Ovule and pollen development in Camelina sativa provides systematic insights

Abstract

Plant sexual reproduction involves highly structured and specialized organs: stamens (male) and gynoecium (female, containing ovules). These organs synchronously develop within protective flower buds. Investigating ovules and pollen is crucial for understanding aspects of fertility and sterility in plants. Research on their development and embryogenesis plays a significant role in determining the taxonomic relationships of various species. Paraffin-embedding associated to examination with light microscope showed the development of ovules and pollen grains in Camelina sativa, a key oilseed crop. The findings indicated that the anthers exhibit tetrasporangiate characteristics, with the anther wall consisting of the epidermis, mechanical layer, transitional layer, and tapetum. The microsporogenesis type is simultaneous and microspore tetrads arrange in tetrahedral tetrads. Scanning electron microscope observations showed that mature pollen grains have a tricolporate aperture and are medium-sized, with microreticulate exine ornamentation on the pollen wall. The gynoecium is characterized as bicarpellate, and the ovule in its mature state is classified as amphitropous and bitegmic. The meiosis division of megasporocytes yields a linear tetrad formation. The eight-nucleate embryo sac following the Polygonum type pattern. With a broader systematic perspective, these embryological and palynological features demonstrate evolutionary conservatism within the Brassicaceae, with minor distinctions potentially representing adaptive changes.

Introduction

Sexual reproduction is a key process in the life cycle of flowering plants, as it is essential for seed production and the maintenance of floral diversity. It relies on highly specialized reproductive organs that are protected by flower buds until flowering¹. Accurate knowledge of the development of reproductive stages of plant species is important for understanding how seed development occurs and for developing effective strategies to increase productivity. Having precise understanding in this area is crucial for evaluating the relative importance and similarities of descriptive characteristics used in plant systematics². Reproductive success in plants that depend on seed production for survival is tied to healthy, viable seeds³. Normal seed development in angiosperms is described as functional in the production of male (pollen grain or microgametophyte) and female (embryo sac or megagametophyte) gametophytes⁴, therefore, a deep understanding of sporogenesis (microsporogenesis and megasporogenesis) and gametogenesis (pollen grain and ovule) is required to further understand other reproductive biological processes such as microspore formation, pollen grain maturation, pollen tube guidance, fertilization, induction of seed development after fertilization, and maternal control of seed development after fertilization⁵. This is particularly evident in seedless cultivars, which have defects in megagametophyte development processes^6,7. The amount of pollen produced can be used to validate the functional status of the microgametophyte⁸, but obtaining phenotypic information from the megagametophyte is a more complex system due to its location at the cellular or subcellular levels, located within the ovule and hidden by coverings⁹. Palynological data also offers accurate information for identifying closely related taxa, while pollen morphology assists in distinguishing taxa at the species level¹⁰. These detailed pollen morphological studies contribute to the enhancement of identification keys, which serve as essential references for botanists, archaeologists, immunologists, and eco-botanists¹¹.

The floral ground plan of Brassicaceae is highly conserved, especially when compared to close relatives Cleomaceae (370 species) and Capparaceae (700 species), this clade (part of core Brassicales, Fig. 1) comprises most of the species in the order Brassicales^12,13. The Brassicaceae family, also known as the Cruciferae family, is a large and economically significant group of plants, consisting of approximately 341 genera and around 3997 species. The tribe Camelineae is part of the Brassicaceae¹⁴ and includes notable genera such as Arabidopsis Heynh. in Holl (& Heynh), Camelina Crantz, and Capsella Medik¹⁵. The genus Camelina is native to the Irano-Turanian floristic region, and identification of members of this genus at the species level based on different life stages can be challenging. Although according to theplantlist (www.theplantlist.org), 54 scientific plant names of species rank for the genus Camelina. have been described in this genus, most of them have been synonymized with previously known species or their taxonomic status is unresolved, and therefore there is considerable uncertainty about the exact number and distribution of these species. Overal, of these 8 are accepted species names^14,16,17. More recently, it is generally accepted that this genus includes 8 species, which differ in their chromosome count and ploidy level. A significant number of species within this genus have not yet been studied, with Camelina sativa being a notable exception, historically recognized for its cultivation as an oil-producing plant. Typically, Camelina species are annuals and biennials herbaceous that overwinter as basal rosettes and flower in the spring¹⁸. The leaves of these species are described as simple, ranging from lanceolate to narrowly ovate in shape. The flowers display hermaphroditic actinomorphic properties, organized in simple racemes of inflorescence, and possess a yellowish pigmentation. The seeds develop within dehiscent siliques¹⁹.

Fig. 1.

Phylogenetic tree of the Brassicales, indicating a position for the Brassicaceae suggested by previous studies²⁷.

Camelina sativa (L.) Crantz, commonly known as false flax or gold of pleasure, is classified as an oilseed crop with a cultivation history extending approximately 6,000 years²⁰. Camelina presents significant potential as a viable alternative oilseed crop for regions characterized by sub-arid climates and water scarcity, including areas reliant on irrigation, and demonstrates considerable resilience against various biological and environmental stressors when contrasted with other oilseed crops^21,22. Its significant agronomic potential is attributed relatively short growing season that requires moderate to low levels of nutrients²³. Despite developmental studies conducted in the Brassicacea family^24–26, bibliographic studies show that plants of the genus Camelina and the species under study have not been studied in terms of reproductive organ development so far. Therefore, in this study, to clarify flower growth patterns among Brassicaseae and related families, the developmental structure of stamens and pistils and the development process of pollen grains and embryo in Camelina are investigated.

Materials and methods

The experimental procedure was executed within a controlled greenhouse environment characterized by a diurnal temperature regime of 25/20^◦C (day/night), a photoperiod of 16 h of light and 8 h of darkness, along with a relative humidity level of 60%. Seeds of Camelina sativa “Soheil cultivar” were supplied from Bisetoon Shafa Co., Kermanshah, Iran. Plants grown in greenhouse and the experiment was designed using a randomized complete block design (RCBD) with three biological replications to ensure statistical reliability and minimize experimental variability.

For histological studies, we collected immature inflorescences in different stages of development (in four diameter sizes: < 2 mm, ~ 2 mm, ~ 4 mm and ~ 8 mm) and mature flower then immediately fixed under vacuum in FAA (formalin, glacial acetic acid, and 70% ethanol, 10:5:85 v/v) and stored in 70% ethanol. The fixed plant material was dehydrated in ascending ethyl alcohol series, embedded in paraffin according to the classical paraffin methods and stored at 4 ^◦C until use²⁸. Consequently, the embedded paraffin samples were sectioned at 7–8 μm with a disposable steel blade on a semiautomatic rotary microtome (ROTO-CUT200, SCILAB Co. UK). Serial slides were stained with Hematoxylin-Eosin and embedded in Entellan, to develop permanent slides. The Photomicrogrphs of key structures and processes involved in the development of male and female reproductive systems were taken with Olympus BX51 microscope attached with Olympus DP72 digital camera.

To conduct a scanning electron microscopy (SEM) investigation, fresh and dehydrated anther and pollen grain samples were mounted on SEM stubs and coated with gold. Micrographs were captured using a MIRA3 SEM (TESCAN, headquartered in Brno, Czech Republic). The analysis of pollen micrographs focused on both qualitative and quantitative attributes, encompassing aperture type, exine ornamentation, pollen dimensions, and lumen size. These characteristics were delineated according to the terminology established by Hesse and his coworkers (Table 1).

Table 1.

Pollen morphology terminology²⁹.

Terminology	Definition
P/E-ratio	Refers to Pollen shape: P/E = 1: spheroidal (or isodiametric), P/E > 1: prolate, P/E < 1: oblate
Pollen size	largest diameter < 10 μm: very small, 10–25 μm: small, 26–50 μm: medium, 51–100 μm: large and > 100 μm: very large
Aperture	A region of the pollen wall that differs significantly from the rest of the wall: inaperturate, (di- tri-tetra-penta- hexa) » (pori, colpi, colpori)
Ornamentation	Applied to surface features: areola, clava, echinus, foveola, fossula, granulum, gemma, plicae, reticulum, rugulae, striae, verruca. can be subdivided into ornamenting elements extending 1 μm in diameter, or if smaller then marked with prefix micro-
Lumen	The space enclosed by exine elements forming the meshes in a reticulum ornamentation

Result

We described here the formation of the flower associated with a series of landmarks that coincide with key developmental events on gametophyte formation. The development of flowers occuring gradually. At this stage, an inflorescence meristem (IM) emerges along with the formation of new lateral primordia, which subsequently develop into floral meristems (FM).

A key early event in these proceses is the increase rate of the cell division in the shoot apical meriste. The rapid and early elongation of rib meristem cells results in a prominent protrusion at the apex, which differentiates into pith cells (Fig. 2a, b). Following this, the establishment of the floral pre-meristem via the corpus and tunica facilitates the development of the receptacle and reproductive structures (stamen and pistil) (Fig. 2c, f).

Fig. 2.

Longitudinal section of reproductive meristem in Camelina sativa; a elongated inflorescence meristem; b the protrusion of the reproductive meristem and the appearance of sepal primordia, c initiate petal primordia; d initiate stamen primordia; e initiate gynoecium primordium; f formation of young flower; an: anther, fi: filament, gyp: gynoecium primordium, im: inflorescence meristem, lb: lateral bud, pe: petal, pep: petal primordium, pi: pistil, se: sepal, sep: sepal primordium, st: stamen, stp: stamen primordium.

Development of male reproductive organ

Anther formation

Stamen, the male reproductive organ, initially emerge as stamen primordia protrusions on the floral meristem below the sepal (Fig. 2d, e). The development of stamens precedes development of the pistil. During ovule primordium formation, anthers with four pollen sacs and the layers forming the pollen sac walls recognized. (Fig. 3e, f).

Fig. 3.

Longitudinal section of a young flower in Camelina sativa.; a The pistil in early developmental stage; b The ovary located above the junction of the sepals and petals, and as a result, the flower is hypogynous (superior ovary); c Each carpel contains numerous ovules on axile placentation; d Transverse section of ovary showed axile placentation; e The formation of stamens were earlier than the appearance of pistil, so during the formation of ovule primordium, following the division of meiosis division and four haploid nuclei create tetrad; f Some cells were differentiated from other cells with a significant increase in volume, which initiated archesporial cells. This was while the immature microspores in the anther were maturing in the pollen sac; an: anther, arc: archesporial cell, ova: ovary, ovu: ovule, pe: petal, pi: pistil, ps: pollen sac, rec: receptacle, se: sepal, sep: septum, sti: stigma, sty: style, tet: tetrad.

The six stamens are tetradynamous, with two shorter lateral stamens and four longer inner stamens (Fig. 4a). The anthers of Camelina sativa are structured as four pollen sacs, or tetrasporangia (Fig. 4b). At the initiation of anther development, each microsporangium consists of a cluster of sub-epidermal cells designated as archesporial cells, which, through periclinal divisions, contribute to the formation of the sporogenous tissue and the microsporangium wall. Following these divisions, the microsporangium wall is established. The anther wall consists of four layers, arranged from the outside to the inside: the epidermis, the endothecium (mechanical layer), the middle layer, and the tapetum (nutrient layer). Each of these layers typically contains a single row of cells (Fig. 4c).

Fig. 4.

Anther and pollen grain development in Camelina sativa; a Longitudinal section of the flower which shows the four anthers and two short anther (red arrow); b Transverse section of the flower, which shows the six stamens and four pollen sacs of each stamen, and the cross section of the pistil can also be seen in the central part; c The pollen sac wall has four layers of epidermis, endothecium layer, middle layer and nutrient layer or tapetum, which are surrounded large, polyhedral cells with thick cytoplasm (sporogenous tissue), d following the division of meiosis I in PMC, a binucleate cell is formed without the formation of a mid-wall; an: anther, bic: binucleate cell, en: endothecium, ep: epidermis, fi: flilament, ml: middle layer, pe: petal, pi: pistil, ps: pollen sac, spt: sporogenous tissue, tap: tapetum layer.

Pollen grain development

Microsporocytes, or pollen mother cells (PMCs), were generated following mitotic divisions within the sporogenous cell mass occupying the center of the pollen sac (Fig. 4c). These cells, characterized by their large volume, dense cytoplasm, and distinct nuclei, entered meiotic division, differentiate them from surrounding cells. At the beginning of meiosis, callose deposition commenced around the microsporocytes. During microsporogenesis, the microsporocytes go through two rounds of meiosis inside the microsporangia to form microspores. Meiosis I and II occur without interruption simultaneously alongside callose wall deposition, and resulting in tetrahedral tetrads (Fig. 4d).

Upon completion of meiosis, the callose wall surrounding the tetrads began to degrade, resulting in the separation of young microspores from each other within the tetrad (Fig. 5a). Each microspore released from the callose cover was enveloped by the primary exine wall. An additional pectocellulosic layer, known as the intine, formed adjacent to the cytoplasm, with increased thickness at the aperture (Fig. 5b). The growth of microspores was evident immediately after their release from the callose cover, marked by the development of a central vacuole, which displaced the nucleus toward the microspore margin (Fig. 5c).

Fig. 5.

pollen grain development in Camelina sativa; a Binucleate cell undergo meiosis II division and four haploid nuclei create (tetrad) in the callus wall. The presence of the tapetum layer is important during divisions; b Each microspore released from the callous cover was surrounded by the primary exine wall, c Immature microspores begin to mature in the pollen sac and take their nutrients from the cells of the secretory; d Immature microspores continued to mature in the pollen sac and take their nutrients from the cells of the plasmodium tapetum layer; cw: callous wall, en: endothecium, ep: epidermis, ip: immature pollen grain, ptap: periplasmodial tapetum, stap: secretory tapetum, tap: tapetum layer, tet: tetrad.

Microspores within the four sporangia (pollen sacs) exhibited synchronized development. The differentiation and specialization of the microsporangium wall layers occurred concurrently with meiosis. Consequently, the tapetal cells, whose primary function is to provide nutrients to the developing microspores, typically reached their maximum size during the tetrad stage. Initially, its constituent cells were uninucleate, even during the meiosis stage in microspore mother cells (MMCs). However, some cells became binucleate or multinucleate due to endomitosis. This layer, characterized by large nuclei, exhibited the highest staining intensity due to its nutritional role compared to other cells of the pollen sac wall (Fig. 5d). In Camelina sativa, the tapetum showed a secretory function in the early stages of development, then it was differentiated into Plasmodium type at the end of anther development, as it moved toward the center of the pollen sac cavity. Prior to anthesis, the tapetal periplasmodia were entirely consumed for pollen grain development. (Fig. 5d). The middle layer gradually underwent degeneration until the final stage of mature pollen grain development (Fig. 4g). Subsequently, the endothecium layer underwent radial elongation and developed fibrous thickenings (Fig. 5c).

Microspore development is completed by mitotic division, which leads to the differentiation of a large vegetative cell that forming the pollen tube and a smaller generative cell. The generative cell experiences a subsequent mitotic division, yielding two sperm cells, thereby rendering the mature pollen grain tricellular (Fig. 6a). The dehiscence of the tetrasporangiate anther occurred through longitudinal splitting in the stomium. At this stage, the tapetum had degenerated, leaving only the endothecium and epidermis layers in the mature anther wall. The released mature pollen grains were oval and tricolporate (Fig. 6b). During the process of anther dehiscence, the anther exhibits twisting from the apex to the base to disperse pollen towards the center of the flower, thereby classifying it as an introrse anther (Fig. 6c).

Fig. 6.

Mature pollen grain and dehiscence in Camelina sativa; a Following the maturation process of microspores. Mitotic division occurs in them and mature pollen grains with two vegetative and reproductive nuclei are produced; b After the maturation of the pollen grains, the endothecium causes the anther dehiscence; c The dehiscence of the tetrasporangiate anther occurred through longitudinal splitting in the stomium; en: endothecium, ep: epidermis, ex: exine, gn: generative nucleus, in: intine, mp: mature pollen grain, sto: stomium, vn: vegetative nucleus.

Scanning electron microscope observation

A scanning electron microscope was employed to examine the ultrastructure of pollen grains and exine ornamentation. In Camelina sativa, the pollen grains were tricolporate (Fig. 7b, e), exhibiting microreticulate exine ornamentation (Fig. 7c, f). The morphology of pollen exhibited significant changes between dry and hydrated states. The average polar axis length of hydrated pollen was 21.28 μm, while the average equatorial axis length was 20.02 μm, resulting in a P/E ratio of 1.07, indicating a tricolporate spheroidal shape. The average lumen length was 1.28 μm (Fig. 7d-f). During dehydration, the colpi fold inward, creating sunken apertures, and the pollen shape becomes more prolate (Fig. 7a-c). The average polar axis length of dry pollen was 29.25 μm, the average equatorial axis length was 18.74 μm, the P/E ratio was 1.56, and the average lumen length was 1.37 μm.

Fig. 7.

Electron micrographs of Camelina sativa pollen grains; a The micrograph of desiccated pollen grains in various orientations; b Equatorial view of a tricolporate desiccated pollen grain; c Micro reticulate exine ornamentation of desiccated pollen grains; d The micrograph of hydrated pollen grains; e Equatorial view of hydrated pollen grains; f Exine ornamentation in hydrated pollen grains.

Development of female organs

Pistil development

After differentiation of the stamen primordia from the lateral expansion of the floral meristematic region, the residual meristematic tissue undergoes elongation, forming the pistil primordium, from which the pistil subsequently emerges (Fig. 3a). The initial plan of the pistil undergoes rapid elongation, resulting in the differentiation of its three constituent parts: ovary, style, and stigma. The vascular architecture of the pistil and the presence of four rows of ovules indicate a syncarpous gynoecium composed of at least two fused carpels that separated by a false septum. The flower exhibits a hypogynous arrangement with a superior ovary (Fig. 3b, d). Each carpel contains numerous ovules on axile placentation (Fig. 3c, d). The formation of pistil was later than the appearance of stamens, so during the formation of ovule primordium, in anther following the division of meiosis division four haploid nuclei create tetrad (Fig. 3e). In ovary some cells were differentiated from other cells with a significant increase in volume, which initiated archesporial cells. At the same time in the anther, the immature microspores matured in the pollen sac (Fig. 3f).

Ovule development and embryo sac formation

The ovule primordium is initiated by periclinal division in the second subsurface layer of the placenta. By rapid cell division, the apical part of the ovule primordium forms the primary body of the ovule, while the ovule funicle is formed in the basal part, which is narrower and connected to the ovary wall (Fig. 3c, d). In the ovule body, some cells are distinguished from other cells by increasing their significant volume and create archesporial cells, while other cells around them make nucellus cells and ovule integument (Figs. 3f and 8a). The mature ovule is amphitropous type, bitegmic, funicular and tenuinucellate (Fig. 9c). Following meiotic division, the megasporocyte (archesporial cell) with its prominent nucleus generates four haploid megaspores in a linear tetrad, of which only a single chalazal megaspore persists. This surviving megaspore subsequently develops into the megagametophyte (embryo sac) through the process of megagametogenesis (Fig. 8b, c). In the functional megaspore rapid enlargement was hapened, its volume increasing significantly in comparison to the surrounding nucellus cells. It developed into a vacuolated megagametophyte with a distinct nucleus, exhibiting the characteristic morphology of a developing embryo sac. An intercellular space formed between the nucellus cells and the developing megagametophyte, within which remnants of degenerated nucellus cells were apparent (Fig. 8d).

Fig. 8.

Ovule development in Camelina sativa; a-b initial of ovule primordium; c division of megaspore mother cell and initials of embryo sac; d single cell remained gave rise to the mother cell of the embryo sac; arc: archesporial cell, dy: dyad, emc: embryo mother cell, fu: funicle, ii: inner integument, mi: micropyle, nu: nucellus, oi: outer integument, ow: ovary wall, pl: Placenta, tet: tetrad.

Fig. 9.

Embryo development in Camelina sativa; a-b Shortly after fertilization, the zygote undergoes a series of divisions, leading to the formation of the proembryo; c-d globular embryo; en: endothelium, end: endosperm, endmc: endosperm mother cell, es: embryo sac, ge: globular embryo, h: hypostase, ii: inner integument, mi: micropyle, nu: nucellus, oi: outer integument, pd: podium, pe: proembryo, su: suspensor.

Three successive mitotic divisions occurred within the functional megaspore, resulting in the formation of an eight-nucleate embryo sac exhibiting the Polygonum type pattern. (Fig. 10a). The megagametophyte displayed an elongated and asymmetric morphology. Additionally, remnants of degraded nucellar tissue were identified at the periphery of the developing embryo sac (Fig. 10b). Following the eight-nucleate stage, nuclear migration and cellularization occurred. The megagametophyte exhibited polar organization with three nuclei positioned at the micropylar pole, differentiating into the egg apparatus consisting of one oosphere (egg cell) with a prominent spherical nucleus and two synergids with elongated stripe structure. Three nuclei were situated at the chalazal pole, forming the antipodal cells. The two polar nuclei were centrally located within the central cell (Fig. 10b). Subsequently, cytokinesis and membrane formation occurred around each nucleus, resulting in the mature seven-celled, eight-nucleate megagametophyte. The egg apparatus, comprising two synergids flanking the oosphere, underwent cellularization prior to the other megagametophyte components. The polar nuclei migrated towards the micropylar end and fused, forming the diploid secondary nucleus of the central cell. The antipodal cells underwent programmed cell death prior to fertilization. The resultant mature megagametophyte was ready for double fertilization. The embryo sac was enveloped by the endothelium, characterized by cells with prominent nuclei, dense cytoplasm, and persistent viability throughout embryogenesis. The chalazal megaspore differentiates into an eight-nucleate embryo sac characterized by the Polygonum type pattern (Fig. 10c). It was observed that in C. sativa, a system of various structures and specialized tissues creates a targeted transport of metabolites in the ovule into embryo sac from vascular bundle through chalaza and then via hypostase, podium and postament (Fig. 10c).

Fig. 10.

Embryo sac development in Camelina sativa; a-b Three consecutive divisions are done in the embryo sac and an eight-nucleate embryo sac is formed; c The embryo sac is Polygonum type, composed of seven cells: one central cell with polar nuclei, two synergids, one oosphere cell, and three antipodal; ant: antipodal cells, en: endothelium, es: embryo sac, fu: funicle, h: hypostase, mi: micropyle, nu: nucellus, oo: oosphere, pd: podium, pn: polar nuclei, ps: postament, sy: synergid cells.

Embryo development

Following successful pollination and the guidance of the pollen tube into the embryo sac, double fertilization occurred. One male gamete (antherozoid cell) fused with the oosphere cell (female gamete) resulting in the formation of a diploid zygote. Simultaneously, the second male gamete combined with the two polar nuclei, resulting in the formation of the triploid endosperm mother cell. This triploid endosperm mother cell produced nucleate endosperm until the final stage of the globular embryo, subsequently partitioning and yielding cellular endosperm, which functions as a nutritive tissue for the developing embryo. Subsequently, the zygote underwent mitotic divisions. Embryogenesis in Camelina was the Onagrad type (Fig. 9a). During embryogenesis, the first asymmetric division of the zygote resulted in the formation of two distinct cells: the apical cell and the basal cell. The basal cell subsequently underwent differentiation to form the suspensor, while the apical cell initiated the development of the proembryo through a series of mitotic divisions (Fig. 9b). During the proembryo development stage, the rate of cell divisions was markedly high, resulting in the rapid formation of a spherical cell mass known as the globular embryo. This structure was accompanied by a multicellular suspensor (Fig. 9c, d).

Discussion

Development of the Male Reproductive Organs

Our studies on Camelina sativa revealed that the stamens exhibit a tetradynamous arrangement, characterized by four long stamens and two short stamens that align with Conner’s report³⁰. The primordia of the four inner stamens are initiated independently from one another and develop slightly earlier than the two lateral, outer stamen primordia. This observation is consistent with the findings reported by Smyth²⁶ in Arabidopsis and is typical of the Brassicaceae family. During the initial stages of stamen differentiation, the sporophytic tissue comprises several multifaceted cells exhibiting high chromatin density, surrounded by a subepidermal layer. This subepidermal layer undergoes division and subsequent differentiation to form the anther wall. Our findings reveal that the stamen of Camelina sativa possesses four microsporangia, a characteristic common within the Brassicaceae family. These observations are consistent with the reports of Owen & Makaroff³¹. The anther wall is composed of four distinct layers: the epidermis, the endothecium, the middle layer, and the tapetum (nutritive layer).

This structural organization is consistent with the findings reported by Ma³². As observed in other angiosperms, two types of tapetum can be identified during anther development in C. sativa: secretory and amoeboid. Initially, during microsporogenesis, the tapetum exhibits a secretory morphology and is clearly discernible at the periphery of the microsporangia. As pollen grain development progresses, the tapetum transitions to an amoeboid type, observed in proximity to the developing microspores. These observations are in accordance with the findings reported by Liu & Fan³³ and Feng & Dickinson³⁴. The endothecium begins lignification at the uninucleate microspore stage, concurrent with tapetal degeneration, which aligns with previous reports^35,36. According to the findings of Hsieh et al.²⁴ and Zhang et al²⁵. in the Brassicaceae family, the middle layer is observed to be e temporary and transient, undergoing degeneration during early stages of anther development.

Following divisions in the sporogenous cells, pollen mother cells formed. At the onset of meiosis I, a callose wall begins to develop around the pollen mother cells and persists until the formation of tetrads. This callose wall serves to protect the dividing cells from potential interactions with neighboring cells. The formation and stability of this wall are consistent with previous findings^35,37. At the conclusion of meiosis II, four haploid cells (tetrads) are enclosed within the callose wall in a tetrahedral arrangement. These characteristics are typical of dicotyledons and align with prior reports^38–40. The simultaneous cytokinesis separates the tetrad cells. This observation aligns with the findings of Reeder et al. in their research on Arabidopsis⁴¹. Concurrent with the differentiation of microsporocytes and their preparation for meiosis, the tapetal cells initiate their secretory function and undergo hypertrophy. As the locule volume increases, these tapetal cells become separated from each other and from the middle layer. Following the binucleate stage, the cytoplasm of these cells begins to degrade, and upon the formation of immature microspores, they commence decomposition. These changes, which emphasis the supportive role of the tapetal cells in pollen grain development, are consistent with the findings of Pacini⁴², Feng & Dickinson³⁴ and Teagen⁴³. The multiple layers of the anther wall appear to function collectively not only in the development of microspores but also in the release of mature pollen grains. The endothecium, by interacting with the middle layer and the tapetum, plays an important role in anther dehiscence and pollen release. After microspore meiosis, in addition to the destruction of the tapetum and middle layers, the endothecium undergoes a specific secondary thickening⁴⁴. The thickening of the endothecium layer occurs before the anther dehiscence and creates tension in the remaining layers of the anther to generate sufficient force to rupture the stomium, contract the anther walls during dehydrating, and disperse the pollen⁴⁵. In addition to aiding anther dehiscence, the endothecium appears to act as the last lipid storage site during the final stages of pollen development, and fatty acids derived from the endothecium are thought to help facilitate pollen hydration^46,47.

Palynology is the scientific study of pollen grains and spores. Palynological investigations can provide valuable insights into the systematic and phylogenetic relationships among various angiosperm taxa. Pollen grains demonstrate significant morphological diversity, characterized by variations in dimensions, shapes, the number and types of apertures, positions of apertures, and patterns of exine ornamentation²⁹.

In our study, the morphology of pollen grains exhibited significant changes between dry and hydrated conditions. Under hydrated conditions, pollen grains assumed a spheroidal shape, whereas in dry conditions, the colpi folded inward, resulting in a more prolate shape. This morphological alteration is attributed to the Harmomegathic Mechanism or Wodehouse Effect⁴⁸, where in the infolding of the pollen wall helps manage osmotic pressure changes within the cytoplasm during hydration and dehydration²⁹.

Camelina sativa is characterized by tricolporate pollen grains, a palynological feature consistent with observations in other Brassicaceae species. The pollen morphology aligns with findings reported for various Brassicaceae taxa in Central Punjab, Pakistan⁴⁹ and for the genus Brassica in Bangladesh⁵⁰. Specifically, C. sativa pollen exhibits a spheroidal shape when hydrated, with a size range of 21–25 μm. The exine ornamentation is reticulate, as observed under light microscopy. These palynological characteristics are typical of the Brassicaceae family and contribute to the taxonomic understanding of Camelina within this clade⁵¹.

According to palynological studies conducted on Thlaspi L. (Brassicaceae) from Turkey and other Brassicaceae taxa, reticulate exine ornamentation is commonly observed^52,53. However, our findings for Camelina sativa diverge from this pattern. The lumen diameter on exine elements in C. sativa is < 1 μm, classifying the exine ornamentation as microreticulate. Umber et al. in their comparative study of pollen morphology in selected Asteraceae and Brassicaceae taxa, reported pollen sizes ranging from small to medium⁵⁴. Our observations indicate that the majority of C. sativa pollen grains fall within the medium size category, aligning with the broader trends observed in the Brassicaceae family^54,55.

Development of the Female Reproductive Organs

The inflorescence of Camelina sativa is characterized as a raceme. Each flower is bisexual and actinomorphic, exhibiting tetramerous symmetry with four distinct sepals and four distinct petals, the latter being yellow in coloration. Following the initiation of the gynoecial primordium from the floral meristem, the preliminary pistil undergoes rapid elongation, differentiating into three distinct regions: the ovary, style, and stigma that aligns with findings of Chang & Sun⁵⁵.

Concurrent with gynoecial development and ovarian differentiation, the initial stages of ovule initiation are observed along the ovary wall. The ovary of C. sativa is superior. The vascular architecture of the Camelina pistil and the presence of four rows of ovules indicate a syncarpous gynoecium composed of at least two fused carpels that aligns with findings of Hill & Lord⁵⁶ and Okada et al.⁵⁷.

This carpel arrangement is consistent with the typical gynoecial structure observed in the Brassicaceae family. The ovule of Camelina sativa exhibits an amphitropous orientation, tenuinucellate structure, and bitegmic organization. These characteristics align with the observations of Shamrov⁵⁸ on Capsella bursa-pastorisovules and seeds, as well as the findings of Yankova-Tsvetkova E et al.⁵⁹. However, they contrast with Shamrov’s report on Arabidopsis thaliana, which described hemicampylotropous and medionucellate ovules⁶⁰. In agreement with Bouman’s observations, we noted that the initiation of both integuments occurs simultaneously on the ovule primordium^61–63. These ovule characteristics contribute to our undersanding of reproductive development in Camelina and its relationship to other Brassicaceae taxa.

Our findings are consistent with Shamrov’s investigations, which demonstrated that the inner and outer integument layers of the ovule are generated through regular cell divisions at the ovular base. The inner integument comprises three cellular layers, whereas the outer integument is constituted of two layers⁶⁴. During the two-nucleate megagametophyte stage, periclinal divisions commence in the inner epidermal cells of the inner integument. This division is frequently associated with the differentiation of an endothelium. These observations are consistent with the research conducted by Bowman et al.⁶⁵; Kapil & Tiwari⁶⁶ and Robinson et al.⁶⁷.

The developmental patterns of megasporogenesis and microsporogenesis are generally not simultaneous. For example, meiosis in anthers was about 19 days earlier than in ovules in lily⁶⁸. Our results also showed that tetrad formation in anthers coincided with the observation of archesporium in the ovule, a developmental pattern that differs from the pattern reported by Yankova-Tsvetkova in Brassica jordanoffi⁵⁹. It has also been reported in Paspalum rufum that the development of reproductive organs in diploids was uniformly synchronized, while the megasporogenesis was delayed relative to microsporogenesis in tetraploids⁶⁹. However, these distinctions between megasporogenesis and microsporogenesis do not subsequently affect natural self-pollination and self-fertilization in camelina, because they are synchronous across the entire flower. The alternating growth of megasporogenesis relative to microsporogenesis is lost at anthesis, when pollen tube growth continues through the transporting tissue in the style, while the megagametophytes are forming in the ovules⁵⁵.

The precise identification of initial archesporial cells during early developmental stages presents challenges, potentially leading to misinterpretation of their position. We noted that the archesporial cell is located beneath two to three layers of nucellar tissue and is entirely enveloped by the nucellar epidermis. A unicellular archesporium was discerned within the ovular primordium, corroborating the findings reported by Shamrov⁶⁰. This contrasts with observations in Paeonia (in Brassicaseae) here a multicellular archesporium has been reported⁷⁰. These variations in archesporial development contribute to our understanding of ovule ontogeny diversity within the Brassicaceae and related families.

The singular archesporial cell differentiates directly into the megaspore mother cell (MMC). Following this, the MMC undergoes meiotic division, culminating in the formation of a linear tetrad of megaspores, as documented in the majority of Brassicaceae^1,59,71.

In C. sativa, similar to most representatives of the Brassicaceae family, the chalazal megaspore enlarges while the other three degenerate. The functional megaspore then undergoes three successive mitotic divisions, forming an eight-nucleate embryo sac (ES). The ES development follows a monosporic, Polygonum-type developmental pattern^1,59,72.

Our results elucidate that in C. sativa, a distinguishing characteristic of the embryo sac is the emergence of hypostasis, an organized tissue located at the base of the nucleus and integument, which typically manifests in the chalaza region following the four-nucleate embryo sac stage, as observed in numerous species within the Brassicaceae family^64,73.

This tissue serves a protective and nutritive function facilitating the transport of nutrients from the vascular bundles through the podium, postament, integumentary tapetum, nucellar epiderm and parietal cells to the embryo sac⁵⁸. The mature ES comprises a three-celled egg apparatus (typically consisting of a pear-shaped egg cell and two synergids), a central cell, and a three-celled antipodal apparatus situated on the podium in the chalazal region of the ES^9,74. In C. sativa, the podium is described as cup-shaped. This structure differentiates early during ovule development and is observable as early as the four-nuclear embryo sac stage, persisting until the formation of the young embryo^75,76.

Post-fertilization, the synergids undergo degeneration. These cells exhibit a hooked morphology and possess a filiform apparatus, characteristics consistent with most Brassicaceae species^1,76. The antipodal cells degenerate rapidly during pre-fertilization stages. In contrast to other cell types within the female gametophyte, the specific function of antipodal cells remains elusive^58,74. It is worth noting that the timing and mechanism of synergid degeneration can be different among species. In some angiosperms, the degeneration of synergids transpires prior to the arrival of the pollen tube, while in others, this degeneration is instigated upon contact with the pollen tube⁷⁷. In Arabidopsis thaliana, for example, synergid degeneration has been observed to start as early as 5 h after pollination, with most embryo sacs showing degeneration by 9 h after pollination. The precise timing of synergid degeneration in relation to pollen tube arrival and discharge may have implications for pollen tube guidance and the fertilization process⁷⁸. The rapid degeneration of antipodal cells in C. sativa aligns with observations in other Brassicaceae species. While the specific role of antipodal cells remains unclear, their transient nature suggests they may serve a temporary function during female gametophyte development or early stages of fertilization.

Our findings demonstrated that following porogamous double fertilization, which entails the degeneration of one synergid upon pollen tube entry via the micropyle, embryo and endosperm development is initiated. The data suggest that embryogenesis initiates subsequent to the formation of the endosperm, and by the time the embryo attains maturity, the endosperm is entirely assimilated by the embryo. These observations align with previous studies on Arabidopsis thaliana, Brassica jordanoffii, and Cardamine parviflora^1,59,60. The results highlight two key points: they reinforce the conserved patterns of embryo and endosperm development across Brassicaceae species and emphasize the crucial role of the endosperm in supporting embryo growth until maturity. Future research could aim to identify key regulatory genes that coordinate embryo and endosperm development in the species of interest.

The proembryo differentiation in C. sativa accordance to the Onagrad developmental pattern as elucidated by Johansen⁷⁹. The ontogenetic sequence observed in C. sativa proembryos exhibits notable similarities to those documented in Capsella⁸⁰, Brassica napus⁸¹, and Arabidopsis⁸². During embryogenesis in C. sativa, the suspensor cell undergoes elongation and continued mitotic divisions, resulting in the formation of an absorptive structure termed the suspensor. This developmental process aligns with observations reported by Kawashima & Goldberg;⁸³ Lau et al.⁸⁴ and Liu et al.⁸⁵. The conserved nature of early embryonic development across diverse taxa in the Brassicales order suggests a shared evolutionary origin for these embryogenic patterns, highlighting their fundamental role in establishing the embryo’s basic body plan. Despite the overall similarity in developmental stages, species-specific variations may occur in timing, cell division patterns, or molecular regulation. The suspensor cells exhibit dual critical functions in embryogenesis Primarily, the suspensor acts as a reservoir for plant hormones and signals that are essential for embryonic development. Additionally, it functions as an absorptive organ, facilitating the uptake of nutrients from the seed’s somatic tissues and their subsequent translocation to the developing embryo proper^84–86.

The order Brassicales includes 17 families, about 400 genera, and about 4,700 species^87–89. This order is highly diverse in habit, floral morphology, and biogeographic distribution. Capparaceae and Brassicaceae have long been recognized as closely related families. Capparaceae traditionally has two main subfamilies: Cleomoideae and Capparoideae. Both of these subfamilies exhibit extraordinary floral diversity, including actinomorphy, asymmetry, zygomorphy, a wide range of stamen numbers (1–250), and elongated stamens. In contrast, the monophyly of Brassicaceae is not in doubt, and the floral background is remarkably stable⁹⁰. Given the central role of Arabidopsis thaliana and other Brassicaceae species as model organisms in various research fields, Brassicaceae has entered contemporary textbooks, overshadowing the sixteen remaining families⁹¹, therfore Brassicaceae is relatively well studied in terms of embryology and pollinology among the main families of Brassicales. Here, we have attempted to illustrate the similarities and differences between these sister families by reviewing the embryo and pollen characteristics of several species of this order and comparing them with Camelina as a representative of Brassicaceae (see Tables 2 and 3). The comparisons show that the Brassicaceae family shares many embryological and pollenological features with the Capparaceae and Cleomaceae families, especially in the traits related to the integuments, embryo sac, type of nucleus and pollen apertures. However, the Brassicaceae family is more closely aligned with the Capparaceae than with the Cleomaceae. In fact, the Brassicaceae, Cleomaceae and Capparaceae families show almost identical features in aspects such as anther structure, microsporangium, nucleus, megagametophyte, pollen polar view and type of pollen aperture type (Tables 2 and 3). Only minor differences in embryology and pollination separate Brassicaceae from Cleomaceae and Capparaceae. For example, Brassicaceae differs from Cleomaceae in placentation and pollen size, and from Capparaceae in features such as archesporium, megaspore tetrads, pollen sculpture, and P/E ratio. These differences may reflect unique evolutionary traits within each family. Close similarities in ovule curvature, endosperm formation, and pollen aperture type, along with minor differences in mating, P/E ratio, and other features, suggest that Brassicaceae, although distinct, are closely related within the Brassicales to Cleomaceae and Capparaceae. Due to the limited number of comprehensive embryological studies on Cleomaceae and Capparaceae, it remains challenging to clearly define the boundaries between these related families using embryological data alone. Thus, detailed descriptions of floral stages are needed for comparative analyses among different plant species. It is recommended that more species in these families be studied in terms of embryology and pollinology to better define their taxonomic boundaries. Detailed descriptions of floral stages are needed for comparative analyses among different plant species.

Table 2.

A summary of embryological data of Camelina sativa, Brassicaseae and related families.

Characters	Camelina sativa	Brassicaseae	Cleomaceae	Capparaceae
Inflorescence	Raceme	Raceme	Raceme	Raceme
No. Microsporangia	4	4	4	4
Anther wall formation	Dicotyledonous-type	Dicotyledonous-type	Dicotyledonous-type	Dicotyledonous-type
No. Middle layer	1	1–2	2	2
Tapetum type	Secretory Periplasmodial	• Secretory • Periplasmodial	Secretory	Secretory
Cytokinesis in meiosis	Simultaneous	Simultaneous	Simultaneous	Simultaneous
Tetrad of microspore	Tetrahedral	• Tetrahedral • Tetragonal	• Tetrahedral • Decussate	• Decussate • Tetrahedral
Mature pollen	Two and three celled	• Three- celled • Two-celled • Two and three celled	Three-celled	• Two-celled • Three-celled
Ovary	Superior	Superior	Superior	Superior
Carpels	2	2	2	2 – (8)
Placentation	Axile	• Axile • Central	Parietal	Axile
Archesperium	Unicellular	Unicellular	Unicellular	• Unicellular • Two and three celled
Megasporocyte	Archesporial becomes MMC	• Archesporial becomes MMC • Archesporial forms primary parietal and primary sporogenous cells	Archesporial forms primary parietal and primary sporogenous cells	Archesporial forms primary parietal and primary sporogenous cells
Megaspore tetrads	Linear	Linear	Linear	Linear, T-shape
Curvature of ovules	Amphitropous	• Amphitropous • Campylotropous	Campylotropous	• Anatropous • Campylotropos to amphitropous
Nucellus type	Tenuinucellate	• Tenuinucellate • Crassinucellate	Crassinucellate	Crassinucellate
No. Integuments	Bitegmic	Bitegmic	Bitegmic	Bitegmic
Embryonic sac	Polygonum type	Polygonum type	Polygonum type	Polygonum type
Antipodal cells	Ephemeral	Ephemeral	Ephemeral	Ephemeral
Hypostase	Formed	Formed	Formed	Formed
Podium	Formed	Formed	No data	Formed
Postament	Formed	rarely Formed	No data	Formed
Endothelium	Formed	Formed	Not formed	Not formed
Endosperm formation	Cellular	Cellular	Cellular	Nuclear

References: ^{33,58–60,65,71,92–97}.

Table 3.

A summary of pollen morphological attributes of Camelina sativa, Brassicaseae and related families.

Characters	Camelina sativa	Brassicaseae	Cleomaceae	Capparaceae
Size	Medium	Small-Medium	Very small	Small
Polar view	Lobate Circular	• Lobate • Circular	• Lobate • Circular • Triangular	• Lobate • Circular • Triangular
Apertures Type	Tricolporate	Tricolporate	Tricolporate	• Most: Tricolporate • Rarely: Tetracolporate
Shape	Prolate-Spheroidal	• Oblate-spheroidal • Prolate-spheroidal • subprolate	• Prolate • Prolate-Spheroidal • subprolate	• Subprolate • Prolate-spheroidal • Oblate-spheroidal
Sculpture elements	Microreticulate	Reticulate	• Microreticulate • Echinate • Striat-faveolate	• Spinulose • Striaterugulate • Rugulate-reticulate
Lumen length (nm)	675.02	525.2–691.3	A	A
PD (µm)	29.25	15.6–23.08	15.3–28.9	19.4–28.2
ED (µm)	18.74	16–22.5	11.75–20.12	11.12–15.6
P/E ratio	1.56	0.94–1.27	0.94–1.58	1.25–1.5

Abbreviations: A = absent; ED = equatorial diameter; PD = polar diameter; µm = micrometer; nm = nanometer.

References: ^49,98–106.

Conclusion

We provide a complete set of morphological landmarks for flower development in Camelina sativa from early flower development to the opening of the flowers. These landmarks also identify key steps in male and female gametophyte development. Camelina as a Brassicaceae members, from the floral meristem initiation to gametophyte differentiation demonstrates both conserved and lineage-specific features in stamen, pistil, and ovule ontogeny. Our findings underscore the systematic importance of key traits such as tetradynamous stamens, microreticulate pollen exine ornamentation, amphitropous bitegmic ovules, and Polygonum-type embryo sacs, which collectively reinforce the phylogenetic placement of Camelina within Brassicaceae while delineating subtle variations that distinguish it from Cleomaceae and Capparaceae. Moreover, the protandrous flowering sequence, hypostase formation, and suspensor-mediated nutrient transfer highlight functional adaptations that optimize reproductive success. From a broader systematic perspective, these embryological and palynological attributes exemplify evolutionary conservatism across Brassicales, with minor differentiations potentially representing adaptive shifts. Altogether, this integrated developmental and comparative analysis provides valuable insights for understanding both the evolutionary relationships and the reproductive strategies of Camelina sativa within its taxonomic context.

Author contributions

S.T., P.J. and M.M. conceived and design the study. S.T., P.J., M.M. and P.H. organized and performed the experiments. S.T., P.J., M.M. and A.M. were involved in data interpretation. S.T., P.J. and M.M. wrote the manuscript. P.J. and M.M. planned and supervised the study and edited the final version of the manuscript. All authors read and approved the final version of the manuscript.

Data availability

The data generated or analyzed in this study are included in this article. Other materials that support the findings of this study are available from the corresponding author upon reasonable request.

Declarations

Competing interests

The authors declare no competing interests.