International Conference on Plant, Marine and Environmental Sciences (PMES-2015) Jan. 1-2, 2015 Kuala Lumpur (Malaysia)
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 Nurkhalida3
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,
inflorescence, sterilization method
Cocos
Nucifera,
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.
immature
II. MATERIAL AND METHODS
I. INTRODUCTION
T
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].
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).
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:
[email protected] ).
http://dx.doi.org/10.15242/IICBE.C0115029
84
International Conference on Plant, Marine and Environmental Sciences (PMES-2015) Jan. 1-2, 2015 Kuala Lumpur (Malaysia)
TABLE 1
METHOD OF STERILIZATION FOR INFLORESCENCE OF COCOS NUCIFERA LINN.
VAR. MATAG
Method A
Thiram 20%
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
Method B
Thiram 15%
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
(DMRT) using the Statistical Package for Social Science
(SPSS) Programme. Data are expressed as mean of three
determinations ± SE.
Method C
Thiram 10%
III. RESULTS & DISCUSSION S
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
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.
A
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).
C
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:
Fig. 1 represents isolation of anther from the male flowers of
inflorescence; (A) close view of the male flower attached to the
Statistical analysis was done by analysis of variance between
groups (ANOVA) and Duncan’s Multiple Ranges Test
http://dx.doi.org/10.15242/IICBE.C0115029
B
85
International Conference on Plant, Marine and Environmental Sciences (PMES-2015) Jan. 1-2, 2015 Kuala Lumpur (Malaysia)
TABLE II
THE EFFECTS OF DIFFERENT CONCENTRATIONS OF 2,4-D
ON CALLUS INDUCTION (%) FOR MATAG
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
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.
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.
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 2,4D, 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 2,4D 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.
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].
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]
[2]
[3]
[4]
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.
http://dx.doi.org/10.15242/IICBE.C0115029
2,4-D (mg/L)
[5]
[6]
86
E. Chan and C. R. Elevitch, “Cocos nucifera (coconut),” no. 2.1, pp. 30–
50, Apr. 2006.
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
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.
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
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
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)
[7]
[8]
[9]
[10]
[11]
[12]
[13]
[14]
[15]
[16]
coconut anthers,” Plant Cell, Tissue and Organ Culture., vol. 96, pp. 171180, 2008. http://dx.doi.org/10.1007/s11240-008-9473-y
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
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
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.
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.
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
M. R. C. Oropeza, “Regeneration of coconut (Cocos nucifera L.) from
plumule explants through somatic embryogenesis,” pp. 515–521, 1998.
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
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
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/s11240012-0161-6
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
http://dx.doi.org/10.15242/IICBE.C0115029
87