Plant Regeneration Through Indirect Somatic Embryogenesis in Coelogyne Cristata Orchid

doi:10.4236/ajps.2011.22028

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American Journal of Plant Sciences, 2011, 2, 262-267

doi:10.4236/ajps.2011.22028 Published Online June 2011 (http://www.SciRP.org/journal/ajps)

Plant Regeneration through Indirect Somatic

Embryogenesis in Coelogyne Cristata Orchid

Aung Htay Naing, Jae Dong Chung, Ki Byung Lim

School of Applied Biosciences, College of Agriculture and Life Science, Kyungpook National University, Daegu, Republic of Korea.

Email: [email protected]

Received April 7th, 2011; revised May 6th, 2011; accepted May 18th, 2011.

ABSTRACT

A newly efficient protocol has been established for high frequency somatic embryogenesis through callus culture of

Coelogyne cristata. The best frequency of callusing was obtained from leaf segments (3 - 5 mm) cultured on the MS

medium supplemented with 2 mg·L–1 2,4-D and 2 mg·L–1 BA combination. A negative effect of coconut water was ob-

served on the callus inductio n medium. When ca llus lines No. 4, 6 and 8 induced from leaf segments were sub-cultured

separately on 1/2 MS and MS media containing AC (1 - 3 g·L–1), formation of somatic embryos was found. However,

percentages of embryo formation and the number of embryos per explants were strongly affected by media and callus

lines used. The effect of 1/2 MS media is definitely be tter than MS medium for somatic embryogenesis from the selected

lines of leaf derived callus. Among the callus lin es, line no. 4 is the best for soma tic embryogenesis fo llowed by line no.

6 and 8. The somatic embryos converted into healthy plants with well developed shoots on the same media. The plant-

lets were transferred to 1/2 MS medium containing 1 g·L–1 AC for plant regenera tion until 8 weeks of culture and suc-

cessfully acclimatized in the greenhouse.

Keywords: Callus Formation, Embryo Formation, Leaf Segment, Plant Growth Regulators, Tropical Orchid

1. Introduction

Coelogyne cristata which belongs to the family Orchi-

daceae has high ornamental value as a cut flower. It pro-

duces its graceful racemes of white flowers with yellow

splotches on the throat, and has a long lasting and fra-

grant scent. In Myanmar, the pseudobulb of Coelogyne

cristata is used in making traditional medicine for dys-

entery and diarrhea. However, wild orchids including C.

cristata in Myanmar are nowadays steadily decreasing

due to over collection of orchid hunters, shifting cultiva-

tion, extension of crop cultivation and urban develop-

ment, which is causing loss of their natural habitats and

is leading to extinction. Thus, it is essential to take

measures to conserve their germplasm and to reveal effi-

cient propagation protocols to allow for the continuous

large supply of materials for medicine. However, there is

no report on plant regeneration through somatic em-

bryogenesis in C. cristata. The process has been induced

in tissue cultures of orchids either directly from the epi-

dermal cells of explants [1-3] or indirectly via interven-

ing callus [4].

The phenomenon of somatic embryogenesis via callus

is rather rare as success of callus formation in orchids has

limitations due to slow growth and a tendency to become

necrotic [5,6]. Especially, callus formation from leaf ex-

plants of orchids has been and is the rarest even now.

Even when callus induction was achieved, the frequency

of callusing might have been ultimately low and the cal-

lus was difficult to maintain and eventually failed to sur-

vive. Even when plant regeneration from callus of or-

chids was achieved, it usually occurred via protocorm-

like-bodies PLBs [6-8]. Therefore, success of somatic

embryogenesis via callus culture is interesting in plant

regeneration of orchids. This study presents optimal con-

centrations of 2,4-D and BA for callus formation and

high formation of somatic embryos from the culture of

leaf derived callus in Coelogyne cristata using different

kinds of media and AC

2. Materials and Methods

Mature capsules of Coelogyne cristata were collected

from Mingalardon Orchid Farm in Yangon, Myanmar.

The capsules were washed thoroughly under running tap

water. Then they were sterilized by immersion in 70%

ethanol for 10 seconds and flamed for a short time. After

they were dissected longitudinally, seeds were isolated

and sown on Hyponex (HP) basal media for in vitro ger-

Plant Regeneration through Indirect Somatic Embryogenesis in Coelogyne Cristata Orchid263

mination for 3 months. The germinated plantlets about

1.0 cm in height were transferred to Murashige and

Skoog (MS) basal media supplemented with NAA 1.0

mg·L–1 and BA 2.0 mg·L–1 for plant growth. After 2

months of culture, leaves of the in vitro plantlets become

the explants source to conduct further experiments.

2.1. Effect of PGRs and Coconut Water on

Callus Induction from Leaf Segments

Leaf segments of about 3 - 5 mm in length were used as

explants. For investigations on the effects of coconut

water (5% v/v) (MB cell, Seoul, Korea) and plant growth

regulators (PGRs) on callus induction, various combina-

tions of 2,4-dichlorophenoxyacetic acid (2,4-D; 0, 1.0,

2.0 and 3.0 mg·L–1) and benzyladenine (BA; 0, 1.0, 2.0

and 3.0 mg·L–1) were added to MS basal media. Each

experiment consisted of 10 explants with three replica-

tions. The culture vessels were placed in dark conditions

for 12 weeks and forming calli were recorded.

2.2. Effect of Media and AC on Somatic

Embryogenesis

To examine the effect of media and AC on somatic em-

bryogenesis, the above leaves derived callus lines [line

No. 4 (26.7%), line No. 6 (40.0%), line No. 8 (36.7%)]

without CW, which showed better callus formation and

originated from the same tissue but cultured on the dif-

ferent media compositions, were chosen. And, each cal-

lus line was dissected into 2 - 3 mm sized callus pieces

and cultured on the different kinds of basal media (MS

and 1/2 MS) containing (1 - 3 g·L–1) activated charcoal

(AC). Each experiment consisted of 10 explants with

three replications. The culture vessels were placed at

room temperature under 16/8 hr day/night photoperiod at

a 20 - 50 μmolm–²s–¹ photon flux density. Data were

recorded after 8 weeks of culture.

2.3. Plant Regeneration

Single plants derived from the above experiments were

transferred to half-Murashige and Skoog 1/2 MS basal

medium supplemented with 1 g·L–1 AC for plant regen-

eration. Regenerated plants were transplanted and accli-

matized in the greenhouse.

For all cultures, percentage of sucrose and agar was

3.0 and 0.8% respectively. The pH of medium was ad-

justed to 5.8 before adding agar. Data were analyzed by

DMRT (P > 0.05).

3. Results and Discussion

Developing somatic embryogenic culture systems with

reliable regeneration capacity from ornamental plants is a

prerequisite for mass propagation and their genetic im-

provement. Studies were being intensively made to de-

velop a protocol for somatic embryogenesis. The process

can be induced in tissue cultures of orchids either directly

from the epidermal cells of explants [1-3] or indirectly via

intervening callus [4 ]. In this study, somatic embryo-

genesis was induced indirectly through callus culture.

In general, formation of callus in orchids is rather dif-

ficult due to its slow growth and necrotic tendency, espe-

cially, success of callus induction from leaf segments is

the rarest. In this study, when leaf segments were cul-

tured on the hormone free medium they became swollen,

and followed by initiation of a callus mass was visible

from the wound edges of leaf segment after 4 weeks of

culture. The calli were granular, whitish to light yellow

and comprised of isodiametric cells. Higher frequencies

of callus induction from explants were obtained on the

basal media supplemented with various concentrations of

2,4-D and BA (Table 1). However, ratios between con-

centrations of 2,4-D and BA were significantly associ-

ated with percentages of survival of explants and callus

formation. On the contrary, either the lowest concentra-

tion of 2,4-D and the highest concentration of BA or a

higher concentration of 2,4-D and lower concentration of

BA showed synergistic effects on callus induction from

leaf segments. Of the combinations of 2,4-D and BA

tested, 2 mg·L–1 2,4-D and 2 mg·L–1 BA were found as

optimal concentrations for the best callus induction

(40.0%). The color of callus induced from each combina-

tion was slightly changed from whitish to yellow and

most yellow callus showed granular type. However, cal-

lus mass obtained from highest concentrations of 3

mg·L–1 2,4-D was reddish yellow in color.

Addition of CW to the same media containing 2,4-D

and BA, frequencies of callusing were relatively low as

compared to those on the media devoid of CW. Almost

all of the leaf segments died on CW containing media.

However, percentage of callusing was stable on the me-

dia containing concentration of 3 mg·L–1 2,4-D and 1

mg·L–1 BA as devoid of CW.

Recently, combinations of 2,4-D and TDZ have been

reported for the callus induction of ornamental plants

including some orchid genera, Cymbidium [7], Vanda

coerulea [9], Cypripedium formosanum [10]. However,

BA alone or in combination with 2,4-D totally inhibited

callus induction in Paphiopedilum hybrid [11]. Similarly,

[12] mentioned that the combination of 2,4-D and BA

could not effectively induce callus from leaf segment in

Phalaenopsis. In the present study, combination of 2,4-D

and BA has successfully induced callus from leaf seg-

ments within a short period of time. However, the pres-

ence of CW inhibited callus induction from leaf segment

in this species. A similar result was observed in Pha-

laenopsis [12]. It is clear that CW has an inhibitory effect

on callus induction from leaf segments of orchids. Ac-

Plant Regeneration through Indirect Somatic Embryogenesis in Coelogyne Cristata Orchid

264

cording to our findings in this study, exogenous hor-

mones are quite important for callus induction and no

effect of CW was observed on callus induction from leaf

segments in this species.

According to the results shown in Table 1, the lines

(No. 4, 6, 8) showed better callus formation. Thus, plant

regeneration through the lines should be critically con-

sidered. Although plant regeneration through organo-

genesis of callus culture has been fully developed in

some orchids, efficient protocol for embryogenesis

through callus culture of orchids still has been quite lim-

ited. Therefore, pieces of callus originating from the

above callus lines (4, 6, 8) without CW were separately

cultured on MS and 1/2 MS basal media supplemented

with different concentrations of AC (1 - 3 g·L–1) to ex-

Table 1. Effect of phytohormones on callus induction from

leaf segments of Coelogyne cristata after 12 weeks of culture

in dark conditions.

PGR (mg·L–1)

2,4-D BA

No. of

inoculated

explants

Survival

rate

Formation

of callus %

0 30 13g 3.3h

1 1 30 10h 5.7h

2 30 20f 13.3f

3 30 63a 26.7d*

2 1 30 43d 26.7d

2 30 46c 40.0a*

3 30 20f 5.3h

3 1 30 40e 36.7b*

2 30 50b 30c

3 30 40e 20e

With coconut water 5%

0 30 0j 0i

1 1 30 0j 0i

2 30 20f 0i

3 30 5i 5h

2 1 30 0j 0i

2 30 0j 0i

3 30 10h 10g

3 1 30 40e 40a

2 30 0j 0i

3 30 0j 0i

Means within the same letter are not significantly different by DMRT (P >

0.05).

amine somatic embryogenesis of the callus lines.

Degrees of somatic embryogenesis were totally af-

fected by callus lines and media. In general, explants

from all callus lines cultured on 1/2 MS basal media re-

sponded with embryo formation well. However, per-

centages of embryo formation and the number of em-

bryos were distinctly different along with the callus lines

used. Line 4 callus responded formation of embryos in all

treatments when they were cultured on 1/2 MS medium.

However, the highest number of embryos per explants

(30.3) was recorded on the media with 2.0 mg·L–1 AC.

When line 6 callus was used, the maximum average

number of embryos completely decreased (15.0/explant)

and the optimum concentration of AC was varied. Cul-

ture of line 8 callus on the same media responded with a

lower average number of embryos than that of line 6.

Although the callus lines originated from the same origin,

the line 8 attained from the highest concentration of

2,4-D (3 mg·L–1) was the lowest capacity for embryo-

genesis and also required the highest concentration of

AC (3 g·L–1) for optimum induction of somatic embryos.

It seems that the effect of higher concentration of 2,4-D

inhibited positively on somatic embryogenesis of later

experiments. Many workers also reported that the higher

concentrations of 2,4-D distinctly suppressed somatic

embryo formation in Oncidium [1,2] and in Phalaenopsis

[13]. The presence of 2,4-D in the culture medium

seemed to inhibit somatic embryogenesis in Citrus [14].

Thus, lower concentration of 2,4-D should be used in

callus induction to occupy higher somatic embryo forma-

tion capacity. As we discussed here, callus obtained from

hormone free medium might have the highest somatic

embryogenesis capacity as compared to those obtained

from 2,4-D containing media.

On MS medium, formation of embryos was rarely ob-

served from line 4 and line 6 calli and formation fre-

quency and number of embryos per callus were ex-

tremely low as compared to those on 1/2 MS medium.

Different capacities of somatic embryogenesis in callus

lines of the same origin were distinctly noticed in each

medium. On MS and 1/2 MS media, capacity of embryo

formation shows line 4 (30%, 5.0) > line 6 (20%, 2.0) >

line 8(0, 0), and line 4 (100%, 30.3) > line 6 (100%, 15.0)

> line 8 (70%, 12.0) (Tables 2, 3 and 4), respectively.

According to our findings herein, 1/2 MS medium in-

duced a higher number of somatic embryos (12 to 30 per

explant) as compared to MS media where the number of

embryos was 2 to 4 per explant. The obtained embryos

passed through the developmental stages, firstly the em-

bryogenic callus produced localized groups of cells, which

differentiated into embryogenic forms on the peripheral

region of the cluster old callus, showed induction of

rhizogenesis and trichome after 20 days of culture

Plant Regeneration through Indirect Somatic Embryogenesis in Coelogyne Cristata Orchid265

Table 2. Effect of different basal media and AC on somatic

embryogenesis of callus line 4 obtained from 1 mg·L–1 2,4-D

and 3 mg·L–1 BA combination.

Basal

medium

Activated

charcoal (g·L–1)

Forming

embryo (%)

No. of

embryos/explant

1.0 100a 5.0c

1.5 100a 5.6c

2.0 100a 30.3a

2.5 100a 8.0b

1/2 MS

3.0 100a 8.0b

1.0 0c 0d

1.5 0c 0d

2.0 30b 5.0c

2.5 0c 0d

3.0 0c 0d

Means within the same letter are not significantly different by DMRT (P >

0.05).

Table 3. Effect of different basal media and AC on somatic

embryogenesis of callus line 6 obtained from 2 mg·L–1 2,4-D

and 2 mg·L–1 BA combination.

Basal

medium Activated

charcoal (g·L–1) Forming

embryo (%) No. of

embryos/ explant

1.0 0d 0d

1.5 0d 0d

2.0 80b 3.3b

2.5 100a 15.0a

1/2 MS

3.0 0d 0d

1.0 0d 0d

1.5 0d 0d

2.0 0d 0d

2.5 20c 2c

3.0 0d 0d

Means within the same letter are not significantly different by DMRT (P >

0.05).

Table 4. Effect of different basal media and AC on somatic

embryogenesis of callus line 8 obtained from 3 mg·L–1 2,4-D

and 1 mg·L–1 BA combination.

Basal

medium Activated

charcoal (g·L–1) Forming

embryo (%) No. of

embryos/explant

1.0 0b 0c

1.5 0b 0c

2.0 0b 0c

2.5 70a 4b

1/2 MS

3.0 70a 13.5a

1.0 0b 0c

1.5 0b 0c

2.0 0b 0c

2.5 0b 0c

3.0 0b 0c

Means within the same letter are not significantly different by DMRT (P >

0.05).

(Figure 1(a)) and finally the process subsequently con-

tinued leading to formation of somatic embryos (Figure

1(b)). After 10 days of culture, the somatic embryos be-

came mature, green plumules emerged and completely

differentiated into individual plants. Roots with clear

visible root- cap and root-hairs were observed (Figures

1(c) and (d)).

Addition of higher concentrations of AC to 1/2 MS

medium showed even root and embryos formation from

the embryogenic clumps. In contrast to lower concentra-

tion of AC, formation of roots was much higher than

formation of embryos and the roots gradually died. No

morphological differences were found among the em-

bryos of callus lines cultured on 1/2 MS medium. How-

ever, there was slight morphological variation between

embryos obtained from MS and 1/2 MS media. It was

found that there was no roots formation from embryo-

genic callus clump cultured on MS medium containing

AC and obtained embryos seemed slow and had poor

growth, and did not differentiate into individual plantlets

until 8 weeks of culture. Plants obtained through somatic

embryogenesis of leaf derived callus cultured on 1/2 MS

media were almost uniform and looked quite healthy.

Recently, AC has been reported on somatic embryo-

genesis of some plants, Castanea dentata [15], Vitis spp.

[16], Myrciaria aureana [17]. However, using AC for

somatic embryogenesis in orchids is considerably rare. In

this study, AC could effectively stimulate formation of

somatic embryos in each line cultured on 1/2 MS media.

Plant Regeneration

The 1.0 cm high individual plants from multiple shoot

clusters were separated after 8 weeks of culture on above

media and transferred to 1/2 MS basal medium supple-

ment with 1 g·L–1 AC for rooting and shoot growth. After

2 weeks, formation of roots from each plant cultured on

the medium was distinctly observed. They grew faster

and became 4-cm-high plants within 8 weeks of culture.

Then, these plantlets that bore over 7 roots were trans-

ferred to small plastic pots containing moss and bark (2:1)

and acclimatized in the greenhouse. Cultural require-

ments such as watering, temperature control and fertili-

zation were done as necessary.

In conclusion, somatic embryos were indirectly in-

duced from leaf derived callus. Application of the callus

was appropriate for somatic embryogenesis in this spe-

cies. Formation of callus was achieved on MS medium

supplemented with 2,4-D and BA combinations. Callus

obtained from lower concentration of 2,4-D combination

was revealed to have a greater potential for the phe-

nomenon of somatic embryogenesis on 1/2 MS medium

with 2 mg·L–1 AC. Healthy plants developed through

somatic embryogenesis survived well when transplanted

Plant Regeneration through Indirect Somatic Embryogenesis in Coelogyne Cristata Orchid

266

Figure 1. Plant regeneration through somatic embryogenesis of leaf derived callus. (a) showing of clusters old callus, rhizogene-

sis and trichomes. (b) formation of somatic embryos. (c) fully developed somatic embryos. (d) conversion of somatic embryos

to shoots.

[2] J. T. Chen and W. C. Chang, “Effects of Auxins and Cy-

tokinins on Direct Somatic Embryogenesis on Leaf Ex-

plants of Oncidium ‘Gower Ramsey’,” Plant Growth

Regulation, Vol. 34, No. 2, June 2001, pp. 229-232.

doi:10.1023/A:1013304101647

in the greenhouse. This protocol is simple, easy to carry

out and can provide a large number of embryos and

plants for mass propagation in a short period of time. We

expect that this ability will also open up the prospect of

using biotechnological approaches for Coelogyne cristata

improvement. [3] H. H. Chung, J. T. Cheng and W. C. Chang, “Plant Re-

generation through Direct Somatic Embryogenesis from

Leaf Explants of Dendrobium,” Biologia Plantarum, Vol.

51, No. 2, June 2007, pp. 346-350.

doi:10.1007/s10535-007-0069-x

4. Acknowledgements

The authors acknowledge the financial support of Bio

Green 21 Program (code # 20070301034033), Rural De-

velopment Administration, Republic of Korea.

[4] J. F. Wu, J. T. Chen and W. C. Chang, “Effects of Auxins

and Cytokinins on Embryo Formation from Root-Derived

Callus of Oncidium ‘Gower Ramsey’,” Plant Cell, Tissue

and Organ Culture, Vol. 77, No. 1, April 2004, pp. 107-

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