PertanikaJ. Trap. ~ric. Sci. 29 (1 & 2): 57-66 (2006)ISSN: 1511-3701

©Universiti Putra Malaysia Press

Paraquat (Methyl viologen) Toxicity in Centella asiatica Callus Cultures

NOR'AINI MOHD FADZILLAH, NORHAYATI YUSUF, IMARZIAH MAHMOOD,MISRI KUSNAN & SITI KHALIJAH DAUD

Department ofBiology, Faculty ofScience, Universiti Putra Malaysia,43400 UPM Serdang, Selangor; Malaysia

lDepartment ofBiochemistry, Faculty ofBiotechnology and Biomolecular Sciences,Universiti Putra Malaysia, 43400 UPM Serdang, Selangor; Malaysia

E-mail: aini@fsas.upm.edu.my

Keywords: Centella asiatica, paraquat, callus growth, cell viability, malondialdehyde, hydrogen peroxide

ABSTRAK

Kajian telah dijalankan untuk mengkaji kesan rawatan paraquat (PQ) ke atas pertumbuhan, viabiliti, kandunganhidrogen peroksida dan malondialdehida di dalam kultur kalus Centella asiatica (CA03 dan CA09). Kalus dirawatdengan 50 J.lM PQ selama 5 hari di dalam medium cecair Murashige dan Skoog (MS). Pertumbuhan, viabiliti kalusdan juga kandungan hidrogen peroksida (H20 2) dan malondialdehida (MDA) ditentukan pada hari 0, 1, 2, 3 dan 5rawatan. Berat basah dan berat kering kalus CA03 yang diberi rawatan adalah lebih rendah berbanding kalus kawalan.Bagi kalus CA09 pula, pertumbuhan kalus rawatan adalah lebih rendah berbanding kalus kawalan pada peringkatakhir tempoh rawatan. Walaupun terdapat perencatan pertumbuhan bagi kedua-dua CA03 dan CA09 yang diberirawatan, perencatan berat basah sebanyak 36%bagi CA 09 berbanding dengan kawalannya pada akhir tempoh rawatanadalah lebih tinggi dari CA03 di mana terdapat perencatan pertumbuhan berat basah sebanyak 18.2%. Penurunanperatus viabiliti sel juga adalah sangat ketara terutama pada kalus CA09 selepas dirawat dengan PQ. Walaupunkandungan MDA adalah lebih tinggi di dalam kalus CA03 berbanding CA09 yang diberi rawatan pada peringkatawal, ia menunjukkan corak perubahan yang menurun mengikut masa manakala kandungan MDA pada CA09 pulamenunjukkan corak perubahan yang meningkat. Pada akhir tempoh rawatan, MDA pada CA09 adalah lebih tinggiberbanding CA03. Tambahan lagi, kandungan H20 2 pada amnya adalah lebih tinggi pada CA09 yang diberi rawatanberbanding CA03 kecuali pada hari ke 3. Kajian ini menunjukkan bahawa rawatan dengan PQ boleh meransangpenghasilan MDA dan H 20 2 dan juga merencatkan pertumbuhan dan juga viabiliti kalus. Kajian juga menunjukkankalus CA03 adalah lebih toleran kepada PQ berbanding CA09.

ABSTRACT

The effect ofparaquat (PQ) treatment on growth, cell viability, hydrogen peroxide (H202Y and malondialdehyde (MDA)levels were investigated in the callus of two Centella asiatica accessions (CA03 and CA09). Callus of C. asiatica weretreated with 50l1M PQforfive days in Murashige and Skoog (MS) liquid medium. Callus growth, viability of the callusas well as H 20 2 content and MDA levels were evaluated at 0, 1, 2,3 and 5 days of treatment periods. Fresh weight anddry weight of treated calli were significantly lower in CA03 as compared to the untreated calli. In CA09, the growth oftreated calli was significantly lower compared to controls at the later stages of the treatment period. Although decreases ingrowth were observedfor both treated CA 03 and CA09, thefinal reduction in fresh weight at 36%for CA 09 compared toits control was much higher compared to CA03 with an 18.2% final reduction in fresh weight. PQ treatment alsoresulted in a marked decrease in the viability of the callus especially in CA09. Although MDA levels were significantlyhigher in treated CA03 as compared to treated CA09 at the early treatment stages, they showed a decreasing trend, whileMDA levels in CA09 showed an increasing trend, which was significantly higher than that of CA03 at the end of thetreatment period. In addition, H 20 2 concentrations were generally higher in treated CA09 compared to treated CA03except at day 3. This study indicated that PQ treatment can induce increases in levels ofMDA and H 20 2, associated withthe decrease in growth and viability of the callus. Results also suggested that CA03 was more tolerant to PQ treatment as

compared to CA09.

OR'AINI MOHD FADZILLAR ITAL.

INTRODUCTIONOxidative stress is very frequent in nature andis due to an increase of the reactive oxygenspecies (ROS). It is induced by several bioticand abiotic factors including phytotoxicchemical agents including non selectiveherbicides (e.g paraquat, PQ). The bipyridylherbicides such as paraquat, also known asmethyl viologen, with its active compound 1,1'­dimethyl-4,4' -bipyridylium dichloride anddiquat are non-selective, quick actingherbicides, effective against grasses as well asmost broad-leaved weed species (Calderbankand Slade, 1976). The major target of thebipyridyl compounds in PQ seems to be thechloroplast; PQ can accept one electron fromphotosystem I and the formed PQ radicals israpidly oxidized under the catalyzation ofmetalions, leading to the formation of superoxideradicals. In further reactions, various ROS e.gH 20 2 and hydroxyl radicals are generated(Lorenzini et. aL, 2002). The hydroxyl radicalgenerated will rapidly react with membraneunsaturated fatty acid leading to membranedamage, reduction in CO 2 uptake anddegradation of chloroplast and pigments(Kirtikara and Talbot, 1996). These ROS areefficiently scavenged by a series ofenzymes andquenching systems such as superoxidedismutase, enzymes in ascorbate glutathionecycle, ascorbate, glutathione and membranebound a-tocopherol (Suntres, 2002).

Oxidative stress is also involved in loss ofviability of plants exposed to a variety ofenvironmental stress. The 2,3,5-triphenyltetrazolium chloride (TIC) assay was used asa viability assay for callus exposed to variousconcentrations ofPQ. The production ofMDAand changes in cell conductivity havefrequently been used as sensitive markers forherbicides' action in plants (Peleg' et aL, 1992).

Centella asiatica is commonly used as avegetable or eaten raw as an 'ulam' (Malaysalad). Apart from being a nutritious plant,' C.asiatica is also believed to have many healingproperties, conferring a wide range ofbeneficial effects and is treated as a valuable

medicinal plant in Chinese traditionalmedicine and classical Indian Ayurvedicmedicine. Research has demonstrated that C.asiatica is a rich source of natural antioxidan ts.These antioxidants are scavengers ofROS andinhibitors of lipid peroxidation and thus, canprotect and defend cells against damage by theROS (Subramaniam et al., 1998).

The main objectives of this study were todetermine the effect of PQ treatments ongrowth, cell viability, H 20 2 and MDA levels oftwo C. asiatica callus cultures i.e CA03 andCA09.

MATERIALS AND METHODSCallus Initiation and MaintainanceSterile leafexplants of C. asiaticawere culturedon MS medium (Murashige and Skoog, 1962)supplemented with 2,4-Dichlorophenoxyaceticacid (2,4-D) and kinetin. The cultures weremaintained by regular subculturing at 10 dayintervals onto fresh medium. All cultures wereincubated in 12h/12h (light/dark)photoperiod under cool, white fluorescentlamps at 27 ± 2°C.

PQ (1,1 '-dimethyl-4, 4 '-bipyridylium dichloride)TreatmentCallus pieces were transferred to MS mediumcontaining 50 JiM PQ. PQ is heat-stable andfor all experiments, was added to the mediumprior to autoclaving. Callus growth, cellviability, H20 2 content and MDA levels wereassayed at 0, 1, 2, 3 and 5 days of treatmentperiods.

Callus Growth and ViabilityTreated callus were washed with distilled waterand weighed immediately for fresh weight. Fordry weight, callus were dried in an oven at 50°Cfor 2 days. The 2,3,5-triphenyltetrazoliumchloride (TIC) assay was used to estimate theproportion ofviable cells after PQ treatments.The absorbance of the supernatant wasdetermined at 485 nm (Towill and Mazur,1974).

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PARAQUAT (METHYL VIOLOGEN) TOXICITY IN CENTELLA ASIATICA CALLUS CULTURES

MDAAssayMDA concentration was determined by thethiobarbituric acid (TBA) reaction, based onthe method by Heath and Packer (1968) withslight modification by Hodges et at. (1999).

H 20 2 DeterminationH 20 2 assay was done following the method ofVelikova et al. (2000).

RESULTS AND DISCUSSIONThe changes in growth of C. asiatica calluscultures treated with 50 jiM PQ are shown inFigs. 1 and 2. Decreases in fresh weight anddry weight were observed 24 hours aftertreatment with PQ in both accessions. Thereduction in growth was greater in CA09 ascompared to CA03 especially after 2 days oftreatment (Figs. 1C and 2C). In CA03, thegrowth of treated callus were significantly lower(p<0.05) than the control (Figs. lA and 2A),but in CA09 the growth of treated callus wereonly significantly lower than its control(p<0.05) at the later stages of treatment periods(Figs. lB and 2B). This however was due to thesharp decrease in fresh weight of the controlCA09 callus at day 2 which increased slightlythereafter. Therefore, although significantdifferences in fresh weight for CA09 onlyoccurred at the later stages of treatment period,this was due to the low fresh weight values ofits control. A comparison of the actual finalreduction in growth between CA03 and CA09showed a 36% reduction in CA09 while CA03showed 18.2% reduction of growth comparedto their respective controls (Figs. lA and lB).The similar dry weigh t values of treated CA03and CA09 showed that there were nodifferences in terms of actual organic mattercontent in the two accessions (Fig. 2C).However, the lower reduction in growth ofCA09 is probably due to the significantly lowerpercentage ofviability compared to CA03 (Fig.3C). Fig. 3 demonstrates that PQ treatmentresulted'in a marked decrease in the viabilityof the callus especially in CA09. Mter 24 hourstreatment with PQ, only 5% ofCA09 callus werestill viable while only 1.5% of the cells were

viable after 5 days of treatment (Fig. 3(;). Theviable proportion of the callus weresignificantly higher (p<0.05) in controls ascompared to the treated callus in bothaccessions (Figs. 3A and 3B). The results ofWong (2000) also suggest that PQ at a lowconcentration (0.02 mg/l) can significantlyinhibit the growth, photosynthetic rates andchlorophyll content of Scenedesmus quadricaudaBerb 614. Clearly, PQ is more effective indecreasing the growth and cell viability ofCA09callus than CA03. Results thus indicate thatPQ could induce oxidative stress in C. asiaticacallus cultures.

The condition of oxidative stress inducedby PQresulting in peroxidation of membranelipids in the C. asiatica cultures is clearlyindicated by the increased MDA levels in bothaccessions compared to their respectivecontrols except for days 1 and 3 in CA09 callus(Figs. 4A and 4B). MDA levels were initiallysignificantly higher (p<0.05) in CA03 ascompared to CA09 especially up to 2 days oftreatment periods. However, longer treatmentperiod decreased the MDA levels in CA03 andincreased the MDA levels in CA09 (Fig. 4C).At the end of the treatment period, MDA levelsin CA09 were significantly higher (p<0.05) thanthat of CA03.

H 20 2 concentrations between control andtreated cultures of CA03 and CA09 were notsignificantly different except for day 3 for CA03where the H20 2 concentrations in the treatedcallus were significantly higher (p<0.05) thanits control (Fig. 5A). The treated CA09 callusdid not show any increases in H 20 2

concentrations after day 2 (Fig. 5B). This couldbe due to a compromised antioxidative defensein CA09 which although may have increasedlevels of superoxide radicals (02-) productiondue to the PQ-induced oxidative stress, was notable to dismutate these radicals to H20 2, CA03on the other hand was probably still capableof modulating its antioxidative defenseresulting in the sudden burst of H 20 2

production which was subsequently neutralizedto less harmful forms. This was reflected inthe decreased levels of H20 2 after day 3 (Fig.

PERTANIKAJ. TROP. AGRIC. SCI. VOL. 29 NOS. 1 & 2 (2006) 59

NOR'AINI MOHD FADZILLAH ET AL.

1.8

1.6 A

1.4

3 1.2

.c00·v~ 0.8

~...<l: 0.6

0.41---.--control --"-PQ

0.2

0

0 2 3 5

days

1.8

1.6 B

1.4

§ 1.2w..c00'v~ 0.8~... 0.6<l:

0.4 I---+-- control ---"'-- PQ

0.2

0

0 2 3 5

days

1.8

1.6 C

§ 1.4

w 1.2..c00'v~

~ 0.8...<l:

0.6

0.4

0.2 I__CA03 ~CA09

00 2 3 5

days

Fig. 1: Fresh weight (g) oJe. asiatica caUus culture:A) CAD] caUusB) CA09 caUusC) CAD] and CA09 caUi (PQ treatment)Vertical bars represent standard errors (n=5)

60 PERTANlKAJ. TROP. AGRIC. SCI. VOL. 29 OS. 1 & 2 (2006)

PARAQUAT (METHYL VIOLOCE ) TOXICITY IN CENTELLA ASIATICA CALLUS CULTURES

B

B

c

5

1---.-CA03 ----- CA09

" 5

"

days

I----+-- control ----- PQ

I----+-- control ~ PQ

days

0.09

0.08

0.07

0.06

:§,;: 0.05

b<l'il

0.0'"~

e:--0 0.03

0.02

0.01

0

0

0.09

0.08

0.07

:§ 0.06

,;:0.05b<l

'il~

e:- 0.0'"

-0

0.03

0.02

0.01

0

0

0.09

0.08

0.07

:§ 0.06

,;:b<l 0.05'il~

e:- 0.04-0

0.03

0.02

0.01

0

0

days

Fig. 2: Dry weight (g) ofe. asiatica callus culture:A) CAO] callusB) CA09 callusC) CAO] and CA09 calli (PQ treatment)Vertical bars represent standard errors (n=5)

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OR'AINI MOHD FADZILlAH ET AL.

I--control --"-PQ I

5

5

3

3

I--+--control ----pQ. I

I--CA03 ---CA09 I

2

2

days

days

120

A100

80

~ 60

.qii 40

'"'S:

20

0

0

120

B100

80

~60

kii 40'"'S:

20

00

120

C100

80~

g 60

ii'"'S: 40

20

00

days

Fig. 3: Viability (%) ofe. asiatica callus culture:A) CA03 callusB) CA09 callusC) CA03 and CA09 calli (PQ treatment)Vertical bars represent standard errors (n=5)

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PARAQUAT (METHYL VIOLOGE ) TOXICITY IN CENTELLA ASIATICA CALLUS CULTURES

4

3.5 A

3

~ 2.5boO"-""0 2EE. 1.5-<0::E

0.5 I--control -a..-PQ I0

0 2 3 5

days

4

3.5B

3

1 2.5boO"-""0 2EE. 1.5-<0::E

0.5 I--control ----PQ I0

0 2 3 5

days

\--CA03 ----CA09

4

3.5

i 3

boO 2.5"-""0 2EE. 1.5-<0::E

0.5

00 2 3 5

c

days

Fig. 4: Malondialdehyde (MDA) concentrations ofc. asiatica callus culture:

A) CA03 callus B) CA09 callusC) CA03 and CA09 calli (PQ treatment)Vertical bars represent standard errors (n=5)

PERTANlKAJ. TROP. AGRIC. SCI. VOL. 29 NOS. 1 & 2 (2006) 63

NOR'A! I MOHD FADZILLAH ET AL.

I-+--control --'--PQ I

0.0025

i 0.002

be"-<l) 0.0015<5E~

0: 0.001

:t

0.0005

00

0.0025

2

days

3 5

A

B

~ 0.002

t"­

<l)

<5 0.0015E~

0' 0.001:i'

0.0005I--+--control -PQ I

O+-----~----~---_~---___.----_l

I-+--CA03 ---""--CA09

o

0.0025

0.002

1be"-

<l) 0.0015<5E~

0' 0.001

:i'0.0005

00

2

days

2

days

3

3

5

5

c

64

Fig. 5: Hydrogen peroxide (HPJ concentrations oJC. asiatica callus culture:A) CA03 callus B) CA09 callusC) CA03 and CA09 calli (PQ treatment)Vertical bars represent standard errors (n=5)

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5C). Accumulation of H 20 2 is potentiallyharmful since it can lead to oxidative damageand loss of structure and function. Thedecrease in H20 2 and MDA concentrations inGA03 callus especially after 3 days of treatmentperiod may be due to the higher antioxidantactivity which had enhanced oxidative stresstolerance. H 20 2 is an active oxygen specieswhich can also react with superoxide radicalsto form more powerful oxygen free radicalsand hydroxyl radical in the presence of traceamounts of Fe or Cu (Bowler et ai., 1992). Thehydroxyl radicals initiate self-propagatingreactions leading to peroxidation ofmembranelipids (Halliwell, 1987). These results are inagreement with the hypothesis that ROS andlipid peroxidation are maJor contributors toPQ toxicity (Hart and DiTomaso, 1994).Hutchison (1979) also demonstrated that PQstimulated both H 20 2 and MDA production inleaf and thylakoids of spinach.

CONCLUSIONSThe results obtained showed that PQ treatmentcan induce oxidative stress in C. asiatica calluscultures. There was a direct relationshipbetween lipid peroxidation and ROSproduction since MDA levels increased inresponse to PQ treatment. In addition, PQinhibited the growth and viability of the callus.Results also suggest that CA03 was moretolerant to PQtreatmentas compared to GA09.

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