In vitro propagation, genetic and phytochemical assessment of Thymus persicus — a medicinally important source of pentacyclic triterpenoids

Abstract

Thymus persicus (Ronniger ex Rech. f.) Jalas is a valuable and endangered natural source of antitumor pentacyclic triterpenoids, i.e., betulinic acid, oleanolic acid and ursolic acid, which grows in northwest Iran. As the plant has a low propagation rate in nature, a suitable method for in vitro-propagation is needed. With the aim of identifying a suitable system for regenerating T. persicus via direct organogenesis, Murashige & Skoog (MS) medium supplemented with different plant growth regulators (PGRs) was tested. In vitro-grown shoot tips were exposed to the cytokinins 6-benzylaminopurine (BAP), kinetin (KN), and thidiazuron (TDZ), alone or in combination with the auxins 1-naphthalene-acetic acid (NAA), 2,4-dichlorophenoxyacetic (2,4-D), indole-3-butyric acid (IBA) and indole-3-acetic acid (IAA). The highest shoot formation (7.1 ± 0.9) was obtained with a medium fortified with 8.9 μM BAP plus 2.7 μM NAA. Regenerated shoots were easily rooted on the different tested media, with the most abundant (16.6 ± 1.4) and strongest roots obtained on half-strength MS medium containing 2.5 μM IBA. The rooted plantlets were successfully acclimatized (76.6%) in a greenhouse before transference to natural conditions. Homogeneity and phytochemical productivity of the in vitro regenerated plantlets were confirmed by random amplified polymorphic DNA (RAPD) profiles and high performance liquid chromatography (HPLC), respectively.

Chemical Compositions, Somatic Embryogenesis, and Somaclonal Variation in Cumin

“…It may be said that the variation is associated with the heterozygous nature of the cumin landrace and its pollination system. Some studies suggest somatic variations in plants regenerated from tissue culture and some others suggest genetic stability [65–67]. Bakhtiar et al [67], in their study on the genetic diversity of Thymus regenerated seedlings and seedlings from mother plant, found that RAPD marker suggests the presence of polymorphism bands among them.…”

“…They also stated that the optimized protocol for Thyme regeneration is a useful tool to protect germplasm and it does not seem that interferences have been made in the genetic uniformity of the regenerated plants [67]. Analysis of Arabidopsis regenerated using RAPD and RFLP markers indicated the lack of diversity in the DNA profile of regenerated plants [68].…”

“…It is also well known that increasing numbers of subcultures increases the likelihood of somaclonal variation [70]. Genetic uniformity of produced plants through tissue culture depends on several factors; the most important ones are the propagation method and genotype [51, 67]. As compared to classical methods of vegetative propagation, the regenerated plants from calluses, depending on the species, origin, and culture age, may show varying amounts of structural or physiological disorders.…”

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

“…Similarly, the addition of BAP at concentrations higher than 2.2 μM resulted in a decrease in the number of shoots [47]. As well, Bakhtiar et al [29] reported that Kin and TDZ are less effective for the micropropagation of Thymus persicus (Ronniger ex Rech.f.) Jalas.…”

“…Thus, a better multiplication of Thymus caespititius Brot. was obtained by combining 1 mg/l BAP with 0.25 mg/l IBA [33], as well as 8.9 μM BAP with 2.7 μM NAA for Thymus persicus [29], as for Sáez et al [43] these cultures developed morphological characteristics of hyperhydricity, with translucency, very short shoots, rigid and abnormal leaf appearance [34]. Also, Fraternale et al [41] reported a maximum biomass increase of Thymus mastichina plantlets, estimated through their fresh and dry weight, on an MS medium supplemented with 0.44 µM BAP, alone or in combination with 10 or 20 μg/l of triacontanol.…”