Thymus vulgaris L. (Thyme)
Thymus vulgaris L. (Thyme): In Vitro Culture and the Production of Secondary Metabolites
Thymus is a perennial herb of the Labiatae family and can grow to a height of some 30 cm. There are more than 100 varieties of Thymus distributed throughout Europe. It has dark green leaves and light mauve-pink flowers in early summer. Thyme is a good rock-garden plant, as it spreads easily. It originates on the coast of the Mediterranean Sea and in the region of Asia Minor. Now it is cultivated in numerous European countries, Russia, Canada, America, and some of the North African countries. Thyme is one of the great culinary and medicinal herbs, and is a classic constituent of a bouquet garni, but only a few species are used in the kitchen, e.g., common thyme (Thymus vulgaris) and lemon thyme (Thymus citriodorus). The essential oil of thyme has also been reported to be effective in food preservatives, antioxidants, antimicrobials, breath-freshening dentifrices, medical disinfectants, etc. In general, the dried leaves contain 0.7-2.5% dry wt. of the essential oil. The major components are thymol (15-62%), carvacrol (2-44%), p-cymene, and y-terpinene.
One of our experiments involved the selection of strains according to visual color differences in the cells. Green, yellow, and white calli, those colors which are independent of the desired metabolite, were determined as the representative colors of thyme callus. Furthermore, we examined the correlation between color difference and the production of characteristic monoterpenes such as thymol and carvacrol. Another experiment was designed to trap the volatiles before these compounds can act as toxic substances. To promote the production of volatiles from tissue culture of the leaves of Thymus vulgaris L., special attention was given to the cell oil, because it has been found that volatiles can sometimes act as toxic substances. The cell has to have some organs, e.g., oil globules, in order to store the toxic materials or to carry the toxic and volatile compounds to the outside of the cells. The effect of several adsorbents on cell growth and the production of secondary metabolites was examined.
Conclusions and Prospects
The volatiles of thyme oil were identified by using retention indices (RI) of GC and GC/MS. The characteristic compounds were determined to be thymol (46.1%), carvacrol (3.4%), linalool (1.7%), l-octen-3-ol (1.2%), trans-sabinene hydrate (1.1%), terpinen-4-ol (0.7%), borneol (0.5%), camphor (0.2%), and 3-octanol (0.1%) from the sensory evaluations.
Thyme callus was induced and subcultured every 30 days on MS medium with 1 ppm NAA and 1 ppm kinetin or 1 ppm 2,4-D and 1 ppm kinetin with 3% sucrose and 0.9% agar at 25 °C under 2000 lx.
In the callus tissue of Thymus vulgaris L., eight terpenes, including p-chamigrene, nerolidol, geranylacetone, p-guaiene, cuparene, y-cuprenene, P-elemene, and thymol, were found. Only thymol and p-elemene were commonly identified in the intact plant and callus tissue. Sesquiterpene synthesis was considered to be superior to monoterpene synthesis due to the detection of seven sesquiterpenes in the volatiles of the callus tissue. This callus tissue produced a flavor similar to overripe, sweet oriental persimmon fruit, therefore, callus tissues are potentially capable of producing new flavors. It is important that the flavor regulates the biosynthetic pathway, thus, leading to monoterpene synthesis.
Some possible methods to regulate the productivity of metabolites in order to change the flavor quality are summarized as follows:
1. The concentrations of auxin and cytokinin in subculture affect the growth factor and productivity of volatiles in the callus. In the previous paper, it was reported that the concentration of p-chamigrene was enhanced 400-fold more than that of subcultured callus by changing the type and concentration of auxin and cytokinin from 1 ppm NAA and 1 ppm kinetin to 10 ppm IAA and 10 ppm kinetin.
2. From the results of transformation of some precursors of thymol, y-terpinene could be biotransferred to p-cymene and then thymol. Therefore, enzymes necessary for the production of thymol were found in the callus systems. In particular, oxidative activity was found to be high. Therefore, the addition of some precursors to the callus tissue may make it possible to use thyme flavors or other chemicals, for industrial purposes.
3. Cell selection is very important for plant cell culture. However, it is difficult to differentiate the high productive cells from the others because of the colorless chemicals. Nevertheless, cell lines of thyme were visually selected according to color differences. Green and yellow calli contained somewhat more thymol than the nonselected subcultured callus, especially in suspension culture. Visual selection of cells seemed to be effective for the production of secondary metabolites.
4. Finally, under two-layer culture conditions, a large amount of the produced ethanol was trapped in organic solution. It might be concluded that since primitive materials (primary metabolites) necessary for the production of secondary metabolites were metabolized and then trapped as ethanol, an increase in the flavor monoterpenes and sesquiterpenes could not be observed. If two-layer culture had been conducted at the final stage of culture to trap the final products, inhibition of toxic activity might have been possible.
To produce thyme flavors by plant tissue culture, yellow or green cells have to be prepared in suspension culture with MS medium with 1 ppm NAA, 1 ppm kinetin, and 3% sucrose. A combination of two-layer culture and additional precursors might be effective at the final stage, to increase the amount of the secondary metabolites.
H. Tamura, T. Takebayashi, and H. Sugisawa, Medicinal and Aromatic Plants IV