synthetic seeds of endangered medicinal orchid species, dendrobium …icmr.crru.ac.th/journal 11/7....

Volume 6 Number 1, January 2018

127

Synthetic Seeds of Endangered Medicinal Orchid Species,

Dendrobium crumenatum Sw.

Sutha Klaocheed1and Suphat Rittirat2 1Faculty of Science and Technology,

Prince of Songkla University, Pattani campus, 2Faculty of Science and Technology,

Nakhon Si Thammarat Rajabhat University, Thailand.

ABSTRACT

Climate change and

anthropogenic pressure severely

threaten plant genetic diversity

worldwide. Numerous species are

described as rare or endangered, and

integrated programs are required to

protect and preserve current

biodiversity. Ex situ conservation

methods played an important role in

the conservation of threatened plants.

The main methods used in ex situ

conservation are maintenance of

living plants through cultivation, in

vitro conservation and encapsulation.

An in vitro plant regeneration protocol

was successfully established for

threatened medicinal species,

Dendrobium crumenatum Sw. by

culturing axillary buds. Protocorm-

like bodies (PLBs) of D. crumenatum

Sw. can be induced from callus

segments cultured on MS (Murashige

and Skoog, 1962) medium

supplemented with 0.5 mg/l

Thidiazuron (TDZ). The synthetic

seed technology is becoming popular

due to its wide application in

germplasm conservation and for

exchanges among countries in the

floriculture trade. In this study, this

method was used to study the bead

formations and the conversion

capabilities of D. crumenatum Sw. For

synthetic seed, the superior gel matrix

for encapsulation of D. crumenatum

Sw. was obtained using 3 % (w/v)

sodium-alginate and 100 mM calcium

chloride for 30 minutes. Successful

storage of capsules, until 105 days,

was achieved at 8 ± 2oC with

conversion frequency of 50.0 % when

culture on MS medium supplemented

with 0.2 % (w/v) activated charcoal

(AC). Well-rooted plantlets derived

from capsules were acclimatized in

the greenhouse with 95 % survival

rate. The regeneration protocol

developed in this study provides a

basis for ex-situ germplasm

conservation of medicinal importance

present in D. crumenatum Sw.


128

KEYWORDS

Dendrobium Crumenatum, Germ-

plasm Conservation, Micro-

propagation, PLB, Synthetic Seed,

Orchid

INTRODUCTION

The genus Dendrobium s.l.

(Epidendroideae) has in excess of

1,100 species of epiphytic orchids

with a wide distribution from Central

Asia throughout Australasia

(Kamemoto et al., 1999; Kumar et al.,

2011). This genus is one of the largest

among the Orchidaceae, the largest

family of angiosperms (Dressler,

2005; Fay and Chase, 2009). Species

within the Dendrobium genus are

highly prized ornamental assets,

primarily as potted plants with showy

flowers that tend to have a long vase

life (Vendrame et al., 2008). But the

most important aspect of many orchid

species, including Dendrobium

species, is their medicinal and

pharmaceutical value, particularly

Dendrobium crumenatum Sw., which

is abundantly used in traditional

medicine.

To counter exploitation from

wild resources, and to bolster

production of clonal material,

biotechnology-specifically micro-

propagation (Teixeira da Silva et al.,

2015), cryopreservation and low-

temperature preservation (Teixeira da

Silva et al., 2014)-serves as an

important tool for propagation and

preservation purposes (Roberts and

Dixon, 2008; Swarts and Dixon,

2009).

Although many commercial

Dendrobium hybrids are propagated

using clonal procedures, asymbiotic

seed propagation in Dendrobium has

major importance for the conservation

and propagation of wild species

because of loss of habitats and

overexploitation due to agriculture,

urbanization, over collection and

medicinal uses. Dendrobium orchids

are commonly used in traditional

medicine and many wild populations,

for example, of Dendrobium

crumenatum Sw., has become

drastically reduced due to over-

exploitation.

D. crumenatum Sw., com-

monly called pigeon orchid, is a

member of the family Orchidaceae. It

is native to India, Indochina, Taiwan,

Philippines, Malaysia, Indonesia,

New Guinea, and Christmas Island. It

is reportedly naturalized in Fiji,

Hawaii, the West Indies and the

Seychelles. It grows in many localities

from full sun to deep shade. D.

crumenatum Sw. produces upright,

sympodial, pseudobulbs that are

swollen at the first 3 or 4 bottom

nodes. The middle portion carries the

leaves of 7 cm long and 2 cm wide that

are very leathery. Top portion of the

pseudobulbs carries the flowers of

about 2.5 cm and of pure white, with

yellow markings on the labellum

(Figure 1). The bloom cycle is

triggered 9 days after a sudden drop in

temperature (at least 5.5°C or 10°F),

usually as a result of rain, although the

same effect can be artificially created.


129

D. crumenatum Sw. flowers are

fragrant, but the scent lasts only for

one day.

The encapsulation technique

for creating synthetic seeds is an

important application for in vitro

culture. Synthetic seeds have been

defined as artificially encapsulated

somatic embryos or non-embryogenic

in vitro-derived propagules and are

used for sowing under in vitro or ex

vitro condition (Murashige, 1977;

Aitkens-Christie et al., 1995;

Standardi and Piccioni, 1998).

Synthetic seed technology combines

the advantages of clonal propagation

with those of seed propagation (i.e.,

storability, easy to handle and

transport, protection against diseases

and pests). The most recent

application foresees the use of

synthetic seeds in medium and long-

term storage. To date, there are only a

few reports on micropropagation of D.

crumenatum Sw. No investigation has

so far been conducted on synthetic

seed production in this plant.

Therefore, the current study is meant

to optimize protocol for synthetic seed

production from D. crumenatum Sw.

Problem

Climate change and anthro-

pogenic pressure severely threaten

plant genetic diversity worldwide.

Numerous species are described as

rare or endangered, and integrated

programs are required to protect and

preserve current biodiversity. Ex situ

conservation methods played an

important role in the conservation of

threatened plants.

THE RESEARCH OBJECTIVES

The goal of this study was to

evaluate the effects of different

storage temperatures and time on

conversion of encapsulated

protocorm-like bodies (PLBs) of

Dendrobium crumenatum Sw., a

highly commercially important and

threatened medicinal orchid.

RESEARCH METHODOLOGY

Plant materials and surface

sterilization

Main shoots of Dendrobium

crumenatum Sw. (15-25 cm long)

were harvested from plants grown in

greenhouse at Faculty of Science and

Technology, Nakhon Si Thammarat

Rajabhat University. The stalks were

cut into the nodal segments each

holding one axillary bud. These nodal

segments (about 3-4 cm in length)

were first washed with tap water and a

few drop of detergent (Teepol), and

then rinsed with water 2-3 times. After

removing their sheaths, they were

surface sterilized with 20 % Clorox®

(5.25 % sodium hypochlorite, NaOCl)

containing 1-2 drops of Tween-20 for

20 minutes. The series of Clorox®

(5.25 % sodium hypochlorite, NaOCl)

percentage were used as 10 %, 5 % for

10 and 5 minutes, respectively. Finally

the excised buds were washed with

sterile distilled water 2-3 times and

cultured on MS (Murashige and

Skoog, 1962) medium supplemented

with 3 % (w/v) sucrose to promote bud

growth. The 4-week-old buds growing


130

on MS medium were transferred to

MS medium supplemented with 3 %

(w/v) sucrose, combination of 1.0

mg/l BA and 0.1 mg/l NAA, 0.2 %

(w/v) peptone and 0.2 % (w/v)

activated charcoal (AC) at pH 5.7 to

initiate callus. The callus proliferation

was observed after one month of

culture. These calli were then

transferred to the same medium. The

subculture monthly was recom-

mended to produce more totipotent

calli than the subsequent experiment.

For protocorm-like bodies (PLBs)

formation, shoot buds-derived calli at

1 month of culture from previous step

(MS medium supplemented with 3 %

(w/v) sucrose, combination of 1.0

mg/l BA and 0.1 mg/l NAA, 0.2 %

(w/v) peptone and 0.2 % (w/v) AC)

were transferred to MS medium

supplemented with 0.5 mg/l

Thidiazuron (TDZ) for 60 days of

culture.

Effects of different storage

conditions and intervals on their

conversion ability of Dendrobium

crumenatum Sw.

Individual protocorm-like

bodies (PLBs) of Dendrobium

crumenatum Sw. derived from MS

medium supplemented with 0.5 mg/l

TDZ for 60 days of culture was dipped

and drenched in 3 % (w/v) sodium-

alginate solution containing MS liquid

medium with 3 % (w/v) sucrose, free

of calcium and plant growth regulator

for 10 minutes. Aliquots of the

alginate solution, each containing one

PLB, were aseptically pipette out and

gently dropped individually with

Pasteur pipette into 100 mM calcium

chloride (CaCl2.2H2O) solution. The

droplets containing a PLB were then

allowed to polymerize for 30 minutes

to harden the alginate beads. The

resulting beads (7 - 8 mm in diameter)

were washed in sterile distilled water

for 3 times to remove the traces of

CaCl2.2H2O and transferred to sterile

filter paper in Petri dishes for 5

minutes under a laminar air-flow

cabinet to eliminate the excess of

water.

The encapsulated PLBs were

then placed in sterile Petri dishes (ten

beads/plate), sealed with parafilm and

in different shelves of a refrigerator at

temperature of 4 ± 2°C,

8 ± 2°C and 25 ± 2°C to be stored for

15, 30, 45, 60, 75, 90, 105 and 120

days. The Petri dishes were incubated

under dark conditions. About 30 beads

from each set stored in each

temperature regime were taken out

and cultured on MS medium

supplemented with 3 % (w/v) sucrose

with 0.2 % (w/v) activated charcoal

(AC) every 15 days. The encapsulated

PLBs grew out in the medium

rupturing the beads and were

maintained there for a development

into complete plantlets. The frequency

of conversion (%) was recorded every

15 days of culture.

The culture media were

solidified with 0.75 % (w/v) agar-agar

(commercial grade). The pH of MS

was adjusted to 5.7 with 1 N KOH or

1 N HCl prior to autoclaving for 15

minutes at 121°C. All cultures were

maintained at 25 ± 2°C under a 16 h

photoperiod with light supplied by


131

cool-white fluorescent lamps at an

intensity of 10 µmol m-2 s-1

photosynthetic photon flux density

(PPFD).

Greenhouse acclimatization

In vitro rooted plantlets

derived from encapsulated PLBs were

taken out from culture bottles and

rinsed thoroughly with tap water to

remove residual nutrients and agar

from the plantlets. The plantlets were

then transplanted to pots containing

sterilized coconut husks. All plantlets

were grown in the greenhouse with 70

- 80 % relative humidity and about 12

h photoperiod, 300 - 400 µmol m-2 s-1

photosynthetic photon flux density

(PPFD) (shaded sunlight) and 33 ±

2°C to 30 ± 2°C day/night

temperature. The young plants were

sprayed with water twice a day for 2

months.

Experimental design and statistical

analysis

All the experiments were

conducted in a completely

randomized design (CRD) with

5 replicates per treatment and the

experiments were repeated three

times. The results are expressed as

mean ± SE of three experiments. The

data were analyzed by ANOVA using

SPSS version 20 and the mean values

were separated using Duncan’s

multiple range test (DMRT) at a 5 %

probability level.

RESULTS AND DISCUSSION

Effects of different storage

conditions and intervals on their

conversion ability of Dendrobium

crumenatum Sw.

Dendrobium crumenatum Sw.

PLBs (2-month-old) of size ranging

from 0.5 - 0.6 cm in diameter were

encapsulated as synthetic seeds

prepared by alginate encapsulation,

and then stored in artificial endosperm

solution at 4 ± 2°C, 8 ± 2°C and 25 ±

2°C conditions in interaction with

different storage intervals of 15, 30,

45, 60, 75, 90, 105 and 120 days to

evaluate the comparative regrowth

capacity of synthetic seeds.

In this study, among the three

temperature regimes, temperature at 8

± 2°C storage gave promising results

for synthetic seeds conversion.

Conversion percentage of synthetic

seeds decreased from 100.00 % to

50.00 % until 105 days of storage at 8

± 2°C under dark conditions.

However, the PLBs stored more than

105 days in such condition gave no

germination (Table 1 and Figure 2). At

120 days of storage, synthetic seeds

that were stored in sterile Petri dishes

at 8 ± 2°C under dark conditions had

dried up and were unable to

germinate. Encapsulated PLBs stored

at 4 ± 2°C lost their viability

completely (Table 1). At 25 ± 2°C, the

encapsulated PLBs germinated when

storing in sterile Petri dishes under

dark conditions (Table 1). Storage at

room temperature (25 ± 2°C)

implemented in this study was

effective for short-term storage and


132

handling without refrigerated

containers, and even storage up to 45

days gave considerable conversion

(100.00 %) in D. crumenatum Sw.

Complete plantlets of D. crumenatum

Sw. developed from each capsules on

conversion medium, were

successfully transferred to ex vitro

conditions. Well-rooted plantlets

derived from capsules were

acclimatized in the greenhouse with

95 % survival. The regeneration

protocol developed in this study

provides a basis for ex-situ germplasm

conservation of medicinal importance

present in D. crumenatum Sw.

Figure 1. Dendrobium crumenatum Sw.

(A); Mature plants of Dendrobium crumenatum Sw. in natural habitat and

(B); flower of Dendrobium crumenatum Sw. shows pure glittering white and a

bright yellow disc on the lip (Scale bar, A = 3 cm and B = 1 cm).

A

B


133

Figure 2. Regeneration of encapsulated PLBs of Dendrobium crumenatum Sw.

for 4 weeks on MS medium supplemented with 0.2 % (w/v) activated charcoal

(AC).

(A); after storing at 8 ± 2°C for 15 days,

(B); 30 days,

(C); 45 days,

(D); 60 days,

(E); 75 days,

(F); 90 days,

(G) 105 days (Scale bar = 1 cm).


134

Table 1. Effects of different storage temperatures and time on conversion of encapsulated PLBs of Dendrobium crumenatum Sw.

Storage temperature Storage duration Conversion (%)

(°C) (days) (Mean ± SE)

4 ± 2°C 15 0.00 ± 0.00h

30 0.00 ± 0.00h

45 0.00 ± 0.00h

60 0.00 ± 0.00h

75 0.00 ± 0.00h

90 0.00 ± 0.00h

105 0.00 ± 0.00h

120 0.00 ± 0.00h

8 ± 2°C 15 100.00 ± 0.00a

30 96.67 ± 3.33b

45 88.33 ± 1.67c

60 78.33 ± 1.20d

75 70.00 ± 1.15e

90 62.33 ± 1.45f

105 50.00 ± 1.15g

120 0.00 ± 0.00h

25 ± 2°C 15 100.00 ± 0.00a

30 100.00 ± 0.00a

45 100.00 ± 0.00a

60 *

75 *

90 *

105 *

120 *

Similar letters within the same columns mean no significant difference at P ≤

0.05 by DMRT.

*Encapsulated PLBs germinated when storing in sterile Petri dishes.

Gantait et al. (2012) and Gantait and

Sinniah (2013) observed that

encapsulated PLBs of Aranda Wan

Chark Kuan ‘Blue’ x Vanda coerulea

Grifft. ex. Lindl. and encapsulated

shoot tips of monopodial orchid

hybrid Aranda Wan Chark Kuan

‘Blue’ x Vanda coerulea Grifft. ex.

Lindl. could generally maintain

maximum germination percentage

when stored at 4°C for 90 and 160

days; however, there is a decrease in

the conversion percentage to plantlets

when the storage at low temperature is


135

prolonged Lambardi et al. (2006). In

this study, among the three

temperature regimes, encapsulated

PLBs stored at 4 ± 2°C lost their

viability completely due to the

fluctuation in temperature and cold

stress during the storage. The failure

of prolonged storage in 4 ± 2°C was

also described in earlier reports

(Pradhan et al., 2014) where, low

temperature (4 ± 2°C) storage of

artificial seeds of Cymbidium

aloifolium was rather short. Similarly,

the germination rate of encapsulated

protocorm of Cymbidium bicolor

Lindl. was reported to be low

(Mahendran, 2014) under storage in

this temperature. Likewise, the

conversion of encapsulated nodal

segments of Punica granatum L. also

showed markedly decline, following

storage at low temperature (Naik and

Chand, 2006). However, the response

of synthetic seeds to storage

temperature appears to be species

specific. Some responds to either 4°C

(Saiprasad and Polisetty, 2003; Lisek

and Olikowska, 2004; Singh et al.,

2010; Sharma and Shahzad, 2012;

Gantait et al., 2012; Gantait and

Sinniah, 2013) or room temperature

(Devi et al., 2000; Mohanraj et al.,

2009; Hung and Trueman, 2011;

Gantait et al., 2012; Gantait and

Sinniah, 2013).

From the success of the present

study, encapsulation of PLBs of this

orchid appears to be a promising tool

for storage and on-demand supply of

plant material for propagation or

germplasm exchange. Similar use of

the encapsulation method for storage

have also been reported earlier in

many other endemic and endangered

orchids like Cymbidium bicolor

(Wood et al., 2011) and Ipsea

malabarica (Hartman et al., 1997).

The in vitro storage achieved for D.

crumenatum Sw. in our study has the

prospective to cut the cost for

maintaining the continuous

proliferating PLB cultures because of

the abridged requirement for manual

labor due to less frequent subculture.

According to Rai et al. (2008), an

important feature of the encapsulated

vegetative propagules is their

capability to retain viability after

storage for a sufficient period required

for exchange of germplasm.

CONCLUSIONS AND RECOM-MENDATIONS

1. Successful storage of

Dendrobium crumenatum Sw.’s

capsules, until 105 days, was achieved

at 8 ± 2oC with conversion frequency

of 50.0 % when culture on MS

medium supplemented with 0.2 %

(w/v) AC.

2. This study developed highly

effective techniques for synthetic seed

production, short-term conservation

and regeneration of plantlets. The

synthetic seed development protocol

illustrated here offers a substitute

scheme for mass propagation and

germplasm distribution of this

commercially important and

threatened orchid species to

laboratories and extension centers in

distant places.


136

ACKNOWLEDGEMENTS

This research was financially

supported by Prince of Songkla

University, Pattani campus, Pattani

94000, Thailand (Grant No. 59005).

The authors would like to thank the

Department of Technology and

Industries, Faculty of Science and

Technology, Prince of Songkla

University, Pattani campus, Pattani

94000, Thailand, for providing

laboratory facilities for this

investigation.

BIBLIOGRAPHY

Aitkens-Christie, J., Kozai, T. and

Takayama, S. 1995. Auto-

mation in plant tissue culture:

General introduction and

overview. In Automation and

Environmental Control in

Plant Tissue Culture; Aitken-

Christie, J., Kozai, T. and

Smith, M.A.L., Eds.; Kluwer

Academic Publication:

Dordrecht, The Netherlands,

p. 1-18.

Devi, M., Sharma, J. and Sarma, A.

2000. In vitro culture of

artificial seeds of Vanda

coerulea an endangered

orchid. Research on Crops.

1(2): 205-207.

Dressler, R.L. 2005. How many

orchid species? Selbyana. 26:

155-158.

Fay, M.F. and Chase, M.W. 2009.

Orchid biology: from

Linnaeus via Darwin to the

21st century. Ann. Bot. 104:

359-364.

Gantait, S., Bustam, S. and Sinniah,

U.R. 2012. Alginate-

encapsulation, short-term

storage and plant regeneration

from protocorm-like bodies of

Aranda Wan Chark Kuan

‘Blue’ x Vanda coerulea

Grifft. ex. Lindl.

(Orchidaceae). Plant Growth

Regul. 68: 303-311.

Gantait, S. and Sinniah, U.R. 2013.

Storability, post-storage

conversion and genetic

stability assessment of

alginate-encapsulated shoot

tips of monopodial orchid

hybrid Aranda Wan Chark

Kuan ‘Blue’ x Vanda coerulea

Grifft. ex. Lindl. Plant

Biotechnol Rep. 7: 257-266.

Hartman, H., Kester, D., Davis, F. and

Geneve, R. 1997. Plant

propagation; Principles and

Practices, 6th ed. Prentice-hall,

New Jersey, p. 125-144.

Hung, C.D. and Trueman, S.J. 2011.

Encapsulation technology for

short-term preservation and

germplasm distribution of the

African mahogany Khaya

senegalensis. Plant Cell

Tissue Organ Cult. 107(3):

397-405.


137

Kamemoto, H., Kuehnle, A.R. and

Amore, T.D. 1999. Breeding

Dendrobium orchids in

Hawaii. University of Hawaii

Press, Honolulu.

Kumar, P., Rawat, G.S. and Wood,

H.P. 2011. Diversity and

ecology of Dendrobiums

(Orchidaceae) in Chotanagpur

plateau, India. Taiwania. 56:

23-36.

Lambardi, M., Benelli, C., Ozudogru,

E.A. and Ozden-Tokatli, Y.

2006. Synthetic seed

technology in ornamental

plants. In: Teixeira da Silva J.

(ed). Floriculture, Ornamental

and Plant Biotechnology, Vol.

II, Global Science Books, UK,

p. 347-354.

Lisek, A. and Olikowska, T. 2004. In

vitro storage of strawberry and

raspberry in calcium-

alginate beads at 4°C. Plant

Cell Tissue Organ Cult. 78:

167-172.

Mahendran, G. 2014. Encapsulation

of Protocorm of Cymbidium

bicolor Lindl. for Short-

Term Storage and Germplasm

Exchange. JOP. 4: 17-27.

Mohanraj, R., Ananthan, R. and Bai,

V.N. 2009. Production and

storage of synthetic seeds

in Coelogyne breviscapa

Lindl. Asian J. Biotechnol. 1:

124-128.

Murashige, T. and Skoog, F. 1962. A

revised medium for rapid

growth and bioassays with

tobacco tissue cultures.

Physiol Plant. 15: 473-497.

Murashige, T. 1977. Plant cell and

organ cultures as horticultural

practices. Acta Hortic. 78:

17-30.

Naik, S.K. and Chand, P.K. 2006.

Nutrient-alginate encapsula-

tion of in vitro nodal segments of pomegranate (Punica

granatum L.) for germplasm

distribution and exchange.

Sci.Hort. 108: 247-252.

Pradhan, S., Tiruwa, B., Subedee,

B.R. and Pant, B. 2014. In

vitro germination and

propagation of a threatened

medicinal orchid, Cymbidium

aloifolium (L.) Sw. through

artificial seed. Asian Pac J

Trop Biomed. 4: 971-976.

Rai, M.K., Jaiswal, V.S. and Jaiswal,

U. 2008. Encapsulation of

shoot tips of guava (Psidium

guajava L.) for short-term

storage and germplasm

exchange. Sci. Hortic. 118: 33-

38.

Roberts, D.L. and Dixon, K.W. 2008.

Orchids. Curr Biol. 18: R325-

R329.

Saiprasad, G.V.S. and Polisetty, R.

2003. Propagation of three

orchid genera using

encapsulated protocorm-like

bodies. In Vitro Cell. Dev.

Biol.—Plant. 39: 42-48.


138

Sharma, S. and Shahzad, A. 2012.

Encapsulation technology for

short-term storage and

conservation of a woody

climber, Decalepis hamiltonii

Wight and Arn. Plant Cell

Tissue Organ Cult. 111: 191-

198.


139

Singh, S.K., Rai, M.K., Asthana, P.

and Sahoo, L. 2010. Alginate-

encapsulation of nodal

segments for propagation,

short-term conservation and

germplasm exchange and

distribution of Eclipta alba

(L.). Acta Physiol. Plant. 32:

607-610.

Standardi, A. and Piccioni, E. 1998.

Recent perspectives on

synthetic seed technology

using non-embryogenic in

vitro-derived explants. Int. J.

Plant Sci. 159: 968-978.

Swarts, N.D. and Dixon, K.W. 2009.

Terrestrial orchid conservation

in the age of extinction. Ann

Bot. 104: 543-556.

Teixeira da Silva, J.A., Zeng, S.J.,

Dobranszki, J., Cardoso, J.C.

and Kerbauy, G.B. 2014.

In vitro flowering of

Dendrobium. Plant Cell

Tissue Organ Cult. 119: 447-

456.

Teixeira da Silva, J.A., Cardoso, J.C.,

Dobranszki, J. and Zeng, S.

2015. Dendrobium

micropropagation: a review.

Plant Cell Rep. 34: 671-704.

Vendrame, W.A., Carvalho, V.S.,

Dias, J.M.M. and Maguire, I.

2008. Pollination of

Dendrobium hybrids using

cryopreserved pollen. Hort

Science. 43: 264-267.

Wood, J.., Beaman, R.S., Repin, R.

and Vermulen, J.J. 2011. The

Orchids of Mount

Kinabalu, Vol II. Natural

history publications, Borneo,

p. 1-436.

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