Microsporogenesis and Male Gametophyte Development

One of the key evolutionary innovations (major evolutionary acquisitions) that allowed seed plants to colonize diverse terrestrial habitats was the emergence of the pollen grain as a protected and effective means of delivering male gametes. In spore plants (ferns, horsetails, clubmosses), as well as in gnetophytes and cycads, male gametes — sperm cells — are flagellated and capable of active movement only in a film of water (Becker et al., 2025). This strictly tied fertilization to moist habitats or rainy periods.

In angiosperms, flagella were completely lost in sperm cells (zooidogamy replaced by siphonogamy). The male gametophyte (pollen grain) lost the ability for independent movement but acquired a fundamentally new quality — desiccation tolerance. Pollen is transported by wind, insects, or other agents in a dry state and germinates only after landing on a receptive stigma, where it receives water and nutrients. Thus, microsporogenesis and subsequent pollen development ensured complete independence of fertilization from the presence of free water on the plant surface (Stern et al., 2021; Becker et al., 2025).

In the evolution of land plants, there is a clear trend toward simplification (reduction) of the haploid generation — the gametophyte — while the diploid sporophyte becomes more complex and dominant. In ferns, the male gametophyte is a small but multicellular and independent prothallus; in gymnosperms it is reduced to a few cells inside the pollen grain; and in angiosperms — to three cells at the time of pollination (one vegetative and two sperm — in most monocots and many dicots) (Lersten, 2004; Wiese et al., 2024).

This reduction has profound biological significance: the life cycle is accelerated, energy costs for building the haploid phase are reduced, and most importantly, the entire process of male gamete formation and transport is maximally protected by sporophyte tissues (anther) and the resilient pollen grain wall. This has enabled angiosperms to become dominant in most terrestrial ecosystems.

For agricultural practice, the flip side of this high specialization is also important. The male gametophyte of angiosperms, despite its protection, is extremely sensitive to abiotic stresses, especially high temperatures (heat stress) and drought. The critical period is precisely microsporogenesis — from the onset of meiosis in microspore mother cells to tetrad formation and subsequent microspore release (Fartyal et al., 2025).

Even a short-term temperature increase during this period can disrupt chromosome pairing in prophase I of meiosis, chromatin fragmentation, and improper chromosome segregation. The consequence is the formation of non-viable microspores, pollen abortion, and ultimately reduced pollen fertility and grain yield (e.g., in wheat, rice, maize). Understanding these evolutionarily determined “weak points” allows agronomists to manage sowing dates, select tolerant varieties, and develop measures to protect reproductive organs during periods of likely heat stress.

Control over male fertility is a powerful tool for breeders. Natural or induced male sterility (e.g., cytoplasmic male sterility, CMS) allows the production of high-yielding heterotic hybrids without labor-intensive manual emasculation. Disruptions in microsporogenesis or gametophyte development underlie many forms of genic male sterility, which are widely used in hybrid seed production of maize, sunflower, rice, and onion (Wiese et al., 2024).

Furthermore, in plant biotechnology, the method of isolated microspore culture enables the production of haploid plants, which, after chromosome doubling, give fully homozygous (pure) lines. This significantly accelerates the breeding process, reducing the time to obtain inbred lines from 5–7 generations to 1–2 years. Thus, deep knowledge of microsporogenesis and microgametogenesis has not only fundamental evolutionary but also important applied significance for agricultural science.

The key difference concerns when cell walls are formed between the four haploid nuclei after meiosis completion (Lersten, 2004; Beck, 2010).

Successive cytokinesis. After the first meiotic division (telophase I), a cell plate is immediately formed, separating two cells — a diad. Each of these then undergoes the second meiotic division, after which partitions are again formed, resulting in a tetrad of four microspores. This type is characteristic of most monocots (including grasses, lilies) and some primitive dicots. In grasses, for example, the tetrad is planar (Lersten, 2004).

Simultaneous cytokinesis. Both meiotic divisions occur without partition formation, resulting in a four-nucleate coenocyte. Only after both divisions are complete, cell walls are formed by centrifugal furrowing, separating the four microspores. This type occurs in approximately 95% of dicot plants and is considered evolutionarily more advanced. Simultaneous cytokinesis is thought to produce a tetrahedral arrangement of microspores in the tetrad, which subsequently determines the typical triaperturate pollen grain structure in eudicots (Beck, 2010).

Despite taxonomic preferences, exceptions are known: some dicots (e.g., Magnoliales) may exhibit successive cytokinesis, while some monocots may show simultaneous cytokinesis.

The tapetum is the innermost layer of the pollen sac wall, directly contacting developing microspores and supplying them with nutrients, enzymes, and sporopollenin precursors. Based on behavior, two main types are distinguished (Lersten, 2004; Moreira et al., 2025; Ali et al., 2025).

Secretory (parietal) tapetum. Tapetum cells remain in place, lining the pollen sac cavity, and secrete substances throughout microspore development via their plasma membrane or by exocytosis of vesicles. This is the most common type, occurring in about 90% of dicot families and all grasses. Secretory tapetum cells often form special bodies — Ubisch bodies (orbicules) — which become coated with sporopollenin and are transferred to the pollen grain surface (Ali et al., 2025).

Invasive (plasmodial, amoeboid) tapetum. Tapetum cells lose their walls, flow into the pollen sac cavity, and fuse into a single multinucleate protoplasmic mass — a plasmodium — that surrounds each microspore. As pollen matures, the plasmodium is gradually consumed by the developing gametophytes. Invasive tapetum is characteristic of most monocots but is also found in some dicots, e.g., all members of the family Asteraceae (sunflower family) (Lersten, 2004). This type ensures maximal contact between the nourishing tissue and the gametophyte.

Classification of tapetum types is important for understanding cytoembryological features of crop plants and for interpreting mutations that lead to male sterility.

Although this trait is more commonly used in systematics (palynology), it is directly related to the microsporogenesis process, as aperture formation begins at the tetrad stage. Based on the number and arrangement of apertures (pores or furrows), several main types are distinguished (Stern et al., 2021):

Thus, the diversity of microsporogenesis and male gametophyte development variants reflects both the evolutionary history of taxa and fine adaptations to pollination modes, environmental conditions, and reproductive strategies. For the agronomist, the most significant classifications are by pollen type (bicellular vs. tricellular) and tapetum type, as they determine the sensitivity of the reproductive system to stresses and the possibilities for biotechnological manipulation.

The released microspores are haploid, still unspecialized cells. Their further fate is transformation into a male gametophyte capable of fertilization. This process includes several key stages: polarization, first mitosis, formation of vegetative and generative cells, and then (in tricellular pollen) the second mitosis of the generative cell to form two sperm cells.

In parallel with gametophyte divisions, the final construction of the pollen wall occurs. The tapetum, which has provided microspores with nutrients and sporopollenin precursors throughout development, undergoes programmed cell death (PCD) by the time of pollen maturation. Its contents (lipids, proteins, pheromones, pigments) are released into the pollen sac cavity and impregnate the exine sculpture, forming the pollen coating (tryphine, or pollenkitt). This coating performs important functions: protecting pollen from desiccation, enabling adhesion to insect bodies, and participating in self/non-self recognition on the stigma (Ali et al., 2025; Moreira et al., 2025). The process concludes with anther dehiscence and shedding of mature pollen.

Below is a generalized scheme of successive stages in the male reproductive sphere of angiosperms (according to Lersten, 2004; Beck, 2010; Wiese et al., 2024).

Understanding the sequence of these stages, each controlled by complex genetic and physiological mechanisms, is a necessary foundation for interpreting fertility disturbances and developing methods to enhance reproductive system resilience in crop plants under adverse environmental conditions.

Water deficit during microsporogenesis and early male gametophyte development is as damaging as extreme temperatures. Water shortage in anther tissues leads to (Fartyal et al., 2025):

Waterlogging and root flooding, indirectly through root inhibition and impaired shoot supply, can also induce drought-like changes in anthers.

Although microsporogenesis does not require direct light (the process occurs inside buds), overall illumination and photoperiod influence reproductive organ development through hormonal regulation.

Photoperiodic sensitivity. In short-day plants (soybean, rice) or long-day plants (spinach, radish), disruption of the accustomed photoperiod during bud initiation and development can induce male sterility. This underlies the creation of photoperiod-sensitive genic male sterility (PGMS) in rice, used in breeding.

Light intensity. Shading during the budding phase reduces photosynthesis and carbohydrate production, affecting the energy supply for microsporogenesis. Light deficiency increases the percentage of aborted pollen in many cereals and legumes.

The most critical elements for successful male gametophyte development are boron (B) and calcium (Ca²⁺), as well as nitrogen and phosphorus at specific phases (Fartyal et al., 2025; Stern et al., 2021).

Boron (B). Essential for cell wall formation (component of rhamnogalacturonans in pectin) and carbohydrate metabolism. Boron deficiency during microsporogenesis leads to tapetum collapse, disrupted intine formation, and massive pollen abortion. Foliar boron application before and during flowering is a standard agronomic practice to improve fruit set (tomato, apple, rapeseed).

Calcium (Ca²⁺). Involved in signaling during pollen tube growth, establishing a concentration gradient at the tip necessary for vesicular transport and polar growth. Calcium deficiency in stigma and style tissues blocks pollen germination even when pollen grains are normally developed.

Nitrogen and phosphorus. Excessive nitrogen nutrition during the budding phase can delay pollen maturation and reduce its viability; phosphorus, conversely, improves energy metabolism (ATP synthesis) in microspores.

Phytohormones act as systemic and local regulators of microsporogenesis. Their balance determines the transition from proliferation to meiosis, polarization, microspore division, and finally the initiation of programmed tapetum death (Fartyal et al., 2025; Wiese et al., 2024).

Auxins (IAA). Required at early stages for archesporial cell specification and for pollen tube growth. A local auxin maximum in microsporogenous cells triggers meiosis. Auxin deficiency under heat stress is one cause of male sterility.

Gibberellins (GA). Control stamen filament elongation, ensuring anther protrusion from the bud for effective pollination. GA deficiency leads to stamen underdevelopment.

Jasmonic acid (JA). Key regulator of anther dehiscence and sporopollenin synthesis. Disruption of JA synthesis results in delayed dehiscence and male sterility.

Brassinosteroids (BR). Involved in pollen wall (exine) formation and coordination of tapetum and microspore development. BRs are required for pollen tolerance to heat stress (increase thermotolerance).

Ethylene and abscisic acid (ABA). At high concentrations (under stress), they can induce premature tapetum death and pollen abortion. However, at low doses, these hormones also participate in normal tapetum senescence and anther opening.

Applied aspect: exogenous treatment of plants with brassinosteroids or auxins at the budding stage can mitigate the negative effects of heat stress on pollen (Fartyal et al., 2025).

Any stress exposure (heat, drought, herbicides) ultimately acts through the generation of reactive oxygen species (ROS) in tapetum cells and developing microspores (Ali et al., 2025; Fartyal et al., 2025). A moderate level of ROS is necessary as a signaling mediator for initiating tapetum PCD at precisely the right time. However, excessive ROS accumulation causes lipid peroxidation of membranes, damage to mitochondria and nuclear DNA, leading to premature or pathological cell death.

Interestingly, anther tissues (especially the tapetum) contain zones of temporary hypoxia (low oxygen). Hypoxia is necessary to maintain meristematic activity and cell differentiation. Disruption of the oxygen regime (e.g., due to waterlogging) can mimic heat stress and cause sterility (Becker et al., 2025).

Successful microsporogenesis and male gametophyte development are possible only when a set of conditions is met simultaneously:

Understanding these factors allows agronomists to manage the production process: select tolerant varieties, adjust sowing dates, apply micronutrients and growth regulators, creating conditions for maximum pollen fertility and high yield.

Pollen of many crops rapidly loses viability after anther dehiscence. Storage ability depends on pollen type (bicellular pollen stores longer than tricellular) and moisture content (Lersten, 2004; Beck, 2010).

Management practices: Drying pollen to a specific moisture level (10–15%) and storing at low temperatures (−20 °C or −196 °C in liquid nitrogen) can maintain fertility for several years. Pollen cryopreservation is used to preserve genetic resources (gene banks) and for hybridizations separated in time or space (e.g., when working with fruit crops flowering at different times).

Limitations: Tricellular pollen (cereals, Asteraceae) is very sensitive to desiccation and difficult to store long-term.

Exogenous treatments can partially compensate for stress impacts and improve pollen fertility (Fartyal et al., 2025).

Brassinosteroids (BR). Seed treatment or plant spraying at the budding stage with 24-epibrassinolide increases pollen thermotolerance in tomato, rice, and cucumber. BRs improve sporopollenin synthesis, maintain tapetum integrity, and reduce reactive oxygen species levels.

Auxins. Under heat stress causing endogenous auxin deficiency in anthers, exogenous treatment with 1-naphthaleneacetic acid (NAA) or indole-3-acetic acid (IAA) restores fertility in barley and rice (Sakata et al., 2010 — cited by Fartyal, 2025).

Salicylic acid (SA). SA treatment reduces heat damage to microspores in rice by activating antioxidant enzymes.

Boron (B). Foliar boron application at the start of budding and early flowering is a standard practice to ensure normal pollen tube formation and improve fruit set in pome fruits, crucifers, and tomato. Boron participates in carbohydrate metabolism and intine pectin synthesis.

Modern methods allow targeted modification of genes controlling male fertility.

Creating artificial male sterility. Knockout of genes required for tapetum or microspore development (e.g., orthologs of MS1, MS2, CYP703A2) using CRISPR/Cas9 yields stable sterile lines for hybrid seed production. Unlike CMS, such lines do not contain foreign mitochondrial genes and are easier to control for fertility (Wiese et al., 2024).

Restoring fertility. In systems such as “promoter-lethal gene” (e.g., Barnase/Barstar technology), sterility is created by expressing a toxic gene under a tapetum-specific promoter. Fertility is restored by suppressing the toxin or introducing an inhibitor gene. CRISPR can also be used to “turn on” fertility in sterile lines, e.g., by deleting a suppressor element of a restorer gene (Wiese et al., 2024).

Improving thermotolerance. Research is underway to identify alleles of genes that ensure stable microsporogenesis at high temperatures (e.g., thermostable variants of TMS10, TMS15 in rice). Introgression of such alleles or their editing can create varieties resistant to heat stress during flowering.

In applied agronomy and breeding, simple methods for assessing pollen quality are used.

Iodine‑potassium iodide staining (Lugol’s solution) allows assessment of starch content in pollen: mature viable pollen stains dark blue, aborted pollen stains yellow or remains unstained.

Fluorochromatic reaction (FCR) using fluorescein diacetate assesses esterase activity in live pollen grains: live grains fluoresce yellow‑green under UV.

In vitro germination on a medium containing sucrose and boric acid is the most reliable method for assessing fertility and pollen tube vigor. It is used in breeding programs to screen for heat stress tolerance and to evaluate pollen quality for hybridization.

Managing microsporogenesis and male gametophyte development has direct applied significance for improving crop yields. From breeding for male sterility (CMS, PTGMS) to biotechnological methods (microspore culture, CRISPR) and agronomic practices (optimizing sowing dates, boron and hormone applications) — all these approaches are grounded in deep fundamental knowledge of the cytological and molecular mechanisms described in the previous sections. Further research in this area will contribute to the development of varieties with controlled fertility and stress resilience under changing climate conditions.