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Megasporogenesis, megagametogenesis, and embryogenesis in Dendrobium nobile (Orchidaceae)

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Abstract

The orchid reproductive strategy, including the formation of numerous tiny seeds, is achieved by the elimination of some stages in the early plant embryogenesis. In this study, we documented in detail the formation of the maternal tissues (the nucellus and integuments), the structures of female gametophyte (megaspores, chalazal nuclei, synergids, polar nuclei), and embryonic structures in Dendrobium nobile. The ovary is unilocular, and the ovule primordia are formed in the placenta before the pollination. The ovule is medionucellate: the two-cell postament and two rows of nucellar cells persist until the death of the inner integument. A monosporic eight-nucleated embryo sac is developed. After the fertilization, the most common central cell nucleus consisted of two joined but not fused polar nuclei. The embryogenesis of D. nobile is similar to the Caryophyllad-type, and it is characterized by the formation of all embryo cells from the apical cell (ca) of a two-celled proembryo. The only exception is that there is no formation of the radicle and/or cotyledons. The basal cell (cb) does not divide during the embryogenesis, gradually transforming into the uninuclear suspensor. Then the suspensor goes through three main stages: it starts with an unbranched cell within the embryo sac, followed by a branched stage growing into the integuments, and it ends with the cell death. The stage-specific development of the female gametophyte and embryo of D. nobile is discussed.

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Acknowledgments

We thank Mr. Paul Girling for grammatically editing the manuscript.

Funding

This study was carried out under Institutional research project No. 118021490111-5 at the Unique Scientific Installation “The Fund Greenhouse” of the Main Botanical Garden of the Russian Academy of Sciences (Moscow, Russia).

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Authors and Affiliations

  1. N.V. Tsitsin Main Botanical Garden of the Russian Academy of Sciences, Botanicheskaya 4, Moscow, Russia, 127276

    Galina L. Kolomeitseva, Alexander V. Babosha & Andrey S. Ryabchenko

  2. Department of Microbiology, Faculty of Biology, Lomonosov Moscow State University, 1-12 Lenin’s Hills, Moscow, Russia, 119234

    Elena A. Tsavkelova

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  1. Galina L. Kolomeitseva
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  2. Alexander V. Babosha
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  3. Andrey S. Ryabchenko
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Contributions

G.L. Kolomeitseva, A.V. Babosha, and A.S. Ryabchenko are responsible for the conceptualization, microscopic observations, formal analysis, investigation, and methodology. E.A. Tsavkelova is responsible for the translation, revision, and editing of the manuscript.

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Correspondence to Elena A. Tsavkelova.

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ESM 1

Megasporogenesis and megagametogenesis in Dendrobium nobile. a A functional chalazal megaspore, and degenerating micropylar and middle megaspores (autofluorescence). b A functional chalazal megaspore before the first nuclear division (dipyridamole staining). c Both dying megaspores fluorescent brightly, the beginning of the death of the nucellar epidermis before megagametogenesis (autofluorescence). d The bright fluorescence of degenerating micropylar megaspores (calcofluor staining). e The embryo sac with an undivided chalazal nucleus (dipyridamole staining). f The attached daughter nuclei formed after the division of the micropylar and chalazal at the fertilization stage (ethidium bromide staining). cn–chalazal nuclei; ec–egg cell; fm–functional megaspore; ilii–inner layer of inner integument; iloi–inner layer of outer integument; mc–micropyle; mm–micropylar megaspore; mn–micropylar nuclei; ne–nucellar epidermis; olii–outer layer of inner integument; ps–postament; s–sperm; v–vacuole. Scale bar: 10 μm (a-e); 5 μm (f). (PDF 35757 kb)

ESM 2

Fertilization and the central cell nucleus. The figure shows the paired optical sections of two different stacks. a, b A central cell nucleus consisting of three attached nuclei (two polar nuclei and one sperm nucleus; ethidium bromide staining). c, d The suspensor, leaving the embryo sac and the inner integument, and the development of the suspensor blades in the micropylar zone of the outer integument. The suspensor nucleus is localized in the suspensor neck (calcofluor staining). bs–suspensor blades; ccn–central cell nucleus; cn–chalazal nuclei; ec–egg cell; ep–embryo proper; ilii–inner layer of inner integument; iloi–inner layer of outer integument; n–nucleus; ne–nucellar epidermis; olii–outer layer of inner integument; oloi–outer layer of outer integument; pn–polar nuclei; ps–postament; s–sperm; sg–synergids; sn–suspensor neck; sp–suspensor. Scale bar: 10 μm (a, b); 30 μm (c, d). (PDF 8372 kb)

ESM 3

The deposition of the secondary material within the cell walls of the inner layer of the inner integument. a Autofluorescence of the secondary material at the stage of a three-celled embryo. b The fluorescence of the secondary material after staining with calcofluor at the stage of a four-celled embryo (100–120 days after pollination). c A multicellular embryo with a well-developed branched suspensor, the inner layer of the outer integument is destroyed (autofluorescence). d Blue fluorescence of the coat (mantle) of a branched suspensor after staining with ethidium bromide. In all the figures, the thickening is absent in the micropylar part of the embryo sac coat. The micropylar end is in the upper left corner. bs–suspensor blades; cb–cb cell; cc–cc cell; cd–cd cell; ep–embryo proper; ilii–inner layer of inner integument; iloi–inner layer of outer integument; olii–outer layer of inner integument; oloi–outer layer of outer integument; mc–micropyle; ps–postament; sp–suspensor. Scale bars: 20 μm (a); 30 μm (b); 100 μm (c); 50 μm (d) (PDF 16772 kb)

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Kolomeitseva, G.L., Babosha, A.V., Ryabchenko, A.S. et al. Megasporogenesis, megagametogenesis, and embryogenesis in Dendrobium nobile (Orchidaceae). Protoplasma 258, 301–317 (2021). https://doi.org/10.1007/s00709-020-01573-2

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