Effect of Plant Growth Regulator and Explant Types on in vitro Callus Induction of Gynura procumbens (Lour.) Merr

Abstract

Gynura procumbens (Lour.) Merr is a medicinal plant of Asteraceae. The plant is a potent compound as pharmaceutical raw material which can be increased through plant tissue culture. This study aimed to determine the effect of various combinations of plant growth regulator and explant types on induction callus. The explants used were leaf, stem node, stem internode and petiole while the plant growth regulator used were 0.1 mg/L 2,4-D and 0.1 mg/L BAP, 0.5 mg/L 2,4-D and 1.0 mg/L Kinetin, 0.5 mg/L NAA and 0.5 mg/L BAP, 5.0 mg/L 2,4 D and 0.5 mg/L BAP, 0.1 mg/L 2,4-D and 0.1 mg/L IAA. The explants were cultured in MS medium supplemented with 30% sucrose and 8% agar for 28 days. The results of this study indicated that the treatment of 0.5 mg/L NAA and 0.5 mg/L BAP on the petiole explants was the best combination of plant growth regulators to produce the highest callus fresh and dry weights (1478.1 mg and 40.0 mg respectively). Callus derived from leaf, petiole and internode explants was friable and compact in texture while node explant-derived callus was compact in texture.

Full text

Research Journal of Biotechnology Vol. 14 (9) September (2019)
Res. J. Biotech
102
Effect of Plant Growth Regulator and Explant Types
on in vitro Callus Induction of Gynura procumbens
(Lour.) Merr
Amin Nurokhman1, Hanik Faizah2, Sugiharto1, Edy Setiti Wida Utami1 and Yosephine Sri Wulan Manuhara1*
1. Department of Biology, Faculty of Science and Technology, Airlangga University, Surabaya, INDONESIA
2. Department of Biology, Faculty of Science and Technology, UIN Sunan Ampel, Surabaya, INDONESIA
*wulanmanuhara@gmail.com
Abstract
Gynura procumbens (Lour.) Merr is a medicinal plant
of Asteraceae. The plant is a potent compound as
pharmaceutical raw material which can be increased
through plant tissue culture. This study aimed to
determine the effect of various combinations of plant
growth regulator and explant types on induction callus.
The explants used were leaf, stem node, stem internode
and petiole while the plant growth regulator used were
0.1 mg/L 2,4-D and 0.1 mg/L BAP, 0.5 mg/L 2,4-D and
1.0 mg/L Kinetin, 0.5 mg/L NAA and 0.5 mg/L BAP, 5.0
mg/L 2,4 D and 0.5 mg/L BAP, 0.1 mg/L 2,4-D and 0.1
mg/L IAA. The explants were cultured in MS medium
supplemented with 30% sucrose and 8% agar for 28
days.
The results of this study indicated that the treatment of
0.5 mg/L NAA and 0.5 mg/L BAP on the petiole
explants was the best combination of plant growth
regulators to produce the highest callus fresh and dry
weights (1478.1 mg and 40.0 mg respectively). Callus
derived from leaf, petiole and internode explants was
friable and compact in texture while node explant-
derived callus was compact in texture.
Keywords: Gynura procumbens, callus induction, plant
growth regulator, explants type.
Introduction
One of the potential plants as pharmaceutical raw materials
in Indonesia is Gynura procumbens. G. procumbens is an
important plant of Asteraceae and widely used in Southeast
Asia especially in Indonesia, Malaysia and Thailand1. The
plant is used as a medication for fever, rash, kidney disease,
migraine, constipation, hypertension, diabetes mellitus and
cancer2. G. procumbens has several bioactive compounds
such as flavonoids, saponins, alkaloids, tannins, terpenoids
and glycoside sterols3. Flavonoids have many benefits,
especially in health such as antioxidants, antiaging,
anticancer, anti-inflammatory, cardiovascular, stroke and
asthma4-9.
Plant growth regulators such as auxin and cytokinin are the
most important supplements to regulate the growth and
development in plant tissue and organ culture10,11.
Modification of plant growth regulator can increase biomass
and secondary metabolite accumulation12. Callus cultured
from leaf explants of G. procumbens in MS medium
supplemented with sucrose, erythrosa-4-phosphate and
phenylalanine contained flavonoids13 but callus biomass
produced from leaf explants was still low, so in this study we
want to optimize plant growth regulators and explant types
to induce callus of G. procumbens on in vitro culture.
Material and Methods
Material: G. procumbens was obtained from Purwodadi
Botanical Gardens, East Java, Indonesia. The plant has been
identified and confirmed by Purwodadi Botanical Garden,
Indonesian Institute of Sciences, Pasuruan, East Java,
Indonesia.
Callus induction: Leaf, internode, node and petiole of G.
procumbens were washed with detergent solution and rinsed
in running water, then sterilized with 10% v/v chlorox for
leaf and petiole and 20% chlorox v/v for internode and node
during 5 minutes and rinsed with sterile distilled water three
times. Leaf explant was cut ± 2 cm2 while internode, stem
nodes and petiole explants were cut 0.5-1 cm and inoculated
in MS medium14 supplemented with 30 g/L sucrose, 8 g/L
agar and various combinations of plant growth regulator: 0.1
mg/L 2.4-dichlorophenoxy acetic acid (2.4-D) and 0.1 mg/L
6-benzyl amino purine (BAP), 0.5 mg/L, 2.4-D and 0.1 mg/L
kinetin (Kn), 0.5 mg/L of naphthalene acetic acid (NAA) and
0.5 mg/L BAP, 5.0 mg/L 2.4-D and 0.5 mg/L BAP, 0.1 mg/
L 2.4-D and 0.1mg / L indole-3-aceticacid (IAA). Cultures
were incubated at 25±3°C under light of 320 lux for 28 days.
Fresh weight, dry weight and morphology of callus were
observed at the end of cultivation.
Results and Discussion
The addition of various combinations of plant growth
regulators on leaf explants showed that the addition of 0.1
mg/L 2, 4-D and 0.1 mg/L BAP was the best treatment
compared to the others. The treatment could induce 144.6
mg fresh weight and 5.0 mg dry weight of callus. All various
combinations of plant growth regulators on leaf explant
formed callus, but the treatment of 0.1 mg/L 2, 4-D and 0.1
mg/L IAA cannot induced callus (Table 1).
Combination of 0.1 mg/L 2.4-D and 0.1 mg/L BAP could
produce high callus fresh weight and all explants formed
callus. This result was also show at Centella asiatica (L.)15,
Achyranthus aspera L.16 and Glinus lotoides (L)17. The
effects of combination of 2.4-D and BAP play an important
role as growth regulators in plant and have a remarkable

Research Journal of Biotechnology Vol. 14 (9) September (2019)
Res. J. Biotech
103
effect on the percentage of callus induction compared to the
other combinations. The interaction effect of 2.4-D and BAP
on callus induction of Tridax procumbens has also been
reported18.
The addition of 0.5 mg/L NAA and 0.5 mg/L BAP on G.
procumbens internode explants obtained the highest callus
biomass. The biomass reached 581.5 mg fresh weight and
15.3 mg dry weight, but the lowest callus biomass (dry
weight) was obtained on treatment of 0.5 mg/L 2.4-D and
0.1 mg/L kinetin (Table 2).
Internode explant-derived callus with a combination of 0.5
mg/L NAA and 0.5 mg/L BAP was the best combination to
produce fresh and dry weight. Similar result was shown in
Cucumis sativus (L.)19 in which the treatment of 1.0 mg/L
NAA and 0.5 mg/L BAP induced 89% callus from stem
explants. The best result of callus induction from Rauvolfia
serpentine stem explants with the addition of various
concentrations of NAA and BAP was 80% while in
Catharanthus roseus stem explant was 85% 20. Kumlay and
Ercisli21 reported that combination of NAA and BAP
produces callus induction 87.5% from node and leaf explants
of Solanum tuberosum L. The combination of NAA and
BAP could also produce 95% callus induction with 4.75 g
fresh weight and 2,45 g dry weight from Tinospora formanii
node explants22.
The same result was reported by Jan et al.23 The addition of
NAA and BAP on MS media produced 100% callus
induction from internode explants of Ajuga bracteosa.
Malayaman et al24 reported that the addition of NAA and
BAP resulted in 60% callus induction of Phyllanthus debilis
from internode explants. The application of balanced
combination of auxin and cytokinin on the media is a factor
in controlling cell division in tissue culture.
The five different treatments of plant growth regulator
combinations on callus induction from G. procumbens node
explants obtained various results. The best result of callus
induction was on the combination of 0.5 mg/L 2.4-D and 1
mg/L kinetin reached 415.8 mg fresh weight and 18.3 mg
dry weight, but lowest callus biomass was shown in
treatment of 5.0 mg/L 2.4-D and 0.5 mg/L that produced
91.0 mg fresh weight and 4.9 mg dry weight (Table 3).
Treatment of 0.5 mg/L 2.4-D and 1.0 mg/L Kn from node
explants obtained the highest fresh weight and dry weight.
The same response was shown at Crescentia alata25, Parkia
biglobosaceae stem explants26, endosperm explants of
Barringtonia racemosa L.27 and node explants of Rauvolfia
serpentina28. Callus induction from petiole explants
produced highest fresh weigth and dry weight on medium
supplemented with 0.5 mg/L NAA and 0.5 mg/L BAP
(Table 4).
Callus induction from petiole explants with various
concentrations of plant growth regulators obtained the best
results on fresh weight, dry weight and percentage of callus.
Similar result was shown in O. tamineus petiole explants29,30,
Rhodiola imbricate leaf explants31, leaf explants of Glinus
lotoides17, leaves and stems explants of Artemisia annua L.32
However, the addition of NAA and BAP could only produce
91.6% callus induction in shoot explants of Ipomoea
obscura L.; this result was lower than leaf explants that
reached 96.9%33. The different results indicated that the
different types of plant tissue had different responses in
callus induction34. Based on the results of this study, the
petiole is very potential as an explant source of in vitro
propagation of G. procumbens.
Based on the best result from each treatment on different
explants it was shown that the highest production of callus
biomass was obtained on petiole explants in medium
supplemented with 0.5 mg/L NAA and 0.5 mg/L BAP which
produced callus 1478.1 mg fresh weight and 40.0 mg dry
weight. Leaf explants showed the lowest production of
callus biomass (Figure 1).
Morphology of the callus from different explants type can be
seen in figure 2. Callus from leaf explants was white color
and green color; white callus has friable texture while green
callus has compact texture (Figure 2A-B). White and green
color with friable and compact texture were also shown in
internode explants (Figure 2C-D) while in stem node
explants callus was yellowish color with compact texture
(Figure 2E-F). Petiole explant-derived callus on MS medium
supplemented with 0.5 mg/L NAA and 0.5 mg/L BAP had
white in color and friable (Figure 2G) while the addition of
0.5 mg/L 2.4-D and 1.0 mg/L gave green and yellowish
compact calli (Figure 2H).
The effect of combination of plant growth regulators on
callus morphology varied. The addition of 2.4-D and BAP in
G. procumbens leaf explants resulted in white friable and
green compact calli. This was similar with the study of
Mungole et al33 that the combination of 2.4-D and BAP
obtained white friable callus in leaf explants of Ipomoea
obscura (L.) and brown compact callus in the treatment of
2.4–D in various concentrations. The combination of NAA
and BAP obtained green compact callus in G. procumbens
leaf explants. Similar result was also reported by
Elangomathavan et al29 that the addition of a balanced
concentration of NAA and BAP produced green compact
callus of O. stamineus. However, the addition of high
concentration of NAA and BAP produced yellowish white
compact callus in leaf explants of Dianthus caryophyllus
L.35
The addition of 0.5 mg/L NAA and 0.5 mg/L BAP in G.
procumbens internode explants produced white friable and
green compact calli. The addition of the same plant growth
regulator and explant type in different plant produced green
compact callus, but when the concentration of auxin was
enhanced, the light green and greenish white friable calli
were formed29. Besides, the combination of 2.4-D and Kn

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104
could induce brownish white compact callus in G.
procumbens internodus.
However, the addition of 2.4-D and Kn in node explants of
Ipomoea obscura (L.) produced greenish white friable
callus28, reported in Thymus persicus callus induction as
well36. It indicated that different plant growth regulators and
tissue types led to different responses in callus morphology.
Callus induction of G. procumbens petiole explants on MS
medium supplemented with 0.5 mg/L NAA and 0.5 mg/L
BAP produced white friable callus. However, the addition of
NAA and BAP in petiole explant produced greenish white
friable callus whereas different concentrations between
NAA and BAP produced light green friable callus36. The
addition of a balanced combination of NAA and BAP
produced light green compact callus37. Color of callus can
change because chlorophyll is altered due to reaction among
endogenous and exogenous hormones, explant sources and
environmental culture conditions such as temperature and
light exposure.38
Table 1
Effect of plant growth regulators on callus induction of G. procumbens from leaf explants
Plant growth regulator (mg/L)
Leaf explants
2.4-D
IAA
NAA
BAP
Kn
Fresh weight (mg)
Dry weight (mg)
% Explants formed
callus
0.1
-
-
0.1
-
144.6±0.00c
5.0±0.00c
100%
0.5
-
-
1.0
59.8±0.00b
3.0±0.00b
100%
-
-
0.5
0.5
-
64.8±0.03b
4.4±0.00bc
100%
5.0
-
-
0.5
-
61.5±0.02b
3.3±0.00b
100%
0.1
0.1
-
-
-
0.0±0.00a
0.0±0.00a
0%
Mean values within a column followed by the same letters are not significantly different at p=0.05 according to Duncan’s Multiple
Range Test
Table 2
Effect of plant growth regulators on callus induction of G. procumbens from internode explants
Plant growth regulator (mg/L)
Internode explants
2.4-D
IAA
NAA
BAP
Kn
Fresh weight
(mg)
Dry weight(mg)
% Explants formed
callus
0.1
-
-
0.1
-
125.3±0.03a
9.3±0.00c
100%
0.5
-
-
1.0
137.3±0.04a
5.4±0.00a
100%
-
-
0.5
0.5
-
581.5±0.10c
15.3±0.00e
100%
5.0
-
-
0.5
-
153.4±0.14 a
7.6±0.00b
100%
0.1
0.1
-
-
-
376.9±0.10b
12.7±0.01d
100%
Mean values within a column followed by the same letters are not significantly different at p=0.05 according to Duncan’s Mult iple
Range Test
Table 3
Effect of plant growth regulators on callus induction of G. procumbens from stem node explants
Plant growth regulator (mg/L)
Stem node explants
2.4-D
IAA
NAA
BAP
Kn
Fresh weight (mg)
Dry weight (mg)
% Explants formed
callus
0.1
-
-
0.1
-
93.8±0.01a
4.8±0.00a
100%
0.5
-
-
1.0
415.8±0.04c
18.3±0.00c
100%
-
-
0.5
0.5
-
287.9±0.07b
8.2±0.00b
100%
5.0
-
-
0.5
-
91.0±0.03a
4.9±0.00a
100%
0.1
0.1
-
-
-
304.9±0.03b
8.3±0.00b
100%
Mean values within a column followed by the same letters are not significantly different at p=0.05 according to Duncan’s Multiple
Range Test

Research Journal of Biotechnology Vol. 14 (9) September (2019)
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105
Table 4
Effect of plant growth regulators on callus induction of G. procumbens from petiole explants
Plant growth regulator (mg/L)
Petiole explants
2,4-D
IAA
NAA
BAP
Kn
Fresh weight (mg)
Dry weight (mg)
% Explants formed
callus
0.1
-
-
0.1
-
52.2±0.03a
2.8±0.00b
100%
0.5
-
-
1.0
196.5±0.11a
8.1±0.00b
100%
-
-
0.5
0.5
-
1478.1±0.41b
40.0±0.00c
100%
5.0
-
-
0.5
-
164.4±0.01a
6.8±0.00b
100%
0.1
0.1
-
-
-
113.2±0.06a
6.9±0.00a
100%
Mean values within a column followed by the same letters are not significantly different at p=0.05 according to Duncan’s Mult iple
Range Test.
Figure 1: Comparison of the best result (fresh weight) from each treatments (combination of growth regulators)
on different explants
Figure 2: Morphology of G. procumbens calli in different growth regulator and explants type after 28 days culture
periode; (A-B) leaf explants, (C-D) internode explants, (E-F) stem node explants and (G-H) petiole exlants.
Bar = 3 mm
0
200
400
600
800
1000
1200
1400
1600
Leaf (2.4-D
0.1+BAP 0.1) Internode (NAA
0.5+BAP 0.5) Stem node (2.4-D
0.5+Kn 1) Petiole (NAA
0.5+BAP 0.5)
Fresh weight (mg)
A
B
C
D
H
G
F
E

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Conclusion
Various concentrations of plant growth regulators (2,4-D,
IAA, NAA, BAP and kinetin) affected callus induction in
leaf, internode, stem node and petiole explants of Gynura
procumbens; of the four combinations of plant growth
regulators and different types of explants, petiole explants
that were cultured in MS medium supplemented with 0.5
mg/L NAA and 0.5 mg/L BAP produced highest biomass.
Callus derived from leaf, internode, stem node and petiole
explants were friable and compact while node explant-
derived callus was compact.
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(Received 18th December 2018, accepted 11th June 2019)