Yeh 

et al. Bot Stud (2021) 62:6

https://doi.org/10.1186/s40529-021-00311-y

ORIGINAL ARTICLE Open Access

Asymbiotic germination of Vanilla planifolia

in relation to the timing of seed collection
and seed pretreatments
Chih‑Hsin Yeh1,2, Kai‑Yi Chen2* and Yung‑I. Lee3,4*   

Abstract 
Background:  Vanilla planifolia is an important tropical orchid for production of natural vanilla flavor. Traditionally,
V. planifolia is propagated by stem cuttings, which produces identical genotype that are sensitive to virulent patho‑
gens. However, propagation with seed germination of V. planifolia is intricate and unstable because the seed coat is
extremely hard with strong hydrophobic nature. A better understanding of seed development, especially the forma‑
tion 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. For the culture of immature seeds, the seed collection 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. Seeds at 60 DAP and subsequent stages germinated poorly. As the seed approached maturity, the cell wall of
the outermost layer of the outer seed coat became lignified and finally compressed into a thick envelope at maturity.
On toluidine blue O staining, the wall of outer seed coat stained greenish blue, indicating the presence of phenolic
compounds. As well, on Nile red staining, a cuticular substance was detected in the surface wall of the embryo proper
and the innermost wall of the inner seed coat.
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 window for successful germination of
culturing immature seed was short. The quick accumulation of lignin, phenolics and/or phytomelanins in the seed
coat may seriously inhibit seed germination after 45 DAP. As seeds matured, the thickened and lignified seed coat
formed an impermeable envelope surrounding the embryo, which may play an important role in inducing dormancy.
Further studies covering different maturity of green capsules are required to understand the optimal seed maturity
and germination of seeds.
Keywords:  Micropropagation, Embryo, Seed coat, Seed pretreatment, Vanilla

Background
*Correspondence: [email protected]; [email protected];
Vanilla planifolia is a vanilla orchid native to Mexico and
[email protected] Central America (Bory et al. 2008), and has been planted
2
Department of Agronomy, National Taiwan University, Taipei, Taiwan, in many tropical regions around the world to produce
ROC
3
Biology Department, National Museum of Natural Science,
natural vanilla flavor (Sreedhar et  al. 2007). Since the
40453 Taichung, Taiwan, ROC mature seeds of V. planifolia hardly germinate, V. plani-
Full list of author information is available at the end of the article folia is usually propagated commercially by vegetative

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Yeh et al. Bot Stud (2021) 62:6 Page 2 of 12

propagation methods, such as cutting stem or callus soaking durations on germination. We also documented
culture regeneration (Palama et  al. 2010; Havkin‐Fren- the morphological, histological, and histochemical
kel and Belanger 2018; Lee 2018). Nevertheless, seed changes of seed development within a defined timescale,
propagation can produce offspring with differing geno- from fertilization to seed maturity. Such information
types that is important to generate novel traits in breed- would provide insights into mass propagation to meet
ing programs. Asymbiotic seed germination technique commercial needs and breeding programs for vanilla
has been widely used for the commercial propagation production.
of many orchids (Knudson 1922; Yam and Arditti 2017).
However, the application of this technique to some tem- Methods
perate terrestrial orchids is complicated, e.g. Cymbidium, Plant material and seed collection
Cypripedium, Epipactis (Kako 1976; Van der Kinderen The mature plants of V. planifolia were maintained in a
1987; Rasmussen 1995; Miyoshi and Mii 1998). In these greenhouse at Taoyuan District Agricultural Research
intricate-to-germinate orchids, the seeds have deep dor- and Extension Station at Taoyuan City, Taiwan. Anthesis
mancy and the germination of mature seeds could be generally occurs in late April each year (Fig. 1a). For the
extremely low in a range from 0 to 5 %. Causes of low pod setting, flowers were hand-pollinated by transferring
germination of some orchids may relate to the imper- the pollinia onto the stigma of the same flower (Fig. 1b).
meability of the seed coat during seed maturation (Lee Developing pods (Fig. 1c) were collected at regular inter-
et al. 2005) or the accumulation of inhibitory substances vals for morphological measurement, histology, and seed
to germination (Van Waes and Debergh 1986; Van der germination experiments. In January the next year, pods
Kinderen 1987; Lee et  al. 2007, 2015). Although Vanilla began to mature and turned yellow (Fig.  1d). In each
is pantropical in distribution, seed germination in vitro of experiment, seeds were collected at least from three pods
V. planifolia is still considered intricate (Knudson 1950). at regular intervals after pollination.
Successful asymbiotic germination may be related
to the timing of seed collection (Arditti 1967; Lee et  al. Evaluation of asymbiotic germination percentage
2005), components of culture medium, such as organic The pods of different developmental stages were surface-
nutrients, carbon sources, plant growth regulators (Lo sterilized with a 1.8% sodium hypochlorite solution with
et  al. 2004; Gayatri and Kavyashree 2005; Dutra et  al one drop of a wetting agent (Tween 20, Sigma-Aldrich)
2008), light intensity and temperature ranges (Knud- for 15 min. After surface sterilization, the capsules were
son 1950; Suzuki et  al. 2012), and seed pretreatments cut open, and seeds were scooped out with forceps onto
(Lee 2011). Like most orchids, V. planifolia produces the culture medium. To ensure the seed quality and devel-
many seeds that contain globular embryos without the opmental stages of each capsule, the remaining seeds of
endosperm (Clements and Molvray 1999). The seed coat each capsule were fixed and examined under a micro-
of V. planifolia is black and hard, which contrasts mark- scope. The culture medium used was 1/2 Murashige and
edly with the thin transparent seed coat of most orchid Skoog (MS) (Murashige and Skoog 1962) supplemented
seeds (Cameron and Chase 1998; Nishimura and Yukawa with 2 mg ­l–1 glycine, 0.5 mg ­l–1 niacin, 0.5 mg ­l–1 pyri-
2010). Mature seeds of V. planifolia usually have very doxine HCl, 0.1 mg ­l–1 thiamine, 1 g ­l−1tryptone, 20 g ­l−1
low germination (Knudson 1950; Menchaca et al. 2011), sucrose, and solidified with 7 g ­l−1 agar (plant cell culture,
which might be due to the impermeability of the hard- tested, powder; all Sigma-Aldrich). The pH value was
ened seed coat (Withner 1955; Lee et al. 2005, 2007, 2015; adjusted to 5.7 before autoclaving at 121  °C and 1.2  kg
Van der Kinderen 1987; Van Waes and Debergh 1986). ­cm2 for 20 min. An amount of 10 ml medium was placed
Despite the low germination of mature seeds, there are into each culture tube (20 × 100 mm). There were ca. 100
some practical advantages of using mature seeds for seeds per culture tube. After sowing, the cultures were
propagation, e.g. the long term storage and the long dis- incubated in a growth room under a 16/8 h photoperiod
tance shipment (Steele 1996). Therefore, it is necessary to with daylight fluorescent lamps (20  W, China Electric,
find out the pretreatment conditions for breaking mature Taipei) at light intensity 30  µmol  ­m−2 ­s−1. Germination
seed dormancy of V. planifolia. percentage was recorded 60 days after sowing.
This study aimed to establish an efficient propagation
method of V. planifolia via asymbiotic germination. We Effect of seed pretreatments on germination
first tested the effect of seed maturity on asymbiotic To examine the effectiveness of different concentrations
germination and determined the optimal timing for cul- and durations of sodium hypochlorite pretreatments
turing immature seeds. In order to improve the germina- in improving germination, mature seeds were collected
tion of mature seeds, we examined the effect of different from capsules at 135 DAP. There were four capsules col-
combinations of sodium hypochlorite concentrations and lected for each pretreatment. Mature seeds were scooped
 Yeh et al. Bot Stud (2021) 62:6 Page 3 of 12

Fig. 1  Flowers and developing pods of V. planifolia. a Flower at the time of anthesis. Scale bar = 1 cm. b Flower was hand-pollinated by transferring
the pollinia onto the stigma of the same flower. Scale bar = 1 cm. c Developing pods at 60 DAP. Scale bar = 1 cm. d Pods turned yellow by 240 DAP
and became black by 300 DAP. Scale bar = 1 cm

into test tubes, then filled with 30 ml sodium hypochlo- acid–Schiff (PAS) procedure for structural carbohydrates
rite solution. The pretreatments included soaking the and counterstained with 1% (w/v) amido black 10B in 7%
seeds in 0.5, 1, 2 or 4% sodium hypochlorite solution with acetic acid for protein (Sigma-Aldrich, St. Louis, MO,
one drop of Tween 20, for 15, 30, 45, 60, 75 or 90  min. USA) or 0.05% (w/v) toluidine blue O (TBO, Sigma-
In control, seeds were soaked only in water. After pre- Aldrich) for general histological staining (Yeung, 1984).
treatments, seeds were washed three times with steri- For detecting the deposition of cuticular material in
lized distilled water, then inoculated on 1/2 MS medium developing seeds, sections were stained with 1  μg  ­ml−1
as described above. There were ca. 300 seeds per culture Nile red (Sigma-Aldrich) for 1 min, then washed in run-
tube. The culture conditions and germination record ning tap water for 3 min. The fluorescence pattern of Nile
were same as asymbiotic germination procedure as red was viewed under an epifluorescence microscope
described above. (Axioskop 2, Carl Zeiss AG, Germany) equipped with the
Zeiss filter set 15 (546/12  nm excitation and 590 emis-
Histological and histochemical studies sion). All images were recorded by using a CCD camera
Developing seeds were collected and fixed in 2.5% glu- attached to the microscope.
taraldehyde and 1.6% paraformaldehyde buffered with
0.1 M phosphate buffer (pH 6.8) for 48 h at room temper- Rooting and acclimatization of in vitro seedlings
ature. Seeds were then dehydrated in an ethanol series, After 150  days of culture, developing protocorms with
then infiltrated gradually (3:1, 1:1, and 1:3 100% etha- roots were transferred onto seedling growth medium: 1/2
nol: Technovit 7100, 24 h each) by using Technovit 7100 MS medium supplemented with 20 g ­l–1 sucrose, 1 g ­l–1
resin (Kulzer & Co., Germany), followed by three changes activated charcoal powder (C9157, Sigma-Aldrich),
of pure resin. Seeds were then embedded in resin, as 20  g  ­l–1 potato homogenate, and 7  g  ­l–1 agar for grow-
described by Yeung and Chan (2015). Sections of 3-μm ing seedlings described by Lee (2011). The potato was
thick were cut using a Reichert-Jung 2040 Autocut rotary boiled for 10  min, then peeled and cut into ca. 1-cm3
microtome. These sections were stained with periodic cubes, then homogenized with a kitchen blender. The
Yeh et al. Bot Stud (2021) 62:6 Page 4 of 12

pH of the medium was adjusted to 5.6 before autoclaving seed germination was observed after 120 DAP up to 300
at 121 °C for 20 min. An amount of 100 ml medium was DAP (data not shown).
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  h photoperiod with daylight Effect of seed pretreatments on germination
fluorescent lamps (20  W, China Electric, Taipei) at light Pretreatment with sodium hypochlorite solutions
intensity 30 µmol ­m−2 ­s−1. After 90 days of culture in the remarkably breaks the situation of no germination of V.
seedling growth medium, seedlings of about 10  cm tall planifolia mature seeds under the asymbiotic germina-
with 4 leaves were taken out of flasks. tion treatments. Seeds collected at 135 DAP were soaked
with a 2% sodium hypochlorite solution over 60  min
before the asymbiotic germination cultures. The pre-
Experimental design and statistical analysis treatment with sodium hypochlorite solutions resulted in
All experiments were performed in a completely rand- over 5% seed germination (Fig.  3). The effect of sodium
omized design and repeated three times; 12 replicates hypochlorite soaking treatment on V. planifolia seed
(culture tubes) were used for each treatment, with one germination increased with higher strength of sodium
explant planted in each plate. Data were analyzed by hypochlorite and longer soaking time. However, the
using analysis of variance (ANOVA) with Fisher’s pro- effect of soaking time reached a maximum at 75  min in
tected least significant difference test at P < 0.05. All data all of the different concentrations of sodium hypochlorite
were analyzed with SAS v9.0 (Cary, NC, USA). solutions (Fig. 3). In addition, V. planifolia mature seeds
treated with the 4% sodium hypochlorite solution for
75 min can reach over 10% seed germination.
Results
Seed germination can be achieved using asymbiotic
germination technique at early seed developmental stages Development of pod, embryo and seed coat
of V. planifolia The main structural changes occurring within develop-
In this study, different stages of developing seeds were ing pods from anthesis until maturity are summarized in
collected in an interval of 15  days from the day of fer- Table 1. Detailed characteristics are described in follows.
tilization to 300 DAP, and their germination percent- Pods are developed from ovaries. After successful hand-
ages under the asymbiotic germination treatments were pollination, ovaries began to enlarge and elongate rapidly
investigated. Seed germination occurred after 30 DAP (Figs.  1 and 4). The pod length increased steadily and
(Fig.  2). The seed germination percentage reached 9.9% reached the maximum size (19.86 ± 1.99 cm) at 35 DAP,
at 45 DAP, the highest germination percentage through while the diameter of the pod reached the maximum size
the whole experiment. The germination percentage then (12.57 ± 1.09  mm) at 49 DAP (Fig.  4). By 240 DAP, the
markedly declined to approximately 2% at 60 DAP. This
low germination percentage lasted to 90 DAP. Almost no

Fig. 3  Effect of different concentrations and durations of sodium

Fig. 2  Mean percent germination of V. planifolia seeds at each hypochlorite pretreatments on seed germination in vitro of V.
successive 15 days after pollination on 1/2 MS medium. Data were planifolia. Data were recorded after 60 days of culture. Data are
scored after 60 days of culture. Data are mean ± SD (n = 10) mean ± SD (n = 3)
 Yeh et al. Bot Stud (2021) 62:6 Page 5 of 12

Table 1  Major microscopic structural events occurring in the developing pods of Vanilla planifolia after hand pollination
DAP 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 s­ plita Black
a
From 105 to 300 DAP, the seed structure did not change, but the pod gradually became ripe and turned yellow by 240 DAP
DAP  days after pollination

Numerous mature embryo sacs could be observed in

a pod before fertilization. At 30 DAP, zygotes and pro-
embryos were present within developing pods, and no
endosperm was observed (Fig. 5b, c). At this stage, the
seeds were white and moist (Fig. 6a). At 45 DAP, addi-
tional cell divisions occurred within the inner tiers
and the surface layer (Fig.  5d, e), thus resulting in the
growth of the embryo proper. Some seeds had turned
light to dark brown (Fig. 6b). At 60 DAP, more cell divi-
sions occurred within the embryo proper, resulting in
an early globular embryo (Fig.  5f ). At this stage, most
seeds had turned black (Fig.  6c). This species lacked a
structurally defined suspensor during embryo develop-
ment (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.  7a). After this stage,
nearly all seeds were black (Fig. 6d–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. 7b). 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. 1d).
Regarding the development of seed coat, the inner
and outer seed coats derived from the inner and outer
integuments of the ovule, respectively (Fig.  5a), and
these two distinct layers of seed coat surrounded the
Fig. 4  Changes in pod length (a) and width (b) of V. planifolia. Data embryo in the mature seed of V. planifolia (Fig.  7b).
are mean ± SD (n = 30) 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. 5b, c,
pod matured and became yellow, then turned black after 8a). As the seeds approached maturity, the inner seed
300 DAP (Fig. 1d). coat gradually compressed (Figs. 5d–f, 8b), and eventu-
Embryos in a pod were developed from zygotes ally became a thin layer at maturity (Fig. 8C). However,
that were combined between sperm cells in pol- the outer seed coat consisted of three to four cell lay-
lens and eggs in embryo sacs. The megaspore mother ers (Fig. 5b, c). Before fertilization, the outer seed coat
cells develop just after pollination in early-mid May. was still growing and had not enclosed the embryo sac
Yeh et al. Bot Stud (2021) 62:6 Page 6 of 12

Fig. 5  The early embryo development of V. planifolia. 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). 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. c Light micrograph showing
a three-celled proembryo (arrow), and a degenerated endosperm nucleus (arrowhead) stayed beside the zygote. d By 45 DAP, an anticlinal
division (arrow) occurred in the outmost cell layer, resulting in the formation of the globular-shape embryo. 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. 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

completely (Fig.  8a). At this stage, the cell wall of the in the cell wall (Fig.  8b, c). In addition, using Nile red
outer seed coat was relatively thin (Fig.  8a), and the staining, the surface wall of the embryo proper and the
cell wall of the outermost layer of the outer seed coat innermost and outermost walls of the inner seed coat
became thickened after fertilization (Fig.  5b). By 60 reacted positively, which suggested the possible accu-
DAP, the thickened wall of the outermost layer of the mulation of a cuticular substance in the wall of these
outer seed coat became sclerified (Figs.  5f and 8b). As two layers (Fig. 7c, d).
the seed approached maturity, the inner layers of the
outer seed coat gradually compressed and attached to Protocorm and seedling growth
the sclerified outermost layer of the outer seed coat For the development of protocorms, most embryos
(Fig. 8c, d). Using TBO staining, the cell wall of the out- were still enveloped by the seed coat at 30  days of
ermost layer of the outer seed coat stained greenish- culture on 1/2 MS medium. Only a few embryos had
blue, indicating the presence of phenolic compounds emerged from the seed coat, which was considered
 Yeh et al. Bot Stud (2021) 62:6 Page 7 of 12

Fig. 6  Morphology of developing seeds of V. planifolia. a White and moist seeds at 30 DAP. b A mixture of white and brown seeds at 45 DAP. cc
A number of seeds had turned black at 60 DAP. d Nearly all seeds turned black at 75 DAP. e Black seeds at 90 DAP. f Black seeds at105 DAP. Scale
bar = 1 mm

seed germination (Fig. 9a). By 60 days of culture, young Discussion

protocorms had turned pale green, and numerous The mature seed of V. planifolia is black and hard, which
rhizoids appeared at the basal protocorms (Fig.  9b). is distinct from the seeds of many orchids (Arditti and
By 90 days of culture, the protocorm had enlarged and Ghani 2000). In the present study, based on a defined
turned dark green to differentiate shoot apical meris- time frame, we investigated the morphological and struc-
tem (Fig.  9c). Subsequently, the developing protocorm tural changes of seeds (Table 1; Figs. 5, 6 and 7). In many
with the differentiation of the first root was observed orchids, the outer seed coat develops from two cell lay-
(Fig. 9d). By 150 days of culture, the protocorm had fur- ers (Yam et al. 2002), whereas in V. planifolia, the outer
ther elongated with prominent root formation (Fig. 9e). seed coat is derived from four cell layers (Figs. 5 and 8).
After transferring onto the seedling growth medium for The histological and histochemical results indicated that
additional 90  days, the seedlings grew into vines with the sclerification primarily occurred in the outermost
several healthy roots that could be taken out of flasks cell layer of the outer seed coat (Figs.  5, 7 and 8). Simi-
(Fig. 9f ). lar hard seeds can be found in a few vanilloid species,
Yeh et al. Bot Stud (2021) 62:6 Page 8 of 12

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 (Fig. 7c, 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.
Asymbiotic germination has been reported to support
the growth of the immature embryo in several terres-
Fig. 7  The late embryo development of V. planifolia. a As the seed
approached maturity, a number of tiny protein bodies (arrows)
trial orchid species (Lee et al. 2005, 2007; Van Waes and
appeared within the embryo proper cells after amido black 10B Debergh 1986). In this study, we investigated the effect
stain. The thickened outer seed coat (OS) became dehydrated and of seed maturity and seed pretreatments on asymbiotic
compressed, with the inner seed coat (IS) compressed into a thin germination. The optimal germination was observed at
layer. b Light micrograph showing a longitudinal section through a 45 DAP (Fig. 3). At this stage, the early globular embryo
mature seed. Several tiny protein bodies (arrows) were found within
the embryo proper cells. In this preparation, the lipid bodies were not
appeared, and the outermost cell layer of the outer seed
preserved; the spaces (arrowheads) between the protein bodies were coat had become thickened but had not been heavily lig-
occupied by storage lipid bodies. c Nile red staining fluorescence nified (Fig.  5d). V. planifolia is an epiphytic orchid spe-
micrograph of an early globular embryo at the stage similar to cies and pantropical in distribution, while its pattern of
Fig. 3E. After Nile red staining, the surface wall of the embryo proper seed germination is similar to those of terrestrial orchids
(arrows) reacted weakly to the stain, and the innermost (arrowhead)
and outermost (double arrowhead) walls of the inner seed coat
from temperate regions, where immature seeds are easier
also reacted positively. d Nile red staining fluorescence micrograph to germinate in vitro than are mature seeds (Linden 1980;
of a mature seed at the stage similar to Fig. 5B. The inner seed coat Zhang et al. 2013). It is also notable that the optimal ger-
compressed into a thin layer and attached the embryo tightly. The mination at 45 DAP only reached 9.9%. The very low ger-
thin inner seed coat (IS, arrowheads) and the surface of the embryo mination ability in immature seeds of V. planifolia could
proper (arrow) reacted positively to the stain. Fluorescence was never
observed in the thickened outer seed coat (OS). Scale bar = 100 μm
be due to the impermeability of thickened outer seed coat
and/or the presence of inhibitors, e.g. ABA or phenolics
during the early stage of seed development. The germina-
tion decreased sharply by 60 DAP, which agreed with the
e.g., Galeola septentrionalis (Suetsugu et al. 2015) and G. initiation of seed coat lignification (Fig.  3). Because the
javanica (Yang and Lee 2014). Their seed coats are multi- outermost cell layer of the outer seed coat had been lig-
layer, and the walls are heavily thickened with lignin nified, a stronger hydrophobic nature was observed dur-
polymers as seeds mature. The cell wall of the outermost ing the operation of seed sowing. As the seed matured,
cell layer of the outer seed coat stained greenish-blue the outer seed coat gradually compressed into a thick and
by TBO indicated the presence of phenolic compounds heavily lignified layer surrounding the embryo (Fig.  8).
(Fig.  8). In the seed coat of many plants, phenolic com- The low permeable seed coat restricted the water uptake
pounds, including caffeic acid, sinapic acid, coumarin, and solute diffusion, which resulted in poor germination
chlorogenic acid, and ferulic acid, are esterified to the of mature seeds.
wall structure (Gubler and Ashford 1985; Pan et al. 2002). Previous reports have indicated that the poor germi-
The accumulation of phenolic compounds in the seed nation of mature seeds in terrestrial orchids from tem-
coat inhibits seed germination (Bewley and Black 1994). perate regions may be attributed to the accumulation
In V. planifolia, further analysis by nuclear magnetic of chemical inhibitors to germination, such as abscisic
 Yeh et al. Bot Stud (2021) 62:6 Page 9 of 12

Fig. 8  The seed coat development of V. planifolia. 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. 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. 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. 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

acid (ABA) in Dactylorhyza and Epipactis (van der Kin- embryo, which still cannot be leached out by hypochlo-
deren 1987), Calanthe (Lee et al. 2007) and Cypripedium rite solutions. The orchid embryo may be highly sensitive
(Lee et  al. 2015) and phenolics in Cymbidium (Kako to the presence of endogenous ABA and possess deep
1976), and/or the formation of an impermeable con- physiological dormancy (Lee et  al. 2015). Furthermore,
tainer in the seed coat that may make an embryo difficult the black seed color of V. planifolia may be due to the
to obtain water and nutrients for germination (Miyoshi accumulation of phytomelanins (Nishimura and Yukawa
and Mii 1998; Lee et  al. 2005). Seed pretreatments may 2010), which has a high molecular weight and is formed
release dormancy and enhance germination by chang- through the process of oxidation and polymerization
ing the physical integrity of seed coverings, allowing the of phenols (Glagoleva et  al. 2020). In our seed pretreat-
embryo to absorb water and nutrients and to uptake ment experiments, the seed coat remained dark black
oxygen (Taylor et  al. 1998). Seed pretreatments using after soaking with strong hypochlorite solutions. Phy-
bleaching solutions, such as calcium hypochlorite and tomelanins are known to provide additional mechani-
sodium hypochlorite, can greatly stimulate seed ger- cal strength to seed coats, protecting the embryos from
mination of several European terrestrial orchids (Lin- damage, but phytomelanins can affect seed dormancy
den 1980; Rasmussen 1995; Steele 1996; Van Waes and and inhibit seed germination (Debeaujon et al. 2000; Gu
Debergh 1986). In this study, the germination of mature et al. 2011). In Cyrtosia and Vanilla species, as their fruits
seeds was enhanced with soaking in 2 and 4% sodium ripen, they usually have vivid colors and/or a heady fra-
hypochlorite solutions from 75 to 90  min. However, no grance to attract animals, such as rats, bats, or birds (Soto
germination was recorded in fully mature seeds (Fig. 2). Arenas and Dressler 2010; Yang and Lee 2014; Suetsugu
Common bleaching agents such as sodium hypochlorite et  al. 2015). The orchid seed with a heavily lignified,
have been widely used for removing residual lignin from hard seed coat is adapted to the seed dispersal strategy
the wood pulp (Holik 2006). From the scanning electron involved in endozoochory by animals (Suetsugu 2018).
microscopic observations, the seed coat was scarred with The thickened lignified seed coat with the accumula-
hypochlorite oxidation, so the seed coat was relatively tion of phytomelanins can protect the embryos when the
more permeable to water (Lee 2011). Of note, as com- seeds pass through the digestive tract of animals.
pared with previous reports of seed pretreatments using
hypochlorite solutions, we used a stronger concentration Conclusions
(4% sodium hypochlorite) and a longer duration (90 min) In this report, we provide reliable protocols for seed-
to pretreat mature seeds. However, the optimal germina- ling production of V. planifolia through asymbiotic
tion of 12.66 ± 1.14% was relatively low. This low germi- seed germination. For the successful culture of imma-
nation may reflect the hard seed coat of V. planifolia with ture seeds, the timing of seed collection is short and
its extremely impermeable nature. critical, and optimal germination could be obtained
In addition to the physical dormancy, the low germina- from immature seeds collected at 45 DAP (before seeds
tion of pretreated seeds may be attributed to the accumu- turned black). For improving the germination of mature
lation of inhibitory substances, such as ABA inside the seeds, pretreatment with 4% sodium hypochlorite
Yeh et al. Bot Stud (2021) 62:6 Page 10 of 12

Fig. 9  Protocorm development and seedling growth of V. planifolia. a By 30 days of culture, a few embryos had emerged from the seed coat.
Scale bar = 1 mm. b Young protocorms with numerous rhizoids appeared at the basal protocorms by 60 days of culture. Scale bar = 1 mm. c The
protocorm had enlarged with the differentiation of a shoot tip by 90 days of culture. Scale bar = 1 mm. d The protocorm had a differentiated first
root by 120 days of culture. Scale bar = 1 mm. 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. f After 90 days of culture on the growth medium, the seedlings grew into vines with several healthy
roots. Bar = 2 cm

solution for 75 to 90 min is recommended. We propose Funding

This work was supported by grants [107AS-7.6.4-YS-Y1 and 108AS-7.6.4-YS-Y1]
that the thickened and lignified seed coat, forming an from the Council of Agriculture, Executive Yuan, R.O.C. to Chih-Hsin Yeh.
impermeable envelope, is responsible for the seed coat-
imposed dormancy. Further work is needed to exam- Availability of data and materials
Not applicable.
ine the changes in levels of endogenous inhibitors in
embryos, such as ABA, during seed development. Such
studies should help to fully explain the low germination Declarations
percentage of V. planifolia seeds. Ethics approval and consent to participate
Not applicable.

Authors’ contributions Consent for publication

CHY and YIL designed the study; CHY and YIL performed experiments; CHY, Not applicable.
KYC and YIL wrote the manuscript; all authors commented on the manuscript.
All authors read and approved the final manuscript.
 Yeh et al. Bot Stud (2021) 62:6 Page 11 of 12

Competing interests germination of a terrestrial orchid, Calanthe tricarinata Lindl. J Am Soc

The authors declare that they have no competing interests. Hortic Sci 132:246–252
Lee YI (2011) In vitro culture and germination of terrestrial Asian orchid seeds.
Author details In: Thorpe TA, Yeung EC (eds) Plant embryo culture; methods and proto‑
1
 Taoyuan District Agricultural Research and Extension Station, Council of Agri‑ cols. Methods in Molecular Biology. Humana Press, New York, pp 53–62
culture, Executive Yuan, Taoyuan 327, Taiwan, ROC. 2 Department of Agronomy, Lee YI (2018) Vegetative propagation of orchids. In: Lee YI, Yeung EC (eds)
National Taiwan University, Taipei, Taiwan, ROC. 3 Biology Department, National Orchid propagation: from laboratory to greenhouses-methods and
Museum of Natural Science, 40453 Taichung, Taiwan, ROC. 4 Department of Life protocols. Springer Protocols and Handbooks. Humana Press, New York,
Sciences, National Chung Hsing University, 40227 Taichung, Taiwan, ROC. pp 403–426
Lee YI, Chung MC, Yeung EC, Lee N (2015) Dynamic distribution and the role of
Received: 23 December 2020 Accepted: 7 April 2021 abscisic acid during seed development of a lady’s slipper orchid, Cypripe-
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Yeung EC, Chan CKW (2015) The glycol methacrylate embedding resins Publisher’s Note
Technovit 7100 and 8100. Plant microtechniques and protocols. Springer Springer Nature remains neutral with regard to jurisdictional claims in pub‑
International Publishing, New York, pp 67–82 lished maps and institutional affiliations.
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