Long-Term Passage and Characteristics of Melissa officinalis L. Callus Cell Cultures

Abstract

The morphological, cytophysiological, and biochemical (formation of phenolic compounds) characteristics of the population of callus cultures cells of lemon balm (Melissa officinalis L.), a valuable medicinal and essential oil plant, were studied. Callus cultures were obtained from explants of hypocotyls and cotyledons of seedlings in vitro and were grown for more than 1.5 years (19 cultivation passages). The increase in callus weight did not differ significantly during the first seven passages; however, with further subcultivation, the intensity of crop growth (callus weight at the end of the growing cycle) increased. The maximum increase in callus was observed in passages 17–19: the growth indices of calli initiated from cotyledons and hypocotyls reached 13.7 and 11.5, respectively, which is 3.0–3.4 times higher than in the first cycles of crop cultivation. The obtained data indicate the possibility of long-term subcultivation of callus cultures of lemon balm cells, during which autoselection of cells occurs based on growth intensity. The dynamics of callus growth, density and viability of the cell population, and the ratio of various cell types in the culture growth cycle were determined for cell culture M. officinalis for the first time. The duration of the main phases of cell population growth has been established: lag phase from 1 to 6 days; growth acceleration phase from 6 to 10 days. The exponential growth phase took place from 10 to 14 days and was characterized by a high specific growth rate µ = 0.21 days^–1 From days 14 to 20, fixed phase of slowing down the growth of the culture (µ = 0.05 days^–1), which gave way to a phase of linear growth (20–30 days, µ = 0.08 days^–1) and stationary phase (30–40 days of the growth cycle). Thus, a “stepwise” character of culture growth has been established, which may be due to the presence in the culture of subpopulations of cells with different growth rates. During the initial screening, flavonoids and phenolcarboxylic acids were identified in calli of leaf origin in quantities comparable to leaves of intact plants, which indicates preservation of in vitro cells ability to form secondary metabolites and the prospects for further research in this direction.