SAJB-01748; No of Pages 6

South African Journal of Botany xxx (2017) xxx–xxx

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South African Journal of Botany

journal homepage: www.elsevier.com/locate/sajb

Effect of germination media on in vitro symbiotic seed germination of

three Dendrobium orchids
B. Mala a,b, K. Kuegkong a, N. Sa-ngiaemsri a, S. Nontachaiyapoom a,⁎
a
School of Science, Mah Fah Luang University, 333 Moo 1, Thasud, Muang District, Chiang Rai 57100, Thailand
b
Queen Sirikit Botanical Garden, The Botanical Garden Organization, PO Box 7, Mae Rim District, Chiang Mai 50180, Thailand

a r t i c l e i n f o a b s t r a c t

Article history: In vitro symbiotic seed germination has become increasingly popular for orchid propagation. With very few
Received 16 August 2016 exceptions, in vitro symbiotic seed germination methods use oat meal agar (OMA) as the medium. However,
Received in revised form 25 April 2017 effects of oat concentrations on symbiotic germination of orchid seeds have not been investigated. In our first
Accepted 11 May 2017
experiment, effectiveness of three concentrations of ground oat (1, 5, or 10 g/l) in OMA inoculated with an orchid
Available online xxxx
mycorrhizal fungus (Tulasnella deliquescens (Juel) Juel), uninoculated OMA (10 g/l of oat) and one fifth Murashige
Edited by J Van Staden and Skoog medium containing 6 g/l of sucrose (1/5MS) on promoting seed germination of Dendrobium lindleyi
Steud. were compared. Germination and protocorm development of D. lindleyi were found to be enhanced by
Keywords: higher oat concentrations. The OMA containing 10 g/l of oat with mycorrhizal inoculation also outperformed
Dendrobium 1/5MS. In our second and third experiments, the use of common orchid cultivation substrates as alternative
Oat meal agar media for in vitro symbiotic orchid seed germination was investigated. For Dendrobium fimbriatum Hook., we
Orchid mycorrhiza demonstrated that sphagnum peat moss inoculated with T. deliquescens either isolate Da-KP-0-1 or Pv-PC-1-1
Symbiotic seed germination in plastic containers could be used to germinate the orchid to advanced seedling stage, although the germination
Tulasnella
percentages were low, probably due to the waterlogged characteristic of the peat moss and low ventilation of the
plastic boxes. For Dendrobium findlayanum C.S.P. Parish & Rchb.f., we used the mixture of peat moss and coir dust
packed in Petri dishes to solve the mentioned problems and compared the effectiveness of this system to OMA
and 1/5MS. The three treatments resulted in similar germination percentages but the symbiotic methods were
more effective than 1/5MS in promoting protocorm development of D. findlayanum. The findings of this study
can be used to improve symbiotic germination media and they encourage the use of symbiotic germination
method as mean for orchid seed propagation.
© 2017 SAAB. Published by Elsevier B.V. All rights reserved.

1. Introduction 2009). Although the asymbiotic germination method has been used suc-
cessfully for many orchid species, symbiotic germination especially
Seed germination is an essential step in orchid conservation and in vitro methods are gaining popularity because they promoted higher
commercial orchid production. In conservation programs, seed propa- germination rates and/or symbiotic protocorms could develop more
gated orchids are used for the plant reintroduction (Stewart and Kane rapidly than asymbiotic protocorms in many studies (e.g., Rasmussen
2006; Paul et al. 2012). In commercial orchid production, germination et al. 1990; Johnson et al. 2007; Øien et al. 2008; Nontachaiyapoom
methods are used to propagate tissue culture-recalcitrant species et al. 2011; Nikabadi et al. 2014). Moreover, the in vitro-grown symbiotic
(Zeng et al. 2014, 2016) and to propagate hybrids after cross- seedlings are likely to be more adaptable to ex vitro conditions than
pollination. As orchid seeds lack endosperms, they contain either small asymbiotic seedlings since orchid mycorrhizal fungi have been reported
levels of food reserves or the forms of food reserves that can hardly be to facilitate the uptake of water and nutrients (Yoder et al. 2000; Smith
metabolized by orchid embryos (Yam and Arditti 2009). In nature orchid and Read 2008), promote photosynthetic performance and increase
seeds depend on orchid mycorrhizal fungi to provide necessary nutrients radiation-use efficiency (Lee et al. 2014). In vitro symbiotic seed germi-
for germination, a process called symbiotic seed germination (Yam et al. nation methods generally use oat meal agar (OMA) with various concen-
2009). Asymbiotic seed germination method, invented by Lewis trations of oat as the culture media, i.e., 2.5 g/l (Øien et al. 2008; Tan et al.
Knudson, requires the addition of soluble sugar which serves as the car- 2014), 3.0 g/l (Zettler et al. 2007), 4.0 g/l (Zhou and Gao 2016), and 10 g/l
bohydrate source for orchid embryos (Knudson 1922; Yam and Arditti (Nontachaiyapoom et al. 2011). Surprisingly, despite its popularity,
no study has specifically investigated effects of oat concentrations
⁎ Corresponding author.
on the performance of symbiotic seed germination of orchids. The first
E-mail addresses: [email protected], [email protected] experiment of this study was aimed to investigate the effects of oat
(S. Nontachaiyapoom). concentrations (i.e., 1, 5, and 10 g/l) in OMA on seed germination and

http://dx.doi.org/10.1016/j.sajb.2017.05.008
0254-6299/© 2017 SAAB. Published by Elsevier B.V. All rights reserved.

Please cite this article as: Mala, B., et al., Effect of germination media on in vitro symbiotic seed germination of three Dendrobium orchids, South
African Journal of Botany (2017), http://dx.doi.org/10.1016/j.sajb.2017.05.008
2 B. Mala et al. / South African Journal of Botany xxx (2017) xxx–xxx

protocorm development of Dendrobium lindleyi Steud., a popular orna- drops of Tween-20 for 10 min and subsequently rinsed in sterilized
mental orchid. water three times. Approximately equal amount of seeds were then
Agar is known to affect growth and development of in vitro plants sprinkled onto the media using a spatula. Mycorrhizal inoculation was
such as causing hyperhydricity (Aggarwal and Nirmala 2012), and done by aseptically placing a 1.2 × 1.2 cm2 block of PDA containing fun-
plantlets from agar-based media require an extra step of washing to re- gal hyphae of the mycorrhizal isolate Da-KP-0-1 at the center of OMA.
move the agar from the roots. Moreover, the price of OMA is increasing For OMA without mycorrhizal inoculation, a 1.2 × 1.2 cm2 block of PDA
dramatically because the global shortage of Gelidium seaweed has was placed at the center of the medium. All Petri dishes were then
pushed up the price of agar (Callaway 2015). Therefore, alternative ger- kept in the dark at 25 °C for 1 week, and then in a 16-h-light/8-h-dark
mination medium for in vitro symbiotic seed germination is desirable. cycle at 25 °C for 11 weeks. During the 16-h light, irradiation with an
Previously, Aewsakul et al. (2013) reported the use of common orchid intensity of 25–32 μmol m− 2 s−1 was provided by 36 W Cool white
cultivation media (i.e., soil, coir dust and sphagnum peat moss) as fluorescent lamps (Philips, Bangkok, Thailand). Seed germination
the germination media for ex vitro symbiotic seed germination of and protocorm developmental stages (i.e., Stage 0, no germination;
Spathoglottis plicata Blume. In the second and third experiments of Stage 1, seed coat ruptured by enlarged embryo; Stage 2, globular
this study, common cultivation substrates reported to be suitable embryo and rhizoids present; Stage 3, appearance of protomeristem;
for ex vitro symbiotic seed germination (Aewsakul et al. 2013) were Stage 4, emergence of first leaf; Stage 5, elongation of first leaf and
tested for in vitro symbiotic seed germination of two Dendrobium further development) were examined weekly. Photos of seeds and
orchids. At generic level, Dendrobium is among the most heavily traded protocorms were acquired at 12 weeks after sowing using a stereomi-
genera and the most important cut-flower orchids in the global trade croscope (Zeiss-Stemi 2000-C, Zeiss, Oberkochen, Germany) attached
(Swangmaneecharern et al. 2012). Moreover, many Dendrobium species with a digital camera (Canon Powershot G9, Tokyo, Japan), 10 photos
have been used in traditional medicines and/or reported to contain at randomly chosen areas were taken for each replicate of each treat-
phytochemicals that have pharmaceutical activities (Hossain 2011; ment. Percentage of seeds or protocorms at each developmental stage
Bhattacharyya et al. 2016a, 2016b). was calculated by dividing the number of seeds or protocorms at each
stage by the total number of seeds and protocorms. Comparisons of per-
2. Materials and methods centages of germination and percentages of seeds/protocorms at each
developmental stage among the treatments were done using analysis
2.1. Orchid seeds of variance (ANOVA) and Tukey's HSD Test with P = 0.05. Randomly
sampled protocorms from all treatments were examined microscopi-
Flowers of D. lindleyi plants grown in a private orchid nursery at cally to verify the presence or absence of pelotons.
Muang District, Chiang Rai were hand-pollinated in March 2014. Four
undehisced mature capsules were harvested on 12 October 2014 and 2.3.2. Symbiotic seed germination of D. fimbriatum
seeds were removed from the capsules on 17 October 2014. All seeds The seed germination experiment of D. fimbriatum was conducted in
were kept in silica-gel desiccated tubes at 4 °C until use. Seeds a completely randomized design with three treatments, i.e., peat moss
of D. fimbriatum were removed from twenty naturally pollinated without fungal inoculation, peat moss inoculated with either one
mature capsules using aseptic technique in April 2013, and seeds of of the two isolates (i.e., Da-KP-0-1 and Pv-PC-1-1) of T. deliquescens.
D. findlayanum were removed from two naturally pollinated mature Preparation of germination substrate was done by soaking the sphag-
dehisced capsules on 14 June 2013. num peat moss (Pindstrup Mosebrug A. S., Ryomgaard, Denmark) in
tap water overnight three times, packed into 473-ml clear round plastic
2.2. Orchid mycorrhiza fungi containers with thickness of about 1.5 cm and subsequently sterilized
by autoclaving for 2 h. Peat moss was selected as germination substrate
Two isolates of Tulasnella deliquescens (Juel) Juel (previously known in this experiment because Aewsakul et al. (2013) showed that peat
as Epulorhiza repens (N. Bernard) R.T. Moore) isolate Da-KP-0-1 and Pv- moss resulted in better germination and protocorm development com-
PC-1-1 (Nontachaiyapoom et al. 2010) were used in this study. Fungal pared to soil and coir dust. Fungal inoculation on peat moss was done by
isolate Da-KP-0-1 was obtained from a root of Dendrobium anosmum aseptically placing a 1 × 1 cm2 block of PDA containing fungal hyphae
Lind. and fungal isolate Pv-PC-1-1 was obtained from a root of in the center of the germination medium. The containers were then
Paphiopedilum villosum (Lindl.) Stein. They were reported to be highly kept at 30 °C in dark for one week and subsequently the agar blocks
effective in seed germination promotion of many Dendrobium orchids were removed. Seeds were mixed to ensure the homogeneity using
(Nontachaiyapoom et al. 2011; Swangmaneecharern et al. 2012). Agar scalpel knife, then approximately equal amount of seeds were sprin-
pieces containing hyphae of this fungus, stored in sterile water at kled onto peat moss. After seed sowing, the edges of the germina-
room temperature at Mae Fah Luang University, Chiang Rai, were used tion containers were sealed with thin polyvinylchloride cling film
for the initial fungal culture. The agar pieces were placed on potato (Aro, Bangkok, Thailand), kept in the dark at 25 °C for 2 weeks, and
dextrose agar (PDA) containing antibiotics (30 μg/ml oxytetracycline, then in a 16-h-light/8-h-dark cycle at 25 °C for 15 weeks. During the
30 μg/ml streptomycin, and 30 μg/ml ampicillin) at 30 °C for one week. 16-h light, irradiation with an intensity of 25–32 μmol m−2 s− 1 was
provided by 36 W Cool white fluorescent lamps (Philips, Bangkok,
2.3. Seed germination experiments Thailand). Seed germination and protocorm developmental stages
were examined weekly. Photos for assessment of seeds and protocorms
2.3.1. Effects of oat concentrations on symbiotic seed germination of in different stages were performed as described above at 17 weeks after
D. lindleyi seed sowing. Randomly sampled protocorms from all treatments were
The seed germination experiment of D. lindleyi was conducted in a examined microscopically to verify the presence or absence of pelotons.
completely randomized design with five treatments and seven repli- Comparison of germination percentages among the treatments was
cates, i.e., one fifth Murashige and Skoog medium (Murashige and done using ANOVA and Tukey's HSD Test with P = 0.05.
Skoog 1962) containing 6 g/l of sucrose (1/5MS), OMA with 10 g/l
of ground oat without mycorrhizal inoculation, OMA with one of the 2.3.3. Symbiotic seed germination of D. findlayanum
three concentrations of ground oat (1, 5, or 10 g/l) with mycorrhizal The seed germination experiment of D. findlayanum was conducted
inoculation. All media were gelled with 0.8% agar (Criterion™ C5001, in a completely randomized design with three treatments, i.e., 1/5MS
Hardy Diagnostics, Santa Maria, CA, U.S.A.). Seeds were surface- containing 1.5% sucrose and 0.8% agar, OMA (10 g/l of oat, 8 g/l of
sterilized in 50 ml of 1% sodium hypochlorite solution containing two agar) pre-inoculated with T. deliquescens isolate Da-KP-0-1, and the

Please cite this article as: Mala, B., et al., Effect of germination media on in vitro symbiotic seed germination of three Dendrobium orchids, South
African Journal of Botany (2017), http://dx.doi.org/10.1016/j.sajb.2017.05.008
 B. Mala et al. / South African Journal of Botany xxx (2017) xxx–xxx 3

Table 1
Effects of media and mycorrhizal inoculation on the developmental stages of seeds and protocorms of D. lindleyi at 12 weeks after seed sowing.

Treatmenta Percentage of seeds and protocormsb % germinationb

Stage 0 Stage 1 Stage 2 Stage 3 Stage 4 Stage 5

1/5MS 69.9 ± 4.1 b 6.1 ± 1.7 b 13.5 ± 7.1 b 3.0 ± 2.3 ab 5.6 ± 5.0 b 2.0 ± 3.5 a 30.1 ± 4.1 a
10 g/l of oat 71.4 ± 5.6 b 0.9 ± 0.4 a 27.1 ± 5.9 c 0.5 ± 0.3 a 0.0 ± 0.0 a 0.0 ± 0.0 a 28.6 ± 5.6 a
1 g/l of oat + Da 75.8 ± 4.7 b 1.4 ± 0.5 a 9.7 ± 2.9 ab 4.4 ± 1.7 bc 4.6 ± 2.6 ab 4.0 ± 2.1 a 24.2 ± 4.7 a
5 g/l of oat + Da 68.7 ± 4.9 ab 1.1 ± 0.4 a 5.6 ± 1.8 a 6.1 ± 1.6 c 5.3 ± 1.8 ab 13.2 ± 4.6 a 31.3 ± 4.9 ab
10 g/l of oat + Da 54.7 ± 16.2 a 0.6 ± 0.7 a 3.3 ± 3.2 a 4.0 ± 2.2 bc 5.4 ± 4.0 ab 32.0 ± 17.4 b 45.3 ± 16.2 b
a
Da represents fungal isolate Da-KP-0-1.
b
Values are means ± standard deviations of seven replicates. Means in the same column marked with different letters are significantly different (P b 0.05).

mixture of peat moss and coir dust pre-inoculated with T. deliquescens rinsed with sterilized water three times. Seeds were dried on the nylon
isolate Da-KP-0-1. The mixture of peat moss and coir dust was prepared net with 33-μm mesh and then a spatula was used to sprinkle seeds
by soaking the sphagnum peat moss and coir dust (1:1 by volume) onto the media. Petri dishes were sealed with the cling film and kept in
in tap water overnight three times and then dried in a hot air oven at the dark at 25 °C for one week and then in a 16-h-light/8-h-dark cycle
80 °C for 45 min. Subsequently, the substrate was packed into Petri at 25 °C for 9 weeks. Irradiation condition was set as described above.
dishes (100 mm × 20 mm) and then sterilized by autoclaving for Seed germination and protocorm developmental stages were exam-
15 min. Fungal inoculation was done by aseptically placing a 1 × 1 cm2 ined weekly. Photos for assessment of seeds and protocorms in differ-
block of PDA containing fungal hyphae in the center of the media. The ent stages were performed as described above at 10 weeks after seed
Petri dishes were then kept at 30 °C in dark for one week. Seeds of sowing. Randomly sampled protocorms from all treatments were ex-
D. findlayanum were surface-sterilized in 0.5% sodium hypochlorite solu- amined microscopically to verify the presence or absence of pelotons.
tion containing two drops of Tween-20 for 10 min and subsequently Statistical analysis was performed as described above.

Fig. 1. Seeds and protocorms of D. lindleyi at 18 weeks after seed sowing on (a) the five asymbiotic and symbiotic media, (b) 1/5MS, (c) OMA containing 10 g/l of oat without mycorrhizal
inoculation, (d, e, f) OMA containing 1, 5, and 10 g/l of oat inoculated with mycorrhizal inoculation, respectively. Scale bars in b, d, e, and f represent 1 cm and scale bar in c represents
0.5 cm.

Please cite this article as: Mala, B., et al., Effect of germination media on in vitro symbiotic seed germination of three Dendrobium orchids, South
African Journal of Botany (2017), http://dx.doi.org/10.1016/j.sajb.2017.05.008
4 B. Mala et al. / South African Journal of Botany xxx (2017) xxx–xxx

Fig. 2. Hyphae and monilioid cells of T. deliquescens in OMA containing 1 g/l of oat (a), 5 g/l of oat (b), and 10 g/l of oat (c) stained with lactophenol blue solution. Scale bars represent
100 μm.

3. Results and discussion observation revealed denser fungal hyphae and larger number of
monilioid cells of T. deliquescens in OMA containing 10 g/l of oat as com-
3.1. Effects of oat concentrations on symbiotic seed germination pared to OMA containing 1 or 5 g/l of oat (Fig. 2). On the contrary, seeds
of D. lindleyi on OMA containing 10 g/l of oat without mycorrhizal inoculation could
germinate but none of the protocorms developed beyond Stage 3
Seed germination (seeds at Stage 1) of D. lindleyi was observed in all (Table 1 and Fig. 1a, c). Examination of randomly sampled protocorms
treatments as early as 10 days after seed sowing. However, rates of seed confirmed the presence and absence of pelotons in protocorms from
germination and protocorm development in different treatments varied the treatments with and without mycorrhizal inoculation, respectively
(Table 1 and Fig. 1). Among the treatments with mycorrhizal inocula- (data not shown). The finding that increase in oat concentration re-
tion, symbiotic germination and protocorm development of D. lindleyi sulted in higher rates of germination and protocorm development
were found to be promoted by higher oat concentration as OMA con- could be used to improve symbiotic germination media since in the pre-
taining 10 g/l of oat resulted in the highest seed germination percentage vious symbiotic germination studies OMA with oat concentrations be-
and the highest percentage of Stage 5 protocorms at 12 weeks after seed tween 2.5 and 4 g/l was generally used (Zettler et al. 2007; Øien et al.
sowing (Table 1). These protocorms could develop to healthy young 2008; Tan et al. 2014; Zhou and Gao 2016). Moreover, in this study sym-
seedlings with leaves and roots 6 weeks later (Fig. 1a, f). Because orchid biotic germination on OMA containing 10 g/l of oat also outperformed
protocorms could not directly translocate/metabolize exogenous starch, 1/5MS (asymbiotic medium) in terms of both germination percentage
the better performance of OMA with higher oat concentration was and percentage of Stage 5 protocorms (Table 1). It is important to
probably due to the better growth and larger quantity of the orchid my- note that the high levels of macronutrients, micronutrients and sucrose
corrhizal fungus that gave rise to more fungus-derived germination- in the 1/5MS did not enhance germination percentage of D. lindleyi
promoting nutrients and/or water and nutrient uptake. Microscopic seeds (30.1%) compared to that of seeds sown on un-inoculated OMA

Fig. 3. A plastic container used in the seed germination of D. fimbriatum (a). Seeds and protocorms of D. findlayanum at 10 weeks after seed sowing on (b) 1/5MS, (c) OMA inoculated with
fungal isolate Da-KP-0-1, and (d) the mixture of peat moss and coir dust inoculated with fungal isolate Da-KP-0-1. Bar in a represents 2 cm and bars in b, c, and d represent 1 cm.

Please cite this article as: Mala, B., et al., Effect of germination media on in vitro symbiotic seed germination of three Dendrobium orchids, South
African Journal of Botany (2017), http://dx.doi.org/10.1016/j.sajb.2017.05.008
 B. Mala et al. / South African Journal of Botany xxx (2017) xxx–xxx 5

Table 2
Effects of media and fungal inoculation on the developmental stages of seeds and protocorms of D. fimbriatum at 17 weeks after seed sowing.

Treatmenta Repb Percentage of seeds/protocormsc % germinationd

Stage 0 Stage 1 Stage 2 Stage 3 Stage 4 Stage 5

PM 3 45.3 ± 5.0 b 52.2 ± 3.4 b 1.4 ± 0.1 a 1.1 ± 1.9 a 0.0 ± 0.0 a 0.0 ± 0.0 a 54.7 ± 5.0 a
PM + Da 5 32.2 ± 6.8 a 28.7 ± 8.8 a 8.6 ± 5.0 b 16.7 ± 6.5 b 4.8 ± 1.5 b 9.1 ± 5.7 b 67.8 ± 6.8 b
PM + Pv 5 34.4 ± 5.1 ab 44.4 ± 4.8 b 5.2 ± 2.8 ab 13.7 ± 6.1 b 1.3 ± 0.9 a 1.0 ± 1.5 a 65.6 ± 5.1 ab
a
PM represents peat moss, Da represents fungal isolate Da-KP-0-1, and Pv represents fungal isolate Pv-PC-1-1.
b
Number of replicates used in the data analysis.
c
Values are means ± standard deviations.
d
Means in the same column marked with different letters are significantly different (P b 0.05).

(28.6 ± 5.6%) but it could promote the protocorm to the most advanced germination of D. findlayanum by mixing peat moss with coir dust and
stage (Table 1 and Fig. 1b). drying the mixture in the hot air oven to reduce the moisture content
of the germination substrate, and by using Petri dishes instead of plastic
3.2. Symbiotic seed germination of D. fimbriatum containers to increase the ventilation of the germination units. Initially,
each treatment in this experiment consisted of six replicates but some
In this experiment, we used the plastic box (Fig. 3a) as the seed ger- contaminated units were excluded from the data analysis. Number of
mination container to provide more room to larger orchid protocorms replicates of each treatment used in the data analysis was shown in
and to make the method more feasible for orchid growers and hobbyists. Table 3. Seeds of D. findlayanum in all treatments started to germinate
Initially, each treatment consisted of nine replicates. However, contami- at one week after sowing. At 10 weeks after sowing, germination per-
nation occurred in some experimental units and the contaminated units centages of seeds in all treatments (77.6–86.1%) were not significantly
were excluded from the data analyses. Number of replicates of each different (Table 3). This result showed that asymbiotic germination
treatment used in the data analysis is shown in Table 2. Seeds of method (1/5MS) and symbiotic methods (either OMA or the mixture
D. fimbriatum sown on peat moss in all treatments were observed to ger- of peat moss and coir dust with mycorrhizal inoculation) in this exper-
minate at two weeks after sowing. At 17 weeks after sowing, germina- iment performed equally well on supporting seed germination of
tion percentages of seeds in the two treatments with mycorrhizal D. findlayanum. However, protocorm development was found to be
inoculation were higher than that of seeds in the un-inoculated control highly affected by the germination methods (asymbiotic vs symbiotic).
but only seeds co-cultured with T. deliquescens isolate Da-KP-0-1 had Both symbiotic methods were more effective than 1/5MS in promoting
significantly higher germination percentage (Table 2). According to protocorm development of D. findlayanum. At 10 weeks after seed
this study, both fungal isolates (i.e., Da-KP-0-1 and Pv-PC-1-1) were sowing, the protocorms on either OMA or the mixture of peat moss
fully compatible with D. fimbriatum since they supported the orchid and coir dust with mycorrhizal inoculation could develop to Stage 5
protocorms to the most advanced seedling stage (i.e., seedlings with (Fig. 3c, d) but the protocorms on 1/5MS did not develop beyond
elongated leaves and roots). Protocorms on peat moss without the my- Stage 3 (Fig. 3b; Table 3). The oat meal agar resulted in higher percent-
corrhizal fungus did not develop beyond Stage 3 (Table 2). Microscopic age of Stage-5 protocorms compared to the mixture of peat moss
observation revealed pelotons in the protocorms co-cultured with and coir dust, although the difference was insignificant (Table 3). Addi-
either fungal isolate Da-KP-0-1 or Pv-PC-1-1 and the absence of pelotons tionally, the sizes of protocorms on OMA were larger than those on the
in protocorms in the un-inoculated treatment (data not shown). This mixture of peat moss and coir dust (Fig. 3c, d). Microscopic observation
experiment demonstrated that peat moss could be used as germina- revealed pelotons in protocorms on media with mycorrhizal inoculation
tion substrate for in vitro symbiotic seed germination of orchid, al- and the absence of pelotons in protocorms on 1/5MS (data not shown).
though the rates of seed germination in all treatments were unusually This experiment showed that the mixture of peat moss and coir dust
low as compared to seed germination rates of Dendrobium orchids was suitable as germination substrate for in vitro symbiotic seed germi-
(Swangmaneecharern et al. 2012), probably due to the waterlogged nation of the orchid resulting in germination percentage comparable
characteristic of the peat moss observed in the germination boxes to OMA. Moreover, the mixture of peat moss and coir dust has advan-
(data not shown). Therefore, in our next experiment we modified the tages over agar-based media in that they are less expensive and the
germination method to solve the mentioned problems and also com- step of washing agar from the roots during ex vitro transplanting can
pared the effectiveness of the method to OMA and 1/5MS. be omitted. Aggarwal and Nirmala (2012) used coir fibers as an eco-
friendly and inexpensive substitute for gelling agents for in vitro seed
3.3. Symbiotic seed germination of D. findlayanum germination. However, the coir fibers must be soaked in Mitra orchid
growing medium to enable seed germination. Our study reported the
Since we hypothesized that low germination percentages of mixture of peat moss and coir dust as substitute for gelling agents and
D. fimbriatum in the second experiment were caused by too high mycorrhizal inoculation as an alternative to asymbiotic media, although
moisture of the peat moss and low ventilation efficiency of the plas- asymbiotic germination media can be added to the symbiotic germina-
tic container, we modified the protocol for in vitro symbiotic seed tion substrate to promote the growth of seedlings. It is also important to

Table 3
Effects of media and fungal inoculation on the developmental stages of seeds and protocorms of D. findlayanum at 10 weeks after seed sowing.

Treatmenta Repb Percentage of seeds and/or protocormc,d % germinationd

Stage 0 Stage 1 Stage 2 Stage 3 Stage 4 Stage 5

1/5MS 6 17.6 ± 6.4 a 9.1 ± 5.4 b 62.7 ± 6.8 b 10.6 ± 3.6 a 0.0 ± 0.0 a 0.0 ± 0.0 a 82.4 ± 6.4 a
OMA + Da 3 13.9 ± 4.1 a 0.0 ± 0.0 a 13.2 ± 15.5 a 12.4 ± 9.8 a 16.2 ± 5.9 b 44.3 ± 33.5 b 86.1 ± 4.1 a
P&C + Da 4 22.4 ± 4.4 a 1.0 ± 1.7 a 20.9 ± 11.1 a 11.7 ± 3.0 a 12.2 ± 1.1 b 31.8 ± 10.4 b 77.6 ± 4.4 a
a
P&C represents the mixture of peat moss and coir dust, and Da represents fungal isolate Da-KP-0-1.
b
Number of replicates used in the data analysis.
c
Values are means ± standard deviations.
d
Means in the same column marked with different letters are significantly different (P b 0.05).

Please cite this article as: Mala, B., et al., Effect of germination media on in vitro symbiotic seed germination of three Dendrobium orchids, South
African Journal of Botany (2017), http://dx.doi.org/10.1016/j.sajb.2017.05.008
6 B. Mala et al. / South African Journal of Botany xxx (2017) xxx–xxx

note that we attempted to use the plastic boxes as the seed germination Bhattacharyya, P., Kumaria, S., Tandon, P., 2016b. High frequency regeneration protocol
for Dendrobium nobile: a model tissue culture approach for propagation of medicinal-
containers in the second experiment; however, we observed that they ly important orchid species. South African Journal of Botany 104, 232–243.
had poor ventilation efficiency. We recommend that for more practical Callaway, E., 2015. Lab staple agar hit by seaweed shortage. Nature News 528, 171–172.
Hossain, M.M., 2011. Therapeutic orchids: traditional uses and recent advances — an
in vitro and ex vitro symbiotic seed germination methods using com- overview. Fitoterapia 82:102–140. http://dx.doi.org/10.1016/j.fitote.2010.09.07.
mon orchid cultivation substrates, the plastic boxes can be used but Johnson, T.R., Stewart, S.L., Dutra, D., Kane, M.E., Richardson, L., 2007. Asymbiotic and
they must be vented with filter or membrane to increase ventilation symbiotic seed germination of Eulophia alta (Orchidaceae) — preliminary evidence
for the symbiotic culture advantage. Plant Cell Tissue and Organ Culture 90:
efficiency. 313–323. http://dx.doi.org/10.1007/s11240-007-9270-z.
Knudson, L., 1922. Nonsymbiotic germination of orchid seeds. Botanical Gazette 73:1–25.
http://dx.doi.org/10.1086/332956.
4. Conclusion
Lee, M.C., Chang, D.C.N., Wu, C.W., Wnag, Y.T., Chang, Y.S., 2014. Phalaenopsis efficiently accli-
mate to highlight environment through orchid mycorrhization. Scientia Horticulturae
This study comprised three experiments. In the first experiment, ef- 179, 184–190.
Murashige, T., Skoog, F., 1962. A revised medium for rapid growth and bioassays with
fectiveness of three concentrations of ground oat (1, 5, or 10 g/l) in OMA tobacco tissue cultures. Physiologia Plantarum 15, 473–497.
inoculated with the orchid mycorrhizal fungus (T. deliquescens), uninoc- Nikabadi, S., Bunn, E., Stevens, J., Newman, B., Turner, S.R., Dixon, K.W., 2014. Germination
ulated OMA (10 g/l of oat) and 1/5MS on promoting seed germination of responses of four native terrestrial orchids from south-west Western Australia to
temperature and light treatments. Plant Cell Tissue and Organ Culture 118, 559–569.
D. lindleyi were compared. Higher oat concentrations were found to en- Nontachaiyapoom, S., Sasirat, S., Manoch, L., 2010. Isolation and identification of Rhizocto-
hance germination and protocorm development of D. lindleyi. The OMA nia-like fungi from roots of three orchid genera, Paphiopedilum, Dendrobium, and
Cymbidium, collected in Chiang Rai and Chiang Mai Provinces of Thailand. Mycorrhiza
containing 10 g/l of oat with mycorrhizal inoculation also outperformed 20:459–471. http://dx.doi.org/10.1007/s00572-010-0297-3.
1/5MS. In the second and third experiments, the use of common orchid Nontachaiyapoom, S., Sasirat, S., Manoch, L., 2011. Symbiotic seed germination of
cultivation substrates as alternative media for in vitro symbiotic orchid Grammatophyllum speciosum Blume and Dendrobium draconis Rchb. F., native orchids
of Thailand. Scientia Horticulturae 130:303–308. http://dx.doi.org/10.1016/j.scienta.
seed germination was investigated. We demonstrated that sphagnum 2011.06.040.
peat moss inoculated with T. deliquescens in plastic containers could Øien, D., O'Neill, J.P., Whigham, D.F., McCormick, M.K., 2008. Germination ecology of the
be used to germinate seeds of D. fimbriatum to advanced seedling boreal-alpine terrestrial orchid Dactylorhiza lapponica (Orchidaceae). Annales
Botanici Fennici 45, 161–172.
stage, although the germination percentages were low, probably due Paul, S., Kumaria, S., Tandon, P., 2012. An effective nutrient medium for asymbiotic seed ger-
to the waterlogged characteristic of the peat moss and low ventilation mination and large-scale in vitro regeneration of Dendrobium hookerianum, a threatened
orchid of northeast India. AoB Plants:plr032. http://dx.doi.org/10.1093/aobpla/plr032.
of the plastic boxes. Therefore, in our last experiment, we used the mix- Rasmussen, H., Andersen, T.F., Johansen, B., 1990. Temperature sensitivity of in vitro
ture of peat moss and coir dust packed in Petri dishes to solve the men- germination and seedling development of Dactylorhiza majalis (Orchidaceae)
tioned problems and compared the effectiveness of this system to OMA with and without a mycorrhizal fungus. Plant Cell & Environment 13:171–177.
http://dx.doi.org/10.1111/j.1365-3040.1990.tb01289.x.
and 1/5MS. The three treatments resulted in similar germination per- Smith, S.E., Read, D.J., 2008. Mycorrhizal Symbiosis. third ed. Academic Press, New York.
centages but the symbiotic methods were more effective than 1/5MS Stewart, S.L., Kane, M., 2006. Symbiotic seed germination of Habenaria macroceratitis
(Orchidaceae), a rare Florida terrestrial orchid. Plant Cell Tissue and Organ Culture
in promoting protocorm development of D. findlayanum. The findings
86, 159–167.
of this study can be used to improve symbiotic germination media and Swangmaneecharern, P., Serivichyaswat, P., Nontachaiyapoom, S., 2012. Promoting effect of
they encourage the use of symbiotic germination method as mean for orchid mycorrhizal fungi Epulorhiza isolates on seed germination of Dendrobium orchids.
Scientia Horticulturae 148:55–58. http://dx.doi.org/10.1016/j.scienta.2012.09.013.
orchid seed propagation. Tan, X.M., Wang, C.L., Chen, X.M., Zhou, Y.Q., Wang, Y.Q., Luo, A.X., Liu, Z.H., Guo, S.X.,
2014. In vitro seed germination and seedling growth of an endangered epiphytic
Acknowledgements orchid, Dendrobium officinale, endemic to China using mycorrhizal fungi (Tulasnella
sp.). Scientia Horticulturae 165, 62–68.
Yam, T.W., Arditti, J., 2009. History of orchid propagation: a mirror of the history of bio-
We thank Mr. Phunsak Kidmung, Miss Daroonsri Maneesorn and technology. Plant Biotechnology Report 3:1–56. http://dx.doi.org/10.1007/s11816-
008-0066-3.
Mr. Auttapon Taluengjit for providing the orchid capsules. This work Yam, T.W., Arditti, J., Cameron, K.M., 2009. The orchids have been a splendid sport — an
was supported by the Thailand Research Fund and Mae Fah Luang alternative look at Charles Darwin's contribution to orchid biology. American Journal
University (RSA5580040). of Botany 96, 2128–2154.
Yoder, J.A., Zettler, L.W., Stewart, S.L., 2000. Water requirements of terrestrial and epi-
phytic orchid seeds and seedlings, and evidence for water uptake by means of
References mycotrophy. Plant Science 156, 145–150.
Zeng, S., Zhang, Y., Teixeira da Silva, J.A., Wu, K., Zhang, J., Duan, J., 2014. Seed biology
Aewsakul, N., Maneesorn, D., Serivichyaswat, P., Taluengjit, A., Nontachaiyapoom, S., and in vitro seed germination of Cypripedium. Critical Reviews in Biotechnology 34:
2013. Ex vitro symbiotic seed germination of Spathoglottis plicata Blume on common 358–371. http://dx.doi.org/10.3109/07388551.2013.841117.
orchid cultivation substrates. Scientia Horticulturae 160:238–242. http://dx.doi.org/ Zeng, S., Huang, W., Wu, K., Zhang, J., Teixeira da Silva, J.A., Duan, J., 2016. In vitro propa-
10.1016/j.scienta.2013.05.034. gation of Paphiopedilum orchids. Critical Reviews in Biotechnology 36:521–534.
Aggarwal, S., Nirmala, C., 2012. Utilization of coir fibers as an eco-friendly substitute for http://dx.doi.org/10.3109/07388551.2014.993585.
costly gelling agents for in vitro orchid seed germination. Scientia Horticulturae Zettler, L.W., Poulter, S.B., McDonald, K.I., 2007. Conservation-driven propagation of an
133, 89–92. epiphytic orchid (Epidendrum nocturnum) with a mycorrhizal fungus. Hortscience
Bhattacharyya, P., Kumaria, S., Job, N., Tandon, P., 2016a. En-masse production of elite 42, 135–139.
clones of Dendrobium crepidatum: a threatened, medicinal orchid used in Traditional Zhou, X., Gao, J.Y., 2016. Highly compatible Epa-01 strain promotes seed germination and
Chinese Medicine (TCM). Journal of Applied Research on Medicinal and Aromatic protocorms development of Papilionanthe teres (Orchidaceae). Plant Cell Tissue and
Plants 3, 168–176. Organ Culture 125, 479–493.

Please cite this article as: Mala, B., et al., Effect of germination media on in vitro symbiotic seed germination of three Dendrobium orchids, South
African Journal of Botany (2017), http://dx.doi.org/10.1016/j.sajb.2017.05.008