Ultraviolet-B (UV-B) Radiation as an Elicitor of Flavonoid Production in Callus Cultures of Jatropha (Jatropha Curcas L.)

8/9/2019 Ultraviolet-B (UV-B) Radiation as an Elicitor of Flavonoid Production in Callus Cultures of Jatropha (Jatropha Curcas L.)

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Ultraviolet -B (UV -B) Radiation as an Elicitor of FlavonoidProduction 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 DilQuezon City, Philippines*Author for correspondence; e-mail: [email protected]; Telefax: (+632) 920-5479

Callus cultures of jatropha ( Jatropha curcas L.) grown in Murashige and Skoog’s (MS) mediumsupplemented with naphthalene - acetic acid (NAA; 20µM) and 6 - furfurylaminopurine (kinetin; 20 µM)were exposed to ultraviolet - B (UV - B) radiation to investigate its potential as an abiotic elicitor offlavonoid production. Prior to irradiation, the levels of the flavonoids, apigenin, vitexin andisovitexin in the leaf and callus extracts were determined through high - performance liquidchromatography (HPLC). Results showed that vitexin and isovitexin were the dominant flavonoids inthe 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 dusing two UV - B doses (12.6 and 25.3 kJ m- 2) induced synthesis of all three flavonoids (up to 780 μ 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 thecontrol and were comparable to concentrations found in leaves while a 10 - fold increase in combinedflavonoid levels was observed in calli irradiated with the lower UV - B dose. Furthermore, randomamplified polymorphic DNA (RAPD) analyses of DNA extracts from the leaves and calli revealed thatUV - B irradiation enhanced flavonoid synthesis without altering DNA sequence. These resultsfurther support the supposed involvement of UV - B in the transcriptional regulation of theexpression of flavonoid biosynthetic genes. Overall, the findings showed that elicitation through UV -

B irradiation is an effective strategy to induce flavonoid production in dedifferentiated J. curcascultures that have lost their capacity to produce the flavonoids normally synthesized in intactorgans.

Key Words: callus culture, dedifferentiation, elicitor, flavonoid, Jatropha curcas , ultraviolet-B radiation

Abbreviations: CHS – chalcone synthase, HPLC – high performance liquid chromatography, MS – MurashiSkoog, NAA – naphthalene-acetic acid, PAL – phenylalanine ammonia lyase, PCR – polymerase chain reacRAPD – Random Amplified Polymorphic DNA, UV-B – ultraviolet-B

INTRODUCTION

Flavonoids comprise a large group of natural phenoliccompounds found in vascular plants as products ofsecondary metabolism. Their therapeutic potential asantioxidants and their promising role in anti-cancer

therapy, along with their manifold applications in thecosmetic and pharmaceutical industry, have arousedinterest in developing alternative systems for increased production. Biotechnological approaches such as cell ortissue cultures, which allow optimization of growthconditions and genetic manipulation to enhance flavonoid production, offer an opportunity to circumvent problemsregarding insufficient yield. Of the various tissue culturemethods, callus induction is frequently used for flavonoid production since extraction procedures fromundifferentiated tissues are easier to perform (Jedinak et

al. 2004). Unfortunately, the dedifferentiation of placells during callus initiation often results in a decreasetheir capacity to synthesize compounds normal produced in the plant (Yeoman and Yeoman 199Biondi et al. 2002). Thus, to exploitin vitro grown callustissues as an alternative source of flavonoids, effecti

strategies to increase yield must be employed.

One approach to enhance flavonoid production through elicitation with ultraviolet radiation in the UV-Brange or of wavelength ranging from 280 to 320 nFlavonoids are protective UV-B absorbing compoundsand their synthesis is highly regulated by UV-B radiation.Irradiation with various doses of UV-B has been shownto stimulate a considerable increase in the amount flavonoids produced by callus cultures (Antognoni et 2007; Hao et al. 2009) and whole plants (Lavola et a 1997; Schnitzler et al. 1997; Tegelberg and Julkunen-

ISSN 0031-7454 PHILIPP AGRIC SCIENTIST Vol. 95 No. 4, 335–343 December 2012

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



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Tiitto 2001; Turtola et al. 2005). This action has beenattributed to the ability of UV-B radiation to inducetranscription of genes encoding enzymes involved in the biosynthesis of flavonoids (Logemann et al. 2000), whichis regarded as a protective mechanism against the potentially damaging effects of irradiation. Hence, as anabiotic stress factor, UV-B radiation may serve as a toolfor improving secondary metabolite production in plantcultures.

Propagation of Jatropha curcas L. through tissueculture has been the focus of a number of studies since itwas discovered that its seed oil can be used as biofueland various medicinally-important compounds can beextracted from its organs (Nath and Dutta 1991; Lin et al. 2003; Muangman et al. 2005). The leaves, which are themost commonly used explants in callus inductionexperiments, have been previously identified as potentsources of the flavonoids, apigenin, vitexin andisovitexin. These compounds are responsible for the anti-

inflammatory activity of J.curcas leaves (Chhabra et al. 1990) and may also account for the effectiveness of leafextracts and decoctions as treatment for wounds,rheumatism and lymphocytic leukemia (Hufford andOguntimein 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 themvaluable in the fields of scientific research and medicine.

The aim of this research was to investigate whetheror not UV-B irradiation of J. curcas callus tissues couldincrease their flavonoid biosynthetic potential by acting

as an abiotic elicitor. The effects of different doses ofUV-B radiation on the amount of flavonoids produced bythe callus cultures and the degree of genetic variabilityamong the cultures were also examined to determineflavonoid production and the levels of genetic variation.

The potential of UV-B irradiation to increaseflavonoid production has not been investigated in J.curcas , particularly in the callus stage. The present work provides an example of how the benefits derived fromelicitation can be utilized to increase production of pharmaceutically important flavonoids in J. curcas calluscultures without the use of expensive and sophisticated biotechnological methods such as genetic transformationor e x vivo chemical synthesis. This approach to flavonoidsynthesis would not only circumvent the problem ofinsufficient production in intact organs and the difficultyof extraction but would also provide a steady source offlavonoids independent of climate changes, geographicallocation and pest damage. Moreover, inducing suchvaluable variations in J. curcas calli may also serve as aninitial step in the propagation of plants with increasedflavonoid production through organogenesis.

MATERIALS AND METHODS

Callus Induction J. curcas leaves (3–5 nodes from the apex) werecollected from a shrub located at Jacinto Street,University of the Philippines (UP), Diliman, QuezonCity. The vegetative organs of the plants from the sitewere previously authenticated by Dr. Daniel Lagunzad,former curator of the J. V. Santos Herbarium, Institute ofBiology, UP Diliman. Voucher specimens from thisshrub are kept at the herbarium. Immediately aftercollection, petioles were removed and the edges of theleaves were trimmed prior to washing with very dilutedetergent. The cut leaves were placed under runningwater for 15 min and subsequently washed with steriledistilled 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 ofTween 20. The leaves were rinsed five times with steriledistilled water after immersion in each of the sterilizingsolutions.

Leaf explants (approximately 1 cm x 1 cm) wereexcised from the midrib area. These were inoculated intotest tubes containing 20 mL MS basal medium (pH 5.6)with sucrose as carbon source at 2 g L-1 and plant tissueculture agar as solidifying agent at 8 g L-1 (Murashigeand Skoog 1962). All leaf pieces were placed onto themedium with the adaxial surface facing the agar. Variouscombinations of equal concentrations of the plant growthregulators, 1-naphthaleneacetic acid (NAA) and 6-furfurylaminopurine (kinetin, Kin) were tested (Table 1)

in order to determine which concentrations can producesufficient 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 orcontamination. Callus formation, morphology, textureand color were monitored by visual inspection over a 1-mo period. Callus growth, in terms of fresh weight, wasmeasured weekly. Among the media that were able to produce sufficient amounts of calli, the one containingthe lowest hormone concentration was utilized toestablish callus cultures that were used in the succeedingexperiments as higher hormone levels were previouslyfound to induce genetic variation in the cultures (Smithand Street 1974; Rao et al. 1992; Kaeppler et al. 2000). Atotal of 60 explants excised from the leaf (including themidrib) were cultured in test tubes containing the selectedgrowth medium. Once established, the calli weresubcultured at monthly intervals on the same medium,under the same conditions.

Ultraviolet-B Radiation as an Elicitor Erika Marie Alvero-Bascos and Lilian B. Ungson




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Ultraviolet -B (UV -B) Irradiation Six-week -old calli were subcultured in petri platescontaining 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-Bradiation was provided by 40-watt UV-B lamps (PhilipsTL40/12, Germany). The lamps were suspended directlyabove the dishes and filtered with 0.13-mm thickcellulose acetate (transmission down to 280 nm) toremove any ultraviolet C component emitted. Two UV-Birradiation doses (12.6 and 25.3 kJ m−2 ) were tested(Antognoni et al. 2007; Hao et al. 2009). The desireddoses were obtained by changing the distance betweenthe lamps and the dishes (Predieri et al. 1993). Thespectral irradiance from the lamp was determined using aUV meter (Model 3D-UVB Solar Light Co.). Controldishes were further covered with 0.13-mm thick polyester

film to exclude UV-B (cut

-off at 318 nm). All culturesalso 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 werecarried out with three replications under a completelyrandomized design.

Random Amplified Polymorphic DNA (RAPD)Analyses DNA was extracted from the control and irradiated calliand from leaves of the parent plant. Genomic DNA wasextracted using Promega™ Wizard Genomic DNAPurification Kit. Forty milligrams of leaf and callus tissuewere 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 etal. 2008). PCR amplification was performed in a totalvolume of 25μ L containing 10 mM Tris/HCl (pH 8.3),50 mM KCl, 200μ M of each dNTPs, 2 mM MgCl2, 2μ M primer, approximately 130 ng of template DNA and1.5 U Taq polymerase using a thermal cycler (LabnetMultigene).

After an initial denaturation step for 3 min at 95 °the amplification reactions were carried out for 40 cyclThe procedures for each cycle were as follows: 1 mdenaturation at 94 °C, 1 min annealing at 35 °C andmin extension at 72 °C. The final elongation step wextended to 5 min. These PCR conditions were based the protocol of Ganesh Ram et al. (2008) with sommodifications. Amplification products were separated 1.2% agarose gels in TAE buffer, stained with ethidiu bromide and photographed under UV light using tUVP® Gel Documentation System.

High Performance Liquid Chromatography (HPLC)Analyses Five grams of fresh callus and leaf samples were driedan oven for 24 h at 60 °C. Dried tissues were weighand homogenized with 10 volumes of methanol. Tmethanolic extracts were placed in an ultrasound bath f30 min. The homogenate was filtered through sever

layers of cheesecloth, and the residue was re-extractedtwice. Filtrates were evaporated to dryness at 40 °Dried residues were weighed and taken up in 10 mmethanol. After filtering through a 0.22-μ m microsyringefilter, 10 μ L of the filtrates were directly injected foHPLC analysis (Antognoni et al. 2007).

The HPLC procedure was adapted from the study Peng et al. (2005) with some modifications. Separationwere performed using an HPLC instrument equippwith a 20μ L loop and UV-variable wavelength detectorset at 280 nm. Methanolic extracts were run in an HPLC18 column (5μ m, 250 × 4.6 mm) at a flow rate of 1.mL min-1. The mobile phases included acetonitril

(solvent A) and 42% (v/v) methanol in water (solvent mixed using a linear gradient starting with 0% A (2 miincreasing to 12% A (15 min), 12% A (3 min) and 0%(5 min). Standard solutions of apigenin, isovitexin avitexin were used to identify and quantify thecompounds in the extracts. Peak areas of increasiconcentrations of vitexin, isovitexin and apigenstandards (Sigma®) were used to construct calibrationcurves. The linear regression equations generated frothese standard curves were used to calculate thflavonoid concentrations of the methanolic extracobtained from fresh leaves, as well as from the untreatand UV-irradiated calli.

Statistical Analyses The bands generated by RAPD-PCR were scored fortheir presence ‘1’ or absence ‘0’ for each primer. Verfaint or unclear bands were not considered. The binadata were subjected to cluster analysis. Similarimatrices were generated by Jaccard’s coefficient similarity using Multivariate Statistical Package 2008.dendrogram was constructed using the unweighted pgroup method with arithmetic average (UPGMA) obtain a representation of genetic relationships

Table 1 . Combinations of the plant growth regulatorsnaphthalene acetic acid (NAA) and kinetin (Kin) testedfor callus induction potential in Jatropha curcas L.

Medium Growth Regulators (µM)

NAA Kin

1 0 0 2 10 10 3 20 20 4 30 30 5 40 40 6 50 50





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revealed by the similarity coefficient. For the othermeasured parameters (fresh weight, flavonoidconcentrations), analysis of variance (ANOVA) andBonferroni test were used in comparing the data (SPSS).P values less than 0.05 were considered statisticallysignificant. Means, standard deviations and standard errorwere computed and corresponding graphs wereconstructed using Graph Pad Prism® Software (2011).

RESULTS

Callus Induction and UV Irradiation In the two trials performed, all the tested culture mediawere able to induce callus formation in more than 60% ofthe leaf explants, except for the medium without plantgrowth regulators (Table 2). Calli were first observed 6–7d after inoculation and were found predominantly on thecut portions of the midrib and secondary veins. Most ofthe calli formed were green in color and exhibited aslightly compact structure. Callus growth rate variedamong the different media but all cultures exhibitedabout 3- to 5- fold increases in a 5-wk period based onfresh weight (Fig. 1). By the end of the fifth week, thecalli grown in medium containing 10μ M NAA + 10μ Mkinetin were found to have significantly lower freshweights than those cultured in other growth media (Fig.2) and reached fresh weights averaging 4 g.

Among the growth media that were able to produceat least 5 g of calli on the fifth week of culture, themedium supplemented with the lowest levels of plantgrowth regulators (20μ M NAA + 20μ M kinetin) was

chosen to initiate and maintain the callus cultures thatwere used in the irradiation experiments in order tominimize the probable effects of high hormone levels onthe degree of genetic variation in the cultures. After 8–9d from the start of culture, 80–83% of the explantscultured in the two trials had formed callus. By the fifthweek, the calli reached an average weight of 5.02 ± 0.432g and were then subjected to the UV treatments. UV-Birradiation had no noticeable effect on the growth andmorphology of the treated calli.Genetic Variation In the two trials performed, sizes of the productsamplified using 10 random primers varied from 150 to4,000 bp (Table 3). The number of bands for each primervaried from 8 (OPAL 8) to 17 (OPAB 5), with an averageof 12.4±4.94 bands per primer. The RAPD analyses ofDNA obtained from the leaf (mother plant), along withthe control and UV-B irradiated calli generated a total of890 bands, all of which exhibited monomorphic patterns.Examples of the monomorphic bands and their intensitiesare shown in Figure 3. These identical banding patternsgenerated by the 10 primers were observed in the twoindependent amplifications performed. The dendrograms

generated by cluster analysis of Jaccard’s similaritycoefficient only had one cluster in both trials and are nolonger shown.

Flavonoid Synthesis As reflected in Figures 4 and 5, only vitexin andisovitexin were detected in the leaf extracts. Extractsfrom the control calli did not contain these twocompounds or their amounts were probably too low to bedetected. In some cases, apigenin was detected in theextracts from the control calli in both experiments. Asreflected in Figure 4, UV-B irradiation was able toenhance production of all three compounds, even thosenot detected in the control calli. The amounts offlavonoids detected in calli irradiated with high levels ofUV-B were all significantly higher than the levels in theuntreated cultures. It is also worth mentioning that afterirradiation of the cultures with a high UV-B dose, thelevels of the flavonoids, vitexin and isovitexin reached

concentrations that were similar to the levels found infresh leaves. The amounts of flavonoids in calli exposedto low UV-B doses did not differ significantly from thecontrol calli (Fig. 4).

A comparison of the substantial increase in thecombined levels of the three flavonoids in UV-Birradiated calli with that of the untreated cultures can beclearly seen in Figure 5. The calli treated with high andlow UV-B doses exhibited a 20-fold and a 10-foldincrease in flavonoid content, respectively, based on thelevels found in the control cultures. In addition, thecombined flavonoid levels in the calli treated with highUV-B doses reached concentrations similar to those

found in whole leaves. The presence of apigenin in thecallus extracts but not in the leaf is clearly a contributingfactor in the observed higher levels of flavonoids in thecalli (Fig. 5).

DISCUSSION

Before embarking on any genetic transformation orelicitation experiment usingin vitro grown plant tissues,the feasibility of establishing tissue cultures must first bedetermined. The experiments conducted in the presentstudy showed that MS medium supplemented with equalconcentrations of NAA and kinetin, ranging from 10 to50 μ M, can induce callus growth on leaf explants of J.curcas. The resulting calli were green and non-embryogenic which is important since chloroplast-containing cells are known to possess flavonoid biosynthetic potential and non-embryogenic calluscultures containing homogenous clumps ofdedifferentiated cells are often used for experiments onsecondary metabolite production (Havsteen 2002;Jedinak et al. 2004).





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0 7 14 21 28 350

1

2

3

4

5

6

7

10 NAA : 10 Kin

20 NAA : 20 Kin

30 NAA : 30 Kin

40 NAA : 40 Kin

50 NAA : 50 Kin

Time in culture (days)

F r e s h W e i g

h t ( g r a m s )

Fig. 1. Growth curve of calli obtained from Jatrophacurcas L. leaf explants grown on Murashige andSkoog’s (MS) medium supplemented withdifferent concentrations (µM) of naphthaleneacetic acid (NAA) and kinetin (Kin). Each value isthe mean ± SD of 10 determinations.

1 0 N A A

: 1 0 K i

n

2 0 N A A

: 2 0 K i

n

3 0 N A A

: 3 0 K i

n

4 0 N A A

: 4 0 K i

n

5 0 N A A

: 5 0 K i

n0

2

4

6

8

a

b bb b

Hormone Combinations

F r e s h W e i g h t ( g r a m s )

Fig. 2. Fresh weights of calli obtained from Jatrophacurcas L. leaf explants grown on Murashige andSkoog’s (MS) basal medium + 2% sucrose +different concentrations ( μ M) of naphthaleneacetic acid (NAA) and kinetin (Kin). Data wererecorded after 35 d of culture. Data are means ±SD of 10 determinations. Different lettersindicate significant differences at P≤0.05.

Fig. 3. Random amplified polymorphic DNA (RAPD) profilesof Jatropha curcas L. field -grown mother (source)plant, control and irradiated callus cultures using thedecamer primers OPAK 14 ( A), OPD 14 ( B) andOPAD 11 ( C). Lane M represents the 10,000 bpDNA ladder. Lane 1 represents the field -grownmother plant. Lanes 2–4, control callus cultures;Lanes 5–7, the callus cultures irradiated with 12.6kJ m −2 UV-B radiation dose; Lanes 8 -10, the calluscultures irradiated with 25.3 kJ m −2 UV-B radiationdose; and Lane 11, a blank sample.

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

Growth Regulators(µM)* Total No.

ofExplants

% CallusInduction

Callus Characteristics

NAA Kin Nature Color Growth Rate**

0 0 30 3.35±4.74 Dry, compact Light green Very slow 10 10 30 63.5±4.95 Dry, compact Light green Slow

20 20 30 80.0±8.90 Slightly compact Green Moderate 30 30 30 83.5±4.95 Slightly compact Green Moderate 40 40 30 76.5±4.95 Slightly compact Green Fast 50 50 30 77.0±14.1 Slightly compact Light green 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 13–15 d), slow (callus first appeared after 10–12 d), moderate (callus first appeared after 7–9d), fast (callus first appeared after 4–6 d).



Table 3. Details of random applied polymorphic DNA(RAPD) primers used and number of scorable bandsobtained from each primer.

Primer Sequence 5’ - 3’ No. of

ScorableBands

Size ofFragments

(bp)OPAK 14 OPD14 OPAW 7 OPAB 5

CTGTCATGCC CTTCCCCAAG

AGCCCCCAAG CCCGAAGCGA

14 12 11 17

200 -3000 300 -1500 500 -3000 150 -2000

OPF 11 TTGGTACCCC 13 250 -3000 OPF 2 CCTGATCACC 12 250 -1000 OPA 4 AATCGGGCTG 13 500 -4000 OPAD 11 CAATCGGGTC 14 400 -2500 OPAL 8 GTCGCCCTCA 8 400 -1500 OPAB 6 GTGGCTTGGA 10 250 -1500

Total : 124



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A number of phytochemical studies reported the presence of the flavones, apigenin, vitexin and isovitexinin J. curcas leaves (Mitra et al. 1970; Khafagy et al. 1977; Hufford and Oguntimein 1987). However, theamounts of these compounds in the leaf extracts, as wellas in the callus extracts, have not been documented.Quantitative HPLC analyses conducted in the presentwork showed that only vitexin and isovitexin are presentin detectable levels in the leaf extracts. Apigenin was

either absent or its concentration was too low to bedetected by simple HPLC. These findings may be aconsequence of the developmental stage of the source plant and the maturity of the leaves used. Studies onflavonoid production in various plant taxa showed thatlevels of flavonoid aglycones, such as apigenin, declinedwith leaf age and the concentrations of flavonoidglycosides vary depending on plant developmental stage(Peñuelas et al. 1999; Valkama et al. 2004; Antognoni etal. 2007). Reduction in synthesis together withsimultaneous degradation of these flavonoids or theirtransformation into other chemical forms, may accountfor these observations. Of note, apigenin, afterderivatization with sugar groups, gives rise to vitexin andisovitexin. These flavoneC -glycosides, due to theirchemical structure, have superior antioxidant activity and provide better UV protection than their aglycones(Graham 1998). Present results showed that in J. curcasleaves, apigenin glycosides are the dominant flavonoids.This result is consistent with findings of previous workon rice whereinC -glycosylated-flavones were found to be the dominant flavonoids due to the action of a recentlycharacterized enzyme that catalyzesC -glucosylation of

L e a f

( - ) U V B

( + ) U V

B l o w

( + ) U V

B h i g h

0

200

400

600

800

1000

1200

1400

a

b

b

a

µ g / g D r y

W e i g h

t

A. Vitexin

L e a f

( - ) U V B

( + ) U V

B l o w

( + ) U V

B h i g h0

200

400

600

800

1000

1200

1400a

b

bc

ac

µ g / g D r y

W e i g h t

B. Isovitexin

L e a f

( - ) U V

B

( + ) U V B

l o w

( + ) U V B

h i g h0

200

400

600

800

1000

1200

1400

a

a

ab

b

µ g / g D r y

W e i g h t

C. Apigenin

Fig. 4 . Vitexin ( A), isovitexin ( B) and apigenin ( C)levels in Jatropha curcas L. fresh leaves,control callus cultures, calli irradiated with lowUV-B dose (12.6 kJ m −2 ) and calli irradiatedwith high UV -B (25.3 kJ m −2 ). Data are means± SE, N=3; different letters indicate significantdifferences at P≤0.05.

Fig. 5 . Combined apigenin, vitexin and isovitexinlevels in Jatropha curcas L. fresh leaves,control callus cultures, calli irradiated with lowUV-B dose (12.6 kJ m −2 ) and calli irradiated

with high UV -B (25.3 kJ m−2

). Data aremeans ± SE, N=3; different letters indicatesignificant differences at P≤0.05.



µ g

g -

1

µ g

g -

1

µ g

g -

1



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apigenin and other aglycones (Hicks et al. 2009; Du et al. 2010). It is possible that a similar system may beoperating in J. curcas .

Analyses of flavonoid production in J. curcas callusextracts revealed that the cultures lost the ability tosynthesize the compounds that are produced by intactorgans. The dedifferentiation or the reversion of themature cells to the meristematic state leading to callusformation may account for the decrease or complete lossof biosynthetic potential (Biondi et al. 2002). The reasonis that, in the dediffentiated cell cultures, there may beupcoupling of the enzymatic machinery, insufficientexpression of developmentally regulated biosyntheticgenes, or lack of environmental stimuli (Matkowski2008). In the present study, concentrations of both vitexinand isovitexin were below detection levels in the callusextracts. However, some cultures were able to produceapigenin, indicating that in dedifferentiated cells,apigenin biosynthetic potential may be retained. This

result may again be related to the degree ofdifferentiation of the tissue. As aforementioned,concentrations of flavonoid aglycones such as apigenin inthe very young leaves are usually higher than those in themature leaves. Perhaps flavonoid biosynthesis in thecallus cultures is comparable to that in the younger, lessdifferentiated tissues of immature leaves.

In the present study, irradiation of J.curcas calli withUV-B was found to significantly induce synthesis offlavonoids, with the higher dose eliciting a greaterincrease in the amounts of apigenin, vitexin andisovitexin. This result concurs with findings of previouswork wherein UV-B radiation induced expression of

flavonoid biosynthetic genes and consequently, flavonoidsynthesis, in intact plants andin vitro cultures(Logemann et al. 2000; Hao et al. 2009). Moreover, themonomorphic RAPD patterns of the UV-B irradiatedcalli, which were identical to those of the untreated calliand the mother plant, showed that UV-B did not enhancegenetic variation in the cultures. This result is in line withthe well-known principle that UV-B induction offlavonoid production is a transcriptional event given thatthe promoters for the genes encoding the key enzymesrequired for flavonoid synthesis, such as phenylalanineammonia lyase (PAL) and chalcone synthase (CHS), areUV-inducible (Logemann et al. 2000). Another perspective is that the presence of high levels offlavonoids in the treated cultures prevented damages oralterations to DNA that commonly result from exposureto 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 establishedrole of flavonoids as the major UV-B absorbingcompounds in plant tissues. This protective function offlavonoids is also important in the defense against UV-Binduced oxidative damage and photosystem II inhibition

(Ryan et al. 2002; Matkowski 2008). Preferentiasynthesis of some flavonoids following UV-B exposurehas been documented in previous work and is mainattributed to the varying efficiency of the compounds free radical scavengers (Ryan et al. 2002). In contrast tothese reports, synthesis of all three flavonoids wupregulated by UV-B irradiation in this study. Albeit pasreports that vitexin and isovitexin possess greatantioxidant potential than apigenin (Graham 2008), preferential synthesis of the former two compounds wobserved in this investigation. Nevertheless, the preseresults suggest that, as far as the flavonoids in J. curcascalli are concerned, irradiation with high UV-B doses isan effective strategy to increase their synthesis.

The process ofin vitro callus induction forces maturetissues to dedifferentiate under conditions that aconsidered mutagenic. Thus, the mere presence ofcallus stage in the course of micropropagation has beregarded as a factor that increases the occurrence

somaclonal variation in the regenerants (Collin aEdwards 1998). In the present investigation, however, tcallus cultures obtained did not vary genetically from tmother plant as evident in their RAPD profiles. Thresult may be attributed to factors such as small numbof subculture cycles, short culture period, and use of lohormone concentrations in callus induction anmaintenance. All of these conditions have been provenminimize the occurrence of genetic variation in tcultures (Kaeppler et al. 2000; Hao and Deng 2002Bordallo et al. 2004; Pontaroli and Camadro 2005).

In all, the findings showed that UV-B irradiation may be utilized to increase production of flavonoids inin vitro

callus cultures. Elicitation through irradiation with UV-Bmay be considered as an alternative to expensiv

approaches to flavonoid production, such as genetransformation and e x vivo chemical synthesis.

ACKNOWLEDGMENTS

We would like to thank the Institute of Biology, Colleof Science, University of the Philippines Diliman f providing us with financial and logistical support. Specthanks also to the Philippine Council for Health ReseaDevelopment (PCHRD)- Department of Science andTechnology (DOST) and to Congressman Michael JoDuavit for the grants awarded to E. M. Alvero-Bascos.We would also like to express our gratitude to Dr. IFontanilla, Dr. Jonas Quilang, Dr. Windell Rivera, DSonia Jacinto, Dr. Ernelea Cao, Dr. Juliana Janet PuzoEdric Adao and Chris Mendoza for all the help that thhave extended to us.





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Ultraviolet-B (UV-B) Radiation as an Elicitor of Flavonoid Production in Callus Cultures of Jatropha (Jatropha Curcas L.)