Dujuanlan (Cremastra appendiculata (D. Don) Makino) is a perennial medicinal herb of Cremastra genus in the family of Orchidaceae with rhizomes and pseudobulbs (Editorial Committee of FRPS, Chinese Academy of Sciences, 1999, Flora Republican Popularis Sinicae, pp.56). Its dry pseudobulbs are also known as Shancigu in Chinese, which has important functions to be taken orally such as hypoglycemic, anti-tumor, and hypotensive effects (Chinese Pharmacopoeia Commission, 2005, Chinese Pharmacopoeia, pp.23; Gao and Feng, 2011; Zhang et al., 2011; Lü et al., 2018), and can be used externally to treat snake and insect bites and pain relief of metastatic bone tumor (Xia et al., 2005; Zhang et al., 2006). However, with the destruction of the natural environment and excessive human excavation, coupled with its low coefficient of vegetative reproduction, natural pollination is difficult. Although artificial pollination can make Cremastra appendiculata bear fruit (Zhang et al., 2010; Tian et al., 2019), its seeds are as small as dust, seed coat is dense, embryo development is poor, and there is no endosperm (Guo and Xu, 1990; Liu et al., 2015; Wang et al., 2017), leading to the depletion of its wild resources. After realizing the artificial pollination and fruiting of Cremastra appendiculata, exploring an effective method to promote its seed germination has become an important problem to break through the sexual reproduction of this species. Through previous studies, we preliminarily determined that the main factor limiting the germination of Cremastra appendiculata seeds is that their embryos are not fully developed, while auxin and cytokinin play an important role in the process of embryo development, and have an important impact on the regulation of embryo growth and development process, organ formation and differentiation direction. The types and concentrations of plant growth regulators required by different plant varieties, immature embryos of the same variety at different development stages and different stages of immature embryo culture vary greatly (Liang et al., 2006).

In view of this, this study started from the role of plant growth regulators on the development of Cremastra appendiculata species embryos by paraffin method, which provides a theoretical basis for the construction of sexual reproduction technology of Cremastra appendiculata.

1 Results and Analysis

1.1 Effects of different plant growth regulators on embryo development

Under the action of different plant growth regulators, the rate of embryo development is obviously different. After 40 d of culture, the seed embryos added with 0.5 mol/L 2,4-D did not develop (Figure 1A), and the seeds did not swell with water. The seed embryo added with 0.5 mol/L IBA began to break through the seed coat (Figure 1B). The seeds added with 0.5 mol/L NAA have formed early hypocotyls, and the development speed is the fastest (Figure 1C), followed by the seeds added with 0.5 mol/L 6-BA, which also formed growth cones (Figure 1D). The seeds with 0.5 mol/L KT and 0.5 mol/L ZT could only absorb water and swell within 40 d, while the embryo was almost undeveloped (Figure 1E; Figure 1F). It can be seen that different plant growth regulators have different effects on the development of C. appendiculata, and NAA and 6-BA have better effects. 

1.2 Effects of different ratios of NAA and 6-BA on the development of seed embryos

The combination of plant growth regulator NAA and 6-BA can significantly accelerate the development of embryos, which can grow into mature embryo structure in about 40 d (Figure 2). The embryo of mature C. appendiculata has large polarity at both ends, and its front end is closely formed by irregular quadrilateral cells, with obvious staining effect and plump cells. The stalk end is composed of large rectangular cells. When the seeds of C. appendiculata are swollen by absorbing water in the culture medium, the seed embryo is observed by sectioning. Because of the swelling by absorbing water, its volume increases and becomes transparent. At the same time, it is observed that the two ends of the embryo body still have polarity. The cytoplasm of the epidermal cells at the front end is dense, closely arranged, the nucleus is clear, and the number of cells increases. And there was no significant change in the suspensor. When the seed embryo continues to absorb water until the embryo breaks through one side of the seed coat, the polarity of the two ends of the embryo increases, and the embryonic stem cells divide slower than the cells at the front of the embryo body, and the volume is larger. With the embryo breaking through the seed coat, the breaking embryo begins to form a growth point at the front of the embryo body, and continues to grow and differentiate. At this time, the suspensor cells gradually incline to the front of the embryo body. In this process, the embryo body gradually completely breaks through the seed coat to form a protocorm. With the further growth and development of the protocorm, a growth cone begins to appear on one side of the protocorm. The growth cone is composed of a large number of cells with large nuclei and dense cytoplasm. With the further growth and development, the protocorm begins to appear "embryonic form" of different parts, forming a special structure of "W" shape on its surface, which is similar to the "top zone" of Poaceae, and the formation zone of future leaf sheaths. The "Organogenesis area", which is similar to the Poaceae, will form embryo buds and hypocotyls in the future. The base of the protocorm could be regarded as the "radicle forming area" and will develop into a root in the future. In previous studies, this development process took three months, but now the interaction of NAA and 6-BA reduces this time to about 40 d. However, the proportion of NAA and 6-BA was different, and the number of seeds that could germinate after 40 d was significantly different. The lowest germination rate was 44.1% with 0.5 mol/L NAA and 1 mol/L 6-BA, and the highest germination rate was 88.11% with 1 mol/L NAA and 1 mol/L 6-BA (Table 1). Finally, the best proportion of plant growth regulators was selected as NAA: 6-BA=1 mol/L: 1 mol/L.

2 Discussion

Auxin and cytokinin are essential to regulate plant cell division and differentiation, and participate in the regulation of the formation of new organs such as lateral roots, nodules, crown galls, etc (Sugiyama et al., 1990; Hou, 2017). The combination of NAA and ZT can significantly improve the development height of mulberry (Morus alba L.). The same results have been obtained in other relevant studies: NAA added to the culture medium can promote the germination of Sutherlandia frutescens buds, and the mixed use of NAA and growth regulator Nijmegen can promote the increase of metabolites in the bud cells (Grobbelaar et al., 2014). Mixed use of low concentration NAA (1.3 μmol/L) and 6-BA (4.4 μmol/L) can improve the proliferation of tissue culture buds of Swerti corymbose (Mahendgan and Bai, 2014). Palovaara and Hakman (2009) found that cytokinin in Arabidopsis can regulate auxin induced root organogenesis by regulating auxin transport. This experiment showed that plant growth regulators have an impact on the development of C. appendiculata embryos. Except 2,4-D, the other five plant regulators have different degrees of promotion on the development of C. appendiculata embryos, and the combination of NAA and 6-BA has the most obvious effect on the promotion of embryo development.

After the mature seeds were artificially cultured, the spherical proembryo developed into a complete embryo structure after pear shaped embryo, and it cannot be simply considered that the germination rate of mature seeds was higher than that of immature seeds, because when the seeds reach a certain maturity, the increase of seed maturity will no longer significantly improve the germination rate, and may even reduce the germination rate (Kitsaki et al., 2004; Lan et al., 2013; Jia et al., 2017; Lin, 2018). After the seed embryo of C. appendiculata breaks through the seed coat to form the original ball diameter, there will be two ways of seedling emergence, one is to directly differentiate into seedlings, and the other is that the original embryo first multiplies into multiple branches of the original ball diameter, and then differentiates into seedlings. This study found that NAA combined with 6-BA can not only sprout directly but also root directly, which may be related to auxin regulating embryonic development at multiple levels (establishment of polarity of zygotes and apical-basal axis, differentiation of protoderm, transition of radial symmetry to bilateral symmetry, formation of hypophysis, root apical meristem and shoot apical meristem, etc.) (Song et al., 2013).

In this study, different plant growth regulators were used to greatly shorten the time required for the development and maturation of C. appendiculata embryos, and the plant growth regulator proportion suitable for the development of C. appendiculata embryos was NAA: 6-BA=1 mol/L: 1 mol/L. The germination time was 6 weeks, and the germination rate was from 32.87% to 88.11% (Wang et al., 2017), which laid a certain technical foundation for mass culture of C. appendiculata plants.

3 Materials and Methods

3.1 Experimental materials

Cremastra appendiculata Makino in this study was collected from Gaopo Township, Huaxi District, Guiyang City, Guizhou Province, and cultivated in the experimental base of Yangniu Village, Huaxi District (N26°27′55″E, 106°39′21″~N26°27′55″E106°39′21″, with average annual precipitation of 108.344, average maximum temperature of 21.66°C, average minimum temperature of 13.85°C, altitude of 1 152.5 m). The seeds of plants obtained by artificial pollination were used as experimental materials.

3.2 Seed germination

The basic medium for seed germination used in this experiment was MS+4 g/L agar+0.5 g/L activated carbon+20 g/L sucrose+75 g/L potato juice, pH 5.6, and NAA, 6-BA, KT, ZT, IBA, 2,4-D were added respectively to screen the appropriate medium for seed embryo development. Wiped the Cremastra appendiculata capsule with 75% alcohol, took out the seeds on the clean bench, soaked them in 75% alcohol for 15~30 s, and then sterilized them with 0.1% HgCl₂ for 6~8 min. After washing with sterile water for three times, used tweezers to take about 100 seeds and evenly spread them on the culture medium. Inoculated 5 bottles of each treatment for 3 times. The culture condition was set as follows: temperature of (24±1)℃, and the light intensity of 18.75~25 μmol·m^-2·s^-1, illumination time of 12 h/d. After 40 d of culture, the germination rate was counted (based on the standard of embryo breaking through the seed coat).

3.3 Observation on the development of seed embryo

Cremastra appendiculata seeds in different culture periods were fixed with 70% FAA fixative for more than 2 d. Then 2% agar was used to make agar blocks, which were dehydrated by alcohol at all levels. And 1/2 alcohol+1/2 xylene and xylene (twice) for clearing. After soaking in 1/2 xylene+1/2 wax, soaked in pure wax for 3 times. After embedding, the embryos were sliced, glued, unfolded, baked, dewaxed, rehydrated, dyed, dehydrated, fixed green and re stained, and observed under microscope after transparency to understand the development of the embryos.

3.4 Statistical analysis

Excel 2016 was used for data sorting, SPSS 22.0 was used for data analysis, and PhotoShop CS6 was used for image processing.

Authors’ contributions

TL was the experimental designer and executor of this study, and was responsible for the writing of the first draft. PSJ participated in artificial pollination of Cremastra appendiculata to obtain experimental seeds. GYY and YNX participated in the paper revision. ZMS guided paper writing and revision. All authors read and approved the final manuscript.

Acknowledgments

This study was supported by National Natural Science Foundation of China (81660627 and 81360613), National Key R&D Program (2016YFC0502604), Science and Technology Program of Guizhou Province (Talents of QKH [2017] 5411-06 and QKH [2017] 5788), Special Fund Project of Guizhou Province for the Construction of Scientific and Technological Innovation Talent Team (Talents of QKH [2016] 5624), High Level Innovative Talents Training Program of Guizhou Province (Talents of QKH [2015] 4031), and Major Research Project of Innovative Groups of Department of Education of Guizhou Province (QJH KY [2016] 023).

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