PROPAGATION AND TISSUE CULTURE HORTSCIENCE 52(8):1111–1116. 2017. doi: 10.21273/HORTSCI10525-17 embryogenesis, especially in species such as the orchid, in which zygotic embryos are inaccessible. Initiation and Cytological Aspects of Although there were a few studies on the zygotic embryogenesis of orchid such as Somatic Embryogenesis in Dendrobium Cymbidium (Huang et al., 1998; Ye and Guo, 1995), Phaius tankervilliae (Ye et al., candidum Wall ex Lindl. 1997), and D. candidum (Xu et al., 1995), how somatic embryos are generated in the Yihui Cui1 orchid and how similar the process might be School of Life Science and Engineering, Southwest Jiaotong University, to zygotic embryogenesis remain unclear. Chengdu 610031, People’s Republic of China We have previously established an efficient somatic embryogenesis system in D. candidum, Peng Zhao1 in which new PLBs would be achieved from School of Life Science and Engineering, Southwest Jiaotong University, the embryonic calli (Zhao et al., 2008). In this article, we described the details on the de- Chengdu 610031, People’s Republic of China; and State Key Laboratory of velopmental process of somatic embryogen- Hybrid Rice, College of Life Sciences, Wuhan University, Wuhan 430072, esis and focus on the formation of initial People’s Republic of China somatic embryo cells and pathways of so- matic embryo’s origin. Hongqiang An, Nan Lv, Zifeng Zhang, Wei Pei, and Wanjun Wang2 School of Life Science and Engineering, Southwest Jiaotong University, Materials and Methods Chengdu 610031, People’s Republic of China Induction and regeneration of somatic Additional index words. somatic embryogenesis, Dendrobium candidum, orchid, calli embryo. Mature seeds of D. candidum were Abstract cultured in an MS medium supplemented . To find the characteristics of somatic embryogenesis of orchids and elucidate m the mechanism, we had previously established an efficient plant regeneration system with 1.08 M 1-naphthaleneacetic acid. The via somatic embryogenesis in Dendrobium candidum Wall ex Lindl. In this study, protocorms, about 3 mm in diameter, were a detailed cytological investigation was carried out on the initiation and developmental longitudinally bisected. The calli were in- process of somatic embryogenesis. Based on our observations, the somatic embryo- duced from longitudinally bisected segments genesis in D. candidum originated from the transition of an embryonic callus cell to the of protocorms and subcultured two times initial somatic embryo cell, and the somatic embryos initiated from those cells. During every 40 d for screening the embryonic calli on a 1/2MS medium supplemented with the transition process, condensation and devacuolation successively occurred in the m cytoplasm of the embryonic callus cells, giving rise to the formation of a typical initial 8.8 M 6-Benzylaminopurine (Zhao et al., somatic embryo cell with dense cytoplasm and a clear nucleus. One of the two 2008). The embryonic calli were then cul- pathways in somatic embryogenesis is the single-cell-derived somatic embryo which is tured for the induction of somatic embryos on generated from an inner initial somatic embryo cell in embryonic callus and develops a modified 1/2MS medium without any plant into a globular somatic embryo in a way similar to zygotic embryogenesis and then growth regulators (mMS) under illumination (30–34 mmol·m–2·s–1) with 12-h photoperiod keeps developing into a protocorm-like body (PLB). The other is a multiple-cell- ° derived somatic embryo which is generated from peripheral grouped initial somatic and a temperature of 25 ± 2 C. cells in embryonic calli and directly forms globular embryo or multicellular somatic Histological study of somatic embryo proembryo, lacking the typical early stages of embryogenesis. Both pathways were development. The embryonic calli on mMS observed in the somatic embryogenesis system, indicating that the culture system in were fixed in FAA solution (formalin: acetic D. candidum acid: 70% ethanol = 1:1:18 v/v/v) every day can be a useful tool for investigating the mechanisms underlying orchid ; embryogenesis. (0 40 d), dehydrated in an ethanol series, and embedded in paraffin wax. The embedded materials were sectioned to a thickness of 8 mm with a Microtome QPJ-1B (TianLi Dendrobium candidum Wall ex Lindl., 1985; Nagmani et al., 1987; Williams and Aviation Electro-Mechanical Co., Ltd, China) a monocotyledon belonging to the family Maheswaran, 1986). Thus, little is known and stained with 1% Safranin 0 (Sigma- Orchidaceae, is distributed in countries of about the process of embryo development in Aldrich, St. Louis, MO) and 0.5% Fast Green South and Southeast Asia. As an endangered Dendrobium, and somatic embryogenesis (Amresco, Solon, OH) according to previously species, mass propagation of this orchid has may provide an alternative system to study reported methods (Jensen, 1962). been actively studied (Shiau et al., 2005; this process in orchids. Observation and image collection. Single- Zhao et al., 2007, 2008), and it is also of Somatic embryogenesis, a regeneration and multiple-cell origination of somatic em- interest in the isolation of new medical technique effective in plant mass propagation, bryos was visualized from sections under compounds (Li et al., 2008, 2009a, 2009b). has been established in many species, e.g., a light microscope (IX71, Olympus, Tokyo, Embryo development in the Orchidaceae Daucus carota (Lee et al., 2001; Nishiwaki Japan) equipped with a charge-coupled device differs from that of all other monocotyle- et al., 2000), Oryza sativa L. (Meneses et al., camera (SPOT Imaging Solutions, Sterling dons and dicotyledons because embryogeny 2005; Raval and Chattoo, 1993), Arabidopsis Heights, MI). ceases at very early stage as the endosperm thaliana (Ikeda-Iwai et al., 2002; Luo and Environmental scanning electron degenerates (Maheswaran and Williams, Koop, 1997), Phalaenopsis (Chen and Chang, microscope (ESEM) study. Embryonic calli 2006; Chung et al., 2006), and Cymbidium at different developmental stages were exam- (Huan et al., 2004). The developmental ined by ESEM (Quanta 200; FEI, Eindhoven, Received for publication 15 Dec. 2015. Accepted process of somatic embryogenesis shares The Netherlands) in their natural form with- for publication 10 May 2016. considerable similarity with that of zygotic out additional sample preparation. This work was supported by the Grant (No. 31371232) from the National Natural Science Foundation of the embryogenesis (Zimmerman, 1993) because People’s Republic of China. of the proposed conservation of molecular Results 1Co-first authors. mechanisms underlying both processes 2Corresponding author. E-mail: wanjunwang@home. (Higashi et al., 1998). Therefore, it provides Cytological characteristics of embryonic swjtu.edu.cn. us a useful model system for studying callus cells. Histologically, embryonic calli HORTSCIENCE VOL. 52(8) AUGUST 2017 1111 were composed of two different types of embryo cell are always located inside the cell was usually elongated and dense in cells. One comprises highly vacuolated cells embryonic callus, whereas those from the cytoplasm (Fig. 3A). The first division of with a very thin layer of cytoplasmic mate- grouped initial somatic embryo cells are often the initial somatic embryo cell was usually rials. The other is relatively cytoplasm-rich located at the peripheral region of the embry- asymmetric and gave rise to two cells of cells with loosened cytoplasmic materials onic calli. Multiple-cell-derived somatic em- different size (Fig. 3B), a smaller ‘‘apical (Fig. 1A). Somatic embryogenesis was initi- bryos were usually generated at the peripheral cell’’ with dense cytoplasm and a larger ated when the embryonic calli were trans- region of embryonic calli, where both the ‘‘basal cell’’ with some vacuoles, which ferred into the mMS medium. Some of the outermost and sublayer cells of the embryonic turned on the development of the somatic embryonic callus cells were undergoing a cy- calli could convert into initial somatic embryo proembryo. The larger ‘‘basal cell’’ divided tological transition, which was indicated cells (Fig. 2A and B). These cells were located obliquely first (Fig. 3C and D), and the by cytoplasmic materials’ condensation fol- in a group and actively dividing. With rapid smaller ‘‘apical cell’’ continued to divide lowed by cytoplasm enrichment. During cell division, the initial somatic embryo cells longitudinally, resulting in the formation of somatic embryo induction, the loosened cy- developed directly into globular somatic em- a four-celled somatic embryo (Fig. 3E). Sub- toplasmic materials of some embryonic cal- bryos. Young globular somatic embryos de- sequently, the newly formed additional basal lus cells (Fig. 1A) gradually gathered to rived from the initial somatic embryo cells on cell divided once, giving rise to a five-celled become concentrated (Fig. 1B and C). Sub- the outermost layer of calli were soon ob- somatic embryo (Fig. 3F). The number of sequently, the cytoplasm of embryonic callus served on the surface (Fig. 2C and I). By cells increased without clearly visible expan- cells became denser and large vacuoles grad- contrast, a multicellular somatic proembryo sion of somatic embryo volume until the five- ually disappeared. In some cases, however, formed from the gathered multiple initial celled somatic embryo stage. Since then, as small vacuoles remained visible in the cyto- somatic embryo cells in the sublayer of the cells divided and somatic embryo grew, it plasm.