Callusogenesis and Organogenesis of Thymus Cultivars In Vitro

. The article is devoted to the in vitro cultivation of three Russian cultivars of two Thymus species ( Th. vulgaris and Th. serpyllum ) in order to study the possibility of induction of callusogenesis and organogenesis. The developed technology opens up prospects for the induction of somaclonal variability, obtaining the source material for breeding, as well as regeneration of thyme plants after genetic transformation. For the study, we used nutrient media based on the mineral composition of the Murashige and Skoog medium with the addition of auxin (indole-3-butyric acid, indole-3-acetic acid) and cytokinin (6-benzyladenine, kinetin) substances. For the induction of callusogenesis, the use of stem explants on nutrient media supplemented with 2,4-dichlorphenoxyacetic acid (1 or 2 mg/L) can be recommended. The efficiency of shoot organogenesis also depends on the type of explant, and its rather low values in most experimental variants are apparently due to the accumulation of endogenous auxins and phenolic compounds. Thus, a cultivar-specific morphogenetic response to the use of various phytohormones and growth regulators was established, as well as the influence of the explant type on the efficiency of morphogenic processes.


Introduction
At present, much attention is paid to fundamental research of natural products of plant metabolism obtained by means of cell culture as well as to their use as an alternative source of various compounds for medicine, perfumery and other sectors of the national economy.This possibility is associated with the fact that cultivated cells, as a rule, retain the ability to synthesize secondary substances specific for the plant species from which they are obtained [1].In turn, the products of plant metabolism, in particular essential oils, can be used in tissue culture as an alternative to the traditional sterilization of plant explants with chemical reagents.Antimicrobial activity of Cistus ladanifer L. and Cistus salvifolius L. essential oils has been reported against microorganisms such as Salmonella choleraesuis, Kregervanrija fluxuum, Bacillus cereus and Priceomyces carsonii [2].And Brazilian scientists have studied the effect of essential oils of plants Origanum vulgare L., Allium sativum L., Cymbopogon nardus L. and Zingiber officinale Rosc on reducing the growth of microbial contamination and on the survival of explants during clonal micropropagation of Myrciaria dubia [3].A group of scientists from Tunisia shown the results of studying the effectiveness of using rosemary (Salvia rosmarinus Spenn.)essential oil to control enzymatic browning and contamination of Aloe vera L. plants in in vitro culture [4].
In vitro culture methods open up a wide range of possibilities for secondary metabolite producers.The researcher is able to change the conditions in which the plant is located, thereby influencing its growth, as well as regulating its metabolic pathways.These conditions include many parameters, such as temperature, light quality and intensity, substrate or atmosphere composition.Mulder-Krieger et al. [5] examined the production of essential oils and aromatics in cell and tissue culture in 70 plant species, including mosses, conifers, monocots, and dicots.There were often significant differences between plant material in vitro and in vivo; several target compounds may even be absent when grown in vitro [5,6].Tamang and co-authors [7] also demonstrated that Picrorhiza kurroa Royle ex Benth.plants obtained in vitro had a reduced content of therapeutic secondary metabolites compared to their wild counterparts.The main condition affecting the accumulation of hepatoprotective substances in R. kurrooa in in vitro culture was the loss of endophytic communities associated with plants.
Differences in the biosynthetic ability of plant tissues indicate, on the one hand, the diverse role of secondary metabolites in the process of cell life, and, on the other hand, depend on the species specificity of the studied plants.In calli of plants of different species, varieties, and even clones, the relationship between the studied processes and the accumulation of phenols may be directly or inversely proportional, or even completely absent.Consequently, it is difficult to obtain an in vitro cell culture that has both a high growth rate of the culture and a high content of secondary metabolic products [8].
In the literature, there are some data on the culture of isolated tissues and organs of different Thymus species.These studies mainly concern clonal micropropagation by in vitro culture with the selection and optimization of nutrient media [9][10][11][12][13], as well as the ex-vitro transfer of plants [14].The influence of environmental conditions and biofertilizers on the accumulation of secondary metabolites in vivo is also being studied [15].
For example, the production of secondary metabolites of Th. vulgaris was the subject of Tamura et al. (1993), who focused on the selection of callus cells and the management of cells in a nutrient medium for the production of secondary metabolites.He established a correlation between the color of callus and the presence of thymol and carvacrol.On agarose media, green and yellow calli were found to produce trace amounts of thymol, while white callus produced ethanol.The addition of mevalonic acid increases the formation of the amount of volatiles up to two times compared to the control.Thus, it was shown that the enzymatic activity of the synthesis of monoterpenoids depends on the type of callus [16][17].
Thus, the decisive factor for the production of secondary metabolites in in vitro culture is the possibility of callusogenesis induction and control of the further development of callus cells: an increasing cell biomass or secondary cell differentiation -regeneration by shoot and root organogenesis.In addition, the production of callus tissue and subsequent regeneration make it possible to induce somaclonal variation, which plays an important role in the process of creating a source material for plant breeding, as well as to increase the multiplication factor of plants by the development of in vitro germplasm collections [9].

Plant materials
Seeds of various Thymus species (Th.vulgaris, Th. serpyllum) were used as the source material for in vitro culture introduction.Th. vulgaris (common thyme) cv.Limonny (from AGRONIKA Ltd.) is a perennial shrub up to 35 cm tall, the plant is highly aromatic, with a spicy warm scent.The leaves contain an essential oil with thymol and carvacrol as the main components.In addition to vitamin C, carotene, trace elements, there are tannins and resinous substances, other useful components.Th. vulgaris Alpiysky med (from "ZTEK AELITA" Ltd.) is a perennial shrub up to 40 cm tall, the stem is highly branched.It has a strong aroma and is an excellent honey plant.Th. serpyllum (creeping thyme) cv.Pikantny (from agrofirm "SeDeK") is a dwarf shrub with creeping stems, up to 20 cm tall, forms small cushion tufts.The flowers have a strong aroma and pleasant honey smell.The plant contains a large amount of vitamin C, and also has a bactericidal and disinfectant effect.
The indicators of the explant state were the frequency of callus formation, the frequency of root and shoot organogenesis.The resulting calli were regularly transplanted onto fresh medium after 28...30 days.The explants were cultured at a 16-hour lighting.The morphogenesis frequency (%) was assessed by the proportion of explants producing morphogenic structures of the total number of explants.All tables show the average values of the parameters with the indication of the confidence interval.

Induction of callusogenesis
As a result of the studies, it was found that when the leaf and stem explants were cultivated on most of the analyzed nutrient media, callusogenesis was induced already on the 10...14th day.Callus obtained from different explant types had morphological differences (Figure 1).A dense beige callus, sometimes with white areas, usually formed from the stem segments (Figure 1a).Callus tissue obtained from leaf explants was also dense and had a green or light green color with beige patches (Figure 1b).
The morphological characteristics of callus did not depend on the modifications of the nutrient medium we used, with the exception of the medium containing 2,4-D.In this case, from the stem and leaf explants, a hydrated loose callus was formed, which easily disintegrates into fragments.
For Th. vulgaris cv.Limonny the maximum frequency of callus formation was observed on stem explants when cultivated on the media containing 1 mg/L(84 %) or 2 mg/L2,4-D (80 %) or 1 mg/LBA (80 %).Leaf explants formed callus with the highest frequency of 74 % on the medium with 2 mg/L2,4-D, but on the most of media leaf explants formed no callus (Table 1).For the cv.Alpiyskiy med, the highest frequency of callusogenesis is observed on stem segments on the media containing 2 mg/L 2,4-D (77.1 %) or on leaf segments on the media containing 3 mg/L BA + 0.5 mg/L IAA (80 %) (Table 1).
For Th. serpyllum cv.Pikantny on a nutrient medium supplemented with 1 mg/L 2,4-D, the highest frequency of callus formation on stem explants was 80 %.On the medium containing 1 mg/L BA + 0,1 mg/LIAA the highest frequency of callus formation on leaf explants were observed (84 %) (Table 1).
It should be noted that none of the cultivars formed callus on a nutrient medium containing kinetin.

Induction of organogenesis
Root organogenesis from stem explants occurs only on nutrient media containing 0.5 mg/L IAA or 1 mg/L IBA by cv.Limonny (78.5 %) and cv.Alpiysky med (71.6 %) (Figure 2).By cv.Pikantny, no root organogenesis occurs on any medium variant.Root organogenesis does not occur from leaf explants on any medium variant.Th. vulgaris cv.Limonny formed shoots with a very low frequency (less 32 %) almost only on stem explants.The same trend is characteristic of cv.Alpiysky med except the variant with 3 mg/L BA + 0.1 mg/L IAA (78 %).Th. serpyllum cv.Pikantny has a high degree of shoot organogenesis also on media containing the combination 3 mg/L BA + 0.1 mg/L IAA (Table 2, Figure 3).Leaf segments were less likely to form regenerants.From the Table 2 it be seen, shoot organogenesis was observed by all 3 cultivars only on the nutrient medium containing 1 mg/L kinetin.
On the nutrient media containing 2,4-D, shoot organogenesis was absent for all three cultivars on both stem and leaf explants.

Discussion
The study shown that the callusogenesis induction for three considered Thymus cultivars depends on both hormonal content of nutrient media and the explant type.Generally, stem explants are more preferable as leaf explants, for Th.serpyllum this trend is not so pronounced.The medium containing kinetin as a cytokinin component does not allow getting callus.As expected, the highest callusogenesis efficiency was observed on the nutrient media with 2,4-D.This auxin provides the callus formation frequency up to 80 %.Th. serpyllum cv.Pikantny has a higher ability to form callus as two Th.vulgaris cultivars.The reason for this may be the reduced content of phenolic compounds, which can disrupt morphogenetic processes.
When the primary callus was passaged on a nutrient medium with a lower auxin concentration, the secondary callus was formed rather quickly.The transplant callus culture did not have morphological differences depending on the explant type.The callus tissue had a rather dense structure; as a result of long-term cultivation of callus on a nutrient medium, an accumulation of phenolic compounds was observed in the callus tissues and in the medium, after two weeks the callus and the nutrient medium acquired a brown, sometimes dark brown color.Phenolic compounds are one of the most common secondary metabolites, the formation of which is characteristic of almost all plant cells [18,19].Thus, in the variant with MS medium containing 1 mg/L BA, during long-term (19 weeks) cultivation of the callus of the cv.Limonny, secondary metabolites were formed, the callus changed color from green to black, and oily droplets formed on the surface, presumably an essential oil.
The low frequency of root formation may be associated with high concentrations of endogenous auxins, which is indirectly evidenced by data on the effectiveness of shoot organogenesis.The fact that root formation occurred only on media with IAA and IBA correlates with the data of Furmanowa & Olszowska [20], which shown that the best rooting was achieved on a nutrient medium containing 0.5 mg/L of IBA.The other authors found that the higher (over 0.05 mg/L) cytokinin concentrations inhibit rooting and shoot growth [21].In our study shoot organogenesis was observed only on the nutrient media with a high ratio of cytokinin to auxin, what may indicate an excess of endogenous auxins.An additional factor in reducing the efficiency of organogenesis by the cv.Limonny could be the accumulation of phenolic compounds described above.

Conclusions
For Th. vulgaris cv.Limonny, the best nutrient medium for the callus formation is the MS medium with 1...2 mg/L 2,4-D and 1 mg/L BAP.Callus is formed to a greater extent from stem segments.For shoot organogenesis, it is preferable to use a medium with kinetin.Root organogenesis occurs on the MS media containing 0.5 mg/L IAA and 1 mg/L IBA.
For Th. vulgaris cv.Alpiysky med, the best medium for the callus formation is the MS medium with 2 mg/L 2,4-D and 3 mg/L BAP + 0.1 mg/L IAA.Shoot organogenesis is induced by a medium supplemented with 3 mg/L BAP + 0.1 mg/L IAA.Roots are formed from stem segments on the media with 0.5 mg/L IAA or 1 mg/L IBA.
Th. serpyllum cv.Pikantny has the highest ability to form callus from both stem and leaf segments.Nutrient media containing 2 mg/L 2,4-D or 1 mg/L BAP + 0.1 mg/L IAA are highly active in the callusogenesis induction.For shoot organogenesis, it is preferable to use a nutrient medium in combination with 1 mg/L BAP + 0.1 mg/L IAA.Root organogenesis does not occur on studied nutrient media.

Table 1 .
Callusogenesis rate from Thymus explants on different nutrient media.

Table 2 .
Shoot organogenesis rate from Thymus explants on different nutrient media.