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Megasporogenesis, megagametogenesis, and embryogenesis in Maxillaria crassifolia (Lindl.) Rchb.f. (Cymbidieae, Orchidaceae)

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G. L. Kolomeitseva at Main Botanical Garden Of Russian Academy of Science
G. L. Kolomeitseva
Alexander V Babosha at Main Botanical Garden Of Russian Academy of Science
Alexander V Babosha
Andrey S. Ryabchenko at Main Botanical Garden Of Russian Academy of Science
Andrey S. Ryabchenko
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Abstract and Figures

Maxillaria crassifolia (Lindl.) Rchb.f. belongs to the polyphyletic genus Maxillaria Ruiz & Pav., which currently is the subject of several taxonomic research. There are conflicting descriptions of megasporogenesis, megagametogenesis, and embryogenesis in orchids from the tribe Cymbidieae, in general, and in the genus Maxillaria, in particular. In the present report, all stages of embryonic development of M. crassifolia were examined using confocal fluorescence microscopy. Some features of the development of the ovule and embryo, which distinguish M. crassifolia from other species of the tribe Cymbidieae were identified. The T-shaped arrangement of megaspores is formed by dividing the micropylar megaspore of the dyad. The megagametophyte develops according to the modified Polygonum-type with an unstable number of nuclei in the embryo sacs. The nucleus of the central cell varies in composition and may include unfused micropylar and chalazal nuclei and daughter nuclei formed during their division. The sequence of embryonal divisions is strictly structured. A special variant of embryogenesis, the Cymbidium-type Maxillaria-variant, has been described. Its characteristic features are the strictly apical nature of embryonic divisions, the absence of basal cell (cb) division, the formation of one to three pairs of tubular suspensor cells, and the localization of all suspensor cells within the inner integument.
Maxillaria crassifolia. a Plant with cleistogamous and normal flowers. b Fruits at different ripening periods. Abbreviations: nf, normal flower; cf, cleistogamous flower
Maxillaria crassifolia. a Plant with cleistogamous and normal flowers. b Fruits at different ripening periods. Abbreviations: nf, normal flower; cf, cleistogamous flower
… 
A nucellar filament formation. a Placental ridges with proliferative papillae (DAPI). b Ovule primordia (DAPI). c Periclinal divisions of subepidermal cells and anticlinal divisions of epidermal cells in ovule primordium (Brb). d The formation of a three-celled axial row: division of a cell located under the archesporium (Brb). e Four-celled axial row (Brb). f The second cell of a four-celled axial row of nucellar filament is divided by an oblique septum, one of the daughter cells shifts to the dermal layer (DAPI). Abbreviations: ac, archesporial cell; ar, axial row of cells; el, epidermal layer; f, funiculus; mp, metaphase plate; hp, hypostase; ne, nucellar epidermis; ov, ovule primordia; pl, placental ridges; pp, proliferative papillae; sl, subepidermal layer; t, trichome. Scale bars: a, b = 100 µm; c, f = 10 µm
A nucellar filament formation. a Placental ridges with proliferative papillae (DAPI). b Ovule primordia (DAPI). c Periclinal divisions of subepidermal cells and anticlinal divisions of epidermal cells in ovule primordium (Brb). d The formation of a three-celled axial row: division of a cell located under the archesporium (Brb). e Four-celled axial row (Brb). f The second cell of a four-celled axial row of nucellar filament is divided by an oblique septum, one of the daughter cells shifts to the dermal layer (DAPI). Abbreviations: ac, archesporial cell; ar, axial row of cells; el, epidermal layer; f, funiculus; mp, metaphase plate; hp, hypostase; ne, nucellar epidermis; ov, ovule primordia; pl, placental ridges; pp, proliferative papillae; sl, subepidermal layer; t, trichome. Scale bars: a, b = 100 µm; c, f = 10 µm
… 
The differentiation of initials of integuments and curvature of the ovule. a The first divisions of the initials of the inner integument (Auto). b The first divisions of the initials of the outer integument; initial curvature of the ovule (Auto). c The archesporial cell turns into a large megaspore mother cell, a two-layer inner integument is formed. When staining with DPM, the thickened cell walls of the cells of the nucellus and inner integument are visible (DPM). d The megaspore mother cell increased before meiosis 1, the outer integument was almost equal in length to the inner integument, and the ovule became anatropous. e An axial section of the anatropous ovule through the apical part of the nucellus and funiculus (Auto). f Anatropous ovules at the stage of megasporogenesis (DPM). Abbreviations: ac, archesporial cell; ar, axial row of cells; f, funiculus; hp, hypostase; ii, inner integument; iii, initials of the inner integument; ilii, inner layer of inner integument; olii, outer layer of inner integument; ioi, initials of the outer integument; m, megaspore mother cell; n, nucleus; ne, nucellar epidermis; oi, outer integument; pt, pollen tube. Scale bars = 10 µm
The differentiation of initials of integuments and curvature of the ovule. a The first divisions of the initials of the inner integument (Auto). b The first divisions of the initials of the outer integument; initial curvature of the ovule (Auto). c The archesporial cell turns into a large megaspore mother cell, a two-layer inner integument is formed. When staining with DPM, the thickened cell walls of the cells of the nucellus and inner integument are visible (DPM). d The megaspore mother cell increased before meiosis 1, the outer integument was almost equal in length to the inner integument, and the ovule became anatropous. e An axial section of the anatropous ovule through the apical part of the nucellus and funiculus (Auto). f Anatropous ovules at the stage of megasporogenesis (DPM). Abbreviations: ac, archesporial cell; ar, axial row of cells; f, funiculus; hp, hypostase; ii, inner integument; iii, initials of the inner integument; ilii, inner layer of inner integument; olii, outer layer of inner integument; ioi, initials of the outer integument; m, megaspore mother cell; n, nucleus; ne, nucellar epidermis; oi, outer integument; pt, pollen tube. Scale bars = 10 µm
… 
Megasporogenesis. a The first meiotic division of the megaspore mother cell (Brb). b Dyad stage (DAPI). c Division of the chalazal dyad with the formation of a functional megaspore (Auto). d Tetrad stage, micropylar megaspores separated by an oblique septum to form a T-shaped tetrad, dying cells fluoresce in red (PI). e T-shaped tetrad of megaspores, three dying cells fluoresce in yellow (Brb). f Linear tetrad of megaspores, ovule at the stage of completion of megasporogenesis (Brb). Abbreviations: cd, chalazal dyad; fm, functional megaspore; hp, hypostase; ii, inner integument; ilii, inner layer of inner integument; iloi, inner layer of outer integument; m, megaspore mother cell; mc, micropyle; mcm, micropylar megaspore; md, micropylar dyad; mdm, middle megaspore; mp, metaphase plate; ne, nucellar epidermis; oi, outer integument; olii, outer layer of inner integument; oloi, outer layer of outer integument. Scale bars = 10 µm
Megasporogenesis. a The first meiotic division of the megaspore mother cell (Brb). b Dyad stage (DAPI). c Division of the chalazal dyad with the formation of a functional megaspore (Auto). d Tetrad stage, micropylar megaspores separated by an oblique septum to form a T-shaped tetrad, dying cells fluoresce in red (PI). e T-shaped tetrad of megaspores, three dying cells fluoresce in yellow (Brb). f Linear tetrad of megaspores, ovule at the stage of completion of megasporogenesis (Brb). Abbreviations: cd, chalazal dyad; fm, functional megaspore; hp, hypostase; ii, inner integument; ilii, inner layer of inner integument; iloi, inner layer of outer integument; m, megaspore mother cell; mc, micropyle; mcm, micropylar megaspore; md, micropylar dyad; mdm, middle megaspore; mp, metaphase plate; ne, nucellar epidermis; oi, outer integument; olii, outer layer of inner integument; oloi, outer layer of outer integument. Scale bars = 10 µm
… 
Megagametogenesis. a The first mitotic division of the functional megaspore, the metaphase plate is visible (Brb). b Binucleated embryo sac (DAPI). c Four-nucleated embryo sac (DAPI). d Eight-nucleated embryo sac (DAPI). e Seven-nucleated embryo sac with three micropylar nuclei (DAPI). f Seven-nucleated embryo sac with three chalazal nuclei (DAPI). Abbreviations: cn, chalazal nucleus; es, embryo sac; fm, functional megaspore; hp, hypostase; ii, inner integument; ilii, inner layer of inner integument; iloi, inner layer of outer integument; mc, micropyle; mcm, micropylar megaspore; mdm, median megaspore; mp, metaphase plate; cn, chalazal nuclei; mn, micropylar nuclei; ne, nucellar epidermis; oi, outer integument; olii, outer layer of inner integument; oloi, outer layer of outer integument; sg, synergids; v, vacuole. Scale bars = 10 µm
+6
Megagametogenesis. a The first mitotic division of the functional megaspore, the metaphase plate is visible (Brb). b Binucleated embryo sac (DAPI). c Four-nucleated embryo sac (DAPI). d Eight-nucleated embryo sac (DAPI). e Seven-nucleated embryo sac with three micropylar nuclei (DAPI). f Seven-nucleated embryo sac with three chalazal nuclei (DAPI). Abbreviations: cn, chalazal nucleus; es, embryo sac; fm, functional megaspore; hp, hypostase; ii, inner integument; ilii, inner layer of inner integument; iloi, inner layer of outer integument; mc, micropyle; mcm, micropylar megaspore; mdm, median megaspore; mp, metaphase plate; cn, chalazal nuclei; mn, micropylar nuclei; ne, nucellar epidermis; oi, outer integument; olii, outer layer of inner integument; oloi, outer layer of outer integument; sg, synergids; v, vacuole. Scale bars = 10 µm
… 
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https://doi.org/10.1007/s00709-021-01710-5
ORIGINAL ARTICLE
Megasporogenesis, megagametogenesis, andembryogenesis
inMaxillaria crassifolia (Lindl.) Rchb.f. (Cymbidieae, Orchidaceae)
GalinaL.Kolomeitseva1 · AlexanderV.Babosha1 · AndreyS.Ryabchenko1
Received: 26 April 2021 / Accepted: 23 September 2021
© The Author(s), under exclusive licence to Springer-Verlag GmbH Austria, part of Springer Nature 2021
Abstract
Maxillaria crassifolia (Lindl.) Rchb.f. belongs to the polyphyletic genus Maxillaria Ruiz & Pav., which currently is the
subject of several taxonomic research. There are conflicting descriptions of megasporogenesis, megagametogenesis, and
embryogenesis in orchids from the tribe Cymbidieae, in general, and in the genus Maxillaria, in particular. In the present
report, all stages of embryonic development of M. crassifolia were examined using confocal fluorescence microscopy. Some
features of the development of the ovule and embryo, which distinguish M. crassifolia from other species of the tribe Cym-
bidieae were identified. The T-shaped arrangement of megaspores is formed by dividing the micropylar megaspore of the
dyad. The megagametophyte develops according to the modified Polygonum-type with an unstable number of nuclei in the
embryo sacs. The nucleus of the central cell varies in composition and may include unfused micropylar and chalazal nuclei
and daughter nuclei formed during their division. The sequence of embryonal divisions is strictly structured. A special vari-
ant of embryogenesis, the Cymbidium-type Maxillaria-variant, has been described. Its characteristic features are the strictly
apical nature of embryonic divisions, the absence of basal cell (cb) division, the formation of one to three pairs of tubular
suspensor cells, and the localization of all suspensor cells within the inner integument.
Keywords Orchidaceae· Embryogenesis· Suspensor· Confocal microscopy
Introduction
The genus Maxillaria Ruiz & Pav. (Cymbidieae, Orchi-
daceae) is polyphyletic and is currently the subject of taxo-
nomic research. The taxonomic affiliation of M. crassifolia
(Lindl.) Rchb.f., as well as other species from the subtribe
Maxillariinae, has been repeatedly revised, and the species
itself has changed both generic and species names (Dressler
1981; Carnevali 1991; Senghas 2002; Christenson 2002;
Chase etal. 2003; Ojeda Alaion 2003; Ojeda Alaion etal.
2003; Cabral etal. 2006; Blanco etal. 2007; Whitten etal.
2007; Szlachetko etal. 2012; Moraes etal. 2016). In par-
ticular, in several modern studies, this species appears as
Heterotaxis sessilis (Sw.) Barros (de Barros 2002) and H.
gatunensis (Schltr.) Szlach. & Sitko (Szlachetko etal. 2012).
In this study, we have investigated the embryonic develop-
ment of Maxillaria crassifolia to determine important diag-
nostic characters that can help in defining this group along
with the molecular data.
Genus Maxillaria belongs to the tribe Cymbidieae, which
consists of 10 subtribes, including the subtribes Coeliopsidi-
nae, Cymbidiinae, Eulophiinae, Maxillariinae, Oncidiinae,
and Stanhopeinae (Chase etal. 2015). It was hypothesized
that embryogenesis in all Cymbidieae species is similar and
corresponds to embryogenesis of Cymbidium-type or V-type
with an irregular division of both basal (cb) and apical (ca)
cells, filamentous proembryo, and multicellular suspensor
with several tubular cells (Swamy 1949b; Clements 1999).
Ovule growth of 16 species of orchids from the tribe Cym-
bidieae is described in the literature (Treub 1879; Afzelius
1916, 1918; Swamy 1942, 1943, 1946; Veyret 1965; Abe
1967; Nagashima 1982; Vij and Sharma 1986; Zee and Ye
1995; Yeung etal. 1994, 1995, 1996; Huang etal. 1998;
Communicated by Handling Editor: Dorota Kwiatkowska.
* Andrey S. Ryabchenko
marchellos@yandex.ru
Galina L. Kolomeitseva
kmimail@mail.ru
Alexander V. Babosha
phimmunitet@yandex.ru
1 N.V. Tsitsin Main Botanical Garden ofRussian Academy
ofSciences, Botanicheskaya 4, 127276Moscow, Russia
/ Published online: 30 September 2021
Protoplasma (2022) 259:885–903
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
... Certain genes, such as CKI1 (histidine kinase), can be expressed during female gametophyte development, restricting the fusion of the polar nuclei and interacting with the antipodals activity (Li and Yang 2020). The occurrence of the Polygonum-type embryo sac was unstable in the sexually reproducing Maxillaria orchid, where micropylar and chalazal nuclei remain unfused (Kolomeitseva et al. 2022). However, studies show that in Tulipa sp. with the tetrasporic Fritillaria-type embryo sac and a pentaploid endosperm, the fusion of micropylar (one maternal) and sperm (one paternal) nuclei results in the formation of a diploid endosperm (Mizuochi et al. 2009). ...
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