In vitro multiplication and essential oil composition of Thymus moroderi Pau ex Martinez, an endemic Spanish plant

Abstract

Thymus moroderi Pau ex Martinez (Lamiaceae) is a species endemic to South-eastern Spain valuable for its use in traditional medicine and in the production of a liqueur called cantueso. We describe a rapid and reliable in vitro propagation protocol for this species, starting from wild adult vegetative explants. Sterilization and establishment of in vitro culture were shown to be extremely difficult because of the high presence of contamination and the inability of non-contaminated explants to restore growth. In just two cases we were able to obtain non-contaminated explants with ability to grow: combining a fungicide dip with the presence of kanamycin in culture medium, and when mother plants were maintained for a week in a growth chamber with occasional fungicide spray. Once established in vitro, explants showed rapid and vigorous growth with spontaneous rooting. The presence of cytokinin alone or in combination with auxin gave worse results than untreated controls, thus revealing Thymus moroderi as a cytokinin-sensitive species. Better multiplication rates were however obtained when double phase system was included in the protocol. Fully acclimatized micropropagated plants were phenotypically indistinguishable from their wild relatives. Essential oils of micropropagated and wild plants were analyzed revealing a slightly different overall terpene profile. Micropropagated plants were abundant in monoterpene and sesquiterpene hydrocarbons but less in oxygenated sesquiterpenes with respect to wild plants. Major compounds were in both cases 1,8-cineole and camphor, although at different relative abundances. Despite these dissimilarities micropropagated plants produced a clearly recognizable and characteristic thyme odor.