Volume 06

Issue 01

p-ISSN 2655-8564, e-ISSN 2685-943, pp. 125-136, 2024

Response of Shoots from Porang Leaf Bulbs to

Cytokinins and IAA in Shoot
Multiplication In Vitro
Lisa Erfa1, *, Desi Maulida1, Rahmadyah Hamiranti1, Ferziana1,
Yuriansyah1, Betari Safitri1, Yeni1
1
Department of Food Plant Cultivation Lampung State Polytechnic, Jln
Soekarno Hatta Rajabasa Bandar Lampung. Indonesia
*
Corresponding Author: [email protected]

(Received 16-11-2023; Revised 13-02-2024; Accepted 29-02-2024)

Abstract
The growth regulators in media have the potential to significantly influence the process
of in vitro plant regeneration. The response of explants in forming shoots also depends
on the type and the combination of growth regulators. This research aimed to identify the
most optimal type and concentration of cytokinin for the multiplication of in vitro porang
shoots. Additionally, it determined whether the addition of cytokinins should be
accompanied by auxin/IAA for porang shoot multiplication. To achieve these objectives,
a completely randomized design with six replications was employed, followed by a 5%
LSD. The tested treatments consisted of various combinations of growth regulators.
Several observations were recorded, including the speed of explants forming prospective
shoots/shoots (days), the age of the explants forming prospective shoots/shoots, the
number of prospective shoots and shoots produced per explant, and shoot height (cm).
The results showed that 1) Tdz 2 and IAA 0.5 mg.l-1 formed the highest shoots candidates
and shoots in porang multiplication in vitro, and 2) the addition of BAP and Tdz 2 mg.l-
1 should be combined with IAA 0.5 mg.l-1, while BAP and Tdz 3 mg.l-1 did not require
IAA.

Keywords: BAP, IAA, multiplication, porang, thidiazuron

1 Introduction
Porang (Amorphophallus muelleri Blume), also recognized as iles-iles, is one of
the three varieties of tubers cultivated for commercial purposes, improving significant
economic value [1]. According to [2], the tuber has a high selling price due to its main
compound, glucomannan [3], functioning as a cholesterol-lowering agent [4], anti-
diabetic [5], anti-colorectal cancer [6], and anti-inflammatory [7].

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According to [8], the propagation of porang plants can be accomplished

generatively or vegetatively. Furthermore, the seeds are not always available [9] and
planting with corms or bulbils takes a long time due to dormancy in the bulbs. To
support the availability of seedlings, the propagation is obtained using tissue culture
methods since it can produce uniform plantlets in large quantities within a relatively
short time and free from diseases [10].
The response of plant tissues/parts cultured in vitro shoot formation depends on
the type of media [11]. As stated by [12] the response depends on the combination of
growth regulators, including auxin [13], cytokinin [14], and the genotype (type and
cultivar) of the plant [15]. Several research [16-18] stated that the highest number of
shoots produced in porang multiplication was achieved using MS media with the
addition of BAP without auxin. According to [19] showed that there was an interaction
between IAA and kinetin (Kin) in shoot multiplication and the best combination was
obtained with 0 mg.l-1 IAA and 2 mg.l-1 Kin. The research conducted by [20] on
Prunus duicis Mill. (Almond) showed that thidiazuron (Tdz) was more effective than
the other cytokinins. Application of Tdz at a concentration of 1 mg.l-1 significantly
increased shoot proliferation. According to [21] on Rhododendron plants, the
frequency of shoot regeneration and the number per explant increase with the
concentration of Tdz. Therefore, this research aimed to determine the effect of
cytokinin types (with or without combination with IAA) and their concentrations in
promoting axillary shoot growth, particularly in the shoot multiplication stage.

2 Material and Methods

This research was conducted between May and October 2022 at the Tissue
Culture Laboratory of Lampung State Polytechnic. The materials used consisted of
propagules obtained from the previous year's initiation, along with Murashige and
Skoog [22] basal media. This was supplemented with vitamins (thiamine-HCl,
pyridoxine-HCl, nicotinic acid), and myo-inositol, as well as growth cytokinin (BAP,
Kin, and Tdz), and auxin (IAA). Additionally, HCl and NaOH, agar, sugar, rubber,
plastic, and aluminum foil were employed. The tools also used encompassed an

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autoclave, laminar air flow cabinet, hand sprayer, and dissection equipment (forceps,
scalpel, petri dish).
The experiment was conducted using a completely randomized design (CRD).
The treatments tested were BAP 2 mg.l-1 + 0 mg.l-1 IAA (P1), BAP 2 mg.l-1 + 0.5
mg.l-1 IAA (P2), BAP 3 mg.l-1 + 0 mg.l-1 IAA (P3), BAP 3 mg.l-1 + 0.5 mg.l-1 IAA
(P4), Kin 2 mg.l-1 + 0 mg.l-1 IAA (P5), Kin 2 mg.l-1 + 0.5 mg.l-1 IAA (P6), Kin 3
mg.l-1 + 0 mg.l-1 IAA (P7), Kin 3 mg.l-1 + 0.5 mg.l-1 IAA (P8), Tdz 2 mg.l-1 + 0
mg.l-1 IAA (P9), Tdz 2 mg.l-1 + 0.5 mg.l-1 IAA (P10), Tdz 3 mg.l-1 + 0 mg.l-1 IAA
(P11), and Tdz 3 mg.l-1 + 0.5 mg.l-1 IAA (P12). The data were subjected to analysis
of variance, and differences between treatments were tested using the LSD test at the
5% level.
This research started by preparing the treatment media and the chemical
materials of Murashige and Skoog basal media were prepared as stock solutions. The
media were prepared by pipetting each stock solution and adding the growth regulators
according to the treatments. Subsequently, 30 g.l-1 of sugar and distilled water were
added, and the pH was adjusted to 5.7 by adding HCl 1 N or NaOH 1 N. The media
were sterilized at a temperature of 121°C for 20 minutes, then incubated for 3-5 days.
Apart from the treatment media, MS0 media (media without growth regulators) were
also prepared to grow to shoot candidates into shoots used as explants. The planted
explants were maintained in a room with a temperature of 25°C - 27°C, with 16 and 8
hours of light and darkness. The observations were made on the following variables 1)
the speed of explant forming shoot candidates/shoots (days), 2) the age of explants
forming shoot candidates/shoots, 3) the number of shoot candidates produced per
explant, and 4) shoot height (cm).

3 Results and Discussions

The shoot candidates were grown into shoots by subculturing the propagules on
Murashige and Skoog media until proliferation occurred, which were used as explants
in the multiplication stage (Fig. 1).
Shoot proliferation for the explants occurred 3-5 weeks after subculturing. The
shoots, which had reached a size of 1 cm, were isolated and planted on the treatment

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media. Generally, the explants showed a response in multiplication with a speed of

forming shoot candidates/shoots ranging from 10 to 20 days after being subcultured
on the treatment media. Meanwhile, the number of shoots on the explants ranged from
1-2 shoots per explant with a shoot height of 0.6-0.8 cm, as shown in Table 1.
The analysis of variance showed that the treatment of cytokinin with or without
auxin significantly influenced the number of shoot candidates produced on the
explants. According to [23], shoot meristem formation requires an increase in
cytokinin and auxin levels. Furthermore, cytokinin and auxin are needed for cell
division and play a role in meristem formation and activity. Cytokinin also plays a
crucial role in shoot organogenesis when the ratio to auxin increases.
Explants planted on treatment media showed different rates of forming shoot
candidates. The LSD test at a 5% significance level showed that explants cultured on
media with the addition of BAP 2 IAA 0,5 mg.l-1, BAP 3 IAA 0 mg.l-1, Tdz 2 IAA
0,5 mg.l-1, and Tdz 3 IAA 0 mg.l-1+ exhibited the fastest growth response in shoot
candidate and took 10-10.2 days (Table 2). Meanwhile, explants cultured on media
with the addition of BAP 3 IAA 0.5 mg.l-1, Kin 2 IAA 0.5 mg.l-1, and Kin 3 IAA 0.5
mg.l-1 showed the slowest growth response in shoot candidate and formation, taking
19.7-20.2 days. The LSD test at a 5% significance level showed that the explants
cultured on media with the addition of Tdz 2 IAA 0.5 mg.l-1 (P10) produced the
highest number of shoot candidates and shoots (31.33), followed by the treatment of
BAP 3 IAA 0 mg.l-1 (P3) at 22.00 (Table 2). The lowest number of shoot candidates
and shoots were obtained from the explants cultured on media with the addition of Kin
3 IAA 0.5 mg.l-1 (P8) at 6.33 (Fig. 2).

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Propagules of shoot candidates Proliferation of shoots on propagules

Porang shoot explants

Figure 1. The stage of preparing shoot explants for multiplication

Tdz 2 IAA 0.5 mg.l-1 (P10) BAP 3 IAA 0 m g.l-1 (P3)

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Kin 3 IAA 0.5 (P8)

Figure 2. Growth of prospective shoots and shoots on explants cultured on treatment
media

The research [16-18] on porang showed that the use of a single cytokinin can
increase the number of formed shoots. Furthermore, [19,24] stated that the
combination of cytokinin with low auxin can improve the percentage of shoot
formation and multiplication. According to [25], the response of plants to the use of
specific types and concentrations of auxin and cytokinin in organ regeneration is
species-specific, depending on the genotype of the cultured plant. [26] stated that the
use of Tdz combined with IAA has been widely reported to promote shoot regeneration
in many plant species. The combination of these two growth regulators with a lower
concentration of IAA plays a crucial role in morphogenesis, such as inducing and
proliferating axillary shoots.

Table 1. Average age of explants forming shoot candidates/shoots, number of shoots,

and shoot height (cm)
Treatment Speed of Explants Number of Shoot
Forming Shoot Shoots Height (cm)
Candidates and
Shoots (days)
P1 (BAP 2 + IAA 0 mg.l-1) 15 1 0.6
P2 (BAP 2 + IAA 0.5 mg.l-1) 10 1 0.7
P3 (BAP 3 + IAA 0 mg.l-1) 10 1.2 0.8
P4 (BAP 3 + IAA 0.5 mg.l-1) 20 1 0.6
P5 (Kin 2 + IAA 0 mg.l-1) 14.7 1 0.7
P6 (Kin 2 + IAA 0.5 mg.l-1) 19.7 1 0.7
P7 (Kin 3 + IAA 0 mg.l-1) 15 1 0.7
P8 (Kin 3 + IAA 0.5 mg.l-1) 20.2 1 0.8

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P9 (Tdz 2 + IAA 0 mg.l-1) 13.2 1 0.8

P10 (Tdz 2 + IAA 0.5 mg.l-1) 10 1 0.7
P11 (Tdz 3 + IAA 0 mg.l-1) 10.2 0.8 0.6
P12 (Tdz 3 + IAA 0.5 mg.l-1) 11 1 0.7

Table 2. LSD test at the 5% significance level for the variable of the speed of explant
forming shoot candidates and shoots, as well as the number of shoot candidates and
shoots formed on the explants
No. Treatment Average Speed of Explants Average of the Number
to Form Shoot Candidates of Shoot Candidates and
& Shoots (days) Shoots
-1
1. P1 (BAP 2 + IAA 0 mg.l ) 15.0 C 13.33 E
2. P2 (BAP 2 + IAA 0.5 mg.l-1) 10 .0 A 20.00 B C
3. P3 (BAP 3 + IAA 0 mg.l-1) 10.0 A 22.00 B
-1
4. P4 (BAP 3 + IAA 0.5 mg.l ) 20.0 D 12.50 E
5. P5 (Kin 2 + IAA 0 mg.l-1) 14.7 C 11.00 E
-1
6. P6 (Kin 2 + IAA 0.5 mg.l ) 19.7 D 14.00 D E
7. P7 (Kin 3 + IAA 0 mg.l-1) 15.0 C 17.67 C D
-1
8. P8 (Kin 3 + IAA 0.5 mg.l ) 20.2 D 6.33 F
9. P9 (Tdz 2 + IAA 0 mg.l-1) 13.2 B C 10.33 E
10. P10 (Tdz 2 + IAA 0.5 mg.l-1) 10.0 A 31.33 A
11. P11 (Tdz 3 + IAA 0 mg.l-1) 10.2 A 18.50 C
-1
12. P12 (Tdz 3 + IAA 0.5 mg.l ) 11.0 A B 12.00 E

Even though the use of a single cytokinin was the primary factor for inducing
and proliferating shoots, this research showed that the addition of both auxin and
cytokinin to the shoot induction media resulted in a higher number of shoots. The
explants showed a faster ability to form shoots compared to the treatment with a single
cytokinin.
The effectiveness of Tdz as cytokinin in shoot induction and multiplication may
be due to its ability to stimulate endogenous cytokinin biosynthesis, which will alter
the metabolism of endogenous cytokinins. The compound is also highly resistant to
degradation by cytokinin oxidase enzymes in plants. Therefore, Tdz exhibits strong
cytokinin-like activity that surpasses other commonly used adenine types such as
benzylaminopurine or Kin [27].
IAA as auxin also affected on number of shoots. This may due of its activity on
cell division and cell differentiation that makes shoots proliferation higher. [28] stated

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that effectivity on cell division of IAA is more powerful than another auxin such as
NAA and IBA.
Based on the results, the response of porang shoot explants to growth regulators
in forming shoot candidates and shoots is best observed with Tdz (Tdz 2 IAA 0.5 mg.l-
1), which outperforms BAP and Kin. This was consistent with [21] in Sutsukui Azalea
(Rhododendron indicum), where Tdz optimally induced shoots compared to BA and
Kin. It also aligned with [20] that Tdz was more effective than the other cytokinins in
enhancing shoot proliferation. According to [29], the use of Tdz in Gymnocladus
assamicus resulted in higher shoot induction and proliferation compared to BA.

4 Conclusions
Based on the research on the effects of cytokinin and IAA treatments in media,
the following conclusions were drawn:
1. The application of Tdz 2 + IAA 0.5 mg.l-1 resulted in the highest formation of
shoot candidates and shoots in in vitro multiplication of porang shoots.
2. The addition of BAP and Tdz 2 mg.l-1 should be combined with IAA 0.5 mg.l-1,
while BAP and Tdz 3 mg.l-1 did not require IAA.

Acknowledgements
The authors would like to acknowledge the Lampung State Polytechnic,
Indonesia, for its financial support.

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