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Comparison of the Micromorphology and Ultrastructure of Pollen Grains of Selected Rubus idaeus L. Cultivars Grown in Commercial Plantation

The genus Rubus is one of the largest taxonomically diverse and complex genera in the family Rosaceae. Morphology of pollen grains (equatorial and polar axes length, shape and size, aperture position, exine sculpture, perforations) is regarded as one of its main diagnostic features for identification of species and varieties. An attempt was made to fill the gap concerning the pollen micromorphology and ultrastructure of R. idaeus L. using light, scanning, and electron transmission microscopy. This study is a comparative analysis of micromorphological and ultrastructural traits of pollen from six raspberry cultivars. The pollen grains were classified as small or medium of shape prolato-spheroids. The parallel striae in the equatorial view in the exine sculpture were sometimes branched dichotomously in ‘Glen Ample’, ‘Polka’, and ‘Polana’, arcuate in ‘Laszka’ and ‘Pokusa’, or irregularly overlapping in ‘Radziejowa’. The width of exine striae of biennial fruiting cultivars was much larger than in repeated fruiting cultivars. In terms of the increasing number of perforations per unit area of the exine surface, the cultivars were ranked as follows: ‘Pokusa’ < ‘Glen Ample’ < ‘Laszka’ < ‘Polka’ < ‘Polana’ < ‘Radziejowa’. The thickest tectum, the highest and thickest columellae with the largest distances between them, and the thicker foot layer were demonstrated in ‘Glen Ample’. The ectoexine constituted on average ca. 78–90% of the exine thickness. The findings may constitute auxiliary traits i.a. for identification of related taxa, interpretation of phylogenetic relationships, and pollination biology.


Genus Rubus in the Flora of Poland
In the flora of Poland, Rubus plants represent several subgenera: Idaeobatus, Cylactis, Chamaerobus, Anoplobatus, and Rubus. The subgenus Idaeobatus comprises, e.g., the raspberry (R. idaeus L.) with red fruits and the Chinese raspberry (R. xanthocarpus Bureau. et Franch.) with yellow fruits. The second and third subgenera mentioned include herbaceous plants. One of the species of the subgenus Cylactis is the stone bramble (R. saxatilis L.) with red fruits. In turn, the genus Chamaerobus is represented by the cloudberry (R. chamaemorus L.) with yellow fruits, which is under strict species protection. Blackberries are representatives of the last two genera mentioned above. The purple-flowered

Micromorphology of Pollen Grains
The anthers of the R. idaeus flowers released pollen grains in the bud burst phase. Most of the anthers released pollen on the first and second day of flowering.

Size and Shape of Pollen Grains
The pollen grains of the R. idaeus cultivars represent the tricolporate type. The length of the polar (P) and equatorial (E) axes in the pollen grains from the biennial fruiting cultivars ranged from 23.8 ('Radziejowa') to 28.3 µm ('Laszka') and from 21.4 ('Radziejowa') to 27 µm ('Radziejowa'), respectively. In the other group of the cultivars, the mean length of the polar axis in 'Pokusa', 'Polana', and 'Polka' was in the range of 25.4 ('Polana')-29.5 µm ('Polka'). In turn, the length of the equatorial axis ranged from 22.8 ('Polana') to 28.6 µm ('Polka'). Based on the size of the longer axis of the sporomorphs in the R. idaeus cultivars, the pollen grains from the first group of cultivars were classified as small and medium, but they were medium in the second group. The mean value of the calculated shape coefficient P/E indicated that the pollen grains of the analyzed cultivars represented prolato-spheroides in terms of the shape (P/E=1.01-1.14). Additionally, as indicated by the value of this coefficient in the subsequent years of the study, a part of prolato-spheroides shape, pollen grains of subprolatum shape (P/E = 1.17) were detected in 'Laszka', 'Polana', and 'Polka' (Table 1). Means followed by the same small letter are not significantly different within the cultivar for the years and means followed by the same capital letter do not differ between the cultivars at a significance level α = 0.05; SD standard deviation.

Exine Sculpture
The examined R. idaeus cultivars had a striated exine with visible perforations ( Figure 1A-F, Figure 2A-F, Figure 3A-F, Figure 4A-F, Figure 5A-F, Figure 6A-F). In the polar view, the pollen grains had a triangular outline. There were colpi and pori in the equatorial plane. The colpi were arranged longitudinally. The striae formed a cultivar-specific pattern. On the surface of the apocolpium, they were arranged side by side with a delicate arcuate curve in 'Glen Ample', 'Radziejowa', and 'Polka' ( Figure 1A, Figure 3A, Figure 6A). They were arranged in parallel in 'Laszka' and 'Polana' (Figure 2A, Figure 5A) or intertwined in 'Pokusa' ( Figure 4A) towards the mesocolpium.
Plants 2020, 9, x FOR PEER REVIEW 6 of 31 'Radziejowa' was significantly higher than the value of this parameter in 'Glen Ample' and 'Laszka'. The value of this parameter in pollen grains of 'Pokusa', i.e., a repeated fruiting cultivar, was significantly lower than in 'Polana' and 'Polka'. The comparison of the biennial and repeated fruiting cultivars showed a significantly lower number of perforations per 10 µm 2 of exine than in the other cultivars (Table 3).  (arrow); E-colpus and porus in the equatorial plane, visible colpus reaching the distal and proximal poles; F-varied diameter of perforations (double-headed arrow), visible muri (arrow). A-F -SEM.         In the equatorial view, the pollen grains had an elliptical shape ( Figure 1C, Figure 2C, Figure 3C, Figure 4C, Figure 5C, Figure 6C). The muri were arranged along the polar axis. On the mesocolpium surface, the striae in the exine sculpture in all cultivars were arranged side by side; they were sometimes dichotomously branched in 'Glen Ample', 'Polka', and 'Polana' ( Figure 1C,D, Figure 4C,D, Figure Figure 3C). The colpi in the analyzed pollen grains were long and almost reached the poles ( Figure 1E, Figure 2E, Figure 3E, Figure 4E, Figure 5E, Figure 6E).
Striae. The width of the striae in the exine varied ( Figure 1D, Figure 2D, Figure 3D, Figure 4D, Figure 5D, Figure 6D). The parameter ranged from 248 ('Radziejowa') to 417 nm ('Glen Ample') in the biennial fruiting cultivars and from 156 ('Polka') to 203 nm ('Polana') in the other group. The largest and smallest distance between muri in the biennial fruiting cultivars was found in 'Radziejowa' (786 nm) and 'Laszka' (574 nm), respectively. This parameter of the exine of cultivars with repeated fruiting ranged from 440 nm in 'Polka' to 686 nm in 'Pokusa'. The width of muri in exine of 'Glen Ample' was significantly larger than in 'Laszka' and 'Radziejowa'. The distance between muri was significantly lower in 'Laszka' than in 'Glen Ample' and 'Radziejowa'. Width of muri in the repeated fruiting cultivars was significantly larger in 'Polana' than in 'Pokusa' and 'Polka', and the distance between muri in 'Pokusa' was significantly greater than in 'Polana' and 'Polka'.
Perforations. The perforations in the exine had a spherical or elliptical outline and a varied diameter ( Figure 1B,D,E, Figure 2B,D,E, Figure 3B,D,E, Figure 4B,D,E, Figure 5B,D,E, Figure 6B,D,E). These perforations were very small, medium-sized, or large. In the biennial fruiting cultivars, the smallest diameter of the large perforations was found in 'Glen Ample' (118-152 nm) and the biggest diameter was exhibited by 'Laszka' (249-324 nm). In turn, the smallest and biggest values of this parameter in the cultivars with repeated fruiting were measured in 'Polka' (131-173 nm) and 'Polana' (194-269 nm), respectively. In the group of the biennial fruiting cultivars, the diameter of perforations in the tectum of 'Glen Ample' was significantly lower than in 'Radziejowa' and 'Laszka'. The value of the parameter in the second group cultivars was significantly lower in 'Polka' than in 'Pokusa' and 'Polana'. As shown by the comparative analysis of the biennial and repeated fruiting cultivars, the 'Glen Ample' cultivar had a comparable diameter of perforations to that in 'Polka' and a significantly lower value than in 'Laszka', 'Radziejowa', 'Pokusa', and 'Polana' (Table 3). Perforations in the colpi were usually arranged in series and sometimes formed two rows or were scattered irregularly ( Figure 1B,D,E, Figure 2B,D,E, Figure 3B,D,E, Figure 4B,D,E, Figure 5B,D,E, Figure 6B,D,E).
The number of perforations in a 2-µm long colpus fragment in the examined cultivars was similar (except in the exine of 'Pokusa' pollen) and ranged from 5 ('Glen Ample') to 7 ('Polka'). In turn, there were 39-92 perforations per 10 µm 2 of the exine surface. Considering the increasing number of perforations per exine unit area, the cultivars were ranked as follows (perforations/10 µm 2 ): 'Pokusa' (92). In the group of the biennial fruiting cultivars, the number of perforations in the tectum of 'Radziejowa' was significantly higher than the value of this parameter in 'Glen Ample' and 'Laszka'. The value of this parameter in pollen grains of 'Pokusa', i.e., a repeated fruiting cultivar, was significantly lower than in 'Polana' and 'Polka'. The comparison of the biennial and repeated fruiting cultivars showed a significantly lower number of perforations per 10 µm 2 of exine than in the other cultivars (Table 3). Table 2. Width of muri, distance between muri, and number of muri per unit area of exine in pollen grains of the examined R. idaeus cultivars. Means followed by the same small letter are not significantly different between cultivars differ at significance level α = 0.05. SD -standard deviation. Designations as in Table 2.

Pollen Grain Cell Wall
The transmission electron microscopy observations revealed morphometric differences in the structure of the elements of the ectoexine (tectum, columellae, foot layer) and the endoexine and intine between some of the R. idaeus cultivars examined.
Tectum. A perforated tectum was observed in the exine of the cell wall of pollen grains in the biennial fruiting cultivars ( Figure 7A, Figure 8A,B, Figure 9A) and in the repeated fruiting cultivars ( Figure 10B, Figure 12B). The thickness of this layer in both groups was in the range of 209 ('Laszka')-431 nm ('Glen Ample') and 228 ('Pokusa')-308 nm ('Polana'), respectively. In the group of the biennial fruiting cultivars, pollen grains of 'Glen Ample' was characterized by a significantly higher value of the thickness of the tectum in comparison with 'Laszka' and 'Radziejowa', whereas no significant differences in this parameter were found in the group of repeated fruiting cultivars. The comparative analysis between the biennial and repeated fruiting cultivars demonstrated that the 'Glen Ample' exine had a significantly higher thickness of the tectum than the other cultivars, with statistically confirmed differences between the 'Polana' and 'Laszka'. The thickness of the tectum was significantly higher in 'Polana' than in 'Laszka' (Table 4). Electron-dense spherical pollenkitt was visible on the surface of the tectum and between the columellae of the middle layer ( Figure 7B, Figure 8B, Figure 10B, Figure 11A, Figure 12A).

Pollen Grain Cell Wall
The transmission electron microscopy observations revealed morphometric differences in the structure of the elements of the ectoexine (tectum, columellae, foot layer) and the endoexine and intine between some of the R. idaeus cultivars examined.
Tectum. A perforated tectum was observed in the exine of the cell wall of pollen grains in the biennial fruiting cultivars ( Figure 7A, Figure 8A,B, Figure 9A) and in the repeated fruiting cultivars ( Figure 10B, Figure 12B). The thickness of this layer in both groups was in the range of 209 ('Laszka')-431 nm ('Glen Ample') and 228 ('Pokusa')-308 nm ('Polana'), respectively. In the group of the biennial fruiting cultivars, pollen grains of 'Glen Ample' was characterized by a significantly higher value of the thickness of the tectum in comparison with 'Laszka' and 'Radziejowa', whereas no significant differences in this parameter were found in the group of repeated fruiting cultivars. The comparative analysis between the biennial and repeated fruiting cultivars demonstrated that the 'Glen Ample' exine had a significantly higher thickness of the tectum than the other cultivars, with statistically confirmed differences between the 'Polana' and 'Laszka'. The thickness of the tectum was significantly higher in 'Polana' than in 'Laszka' (Table 4). Electron-dense spherical pollenkitt was visible on the surface of the tectum and between the columellae of the middle layer ( Figure 7B, Figure  8B, Figure 10B, Figure 11A, Figure 12A).  Middle layer. The height of columellae in the ectoexine in the six analyzed raspberry cultivars ranged from 299 ('Laszka') to 403 nm ('Glen Ample'). The thickness of the columellae was in the range from 208 ('Pokusa') to 261 nm ('Polana'). The distance between adjacent columellae in the biennial fruiting cultivars group ranged from 297 ('Laszka') to 407 nm ('Glen Ample') nm, while in the group of cultivars with repeating fruits from 321 ('Pokusa') to 339 nm ('Polana'). In the group of the biennial fruiting cultivars, 'Glen Ample' had a significantly greater distance between the columellae than 'Laszka' and 'Radziejowa' (Table 4). Middle layer. The height of columellae in the ectoexine in the six analyzed raspberry cultivars ranged from 299 ('Laszka') to 403 nm ('Glen Ample'). The thickness of the columellae was in the range from 208 ('Pokusa') to 261 nm ('Polana'). The distance between adjacent columellae in the biennial fruiting cultivars group ranged from 297 ('Laszka') to 407 nm ('Glen Ample') nm, while in the group of cultivars with repeating fruits from 321 ('Pokusa') to 339 nm ('Polana'). In the group of the biennial fruiting cultivars, 'Glen Ample' had a significantly greater distance between the columellae than 'Laszka' and 'Radziejowa' (Table 4).
Foot layer. The foot layer had an irregular outline on the side of columellae and tightly adhered to the endoexine ( Figure 7B, Figure 8A, Figure 9B, Figure 10A, Figure 11A, Figure 12F). This band was the thinnest of the three structural ectoexine layers. Its thickness in the wall of pollen grains of the six analyzed cultivars varied. The value of this parameter ranged from 78 ('Laszka') to 149 nm ('Glen Ample') in the first group of cultivars and from 109 ('Pokusa') to 133 nm ('Polka') in the other group. As shown by the statistical analysis performed in the group of the biennial fruiting cultivars, 'Glen Ample' was characterized by a significantly higher value of the thickness of the foot layer in comparison with 'Radziejowa'. In turn, the value of this parameter in 'Radziejowa' was significantly higher than in 'Laszka'. In the group of the repeated fruiting cultivars, the thickness of the foot layer in the 'Polka' cultivar was significantly higher than the value of this parameter in 'Pokusa' and 'Polana'. The comparative analysis of the biennial and repeated fruiting cultivars revealed that the thickness of the foot layer in 'Glen Ample' and 'Polka' was significantly lower than in 'Radziejowa', 'Pokusa', and 'Polana', but the value of the parameter in these three cultivars was significantly higher than that in 'Laszka' (Table 4). Foot layer. The foot layer had an irregular outline on the side of columellae and tightly adhered to the endoexine ( Figure 7B, Figure 8A, Figure 9B, Figure 10A, Figure 11A, Figure 12F). This band was the thinnest of the three structural ectoexine layers. Its thickness in the wall of pollen grains of the six analyzed cultivars varied. The value of this parameter ranged from 78 ('Laszka') to 149 nm ('Glen Ample') in the first group of cultivars and from 109 ('Pokusa') to 133 nm ('Polka') in the other group. As shown by the statistical analysis performed in the group of the biennial fruiting cultivars, 'Glen Ample' was characterized by a significantly higher value of the thickness of the foot layer in comparison with 'Radziejowa'. In turn, the value of this parameter in 'Radziejowa' was significantly higher than in 'Laszka'. In the group of the repeated fruiting cultivars, the thickness of the foot layer in the 'Polka' cultivar was significantly higher than the value of this parameter in 'Pokusa' and 'Polana'. The comparative analysis of the biennial and repeated fruiting cultivars revealed that the thickness of the foot layer in 'Glen Ample' and 'Polka' was significantly lower than in 'Radziejowa', 'Pokusa', and 'Polana', but the value of the parameter in these three cultivars was significantly higher than that in 'Laszka' (Table 4). The tectum layer, columellae, and foot layer described in the six analyzed cultivars formed an ectoexine with a thickness in the range from 586 nm ('Laszka') to 982 nm ('Glen Ample'). These values accounted for on average 89-92% of the exine thickness (662-1072 nm). As indicated by the statistical analysis performed in the group of the biennial fruiting cultivars, 'Glen Ample' exhibited a significantly higher value of the ectoexine and exine thickness than 'Laszka' and 'Radziejowa'. In turn, in the group of the repeated fruiting cultivars, 'Pokusa' had a significantly lower exine thickness value than 'Polana' and 'Polka'. The comparative analysis of both cultivar groups revealed a significantly higher value of the ectoexine and exine thickness in 'Glen Ample' than that in the other cultivars. 'Laszka', 'Radziejowa', and 'Pokusa' were characterized by significantly lower ectoexine thickness than 'Polana' and 'Polka'. Furthermore, the 'Polana' and 'Polka' cultivars had a significantly higher value of exine thickness than 'Laszka' and 'Radziejowa', and the exine thickness in 'Polka' was higher than in 'Pokusa'. Simultaneously, the endoexine thickness in 'Polka' was significantly higher than in the other cultivars (Table 5).  Endoexine. The endoexine layer in the pollen grains of the studied cultivars ranged from 76 nm 'Laszka' to 108 nm 'Polka'. The ultrastructural study of the cross-sections of the pollen grains of the analyzed cultivars showed that the layer had a regular outline. This electron dense layer was darker than the lighter structural elements of the ectoexine and the unstained intine ( Figure 7A,B, Figure 8A,B, Figure 9B, Figure 10A, Figure 12B). The increasing thickness of the exine in the pollen grains ranked the cultivars as follows: 'Laszka' < 'Radziejowa' < 'Pokusa' < 'Polka' < 'Polana' < 'Glen Ample'. As indicated by the statistical analysis performed in the group of the biennial fruiting cultivars, 'Glen Ample' exhibited a significantly higher value of the ectoexine and exine thickness than 'Laszka' and 'Radziejowa'. In turn, in the group of the repeated fruiting cultivars, 'Pokusa' had a significantly lower exine thickness value than 'Polana' and 'Polka'. The comparative analysis of both cultivar groups revealed a significantly higher value of the ectoexine and exine thickness in 'Glen Ample' than that in the other cultivars. 'Laszka', 'Radziejowa', and 'Pokusa' were characterized by significantly lower ectoexine thickness than 'Polana' and 'Polka'. Furthermore, the 'Polana' and 'Polka' had a significantly higher value of exine thickness than 'Laszka' and 'Radziejowa', and the exine thickness in 'Polka' was higher than in 'Pokusa'. Simultaneously, the endoexine thickness in 'Polka' was significantly higher than in the other cultivars (Table 5).  Intine. The thickness of the intine layer in the pollen grains of the studied cultivars varied. This layer in the biennial fruiting cultivars was the thinnest in 'Laszka' (317 nm) and the thickest in 'Glen Ample' (498 nm). In turn, in the group of repeated fruiting cultivars, the thickness of this layer ranged from 229 nm ('Polka') to 541 nm ('Polana'). The thickness of the intine layer accounted for 21-38% of the thickness of pollen grains cells (979-1570 nm). In the group of the biennial fruiting cultivars, pollen grains of 'Glen Ample' exhibited a significantly higher value of intine and cell wall thickness in comparison with 'Radziejowa' and 'Laszka'. In the repeated fruiting cultivars, the intine and cell wall thickness was significantly higher than these parameters in the 'Pokusa' and 'Polka'. The comparative analysis of the biennial and repeated fruiting cultivars revealed a significantly higher value of the intine and cell wall thickness in 'Glen Ample' and 'Polana' in comparison with the other cultivars. The intine thickness in 'Radziejowa' and 'Laszka' was significantly higher than in 'Pokusa' and 'Polka' (Table 5). The tectum layer, columellae, and foot layer described in the six analyzed cultivars formed an ectoexine with a thickness in the range from 586 nm ('Laszka') to 982 nm ('Glen Ample'). These values accounted for on average 89-92% of the exine thickness (662-1072 nm). As indicated by the statistical analysis performed in the group of the biennial fruiting cultivars, 'Glen Ample' exhibited a significantly higher value of the ectoexine and exine thickness than 'Laszka' and 'Radziejowa'. In turn, in the group of the repeated fruiting cultivars, 'Pokusa' had a significantly lower exine thickness value than 'Polana' and 'Polka'. The comparative analysis of both cultivar groups revealed a significantly higher value of the ectoexine and exine thickness in 'Glen Ample' than that in the other cultivars. 'Laszka', 'Radziejowa', and 'Pokusa' were characterized by significantly lower ectoexine thickness than 'Polana' and 'Polka'. Furthermore, the 'Polana' and 'Polka' cultivars had a significantly higher value of exine thickness than 'Laszka' and 'Radziejowa', and the exine thickness in 'Polka' was higher than in 'Pokusa'. Simultaneously, the endoexine thickness in 'Polka' was significantly higher than in the other cultivars (Table 5).   Designations as in Table 2.
Size of grains. The pollen grains in the analyzed R. idaeus cultivars were classified as small or medium, as indicated by the length of the longer axis of the sporomorphs. A similar size of pollen grains has been described in other species of the genus Rubus, e.g., in R. acuminatissimus, R. chrysogaeus, R. clementis, R. diclinis, R. ellipticus, R. ferdinandi-muelleri, R. fraxinifolius, R. megacarpus, R. montis-wilhelmi, R. niveus, R. novoguineensis, R. papuanus, R. pectinellus, R. royenii var. ikilimbu, and R. trigonus. Medium-sized pollen grains were detected in e.g., R. copelandii, R. chamaemorus, R. calycinus, R. alpestris, and R. archboldianus [74,75,98]. The variation in the pollen grain size may result from the pollination and habitat conditions, whereas the pollen grain transport conditions have been found to play a negligible role in the evolution of the pollen size in species pollinated by bees and birds. The change in the grain size has been shown to influence pollen germination, pollen tube growth, and fertilization [107,108]. The pollen grain size may be modified by the content of nitrogen and phosphorus in the soil. Higher concentrations of these elements have been shown to increase the size, yield, and germinability of grains and enrich their chemical composition. It was also found to have a positive effect on the number of produced seeds [109]. In turn, it has been demonstrated that defoliation of shoots reduces pollen grain size, inhibits the growth of pollen tubes, and reduces the number of seeds [110]. Pollen morphology is affected by climate change. There is a documented relationship between reduced water availability and an increased grain size, which is regarded as an indicator of long-term climate change [111].
Drought stress during flowering of taxa from the family Rosaceae was reported to reduce the amount of produced pollen, increase the pollen grain size, and slightly change their shape. In such conditions, the pollen grains of Rubus and Rosa plants were elongated and larger. Such features enhance the chances of survival and increase pollen germination rates [112].
Shape of pollen grains. The pollen grains in the analyzed cultivars had the shape of prolato-spheroides; or occasionally subprolatum. The first type of shape was also described in R. chrysoageus, R. macgregrii, R. moluccanus var. trilobus, R. montis-wilhelmi, R. novoguineensis, R. papuanus, R. royenii, and R. trigonus [75]. Subprolatum and spheroides types were identified frequently in Rubus plants, whereas the oblato-spheroides shape was rarely observed [79]. The shape and brightness of grains have been used as descriptors in the computer classification of pollen based on their morphology and ornamentation. The size and shape of grains are palynological data used, e.g., for automatic identification of a given taxon, often in combination with sculpture analysis [113,114].
Striae. The striae in the exine sculpture in the pollen grains of the biennial fruiting cultivars ('Glen Ample', 'Laszka', and 'Radziejowa') were approximately 1.5-2 fold wider than in the repeated fruiting cultivars. The width of the exine striae in 'Radziejowa' and 'Polana' was similar to that reported in the literature for R. alpestris and R. pedemontanus (200-250 nm), whereas in the range specified for R. macgregorii and R. nessensis was 100-380 nm [64,75]. The value of this parameter in the exine of 'Polka', 'Pokusa', and 'Laszka' was similar but 'Glen Ample' higher to that of R. macgregorii and R. nessensis (100-380 nm) but lower than in R. megacarpus (600-800 nm) [64,74]. In turn, the width of the striae in the other cultivar group was similar to that reported for R. acuminatissimus, R. armeniacus, R. laciniatus, and R. fabrimontanus (160-180 nm) [64,75]. There were 8-12 striae per 10 µm of exine surface area in the cultivars examined in this study. The longitudinal axis of adjacent striae was located at a distance of 440-786 nm. As reported by Tomlik-Wyremblewska et al. [64], the width of colpi between striae in the exine of R. armeniacus, R. hirtus, and R. pedemontanus ranged between 100 and 400 nm.

Ultrastructure of Pollen Grains
Using a transmission electron microscope, a detailed comparison of the cell wall of pollen grains of the six studied R. idaeus cultivars was carried out for the first time. The tectum in the analyzed cultivars was perforated and had a thickness of 209-431 nm. A perforated tectum was observed in other Rubus species as well [66,74,75,[115][116][117][118][119][120][121][122].
The columellae present between the tectum and the foot layer in the examined grains were located at a distance of 297-407 nm. They were between 299 and 403 nm high and 208-261 nm thick. Ulrich et al. [123] have reported that the ectoexine is composed of the tectum, infratectum, and foot layer. As shown by Doyle and Endress [124], the infratectum can be granular, intermediate, and columellar. The first form is characteristic for pollen grains of gymnosperms but also occurs in some angiosperm species, mainly as the columellar type [125]. The structural part of the ectoexine, i.e., the foot layer, in the grains of the analyzed cultivars formed a 78-149 nm thick continuous layer. As reported by Ulrich et al. [123], this layer can be either continuous, discontinuous, or absent. The structureless ectoexine in their study formed a continuous layer, and its thickness constituted 8-13% of the thickness of the exine layer. These authors indicate that the endoexine may be continuous or discontinuous, spongy or compact, sometimes present only in apertures, or absent.
The thickness of the exine in the grains was lower in 'Laszka', 'Radziejowa', 'Pokusa', and 'Polka' were close to the values, but in the case of 'Polana' it was in the range of values determined in other Rubus species (0.9-2.4 µm) [64,73,74,78,79,105]. Sporopollenin contributes to high exine stability. It consists of oxidizing polymers of carotenoids, polyunsaturated fatty acids, and phenolic compounds [126]. An analysis of the two structural layers of the exine revealed that the endoexine is more resistant to oxidation than the ectoexine. This is determined primarily by the reduction-resistant sporopollenin accumulated on tuft units originating from plasma membrane glycocalyx rather than the secondary sporopollenin accumulated in the ectoexine [127]. Sporopollenin has a common aliphatic core and various taxon-dependent aromatic side chains [126]. Genes responsible for acyl-CoA-derived tetraketide components are involved in its synthesis [128]. One of them is the LAP3 gene involved in the formation of the normal exine structure and development of pollen. A mutation in this gene was found to lead to formation of a thin exine, disturbances in its sculpture, and metabolic changes in pollen grains [129].
The intine layer in the pollen of the examined cultivars constituted 21-38% of the thickness of the grain cell wall. The intine is usually thicker and composed of two layers at the aperture site [123]. The pectate lyase-like 9 gene (BcPLL9) is involved in intine formation [130]. The structure of intine comprises mainly cellulose, pectins, and callose [123,[131][132][133]. The former polysaccharide, which is evenly distributed on the intine surface, is a pollen tube wall progenitor [132,133]. It is formed with the involvement of intine components, e.g., hydrolytic enzymes and hydrophobic proteins maintaining the structural compactness of pollen grains. These substances are necessary for recognition of the receptivity of stigmata and further penetration of the pollen tube through the pistil style [134][135][136].
Numerous plastids, sometimes containing starch granules, were observed in the intine-adhering protoplast of the pollen of the analyzed R. idaeus cultivars. Plastoglobules, mitochondria, and Golgi apparatus were visible in the electron-dense cytoplasm. As reported by Pacini et al. [137], this polysaccharide accumulates in pollen grains during development and persists until the last phase of pollen maturation. Next, before anther dehiscence, it is converted into pectin, glucose, fructose, sucrose, and other sugars. The function of starch is associated with the use of pollen by pollinators. Pollen grains with amyloplasts contain lower content of lipid compounds than starchless pollen. Starch also serves a protective function against pollen grain drying [138].
However, the carbohydrate metabolic process varies: the compounds can be utilized directly, transformed into other molecules, or polymerized to form intine, or stored as starch. During pollen development, the amylogenesis cycle may be specific for each species. Starch in pollen grains can be used as a reserve formed through partial hydrolysis. Carbohydrates are utilized as a raw material necessary during pollen development. In the final phase, they accumulate, thus contributing to the ability of pollen to spread spontaneously [136].

Materials and Methods
The research conducted in 2017, 2018, and 2019 involved six cultivars of R. idaeus that are commonly produced in commercial plantations and have been included in the national register. The investigations were conducted on three biennial fruiting cultivars: 'Glen Ample', 'Laszka', and 'Radziejowa' and three repeated fruiting cultivars: 'Polka', 'Polana', and 'Pokusa'.

Study Area and Plant Material
The raspberries were grown on a commercial plantation in Blinów II (50 • 52 57.03" N; 22 • 23 2.663" E), Szastarka commune, Lublin Province, in the south-eastern part of Poland. The shrubs grew at a 0.5 × 2.8 m spacing. Fertilization and protection agents were used in accordance with the raspberry cultivation recommendations. Flowers, i.e., the source of the pollen grains, were collected three times from each cultivar at the initial stage of flowering to determine the morphological, micromorphological, and ultrastructural characteristics of pollen.

Fixation of the Material
Stamens with pollen were collected from the flowers at the initial stage of flowering (the bud burst phase). The anthers were fixed in 4% glutaraldehyde for 6 h at room temperature. Afterwards, the samples were treated with 0.01 M phosphate buffer, pH 7.0, for 48 h at 4 • C.
Different methods were employed depending on the intended use of the sample for (i) light field light microscopy, (ii) scanning electron microscopy, and (iii) transmission electron microscopy.

Light Microscopy (LM)
Pollen for the analyses was sampled three times from 30 randomly flowers of the six cultivars in the consecutive study years. After washing out from anthers with 70% ethyl alcohol, the pollen grains were stained with a 1% safranin solution and sealed in a 50% glycerin solution [139,140]. The microscopic slides were analyzed with a light microscope by measuring the equatorial (E) and polar (P) axes in the light microscope. Comparative observations of the morphology of the pollen grains were carried out using a Nikon Eclipse 90i bright field microscope (Nicon Instruments INC, Tokyo, Japan).

Scanning Electron Microscopy (SEM)
Fixed samples of anthers with pollen grains were dehydrated in a successive 15, 30, 50, 70, 90, and 99.5% acetone series for 15 min at room temperature and twice in anhydrous acetone. Next, the pollen grains were critical point dried in liquid CO 2 using an Emitech K850 dryer (Quorum Technologies Ltd., Ashford, United Kingdom). Dried grains were transferred onto the microscope stage and sputter-coated with gold using an Emitech K550X device (Quorum Technologies Ltd., Ashford, United Kingdom). Observations of the surface of pollen grain sculpture (striae, perforations, microstriae) and photographic documentation were made using a Tescan Vega II LMU scanning electron microscope (SEM) (Tescan Orsay Holding, Brno, Czech Republic). The microscope was used for analysis of 100-150 pollen grains from each cultivar.

Transmission Electron Microscopy (TEM)
The fixed anthers with pollen grains were contrasted in a 1.5% osmium tetroxide solution for 1.5 h. After rinsing with distilled water, a 0.5% aqueous uranyl acetate solution was applied for 2 h at room temperature. After rinsing with distilled water twice, fragments of anthers and pollen grains were dehydrated for 15 min in a series of ethyl alcohol at subsequent concentrations of 15,30,50,70,90,96, and 99.8% and twice in anhydrous ethanol. Dehydrated samples were embedded in Spurr Low Viscosity resin and polymerized at 60 • C for 48 h. The resin-embedded plant material was cut with a glass knife into half-thin sections with a thickness of 1 µm using a Reichert Ultra Cut S microtome; next, the material was contrasted with osmium tetroxide and mounted in Eukit.
In turn, ultrathin sections of the anther with pollen grains with a thickness of 70 nm were stained with an 8% solution of uranyl acetate in 0.5% acetic acid for 40 min.
After rinsing with distilled water twice for 5 min), Reynolds reagent was applied for 15 min [141]. The sections were rinsed with water again (twice for 5 min) and dried. The structural elements of the ectoexine (tectum, middle, and foot layers), endoexine, intine, and protoplast walls were carried out with the use of the FEI, Tecnai G2 Spirit transmission electron microscope (TEM) (FEI Company, Hillsboro, OR, USA). The comparative analyses of the pollen grain ultrastructure were performed on live images and microphotographs of 3-4 ultrathin sections from each of the 12 blocks in each of the six cultivars. In each block, 2-4 stamen heads were embedded.

Morphometric Measurements
The pollen grains of the R. idaeus cultivars were characterized by assessment of (1) the length of the equatorial (E) and (2) polar axes (P) (n = 150). Based on these values, (3) the shape index (P/E) was calculated, and (4) the grain shape and (5) size were determined following the classification proposed by Erdman [142] as well as Dybova-Jachowicz and Sadowska [143]. The study of the micromorphology of the exine sculpture in the pollen grains consisted in comparison of (6) the width of striae, and (7) colpi (n = 30). The number of (8) striae and (9) perforations per unit area (10 µm 2 ) as well as (11) the number of perforations per 2-µm colpus fragment were calculated. The assessment of the cell wall ultrastructure consisted in measurements of the thickness of the following elements: (12) tectum, (13) middle layer, (14) foot layer, (15) ectoexine, (16) endoexine, (17) exine, (18) intine, and (19) cell wall. Additionally, the thickness (20) and height (21) of columellae and (21) the distance between adjacent columellae (n = 30) were measured. The morphometric measurements were carried out using Nikon NIS-Elements version 3.0 Advance Research microscopic image analysis software.

Statistical Analysis of Results
The mean results of the morphometric measurements of the micromorphological parameters of the exine sculpture and ultrastructure of pollen grains as well as the standard deviation were calculated using the Microsoft Excel 2013 program. The significance of differences between the examined pollen characteristics was statistically analyzed using the statistical software Statistica 6.0. The differences between the selected traits were evaluated using one-way ANOVA analysis of variance. Statistical inference was carried out at the significance level α < 0.05.

Conclusions
The pollen grains of the six R. idaeus cultivars studied were classified as small or medium, and their shape was classified as prolato-spheroides or subprolatum. In the striated grain sculpture, adjacent striae in the equatorial position were sometimes dichotomously branched in 'Glen Ample', 'Polka', and 'Polana', arcuate in 'Laszka' and 'Pokusa', and irregularly arranged and overlapping in 'Radziejowa'. In comparison with the pollen grains of the biennial fruiting cultivars as compared to the repeated fruiting cultivars, the width of striae and colpi on the exine was 46% and 23%, larger, respectively, and the number of muri per unit area was 14% higher. In terms of the increasing number of perforations per unit area, the cultivars are ranked as follows: 'Pokusa' < 'Glen Ample' < 'Laszka' < 'Polka' < 'Polana' < 'Radziejowa'. The ultrastructure analysis demonstrated the thickest tectum in 'Glen Ample', the highest and thickest columellae with the largest distance between them in 'Polka', and the thickest foot layer in 'Polana'. The ectoexine in the pollen grains accounted for approximately 89-92% of the thickness of the exine, whereas the intine layer represented 21-38% of the thickness of the grain wall. The present investigations may be helpful indicators for identification of related taxa of the genus Rubus.
The micromorphological and ultrastructural traits of the exine in the pollen of the six cultivars of R. idaeus documented in the present study may be useful for not only botany, biology, and palynology specialists but also for a wider audience in the field of forestry, horticulture, medicine, apiculture, and sciences related to nutrition and environmental protection.
The parameters of exine sculpture can be used as taxonomic indicators in many fields of science, e.g., in paleoecology and biogeography, archaeological excavations, and interpretation of phylogenetic relationships in the genus Rubus. They can also be used in innovative research programs to identify pollen supported by chemotaxonomy and DNA barcoding of plant genomes. These indicators can be useful in practice, e.g., in forensics, biochemistry, biology of the pollination process, assessment of mutualistic relationships, monitoring pollen allergen concentrations, and identification of pollen-based dietary supplement products. They can also be applied in innovative nanotechnology to form microcapsules and natural polymers.
Future research should be focused on micromorphological and ultrastructural analysis as well as microanalysis of pollen in many groups of Rubus cultivars in view of their use in cultivation work on, e.g., genetic diversity and resistance to biotic factors and climate change. Investigations should also focus on invasive food processing and nanotechnology as carriers of phytochemicals in pharmacy and cosmetology.