J. AMER.SOC.HORT.SCI. 141(3):275–284. 2016. Pollen Morphology of Herbaceous Peonies with Different Ploidy Levels Lihong Hao and Hui Ma College of Landscape Architecture, Beijing Forestry University, Beijing, 100083, P.R. China Jaime A. Teixeira da Silva P.O. Box 7, Miki-cho post office, Ikenobe 3011-2, Kagawa-ken, 761-0799, Japan XiaoNan Yu1 College of Landscape Architecture, Beijing Forestry University, Beijing, 100083, P.R. China; and Beijing Key Laboratory of Ornamental Plants Germplasm Innovation and Molecular Breeding, National Engineering Research Center for Floriculture, Beijing, 100083, P.R. China ADDITIONAL INDEX WORDS. Paeonia ABSTRACT. The pollen morphology of 26 cultivars of herbaceous peony (Paeonia L.) was examined by scanning electron microscopy (SEM). Pollen grains of the cultivars were mostly subprolate to perprolate, tricolporate, or ellipsoidal in equatorial view, but rounded-trifid in polar view. The poles of pollen grains were mainly flat when viewed in cross-section and only a few appeared to have a circular or arc shape. In addition, the surface of the pollen grains was psilate, and the muri were partly or fully protuberant with a reticulate surface sculpture. However, the shapes and sizes of pollen grains as well as surface ornamentation varied significantly among cultivars with different chromosome numbers. The pollen grains from diploid cultivars were regular and full, and the reticulation holes were evenly distributed, while most of the pollen grains from triploid cultivars were empty flat shells with some heteromorphic pollen whose surface ornamentation was mostly rugulate-reticulate exine. The pollen grains from tetraploid cultivars were the largest although pollen morphology and surface ornamentation differed between Athena and Cream Delight. Six indices [polar axis length (P), equator axis length (E), P/E, pollen perforation diameter (D), ridge width (W), and D/W] were used in Q cluster analysis, which divided the 26 cultivars into three groups. Group I included eight diploid, four triploid, and one tetraploid cultivar. Group II included 12 triploid cultivars with complex genetic backgrounds. Group III included only one tetraploid cultivar Cream Delight. This work may provide an important palynological basis for studying taxonomy and hybrid breeding of herbaceous peony cultivars. Herbaceous peonies, famous ornamental plants of the genus herbaceous peony pollen is thus likely be more stable, and can Paeonia (Paeoniaceae), have more than 1000 years of cultiva- reflect common characteristics of a genus or family and define tion history around the world. Herbaceous peonies are usually plant origin and evolutionary relationships (Zhang et al., 2014). divided into three distinct groups based on their provenance Wang (2006) indicated, in studies of tree peony (Paeonia around the world, the Chinese Peony Cultivar Group [Lactiflora decomposita Hand.-Mazz.) and herbaceous peony (Paeonia Group (LG)], the European Peony Cultivar Group [Officinalis intermedia C.A. Mey.), that pollen sterility is directly related Group (OG)], and the Hybrid Peony Cultivar Group to its size. In the genus Rosa L., Ma and Chen (1991) [Hybrid Group (HG)] (Li, 1999; Qin, 2004). Paeonia lactiflora confirmed the genetic relationship between hybrid offspring Pall. is the parental species of LG, which primarily includes and their parents by comparing pollen exine ornamentation. diploid cultivars (2n =2x = 10). The parents of OG are mainly To date, many researchers have paid more attention to tree Paeonia officinalis L. (2n =4x =20),Paeonia tenuifolia L. (2n = peony than to herbaceous peony. As a result, there is little 2x = 10), and Paeonia anomala L. (2n =2x = 10); whereas the research on the genetic diversity in section Paeonia (Yu et al., parents of HG includes P. officinalis (2n =4x = 20), Paeonia 2012). Xi (1984) observed few differences in the pollen macrophylla (Albov) Lomakin (2n =4x = 20), and Paeonia morphology of nine Paeonia species (Paeonia delavayi coriacea Retz. (2n =4x = 20), among others (Hong et al., 2010). Franch., Paeonia szechuanica Fang., Paeonia mairei H. Lev., As the structure of angiosperm pollen is weakly affected by the Paeonia anomala, P. lactiflora, Paeonia suffruticosa Andr., environment (Salmaki et al., 2008; Walker, 1974) the structure of Paeonia veitchii Lynch., Paeonia obovata Maxim., and Paeonia sinjiangensis K.Y. Pan.) when examined by light microscopy (LM) and SEM. In palynological studies, exine ornamentation Received for publication 28 Dec. 2015. Accepted for publication 1 Mar. 2016. examined by SEM is usually used to identify a species. Guo et al. This work was financially supported by the National Natural Science Founda- (2005) found a close relationship between P. veitchii, P. obovata, tion of China (31400591) and Co-building Program of Beijing Municipal Commission of Education (BLCXY201527). P. sinjiangensis,andP. anomala var. intermedia. Jin et al. (2005) We thank Prof. David Byrne from Texas A&M University for providing helpful found that each of 15 P. lactiflora cultivars showed distinct advice and edits about this paper. pollen morphology when examined with SEM. Jaime A. Teixeira da Silva and Xiaonan Yu planned and devised the Three methods are most frequently used to study pollen experiments. Lihong Hao and Hui Ma analyzed the pollen. All authors developed all drafts of the manuscript, approved it for publication, and take morphology: LM, SEM, and transmission electron microscopy public responsibility for its content. (TEM). TEM is often used to observe the ultrastructure to 1Corresponding author. E-mail: [email protected]. explore the structure and composition of the pollen wall. For J. AMER.SOC.HORT.SCI. 141(3):275–284. 2016. 275 Table 1. Chromosome number, flower color, blooming date (2013), group, and origin of the 26 herbaceous peony cultivars used in this pollen morphology research. Code Cultivar Ploidyz Flower color Blooming date Group Origin, country A Scarlett O’Hara 2n =2x = 10 Red 31 Apr. Hybrid Glasscock Falk, the United States B Paula Fay 2n =2x = 10 Pink 5 May Hybrid Fay, the United States C Alexander Fleming 2n =2x = 10 Pink 2 May Lactiflora Blonk, The Netherlands D Nippon Beauty 2n =2x = 10 Dark red 10 May Lactiflora Auten, the United States E Sarah Bernhardt 2n =2x = 10 Pink 9 May Lactiflora Lemoine, France F Kansas 2n =2x = 10 Bright red 11 May Lactiflora Bigger, the United States G Charle’s White 2n =2x = 10 White 8 May Lactiflora Klehm the United States H White Wings 2n =2x = 10 White 11 May Lactiflora Hoogendoorn, The Netherlands I Buckeye Belle 2n =3x = 15 Dark red 6 May Hybrid Walter Mains, the United States J Brightness 2n =3x = 15 Brilliant red 15 May Hybrid Glasscock, the United States K Red Red Rose 2n =3x = 15 Red 27 Apr. Hybrid Saunders, the United States L Flame 2n =3x = 15 Coral 28 Apr. Hybrid Glasscock, the United States M Halcyon 2n = 3x = 15 Pure white 11 May Hybrid Saunders, the United States N Apache 2n =3x = 15 Red 20 May Hybrid Wolfe-Bigger, the United States O Nosegay 2n =3x = 15 Rose pink 7 May Hybrid Saunders, the United States P Roselette 2n =3x = 15 Pink 8 May Hybrid Saunders, the United States Q Old Faithful 2n =3x = 15 Dark red 10 May Hybrid Glasscock, the United States R Picotee 2n =3x = 15 White 28 Apr. Hybrid Saunders, the United States S Pink Teacup 2n =3x = 15 Pink 30 Apr. Hybrid Hollingsworth, the United States T Coral ’n Gold 2n =3x = 15 Rose coral 5 May Hybrid Klehm, the United States U Laddie 2n =3x = 15 Bright red 3 May Hybrid Glasscock, the United States V John Harvard 2n =3x = 15 Dark red 4 May Hybrid Auten, the United States W Paladin 2n =3x = 15 Pink 5 May Hybrid Saunders, the United States X Pink Hawaiian Coral 2n =3x = 15 Pink coral 5 May Hybrid Klehm, the United States Y Athena 2n =4x = 20 Ivory 5 May Hybrid Saunders, the United States Z Cream Delight 2n =4x = 20 Cream 10 May Hybrid Reath, the United States zPublished in Ma (2014). example, Tellerıa et al. (2013) and Xu and Ronse de Craene 2010 (Autodesk, San Rafael, CA). Measurements included P, (2013) used TEM to study the ultrastructure of the pollen wall E, P/E, D, W, and D/W. Experimental data were analyzed with in Compositae and Annonaceae, respectively. A limitation of SPSS (version 18.0; IBM, Armonk, NY), and the Duncan’s LM observations is the level of detail in pollen grain surface multiple range test (P < 0.05) was used for mean separation. In sculpture that is discernable. The detail that is available from addition, the partial correlation I between these six indexes was SEM is often required for taxonomic studies (Kodela, 2006). In analyzed. this study, the pollen morphology, including shape, size, and The P · E value represents the size of pollen grains, whereas surface sculpture, of 26 herbaceous peony cultivars with P/E represents pollen shape. If P/E > 2, it is perprolate, and if different ploidy levels were examined to provide a useful 1.14 < P/E < 2, it is subprolate (Wang and Wang, 1983). Pollen palynological basis for studying kinship and taxonomy among characteristics were observed in equatorial view as well as in peonies and to advance hybrid peony breeding. polar view, including pollen shape, the type of perforations and exine ornamentation, which were all based on SEM images. Materials and Methods Descriptive palynological terminology follows that used by Erdtman (1943, 1952, 1969) and Punt et al. (2007). Finally, PLANT MATERIALS. Fresh pollen grains of the 26 cultivars based on the data of six indices (P, E, P/E, D, W, and D/W), cultivated at the experimental farm of the Beijing Forestry Q cluster analysis within SPSS was used to develop a University were collected from mature stamens in Apr. and dendrogram. May 2013 (Table 1). Most of the cultivars introduced from United States were cultivated in Beijing for more than 3 years.