embryogenic structures. m Three different types of embryogenic structures. n Detail of the area between two cells of a callus-like structure where the exine has ruptured; o Same area in n. with higher magnification, showing that only a few fibers connect adjacent cells. p Exine-enclosed embryo where the exine has started to rupture. q Detail of the exine rupture site in an exine-enclosed embryo. r Same area in q, with higher maginification, showing that many fibers connect adjacent cells. Labels: ee, exine-enclosed embryo; cc, compact callus; lc, loose callus; e, exine. s–v Pectin deposition in embryogenic structures. Whole-mount samples stained with Ruthenium Red. s, exine-enclosed embryo; t, suspensor-bearing embryo; u, compact callus; v, loose callus. Label: e, exine; sus, suspensor; ep, embryo proper; arrowhead, area with pectin staining; asterisk, area without pectin staining. w. Proportion of different embryogenic structures found in 5-day old cultures induced by heat stress (HS), a combined heat stress and 0.05 µM TSA treatment and a combined heat stress and 0.5 µM TSA treatment. x Proportion of histodifferentiated embryos with or without a suspensor in 10-day-old cultures found in the different culture treatments described in (w). ‘Suspensor embryo’ refers to both suspensors and suspensor-bearing embryos, i.e., a suspensor with an embryo proper. Scale bars: a–d 10 μm; e–l 25 μm; m 10 μm; n 1 μm; o 300 nm; p 10 μm; q 1 μm; r 300 nm; s–v, 25 μm