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PHILIPP AGRIC SCIENTIST

Vol. 95 No. 4, 335343


December 2012

ISSN 0031-7454

Ultraviolet-B (UV-B) Radiation as an Elicitor of Flavonoid


Production in Callus Cultures of Jatropha (Jatropha curcas L.)
Erika Marie Alvero-Bascos* and Lilian B. Ungson
Plant Tissue Culture Laboratory, Institute of Biology, College of Science, University of the Philippines, 1101 Diliman,
Quezon City, Philippines
*
Author for correspondence; e-mail: khai_213@yahoo.com; Telefax: (+632) 920-5479
Callus cultures of jatropha (Jatropha curcas L.) grown in Murashige and Skoogs (MS) medium
supplemented with naphthalene-acetic acid (NAA; 20M) and 6-furfurylaminopurine (kinetin; 20 M)
were exposed to ultraviolet-B (UV-B) radiation to investigate its potential as an abiotic elicitor of
flavonoid production. Prior to irradiation, the levels of the flavonoids, apigenin, vitexin and
isovitexin in the leaf and callus extracts were determined through high-performance liquid
chromatography (HPLC). Results showed that vitexin and isovitexin were the dominant flavonoids in
the leaves while only apigenin was detected in the calli, suggesting a correlation between the degree
of differentiation and biosynthesis of flavonoids in plant tissues. Irradiation of callus cultures for 7 d
using two UV-B doses (12.6 and 25.3 kJ m-2) induced synthesis of all three flavonoids (up to 780 g
g-1 dw increase) to levels similar to or higher than those found in whole leaves. The combined levels
of the three flavonoids in the cultures treated with the higher UV-B dose were 20-fold higher than the
control and were comparable to concentrations found in leaves while a 10-fold increase in combined
flavonoid levels was observed in calli irradiated with the lower UV-B dose. Furthermore, random
amplified polymorphic DNA (RAPD) analyses of DNA extracts from the leaves and calli revealed that
UV-B irradiation enhanced flavonoid synthesis without altering DNA sequence. These results
further support the supposed involvement of UV-B in the transcriptional regulation of the
expression of flavonoid biosynthetic genes. Overall, the findings showed that elicitation through UVB irradiation is an effective strategy to induce flavonoid production in dedifferentiated J. curcas
cultures that have lost their capacity to produce the flavonoids normally synthesized in intact
organs.

Key Words: callus culture, dedifferentiation, elicitor, flavonoid, Jatropha curcas, ultraviolet-B radiation
Abbreviations: CHS chalcone synthase, HPLC high performance liquid chromatography, MS Murashige and
Skoog, NAA naphthalene-acetic acid, PAL phenylalanine ammonia lyase, PCR polymerase chain reaction,
RAPD Random Amplified Polymorphic DNA, UV-B ultraviolet-B

INTRODUCTION
Flavonoids comprise a large group of natural phenolic
compounds found in vascular plants as products of
secondary metabolism. Their therapeutic potential as
antioxidants and their promising role in anti-cancer
therapy, along with their manifold applications in the
cosmetic and pharmaceutical industry, have aroused
interest in developing alternative systems for increased
production. Biotechnological approaches such as cell or
tissue cultures, which allow optimization of growth
conditions and genetic manipulation to enhance flavonoid
production, offer an opportunity to circumvent problems
regarding insufficient yield. Of the various tissue culture
methods, callus induction is frequently used for flavonoid
production
since
extraction
procedures
from
undifferentiated tissues are easier to perform (Jedinak et
The Philippine Agricultural Scientist Vol. 95 No. 4 (December 2012)

al. 2004). Unfortunately, the dedifferentiation of plant


cells during callus initiation often results in a decrease in
their capacity to synthesize compounds normally
produced in the plant (Yeoman and Yeoman 1996;
Biondi et al. 2002). Thus, to exploit in vitro grown callus
tissues as an alternative source of flavonoids, effective
strategies to increase yield must be employed.
One approach to enhance flavonoid production is
through elicitation with ultraviolet radiation in the UV-B
range or of wavelength ranging from 280 to 320 nm.
Flavonoids are protective UV-B absorbing compounds
and their synthesis is highly regulated by UV-B radiation.
Irradiation with various doses of UV-B has been shown
to stimulate a considerable increase in the amount of
flavonoids produced by callus cultures (Antognoni et al.
2007; Hao et al. 2009) and whole plants (Lavola et al.
1997; Schnitzler et al. 1997; Tegelberg and Julkunen335

Ultraviolet-B Radiation as an Elicitor

Tiitto 2001; Turtola et al. 2005). This action has been


attributed to the ability of UV-B radiation to induce
transcription of genes encoding enzymes involved in the
biosynthesis of flavonoids (Logemann et al. 2000), which
is regarded as a protective mechanism against the
potentially damaging effects of irradiation. Hence, as an
abiotic stress factor, UV-B radiation may serve as a tool
for improving secondary metabolite production in plant
cultures.
Propagation of Jatropha curcas L. through tissue
culture has been the focus of a number of studies since it
was discovered that its seed oil can be used as biofuel
and various medicinally-important compounds can be
extracted from its organs (Nath and Dutta 1991; Lin et al.
2003; Muangman et al. 2005). The leaves, which are the
most commonly used explants in callus induction
experiments, have been previously identified as potent
sources of the flavonoids, apigenin, vitexin and
isovitexin. These compounds are responsible for the antiinflammatory activity of J.curcas leaves (Chhabra et al.
1990) and may also account for the effectiveness of leaf
extracts and decoctions as treatment for wounds,
rheumatism and lymphocytic leukemia (Hufford and
Oguntimein 1987; Gubitz et al. 1999). In their pure form,
these flavonoids were found to exhibit strong antioxidant,
anti-irritant, anti-cancer and photo-protective activities
(Lepley et al. 1996; Mc Vean et al. 2000; Lin et al. 2002;
Lin et al. 2005; Szliszka et al. 2008), making them
valuable in the fields of scientific research and medicine.
The aim of this research was to investigate whether
or not UV-B irradiation of J. curcas callus tissues could
increase their flavonoid biosynthetic potential by acting
as an abiotic elicitor. The effects of different doses of
UV-B radiation on the amount of flavonoids produced by
the callus cultures and the degree of genetic variability
among the cultures were also examined to determine
flavonoid production and the levels of genetic variation.
The potential of UV-B irradiation to increase
flavonoid production has not been investigated in J.
curcas, particularly in the callus stage. The present work
provides an example of how the benefits derived from
elicitation can be utilized to increase production of
pharmaceutically important flavonoids in J. curcas callus
cultures without the use of expensive and sophisticated
biotechnological methods such as genetic transformation
or ex vivo chemical synthesis. This approach to flavonoid
synthesis would not only circumvent the problem of
insufficient production in intact organs and the difficulty
of extraction but would also provide a steady source of
flavonoids independent of climate changes, geographical
location and pest damage. Moreover, inducing such
valuable variations in J. curcas calli may also serve as an
initial step in the propagation of plants with increased
flavonoid production through organogenesis.

336

Erika Marie Alvero-Bascos and Lilian B. Ungson

MATERIALS AND METHODS


Callus Induction
J. curcas leaves (35 nodes from the apex) were
collected from a shrub located at Jacinto Street,
University of the Philippines (UP), Diliman, Quezon
City. The vegetative organs of the plants from the site
were previously authenticated by Dr. Daniel Lagunzad,
former curator of the J. V. Santos Herbarium, Institute of
Biology, UP Diliman. Voucher specimens from this
shrub are kept at the herbarium. Immediately after
collection, petioles were removed and the edges of the
leaves were trimmed prior to washing with very dilute
detergent. The cut leaves were placed under running
water for 15 min and subsequently washed with sterile
distilled water. Further surface sterilization was done by
brief immersion in 70% ethanol twice for 3 min. This
procedure was followed by a 7-min immersion in 20%
commercial bleach solution containing a few drops of
Tween 20. The leaves were rinsed five times with sterile
distilled water after immersion in each of the sterilizing
solutions.
Leaf explants (approximately 1 cm x 1 cm) were
excised from the midrib area. These were inoculated into
test tubes containing 20 mL MS basal medium (pH 5.6)
with sucrose as carbon source at 2 g L-1 and plant tissue
culture agar as solidifying agent at 8 g L-1 (Murashige
and Skoog 1962). All leaf pieces were placed onto the
medium with the adaxial surface facing the agar. Various
combinations of equal concentrations of the plant growth
regulators, 1-naphthaleneacetic acid (NAA) and 6furfurylaminopurine (kinetin, Kin) were tested (Table 1)
in order to determine which concentrations can produce
sufficient calli for the succeeding experiments.
Fifteen explants were used per treatment. Test tubes
were incubated in a lighted growth chamber at 26 C.
Cultures were observed daily for any sign of growth or
contamination. Callus formation, morphology, texture
and color were monitored by visual inspection over a 1mo period. Callus growth, in terms of fresh weight, was
measured weekly. Among the media that were able to
produce sufficient amounts of calli, the one containing
the lowest hormone concentration was utilized to
establish callus cultures that were used in the succeeding
experiments as higher hormone levels were previously
found to induce genetic variation in the cultures (Smith
and Street 1974; Rao et al. 1992; Kaeppler et al. 2000). A
total of 60 explants excised from the leaf (including the
midrib) were cultured in test tubes containing the selected
growth medium. Once established, the calli were
subcultured at monthly intervals on the same medium,
under the same conditions.

The Philippine Agricultural Scientist Vol. 95 No. 4 (December 2012)

Ultraviolet-B Radiation as an Elicitor

Erika Marie Alvero-Bascos and Lilian B. Ungson

Table 1. Combinations of the plant growth regulators


naphthalene acetic acid (NAA) and kinetin (Kin) tested
for callus induction potential in Jatropha curcas L.
Medium
1
2
3
4
5
6

Growth Regulators (M)


NAA

Kin

0
10
20
30
40
50

0
10
20
30
40
50

Ultraviolet-B (UV-B) Irradiation


Six-week-old calli were subcultured in petri plates
containing 20 mL of growth medium. The lids of the
petri dishes were removed and replaced with plastic film
(Reynolds, USA), which is 85% transparent to UV-B
radiation 300 nm (Antognoni et al. 2007). UV-B
radiation was provided by 40-watt UV-B lamps (Philips
TL40/12, Germany). The lamps were suspended directly
above the dishes and filtered with 0.13-mm thick
cellulose acetate (transmission down to 280 nm) to
remove any ultraviolet C component emitted. Two UV-B
irradiation doses (12.6 and 25.3 kJ m2) were tested
(Antognoni et al. 2007; Hao et al. 2009). The desired
doses were obtained by changing the distance between
the lamps and the dishes (Predieri et al. 1993). The
spectral irradiance from the lamp was determined using a
UV meter (Model 3D-UVB Solar Light Co.). Control
dishes were further covered with 0.13-mm thick polyester
film to exclude UV-B (cut-off at 318 nm). All cultures
also received white light from fluorescent lamps.
Cultures were exposed to the two UV-B doses for 7 d
(Antognoni et al. 2007; Hao et al. 2009). Two trials were
carried out with three replications under a completely
randomized design.
Random Amplified Polymorphic DNA (RAPD)
Analyses
DNA was extracted from the control and irradiated calli
and from leaves of the parent plant. Genomic DNA was
extracted using Promega Wizard Genomic DNA
Purification Kit. Forty milligrams of leaf and callus tissue
were used. Polymerase chain reaction (PCR) was
performed using 10 random decamer primers which have
been used in an earlier study wherein the variability
between and within the genomes of different cultivars of
J. curcas were distinctly differentiated (Ganesh Ram et
al. 2008). PCR amplification was performed in a total
volume of 25 L containing 10 mM Tris/HCl (pH 8.3),
50 mM KCl, 200 M of each dNTPs, 2 mM MgCl2, 2
M primer, approximately 130 ng of template DNA and
1.5 U Taq polymerase using a thermal cycler (Labnet
Multigene).
The Philippine Agricultural Scientist Vol. 95 No. 4 (December 2012)

After an initial denaturation step for 3 min at 95 C,


the amplification reactions were carried out for 40 cycles.
The procedures for each cycle were as follows: 1 min
denaturation at 94 C, 1 min annealing at 35 C and 2
min extension at 72 C. The final elongation step was
extended to 5 min. These PCR conditions were based on
the protocol of Ganesh Ram et al. (2008) with some
modifications. Amplification products were separated on
1.2% agarose gels in TAE buffer, stained with ethidium
bromide and photographed under UV light using the
UVP Gel Documentation System.
High Performance Liquid Chromatography (HPLC)
Analyses
Five grams of fresh callus and leaf samples were dried in
an oven for 24 h at 60 C. Dried tissues were weighed
and homogenized with 10 volumes of methanol. The
methanolic extracts were placed in an ultrasound bath for
30 min. The homogenate was filtered through several
layers of cheesecloth, and the residue was re-extracted
twice. Filtrates were evaporated to dryness at 40 C.
Dried residues were weighed and taken up in 10 mL
methanol. After filtering through a 0.22-m microsyringe
filter, 10 L of the filtrates were directly injected for
HPLC analysis (Antognoni et al. 2007).
The HPLC procedure was adapted from the study of
Peng et al. (2005) with some modifications. Separations
were performed using an HPLC instrument equipped
with a 20 L loop and UV-variable wavelength detector
set at 280 nm. Methanolic extracts were run in an HPLC
C18 column (5 m, 250 4.6 mm) at a flow rate of 1.2
mL min-1. The mobile phases included acetonitrile
(solvent A) and 42% (v/v) methanol in water (solvent B)
mixed using a linear gradient starting with 0% A (2 min),
increasing to 12% A (15 min), 12% A (3 min) and 0% A
(5 min). Standard solutions of apigenin, isovitexin and
vitexin were used to identify and quantify these
compounds in the extracts. Peak areas of increasing
concentrations of vitexin, isovitexin and apigenin
standards (Sigma) were used to construct calibration
curves. The linear regression equations generated from
these standard curves were used to calculate the
flavonoid concentrations of the methanolic extracts
obtained from fresh leaves, as well as from the untreated
and UV-irradiated calli.
Statistical Analyses
The bands generated by RAPD-PCR were scored for
their presence 1 or absence 0 for each primer. Very
faint or unclear bands were not considered. The binary
data were subjected to cluster analysis. Similarity
matrices were generated by Jaccards coefficient of
similarity using Multivariate Statistical Package 2008. A
dendrogram was constructed using the unweighted pair
group method with arithmetic average (UPGMA) to
obtain a representation of genetic relationships as
337

Ultraviolet-B Radiation as an Elicitor

revealed by the similarity coefficient. For the other


measured parameters (fresh weight, flavonoid
concentrations), analysis of variance (ANOVA) and
Bonferroni test were used in comparing the data (SPSS).
P values less than 0.05 were considered statistically
significant. Means, standard deviations and standard error
were computed and corresponding graphs were
constructed using Graph Pad Prism Software (2011).

RESULTS
Callus Induction and UV Irradiation
In the two trials performed, all the tested culture media
were able to induce callus formation in more than 60% of
the leaf explants, except for the medium without plant
growth regulators (Table 2). Calli were first observed 67
d after inoculation and were found predominantly on the
cut portions of the midrib and secondary veins. Most of
the calli formed were green in color and exhibited a
slightly compact structure. Callus growth rate varied
among the different media but all cultures exhibited
about 3- to 5- fold increases in a 5-wk period based on
fresh weight (Fig. 1). By the end of the fifth week, the
calli grown in medium containing 10 M NAA + 10 M
kinetin were found to have significantly lower fresh
weights than those cultured in other growth media (Fig.
2) and reached fresh weights averaging 4 g.
Among the growth media that were able to produce
at least 5 g of calli on the fifth week of culture, the
medium supplemented with the lowest levels of plant
growth regulators (20 M NAA + 20 M kinetin) was
chosen to initiate and maintain the callus cultures that
were used in the irradiation experiments in order to
minimize the probable effects of high hormone levels on
the degree of genetic variation in the cultures. After 89
d from the start of culture, 8083% of the explants
cultured in the two trials had formed callus. By the fifth
week, the calli reached an average weight of 5.02 0.432
g and were then subjected to the UV treatments. UV-B
irradiation had no noticeable effect on the growth and
morphology of the treated calli.
Genetic Variation
In the two trials performed, sizes of the products
amplified using 10 random primers varied from 150 to
4,000 bp (Table 3). The number of bands for each primer
varied from 8 (OPAL 8) to 17 (OPAB 5), with an average
of 12.44.94 bands per primer. The RAPD analyses of
DNA obtained from the leaf (mother plant), along with
the control and UV-B irradiated calli generated a total of
890 bands, all of which exhibited monomorphic patterns.
Examples of the monomorphic bands and their intensities
are shown in Figure 3. These identical banding patterns
generated by the 10 primers were observed in the two
independent amplifications performed. The dendrograms

338

Erika Marie Alvero-Bascos and Lilian B. Ungson

generated by cluster analysis of Jaccards similarity


coefficient only had one cluster in both trials and are no
longer shown.
Flavonoid Synthesis
As reflected in Figures 4 and 5, only vitexin and
isovitexin were detected in the leaf extracts. Extracts
from the control calli did not contain these two
compounds or their amounts were probably too low to be
detected. In some cases, apigenin was detected in the
extracts from the control calli in both experiments. As
reflected in Figure 4, UV-B irradiation was able to
enhance production of all three compounds, even those
not detected in the control calli. The amounts of
flavonoids detected in calli irradiated with high levels of
UV-B were all significantly higher than the levels in the
untreated cultures. It is also worth mentioning that after
irradiation of the cultures with a high UV-B dose, the
levels of the flavonoids, vitexin and isovitexin reached
concentrations that were similar to the levels found in
fresh leaves. The amounts of flavonoids in calli exposed
to low UV-B doses did not differ significantly from the
control calli (Fig. 4).
A comparison of the substantial increase in the
combined levels of the three flavonoids in UV-B
irradiated calli with that of the untreated cultures can be
clearly seen in Figure 5. The calli treated with high and
low UV-B doses exhibited a 20-fold and a 10-fold
increase in flavonoid content, respectively, based on the
levels found in the control cultures. In addition, the
combined flavonoid levels in the calli treated with high
UV-B doses reached concentrations similar to those
found in whole leaves. The presence of apigenin in the
callus extracts but not in the leaf is clearly a contributing
factor in the observed higher levels of flavonoids in the
calli (Fig. 5).

DISCUSSION
Before embarking on any genetic transformation or
elicitation experiment using in vitro grown plant tissues,
the feasibility of establishing tissue cultures must first be
determined. The experiments conducted in the present
study showed that MS medium supplemented with equal
concentrations of NAA and kinetin, ranging from 10 to
50 M, can induce callus growth on leaf explants of J.
curcas. The resulting calli were green and nonembryogenic which is important since chloroplastcontaining cells are known to possess flavonoid
biosynthetic potential and non-embryogenic callus
cultures
containing
homogenous
clumps
of
dedifferentiated cells are often used for experiments on
secondary metabolite production (Havsteen 2002;
Jedinak et al. 2004).

The Philippine Agricultural Scientist Vol. 95 No. 4 (December 2012)

Ultraviolet-B Radiation as an Elicitor

Erika Marie Alvero-Bascos and Lilian B. Ungson

Table 2. Callusing response of Jatropha curcas L. leaf explants in different media.


Growth Regulators
(M)*
NAA

Kin

Total No.
of
Explants

0
10
20
30
40
50

0
10
20
30
40
50

30
30
30
30
30
30

% Callus
Induction
3.354.74
63.54.95
80.08.90
83.54.95
76.54.95
77.014.1

Callus Characteristics
Nature

Color

Dry, compact
Dry, compact
Slightly compact
Slightly compact
Slightly compact
Slightly compact

Light green
Light green
Green
Green
Green
Light green

Growth Rate**
Very slow
Slow
Moderate
Moderate
Fast
Fast

MS basal medium + 2% sucrose + different combinations of naphthalene acetic acid (NAA) and kinetin (Kin). Data were recorded after 20 d of culture.
Each treatment was repeated twice and each replicate consisted of 15 explants. Callus induction data are means SD.
**
Callus growth rate: very slow (callus first appeared after 1315 d), slow (callus first appeared after 1012 d), moderate (callus first appeared after 79
d), fast (callus first appeared after 46 d).

Table 3. Details of random applied polymorphic DNA


(RAPD) primers used and number of scorable bands
obtained from each primer.
No. of
Size of
Primer Sequence 5-3 Scorable
Fragments
Bands
(bp)

10 NAA :
20 NAA :
30 NAA :
40 NAA :
50 NAA :

Fresh Weight (grams)

10 Kin
20 Kin
30 Kin
40 Kin
50 Kin

OPAK 14
OPD14
OPAW 7
OPAB 5
OPF 11
OPF 2
OPA 4
OPAD 11
OPAL 8
OPAB 6

0
0

14

21

28

35

Time in culture (days)

Fig. 1. Growth curve of calli obtained from Jatropha


curcas L. leaf explants grown on Murashige and
Skoogs (MS) medium supplemented with
different concentrations (M) of naphthalene
acetic acid (NAA) and kinetin (Kin). Each value is
the mean SD of 10 determinations.

CTGTCATGCC
CTTCCCCAAG
AGCCCCCAAG
CCCGAAGCGA
TTGGTACCCC
CCTGATCACC
AATCGGGCTG
CAATCGGGTC
GTCGCCCTCA
GTGGCTTGGA

14
12
11
17
13
12
13
14
8
10
Total: 124

200-3000
300-1500
500-3000
150-2000
250-3000
250-1000
500-4000
400-2500
400-1500
250-1500

Fresh Weight (grams)

8
b

a
4
2

in
:5
0

in
50

N
A

:4
0

in
A
N
A
40

30

N
A

:3
0

in
K
:2
0
A

N
A
20

10

N
A

:1
0

in

Hormone Combinations

Fig. 2. Fresh weights of calli obtained from Jatropha


curcas L. leaf explants grown on Murashige and
Skoogs (MS) basal medium + 2% sucrose +
different concentrations (M) of naphthalene
acetic acid (NAA) and kinetin (Kin). Data were
recorded after 35 d of culture. Data are means
SD of 10 determinations. Different letters
indicate significant differences at P0.05.
The Philippine Agricultural Scientist Vol. 95 No. 4 (December 2012)

Fig. 3. Random amplified polymorphic DNA (RAPD) profiles


of Jatropha curcas L. field-grown mother (source)
plant, control and irradiated callus cultures using the
decamer primers OPAK 14 (A), OPD 14 (B) and
OPAD 11 (C). Lane M represents the 10,000 bp
DNA ladder. Lane 1 represents the field-grown
mother plant. Lanes 24, control callus cultures;
Lanes 57, the callus cultures irradiated with 12.6
kJ m2 UV-B radiation dose; Lanes 8-10, the callus
cultures irradiated with 25.3 kJ m2 UV-B radiation
dose; and Lane 11, a blank sample.
339

Ultraviolet-B Radiation as an Elicitor

Erika Marie Alvero-Bascos and Lilian B. Ungson

A. Vitexin

1200
1000
800

600
400

200

lo
w

VB
U

VB
(+
)

(+
)

(-)

Le
af

B. Isovitexin
a

1200
1000

600

bc

400
200

lo
w

VB
U

VB
(+
)

(+
)

(-)

Le
af

hi
gh

C. Apigenin

1200

1000
800
ab

600
400
a

200

hi
gh

lo
w

VB

(+
)

VB

VB
U
(+
)

(-)

Le
af

g/g
g-1 Dry Weight
g

1400

Fig. 4. Vitexin (A), isovitexin (B) and apigenin (C)


levels in Jatropha curcas L. fresh leaves,
control callus cultures, calli irradiated with low
UV-B dose (12.6 kJ m2) and calli irradiated
with high UV-B (25.3 kJ m2). Data are means
SE, N=3; different letters indicate significant
differences at P0.05.

340

Fig. 5. Combined apigenin, vitexin and isovitexin


levels in Jatropha curcas L. fresh leaves,
control callus cultures, calli irradiated with low
UV-B dose (12.6 kJ m2) and calli irradiated
with high UV-B (25.3 kJ m2). Data are
means SE, N=3; different letters indicate
significant differences at P0.05.

ac

800

VB

g-1 Dry Weight


g
g/g

1400

hi
gh

VB

g-1 Dry Weight


g
g/g

1400

A number of phytochemical studies reported the


presence of the flavones, apigenin, vitexin and isovitexin
in J. curcas leaves (Mitra et al. 1970; Khafagy et al.
1977; Hufford and Oguntimein 1987). However, the
amounts of these compounds in the leaf extracts, as well
as in the callus extracts, have not been documented.
Quantitative HPLC analyses conducted in the present
work showed that only vitexin and isovitexin are present
in detectable levels in the leaf extracts. Apigenin was
either absent or its concentration was too low to be
detected by simple HPLC. These findings may be a
consequence of the developmental stage of the source
plant and the maturity of the leaves used. Studies on
flavonoid production in various plant taxa showed that
levels of flavonoid aglycones, such as apigenin, declined
with leaf age and the concentrations of flavonoid
glycosides vary depending on plant developmental stage
(Peuelas et al. 1999; Valkama et al. 2004; Antognoni et
al. 2007). Reduction in synthesis together with
simultaneous degradation of these flavonoids or their
transformation into other chemical forms, may account
for these observations. Of note, apigenin, after
derivatization with sugar groups, gives rise to vitexin and
isovitexin. These flavone C-glycosides, due to their
chemical structure, have superior antioxidant activity and
provide better UV protection than their aglycones
(Graham 1998). Present results showed that in J. curcas
leaves, apigenin glycosides are the dominant flavonoids.
This result is consistent with findings of previous work
on rice wherein C-glycosylated-flavones were found to
be the dominant flavonoids due to the action of a recently
characterized enzyme that catalyzes C-glucosylation of

The Philippine Agricultural Scientist Vol. 95 No. 4 (December 2012)

Ultraviolet-B Radiation as an Elicitor

apigenin and other aglycones (Hicks et al. 2009; Du et al.


2010). It is possible that a similar system may be
operating in J. curcas.
Analyses of flavonoid production in J. curcas callus
extracts revealed that the cultures lost the ability to
synthesize the compounds that are produced by intact
organs. The dedifferentiation or the reversion of the
mature cells to the meristematic state leading to callus
formation may account for the decrease or complete loss
of biosynthetic potential (Biondi et al. 2002). The reason
is that, in the dediffentiated cell cultures, there may be
upcoupling of the enzymatic machinery, insufficient
expression of developmentally regulated biosynthetic
genes, or lack of environmental stimuli (Matkowski
2008). In the present study, concentrations of both vitexin
and isovitexin were below detection levels in the callus
extracts. However, some cultures were able to produce
apigenin, indicating that in dedifferentiated cells,
apigenin biosynthetic potential may be retained. This
result may again be related to the degree of
differentiation of the tissue. As aforementioned,
concentrations of flavonoid aglycones such as apigenin in
the very young leaves are usually higher than those in the
mature leaves. Perhaps flavonoid biosynthesis in the
callus cultures is comparable to that in the younger, less
differentiated tissues of immature leaves.
In the present study, irradiation of J.curcas calli with
UV-B was found to significantly induce synthesis of
flavonoids, with the higher dose eliciting a greater
increase in the amounts of apigenin, vitexin and
isovitexin. This result concurs with findings of previous
work wherein UV-B radiation induced expression of
flavonoid biosynthetic genes and consequently, flavonoid
synthesis, in intact plants and in vitro cultures
(Logemann et al. 2000; Hao et al. 2009). Moreover, the
monomorphic RAPD patterns of the UV-B irradiated
calli, which were identical to those of the untreated calli
and the mother plant, showed that UV-B did not enhance
genetic variation in the cultures. This result is in line with
the well-known principle that UV-B induction of
flavonoid production is a transcriptional event given that
the promoters for the genes encoding the key enzymes
required for flavonoid synthesis, such as phenylalanine
ammonia lyase (PAL) and chalcone synthase (CHS), are
UV-inducible (Logemann et al. 2000). Another
perspective is that the presence of high levels of
flavonoids in the treated cultures prevented damages or
alterations to DNA that commonly result from exposure
to UV-B light; thus, no changes in genetic structure have
been detected by RAPD analysis in the irradiated calli.
This result is again in accordance with the established
role of flavonoids as the major UV-B absorbing
compounds in plant tissues. This protective function of
flavonoids is also important in the defense against UV-B
induced oxidative damage and photosystem II inhibition

The Philippine Agricultural Scientist Vol. 95 No. 4 (December 2012)

Erika Marie Alvero-Bascos and Lilian B. Ungson

(Ryan et al. 2002; Matkowski 2008). Preferential


synthesis of some flavonoids following UV-B exposure
has been documented in previous work and is mainly
attributed to the varying efficiency of the compounds as
free radical scavengers (Ryan et al. 2002). In contrast to
these reports, synthesis of all three flavonoids was
upregulated by UV-B irradiation in this study. Albeit past
reports that vitexin and isovitexin possess greater
antioxidant potential than apigenin (Graham 2008), no
preferential synthesis of the former two compounds was
observed in this investigation. Nevertheless, the present
results suggest that, as far as the flavonoids in J. curcas
calli are concerned, irradiation with high UV-B doses is
an effective strategy to increase their synthesis.
The process of in vitro callus induction forces mature
tissues to dedifferentiate under conditions that are
considered mutagenic. Thus, the mere presence of a
callus stage in the course of micropropagation has been
regarded as a factor that increases the occurrence of
somaclonal variation in the regenerants (Collin and
Edwards 1998). In the present investigation, however, the
callus cultures obtained did not vary genetically from the
mother plant as evident in their RAPD profiles. This
result may be attributed to factors such as small number
of subculture cycles, short culture period, and use of low
hormone concentrations in callus induction and
maintenance. All of these conditions have been proven to
minimize the occurrence of genetic variation in the
cultures (Kaeppler et al. 2000; Hao and Deng 2002;
Bordallo et al. 2004; Pontaroli and Camadro 2005).
In all, the findings showed that UV-B irradiation may
be utilized to increase production of flavonoids in in vitro
callus cultures. Elicitation through irradiation with UV-B
may be considered as an alternative to expensive
approaches to flavonoid production, such as genetic
transformation and ex vivo chemical synthesis.

ACKNOWLEDGMENTS
We would like to thank the Institute of Biology, College
of Science, University of the Philippines Diliman for
providing us with financial and logistical support. Special
thanks also to the Philippine Council for Health Research
Development (PCHRD) - Department of Science and
Technology (DOST) and to Congressman Michael John
Duavit for the grants awarded to E. M. Alvero-Bascos.
We would also like to express our gratitude to Dr. Ian
Fontanilla, Dr. Jonas Quilang, Dr. Windell Rivera, Dr.
Sonia Jacinto, Dr. Ernelea Cao, Dr. Juliana Janet Puzon,
Edric Adao and Chris Mendoza for all the help that they
have extended to us.

341

Ultraviolet-B Radiation as an Elicitor

Erika Marie Alvero-Bascos and Lilian B. Ungson

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