The effect of varying levels of Gamborg's B5 salts and temperature on the accumulation of starch and hyoscyamine in batch cultures of transformed roots of Datura stramonium

Abstract

The partitioning of carbon between reserve polysaccharide and alkaloid secondary products was investigated in batch cultures of transformed roots of Datura stramonium grown in media in which the carbon substrate concentration was held constant and the level of mineral nutrients was varied. The growth and accumulation of starch and hyoscyamine was examined in roots grown at temperatures of 20°C, 25°C or 30°C in media containing 5% sucrose and levels of mineral nutrients varying from ¼ to twice the standard level of Gamborg's B5 salts. The dry matter content was highest (up to 15% w/w) in roots grown at either 20°C or 25°C in medium of the lowest ionic strength (¼ B5 salts) and decreased as the ionic strength was raised (down to 7% w/w with 2 B5 salts). Up to half of this decrease could be accounted for by loss of starch from the roots. At 20°C and 25°C, the starch content of the roots grown in medium of the lowest ionic strength (1 B5) was 40 mg g-l and 22 mg g-1 fresh weight respectively but decreased to less than 1 mg g-1 weight at either temperature when the ionic strength of the medium was raised to 2 B5. At 30°C, starch accumulation was severely inhibited in all media. In contrast, varying either the temperature or the ionic strength of the medium had only a small effect on hyoscyamine accumulation which remained at between 0.4-0.6 mg g-1 fresh weight. Although increases in the level of mineral salts had little effect on the hyoscyamine content of the roots, total yields however, increased due to stimulation of growth. Time course experiments showed that cultures grown at either 20°C or 25°C continued to accumulate both starch and hyoscyamine into late stationary phase.

Influence of the ion-composition of the medium on alkaloid production by “hairy roots” ofDatura stramonium

“…From these results, it can be concluded that hyoscyamine yields increased for the duration of the experiment (Figure 1). Robins et al (1990) and Hilton and Rhodes (1994) had maximal yields after 4 weeks of cultures. These differences can be explained by the greater volume of culture medium, the small inoculum (2 root tips/flask) and by the sucrose concentration (2%) used.…”

Hairy Root Culture for Mass-Production of High-Value Secondary Metabolites

“…Variables examined for their influence on growth and secondary Bonhomme et al, 2000 A. belladonna Atropine and hysocyamine Jung and Tepfer, 1987 Cinchona ledgeriana Quinine, quinidine, and cinchonidine Hamill et al, 1989 Datura innoxia Hysocyamine and scopolamine Shimomura et al, 1991a D. quercifolia Scopolamine and hysocyamine Dupraz et al, 1994 D. candida Scopolamine and hysocyamine Christen et al, 1991 Valpotriates Granicher et al, 1992 metabolite production from hairy roots include different basal media , sucrose level (Uozumi et al, 1993), exogenous supply of growth hormone (Bais et al, 2001), nature of the nitrogen source and their relative amounts (Norton and Towers, 1986), and phosphate concentration (Taya et al, 1994). Physical factors including light (Hirata et al, 1991;Yu et al, 2005), temperature (Hilton and Rhodes, 1994;Yu et al, 2005), presence of chemicals inducing physical stress (Sim et al, 1994), and magnetic field induction (Kato et al, 1989) have also been reported to affect secondary metabolite production from hairy roots. Betacyanin release from hairy roots of Beta vulgaris was achieved by oxygen starvation .…”