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.
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 main active compounds
Noticeable interest was given to the study of EO in different
Carvacrol and thymol have
Linalool and geraniol (Figure 1, compounds 3 and 4, respectively) are monoterpene alcohols, which characterize several chemotypes of
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
From the class of flavones, luteolin and apigenin (Figure 1, compounds 8 and 11, respectively) are the most important in
3. Micropropagation of
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
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
|Species||Achievements||Growth regulators or others||References|
|Propagation of thyme from mature field-grown plants||Nodal segments—MS + 0.1 mg L−1 BAP|
Roots—MS + 1 mg L−1 NAA
|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|||
|Propagation protocol usingseeds as explants||MS|
BAP induce high % of hyperhidric shots
|Shoots with high proliferation capacity||MS + 0.4 mg L−1 BA + 0.1 mg L−1 IBA|||
|System for regeneration via direct organogenesis||Shoot, 8.9 μM BAP + 2.7 μM NAA; roots, 1/2 MS + 2.5 μM IBA|||
|Regeneration of plants through somatic embryogenesis||MS + 4.44 μM BAP, 0.54 μM NAA, and 4.65 μM KIN|||
|Colloidal silver nanoparticles reduce hyperhydricity||2.5 mg L−1AgNPs (silver nanoparticles)|||
|Propagation disinfection process|
Double phase culture system
|growth regulators did not improve the morphogenic response|||
|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
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
|Identification of terpene synthasegenes in ||TPS gene|||
|Assessment of genetic diversity and relationships||ISSRs|||
|Assessment of genetic diversity andgeographic differentiation||ISSRs|||
|Development of 23 microsatellite primerpairs for ||Microsatellites|||
|Confirmation of genetic homogeneity in invitro regenerated plants||RAPDs|||
|Assessment of genetic diversity of wild populations||RAPDs|||
|Assessment of genetic diversity andchemical polymorphism of ||RAPDs|||
|Assessment of thyme genetic diversity in Palestine||RAPDs|||
|Correlation between the chemical andgenetic relationships among commercial ||RAPDs|||
|Essential oil composition and molecular analysis||RAPDs|||
|Assessment of genetic and chemical variability||ISRRs|||
|Assessment of genetic diversity and relationships among species of thegenus ||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
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
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
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
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].
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) .
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.
Many studies have shown that thyme (
The fungal activity of
|Thymus plant||Inhibited microorganisms||References|
|[72, 86, 94, 99, 100]|
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.
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