In recent years, along with the use of essential oil and medicinal plants traditional for the south of Russia, there have been an active introduction of new species, including representatives of Lamiaceae family such as Melissa officinalis and Origanum vulgare, into production. These plant species are widely used in perfumery, cosmetics and food industries, as well as in medicine, because of the number of biologically active compounds. Therefore breeding work is being carried out to create cultivars with a high content of essential oil and valuable components in its composition. Currently, biotechnological techniques are used to address a wide range of challenges of ecology, plant breeding and seed production. One of the most popular methods is clonal micropropagation. Biotechnologies of micropropagation in vitro have been developed for many agricultural and flowering ornamental plants. However, for lemon balm and oregano, many issues relating to propagation in tissue and organ culture have not been studied enough. In particular, this applies to the study of long-term propagation in vitro. The aim of our investigation was to study the morphometric parameters of explant development during long-term subcultivation of cultivars and breeding samples of M. officinalis and O. vulgare at the second stage of clonal micropropagation. For studies, we used three cultivars of Melissa officinalis L. (Tsitronella, Sobornaya, Krymchanka) and breeding sample No 10 of Origanum vulgare L. When introduced in vitro, axillary meristems with 2 leaf primordia were isolated from plants. At the second stage (micropropagation itself), stem segments (5-8 mm) with one node, obtained by microcutting of shoots developed from meristems, were used as explants. Explants were cultivated on Murashige and Skoog (MS) culture medium (which we modified earlier for oregano and lemon balm) supplemented with BAP and GA(3). The meristems and shoots were cultivated at 26 +/- 2 degrees C, 70% air humidity, 2-3 klx light intensity and 16-h photoperiod. After 30-35 days of cultivation, we determined the frequency of multiple shoot formation, the number and length of shoots, the number of nodes on the shoot, the frequency of rhizogenesis and hyperhydric shoots. The multiplication index was calculated as the number of microcuttings that were obtained after one subculture. To do this, the number of shoots formed on explants was multiplied by the number of nodes on a shoot. Hyperhydric microshoots were not taken into account. Then, we divided the shoots into microcuttings and transferred to a fresh culture medium. Experiments were repeated three times, and at least 20 explants were analyzed in each variation. The confidence of differences was assessed using Student's t-test, p<0.05. Mean values and standard errors are shown in tables and graphs. As a result of the research, we showed that with the introduction of M. officinalis meristems to the MS culture medium with 1.0 mg/1 BAP and 0.5 mg/1 GA(3), the development of the main and sometimes adventitious shoots occurred on the 7th-10th day of cultivation. After 35 days of cultivation, 1.3 (Tsitronella cultivar) to 4.2 (Sobornaya cultivar) shoots were formed from one explant, and their length varied from 5.8 to 8.2 mm. For further micropropagation of lemon balm, microshoots obtained at the first stage were divided into one node segments and transferred to the MS medium with 0.5 mg/lBAP. Cultivation of microcuttings during seven passages led to the formation of axillary and adventitious shoots in the studied cultivars (See Fig. 1, A). The frequency of multiple shoot formation, depending on the cultivar and passage, varied from 32.0% to 93.3% (See Table 1). In some variants of the experiment, hyperhydric microshoots were formed with a frequency of 15.1-23.4%. The morphometric parameters of Sobornaya and Krymchanka cultivars were 1.3-2.5 times higher than those of Tsitronella. During successive subcultivations in all cultivars, we revealed an increase not only in multiple shoot formation frequency but also in the length of shoots in some variants. It should be noted that at the second stage of micropropagation on a culture medium with BAP, many shoots developed roots (See Fig. 1, A). The rhizogenesis frequency, depending on the cultivar and passage, reached 44.4-92.7%. The revealed spontaneous rooting of M. officinalis cultivars on a nutrient medium for micropropagation allows, at a high rhizogenesis frequency, excluding the third stage of propagation and immediately transferring microplants with roots for adaptation ex vitro. When comparing the morphometric parameters of the microcuttings development at different passages, we found that in the studied cultivars the number of shoots varied from 2.2 to 5.7 pcs./explants, and the number of nodes varied from 2.2 to 4.1 pcs./shoot. The length of the shoots reached 55.4-73.8 mm. In Tsitronella cultivar, the maximum value of the studied parameters (See Table 1) and the multiplication index (up to 12.0) (See Fig. 2) were noted in the 3rd-5th subcultivations. For Sobomaya and Krymchanka cultivars - in the fifth passage, in which the multiplication index was 14.2 and 17.6, respectively. In the seventh subcultivation, the multiplication indexes of all studied cultivars decreased to 6.3-8.9, depending on the genotype. When meristems of O. vulgare were placed on MS culture medium, containing 1.0 mg/1 BAP, after a month of cultivation, they developed up to 4.5 shoots per explant of 17.0 mm long. The frequency of multiple shoot formation was 66.6%. At the second stage of micropropagation, explants (stem segments with one node) isolated from microshoots, developed from meristem, were cultured on MS medium with 0.5 mg/1 BAP. The growth of the main shoot started on the 5th-7th day, and the development of adventitious shoots - in 2-3 weeks (See Fig. 1 B). On the 35th day of cultivation, the frequency of multiple shoot formation varied from 75.0 to 95.2%, and the number of shoots ranged from 9.5 to 54.8 per explant depending on the number of subcultures (See Table 2). During micropropagation, individual shoots (from 5.2 to 28.5%, in different passages) formed 2-3 roots up to 2-3 cm in length. Sometimes, when oregano was cultivated, the hyperhydric microshoots with thickened vitreous anomalous stems and leaves were noted; they cannot be used for further propagation. The frequency of vitrified microshoots was 16.7-25.8%. However, it reached 44.5% in the fifth passage. When studying the effect of cultivation duration on micropropagation of 0. vulgare in vitro, 13 passages were conducted. The maximum number of shoots (54.8 pcs / explants) and the multiplication index (74.1) were obtained in the fifth subcultivation (See Fig. 3). With further subcultivations, the multiplication index decreased in the 10th passage up to 43.0. The lowest values of this parameter (16.9-18.8) were noted in the late 12th and 13th passages. Thus, the conducted studies indicate the possibility of long-term micropropagation of 0. vulgare and M. officinalis in vitro (at least for 1-1.5 years), while the maximum efficiency of this process was observed during the fifth subcultivation.
