The combination effect of auxin and

cytokinin on in vitro callus formation of

Physalis angulata L. – A medicinal plant
Cite as: AIP Conference Proceedings 1908, 040007 (2017); https://doi.org/10.1063/1.5012721
Published Online: 29 November 2017

Retno Mastuti, Aminatun Munawarti, and Elok Rifqi Firdiana

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AIP Conference Proceedings 1908, 040007 (2017); https://doi.org/10.1063/1.5012721 1908, 040007

© 2017 Author(s).
The Combination Effect of Auxin and Cytokinin on In Vitro
Callus Formation of Physalis angulata L. – A Medicinal
Plant

Retno Mastuti1, a) Aminatun Munawarti2, b) and Elok Rifqi Firdiana3, c)

1,2
Department of Biology, Faculty of Mathematics and Natural Sciences, University of Brawijaya, Veteran Street,
Malang, East Java, Indonesia 65145
3
Laboratory of Plant Physiology, Tissue Culture & Microtechnique, Department of Biology, Faculty of Mathematics
and Natural Sciences University of Brawijaya, Veteran Street, Malang, East Java, Indonesia 65145

a)
Corresponding author: mastuti7@ub.ac.id
b)aminatun@ub.ac.id; c)elok.firdiana@gmail.com

Abstract. Physalis angulata L. (Ciplukan) is one member of Solanaceae that has a potential as herbal medicine. This
plant grows wild in the crop fields, forest edges, etc. However, ciplukan is increasingly difficult to find recently. In vitro
callus is an alternative source to produce secondary metabolite production as well as to regenerate plants through indirect
organogenesis. This study aims to identify the response of hypocotyl explants on in vitro callus formation induced by a
combination of auxin and cytokinins. Two types of cytokinins, Kinetin and BAP (0.5 ppm) were combined with three
types of auxin, i.e. 2.4-D, IBA and IAA, at three concentrations 0.5, 1.0 and 1.5 ppm. In all combinations of cytokinin
and auxin, 50–100% of hypocotyl explants derived from in vitro seedling were able to produce callus either in a compact
or watery friable texture. In MS medium supplemented with 2.4-D, callus FW (fresh weight) began to decline in the
fourth week after culture. Callus FW that increased until 5 weeks of culture was obtained in medium IAA 0.5 + Kin 0.5,
IBA 1.0 + Kin 0.5 and IBA 1 + BA 0.5. Almost all calli induced on a medium + Kinetin also produced roots. While
medium + BAP was able to induce shoots regeneration.

Keywords: auxin, cytokinin, in vitro culture, callus formation, Physalis angulata L., medicinal plant

INTRODUCTION
Ciplukan (Physalis angulata L.) is one member of Solanaceae that has medicinal properties. Currently, ciplukan
is being developed as an herbal medicine that is produced in industrial scale. Therefore, the availability of ciplukan
plants as a continuous raw material of drug is needed. However, so far ciplukan is considered as a weed that has not
been widely cultivated. Generally, ciplukan grow wild in corn fields or forest edges.
Callus is an amorphous tissue composed of actively dividing parenchyma cells. In vivo callus is formed as the
initial growth response to a wound (or wounding or injury) to protect the tissue from further damage. All in vitro
cultures are started by cutting the explant tissue. Therefore the wounding response must occur. In the most of the
dicot the process that occurs in wounding area surfaces is a combination of cell division and cell wall
reinforcement.1 Callus initially consists of a morphogenic cells that will cover the wound area. Subsequently, they
differentiate into the type of cells or tissues origin that have been damaged. Most of the biochemical activity to heal
this wound is the synthesis of phenolic compounds to strengthen the cell wall through induction of phenylpropanoid
pathway. So the wounding will initiate a complex developmental process, i.e., the de-differentiation of tissues had
already in the quiescent stage, induction of cell division, redifferentiation and finally back to the quisence.2 This
series of the wound-treading process (de-differentiation and callus proliferation) is needed during initiation of callus
cultures. Wounding callus and callus derived culture have the same morphology. Several previous studies 3,4.5,6

8th International Conference on Global Resource Conservation (ICGRC 2017)

AIP Conf. Proc. 1908, 040007-1–040007-6; https://doi.org/10.1063/1.5012721
Published by AIP Publishing. 978-0-7354-1600-0/$30.00

040007-1
suggest that both types of callus have the potential for a shoot or root organogenesis. In contrast, monocots lack or
does not show cell proliferation to the wounding response.
The type of wounding response given by explant depends on species, cultivars, tissue type and tissue age. 1 The
condition of both physical and chemical culture affects the developing regeneration system. Optimum culture
conditions for one species or genotype are not necessarily optimal for other species or genotypes. Therefore, the
optimization of culture conditions should be empirically applied to each individual genotype or type of explant. 7
Of all the medium composition culture conditions consisting of macro and micronutrients, reduced nitrogen,
carbon source, and growth regulator are the most important factors. Among these components, the plant growth
regulators are the most critical for the initiation of culture and morphogenesis. The intermediate ratio of auxin and
cytokinin promotes callus induction.8 The meaning of 'balance of concentration' should be understood not as an
exogenous concentration of applied hormones but rather a balance of combined total concentrations between
endogenous and exogenous hormones.
Callus cultures in plant regeneration systems are used for conservation. Callus mediated by regeneration system
can also be used to exploit secondary metabolite production. Therefore the objective of this research was to identify
the various responses of callus formation-derived in vitro hypocotyls explants in medium containing some
combinations of cytokinins: auxin. This is the first paper reporting callus induction of P. angulata.

EXPERIMENTAL DETAILS

Plant Materials and Seed Germination

Wild ciplukans grown in the farmland in rural areas of Gondanglegi, Southern Malang were used for plant
material source. Seeds from ripe yellow fruits were taken and kept at room temperature until dried. The dried seeds
were surface sterilized with ethanol 70% for 30 sec, then 20% commercial bleaching (containing sodium
hypochlorite 5.25%) for 10 min and followed by 3 rinsed in sterile distilled water for 5 min each. Finally, the
sterilized seeds were germinated in agar medium.

Media and Callus Induction

Callus induction was carried out on MS media supplemented with 3% sucrose and various combinations of plant
growth regulators, namely auxins (IAA, IBA, and 2-4, D), 3 levels of concentrations (0.5; 1; and 1.5 mg/l), and
cytokinins (BAP and kinetin 0.5 mg/l). Media were solidified with 1% commercial agar. The hypocotyls of 8-day-
old in vitro seedlings were used as explants for callus induction. Approximately 1 cm long segment of hypocotyls
was cultured in callus induction medium. The cultures were incubated under continuous 40 W cool-white
fluorescent lamps for five weeks at 25 r 1qC. All experiments were repeated thrice with 2 replicates for each
treatment.

Callus Growth Measurement

The effect of each combination of plant growth regulator to induce callus formation was determined by
calculating the percentage of callus formed in every piece of the explant. The morphology of callus resulted in every
treatment was observed in the fifth week. In order to find out the effect of those combinations on the growth of
callus yielded, the fresh weight and dry weight of callus were observed every week for five weeks. The percent
explants segment forming callus were observed after 1 week of culture while the morphology of callus and the fresh
weight of formed callus were recorded after 5 weeks. The quantitative data were analyzed using ANOVA, and the
means were compared using Duncan's multiple range test (DMRT) at 5% level of significance (P < 0.05).

RESULT AND DISCUSSION

Callus Induction
One week after culture the most of the hypocotyl explants has produced callus with the percent callus formed
per explants segment ranging from 48-100% (Figure 1). All combination of 2.4-D with kinetin or BAP was able to
induce > 80% segment of hypocotyl explants to be dedifferentiated to produce callus. The same pattern was shown
in a medium containing a combination of IAA with kinetin or BAP except at IAA 1 mg / l + Kin 0.5 mg / l which
could only induce approximately 63 % of hypocotyl segments to produce callus. The lowest response was shown in

040007-2
the medium containing IBA and Kinetin, which was only able to induce 48–73% of hypocotyl segments to form
callus. The results showed that 2.4-D is more appropriate to induce a de-differentiation process in hypocotyl
explants.

120
de
Callus formed per explant segment (%) e e de de cde de
100 cde cde de de cde bcd cde de
abc
80
ab
a
60

40

20

0
D1 B0,5

IA0,5 K0,5

IA1,5 K0,5

IA1 B0,5

IB1 K0,5
IA1 K 0,5

IA0,5 B0,5

IA1,5 B0,5
D0,5 K0,5

D1,5 K0,5

IB0,5 K0,5

IB1,5 K0,5

IB1 B0,5
DI K0,5

D0,5 B0,5

D1,5 B0,5

IB0,5 B0,5

IB1,5 B0,5
2,4-D IAA IBA

Medium

FIGURE 1. The effect of different auxin inducing hypocotyl segments of P. angulata in vitro seedling for callus formation one
week after culture. Note D: 2.4-D, IA: IAA; IB: IBA; B: BAP; K: Kinetin

Callus formation of differentiated tissue begins with a de-differentiation process to produce meristematic and
actively proliferated cell. This process causes the explants tissues to become visibly thick, stiff and swollen.
Actively divided cells produce unorganized cell masses on the explants surfaces. In the results the combination of
2.4-D 0.5 and 1 mg/l with kinetin 0.5 mg/l induced the highest segment part of hypocotyl to produce callus. This
suggests that 2.4-D is more effective to initiate callus formation from hypocotyl explants of P. angulata than IAA
and IBA. A 2.4-di-chlorophenoxy-acetic acid (2,4-D) is the most common auxin applied to initiate callus growth
since it can revert explant cells to a dedifferentiated state and begin to actively proliferate. 9
A combination of 2,4-D with kinetin promoted the cytoplasm of the vacuolated parenchyma cells (derivative
cells) of petiole cells in Arabidopsis thaliana become denser.10 2.4-D and kinetin also gave better callusing in B.
racemosa endosperms culture.11 In contrast, kinetin alone did not induce the cytoplasm of the derivative
parenchyma cells to become denser but remained highly vacuolated. The hormonal balance had been proposed by
Skoog and Miller8 to the concept of organ formation or unorganized growth of the tissue.
The morphology of callus derived from various combinations of auxins and cytokinins showed different color
and texture after two weeks of culture. The watery friable white translucent callus was more generated in 2.4-D +
Kinetin rather than 2.4-D + BAP (Figure 2). The combination of IAA and kinetin produced more callus than those
between IAA and BAP. Root regeneration was observed in medium containing IAA 1.5 mg/L + kinetin 0.5 mg/L.
In the medium supplemented with IBA + BAP/kinetin, the globular white greenish nodules were observed in the
cutting site of mostly explants. These results show that a combination of all kinds of auxin with BAP produced
greenish white compact callus while a combination of all kinds of auxin with kinetin produced watery friable callus
and was able to induce root regeneration.

040007-3
 FIGURE 2. Morphology of callus in the different auxin-containing medium after two weeks of culture. Note (o) : friable
watery callus. (*) : regenerated root.

Callus Growth
The three types of auxin (2.4-D, IBA, IAA) combined with cytokinin resulted in different callus growth
patterns for 5 weeks of culture. Applying 2.4-D in culture medium was able to increase the fresh weight of callus
until the fourth week of culture (Figure 3). Callus fresh weight increased until the fourth week on all 2.4-D
combinations with BAP/kinetin except in combination of 2,4-D 0.5 mg / l + Kinetin 0.5 mg / l which decreased
sharply. However, in the fifth week of culture, the callus fresh weight on all auxin: cytokinin combinations tend to
decrease.

0.7
a
Callus fresh weight (FW) (g)

0.6 a
0.5 c
ab abc c a
0.4 bc a
0.3 ab ab b
b
0.2 a
ab ab ab ab b a a a
0.1 a
a
0.0
DI Kin0.5

DI Kin0.5

DI Kin0.5

DI Kin0.5
D1 BAP0.5

D1 BAP0.5

D1 BAP0.5

D1 BAP0.5
D0.5 BAP0.5

D1.5 BAP0.5

D0.5 BAP0.5

D1.5 BAP0.5

D0.5 BAP0.5

D1.5 BAP0.5

D1.5 BAP0.5
D0.5 BAP0.5
D0.5 Kin0.5

D1.5 Kin0.5

D0.5 Kin0.5

D1.5 Kin0.5

D0.5 Kin0.5

D1.5 Kin0.5

D0.5 Kin0.5

D1.5 Kin0.5

2 3 4 5

week(s) of culture

FIGURE 3. The effect of 2,4-D on callus fresh weight of P. angulata during five weeks of culture. Means denoted by different
letters at the same week of culture are significantly different at P < 0.05 according to Duncan’s Multiple Range Test.

In medium IAA 1.5 mg / l + kinetin 0.5 mg / l, the callus fresh weight continued to increase until the fifth week
of culture (Figure 4). Meanwhile, in medium IAA 1.0 mg/l + BAP 0.5, mg/l callus fresh weight reached a maximum
at four weeks after culture and then decreased at five weeks after culture.

040007-4
 0.90 ab
0.80

Callus fresh weight (FW) (g)

0.70 b
0.60
0.50 ab
0.40 ab a ab b
bc bc ab
0.30 a a
bc a a
0.20 ab ab ab
0.10 a a a a a a
0.00
IAA 1 Kin 0.5

IAA 1 Kin 0.5

IAA 1 Kin 0.5

IAA 1 Kin 0.5

IAA 1.5 Kin 0.5

IAA 0.5 Kin 0.5

IAA 1.5 Kin 0.5

IAA 0.5 Kin 0.5

IAA 1.5 Kin 0.5

IAA 0.5 Kin 0.5

IAA 1.5 Kin 0.5

IAA 1 BAP 0.5

IAA 1 BAP 0.5

IAA 1 BAP 0.5

IAA 1 BAP 0.5

IAA A 0.5 Kin in 0.5

IAA 0.5 BAP 0.5

IAA 1.5 BAP 0.5

IAA 0.5 BAP 0.5

IAA 1.5 BAP 0.5

IAA 0.5 BAP 0.5

IAA 1.5 BAP 0.5

IAA 0.5 BAP 0.5

IAA 1.5 BAP 0.5

2 3 4 5

week(s) after culture

FIGURE 4. The effect of IAA on callus fresh weight of P. angulata during five weeks of culture. Means denoted by different
letters at the same week of culture are significantly different at P < 0.05 according to Duncan’s Multiple Range Test.

Five weeks of culturing the medium IBA 1.5 mg/L + BAP 0.5 mg/L tends to produce the highest callus fresh
weight (Figure 5). While in the other combinations of IBA + BAP/kinetin the callus fresh weight decreased.

0.60 a
0.50
Callus fresh weight (FW) (g)

ab
0.40 b a
a
0.30
bc ab ab a
0.20 b ab a a
ab ab a
0.10 a a a a a a a a
0.00
IBA0.5 BAP0.5
IBA1 BAP0.5
IBA1.5 BAP0.5

IBA0.5 BAP0.5
IBA1 BAP0.5
IBA1.5 BAP0.5

IBA0.5 BAP0.5
IBA1 BAP0.5
IBA1.5 BAP0.5

IBA0.5 BAP0.5
IBA1 BAP0.5
IBA1.5 BAP0.5
IBA1 Kin0.5

IBA1 Kin0.5

IBA1 Kin0.5

IBA1 Kin0.5
IBA0.5 Kin0.5

IBA1.5 Kin0.5

IBA0.5 Kin0.5

IBA1.5 Kin0.5

IBA0.5 Kin0.5

IBA0.5 Kin0.5
IBA1.5 Kin0.5

IBA1.5 Kin0.5

2 3 4 5
week(s) after culture

FIGURE 5. The effect of IBA on callus fresh weight of P. angulata during five weeks of culture. Means denoted by different
letters at the same week of culture are significantly different at P < 0.05 according to Duncan’s Multiple Range Test.

Callus formation promoted by callus induction has been reported in P. minima12,13, P. pubescen14, and P.
peruviana.15 However, there is no report about callus induction of P. angulata. The results of previous studies show
that the best medium for callus formation varies which is also influenced by other factors, such as genotype and
explant type. Producing callus is of interest for studying the second metabolites compounds. A group of steroids,
known as physalins, are found in P. angulata stems and leaves.16

SUMMARY
These results show that hypocotyl explants of P. angulata derived from in vitro seedling were able to produce
either friable watery or hard compact callus. 2,4-D was better to induce dedifferentiation (percentage of explants

040007-5
part that produced callus) than IAA and IBA however for better growth it should be regularly subcultured after 4
weeks of culture. Exogenously applied kinetin and auxin have synergistic effect on root formation while BAP and
auxin have synergistic effect on shoot formation. IAA 1.5 + Kin 0.5 (0.42 g FW) promote better callus growth until
five weeks of culture than IBA 1.5 + BAP 0.5 (0.27 g FW).

ACKNOWLEDGMENT
This research was supported by Directorate of Research and Community Service, Directorate General for
Research and Development at the Ministry of Research, Technology and Higher Education No.:
063/SP2H/LT/DRPM/IV/2017

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