Introduction

Brachiaria is an economically important genus of the family Poaceae, a forage grass cultivated in tropical countries, especially in Brazil, where breeding programs aim to obtain better forage quality, resistance to pests and diseases, and high seed production, among other traits (Valle et al. 2009). Brachiaria brizantha (syn. Urochloa brizantha) is one of the species with great importance for breeding purposes. In this and other species, such as B. decumbens (syn. U. decumbens) the cultivated plants are tetraploids and reproduce by apomixis. Apomixis is an asexual mode of reproduction through seeds (Nogler 1984), which generate plants that are clones of the mother plant. As a consequence of apomixis, cultivated areas with apomictic plants of B. brizantha or B. decumbens are wide monocultures, implying potential economic and environmental risks (Dusi 2001). Sexual diploid noncultivated genotypes are available for both species; however, the difference in levels of ploidy among sexual and apomictic plants impairs breeding (Valle et al. 2009). Approaches to producing hybrids include interspecific and intraspecific crosses between apomicts and artificial tetraploid sexual plants (Miles and Valle 1996; Monteiro et al. 2016), followed by a selection of desired traits in the progeny.

In Brachiaria spp., apomixis occurs by apospory with pseudogamy, meaning—the fertilization of the central cell by the male reproductive cell is necessary for endosperm development (Alves et al. 2001; Dusi 2001). Brachiaria spp. apomictic plants produce viable pollen grains (Dusi et al. 2010; Dusi and Willemse 1999a). In some genotypes of B. decumbens, pollen grains follow the same pattern of development in sexual and apomictic plants, resulting in the production of viable pollen grains, varying in the degree of abortion of microspores and mature pollen grains, which is higher in apomictics (Dusi et al. 2010; Dusi and Willemse 1999a). In another genotype, correspondent to the apomictic B. decumbens cv. Basilisk, sterile pollen grains resulted from abnormal pollen mitoses (Junqueira Filho et al. 2003). Synchrony among male and female development was observed (Dusi and Willemse 1999a), thus anther and pistil morphology should be a good indicator for the stage of pollen development.

The cytogenetic and morphological characterizations of microsporogenesis or microgametogenesis in diploid, tetraploid, and induced tetraploid plants of Brachiaria spp. (Araujo et al. 2005; Dusi et al. 2010; Dusi and Willemse 1999a; Mendes-Bonato et al. 2002, 2009; Risso-Pascotto et al. 2006) assist the selection of genotypes that produce high amounts of viable pollen that could be used as male progenitors in conventional breeding programs or for in vitro fertilization.

Haploids and doubled haploids are important sources for the generation of new genotypes with potential use in intraspecific crosses (Weyen 2021), which may contribute to the study of sexual and apomictic reproduction in Brachiaria spp. A key factor for the success of haploid production through in vitro culture is to identify the adequate microspore developmental stages for efficient embryogenesis induction, with morphological markers being very helpful to choose the best explant stage to be introduced in vitro. The knowledge of morphological characteristics of pistil, spikelet, and flower sizes according to stages of the ovule, embryo sac, and pollen development, has proven to be very useful for molecular and cellular studies of Brachiaria spp. reproduction (Alves et al. 2007; Dusi and Willemse 1999a, b; Dusi 2001; Guimarães et al. 2013; Lacerda et al. 2012; Rodrigues et al. 2003; Silveira et al. 2012; Worthington et al. 2016). Nevertheless, few reports emphasize the pollen grain development, and the detailed events during anther development are yet to be described in this genus. Histological characterization of anther and microspore/pollen grain development in B. brizantha will increase the possibility of application of biotechnological techniques in this forage. The objective of this work was to characterize anther development stages in B. brizantha, providing histological and ultrastructural details, and correlating them with morphological characteristics. BbrizSERK expression was found to be a marker in the process of somatic embryogenesis in B. brizantha (Koehler et al. 2020) in vitro culture and the possibility of using the expression of this gene as a marker to track embryogenesis inside the anther should be investigated.

The inflorescence of Brachiaria is a panicle with racemes that support the spikelets placed in two series on the raceme (Skerman and Riveros 1989). Each spikelet has two flowers, a male and a hermaphrodite flower (Dusi 2001; Ndikumana, 1985). Both types of flowers have three stamens each. Each anther has a connective tissue (with vascular bundle) with two thecae and four locules, and dehiscence is longitudinal. In this article, we report the histological, cytological, and ultrastructural characterization of anthers of sexual accession of B. brizantha and correlate the stages of development to morphological markers. In addition, we present a first impression of the response of anthers cultured in vitro using histology and the expression of BbrizSERK to observe the transition of microspore development to an embryogenic route.

Material and methods

Plant material

Two diploid (2n = 2x = 18) sexual accessions, B. brizantha BRA 002,747, and B. decumbens BRA 004,430, were used, from the germplasm collection of Embrapa, cultivated in the experimental field at Embrapa Genetic Resources and Biotechnology.

Structural and ultrastructural analysis of the in vivo anther development

Developmental stages were associated with the following morphological parameters: in the early stages of development, the length of spikelet or hermaphrodite flower, and in later stages, the length and coloration of anthers and pistil morphology. Pistil morphology was annotated according to Dusi and Willemse (1999a), based on the aspect of the stylodia, which in grasses constitute the so-called stigma (Heslop-Harrison 1982).

Spikelets, hermaphrodite flowers, and anthers from the hermaphrodite flowers of B. brizantha were collected from plants growing in the field, at different stages of development. For transmission electron microscopy (TEM), explants were fixed in a modified Karnovsky solution (2% glutaraldehyde, 2% paraformaldehyde, and 0.001 M CaCl2 in 0.2 M sodium cacodylate buffer, pH 7.2), for 48 h, under refrigeration and post fixed in osmium tetroxide (1% in 0.1 M sodium cacodylate buffer, pH 7.2), for 1 h, at room temperature. Samples were then dehydrated in an acetone series (30–100%) and infiltrated and embedded in Spurr’s (1969) low viscosity resin (EMS, Electron Microscopy Sciences, Hatfield, PA, USA), for 48 h. Semithin Sects. (120–200 nm) for LM were collected in glass slides, stained with toluidine blue (2,5% in water) for 5 min, rinsed in distilled water, and air-dried. The sections were permanently mounted in Entellan®. The sections were then analyzed, and digital images were obtained using an upright microscope (Axioskop 2 photomicroscope, Carl Zeiss, Jena, Germany). For TEM analyses, ultrathin Sects. (60–90 nm) were obtained using a diamond knife in an ultramicrotome (Porter Blum MT2, Dupont-Sorvall), collected on copper grids (300 mesh), and poststained with aqueous uranyl acetate (2.5%), followed by lead citrate (0.1%) (Reynolds 1963). Sections were examined at 80 kV under a transmission electron microscope (JEM1400 JEOL, Tokyo, Japan), and the images were digitalized. For light microscopy (LM), explants were fixed in a modified Karnovsky solution for 1 h under vacuum at room temperature. The fixative was refreshed, and after 36 h without vacuum, the samples were dehydrated through an ethanol series (from 10 to 100% v/v). Samples were embedded in Historesin (Leica). Semi-thin sections of 5 µm, were obtained and stained with 0.05% toluidine blue. Results were photo-documented using a Zeiss Axioskop 2 HBO 100 w/2 or Zeiss Axiophot. Fresh dissected flower, anthers and pistils were observed with a Zeiss Discovery V8 stereomicroscope and photo-documented using a Zeiss AxioCam MRc.

In vitro culture of anthers—an assay to observe changes in microspore development

Inflorescence of B. brizantha, BRA 002,747, and B. decumbens, BRA 004430, were collected from the field. Racemes were excised and surface sterilized by immersion in 70% ethanol, for 5 min, followed by immersion in a commercial bleach solution (2.5% active chlorine), for 20 min. The racemes were then rinsed three times with autoclaved water. B. brizantha stage 8 anthers were isolated and inoculated in Petri dishes containing YP medium supplemented with the antiauxin 2,3,5-triiodobenzoic acid, TIBA 0.2 μM, and activated charcoal, at pH 5.8 (Genovesi 1990). Three Petri dishes with 20 anthers per dish were prepared. Cultures were maintained in a growth chamber at 25 ± 1 °C, in the dark, for 75 days. Anthers were subcultured every 30 days. Anthers of B. decumbens at a stage equivalent to stage 7 were placed in liquid N6 medium (Chu 1978) pH 5.8, with a total of 50 anthers per Petri dish and three Petri dishes.

Histology of B. brizantha cultivated anthers

To characterize microspore development, anthers were collected at 45 and 75 days, fixed in a solution of 2% glutaraldehyde, 2% paraformaldehyde, 0.001 M CaCl2 in 0.2 M sodium cacodylate buffer, pH 7.2, for 1 h under vacuum at room temperature followed by 36 h at 4 °C. The samples were then dehydrated in an ethanol series (35 to 100%) and embedded in Historesin (Leica) and polymerized at room temperature. Histological Sects. (5 μm) were obtained using a rotary microtome (Leica RM2155); stained with 0.05% toluidine blue in water and mounted using Entellan® (Merck). The analysis was performed in the light microscope Zeiss Axioskop2 HBO 100 w/2 and digital images were obtained.

Localization of SERK transcripts in cultivated anthers of B. decumbens

In situ hybridization (ISH) was performed as described by Dusi (2015) and Koehler et al. (2020). Briefly, anthers of B. decumbens at the stage equivalent to stage 8 were collected and fixed overnight in 4% paraformaldehyde and 0.25% glutaraldehyde in phosphate buffer, at pH 7.2. Thereafter, they were rinsed, dehydrated in an ethanol series, and infiltrated in butyl methyl methacrylate (BMM). Sections of 0.4 µm were obtained and deposited on glass slides. After BMM removal, tissues were used for ISH. BbrizSERK sense and antisense probes labeled with digoxigenin, were prepared according to Koehler et al. (2020), and used at a final concentration of 0.6 ng/μL hybridization solution. ISH was performed overnight at 42 °C. After rinsing, blocking, and immunodetection typical violet precipitation, indicating hybridization, was observed under a light microscope with differential interference contrast (DIC).

Results

Stages of anther development analyzed using light (LM) and transmission electron microscopy (TEM)

The hermaphrodite flower of Brachiaria has three stamens, one pistil with one ovary and two styles with an enlarged feathery stigma. This stigma can cover a large part of the style and is also called a stylodium (Fig. 1a). From the observation of the archesporial cells to the anther opening, 11 stages are distinguished with the following characteristics:

Fig. 1
figure 1

Anther development in B. brizantha, stages 1 to 2. b–f LM images, g, h TEM image. a Schematic representation of a dissected hermaphrodite flower of Brachiaria showing the three anthers (arrows) and in the pistil, the ovary, the two styles (arrowhead), and the stylodia. b Stage 1 anther. c Cross-section showing epidermis and archesporial cell (arrow). d Anther at stage 2. e Cross-section showing the four locules anther with the connective tissue (arrowhead). f Anther in stage 2 showing epidermis, outer (arrowhead) and inner (arrow) subepidermal layers and sporogenous cells. g, h Detail of the inner subepidermal layer and sporogenous cells from anther at stage 2; the arrow points to mitochondria/plastids. Abbreviations: cn, connective tissue; ct, cuticle; cy, cytoplasm; ep, epidermis; isl, inner subepidermal layer; n, nucleus; nu, nucleolus; ov, ovary; sc, sporogenous cells; v, vacuoles. Scale bars = 0.2 mm (b), 25 µm (c), 0.2 mm (d), 50 µm (e), 20 µm (f), and 8 µm (g, h)

Full size image

Stage 1 represents the beginning of anther development, when the spikelets are very tender, with well-developed glumes, lemma, and palea. The translucent anther (Fig. 1b) shows bilateral symmetry. In the cross section, the epidermis has elongated cells with anticlinal divisions. The differentiation of the thecae and the delimitation of the locules are not yet visible. Subepidermally, the archespore cell is distinguished (Fig. 1c). When the spikelets reach 1 to 2 mm in length, in elongating anthers (Fig. 1d), the connective tissue differentiates the vascular tissue joining the four defined locules (Fig. 1e). The anther wall is composed of the epidermis with a visible cuticle and the outer and the inner subepidermal layers; centrally in the locule, the sporogenous cells differentiate and enlarge (Fig. 1f). All cells present a dense cytoplasm. The ultrastructure of the parietal and sporogenous cells are comparable in cytoplasmic density and vacuolation; mitochondria/plastids are more evident in the sporogenous cell (Fig. 1g, h). At stage 3, hermaphrodite flowers are approximately 1 mm in length and contain small pistils with a very short style and stylodium, with anthers exhibiting a pale green color (Fig. 2a). The anther wall is fully formed with four layers of cells: epidermis, endothecium, middle layer, and tapetum (Fig. 2b). Inside the locule, in stage 4, prominent nucleus and nucleolus are found in pollen mother cells (Fig. 2c, d), with yellow translucent anthers, and callose start to be deposited around the cells from the center of the anther locule (Fig. 2e), preceding the first meiotic division (Fig. 2f, g). Highly vacuolated cells are observed, especially in the epidermis, endothecium, and middle layer, while tapetum cells have smaller vacuoles (Fig. 2h). At this stage, the tapetum cells present a conspicuous nucleus and a prominent nucleolus (Fig. 2i) and plasmodesmatal connections are observed between tapetum cells (Fig. 2j). After meiosis, the important tetrad period follows when callose envelopes every microspore and the entire tetrad. When pale-yellow anthers are approximately 2.5 mm in length, the pistil has an elongated stylodial axis (Fig. 3a), at stage 5, marked by the late tetrad stage (Fig. 3b, c, d), with the dissolution of the callose wall, the isobilateral tetrad enters a disintegration process (Fig. 3c, d). At this stage, the tapetum cells show electron-dense particles, with a lobate nucleus and prominent nucleolus, cytoplasm with many lipid bodies (Fig. 3e), and initial signs of Ubisch bodies (UB) (Fig. 3f). Finally, the callose disappears and free microspores are released inside the locule (Fig. 3g). Subsequently, at stage 6, anthers are yellow, 3 mm in length, and stigmatic hairs develop in pistils (Fig. 4a). Microspores have migrated toward the anther wall (Fig. 4b, c), some of them observed with the microspore pore against the turgid tapetum cell, which presents a dense cytoplasm. Microspores present exine and small vacuoles (Fig. 4d) and are against the Ubisch bodies secreted by the tapetal cells (Fig. 4e). Finally, the tapetum shows initial signs of degeneration (Fig. 4f). At stage 7, anthers are 3.5 to 4 mm in length; the pistil shows a white feathering-like stylodium with long white hairs (Fig. 4g). At this stage, microspores have a single large vacuole that presses the cytoplasm and the nucleus toward the cell wall (Fig. 4h). A sign of advanced degeneration of the anther wall was observed, tapetum cells have dense cytoplasm, and the middle layer is almost imperceptible (Fig. 4i, j). At stage 8, anthers are 4 to 5 mm in length, and the pistil has elongated red/purple stylodium (Fig. 5a). Starch grains accumulate in the microspores, and the pore develops in the same position (Fig. 5b, c). Anthers finally reach the final length of 5 mm, and the pistil has stylodium with dark hairs at stage 9 (Fig. 5d). A two-locule anther is a result of the septum rupture (Fig. 5e). Microspores divide by mitosis and cytokinesis results in a smaller generative and a larger vegetative cell (Fig. 5f, g). Exine and intine are very well defined (Fig. 5h). Remnants of the tapetum and Ubisch bodies are present in the locule (Fig. 5i). During anthesis (Fig. 6a), at stage 10, anthers have a deep yellow color with a touch of red and a very fragile cell wall, while pistil presents a very dark stylodium. Anther dehiscence has occurred (Fig. 6b) and, with the opening of the stomium (Fig. 6c), mature tricellular pollen grains are present with the vegetative cell and two generative cells (Fig. 6d). The mature pollen grain contains starch and lipids as storage compounds, the exine and intine, as well as the pore, are fully developed (Fig. 6e). The cytoplasm of the vegetative cell has small osmiophilic lipid bodies, and a high number of small vesicles (Fig. 6f). An operculum covers the pollen grain aperture and the annulus surrounding the operculum is elevated (Fig. 6g). The exine is composed of foot layer, baculum, and tectum (Fig. 6h). Finally, at stage 11, the anthers are wilted and empty (Fig. 6i, j). In the dehiscent anther, after pollen grains are released, only two of the four original cell layers remain: the endothecium and epidermis (Fig. 6k).

Fig. 2
figure 2

Anther development in B. brizantha, stages 3 and 4. a–d, f–g LM images, e, h–j TEM images. a Dissected flower with anthers at stage 3 showing the pistil with very small stylodia (arrow). b Anther wall formed by four layers and the pollen mother cells. c Dissected flower with anthers at stage 4 showing anthers and stylodium beginning to elongate. d Detail of an anther showing PMC. e Callose deposition in the inner part of the locule of the anther, surrounding pollen mother cells. f Meiocytes at prophase (arrowheads). g Meiocyte at anaphase (arrowhead). h Detail of the anther wall with highly vacuolated cells of epidermis, endothecium, and middle layer. i Detail of a tapetum cell showing a conspicuous nucleus with an evident nuclear envelope (arrowhead) and a prominent nucleolus. j Detail of the tapetum cells connected by plasmodesmata (arrowhead). Abbreviations: an, anther; ca, callose; en, endothecium; ep, epidermis; ml, middle layer; ov, ovary; pmc, pollen mother cell; ta, tapetum; v, vacuole. Scale bars = 0.5 mm (a), 50 µm (b), 0.8 mm (c), 20 µm (d), 15 µm (e), 10 µm (f, h), 5 µm (g), and 2.5 µm (i), 1 µm (j)

Full size image
Fig. 3
figure 3

Anther development in B. brizantha, stage 5. a–b, g LM images, c–f TEM images. a Pistil and anther at stage 5 showing stylodium (arrow). b Tetrad of microspores (arrow). c Anther wall layers and degrading callose wall surrounding microspores of the tetrad (arrow), near the tapetum. Note the highly vacuolated epidermis and endothecium and the very thin middle layer. d Detail of a microspore from the tetrad showing condensed chromatin. e Tapetum cells showing a lobated nucleus and prominent nucleolus (arrow) and lipid bodies (arrowhead). f Detail of tapetum cells, note a number of initial Ubisch bodies secreted by the tapetum cells (white arrowheads). g Free microspores. Abbreviations: an, anther; en, endothecium; ep, epidermis; ml, middle layer; mp, microspores; n, nucleus; pp, plastids; ta, tapetum; te, tetrads. Scale bars = 0.3 mm (a), 5 µm (b, c, e, f), 2 µm (d) and 50 µm (g)

Full size image
Fig. 4
figure 4

Anther development in B. brizantha, stages 6 (a–f) and 7 (g–j). a, b, f–h LM images, c–e, i–j TEM images. a Dissected hermaphrodite flower indicating an anther at stage 6 and one stylodium (arrowhead). b Microspores organized against the tapetum cells (arrow), some with visible pores (arrowheads) and large tapetum cells. c Microspores positioned in contact with the tapetum cells. d Detail of the microspore showing small vacuoles and exine. e Detail of the tapetum cells next to microspores and Ubisch bodies (arrowheads), which contribute to exine formation. f Beginning of tapetum degeneration (arrow), showing microspore pores facing the tapetum (arrowhead); at this stage, the middle cell layer is crushed. g Pistil and anthers at stage 8, showing feathered stylodium with long white hairs (arrowhead). h Microspores containing a single large vacuole pushing the cytoplasm and nucleus (arrow) toward the cell wall. i Degenerating anther wall. Note the middle cell layer crushed (arrow). j Detail of a tapetum cell in degeneration still showing Ubisch bodies (arrowheads). Abbreviations: cw, cell wall; en, endothecium; ex, exine; go, Golgi complex; ml, middle layer; mp, microspore; mt, mitochondria; n, nucleus; ta, tapetum; v, vacuole. Scale bars = 0.6 mm (a), 40 µm (b), 10 µm (c, h), 3 µm (d, e), 50 µm (f), 1.3 mm (g), 5 µm (i), and 1 µm (j)

Full size image
Fig. 5
figure 5

Anther development in B. brizantha, stages 8 and 9. a–b, d–f LM images, c, g–i TEM images. a–c Anthers at stage 8 and pistil showing a red stylodium (arrowhead). b Microspores are mostly uninucleate and vacuolated, they accumulate starch and the position of the pore remains stable. c Detail of the vegetative cell with starch grains in the cytoplasm (arrows). d Flower with anther at stage 9, and stylodium with very dark hairs (arrow). e The septum rupture results in a two locular anther. f Microspores pursue a vegetative (arrow) and a generative (arrowhead) nucleus. g Pollen showing the nucleus in the center of the generative cell and a vacuole. h The nucleus of the vegetative cell and pollen exine and intine with the Ubisch bodies (arrowheads). i Detail of the Ubisch bodies (arrows). Abbreviations: ep, epidermis; ex, exine; GC, generative cell; in, intine; mp, microspores; n, nucleus; nu, nucleolus; po, pore; v, vacuole. Scale bars = 2 mm (a), 20 µm (b), 5 µm (c), 3 mm (d), 50 µm (e), 10 µm (f), 2.5 µm (g, h), and 0.5 µm (i)

Full size image
Fig. 6
figure 6

Anther development in B. brizantha, stages 10 and 11. a–d, i–j LM images, c–f TEM images. a–b At anthesis, anthers at stage 10. c Section of the apex of the anther showing stomium opening (arrowheads) and releasing the pollen grains. d Pollen grain with the generative nucleus (arrow) and two sperm nuclei (arrowhead). e Mature pollen grain with starch and lipids, intine and exine, and a well-developed pore (arrowheads). f Cytoplasm of the vegetative cell of a mature pollen grain showing small osmiophilic lipid bodies (arrowheads) and small vesicles (arrows). g Detail of the elevated annulus surrounding the operculum that covers the aperture. h Detail of the pollen wall showing intine and the exine layers, tectum, baculum or columella, and foot layer. i Stage 11: wilted and empty anther. j Section of apex of the empty anther. Note that the connective tissue is not present. k Wall of dehiscent anther after pollen grains release, only two of the original four layers remains: the endothecium and the epidermis. Abbreviations: an, annulus; ba, baculum; en, endothecium; ep, epidermis; ex, exine; fl, foot layer; in, intine; op, operculum; te, tectum. Scale bars = 1 mm (a), 2 mm (b), 200 µm (c, j), 10 µm (d), 5 µm (e), 2 µm (f, g), and 0.5 µm (h, i), 10 µm (k)

Full size image

Table 1 summarizes the characteristics of the 11 cytological stages of sexual B. brizantha anther development showing the morphological markers linked to the stages.

Table 1 Events observed during anther development in Brachiaria brizantha
Full size table

Histology of anthers after embryogenesis induction

Stage 8 anthers of sexual B. brizantha (Fig. 7a), characterized by mostly late-stage uninucleated microspores with one large vacuole (Fig. 7b), were cultured in YP medium, a medium used originally for maize anther tissue culture (Genovesi 1990). After 30 days in culture, a mass of cells proliferating from the stomium could be seen. Histology of cultured anthers after 75 days in culture showed microspores in equal divisions, with two equally sized nuclei, indicating a possible embryogenic route (Fig. 7c, d, e). Two types of nuclei division were observed. Nuclei side by side in the microspore (Fig. 7c, d), nuclei separated by each other by the vacuole (Fig. 7e). Cytokinesis was not observed. Only in a few cases in anthers cultured for 45 days, multicellular structures could be observed inside the anther locules (Fig. 7f, g).

Fig. 7
figure 7

Flower with anthers at stage 7 of B. brizantha. a Dissected flower showing stage 3 pistil and the three anthers at stage 8. b Transverse section of the anther at stage 8 with the microspores with a single nucleus and a large vacuole (arrows). c–e Binucleate microspores after mitosis without cytokinesis, from 75 days cultured anthers, stained with toluidine blue, with two equally sized nuclei (arrows). f–g Multicellular structures inside the locules in anthers after 45 days in culture. h–i In situ hybridization in sections of anther cultured for 30 days with sense probe (h) without hybridization signal, and with the antisense probe (i) showing high signal in vacuolated microspores and binucleate microspores after mitosis. Scale bars = 1 mm (a), 25 µm (b), 10 µm (c–f), and 20 µm (g–i)

Full size image

SERK expression in cultivated anthers of B. decumbens

After 30 days in culture, B. decumbens anthers presented a mass of cells proliferation from the stomium. At this stage, while SERK ISH with the sense probe did not produce a signal (Fig. 7h), hybridization with the antisense probe showed the characteristic ISH signal in some vacuolated microspores (Fig. 7i).

Discussion

Anther development is an essential step of plant reproduction and comprises two phases: the sporophytic and the gametophytic phases. The sporophytic phase includes stamen meristem specification followed by archesporial cells and sporogenous cells formation, differentiation of microsporocytes which undergo meiosis, finishing with the resulting haploid cells, the microspores (microsporogenesis). The gametophytic phase (microgametogenesis), comprises the microspores entering mitosis to produce the microgametophytes, or pollen grains, and their maturation. (Åstrand et al. 2021, Liu et al. 2012, Pandey et al 2022; Scott et al. 2004; Yang et al 2003).

In this work, the development of the anthers from the hermaphrodite flower of B. brizantha was categorized in 11 stages that were related to morphological and cytological markers (see Table 1) and B. brizantha anther cell wall differentiation, development, and degeneration were closely observed. Anther wall layers play a role during anther development (Ma 2005; Sun et al. 2018). The establishment of the anther wall layers, as we observed in Brachiaria sp., followed the pattern of other members of the family Poaceae and other monocots (Gómez et al. 2015; Nakamura et al. 2010). The outer subepidermal layer differentiates into endothecium and the inner subepidermal layer giving rise to the middle layer and the tapetum (Åstrand et al. 2021). In Brachiaria sp. this differentiation occurs between stage 2, when the difference between the outer and inner subepidermal layers are obvious, and stage 3 when the four layers of the anther wall are visible is a specialized layer, which functions in the nutrition of meiocytes and microspores (Pacini 2010; Pacini and Franchi 1993; Pacini et al. 1985). Alterations in the pattern of anther wall with the absence of tapetum may result in male sterility (Canales et al. 2002; Walbot and Egger 2016; Yang et al. 2003). In A. thaliana, double mutants of SERK1 and SERK2, genes expressed in the tapetum and middle layer precursors, resulted in failures of tapetum specification leading to male sterility (Albrecht et al. 2005; Colcombet et al. 2005). In sexual and apomictic B. brizantha genotypes that produce viable pollen, SERK expression was observed in PMC and tapetum (Koehler et al. 2020). It is possible that SERK genes also play a role in the tapetum specification controlling sporophytic development in Brachiaria.

Microsporogenesis phase is marked by the enlargement of tapetum cells and callose deposition in the wall of microsporocytes, initiating from the center of the anther locule, similarly to what was observed in B. decumbens (Dusi and Willemse 1999a). B. brizantha, like other species of Poaceae (Barnes and Blackmore 1992; Pacini 2010) has the parietal type of tapetum. It is attributed to the tapetum, the production of locular fluid, production and secretion of callase, a β-1,3 glucanase. In Brachiaria sp., as in other plants, callose digestion led to the release of microspores, ending microsporogenesis.

Microgametogenesis starts at stage 6 up to stage 10 with mature pollen formation. The uninucleate microspore phase starts at the very end of stage 5 and ends at stage 8, a period in which the events of exine and intine formation occur. The pore is visible in the early stages of B. brizantha microsporogenesis, a feature also observed in Brachypodium distachyon (Sharma et al. 2015a). Meanwhile, microspores migrate toward anther wall, near the tapetum that provides material like. Ubisch bodies (sporopollenin orbicules), for microspore development (Heslop-Harrison and Dickinson 1969). In contrast to what was described for B. distachyon (Sharma et al. 2015b), in which Ubisch bodies were observed initially at the free microspore stage, in B. brizantha, they were first observed between the tapetum cells at stage 5, when dissolving tetrads were visible. The appearance of the Ubisch bodies secreted from the fully developed tapetum cells toward the microspore wall at stage 6, as observed in this work, is consistent with the exine formation.

Reprogramming microgametogenesis to a sporophytic pathway in Brachiaria spp. will benefit from the association of morphological characteristics of anther development in which uninucleate microspores were identified. Aiming embryogenesis in B. brizantha, it will be important to establish which one of the anther developmental stages presenting uninucleate microspores, from stage 5 (young microspores) to 8 (microspores entering mitosis), will respond better when culture in vitro. During microspore development, the uninucleate microspore phase is the longer one. Before the inequal division microspores go through structural changes such as a large vacuole formation, nucleus polarization, and exine development. Embryo formation from microspores can follow different pathways, but the competence for embryo induction for most species are the stages just before or after pollen grain mitoses I, as reviewed by Soriano et al. (2013). In some species of the Poaceae family (Silva 2012; Ślusarkiewicz-Jarzina et al. 2017; Maluszynski et al. 2003), microspore embryogenesis is successful when the explants are anthers with uninucleate microspores or the isolated microspores. Taking this into account, in B. brizantha, the phase that should be more adequate to androgenesis induction would be amongst the stages 5 to 8. In this work, stage 7 anthers, characterized by vacuolated microspores with one polarized nucleus, were used to evaluate the capacity of their microspores to enter the sporophytic pathway that could result in somatic embryos. While the gametophytic pathway is characterized by inequal divisions, equal divisions could, in some cases, indicate the shift of the gametophytic to sporophytic program characterizing the beginning of the somatic embryogenesis pathway in microspores as observed in species like, Olea europaeae L. (Solís et al. 2008) and Brassica napus (Zaki and Dickinsonn 1991). In this work, two types of equal binucleate microspores were observed according to the position of a vacuole, a feature that could be related to cytoskeleton rearrangements as observed in Brassica napus, in which microtubules rearrangements play a role in microspore equal divisions (Hause et al. 1993). However, for species and genotypes for which information about microspore development in vitro is missing, it is not possible to sustain that symmetrical divisions are a sign of the entrance into an embryogenic route. Actually, there are not enough evidence to confirm that microspores in equal divisions would further develop into embryos (Soriano et al. 2013). In this respect, the detection of SERK gene expression in anthers’ cultures contributes to establishing if microspores enter an embryogenic route. We previously determined that expression of SERK is observed in anthers of B. brizantha during microsporogenesis (Koehler et al. 2020) and not during microgametogenesis (data not shown). Here, the expression of SERK in uninucleate and equal binucleate microspores of B. decumbens observed after 30 days in culture indicates that they are able to enter the embryogenic route. In B. brizantha, with few exceptions when multicellular structures were observed, the sporophytic development ceased in the first equal division. Tissue culture conditions have to be optimized to allow the induced microspores to continue the embryogenic route. In addition, other genotypes and stages of anther development should be tested and SERK expression can be used to assist experiment evaluation.

Different approaches have been used to analyze the development of anther and pollen wall (Jaffri and MacAlister 2022; Xue et al. 2021) and pollen development (Gómez-Mena et al. 2022), but morphological markers of the stage of pollen development remain important as a base for further studies (Jaffri and MacAlister 2022). The association of morphological characteristics, identifying the stages to anther and pollen grain or ovule and embryo sac development, is the basis for studying and unrevealing the genes and metabolic pathways involved in the process of reproduction, as known from monocotyledonous such as B. decumbens (Dusi and Willemse 1999a, b), Hordeum vulgare (Gómez and Wilson 2012), B. distachyon (Sharma et al. 2015a, 2015), and Triticum aestivum (Browne et al. 2018). Up to now, four stages of pistil morphology have been used as a parameter to isolate anthers of B. brizantha. The aspect of the stylodia provides a good marker to establish the stage of anther development due to the evident modification in morphology that occurs during flower growth. The data collected and analyzed here, which defined and characterized 11 stages during anther development, gives a much more precise indication of male development and more than one morphological characteristic might be used to select the stage. In addition to the benefits for haploid culture, molecular studies will profit from the knowledge provided here. Brachiaria is an open pollination plant, and breeding could benefit from a male-sterile plant in intraspecific crosses between sexual plants, that went through an artificial polyploidization, and the polyploid apomicts.

Conclusions

The dynamics of the in vivo development of sporophytic tissues and gametophytic cells of B. brizantha was described in this work by morphological and cytological analyses, reaching our objective. This work provides a rapid assessment of the stages of anther and pollen development in B. brizantha, using as markers the length of spikelet, hermaphrodite flower, length, and coloration of anthers, and pistil morphology. It can be used to direct cellular and molecular studies that rely on specific stages of anther development, such as pollen embryogenesis, haploid and double haploid production, identification of genes related to the events of anther development. The detailed morphological and cytological characterization of 11 stages of development presented here adds important knowledge to B. brizantha reproductive development and complements the information presented in the reproductive calendar of B. decumbens (Dusi and Willemse 1999a). This is a guideline for Brachiaria sp. for anther and pollen development to find the suitable stage for haploid cell culture.