Plant Biotechnology Reports (2023) 17:369–378 Online ISSN 1863-5474

https://doi.org/10.1007/s11816-023-00838-5 Print ISSN 1863-5466

ORIGINAL ARTICLE

In vitro rapid propagation technology system of Dendrobium

moniliforme (L.) Sw., a threatened orchid species in China
Xu Liu1 · Liyong Sun1 · Tangjie Nie1 · Yao Chen1 · Zengfang Yin1

Received: 20 August 2022 / Revised: 26 March 2023 / Accepted: 25 April 2023 / Published online: 3 May 2023
© Korean Society for Plant Biotechnology 2023

Abstract
Dendrobium moniliforme is a threatened and medicinal orchid species in China. The establishment of in vitro rapid propa-
gation technology system will facilitate preservation and utilization of germplasm resources in D. moniliforme. In this
study, the effects of different plant growth regulators (PGRs) on seed germination, proliferation of cluster shoots, rooting
of shoots were tested under asymbiotic culture condition, and the best acclimatization condition of plantlets was screened.
Our research results were shown as follows: germination rate (GR) was significantly increased by adding N6-benzyladenine
(BA, 0.1 mg/L) during asymbiotic culture. In Murashige and Skoog (MS) medium supplemented with α-naphthaleneacetic
acid (NAA, 1.0 mg/L) and BA (1.0 mg/L), the proliferation coefficient of cluster shoots was 6.1 times comparing with the
control group. The MS medium with NAA (0.5 mg/L) and indole-3-butyric acid (IBA, 0.5 mg/L) promoted the highest
rooting rate (RR). For plantlet transplanting, the equal mixing volume ratio of pine bark, turfy soil, and peanut shells was
the best substrate. In a word, this rapid propagation system provides strong technical support for D. moniliforme to expand
proliferation and germplasm resource protection.

Keywords Dendrobium moniliforme · Seed germination · Rapid propagation technology system · Plant growth regulators ·
Asymbiotic culture

Introduction reduces habitat pressure (Kang et al. 2020); (Arcidiacono

et al. 2021).
Orchidaceae is one of the largest families of angiosperms, as Dendrobium moniliforme is a perennial epiphytic herb
well as one of the most threatened taxa in the world (Wraith and one of the most widely distributed species in the genus
and Pickering 2018). With low natural reproduction rates Dendrobium of the Orchidaceae family, mainly in East Asia,
and over exploitation to satisfy various purposes, the endan- including China, Japan, Korea, and Nepal (Ye et al. 2017);
gered status of many orchids remains unimproved (Pujasa- (Meng et al. 2019); (Shah et al. 2019). It has important eco-
tria et al. 2020). Since in situ conservation and manage- nomic values such as wide applications in ornamental horti-
ment of rare populations alone are not sufficient to ensure culture, modern medicine, and cosmetic industries (Lee et al.
species survival, relocation and proliferation are the key to 2012a; Lee et al. 2012b); (Baek et al. 2016). Due to over-
conserving their germplasm resources (Seaton et al. 2010); exploitation, habitat destruction and shrinkage, the distribu-
(Arcidiacono et al. 2021). The natural propagation of orchids tion of D. moniliforme in China is threatened with extinction.
often hindered by low seed germination and slow growth, Therefore, it is listed in List of National Key Protected Wild
therefore, in vitro tissue culture is a reliable conservation Plants (new edition) in China and Appendix II of the CITES
strategy that not only provides commercial material but also list. The conservation of D. moniliforme has attracted a lot of
attention from scholars. (Lo et al. 2008) screened the suitable
basic medium for D. moniliforme seed germination and tested
the effect of sucrose concentration and organic additions on
* Zengfang Yin
zfyin@njfu.edu.cn plant growth. There are also research data to confirm activated
charcoal (AC) and sucrose can enhance shoot proliferation
1
Co‑Innovation Center for Sustainable Forestry in Southern and rooting (Bae et al. 2014). Using the mixture of Hyponex,
China, College of Biology and the Environment, Nanjing peptone, sucrose and charcoal, (Kim et al. 2016) cultivated
Forestry University, Nanjing 210037, China

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370 Plant Biotechnology Reports (2023) 17:369–378

plantlets under asymbiotic culture condition, and eventually times with running water. In a super-clean bench, the capsules
succeeded in restoring the natural planting status of D. mon- were sterilized with 75% alcohol for 3 min followed by three
iliforme. For the effect of plant growth regulators (PGRs) on times rinse in sterile water. Then, the capsules were transferred
propagation, only protocorm-like body (PLB) formation was into 2% NaOCl for 10 min followed by five times rinse in
involved (Bae et al. 2014). PGRs have been widely used in sterile water. Under aseptic conditions, one end of a capsule
rapid propagation techniques for orchids and other ornamental was cut by aseptic scalpel. And then the seeds were poured
species (Rademacher 2015); (Kumar et al. 2022). However, into a 2 mL centrifuge tube and made a suspension with 2 mL
the effect of PGRs on seed germination of D. moniliforme and sterile water followed by tenfold dilutions. Using a sterilized
their effects on plant growth and development has not been pipette, 10 μL of the seed suspension as a sample drop was
reported. Using asymbiotic culture condition, our group have transferred onto the medium surface. There were 20 drops in
already observed the post-embryonic development and seed- per petri dish. The cultures were carried on a growth chamber
ling morphogenesis (Liu et al. 2022). In this study, we focus with a light intensity of 20 μE, 16 h light/8 h dark under long
on the effects of different PGRs on seed germination, cluster daylight conditions and a controlled temperature of 26 ± 1 °C.
shoots induction, rooting, and screen the best acclimatization
condition of plantlets. This study successfully established a Screening of PGRs components on promoting seed
technical system for the rapid propagation of D. moniliforme, germination
which is not only beneficial to the conservation of germplasm
resources, but also has great application value in the produc- Four concentration gradients (0.1 mg/L, 0.5 mg/L, 1.0 mg/L,
tion practice of D. moniliforme. 2.0 mg/L) were set with gibberellic acid (­ GA3), indole-3-acetic
acid (IAA) and N6-benzyladenine (BA) respectively, while
Materials and methods no PGR as control. The culture medium was Murashige and
Skoog (MS), which contained 3% sucrose and 0.7% agar (pH
Collection of plant material and seed viability 5.8). According to the method mentioned above, seeds were
determination sown with 10 drops per treatment, each drop contained at least
10 seeds, and three times replicated. The seeds were observed
D. moniliforme capsules were harvested in six months after with a stereomicroscope on 20 days after culture (DAC), while
pollination from Huoshan County, Anhui Province, China. The germination rate (GR) of seeds was calculated. Also, the num-
indehiscent mature fruits were yellowish green. Then, they ber of days with > 50% of germination seeds was recorded. The
were kept in the refrigerator at 4 °C for the next experiment. formula for calculating GR is as follows:
The viability of D. moniliforme seeds was evaluated using
Number of germinated seeds
improved Vujanovic’s method of 2,3,5-triphenyltetrazolium Germination rate(%) = × 100%
chloride (TTC) test (Vujanovic et al. 2000) before asymbiotic Total number of seeds × SV
culture. Seeds were taken from three capsules randomly, then
stained with 1% (w/v) TTC in phosphate-buffered saline (PBS, Selection of the optimum combination of different
pH 6.5) at 30 ± 1 °C for 30 h in dark condition. After rinsed PGRs for proliferation of cluster shoots
three times with sterile water, the seeds were transferred to
concave slides and observed under a stereomicroscope (Olym- Plantlets of about 1 cm in height and 2–3 leaves were
pus SX7, Tokyo, Japan). The experiment was repeated three selected for this test. MS was used as the basic medium
times. The number of red seed embryos and the total number and no PGR was added as control. Then the treat meant
of seed embryos in each field of view were counted, and then was carried out with four gradients of 0.1 mg/L, 0.5 mg/L,
the seed viability (SV) was calculated. The formula for calcu- 1.0 mg/L and 2.0 mg/L for α-naphthaleneacetic acid
lating SV is as follows: (NAA) and BA, respectively, with 20 samples per group
Number of red seed embryos and three replications. On 40 DAC, plant height and num-
Seed viability(%) = × 100% ber of shoots were measured. The proliferation coefficient
Total number of seed embryos
of cluster shoots on 40 DAC were calculated based on the
new shoots number divided by the plantlet number at the
Surface disinfection of capsules and culture start of culture. Three times repeated.
conditions
Number of new shoots
Proliferation coefficient of cluster shoots =
Total number of plantlets
Surface sterilization of capsules and inoculation of the seeds
have been done using improved Tan’s method (Tan et al. 2014).
The capsules were washed with detergent and then rinsed three

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Screening of the optimum combination of PGRs Seeds were taken from the mature capsules (Fig. 1a). Before
for rooting TTC staining, the color of seeds was almost pale yellow
(Fig. 1a1), and the immature embryo was ellipsoidal sur-
The cluster shoots of approximately 3–4 cm in height were rounded by membranous seed coat (Fig. 1c). After 30 h,
selected for rooting medium screening. Two PGRs, NAA some seeds stained red, it meant the embryo has viability.
and indole-3-butyric acid (IBA), were mixed in MS medium Normally, 57.06% of the seeds in capsules were viable
containing 2% carbon powder, and also were set with four (Fig. 1b). Under in vitro culture condition, the viable seed
concentration gradients (0.5 mg/L, 1.0 mg/L, 1.5 mg/L, size enhanced gradually, the embryo grew into sphere, and
2.0 mg/L). After 60 d of incubation, the length, number, then the seed coat broke (Fig. 1d, e).
and rooting rate (RR) were measured. Three times repeated.
The formula for calculating RR is as follows:
The effect of PGRs on seed germination
Number of rooting plantlets
Rooting rate (%) = × 100%
Total number of plantlets Using ­GA3, IAA and BA, we investigated the effect of PGRs
on seed germination. The results are shown in Table 1. ­GA3
and IAA had poor promoting effects, and GR of seeds was
Acclimatization and transplant
lower than that of the control group. On the contrary, the
promoting effect of BA was the best one. When BA concen-
After 7 d of acclimatization under natural conditions,
tration was 0.1 mg/L, GR of D. moniliforme was 99.39%
in vitro plantlets about 5 cm in height with healthy roots
on 7 DAC. However, GR showed a decreasing trend with
were washed with tap water to remove the rooting medium,
the increase of BA concentration. Figure 2 shows the sta-
then they were transplanted into plastic boxes with 6 cells in
tus of seed germination under different concentration of
each one at a controlled temperature of 26 ± 1 °C. The boxes
BA treatment. Under the condition of BA 0.1 mg/L, the GR
were covered with clear plastic lids to prevent drying in the
was the highest one, also the spherical embryos in the seed
early stages of acclimatization, and the lids were gradu-
were yellow-green and were almost equal in size, indicating
ally opened each week until they were completely removed
relatively synchronous development (Fig. 2a). The 0.5 mg/L
after 4 weeks. From the fifth week onward, the boxes were
BA treatment was similar with the 0.1 mg/L BA, but the
watered every two days. The transplanting substrates were
growth status of embryo was less synchronous (Fig. 2b).
selected from pine bark, turfy soil and peanut shells and
Also 1.0 mg/L BA treatment was similar with 2.0 mg/L BA,
mixed in varied ratios and divided into 7 groups as follows:
the spherical embryos were vitrified and did not develop
(a) 1:0:0, (b) 0:1:0, (c) 0:0:1, (d) 1:1:0, (e) 0:1:1, (f) 1:0:1,
synchronously (Fig. 2c, d). In addition, with GR > 50% as
(g) 1:1:1. Two plantlets were planted in each cell and 24
node, the culture time was recorded one by one except the
plantlets were planted in each group with three replications.
treatment which was never reached 50%. It is obviously that
The survival rate, number of new shoots and height of plant-
the shortest germination time is 7 days, and GR is also the
lets were counted after 6 weeks.
highest one accordingly (Table 1).
Number of surviving plantlets
Survival rate (%) = × 100%
Number of transplanted plantlets
Effect of PGRs on the proliferation of cluster shoots
Statistics The results of the proliferation of cluster shoots under dif-
ferent combination of NAA and BA are shown in Fig. 3. As
The data were analyzed for significant differences. One-way a general rule, it is not conducive to the growth of cluster
analysis of variance (ANOVA), Duncan's test, and homoge- shoots that the BA and NAA are too much lower or higher
neity of variance tests were analyzed with IBM SPSS Sta- in D. moniliforme. Figure 3a is showing the development
tistics 21 software. Means were separated using Duncan’s status of cluster shoots. The number of shoots in 0.5 mg/L
multiple range test (DMRT) at p < 0.05. BA + 2.0 mg/L NAA culture condition was the worst one
comparing with the control group and the other treatments
Results (Fig. 3a-c). When the concentration of NAA was 1.0 mg/L,
the number of cluster shoots increased with the increase of
Seed viability and germination BA concentration and then decreased, the maximum prolif-
eration coefficient was 6.1 when BA was 1.0 mg/L (Fig. 3b).
Seed viability test has been done because it will be avoided Simultaneously, the growth of cluster shoots was the best
effect the statistic results of the following seed germination. one (Fig. 3a), and the length of shoots could reach 2.205 cm

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Fig. 1  Seed morphology, viability and germination. a The mature Expanded seed embryo not separated from seed coat. e Spherical
capsule of D. moniliforme. a1 The seed in mature capsule. b Via- embryo breaking through seed coat after germination. Scale bar: a 1
ble seeds, showing in red color. c Seed morphology on 0 DAC. d cm, a1 500 μm, b 200 μm, c-e 100 μm

Table 1  Effect of PGRs on seed germination Effect of PGRs on rooting

PGRs (mg/L) GR (%) Time of ger-
mination (d, The effects of the PGRs on rooting ratios (RR) and the plant-
GA3 IAA BA GR > 50%) let growth status are shown in Fig. 4. Overall, appropriate
0 0 0 73.37 ± 0.86b 9
auxin and cytokinin ratio is beneficial to cluster shoots root-
0.1 57.26 ± 0.95b 14
ing in D. moniliforme. The highest RR could be achieved
0.5 67.80 ± 1.21b 10
93.33% when NAA was 0.5 mg/L and IBA was 0.5 mg/L,
1.0 52.22 ± 0.71d 18
likewise, the number of roots was higher with an average of
2.0 45.68 ± 0.43d /
5.9 (Fig. 4b–d). In particular, the longest roots were induced
0.1 38.33 ± 0.96d /
on the medium with NAA 1.5 mg/L and IBA 0.5 mg/L, with
0.5 61.42 ± 1.36b 13
a length of 1.692 cm (Fig. 4c), but the RR was only 46.67%
1.0 53.40 ± 1.48c 17
(Fig. 4d), and the number of roots was also lower with an
2.0 45.45 ± 0.69d /
average of 3.7 (Fig. 4b). Therefore, NAA at 0.5 mg/L plus
0.1 99.39 ± 0.19a 7
IBA at 0.5 mg/L was the best combination for the plantlet
0.5 95.25 ± 0.64b 8
rooting of D. moniliforme. Accordingly, the plantlet growth
1.0 66.98 ± 1.40b 12
was the best one comparing with other culture conditions
2.0 59.12 ± 0.50b 13
(Fig. 4a).

Data indicate the mean ± SD of three replicates and means with the The effect on acclimatization and transplanting
same letter are not significantly different (p < 0.05) according to
DMRT
The transplanting conditions and the plantlets growth sta-
tus of D. moniliforme are shown in Fig. 5. There was no
(Fig. 3c). As a whole, the optimal concentration of PGRs is doubt that the single pine bark and peanut shells were not
BA 1.0 mg/L + NAA 1.0 mg/L on the proliferation of cluster a suitable substrate for the growth of plantlets comparing
shoots. with turfy soil (Fig. 5a–c, h). Although the survival rate was

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Fig. 2  Effects of BA on seed germination. a BA 0.1 mg/L, b 0.5 mg/L, c 1.0 mg/L, d 2.0 mg/L. Scale bar: 200 μm

Fig. 3  Effects of PGRs on cluster shoots proliferation. a The cluster of shoots length. Note: The bars represent the standard error, and the
shoots morphology under different PGRs combination, Scale bar: different letters on the top of the bars represent significant differences
2 cm. b The difference of proliferation coefficient. c The difference in individual traits analyzed by DMRT at p < 0.05

higher in the single substrate of turfy soil, the number of new soil was less than that of peanut shells and turf soil substrate
shoots was lower, and the plants grew poorly (Fig. 5b). It (Fig. 5d, e, h). Although the survival rate was slightly lower,
was worth noting that the survival rate of the plantlets could the plantlet was higher when it was planted in a mixture of
reach more than 90% in all combinations containing turfy pine bark and peanut shells, but the new shoot number was
soil, and the new shoot numbers and plantlet heights were a little bit lower (Fig. 5f, h). In particular, plantlets trans-
much better (Fig. 5h). The new shoot number of plantlet planted in a substrate of peanut shells: turfy soil: pine bark
which was planted in the mix substrate of pine bark and turf (1:1:1) had the highest survival rate of 93.06%, and their

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Fig. 4  Effects of PGRs on rooting. a The rooting plantlet morphology ence of RR. Note: The bars represent the standard error, and the dif-
under different PGRs combination, Scale bar: 2 cm. b The difference ferent letters on the top of the bars represent significant differences in
of root number. c The difference of average root length. d The differ- individual traits analyzed by DMRT at p < 0.05

plant heights was also the highest at 5.629 cm, with a higher Discussion
number of new shoots (Fig. 5g, h). Therefore, the transplant-
ing substrate with a mixture of pine bark, turfy soil and pea- Under asymbiotic culture, appropriate BA concentration has
nut shells were more favorable for the transplanting culture been shown to promote seed germination in orchids. It was
of D. moniliforme. suggested that the addition of BA in the medium could accel-
erate GR of Calanthe tricarinata (Godo et al. 2010). (Pierce

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Fig. 5  Effects of different substrate composition on acclimatization 1:1:1. h The relative data of plantlet growth. Note: The different let-
and transplanting. a-g The different plantlet growth status, growing ters on the top of the error bars represent significant differences in
in the composition of mix substrates which is pine bark: turfy soil: individual traits analyzed by DMRT at p < 0.05. Scale bar: 2 cm
peanut shells, i.e. a 1:0:0, b 0:1:0, c 0:0:1, d 1:1:0, e 0:1:1, f 1:0:1, g

and Cerabolini 2011) found that BA, ­GA3, kinetin and thidi- Phaleonopsis hybrid ‘Little gem’ (Chung et al. 2016). Pres-
azuron (TDZ) all significantly increased GR of immature ently study has confirmed that BA played an important role
seeds in Pseudorchis albida, among which BA was the most during proliferation of protocorms or in vitro multiplication,
effective (50.5%). The same result was confirmed in the seed thus resulted in maximum number of shoots, root length
germination in Cypripedium candidum seeds (Pauw et al. on MS medium with 1.0 mg/L BA in Cymbidium aloifo-
1995) and Calanthe tricarinata (Godo et al. 2010). As we lium (Kumar et al. 2022). Similarly, the highest number of
all known, cytokinin is involved in maintaining meristematic cluster shoots was produced when the MS medium contains
tissue function (Kurakawa et al. 2007). Meanwhile, it is also 15 μM BA and 15 μM NAA in D. longicornu (Dohling et al.
known from morphological and anatomical analyses that the 2012). In this study, the highest number of cluster shoots
embryo is undifferentiated in orchid seeds. The embryo has and the most increased shoot length of D. moniliforme was
not yet established axial tissues when a seed has germinated recorded when MS medium contained 1.0 mg/L BA and
(Liu et al. 2022). Therefore, the immature embryo needs 1.0 mg/L NAA. It’s worth noting that the induction rate by
cell division and differentiation by increasing its size rather meta-topolin (mT) in combination with NAA was superior
than radicle elongation to break out of the seed coat (Zhang to that of by BA in combination with NAA in the induction
et al. 2015); (Kunakhonnuruk et al. 2018). These findings of cluster shoots in D. nobile (Bhattacharyya et al. 2016).
may be able to explain the reason why BA promotes the In recent years, a suggestion has been made to utilize mT
seed germination of orchids. Our research work confirmed instead of BA for greater efficiency (Saeiahagh et al. 2019).
this view by showing that the highest GR (99.39%) and the Although mT improves the adaptability and high genetic
shortest germination time (7 DAC) of D. moniliforme seeds fidelity of plantlets (Lata et al. 2016), and BA has the poten-
in the medium supplemented with 0.1 mg/L BA. tial to induce variation in vitro clones (Biswas et al. 2009),
PGRs significantly influence the induction of cluster undeniably, BA is still widely used in the tissue culture of
shoots, and the selection of suitable PGRs is particularly orchids (Chung et al. 2016); (Kumar et al. 2022). Further-
crucial in the culture process. The main goal of plant tissue more, considering of the cost it should be spent, BA is more
culture is to produce the maximum number of cluster shoots readily available and affordable in the rapid propagation
during clonal propagation (Bhattacharyya et al. 2016). Previ- technology systems which are utilized widely (Ivanova and
ous studies proved that the regeneration and proliferation of Staden 2008).
cluster shoots are closely related to the type and concentra- Two effect factors of auxin are noticed mostly during
tion of cytokinin (Amoo et al. 2014). Actually, adding BA cluster shoot rooting, i.e., IBA and NAA. IBA has been
into MS medium not only induced more shoots, but also, widely utilized in the rooting culture of orchids (Bhattacha-
they were healthier than those one induced by other PGRs in ryya et al. 2016). In Bulbophyllum odoratissimum, different

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types of auxins reflected a wide range of variations in its Acknowledgements The authors would like to thank Deputy General
rooting process. The highest number of roots per explant was Manager Daxue Sun from Chinese traditional Chinese medicine Dend-
robium Huoshanense Science And Technology co., LTD for providing
found in 0.5 mg/L IBA with better root length after 8 weeks seed materials for this study.
of inoculation comparing with NAA enriched medium
(Prasad et al. 2021). Similarly, (Bhattacharyya et al. 2018) Author contribution XL and LS were responsible for the design of the
found that using IBA alone in D. aphyllum could induce experiment, XL and TN were responsible for the implementation of the
experiment, YC was responsible for the statistics of the data, and XL
higher RR and longer roots than that of NAA. In particu- and ZY were responsible for the writing of the manuscript.
lar, the effects of IBA better than other auxin in promoting
orchid rooting have been discussed adequately (Mahendran Funding (1) Investigation, Collection and Monitoring of Wild Agri-
and Bai 2009); (Mohanty et al. 2012). In contrast to the cultural Plant Resources (Wild Aquatic Plants, Wild Soybeans, etc.)
in Typical Regions (13200355); (2) The Priority Academic Program
above findings, we found that the highest rooting percent- Development of Jiangsu Higher Education Institutions (PAPD).
age (93.33%) and the number of roots (5.9) were achieved
when the IBA concentration was 0.5 mg/L, and the NAA Declarations
was 0.5 mg/L. It meant that the proper combination of NAA
and IBA were necessary for getting the maximum RR and Conflict of interest The authors declare that they have no conflict of
interest.
the highest number of roots during the rooting culture of D.
moniliforme.
In orchid species cultivation, the substrate is essential
since it influences the quality of the final plantlet, mainly
performing the supportive function of the root system (Faria
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