Asymbiotic Germination of Mature Seeds and the Seed Development of Vanilla Planifolia

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Traditionally, V. planifolia is propagated by stem cuttings, which produces identical genotype that are sensitive to virulent pathogens. However, sexual propagation with seed germination of V. planifolia is intricate and unstable because of the extremely hard seed coat. A better understanding of seed development, especially the formation of impermeable seed coat would provide insights into seed propagation and conservation of genetic resources of Vanilla . Results: We found that soaking mature seeds in 4 % sodium hypochlorite solution from 75 to 90 min significantly increased germination and that immature seeds collected at 45 days after pollination (DAP) had the highest germination percentage. We then investigated the anatomical features during seed development that associated with the effect of seed pretreatment on raising seed germination percentage. The 45-DAP immature seeds have developed globular embryos and the thickened non-lignified cell wall at the outermost layer of the outer seed coat. After 60 DAP, the cell wall of the outermost layer of the outer seed coat became lignified and finally compressed into a thick envelope. These features matches the significant decreases of immature seed germination percentage after 60 DAP. Conclusion: We report a reliable protocol for seed pretreatment of mature seeds and for immature seeds culture based on a defined time schedule of V. plantifolia seed development. The thickened and lignified seed coat formed an impermeable envelope surrounding the embryo, and might play an important role in seed dormancy of V. plantifolia . Plant Physiology and Morphology Plant Molecular Biology and Genetics Botany micropropagation embryo seed coat seed pretreatment Vanilla Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Background Vanilla planifolia is a vanilla orchid native to Mexico and Central America (Bory et al. 2008 ), and has been planted in many tropical regions around the world to produce natural vanilla flavor (Sreedhar et al. 2007 ). Since the mature seeds of V. planifolia hardly germinate, V. planifolia is usually propagated commercially by vegetative propagation methods, such as cutting stem or callus culture regeneration (Palama et al. 2010 ; Havkin-Frenkel and Belanger 2018 ; Lee 2018 ). Nevertheless, seed propagation can produce offspring with differing genotypes and is important to generate novel traits in breeding programs. Asymbiotic germination technique has been widely used for the commercial propagation of many orchids by seeds (Knudson 1922 ; Yam and Arditti 2017 ). However, the application of this technique to temperate terrestrial orchids, including V. planifolia , is complicated (Rasmussen 1995 ; Miyoshi and Mii 1998 ). Successful asymbiotic germination depends on seed maturity (Arditti 1967 ; Lee et al. 2005 ), growth medium components such as organic nutrient, carbon source, plant growth regulators (Lo et al. 2004 ; Gayatri and Kavyashree 2005 ; Dutra et al 2008 ), light and temperature (Knudson 1950 ; Suzuki et al. 2012 ), and pretreatment (Lee 2011 ). Like most orchids, V. planifolia produces many seeds that contain an embryo without endosperm (Clements and Molvray 1999 ). However, the seed coat of V. planifolia is black and hard, which contrasts markedly with the thin transparent seed coat of most orchid seeds (Cameron and Chase 1998 ; Nishimura and Yukawa 2010 ). Sib-crossed or self-pollinated seeds of V. planifolia usually showed very low germination (Knudson 1950 , Menchaca et al. 2011 ), which might result from the impermeability of the hardened seed coat (Withner 1955 ; Lee et al. 2005 ; Lee et al. 2007 ; Lee et al. 2015 ; Van der Kinderen 1987 ; Van Waes and Debergh 1986 ). This study aimed to establish an effective propagation method of V. planifolia via asymbiotic germination. We first tested the effects of asymbiotic germination at different seed developmental stages. We then examined the effect of different combinations of sodium hypochlorite strengths and soaking time on the germination of mature seeds under asymbiotic germination treatments. We also investigated the morphological, histological, and histochemical changes of seed development within a defined timescale, from fertilization to seed maturity. Based on this time frame, we determined the optimal timing for collecting seeds to inoculate in vitro . Such studies would benefit mass propagation to meet commercial needs and breeding programs for vanilla production. Methods Plant material and seed collection The mature plants of V. planifolia were maintained in a greenhouse at Taoyuan District Agricultural Research and Extension Station at Taoyuan City, Taiwan. Anthesis generally occurs in late April each year (Fig. 1 A). For the pod setting, flowers were hand-pollinated by transferring the pollinia onto the stigma of the same flower (Fig. 1 B). Developing pods (Fig. 1 C) were collected at regular intervals for morphological measurement, histology, and seed germination experiments. In January the next year, pods began to mature and turned yellow (Fig. 1 D). In each experiment, at least three pods were collected at regular intervals after pollination. Evaluation of asymbiotic germination percentage The pods of different developmental stages were surface-sterilized with a 1.8% sodium hypochlorite solution with one drop of a wetting agent (Tween 20, Sigma-Aldrich) for 15 min. After surface sterilization, the capsules were cut open, and seeds were scooped out with forceps onto the culture medium. To ensure the seed quality and developmental stages of each capsule, the remaining seeds of each capsule were fixed and examined under a microscope. The culture medium used was 1/2 Murashige and Skoog (MS) (Murashige and Skoog 1962 ) supplemented with 2 mg l − 1 glycine, 0.5 mg l − 1 niacin, 0.5 mg l − 1 pyridoxine HCl, 0.1 mg l − 1 thiamine, 1 g l − 1 tryptone, 20 g l − 1 sucrose, and solidified with 7 g l − 1 agar (plant cell culture, tested, powder; all Sigma-Aldrich). The pH value was adjusted to 5.7 before autoclaving at 121 °C and 1.2 kg cm 2 for 20 min. An amount of 10 ml medium was placed into each culture tube (20 × 100 mm). After sowing, the cultures were incubated in a growth room under a 16/8 hours photoperiod with daylight fluorescent lamps (20W, China Electric, Taipei) at light intensity 30 µmol m − 2 s − 1 . Germination percentage was recorded 60 days after sowing. Effect of seed pretreatments on germination To examine the effectiveness of different concentrations and durations of sodium hypochlorite pretreatments in improving germination, mature seeds at 135 DAP were collected. The pretreatments included soaking the seeds in 0.5%, 1%, 2% or 4% sodium hypochlorite solution with one drop of Tween 20, for 15, 30, 45, 60, 75 or 90 min. In control, seeds were soaked only in water. After pretreatments, seeds were washed three times with sterilized distilled water, then inoculated on 1/2 MS medium as described above. Histological and histochemical studies Developing seeds were collected and fixed in 2.5% glutaraldehyde and 1.6% paraformaldehyde buffered with 0.1M phosphate buffer (pH 6.8) for 48 h at room temperature. Seeds were then dehydrated in an ethanol series, then infiltrated gradually (3:1, 1:1, and 1:3 100% ethanol: Technovit 7100, 24 hr each) by using Technovit 7100 resin (Kulzer & Co., Germany), followed by three changes of pure resin. Seeds were then embedded in resin, as described by Yeung and Chan ( 2015 ). Sections of 3-µm thick were cut using a Reichert-Jung 2040 Autocut rotary microtome. These sections were stained with periodic acid–Schiff (PAS) procedure for structural carbohydrates and counterstained with 1% (w/v) amido black 10B in 7% acetic acid for protein (Sigma-Aldrich, St. Louis, MO, USA) or 0.05% (w/v) toluidine blue O (TBO, Sigma-Aldrich) for general histological staining (Yeung, 1984). For detecting the deposition of cuticular material in developing seeds, sections were stained with 1 µg ml − 1 Nile red (Sigma-Aldrich) for 1 min, then washed in running tap water for 3 min. The fluorescence pattern of Nile red was viewed under an epifluorescence microscope (Axioskop 2, Carl Zeiss AG, Germany) equipped with the Zeiss filter set 15 (546/12 nm excitation and 590 emission). All images were recorded by using a CCD camera attached to the microscope. Rooting and acclimatization of in vitro seedlings After 150 days of culture, developing protocorms with roots were transferred onto seedling growth medium: 1/2 MS medium supplemented with 20 g l − 1 sucrose, 1 g l − 1 activated charcoal, 20 g l − 1 potato homogenate, and 7 g l − 1 agar for growing seedlings described by Lee ( 2011 ). The potato was boiled for 10 min, then peeled and cut into about 1-cm 3 sections, then homogenized with a kitchen blender. The pH of the medium was adjusted to 5.6 before autoclaving at 121 °C for 20 min. An amount of 100 ml medium was dispensed into a 500-mL culture flask. After transferring to the seedling growth medium, flasks were placed in a growth room under a 16/8 hours photoperiod with daylight fluorescent lamps (20W, China Electric, Taipei) at light intensity 30 µmol m − 2 s − 1 . After 90 days of culture in the seedling growth medium, seedlings of about 10 cm tall with 4 leaves were taken out of flasks. Experimental design and statistical analysis All experiments were performed in a completely randomized design and repeated three times; 12 replicates (culture tubes) were used for each treatment, with one explant planted in each plate. Data were analyzed by using analysis of variance (ANOVA) with Fisher’s protected least significant difference test at P < 0.05. All data were analyzed with SAS v9.0 (Cary, NC, USA). Results Seed germination can be achieved using asymbiotic germination technique at early seed developmental stages of V. planifolia It is known that asymbiotic germination technique can support the growth of the immature embryo in other terrestrial orchid species (Lee et al. 2005 ; Lee et al. 2007 ; Van Waes and Debergh 1986 ), so different stages of developing seeds were collected in an interval of 15 days from the days to pollination to 300 DAP and their germination percentages under the asymbiotic germination treatments were investigated. Seed germination occurred after 30 DAP (Fig. 2 ). The seed germination percentage reached 9.9% at 45 DAP, the highest germination percentage through the whole experiment. The germination percentage then markedly declined to approximately 2% at 60 DAP. This low germination percentage lasted to 90 DAP. Almost no seed germination was observed after 120 DAP up to 300 DAP (data not shown). Effect of seed pretreatments on germination Pretreatment with sodium hypochlorite solutions remarkably breaks the situation of no germination of V. planifolia mature seeds under the asymbiotic germination treatments. Seeds collected at 135 DAP were soaked with a 2% sodium hypochlorite solution over 60 min before the asymbiotic germination cultures. The pretreatment with sodium hypochlorite solutions resulted in over 5% seed germination (Fig. 3 ). The effect of sodium hypochlorite soaking treatment on V. planifolia seed germination increased with higher strength of sodium hypochlorite and longer soaking time. However, the effect of soaking time reached a maximum at 75 minutes in all of the different concentrations of sodium hypochlorite solutions (Fig. 3 ). In addition, V. planifolia mature seeds treated with a 4% sodium hypochlorite solution for 75 minutes can reach over 10% seed germination. A higher concentration of sodium hypochlorite solution treatment is not recommended because a high concentration of sodium hypochlorite may destroy cell membranes and cause seed death. Development of pod, embryo and seed coat To understand the possible causes for the effect of pretreatment of sodium hypochlorite, morphological, histological, and histochemical changes of seed development were investigated. The main structural changes occurring within developing pods from anthesis until maturity are summarized in Table 1 . Detailed characteristics are described in follows. Table 1 Major microscopic structural events occurring in the developing pods of Vanilla planifolia after hand pollination. DAP z Developmental Stage Seed color 30 Fertilization and the formation of zygote White 45 Proembryo A mixture of white and brown seeds 60 Proembryo and the developing of early globular embryo Most seeds turned black 75 Early globular embryo Black 90 Globular embryo Black 105 Late globular embryo Black 300 Pod ripe and split y Black z DAP = days after pollination. y From 105 to 300 DAP, the seed structure did not change, but the pod gradually became ripe and turned yellow by 240 DAP. Pods are developed from ovaries. After successful hand-pollination, ovaries began to enlarge and elongate rapidly (Figs. 1 and 4 ). The pod length increased steadily and reached the maximum size (19.86 ± 1.99 cm) at 35 DAP, while the diameter of the pod reached the maximum size (12.57 ± 1.09 mm) at 49 DAP (Fig. 4 ). By 240 DAP, the pod matured and became yellow, then turned black after 300 DAP (Fig. 1 D). Embryos in a pod were developed from zygotes that were combined between sperm cells in pollens and eggs in embryo sacs. The megaspore mother cells develop just after pollination in early-mid May. Numerous mature embryo sacs could be observed in a pod before fertilization. At 30 DAP, zygotes and proembryos were present within developing pods, and no endosperm was observed (Figs. 5 B and C). At this stage, the seeds were white and moist (Fig. 6 A). At 45 DAP, additional cell divisions occurred within the inner tiers and the surface layer (Figs. 5 D and E), thus resulting in the growth of the embryo proper. Some seeds had turned light to dark brown (Fig. 6 B). At 60 DAP, more cell divisions occurred within the embryo proper, resulting in an early globular embryo (Fig. 5 F). At this stage, most seeds had turned black (Fig. 6 C). This species lacked a structurally defined suspensor during embryo development (Figs. 5 and 7 ). As the embryo developed to the globular stage at 75 DAP, the embryo proper had filled the cavity of the embryo sac (Fig. 7 A). After this stage, nearly all seeds were black (Figs. 6 D, E, and F). By 105 DAP, the mature embryo was about 11 cells long and 7 cells wide without the formation of shoot apical meristem and cotyledon (Fig. 7 B). At this stage, the cytoplasm of the embryo proper cell was filled with a large number of storage products (e.g., protein bodies and lipid bodies), and starch grains had disappeared. By 300 DAP, the pod had fully matured and desiccated. Then, the pod split, and the mature seeds were released (Fig. 1 D). Regarding the development of seed coat, the inner and outer seed coats derived from the inner and outer integuments of the ovule, respectively (Fig. 5 A), and these two distinct layers of seed coat surrounded the embryo in the mature seed of V. planifolia (Fig. 7 B). The inner seed coat was two cells thick, and the cell wall of the inner seed coat remained primary in nature during the early stage of seed development (Figs. 5 B and C; Fig. 8 A). As the seeds approached maturity, the inner seed coat gradually compressed (Figs. 5 D, E and F; Fig. 8 B), and eventually became a thin layer at maturity (Fig. 8 C). However, the outer seed coat consisted of three to four cell layers (Figs. 5 B and C). Before fertilization, the outer seed coat was still growing and had not enclosed the embryo sac completely (Fig. 8 A). At this stage, the cell wall of the outer seed coat was relatively thin (Fig. 8 A), and the cell wall of the outermost layer of the outer seed coat became thickened after fertilization (Fig. 5 B). By 60 DAP, the thickened wall of the outermost layer of the outer seed coat became sclerified (Figs. 5 F and 8 B). As the seed approached maturity, the inner layers of the outer seed coat gradually compressed and attached to the sclerified outermost layer of the outer seed coat (Figs. 8 C and D). Using TBO staining, the cell wall of the outermost layer of the outer seed coat stained greenish-blue, indicating the presence of phenolic compounds in the cell wall (Figs. 8 B and C). In addition, using Nile red staining, the surface wall of the embryo proper and the innermost and outermost walls of the inner seed coat reacted positively, which suggested the possible accumulation of a cuticular substance in the wall of these two layers (Figs. 7 C and D). Protocorm and seedling growth For the development of protocorms, most embryos were still enveloped by the seed coat at 30 days of culture on 1/2 MS medium. Only a few embryos had emerged from the seed coat, which was considered seed germination (Fig. 9 A). By 60 days of culture, young protocorms had turned pale green, and numerous rhizoids appeared at the basal protocorms (Fig. 9 B). By 90 days of culture, the protocorm had enlarged and turned dark green to differentiate shoot apical meristem (Fig. 9 C). Subsequently, the developing protocorm with the differentiation of the first root was observed (Fig. 9 D). By 150 days of culture, the protocorm had further elongated with prominent root formation (Fig. 9 E). After transferring onto the seedling growth medium for additional 90 days, the seedlings grew into vines with several healthy roots that could be taken out of flasks (Fig. 9 F). Discussion The mature seed of V. planifolia is black and hard, which is distinct from the seeds of many orchids (Arditti and Ghani 2000 ). In the present study, based on a defined time frame, we investigated the morphological and structural changes of seeds (Table 1 ; Figs. 5 , 6 and 7 ). In many orchids, the outer seed coat develops from two cell layers (Yam et al. 2002 ), whereas in V. planifolia , the outer seed coat is derived from four cell layers (Figs. 5 and 8 ). The histological and histochemical results indicated that the sclerification primarily occurred in the outermost cell layer of the outer seed coat (Figs. 5 , 7 and 8 ). Similar hard seeds can be found in a few vanilloid species, e.g., Galeola septentrionalis (Suetsugu et al. 2015 ) and G. javanica (Yang and Lee 2014 ). Their seed coats are multi-layer, and the walls are heavily thickened with lignin polymers as seeds mature. The cell wall of the outermost cell layer of the outer seed coat stained greenish-blue by TBO indicated the presence of phenolic compounds (Fig. 8 ). In the seed coat of many plants, phenolic compounds, including caffeic acid, sinapic acid, coumarin, chlorogenic acid, and ferulic acid, are esterified to the wall structure (Gubler and Ashford 1985 ; Pan et al. 2002 ). The accumulation of phenolic compounds in the seed coat inhibits seed germination (Bewley and Black 1994 ). In V. planifolia , further analysis by nuclear magnetic resonance spectroscopy revealed the heavy deposition of catechyl units during lignification of the cell wall of the seed coat (Chen et al. 2012 ). Also, the presence of a cuticular substance or suberin has been detected in the cell wall of the inner seed coat (also known as a carapace) and/or outer seed coat of some orchids, such as Cephalanthera , Cymbidium , and Cypripedium (Carlson 1940 ; Yeung et al. 1996 ; Lee et al. 2005 ; Yamazaki and Miyoshi 2006 ). In V. planifolia , after Nile red staining, a thin fluorescent layer was observed in the innermost layer of the inner seed coat cells and the surface wall of the globular embryo, which suggests the accumulation of cuticular substance in walls. However, the fluorescence pattern was absent in the walls of the outer seed coat (Figs. 7 C and D). In orchids, the deposition of cuticular substance on the surface of the embryo proper and in the innermost layer of the seed coat may provide physical protection and ensure moisture retention to developing embryos. During the same time, we investigated the effect of seed maturity and seed pretreatments on asymbiotic germination. The optimal germination was at 45 DAP (Fig. 3 ). At this stage, the early globular embryo appeared, and the outermost cell layer of the outer seed coat had become thickened but had not been heavily lignified (Fig. 5 D). The germination decreased sharply by 60 DAP, which agreed with the beginning of seed coat lignification (Fig. 3 ). Because the outermost cell layer of the outer seedcoat had been lignified, a stronger hydrophobic nature was observed during the operation of seed sowing. As the seed matured, the outer seed coat gradually compressed into a thick and heavily lignified layer surrounding the embryo (Fig. 8 ). The low permeable seed coat restricted the water uptake and solute diffusion, which resulted in poor germination. Although V. planifolia is an epiphytic orchid species and pantropical in distribution, this pattern of seed germination is similar to those of terrestrial orchids from temperate regions, where immature seeds are easier to germinate in vitro than are mature seeds (Linden 1980 ; Zhang et al. 2013 ). The poor germination of mature seeds in terrestrial orchids from temperate regions may be attributed to the accumulation of chemical inhibitors to germination, such as abscisic acid in Dactylorhyza and Epipactis (van der Kinderen 1987 ), Calanthe (Lee et al. 2007 ) and Cypripedium (Lee et al. 2015 ) and phenolics in Cymbidium (Kako 1976 ), and/or the formation of an impermeable container in the seed coat that may make an embryo difficult to obtain water and nutrients for germination (Miyoshi and Mii 1988; Lee et al. 2005 ). Seed pretreatments may release dormancy and enhance germination by changing the physical integrity of seed coverings, allowing the embryo to absorb water and nutrients and to uptake oxygen (Taylor et al. 1998 ). Seed pretreatments using bleaching solutions, such as calcium hypochlorite and sodium hypochlorite, can greatly stimulate seed germination of several European terrestrial orchids (Linden 1980 ; Rasmussen 1995 ; Steele 1996 ; Van Waes and Debergh 1986 ). In this study, the germination of mature seeds was enhanced with soaking in 2% and 4% sodium hypochlorite solutions from 75 to 90 min. However, no germination was recorded in fully mature seeds (Fig. 4 ). Common bleaching agents such as sodium hypochlorite have been widely used for removing residual lignin from the wood pulp (Holik 2006 ). From the scanning electron microscopic observations, the seed coat was scarred with hypochlorite oxidation, so the seed coat was relatively more permeable to water (Lee 2011 ). Of note, as compared with previous reports of seed pretreatments using hypochlorite solutions, we used a stronger concentration (4% sodium hypochlorite) and a longer duration (90 min) to pretreat mature seeds. However, the optimal germination of 12.66 ± 1.14% was relatively low. This low germination may reflect the hard seed coat of V. planifolia with its extremely impermeable nature. The relative low germination of pretreated seeds may be attributed to the accumulation of inhibitory substances, such as abscisic acid inside the embryo, which still cannot be leached out by hypochlorite solutions. The orchid seed with a heavily lignified, hard seed coat is adapted to the seed dispersal strategy involved in endozoochory by animals (Suetsugu 2018 ). In Cyrtosia and Vanilla species, as their fruits ripen, they usually have vivid colors and/or a heady fragrance to attract animals, such as rats, bats, or birds (Soto Arenas and Dressler 2010 ; Yang and Lee 2014 ; Suetsugu et al. 2015 ). The thickened lignified seed coat can protect the embryos when the seeds pass through the digestive tract of animals (Suetsugu 2018 ). Conclusions In this report, we provide reliable protocols for seedling production of V. planifolia through asymbiotic seed germination. For the successful culture of immature seeds, the timing of seed collection is short and critical, and optimal germination could be obtained from immature seeds collected at 45 DAP (before seeds turned black). For improving the germination of mature seeds, pretreatment with 4% sodium hypochlorite solution for 75 to 90 min is recommended. We propose that the thickened and lignified seed coat, forming an impermeable envelope, is responsible for the seed coat-imposed dormancy. Further work is needed to examine the changes in levels of endogenous inhibitors in embryos, such as abscisic acid, during seed development. Such studies should help to fully explain the low germination percentage of V. planifolia seeds. Declarations Author contributions CHY, KYC and YIL designed the study; CHY and YIL performed experiments; CHY, KYC and YIL wrote and edited the manuscript; all authors commented on the manuscript. Author details 1 Taoyuan District Agricultural Research and Extension Station, Council of Agriculture, Executive Yuan, Taoyuan 327, Taiwan, ROC. 2 Department of Agronomy, National Taiwan University, Taipei, Taiwan, ROC. 3 Biology Department, National Museum of Natural Science, 40453 Taichung, Taiwan, ROC. 4 Department of Life Sciences, National Chung Hsing University, 40227 Taichung, Taiwan, ROC. Competing interests. The authors declare that they have no competing interests. Availability of data and material. Not applicable. Consent for publication. Not applicable. Ethics approval and consent to participate. Not applicable. Funding. This work was supported by grants [107AS-7.6.4-YS-Y1 and 108AS-7.6.4-YS-Y1] from the Council of Agriculture, Executive Yuan, R.O.C. to Chih-Hsin Yeh. 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Kluwer Academic Publisher, Dordrecht, pp 287-385 Yam TW, Arditti J (2017) Micropropagation of orchids, 3rd ed. John Wiley and Sons, Inc., New York Yamazaki J, Miyoshi K (2006) In vitro asymbiotic germination of immature seed and formation of protocorm by Cephalanthera falcata (Orchidaceae). Ann Bot 98:1197-1206 Yang CK, Lee YI (2014) The seed development of a mycoheterotrophic orchid, Cyrtosia javanica Blume. Bot Stud 55:44 Yeung EC, Zee SY, Ye XL (1996) Embryology of Cymbidium sinense : embryo development. Ann Bot 78:105-110 Yeung EC, Chan CKW (2015) The glycol methacrylate embedding resins Technovit 7100 and 8100. Plant microtechniques and protocols. Springer International Publishing, New York, pp 67-82 Zhang Y, Lee YI, Deng L, Zhao S (2013) Asymbiotic germination of immature seeds and the seedling development of Cypripedium macranthos Sw., an endangered lady's slipper orchid. Sci Hortic 164:130-136 Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Minor revision 30 Jan, 2021 Review # 3 received at journal 29 Jan, 2021 Review # 1 received at journal 11 Jan, 2021 Review # 2 received at journal 10 Jan, 2021 Reviewer # 3 agreed at journal 04 Jan, 2021 Reviewer # 2 agreed at journal 03 Jan, 2021 Reviewers invited by journal 02 Jan, 2021 Reviewer # 1 agreed at journal 02 Jan, 2021 Editor assigned by journal 23 Dec, 2020 Submission checks completed at journal 23 Dec, 2020 Editor invited by journal 23 Dec, 2020 First submitted to journal 22 Dec, 2020 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-138206","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Original Article","associatedPublications":[],"authors":[{"id":7342879,"identity":"cea4425e-646d-45cc-9d43-12c63c109b64","order_by":0,"name":"Chih-Hsin Yeh","email":"","orcid":"","institution":"National Taiwan University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Chih-Hsin","middleName":"","lastName":"Yeh","suffix":""},{"id":7342880,"identity":"f7cb17c3-f716-4f65-97f8-4d13135634c2","order_by":1,"name":"Kai-Yi Chen","email":"","orcid":"","institution":"National Taiwan University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Kai-Yi","middleName":"","lastName":"Chen","suffix":""},{"id":7342881,"identity":"ab1ece50-f068-4779-854e-9d7ad42c9885","order_by":2,"name":"Yung-I Lee","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABBUlEQVRIiWNgGAWjYBACPijNww8kDjyACfPg0cIGUyPZANSSgKRFgpAWBoMDQII4LezNx6R5d9TKGF87/BBoy51oc/4DjA/etjHUgQ3BpoXnWJo075njPGa30wyAWp7l7pyRwGw4t41BAqcWiRwzad62Y0AtCSAth3M33GBgA4owSJjh0iL//htYi/Hs9A8QLecPsP/Gq0WCB2RmDY+BdA7UlgMJbMx4tfCkGVvObTvAI3E7p+BAggHIYYnNknPOSUjux6GFn/3wwxtv2+rs+Wenb/7woQLksMMHP7wps+EHxRQOwAKMgcNQtgGIYASpxRktIMD8gYGhDp+CUTAKRsEoGOkAABBcW51K+CKsAAAAAElFTkSuQmCC","orcid":"https://orcid.org/0000-0001-9994-2003","institution":"National Museum of Natural Science","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Yung-I","middleName":"","lastName":"Lee","suffix":""}],"badges":[],"createdAt":"2020-12-30 10:19:52","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-138206/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-138206/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":4725039,"identity":"45e62f3c-10fb-49c1-9496-b1544f656169","added_by":"auto","created_at":"2021-01-05 19:27:11","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":886034,"visible":true,"origin":"","legend":"Flowers and developing pods of V. planifolia.\n(A) Flower at the time of anthesis. Scale bar = 1 cm.\n(B) Flower was hand-pollinated by transferring the pollinia onto the stigma of the same flower. Scale bar = 1 cm.\n(C) Developing pods at 60 DAP. Scale bar = 1 cm.\n(D) Pods turned yellow by 240 DAP and became black by 300 DAP. Scale bar = 1 cm.\n","description":"","filename":"Fig.1.poddevelopment.jpg","url":"https://assets-eu.researchsquare.com/files/rs-138206/v1/26fcc0b25b75b3198b28f707.jpg"},{"id":4725040,"identity":"e792b4ec-f0d6-4202-8cb7-3cb2899c17c2","added_by":"auto","created_at":"2021-01-05 19:27:11","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":158743,"visible":true,"origin":"","legend":"Mean percent germination of V. planifolia seeds at each successive 15 days after pollination on 1/2 MS medium. Data were scored after 60 days of culture. Data are mean ± SD (n = 10).","description":"","filename":"Fig.2.Germination.jpg","url":"https://assets-eu.researchsquare.com/files/rs-138206/v1/b84fd38ee156970da0c56360.jpg"},{"id":4725037,"identity":"0e991089-73bb-4e34-8d74-8b97152f91d4","added_by":"auto","created_at":"2021-01-05 19:27:11","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":219453,"visible":true,"origin":"","legend":"Effect of different concentrations and durations of sodium hypochlorite pretreatments on seed germination in vitro of V. planifolia. Data were recorded after 60 days of culture. Data are mean ± SD (n = 3).","description":"","filename":"Fig.3.Seedtreatment.jpg","url":"https://assets-eu.researchsquare.com/files/rs-138206/v1/59f7d179c94965ae38bc6b03.jpg"},{"id":4725038,"identity":"6dcd0c26-74e0-4bc1-85a0-61fade5eef64","added_by":"auto","created_at":"2021-01-05 19:27:11","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1506109,"visible":true,"origin":"","legend":"Changes in pod length (A) and width (B) of V. planifolia. Data are mean ± SD (n = 30).","description":"","filename":"Fig.4.Podsize.jpg","url":"https://assets-eu.researchsquare.com/files/rs-138206/v1/0745f3fb82f8713b40f16442.jpg"},{"id":4724832,"identity":"819c4615-1d1b-41e3-927c-5d69181d6b54","added_by":"auto","created_at":"2021-01-05 19:24:11","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":2380745,"visible":true,"origin":"","legend":"The early embryo development of V. planifolia.\n(A) A mature embryo sac showing the egg apparatus, including the egg cell (arrow) and two synergids (arrowheads). The outer seed coat (OS) was elongating and had not enclosed the inner seed coat (IS).\n(B) At 30 DAP, after fertilization, zygote (arrow) had a dense cytoplasm, and the degenerated antipodal cells (arrowhead) at the chalazal end were densely stained. In this species, the endosperm failed to develop, and a degenerated endosperm nucleus (double arrowhead) could be observed.\n(C) Light micrograph showing a three-celled proembryo (arrow), and a degenerated endosperm nucleus (arrowhead) stayed beside the zygote.\n(D) By 45 DAP, an anticlinal division (arrow) occurred in the outmost cell layer, resulting in the formation of the globular-shape embryo.\n(E) Light micrograph showing an early globular embryo with an additional anticlinal division (arrow). This species did not have a distinct suspensor during embryo development.\n(F) Light micrograph showing a longitudinal section through a globular embryo at 60 DAP. The outer most layer of outer seed coat (OS) became lignified. Scale bar = 100 μm.\n","description":"","filename":"Fig.5.Vanillaearlyembryo.jpg","url":"https://assets-eu.researchsquare.com/files/rs-138206/v1/c06c1d75e2c927f77761991b.jpg"},{"id":4725041,"identity":"fe747eb0-f780-4c3b-9d0d-0e88b8d8a410","added_by":"auto","created_at":"2021-01-05 19:27:11","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":2631006,"visible":true,"origin":"","legend":"Morphology of developing seeds of V. planifolia.\n(A) White and moist seeds at 30 DAP.\n(B) A mixture of white and brown seeds at 45 DAP.\n(C) A number of seeds had turned black at 60 DAP.\n(D) Nearly all seeds turned black at 75 DAP.\n(E) Black seeds at 90 DAP.\n(F) Black seeds at105 DAP. Scale bar = 1 mm.\n","description":"","filename":"Fig.6.Vanillaseedslabel.jpg","url":"https://assets-eu.researchsquare.com/files/rs-138206/v1/618f615376d743022bc78f21.jpg"},{"id":4724835,"identity":"8e284db5-5e0e-44b6-81e1-046f30d46a52","added_by":"auto","created_at":"2021-01-05 19:24:11","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":710346,"visible":true,"origin":"","legend":"The late embryo development of V. planifolia.\n(A) As the seed approached maturity, a number of tiny protein bodies (arrows) appeared within the embryo proper cells after amido black 10B stain. The thickened outer seed coat (OS) became dehydrated and compressed, with the inner seed coat (IS) compressed into a thin layer.\n(B) Light micrograph showing a longitudinal section through a mature seed. Several tiny protein bodies (arrows) were found within the embryo proper cells. In this preparation, the lipid bodies were not preserved; the spaces (arrowheads) between the protein bodies were occupied by storage lipid bodies.\n(C) Nile red staining fluorescence micrograph of an early globular embryo at the stage similar to Figure 3E. After Nile red staining, the surface wall of the embryo proper (arrows) reacted weakly to the stain, and the innermost (arrowhead) and outermost (double arrowhead) walls of the inner seed coat also reacted positively.\n(D) Nile red staining fluorescence micrograph of a mature seed at the stage similar to Figure 5B. The inner seed coat compressed into a thin layer and attached the embryo tightly. The thin inner seed coat (IS, arrowheads) and the surface of the embryo proper (arrow) reacted positively to the stain. Fluorescence was never observed in the thickened outer seed coat (OS). Scale bar = 100 μm.\n","description":"","filename":"Fig.7.Vanillalateembryo.jpg","url":"https://assets-eu.researchsquare.com/files/rs-138206/v1/ec5a4af63aca4e009c244674.jpg"},{"id":4725405,"identity":"798fc18b-129b-44b3-876b-d103c2b53a79","added_by":"auto","created_at":"2021-01-05 19:33:11","extension":"jpg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":478651,"visible":true,"origin":"","legend":"The seed coat development of V. planifolia.\n(A) The seed coat consisted of the inner seed coat (IS, two cells thick) and outer seed coat (OS, three to four cells thick). At the time of fertilization, the cell wall of the outermost layer of outer seed coat still remained primary in nature.\n(B) At the globular embryo stage, the cell wall of the outermost layer of outer seed coat (OS) had become thickened, and the inner seed coat (IS) was dehydrating and compressing.\n(C) As the seed approached maturity, the thickened outermost layer of the outer seed coat had dehydrated and compressed, and the inner layers of the outer seed coat were gradually dehydrating and compressing. At this stage, the inner seed coat (IS) had compressed into a thin layer.\n(D) At maturity, both the thin inner seed coat (IS) and the thickened outer seed coat (OS) compressed and enveloped the embryo (E) tightly. Scale bar = 20 μm.\n","description":"","filename":"Fig.8.Vanillaseedcoat.jpg","url":"https://assets-eu.researchsquare.com/files/rs-138206/v1/0e26e6b857ec8c0de82fac86.jpg"},{"id":4725215,"identity":"64a2a7fc-42e7-47ee-92b0-f091e5d78015","added_by":"auto","created_at":"2021-01-05 19:30:12","extension":"jpg","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":683132,"visible":true,"origin":"","legend":"Protocorm development and seedling growth of V. planifolia.\n(A) By 30 days of culture, a few embryos had emerged from the seed coat. Scale bar = 1 mm.\n(B) Young protocorms with numerous rhizoids appeared at the basal protocorms by 60 days of culture. Scale bar = 1 mm.\n(C) The protocorm had enlarged with the differentiation of a shoot tip by 90 days of culture. Scale bar = 1 mm.\n(D) The protocorm had a differentiated first root by 120 days of culture. Scale bar = 1 mm.\n(E) By 150 days of culture, the protocorm had elongated with the prominent root formation, ready to transfer to the growth medium. Bar = 2 mm.\n(F) After 90 days of culture on the growth medium, the seedlings grew into vines with several healthy roots. Bar = 2 cm.\n\n","description":"","filename":"Fig.9.Seedlings.jpg","url":"https://assets-eu.researchsquare.com/files/rs-138206/v1/78c1783c048bf6e4961d5d23.jpg"},{"id":13643148,"identity":"53f168f7-d094-4913-a1a9-e1bbf78b9738","added_by":"auto","created_at":"2021-09-17 09:10:27","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1516379,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-138206/v1/7fae92c9-91b0-47ac-b1ee-220d4ca9bda7.pdf"}],"financialInterests":"","formattedTitle":"\u003cp\u003eAsymbiotic Germination of Mature Seeds and the Seed Development of Vanilla Planifolia\u003c/p\u003e","fulltext":[{"header":"Background","content":" \u003cp\u003e \u003cem\u003eVanilla planifolia\u003c/em\u003e is a vanilla orchid native to Mexico and Central America (Bory et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2008\u003c/span\u003e), and has been planted in many tropical regions around the world to produce natural vanilla flavor (Sreedhar et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). Since the mature seeds of \u003cem\u003eV. planifolia\u003c/em\u003e hardly germinate, \u003cem\u003eV. planifolia\u003c/em\u003e is usually propagated commercially by vegetative propagation methods, such as cutting stem or callus culture regeneration (Palama et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Havkin-Frenkel and Belanger \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Lee \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Nevertheless, seed propagation can produce offspring with differing genotypes and is important to generate novel traits in breeding programs. Asymbiotic germination technique has been widely used for the commercial propagation of many orchids by seeds (Knudson \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e1922\u003c/span\u003e; Yam and Arditti \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). However, the application of this technique to temperate terrestrial orchids, including \u003cem\u003eV. planifolia\u003c/em\u003e, is complicated (Rasmussen \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e1995\u003c/span\u003e; Miyoshi and Mii \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e1998\u003c/span\u003e). Successful asymbiotic germination depends on seed maturity (Arditti \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1967\u003c/span\u003e; Lee et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2005\u003c/span\u003e), growth medium components such as organic nutrient, carbon source, plant growth regulators (Lo et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2004\u003c/span\u003e; Gayatri and Kavyashree \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2005\u003c/span\u003e; Dutra et al \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2008\u003c/span\u003e), light and temperature (Knudson \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e1950\u003c/span\u003e; Suzuki et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2012\u003c/span\u003e), and pretreatment (Lee \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). Like most orchids, \u003cem\u003eV. planifolia\u003c/em\u003e produces many seeds that contain an embryo without endosperm (Clements and Molvray \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e1999\u003c/span\u003e). However, the seed coat of \u003cem\u003eV. planifolia\u003c/em\u003e is black and hard, which contrasts markedly with the thin transparent seed coat of most orchid seeds (Cameron and Chase \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e1998\u003c/span\u003e; Nishimura and Yukawa \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). Sib-crossed or self-pollinated seeds of \u003cem\u003eV. planifolia\u003c/em\u003e usually showed very low germination (Knudson \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e1950\u003c/span\u003e, Menchaca et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2011\u003c/span\u003e), which might result from the impermeability of the hardened seed coat (Withner \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e1955\u003c/span\u003e; Lee et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2005\u003c/span\u003e; Lee et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Lee et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Van der Kinderen \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e1987\u003c/span\u003e; Van Waes and Debergh \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e1986\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThis study aimed to establish an effective propagation method of \u003cem\u003eV. planifolia\u003c/em\u003e via asymbiotic germination. We first tested the effects of asymbiotic germination at different seed developmental stages. We then examined the effect of different combinations of sodium hypochlorite strengths and soaking time on the germination of mature seeds under asymbiotic germination treatments. We also investigated the morphological, histological, and histochemical changes of seed development within a defined timescale, from fertilization to seed maturity. Based on this time frame, we determined the optimal timing for collecting seeds to inoculate \u003cem\u003ein vitro\u003c/em\u003e. Such studies would benefit mass propagation to meet commercial needs and breeding programs for vanilla production.\u003c/p\u003e "},{"header":"Methods","content":"\u003ch2\u003ePlant material and seed collection\u003c/h2\u003e\n\u003cp\u003eThe mature plants of \u003cem\u003eV. planifolia\u003c/em\u003e were maintained in a greenhouse at Taoyuan District Agricultural Research and Extension Station at Taoyuan City, Taiwan. Anthesis generally occurs in late April each year (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eA). For the pod setting, flowers were hand-pollinated by transferring the pollinia onto the stigma of the same flower (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eB). Developing pods (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eC) were collected at regular intervals for morphological measurement, histology, and seed germination experiments. In January the next year, pods began to mature and turned yellow (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eD). In each experiment, at least three pods were collected at regular intervals after pollination.\u0026nbsp;\u003c/p\u003e\n\u003ch2\u003eEvaluation of asymbiotic germination percentage\u003c/h2\u003e\n\u003cp\u003eThe pods of different developmental stages were surface-sterilized with a 1.8% sodium hypochlorite solution with one drop of a wetting agent (Tween 20, Sigma-Aldrich) for 15\u0026nbsp;min. After surface sterilization, the capsules were cut open, and seeds were scooped out with forceps onto the culture medium. To ensure the seed quality and developmental stages of each capsule, the remaining seeds of each capsule were fixed and examined under a microscope. The culture medium used was 1/2 Murashige and Skoog (MS) (Murashige and Skoog \u003cspan class=\"CitationRef\"\u003e1962\u003c/span\u003e) supplemented with 2\u0026nbsp;mg l\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e glycine, 0.5\u0026nbsp;mg l\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e niacin, 0.5\u0026nbsp;mg l\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e pyridoxine HCl, 0.1\u0026nbsp;mg l\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e thiamine, 1\u0026nbsp;g l\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003etryptone, 20\u0026nbsp;g l\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e sucrose, and solidified with 7\u0026nbsp;g l\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e agar (plant cell culture, tested, powder; all Sigma-Aldrich). The pH value was adjusted to 5.7 before autoclaving at 121\u0026nbsp;\u0026deg;C and 1.2\u0026nbsp;kg cm\u003csup\u003e2\u003c/sup\u003e for 20\u0026nbsp;min. An amount of 10\u0026nbsp;ml medium was placed into each culture tube (20\u0026thinsp;\u0026times;\u0026thinsp;100\u0026nbsp;mm). After sowing, the cultures were incubated in a growth room under a 16/8 hours photoperiod with daylight fluorescent lamps (20W, China Electric, Taipei) at light intensity 30\u0026nbsp;\u0026micro;mol m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. Germination percentage was recorded 60 days after sowing.\u003c/p\u003e\n\u003ch2\u003eEffect of seed pretreatments on germination\u003c/h2\u003e\n\u003cp\u003eTo examine the effectiveness of different concentrations and durations of sodium hypochlorite pretreatments in improving germination, mature seeds at 135 DAP were collected. The pretreatments included soaking the seeds in 0.5%, 1%, 2% or 4% sodium hypochlorite solution with one drop of Tween 20, for 15, 30, 45, 60, 75 or 90\u0026nbsp;min. In control, seeds were soaked only in water. After pretreatments, seeds were washed three times with sterilized distilled water, then inoculated on 1/2 MS medium as described above.\u003c/p\u003e\n\u003ch2\u003eHistological and histochemical studies\u003c/h2\u003e\n\u003cp\u003eDeveloping seeds were collected and fixed in 2.5% glutaraldehyde and 1.6% paraformaldehyde buffered with 0.1M phosphate buffer (pH 6.8) for 48\u0026nbsp;h at room temperature. Seeds were then dehydrated in an ethanol series, then infiltrated gradually (3:1, 1:1, and 1:3 100% ethanol: Technovit 7100, 24 hr each) by using Technovit 7100 resin (Kulzer \u0026amp; Co., Germany), followed by three changes of pure resin. Seeds were then embedded in resin, as described by Yeung and Chan (\u003cspan class=\"CitationRef\"\u003e2015\u003c/span\u003e). Sections of 3-\u0026micro;m thick were cut using a Reichert-Jung 2040 Autocut rotary microtome. These sections were stained with periodic acid\u0026ndash;Schiff (PAS) procedure for structural carbohydrates and counterstained with 1% (w/v) amido black 10B in 7% acetic acid for protein (Sigma-Aldrich, St. Louis, MO, USA) or 0.05% (w/v) toluidine blue O (TBO, Sigma-Aldrich) for general histological staining (Yeung, 1984). For detecting the deposition of cuticular material in developing seeds, sections were stained with 1\u0026nbsp;\u0026micro;g ml\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e Nile red (Sigma-Aldrich) for 1\u0026nbsp;min, then washed in running tap water for 3\u0026nbsp;min. The fluorescence pattern of Nile red was viewed under an epifluorescence microscope (Axioskop 2, Carl Zeiss AG, Germany) equipped with the Zeiss filter set 15 (546/12\u0026nbsp;nm excitation and 590 emission). All images were recorded by using a CCD camera attached to the microscope.\u003c/p\u003e\n\u003ch2\u003eRooting and acclimatization of in vitro seedlings\u003c/h2\u003e\n\u003cp\u003eAfter 150 days of culture, developing protocorms with roots were transferred onto seedling growth medium: 1/2 MS medium supplemented with 20\u0026nbsp;g l\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e sucrose, 1\u0026nbsp;g l\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003eactivated charcoal, 20\u0026nbsp;g l\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e potato homogenate, and 7\u0026nbsp;g l\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e agar for growing seedlings described by Lee (\u003cspan class=\"CitationRef\"\u003e2011\u003c/span\u003e). The potato was boiled for 10\u0026nbsp;min, then peeled and cut into about 1-cm\u003csup\u003e3\u003c/sup\u003e sections, then homogenized with a kitchen blender. The pH of the medium was adjusted to 5.6 before autoclaving at 121\u0026nbsp;\u0026deg;C for 20\u0026nbsp;min. An amount of 100\u0026nbsp;ml medium was dispensed into a 500-mL culture flask. After transferring to the seedling growth medium, flasks were placed in a growth room under a 16/8 hours photoperiod with daylight fluorescent lamps (20W, China Electric, Taipei) at light intensity 30\u0026nbsp;\u0026micro;mol m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. After 90 days of culture in the seedling growth medium, seedlings of about 10\u0026nbsp;cm tall with 4 leaves were taken out of flasks.\u003c/p\u003e\n\u003ch2\u003eExperimental design and statistical analysis\u003c/h2\u003e\n\u003cp\u003eAll experiments were performed in a completely randomized design and repeated three times; 12 replicates (culture tubes) were used for each treatment, with one explant planted in each plate. Data were analyzed by using analysis of variance (ANOVA) with Fisher\u0026rsquo;s protected least significant difference test at \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05. All data were analyzed with SAS v9.0 (Cary, NC, USA).\u003c/p\u003e"},{"header":"Results","content":"\u003ch2\u003eSeed germination can be achieved using asymbiotic germination technique at early seed developmental stages of \u003cem\u003eV. planifolia\u003c/em\u003e\u003c/h2\u003e\n\u003cp\u003eIt is known that asymbiotic germination technique can support the growth of the immature embryo in other terrestrial orchid species (Lee et al. \u003cspan class=\"CitationRef\"\u003e2005\u003c/span\u003e; Lee et al. \u003cspan class=\"CitationRef\"\u003e2007\u003c/span\u003e; Van Waes and Debergh \u003cspan class=\"CitationRef\"\u003e1986\u003c/span\u003e), so different stages of developing seeds were collected in an interval of 15 days from the days to pollination to 300 DAP and their germination percentages under the asymbiotic germination treatments were investigated. Seed germination occurred after 30 DAP (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e). The seed germination percentage reached 9.9% at 45 DAP, the highest germination percentage through the whole experiment. The germination percentage then markedly declined to approximately 2% at 60 DAP. This low germination percentage lasted to 90 DAP. Almost no seed germination was observed after 120 DAP up to 300 DAP (data not shown).\u0026nbsp;\u003c/p\u003e\n\u003ch2\u003eEffect of seed pretreatments on germination\u003c/h2\u003e\n\u003cp\u003ePretreatment with sodium hypochlorite solutions remarkably breaks the situation of no germination of \u003cem\u003eV. planifolia\u003c/em\u003e mature seeds under the asymbiotic germination treatments. Seeds collected at 135 DAP were soaked with a 2% sodium hypochlorite solution over 60\u0026nbsp;min before the asymbiotic germination cultures. The pretreatment with sodium hypochlorite solutions resulted in over 5% seed germination (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e). The effect of sodium hypochlorite soaking treatment on \u003cem\u003eV. planifolia\u003c/em\u003e seed germination increased with higher strength of sodium hypochlorite and longer soaking time. However, the effect of soaking time reached a maximum at 75 minutes in all of the different concentrations of sodium hypochlorite solutions (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e). In addition, \u003cem\u003eV. planifolia\u003c/em\u003e mature seeds treated with a 4% sodium hypochlorite solution for 75 minutes can reach over 10% seed germination. A higher concentration of sodium hypochlorite solution treatment is not recommended because a high concentration of sodium hypochlorite may destroy cell membranes and cause seed death.\u0026nbsp;\u003c/p\u003e\n\u003ch2\u003eDevelopment of pod, embryo and seed coat\u003c/h2\u003e\n\u003cp\u003eTo understand the possible causes for the effect of pretreatment of sodium hypochlorite, morphological, histological, and histochemical changes of seed development were investigated. The main structural changes occurring within developing pods from anthesis until maturity are summarized in Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e. Detailed characteristics are described in follows.\u003c/p\u003e\n\u003cdiv class=\"gridtable\"\u003e\n\u003ctable id=\"Tab1\" border=\"1\"\u003e\u003ccaption\u003e\n\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\n\u003cdiv class=\"CaptionContent\"\u003e\n\u003cp\u003eMajor microscopic structural events occurring in the developing pods of \u003cem\u003eVanilla planifolia\u003c/em\u003e after hand pollination.\u003c/p\u003e\n\u003c/div\u003e\n\u003c/caption\u003e\n\u003cthead\u003e\n\u003ctr\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003eDAP\u003csup\u003ez\u003c/sup\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003eDevelopmental Stage\u003c/strong\u003e\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003eSeed color\u003c/strong\u003e\u003c/p\u003e\n\u003c/th\u003e\n\u003c/tr\u003e\n\u003c/thead\u003e\n\u003ctbody\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e30\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eFertilization and the formation of zygote\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eWhite\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e45\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eProembryo\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eA mixture of white and brown seeds\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e60\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eProembryo and the developing of early globular embryo\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eMost seeds turned black\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e75\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eEarly globular embryo\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eBlack\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e90\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eGlobular embryo\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eBlack\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e105\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eLate globular embryo\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eBlack\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e300\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003ePod ripe and split \u003csup\u003ey\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eBlack\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tbody\u003e\n\u003ctfoot\u003e\n\u003ctr\u003e\n\u003ctd colspan=\"3\"\u003e\u003csup\u003ez\u003c/sup\u003eDAP = days after pollination.\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd colspan=\"3\"\u003e\u003csup\u003ey\u003c/sup\u003e From 105 to 300 DAP, the seed structure did not change, but the pod gradually became ripe and turned yellow by 240 DAP.\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tfoot\u003e\n\u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003ePods are developed from ovaries. After successful hand-pollination, ovaries began to enlarge and elongate rapidly (Figs.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e and \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e). The pod length increased steadily and reached the maximum size (19.86\u0026thinsp;\u0026plusmn;\u0026thinsp;1.99\u0026nbsp;cm) at 35 DAP, while the diameter of the pod reached the maximum size (12.57\u0026thinsp;\u0026plusmn;\u0026thinsp;1.09\u0026nbsp;mm) at 49 DAP (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e). By 240 DAP, the pod matured and became yellow, then turned black after 300 DAP (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eD).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eEmbryos in a pod were developed from zygotes that were combined between sperm cells in pollens and eggs in embryo sacs. The megaspore mother cells develop just after pollination in early-mid May. Numerous mature embryo sacs could be observed in a pod before fertilization. At 30 DAP, zygotes and proembryos were present within developing pods, and no endosperm was observed (Figs.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eB and C). At this stage, the seeds were white and moist (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eA). At 45 DAP, additional cell divisions occurred within the inner tiers and the surface layer (Figs.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eD and E), thus resulting in the growth of the embryo proper. Some seeds had turned light to dark brown (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eB). At 60 DAP, more cell divisions occurred within the embryo proper, resulting in an early globular embryo (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eF). At this stage, most seeds had turned black (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eC). This species lacked a structurally defined suspensor during embryo development (Figs.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e and \u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003e). As the embryo developed to the globular stage at 75 DAP, the embryo proper had filled the cavity of the embryo sac (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003eA). After this stage, nearly all seeds were black (Figs.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eD, E, and F). By 105 DAP, the mature embryo was about 11 cells long and 7 cells wide without the formation of shoot apical meristem and cotyledon (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003eB). At this stage, the cytoplasm of the embryo proper cell was filled with a large number of storage products (e.g., protein bodies and lipid bodies), and starch grains had disappeared. By 300 DAP, the pod had fully matured and desiccated. Then, the pod split, and the mature seeds were released (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eD).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eRegarding the development of seed coat, the inner and outer seed coats derived from the inner and outer integuments of the ovule, respectively (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eA), and these two distinct layers of seed coat surrounded the embryo in the mature seed of \u003cem\u003eV. planifolia\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003eB). The inner seed coat was two cells thick, and the cell wall of the inner seed coat remained primary in nature during the early stage of seed development (Figs.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eB and C; Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e8\u003c/span\u003eA). As the seeds approached maturity, the inner seed coat gradually compressed (Figs.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eD, E and F; Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e8\u003c/span\u003eB), and eventually became a thin layer at maturity (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e8\u003c/span\u003eC). However, the outer seed coat consisted of three to four cell layers (Figs.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eB and C). Before fertilization, the outer seed coat was still growing and had not enclosed the embryo sac completely (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e8\u003c/span\u003eA). At this stage, the cell wall of the outer seed coat was relatively thin (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e8\u003c/span\u003eA), and the cell wall of the outermost layer of the outer seed coat became thickened after fertilization (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eB). By 60 DAP, the thickened wall of the outermost layer of the outer seed coat became sclerified (Figs.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eF and \u003cspan class=\"InternalRef\"\u003e8\u003c/span\u003eB). As the seed approached maturity, the inner layers of the outer seed coat gradually compressed and attached to the sclerified outermost layer of the outer seed coat (Figs.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e8\u003c/span\u003eC and D). Using TBO staining, the cell wall of the outermost layer of the outer seed coat stained greenish-blue, indicating the presence of phenolic compounds in the cell wall (Figs.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e8\u003c/span\u003eB and C). In addition, using Nile red staining, the surface wall of the embryo proper and the innermost and outermost walls of the inner seed coat reacted positively, which suggested the possible accumulation of a cuticular substance in the wall of these two layers (Figs.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003eC and D).\u0026nbsp;\u003c/p\u003e\n\u003ch2\u003eProtocorm and seedling growth\u003c/h2\u003e\n\u003cp\u003eFor the development of protocorms, most embryos were still enveloped by the seed coat at 30 days of culture on 1/2 MS medium. Only a few embryos had emerged from the seed coat, which was considered seed germination (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e9\u003c/span\u003eA). By 60 days of culture, young protocorms had turned pale green, and numerous rhizoids appeared at the basal protocorms (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e9\u003c/span\u003eB). By 90 days of culture, the protocorm had enlarged and turned dark green to differentiate shoot apical meristem (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e9\u003c/span\u003eC). Subsequently, the developing protocorm with the differentiation of the first root was observed (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e9\u003c/span\u003eD). By 150 days of culture, the protocorm had further elongated with prominent root formation (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e9\u003c/span\u003eE). After transferring onto the seedling growth medium for additional 90 days, the seedlings grew into vines with several healthy roots that could be taken out of flasks (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e9\u003c/span\u003eF).\u0026nbsp;\u003c/p\u003e"},{"header":"Discussion","content":" \u003cp\u003eThe mature seed of \u003cem\u003eV. planifolia\u003c/em\u003e is black and hard, which is distinct from the seeds of many orchids (Arditti and Ghani \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2000\u003c/span\u003e). In the present study, based on a defined time frame, we investigated the morphological and structural changes of seeds (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e; Figs.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e, \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e and \u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). In many orchids, the outer seed coat develops from two cell layers (Yam et al. \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2002\u003c/span\u003e), whereas in \u003cem\u003eV. planifolia\u003c/em\u003e, the outer seed coat is derived from four cell layers (Figs.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e and \u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e). The histological and histochemical results indicated that the sclerification primarily occurred in the outermost cell layer of the outer seed coat (Figs.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e, \u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e and \u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e). Similar hard seeds can be found in a few vanilloid species, e.g., \u003cem\u003eGaleola septentrionalis\u003c/em\u003e (Suetsugu et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2015\u003c/span\u003e) and \u003cem\u003eG. javanica\u003c/em\u003e (Yang and Lee \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Their seed coats are multi-layer, and the walls are heavily thickened with lignin polymers as seeds mature. The cell wall of the outermost cell layer of the outer seed coat stained greenish-blue by TBO indicated the presence of phenolic compounds (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e). In the seed coat of many plants, phenolic compounds, including caffeic acid, sinapic acid, coumarin, chlorogenic acid, and ferulic acid, are esterified to the wall structure (Gubler and Ashford \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e1985\u003c/span\u003e; Pan et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2002\u003c/span\u003e). The accumulation of phenolic compounds in the seed coat inhibits seed germination (Bewley and Black \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e1994\u003c/span\u003e). In \u003cem\u003eV. planifolia\u003c/em\u003e, further analysis by nuclear magnetic resonance spectroscopy revealed the heavy deposition of catechyl units during lignification of the cell wall of the seed coat (Chen et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2012\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAlso, the presence of a cuticular substance or suberin has been detected in the cell wall of the inner seed coat (also known as a carapace) and/or outer seed coat of some orchids, such as \u003cem\u003eCephalanthera\u003c/em\u003e, \u003cem\u003eCymbidium\u003c/em\u003e, and \u003cem\u003eCypripedium\u003c/em\u003e (Carlson \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e1940\u003c/span\u003e; Yeung et al. \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e1996\u003c/span\u003e; Lee et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2005\u003c/span\u003e; Yamazaki and Miyoshi \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). In \u003cem\u003eV. planifolia\u003c/em\u003e, after Nile red staining, a thin fluorescent layer was observed in the innermost layer of the inner seed coat cells and the surface wall of the globular embryo, which suggests the accumulation of cuticular substance in walls. However, the fluorescence pattern was absent in the walls of the outer seed coat (Figs.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eC and D). In orchids, the deposition of cuticular substance on the surface of the embryo proper and in the innermost layer of the seed coat may provide physical protection and ensure moisture retention to developing embryos.\u003c/p\u003e \u003cp\u003eDuring the same time, we investigated the effect of seed maturity and seed pretreatments on asymbiotic germination. The optimal germination was at 45 DAP (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). At this stage, the early globular embryo appeared, and the outermost cell layer of the outer seed coat had become thickened but had not been heavily lignified (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD). The germination decreased sharply by 60 DAP, which agreed with the beginning of seed coat lignification (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Because the outermost cell layer of the outer seedcoat had been lignified, a stronger hydrophobic nature was observed during the operation of seed sowing. As the seed matured, the outer seed coat gradually compressed into a thick and heavily lignified layer surrounding the embryo (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e). The low permeable seed coat restricted the water uptake and solute diffusion, which resulted in poor germination. Although \u003cem\u003eV. planifolia\u003c/em\u003e is an epiphytic orchid species and pantropical in distribution, this pattern of seed germination is similar to those of terrestrial orchids from temperate regions, where immature seeds are easier to germinate \u003cem\u003ein vitro\u003c/em\u003e than are mature seeds (Linden \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e1980\u003c/span\u003e; Zhang et al. \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2013\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe poor germination of mature seeds in terrestrial orchids from temperate regions may be attributed to the accumulation of chemical inhibitors to germination, such as abscisic acid in \u003cem\u003eDactylorhyza\u003c/em\u003e and \u003cem\u003eEpipactis\u003c/em\u003e (van der Kinderen \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e1987\u003c/span\u003e), \u003cem\u003eCalanthe\u003c/em\u003e (Lee et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2007\u003c/span\u003e) and \u003cem\u003eCypripedium\u003c/em\u003e (Lee et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2015\u003c/span\u003e) and phenolics in \u003cem\u003eCymbidium\u003c/em\u003e (Kako \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e1976\u003c/span\u003e), and/or the formation of an impermeable container in the seed coat that may make an embryo difficult to obtain water and nutrients for germination (Miyoshi and Mii 1988; Lee et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2005\u003c/span\u003e). Seed pretreatments may release dormancy and enhance germination by changing the physical integrity of seed coverings, allowing the embryo to absorb water and nutrients and to uptake oxygen (Taylor et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e1998\u003c/span\u003e). Seed pretreatments using bleaching solutions, such as calcium hypochlorite and sodium hypochlorite, can greatly stimulate seed germination of several European terrestrial orchids (Linden \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e1980\u003c/span\u003e; Rasmussen \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e1995\u003c/span\u003e; Steele \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e1996\u003c/span\u003e; Van Waes and Debergh \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e1986\u003c/span\u003e). In this study, the germination of mature seeds was enhanced with soaking in 2% and 4% sodium hypochlorite solutions from 75 to 90\u0026nbsp;min. However, no germination was recorded in fully mature seeds (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Common bleaching agents such as sodium hypochlorite have been widely used for removing residual lignin from the wood pulp (Holik \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). From the scanning electron microscopic observations, the seed coat was scarred with hypochlorite oxidation, so the seed coat was relatively more permeable to water (Lee \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). Of note, as compared with previous reports of seed pretreatments using hypochlorite solutions, we used a stronger concentration (4% sodium hypochlorite) and a longer duration (90\u0026nbsp;min) to pretreat mature seeds. However, the optimal germination of 12.66\u0026thinsp;\u0026plusmn;\u0026thinsp;1.14% was relatively low. This low germination may reflect the hard seed coat of \u003cem\u003eV. planifolia\u003c/em\u003e with its extremely impermeable nature. The relative low germination of pretreated seeds may be attributed to the accumulation of inhibitory substances, such as abscisic acid inside the embryo, which still cannot be leached out by hypochlorite solutions. The orchid seed with a heavily lignified, hard seed coat is adapted to the seed dispersal strategy involved in endozoochory by animals (Suetsugu \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). In \u003cem\u003eCyrtosia\u003c/em\u003e and \u003cem\u003eVanilla\u003c/em\u003e species, as their fruits ripen, they usually have vivid colors and/or a heady fragrance to attract animals, such as rats, bats, or birds (Soto Arenas and Dressler \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Yang and Lee \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Suetsugu et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). The thickened lignified seed coat can protect the embryos when the seeds pass through the digestive tract of animals (Suetsugu \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2018\u003c/span\u003e).\u003c/p\u003e "},{"header":"Conclusions","content":" \u003cp\u003eIn this report, we provide reliable protocols for seedling production of \u003cem\u003eV. planifolia\u003c/em\u003e through asymbiotic seed germination. For the successful culture of immature seeds, the timing of seed collection is short and critical, and optimal germination could be obtained from immature seeds collected at 45 DAP (before seeds turned black). For improving the germination of mature seeds, pretreatment with 4% sodium hypochlorite solution for 75 to 90\u0026nbsp;min is recommended. We propose that the thickened and lignified seed coat, forming an impermeable envelope, is responsible for the seed coat-imposed dormancy. Further work is needed to examine the changes in levels of endogenous inhibitors in embryos, such as abscisic acid, during seed development. Such studies should help to fully explain the low germination percentage of \u003cem\u003eV. planifolia\u003c/em\u003e seeds.\u003c/p\u003e "},{"header":"Declarations","content":"\u003ch2\u003eAuthor contributions\u003c/h2\u003e\n\u003cp\u003eCHY, KYC and YIL designed the study; CHY and YIL performed experiments; CHY, KYC and YIL wrote and edited the manuscript; all authors commented on the manuscript.\u0026nbsp;\u003c/p\u003e\n\u003ch2\u003eAuthor details\u003c/h2\u003e\n\u003cp\u003e\u003csup\u003e1\u003c/sup\u003eTaoyuan District Agricultural Research and Extension Station, Council of Agriculture, Executive Yuan, Taoyuan 327, Taiwan, ROC. \u003csup\u003e2\u003c/sup\u003eDepartment of Agronomy, National Taiwan University, Taipei, Taiwan, ROC. \u003csup\u003e3\u003c/sup\u003eBiology Department, National Museum of Natural Science, 40453 Taichung, Taiwan, ROC. \u003csup\u003e4\u003c/sup\u003eDepartment of Life Sciences, National Chung Hsing University, 40227 Taichung, Taiwan, ROC.\u0026nbsp;\u003c/p\u003e\n\u003ch2\u003eCompeting interests.\u003c/h2\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u0026nbsp;\u003c/p\u003e\n\u003ch2\u003eAvailability of data and material.\u003c/h2\u003e\n\u003cp\u003eNot applicable.\u0026nbsp;\u003c/p\u003e\n\u003ch2\u003eConsent for publication.\u003c/h2\u003e\n\u003cp\u003eNot applicable.\u0026nbsp;\u003c/p\u003e\n\u003ch2\u003eEthics approval and consent to participate.\u003c/h2\u003e\n\u003cp\u003eNot applicable.\u0026nbsp;\u003c/p\u003e\n\u003ch2\u003eFunding.\u003c/h2\u003e\n\u003cp\u003eThis work was supported by grants [107AS-7.6.4-YS-Y1 and 108AS-7.6.4-YS-Y1] from the Council of Agriculture, Executive Yuan, R.O.C. to Chih-Hsin Yeh.\u0026nbsp;\u003c/p\u003e\n\u003ch2\u003ePublisher\u0026rsquo;s Note\u0026nbsp;\u003c/h2\u003e\n\u003cp\u003eOpen Access\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eArditti J (1967) Factors affecting the germination of orchid seeds. 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In: Thorpe TA, Yeung EC (eds) Plant embryo culture; methods and protocols. Methods in Molecular Biology. Humana Press, New York, pp 53-62\u003c/li\u003e\n\u003cli\u003eLee YI (2018) Vegetative propagation of orchids. In: Lee YI, Yeung EC (eds) Orchid propagation: from laboratory to greenhouses-methods and protocols. Springer Protocols and Handbooks, Humana Press, New York, pp 403-426\u003c/li\u003e\n\u003cli\u003eLee YI, Chung MC, Yeung EC, Lee N (2015) Dynamic distribution and the role of abscisic acid during seed development of a lady\u0026rsquo;s slipper orchid,\u003cem\u003e Cypripedium formosanum\u003c/em\u003e. Ann Bot 116:403-411\u003c/li\u003e\n\u003cli\u003eLinden B (1980) Aseptic germination of seeds of Northern terrestrial orchids. 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New Phytol 217:828-835\u003c/li\u003e\n\u003cli\u003eSuzuki RM, Moreira VC, Pescador R, Ferreira WM (2012) Asymbiotic seed germination and in vitro seedling development of the threatened orchid \u003cem\u003eHoffmannseggella cinnabarina\u003c/em\u003e. In Vitro Cell Dev Biol - Plant 48:500-511\u003c/li\u003e\n\u003cli\u003eTaylor AG, Allen PS, Bennett MA, Bradford KJ, Burris JS, Misra MK (1998) Seed enhancements. Seed Sci Res 8:245-256\u003c/li\u003e\n\u003cli\u003eVan der Kinderen G (1987) Abscisic acid in terrestrial orchid seeds: a possible impact on their germination. Lindleyana 2:84-87\u003c/li\u003e\n\u003cli\u003eVan Waes JM, Debergh PC (1986) In vitro germination of some Western European orchids. Physiol Plant 67:253-261\u003c/li\u003e\n\u003cli\u003eWithner CL (1955) Ovule culture and growth of \u003cem\u003eVanilla\u003c/em\u003e seedlings. Amer Orchid Soc Bull 51:380-392\u003c/li\u003e\n\u003cli\u003eYam TW, Yeung EC, Ye XL, Zee SY, Arditti J (2002) Orchid embryos. In: Kull T, Arditti J (eds) Orchid biology: reviews and perspectives. VIII. Kluwer Academic Publisher, Dordrecht, pp 287-385\u003c/li\u003e\n\u003cli\u003eYam TW, Arditti J (2017) Micropropagation of orchids, 3rd ed. John Wiley and Sons, Inc., New York\u003c/li\u003e\n\u003cli\u003eYamazaki J, Miyoshi K (2006) In vitro asymbiotic germination of immature seed and formation of protocorm by \u003cem\u003eCephalanthera falcata \u003c/em\u003e(Orchidaceae). Ann Bot 98:1197-1206\u003c/li\u003e\n\u003cli\u003eYang CK, Lee YI (2014) The seed development of a mycoheterotrophic orchid, \u003cem\u003eCyrtosia javanica\u003c/em\u003e Blume. Bot Stud 55:44\u003c/li\u003e\n\u003cli\u003eYeung EC, Zee SY, Ye XL (1996) Embryology of \u003cem\u003eCymbidium sinense\u003c/em\u003e: embryo development. Ann Bot 78:105-110\u003c/li\u003e\n\u003cli\u003eYeung EC, Chan CKW (2015) The glycol methacrylate embedding resins Technovit 7100 and 8100. Plant microtechniques and protocols. Springer International Publishing, New York, pp 67-82\u003c/li\u003e\n\u003cli\u003eZhang Y, Lee YI, Deng L, Zhao S (2013) Asymbiotic germination of immature seeds and the seedling development of \u003cem\u003eCypripedium macranthos\u003c/em\u003e Sw., an endangered lady's slipper orchid. Sci Hortic 164:130-136\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"botanical-studies","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"bost","sideBox":"Learn more about [Botanical Studies](http://as-botanicalstudies.springeropen.com)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/bost/default.aspx","title":"Botanical Studies","twitterHandle":"@SpringerOpen","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"micropropagation, embryo, seed coat, seed pretreatment, Vanilla","lastPublishedDoi":"10.21203/rs.3.rs-138206/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-138206/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground: \u003c/strong\u003e\u003cem\u003eVanilla planifolia\u003c/em\u003e is an important tropical orchid for production of natural vanilla flavor. Traditionally, \u003cem\u003eV. planifolia \u003c/em\u003eis propagated by stem cuttings, which produces identical genotype that are sensitive to virulent pathogens. However, sexual propagation with seed germination of \u003cem\u003eV. planifolia \u003c/em\u003eis intricate and unstable because of the extremely hard seed coat. A better understanding of seed development, especially the formation of impermeable seed coat would provide insights into seed propagation and conservation of genetic resources of \u003cem\u003eVanilla\u003c/em\u003e.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eResults: \u003c/strong\u003eWe found that soaking mature seeds in 4 % sodium hypochlorite solution from 75 to 90 min significantly increased germination and that immature seeds collected at 45 days after pollination (DAP) had the highest germination percentage. We then investigated the anatomical features during seed development that associated with the effect of seed pretreatment on raising seed germination percentage. The 45-DAP immature seeds have developed globular embryos and the thickened non-lignified cell wall at the outermost layer of the outer seed coat. After 60 DAP, the cell wall of the outermost layer of the outer seed coat became lignified and finally compressed into a thick envelope. These features matches the significant decreases of immature seed germination percentage after 60 DAP. \u003c/p\u003e\u003cp\u003e\u003cstrong\u003eConclusion: \u003c/strong\u003eWe report a reliable protocol for seed pretreatment of mature seeds and for immature seeds culture based on a defined time schedule of \u003cem\u003eV. plantifolia\u003c/em\u003e seed development. The thickened and lignified seed coat formed an impermeable envelope surrounding the embryo, and might play an important role in seed dormancy of \u003cem\u003eV. plantifolia\u003c/em\u003e.\u003c/p\u003e","manuscriptTitle":"Asymbiotic Germination of Mature Seeds and the Seed Development of Vanilla Planifolia","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2021-01-05 19:24:09","doi":"10.21203/rs.3.rs-138206/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Minor revision","date":"2021-01-31T00:00:00+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2021-01-30T00:00:00+00:00","index":3,"fulltext":"Recommendation: Reviewer's comments unavailable due to the journal's policy.\n"},{"type":"editorInvitedReview","content":"","date":"2021-01-12T00:00:00+00:00","index":1,"fulltext":"Recommendation: Reviewer's comments unavailable due to the journal's policy.\n"},{"type":"editorInvitedReview","content":"","date":"2021-01-11T00:00:00+00:00","index":2,"fulltext":"Recommendation: Reviewer's comments unavailable due to the journal's policy.\n"},{"type":"reviewerAgreed","content":"","date":"2021-01-05T00:00:00+00:00","index":3,"fulltext":""},{"type":"reviewerAgreed","content":"","date":"2021-01-04T00:00:00+00:00","index":2,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2021-01-03T00:00:00+00:00","index":"","fulltext":""},{"type":"reviewerAgreed","content":"","date":"2021-01-03T00:00:00+00:00","index":1,"fulltext":""},{"type":"editorAssigned","content":"","date":"2020-12-24T00:00:00+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2020-12-23T23:00:00+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2020-12-23T23:00:00+00:00","index":"","fulltext":""},{"type":"submitted","content":"","date":"2020-12-23T00:00:00+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"botanical-studies","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"bost","sideBox":"Learn more about [Botanical Studies](http://as-botanicalstudies.springeropen.com)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/bost/default.aspx","title":"Botanical Studies","twitterHandle":"@SpringerOpen","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"43e02621-a090-4e22-948f-8edabaad1007","owner":[],"postedDate":"January 5th, 2021","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":1701676,"name":"Plant Physiology and Morphology"},{"id":1701677,"name":"Plant Molecular Biology and Genetics"},{"id":1701678,"name":"Botany"}],"tags":[],"updatedAt":"2021-06-03T12:16:42+00:00","versionOfRecord":[],"versionCreatedAt":"2021-01-05 19:24:09","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-138206","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-138206","identity":"rs-138206","version":["v1"]},"buildId":"7rjqhiLT3MXkJMwkYKINL","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}