Thirty pistils were, respectively, sampled at 0.5, 1, 2, 4, 6, 8, 10, and 12h after pollination and fixed and stored in FAA solution (5:5:90 of formalin, acetic nothing acid and 70% ethanol) at 4��C until use. The samples were softened overnight in 1molL?1 NaOH, rinsed in water and mounted on a microscope slide with a drop of 0.1% aniline blue (0.1molL?1 K3PO4 supplemented with 18% glycerol), and then observed under a fluorescence microscope (Zeiss Axioskop 40; Carl Zeiss Shanghai Company Ltd, Shanghai, China) with excitation filter BP 395�C440, chromatic beam splitter FT 460, and barrier filter LP 470. Digital images were captured using an Axiocam MRC camera [12]. In addition, some pistils were fixed in 2.5% glutaraldehyde (0.1M phosphate buffer, pH 7.2).

After rinsing in buffer, the samples were postfixed overnight in 1% (w?v) buffered osmium tetroxide, washed in buffer, dehydrated in an ethanol series (40, 70, 90 and 100%, 15min each time), and then critical-point-dried using liquid CO2. The pistils were then mounted on aluminum specimen stubs with adhesive tabs. After coating with gold, they were examined using a Philips XL-30 environment scanning electron microscope (SEM) (Hitachi S-3000N) [13].2.5. Embryo Development after Pollination and Seed SetAbout 80 ovaries at 1 and 2d after pollination were, respectively, collected and then immersed in FAA at 4��C until use for examination of embryo development. The ovules were dehydrated through a graded series of alcohol solutions (70, 85, 95, and 100%, 5min each time), infiltrated with xylene, and embedded in paraffin wax [11].

Sections were cut to a thickness of 8�C10��m using a Leica RM2016 microtome (Shanghai Leica Instruments Co Ltd, China), stained in Heidenhain’s haematoxylin, and were observed and photographed under the Zeiss Axioskop 40 microscope. In addition, we collected about 80 ovaries at different periods after pollination in order to examine the percentage of normal embryos, and the samples were observed under a stereomicroscope equipped with a digital camera.One month after artificial pollination, about 30 lotus seed pods were randomly chosen and plump seeds were collected from them. Then seed set in each cross was calculated with the following formula: seed set = (number of plump seeds/total number of pollinated stigmas) �� 100% [11].2.6.

Statistical AnalysisThe AV-951 data were analysed by a one-way analysis of variance using the SPSS software 16.0 (SPSS Inc, Chicago, IL, USA). Tukey’s Honestly Significant Difference (HSD) (P �� 0.05) was used to discriminate the values.3. Results3.1. Viability of Pollen Grains Collected at Different Times The collection time of lotus pollen grains significantly affected pollen viability (Figure 1): viabilities of ��Jinsenianhua�� pollen grains collected at 05:00-06:00hrs, 06:00-07:00hrs, and 07:00-08:00hrs were 20.6, 12.1, and 6.