An investigation on the distribution and genetic diversity of Eurycoma longifolia Jack, and in vitro conservation of this valuable medicinal tree in Thua Thien Hue, Vietnam

Abstract

E. longifolia is a valuable medicinal tree and has a long history of usage in traditional medicine in
Asia. However, overexploitation is currently causing the depletion of this species in many places. A
preliminary study was carried out to get an idea on the distribution and genetic diversity of
E. longifolia in Thua Thien Hue Province, Vietnam, and in vitro culture of this valuable medicinal
species for conservation. The results indicate that E. longifolia distributes in two main site types:
sandy soil and mountains. In mountains, they can live at an elevation of over 1000 m, though mainly
focused at 300-700 m. RAPD markers were used to assess the genetic diversity of E. longifolia
collected from 3 natural populations. A total of 13 individuals were analyzed using 10 RAPD
primers and produced 73/394 (18.5%) polymorphic DNA fragments. The UPGMA dendrogram
formed 4 significant clusters (Phu Loc at an elevation <100 m, Nam Dong >200 m, Phong Dien, and
Nam Dong <200 m). Micropropagation through in vitro germination of seeds and direct shoot
regeneration from in vitro explants was conducted. About 50% of seeds germinated after 11 days of
culture and the highest percentage (70%) was obtained from seeds of yellow fruits. The stem
segments from in vitro seedlings had the highest number of regenerated shoots (4.8 shoots/explant)
with 1.2 cm in height on the medium MS supplemented with 0.5 mg/L BAP.

Full text

Plant Cell Biotechnology and Molecular Biology XX(X&X):XX–XX;20YY ISSN: 0972-2025
AN INVESTIGATION ON THE DISTRIBUTION AND
GENETIC DIVERSITY OF Eurycoma longifolia JACK, AND
in vitro CONSERVATION OF THIS VALUABLE
MEDICINAL TREE IN THUA THIEN HUE, VIETNAM
NGUYEN HOANG LOC
*
, PHAM THI NGOC LAN, LE THI HA THANH,
NGUYEN VIET THANG, NGUYEN NGOC LUONG, TRAN MINH DUC,
VAN THI YEN, NGUYEN HOI, HO THI NGOC TU AND PHAM HOAI DOANH
College of Sciences, Hue University, Hue 530000, Vietnam [NHL, PTNL, LTHT, NVT, NNL,
HTNT, PHD]
College of Agriculture and Forestry, Hue University, Hue 530000, Vietnam [TMD, VTY, NH]
[*For Correspondence: E-mail: nhlocs@gmail.com]
ABSTRACT
E. longifolia is a valuable medicinal tree and has a long history of usage in traditional medicine in
Asia. However, overexploitation is currently causing the depletion of this species in many places. A
preliminary study was carried out to get an idea on the distribution and genetic diversity of E.
longifolia in Thua Thien Hue Province, Vietnam, and in vitro culture of this valuable medicinal
species for conservation. The results indicate that E. longifolia distributes in two main site types:
sandy soil and mountains. In mountains, they can live at an elevation of over 1000 m, though mainly
focused at 300-700 m. RAPD markers were used to assess the genetic diversity of E. longifolia
collected from 3 natural populations. A total of 13 individuals were analyzed using 10 RAPD
primers and produced 73/394 (18.5%) polymorphic DNA fragments. The UPGMA dendrogram
formed 4 significant clusters (Phu Loc at an elevation <100 m, Nam Dong >200 m, Phong Dien, and
Nam Dong <200 m). Micropropagation through in vitro germination of seeds and direct shoot
regeneration from in vitro explants was conducted. About 50% of seeds germinated after 11 days of
culture and the highest percentage (70%) was obtained from seeds of yellow fruits. The stem
segments from in vitro seedlings had the highest number of regenerated shoots (4.8 shoots/explant)
with 1.2 cm in height on the medium MS supplemented with 0.5 mg/L BAP.
Keywords: Eurycoma longifolia; in vitro conservation; medicinal plant; genetic diversity; distribution.
INTRODUCTION
Eurycoma longifolia Jack is a popular medicinal
plant, member of Simaroubaceae, widely
distributed in South East Asian countries such as
Indonesia, Malaysia, Thailand, Vietnam, and Laos
(Rosmaina and Zulfahmi 2013). E. longifolia is
generally found in shrub or tree forms of which
the height can reach up to 10 m (Kartikawati et al.
2014) and the diameter of the stem could be
approximately 15 cm (Burkill 1966). The root of
E. longifolia has been used in traditional medicine
of many Asian countries to restore energy and
vitality, enhance blood flow and have function as
herbal ingredient for women after child birth
(Ismail et al. 1999). Due to its therapeutic benefits,
E. longifolia was overexploited causing the
depletion of this medicinal species in nature
(Rosmaina and Zulfahmi 2013).
Nordin (2014) investigated the distribution of
Eurycoma spp. populations in Malaysia.
Kartikawati et al (2014) also studied the habitat
preferences, distribution pattern, and root weight
estimation of E. longifolia in Indonesia. Osman et
al (2003) and Razi et al (2013) estimated genetic
diversity of some E. longifolia populations in
Malaysia by single nucleotide polymorphism
(SNP) and random amplified polymorphic DNA
(RAPD) analysis. Rosmaina and Zulfahmi (2013)

Loc et al.
analyzed genetic diversity of E. longifolia
populations in Riau Province, Indonesia.
There were some reports on E. longifolia tissue
culture such as shoot multiplication from in vitro
germinated seedlings (Hasnida et al. 2001),
somatic embryogenesis and shoot regeneration
from embryo (Hussein et al. 2005a, Dalila et al.
2015), micropropagation from different parts of
plant (Hussein 2005b and 2006, Hassan et al.
2012), induction of adventitious roots from in
vitro plantlets (Hussein et al. 2012), and
acclimation of E. longifolia plantlets in ex vitro
conditions (Yahya et al. 2015).
However, there is very little information on
distribution and genetic variation of E. longifolia
species in Vietnam. Until now, we only found a
report of Men et al (2014) on the populations of
this species in Lam Dong Province. In this work,
we preliminary investigated the distribution and
genetic diversity of E. longifolia species in various
ecological systems in Thua Thien Hue Province
(Vietnam), and conducted micropropagation to
conserve this valuable medicinal plant under in
vitro condition.
MATERIALS AND METHODS
The studies on Eurycoma longifolia species (Fig.
1) were conducted in the mountains of Phu Loc
and Nam Dong Districts, and the sandy area of
interior field of Phong Dien District in Thua Thien
Hue Province, Vietnam. The mountain area has an
elevation that reached over 1000 m asl.
Fig. 1. E. longifolia tree distributes in Bach Ma
National Park (Phu Loc)
Data Collection
Data collection to assess the distribution of E.
longifolia was performed using standard plot
survey method (20 for sandy soil areas and 36 for
mountain areas). Each plot has the area of 500 m
2
(25×20 m), and the data recorded in each plot
consisted of number of individuals, height (H
vn
),
stem diameter (D
0
), and branching.
There are three standard plots on a survey line
(approximately 1 km), and the distance between
each plot is 300 m. Sampling locations of E.
longifolia was determined by GPS navigation.
RADP Analysis
Genomic DNA of E. longifolia was isolated by the
CTAB (cetyltrimethylammonium bromide)
method from Babu et al (2014) with slight
modification. For each of 13 individuals (Table 1),
5 g of young leaf was homogenized using liquid
nitrogen with porcelain mortar and pestle to a fine
powder.
All fine powder and 5 ml of 1.5× CTAB were
placed into a 50 mL-Falcon tube and mixed well
for 15 s, followed by adding 2% β-
mercaptoethanol and mixing well for 10 s. The
sample was incubated at 60°C for 1 h with
occasional mixing, and then centrifuged at 13,000
rpm at 4
o
C for 20 min to obtain supernatant. An
equal volume of chloroform/isoamyl alcohol (24:1
v/v) was added to each sample. The sample was
vortexed briefly and then centrifuged for 10 min at
13,000 rpm/4
o
C. The supernatant was transferred
to a new tube, an equal volume of ice-cold
isopropanol was added to the tube, the tube was
then inverted 5 times to precipitate the nucleic
acid at -20
o
C for 1 h. The sample was centrifuged
at 13,000 rpm for 10 min at 4
o
C, and the
supernatant was discarded. The pellet was washed
by 700 µL of 70% cold ethanol and air-dried for
15 min and then resuspended in double-distilled
water and stored at -20
o
C.
DNA extracted from the 13 individuals of E.
longifolia was used for PCR. The sequence of the
primers is indicated in Table 2. PCR amplification
was performed in 20 µL reactions using 50 ng
genomic DNA, 10 µ L master mix solution (2×

Loc et al.
GoTaq® Green Master Mix, Promega, Cat No:
M7122) and 20 pmol each primer. PCR conditions
were 94°C for 3 min; 94°C for 1 min, 36°C for 1
min, and 72°C for 2 min, for 43 cycles; and a final
extension step at 72
o
C for 6 min. After PCR, 10
µL of the amplified DNA was separated on a 1.5%
agarose gel at 40 V, and DNA bands were
visualized by ethidium bromide staining.
Results of RAPD was analyzed by NTSYS-pc
program (ver. 2.1) with NEI72 coefficient (Nei
1972). Then, cluster analysis was performed by
means of SAHN procedure (Rohlf 1994).
Plant Tissue Culture
The source of E. longifolia used in this study was
obtained from Bach Ma National Park (Phu Loc
District).
Seeds of E. longifolia with the endocarp removed
were initiated by washing under running tap water
for 30 min followed by surface sterilization using
70% ethanol and 1 drop of Tween 20 for 1 min,
and then by 1% AgNO
3
for 20 min. Seeds were
rinsed 5 times with sterile distilled water and then
transferred to the solid nutrient medium [MS
medium (Murashige and Skoog 1962)
supplemented with 2 mg/L BAP
(benzylaminopurine), 6 mg/L AgNO
3
, 30 g/L
sucrose, 8 g/L agar, and pH of 5.8] according to
Hassan et al (2012) with slight modification for
germination. After 2 months, in vitro shoots were
cut into shoot tips, stem segments and stem base
(about 2 cm in length) to subculture on shoot
multiplication medium containing 0.5 mg/L BAP
(Hassan et al. 2012).
The seedlings (20-30 cm in height) were initially
washed under running tap water for 30 min. The
explants were then cut into shoot tips (about 3 cm)
followed by treatment with 3% NaClO for 5 min
and then 0.1% HgCl
2
and 1 drop of Tween 20 for
6.5 min. The explants were washed 5 times with
sterilized water and cultured on the MS medium
supplemented with 0.5 mg/L BAP, 30 g/L sucrose,
8 g/L agar, and pH of 5.8 for shoot multiplication
(Hassan et al. 2012).
In vitro culture condition: temperature of 25±2
o
C,
light intensity of 2000-3000 lux with a
photoperiod of 12 h/day.
Table 1. E. longifolia sampling locations in Thua Thien Hue
No Coordinates Altitude (m) Locations
Latitude (X) Longitude (Y) Error (m)
1 16.248907 107.867544 ± 4 64 Bach Ma National Park (Phu Loc)
2 16.219565 107.723730 ± 2 165
Huong Phu Commune (Nam
Dong)
3 16.192874 107.736579 ±5 268
4 16.224731 107.730102 ± 3 333
5 16.228115 107.729569 ± 2 468
6 16.229425 107.733027 ± 3 565
7 16.230636 107.733698 ± 4 630
8 16.235571 107.732691 ± 3 755
9 16.236840 107.732326 ± 5 823
10 16.238125 107.732166 ± 5 855
11 16.638442 107.371444 ± 4 30-40 Phong Binh Commune (Phong
Dien) 12 16.638897 107.370428 ± 2 30-40
13 16.639857 107.370946 ± 1 30-40
Table 2. Random primers use for RAPD-PCR (Operon Technologies, Alameda, CA, USA).
No Primers Sequences (5'-3')
1 OPA-02 TGCCGAGCTG
2 OPA-11 CAATCGCCGT
3 OPA-17 GACCGCTTGT
4 OPB-05 TGCGCCCTTC
5 OPB-10 CTGCTGGGAC
6 OPC-02 GTGAGGCGTC
7 OPL-14 GTGACAGGCT
8 OPM-18 CACCATCCGT
9 OPM-10 TCTGGCGCAC
10 OPAD-10 AAGAGGCCAG

Loc et al.
Statistical Analysis
Each experiment was repeated three times with at
least ten replicates to calculate the mean and
standard error.
RESULTS AND DISCUSSION
Distribution of E. longifolia
The study showed that E. longifolia species was
distributed in two main site types in Thua Thien
Hue: sandy soil and mountain. The data in Table 3
indicate that the density of mature E. longifolia in
sandy soil area (164 trees/ha) is higher than in
mountain areas (150 trees/ha). Meanwhile the
density of natural regenerated trees exhibited an
opposite pattern (109 trees/ha vs 373.33 trees/ha).
In the sandy soil area, D
0
of trees (6.34 cm) is
wider than that of the trees distributed in
mountains (5.03 cm). E. longifolia trees in the
mountains are often non-branched and their size
reached approximately 3.2 m in height. However,
trees growing in sandy soil had approximately 5
branches with an average height of 2.96 m.
The distribution and the growth status of E.
longifolia were also investigated at different
elevations. Table 4 shows this species mainly
focuses at elevations below 1000 m (110-171
trees/ha), and their density only remains about 20
trees/ha at an elevation above 1000 m. At 300-700
m, the growth of E. longifolia is relatively stable,
but below 100 m, the trees are already exploited or
destroyed indiscriminately due to distribution near
the residential area. Individuals that have already
been felled may stimulate new growth, so the
number of the branches increased 6 times compare
to the normal trees, but their D
0
and H
vn
decreased
obviously.
Genetic Diversity of E. longifolia
Out of 10 primers, 9 primers showed good
amplifications and 1 random primer (OPM-18) did
not give any amplification products. Of 9 primers
which showed amplifications, the intensity of two
primers (OPM-10 and OPL-14) was low and only
seven primers had good fragment intensity (OPA-
11, OPB-10, OPA-02, OPA-10, OPA-17, OPB-05,
and OPC-02). OPA-11 had the highest number of
polymorphic DNA bands (11 bands) and OPL-14
the lowest with only 5 bands. 321 (81.5%) of the
total 394 DNA-fragments were found to be
monomorphic. The remaining 73 (18.5%) were
polymorphic (Fig. 2).
A dendrogram of 13 E. longifolia individuals was
generated by the NTSYSpc 2.1 program based on
the results of RAPD-PCR. This showed a close
relationship of the individuals in the same location
of distribution (sandy soil or mountains) with four
significant clusters (Fig. 3). This result indicates
the genetic differences between the populations in
various ecosystems or in the same ecosystem but
at different elevation (<200 m and >200 m).
Study of Rosmaina and Zulfahmi (2013) on
genetic diversity of E. longifolia populations in 5
districts of Riau Province (Indonesia) showed that
44 scored DNA bands with two significant
clusters. Razi et al (2013) analyzed RAPD of E.
longifolia cultivars from 7 different locations in
Malaysia. The results indicated 249 of 320 DNA
fragments (78%) were polymorphic. The
dendrogram showed that E. longifolia cultivars
can be clustered into three main clusters.
In vitro Culture of E. longifolia
Nearly 50% of E. longifolia seeds germinated
after 11 days of culture on the MS medium
supplemented with 2 mg/L BAP. The study results
presented in Table 5 shows that the seed
physiology influenced the germination. The
germination rate of seeds without endocarp from
yellow fruits is the highest (70%), followed by
young green fruits (50%) and the lowest rate for
seeds from ripe blackish-red fruits (34.8%). In our
thinking when the fruits are too ripe (about to
fall), their seeds with very thick and hard coats
will be difficult to germinate. While in young
fruits, because their embryos are immature so
germination rate is also lower. Our results on in
vitro germination of E. longifola are similar to
results of Keng et al (2002). According to
Broschat and Donselman (1987), the seeds of
Roystonea regia from ripe fruits germinated more
slowly than seeds from half-ripe or green fruits,
but fewer of the unripe seeds ultimately
germinated.

Loc et al.
Table 3. Characteristics of E. longifolia species distributed in two site types in Thua Thien Hue
Region Number of mature
trees (>1 m)/ha
D
0
(cm) H
vn
(m) Number of
branches/tree
Regenerated trees
(0.2-1 m)/ha
Sandy soil 164 6.34 2.96 4.70 109.00
Mountain 150 5.03 3.20 1.56 373.33
Table 4. Characteristics of E. longifolia species distributed by altitude in Thua Thien Hue
Characteristics Elevation (m)
<100 100-300 300-500 500-700 700-1000 >1000
Number of trees/ha
140 156 170 171 110 20
Number of branches/tree 6.71 2.54 1.05 1.00 1.00 1.00
D
0
(cm) 1.20 5.13 5.72 4.85 5.05 3.73
H
vn
(m) 1.50 3.03 3.45 3.47 2.31 3.07
Fig. 2. RAPD analysis of 13 E. longifolia individuals distributed in Thua Thien Hue.

Loc et al.
Fig. 3. Dendrogram of 13 E. longifolia individuals was established by UPGMA cluster analysis based
on NEI’s genetic distance (1972)
Table 5. In vitro germination of E. longifolia seeds (n=94)
Young green fruit (n=18) Yellow fruit (n=30) Ripe blackish-red fruit (n=46)
9 (50%) 21 (70%) 16 (34.8%)
Total: 46 (48.9%)
After 2 months of culture, the in vitro seedlings
reached a height of approximately 4 cm and
looked healthy (Fig. 4). The shoot tips, stem
segments and stem base (about 1 cm in length)
were excised from in vitro seedlings and
subcultured on the medium of shoot
multiplication.
After 15 days of culture, the shoot tips were
isolated from E. longifolia growing under natural
condition began shoot elongation and new shoot
formation. Table 6 presents the results of 2 months
of culture. In general, the shoot regeneration
seemed not strongly, perhaps due to E. longifolia
is a recalcitrant tropical woody plant (Hussein et
al. 2012).
These in vitro shoots and other explant types
(shoot tips, stem segments and stem base) from in
vitro seedlings were subcultured on the MS
medium with 0.5 mg/L BAP for shoot
multiplication. As can be seen from Table 7 and
Fig. 5, the stem segments had the highest number
of regenerated shoots (4.8 shoots) with 1.2 cm in
height, shoot tips with 3.3 shoots/2.8 cm, while
stem base had only 1.3 shoots/1.7 cm.
Table 6. Shoot regeneration from shoot tips (n = 98) after 8 weeks of culture
Number of shoot regeneration explants Number of shoots/explant Shoot height (cm)
15 (15.3%) 2.1 ± 0.5 0.9 ± 0.2
Table 7. Shoot multiplications from in vitro shoots (after 8 weeks of culture)
Stem segments Shoot tips Stem base
Number of shoots/explant 4.8 ± 2.6 3.3 ± 0.9 1.3 ± 0.3
Height (cm) 1.2 ± 0.3 2.8 ± 0.7 1.7 ± 0.5

Loc et al.
Fig. 4. E. longifolia plantlets germinated from seeds after 2 months of culture
Fig. 5. Shoot regeneration from shoot tip
culture.
Keng et al (2002) found that seeds, of which the
endocarp were removed, have germinated after 2
weeks of culture on the MS medium. According to
Hassan et al (2012), the germination of E.
longifolia seeds on the WPM (woody plant
medium) or MS medium supplemented with 2
mg/L BAP can reach to 1.5 or 2.75 shoots/explant,
respectively. The ratio of shoot multiplication is
approximately 1.82 on the MS medium with 0.5
mg/L BAP. The study of Hussein et al (2005b)
obtained the highest regeneration percentage
(90%) with 4 shoots/explant on the MS medium
containing 5 mg/L kinetin.
CONCLUSION
In conclusion, the present work indicates that E.
longifolia distributes in two main site types in
Thua Thien Hue Province, Vietnam: sandy soil
and mountains. In mountains, they mainly focused
at an elevation of 300-700 m. A total of 13
individuals from 3 natural populations were
analyzed using 10 RAPD primers and produced
73/394 (18.5%) polymorphic DNA fragments. The
UPGMA dendrogram formed 4 significant
clusters. About 50% of seeds germinated after 11
days of in vitro culture and the highest percentage
(70%) was obtained from seeds of yellow fruits.
The stem segments from in vitro seedlings had the
highest number of regenerated shoots (4.8
shoots/explant) on the medium MS supplemented
with 0.5 mg/L BAP.
ACKNOWLEDGEMENTS
This work was supported by Vietnam Ministry of
Education and Training (Code No. B2015-15-
08GEN).
COMPETING INTERESTS
Authors have declared that no competing interests
exist.
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