American Journal of Plant Sciences, 2012, 3, 1562-1567
http://dx.doi.org/10.4236/ajps.2012.311188 Published Online November 2012 (http://www.SciRP.org/journal/ajps)
Season, Environment Stress and Refrigerated Storage
Affect Genomic DNA Isolation of Tung Tree
Lingling Zhang1,2,3, Yue Pan1,2,3, Jinm in Fu1,2, Junhua Peng1,2*
1Wuhan Botanical Garden, Chinese Academy of Sciences, Wuhan, China; 2Key Lab of Plant Germplasm Enhancement and Specialty
Agriculture, Chinese Academy of Sciences, Wuhan, China; 3Graduate University of Chinese Academy of Sciences, Beijing, China.
Received August 11th, 2012; revised September 19th, 2012; accepted October 6th, 2012
ABSTRACT
Many metabolites in leaf tissue disturbed plant genomic DNA isolation and they always varied when leaf was harvested
from different environments. Objective of this study was to investigate whether season, environment stress and refriger-
ated storage affect genomic DNA isolation of tung tree leaves. Five types of young leaves and two DNA isolation pro-
tocols, the recycling CTAB protocol I and II, were adopted to carry out the experiment. Our results showed that both
leaf type and protocol affected DNA isolation of tung tree. Using the recycling CTAB protocol II, though little DNA
were obtained from three types of young leaves, though the other two have satisfying results. Whereas the recycling
CTAB protocol I could produce high yield genomic DNA from all the five types of young leaves. All the detectable
DNA samples in agarose gel electrophoresis were good templates for PCR reaction. Season, environment stress and
refrigerated storage had a big effect on genomic DNA isolation of tung tree. The recycling CTAB protocol I was proved
to be an effective and universal protocol for DNA isolation of tung tree. Five types of young leaves could all act as the
tissue for isolation of genomic DNA, but the summer healthy young leaves without long-time refrigerated storage are
the best. The optimal leaf tissue will benefit DNA isolation of plant species.
Keywords: Tung Tree; Vernicia fordii; DNA Isolation; Season; Environment Stress; Storage
1. Introduction
Plant often produces many metabolites, such as polysac-
charides, polyphenols, protein and tannin. These com-
pounds always interfere with DNA isolation and down-
stream DNA-based experiments [1-6]. To date, lots of
DNA isolation protocols have been published and aimed
at eliminating the negative effects of biochemical com-
ponents on DNA isolation [7-17]. However, DNA isola-
tion is still a challenge for many plant species.
Chemotypic heterogeneity among different species did
not allowed the direct application of an extraction proto-
col for a species to other species [17-19]. Metabolites in
plant leaves were not stable, changing with many factors,
such as leaf age [19-21], season [22-24], light intensity
[25] and environmental stress [26,27]. Leaf age was re-
ported to affect the properties of extracted DNA, which
was inferred to be related with the accumulation of de-
fense compounds during the leaf development [19]. En-
vironmental factors such as light intensity, temperature
and seasonal variance were reported to affect the produc-
tion of polysaccharide [25], phenolic compounds [28,29]
and other metabolites in leaves. However, up to now,
little reports were about the effect of theses environ-
mental factors on DNA isolation of plant species.
Tung tree (Vernicia fordii), belonging to Euphor-
biaceae, is an oil-producing plant with multiple uses es-
pecially its potential in biodiesel production [30-32].
DNA extraction is essential for molecular genetic analy-
ses and marker-assisted improvement of this oil-pro-
ducing species. In present study, we found that season,
environment stress and refrigerated storage all had big
effect on genomic DNA isolation of tung tree. Besides,
the recycling CTAB protocol I was proved to be a uni-
versal method for DNA isolation of tung tree.
2. Materials and Protocols
2.1. Materials
Young leaves were harvested from healthy tung tree at
different seasons, in the autumn (early November in
2009), spring (early April in 2010) and summer (July in
2010), respectively (hereafter called autumn healthy
leaves, spring healthy leaves and summer healthy leaves
for short). Besides, leaves were also harvested from the
tung tree that suffered from both transplant and water-
drown stress in the summer of 2010 (hereafter called
*Corresponding author.

Season, Environment Stress and Refrigerated Storage Affect Genomic DNA Isolation of Tung Tree 1563
summer stressed leaves for short). These four types of
leaf tissues were ground into fine powder in liquid nitro-
gen, and then 0.3 g of powders for each leaf tissue type
were immediately used to isolate genomic DNA. Besides,
some other summer healthy leaf tissues were stored in
freezer for three months and then was used to isolate
genomic DNA. Tissue powder was a mixture of several
genotypes. Unless state, the leaf referred to the young
leaf without the refrigerated storage in the text below.
2.2. Protocols
Two protocols (Lingling Zhang et al., unpublished) were
adopted to extract DNA from the five types of tung tree
young leaves. The main procedures of recycling CTAB
protocol I was followed. 1) Add 1.5 mL of wash buffer to
0.3 g of ground leaf sample. Mix well and place at the
low temperature with occasional swirling for at least 15
min. Then centrifuge at 12,000 rpm for 15 min. Discard
the supernatant. The tissue pretreatment was carried out
for three times; 2) Add 0.5 mL of CTAB extraction buffer
and re-suspend tissue pellet. Incubate at 65˚C for 40 min.
Centrifuge at 12,000 rpm for 15 min. Transfer the super-
natant to a new tube; 3) Add 200 μL Chloroform-isoa-
mylalcohol (24:1). Mix well and let stand for several
minutes. Centrifuge at 12,000 rpm for 15 min and trans-
fer the supernatant to a new 1.5 mL tube carefully; 4)
Precipitate the DNA by adding two volumes of cold ab-
solute ethanol and incubate for 30 min at −20˚C. Transfer
the DNA precipitation with a tip and wash it with 70%
ethanol for 30 min, better for overnight. Then pour off
the ethanol; 5) Dry the DNA and dissolve it in 100 μL
T0.1E. Add 1 µL of 10 μg/mL ribonuclease. Incubate the
DNA solution at 37˚C for 30 min, and then incubate at
65˚C for 15 min. This DNA sample was termed as the
first DNA (1st DNA); 6) Similarly, treat the residual pel-
lets in the above step 2) for other three cycles of the step
2) to the step 5). Correspondingly, the resulted DNA
samples were termed as the second DNA (2nd DNA),
third DNA (3rd DNA) and fourth DNA (4th DNA), re-
spectively. The recycling CTAB protocol II shared all the
procedure of the recycling CTAB protocol I except that
the tissue pretreatment was only carry out for one time.
2.3. DNA Isolation and Assessment
The quality and yield of extracted DNA were determined
by agarose gel electrophoresis. Concentration of DNA
samples was assessed as the following. 2 μL of DNA
solution for each sample was added into 48 μL of steril-
ized water (mix well). Then 5 μL of the diluted DNA
solution plus 5 μL of 2.5X loading buffer was loaded into
0.8% agrose gel and run in 0.5X TBE buffer (pH 8.0) at
room temperature. The gel was visualized with ethidum
bormide staining. DNA quantity for each lane/sample
was estimated by comparing band intensity with the
concentration-known λ DNA standards (Promega, Madi-
son, WI, USA). DNA concentration was calculated using
formula, concentration = [quantity (ng) of 5 μL diluted
DNA/5 μL × dilution factor (50/2 = 25)].
2.4. Polymerase Chain Reaction Amplification
Coding region cloning of fad2 gene was performed using
PCR approach. On the basis of GenBank sequence
AF525534, sequences of primers for PCR amplification
are 5’-GATGGGTGCTGGTGGCAGAATGTCA-3’ and
5’-CCAGAACTTCCAAGCCCTTCACTTTTG -3’ [33],
which amplify an approximately 1.2 kb fragment encom-
passing the entire coding region of fad2 gene. The PCR
were performed in a 25 μL reaction volume (60 ng tem-
plate DNA, 0.4 μM specific primers, 1 U Taq DNA po-
lymerase, 0.2 mM dNTP mixture, 1 × PCR buffer, 0.15
mM Mg2+) using BIORAD-My Cycle Thermocyclers
(Bio-Rad Laboratories, Inc., California, USA) with the
following program: pre-denaturation at 94˚C for 4 min;
30 cycles of denaturation at 94˚C for 45 s, annealing at
65˚C for 45 s, extension at 72˚C for 60 s; and a final ex-
tension step at 72˚C for 10 min, then hold on for 15 min
at 4˚C. Then PCR products were separated in 1% agarose
gels and the gel was visualized with ethidium bromide
staining.
3. Results
3.1. Comparisons of DNA Samples Isolated
From Five Types of Leaves Using the
Protocol I
Using agarose gel electrophoresis and λDNA of known
concentration as standards, concentration of DNA sam-
ples produced by the protocol I were determined. As
shown in table 1, yield of the 1st DNA varied for the five
types of young leaves, about 200 ng/μL both for spring
healthy leaf and summer healthy leaf, 100 ng/μL for
summer stressed leaf, but little DNA for summer healthy
leaf with three-month refrigerated storage and autumn
healthy leaf (Figure 1). As shown in Figure 1, the 2nd,
3rd and 4th DNA for each type of leaf were all obviously
detectable in agrose gel electrophoresis, the concentra-
tion of these DNA samples ranging from 300 ng/μL to
500 ng/μL (Table 1). These data indicated that the pro-
tocol I could be successfully applied to different types of
young leaves without significant differences.
3.2. Comparisons of DNA Samples Isolated from
Five Types of Leaves Using the
Protocol II
However using the recycling CTAB method II, DNA
samples extracted from the fve types of leaves greatly i

Season, Environment Stress and Refrigerated Storage Affect Genomic DNA Isolation of Tung Tree 1564
Figure 1. Agarose gel electrophoresis of genomic DNA isolated from five types of young leaves using two protocols. (a) Au-
tumn healthy leaf; (b) Spring healthy leaf; (c) Summer healthy leaf; (d) Summer stressed leaf; (e) Summer healthy leaf with
three-month refrigerated storage.
Table 1. Concentration of DNA samples for the five types of young leaves using the two protocols.
C1 (ng) C2 (ng/μL)
Protocol Leaf Type
1st 2nd 3rd 4th 1st 2nd 3rd 4th
a \ 100 100 100 \ 500 500 500
b 40 100 100 100 200 500 500 500
c 40 100 100 100 200 500 500 500
d 20 60 100 80 100 300 500 400
Recycling CTAB
Protocol I
e \ 60 60 60 \ 300 300 300
a \ \ \ 20 \ \ \ 100
b \ 60 60 60 \ 300 300 300
c 60 150 80 60 300 750 400 300
d \ 10 20 \ \ 50 100 \
Recycling CTAB
Protocol II
e \ \ \ 10 \ \ \ 50
C1, DNA quantity (ng) in 5 μL of diluted solution loaded into agaroe gel estimated by comparison with the standard λDNA of known content, 50 ng; C2, DNA
concentration (ng/μL) estimated using agarose gel electrophoresis and calculated as C2/5 μL (volume of the loaded DNA dissolution) × 25 (dilution factor).
Four DNA samples were isolated from a single sample. 1st, 2nd, 3rd and 4th represent the first DNA samples, the secondary DNA samples, the third DNA
samples and the four DNA samples, respectively. Lowercase letter a, b, c, d and e represent the autumn healthy leaf, spring healthy leaf, summer healthy leaf,
summer stressed leaf and summer healthy leaf with three-month refrigerated storage, respectively. The slashes in the tables represent the DNA samples which
could not be detectable in agarose gel electrophoresis.
summer leaf with three months refrigerated storage, only
the 4th DNA of the four samples was detectable but the
concentration was relatively low, about 50 ng/μL. These
data indicated that the protocol II could not be applied to
the different types of young leaf and also demonstrate the
biochemical components for the five types of young leaf
tissues were greatly varied.
varied in yield. First, for autumn healthy leaf, the first
three of the four DNA samples were not detectable and
concentration of the 4th DNA was about 100 ng/μL (Fig-
ure 1 and Table 1). Secondly, for spring healthy leaf, the
1st DNA was not detectable but 2nd, 3rd and 4th DNA-
samples were all detectable, each about 300 ng/μL. Thirdly,
all the four DNA samples for summer healthy leaf were
detectable, and concentration of 1st to 4th DNA was
about 300 ng/μL, 750 ng/μL, 400 ng/μL, and 300 ng/μL,
respectively (Tabl e 1). Fourthly, for the summer stressed
leaf, the 1st and 4th DNA samples was not detected in
agarose gel while the band of the 2nd and 3rd DNA sam-
ples were very weak, apparently different from the four
bright bands of summer healthy leaf above. Last, for the
3.3. Analysis of DNA Quality by PCR
Amplifycation of Fad2 Gene
For the four DNA samples of a tissue sample, the DNA-
detectable samples were mixed into one DNA sample
with an exception for the four DNA samples of the
summer healthy leaves. Thus, using the two protocols,

Season, Environment Stress and Refrigerated Storage Affect Genomic DNA Isolation of Tung Tree 1565
eight DNA-incorporated samples were obtained from the
four types of leaves and eight DNA samples were got
from the summer healthy leaf. These sixteen DNA sam-
ples were all used as the templates to amplify fad2 gene.
As shown in Figure 2, the target bands for all the sixteen
DNA samples were visible and distinct, and were the
expected size of fad2 gene fragments (1.2 kbp) targeted
by the primers used, indicating that all the sixteen DNA
samples had enough purity for fad2 gene cloning.
4. Discussion
4.1. Factors Affecting Genomic DNA Isolation of
Tung Tree
Leaves were the main source of DNA isolation. Many
metabolites in leaves interfere with isolation of clean
DNA. These metabolites in leaves were in mobile droved
by the exogenous and endogenous signals. Defense
compounds like polyphenols and tannin were reportedly
largely produced and accumulated when plants were in
the stress [28,29]. Leave development also had a big ef-
fect on the composition of biochemical components in
leaves [19-22,34]. The matured tree leaves were reported
to be difficult in DNA isolation for its thick cell wall, and
high content secondary metabolites [18]. Leaf age of
Fabaceae (Dimorphandra mollis) was reported to affect
DNA isolation, in which DNA was successfully isolated
from the young but failed from the old leaves, which was
inferred to be attributed to the differently cumulative
amount of chemical defenses such as tannins and phenols
during leaf development [19]. In this study, five types of
young leaves of tung tree were harvested in different
seasons or environments. Using the recycling CTAB
method II, the greatly varied DNA yield indicated the
season for leaf harvest, environment stress and refriger-
ated storage all had big effect on DNA isolation. In con-
clusion, it will benefit DNA isolation if taking the envi-
ronments, such as seasons into account at the step of leaf
harvesting.
4.2. An Effective and Universal Method for DNA
Isolation of Tung Tree
Chemotypic heterogeneity in different species didn’t
allow the direct application of an extraction protocol for
a specific species to other species [19,35]. In present
study, chemotypic heterogeneity did not affect the appli-
cation of protocol I among the five types of young leaf,
but seriously disturb the application of the protocol II. It
could conclude that the protocol I is an effective and
universal method for DNA isolation of tung tree. The
only difference between the two protocols is focused on
the time of tissue pretreatment, which was carried out for
three times for protocol I but one once for protocol II.
Hence, it highlighted the importance of efficient elimi-
2kbp
1kbp
Figure 2. Agarose gel electrophoresis of fad2 gene fragment.
Lowercase letter a, b, c, d and e represent the autumn
healthy leaf, spring healthy leaf, summer healthy leaf, sum-
mer stressed leaf and summer healthy leaf with three-
month refrigerated storage, respectively.
nating the secondary compounds on DNA isolation. As
discussed above, we choose the optimal leaves for DNA
isolation to reduce difficulties in DNA isolation. How-
ever, the leaves available were always limited by various
reasons and we have to isolate the DNA from the leaves
with various backgrounds. Therefore, a universal DNA
isolation protocol is expected for the species of inter-
ested.
5. Acknowledgements
This work was supported by the National Natural Sci-
ence Foundation of China (NSFC) Grant Nos. 31030055
and 30870233, Chinese Academy of Sciences under the
Important Directional Program of Knowledge Innovation
Project Grant No. KSCX2-YW-Z-0722.
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