Galanthamine, an isoquinoline alkaloid acetylcholinesterase inhibitor, is an important agent used all around the world for the symptomatic treatment of senile dementia of the Alzheimer's type. The production of this metabolite and the availability of the plant are limited and prompted the search for an alternative way to obtain this valuable metabolite using in vitro cultures of Leucojum aestivum L. It is known that cell differentiation level shows a major influence upon the accumulation of alkaloids. For this reason, tissue cultures of L. aestivum showing different stages of morphogenesis controlled by exogenous growth regulators were established. Agrobacterium rhizogenes strain LBA 9402 has been tested for its capacity to induce hairy roots of this monocotyledonae plant.

In vitro cultures, at different stages of morphogenesis, were established from leaves of Leucojum aestivum (Amaryllidaceae) for determination of their galanthamine content, an alkaloid that possesses cholinesterase inhibitory activity and which is used for the treatment of Alzheimer's disease. A suitable HPLC method for qualitative and quantitative determination of galanthamine in both in vitro and in vivo extracts has been developed. Confirmation of the identity of galanthamine in sample extracts was achieved using LC-MS-MS. A correlation was observed between the state of differentiation and the galanthamine content of the tissue cultures. No galanthamine was detected in the roots grown in vitro, while all bulblets grown in vitro showed the presence of this alkaloid, with dramatic variations in concentration levels, according to the growth substance balance. The best result (6.79 x 10-3 % of D.W.) was obtained with bulblets initiated with NAA (10 µM) combined with BA (0.5 µM).