Germination response of coconut (Cocos nucifera L.) zygotic embryo

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*Corresponding Email: beckley.ikhajiagbe@uniben.edu
JASEM ISSN 1119-8362
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J. Appl. Sci. Environ. Manage. October 2017
Vol. 21 (6) 1019-1021
Full-text Available Online at
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www.bioline.org.br/ja
Germination response of coconut (Cocos nucifera L.) zygotic embryo
*1
PEACE A. NWITE;
1
BECKLEY IKHAJIAGBE;
2
IDABA OWOICHO
1
Environmental Biotechnology Sustainability Research Group
Dept. of Plant Biol. and Biotechnology, University of Benin, Benin City
2
Nigeria Institute for Oil Palm Research, Benin City
*Corresponding Email: beckley.ikhajiagbe@uniben.edu
ABSTRACT:
The study investigated the effects of liquid and solid media in the propagation of coconut (Cocos
nucifera) zygotic embryos at initiation stage. Eeuwen’s medium supplemented with growth hormones naphthalene
acetic acid ( NAA) and indole butyric acid (IBA) at different concentrations (0.5, 1.0, 1.5, 2.0 and 2.5mg/l) were used
for this experiment in both liquid and solid states. Results showed that liquid state medium was better compared to solid
state, with a successful shoot emergence of 46.7% of inoculants, compared to 13.3% in the solid state. Within 2 – 4
weeks, 77.8% of germinated zygotic embryo developed shoots in the liquid medium compared to 50% rate in the solid
medium. Comparing zygotic embryo development in NAA and IBA, results showed that root yields were better in
NAA, with inoculants in 1.5 mg/l NAA showing profuse rooting compared to 0.5 – 2.0 mg/l IBA with no root
development.
© JASEM
https://dx.doi.org/10.4314/jasem.v21i6.3
Keywords: Coconut, Eeuwen’s medium, hormones, root yield, zygotic embryo
The coconut palms (Cocos nucifera L.), usually
termed as a “tree of life”, is grown throughout the
tropic regions (Waaijenberg, 1994). Virtually all
parts of the coconut tree are utilized; from the nuts,
husks, inflorescences, stems, and roots. The leaves
are used for roofing and brooms, trunks are used for
construction, and the copra can be processed into oil
mainly for the soap industry, cosmetics, and candle
wax (Campbell et al., 2000). Coconut cultivars are
generally classified into the Tall and Dwarf types.
Tall coconuts grow to a height of about 20-30 m and
are allogamous, late flowering, and their nuts are
medium to large in size; they are hardy and thrive in
a wide range of environmental conditions (Child,
1974). Dwarf coconuts grow to a height of about 10 -
15 m and are autogamous, early flowering, and
generally produce a large number of small nuts with
distinctive colour forms (Child, 1974).
The coconut palm 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 (Muhammed, 2013). Therefore,
there is an urgent need to implement efficient
coconut germplasm via in vitro technique of tissue
culture that allows germination and conversion into
plantlets in a controlled environment.
Embryo culture micropropagation has several
advantages over conventional propagation methods;
for instance, the production of disease free, high
quality planting materials and the rapid production of
many uniform plantlets in a limited space area are
achievable by this technology. In the present study,
West African tall and Malayan orange dwarf zygotic
embryos were used as the explants. Thus, the aim of
this study was to understudy the germination
responses of coconut (Cocos nucifera L.) zygotic
embryo via liquid and solid nutrient media.
MATERIALS AND METHODS
This study was carried out using the facilities of
Tissue Culture Laboratory of the Nigerian Institute
for Oil Palm Research (NIFOR), Benin City, Nigeria.
Source of explants: Embryos were obtained from10
to 12 month old nuts, from Nigerian institute for oil
palm research (NIFOR) substation Badagry in Lagos
state. Harvested from mother plant of the West
African Tall (WAF) and Malayan orange Dwarf
(MOD), coconut palms which had been identified to
be highly productive by the plant breeders.
Preparation of culture medium: The basal mediums
used for this experiment were Eeuwens (1976)
medium. The basal mediums were supplemented with
growth hormones (auxins) naphthalene acetic acid
(NAA) and indole butyric acid (IBA) at different
concentrations 0.5, 1.0, 1.5, 2.0 and 2.5mg/l. The
medium contained 30g/l sucrose plus 2.5 g/l activated
charcoal, pH was adjusted to 5.7, medium was
prepared without or with agar autoclaved at a
temperature of 121°C for 20 min before use.
Embryo Extraction: The collected nuts (10 to 12
months old) were split transversely using a machete
and endosperm surrounding embryo was excised
from the split nuts using a clean knife, with a method
adapted by (Molla et al., 2004).

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NWITE, P.A; IKHAJIAGBE, B; OWOICHO, I
Surface sterilization of endosperm extracts: The
endosperm extracts were washed using detergents
and rinsed under a running tap for 10 minutes, they
were further rinsed in the laminar flow chamber with
distilled water and sterilized for 3 minutes in 0.1%
mercuric chloride. The endosperm were thoroughly
rinsed with sterile distilled water, the embryos were
then excised from endosperm using sterile scalpel. A
method adapted by (cutter et al 1954) Cutter.
Thereafter, embryos were then sterilized in 0.2%
sodium hypochlorite for 3minutes, rinsed thrice in
sterile distilled water and inoculated in a liquid
medium and solid medium.
Culture conditions: After two weeks of culture, the
liquid medium was decanted out and the embryos
were transfer into a fresh medium and incubated in
darkness for additional two weeks before transferring
to light. All subsequent in vitro culture was carried
out under dark and light photoperiod, 12-14 and 8-10
hours respectively. After six weeks, embryos that
had started forming shoot were transferred onto semi-
solid medium supplemented with the following plant
growth regulators, naphthalene acetic acid (NAA)
(0.5 – 2.5 mg/l) and indole butyric acid (IBA) (0.5 –
2.5 mg/l). Plantlets were transferred onto fresh
medium every four weeks until they developed leaves
and secondary roots.
RESULTS AND DISCUSSION
The culture of zygotic embryos of coconut as
described in this report hold great potential for use in
propagation work. In numerous other difficult-to-
culture crops, using immature embryos and growth
hormones lead to success in the propagation of the
former.
Results showed that the better media state for
inoculation of coconut zygotic embryo was liquid
(Table 1, Plates 1 and 2). Out of the 15 test tubes
inoculated, 60% successfully germinated. This was
determined on the basis of initial root emergence. In
the solid medium, only 26.7% of the inoculated
embryos germinated. Further, 46.7% of the
germinated rooting embryos successfully developed
shoots within 2 – 4 weeks, amounting to a 77.8%
rate, compared to 50% shooting success rate in the
solid medium.
The findings of the present study are similar to the
earlier report of El Rosario and De Guzman (1976)
on the comparative outcome of the use of liquid and
solid media to initiate Makapuno embryos. Similarly,
Thuzar et al. (2012) reported the use of MS medium
for rapid plant regeneration of oil palm zygotic
embryos.
Table 1: Effect of culture in Eeuwems medium for coconut embryos germination between
Medium Total number of
test tube
inoculated
Percentage zygotic
embryo
germination at 2 –
4 wks (%)
Successful shoot
emergence of
zygotic embryos
b/w 2- 4 weeks
(%)
Successful shoot
establishment (%)
No. of contaminated
culture (percentage, %)
Liquid
medium
15 60.0 46.7 77.8 2 (13.3)
Solid medium 15 26.7 13.3 50.0 8 (53.2)
Plate 1: Zygotic embroyo in liquid medium
Plate 2: Zygotic embryo in solid medium
As presented on Table 2 and Plates 3 – 5, there were
shoot formations from immature zygotic embryos in
some treatments between 4 – 6 weeks. The shoots
were induced in liquid medium containing 1.5 mg/l
of NAA, with the most shoot presence (60%) and the
least shoot presence (20%) at 1.0mg/l concentration.
There were root formations in all the treatments
except 2.5mg/l. The zygotic embryo cultures in IBA
hormone with 2.5mg/l concentration produced shoots
with roots (20%); while those in 0.5, 1.0, 1.5 and 2.0
mg/l had no shoot and root formation. A similar
observation was reported by Venkatesh et al. (2009)
on the effects of auxins and concentration on
groundnut.

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NWITE, P.A; IKHAJIAGBE, B; OWOICHO, I
These results probably suggest that the liquid
medium provided optimum uptake of nutrients by the
germinating embryos and also improved gaseous
exchange than solid medium. Embryos were
considered germinated when the plumule sprouts and
the radicle shows signs of emergence as reported by
Danson et al. (2009). By way of comparing; NAA
was more preferred in shoot and root formation from
1.0 -1.5mg/l concentration than IBA.
Table 2: Effect of NAA and IBA concentrations on embryos germination in
liquid medium between 4-6 weeks
Media Total number of
embryos inoculated
Percentage (%)
successful of Shoot development
Root yields
NAA mg/l
0.5 15 0.0 +
1.0 15 20.0 ++
1.5 15 60.0 +++
2.0 15 0.0 +
2.5 15 0.0 -
IBA mg/l
0.5 15 0.0 -
1.0 15 0.0 -
1.5 15 0.0 -
2.0 15 0.0 -
2.5 15 20.0 ++
+ scanty roots, ++ slightly profuse roots, +++ presence of highly profuse roots. – absence of roots
Conclusion: The preference of liquid media in embryonic growth
initiation has been established in this study. Media
supplementation with 1.0 -1.5mg/l NAA is also preferred.
Germinated embryos may then be transferred onto solid medium.
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Plat
e 3:
Plant from zygotic
embryo in liquid medium
containing NNA
(Tall)
Plate 4
:
Plant from zygotic
embryo
in liquid medium
containing NNA
(Dwarf
)
Plate 5
:
Plant from zygotic
embryo in
liquid
medium
containing IB
A
(Tall
)