In  vitro  studies  with  rhizome  explants  of Vetiveria  zizanioides  obtained  through  organogenetic pathway was focused on the difference in essential oil content with that of control plants. There was variation exhibited by

In  vitro  regenerated  plants  in  their  morphological  growth  and  were  referred  as  “morphotypes”. Two types of morphotypes were identified as long and short based on differences in their height. Essential oil was extracted and analyzed by Gas chromatography. The two morphotypes showed varied essential oil content of 2.1% (long), 1.9% (short) with the control showing 1.8% and also the plants showed marked differences in percentage  composition  of  essential  oil  with  respect  to  important  compounds  like  khusimol,  valencene, vetiverol, vetivone, vetivenene, vetiselinol and nootketone. Further studies on the screening and selection of the chemotypes for high producing essential oil content and composition are under progress

Jayalakshmi, N.R.

Shiva Kameshwari, M.N.

Thara Saraswathi, K.J.

Vyshali, P.

ePrints@Bangalore University

American-Eurasian J. Agric. & Environ. Sci., 10 (3): 458-463, 2011ISSN 1818-6769© IDOSI Publications, 2011Corresponding Author: N.R. Jayalakshmi, Department of Microbiology and Biotechnology, Bangalore University,Jnana Bharathi Campus, Bangalore-560056, India,  E-mailL jb-urs@rediffmail.com.458Comparitive Study on Essential Oil in Natural and In vitro Regenerated Plants of Vetiveria zizanioides (Linn.) NashK.J. Thara Saraswathi, N.R. Jayalakshmi,1 1P. Vyshali and M.N. Shiva Kameshwari1 2Department of Microbiology and Biotechnology, Bangalore University,1Jnana Bharathi Campus, Bangalore-560056, IndiaDepartment of Botany, Bangalore University, Bangalore, India2Abstract: In vitro studies with rhizome explants of Vetiveria zizanioides obtained through organogeneticpathway was focused on the difference in essential oil content with that of control plants. There was variationexhibited by In vitro regenerated plants in their morphological growth and were referred as “morphotypes”.Two types of morphotypes were identified as long and short based on differences in their height. Essential oilwas extracted and analyzed by Gas chromatography. The two morphotypes showed varied essential oil contentof 2.1% (long), 1.9% (short) with the control showing 1.8% and also the plants showed marked differences inpercentage composition of essential oil with respect to important compounds like khusimol, valencene,vetiverol, vetivone, vetivenene, vetiselinol and nootketone. Further studies on the screening and selection ofthe chemotypes for high producing essential oil content and composition are under progress.Key words: Morphotypes  Aroma therapy  Chemotypic variation  Gas chromatographyINTRODUCTION smell and very pleasant odor when diluted. It blends wellThe pharmacological properties of essential oils patchouli, oakmoss, lavender, clary sage, mimosa, cassiaextracted from plants have received the great interest of and ylang ylang [14]. Vetiver oil is high-priced andacademic institutes and pharmaceutical companies [1-4]. extensively used as a cosmetic product for pharmaceuticalMany investigations and new findings have significantly companies as anti-microbial and anti-fungal agent [8].prompted and expanded novel applications of essential oil Chemically  vetiver  oil  is  extremely complex; overwhich are now been widely used as pharmaceutical and 150  compounds  have  been  isolated and characterized.aroma therapeutic agents [5]. A major portion of oil consists of sesquiterpene alcoholOne such plant that is extensively used is vetiver and their derivatives [6, 7]. Among them the ketones like(Vetiveria zizanioides L.) recently reclassified as  and -vetivone (10%), khusimol (15 %) are the majorChrysopogon zizanioides (L.) Roberty, originated from constituents and their presence is often considered asIndia. Vetiveria was used in ancient times as fragrant fingerprint of the oil contributing significantly to its odourmaterial   and   in  traditional  medicine for its essential oil [9, 10]. The oil fraction is also rich in vetivones and[6, 7]. Vetiver oil also called vetivert, khus-khus, khas- vetiverol [8].khas or 'oil of tranquillity', is a fragrant in its own right for Since synthetic vetiver oil cannot yet bewhich no synthetic substitute is available. It has been manufactured  [15, 16], there is an interest and need forutilized as a raw material for products like perfumes, the perfume industry in obtaining new variants withdeodorants, lotions, soaps etc., additionally playing an higher oil yield and different odor tonalities. Therefore,important role in aromatherapy [8] and is one of the finest the present study has been made to generate variationfixatives known [9, 10]. Vetiver oil is the basis of Indian under In vitro culture to further select the variants withand western (36%) perfumery [11-13], gives a powerful desirable qualities.with oils of sandal wood, rose, violet, jasmine, opapanax,Am-Euras. J. Agric. & Environ. Sci., 10 (3): 458-463, 2011459MATERIALS AND METHODSThe In vitro generated plants of V. zizanioidesobtained from rhizome explants were maintained underuniform conditions in the field along with the controlplants. After  one  year,  the  roots  were  collected fromIn vitro regenerated plants (long and short morphotypes)and control plants.The roots collected were thoroughly washed withrunning  tap water for 30 minutes, cut into small piecesand used for the studies. Approximately 500g of the rootswere weighed and subjected to extraction of essential oilusing Clavenger’s apparatus. The essential oils werecollected in a separator by downward displacement ofwater.Gas chromatography of the essential oil wasperformed on a Perkin - Elmer Gas chromatography model8500 using glass capillary column BA-1 (coated with di-methyl silaxane) of the size 25m x 0.5mm. Nitrogen wasused as a carrier gas at 10psi inlet pressure. Temperatureprogramming was performed from 60-220° at 5°C/ min. Thesamples were injected by splitting the split ratio 1:80.The compounds of essential oil were identified byrelative retention time with standard reference compoundsrun under the same conditions by comparing the Kovat’sretention indices of the peaks and also by peakenrichment on co- injection with authentic compounds[17-19].RESULTSMultiple shoots were obtained from the auxiliarybuds of rhizome explants grown in MS medium and wellestablished regenerated plants were obtained of long andshort morphotypes (Fig. 1, 2, 3). A good yield of brightyellowish essential oil was obtained from the In vitroregenerated long morphotype when compared to the shortmorphotype  with light yellow color, along with thecontrol plants. The long and short morphotypes yielded2.1 and 1.9% of oil, respectively, whereas the control plantyielded 1.8%. The GC analysis results obtained aretabulated in Table 1 and shown in graphs (1, 2, 3). Thelong  morphotype in addition to possessing an increasein percentage yield of oil also showed enhancedcomposition of oil with respect to important componentslike - thujene, camphene, linalyl acetate, bornyl acetate,geranyl  acetate, - elemene, valencene, -eudesmol and- cardinal in the long morphotype compared to controlplants.Table 1: Percentage composition of essential oil in natural plant and invitro generated morphotypes of V. zizanioidesIn vitro generated morphotypes-----------------------------------------Compounds Natural plant Long Short-thujene 0.15 0.72 0.55-pinene 0.18 0.10 0.23Camphene 0.68 1.09 2.95Myrcene 0.10 - 0.05- terpinene 0.06 - -P-cymene - - -Limonene 0.64 0.34 0.89(Z)- - ocimene 0.08 - -Linalool 0.47 - 0.20(Z)- - terpineol 0.16 - 0.09(E)- - terpineol 0.09 - -Borneol 1.51 0.25 0.36Terpinen-4-ol 0.11 - 0.05- terpineol 0.36 - 0.05Nerol 0.21 0.21 0.58Geraniol 0.15 - -Linalyl acetate 0.08 0.65 0.59Bornyl acetate 0.18 0.92 1.66Neryl acetate 0.05 - 0.05Geranyl acetate 0.10 0.21 -- copaene 0.15 0.10 0.18-bourbonene 0.17 - 0.08-caryophyllene 0.12 0.34 0.42-elemene 0.25 0.30 0.17Valencene 0.73 1.40 1.29Salina-4(14),17-diene 0.20 0.14 0.07Zizaene 1.24 0.70 0.68Nootkatene 0.65 0.27 0.25-bisabolene 1.19 0.97 1.38-cardienene 3.61 0.68 0.62Elemol 1.60 0.36 0.46-vetivenene 2.99 2.46 2.8210-epi- -eudesmol 0.66 0.42 0.42-eudesmol 0.12 0.30 0.27-cardinol 1.73 2.57 2.49-bisabolol 4.70 3.06 0.70Vetiverol 2.26 2.31 1.94-bisabolol 1.06 0.65 0.70Vetiselinol 5.60 2.16 2.74Khusimol 21.45 24.69 20.72-vetivone 4.30 4.27 3.77-vetivone 8.29 10.23 7.44Nootketone 4.13 2.45 2.37Am-Euras. J. Agric. & Environ. Sci., 10 (3): 458-463, 2011460(Fig .1)   (Fig. 2)     (Fig. 3)Fig. 1-3: Natural plant of V.zizanioides, Multiple shoots from auxilllary buds in MS medium and well establishedregenerated plants, long and short morphotypesDISCUSSION The  variations  generated  in  the  In vitroThe present study on V. zizanioides showedconsiderable variations under In vitro condition in theregenerated plants with respect to essential oil contentand composition. Long morphotypic plants expressedsuperior essential oil compared to the control plants aswell as short morphotypes studied, similar to the earlierstudies made by Sreenath, Jagadishchandra [20] andThierry et al. [21] in Cymbopogon sps.regenerated  plants  could  be due to the cultureconditions  and  culture  type which could alter thecontent and  composition  of  essential  oil  [22,  23].Related studies in plant cell cultures by Spreitzer et al.[24] and Yamamoto [25] also support that the In vitroconditions provided for the culture triggers theproduction of secondary metabolites there by favoringgreater essential oil production and accumulation indifferent morphotypes.Am-Euras. J. Agric. & Environ. Sci., 10 (3): 458-463, 2011461Graph 1: GC analysis of essential oil from natural plant of Graph 3: GC analysis of essential oil of in vitro derivedV. zizanioidesGraph 2: GC analysis of essential oil of in vitro derivedlong morphotype of V. zizanioidesshort morphotype of V. zizanioidesThe components - thujene, camphene, linalylacetate, bornyl acetate, geranyl acetate, - elemene,valencene, -eudesmol and - cardinal are goodtherapeutic agents, psychoactive in nature and are highlyanti-microbial and anti-fungal, hence can be used fortreating many ailments [26].There is very less study on involvement of geneactivation in essential oil production, but thebioproductivity characteristics of essential oils aspresented by Encheva [27] and Theirry et al. [21] signifiesthat the manifestation of phenotypical characters isdetermined by genotypic expressions. Hence, it is veryessential to study the In vitro derived genotypic variantsfor their varied secondary metabolite production.CONCLUSIONAs the vetiver oil is very complex and cannot beproduced artificially, the present study opens up newvistas to take up a systematic approach for selecting,screening of In vitro derived variants of V. zizanioideswith varied secondary compounds for their utilization inaromatic and pharmaceutical industries.Am-Euras. J. Agric. & Environ. Sci., 10 (3): 458-463, 2011462REFERENCES 14. Lawless, J., 1995. The illustrated encyclopedia of1. Ryman, D., 1992. Aromatherapy, the encyclopedia ofplants and oils and how they help you. Judy Piatkus.London.2. Benencia, F. and M.C. Courreges, 2000. In vitro andIn vivo antiviral activity of eugenol on humanherpesvirus. Phytoterapy Res., 14: 495-500.3. Carnesecchi, S.,  K.  Langley, F. Exinger, Gosse andF. Raul, 2002. 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Comparitive Study on Essential Oil in Natural and 

In vitro Regenerated Plants of Vetiveria zizanioides (Linn.) Nash

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Comparitive Study on Essential Oil in Natural and In vitro Regenerated Plants of Vetiveria zizanioides (Linn.) Nash

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