Effects of Xenia on Fruit and Seed in Vernicia fordii

Tung tree, Vernicia fordii, is a plant species producing industrial oil (tung oil). Although the cultivation of the tung tree produces great economic value, some important genetic and physiological traits in V. fordii have not been fully recognized. As one of them, the effect of pollen on the maternal plant (xenia) is unknown in V. fordii, which is an important part of the efficient cultivation system of many crops. This study performed hybridization with three important tung cultivars (Dami, Xiaomi and Putao) to evaluate the influence of pollen source on fruit and seed development. The results revealed that xenia is present in V. fordii, which influences fruit setting, fruit size, seed weight and oil content. Among the cultivars investigated, the hybridization combination with Putao as a female parent and Dami as male parent showed significant improvement of seed yield and oil content than self-pollination, which could be considered to apply in practice.

V. fordii have not been understood in terms of breeding and utilization, in which xenia has not been paid attention to for a long time.
It has been well known that pollen sources could have a direct influence on fruit and seed features in maternal plants, which is defined as xenia (effects on the endosperm and embryo or even effects on tissues around embryo) [4] [5].
The effects of xenia on fruit and seed characteristics have been revealed in a large number of plant species, such as maize [6], cotton [7], cucumber [8], grape [5], and almond [9], etc. Furthermore, there are available reports demonstrating that xenia could improve oil content in maize [10] and almond [9]. Whether xenia affects oil content and oil quality in V. fordii is an interesting question and is of great importance in the application.

Materials and Methods
The V. fordii trees (planted in 2012) were growing in a plantation in Kaizhou District, Chongqing, China (N31˚07', E108˚23'), which is one of the natural dis- was open but that of female flower was not (April 10th to 15th), pollens from male flower were collected and painted on stigma of female flower, then the female flower was wrapped around by plastic bags to avoid the influence of other pollens. The pollen activity was assayed according to the report of Trognitz [11], specifically, iodine-potassium iodide test, 2-3-5-triphenyle tetrazolium chloride test and in vitro pollen germination were performed.
In 2018, the cultivar Putao acted as female parent and Putao, Xiaomi and Dami as male parents. In the hybridization experiment of 2019, six combinations were set with the cultivars Xiaomi and Putao as female parents and Xiaomi, Putao and Dami as male parents. Each combination included at least 400 flowers and was repeated for three times. One month after the hybridization operation, fruit setting rate was counted. On October 30th, fruits were collected to measure fruit size (the transverse diameter and longitudinal diameter), fresh fruit weight, number of seed in fruit, dry weight of single seed. The kernel ratio was calculated with seed weight divided by fruit weight.
Lipids in seed were extracted according to the method of Shockey et al. [12].
Specifically, kernels were dried at 80˚C for 48 h, then were ground into powder.  sulphate. The organic phase was collected and the aqueous phase was back-extracted with 15 ml hexane: 2-propanol (7:2, v/v). Kernel oil content was calculated as the quality of oil extracted divided by dry weight of seeds. In addition, the acid value, saponification value and iodine value of extracted oil were determined by standard procedures described by Ajiwe et al. [13].

The Pollen Viability of Different Cultivars
Pollen quality is important for successful fertilization in plants. In this study we examined the pollen viabilities and germination rates of three different cultivars (Table 1). Although the results of I 2 -KI staining and TTC staining varied, there was no significant difference of pollen viability among the cultivars Xiaomi, Putao and Dami shown by certain a method (Table 1). It is noteworthy that the pollen germination rate of Dami was lower than those of Xiaomi and Putao ( Figure 1).

The Influence of Pollen Source on Fruit Setting in V. fordii
According to long-term practical experience, Putao produces most fruits, but

The Influence of Pollen Source on Fruit Development
In 2018, we observed that pollen source had influence on fruit in female parent plant ( Figure

The Influence of Pollen Source on Seed Development
The number of seed in single fruit and the dry weight of single seed are important indicators for oil production. In 2018, we found that the number of seed in fruit was not affected by pollen source when only Putao was used as female parent ( Figure S2). However, the dry weight of single seed was promoted by the  pollen from Dami. In 2019, it was found that heterogenous pollen did not affect the number of seed in single fruit in both plants of Xiaomi and Putao ( Figure   4(a)). However, the cultivar Putao tended to produce more seeds in fruit than Xiaomi. In terms of dry weight of each seed, there was no change resulted from pollen source in plant of Xiaomi, but a significant increase of dry weight of single seed was observed in plant of Putao pollinated by Dami (Figure 4(b)). Furthermore, we analyzed the kernel ratio (kernel weight/seed weight) to find that different pollen source did not affect the kernel ratio in plant of xiaomi, while the pollen from Dami led to a higher kernel ratio in plant of Putao ( Figure   4(c)).
S. F. Qin et al.

The Influence of Pollen Source on Kernel Oil Content and Oil Characteristics
Whether oil content is affected by pollen source is the most interested question.
The experimental results in 2018 showed that the pollen from Dami could improve the oil content in seed in plant of Putao ( Figure S3). In 2019, we found that the pollen from Putao declined the oil content in kernel in plant of Xiaomi, while the pollen from Dami promoted it ( Figure 5(a)). In the plants of Putao, both the pollens from Xiaomi and Dami improved the oil content in kernel compared to the pollen from Putao itself ( Figure 5(a)).

Discussion and Conclusion
To date, it has been well known that xenia, an important genetic phenomenon, is widespread in planta, which brings about changes in various aspects. To our knowledge, so far there is no effect of xenia found in plants of the Euphorbiaceae family. This study revealed that xenia is present in tung tree for the first time, which affects fruit setting, fruit size, seed formation and seed oil content. In this study, the effect of xenia was demonstrated only by a specific combination of male and female parent, although it is unknown why xenia affects fruit and seed in a species-dependent manner in tung tree, similar phenomena were reported in many plant species, e.g. Citrus mangshanensis [14], Actinidia arguta [15] and Macadamia [16]. In this study, we found that the pollen germination rate of the cultivar Dami was lower than those of the cultivars Xiaomi and Putao, but pollens from Dami did improve fruit setting rates in plans of Xiaomi and Putao.
The reason is probably that excessive pollens were used in the hybridization operation, which offset the drawback of pollen germination rate. The study showed that heterologous pollens are beneficial for promoting fruit setting rate, which has been revealed in many plant species, such as pummel [14]. There are two hypotheses to explain the effect of xenia on fruit setting: the hormonal hypothesis and the mobile mRNAs hypothesis. They both propose that heterologous pollens induce more physiological processes associated with seed formation (pollen tube growth and pollen-pistil interaction, etc.) [17]. synthesis in the process of rapid oil accumulation in seeds [21]. So far, the process of oil synthesis in V. fordii has been well revealed [22] [23], whether it is enhanced by heterologous pollens in V. fordii needs to be proven in future.
In this study, we observed that only one or a few traits of fruit or seed were influenced by pollen source, this phenomenon is also shown by a lot of studies in various plant species. It could be affirmed that the newly-formed embryo after pollination with heterogenous pollen could bring about stimulus to seed and fruit whose development are controlled by the genetic character of cultivar.
However, the mechanism underlying it is still not clear. When