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Indian Journal of BiotechnologyVol 7, July 2008, pp 383-387

In vitro propagation and quercetin quantification in callus cultures of Rasna

(Pluchea lanceolata Oliver & Hiern.)

Deepika Arya, Vidya Patni* and Uma Kant

Plant Pathology, Tissue Culture and Biotechnology Laboratory, Department of BotanyUniversity of Rajasthan, Jaipur 302 004, India

Received 11 April 2007; revised 19 September 2007; accepted 15 December 2007

A protocol for micropropagation of Pluchea lanceolata, an important medicinal herb was developed. Leaf explantsobtained from field grown plants when tested for callus induction on Murashige and Skoogs (MS) medium, supplemented

with NAA in combination with BAP, produced the best callus. Maximum number of multiple shoots from the callus

(26.60.67) was obtained on MS medium supplemented with BAP (1.0 mg/L) and Kn (1.0 mg/L). More or less uniformelongation of multiple shoots was obtained on MS medium with lower concentrations of cytokinins, i.e., BAP (0.25 mg/L)and Kn (0.5 mg/L). Further elongation and profuse rooting were achieved when the well-grown shoots were cultured on half

strength MS medium supplemented with IBA (1.0 mg/L). The regenerated plantlets were hardened and established at 70%survival rate in pots. The bioactive secondary metabolite, quercetin, was isolated from callus tissues of different age groups

and its identification and confirmation was carried out by the colour reaction, TLC behaviour, IR spectrum and HPLCtechniques. Maximum quercetin content (0.23 mg/g dry wt of tissue) was obtained in 6-wk-old callus tissues.

Keywords:In vitropropagation, quercetin; callus,Pluchea lanceolataIntroduction

Pluchea lanceolata (Asteraceae) commonly known

as Rasna is an important xerophytic medicinal herb.

All parts of the plant are extensively used in indigenous

system of medicine. It has anti-inflammatory andanalgesic activities1,2 and is extensively used in drug

formulations given in rheumatoid arthritis, bronchitis,

dyspepsia, sciatica, edema, neuralgic disorders, etc.3

The plant contains high amounts of medicinally

important secondary metabolites (quercetin, -

sitosterol, triterpenol, etc., which gives it anti-

inflammmatory and analgesic properties4,5. Of these,

quercetin (flavonoid) is considered as active ingredient

which has many biological roles including anti-

inflammatory, anti-cancerous, antibacterial, antiviral,

antigonadotropic and antihepatotoxic activities6.

Due to unviable seed production and unscrupulouscollection, the wild population of plant species has

become vulnerable to extinction. However, the plant

species has been listed as a priority species by

Ministry of Health and Family Welfare, Govt. of

India7, therefore measures are needed to conserve it

using tissue culture techniques. Though in vitro

propagation of P. lanceolata via callus regeneration

from leaf explant has been reported earlier8, the

maximum number of multiple shoots and roots

obtained were 14 and 10 per culture, respectively.

Culture medium used for callus induction (MSmedium)9 and regeneration (WB medium)10 were

different; concentration of plant growth regulator

(Kn) in regeneration medium was 5.0 mg/L and in

vivo explant was not significant over the in vitro leaf

explant. This paper describes rapid, highly efficient

and easier technique for multiple shoot proliferation

and large-scale production of this medicinally

important priority species. The present study is an

advancement over the earlier protocol because it

describes multiple shoot proliferation from in vivo

leaf explants on simpler culture medium with lower

concentrations of plant growth regulators as well asdetermination of the level of bioactive secondary

metabolite, quercetin in non-differentiated callus

cultures of different age groups. Results of this study

may provide useful information for conservation of

this invaluable chemical factory in its natural habitat.

Materials and Methods___________________________*Author for correspondence:

Tel: 0141-2711654; 09829059868E-mail: patni-vidya@uniraj.ernet.in

Plant Material and Culture Establishments

P. lanceolata was collected from the wild regions

of Jaipur. Leaf explants were excised from the young

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INDIAN J BIOTECHNOL, JULY 2008384

plants, washed in running tap water for 30 min and

then in 2% liquid detergent solution (Extran E.

Mercka commercial grade detergent) for 5 min.

They were then surface disinfected (mercuricchloride, 0.5% for 1-3 min) and thoroughly washed

with sterile distilled water, 5 min for each wash. The

explants were dissected into small pieces and cultured

on MS medium15 consisting of basal salts and

vitamins with 3% (w/v) sucrose and 0.8% agar with

and without growth regulators. The concentrations

and combinations of different plant growth regulators

used for callus induction and establishment were: -

Naphthalene acetic acid (NAA), Indole-3-acetic acid

(IAA), Indole-3-butyric acid (IBA), 2,4-

Dichlorophenoxy acetic acid (2,4-D)- (0.25, 0.5, 1.0,

1.5, & 2.0 mg/L), and *NAA (*optimum

concentration suitable for callus induction)+6-Benzyl-

aminopurine (BAP)/6-Furfuryl-aminopurine (Kn)

(0.25, 0.5, 1.0, 1.5, & 2.0 mg/L). The pH of the

medium was adjusted to 5.8 and autoclaved at 15 psi

for 20 min. Cultures were maintained at 262C

under 16 h photoperiod illuminated by fluorescent

light (2000-3000 lux) and 555% relative humidity.

For inducing multiple shoot buds, the proliferated

calli were divided into small pieces (~500 mg fresh

wt) and subcultured on MS medium with different

concentrations and combinations of BAP (0.25-3.0

mg/L) and Kn (0.25-3.0 mg/L).

For rooting, the in vitro regenerated shoots were

transferred on half strength MS medium with NAA

(0.5-2.0 mg/L), IAA(0.5-2.0 mg/L) and IBA (0.5-2.0

mg/L). All the experiments were repeated thrice and

results were recorded after 4 wk of culture.

Callus Culture and Quercetin Synthesis

Growth and production of quercetin by callus

cultures were studied after 2, 4, 6 and 8 wk of

subculture. Established unorganised callus cultures

grown on MS medium with NAA (1.0 mg/L) and

BAP (0.5 mg/L) at different growth periods were

taken up for determination of quercetin contents.

Extraction of Harvested Callus Cultures

Dried callus (1.0 g) of different growth periods was

powdered and soxhlet extracted with 80% methanol

(100 mL/g sample) for 24 h on a water bath 11. Filtrate

was subsequently extracted in separating funnel with

petroleum ether, ether and ethyl acetate. Ether and

ethyl acetate fractions were analysed for free andbound quercetin content, respectively. Ethyl acetate

fraction was further hydrolysed by 7% H2SO4 for 2 h

and re-extracted with ethyl acetate. The fraction was

washed with distilled water till neutrality and dried.

Identification of QuercetinThe extracts were applied on TLC (silica gel G

coated plates) along with standard reference

compound of quercetin developed in air tight chamber

containing n-butanol, acetic acid and water (4:1:5)11.

Identification of compound was carried out by

spraying with 5% ethanolic FeCl3 solution, 1%

alcoholic AlCl3 solution separately and also by

exposing the plates in I2 vapours, NH3 vapours and

UV light chambers. Rf values of standard and samples

were also calculated.

Further analysis of compound was confirmed byhigh Performance Liquid Chromatography (HPLC)

and Infra-red (IR) spectral methods in comparison

with the authentic samples of quercetin. In the HPLC

analysis12 the elution was monitored at 254 nm and

peak identification was carried out on the basis of an

authentic sample of quercetin.

Quantification of Quercetin

Quantification of the isolated and identifiedquercetin was carried out colorimetrically11. The

amount of quercetin in various test samples was then

determined (mg/g dry wt) by comparing with those of

their respective standard regressive curve. Five such

replicates were examined and mean values werecalculated.

Results and Discussion

During the present set of experiments, different

auxins (NAA, IAA, IBA, 2,4-D) singly or in

combination with cytokinins (BAP, Kn) on MS

medium were tried for callus induction from leaf

explants. Among various auxins tried, NAA was

found to be best for producing creamish green, fast

growing and friable callus, the optimal concentration

being 1.0 mg/L. IAA and IBA gave brown, slow

growing and rhizogenic callus. 2,4-D showed nosignificant effect on callus induction. When optimal

concentration of NAA (1.0 mg/L) was combined with

BAP (0.25-2.0 mg/L) or Kn (0.25-2.0 mg/L), 0.5

mg/L concentration of BAP was found to be suitable

for callus induction. The callus so produced on the

medium was green, compact, healthy, fast growing

and organogenic (Fig. 1A). Similar results were

obtained inMentha arvensis13.

For differentiation, this stock callus was inoculated

on MS medium with different concentrations and

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ARYA et al: In vitro PROPAGATION & QUERCETIN QUANTIFICATION IN RASNA 385

Table 1Effect of cytokinins on multiple shoot bud developmentfrom callus in P. lanceolata on MS medium fortified withBAP/Kn/BAP+Kn (0.25-3.0 mg/L) after 4 weeks of callusinoculation

No. of shoot buds initiated perunit callus

*MeanS.E.

Control: MS basal medium Nil

Cytokinin/s conc. (mg/L)

BAP Kn0.25 - 7.40.590.5 - 10.60.67

1.0 - 16.80.77

1.5 - 18.20.56

2.0 - 13.60.75

2.5 - 11.80.56

3.0 - 9.40.69

- 0.25 5.20.78- 0.5 7.20.93

- 1.0 8.60.67- 1.5 10.40.53

- 2.0 7.80.77

- 2.5 7.00.28

- 3.0 6.40.59

0.5 0.5 19.20.77

1.0 1.0 26.60.67

1.5 1.5 21.40.692.0 2.0 18.80.56

2.5 2.5 14.60.753.0 3.0 10.00.48

* Values are 95% confidence limits for mean

Fig. 1In vitro propagation ofP. lanceolata from leaf explant: A,

Callus induction and establishment on MS medium; B, Multipleshoot induction from callus after 3 wk; C, Elongation of in vitroregenerated multiple shoots; D, Rooting and further elongation of

regenerated shoots on half strength MS medium; E, In vitroregenerated plantlet with well developed roots; & F,Acclimatization of micropropagated plantlet in pot containingnormal garden soil.

combinations of BAP (0.25-3.0 mg/L) and Kn (0.25-

3.0 mg/L). Results indicated that neither BAP nor Knalone proved beneficial in organogenesis from callus

(Table 1). Maximum shoot bud induction (26.60.67)

was obtained on MS medium with BAP (1.0 mg/L)

and Kn (1.0 mg/L) (Fig. 1B). Synergistic effect of

cytokinins on organogenesis from callus was reported

earlier14,15. More or less uniform elongation was

obtained on MS medium fortified with lower

concentrations of BAP (0.25 mg/L) and Kn (0.5

mg/L) (Fig. 1C). Similar findings have also been

reported forAcaciacatechu andA. senegal16,17.

Best rooting in the in vitro regenerated shoots was

observed on half strength MS medium with 1.0 mg/L

IBA (Fig. 1D,E). On this auxin, number of roots

(16.40.53) and rooting percentage (90-100%) were

highest and elongation of regenerated shoots also

occurred (Fig. 1D,E). IAA and NAA showed lesser

numbers of roots with higher amount of intervening

callus. Similar effect of IBA on in vitro rooting of

regenerated shoots has also been reported in Centella

asiatica18.After successful root establishment, in vitro

developed rooted plantlets were grown in plastic potsfilled with sterile vermiculite and soil (1:3) kept in the

growth room (262C, 555% humidity and 2000 lux

intensity), initially covered with polybags to control

humidity. The hardened plants were acclimatized to

natural conditions and then finally transferred to pots

containing normal garden soil (Fig. 1F). Survival rate

of plants was 70%.

The present tissue culture study is significant over

the earlier report of in vitro propagation of

P. lanceolata through callus mediated regeneration8.

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INDIAN J BIOTECHNOL, JULY 2008386

In previous report of micropropagation of P.

lanceolata, maximum number of shoots (14) and

roots (10) were obtained from in vitro leaf explants on

WB medium supplemented with higherconcentrations of Kn (5.0 mg/L), while the present

study comprises a technique to multiply plant species

quickly and possibly less expensive with more

number of multiple shoots (26.60.67) and roots

(16.40.53) on simpler culture medium (MS)

supplemented with lower concentrations of growth

regulators (BAP 1.0 mg/L and Kn 1.0 mg/L) as

compared to previous report. Number of multiple

shoots obtained in present study (26.60.67) was

almost twice the number obtained in earlier report (14

shoots per culture).

Active principle isolated was identified andconfirmed as quercetin [Rf 0.82; colour in UV

fluorescent (bluish yellow), ammonia vapour

(yellow), iodine vapour (yellowish brown); colour after

spraying with 5% ethanolic Fecl3 (bluish grey) and 1%

alcoholic AlCl3 (dull yellow)]. IR spectrum and HPLC

analysis of isolated quercetin along with authentic

samples also showed the presence of quercetin (Fig. 2

A,B,C). HPLC peak of isolated quercetin was

identified on the basis of retention time (tR~2.9 min) of

authentic sample of quercetin (Fig 2 B,C). Some

unknown peaks were also observed in extract.

Quantitative estimation of total quercetin contentalso showed maximum amount in 6-wk-old callus

tissues (0.23 mg/g dry wt), followed by 8-wk-old (0.20

mg/g dry wt), 4-wk-old callus (0.17 mg/g dry wt) and

2-wk-old (0.12 mg/g dry wt). In the same way, free and

bound quercetin was also maximum in 6-wk-old callus

tissues, i.e., 0.08 and 0.15 mg/g dry wt, respectively.

Quercetin production in cell cultures was also observed

inBalanites aegyptiaca19 and Cassia angustifolia.

20

In the present study, the bioactive secondary

metabolite quercetin accumulated in appreciable

amounts (0.23 mg/g dry wt) in callus cultures of P.

lanceolata, which increased further by theincorporation of phenylalanine and cinnamic acid as

reported earlier by authors21. In the previous report,

quercetin has been quantified from in vivo and in vitro

grown shoots ofP. lanceolata8. However, the present

study is significant because no attempts have been

made previously on systematic monitoring of quercetin

in static callus cultures of different age groups. The

present finding is also significant as the quercetin

accumulated in higher amounts in 6-wk-old callus

tissues could be attributed to the fact that stationary

phase of callus growth shows accumulation of

secondary metabolites22.

Fig. 2Isolation and identification of quercetin in 6-wk-old static

callus tissues of P. lanceolata: A, Superimposed IR spectra ofisolated and authentic samples of quercetin; and B & C, HPLCchromatograms of authentic and isolated quercetin showing

retention time (tR~ 2.9 min).

Therefore, the protocol developed is superior to the

method already reported on P. lanceolata for defining

easier approaches of high rate multiplication

(26.60.67), which can be used for conservation of this

medicinally important priority species. Accumulation

of quercetin in callus cultures can be exploited for their

large-scale production in a wide array of health

promoting benefits as antioxidant, antiinflammatory

and anticancerous agents. Thus, results of this study

when applied on a large-scale will help in the

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ARYA et al: In vitro PROPAGATION & QUERCETIN QUANTIFICATION IN RASNA 387

conservation of this invaluable chemical factory in its

natural habitat.

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