Micropropagation of Sumatra Benzoin (Styrax benzoin Dryander) to

Obtain Plant Seedling

Isnaini Nurwahyuni

and Riyanto Sinaga

Departement of Biology, Faculty of Mathematics and Natural Science, Universitas Sumatera Utara, Jl. Bioteknologi No.1

Padang Bulan, Medan, North Sumatera, Indonesia

Keywords: The paper Micropropagation, Sumatra Benzoin, Styrax benzoin Dryander, good seedling.

Abstract: The strategy to produce high quality seedling of Sumatra Benzoin (Styrax benzoin Dryander) is very urgent

as a technique to provide seedling for industry and forestry purposes in Indonesia. The limitation number of

good quality seedling becomes the main problem because generative propagation is the only strategy that has

been conducted to produce Styrax benzoin seedling, which are obtained naturally grown from the seed around

the mature styrax trees. Micropropagation is an alternative to produce good quality seedling of Sumatra

Benzoin. The study is aimed to obtain optimum conditions for micropropagation of Sumatra Benzoin as a

step in the production of styrax seedling for forestry purposes. The study is performed in a completely

randomized design with using the concentrations of growth stimulators NAA (0 - 3 mg/L) and BAP (0 - 3

mg/L). The results showed that growth stimulator influenced the growth of the calli. The NAA and BAP was

effective to induce calli of the benzoin in culture medium. The concentration combination of 3 mg/L NAA

and 3 mg/L BAP was the best condition to obtain the heaviest callus. The micropropagation step obtained in

this study is promising for mass production of Styrax benzoin seedling.

1 INTRODUCTION

The conservation of forest by using woody plants

that containing bioactive compounds is very strategic

in Indonesia to preserve the trees as well as avoid the

logging since the tree can be used as a source of

income to the people around the forest. One of the

potential trees is Sumatra Benzoin that are grown well

in Indonesia. The skin saps of Styrax produce incense

containing bioactives that can be used in medical and

cosmetics purposes (Singh, et al. 2017). Sumatra

Benzoin is a Divisio of Spermatophyta, sub Divisio

of Angiospermae, where the Class is Dicotyledonae,

Ordo is Ebenales, the Family is Styraceae, Genus is

Styrax, and the Spesies Styrax benzoin Dryander

(Nurwahyuni and Elimasni, 2006). Sumatra Benzoin

grown well in North Sumatera Indonesia and become

forest commodity, and it becomes income generate to

the people around the forest in many regencies in

Indonesia (BPS, 2017). The insence of benzoin was

commonly use as traditional and modern medicine

(Fan, et al. 2017; van Wyk, 2009; Sianipar and

Simanjuntak, 2000). The Sumatra Benzoin has been

known to be valuable tree and have high prospect,

however, the cultivation of Sumatra Benzoin has not

been done properly in Indonesia. Production of high

quality styrax seedling become a problem since the

peasants in North Sumatera are still depend on a

generative propagation for Benzoin seedling.

Therefore, the existence of Sumatra Benzoin was

merely obtained from naturally grown in the forest

due to the difficulty to obtain good quality of styrax

seedling. It has been predicted that the existence of

Sumatra Benzoin will be lower down in the future if

there is no action being carried out to plant the

Sumatra Benzoin in North Sumatera Indonesia.

Micropropagation technique is a good alternative

to produce high quality seedling of wood trees. Mass

production of the seedlings similar to its parent plant

can be provided with this technique (Mirani, et al.

2017; Pniewski, et al. 2017; Martínez-Estrada, et al.

2017; Gashi, et al. 2015). The source of explant for

micropropagation can be come from seed, leaf blade,

petiole, stem segment, axilar shoot, and meristemic

axilar from young plant with active cell (Panigrahi, et

al. 2017; Pedro, et al. 2017; Ahmed and Anis, 2014;

Chen, et al. 2014). The strategy was made with plated

the explant in MS medium containing mineral salts,

amino acid, vitamin, glucose and growth stimulator at

certain composition (Zhang, et al. 2016; Murashige

and Skoog, 1962; Murashige and Tucker, 1969).

Various types of growth stimulators have been

introduced such as auxin (NAA, 2,4-D, IBA, etc.) and

Nurwahyuni, I. and Sinaga, R.

Micropropagation of Sumatra Benzoin (Styrax benzoin Dryander) to Obtain Plant Seedling.

DOI: 10.5220/0010067809570963

In Proceedings of the International Conference of Science, Technology, Engineering, Environmental and Ramiﬁcation Researches (ICOSTEERR 2018) - Research in Industry 4.0, pages

957-963

ISBN: 978-989-758-449-7

957

cytokinin (BA, kinetin, dan zeatin) for plants

propagation (Trettel, et al. 2017; Quiroz, et al. 2017;

Yakhin, et al., 2017). It has been obtained that the

growth stimulators influenced the development of the

plants (Quiroz, et al. 2017; Shinde, et al. 2016). It has

been known that low concentration cytokinine is

found to be effective to generate the callus and the

shoot from stem segment (Nurwahyuni, et al. 2017;

Nurwahyuni, et al. 2015), and the auksin to

regenerate the roots (Shukla, et al. 2017; Alatar, et al.

2017). Therefrore, the strategy has to be made to

optimise the concentration of growth stimulators in

the regeneration of new target plants (Thiem, et al.

2017); Nurwahyuni and Sinaga, 2014; Soni and Kaur,

2014). Based on literature studies, the paper publish

on propagation of benzoin is very limited, only to

study the activity of cytotoxisity (de Oliveira, et al.

2016) and in vitro propagation (Nurwahyuni and

Elimasni, 2006; Nurwahyuni, 2005). Therefore,

Benzoin micropropagation techniques applied in this

study was carried out followed the success for

propagation of medicinal woody plants (Baskaran, et

al. 2017; Cardoso, et al. 2017; Wu, et al. 2014). This

research was aimed to implement micropropagation

technique to propagate Sumatra Benzoin (Styrax

benzoin Dryander) as a strategy to obtain good quality

of styrax seedling.

2 RESEARCH METHODS

2.1 Materials and Method

The study was conducted at Universitas Sumatera

Utara, in the Department of Biology. Mother plant of

Sumatra Benzoin (Styrax benzoin Dryander) was

obtained from the forest at Kecamatan Pergetteng-

Getteng Sengkut, Kabupaten Pakpak Bharat, North

Sumatera, Indonesia. The procedures carried out in

this study are consisted of sterilization of equipments,

preparation of medium solution, micropropagation,

and data collections followed the micropropagation

procedures explained previously (Nurwahyuni and

Sinaga 2018; Nurwahyuni, 2016).

2.2 Sterilization of Equipment

The equipments were sterilized by soaking them in

hot detergent, followed by rinsing with sterile water

flow and keep to dry. They are then wrapped in

aluminium foil and sterilized in oven at 180

C for 2

hours. Glass wares are sterilized by using autoclave

at 121

C, 15 psi for 15 minutes. The laminar air flow

cabinet (LAFC) was sterilized using UV light and

alcohol (70%).

2.3 Preparation of Medium Culture

Medium culture used in this study was MS medium

containing sugar, nutrients (macro, micro, and trace),

with supplementation of -napthaleneacetic acid

(NAA) and benzyl amino purin (BAP) in various

combination concentrations (Nurwahyuni and Sinaga

2018; Nurwahyuni, 2016). The solution was prepared

in sterile water and transferred in to an erlenmeyer

and the stock solution was stored in the fridge when

not in use. The buffer solutions (pH 5.8 - 6.8) were

prepared and they were sterilized (121

C, 15 lb) for

20 minutes. The variation compositions of growth

stimulator (0 - 3 mg/L) BAP and (0 - 3 mg/L) NAA

were prepared with five replicates (Zar, 1996) as

summarized in Table 1. The medium consisted of

sucrose that are enriched with growth stimulator of

NAA and BAP. Callus initiation was performed in

MS culture medium enriched with growth stimulator

(Murashige and Skoog, 1962).

Tabel 1: Experimental design for micropropagation of

Sumatra Benzoin (Styrax benzoin Dryander) in MS medium

containing of -napthaleneacetic acid (P) and benzyl amino

purin (Q). The experiments are carried out with 5 replicates.

Citokinine BAP

(

)

Auxine

NAA (P)

01 2 3

= Naphthalene acetic acid 0.0 mg/L; P

= Naphthalene

acetic acid 0.5 mg/L; P

= Naphthalene acetic acid 1.0

mg/L; P

= Naphthalene acetic acid 3.0 mg/L; Q

= Benzyl

Amino Purin and BAP 0.0 mg/L; Q

= Benzyl Amino Purin

and BAP 0.5 mg/L; Q

= Benzyl Amino Purin and BAP 1.0

mg/L; Q

= Benzyl Amino Purin and BAP 3.0 mg/L.

2.4 Micropropagation Procedures

The explants used in the study are obtained from

mature seeds of Sumatra Benzoin (Styrax benzoin

Dryander) that was taken from good quality benzoin

(Fig. 1a). The seed is then opened and the embryo is

taken (Fig. 1b). The explants for micropropagation is

prepared from healthy benzoin seeds (Fig. 1c) which

are then sterilized, and were then cut (0.5 - 1.0 cm

long), successively washed in water and detergent,

followed by immersion in 70% (v/v) ethanol for 5

ICOSTEERR 2018 - International Conference of Science, Technology, Engineering, Environmental and Ramiﬁcation Researches

958

min, in a solution of 20% (v/v) sodium hypoclorate

(0.8 %w/v NaClO) for 20 min, followed by rinsed

with sterile water three times. After sterilization

process have been conducted, the explant was cut and

inoculated in culture medium with variation in the

concentration of 2,4-D and BAP in the culture

medium (Fig. 1d). The incubation of cultures were

then kept at 25±2 °C. The culture was daily

illumination with UV light for 16-h and spray with

alcohol 70% regularly.

Figure 1. Preparation of healthy benzoin seeds as sources of

explants for micropropagation: (a) A mature seed obtained

from good quality Sumatra Benzoin (Styrax benzoin

Dryander), (b) Typical inner side of Sumatra Benzoin seed,

of explant, (d) The explant is planted in cultur media for

incubation.

3 RESULTS AND DISCUSSION

3.1 Callus Induction of Sumatra

Benzoin

Induction of callus is very important in the

propagation of Sumatra Benzoin as it was known that

the ability of cell multiplication and differentiation

are influenced various factors such as culture medium

components, the concentration of growth stimulators,

and the intensity of UV light (Nurwahyuni and Sinaga

2018; Nurwahyuni, 2016). Incubation of Sumatra

Benzoin (Styrax benzoin Dryander) culture has been

carried out at various treatment conditions (Figure

2a). It has been observed that the callus induction was

starting after 20 days, that was begin with the cell

multiplication to become bigger explant until the

callus has grown at week three. The culture

regenerated to become callus. Growth intensity of the

callus are vary depends on the variation of the

concentration of growth stimulator in culture

medium. Typical growth of the culture is presented in

Figure 2b where the callus started to grow from the

edge of the explant (Figure 2c-d).

Figure 2. Typical growth of callus in micropropagation of

Sumatra Benzoin (Styrax benzoin Dryander): (a) Callus

initiation of the benzoin in culture medium at various

treatment conditions at room temperature; (d) The

development of explant to become callus in cultur media,

closed view of the callus,

Micropropagation condition has been optimised

to obtain the best condition in the callus induction.

The growth of the culture of Sumatra Benzoin with

the variation of BAP and NAA in MS culture medium

is observed after seven weeks as presented in Table 1.

It was observed that in the first and second weeks, the

explants are all transformed to bigger explant

condition, and some callus are grown after three

weeks. The growth intensity of the callus are assigned

to be low to high intensity, depends on the variation

of growth stimulator supplemented in the culture

media. The formation of callus have covered the

explants in some treatment conditions, however,

some of the explants are having callus in low and

medium growth intensity after seven weeks while

many of the callus growth are in high intensity (see

the results in Table 2). Some cultures are browning

without callus due to contamination.

Micropropagation of Sumatra Benzoin (Styrax benzoin Dryander) to Obtain Plant Seedling

959

Table 2: Typical growth development of the callus culture on micropropagation of Sumatra Benzoin (Styrax benzoin

Dryander) in medium culture after incubation for seven weeks.

Experimen

tal Treatment

Growth of the callus (week)

1 2 3 4 5 6 7

* * * * * * *

* * + + + + +

* * + + ++ ++ ++

* * ++ ++ +++ +++ +++

* * + + + + +

* * + + ++ ++ ++

* * ++ ++ +++ +++ +++

* * + + + ++ ++

* * * * + + +

* * + + ++ ++ ++

* * + + + + +

* * + + ++ ++ ++

* * ++ ++ +++ +++ +++

* * + + + + +

* * + + ++ ++ ++

* * ++ ++ +++ +++ +++

= Naphthalene acetic acid 0.0 mg/L; P

= Naphthalene acetic acid 0.5 mg/L; P

= Naphthalene acetic acid 1.0 mg/L; P

Naphthalene acetic acid 3.0 mg/L; Q

= Benzyl Amino Purin and BAP 0.0 mg/L; Q

= Benzyl Amino Purin and BAP 0.5

mg/L; Q

= Benzyl Amino Purin and BAP 1.0 mg/L; Q

= Benzyl Amino Purin and BAP 3.0 mg/L. (*) The explant grow

bigger, (+) low callus growth intensity, (++) medium callus growth intensity, (+++) high callus growth intensity,

3.2 Optimization of Growth Stimulator

The weight of the callus in the culture media have

been observed with results from the variation of

growth stimulator supplemented in the study. The

growth development of the culture was categorised

very slow. The weight of the callus obtained at

different experimental treatments is presented in

Table 3. The results showed that variation of the

experimental conditions influenced the weight of

callus. The percentage of styrax culture survive until

week seven are lies between 50-83%, where

treatment condition on P

are only survive 50%,

while P

and P

are 83%. It has been observed

that the weight of callus increased as increasing the

growth stimulator in the culture medium. The

heaviest callus is obtained at P

(1.275 gram), that

is in the experiment condition with combination of 3

mg/L NAA and 3 mg/L BAP. The growth trend has

showed in the data that the concentration of BAP is

significantly improved the growth of the callus. Data

analysis has revealed that high concentration of NAA

and BAP influenced growth intensity of Sumatra

Benzoin callus (F

count

27.39 > F

table

2.44), with

significant level at 0.01. The effect of each of the

growth stimulator onto the growth intensity of the

callus of Sumatra Benzoin has been observed. It was

found that the concentration of the NAA in MS media

highly influenced the growth intensity of the callus

count

113.45 > F

tablet

4.23). The results also revealed

that the concentration of BAP influenced the growth

intensity of the Benzoin callus (F

count

11.70 > F

crit

4.23). The interaction effects for both the NAA and

BAP are also found influenced the growth intensity

of styrax callus (F

count

3.94 > F

table

2.44). The

concentration of growth stimulators that are

supplemented in the culture media become crucial

factor in the micropropagation of the Sumatra

Benzoin.

ICOSTEERR 2018 - International Conference of Science, Technology, Engineering, Environmental and Ramiﬁcation Researches

960

Table 3: The influences of the concentration variation of the growth stimulators (NAA and BAP) onto the growth intensity

of the callus of Sumatra Benzoin (Styrax benzoin Dryander). The results are obtained after incubation seven weeks.

Experimental

Treatment

Weight of Callus (Gram) / Replication

Total Average*

I II III IV V

0.016 0.018 0.017 0.015 - 0.066 0.017

0.028 0.030 - 0.028 0.027 0.113 0.028

0.030 - 0.030 0.030 0.030 0.120 0.030

0.031 0.031 0.031 0.031 - 0.124 0.031

0.040 0.039 0.040 0.060 0.031 0.210 0.042

0.042 0.042 0.041 0.042 - 0.167 0.042

0.050 0.049 0.051 0.050 - 0.200 0.050

0.053 0.054 0.053 0.052 - 0.212 0.053

0.065 0.070 0.060 0.065 0.065 0.325 0.065

0.067 0.068 0.065 0.070 - 0.270 0.068

0.073 0.070 0.075 0.073 - 0.291 0.073

0.088 0.093 0.090 0.102 - 0.373 0.093

0.147 0.141 0.150 0.147 - 0.585 0.146

0.160 0.165 0.158 - - 0.483 0.161

0.180 - 0.202 0.191 0.185 0.758 0.190

0.186 - 0.275 0.302 0.512 1.275 0.319

Total 1.256 0.870 1.338 1.258 0.850 5.572 1.114

Avera

e 0.079 0.067 0.089 0.084 0.142 0.343 0.086

= Naphthalene acetic acid 0.0 mg/L; P

= Naphthalene acetic acid 0.5 mg/L; P

= Naphthalene acetic acid 1.0 mg/L; P

Naphthalene acetic acid 3.0 mg/L; Q

= Benzyl Amino Purin and BAP 0.0 mg/L; Q

= Benzyl Amino Purin and BAP 0.5

mg/L; Q

= Benzyl Amino Purin and BAP 1.0 mg/L; Q

= Benzyl Amino Purin and BAP 3.0 mg/L. (-) The culture was not

grown.

*Data shown are mean of five experiments followed by notation letter are significant according to Duncan’s multiple range

analysis (P = 0.05)

4 CONCLUSIONS

Micropropagation of Sumatra Benzoin (Styrax

benzoin Dryander) has successfully conducted to

produce callus as a primary step in the propagation of

local benzoin. The effect of NAA and BAP onto the

growth of the styrax callus has been formulated to

obtain optimum condition in the propagation

procedures, and become a good step in the production

of benzoin seedling. The results have shown that the

concentration variation of growth stimulators alone

(NAA or BAP), and the combination of both the NAA

with BAP, are all acted to induce the callus of

Sumatra Benzoin

in MS medium, and showed

significantly effect to the weight intensity of the

Benzoin callus. The best condition for

micropropagation of the

Sumatra Benzoin was

obtained when using combination concentration of 3

mg/L NAA and 3 mg/L BAP (P

) that was

produced 1.275 gram callus. Supplementation of high

concentration of NAA and BAP is effective to

improve the growth of callus of Sumatra Benzoin

Micropropagation of Sumatra Benzoin (Styrax benzoin Dryander) to Obtain Plant Seedling

961

ACKNOWLEDGMENTS

This funding support for this research is obtained

from Universitas Sumatera Utara, Under TALENTA

Basic Research, Financial Year 2018, Contract no

174/UN5.2.3.1/PPM/KP-TALENTA USU/2018, 16

March 2018. The help from Hotdi Brutu and Hamda

Situmorang at Pergetteng-Getteng Sengkut,

Kabupaten Pakpak Bharat, to provide Sumatra

Benzoin for the study is also acknowledged.

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