~ Pergamon PIh S0031-9422(97)00934-5 Phytochemistry, Vol. 48, No. 5. pp. 79%799, 1998 c 1998 Elsevier Science Ltd. All rights reserved Printed in Great Britain 0031 9422/98 $19.00+0.00 ESSENTIAL OILS AND HYDROCARBONS FROM LEAVES AND CALLI OF ORIGANUM VULGARE SSP. VIRENS ISABELM. S. ALVES-PEREIRA*and MANUEL FERNANDES-FERREIRAt * Dep. de Fitotecnia, Universidade de l~vora, Apartado 94, 7001 l~vora Codex, Portugal; 1 Dep. de Biologia, Universidade do Minho, Largo do Paso, 4709 Braga Codex, Portugal (Received 15 September 1997) Key Word Index--Oriyanum vulyare ssp. virens; Lamiaceae calli; essential oils; hydrocarbons; n-alkanes; monoterpenoids; sesquiterpenoids; naphthalene. Abstract--Green friable calli (G-calli) and dark abnormal root primordia containing calli (R-calli) of Origanum vulgate ssp. virens were induced from leaves and established in the presence of 0.25 mg and 1 mg 1-~ (2,4-D), respectively. Leaves, of the same type of those used in the calli induction, G-calli and R-calli were submitted to hydrodistillation and the respective hydrodistillates were analysed by GC and GC-mass spectrometry. The hydrodistillate from leaves consisted of ca 50% monoterpenoids (35.2 % oxygenated monoterpenes and 14.3 % monoterpene hydrocarbons) and ca 40% of sesquiterpenoids (2.5% oxygenated sesquiterpenes and 37.4% sesquiterpene hydrocarbons). Linalool (16.4%) and (E)-fl-ocimene (6.6%) were the major oxygenated mon- oterpene and monoterpene hydrocarbon, respectively. Globulol (0.94%) and 6-elemene (12.85%) were the major oxygenated sesquiterpene and sesquiterpene hydrocarbon, respectively, n-Alkanes, namely pentacosane (0.97%), heptacosane (0.9%) and nonacosane (1.0%), were also present. Hydrodistillates from G- and R-calli did not contain either mono- or sesquiterpenoids; n-alkanes were the main compounds found. The alkane concentration in R-calli was more than twice that of G-calli. Naphthalene (0.5/~gg-~ dry wt) and eicosane, (0.9 #g g-~ dry wt) produced by R-calli were absent in G-calli. Squalene concentration in G-calli was 6.7 fold greater than that found in R-calli. ~? 1998 Elsevier Science Ltd. All rights reserved. INTRODUCTION The composition of the essential oils of Origanum species have been studied by several authors [1-6]. Some have reported variations in yield and com- position according to genetic characteristics [7], phase of life-cycle [8, 9] and geographic origin [5, 6]. Studies on production of essential oils by in vitro cultures of Origanum species are scarce. Although in vitro cultures of O. vulgare shoots have been established [10, 11], reports on the production of essential oils by such systems are absent. However, according to Svoboda et al.[12], accumulation of small amounts of volatile oil, not associated to differentiation of compartments, occurs in O. vulyare calli. Origanum vulgare L. grows wild in the National Park of Peneda Germs in the north of Portugal and has been used for a long time by people of that region as an aromatic and medicinal plant. To our knowl- edge, determination of the composition of essential oils, as well as the establishment of in vitro cultures from O. v. ssp. virens plants of this ecotype has not yet been performed. In the present paper, we compare the composition of hydrodistillates from leaves of in vivo O. v. ssp. virens plants with those of calli induced from the same type of leaves. RESULTS AND DISCUSSION The essential oil isolated from leaves of O. v. ssp virens was a complex mixture with more than 70 com- ponents, 50 of which were identified, corresponding to 80% of the total oil. The identified components with their percentages and retention times on a DB-5 column are listed in Table 1. Most of the compounds were hydrocarbons (14.3% monoterpene hydro- carbons; 37.4% sesquiterpene hydrocarbons and 2.8% alkanes). The mass spectra of compounds 22 and 30 are both consistent with sesquiterpene hydro- carbons. (E)-fl-Ocymene (6.57%) was the major mon- oterpene hydrocarbon and 6-elemene (12.85%), tog- ether with fl-caryophyllene (11.07%), were the major sesquiterpene hydrocarbons. Similar total relative amounts of monoterpene hydrocarbons are reported for flowering parts of O. majorana [1] and O. onites [3]. Higher relative amounts of these type of com- pounds were reported, however, for O. vulgare ssp. 795