Asparagus officinalis L.(asparagus) is a typical dioecious plant with the genetic research value. Its gender has a variety of diversity, including female strain, male strain and hermaphroditic strain (containing sound and complete male buds, ovaries and stigmas) and supermale strain. In addition, asparagus is a nutritional and health-care vegetable and has important economic value in the agricultural production (Chen, 2013).

There is a significant correlation between sex and sex chromosomes of asparagus, and the external environment and exogenous hormones have little effect on asparagus organ development. It is found that the sex of asparagus is controlled by sex-determining genes on homologous L₅ sex chromosomes (Zhou et al., 2010; Norup et al., 2015). By studying the evolutionary process of sex chromosome of the dioecious plant, the investigator discovered that asparagus sex chromosomes were in the early stage of sex evolution and contained more primitive X and Y chromosomes, and the recombination of male and female sterile sites was inhibited on sex chromosomes, which could form normal supermale and bisexual plants (Ray et al., 2011). It was found that the male and female sex of asparagus were controlled by the interaction between male inhibitory gene TDF1 and female inhibitory gene SOFF (Harkess et al., 2017).

At present, little is known about the regulation network of asparagus sex determination, the mechanism of sex differentiation, the evolutional motive force of sex chromosome and so on. It has been reported that the observation of the paraffin section is an important means to understand the changes of the structure of the plant organs (Zhang et al., 2015). Therefore, in order to furthermore promote the study of sex determination and differentiation mechanism of asparagus, the internal relationship between different flower bud size and flower bud morphology was studied by paraffin section technique and morphological observation, which provided a reference for the selection of materials at different developmental stages for further research.

1 Results and Analysis

Observation on the growth and development process of asparagus, we could find that asparagus is a typical dioecious plant, and the sex difference is mainly reflected in the flower structure. Normally, the male plants only bloom male flowers, the female plants bloom female flowers, and the flower structure was radially symmetrical. Observation on the structure from the outside to the inside, we found that the node of the petiole was located in the upper part of the flower, with the bell-shaped perianth. The calyx and the petal were metamorphosed into tepal, and the inner and outer perianth was symmetrically distributed. The inner perianth was wider than the outer, 3 pieces per round. The base was symbiotic, and there were nectaries inside. Perianth of male flower is longer than perianth of female flower, stamen is hidden in perianth, with total of 6, the same number as perianth. Its opposite is rectangular round, the back is normal, the base is two-lobed. The medicine septum distally has triangle tip-shaped appendage, with 2 locules, introversion is dehiscent. Below the middle part of filament is adnate to perianth base. The pistil is tricarpellary and syncarpous, and is composed of an ovary and a stigma. The ovary is obovate and superior with axile placentation and trilocular with rows of anatropous ovules in each locule. The ventral suture is depressed. The stigma is obvious with three-lobed.

Morphology and anatomy research showed that the flower bud of Asparagus protruded in leaf axils when the length of pistil and stamen was 0.4 mm, both of them contained the pistil and stamen primordium, and there was no significant difference between them (Table 1; Figure 1A; Figure 2A).

Table 1 Developmental stages of female and male flowers in Asparagus officinalis L.

Figure 1 Dynamic developmental stages of female asparagus Note: st: stamen; C: carpel; A: the length of flower buds in “Atlas”(0.4 mm, ×40); B: the length of flower buds in “Atlas”(0.8 mm, ×40); C: the length of flower buds in “Atlas”(1.2 mm, ×40); D: the length of flower buds in “Atlas”(1.6 mm, ×40); E: the length of flower buds in “Atlas”(2.0 mm, ×40); F: A:the length of flower buds in “Atlas”(2.4 mm, ×10)

Figure 2 Dynamic developmental stages of male asparagus Note: st: Stamen; C: Carpel; A: The length of flower buds in “Atlas”(0.4 mm, ×40); B: The length of flower buds in “Atlas”(0.8 mm, ×40); C: The length of flower buds in “Atlas”(1.2 mm, ×40); D: The length of flower buds in “Atlas”(1.6 mm, ×40); E: The length of flower buds in “Atlas”(2.0 mm, ×40); F: The length of flower buds in “Atlas”(2.4 mm, ×10)

When the length of flower buds was 0.8 mm, the center of flower bud primordium is inhibited, and the perianth floral primordium was differentiated. Stamen and pistil continued to grow and develop (Figure 1B; Figure 2B).

When the length of flower buds was 1.2 mm, in female flowers, pistil denegration appeared, the flower bud center denegration formed the lower ovary, the placenta is differentiated, and the ovule appeared (Figure 1C). In male flowers, the anther primordia had been swelled, but the development of styles in female organs was blocked (Figure 2C).

When the length of flower buds was 1.6 mm, the stamen in the female plant stopped developing, ovary continued to develop to form 3 locules, and megaspore mother cells result in meiosis (Figure 1D). Filaments and anthers in male plant continued to develop, pollen mother cell meiosised. Obvious pollen grains could be observed, while the stigma stopped developing (Figure 2D).

When the flower bud length was 2.0 mm, the female structure was observed to change in the female flower, such as ovary enlargement, mitosis, and elongation of the style (Figure 1E). In the male flower, microspores were undergoing mitosis and pollen grains were further developed. However, the stigma stopped developing (Figure 2E).

When the length of the flower bud was 2.4 mm, the ovary and style of the female flower were basically mature. It can be observed that the female bud had a blooming phenomenon, and only a small amount of residual remained in the filament (Figure 1F). In the male flower, the pollen grains were mature and golden yellow. They were about to sprout. The remaining styles of varying lengths couldn’t develop into a complete female organ (Figure 2F).

With the extension of the length of the flower buds, the male and female flower buds were further specialized to the typical male and female characters. The female flowers and the male flowers were elongated. Finally, the open male flower could be up to 6 mm in length, grown in a pale yellow bell-shaped shape with a diameter of 2.4 mm. While the open female flower had a short round bell-shaped shape with a length of 3.0 mm and a diameter of 2.0 mm, which is greenish-white.

2 Discussion

In this study, it was found that the process of asparagus flower development was accompanied by the determination of the sex of asparagus. During the development of male and female flowers, there is a critical period from the transition from bisexual to monosexual. The transition period appears in the early and middle stages of its development. And then, they develop to the male and female genders separately. In the early stage of their dynamic development, there is no significant difference between them. This phenomena was also observed by Qin Li (2015) and Hu Shuming et al. (2013). It is believed that the sex of asparagus is not determined from the beginning. The sex of asparagus is not determined at the beginning, but is gradually developed. However, there are differences in the developmental stages of male and female differentiation, which is presumed to be related to various factors such as the age of the asparagus material itself, the physiological state, and the external environment. Therefore, to have a more detailed understanding of the dynamic process of asparagus development, it is necessary to make a staged dynamic morphological anatomy and scanning electron microscopy of the asparagus flower development process. According to the study of Caporali et al. (1994), flower development is divided into 13 periods. A horizontal and vertical comparison of the developmental time of the male and female buds is made, and it is concluded that gender determination occurs during a specific period of flower development.

In this study, the development of asparagus flower was divided into six sampling periods according to the different growth length of the same asparagus flower bud. It was found that when the length was 0.4 mm, it was the period of hermaphroditism, both having primordium of androgyny differentiation, and there was no significant difference between them. When the length was 1.2 mm, it was the period of determining hermaphroditism, and the hermaphroditism and pistil differentiated and developed in their respective gender direction. Then the stamen and pistil were developed and matured respectively until complete mature unisexual flowers are formed. Therefore, the difference of gene expression of male and female development mainly occurs in the latter two periods, which provides a reference for the study of dynamic development of asparagus flower structure, the sampling period of sex determination and differentiation, and the sampling period of sex chromosome evolution.

In addition, in the process of sex determination and differentiation and development of asparagus, our team has isolated the double gene TDF1-SOFF system to control the sex of asparagus in the early cooperation research, but it is also affected by a large number of development regulatory genes, and then forms different types of asparagus plants with different sex and various male and female organ development degree, which is also consistent with the various genetic phenomena observed in the asparagus population in nature (Harkess et al., 2017). At present, MADS box gene, which is homologous to ABC model related to the development of flower structure, has also been isolated, which is an important evidence at the molecular level (Park et al., 2004). Therefore, it is of great significance to analyze the mechanism of sex development of asparagus through reasonable sampling of flower buds in different development stages.

3 Materials and Methods

3.1 Experimental materials

In the experiment, five-year-old male and female variety "Atlas" was selected as the test material in spring of 2018. The buds of female and male plants at different stages of development were graded according to different sizes of bud length (divided into 6 stages: Level 1: 0.4mm; Level 2: 0.8mm; Level 3: 1.2mm; level 4: 1.6mm; level 5: 2.0mm; level 6: 2.4mm). Each stage had 20 buds, sampling with a size difference of no more than ± 0.1mm.

3.2 Experimental methods

According to the improved paraffin section method of Yang (2006), the experimental samples were fixed by FAA, dehydrated, paraffin embedded after transparency, conventional paraffin section, saffron green stained, sealed, and microscopic observed and photographed to compare the development process of male and female flower buds of asparagus and the morphological differences between them.

Authors’ Contribution

Wentao Sheng is responsible for experiment design, thesis writing and revision; Yongping Tang is responsible for experiment and image analysis. Wentao Sheng and Yongping Tang read and agreed to the final text.

Acknowledgement

The research was jointly supported by the key support project of science and technology in Jiangxi Province, "Construction of asparagus microspore culture technology system and creation of excellent germplasm" (20141BBF60012), Jiangxi Natural Science Youth Fund (20171BAB214024), Nanchang Normal University "11531" project and Nanchang Normal University Doctoral Fund Project (NSBSJJ2015036).

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