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Reproductive barriers exist in some water lily crosses that result in low seed set and low breeding efficiency. We investigated pollen morphology, pollen viability, microspore development, pistil receptivity, and embryo and endosperm development in six water lily crosses using paraffin section as well as light and scanning electron microscopy (SEM) techniques. The results indicated that the percentage of pollen with normal morphology ranged from 8.9% to 55.2%. The pollen viabilities of ‘Fen Zhuang’, ‘Bai Lu’, and ‘Hong Ying’ were 33.9%, 3.3%, and 20.7%, respectively. Stigmatic pollen germination peaked at 12 h after pollination and varied from 0.3 to 65.7 grains per stigma among the crosses. The production of embryos with normal morphology ranged from 0% to 43.6% at 5 days after pollination, from 0% to 31.4% at 15 days after pollination, and from 0% to 19.7% by 20 days after pollination. The seed sets of the six crosses were from 0% to 10.9%. Our results suggest that the low seed set in some crosses is the result of low pollen viability, low pistil receptivity, and embryo abortion. Water lilies (Nymphaea spp.) are important ornamental flower plants that are widely cultivated in gardens for the purpose of environment beautification as well as water purification. According to their ecological characteristics, water lilies can be divided into two categories: hardy water lilies and tropical water lilies. Hardy water lilies are divided into five types according to their tubers: marliac, odorata, tuberosa, and finger types as well as underground rhizome (Huang et al., 2009). As the Chinese economy and living standards have rapidly improved, the demand for water lilies with novel traits has increased. Therefore, Chinese breeders have made efforts to develop new cultivars using methods such as artificial interspecific hybridization, mutation techniques, and multiploid approaches. The most widely used method currently is hybridization when developing water lily cultivars. However, the existence of incongruity barriers often makes many water lily crosses difficult, which reduces the breeding efficiency. To date, only a few previous studies have been carried out to investigate this problem, and thus the factors that influence the breeding efficiency of water lily remain unclear. To obtain an understanding of the crossability of water lily cultivars, we studied the chromosome number of three water lily cultivars. The results indicated that they all have a chromosome number of 112 (2n = 8x = 112). In addition, 33 morphological characters of the three water lily cultivars were investigated with the combination of principal component analysis and cluster analysis methods as presented by Zhang et al. (2009). Their results showed that the three cultivars were located close to each other in a phylogenetic tree based on morphological characters obtained through the use of the squared Euclidean distances method, suggesting that they have a close genetic relationship. Thus, the poor seed setting cannot be attributed to the genetic distance among the cultivars. It may be related to pollen quality, pollen germination, or embryo abortion. Pollen quality, pistil receptivity, and embryo and endosperm development have been widely examined in many crops, for example, Dendranthema grandiflorum, Nelumbo nucifera, Phaseolus vulgaris, and Fragaria ananssa (Marta et al., 2004; Ndoutoumou et al., 2007; Sun et al., 2010; Teng et al., 2012), and many of these studies successfully revealed the causes of the reduced seed production and breeding efficiency. For instance, in a study of the factors that affect seed set in several chrysanthemum crosses, it was found that a low number of germinated pollen grains and the abnormal growth of most pollen tubes were the main causes of the failure of the cross between D. grandiflorum ‘Yuhuaxingchen’ and D. nankingense (Sun et al., 2010). In the present study, we screened the three cultivars Fen Zhuang, Bai Lu, and Hong Ying and systematically investigated factors that may affect breeding efficiency of water lily. These factors included pollen morphology, pollen viability just before artificial pollination, germination behavior of pollen grains on stigmas, and embryo development after fertilization. Materials and Methods Experimental materials. Three cultivars, Bai Lu, Fen Zhuang, and Hong Ying, were used in this study (Fig. 1; Table 1). They are hardy water lilies with chromosome number of 112 (2n = 8x = 112). The plants were grown in ponds in Xingxiang town, Zhenjiang, China. Reciprocal crosses were made among three cultivars where ‘Fen Zhuang’ · ‘Hong Ying’ was denoted as FH and its reciprocal cross was referred to as HF; ‘Bai Lu’ · ‘Hong Ying’ as BH and its reciprocal cross as HB; and ‘Fen Zhuang’ · ‘Bai Lu’ as FB and its reciprocal cross as BF. The cultivars used in this work were selected for two main reasons. First, the ornamental values of ‘Fen Zhuang’ and ‘Bai Lu’ are complementary; for example, the flower color of ‘Fen Zhuang’ is pink, whereas that of ‘Bai Lu’ is white. If these two cultivars could be crossed successfully, it would be possible to obtain genotypes with novel traits. Second, preliminary studies indicated that seed set differed among the crosses, which motivated us to examine the factors that influence the fecundity of these crosses. Flowers from multiple plants of each cultivar were used in this study, which was because one plant was unable to produce a sufficient number of flowers for the experiment. Fig. 1. Morphology of the inflorescence. (A) Nymphaea ‘Fen Zhuang’. (B) Nymphaea ‘Bai Lu’. (C) Nymphaea ‘Hong Ying’. Received for publication 22 July 2014. Accepted for publication 4 Nov. 2014. This work was supported by the Fund of Independent Innovation of Agricultural in Jiang Su Province of China [CX(11)4005]. To whom reprint requests should be addressed; e-mail panyueping1224@163.com. 30 HORTSCIENCE VOL. 50(1) JANUARY 2015 Determination of pollen viability. On sunny days, fresh pollen grains were collected from 0900 to 1000 HR. The pollen grains were distributed on a glass slide coated with culture medium comprising 300 g·L sucrose, 10 mg·L H3BO3, 0.8 g·L –1 CaCl2·2H2O, 0.9 g·L KNO3 and 100 g·L –1 PEG4000 and then incubated at 20 C. After 6 h of culturing, the germination rates of the pollen grains were determined from 10 optical fields (at least 60 pollen grains in each optical field) with an Olympus BX41 microscope (Olympus Co. Ltd., Shanghai, China). Pollen grains were scored as viable when the pollen tube length was longer than the diameter of the pollen grain (Tang et al., 2009). Each experiment was repeated three times and statistical analysis was performed according to Tukey’s test. Artificial pollination. Water lilies are insect-pollinated and their pistil matures 1 or 2 d before the stamen; thus, we did not emasculate the flowers of the female parent and only bagged them 2 d before their pistils matured (Huang et al., 2009). The best time for pollination ranges from 0800 to 1000 HR on sunny days (Huang et al., 2009). When the flowers opened and the stigmas were full of bright mucus, the pollen grains of the bagged male flowers were collected for artificial pollination. The stigmas were pollinated with fresh pollen grains from male parents. After artificial pollination, the female flowers were bagged again. The crosses were performed from late September to early Oct. 2013, when the average temperature was 27 C with a range of 26 to 30 C. Pollen morphology and behavior on stigmas after pollination. The pollen samples were collected from fresh flowers fixed in 2.5% glutaraldehyde (0.1 M phosphate buffer, pH = 7.2) and stored at 4 C until use. The detailed surface ornamentation and aperture characteristics were examined under a SEM and photomicrographs were taken. Minor modifications to the method of Sun et al. (2010) were made and it was used to monitor the in vivo pollen germination on the stigmas. Twenty pistils at each time point per cross were fixed in 2.5% glutaraldehyde (0.1 M phosphate buffer, pH = 7.2) 2, 6, 12, and 24 h after pollination. After that, the pollen and pistil samples were dehydrated in an ethanol series (40%, 70%, 90%, and 100%, 15 min each time), subjected to critical point drying, and coated with gold for SEM (Hitachi S-3000N; Hitachi Co. Ltd., Beijing, China) analysis, and images were processed digitally (Jin et al., 2010). Examination of microspore development, embryo development, endosperm development, and seed set. Floral buds 0, 5, 10, 15, 20, and 25 d before blossom were fixed in FAA (5:5:90 formalin: acetic acid:70% ethanol) and stored at room temperature until they were used for the examination of microspore development. Approximately 200 ovaries were collected from 10 flowers on five plants (two flowers per plant) for each parental combination at 2, 5, 8, 12, 15, and 20 d after pollination and stored in FAA at 4 C for the examination of embryo and endosperm development. These samples were dehydrated through a graded series of ethanol solutions (70%, 85%, 95%, and 100%, 5 min in each solution), infiltrated with xylene, and embedded in paraffin wax. Sections were cut to a thickness of 8 to 10 mm using a Leica RM2016 microtome (Shanghai Leica Instruments Co. Ltd., Shanghai, China), stained in Heidenhain’s hematoxylin, and were observed and photographed under a Zeiss Axioskop 40 microscope (Carle Zeiss Management Co. Ltd., Shanghai, China) (Deng et al., 2010a). In addition, 200 ovaries per cross per time point (2, 5, 8, 12, 15, and 20 d after pollination) were examined using a stereomicroscope (Nanjing Jiangnan Yongxin Optical Co. Ltd., Nanjing, China) with digital camera, and the percentage of normal embryos was calculated. One month after artificial pollination, 30 water lily seed pods were randomly chosen and plump seeds were collected from them. Then the seed set in each cross was calculated using the following formula: seed set = (number of p

Cellular Mechanisms of Reproductive Barriers in Some Crosses of Water Lily (Nymphaea spp.) Cultivars