Palynological variability study among strawberry (Fragaria × Ananassa Duch) cultivars for pollination and breeding

Introduction

Strawberry (Fragaria × ananassa Duch.) is a high-value commercial fruit cultivated extensively in temperate, subtropical, and protected production systems, prized for its sensory quality, nutritional profile, and dual suitability for fresh and processed markets. Despite advances in protected cultivation and breeding technologies, sustaining high marketable yields and consistent fruit quality remains a primary production challenge (Hernández-Martínez et al., 2023). Productivity is uniquely constrained by pollination and fertilization dynamics due to the plant's aggregate floral biology. The commercial "fruit" develops from an enlarged receptacle whose expansion is directly governed by surrounding true fruits the achenes each derived from an individually fertilized ovary. Post-fertilization, developing achenes synthesize key phytohormones, notably auxin, which trigger and coordinate early receptacle hypertrophy (Tian et al., 2022). Consequently, sub-optimal or spatially heterogeneous fertilization across the carpellary collective leads to localized developmental arrest, yielding misshapen or unmarketable fruit. Fertilization success requires mutual functional competence of the staminate and pistillate structures. Pollen viability and in vitro germination capacity determine male gametophytic fitness and pollen-tube elongation rates toward the ovule, while stigma receptivity defines the temporal window permitting pollen adhesion, hydration, and style penetration. Recent evidence confirms that floral architecture, pollen load, and viability are directly proportional to the percentage of fertilized achenes and final fruit mass (Lee et al., 2026). Genotypic variation in male fertility and female receptivity is therefore a principal driver of divergence in reproductive efficiency and yield architecture among cultivars. While cultivated strawberry is generally considered self-compatible, autonomous self-pollination often fails to deliver adequate, uniform pollen across the numerous pistils of a receptacle, directly limiting fruit set and commercial quality. Klatt et al. (2014) showed that insect pollination significantly reduces malformation while enhancing fruit mass and market grade relative to wind-or self-pollination. This is corroborated by Wietzke et al. (2018), who linked efficient pollination to increased fertilized achene set and physiological improvements during receptacle development. Furthermore, Lee et al. (2026) demonstrated that pollen load and viability directly govern achene fertilization rates and ultimate fruit weight. Collectively, these data confirm that floral reproductive efficiency is a critical physiological determinant of marketable yield. Pollen viability and germination are strongly governed by microclimatic conditions during microsporogenesis and post-anthesis exposure. Ambient temperature and relative humidity modulate pollen longevity, germination capacity, and tube elongation kinetics, with the magnitude of susceptibility varying genotypically. Leech et al. (2000) observed cultivar-dependent declines in strawberry pollen germination under prolonged post-anthesis exposure, which were exacerbated by concurrent thermal and hygric stress. Similarly, elevated temperatures suppressed pollen germination and tube growth in everbearing genotypes, ultimately reducing yield (Karapatzak et al., 2012). Because male gametophytic fitness represents a genotype-by-environment interaction, multi-season phenotyping is essential to reliably characterize reproductive stability across cultivars. Successful fertilization requires strict coordination between pollen quality and pistillate competence. Effective pollen adhesion, hydration, and germination demand that viable pollen deposition coincides with peak stigma receptivity, a primary determinant of reproductive success and optimal timing for controlled hybridization. Stigmatic receptivity is functionally marked by surface physiological alterations and elevated enzymatic activity (Dafni and Maués, 1998). In strawberry, this receptivity window is highly transient, Lata et al. (2018) identified peak receptivity at anthesis, followed by a rapid post-anthesis decline. Consequently, temporal asynchronous overlap between viable pollen release and stigma receptivity risks incomplete fertilization, regardless of pollen load. Pollen viability, in vitro germination capacity, and stigma receptivity represent distinct yet functionally complementary components of floral reproductive performance. Evaluating any single parameter in isolation provides an incomplete assessment of overall reproductive competence. While previous investigations documented cultivar-specific variation in pollen viability (Żebrowska, 1997), environmental modulation of pollen germination (Leech et al., 2000;Karapatzak et al., 2012), and floral phenology (Lata et al., 2018), substantial knowledge gaps remain. Specifically, data are limited regarding the temporal stability of these traits across consecutive field seasons and the extent to which superior male gametophytic traits correlate with high stigma receptivity within a given genotype. Resolving these relationships is critical for breeding programs, where optimizing parental selection and cross-synchronization dictates hybridization efficiency. Accordingly, this study evaluated six strawberry cultivars (Chandler, Kimberly, Larson, Sweet Charlie, Winterdawn, and Selva) over two consecutive seasons (2024-25 and 2025-26) under the temperate conditions of Kashmir to quantify genotypic variation in pollen viability, in vitro germination, and stigma receptivity. Specifically, we examined the concordance among these reproductive components to determine whether high performance in a single floral trait predicts overall reproductive competence. These findings aim to establish reliable reproductive benchmarks for parental selection and targeted hybridization in strawberry improvement programs.

Materials and Methods

Experimental site and study period

The investigation was conducted during two consecutive strawberrygrowing seasons, 2024-25 and 2025-26, at the Strawberry Germplasm Block, Division of Fruit Science, Faculty of Horticulture, Sher-e-Kashmir University of Agricultural Sciences and Technology of Kashmir (SKUAST-K), Shalimar, Srinagar, Jammu and Kashmir, India. The experimental site is located at approximately 34°09′22″ N latitude and 74°52′55″ E longitude, at an elevation of 1685 m above mean sea level. The same experimental site and observational procedures were used during both seasons.

Plant material and experimental design

Six strawberry (Fragaria × ananassa Duch.) cultivars, namely Chandler, Kimberly, Larson, Sweet Charlie, Winterdawn and Selva, maintained in the Strawberry Germplasm Block of SKUAST-K, were used in the study. The experiment was conducted in a randomized complete block design (RCBD), with cultivar considered as the treatment. Plants were maintained following the recommended cultural practices for strawberry cultivation under local conditions. Freshly opened, healthy and morphologically normal flowers were selected at anthesis for assessment of pollen viability, pollen germination and stigma receptivity. Flowers showing visible injury, disease symptoms or abnormal development were excluded from sampling. Observations were recorded separately for each cultivar and replication during both cropping seasons.

Pollen viability

Pollen viability was determined using the 0.1% erythrosin B staining test, following the general staining procedure used for strawberry pollen assessment (Lata et al., 2018). A 0.1% erythrosin B solution was prepared by dissolving 100 mg erythrosin B in distilled water and making the final volume to 100 mL. Fresh pollen grains collected from flowers at anthesis were dusted onto clean glass slides. One to two drops of erythrosin B solution were added and the preparation was covered with a coverslip. The slides were allowed to stand for approximately 5 min to permit staining and were subsequently examined under a microscope. With erythrosin B, pollen grains that remained unstained or colourless were considered viable, whereas grains that absorbed the stain and appeared red or pink were considered non-viable, following the criterion used in previous pollen studies (Sharma et al., 2025;Lata et al., 2018). Pollen grains were counted for each replication and cultivar, and pollen viability was calculated as:

Pollen viability (%) = Number of viable (unstained) pollen grains Total number of pollen grains examined × 100 (A1)

Pollen germination was assessed in vitro using a sucrose-boric acid medium, following the general procedure described for strawberry pollen germination by Lata et al. (2018), with appropriate modification. Freshly collected pollen grains were placed in small drops of 15% sucrose solution supplemented with 0.01 g boric acid on cavity slides. The preparations were covered carefully with coverslips. The margins of the coverslips were sealed with a thin layer of petroleum jelly (Vaseline) to reduce evaporation of the germination medium. The prepared slides were placed in Petri dishes lined with moist filter paper to maintain a uniformly high relative humidity during incubation. The slides were maintained at room temperature and pollen germination was examined microscopically after 24, 48 and 72 h.

A pollen grain was considered germinated when the length of its pollen tube exceeded the diameter of the pollen grain. The numbers of germinated and non-germinated pollen grains were recorded for each cultivar and replication. Pollen germination was calculated as:

Pollen germination (%) = Number of germinated pollen grains Total number of pollen grains examined × 100

Stigma receptivity

Stigma receptivity was evaluated using the hydrogen peroxide (H 2 O 2 ) test, based on the principle described by Dafni and Maués (1998) and the scoring procedure subsequently employed by Crispim et al.

(2017) [1] . This test is based on the reaction of hydrogen peroxide with enzymes, particularly peroxidases, present on receptive stigmatic surfaces; visible effervescence is therefore used as an indicator of re ceptivity. The H 2 O 2 method and its relationship with stigmatic enzymatic activity have been widely used for rapid assessment of stigma receptivity. For each cultivar and replication, ten freshly opened flowers at anthesis were selected. A 3% H 2 O 2 solution was applied directly to the stigmatic surface, and the resulting reaction was examined under a stereoscopic microscope. Stigma receptivity was assessed according to the intensity and extent of bubble formation on the stigmatic surface. Following Crispim et al. (2017) [1] , receptivity was scored on a scale of 0-3, corresponding to the approximate proportion of the stigmatic surface showing bubbling: 0 = 0%, 1 = 30%, 2 = 60%, and 3 = 100% bubble coverage. This scoring system is consistent with the hydrogen-peroxide-based assessment reported by Crispim et al. (2017) [1] . Greater bubble formation was interpreted as greater stigmatic receptivity. For each cultivar, the percentage value corresponding to the score of each stigma was recorded, and mean stigma receptivity was calculated as:

Stigma receptivity (%) = ∑bubble-coverage values of the stigmas examined Total number of stigmas examined (A3)

Statistical analysis

Data recorded during the two cropping seasons were subjected to analysis of variance (ANOVA) appropriate to a randomized complete block design. Cultivar effects were tested for each reproductive trait, and the data from the two seasons were subsequently used to obtain pooled estimates. Statistical analyses were performed using OPSTAT, a statistical software package developed by the Department of Mathematics and Statistics, CCS Haryana Agricultural University, Hisar, India. OPSTAT is commonly used for analysis of agricultural experiments based on randomized experimental designs. Where the cultivar effect was significant, treatment means were compared using the critical difference (CD) at the 5% probability level (P ≤ 0.05).

Results are presented as cultivar means together with the corresponding measures of variability.

Results and Discussion

Pollen fertility

Pollen viability

Pollen viability varied significantly (P < 0.05) among the evaluated strawberry cultivars across both growing seasons ( ), with no significant differences among these three top performing cultivars (CD = 4.291). Representative light micrographs illustrating differential stain uptake by viable versus non-viable pollen grains following 0.1% erythrosin B staining are presented in Figure 1. Across both study years, Chandler and Winterdawn maintained superior viability, whereas Larson consistently exhibited inferior performance with minimal inter-annual numerical fluctuation. However, because multi-factor analysis of variance was not performed to formally partition the season and cultivar \times and season interaction effects, these cross-year trends remain descriptive rather than statistically confirmed benchmarks of phenotypic stability.

Pollen germination

In vitro pollen germination varied significantly (P < 0.05) among the evaluated cultivars across both cropping seasons (Table 1). In 2024-25, germination ranged from 51.00% in Larson to 67.67% in Selva; Selva was statistically comparable to Sweet Charlie (64.33%) and Chandler (63.00%). A similar pattern occurred in 2025-26, with Selva maintaining the highest germination capacity (70.33%), followed closely by Sweet Charlie (67.67%) and Chandler (66.33%), with no significant divergence among these top three performers. Larson exhibited the lowest germination capacity in both seasons (51.00% and 55.00%, respectively). Representative light micrographs illustrating in vitro pollen germination and subsequent tube elongation are presented in Figure 2.

Pollen grains were scored as germinated when pollen tube length exceeded grain diameter. Pooled analysis resolved clear genotypic distinctions (pooled CD = 3.726): Selva achieved the highest overall germination (68.67%), which was statistically comparable only to Sweet Charlie (66.00%). Chandler (64.67%) and Winterdawn (62.17%) displayed intermediate germination, whereas Kimberly (57.67%) and Larson (53.00%) exhibited the lowest performance. Consequently, the genotypic hierarchy for germination diverged from that observed for viability.

A notable physiological discordance emerged between Selva and Chandler. Although Chandler produced the highest proportion of viable pollen, it did not maximize germination; conversely, Selva yielded lower stainability yet achieved the highest in vitro germination. This discrepancy underscores that cytochemical staining and functional germination are not interchangeable proxies for male gametophytic fitness. Staining reflects cellular integrity and membrane intactness, whereas in vitro germination functionally evaluates physiological activation, metabolic competence, and tube emergence kinetics under specific osmotic and nutritional conditions. These genotypic differences align with Lata et al. (2018), who observed significant variation in strawberry pollen germination using a standard 15% sucrose medium. Moreover, functional germination is highly sensitive to microclimatic conditions during microsporogenesis and anthesis. terms, these inter-annual differences remain descriptive rather than statistically verified.

Stigma receptivity

Stigma receptivity varied significantly (P < 0.05) among the evaluated cultivars across both cropping seasons (Table 2). In 2024-25, receptivity ranged from 69.00% in Sweet Charlie to 89.33% in Winterdawn. Winterdawn was statistically comparable to Kimberly (87.33%), while the remaining cultivars exhibited significantly lower values. During 2025-26, Winterdawn maintained the highest receptivity (92.33%), followed by Kimberly (90.33%) and Chandler (87.00%), with no significant divergence among these top three cultivars (CD = 6.305). Across both seasons, numerical pooled means ranked in descending order, Winterdawn (90.83%), Kimberly (88.83%), Chandler (83.67%), Larson (78.50%), Selva (72.50%), and Sweet Charlie (71.67%). Because a pooled critical difference was not computed for this parameter, these pooled values are reported descriptively to delineate overall cross-season trends without formal mean separation.

Conclusion

The evaluated strawberry cultivars exhibited significant genotypic divergence across pollen viability, in vitro germination capacity, and stigma receptivity. Chandler and Winterdawn demonstrated superior pollen viability, Selva and Sweet Charlie maximized pollen germination, and Winterdawn and Kimberly displayed the highest stigmatic receptivity; conversely, Larson consistently underperformed across both male gametophytic traits.

No single cultivar maximized all three reproductive attributes, demonstrating that pollen viability, germination capacity, and pistillate receptivity are physiologically uncoupled traits that cannot serve as reciprocal proxies for reproductive competence. However, Winterdawn emerged as the most balanced genotype, combining high pollen viability with peak stigma receptivity, underscoring its high potential as a parental candidate for controlled hybridization schemes.

Collectively, these findings demonstrate that multi-trait phenotyping encompassing both staminate and pistillate fitness parameters is critical for accurately characterizing strawberry floral biology, identifying elite parental material, and optimizing reproductive success in temperate breeding programs.

References

1. Crispim JG, Rêgo ER, Rêgo MM, Nascimento NFF, Barroso PA. Stigma receptivity and anther dehiscence in ornamental pepper. Horticultura Brasileira. 2017;35(4):609-612.
2. Areia M, Crispim JG. Stigma receptivity and anther dehiscence in ornamental pepper. Horticultura Brasileira. 2017;35:609-612.
3. How to cite this article: Dhar S, Sundouri A, Kumar A, Masoodi K, Ali G, Singh P, et al. Palynological variability study among strawberry (Fragaria × ananassa Duch.) cultivars for pollination and breeding. International Journal of Advanced Biochemistry Research. 2026;SP-10(10):437-442. doi: https://w ww.doi.org/10.33545/26174693.2026.v10.i10Sf.10318