RESEARCH ARTICLE | FEBRUARY 21 2024

Effect of sucrose concentration on porang (Amorphophallus

muelleri Blume) in vitro micro tubers induction 
A. Rahma; A. S. P. Pratama; M. N. Fikri; R. Azrianingsih 

AIP Conf. Proc. 3001, 030015 (2024)

https://doi.org/10.1063/5.0197055

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25 February 2024 12:21:49

 Effect of sucrose concentration on porang (Amorphophallus
muelleri Blume) in vitro micro tubers induction
A Rahma1, A S P Pratama1, M N Fikri1 and R Azrianingsih1,2, a)
1
Department of Biology, Faculty of Mathematics and Natural Sciences, Brawijaya University, Malang 65145,
Indonesia
2
Porang Research Centre, Brawijaya University, Malang 65145, Indonesia
a)
Corresponding author: rodiyati@ub.ac.id

Abstract. Porang (Amorphophallus muelleri Blume) is a commodity that is currently on high demand, therefore its
plantations expanse widely and require huge seed provision. In vitro propagation is a strategic way to provide the seeds
shortly, hence this study aimed to determine appropriate sucrose concentration for in-vitro micro tubers induction and to
analyze sucrose concentration effect on it’s quality and quantity. The research was a complete randomized design with 5
sucrose concentration treatments (0, 30, 60, 90, and 120 mg/L) in 5 replications. Micro tubers number, weight, diameter,
days of micro tubers break after planting (DAP) and morphological characteristic was observed. Effect of sucrose

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concentration was analyzed by analysis of variance (ANOVA) (p = 0,05) followed by Tukey HSD test. The micro tubers
broke earliest on 23 DAP in the 30 g/L treatment. Highest petiole swelling percentage (80%) occurred on 30 g/L
treatment. Highest micro tubers number and weight was also found on 30 g/L treatment (3 micro tubers, 0,0312 g), while
highest diameter was found on 60 g/L treatment (3,1 mm). Both 30 and 60 g/L treatments were recommended to be
potential concentrations to induce in vitro micro tubers formation in porang.

INTRODUCTION

Porang (Amorphophallus muelleri Blume) is one of plant species belong to Araceae family. Porang have a stem
tubers growing in the base of petiole that has high glucomannan content (35-29%) in one period of tubers growth
and increase in the next period. Glucomannan is a polysaccharide that commonly used as food source, thickener,
emulsifier on pharmaceutical and chemical industry [1]. Highest porang export demand come from China, Vietnam
and thailand that reach 14 million ton [2]. Porang fresh stem tubers sold for Rp. 2,500 - Rp. 10,000 per kilogram,
tubers chips sold for Rp. 27,000 - Rp. 35,000 per kilogram and tubers flour even reach Rp. 150,000 - Rp. 250,000
per kilogram [3]. This high selling price and high export demand cause the occurrence of porang farming trend in
Indonesia therefore it also causes high Porang seeds demand.
Generally, porang plant nurseries are carried out generatively or vegetatively. The generative nursery technique
used polyembryonic seed germination which in one seed can produce more than one plant because it contains many
embryos [4]. It required the occurrence of flower that only bloom when the plant reach 4 years old [5]. Vegetative
nurseries use stem tubers and leaf bulbs [6]. Bulbs required 2 months to grow in leaf and need more time until the
bulbs fall by itself then it can be used as porang seed. Stem tubers provide faster plant growth and can be obtained
after harvest. But, stem tubers usage cause decrease on total yields obtained because stem tubers are the harvested
parts. Both methods also have a low and slow seeds production. In fact, the demand for seeds increases by around
three million tons/year, so to overcome this, an appropriate method is needed for large-scale production of porang
seedlings in a short time.
In vitro tissue culture is a method to propagate plant in a sterile culture condition that can produce huge number
of identical plant in a short time. It is done by excising one part of mother plant and cultured in sterile medium
which will regenerate into complete and healthy plant. This method has been studied on other Amorphophallus
genus like A.titanum [7], A. albus [8], and A. campanulatus [9]. There are several factors that affect in vitro culture

International Conference on Environmental, Mining, and Sustainable Development 2022

AIP Conf. Proc. 3001, 030015-1–030015-6; https://doi.org/10.1063/5.0197055
Published by AIP Publishing. 978-0-7354-4850-6/$30.00

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such as mother plant genotype, explant condition, medium composition (plant growth regulator, sucrose, agar
concentration), temperature and light intensity light. One factor that have huge effect and can regulate in vitro plant
development is medium composition. Medium consist of several compound like macro- and micronutrient, plant
growth regulator, and agar. Sugar is a source of in vitro culture macronutrient. Sugar have important role to provide
energy and maintain osmotic stability of in vitro culture. It can also be used for several in vitro culture technique.
One of in vitro culture technique is micro tuber induction. Micro tuber induction is a technique in in vitro tissue
culture to produce small corms that can be grown outside become a whole plant. In vitro micro tuber induction is
affected by some factor like sucrose concentration. Early research on potato plant (Solanum tuberosum L.) showed
that 60 g/L sucrose was optimum concentration to induce in vitro micro tuber [10]. Early study of in vitro micro
tuber induction on Porang showed that optimum hormone concentration for micro tuber induction is 2,69 µM NAA
and 4,4 µM BA, but sucrose concentration has never been known before [11]. This study aimed to determine
appropriate sucrose concentration for in-vitro micro tubers induction and to analyze sucrose concentration effect on
it’s quality and quantity.

MATERIALS AND METHODS

This research was conducted at Physiology, Tissue Culture and Microtechnique Laboratory, Brawijaya
University and Porang Research Centre Laboratory, Brawijaya University from June to August 2021. Material and
instrument used in this research were Murashige-Skoog (MS) medium, in vitro grown plantlets, naphthalene acetic
acid (NAA), benzylaminopurine (BAP), sucrose, aquades, alcohol, myo-inositol, NaOH, HCl, agar, culture bottles,
micropipette, autoclaves and laminar air flow cabinet. In vitro grown plantlet was obtained from Physiology, Tissue
Culture and Microtechnique Laboratory. Research stage consisted of medium preparation, plantlet multiplication,
and micro tubers induction.
Medium used in this research consisted of multiplication medium and micro tubers induction medium.
Multiplication medium used are Murashige-Skoog (MS) media +8,8 µM BA added with 30 g/L sucrose, 9 g/L agar,

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with pH adjusted to 5,6-5,8 prior to autoclave. Micro tubers induction medium used are Murashige-Skoog (MS)
media + 4,4 µM BA + 2,69 µM NAA added with sucrose treatment (0, 30, 60, 90, and 120 mg/L), 9 g/L agar with
pH adjusted to 5,6-5,8 prior to autoclave.
Multiplication stage was done by subculturing in vitro grown plantlets into multiplication medium. Plantlets was
subcultured every 3 weeks into fresh multiplication medium until there were enough plantlets number with uniform
height. Multiplication purpose is to obtained cultures stock. Culture was incubated at 20 °C under 24 hours light.
Micro tubers induction stage was done by subculturing in vitro grown plantlet into micro tubers induction
medium. In vitro grown plantlet with 5-6 cm height was excised for about 2-3 cm with 3-4 leaf lamina and cultured
into induction media. Culture then incubated at 21°C under dark condition for 8 weeks.
The research was a complete randomized design with 5 sucrose concentration treatment (0, 30, 60, 90, and 120
mg/L) in 5 replications. Micro tubers number, weight, diameter, days of micro tubers break after planting (DAP) and
morphological characteristic was observed after the end of the observation. Effect of sucrose concentration was
analyzed by analysis of variance (ANOVA) (p = 0,05) followed by Tukey HSD test.

RESULTS

Effect of sucrose concentration on leaf morphology

Morphological observation showed that micro tubers was formed in 30 and 60 g/L. On 90 g/L treatment, the base
petiole were swelled but did not form a micro tubers. Micro tubers formed earliest on 30 g/L treatment (23 days) and
the late micro tubers formed in 60 g/L treatment. (27 days). The difference time in the formation of micro tubers
between 30 g/L and 60 g/L treatment were only 4 days. Base petiole morphology on 0 g/L treatment showed no sign
of swelling and the leaf lamina turned from green to yellow. 30, 60 and 90 g/L treatment of sucrose resulted
swelling on it's base petiole and the leaf lamina was green. On 120 g/L treatment, base petiole showed no sign of
swelling, the leaf was dried, and the lamina turn to yellow (Table 1).

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 TABLE 1. Effect of sucrose concentration on leaf morphology.

Average time for

Sucrose Concentration Base petiole
microtubers formation Leaf morphology
(g/L) morphology
(day after planting)

0 No swelling - Green to yellow

30 Swelling 23 Light green
60 Swelling 27 Light green
90 Swelling 35 Dark green
120 No swelling - Yellow

Effect of sucrose concentration on base petiole swelling

Micro tubers in A. muelleri was formed on it's base petiole. Observation result showed that base petiole swelling
occurred on 30, 60 and 90 g/L. Highest swelling ratio between swelled and non swelled petiole occurred in 30 and
60 g/L treatment where 80% of the total petiole are swelled followed by 90 g/L where 40% of the total petiole are
swelled. On 0 and 120 g/L, the base petiole swelling did not occur (Figure 1).

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FIGURE 1. Base petiole swelling percentage (%) 8 weeks after micro tubers induction with various sucrose concentration.

Effect of sucrose concentration on base petiole diameter

Result showed that highest base petiole diameter was found on 60 g/L treatment (3,1 mm) followed by 30 g/L
treatment (2,7 mm), 90 g/L treatment (1,8 mm), 0 g/L (1,8 mm) and 120 g/L (1,6 mm). The difference of diameter
between control treatment and 30 g/L treatment was 1,3 mm. Base petiole diameter with 60 g/L treatment showed
significantly diameter compared with 0, 90 and 120 g/L while 60 and 30 g/L treatment has no significant different.
Swelling was determined by how much the diameter increased compared with it's diameter before treatment (Figure
2).

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 FIGURE 2. Base petiole diameter 8 weeks after micro tubers induction with various sucrose concentration.

Effect of sucrose concentration on micro tubers number and weight

Micro tubers formation involved base petiole swelling which occurred on 30, 60 and 90 g/L. But, result showed
that only 30 and 60 g/L treatment that formed microtubers. 30 g/L treatment formed 3 micro tubers while 60 g/L
treatment formed 1 micro tuber. Observation on micro tubers weight showed that 30 and 60 /L treatments have
microtubers weight that are significantly different with 0, 90 and 120 g/L treatment, but micro tubers weight on 30
and 60 g/L treatments showed no significant different. Highest micro tuber weight formed on 30 g/L treatment
(0.0312 g) while lowest micro tuber weight formed on 60 g/L treatment (0.0288 g). On 90 g/L treatment, base
petiole swelling was measured (Figure 3).

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FIGURE 3. Micro tubers weight 8 weeks after micro tubers induction with various sucrose concentration.

DISCUSSION
As shown by the research result, various sucrose treatment give various effect on A.muelleri leaf morphology,
day needed to micro tubers formation, base petiole diameter, micro tubers number and weight. A. muelleri leaf and
petiole which treated 0 g/L sucrose concentration showed green to yellow colour that indicated chlorosis. Chlorosis
is a phenomenone where low energy source and light intensity cause plant unable to form chlorophyll properly [12].
On the other side, 120 g/L sucrose concentration treatment showed yellow and dried leaf which is caused by osmotic
unstability as the side effect of high sucrose concentration. High sucrose concentration caused water inside the leaf
and petiole cell to moved outside of cell in response to high osmotic pressure environment [13]. Similar result also
shown in other parameter such as day to microtubers formation, base petiole swelling percentage, base petiole

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diameter, micro tubers number and weight where 0 and 120 g/L sucrose concentration treatment showed no sign of
micro tubers formation and even showed growth decline.
Micro tubers formation pathway was affected by several factor, such as plant growth regulator (PGR), light
intensity and sucrose concentration. Sucrose concentration was one important factor that play role in micro tubers
formation pathway. Compared to other sugar type like glucose or fructose, sucrose is the main sugar that was
transported to micro tubers during the micro tubers formation. Recent research on Potato (Solanum tuberosum)
micro tubers induction also showed that sucrose and cytokinin was important factor to induce microtubers in several
signaling pathway. Analysis on S. tuberosum microtubers pathway showed that two-component signaling system
(StHK1) and cytokinin signaling (StHK3, StHP4, StRR1) was the main signaling pathway involved in microtubers
induction. It is also shown that medium with high sucrose concentration (5-8%) was optimum to induce micro tubers
formation without interfering it's osmotic homeostasis [14].
This study also showed that micro tubers diameter and weight is not always correlated to each other. In this
study, the highest base petiole diameter was found in 60 g/L sucrose treatment while the highest micro tubers weight
was found in 30 g/L sucrose treatment. It is maybe caused by irregular shape of A. muelleri micro tubers. Since it's
shape was irregular, it is possible to have oval micro tubers or sphere micro tubers where it could affect micro tubers
diameter [15].

CONCLUSION

It can be concluded that 30 and 60 g/L sucrose concentration was a potential optimum concentration for A.
muelleri micro tubers induction. Those concentrations can maximized A. muelleri micro tubers quality and quantity.
Various sucrose concentration also affected leaf and petiole morphology where low sucrose concentration can cause
chlorosis while higher sucrose concentration can cause osmotic unstability.

ACKNOWLEDGMENTS

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The author would like to thank Mrs. Rodiyati Azrianingsih, S.Si., M.Sc., Ph.D. for the guidance and reviewing
this manuscript. Also the author would like to thank to Porang Research Centre and Biology Department, Faculty of
Mathematic and Natural Science, Brawijaya University for the research facility. This research was funded by
Indonesian Ministry of Culture and Education 2021.

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