Hydrangea macrophylla is a deciduous shrub of Hydrangea genus in the family of Saxifragaceae, which is also known as Xiuqiuhua and Ziyanghua in Chinese. The inflorescence of Hydrangea macrophylla is large and colorful, which is of ornamental value. It can be used as a bouquet of flowers or dried flowers decoration. It is also an excellent green tree species. In recent years, it has been introduced and cultivated in large numbers in China, and has gradually become a "popular" plant, which is used in all kinds of theme flower shows.

At present, the research of Hydrangea macrophylla mainly focuses on in vitro culture, chemical composition separation and identification, cultivation management and so on (Ren, 2018, Journal of Green Science and Technology, 56(13): 165-167). However, there are few reports on the pollen morphological characteristics of Hydrangea varieties. It is recorded that there are more than 600 Hydrangea varieties, and many hybrids. It is difficult to verify their parents after multiple generations of reproduction, and even the classification of some Hydrangea varieties (such as Hydrangea paniculata 'Grandiflora' and Hydrangea serrata Ser.) has been controversial, resulting in confusion in plant naming and defects in the classification system (Zeng et al., 2018). The palynology study on the interspecific relationship of Hydrangeoideae indicated that the morphological traits of pollen, the ornamentation of pollen outer wall surface and secondary ornamentation of Hydrangeoideae verities had great reference value for species classification, and the study on pollen outer wall hierarchical structure and germination pores was helpful to further understand the evolution of pollen traits and intrageneric grouping of this subfamily (Chen, 1954; Hao and Hu, 1996; Zhang et al., 2019). Studies on pollen morphology of Hydrangea genus and its related genera showed that the taxonomic value of pollen morphology of Hydrangea genus and its related genera is mainly reflected in the definition of species (Zhang et al., 2019). Therefore, in this study, ten Hydrangea varieties were used as materials to observe the differences in morphological characteristics of pollen grains of different varieties and different fertility with scanning electron microscope (SEM), so as to further clarify the significance of morphological characteristics of pollen grains of Hydrangea macrophylla in taxonomy and provide scientific basis for the study of genetic diversity of Hydrangea macrophylla.

1 Results and Analysis

1.1 Population characteristics of pollen grains of ten Hydrangea varieties

The results of SEM (Figure 1; Figure 2; Figure 3) showed the number of pollen grains of Hydrangea macrophylla is single, and the number of pollen grains of different Hydrangea varieties is different. Pollen grains could be observed in all varieties of fertile flowers, but the sterile pollen grains of varieties ‘Preziosa’, ‘Mona Lisa’, ‘Headdress flower’ and ‘Komachi’ cannot be observed, which may be related to the small sterile buds of varieties. The pollen grains of fertile flowers of ‘Komachi’ were very few. The pollen grains of ‘Preziosa’, ‘Headdress flower’, and ‘Porpcorn’ were also relatively small, which may be related to flower type.

In the field of vision, there were different number of abnormal pollen grains in different varieties of Hydrangea macrophylla. The number of abnormal pollen grains in fertile flowers ‘Maman rose’, ‘Endless Summer’, and ‘Mona Lisa’ were the largest, which may be related to the small anthers and less nutrients for their growth and development. Abnormal pollen grains in the field of vision showed the irregular shape of shriveled and wrinkled, or the germination ditch was deeply depressed downward, which was the phenomenon of pollen degradation or pollen abortion, and may be one of the reasons for male sterility of Hydrangea macrophylla.

The normal pollen morphology of ten Hydrangea varieties has the following common characteristics: the appearance is prolate, and the equatorial plane has midday germination ditch. However, the size of pollen grains, germination ditch depth and ridge shape of different varieties were different.

1.2 Analysis of pollen characteristics of fertile flowers of ten Hydrangea varieties

1.2.1 Morphological characteristics of pollen grains of ten fertile varieties

The pollen appearance of Hydrangea macrophylla is prolate, and isopolar, radially symmetrical, with equatorial three-hole ditch, and are midday long groove, groove extends to the two extremes do not converge on the polar plane. Pollen outer wall have hole pattern, according to NPC classification system, it belongs to N3P4C5 type pollen. Based on the L/W ratio, the pollen grains of the tested varieties were prolate and the pollen grains of ‘Porpcorn’ and 'Otaksa' were perprolate, which were significantly different from other varieties.

There were significant differences in pollen grain size and morphology among varieties (Table 1). The pollen grains of Hydrangea macrophylla belong to small pollen grains, with length of 16.10~25.50 μm and an average of 17.34~21.74 μm; The width was 7.94~16.90 μm, with an average of 9.61~12.05 μm. Pollen size was expressed by the length of pollen. The order of pollen grains was as follows: ‘Maman rose’ > ‘Porpcorn’ > ‘Headdress flower’ > ‘First love’ > ‘Lanarth’ > 'Otaksa' > ‘Mona Lisa’ > ‘Endless Summer’ > ‘Preziosa’ > ‘Komachi’. The pollen grains of 'Maman rose' and ‘Porpcorn’ were larger than other 8 varieties, and the pollen grains of ‘Komachi’ and ‘Preziosa’ were the smallest, and there were significant differences with other varieties. And there were no significant differences among the other six varieties. The uniformity of pollen grains was expressed by the amplitude of length change. The pollen grains of hydrangea serrata ‘Preziosa’ were the most uniform, and the variation of pollen grain size of 'Maman rose' was the largest among individuals.

The polar shapes of pollen grains were different from each other (Figure 2). The polar shapes of ‘Endless Summer’, 'Otaksa', 'Mona Lisa', ‘Headdress flower’ and 'First love' were three-lobed round, while the other varieties were blunt triangle.

1.2.2 Pollen germination groove characteristics of ten fertile Hydrangea varieties

Pollen grains of ten Hydrangea varieties have 3 germination grooves, parallel to the equator and equidistant distribution. The grooves are long splitting into to the germination grooves of the poles., and no furrows are formed at polar face. The germination groove length and germination groove width of the tested varieties were significantly different (Table 2). The pollen germination groove of ‘Maman rose’ was the longest, reaching 25.60 μm, while the ‘Preziosa’ and ‘Komachi’ were the shortest, which were significantly different from other varieties. But there was no significant difference between ‘Preziosa’, 'Endless summer', 'Otaksa', 'Mona Lisa', ‘Komachi’ and 'First love'. The germination groove width of ‘Maman rose’ were the widest, nearly twice as wide as ‘Preziosa’, which were different from other varieties. While there were no significant differences between 'Endless summer', ‘Headdress flower’, ‘Porpcorn’, ‘Komachi’ and 'Lanarth'.

It could be observed that there were differences in morphological characteristics of germination groove type (Figure 2). The germination groove of hydrangea serrata ‘Preziosa’ was the widest, grooved and deep inward. The germination grooves of the other nine varieties were slit-shaped and deep inward, in which ‘Maman rose’, 'Otaksa', 'Mona Lisa' and ‘Porpcorn’ extending to the middle of the pole.

1.2.3 Pollen pattern features of ten fertile Hydrangea varieties

The outer wall decoration of pollen grains of ten Hydrangea varieties were hole pattern, but the detail patterns of outer wall decoration between different varieties were different, which showed that the perforation distribution density, size and shape were different (Table 3; Figure 2).

From the point of view of the density of the perforation distribution, ‘Maman rose’, 'Otaksa', 'Mona Lisa', ‘Headdress flower’ and ‘Komachi’ have more holes and closely arranged, while 'Lanarth' has the least holes and the sparsest distribution.

Different varieties of pollen outer wall decoration also have differences. Among them, the pollen grain outer wall of ‘Maman rose’, ‘Preziosa’, 'Endless summer' and 'Lanarth' was smooth, while 'Mona Lisa' and ‘Porpcorn’ were wrinkled. According to the different pollen grain perforation shapes, the tested varieties were divided into three categories: (1) Hole pattern, round eyelet: ‘Maman rose’, hydrangea serrata ‘Preziosa’, 'Endless summer', ‘Porpcorn’, ‘Lanarth’ and ‘First love’; (2) Hole pattern, thick stripe eyelets: 'Mona Lisa' and ‘Headdress flower’; (3) Hole pattern, irregular eyelet: 'Otaksa' and ‘Komachi’.

1.3 Analysis of pollen characteristics of infertile flowers of six Hydrangea varieties

1.3.1 Pollen morphological characteristics of infertile flowers of six Hydrangea varieties

The results of SEM showed that only six Hydrangea varieties pollen grains could be observed. The pollen morphology of infertile flowers of six varieties was prolate or ultra-prolate, and the germination groove was equatorial three-hole ditch. There were also significant differences in pollen grain size (represented by length) and morphology of infertile flowers (Table 4). ‘Maman rose’ was the largest, 'Endless summer' was the smallest, which was consistent with the results of fertile flowers. According to the analysis of pollen grain shape by L/W value, it was found that 'Endless summer' and 'Otaksa' were ultra-prolate, and the others were prolate, while the L/W value of ‘Maman rose’ was the smallest, which was significantly different from that of other varieties.

It could be observed that there were differences in the outer wall morphology, perforation shape and density, and germination groove characteristics of pollen grains among the six infertile varieties (Table 5; Figure 3). In which, the perforation of 'Otaksa' was irregular, which was significantly different from other varieties. The characteristics of pollen germination groove of 6 infertile varieties were consistent with that of fertile flowers.

1.3.2 Comparison of the pattern features of fertile and infertile flowers on the six Hydrangea varieties

Among the six Hydrangea varieties, the pollen grains of infertile flowers 'Endless summer' and 'Otaksa' were ultra-prolate, which were different from those of fertile flowers. Analyzed the comparison of the length and width of germination groove, we found that there were significant differences between different fertile flowers of ‘Maman rose’. The outer wall decoration shape and distribution density of the perforation of the same variety were similar, but the polar characteristics were different. Fertile flowers of ‘Maman rose’ have no holes on the extreme surface, but infertile flowers have larger holes, with the large number is and close, and other varieties have no significant differences, indicating that the morphological characteristics of pollen grains of the same variety have little correlation with fertility.

2 Discussion

Pollen is one of the more conservative organs of plants.

Studies have shown that the morphological characteristics of pollen grains, especially the outer wall decoration, size, the length of germination groove, and the size of perforation, could be used as one of the indicators to calculate the evolution degree of species (Ertman, 1978), and could also be used as the basis for inter-species identification. It has been applied to the variety identification of Eucalyptus robusta (Wu et al., 2012), Actinidia chinens (Qi et al., 2017), Paeonia suffruticosa (Wei, 2007), Lonicera japonica (Zhou et al., 2016), Malus spectabilis (Zhang et al., 2018), and Cerasus avium (Wei et al., 2014). Studies on pollen morphology of Hydrangea genus and its related genera showed that the pollen morphology of Hydrangea is complex, and the variation range of pollen morphology covers the variation range of pollen morphology of its related genera. The variation of pollen shape, equatorial surface characteristics, pollen outer wall decoration and pollen size in the species of Hydrangea is relatively stable, while interspecies are obvious (Zhang et al., 2019). Therefore, it is possible to identify the species or varieties of plants in Hydrangea according to the morphological characteristics of pollen.

The study of Lu and Xiong (2010) showed that the perforation size, germination groove length, pollen grain size and decoration characteristics were important shape indexes in the identification and analysis of Chimonanthus praecox varieties by pollen. Chen et al. (2007) suggested that the pollen evolution trend was from small to large, shape evolution was from prolate to ultra-prolate, the longer the pollen grains, the higher the degree of evolution. In this study, we found that the pollen grains of Hydrangea macrophylla were prolate with the help of SEM. Although the pollen grains of infertile flowers 'Endless summer' and 'Otaksa' were ultra-prolate, there was no significant difference, indicating that Hydrangea macrophylla was one of the highly evolved species, and the shape of pollen grains cannot be used as a basis for the identification of these ten Hydrangea varieties. This was inconsistent with previous studies that pollen morphology and size characteristics can be used as a basis for variety identification (Xue et al., 2012; Xiao et al., 2014). This may be due to the complex genetic background of the Hydrangea macrophylla, and the current horticultural varieties are obtained through multiple generations of hybridization.

Many studies have shown that the development process of the pollen outer wall is smooth → small perforation → perforation → retiform → coarse retiform → wrinkled, and the germination groove is developed from three grooves to multi-grooves, to scattered perforation, and whether there are granular substances in the perforation. The data analysis and research are carried out according to the size, shape and decoration of pollen grains, which provides strong evidence for plant classification (Yuan and Wang, 2002; Liu et al., 2013; Chen et al., 2019). In this study, we found that the outer walls of pollen grains of ten Hydrangea varieties were all hole pattern, in which the outer walls of ‘Lanarth’ pollen grains were smooth and sparse. Relatively, the outer walls of ‘Otaksa’, ‘Mona Lisa’, ‘Headdress flower’ and ‘Porpcorn’ pollen grains were dense and wrinkled. Therefore, it was considered that 'Lanarth' is one of the original varieties with thick leaves, strong plants and strong resistance, which is suitable for cross parent.

As far as the width of germination groove is concerned, the germination groove of ‘Preziosa’ is wider, and it is sunken into a boat type, which is significantly different from other varieties. This may be because that the ‘Preziosa’ is the hybrid offspring of Hydrangea serrata Ser. and Hydrangea paniculata 'Grandiflora', and has a distant genetic relationship with other Hydrangea paniculata 'Grandiflora'.

In conclusion, the pollen grain outer wall decoration of different varieties of Hydrangea macrophylla is very different and has a certain correlation with plant morphological characteristics and stress resistance. Although some scholars insisted that pollen morphology cannot be used as a basis for cultivar identification, it must be combined with other traits of plants or biotechnology to obtain the correct results (We, 2007), palynology also has certain reference significance in systematic classification or cultivar identification, which can be used as one of the bases for cultivar identification.

3 Materials and Methods

3.1 Experimental materials

In this study, fresh pollen grains of Hydrangea varieties from the Flower Base of Hunan Agricultural University were used as materials (Table 6).

3.2 Experimental methods

Flower buds that are about to mature and open were collected on a sunny morning and placed in a petri dish with dry filter paper, and the variety name was marked. Then anthers were peeled, and pollen was removed. Fresh pollen was placed under JSM-6390LV scanning electron microscope for photograph observation, photographing, measurement and statistics.

Representative visual fields were selected and photographed with 1 000× (group), 5 000× (individual form, equatorial plane, germinal groove), 10 000× (local, decoration and polar view) and Excel was used to collect data. The length (L), width (W), germination groove length and width were measured by 10 pollen grains of each variety, and the maximum and minimum values were taken. SPSS was used to calculate the average value, standard deviation and DUNCAN multiple comparison analysis. Pollen grain size was represented by length and width product (L×W); the shape of pollen grains was represented by the ratio of length to width (L/W). L/W≥2 was ultra-prolate, 2>L/W>1.5 was prolate, and 1Zhang et al., 2019). Pollen size and morphology, polar characteristics, germination groove characteristics (germination groove length and width of 3 pollen grains per variety) and outer wall decoration were described.

Authors’ Contributions

CHX and ZJW were the experimental designers and executors of this study. CHX and ZJW completed the data analysis, the paper first draft writing. WXM, ZHJ, and ZJF participated in the experimental design and the analysis of experimental results. CHX was the project designer and director, guiding experimental design, data analysis, manuscript writing and revision. All authors read and approved the final manuscript.

Acknowledgments

This study was jointly funded by the National Natural Science Foundation of China (31201656) and the Natural Science Foundation of Hunan Province (2017JJ3105).

References

Yuan T., and Wang LY., 2002, Discussion on the origination of Chinese tree-peony cultivars according to pollen grain morphology, Beijing Linye Daxue Xuebao (Journal of Beijing Forestry University), 24(1): 5-11

Chen H.Y., 1954, Research on the information of Hydrangeoideae, Zhiwu Fenlei Xuebao (Acta Phytotax Sin), 3(2): 108-161

Chen W.W., He X.J., Zhang X.M., and Pu J.X., 2007, Pollen morphology of the genus angelica from Southwest China and its systematic evdution analysis, Xibei Zhiwu Xuebao (Northwestern Journal of Botany), 27(7): 1364-1372

Ertman G., ed., 1978, Handbook of palynology, Paleobotany Research Office, Sporopollen Group, Institute of Botany, Chinese Academy of Sciences, trans., Science Press, Beijing, China, pp.10-60

Hao G., and Hu Q.M.., 1996, A study of pollen morphology of Hydrangeoideae (Hydrangeaceae), Redai Yaredai Zhiwi Xuebao (Journal of Tropical and Subtropical Botany), 4(3): 26-31

Liu Z.H., Xie Q., and Li Z.Q., 2013, Taxonomical status of Pinus tabulaeformis f. shekannesis Yao et Hsü based on morphological characteristics of pollen grain under SEM, Xiebei Linxueyuan Xuebao (Journal of Northwest Forestry University), 28(2): 61-65

Chen Y., Mou Z.S., Zhang Y.H., Yu Y., Wang N., Wang F., Huo Y.L.,Sun K., and Gong G.H., 2019, Discussion on the pollen morphology, evolution and classification of Allium spp. in Changbai Mountains, Jilin Agriculture, (15): 52, 102

Lu J.G., and Xiong G., 2010, Pollen grain characteristics under SEM for classification of Chimonanthus praecox cultivars, Nanjing Linye Daxue Xuebao (Journal of Nanjing Forestry University (Natural Science Edition)), 34(4): 145-148

Qi X.J., Wang R., Lan Y.P., Chen J.Y, Gu H., and Fang J.B., 2017, Morphologic study of pollens of three cultivated Actinidia species by scan-ning electron microscopy, Guoshu Xuebao (Journal of Fruit Science), 34(11): 1365-1373

Wei G.Q., Li F.D., Sun Y.G., Sun Y., and Yang X.H., 2014, Observation of pollen grains of twenty sweet cherry cultivars with scanning electron microscope, Guoshu Xuebao (Journal of Fruit Science), 31(S): 41-47

Wei L., 2007, Pollen morphological comparison of three subshrubby peony species in Paeonia L., Qinghai Daxue Xuebao (Journal of Qinghai University (Nature Science)), 25(6): 52-53

Wu H.X., Liu L.T., Liao B.Y., and Mo X.Y., 2012, Pollen morphology and resuting taxonomic implications of 20 Eucalyptus species and progenies, Zhongnan Linye Keji Daxue Xuebao (Journal of Central South University of Forestry & Technology), 32(3): 29-36

Xiao Y.H., Fu C.N., Ou S.H., Zhang S.N., and Mei Z.M., 2014, Pollen morphology of 6 kinds of pummelo (Citrus grandis L.), Nanfang Nongye Xuebao (Journal of Southern Agriculture), 45(9): 1616-1620

Xue X.M., Wang J.Z., Lu C., and Wang G.P., 2012, SEM observation on the pollen morphology of pollinated species and cultivars in apple, Huanan Nongye Daxue Xuebao (Journal of South China Agricultural University), 33(2): 188-191

Zeng Y., Yang Q.W., and Yu S.J., 2018, Research progress of Hydrangea breeding, Guangdong Nongye Kexue (Guangdong Agricultural Sciences), 45(6): 36-43

Zhang M., Xia C.Y., Fu L.Z., Liu Z.B., and Yu S.X., 2019, Pollen morphology of Hydrangea L. (Hydrangeaceae) and its related genera, Guangxi Zhiwu (Guihaia), 39(3): 297-311

Zhang R., Mao M.M., Liu Z.L., Yang J.M., and Zhang G.J., 2018, SEM observation of pollen morphology of different ornamental crabapple varieties, Fenzi Zhiwu Yuzhong (Molecular Plant Breeding), 16(16): 5407-5414

Zhou J., Bian L.H., Zou L., Zou T.W., Li S.B., and Wang X., 2016, Micro-morphology characteristics of pollen from different strains of Lonicera japonica Thunb., Zhonggua Zhongyiyao Zazhi (Chinese Journal of Traditional Chinese Medicine), 31(2): 484-486)