Plant Tissue Cult. & Biotech.

20(2): 127-131, 2010 (December)

PTC&B

InvitroMassPropagationfromShootTipExplantsof Vernoniacinerea(L.)Less.AnAntioxidant,Anti inflammatoryMedicinalPlant

M.Maharajan1,AbdulBakrudeenAliAhmed*,RosnaMatTaha, S.Jawahar2,R.RaviPaul2andM.Jayaseelan3
InstituteofBiologicalSciences,FacultyofScience,UniversityofMalaya,50603,Kuala Lumpur,Malaysia Key words: Vernonia cinerea, Mass propagation, Shoot tip explants, Antioxidant, Medicinalplant

Abstract
Plantlets were regenerated from the shoot tip explants of Vernonia cinerea (L.) Less.inMSsupplimentedwithBAandKn.MaximumnumberofshootsinBA (13.32 mg/l) and roots in IBA (7.38 mg/l) developed. The rooted plantlets were successfullyestablishedinthefield.

Introduction
Fatty oil, amyrin acetate, amyrin benzoate, sitosterol, stigmosterol, spinnasterol (+)lirioresinol B, stigmasterol, stigmasterol3ODglucoside, 4 sulfobenzocyclobutene compounds inducing NGF activity are extracted from Vernonia cinerea (L.) Less. (Asteraceae) (Zhu et al. 2008). It also contains medicinal properties for eczema, ringworm, elephantiasis, conjunctivitis, diarrhea, leucoderma, dysiria, skin diseases, leprosy, fevers, anticancer, anti oxidant and antiinflammatory (Kumar and Kuttan 2009). Nowadays, tissue culture techniques are actively employed biotechnological methods to improve the metabolites and medicinal plants in a large scale. Here, to develop an effectiveinvitromethodforplantregenerationofV.cinereaandthepropagated plantsweresuccessfullyestablishedinfieldconditions.
*Authorforcorrespondence.<bakru24@yahoo.co.in>.1DepartmentofBotany,SchoolofLifeSciences, Bharathiar University, Coimbatore, Tamil Nadu, India. 2Department of Biotechnology, Bharath College of Science and Management, Thanjavur, Tamil Nadu, India. 3Department of Biotechnology, PonnaiyahRamajayamCollege,TamilNadu,India.

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MaterialsandMethods
Shoot tips of Vernonia cinerea (L.) Less. were collected from field grown plants (Fig. 1a). The explants were washed with 70% ethanol, 0.1% HgCl2 (w/v) for 3 minandrinsedwithsteriledistilledwaterforthreetofourtimes.Explantswere culturedonMScontaining3.0%sucrose(w/v)andBA(2.22to22.20M/l)and Kn (2.32 to 23.20 M/l) for multiplication of plants with optimum pH (5.8 1), light intensity (85 mol/m/s), 16 h. The regenerated shoots were kept into the rootingmediumwithIBA(2.46to14M/l).Plantletsweretransferredtoplastic cups containing sterile soil, sand, vermiculite (1: 1:1), and then transferred to field. All the treatments were statistically analyzed by DMRT) (Gomez and Gomez1976).

ResultsandDiscussion
Table 1 shows the successful results. Shoot tips were cultured on MS supplemented with different concentrations of BA (2.22 to 22.20 M/l) and Kn (2.32to23.20M/l).Highernumberofmultipleshootinductionwasobservedin 30 days and significantly higher in BA than Kn (Fig. 1b, c). The maximum number of multiple shoots (148.2/shoot tip) was obtained in MS with BA 13.32 M/l (Table 1; Fig. 1d, e). Similar results were reported in Emblica offcinalis (VermaandKant1996)andWithaniasominifera(Dekaetal.1999).

Table 1. Effect of Kn and BA on multiple shoot induction from shoot tip explants of Vernoniacinerea(L.)Less.

Plantgrowth regulators(M/l) Kn 2.32 4.64 9.28 13.92 18.56 23.20 BA 2.22 4.44 8.88 13.32 17.76 22.20

Regenerating shoots(%) 33.83.7f 40.32.5e 48.02.6c 69.01.0a 62.62.5b 46.61.5cd 38.10.7f 46.31.5e 57.32.5bc 73.02.6a 59.62.0b 52.02.0d

No.ofshoots/ explants 39.03.6f 69.03.6de 83.07.2b 131.610.4a 81.03.6bc 71.33.2d 46.61.5f 70.01.0e 101.67.6b 148.32.8a 91.65.6c 88.37.6cd

Shoot length(cm) 0.80.2f 2.60.5de 4.01.0c 6.50.5a 4.60.5b 3.01.0d 1.60.5f 3.30.5de 5.60.5c 9.01.0a 6.30.5b 4.01.0d

Valuesaremeanof30replicatespertreatmentandrepeatedthrice.Valueswiththesame superscriptarenotsignificantlydifferentat5%probabilityaccordingtoDMRT.

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PatnaikandChand(1996)reportedBAwassuperiorformultipleshootinduction thanothercytokininsinshoottipexplants.Beenaetal.(2003)reportedthatthe KndidnotsupporttheproliferationofmultipleshootsinCeropegiacandelabrum, buttheKnalsofavouredforhighrateofmultipleshootinduction.

Fig. 1. a. Habitat, bc. Multiple shoot initiation, de. Multiple shoots, f. Rooting andg,h.Hardening.

The well developed shoots were transferred for root induction in medium containing IBA (2.46 to 14.76 M/l) and IAA (2.85 to 17.13 M/l) at 25 days. Maximum number of roots per shoot was observed in IBA 7.38 M/l (Table 2; Fig.1f),whereas,thehigherlevelofIBAshowedlowfrequencyrootinduction.

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Patnaik and Chand (1996) suggested that the best rooting medium contained IBA. Welldeveloped rooted plantlets were transferred to plastic cups and coveredwithpolytheneandmaintainedintissuecultureconditions(Fig.1g,h). Finally,thedevelopedplantletswerekeptingreenhouse,andthentransferredto field.Thesurvivalratewas55%.

Table2.EffectofIAAandIBAonrootinductionfromshoottipsderivedplantletsof Vernoniacinerea(L.)Less.

Plantgrowth regulators(M/l) IAA 2.85 5.71 8.56 11.42 17.13 IBA 2.46 4.92 7.38 9.84 14.76

Rootformation (%) 32.04.89d 54.01.63b 62.32.05a 50.01.23bc 30.04.89cd 32.04.89d 56.62.62bc 70.64.08a 62.32.05b 30.04.89de

AverageNo.of roots/shoot 1.60.47e 2.90.34c 6.00.81a 4.30.47b 2.30.37cd 1.60.47e 3.30.27c 8.00.81a 6.61.24b 3.00.81cd

Valuesaremeanof30replicatespertreatmentandrepeatedthrice.Valueswiththesame superscriptarenotsignificantlydifferentat5%probabilityaccordingtoDMRT.

Inconclusion,themicropropagationprotocolwasestablishedfromshoottip explants of V. cinerea. The rehabilitation micropropagation development total processwascompletedin60days.Thisefficientmicropropagationprotocolwill be useful to conservation, and in the improvement of V. cinerea using genetic transformation.

References
Beena MR, Martin KP, Kirti PB and Hariharan M (2003) Rapid in vitro propagation of medicinallyimportantCeropegiacandelabrum.PlantCellTiss.Org.Cult.72:285289. DekaAC,KalitaMLandBaruahA(1999)Micropropagationofapotentherbalmedicinal plant,Withaniasominiferea.Environ.Ecology.17:596. Gomez KA and Gomez AA (1976) Statistical procedures for agricultural research with emphasis on rice. International Rice Research Institute. Los Banos, Philippines, p. 264. Kumar P and Kuttan G (2009) Vernonia cinerea (L.) Less. scavenges free radicals and regulatesnitricoxideandproinflammatorycytokinesprofileincarrgeenaninduced pawedemamodel.Immunopharma.Immnotoxicol.31:94102.

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Patnaik S and Chand PK (1996) In vitro propagation of the medicinal herbs Ocimum americanumL.Syh.Ocimumsims(Hoarybasil)andOcimumsanctumL.(Holybasil). PlantCellReports15:846850. VermaBandKantU(1996)MicropropagationofEmbilicaofficinaleGaertzthroughmature nodalexplant.J.Phytal.Res.9:107109. ZhuHX,TangYP,PanLMandMinZD(2008)Studiesonbioactiveconstituentsofwhole herbsofVernoniacinerea.ZhongguoZhongyaoZazhi.33:19861988.