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

Abstract

Thymus moroderi Pau ex Martínez (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.

Effect of Macronutrients, Cytokinins and Auxins, on <i>in Vitro</i> Organogenesis of <i>Thymus vulgaris</i> L.

“…Moreover, the difficulty of establishing in vitro culture from acclimatized plants did not show up only in the high levels of contamination obtained, but also in ensuring a high rate of survival uncontaminated plantlets. This remark was also reported in the case of other Thymus species, namely Thymus moroderi [28] with a survival rate not exceeding 28.6%, Thymus bleicherianus with 36% [30], Thymus caespititius with 8% [33] and Thymus longicaulis with 29.4% [17], as well as other Lamiaceae such as Lavandula viridis [64], Salvia pratensis and S. nemorosa [65] with about 30%. Actually, explants from field grown plants contain many contaminants that affect the survival and growth of vitroplants, bacte-ria in most cases.…”

Effect of cytokinins on in vitro multiplication, volatiles composition and rosmarinic acid content of Thymus leucotrichus Hal. shoots

“…The efficiency of MS was expected based on earlier experiment using tissue cultures of many Thymus species (Furmanowa and Olszowska 1992). Since then many researchers have studied MS basal medium (Ozudogru et al 2011;Coelho et al 2012;Bakhtiar et al 2014;Marco-Medina and Casas 2015).…”

Clonal micropropagation of Thymus vulgaris L.

“…According to the literature sources, some thyme species at the second stage of micropropagation developed well on a hormone-free culture medium [3]. However, addition of growth regulators to the culture medium is required for most species.…”

“…There are no clear and effective methods of clonal micropropagation for most Thyme species. During micropropagation of thyme, researchers often faced such problems as a high frequency of explant contamination, explant darkening due to a high concentration of phenolic compounds inhibiting their development in vitro [3][4], low survival rate or shoot vitrification [5][6], low multiplication index. Only a few scientific works were devoted to studying the features of the morphogenetic potential of T. vulgaris in vitro [7].…”