Megasporogenesis and Female Gametophyte Development in Plants

In the evolutionary lineage of plants, there is a persistent trend towards reduction of the haploid phase (gametophyte) in favor of the diploid phase (sporophyte). Whereas in mosses and ferns the female gametophyte (prothallus) is an independent, multicellular, long-lived green plant, in gymnosperms the gametophyte is greatly reduced and develops on the sporophyte, and in angiosperms it reaches extreme miniaturization (Raven et al., 2013; Becker et al., 2025).

The female gametophyte of angiosperms—the embryo sac—typically consists of only seven cells and eight nuclei. This results from a long evolutionary reduction that has led to several key advantages:

Female gametophyte development in flowering plants is inextricably linked with two major evolutionary innovations—the ovule (integumented megasporangium) and double fertilization.

The ovule is a modified megasporangium surrounded by one or two envelopes—integuments. The presence of integuments protects the developing female gametophyte and, after fertilization, forms the seed coat (Beck, 2010). It is important to note that unlike in gymnosperms, where ovules are exposed on megasporophylls, in angiosperms they are enclosed within the ovary of the pistil. This provides additional protection and creates conditions for directed pollen tube growth through the stigma and style tissues toward the ovule.

The second and most significant evolutionary acquisition is double fertilization. One sperm fertilizes the egg cell, giving rise to the zygote (future embryo). The second sperm fuses with the two polar nuclei of the central cell, forming triploid endosperm—a unique storage tissue that nourishes the developing embryo (Raven et al., 2013; Su et al., 2025). This mechanism ensures that mobilization of the maternal plant’s nutritional resources occurs only upon successful fertilization, thereby increasing reproductive efficiency.

Understanding the biological role of megasporogenesis and female gametophyte development has direct applied significance in agriculture:

Thus, the evolutionary reduction of the female gametophyte in angiosperms did not lead to simplification but rather to the creation of a highly efficient and protected reproductive system, in which all stages—from meiosis in the megasporocyte to cellular differentiation in the embryo sac—are under strict genetic and epigenetic control. This system underlies the success of flowering plants in colonizing land and supports modern productive seed production.

This is the most common type, found in approximately 70% of flowering plants (Beck, 2010). Its key feature is that only one of the four megaspores resulting from megasporocyte meiosis participates in embryo sac development.

The process occurs as follows: upon completion of meiosis, a linear tetrad of haploid megaspores forms. Three megaspores (usually located on the micropylar side) undergo programmed cell death and degenerate. One, most often the lowest, chalazal megaspore, survives and becomes the functional megaspore. It is from this megaspore, through three successive mitotic divisions, that the eight-nucleate and subsequently seven-celled embryo sac forms. This classical pathway was first described in detail for the genus Polygonum (knotweed), hence its alternative name—the Polygonum type (Lersten, 2004; Raven et al., 2013).

The mature female gametophyte of the monosporic type has the standard structure: three antipodal cells, a central cell with two polar nuclei (which often fuse before fertilization), an egg cell, and two synergids (Huang et al., 2025). This developmental type is characteristic of most agricultural crops, including wheat, maize, tomatoes, and legumes (Su et al., 2025).

The bisporic type is significantly rarer, found in about 12% of angiosperm species. Classic examples are species of the genus Allium (onion), leading to its common name—the Allium type (Bell, 1991; Kaur et al., 2024).

The fundamental difference from the monosporic type is that cytokinesis (cell plate formation) does not occur after the first meiotic division. Consequently, a binucleate dyad forms instead of a tetrad. Each of the two diploid cells of the dyad (each containing two haploid nuclei) then undergoes the second meiotic division. Cytokinesis does not follow the second division either, or occurs only partially. The end result is a single two-nucleate megaspore (the functional one), where each nucleus is a product of one of the two meiotic divisions. Thus, two nuclei originating from two different megaspores of the original dyad participate in forming the female gametophyte. Subsequent gametophyte development follows the same scenario as the monosporic type: two mitotic divisions yield eight nuclei, which then organize into the standard seven-celled structure (Lersten, 2004).

The tetrasporic developmental type is the most evolutionarily ancient among angiosperms, but in modern flowering plants it is found only in about 5-10% of species. The name derives from the genus Fritillaria (fritillary), where it was first studied in detail (Kaur et al., 2024).

In this case, cytokinesis is absent throughout the entirety of meiosis. The megasporocyte undergoes the two meiotic divisions, but cell walls form neither after the first nor after the second division. As a result, a single, large, tetranucleate megaspore forms, containing all four haploid nuclei. This megaspore becomes the functional one. Subsequently, the nuclei undergo one mitotic division (increasing their number to eight), followed by nuclear migration and cellular differentiation (Raven et al., 2013). However, unlike the monosporic type, the genetic material distribution in such a gametophyte can be more complex.

A characteristic example of the tetrasporic type is embryo sac development in most lily species. After meiosis, the four nuclei separate: one moves to the micropylar pole, three to the chalazal pole. Then the one nucleus at the micropylar end divides, producing two haploid nuclei, while the three nuclei at the chalazal pole fuse and divide, forming two triploid nuclei. Thus, a four-nucleate stage forms, consisting of two haploid and two triploid nuclei. This is followed by another mitotic division, resulting in eight nuclei that organize into the seven-celled embryo sac (Lersten, 2004).

Besides the three main types, other, less common developmental variants exist. For example, in the families Onagraceae and Asteraceae, the Oenothera type occurs, often considered a modification of the monosporic type. Its peculiarity is that only four nuclei participate in embryo sac formation, not eight: the first mitotic division of the functional megaspore yields two nuclei, which then migrate to the poles, while the second division does not occur (Bell, 1991).

It is important to note that in the most ancient (basal) groups of angiosperms—such as Amborellaceae, Nymphaeales, and Austrobaileyales—the female gametophyte structure differs from the classical Polygonum type. These plants often have embryo sacs with four cells and four nuclei (Oenothera-like), and in Amborella, a unique structure with nine nuclei in eight cells has been described (Beck, 2010; Becker et al., 2025). This indicates that the Polygonum type, despite its current prevalence, is evolutionarily derived rather than the ancestral condition for all flowering plants.

In practical agricultural work, knowledge of the female gametophyte developmental type is important for breeding (e.g., overcoming incompatibility) and for understanding the causes of sterility. For instance, bisporic and tetrasporic types can be associated with the formation of unreduced gametes, which is used to obtain polyploid and haploid forms (Liu et al., 2025; Su et al., 2025).

Summary table of main female gametophyte developmental types

Megasporocyte differentiation

Development begins when one cell of the subepidermal layer (L2) in the apical part of the ovule nucellus stops mitotic divisions, enlarges, and differentiates into a megasporocyte (also called the megaspore mother cell). This is the first cell of the female germline. The megasporocyte has a large nucleus, dense cytoplasm, and a prominent nucleolus (Huang et al., 2025; Jiang & Zheng, 2022). At this time, callose (β-1,3-glucan) is deposited around the megasporocyte, isolating it from surrounding somatic cells (Lersten, 2004).

Meiosis and megaspore tetrad formation

The megasporocyte (2n) enters meiosis. First, DNA replication occurs, followed by two successive meiotic divisions (meiosis I and meiosis II). As a result, four haploid (n) cells—megaspores—are formed. In most plants, cytokinesis (cell plate formation) occurs sequentially after each division, and the four megaspores align in a linear tetrad along the micropyle-chalaza axis (Raven et al., 2013; Cao et al., 2018).

It is important to note that the completion of meiosis and tetrad formation is accompanied by callose deposition in the cell walls of the newly formed septa. This is readily detectable with aniline blue and serves as a stage marker (Lersten, 2004).

Functional megaspore selection

Immediately after tetrad formation, programmed cell death of three of the four megaspores is initiated. In typical monosporic development, only the lowest, chalazal megaspore survives (becomes functional), while the three megaspores located closer to the micropyle degenerate (Lersten, 2004; Kaur et al., 2024).

The mechanisms determining megaspore fate are actively studied. Current data indicate that key roles in selection are played by:

Interestingly, in some plants (e.g., members of the Onagraceae), the functional megaspore is the micropylar one, not the chalazal one. In the tetrasporic developmental type (e.g., in lily), no megaspore degeneration occurs at all—all four nuclei participate in building the embryo sac (Raven et al., 2013; Bell, 1991).

To better understand the specificity of the female gametophyte, it is useful to compare its development with that of the male (Raven et al., 2013):

Female gametophyte development is not an autonomous process. Embryo sac cells constantly interact with surrounding sporophytic tissues (nucellus, integuments). For example, in Arabidopsis thaliana, β-1,3-glucanases synthesized in nucellar cells control callose deposition in the developing megaspore wall, affecting the intercellular transport of signals and nutrients (Huang et al., 2025; Kaur et al., 2024).

Key stages—meiosis initiation, functional megaspore selection, mitosis initiation and arrest, cellular differentiation—are regulated by a complex network of genes, phytohormones, small RNAs, and transcription factors. Detailed analysis of these regulatory mechanisms is beyond the scope of this article and will be addressed in specialized sections on plant genetics and physiology.

Specific genes, transcription factors, and epigenetic mechanisms play a fundamental role in controlling female gametophyte development (see sections 2 and 3). Disturbances in their function lead to characteristic phenotypes: formation of additional megasporocytes, multiple functional megaspores, or complete absence of the embryo sac (Huang et al., 2025; Jiang & Zheng, 2022).

Epigenetic disturbances (e.g., changes in DNA methylation or histone modifications at loci controlling gametophyte development) can lead to similar anomalies, especially under stress (Jiang & Zheng, 2022; Su et al., 2025).

Female gametophyte development is sensitive to the balance of phytohormones. Although the full picture is not yet complete, the following hormones are known to play significant roles:

The female gametophyte is particularly sensitive to abiotic stresses, which is a major cause of yield loss under unfavorable conditions. The most critical are:

Temperature. The period from megasporocyte meiosis to fertilization is one of the most thermosensitive phases. Short-term exposure to high or low temperatures during this period can lead to:

Water deficit (drought). Water deficit during the embryo sac development stage causes delays in mitotic divisions of the functional megaspore, anomalies in nuclear size and position, as well as premature degeneration of egg apparatus cells. This leads to reduced seed set (Lersten, 2004).

Nutrition (mineral elements). Boron (B) deficiency is critical for pollen tube development but also affects embryo sac formation: under B deficiency, cell elongation, synergid and central cell formation are disrupted. Calcium (Ca²⁺) deficiency can disrupt cell wall formation and egg cell polarity (Lersten, 2004; Kaur et al., 2024).

Other stresses. Soil salinity, heavy metals, and ozone also negatively affect female gametophyte development, causing an increase in the number of aborted embryo sacs and reduced fertility.

The following deviations from normal development can be observed in experiments and in natural populations:

Thus, normal female gametophyte development is the result of precise tuning of genetic programs, epigenetic landscape, hormonal balance, and favorable environmental conditions. Any significant deviation in these factors leads to impaired fertility and reduced plant productivity, which must be taken into account in agronomic practice and breeding programs.

Understanding the critical stages of female gametophyte development provides tools for controlling seed productivity.

Stress protection. As indicated in Section 4, the female gametophyte is extremely sensitive to high temperature, drought, and nutrient deficiency. Agronomic practices (irrigation, adjusting sowing dates to avoid heat peaks, optimizing mineral nutrition, especially with boron and calcium) can minimize embryo sac abortion and increase seed set (Lersten, 2004; Beck, 2010).

Creating male-sterile lines. Although classical cytoplasmic male sterility (CMS) is associated with the male gametophyte, forms of female sterility caused by disrupted megasporogenesis or embryo sac development also exist. Studying these forms allows the creation of fertility restorer lines for hybrid seed production (Kaur et al., 2024; Su et al., 2025).

One of the most significant applied aspects is the use of unfertilized ovule and embryo sac culture to obtain haploid plants. Haploids (2n = x) are extremely valuable in breeding because, after chromosome doubling (e.g., with colchicine), they yield fully homozygous doubled haploid lines within one or two generations instead of 6-7 generations of inbreeding (Liu et al., 2025; Su et al., 2025).

The technology is based on the ability of unfertilized embryo sac cells (most often the egg cell, but sometimes synergids or antipodals) to undergo androgenesis in vitro—develop into a haploid plant without fertilization. Method steps:

The method is successfully applied to many crops: rice, barley, wheat, maize, tobacco, sugar beet, onion (where the bisporic developmental type is efficient), and rapeseed. In maize, a different approach—haploid induction via an unusual male gametophyte (chromosomal inducer lines)—is often used, but knowledge of the female side of the process aids understanding of interactions (Liu et al., 2025; Yu et al., 2025).

In distant hybridization (between different species or genera), postzygotic barriers often arise: endosperm fails to develop normally, and the embryo dies at early stages due to lack of nutrition. Knowledge of the timing and stages of female gametophyte development allows the application of embryo culture (embryo rescue). Ovules at the early embryo stage (before its degeneration) are excised and transferred to an artificial nutrient medium, where the embryo continues development without endosperm. This is widely used in breeding fruit, grain, and ornamental crops (Beck, 2010; Bell, 1991).

Cytological analysis of embryo sac development (using ovule clearing, fluorescence microscopy with aniline blue for callose detection, immunocytochemistry) is an important tool in seed production and scientific research. It allows:

Apomixis—asexual reproduction through seeds—allows fixing and propagating heterotic hybrids. In most apomicts, the female gametophyte forms without meiosis (unreduced) and the egg cell develops into an embryo without fertilization. Studying the genes controlling functional megaspore selection (e.g., AGP18, miR822), as well as regulators of the mitosis-to-meiosis transition (CDK inhibitors, RBR1), opens the way to engineering apomixis in hybrid crops. Experimental "MiMe" (mitosis instead of meiosis) systems have already been obtained in rice and maize, which, combined with a mutation conferring parthenogenesis, yield apomictic clones (Cao et al., 2018; Yu et al., 2025; Su et al., 2025).

Thus, detailed knowledge of megasporogenesis and female gametophyte development serves as the foundation for a range of key biotechnological and breeding techniques, enabling not only the diagnosis of reproductive disturbances but also the targeted manipulation of reproduction to obtain new genotypes and enhance the productivity of agricultural crops.