Abstract
In this study, we document the primary structural changes that occur during the seed development of Paphiopedilum spicerianum (Rchb.f.) Pfitzer, an endangered species with high horticultural value. Within a defined timeline, our results offer insights into the connection between these structural changes in seeds and their germination percentage. The optimum germination was recorded for immature seeds collected at 180 to 210 days after pollination (DAP), during which the embryos are in the late globular stage and the suspensor begins to degenerate. As seeds continued to mature by 240 DAP, there was a gradual decline in germination. Histochemical staining of mature seeds reveals that only the inner seedcoat and the surface of the embryo exhibit positive reactions to the Nile red stain, suggesting a relatively weak coat-imposed dormancy. This weaker dormancy may contribute to the higher germination observed in mature seeds of P. spicerianum compared with other challenging-to-germinate species. Of the cytokinins examined, 6-(γ,γ-dimethylallylamino)purine (2iP), kinetin (KN), and 6-benzylaminopurine (BA) exhibited a stimulating effect on germination, concurrently enhancing the formation of amorphous protocorms.
The genus Paphiopedilum Pfitzer comprises several horticulturally important species that have been broadly cultivated in the world and produces a wide range of attractive cultivars and hybrids (Cribb 1998). Paphiopedilum spicerianum (Rchb.f.) Pfitzer is a terrestrial orchid that is distributed in Assam, Bhutan, northwestern Myanmar, and southwestern China (Liu et al. 2009; Rankou and Molur 2015). This species has been introduced to the horticultural market since 1878 and used frequently as a parent in the breeding program. Nevertheless, it is a critically endangered species in the wild because of over-collection and habitat destruction. In China, only a small population of ≈10 mature plants in the Pu’er Prefecture of Yunnan Province has been recorded (Ye and Luo 2006). Because of its critically endangered situation in the wild, the integrated approach has been used for conservation works, including seed germination, mycorrhizal symbiosis (Han et al. 2016), reintroduction, and pollination ecology (Chen et al. 2015; Gao et al. 2018).
Under natural conditions, the successful germination of most orchids typically depends on a symbiotic relationship with mycorrhizal fungi because their seeds are minute and lack stored nutrients (Arditti 1992). Following Knudson's discovery (1922) that orchid seeds are capable of germinating in a culture medium without mycorrhizal fungi, the technique has been termed asymbiotic germination. This method has grown into an important propagation strategy for many orchid species. Nonetheless, as in many terrestrial orchids, the seed germination of Paphiopedilum species is intricate (Pierik et al. 1988; Stimart and Ascher 1981). There are several reports of asymbiotic seed germination in Paphiopedilum (Chen 1996; Lee 1998; Yao et al. 2021; Zeng et al. 2012, 2016). Various strategies, including the ultrasonication pretreatment (Chen 1996), presoaking seeds in NaOCl solution (Diengdoh et al. 2017; Lee 2007; Zeng et al. 2012), and the application of phytohormones (Diengdoh et al. 2017; Yao et al. 2021; Zeng et al. 2013) have been used to improve the seed germination and/or protocorm formation of various Paphiopedilum species. For the conservation and commercial production of this endangered species, information concerning its reproductive biology and improved methods of in vitro propagation are thus of great importance (Arditti and Ernst 1993). Basic knowledge of embryo and seed development will aid in the design of experiments for asymbiotic seed germination studies, as shown in our previous studies for Cypripedium L. species (Hsu and Lee 2012; Jiang et al. 2017; Lee et al. 2005; Perner et al. 2022; Zhang et al. 2013). At present, the detailed information concerning embryo development in Paphiopedilum species is limited, except for the work on Paphiopedilum insigne var. sanderae (Wall. ex Lindl.) Pfitzer (Nagashima 1982), Paphiopedilum delenatii Guillaumin (Lee et al. 2006), and Paphiopedilum armeniacum S.C.Chen & F.Y.Liu (Xu et al. 2020). In P. spicerianum, Chen et al. (2015) investigated the effects of medium composition and seed pretreatment on the germination of mature seeds. Afterward, Fang et al. (2021) examined changes in lignin content and endogenous hormone levels during embryo and protocorm development and conducted transcriptome profiling of developing protocorms. Yet, the structure of embryo development in P. spicerianum, as observed through laser scanning confocal microscopy, did not provide high-resolution images within tissues. In this study, we focused on the structural pattern of embryo development in P. spicerianum from fertilization to seed maturity, as well as histochemical changes during seed development, providing complementary information to the study by Fang et al. (2021). In addition, we evaluated the effect of various cytokinins added to the medium on the germination of mature seeds and the growth of protocorms.
Materials and Methods
Plant material and the timing of capsule collection.
Plants of P. spicerianum were artificially propagated and maintained in a greenhouse at Horticulture Technology Center, National Chiayi University, Chiayi, Taiwan. Plants were grown in pine bark medium (Orchiata; Besgrow, Christchurch, New Zealand) under 70% shade. Anthesis occurs during middle September to late October. To ensure a good fruit set and seed quantity, flowers were self-pollinated manually. Developing fruits were harvested at regular intervals after pollination. Approximately 80 developing fruits were gathered for the subsequent experiments.
Light microscopy.
Seeds and protocorm samples were fixed with 2.5% glutaraldehyde in 0.1 M sodium phosphate buffer, pH 6.8, at 4 °C overnight. After three rinses with the buffer for 15 min each, the specimens underwent dehydration in a series of ethanol solutions before being processed for embedding in Historesin following the protocol reported by Yeung and Chan (2015). Sections of 3-μm thickness were cut using glass knives on a Leica RM2135 microtome. These sections were subjected to periodic acid–Schiff (PAS) staining and counterstained with toluidine blue O (TBO) for general histological observations, following the procedure by Yeung (1984). Examination and imaging of these preparations were conducted using a Carl Zeiss Imager A1 light microscope (Carl Zeiss AG, Jena, Germany). The presence of the cuticular material was determined using Nile red according to the procedure described by Yeung et al. (1996). The Historesin embedded tissues were stained with 1 μg·mL−1 of Nile red (Sigma Chemical Co., St. Louis, MO, USA) for 5 min, then briefly washed in distilled water and mounted in water containing 0.1% n-propyl gallate (Sigma Chemical Co.), an antifading agent. The fluorescence pattern was examined using an epifluorescence microscope (Imager A1, Carl Zeiss AG) equipped with the Zeiss filter set 15 (546/12 nm excitation filter and 590-nm emission barrier filter).
Seed germination.
Developing capsules were collected and taken to the laboratory for further analysis. They underwent surface sterilization using a 1% sodium hypochlorite (NaOCl) solution with one drop of Tween 20 (Sigma-Aldrich, St. Louis, MO, USA) for 30 min. Following surface sterilization, the capsules were carefully opened, and the seeds were extracted using forceps onto the culture medium. To ensure the quality and developmental stage of each seed within the capsule, the remaining seeds were fixed and examined under a microscope. Approximately 90% of the seeds exhibited fully developed embryos, as confirmed through compound microscopy. The culture medium used in this experiment was the 1/4 Murashige and Skoog (MS) medium (Murashige and Skoog 1962), which was supplemented with 1 g·L−1 tryptone (Sigma-Aldrich), 20 g·L−1 sucrose (Sigma-Aldrich), 100 mL·L−1 coconut water, 1 g·L−1 activated charcoal (Sigma-Aldrich), and solidified with 7 g·L−1 agar (Sigma-Aldrich). Before autoclaving at 121 °C for 15 min, the pH of the medium was set to 5.6. Each culture tube (20 × 100 mm) was then filled with 10 mL of this medium. Following sowing, the cultures were placed in a growth room and incubated at 25 ± 1 °C in complete darkness.
Effect of cytokinins on asymbiotic germination and protocorm development.
Mature seeds (330 DAP) were collected and used to investigate the effect of cytokinins on asymbiotic germination. Following surface sterilization, capsules were cut open, and then mature seeds were scooped out with forceps onto the 1/4 MS medium, supplemented with either 6-(γ,γ-dimethylallylamino)purine (2iP, Sigma-Aldrich), 6-benzylaminopurine (BA, Sigma-Aldrich) or kinetin (KN, Sigma-Aldrich) at concentrations of 1, 2, 4, and 8 μM. 1/4 MS medium without cytokinin supplementation was used as a control.
Experimental design and statistical analysis.
The experiments were conducted using a completely randomized design and replicated three times. For each treatment, 12 replicates (culture tubes) were used. Statistical analysis of the data was performed using analysis of variance alongside Fisher’s protected least significant difference test, with a significance level set at P < 0.05. All statistical computations were carried out using SAS software (version 9.0; SAS Institute, Cary, NC, USA).
Results
Embryo development.
The major structural changes occurring within the ovary from ovule development until seed maturity at 330 DAP are listed in Table 1. Fertilization occurred ∼60 DAP, and the zygote could be observed in most embryo sacs at 75 DAP (Fig. 1A). By 90 DAP, the zygote began to elongate (Fig. 1B). The zygote appeared as a highly polarized cell. The nucleus of the elongated zygote was located toward the chalazal end, and a prominent vacuole was found toward the micropylar end. Like most orchid species, seeds of P. spicerianum do not possess endosperm. The polar nuclei did not divide within the endosperm cavity, then finally disintegrated during the embryo development (Fig. 1E and F). The first division of the zygote was unequal, producing a smaller terminal cell and a larger basal cell (Fig. 1C). In the five-celled embryo (Fig. 1D), the terminal tier of cells toward the chalazal end further divided and resulted in the formation of early globular embryo proper (Fig. 1E). Mitotic figures were readily observed during the early stages of formation of the embryo proper (Fig. 1F). By 90 DAP, additional cell divisions occurred in the inner tiers as well as in the surface layer of the early globular embryo resulting in the growth of the embryo proper.
Major developmental events occurring in developing seeds of Paphiopedilum spicerianum after pollination.
Light micrographs of the early stages of Paphiopedilum spicerianum embryo development. (A) Light micrograph of the zygote (arrow) after fertilization at 75 d after pollination (DAP). As comparing the entire space within the embryo sac, the volume of the zygote is quite small. Scale bar = 50 μm. (B) Light micrograph of an elongated zygote (arrow) at 105 DAP. The zygote exhibits significant polarization, characterized by a nucleus located chalazally and a prominent vacuole occupying the micropylar end. The endosperm fails to develop in this species and the degenerated polar-chalazal complex (arrowhead) can be observed. Scale bar = 50 μm. (C) The initial cell division of the zygote is asymmetric, leading to the formation of a smaller terminal cell and a larger basal cell. Scale bar = 50 μm. (D) Subsequent cell divisions in both the terminal and basal cells give rise to a proembryo. Scale bar = 50 μm. (E) The development of the embryo proper progresses through periclinal (arrow) and anticlinal (arrowhead) divisions, leading to the formation of a globular-shaped embryo. At this stage, the suspensor (S) comprises two cells. Scale bar = 50 μm. (F) By the early globular stage (135 DAP), the anticlinal division (arrow) takes place in the outer layer of cells, leading to an enlargement of the embryo. Scale bar = 50 μm. IS = the inner seedcoat; OS = the outer seedcoat.
Citation: HortScience 59, 8; 10.21273/HORTSCI17917-24
Periclinal divisions from the surface layer of cells gave rise to the protoderm as well as contributing additional cells to the embryo proper. A distinct protoderm layer could be found ∼105 DAP (Fig. 2A). At the early globular stage (135 DAP), starch granules and vacuoles of variable sizes could be observed within embryo proper cells (Fig. 2B). As the embryo reached the mature globular stage (150 DAP), mitotic activity had ceased. Starch granules gradually disappeared within the cytoplasm and the large vacuoles became smaller (Fig. 2C). At maturity, the embryo was only nine cells long and six cells wide (Fig. 2D). The embryo cells had abundant storage materials deposited in the form of lipid and protein bodies in the cytoplasm.
Light micrographs of the late stages of Paphiopedilum spicerianum embryo development. (A) At 150 d after pollination (DAP), a globular embryo becomes visible. The suspensor has expanded and elongated toward the micropylar end of the embryo sac. Scale bar = 50 μm. (B) Within a globular embryo, the transition from large vacuoles to smaller ones begins, accompanied by the emergence of storage materials (arrow). In addition, the suspensor is visible, consisting of three cells (arrowheads). Scale bar = 50 μm. (C) As the seed approaches maturity (180 DAP), most cells of the embryo proper have filled with the storage materials (arrows). Scale bar = 50 μm. (D) Light micrograph showing a longitudinal section through a mature seed (240 DAP). The embryo proper is approximately nine cells long and six cells across at its broadest point. The suspensor has degenerated, and the mature embryo is enclosed by the withered inner seedcoat (arrow). Scale bar = 50 μm. (E) In some sections of mature seeds, the embryo is absent, revealing the clear outlines of both the outer seedcoat (arrowhead) and the inner seedcoat (arrow, also known as the carapace). Scale bar = 50 μm. (F) Nile red staining fluorescence microscopy image of a matured seed. The inner seedcoat (arrow) and the outer surface wall of the embryo proper (arrowhead) react positively to the stain. Scale bar = 50 μm. IS = the inner seedcoat; OS = the outer seedcoat.
Citation: HortScience 59, 8; 10.21273/HORTSCI17917-24
The suspensor consisted of three cells in this species, and it originated from the larger basal cell after the first division of the zygote (Fig. 1C). At the five-celled embryo stage, the cell nearest to the micropyle further divided and finally gave rise to a three-celled suspensor (Fig. 1D). Throughout the embryo development, the suspensor cells were more vacuolated when compared with cells of the embryo proper. As the seed approached maturity, the suspensor began to degenerate (Fig. 2C) and finally compressed (Fig. 2D).
Seedcoat development.
The seedcoat of P. spicerianum was derived from the inner and outer integuments of the ovule. The inner seedcoat (derived from the inner integument) consisted of only one cell layer, and the outer seedcoat (derived from the outer integument) had two cell layers. As the seed matured, the inner and outer seedcoats began to compress and gradually shrivel (Fig. 2C). Both the inner and outer seedcoats became dehydrated and constricted, forming two distinct layers that enclosed the embryo upon reaching maturity (Fig. 2D and E). The cell wall of the outer seedcoat looked thicker than that of the inner seedcoat (Fig. 2E). The walls of the seedcoat were stained greenish blue with TBO stain (Supplemental Fig. 1), suggesting the presence of phenolic compounds in their walls. Using Nile red stain, prominent fluorescence could be detected on the surface wall of the mature embryo, suggesting the accumulation of cuticular material in the wall (Fig. 2F). As compared with the surface wall of the embryo, the fluorescence was less intense in the inner seedcoat. In the outer seedcoat, no fluorescent signal could be observed, indicating the lack of the cuticular material in the wall (Fig. 2F).
Seed germination in vitro.
The timing of seed collection obviously affected the germination percentage of P. spicerianum (Fig. 3). Higher germination was found at 180 and 210 DAP (ranged from 80.1% to 87.8%). No seed germination was found from 60 to 90 DAP. A noticeable increase in germination was recorded at 150 DAP. After 240 DAP, germination gradually decreased (from 50.8% to 32.4%).
The mean percent germination of Paphiopedilum spicerianum seeds on 1/4 Murashige and Skoog medium, collected at 15-d intervals beginning 60 d after pollination. Data were scored after 6 months of culture. Error bars represent SE (n = 3).
Citation: HortScience 59, 8; 10.21273/HORTSCI17917-24
Influence of cytokinins on asymbiotic germination and protocorm development.
The addition of 2iP, KN, and BA promoted the germination percentage of mature seeds significantly compared with the control without cytokinin after 4 months in culture (Fig. 4). For 2ip, the germination percentage increased with concentration until 8 μM. For both KN and BA, the germination percentage increased with concentration until 4 μM. The higher concentration at 8 μM of KN and BA slightly reduced the germination percentage. Although the addition of cytokinins promoted the germination, it also increased the formation of amorphous protocorms with cells proliferating in an unorganized and undifferentiated manner (Table 2). Histological investigations were also conducted to observe the formation of amorphous protocorms during germination. In the normal protocorm, the embryo became swollen and ruptured the seedcoat. The distinct meristemoid appeared at the terminal region (Fig. 5A). As the protocorm further developed, an obvious gradient of cell size with the smaller cytoplasmic cells at the future shoot pole and the larger, highly vacuolated cells at the basal region could be observed (Fig. 5B). On the contrary, at the early stage of amorphous protocorm development, the terminal region remained vacuolated, and there were fewer starch grains within the protocorm cells (Fig. 5C). As the amorphous protocorm further enlarged, a distinct protoderm could not be observed. Within the amorphous protocorm, several small meristemoids appeared, and most cells were highly vacuolated without the accumulation of storage products, such as starch grains (Fig. 5D).
Effects of different cytokinins at various concentrations on the seed germination of Paphiopedilum spicerianum after 6 months of culture. Error bars represent SE (n = 3).
Citation: HortScience 59, 8; 10.21273/HORTSCI17917-24
Table 2. Effect of cytokinins on protocorm development of Paphiopedilum spicerianum after seed germination.
Light micrographs showing the developmental stages of Paphiopedilum spicerianum from seed germination to protocorm formation. (A) The enlarged embryo after 3 weeks of inoculation. Mitotic divisions (arrow) have begun at the apical region. The cells of the embryo expand due to vacuolar expansion, and the storage nutrients present at seed maturity, such as protein bodies and lipid bodies, have disappeared. Numerous starch grains (arrowheads) appear within most of the embryo cells. Scale bar = 100 μm. (B) By 6 weeks of inoculation, the protocorm has elongated. The cells at the future shoot pole (arrow) can be recognized by their smaller size and a high nucleus-to-cytoplasm ratio. Scale bar = 100 μm. (C) After 3 weeks of inoculation in culture media with cytokinins, some embryos have enlarged but show fewer mitotic divisions in the apical region. Scale bar = 100 μm. (D) At 6 weeks of inoculation, the protocorm developing in culture media with cytokinins lacks a prominent shoot pole and exhibits mitotic divisions (arrows) at both the upper and basal regions, leading to the formation of an amorphous protocorm. Scale bar = 100 μm.
Citation: HortScience 59, 8; 10.21273/HORTSCI17917-24
Discussion
In this study, we report the main structural changes during the seed development of P. spicerianum based on the defined time frame. In addition, we investigated the effect of seed maturity on asymbiotic germination along the timeline. Our results provide information about the interrelation between the structural changes of seeds and germination percentage. The duration of the fruiting period varies a lot among Paphiopedilum species. P. spicerianum and several species in Indochina and southwestern China usually have a long fruiting period (more than 360 DAP), whereas a relatively short fruiting period is observed in the species from the tropical lowland of southeast Asia, for example Paphiopedilum godefroyae (God.-Leb.) Stein (≈210 DAP) and Paphiopedilum philippinense (Rchb.f.) Stein (≈180 DAP) (Chen 1996). The diverse fruiting period may reflect the climate of their natural habitats. It is notable that the fertilization of P. spicerianum took place ≈60 DAP, but the fist cell division of the zygote occurred at 105 DAP (Table 1; Fig. 1A–C). In angiosperms, following double fertilization, the endosperm begins cell division rapidly, while the cell division of the zygote is delayed for a few days. During this period, the nonproliferative state of the zygote is transcriptionally quiescent (Kao and Nodine 2019). The transcriptional quiescence of the plant zygote may provide the opportunity for the endosperm–embryo interactions and for the extensive genome reprogramming (Pillot et al. 2010). In P. spicerianum, it is interesting to note that the degenerated endosperm leaves behind a relatively large embryo sac, measuring ∼125 μm in width and 250 μm in length, compared with other epiphytic orchids after fertilization (Yeung and Law 1997). The long period (≈45 d) of the quiescent state of the zygote in P. spicerianum may be required for the extensive reorganization of the zygote to adjust the large empty cavity in the embryo sac.
The timing of seed collection is known to significantly affect the asymbiotic seed germination in many terrestrial orchids (De Pauw and Remphrey 1993; Jiang et al. 2017; Lee et al. 2005; Zhang et al. 2013). In Cypripedium species, the seeds at the proembryo stage are capable of continuous development and germination when placed on culture media (Lee et al. 2005; Light and MacConaill 1990). In P. spicerianum, although seeds at the proembryo stage exhibit low germination percentage, a noticeable increase in germination is observed by the early globular stage (135 DAP). Seeds collected from 180 to 210 DAP represent the optimum period for maximizing germination (Fig. 3), coinciding with the early globular to globular stages of embryo development. As shown in Fig. 2A and B, a three-celled suspensor is present, accompanied by cell division within the embryo proper. These findings align with previous observations, suggesting few barriers to seed germination in some terrestrial orchids when seeds are excised while the suspensor is still active, and the seedcoat has not yet acquired moisture-repellent qualities (Rasmussen 1995). In this study, for the seeds collected after 240 DAP, the germination gradually decreased (Fig. 3). Difficulties encountered in the germination of fully mature seeds may be due to various factors, including the physical constraint, such as the thick and impermeable seedcoat (Rasmussen 1995; Yamazaki and Miyoshi 2006), and/or physiological factors, such as the presence of inhibitors [e.g., abscisic acid (ABA)] (Lee et al. 2007, 2015; van der Kinderen 1987; Van Waes and Debergh 1986).
In addition to the outer seedcoat, P. spicerianum possesses a thin layer of inner seedcoat (also known as carapace) that encloses the embryo at maturity (Fig. 2E). In several terrestrial orchids, the seed with a firm and thick inner seedcoat is more difficult to germinate than those with an incomplete inner seedcoat or those without an inner seedcoat (Lee et al. 2005; Veyret 1969). Earlier studies have indicated that seeds of terrestrial orchids, like those of Cypripedium formosanum Hayata, which have a closely adhering inner seedcoat filled with heavy hydrophobic materials, are challenging to germinate in vitro (Lee et al. 2005). In contrast, mature seeds of Cypripedium debile Rchb.f., which possess a looser inner seedcoat with less hydrophobic material, tend to germinate more easily (Hsu and Lee 2012).
The germination percentage of mature seeds shows significant variation among Paphiopedilum species. For example, P. armeniacum exhibits a low germination percentage of only 2% (Zhang et al. 2015), whereas P. spicerianum has a notably higher germination range of 32.4% to 37.5%. This disparity might be attributed to the differences in the seedcoat thickness between the two species. Specifically, the seedcoat of P. spicerianum (Fig. 2E) is thinner and potentially less rich in lignin deposition compared with that of P. armeniacum (Fang et al. 2020, 2021). In P. armeniacum, the seedcoat tends to thicken at the early globular stage, with walls forming an unevenly thickened barrier and turning a dark black color as the seeds approach maturity (Zhang et al. 2015). This thickened seedcoat likely acts as a physical barrier to water and gas exchange, contributing to the low germination percentage observed. In the present study, we used a hydrophilic glycol methacrylate embedding resin (Yeung and Chan 2015) to obtain high-resolution, serial sections of developing embryos. During the embryo development of P. spicerianum (Figs. 1 and 2), the seedcoat walls did not become as thick as those in P. armeniacum. Moreover, the seedcoat of P. spicerianum turned greenish blue with TBO stain (Supplemental Fig. 1), indicating the accumulation of lignin in their walls. Our histochemical analysis using Nile red stain revealed minimal cuticular material in the inner seedcoat walls and none in the outer seedcoat walls (Fig. 2F), suggesting a less hydrophobic nature in the mature seeds. This characteristic likely contributes to the higher germination percentage observed in the mature seeds of P. spicerianum.
In several terrestrial orchids, the decrease in germination of mature seeds could be explained in part by the accumulation of ABA as the seed matured (Lee et al. 2007, 2015; Van der Kinderen 1987; Yan et al. 2017). ABA is one of the most important phytohormones that regulates embryonic maturation, seed dormancy, and germination (Bewley and Black 1985). The accumulation of high ABA levels in mature seeds likely helps maintain their dormancy, leading to lower germination. In P. armeniacum, the addition of exogenous ABA suppresses the asymbiotic germination of immature seeds (Xu et al. 2020). Conversely, P. spicerianum seeds initially have a relatively low ABA concentration (0.4 ng/g), which significantly rises to 2.1 ng/g as the seeds mature, suggesting a critical role in regulating seed development and germination regulation (Fang et al. 2021).
Seed germination is affected by cytokinins, but the requirements for exogenous cytokinins vary greatly among orchid species (Yao et al. 2021). The seed germination of Epidendrum fulgens Brongn. does not require additional cytokinins because of the sufficient endogenous cytokinins in the seeds (Mercier and Kerbauy 1991). On the contrary, the requirement for cytokinins in seed germination has been reported in several Cypripedium species (De Pauw et al. 2015; Perner et al. 2022). Although mature seeds of P. spicerianum could germinate on medium without cytokinins, the application of 2iP and KN promoted the germination of mature seeds (Fig. 4). This may be because of the presence of an inadequate level of endogenous cytokinins in those ungerminated seeds. In P. spicerianum, the accumulation of a higher level of ABA was observed in mature seeds (Fang et al. 2021). It has been suggested that cytokinins may enhance seed germination by counteracting the inhibitory effects of ABA (Davies 2004; Thomas et al. 1997). The supplement of cytokinins in the culture medium may overcome the inhibitory influence of ABA and consequently accelerate germination. After germination, the proliferation of protocorms or callus induction from developing protocorms of Paphiopedilum can be induced by the supplementation of cytokinins or in combination with auxins (Nhut et al. 2005; Zeng et al. 2013), but the histological pathway for the formation of multiple protocorms is scarce. In this study, the presence of some meristemoids in the amorphous protocorm could be frequently observed on the medium with exogenous cytokinins (Fig. 5). The proliferation and further differentiation of these meristemoid cells can result in the formation of multiple protocorms. In the control or at low cytokinin concentrations, a single protocorm with a distinct shoot apical meristem elongates and then eventually forms a shoot.
Conclusions
In conclusion, our study reveals the crucial anatomical events occurring during the seed development of P. spicerianum, spanning from fertilization to seed maturity, within a defined timeframe. Notably, we observed a prolonged quiescent period, ∼45 d at the zygote stage, which likely contributes to the extended fruiting period characteristic of this species. By providing a precise timeline of developmental stages, our findings offer valuable insights into determining the optimal stage for seed collection and enhancing seed germination efficiency. Furthermore, our investigation reveals that the addition of cytokinins not only facilitated germination but also led to an increase in the formation of amorphous protocorms. Overall, our study contributes fundamental knowledge essential for the mass propagation of P. spicerianum to meet commercial demands and aid in the conservation efforts of this endangered orchid species.
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