Plant materials and pollination.

This study was conducted in two successive years (2015 and 2016) at the research institute of the Landscape Architecture Department, Northwest Agriculture & Forestry University, Yangling, Shaanxi province, China. The flowering times of 55 cultivars were recorded, and their pollen viability was investigated by the method of in vitro germination (Shivanna and Heslopharrison, 1981) in 2015. Finally, 15 cultivars that had high pollen germination rates (more than 30%) and similar flowering dates to those of P. ostii ‘Feng Dan’ were selected as pollen donors (Table 1).

Table 1.

Fifteen cultivars used as pollen sources in this study.

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The effects of different pollination treatments on the fruit and seed traits of P. ostii ‘Feng Dan’ were investigated by applying cross-, open-, and self-pollination on field-grown plants. Five uniform 8-year-old trees were selected, and in each tree, four flowers were selected for each pollination treatment. For the self-pollination group, 20 flowers were self-pollinated by hand and covered with waxed paper bags. For the open-pollination group, flowers were left without cover. For the cross-pollination group, the selected flowers were emasculated using forceps ≈3 d before blooming and covered with waxed paper bags. These flowers were regularly checked for the appearance of a bead of moisture on the stigma, indicating that the female parts had attained peak receptivity. As soon as emasculated flowers reached peak receptivity, dried pollen, which had been collected from another tree of the same population and stored earlier, was applied to the stigmas. After pollination, all of the pollinated flowers were again bagged to avoid contamination by other pollen and tagged. The bags were removed after 10 d.

The significance of pollen sources for the fruit and seed traits of P. ostii ‘Feng Dan’ was evaluated by pollinating with pollen from different tree peony cultivars under field conditions. Pollen was collected from selected pollen donors when the first flowers began to dehisce and dried on paper at room temperature. The pollination method was the same as that applied in the cross-pollination group in the pollination treatment experiment.

Measurements and data collection.

Two months after pollination, the percentage of fruit set was counted for each combination. Fruit were harvested on 1 Aug. The diameter of each fruit was measured with digital calipers. Then, seeds were extracted from mature fruits, and the number of seeds was counted. Fifty seeds per combination were randomly selected and measured for volume and weight. Seeds were prepared from five individual pods and dried at 60 °C in an oven for 48 h to a constant weight. Then, the seedcoats were removed, and the kernel weight was measured.

The kernels were ground into small pieces and sieved (40 mesh). The extraction of seed oil was performed on a supercritical CO₂ extraction apparatus (SFE-2 model; Applied Separations, Allentown, PA). Samples (10 g) of grounded peony kernel were placed into the extraction vessel. The extraction conditions were as follows: extraction time, static extraction for 30 min followed by dynamic extraction for 2 h; temperature, 45 °C; pressure, 300 bar; and flow rate of carbon dioxide (gaseous fluid), 30 L/h. The extracts were collected in a glass vial (50 mL). When the designated time was reached, the extraction vessel was depressurized, and the obtained oil was used for the following tests. The amount of extracted oil was gravimetrically determined after collection, and the extraction yield was expressed as the percent ratio of the mass of extracted oil to the mass of peony seed loaded into the extraction vessel, as follows:

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Seed oil (0.2 g) was added into a capped glass vial (20 mL) and mixed with 4 mL of a methanol solution containing 2% sodium hydroxide. Then, the vial was placed in a water bath at 60 °C for 30 min. After reaction, the mixture was allowed to cool to room temperature and was treated with 2 mL of a boron trifluoride-methanol solution at a concentration of 14% wt. The vial was then heated in a 60 °C water bath for another 10 min. The mixture was mixed with 2 mL of n-hexane and vigorously shaken, then brought to room temperature. Next, 2 mL of saturated sodium chloride was added. After standing for 5 min, the organic phase was separated. Then, 500 µL of supernatant was transferred to a 2 mL vial and capped with a plastic cap having a polytetrafluoroethylene septum. In addition, 150 µL of tridecane acid methyl ester (10.0 mg·ml⁻¹ in n-hexane) was added as the internal standard for this work via a 200 µL pipette. After filtering, the solution was analyzed by gas chromatography–mass spectrometry.

The analysis of FAs was carried out by using a Thermo TRACE 1310 GC-ISQ and Thermo Scientific TriPlus RSH robotic sampler. A TG-WaxMS (30 m × 0.25 mm internal diameter, 0.25 μm film thickness; Thermo Fisher Scientific, Waltham, MA) capillary column was used in the experiment with helium as the carrier gas. The column temperature was initially 80 °C, was held for 1 min, then increased to 175 °C at 50 °C/min, was held for 1 min, and was finally increased to 210 °C at 3 °C/min (held for 5 min). Ultrahigh purity helium was used as the carrier gas at a flow rate of 1.0 mL/min. The temperatures of the ion source and transfer line were 280 and 250 °C, respectively, whereas the injector temperature was set at 250 °C for split injection at a split ratio of 20:1. The inject volume was 1 μL. The mass spectrometer was operated in positive ion mode with ionization energy of 70 eV and the scan range from 30 to 450 amu.

The oil components were identified by comparing their retention times with external standards and a mass spectra database search (NIST05 Library). Five major FA methyl esters in each sample were quantified using an internal standard method. A standard curve was derived from the total ionic chromatogram of different concentration standards, where the x-coordinates and y-coordinates represent the concentration ratio and peak area ratio of external and internal standards, respectively.

Data analysis.

Statistical analysis was performed using SPSS version 18.0 (SPSS Inc., Chicago, IL). One-way analysis of variance was used to analyze the effect of different pollination treatments and pollen sources on the fruit set percentages, number of seeds per fruit, seed and kernel weights, oil extraction ratios, and quantity of five major FAs and total FA. Statistical differences at P ≤ 0.05 were considered significant, and the means were compared using Duncan’s multiple range test at 0.05.

Fruit setting and number of seeds per fruit.

According to the present results, fruit setting and number of seeds per fruit were significantly affected by different pollen sources. The results showed that the percentage of fruit set ranged between 44.33% and 87.50% (Fig. 1). ‘Yinhong Qiaodui’ produced the highest fruit set (87.50%), which was followed by ‘Zi Ju’ (86%) and ‘Cong Zhong Xiao’ (85%). On the other hand, ‘Mo Run Jue Lun’ and ‘Gejin Zi’ produced the lowest fruit set (44.33% and 55.83%, respectively).

Effects of pollen sources on fruit set and seed number per fruit.

Citation: HortScience horts 52, 5; 10.21273/HORTSCI11803-17

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Effects of pollen sources on fruit set and seed number per fruit.

Citation: HortScience horts 52, 5; 10.21273/HORTSCI11803-17

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Effects of pollen sources on fruit set and seed number per fruit.

Citation: HortScience horts 52, 5; 10.21273/HORTSCI11803-17

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The number of seeds per fruit was also affected by different pollen sources, ranging from 36.25 to 50.86 (Fig. 1). ‘Mochi Jin Hui’, ‘Dahong Baozhu’, and ‘Hongxia Yingyue’ produced more seeds per fruit (>45) than the other pollen sources. The fewest number of seeds per fruit was produced by ‘Sai Huanghou’, which was followed by ‘Dao Jin’.

Pollination treatment effects.

The breeding system plays a crucial role in determining seed and oil yield. Our results showed a certain preference to cross-pollination in fruit setting and seed number per fruit, which were in accordance with the findings of other authors in P. jishanensis (Luo et al., 1998; Zhou et al., 1999) and P. delavayi (Li et al., 2014). The decrease in fruit setting and number of seeds per fruit from self-pollination could be explained by self-incompatibility (SI). The SI response comprises a self- and nonself-recognition process between pollen and pistil, which is followed by inhibiting the development of self-pollen tubes (Takayama and Isogai, 2005). Moreover, P. ostii ‘Feng Dan’ is partially self-compatible, with 29.50% of fruit set producing 31.66 seeds per fruit. The existence of partial self-compatibility is also very important for plants to reduce extinction vulnerability (Bond, 1995).

Open-pollination produced less fruit and seeds than cross-pollination. Because bees are considered to be responsible for successful pollination (Li et al., 2014; Sanchez-Lafuente and Valera, 1999), bee behavior may have a great influence on pollination. Naturally, the activities of bees would be easily affected by environmental conditions (unfavorable weather and temperature) and the color of flowers (Koffi et al., 2013). In addition, pollen from self and crosses could germinate together in the same stigma. Each pollen type caused an opposite effect, which is another reason for the decrease in fruit and seed production by open-pollination.

The results also showed that the values of the studied physical traits of the fruits and seeds produced by self-pollination always remained at a lower level. Fruits harvested from cross-pollination are larger and contain more, larger, and heavier seeds than those from self-pollination. The decrease in seed and kernel weights when comparing self- with cross-pollination has been attributed to inbreeding depression effects (Oukabli et al., 2002; Torre-Grossa et al., 1994). Inbreeding depression is defined as the reduction in biological fitness in a given population as a result of inbreeding or breeding of related individuals. Similar results were found in almonds (Torre-Grossa et al., 1994), hazelnuts (Fattahi et al., 2014), and pecans (Marquard, 1988), where kernels from self-pollination were smaller and lighter than those from cross-pollination.

The contents of oil and five major FAs were also influenced by different pollination treatments. An increase in OA content and decrease in LA were observed in seed oil produced by self-pollination, which was in accordance with the findings of other authors in several almond cultivars (Kodad, 2008; Kodad et al., 2009) and Jatropha curcas L. (Samocha et al., 2014). However, whereas the opposite was also observed in some almond cultivars by Kodad et al. (2009), the mechanism for the impact of cross- and self-pollination on the concentrations of OA and LA is still unknown. The decrease of LA in the self-pollination treatment and its increase in the cross-pollination treatment may be explained by the negative correlation between the percentages of OA and LA (Abdallah et al., 1998). A high OA (monounsaturated fatty acid) to LA (an omega-6 polyunsaturated fat) ratio in self-pollinated kernels is desirable as it improves the stability of the fats against rancidity and increases the kernels’ shelf life (Kodad, 2008).

Pollen source effects.

Pollen, which was obtained from different cultivars, caused wide ranges of fruit setting and number of seeds per fruit in P. ostii ‘Feng Dan’. This is in accordance with the results of a previous study (Xin et al., 2014). ‘Yinhong Qiaodui’ and ‘Zi Ju’ produced higher fruit setting than free-pollination, whereas more seeds per fruit were produced by ‘Mochi Jin Hui’. Both the percentage of fruit setting and seed number per fruit produced by ‘Dahong Baozhu’ remained at high levels, which demonstrates that choosing suitable tree peony cultivars could enhance the diversity of color in a plantation while maintaining its seed yield. Effects of different pollens on fruit set or the number of seeds per fruit have been reported in many fruit crops, such as blueberries (Gupton and Spiers, 1994), cherimoyas (Kahn et al., 1994), Hylocereus polyrhizus (Mizrahi et al., 2004), mandarins (Wallace and Lee, 1999), and Olea europaea L. (Farinelli et al., 2012). The differences in fruit setting and seed number per fruit could be explained by the growth of the pollen tube, the development of fertilized ovules (Hao et al., 2013), or metaxenia.

Significant differences were also found in the phenotypic traits of fruits and seeds as an effect of pollination with pollen grains from different cultivars. These results are in agreement with the findings of other researchers, who reported that the physical traits of fruits and seeds were affected by different pollen sources in date palms (Rezazadeh et al., 2013) and lychees (Degani et al., 1995). Therefore, it is possible that the size and weight of seeds could be enhanced by different pollen donors. However, the reason why the physical traits of fruits and seeds were influenced by pollen genotype remains poorly understood. Swingle (1928) reported that endogenous phytohormones are responsible for the xenia, whereas Liu (2008) proposed that the signal to trigger the xenia effect may be mRNAs.

Previous studies have described that oil content in rapeseed (Wang et al., 2010) and maize (Weingartner et al., 2004) could be affected by pollen source. Our results showed that oil extraction ratio varied among pollen sources, with ‘Gejin Zi’ (26.0%) as the lowest and ‘Caozhou Hong’ (31.6%) as the highest. When we estimated oil yield per fruit, certain differences were revealed (Fig. 4). The oil content per fruit when pollinated by ‘Dahong Baozhu’ was more than twice that when pollinated by ‘Dao Jin’. These results emphasize the crucial effects of the pollen source in determining the oil yield in tree peony plantations. The variability in the oil content has been considered to be mostly dependent on the mother plant genotype, whereas xenia effects also had a certain impact on the oil content of hybrid seeds (Wang et al., 2010). During the development of fruit, the nutrients are not only supplied by the mother plant but also synthesized and stored by its own embryo (Dure, 1975). Therefore, the genotype of the embryo might also affect the level of oil content in the kernel. The fact that there was no significant difference in the oil extraction ratio between self- and cross-pollinated kernels could also be explained by this result.

The FA profile showed significant differences in the PA, OA, LA, and ALA among pollen sources, which reveals a great potential for improving the seed oil composition of P. ostii ‘Feng Dan’ by applying proper pollen. Similar results were observed by Kodad et al. (2009) in almonds and Samocha et al. (2014) in Jatropha curcas L. Previous studies indicated that some fruit chemical compositions in other species could also be influenced by different pollen sources. The pollen source affected the total soluble solids of the fruit in date palms (Phoenix dactylifera L.) (Rezazadeh et al., 2013) and the amygdalin content in almonds (Sanchez-Perez et al., 2012). The variation among pollen sources in oil composition could be contributed by its own embryo, which is affected by both the mother and father genotypes (Kodad et al., 2009).