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American-Eurasian J. Agric. & Environ. Sci., 10 (3): 458-463, 2011 ISSN 1818-6769 © IDOSI Publications, 2011

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

11K.J. Thara Saraswathi, N.R. Jayalakshmi, 12P. Vyshali and M.N. Shiva Kameshwari

1Department of Microbiology and Biotechnology, Bangalore University, Jnana Bharathi Campus, Bangalore-560056, 2Department of Botany, Bangalore University, Bangalore, India

Abstract: In vitro studies with explants of Vetiveria zizanioides obtained through organogenetic pathway was focused on the difference in 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.

Key words: Morphotypes Aroma therapy Chemotypic variation Gas chromatography

INTRODUCTION smell and very pleasant odor when diluted. It blends well with of sandal wood, rose, violet, jasmine, opapanax, The pharmacological properties of essential oils , oakmoss, lavender, clary sage, mimosa, cassia extracted from plants have received the great interest of and ylang ylang [14]. Vetiver oil is high-priced and academic institutes and pharmaceutical companies [1-4]. extensively used as a cosmetic product for pharmaceutical Many 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; over which 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 alcohol One such 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 major zizanioides (L.) Roberty, originated from constituents and their presence is often considered as India. Vetiveria was used in ancient times as fragrant fingerprint of the oil contributing significantly to its odour material 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 be which no synthetic substitute is available. It has been manufactured [15, 16], there is an interest and need for utilized as a raw material for products like , the industry in obtaining new variants with deodorants, 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 variation fixatives known [9, 10]. Vetiver oil is the basis of Indian under In vitro culture to further select the variants with and western (36%) perfumery [11-13], gives a powerful desirable qualities.

Corresponding Author: N.R. Jayalakshmi, Department of Microbiology and Biotechnology, Bangalore University, Jnana Bharathi Campus, Bangalore-560056, India, E-mailL [email protected]. 458 Am-Euras. J. Agric. & Environ. Sci., 10 (3): 458-463, 2011

MATERIALS AND METHODS Table 1: Percentage composition of essential oil in natural plant and in vitro generated morphotypes of V. zizanioides The In vitro generated plants of V. zizanioides In vitro generated morphotypes obtained from rhizome explants were maintained under ------uniform conditions in the field along with the control Compounds Natural plant Long Short plants. After one year, the were collected from -thujene 0.15 0.72 0.55 In vitro regenerated plants (long and short morphotypes) -pinene 0.18 0.10 0.23 and control plants. Camphene 0.68 1.09 2.95 The roots collected were thoroughly washed with Myrcene 0.10 - 0.05 running tap for 30 minutes, cut into small pieces - terpinene 0.06 - - and used for the studies. Approximately 500g of the roots P-cymene - - - were weighed and subjected to extraction of essential oil Limonene 0.64 0.34 0.89 using Clavenger’s apparatus. The essential oils were (Z)- - ocimene 0.08 - - collected in a separator by downward displacement of Linalool 0.47 - 0.20 water. (Z)- - terpineol 0.16 - 0.09 Gas chromatography of the essential oil was (E)- - terpineol 0.09 - - performed on a Perkin - Elmer Gas chromatography model 1.51 0.25 0.36 8500 using glass capillary column BA-1 (coated with di- Terpinen-4-ol 0.11 - 0.05 methyl silaxane) of the size 25m x 0.5mm. Nitrogen was - terpineol 0.36 - 0.05 used as a carrier gas at 10psi inlet pressure. Temperature Nerol 0.21 0.21 0.58 programming was performed from 60-220° at 5°C/ min. The Geraniol 0.15 - - samples were injected by splitting the split ratio 1:80. Linalyl acetate 0.08 0.65 0.59 The compounds of essential oil were identified by 0.18 0.92 1.66 relative retention time with standard reference compounds Neryl acetate 0.05 - 0.05 run under the same conditions by comparing the Kovat’s Geranyl acetate 0.10 0.21 - retention indices of the peaks and also by peak - copaene 0.15 0.10 0.18 enrichment on co- injection with authentic compounds -bourbonene 0.17 - 0.08 [17-19]. -caryophyllene 0.12 0.34 0.42 -elemene 0.25 0.30 0.17 RESULTS Valencene 0.73 1.40 1.29 Salina-4(14),17-diene 0.20 0.14 0.07 Multiple were obtained from the auxiliary Zizaene 1.24 0.70 0.68 buds of rhizome explants grown in MS medium and well Nootkatene 0.65 0.27 0.25 established regenerated plants were obtained of long and -bisabolene 1.19 0.97 1.38 short morphotypes (Fig. 1, 2, 3). A good yield of bright -cardienene 3.61 0.68 0.62 yellowish essential oil was obtained from the In vitro Elemol 1.60 0.36 0.46 regenerated long morphotype when compared to the short -vetivenene 2.99 2.46 2.82 morphotype with light yellow color, along with the 10-epi- -eudesmol 0.66 0.42 0.42 control plants. The long and short morphotypes yielded -eudesmol 0.12 0.30 0.27 2.1 and 1.9% of oil, respectively, whereas the control plant -cardinol 1.73 2.57 2.49 yielded 1.8%. The GC analysis results obtained are -bisabolol 4.70 3.06 0.70 tabulated in Table 1 and shown in graphs (1, 2, 3). The Vetiverol 2.26 2.31 1.94 long morphotype in addition to possessing an increase -bisabolol 1.06 0.65 0.70 in percentage yield of oil also showed enhanced Vetiselinol 5.60 2.16 2.74 composition of oil with respect to important components Khusimol 21.45 24.69 20.72 like - thujene, camphene, linalyl acetate, bornyl acetate, -vetivone 4.30 4.27 3.77 geranyl acetate, - elemene, valencene, -eudesmol and -vetivone 8.29 10.23 7.44 - cardinal in the long morphotype compared to control Nootketone 4.13 2.45 2.37 plants.

459 Am-Euras. J. Agric. & Environ. Sci., 10 (3): 458-463, 2011

(Fig .1) (Fig. 2)

(Fig. 3)

Fig. 1-3: Natural plant of V.zizanioides, Multiple shoots from auxilllary buds in MS medium and well established regenerated plants, long and short morphotypes

DISCUSSION The variations generated in the In vitro regenerated plants could be due to the culture The present study on V. zizanioides showed conditions and culture type which could alter the considerable variations under In vitro condition in the content and composition of essential oil [22, 23]. regenerated plants with respect to essential oil content Related studies in plant cell cultures by Spreitzer et al. and composition. Long morphotypic plants expressed [24] and Yamamoto [25] also support that the In vitro superior essential oil compared to the control plants as conditions provided for the culture triggers the well as short morphotypes studied, similar to the earlier production of secondary metabolites there by favoring studies made by Sreenath, Jagadishchandra [20] and greater essential oil production and accumulation in Thierry et al. [21] in sps. different morphotypes.

460 Am-Euras. J. Agric. & Environ. Sci., 10 (3): 458-463, 2011

Graph 1: GC analysis of essential oil from natural plant of Graph 3: GC analysis of essential oil of in vitro derived V. zizanioides short morphotype of V. zizanioides

The components - thujene, camphene, linalyl acetate, bornyl acetate, geranyl acetate, - elemene, valencene, -eudesmol and - cardinal are good therapeutic agents, psychoactive in nature and are highly anti-microbial and anti-fungal, hence can be used for treating many ailments [26]. There is very less study on involvement of gene activation in essential oil production, but the bioproductivity characteristics of essential oils as presented by Encheva [27] and Theirry et al. [21] signifies that the manifestation of phenotypical characters is determined by genotypic expressions. Hence, it is very essential to study the In vitro derived genotypic variants for their varied secondary metabolite production.

CONCLUSION

As the vetiver oil is very complex and cannot be produced artificially, the present study opens up new vistas to take up a systematic approach for selecting, screening of In vitro derived variants of V. zizanioides Graph 2: GC analysis of essential oil of in vitro derived with varied secondary compounds for their utilization in long morphotype of V. zizanioides aromatic and pharmaceutical industries.

461 Am-Euras. J. Agric. & Environ. Sci., 10 (3): 458-463, 2011

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