Leucojum aestivum, a threatened Amaryllidaceae plant species, used as raw material for commercial production of galanthamine-based medicines in Bulgaria, has been multiplied in vitro by bulb regeneration. Twenty clones were initiated in 2003 from plants of different chemo-types and alkaloid contents obtained from the living collection of the Institute of Botany, Sofia. They were subcultured for 6 years under equal conditions on agar MS medium supplemented with BAP and NAA. The cultures kept the inherent alkaloid profiles and propagation coefficients of the mother plants. Galanthamine contents of the selected high-productive in vitro clones exceeded 1 mg/g DW and were comparable with those of the exploited Bulgarian populations of L. aestivum. However, dynamics in the biosynthetic activity were observed in all clones studied for at least 24 months. Different correlations were established between the dynamics of the main alkaloids galanthamine and lycorine, and the dry matter of the cultures of some clones and between clones. Secondary metabolic processes were stimulated by different means: elicitation with jasmonic acid, addition of alkaloid precursors in the medium, or musical treatment. However, the success of the biosynthetic stimulation was variable and depended on the course of the clone-specific dynamics during the period of treatment. The genotype was established as the crucially important factor for the biosynthetic capacity of the L. aestivum cultures, followed by the dynamics of the biosynthetic activity, and the application of external chemical or physical stimuli as modifications of the medium composition and consistence, and acoustic waves.