Approaches in Thymus plant micropropagation.
Medicinal and aromatic plants are important sources for plant secondary metabolites. The genetic manipulation of plants associated with in vitro plant regeneration systems facilitates efforts to engineer secondary product metabolic pathways. The fungal infections have been increasing in recent years due to several factors, namely, the increased incidence of high-risk patients, particularly immunocompromised hosts. Aromatic plants have been empirically used as antimicrobial agents, but the mechanisms of action are still unknown. Thyme has a great interest due to the possibility of its use in different applications, in medicine, in the cosmetic industry, or as food additives. Several studies have shown that thyme oils possess antimicrobial activity. Increasingly, plant breeding has taken advantage of molecular biology developments in order to genotype the species of interest to accelerate their selection. These approaches consist in choosing desired genotypes based on molecular markers or the knowledge of the genes involved in a particular trait. The in vitro culture techniques can be used to multiply plants selected after molecular and antifungal studies. The course of the investigation and the current state in relation to micropropagation, molecular studies, and antifungal action of the Thymus genus plants will be presented.
The genus Thymus L. belongs to the Lamiaceae family and consists of over 400 species of herbaceous annuals and perennial plants that are extensively used, for medicinal and non-medicinal purposes. These plants are widely distributed throughout the Old World [1, 2] and have been used for many centuries in traditional medicine due to their antiseptic, carminative, antiviral, and antioxidant properties . Thymus species are also interesting as a source of pentacyclic triterpenoids with several properties, as anti-inflammatory, hepatoprotective, antimicrobial, anti-HIV-1 activity, antiulcer, gastroprotective, hypoglycemic, antihyperlipidemic activity, and specific cytotoxicity against a variety of tumor cell lines [4–6]. By all this it becomes increasingly important to know the bioactive components of the Thymus.
Furthermore, interests focusing mainly on few selected chemotypes for the cosmetic and food industries, among others, lead to the loss of other species in nature, such as Thymus cariensis Hub.-Mor. & Jalas, Thymus cilicicus Boiss. & Balansa, Thymus sipyleus Boiss., Thymus pulvinatus Čelak., and Thymus cherlerioides Vis. . These species should be preserved to make available the access to a wide range of genetic diversity. On the other hand, as the plant has a low propagation rate in nature, a suitable method to obtain a high number of plants is needed .
2. Thymus main active compounds
Among the Lamiaceae family, the genus Thymus is one of the most studied genera, due to the use of these plants as a remedy in folk medicine and as a condiment, mainly in the Mediterranean zone . High bioactivity (e.g., antioxidant, anti-inflammatory, antiproliferative, and antimicrobial effects) is linked to the considerable content in phytochemicals of different Thymus species. Volatile compounds are extracted in essential oils (EOs), while nonvolatile compounds are found in alcoholic and aqueous extracts obtained by maceration, decoction, or infusion .
Noticeable interest was given to the study of EO in different Thymus species. High intraspecific chemical polymorphism was reported, especially in Thymus vulgaris L., which was shown to have a polymorphic variation in monoterpene production [10, 11]. Phenolic monoterpenes, such as carvacrol and thymol (Figure 1, compounds 1 and 2), seem to explain the important biological activity of the majority of Thymus spp. essential oils characterized by phenolic chemotypes. However, the presence of other terpenes in the EO results in the enhancement or reduction of EO bioactivity due to synergistic or antagonistic effects . In the Lamiaceae family, EOs are produced in the glandular trichomes and stored in their cavities . Several enzymes are involved in the biosynthetic pathways of terpenes, such as the terpene synthases enzyme class and the cytochrome P450s that are involved in the core terpene molecule functionalization .
Carvacrol and thymol have p-cymene as common precursor (Figure 1, compound 5). Carvacrol is frequent in some Lamiaceae aromatic plant genera like Thymus, Origanum, and Satureja , while thymol is mainly frequent in Thymus plants. Phenolic chemotypes are characteristic of T. vulgaris , Thymus hyemalis Lange , Thymus capitatus L. , Thymus zygis L. , and some populations of Thymus pulegioides L. . Thymol’s antibacterial effect is enhanced by the presence of carvacrol in the essential oil . Both thymol and carvacrol have important antifungal effect; they act by causing damage in the cell membrane and interacting with ergosterol [18, 20–22]. This mechanism was also proved with other monoterpenes, namely, linalool (Figure 1, compound 3), which acts also by interfering with biofilm formation and stability .
Linalool and geraniol (Figure 1, compounds 3 and 4, respectively) are monoterpene alcohols, which characterize several chemotypes of Thymus species such as Thymus pulegioides, T. x. citriodorus (Pers.) Schreb., Thymus algeriensis Boiss. & Reut., and some populations of T. vulgaris [11, 19, 24, 25]. Eucalyptol, or 1,8-cineole (Figure 1, compounds 6), a monoterpene cyclic ether monoterpene that is mainly present in Eucalyptus genus, is the major component of Thymus mastichina (L.) L. EO  and was also reported in populations of T. algeriensis, Thymus hirtus Raf., and Thymus glabrescens [27, 28].
Although more interest has been given to study thyme essential oils, nonvolatile extracts contain highly active secondary metabolites which are mainly phenolic compounds. Aromatic amino acids L-phenylalanine and L-tyrosine, produced by the shikimic acid pathway, are the precursors of the biosynthesis of polyphenols, namely, phenolic acids and flavonoids [29, 30]. Rosmarinic acid and luteolin (Figure 1, compounds 7 and 8, respectively) are the main frequent phenolic compounds found in thyme plants and which are related to their extracts’ biological activity. Rosmarinic acid, caffeic acid esterified with 3,4-dihydroxyphenyllactic acid, is the most abundant phenolic acid in several Thymus species: T. vulgaris, T. pulegioides, T. zygis, T. mastichina, T. capitatus, Thymus longicaulis C. Presl, and T. x. citriodorus [9, 31–36].
From the class of flavones, luteolin and apigenin (Figure 1, compounds 8 and 11, respectively) are the most important in Thymus. Luteolin is frequently present also in fruits and other Lamiaceae genera like Mentha. Its glycosylated derivatives such as luteolin-O-glucoside (Figure 1, compound 9) are also frequent in thyme species. Apigenin and its derivatives (Figure 1) are reported in some species such as T. x. citriodorus, T. vulgaris, and Thymus herba-barona Loisel. . Eriodictyol and naringenin (Figure 1, compounds 15 and 18) are flavanones which were described as well as their glucoside and glucuronide derivatives (Figure 1, compounds 16 and 19 and 17, respectively) in some Thymus species such as T. x. citriodorus and T. vulgaris .
3. Micropropagation of Thymus
Advances in biotechnological approaches provide a set of techniques that contribute to solving problems of extinction or genetic erosion in particular of plants. Alternatives for fast multiplication, like “in vitro micropropagation” that enables propagation of plants under controlled environmental conditions, can help in multiplying selected plants after molecular and antifungal studies or subjected to excessive demand by the people.
Furthermore, it was also possible to develop techniques that allow the maintenance of germplasm for a long time, like “cryopreservation” that make available long-term storage of Thymus germplasm at ultra-low temperatures .
Optimizations for micropropagation process involve the use of different growth regulators like cytokinins, auxins, or gibberellic acid for the induction of multiple shoots. The first works in Thymus micropropagation are from Furmanowa and Olszowska dated from 1980  to 1992 . Since that date, a lot of works for the micropropagation of different species of Thymus (Table 1), as well as the cryopreservation of the same plants, were developed (Figure 2).
|Species||Achievements||Growth regulators or others||References|
|Thymus mastichina||Propagation of thyme from mature field-grown plants||Nodal segments—MS + 0.1 mg L−1 BAP|
Roots—MS + 1 mg L−1 NAA
|Thymus vulgaris and Thymus longicaulis||In vitro propagation protocol||Shoots, semisolid MS + 1 mg L−1 KN and 0.3 mg L−1 GA3; roots, MS + 0.05 mg L−1 2,4-D|||
|Thymus lotocephalus||Propagation protocol usingseeds as explants||MS|
BAP induce high % of hyperhidric shots
|Thymus caespititius||Shoots with high proliferation capacity||MS + 0.4 mg L−1 BA + 0.1 mg L−1 IBA|||
|Thymus persicus||System for regeneration via direct organogenesis||Shoot, 8.9 μM BAP + 2.7 μM NAA; roots, 1/2 MS + 2.5 μM IBA|||
|Thymus hyemalis||Regeneration of plants through somatic embryogenesis||MS + 4.44 μM BAP, 0.54 μM NAA, and 4.65 μM KIN|||
|Thymus daenensis||Colloidal silver nanoparticles reduce hyperhydricity||2.5 mg L−1AgNPs (silver nanoparticles)|||
|Thymus moroderi||Propagation disinfection process|
Double phase culture system
|growth regulators did not improve the morphogenic response|||
|Thymus persicus||Callus induction and micropropagation||Callus, MS + 2.0 mg L−1 NAA and 0.5 mg L−1 KN|
Shoot, MS + 2.0 mg L−1 BAP + 1.0 mg L−1 NAA
Cryopreservation procedures, PVS2 vitrification, encapsulation—vitrification and droplet—and freezing methods showed to be effective to induce cryotolerance and long-term conservation of thyme shoot tips obtained from in vitro propagated plantlets : Different Thymus species have already been studied, Thymus moroderi , T. vulgaris and T. longicaulis , T. cariensis and T. vulgaris , and Thymus lotocephalus .
4. Genetic analysis
Researchers, from different areas, use the genetic analysis of plants as the basis of their work in order to identify and to characterize plant materials in nature, to detect genetic diversity or the genetic homogeneity, and to select plants with desired compounds.
The pool of genetic variation in plants, namely, the medicinal and the aromatic ones, serves as the base for plant breeding as well as for selection. Molecular markers are very useful in breeding program allowing germplasm screening independent to the developmental stage of the plants and/or environmental factors .
Applications of DNA methods, with different purposes, in Thymus genus are few when compared with other economically important plant species. A few reports are available about genetic characterization of species of Thymus genus (Table 2), using different molecular markers, being the techniques of random amplified polymorphic DNA markers (RAPD) and inter-simple sequence repeat (ISSR) the most commonly used. Amplified fragment length polymorphisms (AFLPs) and microsatellites are also utilized for genetic analysis and genetic relationships in Thymus species [56, 63].
|Thymus caespititius||Identification of terpene synthasegenes in Lamiaceae||TPS gene|||
|Assessment of genetic diversity and relationships||ISSRs|||
|Thymus daenensis subsp. daenensis||Assessment of genetic diversity andgeographic differentiation||ISSRs|||
|Development of 23 microsatellite primerpairs for Thymus genus and assessment of genetic diversity and relationships of 48 samples representing nine species and subspecies of the genus Thymus||Microsatellites|||
|Thymus persicus||Confirmation of genetic homogeneity in invitro regenerated plants||RAPDs|||
|Thymus kotschyanus||Assessment of genetic diversity of wild populations||RAPDs|||
|Assessment of genetic diversity andchemical polymorphism of Thymusspecies||RAPDs|||
|Assessment of thyme genetic diversity in Palestine||RAPDs|||
|Thymus vulgaris||Correlation between the chemical andgenetic relationships among commercial Thymus cultivars||RAPDs|||
|Essential oil composition and molecular analysis||RAPDs|||
|Thymus caramanicus||Assessment of genetic and chemical variability||ISRRs|||
|Assessment of genetic diversity and relationships among species of thegenus Thymus L. (section Serpyllum)||AFLPs|||
Beyond the genetic characterization of different species, the knowledge of genetic diversity within species is necessary for any improvement of cultivars and biodiversity maintenance and restoration . Yousefi et al.  studied ecotypes grown in different parts of Iran using ISSRs and verified that the accessions were relatively grouped according to their location and conclude that ISSRs provided a powerful and reliable molecular tool for detecting genetic variation and relationships. A similar study was done by Rahimmalek et al.  with the purpose to assess the genetic diversity of Thymus daenensis Celak accessions toward the conservation of the endangered aromatic species. Solyman and Alkowni  studied genetic diversities of five Palestinian Thymus species using RAPD markers and concluded that it could be useful tools for identifying Thymus species in any putative breeding programs that will be carried in the country. A study, with AFLPs, in Thymus, section Serpyllum , demonstrated that this markers could be suitable for complex genetic relationships analysis, including frequent interspecies hybridization events. Recently Karaca et al.  present the first report of genomic microsatellite markers for the genus Thymus, used in 48 individuals representing 9 species and subspecies of Thymus.
The overexploitation of wild plants for commercial purposes (and consequent decreased of populations) associated to the increasing demand for secondary metabolites has intensified the application of biotechnological methods to propagate and reproduce high-yielding plants under controlled growing conditions and/or to obtain homogenous and stable genotypes. Other application of molecular markers in this genus is the analysis of the reliability of the in vitro propagation regarding the genetic homogeneity, most of times associated to the phytochemical productivity of the produced plantlets, as the experiment reported by Bakhtiar et al.  using RAPDs. In this work RAPD profiles confirmed the homogeneity and high-performance liquid chromatography (HPLC) confirmed the phytochemical productivity of the in vitro regenerated plants.
Mendes et al. , using in vitro genotypes, characterized Thymus caespititius terpene synthase 2 (Tctps2) gene and identified other terpene synthase genes responsible for the chemical polymorphism observed in T. caespititius essential oils.
Another genetic approach is the analysis of the chemical and the genetic relationships among species as the study described by  that also determinate the correlation between these two sets of data, the essential-oil composition and genetic variability of six populations of Thymus. RAPD markers allowed a perfect distinction between the six species, based on their distinctive genetic background: however, they did not show identical clustering with the volatile-oil profiles . Contrary Echeverrigaray et al. , also based in RAPD profiles, observed that the cultivars (T. vulgaris L.) could be divided into two clusters, which coincided with results obtained by oil GS-MS analysis. Chemical and genetic differences of four Thymus species were studied by Pluhár et al.  in order to determine whether molecular characters (RAPDs) and essential oil components could be used as taxonomic markers and obtained a partial correlation between molecular and chemical assessments. In Thymus caramanicus Jalas, Hadian et al.  assessed the genetic (using ISSRs) and chemical variability and observed a relationship between genetic and chemical variability and geographic distribution.
5. Fungal infections
Fungal infections are a serious problem of public health concern and have been increasing in recent years due to several factors given increased international travel, immigration, changing climate conditions, and the increased incidence of high-risk patients . Invasive mycoses are especially problematic for immunocompromised individuals and patients in intensive care units, and some conditions can predispose as organ transplantation, the use of drugs in treatments as corticosteroids and antineoplastics, and complex surgical procedure acts [66, 67]. Other cases may be found in patients suffering from diabetes mellitus, patients with human immunodeficiency virus infection, patients with neoplasias after receiving chemotherapy, patients with transplantation surgeries, or those with prolonged antibiotherapy .
Oral and vulvovaginal candidiases caused by Candida albicans are the most common fungal diseases . Invasive fungal diseases can be less frequent but much more severe. Other mycotic diseases or complications associated included asthma, bronchopulmonary and invasive aspergillosis, pneumocystosis, meningeal cryptococcosis, mucormycoses, or invasive candidiasis . Deaths related with fungal infections are mostly associated with Cryptococcus, Candida, Aspergillus, and Pneumocystis .
Aromatic plants have been empirically used as antimicrobial compounds, but the mechanisms of action are still under study . Inhibitory action of aromatic plants possibly includes cytoplasm granulation, cytoplasmic membrane lesion, and inhibition and/or inactivation of extracellular and intercellular enzymes [72, 73] and might be due to different compounds, including phenolics, terpenoids, and alkaloids. These compounds together or independently use different levels of antifungal effect ending with mycelium germination inhibition . Also, it is described that plant lytic enzymes act in the fungal cell wall causing breakage of β-1,6 glycan, β-1,3 glycan, and chitin polymers . The antimicrobial activity of the aqueous extracts could be due to the anionic components such as chlorides, thiocyanate, nitrate, and sulfates besides other water-soluble constituents which are naturally occurring in the plant material .
6. Antifungal activity
Only limited numbers of new antifungal drugs were developed in recent years, and there are only small numbers of drugs available for their treatment . Toxicity and drug resistance have become an increasing problem. The resistance to antifungal drugs, the high costs associated with treatment, and the fungistatic activity of most of the antifungal drugs are problems making their treatment difficult and expensive . So, alternatives for treating invasive fungal infections are necessary .
The spread of multidrug-resistant strains of fungi is a medical problem worldwide, and the reduced number of drugs available led to a search for therapeutic substitutes, namely, among aromatic and medicinal plants and compounds isolated from them used for their antifungal [76, 77].
Numerous molecules obtained from the natural environment are investigated and described in bibliography with antimycotic activity. Several extracts are investigated for antifungal activities like crude extracts or isolated constituents as essential oils, saponins, terpenoids, alkaloids, phenolic compounds, peptides, and proteins [78, 79].
The in vitro evaluation methods of antifungal activity can be divided into diffusion methods and dilution methods. The diffusion methods yielding to inhibition diameters are used mostly for the qualitative screening. Examples are the agar overlay technique . Dilution methods offer more quantitative results regarding the action of essential oils. Serial dilutions are used for detecting the minimal inhibitory concentration (MIC in mg ml−1) of an essential oil in a liquid medium its antimicrobial properties and rank it among the most potent essential oils in this respect .
A wide range of aromatic and medicinal plants with therapeutic properties have been explored and used for the extraction of essential oils all over the world due to their antimicrobial capacity against fungal pathogens . Several studies have shown that thyme oils possess antimicrobial activity [82–84].
Thymus spp., most of them possessing a large quantity of phenolic monoterpenes, revealed activity against fungi .
Medicinal plants have the capacity to inhibit the growth of a wide range of opportunistic or pathogenic microorganisms due to the presence of essential oils . Essential oils are natural, volatile liquid, complex compounds characterized by rarely colored and a strong odor, soluble in lipid and organic solvents . About 60% of essential oils show antifungal activity .
The essential oils and their components have been used broadly against molds . The essential oil extracts from many plants have shown their considerable antifungal activity against the wide range of fungal pathogens .
Thyme essential oils are apparently among the greatest inhibitors of fungal microorganisms because of the presence of the phenolic compounds such as thymol as main components which might disrupt the fungal cell membrane . Another component that appears to show antimicrobial activity is terpene hydrocarbons (γ-terpinene) . p-Cymene is a compound of essential oil that does not show antibacterial efficacy when used alone which suggest a synergistic effect of the compounds [86, 87].
Thyme essential oils may in the future represent a new source of natural antiseptics with applications in industry of pharmaceutics and food . The essential oils have the ability to penetrate and disrupt the fungal cell wall and cytoplasmic membranes, permeabilizing them and finally causing damage to mitochondrial membranes .
Variability in essential oil compounds might be linked to differences in concentration and amount recovered based on several factors, including species of plant used, method of extraction, solvents, and extraction time, which in turn may differ in their antifungal potency [68, 89]. Differences can also be attributed to raw materials used (dried or fresh), types of soils used for cultivation, the harvesting time in the year, or differences in oil extraction techniques .
These oils have been used in folk medicine in different communities for patients suffering from mycotic infections .
Differences in essential oil compounds might be related to variability in the dried or fresh materials used, to the harvesting time in the year, to types of soils used for cultivation, or to differences in oil extraction techniques. T. vulgaris has medicinal properties and is widely used in traditional medicine for its expectorant, antispasmodic, antibronchiolitic, antitussive, anthelmintic, carminative, and diuretic properties .
Many studies have shown that thyme (T. vulgaris) has antifungal activities and have suggested their integration into pharmaceutical preparations in the treatment of candidiasis [72, 86, 93]. Previous studies showed fungistatic activity of the essential oils of T. vulgaris as carvacrol, p-cymene, and thymol . The antifungal mechanism of action by which thymol or carvacrol acts is actually not well understood, although the antifungal activity is probably because thymol is lipophilic and together with carvacrol can act in the fatty acyl chains of membrane lipid bilayers, and alters the fluidity and permeability of cell membranes . Other mechanisms have been theorized as damaged of membrane and cell wall with disruption associated with morphological deformation, deterioration, collapse, and of the conidia and/or hyphae . Thymol and carvacrol have a stronger antifungal capacity, indicating more susceptibility of Aspergillus spp. than that of Penicillium spp. . Thyme essential oil showed its capabilities of inhibiting fungal development causing leakage of the cytoplasm of Aspergillus flavus and was responsible for degenerative alterations in hyphae alterations, which appeared degraded or with the complete absence of conidia . A previous report showed capabilities of inhibiting aflatoxin production .
Thymus essential oil causes irreversible damage to cell wall, cell membrane, and cellular organelles which affects Aspergillus niger growth and morphology .
The fungal activity of Thymus plants has been summarized in Table 3.
|Thymus plant||Inhibited microorganisms||References|
|Thymus vulgaris||Candida albicans||[72, 86, 94, 99, 100]|
|Thymus vulgaris||Aspergillus flavus|||
|Thymus vulgaris||Aspergillus ochraceus|||
|Thymus vulgaris||Penicillium chrysogenum|||
|Thymus schimperi||Penicillium chrysogenum|||
|Thymus schimperi||Verticillium sp.|||
|Thymus schimperi||Aspergillus tubingensis|||
|Thymus schimperi||Aspergillus minutus|||
|Thymus schimperi||Beauveria bassiana|||
|Thymus schimperi||Microsporum gypseum|||
Thymus plants have been playing, in recent years, an increasingly important role in the intense study being targeted to run through the different areas of biotechnology. So here we intend to present the latest developments involving this plant in such diverse areas as active compound analysis and their effect on antifungal activity, genetic analysis, micropropagation, and finally cryopreservation techniques, one of the most recent methodologies to plant preservation.
The utilization of new plant breeding technologies will be the future in aromatic and medicinal plant manipulation and production, in order to obtain a multitude of valuable characteristics like increased nutrient and metabolite production and resistance to different stresses. Next-generation sequencing (NGS) technology and the associated bioinformatics tools will allow general profiling of RNA expression in plant species with limited molecular genetics studies as the majority of aromatic and medicinal plants.