The Nelumbonaceae is a family of perennial, aquatic, herbaceous angiosperms, consisting of the two species Nelumbo nucifera Gaertn. and N. lutea (Willd.) Pers. Nelumbo nucifera, also called the Indian or sacred lotus, is found throughout Asia and Australia, whereas N. lutea, also known as the American lotus or water chinquapin, occurs in eastern and southern North America [1, 2]. Fossilized leaves of N. nucifera show that wild populations were distributed previously in eastern Asia including China, India and Japan about 50 million years ago. Wild populations of N. nucifera in China are now mainly distributed in the provinces of Helongjiang, Jilin, and Sandong. Nelumbo nucifera was cultivated in China more than 2000 years ago. The current main cultivation areas in China comprise the provinces of Jiangsu, Zhejiang, Hubei, Anhui, Guangdong, Hunan, Fujian, and Jiangxi. This species is propagated vegetatively by rhizomes in commercial cultivation, and by seeds for breeding purposes [3]. As an economically important aquatic plant in China, N. nucifera cultivars are classified into three groups: rhizome lotus, seed lotus and water lotus or ornamental lotus according to their use and morphological features [4, 5]. Water lotus is among the 10 most popular traditional flowers in China, and is an important ornamental plant, widely cultivated in gardens prized for the beauty of its flowers as well as its ability to purify pond water [6, 7]. With the recent rapid growth in the Chinese economy and increased standard of living, the demand for new lotus cultivars with novel characteristics has also increased. Therefore, Chinese breeders are making great efforts to develop new cultivars using methods such as artificial interspecific hybridization, mutation techniques, and multiploid approaches [7–9]. Hybridization is the most widely used breeding technique and the most efficient method by which to produce new water lotus cultivars. However, the existence of incongruity barriers often makes many water lotus crosses difficult, and thus raising hybrids may be hard to achieve, thereby seriously reducing the breeding efficiency [7]. Few previous studies have been carried out to investigate this problem, and thus the factors that influence the breeding efficiency of water lotus remain unclear.

In general, the characteristics of parental reproductive systems and their interactions are closely related to the breeding efficiency of plant hybridization. Consequently, parental reproductive systems and post-pollination phenomena have been examined in many crops, including Chrysanthemum grandiflorum Leymus chinensis Phaseolus vulgaris, and Fragaria ananassa[10–13]. Most of these studies have successfully unraveled the factors that influence seed production and breeding efficiency. Following on from these previous studies, we set out to investigate systematically the reproductive processes following artificial pollination in two reciprocal crosses between water lotus cultivars, including pollen viability of the male parents, germination behavior of pollen grains on the stigma, and embryo development. The aim of the present study was to reveal the main causes that lead to low seed set in water lotus crosses, to overcome reproductive barriers and improve the efficiency of water lotus cross-breeding in the near future.

The cultivars used in the present study were developed about 10 years ago through cross-breeding. Anthesis of lotus flowers is very specific and complex. The pistil of N. nucifera flowers is shaped like a flat-topped cone and contains 20–35 ovules when fully developed. Surrounding the pistil are 200–300 yellow stamens, and both the male and female reproductive organs are protected by 10–35 petals and sepals. Anthesis of individual flowers usually lasts about three days on sunny days. On the first day, the flower begins to open at about 03:00 in the morning and attains its maximum diameter (about 6 cm) at about 05:30. The flower remains at this diameter until about 09:00, after which the flower gradually closes and is completely closed at about 15:00. Substantial fluid is secreted on the stigma surface from 04:00 to 06:00 and the stigma is a bright yellow color, which indicates that the stigma receptivity is high during this period. The anthers do not dehisce on the first day, therefore the pistils mature before the stamens in an individual flower (termed ‘protogyny’). On the second day of anthesis, the process of flower opening is very similar to that on the first day and a maximum diameter of about 10 cm is attained. The anthers dehisce to release quantities of mature pollen from about 04:00 to 05:30. The stigma surface becomes dry and brown, which indicates the loss of stigma receptivity. On the third day of anthesis, the flower begins to open at about 05:00 and is fully open with a maximum diameter of more than 12 cm in diameter at about 10:00. Subsequently, the petals and stamens abscise. The stigma becomes severely dry and brown, and its receptivity is completely lost. An individual flower usually produces about one million pollen grains. Thus the pollen:ovule ratio (P/O) is 30,000-50,000.

The study site is about 33 hectares in area and contains many pools. Many lotus cultivars, including the four included in the present study, were planted in the pools. Therefore, all of the lotus cultivars growing in at study site were possible sources of pollen for “naturally pollinated flowers” in the present study. Bees, flies and beetles may be effective pollinators at the study location.

Many factors affect seed set of angiosperms. Pollen viability is often considered to be an important factor that influences seed or fruit production [13, 15–18]. When pollen grains with low viability are placed on a stigma, the probability of pollination failure usually increases, resulting in reduced seed production [17, 19, 20]. For example, in Bambusa vulgaris, low pollen viability is one of main factors that causes low seed set [21]. In an investigation into the relationship between soybean production and pollen sterility, it was noticed that soybean production significantly decreased when more than 60% of the pollen grains were sterile [19]. In the present study, the time of lotus pollen grain collection significantly influenced pollen viability. Pollen grains collected between 05:00 and 06:00 contained the highest percentage of viable grains, and consequently these grains were used for the artificial pollinations. The viability of pollen of the four cultivars collected between 05:00 and 06:00 ranged from approximately 10% to 20%, which might be somewhat lower than the actual values. A possible explanation is that the pollen viability of N. nucifera quickly decreases after anther dehiscence [3], and it usually took about 2 h to transport the samples from the study site to our laboratory. It is possible that a portion of the pollen grains lost viability during transport. Even if the actual pollen viability of the four cultivars was slightly higher than 20%, seed set may be related to low parental pollen viability.

Pollination is the first step in the sexual reproduction process for most of higher plants, and the interaction between pollen grains and the pistil after pollination is an important factor that influences seed output [22–27]. Pollination failure is widespread in plants, and is a common cause of low seed production. It is often characterized by inhibition of pollen germination and pollen tube growth, and abnormal growth of pollen tubes [13, 17, 26, 27]. For example, low stigma receptivity is considered to be one cause of low seed set in some sorghum crosses [28]. More recently, in a study of the factors that affect seed set in several chrysanthemum crosses, we 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[13]. The present results show very slight differences in pollen viability among the four cultivars, but significant differences in the number of germinated pollen grains on the stigma at 4 h after pollination among the four crosses. In addition, the number of germinated pollen grains on the stigma in each cross was positively correlated with the percentages of normal globular embryos at 1 day after pollination and seed set. Moreover, in the reciprocal crosses of M and Q, large amounts of callose were deposited in some pollen tubes, and some pollen tubes were ruptured, which resulted in failure of the pollen tubes to penetrate the stigma. Therefore it is suggested that low seed set in the reciprocal crosses of M and Q may be largely attributable to low numbers of germinated pollen grains and subsequent abnormal growth of their pollen tubes on the stigma. Because pollen–pistil interactions are complex, usually including cell–cell recognition and cellular signaling processes [19, 26], further investigation into the causes of pollen germination inhibition and pollen tube growth are necessary in order to increase stigmatic receptivity in water lotus crosses.

In addition to pollen viability and pollen–pistil interactions, seed set is closely related to embryo development. Reduced seed production is often caused by degeneration or abortion of a large number of embryos during their development [12, 29–32]. For example, Ndoutoumou et al. [12] found that low seed set in reciprocal crosses between Phaseolus vulgaris and P. coccineus was largely because of embryo abortion after fertilization. Similarly, abortion of immature embryos is considered to be the main factor restricting the transfer of desired traits into cultivated chickpea from wild Cicer relatives [31]. Recently, similar phenomena have been observed in wide crosses of chrysanthemum [13]. Likewise, embryo abortion is also a factor affecting seed set in water lotus crosses, although the proportion of aborted embryo is variable in these crosses. Compared with the three other crosses performed in the present study, GA had a relatively lower ratio of embryo abortion and a higher seed set (23.33%). If seed set of over 10% is regarded as a criterion for a successful water lotus cross, the three crosses, AG, MQ, and QM were all unsuccessful. We suggest that embryo abortion is an important factor leading to the failure of the AG, MQ, and QM crosses and influences the breeding efficiency of GA. The reasons for embryo abortion, however, are still unclear and require further investigation.

Seed set in GA was 23.3% but was only 9.2% in AG, which was similar to the seed set in the reciprocal crosses of M and Q. This difference in seed set in one reciprocal cross may be mainly attributable to differences in pistil receptivity. For example, the number of germinated pollen grains on the stigma at 4 h after pollination was 60.6 in GA and 25.8 in AG, which is a 2.35–fold discrepancy. The percentage of normal embryos at 1 d after pollination in GA was 58.8%, which was 2.21–fold higher than that in AG (26.6%), and seed set in GA was 2.53–fold higher than that in AG. The reasons for the differences in pistil receptivity in one reciprocal cross remain unclear.

In this paper, we carefully studied the factors that govern fecundity in two lotus reciprocal crosses. Three findings in the present study are of note: i) low pollen viability impacts on seed set in water lotus crosses; ii) low stigma receptivity is a vital factor leading to low fecundity of water lotus crosses; and iii) embryo abortion is an additional factor leading to reduced fecundity of lotus crosses. However, the mechanisms involved in low stigma receptivity and embryo abortion remain unknown, and further studies are required to better understand these factors in water lotus, and indeed in other plants as well.