Mercè Bonfill

Ginsenoside production in different phenotypes of Panax ginseng transformed roots.

Transformed roots were obtained after the inoculation of sterile root discs of Panax ginseng C.A. Meyer with Agrobacterium rhizogenes A4. The established hairy root lines displayed three morphological phenotypes when cultured on hormone-free liquid Schenk and Hildebrandt medium. Most of the cultures showed the characteristic traits of hairy roots (HR-M), while others were either callus-like (C-M) or thin (T-M) without branching. The growth rate of the transformed root lines was always higher than that of untransformed roots, showing that the genetic changes caused by the A. rhizogenes transformation conditioned a higher biomass formation. When considering the different transformed root phenotypes, we can observe that the highest ginsenoside production was achieved by HR-M root lines, closely followed by C-M ones, whereas the lowest yield was reached by T-M root phenotype. The study of the integration of the TL-DNA and TR-DNA fragments of the pRiA4 in the root genome showed that the aux1 gene was always detected in HR-M and C-M root phenotypes which presented the highest biomass and ginsenoside productions. This fact suggests a significant role of aux genes in the morphology of Panax ginseng transformed roots. The ginsenoside pattern of transformed roots varied according to their morphology, although the ginsenoside contents of the Rg group was always higher than that of the Rb group. From our results, we can infer the potential of some root phenotypes of Panax ginseng hairy root cultures for an improved ginsenoside production.

Relationship between peroxidase activity and organogenesis in Panax ginseng calluses

The changes of redox status during culture and their effect on organogenesis were investigated in ginseng callus cultures. Three kinds of Panax ginseng calluses (non-organogenic calluses, root-forming and bud-forming calli) 21 were obtained from each of the three examined auxins (2,4-D, IBA and NAA) at 2 mg l−1 (9.05 μM 2,4-D; 9.84 μM IBA; 10.74 μM NAA) concentration and 0.1 mg l−1 (0.46 μM) kinetin over 5 weeks. Peroxidase in the soluble fraction, which indicates oxidative status and shows a correlation with differentiation, was higher in bud-forming calluses cultured in the three types of auxins. On the other hand, reducing capacity (overall reducing capacity of glutathione, ascorbic acid, tocoferol, carotenoids and phenol) which indicates reductive status, was higher when these calluses grew on media containing 2,4-D but specially so in the case of non-organogenic calluses. With the three types of auxin used, the reducing capacity was always higher in non-organogenic calluses and lower in calluses with more organogenic capacity. It seems that organogenesis of calluses is conditioned by a high peroxidase activity in soluble fraction (equal to or more than 0.8 A430/mg protein per min) and therefore a low reducing capacity (less than 0.3 A760/g DW). On the other hand 2,4-D promotes proliferation whereas NAA promotes morphogenesis.

Immobilization of Galphimia glauca Plant Cell Suspensions for the Production of Enhanced Amounts of Galphimine-B

We tested the capacity of Galphimia glauca cells to produce galphimine-B (G-B) when under the effects of a two-stage culture system: cell immobilization in Ca2+-alginate beads and culture scale-up from shake-flask to two different types of bioreactor (stirred and airlift). In the shake-flask culture, using optimum media for cell growth (first stage) and G-B production (second stage), the G-B yield was similar in both immobilised and free cells. However, while the free cells accumulated G-B within cytoplasmatic compartments, where it could not be recovered without cell disruption, immobilized cells excreted up to 100 % of the G-B produced. Immobilized cells grown in bioreactors running for 14 days with growth medium and an additional 26 days with production medium in batch mode showed a high G-B yield. The stirred bioreactor was the most efficient with a G-B content in the culture medium of 1381 microg.L (-1) at day 24 of culture.

Influence of hairy root ecotypes on production of tropane alkaloids in Brugmansia candida

Hyoscyamine and scopolamine are tropane alkaloids widely applied in medicine. Differences in alkaloid production and growth kinetics have been observed in Argentinian and Colombian ecotypes of Brugmansia candida hairy roots. The aim of this work was to analyze the production of key intermediates in tropane alkaloid synthesis in both ecotypes to determine differences in the biosynthetic pathway. Additionally, rolC gene expression was analyzed to determine its correlation with hairy root growth. The results showed a higher accumulation of polyamines in Colombian hairy roots, suggesting that there may be a rate-limiting enzyme in the last steps of hyoscyamine biosynthesis. Additionally, rolC gene expression was correlated with an improvement in hairy root growth, which supports the function of rol genes as growth modulators and suggests that metabolic engineering approaches involving rolC manipulation may be useful for the development of more efficient B. candida hairy root cultures for biotechnological applications.