Introduction
Sour cherries (Prunus cerasus L.) are a unique fruit crop widely cultivated for their distinctive tart flavour and versatile use. They are also highly valued for their potential health benefits, such as their high antioxidant content and anti-inflammatory properties (Khadivi et al., Reference Khadivi, Mohammadi and Asgari2019). However, despite their popularity and importance in the fruit industry, there is still much to discover about the genetic diversity present among different sour cherry genotypes (Sokół-Łętowska et al., Reference Sokół-Łętowska, Kucharska, Hodun and Gołba2020).
Variances in pollen size, shape, or surface ornamentation among different genotypes or varieties within a species serve as indicators of underlying genetic relationships, potential for hybridization and reproductive barriers among populations or cultivars (Toledo et al., Reference Toledo, Rossi, de Andrade Bressan, Shirasuna, Martinelli and Oliveira2022). Investigating pollen morphology entails a meticulous examination and analysis of the physical attributes of pollen grains, which are fundamental as the male reproductive units in flowering plants, essential for plant reproduction (Amina et al., Reference Amina, Ahmad, Bhatti, Zafar, Sultana, Butt and Ashfaq2020). This scrutiny not only reveals the inherent traits of individual plant species but also unveils broader patterns of genetic variation within and among populations. Our research contributes to bridging gaps in the existing scientific literature by offering novel insights into the intricate nuances of pollen morphology and its implications for plant genetic diversity and reproductive dynamics. Studying pollen morphology is relevant to genetic diversity because it provides a window into the genetic makeup of plant populations. Variations in pollen traits reflect underlying genetic diversity, arising from factors such as genetic mutations, gene flow and natural selection. By examining pollen morphology, researchers can assess genetic diversity within populations, identify unique traits, and explore evolutionary relationships among different plant species. Moreover, pollen morphology informs breeding programs aimed at developing plant varieties with desirable traits, thereby contributing to our understanding of genetic diversity and facilitating efforts in plant breeding and conservation.
Pollen morphology serves as an indispensable asset in plant research, offering unparalleled insights into species delineation, reproductive dynamics, genetic variability and evolutionary trajectories. The examination of pollen characteristics not only enhances our comprehension of plant biology but also facilitates diverse applications spanning taxonomy, ecology, reproductive biology and plant breeding. While previous studies have utilized pollen morphology for genotype screening in various plants such as olive (Khaleghi et al., Reference Khaleghi, Karamnezhad and Moallemi2019), Crotalaria juncea (Rajagopal et al., Reference Rajagopal, Ganesan, Anantharaju and Gnanam2021), cashew (Mog et al., Reference Mog, Veena, Adiga, Hebbar, Shamsudheen, Manjesh and Yadav2023) and Abies alba (Wrońska-Pilarek et al., Reference Wrońska-Pilarek, Dering, Bocianowski, Lechowicz, Kowalkowski, Barzdajn and Hauke-Kowalska2020), no studies on pollen morphology of sour cherry genotypes have been undertaken. This study aims to address this gap by undertaking a comprehensive morphological analysis of pollen grains from different elite sour cherry genotypes.
Iran has been recognized as a significant hub of plant species diversity, particularly within the realm of sour cherry. The indigenous sour cherry germplasm in Iran, P. cerasus, showcases an exceptional spectrum of genetic variability (Momeni et al., Reference Momeni, Bouzari, Zeinolabedini and Jahromi2023). This study endeavours to meticulously investigate and characterize the genetic variations inherent among 10 distinct sour cherry genotypes, focusing on their pollen morphology in the Iranian context. Unlike previous studies, which have primarily centred on macroscopic traits or molecular markers, our research uniquely emphasizes the microscopic examination of pollen morphology to unveil nuanced genetic differences.
Specifically, our study aims to characterize genetic diversity harboured within these genotypes, delineating precise patterns of variation in pollen size, shape and ornamentation. The investigation has implications for breeding programs, genetic resource conservation and the targeted development of superior sour cherry cultivars endowed with desired traits such as disease resistance, yield potential and fruit quality. By employing pollen morphology as a diagnostic tool, we not only deepen our understanding of sour cherry genetics but also enrich the broader field of fruit crop research and breeding endeavours.
Furthermore, the significance of our research transcends the confines of the sour cherry industry. The methodologies and findings gleaned from this study can be extrapolated to inform and enhance breeding programs for other fruit crops. Moreover, our investigation underscores the utility of pollen morphology as a pivotal instrument in unravelling the intricacies of plant evolution and genetic diversity, thereby contributing to the broader landscape of plant genetics research.
Materials and methods
Plant materials
The trees selected in this research belong to the national collection of native sour cherry genotypes in a research farm of 100 ha in Meshkin Dasht City(35.7518175, 50.955513), Iran during two years (2020 and 2021). The trees were 8-year-old with 5 × 4 distances. The sampling was done based on completely randomized design (CRD) in four replicates. Totally, 150 were selected for this experiment. Ten genotypes that were previously identified as superior genotypes were selected from the genotypes in this collection. These genotypes have been selected during the last few years and are introduced as promising genotypes. Before the blooming at the early April, several cloth envelopes were placed on the branches in different directions of the trees to prevent possible pollen interference. When the flowers reached the balloon-shaped or popcorn stage (during which flowers are fully swollen and ready to bloom), 300 flowers were collected from different directions of the tree from cloth envelopes and placed in paper envelopes. After transferring the flowers to the laboratory of the Temperate Fruits Research Institute, the anthers were separated from the flowers and dried in a petri dish at ambient temperature for 24 h. To avoid the likely spoilage of pollen because of possible moisture, the pollens were separated from the anthers and refrigerated in glass containers with a lid (with moisture absorbants) at 3–5°C until scanning electrode microscope (SEM) examination.
Preparing pollen grains for SEM
First, the pollen grains were covered with gold particles (20 nm in diameter) using a gold-coating device. Then, the bases carrying the particles were covered with a transparent tape, the pollen grains were covered on this tape and scanned by a SEM TESCAN-Vega 3 device (Brno, Czech Republic) with scales of 10 μm (for the appearance of the pollen grain) and 2 μm (to examine the exine pattern). It should be noted that the pollen grains with unconventional shapes or still immature were not scanned and examined here (Khosropour et al., Reference Khosropour, Attarod, Shirvany, Pypker, Bayramzadeh, Hakimi and Moeinaddini2019). The attained images were studied via Dgimizer software version 5/7/2.
Experimental variables
The measured traits were pollen length and width, colpus length, mesocolpium width (horizontal diameter) and exine characteristics (ornamentation, number of ridges (ridge width), number of mesocolpium in 50 μm, Mesocolpium width, furrow width, number of cells per 50 μm, vertical view area (Polar area) and horizontal view area (tropical area), the diameter of the circle of the ridge (diameter of the circle between the mesocolpium is polar), and the area of the circle between the furrows (the area of the circle between the mesocolpium is polar). To examine the morphological, pomological and phenological traits of the mentioned genotypes and the correlations of these traits with pollen grain morphology traits, the weight traits, core weight, tail weight, tail length, width and length, diameter, tail thickness, leaflets on the tail, kernel size, petal shape, petal diameter, flowering time and full flower time of the fruit were measured using the Sour Cherry International Descriptor.
Results
Morphological traits of pollen grains
The study revealed a broad spectrum of pollen shapes among the examined sour cherry genotypes. Analysis confirmed that all genotypes displayed unipolar and radially symmetrical pollen grains, each possessing three colpates. Polar examination of pollen grains consistently depicted a circular morphology (Fig. 1a), while the equatorial view showcased an elongated oval shape (Fig. 1b). These observations elucidate the structural attributes of sour cherry pollen, furnishing critical groundwork for advancing research in plant breeding and reproductive biology.
Figure 1. The polar view (a) and the equatorial view (b) of sour cherry pollen grains native to Iran. c and d images show striated exines, with the lowest (183608-440) and the highest (511-121674) amount of holes depicted on the c and d figures, respectively. The difference in length, width, and the number of furrows between genotypes is quite clear in the images.
The study delineated the range of pollen grain dimensions across the examined sour cherry genotypes. Pollen grain lengths varied from a maximum of 57.57 μm (genotype 112-183610) to a minimum of 42.18 μm (genotype 121671-129), while pollen grain widths ranged from a maximum of 28.13 μm (genotype 183067-183067) to a minimum of 20.28 μm (genotype 183067-307). Additionally, the length-to-width ratios of pollen grains exhibited genotype-dependent variation, with the highest average ratio of 2.13 μm observed in genotype 121-183066 and the lowest average ratio of 1.64 μm recorded in genotype 183067-416.
According to ERDTMAN's (1971) classification, all studied sour cherry genotypes were characterized by large-sized pollen grains. These precise measurements and classifications of pollen grain characteristics contribute to a comprehensive understanding of sour cherry reproductive biology and hold implications for future breeding endeavours. The length of the colpus, an aperture on the pollen grain, exhibited variation across the studied sour cherry genotypes. The longest recorded colpus length was 50.53 μm in genotype 117-183610, whereas the shortest colpus length observed was 38.18 μm in genotype 121671-121. Similarly, the width of the mesocolpium, the region surrounding the colpus, displayed variability among the genotypes. The widest mesocolpium width measured was 19.69 μm in genotype 12671-129, while the narrowest width recorded was 11.68 μm in genotype 121672-418. On average, the mesocolpium width was 14.28 μm, with the colpus length averaging 24.17 μm.
Examination of the exine, the outer layer of the pollen grain, revealed a consistent striated (semitectate) ornamentation across all studied sour cherry genotypes. Furthermore, certain genotypes exhibited distinct cell-like depressions on the exine surface (Fig. 1c), with the number of these depressions varying among genotypes. The genotype with the highest count of depressions, 51 cells per 50 μm square, was recorded in genotype 121674-511, while the genotype with the lowest count, 4 cells per 50 μm square, was genotype 183608-440.
Moreover, the highest number of exine furrows, indicative of grooves on the exine surface, was observed in genotype 121675-307, with 18.5 furrows per 50 μm square. In contrast, the lowest count of furrows, 8.5 furrows per 50 μm square, was recorded in genotype 183609-405.
The width of the ridges on the exine surface of sour cherry pollen grains exhibited a wide range of values, from 0.33 μm in genotype 121674-511 to 67 μm in genotype 121672-418. On average, the ridges had a width of 0.47 μm across all the genotypes studied. In terms of the distance between the ridges, also known as furrow width, it varied from 0.17 μm in genotypes 121675-121675 and 121671-129 to 0.56 μm in genotype 183066-121. The average furrow width across all genotypes was measured to be 0.36 μm. These observations highlight the significant variation in both ridge width and furrow width within the exine ornamentation of sour cherry pollen grains. These structural characteristics are essential for various aspects of the pollen's reproductive functions, such as recognition, adhesion and germination (Fig. 2).
Figure 2. Images of pollen grains of 10 Iranian sour cherry genotypes from different angles.
Genotype clustering
During the clustering analysis, the examined genotypes initially segregated into three subgroups, subsequently further classified into two primary groups based on their distinctive characteristics. The first primary group comprised genotypes 183608, 183610 and 183609, while the second primary group encompassed genotypes 121674 and 183066. The third primary group included genotypes 121673, 183067, 183067, 121672, 121671 and 121675.
The pivotal traits contributing to the classification of these genotypes were identified as the length and width of the pollen grain, along with the length of the colpus. Cluster one, represented by genotypes 183608, 183610 and 183609, exhibited the highest values for these traits. In contrast, clusters two (genotypes 121674 and 183066) and three (genotypes 121673, 183067, 183067, 121672, 121671 and 121675) displayed comparatively smaller lengths and widths of the pollen grain, as well as colpus length. These delineations, illustrated in Table 1 and Fig. 3a, underscore the significance of these specific morphological attributes in discerning and categorizing sour cherry genotypes through clustering analysis.
Table 1. Some morphological characteristics of pollen grains of superior sour cherry genotypes originated in Iran
- Means with the same letter (s) are not significantly different from each other (P < 0.05).
Figure 3. Clustering of superior Iranian sour cherry genotypes based on pollen grain morphological traits (a). Heatmap of Spearman's correlations between some morphological traits, pomological traits, and the phenological traits of sour sherry genotypes (b).
Correlations between variables
The correlation between the morphological traits of pollen grains and the pomological and phenological traits of the studied genotypes was thoroughly examined. Data analysis and subsequent clustering of these traits revealed a perfect agreement of 100% between them. This correlation is visually represented in Table 2, as well as Fig. 4, which illustrate the results of the data analysis and clustering, showcasing the harmony between the morphological, pomological and phenological traits of the studied genotypes.
Table 2. Some phenological and pomological characteristics of superior sour cherry genotypes originated in Iran
Figure 4. Clustering of superior Iranian sour cherry genotypes based on pollen grain traits (a) and pollen and pomological traits (b).
The substantial agreement observed among these traits suggests a significant relationship between the morphological characteristics of pollen grains and the pomological and phenological traits of the genotypes. This implies that the morphology of pollen grains could serve as a reliable predictor for comprehending and anticipating the pomological and phenological characteristics of the studied genotypes.
Upon scrutinizing the correlation between pollen grain morphological and pomological traits, direct correlations were detected between the fruit tail diameter and various pollen grain morphological traits, including pollen length, pollen width, colpus length, horizontal viewing area, width of exine furrows, and the number of cells. These correlations were particularly pronounced in the pollen grain morphology traits associated with the overall size of the pollen grain. Consequently, it can be inferred that a direct correlation coefficient exists between the size of the pollen grain and the diameter of the fruit tail, as depicted in Fig. 3b.
On the other hand, a negative correlation coefficient was observed between the ridge width (exine ridge width) and the fruit diameter and fruit weight traits. This suggests that as the ridge width of the pollen grain increases, the fruit diameter and fruit weight decrease. Further analysis is required to fully understand the underlying mechanisms and implications of this negative correlation. These findings highlight the potential relationship between pollen grain morphological traits and pomological characteristics, particularly in terms of fruit tail diameter and fruit weight. Further research is needed to delve deeper into these associations and ascertain their significance in understanding and improving fruit quality (Fig. 4).
Discussion
The investigation of sour cherry genotypes in this study revealed a consistent unipolar and radially symmetrical morphology, characterized by three-colpates (tricolpate). The morphological traits of pollen grains, including length and width, observed in superior sour cherry genotypes indicate underlying genetic diversity. This relationship suggests a genetic basis for morphological traits that are similar to patterns observed in other species like cotton (Gossypium spp.), where genetic diversity directly influences phenotypic traits (Sahar et al., Reference Sahar, Zafar, Razzaq, Manan, Haroon, Sajid, Rehman, Mo, Ashraf, Ren and Shakeel2021). These traits are influenced by intricate biochemical processes such as cell division, expansion, hormone signalling, and metabolic pathways involving carbohydrates and lipids (Raja et al., Reference Raja, Vijayalakshmi, Naik, Basha, Sergeant, Hausman and Khan2019). Genetic variations within these pathways contribute to the observed variations in pollen size and width. Additionally, environmental factors also modulate these morphological traits, indicating that both genetic makeup and environmental conditions play a significant role in shaping these characteristics (Fahad et al., Reference Fahad, Ihsan, Khaliq, Daur, Saud, Alzamanan, Nasim, Abdullah, Khan, Wu and Wang2018). This dual influence is important for understanding how morphological traits can be optimized through breeding under varying environmental conditions (Pacini and Dolferus, Reference Pacini and Dolferus2019). To elucidate the precise biochemical mechanisms governing these traits, future research employing advanced techniques like transcriptomics, proteomics and metabolomics is warranted.
The prolate length of pollen grains in sour cherry genotypes mirrored observations in other fruit species such as apricots, almonds, peaches and plums, suggesting a conserved evolutionary trait among these taxa. This morphological consistency, documented across diverse species (Punt et al., Reference Punt, Hoen, Blackmore, Nilsson and Le Thomas2007; Diamantino et al., Reference Diamantino, Costa, Soares, Morais, Silva and Souza2016; Caliskan et al., Reference Caliskan, Bayazit, Kilic, Ilgin and Karatas2021), points to a potential adaptive significance of this pollen shape in facilitating reproductive success under similar ecological conditions. Moreover, the variability in pollen colpus length we observed among the sour cherry genotypes aligns with recent research by Pacini and Franchi (Reference Pacini and Franchi2020), highlighting its utility as a marker for genetic diversity. This trait's variation underscores its potential in genetic screening and breeding programs, suggesting that pollen colpus length could be crucial for selecting genotypes with desirable agricultural characteristics (Awika et al., Reference Awika, Bedre, Yeom, Marconi, Enciso, Mandadi, Jung and Avila2019).
The variation in apertures – holes or cell-like structures on the exine of pollen grains – emerged as a critical characteristic distinguishing among sour cherry genotypes. These apertures play a crucial role in pollen germination and fertilization, making their study important for understanding reproductive strategies. We observed significant differences in the number, size and distribution of these apertures across the genotypes, directly reflecting the genetic diversity within this species. The distinct patterns of aperture variations not only highlight genetic diversity but also suggest specific adaptive reproductive features (Albert et al., Reference Albert, Matamoro-Vidal, Prieu, Nadot, Till-Bottraud, Ressayre and Gouyon2022). Exploring these variations further through biochemical approaches can shed light on the underlying genes and molecular pathways governing aperture formation (Breygina et al., Reference Breygina, Klimenko and Schekaleva2021).
Our investigation into sour cherry genotypes has revealed significant variability in the ridge width of pollen grains, reflecting distinct genetic backgrounds among the genotypes studied. This variability in ridge width, characterized by differing dimensions and patterns of ridges and furrows, plays a crucial role in pollen-stigma interactions, potentially influencing the efficiency of pollen tube germination and fertilization success (Pacini and Franchi, Reference Pacini and Franchi2020). These morphological characteristics are not just variations but represent adaptive traits that may enhance reproductive success under diverse environmental conditions, similar to findings in other species such as cotton (Gossypium spp.) where pollen traits have been linked to environmental adaptability and fertilization efficiency (Cai et al., Reference Cai, Hou, Muhammad, Wang, Xu, Zheng, Wang, Liu, Zhou, Hua and Wang2023). In sour cherries, the detailed study of these ridge patterns, as facilitated by advanced imaging techniques, could inform breeding programs by identifying genotypes that possess optimal pollen traits for improved fruit set and quality (Diamantino et al., Reference Diamantino, Costa, Soares, Morais, Silva and Souza2016). Moreover, the specificity of these traits suggests that they could serve as reliable markers for genetic screening. As noted by Khaleghi et al. (Reference Khaleghi, Karamnezhad and Moallemi2019), such morphological markers are invaluable for breeding strategies that aim to combine desirable traits for enhanced agronomic performance. By focusing on the genetic underpinnings that govern these variations, as suggested by recent advances in genomics and proteomics, we can better understand the molecular pathways that influence these critical pollen characteristics (Breygina et al., Reference Breygina, Klimenko and Schekaleva2021).
Our study distinctly highlights how specific pollen morphological traits, particularly the ridge width of pollen grains, differentiate among superior sour cherry genotypes. These morphological markers are directly linked to genetic diversity, offering a practical method for identifying genotypes with optimal reproductive traits (Booy et al., Reference Booy, Hendriks, Smulders, Van Groenendael and Vosman2000). This approach is pivotal for breeding programs, as it allows for the selection of sour cherry cultivars that are likely to produce higher yields and superior fruit quality (Khaleghi et al., Reference Khaleghi, Karamnezhad and Moallemi2019; Pacini and Franchi, Reference Pacini and Franchi2020).
Our research establishes a significant correlation between pollen grain morphology and fruit yield characteristics in sour cherry genotypes, underscoring the utility of pollen traits as markers of reproductive and agronomic potential (Fig. 3b). Specifically, genotypes featuring larger pollen grains and increased ridge widths demonstrated enhanced fruit weight and dimensions, a finding that aligns with broader trends observed in agricultural genetics (Fuller, Reference Fuller2018). This correlation is particularly notable for its implications in selective breeding programs; larger pollen grains are often associated with improved pollen-stigma interactions, leading to higher rates of successful fertilization and, consequently, more robust fruit development (Althiab-Almasaud et al., Reference Althiab-Almasaud, Teyssier, Chervin, Johnson and Mollet2023). Moreover, the variability in these pollen traits across different genotypes suggests a genetic basis that could be exploited to optimize fruit yield and quality. By integrating detailed phenotypic data with genetic analysis, breeders can identify and propagate genotypes that exhibit both superior pollen characteristics and desirable fruit qualities (De Moraes et al., Reference de Moraes, Geraldi, de Pina Matta and Vieira2005).
Our results demonstrate the value of pollen morphology in differentiating superior sour cherry genotypes, offering novel insights into the genetic diversity and reproductive biology of these valuable fruit crops. By delving into the intricate details of pollen characteristics, such as ridge width, we illuminate previously unexplored dimensions of genetic variability within sour cherry populations. This knowledge is important for advancing breeding programs aimed at developing cultivars with desirable traits for enhanced fruit yield and quality.
Supplementary material
The supplementary material for this article can be found at https://doi.org/10.1017/S1479262124000303
