Kinetin Enhanced Linalool Production by in vitro Plantlets of Lippia alba

Kinetin Enhanced Linalool Production by in vitro Plantlets of Lippia alba

Tavares, Eliana S


Lippia alba Mill. N. E. Br (Verbenaceae) is an aromatic shrub widely distributed in Brazil where the infusion of its leaves is used in folk medicine due to sedative and antispasmodic properties. This study presents data about quantitative variation of the major volatile constituents of the linalool producing L. alba chemotype. The volatiles were extracted by solid phase microextraction (SPME) from plants cultivated in soil (mother plants), from in vitro plantlets grown in Murashige & Skoog (MS) medium and from in vitro plantlets grown in MS medium with growth regulators. The content of [alpha]-pinene, (Z)-3-hexenyl acetate and [alpha]-gurjunene was greater in the mother plants than in plantlets, whereas the content of sabinene, myrcene, 1,8-cineole and p-mentha-l,5,8-triene was lower. The addition of 0.2.3 µM of IAA to the medium significantly enhanced sabinene and myrcene contents. The addition of 0.92 µM of kinetin significantly increased the 3(S)-( + )-linalool level.

Key Word Index

Lippia alba, Verbenaceae, essential oil composition, 3(S)-( + )-linalool, 1,8-cineole, SPME headspace volatiles, tissue culture.


Lippia alba Mill. N. E. Br. (Verbenaceae) is an aromatic shrub, widely distributed in Brazil where it’s commonly known as ‘erva-cidreira.’ An infusion of its leaves is used in folk medicine due to its sedative and antispasmodic properties. This species shows some diversity in the main constituent of its essential oil. Recently, a chemotype originating from Valinhos, State of São Paulo, Brazil, yielded a high content of linalool in the oil (1). Linalool is one of the most useful monoterpene alcohols for the perfumery industry, as well as in the large scale synthesis route to vitamin E (1). It has sedative dose dependent effect on CNS (central nervous system), including hypnotic, hypothermie and anticonvulsant properties (2-4). In order to identify possible factors affecting the oil production and its quality, this species was planted in the soil (in the garden of the Biological Institute, Federal University of Rio de Janeiro) and further cultivated in vitro and the effects of growth regulators on the oil yield and composition were evaluated.


Plant material: Plants of linalool-rich L. alba chemotype (RFAno. 29423) originally collected from the city of Valinhos, São Paulo State (Brazil), were cultivated in soil in the campus of the Biology Institute, Federal University of Rio de Janeiro, RJ, under daily irrigation. These plants (mother plants) were used as source of nodal segment for in vitro introduction.

In vitro introduction: Shoots of L. alba were sterilized by immersion in commercial detergent solution (30 min), 70% (v/ v^sup -1^) ethanol (5 min), 50% (v/v^sup -1^) commercial bleach solution (8 min), always alternated with three immersions in sterile distilled water (1 min). Nodal segments, with two axillary buds without leaves or petioles were cultured on Murashige & Skoog medium (MS) (5), supplemented with 30g/L sucrose, 1.4µM thiamine-HCl, 2.43 µM pyridoxine-HCl, 4.10 µM nicotinic acid and 0.60 µM myo-uiositol. pH was adjusted to 5.8 ±0.1 before addition of 8g/L agar, and media were sterilized by autoclaving(120°Candl.l^sup -2^ for 15min). Cultures we re grown in glass tubes (150 x 25 mm) containing 15 mL of medium. The in vitro cultures were kept under white light illumination (Sylvania fluorescent tubs), at an intensity of 1.6 Wm^sup -2^, 23 µMm^sup -2^/5 daily photoperiod of 16 h at 25 ± I°C. For shoot proliferation the plantlets were subcultured every 40 days. Plantlets at fourth subcultivation were used as expiants donors for tests with growth regulators.

Treatment with growth regulators: Nodal segments of in vitro plantlets were cultured in four different media: a- MS; b- MS + 0.23 µM Indolacetic acid (IAA); c- MS + 0.92 µM of kinetin; d- MS + 0.23 µM of IAA + 0.92 µM of kinetin. The experimental design was fully randomized with five replicates per treatment and six expiants per replicate. The cultures were grown in 500 mL glass flasks containing 50 mL of medium and incubated in a growth room (photoperiod and temperature as previously described) for 40 days.

Simultaneous distillation and extraction (SDE): Three g of comminuted fresh leaves of the mother plants and of the in vitro plantlets were extracted by SDE (6) for 1 h. The solvent utilized was dicloromethane (1 mL), containing 0.24 µg of tridecane as internal standard for GC quantification. The oil yield was expressed on a wet weight basis.

Solid phase microextraction (SPME): The leaf volatiles of the mother plants and the in vitro plantlets were extracted using a manual SPME syringe equipped with a 100 µm polydimethylsiloxane (PDMS) coated fibre (Supelco-USA) (7). Comminuted fresh leaves (50 mg) were sealed in a 3 mL headspace vial with phenolic cap and PTFE/silicone septum and allowed to equilibrate for l h at room temperature before analysis. The SPME fibre was exposed to each sample for 3 min. Extractions were made in triplicate for each treatment.

GC and GC/MS analysis: The SPME fibre was inserted into the inlets of the GC and GC/MS and desorbed for 2 mm under splitless injection condition. The GC analysis was performed using an HP 5890 Series II gas Chromatograph fitted with a PE-5 (5% phenylmethyl silicone) fused capillary column (20 m x 0.18 mm, film thickness 0.4 µm) using hydrogen as the carrier gas (ImL/min). The injector temperature was 250°C and column oven program was 60°0 -240°C at 3°C/min. Detector (FID) was at 300°C.

The GC/MS system was an HP 5973 MSD coupled with a 6890 gas Chromatograph using helium as carrier gas and the same column and conditions as above. Transfer line was at 240°C, ion source was at 230°C, EIMS, 70 eV. Results were compared with the Wiley library of spectra.

Enantiomeric analysis of linalool: The enantiomeric analysis was carried out in a Hewlett-Packard 5890 series II gas Chromatograph fitted with a 50 m [chi] 0.25 mm OV-1701 capillary column coated with heptakis-(2,6-di-O-methyl-3-O-pentyl)-[beta]-cyclodextrin (2,6-Me-3-Pe-[beta]-CD) chiral stationary phase. The column temperature was programmed from 40°-200°C (0.7°C/min). Helium at a flow rate of 1 mL/min, split ratio 20:1, was used as the carrier gas. The injector temperature was 230°C and detector was at 250°C.

Co-injections with standards: Racemic and 3(R)-(-)-linalool samples commercially available from Aldrich were used as standards. Stock solutions of 50 µL/2.0 mL ethanol were prepared. A standard solution was made by taking 10 µL of stock solution diluted with 10 mL H2O mixed with 3 g NaCl. For headspace sampling 0.5 mL of a standard solution was placed in a 3 mL vial, where it equilibrated for l h with 50 mg of comminuted leaves, prior to 3 min of SPME headspace sampling under stirring. Fibre dessorption into the injector was performed for 2 min with the split vent closed.

Statiscs; Dataobtained were analyzedusing STATISTICA 6.0 software for Microsoft Windows. The Student T test was performed to determine the significance of differences between means obtained from the plantlets cultivated in MS medium without growth regulators (control) and different treatments.

Results and Discussion

The addition of 0.23 µM IAA to the MS medium resulted in a significant decrease in plant regeneration, a decrease in the number of shoots per expiant and number of nodes per plantlet, when comparing MS (control) with the growth regulators treatments (Table I). The oil yield, however, was not significantly affected by different media composition (Table I). The correlation between growth and oil formation has been found in Lippiajunelliana (8) but an attempt to establish any relationship between changes in Salvia officinalis oil yield and alteration in growth has failed (9).

The volatiles produced by the mother plants and by the in vitro control plantlets, extracted by SPME, contained 37% and 39% of linalool, respectively, showing no significant difference.

The enantiomeric analyses of linalool produced by the mother plants and the in vitro plantlets confirmed the enantiomeric excess of 3(S)-( + )-linalool in the samples (> 98%). These results suggest that environmental factors and the stage of development have minor influence in L. alba linalool enantiomeric distribution.

Constituents representing more than 1% ofL. alba volatiles extracted by SPME are shown in Table II. Quantitative variation was observed when comparing volatiles obtained from the mother plants with those obtained from the in vitro plantlets.

Plantlets cultivated in MS medium without growth regulators produced lower quantities of oc-pinene, (Z)-S-hexenyl acetate and ce-gurjunene, but produced more sabinene, myrcene, 1,8-cineole andp-mentha-l,5,8-triene than mother plants (Table II). Compositional variation of the oil related to leaf maturity has been reported for Mentha x piperlta (10), Micromeria fruticosa (11), and Melaleuca alternifolia (12). These results probably reflect biosynthetic pathway changes in leaf maturation that could be related to the ecological role played by the oil accumulation during different periods of growth.

The comparison between in vitro plantlets cultivated in MS medium without growth regulators (control) and in MS medium plus different concentrations of growth regulators shows that the presence of 0.23 µM IAA in the medium resulted in a significant increase in sabinene and myrcene levels and a decrease of linalool level (Table II). The quantitative variation caused by the addition of auxin can be explained by the effect of auxin on gene activity, especially in the epidermis (13). Auxins may affect the kind of proteins formed in a plant cell before or as soon as growth promotion starts, which could explain changes in the level of some substances through the modification of the cell enzymatic pattern.

Kinetin (0.92 µM) added to the MS medium increased in ca. 11% the linalool content in the headspace volatiles (Table II). The level of 1,8-cineole decreased. MS with 0.23 µM of IAA and 0.92 µM of kinetin increased the linalool level, when compared to MS added 0.23 µM of IAA alone (Table II).

The effect of cytokinins on the levels of secondary metabolites has been observed: cytokinins are able to stimulate the synthesis of betacyanins (14) and indolic alkaloids (15). The decrease of 1,8-cineole level was observed in the oil isolated from plantlets of Lavandula dent at a cultivated in Linsmaier & Skoog medium added of a cytokinin, although the authors used 6-benziladenine instead of kinetin (16).

The decrease of linalool level by addition of auxin suggests that cytokinin participates in the linalool production since it has been shown that auxin stimulates both oxidative breakdown of cytokinin andinactivation of cytokinins by glucosylation in some plant tissue (17).

The results obtained in the present study suggest that growth regulators can influence L. alba oil composition presumably by direct action on terpene metabolism independently of growth and development efficiency.

The in vitro enhancement of linalool with the addition of 0.92 µM of kinetin could be obtained in the field because the primary site of monoteqDene biosynthesis are the epidermal secretory trichomes, which are exposed structures more sensitive to the effects of foliar applied substances than other parts of the plant.

The enhancement in the linalool production in /_,. alha with the use of exogenous kinetin highlights this species as a potential source of 3S-(+)-linalool. Additionally, L. alha requires small areas for cultivation, grows easily and is a sustainable resource because its oil is obtained from the leaves rather than the wood.


We thank Benjamin Gilbert, from Farmanguinhos/FlOCRUZ for the donation of the plant material and Fernanda Reinert for English revision.


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Eliana S. Tavares*

Intituto de Biologin, UFRJ, CCS, Eloco A, Cidade Universitaria, llha do Fundatilde;o, 21952-590, Rio de Janeiro, Brazil

Daíse Lopes and Humberto R. Bizzo

EMBRAPA Agroindustria de Alimentas, Avenida dos Américas 29501 Rio de Janeiro RJ, Brazil

Celso L.S. Lage

Institute de Biofisica, UFRJ, CCS, Bloco G, llha do Fundão, 21952-590, Rio de Janeiro, Brazil

Suzana G. Leitao

Faculdade de Farmacia, UFRJ, CCS, Bloco A, llha do Fundão, 21952-590, Rio de Janeiro, Brazil

* Address for correspondence

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