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Article

Cytological Characterization of vrnp 1, a Pollen-Free Male Sterile Mutant in Mung Bean (Vigna radiata)

by
Yuxin Cheng
,
Tianjiao Lan
,
Kunpeng Deng
,
Minghai Wang
,
Shuying Bao
,
Dan Han
,
Yapeng Xu
,
Han Wang
,
Ning Xu
* and
Zhongxiao Guo
*
Institute of Crop Germplasm Resources, Jilin Academy of Agricultural Sciences (Northeast Agricultural Research Center of China), Gongzhuling 136100, China
*
Authors to whom correspondence should be addressed.
Agronomy 2025, 15(2), 312; https://doi.org/10.3390/agronomy15020312
Submission received: 11 December 2024 / Revised: 21 January 2025 / Accepted: 24 January 2025 / Published: 26 January 2025
(This article belongs to the Section Plant-Crop Biology and Biochemistry)
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Abstract

:
Mung bean (Vigna radiata) plays a significant role in agricultural trade, food processing and utilization, and cropping structure adjustment due to its abundant nutritional components, medicine-food homology, capacity for nitrogen fixation, and soil improvement. The low yield level is a crucial limitation factor in the mung bean industry, while heterosis is an efficient path for increasing crop yields. The flexible utilization of male sterile mung bean materials may solve this pressing demand in the industry. This study identified a completely male-sterile mutant, vrnp 1, in the EMS-mutagenized mung bean cultivar Jilv 10 population, which is controlled by a single recessive nuclear gene. Furthermore, we employed a series of microscopical and histological techniques and observed that the tapetal cells in the vrnp 1 mutant did not perform as expected when reaching stage 8 of anther development, notably exhibiting a delay in entering PCD. This was accompanied by a failure to deposit cell wall components onto the pollen wall, culminating in a complete absence of mature pollen and the manifestation of male sterility. In conclusion, the vrnp 1 mutant could potentially serve as a promising candidate for male sterility in exploiting hybrid vigor in mung bean. Our research may elucidate how the delayed initiation of programmed cell death in tapetal cells contributes to a factor implicated in mung bean male sterility. Furthermore, the phenotypic data collected during pivotal developmental phases may have contributed to a better grasp of mung bean microspores and anther development.

1. Introduction

Mung bean (Vigna radiata L. Wilczek) is a self-pollinating legume that serves as a multi-purpose grain crop, known for its advantages in drought and barren tolerance, nitrogen fixation, and soil nourishment, all within a short growth period [1,2,3,4,5]. Mung beans are abundant in nutrients, such as starch, protein, B vitamins, minerals, alkaloids, saponins, and plant sterols [6,7,8]. The increasing popularity of mung bean consumption reflects its widespread acceptance by the general public [9]. However, its lower yield has become a significant limiting factor for the development of the mung bean industry [4,9]. Utilizing heterosis can effectively improve the yields of various crops, including mung bean [3,10,11,12,13,14,15]. Male sterility, a crucial agronomic trait, serves as the foundation for hybrid seed production in agriculture [16]. Therefore, the identification and characterization of male sterile materials and the utilization of heterosis have become crucial demands of the mung bean industry and the technical support of breeding practices [11].
In the plant reproductive process, following pollination, the interaction between pollen and the papilla cells on the stigma initiates pollen hydration and germination. The emerging pollen tube then extends through the stigma and style, navigating towards the ovule, where it delivers sperm cells to achieve fertilization. Subsequently, the ovules within the ovary can develop into seeds [17,18]. The normal development of each floral organ is crucial for the reproduction of angiosperms [19]. Particularly, the accurate development of the anther is essential, as it plays a critical role in successful fertilization and serves as a key site within the floral organs [20,21,22]. Pollen develops to maturity within the anther, and Sanders et al. (1999) identified that anther development can be categorized into 14 distinct stages based on the cellular structural characteristics [20]. Furthermore, a multitude of experimental articles has substantiated the claim that anomalies and deviations in anther development can lead to the failure of pollen dispersal and the impairment of pollen grain fertility [22,23,24,25,26,27,28].

1.1. The Significance of the Tapetum and Pollen Wall for Plant Fertility

Callose accumulates on the surface during meiosis in microspore mother cells [29]. Once meiosis is complete, a tetrad structure enclosed by a callose wall forms, marking stage 7 of anther development [30]. In a butterfly-shaped typical anther, microspore mother cells are surrounded by an orderly sequence of cell layers, with the tapetum, middle layer, endothecium, and epidermis cells arranged from the inside out [20]. Among these, the tapetum, abundant in RNA, proteins, enzymes, lipids, carotenoids, and sporopollenin precursors, plays an essential role, performing crucial functions in plant reproduction [31,32]. It includes several key processes: the contribution to releasing individual microspores from the tetrad, the deposition of starch-rich substances into the anther locule, and the provision of tryphine for pollen wall formation [33,34,35,36,37] The tapetum and pollen wall play interrelated roles in anther development. The pollen wall, a hard and sticky structure on the surface of pollen grains, is among the most complex cell walls [38]. Comprising two distinct layers—the intine and the exine—it is enveloped by a lipid complex known as the pollen coat or tryphine [38,39]. The inner wall of the pollen grains is primarily composed of cellulose, hemicellulose, pectin, and structural proteins derived from the pollen itself [38].
In genetic studies of plants, numerous genes have been identified associated with fertility impairments due to disruptions in tapetal programmed cell death [28]. Xiong et al. (2016) identified that the knockout of tapetal-specific transcription factor gene MS188/MYB103/MYB80 leads to early tapetal degeneration and premature pollen degradation [40]. Zhang et al. (2006) noted that DYT1 has a function during the early stage of tapetum development, and Gu et al. (2014) demonstrated that DYT1 interacts directly with the promoter of Defective in Tapetal Development and Function 1, TDF1, a key transcription factor for tapetum development. Research has been increasingly demonstrating that the DYT1-TDF1-AMS-MS188-MS1 genetic pathway is crucial for regulating tapetum development and the shaping of pollen walls [23,40,41,42]. Although sterility genes impair the function of the tapetum, resulting in male sterility, the original causes and the timing of these differences vary significantly.
The development of the pollen wall, which involves establishing its pattern and accumulating various constituent substances within the exine, begins with the emergence of the microspore mother cell [43,44,45]. Over time, the surface structure is progressively refined [46]. Once the microspores are released, the deposition is initially completed, fully encapsulating the microspores [43]. In contrast, the pollen exine formation results from the joint contribution of the tapetum and microspores, creating a multilayered, reticulate structure predominantly made up of chemically stable sporopollenin [33]. The pollen coat renders the compactness and stability of the surface structure of the mature pollen grains [40].
Let us take ABCG subfamily genes as an example. They are part of the ABC transporter family and play a crucial role in the transport of various lipids, proteins, sporopollenin precursors, and other components of pollen coats from the tapetal layer into the anther loculus during the formation of the pollen grain cell wall [33,47]. The reticulated cavities of mature pollen grains are filled with pollen coats originating from the tapetum [46]. Studies by Quilichini et al. [48,49] and Dou et al. [50] have identified numerous loss-of-function mutants of ABCG genes, which typically exhibit collapsed pollen morphology and male sterility in Arabidopsis. It has been observed that alterations in the deposition pattern of the pollen exine can result in the failure of pollen to develop into viable and mature pollen grains [51,52,53].

1.2. Investigation on Male Sterility and Hybrid Vigor in Mung Bean Mutants

Research on male sterility traits, a vital basis for harnessing hybrid vigor in the future, has been conducted across various crops [12,54,55,56,57]. Current research on mung bean male sterility materials and the exploitation of hybrid vigor is progressively advancing [58]. Chen et al. [59] identified that deleting a single base pair in Vradi06g12650 resulted in a change from the original closed-flower pollination characteristics to chasmogamous flowering in the cm mutant. Lin et al. [4] identified that VrSE1 (LOC106777793), which regulates cell division in petals and cell expansion in styles, is associated with the phenotype of the se1 mutant, characterized by wrinkled petals and an exposed stigma. Both Vradi06g12650 and VrSE1 possess considerable potential for application in increasing outcrossing rates in mung bean hybrid breeding [60]. VrCYCA1, identified by Liu et al. [61] as a candidate gene, is associated with the mung bean male sterility mutant ‘ms’, which exhibits abnormal pollen morphology and nearly zero pod-setting rates. Nevertheless, reports on entirely male-sterile mutants in mung bean are rare.
To clarify the cellular and microscopic structure, we employed a suite of histological and microscopic techniques, on an entirely male-sterile mutant, vrnp 1. We identified that the delay in programmed cell death (PCD) within tapetal cells significantly impacted the deposition of surface components on the pollen grains. This delay may be the underlying cause of the observed absence of pollen.

2. Materials and Methods

2.1. Plant Materials and EMS Mutagenesis

These mutagenized seeds were obtained by treating dry seeds of the Jilv 10 variety with the chemical reagent ethyl methanesulfonate (EMS) at a concentration of 0.8% for 12 h in 2018. The background material, Jilv 10, is a cultivar developed by our research team in 2014 and served as the control. The stably inherited sterile progenies, acquired in 2022, were named vrnp 1 (vigna radiata no pollen 1).
All mung bean materials, encompassing both mutagenized and hybrid progeny, were cultivated and bred using standard field practices under local growing conditions in open-air experimental fields of the Institute of Crop Germplasm Resources, Jilin Academy of Agricultural Sciences (Northeast Agricultural Research Center of China), from 2018 to 2023. Specifically, summer cultivation took place in Gongzhuling from May to August, and winter breeding occurred in Hainan from November to January. Each ridge, measuring 2.5 m in length, was planted with 12–15 cm intra-row and a 60 cm inter-row spacing. Measurements of agronomic traits, including the length of flower stalks, were conducted in twelve replicates to ensure the reliability and accuracy of the data under the experimental conditions.

2.2. Genetic Analysis of Male Sterile Mutants

The initial male sterile mutant was identified among the M2 progeny resulting from EMS mutagenesis. We then hand-crossed the mutant with its background variety, Jilv 10, to preserve this trait of complete sterility. After harvesting and planting the F2 hybrid seeds, we proceeded to cross the progeny utilizing the heterozygous fertile and sterile plant lines as parents to breed the generations. Following the identification and removal of the non-segregated F2 individuals, we established a genetic population from the segregated F2 population for genetic analysis. We statistically analyzed the number of fertile and sterile individuals in the segregation of heterozygous fertile plants in the F2 generation and analyzed the data using the chi-square test. For the flower stalk lengths, we applied a t-test for statistical analysis (WPS Office, Kingsoft Corporation, Beijing, China). Statistical significance was set at a p-value below 0.05.

2.3. Observation of Anther Paraffin Sections

For histological analysis, anther samples were quickly immersed in FAA fixative solution (70% ethanol 90 mL + glacial acetic acid 5 mL + formalin 5 mL) for more than 24 h to ensure adequate fixation, preventing dehydration and the distortion of anther structures. The paraffin sectioning materials were meticulously selected and sorted based on the size of the flower buds. The samples were then carefully extracted and trimmed to a thickness not exceeding 3 mm to facilitate dehydration. Dehydration was performed through a gradient series of six alcohol solutions, with concentrations escalating from 75% to 100%, followed by three treatments with dimethylbenzene to achieve transparency. The samples were subjected to paraffin impregnation in 2–3 cycles, each lasting 1–2 h, over a total period of 3–4 h. After embedding and trimming, the samples were cooled and sectioned to a precise 4 µm using a pathology microtome (RM2016, Shanghai Leica Instruments Co., Ltd., Shanghai, China). The resulting sections were mounted on slides and baked. They were stained with Safranin O for 1–2 h and with Fast Green for no longer than 30 s. Following staining, the sections were dehydrated and secured with neutral balsam. Finally, the sections were analyzed under an optical microscope (Nikon Eclipse E100, Tokyo, Japan) equipped with an imaging system (Nikon DS-U3, SlideViewer 2.5) during the year 2023. For each developmental stage, three buds were compared through sectioning and staining for observation.

2.4. Observation of Pollen Grains

During the peak flowering period, complete mung bean inflorescences were collected in the field at around 9 a.m. and stored in containers with 75% ethanol solution at 4 °C. The anthers were carefully extracted from the flower buds, crushed with tweezers on a slide, and then stained with 1–2 drops of 2% (w/v) potassium iodide (I2-KI) or an acetocarmine staining solution for 2–3 min to ensure thorough staining. After staining, pollen grains were observed under an optical microscope (Nikon Eclipse E100, Tokyo, Japan) once covered with a cover slip. Developmental stages were strictly classified based on the size of the flower buds. All compared materials were from the same batch of plants grown under identical environmental conditions. For each development stage, three sets of flower buds were sampled for staining and observation during the year 2024.

2.5. Observation of Pollen Wall

The anthers were examined by scanning electron microscopy analyses to investigate the detailed morphology of the pollen wall. Anthers from Jilv 10 and the vrnp 1 mutant were fixed in a glutaraldehyde solution, following the detailed experimental procedures in this literature [61]. The slice samples were photographed under a scanning electron micrograph (TEM, SU8100, Hitachi, Tokyo, Japan).

3. Results

3.1. Male Sterility in the vrnp 1 Mutant Is Controlled by a Single Gene

To analyze the segregation and preserve the sterility trait, we crossed the M2-sterile mutant with Jilv 10. It was observed that all the F1 individuals were fertile (the phenotype is consistent with Jilv 10), and the F2 plants exhibited a 3:1 segregation ratio between the fertile and sterile offspring (Table 1). These results may confirm that the trait is controlled by a single recessive nuclear gene. Moreover, due to the complete sterility characteristic of vrnp 1, we conducted crosses between non-segregated fertile and sterile individuals from each generation to maintain our genetic population in the experimental fields of Gongzhuling and Hainan. Notably, no fertility restoration was observed in vrnp 1, thereby confirming the stable, pollen-free male sterility trait.

3.2. The vrnp 1 Mutant Is Completely Sterile

During the vegetative growth stage, the vrnp 1 mutant and Jilv 10 variety were quite similar, showing no significant differences in growth rate, plant architecture, or stature (not indicated). However, as the transition into the reproductive growth stage occurred, subtle distinctions began to emerge. While the flower buds, standard petals, wing petals, keel petals, and pistil of the vrnp 1 mutant maintained a normal morphology, the stamen exhibited a noticeable difference compared to that of Jilv 10 (Figure 1A).
As maturity approached approximately 70 days after sowing, the Jilv 10 plants were characterized by their abundant, ripe pods, some of which were nearly black with full ripeness (Figure 1B). At this stage, there was a rare flower bud on the inflorescence, and the remaining leaves were either yellowing or had already fallen. In contrast, the vrnp 1 mutant maintained its vigor, continued to produce buds, and kept its leaves green (Figure 1B).
Upon closer examination, while the Jilv 10 plants were laden with plump pods, the vrnp 1 mutants only had fleshy pods or buds during the same growth stage (Figure 1C). This occurred because the unfertilized ovules could not mature into beans, even though the ovary developed and swelled to form a young pod. Notably, vrnp 1 boasted a more extended flowering period and featured longer flower stalks compared to Jilv 10 (Figure 1D,E). Dissection revealed that although the vrnp 1 mutant may have developed small fleshy pods 3–4 days after flowering, the beans inside were diminutive and failed to mature, demonstrating complete sterility (Figure 1F).

3.3. Anther Development in the vrnp-1 Mutant Is Affected

As crucial sites for pollen development, the anther sections were stained with Safranine O-fast Green staining solution for observation. Notably, butterfly-shaped anthers with four locules could be observed in both the vrnp 1 mutant and Jilv 10 during stage 6. The four layers—the epidermis, the endothecium, the middle layer, and the tapetum—were clearly distinguishable around the locules (Figure 2A). At this stage, the tapetal cells had completed their transformation into a distinct secretory morphology, and the microspore mother cells had emerged within the loculus cavity. The cells secreted and accumulated callose to encapsulate themselves in preparation for meiosis. Significantly, it could be observed that tetrad cell structures enclosed by a callose wall were exhibited both in Jilv 10 and vrnp 1 during stage 7 (Figure 2B). In the microspores, the cell nuclei were distinctly visible, while the epidermis, endothecium, and middle layer exhibited a neat arrangement. Furthermore, the tapetum had reached its developmental peak, characterized by a highly concentrated cytoplasm (Figure 2B). Up to this stage, no notable developmental differences appeared to be observed between the vrnp 1 mutant and Jilv 10 plants.
By stage 8, single-nucleate pollen grains were released from the tetrad structures (Figure 2C), marking a remarkable completion of the first phase of microspore development. Subsequently, the microspores gradually increased in volume, with their pollen walls thickening synchronously. During stage 9, which is not indicated, the enlargement of the anthers coincided with several developmental changes in the microspores, such as an increased cytoplasm concentration, rapid volume expansion, and the vacuolation of the inner wall. Concurrently, the microspores developed distinct cell walls, exhibited uniformly dense cytoplasm, positioned their nuclei centrally within the cells, and the tapetal layer of the anthers initiated programmed cell death. By stage 10, the tapetal cells of Jilv 10 had become increasingly insubstantial and partially degraded, while the middle layer had been wholly degraded (Figure 2D). As the tapetal layer underwent apoptosis, it released cytoplasmic contents that contributed indeed to the formation of the pollen wall, thereby facilitating the gradual maturation of the pollen grains.
During stage 12, the cytoplasm of the microspores exhibited deeper staining as expected, and the nucleus became clearly visible. In typical anthers, the endothecium and connective layer thicken progressively due to fiber deposition, enhancing the fibrous tissue within the endothecium. The septal slits beneath the septum degraded and ruptured, leading to the four micro locules gradually coalescing into two larger anther cavities by stage 12 (Figure 2E). The tapetal layer of Jilv 10 had entirely disintegrated (Figure 2E). The plump pollen grains demonstrated the necessary nutrients supplied from the tapetal layer for pollen maturation. Consequently, by stage 13 of the development, it was observed that the tapetal cells had mostly degraded. The pollen grains were uniformly and deeply stained within the anther cavity. The structure of the anther cavity was optimized to facilitate the release of the pollen grains (Figure 2F). At the final stage, anthers entering senescence underwent gradual contraction and abscission, with the pollen grains dispersed from the anther cavity. The pollen grains of Jilv 10 exhibited a regular shape and were rich and abundant in content. Only the strip-like remnants of the anther wall were left (Figure 2G). At this stage, the anthers of Jilv 10 had completed a standard developmental process, producing plump and fertile pollen grains.
However, from stage 8, in the vrnp 1 mutant, the release of haploid microspores was inefficient, and the morphology of the cavity was indistinct (Figure 2C). By stage 10, it was observed that the individual microspore structures of the vrnp 1 mutant were also indistinct, and the tapetal layer exhibited irregular morphology along with a delay in PCD (Figure 2D). Excessive hypertrophy of the tapetal layers in the vrnp 1 mutant anthers was observed.
Ultimately, this led to the accumulation of immature microspore cells with irregular traits, and the cells could not disperse as individual entities (Figure 2E). The development and outer wall packaging of the microspores were restricted, and the cytoplasm became increasingly concentrated. As a result, the entire anther of the vrnp 1 mutant appeared shriveled than that of the Jilv 10 variety by stage 12. At stage 13, the tapetal layer cells appeared to have degenerated, characterized by distinct cellular outlines and the presence of disintegration debris within the anther locule in the vrnp 1 mutant (Figure 2F). It was observed that the microspores exhibited irregular morphologies and pronounced vacuolation, with an aggregation propensity, failing to attain the canonical spherical and plump form (Figure 2F). The anther cavity contained substantial debris, which was hypothesized to result from the incomplete and retarded degradation of the tapetal layer. The staining results indicated that the anther sacs of the vrnp1 mutant had begun to exhibit pink keratinized cell walls. Near stage 14, the anther presented a shriveled appearance and failed to dehisce in the vrnp 1 mutant. We found that the microspores formed aggregated structures with abnormal staining (Figure 2G). Ultimately, the anther cavity of the vrnp 1 mutant lacked normal microspores as they failed to complete maturation.

3.4. Pollen Development in the vrnp 1 Mutant Is Affected at the Early Uninucleate Stage

To elucidate the disparities in pollen grains between the vrnp 1 mutant and Jilv 10, we collected and examined the inflorescences from both, categorizing the materials and strictly grading the buds by different sizes separately. Our findings revealed that the differences in microspore development between vrnp 1 and Jilv 10 coincided with the period when differences in anther development occurred. During the tetrad phase, both the Jilv 10 and vrnp 1 mutant microspores were observed to secrete callose for self-encapsulation (Figure 3A). Up until this stage, no significant differences in the microspore cells were detected between the vrnp 1 mutant and Jilv 10.
At the early uninucleate stage, the haploid microspore of Jilv 10 was released from the tetrad structure and gradually increased in volume while maintaining its shape (Figure 3B). With the cell nuclei centrally positioned, the microspores of Jilv 10 progressed to the mid-uninucleate stage (Figure 3C). Subsequently, as the volume expanded, a large central vacuole was pushed to the cell periphery. At the late uninucleate stage, the pollen wall developed into a distinct reticulated pattern, particularly evident in Jilv 10, marking the exine formation (Figure 3D). Furthermore, as the microspore cytoplasm condensed and the staining intensified, the cell nucleus underwent division, resulting in binucleate pollen (Figure 3E). The developed, enclosed mature pollen grains were stained brown–black with I2-KI, indicating their advanced maturing phase (Figure 3F–H). Concurrently, the degeneration of the tapetum layer and the deposition of the pollen exine signaled the maturation of Jilv 10 pollen grains.
However, in the vrnp 1 mutant, during the early uninucleate stage, the individual microspore released from the tetrad structure exhibited irregular shapes (Figure 3A). In comparison, the microspores could still be released as individual entities, albeit with a notably delayed development at stage 8 (Figure 2C). At the mid-uninucleate stage and the late uninucleate stage, the vrnp 1 mutant microspores showed no significant change in cell wall thickness; the cells were translucent, and the staining was lighter (Figure 3C,D). By the binucleate stage, although the microspores of the vrnp 1 mutant had smoothly reached the binucleate phase, they exhibited no significant changes in cell wall thickness and still lacked the characteristic reticulated structure of the pollen wall (Figure 3E). In the maturing phase, the pollen grains of the vrnp 1 mutant could not mature or could not be deeply stained (Figure 3F). The mature pollen showed no noticeable morphological changes during maturation. The microspore cells of vrnp 1 had thinner cell walls and lacked the reticulated structure on the surface typical of mature pollen grains (Figure 3G). The internal cytoplasm condensed into small spheres, and the microspore cells tended to aggregate, seldom dispersing freely within the anther cavity (Figure 3H). The immature microspores in the vrnp 1 mutant failed to develop properly within the anther. Consequently, we observed that the anthers did not dehisce to release pollen (Figure 1A).

3.5. Scanning Electron Microscopy Observations of the Anther Interior

To enhance our clear understanding of pollen grain morphology, we conducted scanning electron microscopy (SEM) on two distinct sets of samples. In the imminently blooming buds, we noted that the plump mature pollen grains of Jilv 10 were nearly spherical, evenly scattered throughout the anther cavity. These spherical grains were characterized by a consistent, reticulate pattern on the surface and featured three germination pores (Figure 4B). The inner wall appeared smooth, with a clear and well-structured surface. In contrast, the anthers of the vrnp 1 mutant contained fewer, smaller, and irregularly shaped pollen grains, which were more likely to cling to the inner wall and failed to distribute evenly within the cavity (Figure 4A). These immature pollen grains lacked the characteristic reticulate pattern on their surface. An irregular configuration marked the inner wall of the matured anther. Furthermore, since the sterility defect occurred after the tetrad stage, upon examination of smaller buds beyond the tetrad stage, we observed that individual pollen grains of Jilv 10 had begun to develop a nascent reticulate pattern on their surfaces toward maturity (Figure 4D). The anther cavities of Jilv 10 were clear and exhibited a more regular arrangement. However, the pollen grains of the vrnp 1 mutant exhibited irregular sizes and lacked the emerging reticulate pattern (Figure 4C).

4. Discussion

Our study focused on characterizing an EMS-induced mung bean mutant exhibiting complete male sterility, notably marked by the absence of mature pollen grains both inside and outside the anther chamber. Through a process of hybridization and rigorous identification, we developed genetically stable male-sterile progenies, which we designated as vrnp 1. Further genetic analyses confirmed that the phenotype of this mutant was determined by a single recessive gene located in the nucleus and associated with affected fertility and flowering duration. Thus, this mutant presents itself as a promising candidate for the utilization of hybrid vigor in mung bean.
In angiosperms, the causes of male sterility are diverse and stem from multiple developmental anomalies. Notably, among these processes, the tapetum plays an essential role, with key functions that include the secretion of callose to release individual microspores from the tetrad, the deposition of starch-rich substances into the anther locule, and the provision of tryphine for pollen wall formation [34,35,36,37,38]. On the other hand, accurate structural development, the adequate accumulation of these substances, and the PCD of tapetum are vital for successful pollen production and plant fertility. Indeed, forming fertile pollen grain is contingent upon the optimal development of pollen wall patterns and the entire accumulation of substances within the outer pollen wall [40]. The development of the surface structure in microspores, coupled with the rigidity of the pollen wall, ultimately results in pollen grains that possess a robust, independent, enduring, and persistent physical structure [38].
Focusing on the sequential stages of anther development, at stage 8, the individual microspores that are released begin to absorb nutrients from the tapetal cells. Concurrently, these cells actively synthesize sporopollenin, which is deposited to strengthen the pollen wall, culminating in the development of the mature pollen wall structure [45,62]. Moving forward to stage 10, it is particularly noteworthy that the tapetum begins to degenerate, thereby initiating the process of PCD [32]. By stage 12, the tapetum has degraded, organelles lyse, lipids aggregate, and a significant accumulation of lipid substances on the pollen surface occurs, leading to pollen coat formation [39,63,64,65]. However, it appears that none of these phenotypes were observed in the vrnp 1 mutant.
In this study, we discovered that the development patterns of mung bean microspores and anthers seem consistent with Arabidopsis thaliana [20]. Through the analysis of microspore staining and paraffin sectioning micrographs, we may have determined that vacuolated microspores accumulate degradation products from tapetal cells undergoing PCD, which contributes to the development of maturing pollen grains. However, in the vrnp 1 mutant, developmental defects in pollen grains emerged after the tetrad stage (Figure 2C and Figure 3B). The cytoplasm of its microspore was under-filled and exhibited significant vacuolation (Figure 2E–F and Figure 3F–H). The inability to thicken the pollen wall was due to the failure of pollen grains to absorb degradation products from the tapetum (Figure 2E). In this research, utilizing various staining techniques, we observed that the microspores in the vrnp 1 mutant failed to develop the typical pollen wall structure (Figure 2G, Figure 3H and Figure 4A). These cells eventually adhered to each other, underwent keratinization, and were attached to the inner wall of the anthers (Figure 2G and Figure 4A). Ultimately, the lack of a properly structured pollen wall impacted pollen maturation and dispersal.
Additionally, the surfaces of these immature microspores (Figure 4C), which previously had a rough texture (Figure 4A), became smoother and exhibited irregular protrusions in the anthers from buds on the verge of blooming. The process causing this transformation remains unclear. We hypothesize that the few lipids and other remnants from the degenerating tapetum still adhered to the surface of the microspores. However, pollen coat formation failed due to the untimely deposition and the absence of a reticulate pattern on the microspore.
We could confirm that the sterility trait results from a recessive mutation in a nuclear gene. Hence, we hypothesize that the gene mutation disrupts the timely onset of programmed cell death in the tapetal cells of the anther. Inadequate substance release (Figure 2D) leads to a deficiency in content within the mature vrnp 1 pollen grains, which inhibits staining intensity (Figure 3F–H), and hinders both the formation of mature pollen and the subsequent dehiscence of anthers.
Beyond exploring the causes of the cytological defects, we were also interested in identifying the genes that may underlie the male sterile phenotype of the vrnp 1 mutant. It has been documented that numerous genes are associated with tapetum development [23,38]. For example, the NEF1 gene is likely to affect the preservation of cell envelope integrity within plastids and facilitate lipid accumulation in the tapetum (yet it does not impact the structure of the tapetum [66]). ROXY1 and ROXY2, GRX proteins in Arabidopsis, have redundant roles in anther tapetum development, and the royx1 roxy2 double mutant exhibits defects from stage 6 of anther development [53]. The HKM gene, allelic to MS1, is associated with incomplete intine and exine deposition, leading to defective pollen wall formation and male sterility [51,52]. This defect is attributed to the lack of lipid accumulation in the tapetum, causing vacuolated tapetal cells to crush the microspores, ultimately leading to microspore degeneration [42,67].
Comparing the cellular biological phenotypes among these mutants revealed that vrnp 1 has a distinct phenotype. The developmental irregularities of the vrnp 1 mutant start from the early uninucleate stage, characterized by a lag in tapetal degradation. Thus, this indicates that the mutated gene in vrnp 1 differs from the previously discussed genes. Mut-map sequencing may be a practical approach for localizing the mutated gene. It is essential to further verify and validate the function via genetic transformation in mung beans. Moreover, considering the tetrad stage is a pivotal phase for imminent phenotypic variation in vrnp 1, it is crucial to undertake transcriptome sequencing of anthers at both the pre- and post-tetrad stages for a comprehensive analysis. Through the RNA-seq analysis, differentially expressed genes crucial for anther and microspore development, as well as downstream of the mutated gene, may be discovered. These discoveries will present a wealth of potential candidate mung bean genes for the regulatory network surrounding the mutated gene. Our team is currently advancing this segment of the research.
A notable limitation of this study is that we were unable to concurrently examine pollen grains, anther cross-sectional structures, and the overall internal anther structure within the same bud. To address this limitation, we conducted at least three replicates of samples from the same developmental stage for each analysis. In future genetic studies, the selection of appropriate plants and the precise timing of sample collection will remain critical considerations.

5. Conclusions

In this study, the vrnp 1 mutant, characterized by a complete male sterility phenotype, was generated through EMS mutagenesis. The genetic analysis indicated that a single recessive nuclear gene was the determinant of this sterility. Furthermore, cytological and histological analyses revealed that the sterility in the vrnp 1 mutant originated post-tetrad stage due to tapetal developmental defects, delayed PCD, and microspores failing to absorb nutrients from the tapetum in a timely manner. These factors led to the disruption of pollen coat deposition and the failure of pollen wall maturation, ultimately culminating in premature microspore degeneration and the complete manifestation of the sterile phenotype in the vrnp 1 mutant.

Author Contributions

The experiments were conceptualized by Y.C., N.X. and Y.C. prepared and revised the manuscript; S.B. and M.W. developed the population; Y.C. and T.L. performed the cytological analysis; T.L., D.H., H.W. and Y.X. performed the field experiments; Z.G. and N.X. reviewed the manuscript and supervised the research; K.D., Y.X. and H.W. conducted the hybrids; Z.G., N.X. and Y.C. secured the research funds. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the Innovation Project of Jilin Academy of Agricultural Sciences (CXGC2023RCB002), the Postdoctoral Financial Assistance Program of Jilin Province (E425422001), and the China Agriculture Research System of MOF and MARA-Food Legumes (CARS-08).

Data Availability Statement

All data are contained within the article.

Acknowledgments

We are thankful for Shucai Wang’s (the Laboratory of Plant Molecular Genetics and Crop Gene Editing, School of Life Sciences, Linyi University, Linyi, China) helpful discussions and critical reading of this manuscript. We thank Wuhan Keruino Biological Technology Limited Company for the technical support.

Conflicts of Interest

The authors declare no conflicts of interest.

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Figure 1. Phenotypic analysis of the vrnp 1 mutant. (A) The morphological phenotype of flora organs of Jilv 10 and vrnp 1 at 60 DAS. Inflorescences were taken at the same stage after flowering. Red arrowheads highlight the pistils with (Jilv 10) or without (vrnp 1) pollen grains. Bar = 1 cm. (B) The plant morphology of the vrnp 1 mutant at 70 DAS. While the Jilv 10 had matured, the vrnp 1 remained in the blooming phase. Bar = 10 cm. (C) Phenotypic analysis of the inflorescences at 70 DAS. The vrnp 1 mutant struggled to develop elongated pods, and the fleshy pods always fell off after reaching 2–2.5 cm in length. Red arrowheads highlight the fleshy pods of the vrnp 1 mutant. Bar = 1 cm. (D) Statistics on the flower stalk lengths of the vrnp 1 mutants. Data represent the mean ± SD of 12 flower stalks. p < 0.05. (E) The phenotype comparison of flower stalks between the vrnp 1 mutant and Jilv 10 at 70 DAS. (F) Morphology analysis of the fleshy pods of the vrnp 1 mutant and plum pods of Jilv 10. The picture displays the largest fleshy pods we could obtain from the vrnp 1 mutant at 70 DAS, measuring about 3.3 cm. The pods of the vrnp 1 mutant and Jilv 10 were taken at the same period. Red arrowheads highlight the beans in the pods. Bar = 1 cm. DAS, day after sowing.
Figure 1. Phenotypic analysis of the vrnp 1 mutant. (A) The morphological phenotype of flora organs of Jilv 10 and vrnp 1 at 60 DAS. Inflorescences were taken at the same stage after flowering. Red arrowheads highlight the pistils with (Jilv 10) or without (vrnp 1) pollen grains. Bar = 1 cm. (B) The plant morphology of the vrnp 1 mutant at 70 DAS. While the Jilv 10 had matured, the vrnp 1 remained in the blooming phase. Bar = 10 cm. (C) Phenotypic analysis of the inflorescences at 70 DAS. The vrnp 1 mutant struggled to develop elongated pods, and the fleshy pods always fell off after reaching 2–2.5 cm in length. Red arrowheads highlight the fleshy pods of the vrnp 1 mutant. Bar = 1 cm. (D) Statistics on the flower stalk lengths of the vrnp 1 mutants. Data represent the mean ± SD of 12 flower stalks. p < 0.05. (E) The phenotype comparison of flower stalks between the vrnp 1 mutant and Jilv 10 at 70 DAS. (F) Morphology analysis of the fleshy pods of the vrnp 1 mutant and plum pods of Jilv 10. The picture displays the largest fleshy pods we could obtain from the vrnp 1 mutant at 70 DAS, measuring about 3.3 cm. The pods of the vrnp 1 mutant and Jilv 10 were taken at the same period. Red arrowheads highlight the beans in the pods. Bar = 1 cm. DAS, day after sowing.
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Figure 2. Histological comparison analysis of anther sections from Jilv 10 and the vrnp 1 mutant from stage 6 to stage 14 of anther development. (A) Transverse section of the anthers at stage 6. The microspore mother cells were surrounded in a butterfly-shaped typical anther. (B) Transverse section of the anthers at stage 7 (the tetrad stage). The images indicated that both microspore mother cells in the vrnp 1 mutant and Jilv 10 could undergo mitosis to form the tetrad stage, with the entire anther structure appearing normal. (C) Transverse section of the anthers at stage 8. The structure of the anther locule in the vrnp 1 mutant was indistinct, particularly regarding the tapetum and individual microspores. (D) Transverse section of the anthers at stage 10. The tapetum failed to undergo PCD as expected, resulting in a swollen appearance. Individual pollen grains of the vrnp 1 mutant were barely visible. (E) Transverse section of the anthers at stage 12. The tapetal layers persisted, and the anther locule contained microspores that were irregularly shaped, clustered, significantly vacuolated, and had limited content. (F) Transverse section of the anthers at stage 13. The tapetum exhibited disintegration, with microspores clustering and showing signs of keratinization. (G) Transverse section of the anthers at stage 14. The anther of the vrnp 1 mutant failed to dehisce, with no mature microspores visible in the locule. Keratinization staining was more pronounced. Over three anthers were observed at each stage. Bar = 50 mm. T, tapetum; Msp, microspore; PG, pollen grain; V, vascular region; C, connective; MC, meiotic cell; Tds, tetrads; E, epidermis; En, endothecium; ML, middle layer; StR, stomium region; St, stomium; Sm, septum; Fb, fibrous bands. Bar = 50 mm.
Figure 2. Histological comparison analysis of anther sections from Jilv 10 and the vrnp 1 mutant from stage 6 to stage 14 of anther development. (A) Transverse section of the anthers at stage 6. The microspore mother cells were surrounded in a butterfly-shaped typical anther. (B) Transverse section of the anthers at stage 7 (the tetrad stage). The images indicated that both microspore mother cells in the vrnp 1 mutant and Jilv 10 could undergo mitosis to form the tetrad stage, with the entire anther structure appearing normal. (C) Transverse section of the anthers at stage 8. The structure of the anther locule in the vrnp 1 mutant was indistinct, particularly regarding the tapetum and individual microspores. (D) Transverse section of the anthers at stage 10. The tapetum failed to undergo PCD as expected, resulting in a swollen appearance. Individual pollen grains of the vrnp 1 mutant were barely visible. (E) Transverse section of the anthers at stage 12. The tapetal layers persisted, and the anther locule contained microspores that were irregularly shaped, clustered, significantly vacuolated, and had limited content. (F) Transverse section of the anthers at stage 13. The tapetum exhibited disintegration, with microspores clustering and showing signs of keratinization. (G) Transverse section of the anthers at stage 14. The anther of the vrnp 1 mutant failed to dehisce, with no mature microspores visible in the locule. Keratinization staining was more pronounced. Over three anthers were observed at each stage. Bar = 50 mm. T, tapetum; Msp, microspore; PG, pollen grain; V, vascular region; C, connective; MC, meiotic cell; Tds, tetrads; E, epidermis; En, endothecium; ML, middle layer; StR, stomium region; St, stomium; Sm, septum; Fb, fibrous bands. Bar = 50 mm.
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Figure 3. Developing pollen grains of the vrnp 1 mutant. (A) Tetrad stage. The tetrad structures were visible, with no significant differences observed between the vrnp 1 mutant and Jilv 10. (B) Early uninucleate stage. The microspores of the vrnp 1 mutant exhibited irregular shapes and weaker staining, whereas those of Jilv 10 displayed an increased intensity of I2-KI staining. (C–E) Mid-uninucleate stage, late uninucleate stage, and binucleate stage. The staining intensity of the vrnp 1 mutant microspores did not increase, and the reticulated structure on the microspores was absent. (F–H) Maturing pollen stage. The irregularly shaped microspores of the vrnp 1 mutant still lacked sufficient content and staining intensity. The microspores observed from stage (A) (tetrad stage) to (H) (maturing stage) were derived and arranged based on the size of the flower buds. Bar = 10 mm.
Figure 3. Developing pollen grains of the vrnp 1 mutant. (A) Tetrad stage. The tetrad structures were visible, with no significant differences observed between the vrnp 1 mutant and Jilv 10. (B) Early uninucleate stage. The microspores of the vrnp 1 mutant exhibited irregular shapes and weaker staining, whereas those of Jilv 10 displayed an increased intensity of I2-KI staining. (C–E) Mid-uninucleate stage, late uninucleate stage, and binucleate stage. The staining intensity of the vrnp 1 mutant microspores did not increase, and the reticulated structure on the microspores was absent. (F–H) Maturing pollen stage. The irregularly shaped microspores of the vrnp 1 mutant still lacked sufficient content and staining intensity. The microspores observed from stage (A) (tetrad stage) to (H) (maturing stage) were derived and arranged based on the size of the flower buds. Bar = 10 mm.
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Figure 4. Scanning electron micrograph of anthers and pollen grains of vrnp 1 and Jilv 10. (A,B) Morphology analysis of dissected anthers from buds on the verge of blooming. The immature microspores clustered and adhered to the endothecium, featuring irregular surfaces. Whereas Jilv 10 already possessed a reticulated structure and visible germination pores. This stage was later than the maturing stage shown in (Figure 3H). (C,D) Morphology analysis of anthers dissected from buds after the tetrad stage. The surface of microspores in the vrnp 1 mutant was rough, while those of Jilv 10 exhibited a well-structured surface. More than three sets of anthers were examined, and the photographs presented represent these observations. The three photographs displayed the same anther at varying magnification levels, with scale bars of 100 mm (upper), 20 mm (middle), and 2 mm or 10 mm (lower). Red arrowheads highlight the microspores within the anther locule.
Figure 4. Scanning electron micrograph of anthers and pollen grains of vrnp 1 and Jilv 10. (A,B) Morphology analysis of dissected anthers from buds on the verge of blooming. The immature microspores clustered and adhered to the endothecium, featuring irregular surfaces. Whereas Jilv 10 already possessed a reticulated structure and visible germination pores. This stage was later than the maturing stage shown in (Figure 3H). (C,D) Morphology analysis of anthers dissected from buds after the tetrad stage. The surface of microspores in the vrnp 1 mutant was rough, while those of Jilv 10 exhibited a well-structured surface. More than three sets of anthers were examined, and the photographs presented represent these observations. The three photographs displayed the same anther at varying magnification levels, with scale bars of 100 mm (upper), 20 mm (middle), and 2 mm or 10 mm (lower). Red arrowheads highlight the microspores within the anther locule.
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Table 1. Genetic analysis of the fertility separation population in the F2 generation.
Table 1. Genetic analysis of the fertility separation population in the F2 generation.
PhenotypeTheoretical NumberObserved NumberX2(3:1)P0.05
Fertile214.52143.8410.0047
Sterile71.572
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MDPI and ACS Style

Cheng, Y.; Lan, T.; Deng, K.; Wang, M.; Bao, S.; Han, D.; Xu, Y.; Wang, H.; Xu, N.; Guo, Z. Cytological Characterization of vrnp 1, a Pollen-Free Male Sterile Mutant in Mung Bean (Vigna radiata). Agronomy 2025, 15, 312. https://doi.org/10.3390/agronomy15020312

AMA Style

Cheng Y, Lan T, Deng K, Wang M, Bao S, Han D, Xu Y, Wang H, Xu N, Guo Z. Cytological Characterization of vrnp 1, a Pollen-Free Male Sterile Mutant in Mung Bean (Vigna radiata). Agronomy. 2025; 15(2):312. https://doi.org/10.3390/agronomy15020312

Chicago/Turabian Style

Cheng, Yuxin, Tianjiao Lan, Kunpeng Deng, Minghai Wang, Shuying Bao, Dan Han, Yapeng Xu, Han Wang, Ning Xu, and Zhongxiao Guo. 2025. "Cytological Characterization of vrnp 1, a Pollen-Free Male Sterile Mutant in Mung Bean (Vigna radiata)" Agronomy 15, no. 2: 312. https://doi.org/10.3390/agronomy15020312

APA Style

Cheng, Y., Lan, T., Deng, K., Wang, M., Bao, S., Han, D., Xu, Y., Wang, H., Xu, N., & Guo, Z. (2025). Cytological Characterization of vrnp 1, a Pollen-Free Male Sterile Mutant in Mung Bean (Vigna radiata). Agronomy, 15(2), 312. https://doi.org/10.3390/agronomy15020312

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