Abstract

Plant cell and tissue culture is an established alternative to the harvest and extraction of whole plant material for the production of valuable secondary metabolites. Colchicine, a secondary metabolite of Colchicum autumnale and Gloriosa superba has anti-mitotic and anti-inflammatory properties, has been used for centuries in the treatment of gout and more recently for familial Mediterranean fever, and has been recognized for some time as an anti-tumour agent. This thesis investigated the biotechnological application of cell and tissue cultures of Colchicum autumnale and Gloriosa superba. Two analytical methods (HPLC and ELISA) have been developed and optimised for determination of the colchicine accumulated in liquid medium and in plant tissue. Callus tissue of Colchicum autumnale and callus and root tissue of Gloriosa superba has been established, growth kinetics and accumulation of colchicine examined, and culture optimized for accumulation of colchicine. Precursor feeding has been investigated and yields of colchicine have been increased in both plant cells and root cultures by the addition of relatively low levels of suitable precursors. Total Colchicine accumulated in Colchicum autumnale culture fed with ImM coumaric acid was 180(ig/g dry weight compared to 63 (_ig/g dry weight for the control and the same treatment to the Gloriosa superba culture accumulated 85|_ig/g dry weight compared to 22(j,g/g dry weight for the control. Continuous extraction of colchicine using Amberlite resin as a solid phase extraction has been demonstrated to increase production. Combination of precursor feeding with in situ extraction led to significant enhancement of colchicine production and recovery using a continuous in situ extraction system. Combination of precursor feeding and adsorption on an Amberlite column significantly enhanced the total colchicine accumulation at the end of the batch culture by 3.5 and 3.7 fold in a culture supplemented with 0.5mM coumaric acid or 3-phenyl propionic acid respectively. The growth rate of Gloriosa superba root culture in liquid medium can be estimated by simple, reliable and non-invasive methods by measuring the conductivity and the amount of soluble carbohydrate in the liquid medium. A simple model for enhanced production of colchicine has been developed. In this study the two types of bioreactor, air-lift bioreactor and trickle column bioreactor systems were examined for growing Gloriosa superba root tissues on a pilot scale.