Effect of Biotic Stress (Aspergillus niger) on the Production and Accumulation of Total Alkaloids in Atropa belladonna L. via Tissue Culture

An efficient protocol for enhancement of total alkaloids production from suspension cultures of A. belladonna L. was established. The effect of MS-medium supplemented with different concentrations of naphthalene acetic acid (NAA) and benzyl adenine (BA) on growth parameters as well as total alkaloid production was investigated. In the same connection, the effect of biotic stress caused by various concentrations of Aspergillus niger on alkaloid accumulation and production was investigated. The optimum values of cell growth parameters and alkaloid production were obtained for leaf, stem and root cell cultures, respectively. The optimum supplementation of liquid MS-medium was 1 mg/l of each of NAA and BA. A. niger extract at 10 % (~ 0.5 mg/ mL) gave the highest value for cell growth and alkaloid accumulation in the different types of cell cultures following 10 days of cultivation. Total alkaloid contents of different cell cultures were identified by HPLC. INTRODUCTION Plant secondary products are used extensively in commerce, particularly in the food and pharmaceutical industries, and many of these compounds have proved to be target compounds for plant cell culture production. There have been a number of excellent publications on this topic within the last decade. Plant cell cultures have the capability of producing a wide range of secondary compounds (Phillipson, 1990). Atropa belladonna L. (Solanaceae) is one of the most important medicinal plants and is a source of tropane alkaloids such as hyoscyamine and scopolamine. Medicinally, A. belladonna is used for the use of its alkaloids in the treatment of Parkinson’s disease for its anti-inflammatory properties, for relief of bronchial asthma and motion sickness and its ability to counteract toxic agents. Belladonna extract is used as an antimuscarinic agent, which accounts for its use as a spasmolytic drug. Also, it is used as a concomitant therapy in the treatment of peptic ulcer and functional digestive disorders, including spastic, mucous, and pancreatitis. Plant growth regulators and nutritional factors affect the production of secondary metabolites, as well as growth, of cell cultures of higher plants (Sakuta and Komamine, 1987; Szoke et al., 1982, 1992; Vida et al., 2000; Laszlo et al., 2001). Also, types and concentrations of growth regulators affect secondary product formation (Bohm, 1980; Krajewska et al., 1987; Bálványos et al., 2001). The presence of 2,4-dichlorophenoxy acetic acid (2,4-D), indole acetic acid (IAA) and (NAA) increased the production of indole alkaloids in cell cultures of Cinchona ledgeriana (Harkes et al, 1985). Moreover, Nussbaumer et al., (1998) reported that, half strength Gamborgs B5 medium supplemented with 5% sucrose and 1 mg/l of each of NAA and BA gave the best value for root culture growth of Datura candida x D. aurea. Meanwhile, full strength B5 medium supplemented with the same concentrations of NAA and BA gave the best results for hyoscyamine and scopolamine content. One of the methods frequently used to increase the productivity of plant cell culture is the use of so-called elicitors (Singh, 1996). Elicitors can be all types of compound that increase the production of phytoalexins (Muller, 1956 and Kuc, 1972). Phytoalexins are antibiotically active compounds and, by that, important factors in the Proc. Int. Conf. on MAP Eds. J. Bernáth et al. Acta Hort. 597, ISHS 2003