In vitro micropropagation of the orchid (dendrobium crystallinum var. alba)

AGU International Journal of Sciences – 2019, Vol 7 (3), 1 – 8

IN VITRO MICROPROPAGATION OF THE ORCHID (DENDROBIUM CRYSTALLINUM
VAR. ALBA)
Tran Thi Ngoc Lan1
1

South Central and Highland Institute of Science

Information:
Received: 07/08/2018
Accepted: 17/10/2018
Published: 11/2019
Keywords:
BA, Dendrobium crystallinum
var. alba., NAA, PLB,
protocorm.

ABSTRACT
Studies of micropropagation of Dendrobium crystallinum var. alba. were
conducted in order to conserve and develop this precious orchid species. The
results showed that protocorms were formed from seeds culture on the ½ MS
medium supplemented with 10% potatoes extract within 8 weeks. Protocorms
which were cultured on the ½ MS medium containing 30 g/l sucrose, 0.5 g/l AC,
7 g/l agar, 0.1 mg/l NAA and 1 mg/l BA were optimal for PLB formation (8.67
PLBs/sample) after 8 weeks culture. Protocorms converted into normal plants
with well-developed shoots and roots on the ½ MS medium supplemented with 20
g/l sucrose, 10% potatoes extract, 0.5 g/l AC, and 7 g/l agar after about 90 days.
PLBs converted into normal plants on the same medium supplemented with 0.5
mg/l NAA and 0.5 mg/l BA, too. No abnormal morphological changes were found
in these Dendrobium seedlings.

Micropropagation provided an important method
for mass propagation of many orchid species.
There
were
perusals
of
Dendrobium
micropropagation on the world and Vietnam.
Sunitibala & Rajkumar (2009) cultured seeds of
Dendrobium transparens L. on the ½ Murashige
and Skoog (½ MS) suplemented with 1 mg/l NAA
and 2 mg/l BA. Nguyễn Văn Song (2011) cultured
seeds of Dendrobium chrysotosum Lindl on MS
medium. Nguyễn Thị Sơn et al. (2014) propagated
Dendrobium officinale Kimura et Migo with
sowing seeds on the Vacin Went and cultured
nodal segment on the MS medium. Vũ Kim Dung
et
al.
(2016)
propagated
Dendrobium
gratiosissimum Rchb.f. Nguyễn Văn Việt (2017)
cultured the Dendrobium lituiflorum Lindley
seeds. Rattana & Sangchanjirade (2017)
propagated Dendrobium signatum Rchb.f.
However, there is no article refering to clonal

1. INTRODUCTION

Vietnam has a tropical monsoon climate that is
appropriate for orchid growth such as the
Dendrobiums. Dendrobiums are not a beautiful
species although they are highly valued in the
flower industry as potted plants and cut flowers.
Even though these wild orchids are diverse, they
have been exploited almost to extinction. The
Dendrobium crystallinum var. alba is a very
graceful wild orchid of Vietnam and is a rare
species. It is a epiphytic, sympodial orchid which
also grows in Hymalayan Myanmar, Thailand and
Vietnam at altitudes of 700 – 1700 m. It has
beautiful, long-lasting flowers, noble aroma and
blooms in April- May every year. It is classified as
a group of rare orchids and an endangered species
(EN, IUCN, Averyanov, 2005).

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AGU International Journal of Sciences – 2019, Vol 7 (3), 1 – 8

propagation of Dendrobium crystallinum var. alba.
This study reports
micropropagation of
Dendrobium crystallinum var. alba in order to
conserve and develop this precious orchid species.

2.1 Materials
Research subject: Den. crystallinum var.alba plant

with fruits were collected in the family garden.
Fruit was harvested 180 days after pollination in
October, 2017. (fig. 1).

2. MATERIALS AND METHODS

Figure 1. Dendrobium crystallinum var.alba. a. Plants with their flowers. b. Plants with their fruits (arrow).

consisting of MS, ½ MS, supplemented with
different organic compound (15% coconut water
(CW) – Thickness of coconut pulp = 3 – 4 mm;
10% potatoes extract – 100g potatoes were boiled,
extract to 1 l solution; 5% mashed banana), 30 g/l
sucrose, 0.5 g/l AC, 7 g/l agar. Seeds were cultured
in the bottles (volume = 500 ml with 70 ml
medium/bottle). Each treatment had 5 bottles. The
percentage of orchid seed germination was
obtained by estimating the surface area of seed
germination in the tissue culture bottle with a
diameter of 6.5 cm. The total surface area of the
tissue culture bottle was defined as 100%. After
cultivation for eight weeks, the percentage of seed
germination was recorded. Observations on the
percentage germination of seeds, protocrom
formation rate, shoot formation rate were recorded
8 weeks after culture.

2.2 Experiment conditions
All tests were kept at 25±1οC under a photoperiod
of 16 h light/8 h dark, light intensity of 2000 lux

and 70% relative humidity.
Basal cultured medium: MS, ½ MS, at pH 5.7, 121
οC, 1 atm, autoclaved for 20 minutes.
2.3 Methods
2.3.1 Effect of different media and organic
supplements on germination of Den.
crystallinum var.alba seeds
The mature capsules of Den. crystallinum var.alba
were collected six months after pollination, soaked
in aqueous solution of commercial detergent
(Sunlight, Vietnam) for 10 min, then rinsed
thoroughly three times with sterile distilled water,
followed by dipping them in 70% ethanol for 20
sec. Capsules were then surface sterilised by
dipping in 70% ethyl alcohol and flamed
immediately four to five times in laminar air flow.
The capsules were then cut longitudinally in a
sterilised petri dish. Seeds were scraped from the
capsules, mixed with 100 ml sterile distilled water
and pipetted into 1ml tubes and then cultured on
the surface of the medium. Two different basal
media were used in the whole experiment

2.3.2 Effect of NAA and BA on protocorm
proliferation of Den. crystallinum var.alba
Protocorms from the above study were used as
culture material (the average weight of 20 ± 5 mg)
were proliferated on ½ MS supplemented with 30
g/l sucrose, 10% potatoes extract, NAA (0; 0.1; 0.2
mg/l), BA (0, 0.5, 1 mg/l), 0.5 g/l AC and 7 g/l agar.

Protocorms were cultured in the bottles (volume =
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AGU International Journal of Sciences – 2019, Vol 7 (3), 1 – 8

500 ml with 70 ml medium/bottle) with 20
explants/bottle. Each treatment had 5 bottles.
Observations on the protocorm-like body (PLB)
formation rate, number of PLB/explants, figure of
PLBs were recorded 8 weeks after culture.

days after culture) on the optical microscope
(Olympus
CX21,
Japan)
with
degrees
magnification of 40 - 100 times.
2.3.5 Statistical analysis
All the experiments were set up in completely
randomised design (CRD). Each treatment
consisted of 3 replicates. The difference among the
treatment means was compared based on Duncan’s
multiple range test (DMRT) analysis (with a level
of significance of P<0.05). Excel 2010 and the
statistical package SPSS (Statistical Program
Scientific System)16.0 was used for the analyses.

2.3.3 Effect of plant growth regulators (PGRs) on

development of protocorms and PLBs Den.
crystallinum var.alba
Protocorms from the study (2.3.1) and PLBs from
the study (2.3.2) with being used as explants (the
average weight of 20 ± 5 mg) were cultured on the
½ MS supplemented with 20 g/l sucrose, 10%
potatoes extract, 0.5 g/l AC, 7 g/l agar, NAA (0;
0.5; 1 mg/l), BA (0; 0.5 mg/l) in examnination the
growth of these protocorms and PLBs. They were
cultured in the bottles (volume = 500 ml with 70
ml medium/bottle) with 20 explants/bottle. Each
treatment had 5 bottles. Observations on figure of
seedlings, the PLB formation rate, the seedling
formation rate, height of shoot, number of leaves,
number of roots and length of root were recorded
90 days after culture. Observation was conducted
on the developing stages of protocorms and PLBs
(4 weeks, 8 weeks and 90 days) after culture.

3. RESULTS AND DISCUSSION
3.1 Effect of different media and organic
supplements on germination of Den.
crystallinum var.alba seeds
Naturally, orchid seeds have a poor germination
rate because of the small size of the seeds and the
lack of cotyledons and endosperm. Seeds can only
germinate with isolated mycorrhizal fungi but
seeds run the risk of being infected and die.
Consequently, the sample orchid seeds were
propagated in asymbiotic seed germination. The

results of the study on germinated seeds of Den.
crystallinum var.alba on the different medium were
observed at 8 weeks after culture (Table 1).

2.3.4 Histological observation
Observation of the germination of orchid seeds and
their developing stages (4 weeks, 8 weeks and 90

Table 1. Effect of different media and organic supplements on germination
of Den. crystallinum var.alba seeds.

Culture medium

Survival and formation
rate of protocorms (%)

Survival and formation
rate of shoots (%)

MS

26.33f*

4.33b

MS + 15% CW

55.67d

7.33a

MS + 10% potatoes extract

80.67b

7.67a

MS + 5% mashed banana

68.67c

6.67a

½ MS

37.33e

4.33b

½ MS + 15% CW

98.67a

1.33c

½ MS + 10% potatoes extract

99.33a

0.33c

½ MS + 5% mashed banana

70.67c

6.67a

*: Different alphabetical letters are significantly different according to Duncan’s multiple range tests at P < 0.05.

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AGU International Journal of Sciences – 2019, Vol 7 (3), 1 – 8

It was observed that seeds had been green at 4
weeks after culture (Fig. 2c), germinated and
formed green protocorms. Protocorm had emerge
from capsules and come on growing at 8 weeks
after culture (Fig. 2a, 2d). The most suitable media,
providing seed germination were ½ MS medium
supplemented with 15% CW and ½ MS medium
supplemented with 10% potatoes extract
(protocorm formation rate corresponding 98.67%
và 99.33%). The less protocorm formation rate
gained 26.33% và 37.33% of seeds which were
sowed on the MS medium and ½ MS medium.
Combinations of organic supplements in the media
enhanced seed germination. It show that the
importance of organic compounds such as coconut
water, potatoes extract… for orchid seeds

germination. Den. crystallinum var.alba is
epiphytic orchid, growth rate is low, so, the high
concentration of N, P, K compounds were not
suitable to culture orchid seeds. However, the
minerals derived from organic compounds such as
coconut, potatoes extract were useful in culture
orchid seeds. The reports on the world were
acknowledged this too (Arditti, 2008; Vũ Ngọc
Lan & Nguyễn Thị Lý Anh, 2013; Parthibhan và
cs., 2015, Nguyễn Văn Việt, 2017).
The best medium for seed germination in treatment
was 10% potatoes extract or 15% CW. Potatoes
extract consists of carbohydrates, amino acids,
important vitamins (C, B1, B6…) and mineral
elements (potassium, iron, magnesium) (Molnár et
al., 2011; Sandoval et al., 2014, Parisa et al., 2014).
In the in vitro culture, potato had useful effects on
some orchid species such as Phalaenopsis and
Dendrobium (Rattana & Sangchanjirade, 2017).
This study found that the nutrients of CW and
potato were suitable for orchid seeds. The
effciency of CW may depend on species, age and
thickness of coconut pulp. At this study, CW was
undertaken of coconut fruits with the thickness of
coconut pulp = 3 – 4 mm was suitable for orchid
seeds germination. However, CW was rather
expensive than potato. Potato was easy to look for,
so, potatoes extract was selected in the following
tests. Unlike the results of Sunitibala & Rajkumar

(2009) for sowing seeds Den. transparent L. with
using PGRs (1 mg/l NAA and 1mg/l BA), in this
experiment only used MS medium and organic
compounds for Den. crystallinum var.alba seeds
gemination.
Beside protocorm formation, there was the low
shoot formation rate from orchid seeds (0,33 –
7,67%). It show that orchid embryos can develop
to the different stages of seeds, accordance with
reports on Dendrobium culture (Arditti, 2008; Vũ
Ngọc Lan & Nguyen Thi Ly Anh, 2013, Vu Kim
Dung và cs., 2016; Rattana & Sangchanjirade,
2017).
3.2 Effect of NAA và BA on protocorm
proliferation of Den. crystallinum var.alba
The results of the study on effect of NAA và BA
on protocorm proliferation of Den. crystallinum
var.alba were observed at 8 weeks after culture
(Table 2).
The protocorm explants had showed the induction
of meristem, parenchyma with PGRs. Protocorm is
the germinated stage of orchid embryos, along with
translation of PGRs sush as NAA, BA to
differentiate to PLBs. PLBs and protocorms are
potential in embryo proliferation. Subculture PLBs
was conducted in micropropagation of orchid such
as Dendrobium transparent L.(Sunitibala &
Rajkumar, 2009), Dendrobium aqueum Lindley
(Parthibhan et al., 2015) or Dendrobium lituiflorum
Lindley (Nguyen Van Viet, 2017).

Adding to NAA, BA, potatoes extract was
effective in forming secondary PLBs. In fact, the
composition of potatoes extract includes inorganic
ions (e.g. phosphorus, potassium), nitrogenous
compounds, amino acids, vitamins…). All these
suplements may be the reason for seed germination
in orchids (Arditti, 2008; Rattana &
Sangchanjirade, 2017). Using BA individually had
not much effective but the addition of BA in
combination with NAA improved ability of cell
division of explants. The optimal concentrations
of NAA (0.1 mg/l ) and BA (1 mg/l) gave the
highest PLB formation rate, the number of
PLBs/explant (corresponding 100%, 8.67
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AGU International Journal of Sciences – 2019, Vol 7 (3), 1 – 8

PLBs/explants). The mean weight of PLBs was
higher the mean weight of protocorm ( 32.4 mg and
25.4 mg) and had deep green (Fig.2b). Compared
to PLB proliferation (multiplication factor: 4.2 in
protocorm proliferation of Dendrobium nobile
Lindl; Vũ Ngọc Lan & Nguyễn Thị Lý Anh, 2013),
the results of this study had higher multiplication
factor (8.67). The combined of auxin and

cytokinins was observed in present study is in
accordance with report on Dendrobium

transparens L. (Sunitibala, 2009), Dendrobium
signatum (Rattana K. & Sangchanjirade, 2017).
Thus, the ½ MS medium suplemented with 0.1
mg/l NAA and 1 mg/l BA is optimal to form the
secondary PLB.

Table 2. Effect of NAA và BA on protocorm proliferation of Den. crystallinum var.alba after 8 weeks culture.

NAA
(mg/l)

BA
(mg/l)

Formation rate
PLB (%)

Number of
PLBs/explant

Figure of PLBs

0

0

72.67c*

2.01d

Small, light green

0.1

0

92.33b

2.69d

big, light green

0

0.5

74.33c

4.45c

Small, light green

0.1

0.5

91.67b

2.23d

Big, dark green

0.1

1

100.00a

8.67a

Big, dark green

0.2

0

91.33b

4.74c

Big, light green

0.2

0.5

92.33b

6.33b

Big, light green

0.2

1

73.33c

4.84c

Big, light green

*: Different alphabetical letters are significantly different according to Duncan’s multiple range tests at P < 0.05.

3.3 Effect of plant growth regulators on development of Den. crystallinum var.alba protocorm and
PLB after 90 days culture
Effect of plant growth regulators (NAA and BA) on development of Den. crystallinum var.alba protocorm
and PLB after 90 days culture showed in table 3 and table 4.
Table 3. Effect of NAA and BA on development of protocorm Den. crystallinum var.alba after 90 days culture.

Medium supplement
with
NAA(mg/l)

Formation
rate PLB
(%)

Formation
seedling

(%)

Average
number
of roots

Average
root
length
(cm)

Average
of shoot
height
(cm)

Average
number
of leaves

BA(mg/l)

0

0

17.67c*

82.33b

2.67b

0.92c

3.18b

2.47b

0.5

0

37.33b

62.67c

2.74b

1.93b

3.15b

2.43b

0.5

0.5

7.67d

92.33a

4.13a

3.61a

4.36a

4.87a

1.0

0.5

66.33a

33.67d

1.13c

0.20d

0.52c

2.05b

*: Different alphabetical letters are significantly different according to Duncan’s multiple range tests at P < 0.05.

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AGU International Journal of Sciences – 2019, Vol 7 (3), 1 – 8
Table 4. Effect of NAA and BA on development of PLB, derived from protocorm Den. crystallinum var.alba
after 90 days culture.

Medium supplement
with
NAA(mg/l)

Formation
rate PLB
(%)

Formation
seedling
(%)

Average
number
of roots

Average
root
length
(cm)

Average
of shoot
height
(cm)

Average
number
of leaves

BA(mg/l)

0

0

38.67b*

23.67c

1.57b

0.63c

0.76c

2.17c

0.5

0

39.33b

60.67b

1.79b

1.85b

2.07b

3.18b

0.5

0.5

9.67c

90.33a

4.22a

3.02a

4.19a

4.59a

1.0

0.5

69.33a

16.67d

0.53c

0.13d

0.55c

2.01c

*: Different alphabetical letters are significantly different according to Duncan’s multiple range tests at
P < 0.05.
growth, lateral growth increases so it reduces
height of shoot as well as root formation. The
combination of BA and auxin (NAA), the cells
expand and differentiate, proliferate effectively
protocorms. In the medium concentraton of NAA
and BA (0.5 mg/l), an increase cell division and
shoot and root morphogenesis took place with
organogenesis (Fig. 2i, 2j.). Thus, the treatment
with culture medium suplemented with 0.5 mg/l
NAA and 0.5 mg/l BA was noted to differentiate
protocorms and PLBs into seedlings.

At treatment devoid of PGRs, there was 82.33% of
protocorms converted into seedlings (Table 3,
Fig.2h) but only 23.67% of PLBs converted into
seedlings (Table 4). Formation rate PLB in this
treament was also rather low (corresponding

17.67%
and 38.67%). This show that the
embryogenesis nature of protocorms. They can
invert into seedlings that no using PGRs. However,
PLBs were not so. PLBs need assisting of NAA
and BA to develop and convert into seedings (the
highest formation seedling at treatment
supplemented with 0.5 mg/l NAA and 0.5 mg/l
BA, 90.33%). Similar to protocorm, the seedling
formation rate was 92.33% in this result, too. Shoot
height, number of roots, number of leaves, root
length of the seedlings in this treatment were
highest. The combination of NAA and BA made
the cells loosen, elongate dimension, improved
ability of cell division, made the cell survive and
convert into seedling. The combination of NAA
and BA in micropropagation is uasually applied in
orchid embryo proliferation or in shoot
proliferation with axenic pseudobulb segment
(Sunitibala & Rajkumar, 2009; Nguyễn Thị Sơn et
al., 2014; Parthibhan et al., 2015) or seedlings
formation of Dendrobium signatum Rchb.f.
(Rattana & Sangchanjirade, 2017).

No morphological variations were observed in this
study through micropropagation Den. crystallium
var. alba with protocorms and PLBs. (Fig. 2i, 2j).
3.4 Histological observation
Protocorm is a earliest structure that is formed in
embryogenesis of orchid seed and is united in all of

Orchidaceae (Batygina et al., 2003; Arditti, 2008).
Observating on optical microscope showed that,
the orchid seeds with green embryos after 4 weeks
culture (Fig. 2c), formed protocorms after 8 weeks
culture (Fig. 2d), after that, formed shoots, roots,
converted into seedlings after 90 days culture (Fig.
2g). PLB derived from protocorm (Fig. 2b, 2e)
developed with the stages similar to protocorm
(Stage 1: PLB formed meristem; Stage 2: PLB
formed leaflets, vascular tissue; Stage 3: formed
seedling with shoot and root). That was confirmed
in this study (Fig. 2f, 2j). Thus, protocorms could
directly convert into seedlings or proliferate,
forming PLBs and PLBs could proliferate and
convert into seedlings, too. This is a effective
orchid micropropagation.

At treatment devoid of PGRs, PLBs still converted
into seedlings (23,67%). It show that PLBs can
develop to the different stages. At treatment
medium suplemented with 1 mg/l NAA and 0,5
mg/l BA, PLB proliferation rate was high
(corresponding 66,33% and 69,33%) but formation
seedling rate was low (33,67% and 16,67%).
Citokinin ( BA) uasually inhibits apical bud
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AGU International Journal of Sciences – 2019, Vol 7 (3), 1 – 8

4. CONCLUSION AND PETITION

Protocorms and PLBs converted into seedlings
(corresponding 92.33% and 90.33%) on the ½ MS
medium (supplemented with 10% potatoes extract,
20 g/l sucrose, 0.5 g/l AC, 0.5 mg/l NAA, 0.5 mg/l
BA and 7 g/l agar) after 90 days culture.

4.1 Conclusion
½ MS medium (suplemented with 10% potatoes
extract, 30 g/l sucrose, 0.5 g/l AC, 7 g/l agar) is the
most suitable for Dendrobium crystallinum var.
alba seed germination (the protocorm formation
rate: 99.33%). PLBs were formed from protocorms
on the medium suplemented with 0.5 mg/l NAA
and 1 mg/l BA (number of PLBs/explant: 8.67).

4.2

Petition

The following studies are necessary to the growth
and development of these seedlings in the ex vitro
condittion for mass production this valuable
species.

Figure 2. Micropropagation Dendrobium crystallinum var.alba. a. Seeds germinated, formed protocorm after 8 weeks
culture; b. PLBs formation from protocorms after 8 weeks culture; c. The seeds with embryos after 4 weeks culture;
d. Protocorms formation from seeds after 8 weeks culture; e. PLB with shoot were formed from protocorms after 8 weeks
culture; f. Seedling formation from PLB, derived from protocorm after 90 days culture; g. Seedlings formation from

protocorm after 90 days culture; h. Seedlings formation on no PGRs medium; i. Seedlings formation derived from
protocorm on the medium supplemented with 0.5 mg/l NAA and 0.5 mg/l BA; j. Seedlings formation derived from
PLB on the medium supplemented with 0.5 mg/l NAA and 0.5 mg/l BA. Acknowledgements.

The authors would like to thank College of Economics and Technical Lam Dong for supporting this study.
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AGU International Journal of Sciences – 2019, Vol 7 (3), 1 – 8

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