Direct organogenesis and in vitro flowering was obtained in Vitex trifolia L an endangered

medicinal plant. High frequency and maximum number of multiple shoots were obtained from shoot tip

explants on MS medium supplemented with BAP (3.96-15.85 µM) and IAA (5.70-22.83 µM). Regenerates,

when transferred to rooting medium with IBA at concentration of 2.46-14.76 µM and IAA at concentration of

2.85-17.13 µM initiated flowering along with rooting. In vitro flowers set viable seeds. Rooted plantlets were

hardened and transferred to green house with 100% survivability. This finding has significant role in

Pharmaceutical industries and in vitro flowering facilities in vitro pollination and fertilization, further it also

facilities in advancing the generation at much faster speed under limited progeny size in the segregating

generation of Vitex trifolia L

Nagaveni, C.

Rajanna, L.

ePrints@Bangalore University

Botany Research International 6 (1): 13-16, 2013ISSN 2221-3635© IDOSI Publications, 2013DOI: 10.5829/idosi.bri.2013.6.1.505Corresponding Author: L. Rajanna, Department of Botany, Bangalore University, Jnanabharathi, Bangalore, 560 056, India. 13In-vitro Flowering in Vitex trifolia L.C. Nagaveni and L. RajannaDepartment of Botany, Bangalore University, Jnanabharathi, Bangalore-560 056, IndiaAbstract: Direct  organogenesis and in vitro flowering was  obtained in Vitex trifolia L an endangeredmedicinal plant.  High  frequency and maximum number of  multiple shoots were obtained from shoot tipexplants  on  MS  medium  supplemented with BAP (3.96-15.85 µM) and IAA (5.70-22.83 µM). Regenerates,when transferred to rooting medium with IBA at concentration of 2.46-14.76 µM and IAA at concentration of2.85-17.13 µM initiated flowering along with rooting. In vitro flowers set viable seeds. Rooted plantlets werehardened and transferred to green house with 100% survivability. This finding has significant role inPharmaceutical industries and in vitro flowering facilities in vitro pollination and fertilization, further it alsofacilities in advancing the generation at much faster speed under limited progeny size in the segregatinggeneration of Vitex trifolia L.Abbreviations: IBA-Indole Butyric Acid  BAP-6-Benzyl adenine  IAA-Indole acetic acidKey words: In-vitro  Flowering  Murashige and Skoog’s mediaINTRODUCTION Vitex trifolia Linn (Simple leaf chaste tree) is widelyVitex trifolia L is a large coastalshrubor smalltree, and Australia. In China, the fruit of simple leaf chaste treeless than 5 m in height with the stems covered by soft is called Man Jing Zi and is used for the treatment ofhairs (tomentose). The leaves are oppositely arranged headaches and colds, alleviation of fever, pain relief,along the stems and are usually compound, composed of sedation, anti-inflammatory treatment, etc. There are3 linear leaflets which range between 1-12 cm in length. reports on use of the plant body or an extract of simpleThe upper surfaces of the leaves are green and the lower leaf chaste tree as an external medicine to be applied tosurface grayish green. The flowers are born inpaniclesor the scalp as hair tonic [1]. However, the external medicine,clusters up to 18cm in length. Individual flowers have such as a hair tonic or hair restorer, does not exhibitpurple to violet two-lippedcorollasthat are approximately satisfactory effects. Vitex trifoliaresults in pregnancy,5mm long. Thestamensare in two pairs and the ovary is there is poor evidence based on theoretical and expertsuperior, or develops above the corolla. The fleshy fruits opinion and in vitro studies state that chaste tree mayare about 6mm in diameter and contain 4 small black have estrogenic and progesteronic activity, uterineseeds. V.  trifolia  is  naturally found along coastlines stimulant activity, emmenagogue activity and preventfrom tropical East Africaas far east as French Polynesia. miscarriages. In lactation, theoretical and expert opinionThe leaves are used to treat female ailments in theCook conflict as to whether chaste tree increases or decreasesIslands and used to relievefeverin Samoa. Additionally in lactation [2].Samoa, the dried leaves are burned to determosquitoes. Study of leaves of V. trifolia isolated threeChemical constituents of V. trifolia are viteosin-A, compounds-viteosin-A, vitexicarpin and vitetrifolin-E.vitexicarpin and vitetrifolin-E. Some provide Vitexicarpin   was    the    most    active    of   the  three.valuablelumber. The flexible limbs of some species are The mechanism of activity seems to be non-competitiveused inbasket weaving. Some of thearomaticspecies are antagonism to histamine and stabilization of mast cellsused medicinally. Recently simple leaf chaste tree is Red membrane function. Leaves yield an essential oil andlisted by IUCN with Low-Risk status. resin.  Fruit  contains an acid resin, an astringent organicdistributed from the Korean Peninsula to Southeast AsiaBot. Res. Intl., 6 (1): 13-16, 201314acid, malic acid, traces of an alkaloid and coloring matter. Multiple Shoot Regeneration: Plant growth regulatorsChemical studies of  leaves and twigs yield an essential viz., BAP (3.96-15.85 µM), IBA (4.92-19.68 µM) and IAAoil, 0.11-0.28%. Chief constituents of  the oil are l-d-pinene (5.70- 22.83 µM) were tried individually or in combinationand camphene (55%); terpinyl acetate (10%); and a to obtain the multiple shoot bud induction. All thediterpene alcohol (20%). Study isolated a new experiments were repeated three times to confirm thebenzofuran-type  lignan,   vitrifol   A,   from  the  fruits of reproducibility.V. trifolia with three known compounds. In a studyinvestigating the inhibitory effect of vitexicarpin on the Root Induction: The well-developed shoots were excisedproliferation of human cancer cells showed it induces and were transferred to half strength MS mediumapoptosis in K562 cells via mitochondria-controlled supplemented  with   IBA  (2.46-14.76  µM)  and IAAapoptotic pathway. (2.85-17.13 µM).In a study of four species of Vitex against Culexquinquefasciatus larvae, the highest larvicidal activity Acclimatization: Rooted plantlets were removed fromwas found with the extract of V. trifolia. The in vitro culture tubes and washed their roots in running tap waterphytochemical  and   pharmacological  investigation of and transferred to plastic cups containing 1:1:1 ratio ofthe non-volatile extracts of five South African Vitex Soil rite: Coco peat:  Vermiculite for about 15days andspecies  (Verbenaceae);    V.    obovata    ssp.   obovata, then transferred to pots containing sand and farm yardV. obovata ssp. wilmsii, V.  pooara,  V.  rehmannii  and mixture (1:1) for a week and subsequently transferred toV. zeyheri are carried out [3]. Many scientific pots. All the tissue culture raised plantlets need gradualinvestigations are done in other species of Vitex such as acclimatization for  their survival in the field conditionV. rotundifolia, V.   cannabifolia,     V.     altissima    and from controlled environment. Instead of transferringV.    negundo.  A   negligible   work   is   carried    out   in directly to the pots, plantlets were left for a week in theV.  trifolia  which is red-listed by IUCN with risk factor plastic cups at controlled temperature (25±2°C) with 60%but conservation status near threatened (NT) which relative humidity. After initiation of new roots, they wereinspired us to select the plant. kept in the moist chamber and grown till maturity.MATERIALS AND METHODS Statistical Analysis: Experiments were setup in aHealthy plants of V. trifolia were collected from experiment  was  repeated twice. Data were recorded onBotanical garden, Department of Botany, Bangalore the percentage of response, number of shoots perUniversity, Jnana Bharathi campus, Bangalore, Karnataka, explants, shoot length and number of roots per shoot.India and used for experimental purpose. Stem cuttings Means and Standard errors were carried out for eachwere grown in pots. Plant is perennial and flowers during treatment.the month of August and October after monsoon.Plant Material: Shoot tips were selected as explants fordirect regeneration. The explants were thoroughly washed Vitex trifolia. L plants were efficiently regeneratedin running tap water for 10 minutes, disinfected by using from shoot tips. Explants were capable of directlyliquid detergent tween-20 (5%v/v) for 15 min and rinsed developing multiple shoots on MS basal mediumwith distilled water. The explants were then treated with containing different concentrations of growth regulators.0.5% (w/v) Sodium hypochlorite solution (5 min) followed Multiple shoot initiation  from  shoot  tip  explants  wasby a rinse in double distilled water. Later the explants were observed within 10-15 days after inoculation  (Fig. 1a).treated with 0.01%(w/v) Mercuric chloride (10 min) and The highest number of shoots (15/ explant) was observed0.1%(w/v) Mercuric chloride solution (5 min), followed by in the medium containing  BAP  9.90µM/l followed by IBABavistin solution (30 min) and washed thoroughly in 14.76µM/l (11/ explant). Of the two growth regulatorsdouble distilled water. The  surface sterilized explants (BAP and IBA), BAP was found to be more suitable thanwere  transferred  aseptically to  pre-sterilized culture IBA for initiation  and   proliferation  of  multiple shoottubes containing MS media [4] fortified with hormones. buds (Table 1).Then, the tubes were incubated in culture room under The elongation of shoots and proliferation of nodescontrolled  conditions   of     White     fluorescent   light were achieved on the same parental medium. In the(16 hours light-8 hours dark), temperature (24±2°C) and present study, the relative effectiveness  of  BAP  andhumidity. IBA  varied  for in  vitro  multiple shoot  regeneration fromRandomized Complete Block Design (RCBD) and eachRESULTS AND DISSCUSSIONBot. Res. Intl., 6 (1): 13-16, 201315Fig. 1: a. Multiple shoots; b. Flowering bud initiation; c. In-vitro flowering; d. Fertilized flower and multiple shoots; e.Root formation and f. Hardening.Table 1: Effect of BAP and IBA on shoot Multiplication from shoot tips explants of Vitex trifolia. L.Growth Regulators µM/l Percentage of cultures with induced shoots Number of shoots per explants Shoot length (cm)BAP3.96 10.8±1.7 4.9±1.4 2.0±0.37.92 21.4±5.2 8.3±1.2 4.0±0.15.94 30.8±2.0 10.9±2.3 5.0±0.29.90 86.2±3.6 18.2±5.1 13.4±1.111.88 74.5±3.4 13.8±3.6 11.5±0.913.87 62.0±6.0 8.8±2.0 8.4±0.515.85 50.8±2.8 4.4±4.2 4.2±0.4IBA4.92 5.1±1.1 2.7±1.6 3.5±0.37.38 16.5±2.4 5.7±2.0 3.7±0.79.84 26.4±2.9 7.2±1.5 4.7±0.412.30 56.5±3.0 10.2±2.2 7.6±0.314.76 91.2±2.7 12.6±4.4 12.8±0.417.22 86.2±4.7 9.0±1.5 11.7±0.219.68 60.7±3.5 5.9±2.1 8.1±0.1Each value represents the mean ± SD of 10 replicates and each experiment was repeated at least thrice. Table 2: Effect of different concentrations of IAA and IBA on rooting ofinvitro regenerated shoots of Vitex trifolia L.Plant Growth Percentage of Root Number of RootsRegulators µM/l Induction per ShootIBA2.46 10.8±2.0 5.7±1.34.92 24.5±3.0 8.5±1.87.38 44.2±6.3 11.6±4.79.84 95.1±3.9 14.3±1.612.30 80.8±1.7 11.8±1.514.76 60.9±1.4 7.4±1.0IAA2.85 15.7±4.1 3.6±1.65.70 20.9±1.9 6.4±2.28.56 42.9±6.7 7.8±1.511.41 85.8±2.5 11.6±1.314.27 70.8±5.0 9.7±2.717.13 55.2±1.7 5.0±0.9Each value represents the mean ± SD of 10 replicates and each experimentwas repeated at least thrice.shoot tip explants. BAP (9.90µM/l) was found to be thebest concentration for regeneration of multiple shootsbuds (13 - 15). Shoots were harvested every 30-40 daysand new shoot lets were harvested periodically. All theplantlets   produced    roots   on   the   rooting   medium(1/2 MS) containing  IBA  and  IAA  after 15days ofincubation (Fig 1e; Table 2).  Highest number of Rootswere produced per shoot at 9.84 µ M/l IBA (11-14) and at11.41 µ M/l IAA (9-12) after a week.In vitro Flowering: Flowering was considered to be acomplex process regulated by both internal and externalfactors and its induction under in vitro culture isextensively rare. Temperature and Photoperiod has a greateffect on In  vitro  flowering  and  was  observed  within20   days   of   culture   during    our   study   (Fig.  1b)  [5].Bot. Res. Intl., 6 (1): 13-16, 201316For in vitro flowering, the response of BAP is better than 3. Nyiligira, E., A.M. Viljoen, Van F.R. Heerden, et al.,IBA. The in vitro regenerated plantlets were successfully 2008. Phytochemistry and in vitro pharmacologicaltransferred to plastic cups and then to the field (Fig. 1f). activities of South African Vitex (Verbenaceae)The capacity of shoot bud differentiation and shoot species. J. Ethnopharmacol., 119: 680-685.proliferation from shoot tip explants of Vitex trifolia. L. 4. Murashige, T. and F. Skoog, 1962. A revised mediumdepended on hormonal variation. There was a good shoot for rapid growth and bioassays with tobacco tissuebud induction and proliferation response only in the cultures. Physiol. Plant., 15: 473-497.presence of auxins and cytokinins and no response in the 5. Moss, G.I., 1969. Influence of temperature andbasal medium. Similar results are well documented in photoperiod of flower induction and inflorescenceseveral  medicinal  plants [6], Bixa ovellana L. [7], development in sweet orange. (Citrus sinensis L.Emblila officinale  [8]  and  Withania  somnifera  [9]. Osbeck). Journal of Horticultural Science, 44: 311-320.From our study it was clear that BAP 9.90µM/l and IBA 6. Pattnaik, S.K. and P.K. Chand, 1996. In vitro14.76µM/l were significantly more effective for inducing propagation of the medicinal herbs Ocimumshoot organogenesis. Well-developed shoot lets when americanum (L.) Sy. Ocimum canum Sim (Loarytransferred  to  rooting  medium  containing 9.84 µ M/l basil) and  O.   santum   (Holly   basil)   Plant   Cell. IBA induced higher frequency of roots than 11.41 µ M/l Rep., 15: 846- 850.IAA (Fig.1e). Similar effect of IBA was reported in 7. Sharon and Marie, 2000. In vitro clonal PropagationOcimum americanum, O. canum and O. sanctum [6] and of Bixa ovellana L. Cur. Sci., 78(12): 1532.also in Heracleum candicans [10]. However, BAP was 8. Verma, B. and U. Kant, 1996. Micropropagation offound to be the best in vitro flowering hormone than Embilica officinale Gaertz through Mature nodel exother Auxins and cytokinins, similar results were plant. J. Playtol. Res., 9(2): 107-109.observed in Sunflower [11], in Mulberry [12] and in 9. Deka, A.C.,  M.C.  Kalita  and  A.  Baruah, 1999.Coriander [13]. Micro propagation of  a potent herbal MedicinalHardening  of  regenerated  plants  was  made on Plant, Withania     somnifera.     Environ.   Ecol.,1:1:1 ratio of Soil rite: Coco peat: Vermiculite and then 17(3): 594-596.transferred to  pots  containing  sand and farm yard 10. Wakhlu, A.K. and R.K. Sharma, 1999.mixture (1:1) in Green house and subsequently to field. Micropropagation of Heracleum candicans Wall.-aThe survival rate was about 90% (Fig. 1 f). rare medicinal herb. In vitro cellular andFrom our experimental data, it is evident that BAP Developmental Biol. Plant., 35(1): 79-81.and IBA are the best suited for inducing multiple shoots 11. Patil, M.S., N.M. Ramaswamy and S.R. Sreeand in vitro flowering and IBA for rooting. In conclusion, Rangasamy,  1993. In   vitro flowing  in  sunflowerthis communication describes an efficient rapid (H. annuus L.) Curr. Sc., 65: 565-566.propagation system of Vitex trifolia. L. 12. Naik, G.R. and K. Lata, 1996. In vitro flowering inREFERENCES Sci., 15: 129-132.1. Chuyaku Jiten, Dictionary  of  Chinese  Medicines; and seed  setting  formation  of  corianderpp: 2456-2458 and Japanese Unexamined Patent (Coriandrum sativum L.). Curr. Sci., 74(3): 195-197.Publication Nos. 1984-116211 and 2000-31512.2. Dugoua, J.J., D. Seely, D. Perri, E. Mills and G. Koren,2008. Safety and efficacy of chastetree (Vitex trifolia)during pregnancy and lactation. Winter; 15(1) Can J.Clin Pharmacol., e80-6. Epub 2008 Jan 18.Morus alba L. (Mulberry) Bull. Of pure and Applied13. Stephen, R. and N. Jayabalan, 1998. In vitro flowering

In-vitro Flowering in Vitex Trifolia L.

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In-vitro Flowering in Vitex Trifolia L.

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