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1Hainan Key Laboratory of Tropical Oil Crops Biology/Coconut Research Institute, Chinese Academy of Tropical Agricultural Science,
Wenchang, Hainan 571339, China
2Department of Agriculture, Abdul Wali Khan University Mardan, Pakistan
3Department of Economics and Management, Huazhong Agricultural University Wuhan, Hubei China
4Department of Botany, Abdul Wali Khan University Mardan, Pakistan
Here we explained a convenient, rapid and
cheap method of gDNA extraction from the hard
and complex tissues of the coconut. The method
has also been verified against betel nut and availa-
ble vegetables (mint, spring onion, radish, ginger,
coriander) at the Coconut Research Institute, Wen-
chang-China. Extraction of gDNA was carried out
through 8 simple steps. The method doesn’t require
intensive cleaning steps as designated to the com-
mercially available kits. The method efficiently
isolated the highly intact gDNA with great purity.
This cheap and reliable method has broad future
prospects in the field of plant and animal genetics,
breeding and pathology.
"
gDNA, nucleic acid extraction, coconut, palmacea, hard
tissues
Coconut () is a perennial trop-
ical fruit that is considered as an oil crop due to
high oil content in their endosperm [1-3]. Coconuts
are widely distributed in the humid and sub-humid
coastal parts of the world. Coconuts are good for
health [4] and have an important role in the sustain-
ability of the grower’s life due to its economic val-
ue, thus it is known as ‘tree of life’ [5]. Coconut has
been cultivated in the tropical regions of the globe
for thousands of years and has been used as medi-
cine, baby food and for cooking purposes [6]. Over
the years, large number of cultivars, varieties and
types were resulted because of natural evolution,
manual selection and domestication. Despite its
agronomic importance, the work regarding
germplasm assessment was revolved around mor-
phological and agronomical traits. Molecular biolo-
gy techniques have been scarcely used in the as-
sessment of genetic resources and for the improve-
ment of important agronomic and quality traits in
this important species [7]. To bring an improvement
in the existing cultivars of the coconuts at molecu-
lar level, the extraction of the genetic material is the
first, but very important step. Certainly, it is well
known that gDNA or genome of the species con-
sists of the whole information related to the all
traits of the species, which gives an overall picture
of the important aspects of the species. The extrac-
tion of high quality gDNA in an optimum amounts
is very important for the successful experiments
(such as, constructing a library, PCR amplification,
enzymatic experiments, etc.) in the field of molecu-
lar biology [8-10]. To achieve the high quality of
the gDNA in greater amounts from the hard tissues
of the coconut in short time is very difficult. To
keep the importance of the gDNA extraction, a
quick, cheap, convenient and effective method has
been the demand of the molecular biologists. Cur-
rently there are several gDNA extraction kits from
various companies are available in the market, such
as Promega Corporation, Qiagen Company and
other Gene companies. The kits are indeed very
expensive and give low yields, which seems to be
impractical, while doing large scale population
analysis. On the contrary, the method we have dis-
covered for the extraction of gDNA from the hard
tissues of coconut is cheap, quick and reliable.
Agrose gel, chloroform, chloroform-isoamyl
alcohol (24:1, i.e. 24 parts of chloroform mix with 1
part of isoamyl alcohol), Acid buffered phenol (pH
4.5), NaOH (1M), isopropanol, ammonium thiocy-
nate, guanidine thiocynate, sodium acetate (3M),
glycerol, β-merceptoethanol, PVP-40, eppendorf
tubes, mortar and pestle, scissor/razor blade, plastic
bags, freezer, spatula, pipette, Tris borate EDTA
(TBE) buffer, ethidium bromide, DNA ladder and
DPEC water. All the chemicals used were of high
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analytical purity and were purchased from Real
Times and Chemical Reagents, China.
/$170$7(5,$/6The coconut tissues of fresh-
ly harvested coconuts were collected at the Coconut
Research Institute, Chinese Academy of Tropical
Agricultural Sciences, Wenchang, Hainan-China.
Similarly, leaf tissues of betel nut, mint, spring on-
ion, radish, ginger, corianderwere taken from the
freshly sown vegetables at the vegetable farm of the
same Institute. All the samples were cut into small
pieces with the help of scissors or razor blade and
transferred to small plastic bags. The plastic bags
were then preserved quickly in liquid nitrogen and
were kept frozen at -80 ºC till further analysis.
8))(5(&,3(
i.Ammonium thiocyanate : 0.3 M
ii.Guanidine thiocyanate : 0.7 M
iii.3M sodium acetate (pH 5) : 3.3 ml
iv.Phenol : 38 ml
v.Glycerol : 5 ml
vi.NaOH (1M) : to make the
buffer pH=8
vii.DEPC water : up to 100 ml
viii.β-merceptoethanol : 2%
ix.PVP-40 : 3%
5(3$5$7,21 Take 30 ml of (DEPC water)
and add the contents to it one by one (except PVP-
40), once dissolved make the buffer pH=8 and ad-
just the volume to 100 ml with DPEC water. The
buffer should be handled in a fume hood as it con-
tains phenol and should be kept refrigerated in a
dark bottle to avoid oxidation. The buffer can be
used within six months of preparation.
5272&2/
1.Transfer the sample containing bag from
the freezer to the lab. Pour liquid nitrogen into a
clean mortar and pestle (previously autoclaved) to
make it cool. Take about 0.08g of the frozen tissue
from the plastic bag and transfer it to the mortar
containing liquid nitrogen and PVP-40 (3%). Grind
the tissue to a fine powder with a pestle, top up the
mortar with liquid nitrogen during grinding to avoid
any thawing. Immediately transfer pulverized tissue
with the help of a frozen spatula to a 1.5 ml tube,
containing 1 ml of extraction buffer, vortex for 15
seconds, and then incubates on ice for 4 minutes.
2.Add 200 µl of chloroform and vortex thor-
oughly for 15 seconds, then incubate on ice for 4
min. Centrifuge it for 4 minutes at 12,000 rpm
at 4 °C.
3.Transfer the supernatant (500 µl) to a new
tube and add an equal volume of chloro-
form/isoamyl alcohol to it and centrifuged at 12,000
rpm for 4 minutes.
4.Carefully transfer the upper aqueous phase
(400 µl) of the solution to a fresh, eppendorf tube,
then add an equal volume of ice cold isopropanol
(500 or 600 µl), mix the solution by inverting the
tubes and incubate for 4 minutes on ice. Centrifuge
for 4 min at 12,000 rpm and 4 °C, after centrifuga-
tion discard the supernatant.
5.Redissolve the palette in 30 μl of DEPC
water by pipetting gently for 1-2 minutes. Add 1 μl
of RNAses to the content of the tube and incubate it
at 37 °C for 18 minutes.
6.Add equal volume of the ice cold isopro-
panol to it mix the solution by inverting the tubes
and incubate for 4 minutes on ice. Centrifuge for 4
min at 12,000 rpm and 4 °C, after centrifugation
discard the supernatant.
7.Add 70% ethanol of the ethanol to the pal-
ette and centrifuge it for 4 minutes at 8000 rpm and
4 °C.
8.Discard the supernatant and re-dissolve the
palette in 30 μl of DEPC water by pipetting gently
till the dissolution of the palette.
5($.832)7,0(
•Pulverization of sample: step 1, 20
minutes for 4 samples
•gDNA extraction: step 2 to 4, took 15
minutes
•gDNA extraction: step 5, took 20 minutes
•gDNA precipitation: step 6 to 8, took 15
minutes
•Qualitative and quantitative analysis of
gDNA took 25 minutes
The newly developed protocol for the extrac-
tion of gDNA from complex matrices, rich in car-
bohydrates, lipids, proteins and polyphenols pro-
vided us with a high quality gDNA in appreciable
quantities. The A260/280 ratio of gDNA extracted
from the coconut leaf bud by the claimed method
was 1.98. Similarly, the concentration of the ex-
tracted gDNA from the coconut leaf bud was 27.52
µg/ 80 mg sample. The gDNA was also successful-
ly isolated from other tissues of the coconut and the
tissues of the other available plant species. From
the electrophoretic results, it was quite clear that the
gDNA extracted with the current method has a
strong band without any contamination (Figure 1-
A, B, C).
Genomic DNA extraction is the most im-
portant step for various investigations in the field of
molecular biology (such as, constructing a library,
PCR amplification, enzymatic experiments, etc.).
To extract high quality gDNA from plant and ani-
mal tissues, various methods have been adopted
over the years. Being, the tropical region of China,
Wenchang is famous for its elite coconuts with di-
verse germplasm. To study the various attribute of
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;75$;7,212)*)5209$5,2867,668(62)&2&2187$1'27+(53/$1763(&,(6
Lane 1 in Figure A,B,C represents the DNA markers/ladder; ‘*’ represents coconut leaf bud in Figure (A); ‘**’ represents
coconut apple in Figure (A); ‘*’ represents coconut leaf in Figure (B); ‘**’ represents betel nut leaf in Figure (B); Lane 2-6 in
Figure (C) represents mint, spring onion, radish, ginger and coriander, respectively.
the palm at the molecular level, one should be cer-
tain of the gDNA that can be used effectively in
studying the whole genome. The Biotechnology
Laboratory at the Coconut Research Institute is
actively involved to extract quality gDNA from the
complex matrices of the coconut species. Currently,
the laboratory is extracting the gDNA from a large
number of samples through CTAB, but the method
is time consuming. Therefore, we have developed a
new method to extract gDNA from the hard tissues
of coconut, which is cost effective, easy and less
time consuming to extract quality gDNA from the
matrices that are high in carbohydrates, lipids, pro-
teins and polyphenols. Certainly, the method has
been validated initially for the extraction of gDNA
from coconut tissues, betel nut, mint spring onion,
radish, ginger and coriander. But, it might be ex-
tended to various other tissues from plant and ani-
mal origins that are rich in macromolecules and
other metabolites.
Researches were supported by the Natural
Science Foundation of China (No. 31870670), the
earmarked fund for the Belt and Road Tropical Pro-
ject (BARTP-06), the Fundamental Scientific Re-
search Funds for Chinese Academy of Tropical
Agricultural Sciences (CATAS-No. 16301520170
19), Central Public-interest Scientific Institution
Basal Research Fund for Innovative Research Team
Program of CATAS (NO.17CXTD-28) and the
Talented Young Scientist Program (TYSP) from
Ministry of Science and Technology of China.
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Hainan Key Laboratory of Tropical Oil
Crops Biology, Coconut Research Institute,
Chinese Academy of Tropical Agricultural Science,
Wenchang, Hainan 571339 – China
e-mail: yyang@catas.cn
