Abstract—Cocos Nucifera Linn. Var. MATAG is a new Dwarf

coconut variety in Malaysia. High demand of this hybrid coconut with low supply makes it insufficient to fulfill the market of coconut industry. Problems encountered with conventional breeding of coconut are its long life span and high heterozygosity that make plant breeding a long, difficult and expensive process. Therefore, a higher efficiency

of plant regeneration via in vitro technique is required for mass micropropagation of this coconut variety. To that, the effect of different concentration of 2,4-D and explants will be determined in order to develop in vitro multiplication of Cocos nucifera Linn. var. MATAG. Sterilization method also was optimized to eliminate contamination problems in cultures. The explants produced off-white, friable callus after 4 weeks of culture. Callus induction frequency varied among treatments. Significant callus formation were observed

in hormone free basal media (45.71%) and 20 mg/L of 2,4-D (43.75%). The results achieved suggested that immature inflorescences of Cocos Nucifera Linn. Var. Matag can be alternative sources of explants for the induction of callus formation and somatic embryogenesis

Keywords—callus induction, Cocos Nucifera, immature

inflorescence, sterilization method

I. INTRODUCTION

HE Cocos nucifera Linn. Var. MATAG, a monocot, is

grown as a plantation crop in more than 90 countries for

many uses. It is native to coastal areas of Malaysia, Indonesia,

Philippines and Melanesia [1]. In the tropics, the coconut plant

has the largest number of uses. Plant parts are used for food, oil

production, as construction material, source of energy, and

cosmetics [2]. Coconut cultivars are generally classified into

the Tall and Dwarf types. Cocos Nucifera Linn. Var. MATAG

is new Dwarf varieties in Malaysia.

Their small stature makes them popular to be planted because

the water from immature nuts is drunk as a beverage and the

jelly-like kernel is eaten. However, this species is suffering

from drastic production constraints, including pests and

diseases and susceptibility to natural disasters. In addition, a

number of aging coconut plantations are now being uprooted in

order to make way for the planting of more portable crops [3].

Zawawi Dhiya Dalila

1, Abu Bakar Mohd Fahmi

2 and Abd Kadir Siti

Nurkhalida3 are with the Faculty of Bioresources and Food Technology,

Universiti Sultan Zainal Abidin, Kampus Tembila, 2220, Besut, Terengganu,

Malaysia (corresponding author’s phone: 60139848426; e-mail:

ddalila@unisza.edu.my ).

These factors are resulting in the loss of traditional, locally

adapted coconut germplasm, and therefore, there is an urgent

need to implement efficient coconut germplasm via in vitro

technique of culture that allows germination and conversion

into plantlets in a controlled environment. To that, many

researches had been done in order to propagate Cocos Nucifera

Linn. from different types of explant such as immature

embryos [4], plumule [5], anthers [6] and ovule [7]. In the

present study, immature inflorescence had been used as the

explant.

The greatest problem in micropropagation is contamination

with fungi and bacteria. Therefore, aseptic technique must be

established to sterilize the explants. Success at this stage firstly

requires high rate of the explants should be transferred to the

cultural environment that is free from obvious microbial

contaminants [8]. Thus, the objectives of this study are to

optimize the sterilization method and to induce high rate of

callusing from inflorescence culture with the addition of

activated charcoal to prevent tissue browning of the explants.

II. MATERIAL AND METHODS

A. Plant Materials

Cultures were initiated from ovary and anthers of Cocos

Nucifera Linn. Var. MATAG. Samples of Cocos Nucifera

Linn. Var. MATAG inflorescence was collected from

Department of Agriculture Terengganu farm. The samples were

stored in ice-box to maintain its freshness prior to culture.

B. Optimization of Sterilization Procedure

The rachillae bearing inflorescence were cut off from the

spadices and then were washed under running water for 15 to

20 minutes to remove dirt and debris. The male inflorescence

was isolated from rachillae and subjected to three different

method of sterilization (as described in Table 1).

T

Optimization of Sterilization Method and

Callus Induction of Cocos Nucifera Linn. Var.

Matag from Inflorescence

Zawawi Dhiya Dalila1, Abu Bakar Mohd Fahmi and

2Abd Kadir Siti Nurkhalida

3

International Conference on Plant, Marine and Environmental Sciences (PMES-2015) Jan. 1-2, 2015 Kuala Lumpur (Malaysia)

http://dx.doi.org/10.15242/IICBE.C0115029 84

A B

C

TABLE 1

METHOD OF STERILIZATION FOR INFLORESCENCE OF COCOS NUCIFERA LINN.

VAR. MATAG

Method A

Thiram 20%

Method B

Thiram 15%

Method C

Thiram 10%

Wash the male

inflorescence under

running tap water

(15-20minutes)

↓

Soak in 20% of

thiram for 5 minutes

↓

Rinse with

distilled water

↓

Under aseptic

conditions, soak the

inflorescence in 70%

ethanol for 1 minute.

↓

Rinsed with sterile

distilled water

↓

Treat with

commercial sodium

hypochlorite 20%

plus tween-20 for 15

minutes

↓

Rinsed with sterile

distilled water

↓

Isolation of

anthers from the

male inflorescence

↓

Culture the

anthers on Y3 media

(Euwens 1976) basal

media

Wash the male

inflorescence under

running tap water

(15-20minutes)

↓

Soak in 15% of

thiram for 5 minutes

↓

Rinse with

distilled water

↓

Under aseptic

conditions, soak the

inflorescence in 70%

ethanol for 1 minute.

↓

Rinsed with sterile

distilled water

↓

Treat with

commercial sodium

hypochlorite 20%

plus tween-20 for 15

minutes

↓

Rinsed with sterile

distilled water

↓

Isolation of

anthers from the

male inflorescence

↓

Culture the

anthers on Y3 media

(Euwens 1976) basal

media

Wash the male

inflorescence under

running tap water

(15-20minutes)

↓

Soak in 10% of

thiram for 5 minutes

↓

Rinse with

distilled water

↓

Under aseptic

conditions, soak the

inflorescence in 70%

ethanol for 1 minute.

↓

Rinsed with sterile

distilled water

↓

Treat with

commercial sodium

hypochlorite 20%

plus tween-20 for 15

minutes

↓

Rinsed with sterile

distilled water

↓

Isolation of

anthers from the

male inflorescence

↓

Culture the

anthers on Y3 media

(Euwens 1976) basal

media

C. Media Compositions and Growth Conditions

The basal media used for this experiment were Y3 media [9].

All the media contained 90g/L sucrose plus 0.1% activated

charcoal and were adjusted to pH 5.8 and then solidified with 4

g/L gelrite. The medium was autoclaved at 121°C for 20 min.

The cultures were maintained in a dark culture room. The room

temperature was 25±2°C. For callus induction of the explant,

the basal media were added with the various concentrations of

2,4-Dichlorophenoxyacetic acid (2,4-D) (0, 10, 20, 30 and 40

mg/L).

D. Data Analysis

Data were collected after 4 weeks of culture. There were five

replicates with 10 explants in each replicate assessed. Explants

that showed intense browning were considered to be oxidized;

usually these explants do not progress toward other in vitro

responses. The Callus Induction (CI) was calculated as follows:

Statistical analysis was done by analysis of variance between

groups (ANOVA) and Duncan’s Multiple Ranges Test

(DMRT) using the Statistical Package for Social Science

(SPSS) Programme. Data are expressed as mean of three

determinations ± SE.

III. RESULTS & DISCUSSION S

A. Optimization of sterilization method

Cocos Nucifera Linn. Var. MATAG young fruits developed

by Department of Agriculture (Malaysia) have high

commercial values. However, shortness of young fruits and

conventional breeding method that takes years before the plants

can produce fruits is the reasons why this exotic fruit cannot be

fully commercialized in Malaysia. The protocol for in vitro

mass propagation of this particular Cocos Nucifera species is

therefore crucial to produce a large amount of plants.

Contamination in cultures is one of the limiting factors in plant

tissue culture protocols. Contaminations by bacteria and fungus

affect the percentage of aseptic cultures. Contaminations by

bacteria and fungus affect the percentage of aseptic cultures.

Based on this experiment, method B (85.1 ± 0.05%)

significantly (P<0.05) gave the higher percentage of aseptic

cultures compared to method A (53.4 ± 0.07%) and method C

(58.4 ± 0.07%). [10] reported explants that were surface

sterilized in series or stages resulted in higher percentage of

aseptic cultures. Direct contact of the explants to the sterilant

during sterilization process increased the percentages of aseptic

cultures. This significant result may be to the direct contact of

the sterilant to the explants (inflorescence) and the use of

higher concentration of thiram compare to the other two

methods. Method B gave the highest percentage of aseptic

cultures and therefore was chosen as the sterilization method

for the callus induction experiment.

Fig. 1 represents isolation of anther from the male flowers of

inflorescence; (A) close view of the male flower attached to the

International Conference on Plant, Marine and Environmental Sciences (PMES-2015) Jan. 1-2, 2015 Kuala Lumpur (Malaysia)

http://dx.doi.org/10.15242/IICBE.C0115029 85

middle portion of rachillae; (B) isolated male flower; (C)

excised anther.

TABLE II

THE EFFECTS OF THREE DIFFERENT STERILIZATION METHODS ON THE

PERCENTAGE OF ASEPTIC CULTURES

Sterilization

Method (SM)

Percentage of aseptic

culture (%)

Percentage of

oversterile (%)

A 53.4 ± 0.07 9.9± 0.05

B 85.1 ± 0.05 3.3± 0.06

C 58.4 ± 0.07 6.6± 0.07

Anthers were cultured on Y3 media (Eeuwens, 1976) supplemented with

different concentration of 2,4-D plus 0.1% activated charcoal at 25 ± 2oC in

dark condition. Data were recorded after 4 weeks of culture incubation. Values

represent the mean ± SE.

B. Callus induction of Cocos Nucifera Linn. Var. MATAG

The effect of growth regulators on callus induction of

coconut using anther as explant was investigated using various

concentrations of 2,4-D (Table 1). Induction of callus could be

observed after one month of culture initiation. Table 3 showed

that callus induction frequencies depended on the concentration

of 2,4-D treatment. The highest percentage of callusing was

observed in hormone free basal media (45.71%). The anther

wall was burst open (Figure 2A) and callus grow were visible

(Figure 2B). Among the auxins, 2,4-D has been employed in

many anther culture system [11] and 2,4-D was the most

suitable auxin that can be used alone [12]. Varying the 2,4-D

concentration may lead to more efficient callus or embryo

formation [13]. However, the callus induction percentage

decreasing when using 2,4-D in our study (refer Table 2).

In most cases, anthers were isolated by removing whole

anthers from their filaments and then culturing them on media

without slicing or treating them [14]. In our study, the whole,

uncut and untreated anther is being cultured on media (Figure

1B). No injuries to anther were involved. Injures to anthers

during excision should be avoided in order to prevent somatic

callus production from anther-wall tissues [15]. The

compositions of macro and micro elements in Y3 media has

been reported to be more suitable for palm species compared to

White, Heller, or Murashige and Skoog [9].

Fig. 2 represents callus induction from the anthers of Cocos Nucifera

Linn. Var. MATAG on Y3 media (Eeuwens, 1976) supplemented with

20 mg/L of 2,4-D after 4 weeks of incubation at 25 ± 2oC in dark

condition. A: Anther wall was break open; B: Callus formation from

the anther.

TABLE II

THE EFFECTS OF DIFFERENT CONCENTRATIONS OF 2,4-D

ON CALLUS INDUCTION (%) FOR MATAG

2,4-D (mg/L) Callus induction (%)

0 (Control) 45.71

10 42.22

20 43.75

30 36.17

40 41.30

Anthers were cultured on Y3 media (Eeuwens, 1976) plus 0.1% activated

charcoal at 25 ± 2oC in dark condition. Data were recorded after 4 weeks of

culture incubation. Values represent the mean ±SE.

IV. CONCLUSION

The sterilization method for inflorescence has been optimized

and the results achieved suggested that inflorescence of Cocos

Nucifera Linn. Var. MATAG can be used as explants for callus

initiation and somatic embryogenesis. From this experiment,

we now know that Y3 media supplemented with 20 mg/L 2,4-

D, and hormone free basal media was suitable for callus

induction from the anthers of Cocos Nucifera Linn. Var.

MATAG. However, further improvement of the callus

induction method is required. Positioning of anther without

adjoining filaments will be applied to make sure that Callus

only will be growing from the anther only. Combination of 2,4-

D with another plant growth regulator such as NAA might be

considered in order to increase the quality and quantity of

embryogenic callus and initiation of somatic embryoids.

ACKNOWLEDGMENT

The authors would like to thank Universiti Sultan Zainal

Abidin (UniSZA) and Ministry of Education Malaysia for the

permission to publish this work and financing this project and

the Department of Agriculture Terengganu farm for their

research collaboration and generous supply of Cocos Nucifera

Linn. Var. MATAG inflorescences.

REFERENCES

[1] E. Chan and C. R. Elevitch, “Cocos nucifera (coconut),” no. 2.1, pp. 30–

50, Apr. 2006.

[2] Y. Koffi, O. N’Nan-Alla, J.-L. Konan, B. Malaurie and F. Engelmann ,

“Morphological and agronomical characteristics of coconut (Cocos

nucifera L.) palms produced from in vitro cultured zygotic embryos,”

Vitro Cellular & Developmental Biology – Plant, vol. 49, no. 5, pp. 599–

604. 2013. http://dx.doi.org/10.1007/s11627-013-9531-y

[3] P. Aké, B. Maust, A. Orozco-Segovia, and C. Oropeza, “The effect of

gibberellic acid on the in vitro germination of coconut zygotic embryos

and their conversion into plantlets,” Vitr. Cell. Dev. Biol. - Plant, vol. 43,

no. 3, pp. 247–253, Mar. 2007.

[4] S. C. Fernando and C.K.A. Gamage, “Abscisic acid induced somatic

embryogenesis in immature embryo explants of coconut (Cocos nucifera

L.). Plant Sci. vol. 151, pp. 193–198, 2000.

http://dx.doi.org/10.1016/S0168-9452(99)00218-6

[5] J. L. Chan, L. Saénz, C. Talavera, R. Hornung, M. Robert and C. Oropeza

“Regeneration of coconut (Cocos nucifera L.) from plumule explants

through somatic embryogenesis” Plant Cell Reports, vol. 17, no. 6-7, pp.

515–521. 1998. http://dx.doi.org/10.1007/s002990050434

[6] P.I.P. Perera, D.M.D. Yakandawala, V. Hocher, J.L. Verdeil, and L.K.

Weerakoon, “Effect of growth regulators on microspore embryogenesis in

International Conference on Plant, Marine and Environmental Sciences (PMES-2015) Jan. 1-2, 2015 Kuala Lumpur (Malaysia)

http://dx.doi.org/10.15242/IICBE.C0115029 86

coconut anthers,” Plant Cell, Tissue and Organ Culture., vol. 96, pp. 171-

180, 2008. http://dx.doi.org/10.1007/s11240-008-9473-y

[7] P.I.P. Perera, V. R. M. Vidhanaarachchi, T. R. Gunathilake, D. M. D.

Yakandawala, V. Hocher, J. L. Verdeil and L. K. Weerakoon. Effect of

plant growth regulators on ovary culture of coconut (Cocos nucifera L.).

Plant Cell, Tissue and Organ Culture, vol. 99, no. 1, pp. 73–81. 2009.

http://dx.doi.org/10.1007/s11240-009-9577-z

[8] E.F. George, A.H. Michael, J.D.K. Geert in Plant Propogation by Tissue

Culture. 3rd ed. vol. 1. Springer. Pp. 134-143, 2008.

http://dx.doi.org/10.1111/j.1399-3054.1976.tb05022.x

[9] C.J. Eeuwens “Mineral requirements for growth and callus initiation of

tissue explants excised from mature coconut palms (Cocos nucifera) and

cultured in vitro” Physiol. Plant. Vol. 36, pp. 23-28, 1976.

[10] B. Ghoreyshi, R. Naderi, S. A. G. Maghami, S. Zeyni, “A

decontamination procedure for in vitro culture of Lilium longiflorum cv.

'Dozzel' scale explants” Acta Horticulturae, vol. 829, pp. 289-293, 2009.

[11] S.T. Ball, H. Zhou, C.F. Konzak, “Influence of 2,4-D, IAA and duration

of callus induction in anther culture of spring wheat” Plant Science, vol.

90, 195-200, 1993. http://dx.doi.org/10.1016/0168-9452(93)90240-Z

[12] M. R. C. Oropeza, “Regeneration of coconut (Cocos nucifera L.) from

plumule explants through somatic embryogenesis,” pp. 515–521, 1998.

[13] P. I. P. Perera, D. M. D. Yakandawala, V. Hocher, J.-L. Verdeil and L. K.

Weerakoon. “Effect of growth regulators on microspore embryogenesis in

coconut anthers” Plant Cell, Tissue and Organ Culture, vol. 96, no. 2, pp.

171–180. 2008. http://dx.doi.org/10.1007/s11240-008-9473-y

[14] P.I.P. Perera, V. Hocher, J.L. Verdeil, H.D.D. Bandupriya, D.M.D.

Yakandawala, and L.K. Weerakoon, “Androgenic potential in coconut

(Cocos nucifera L.)” Plant Cell, Tissue and Organ Culture, vol. 92, pp.

293-302, Jan. 2008. http://dx.doi.org/10.1007/s11240-008-9337-5

[15] B. Winarto and J.A. Teixeira da Silva, “Influence of isolation technique

of half anthers and initiation culture medium on callus induction and

regeneration in Anthurium andreanum” Plant Cell, Tissue and Organ

Culture. vol.110, pp. 401-411, 2011. http://dx.doi.org/10.1007/s11240-

012-0161-6

[16] J. Reinert and Y.P.S. Bajaj “Anther culture: haploid production and its

significance. In: Reinert J., and Bajaj Y.P.S. (eds) Applied and

fundamental aspects of plant cell, tissue and organ culture. Springer,

Berlin, pp. 251-267. 1977. http://dx.doi.org/10.1007/978-3-662-02279-5

International Conference on Plant, Marine and Environmental Sciences (PMES-2015) Jan. 1-2, 2015 Kuala Lumpur (Malaysia)

http://dx.doi.org/10.15242/IICBE.C0115029 87