[P-013]
GENETIC TRANSFORMATION IN MEDICINAL PLANTS AND THE POWER OF AGROBACTERIUM-MEDIATED TRANSFORMATION FOR PRODUCTION
OF BIOLOGICALLY ACTIVE COMPOUNDS

Iliana Ionkova1 and A. Alfermann2
1Faculty of Pharmacy, Department of Pharmacognosy, Dunav 2 Str., 1000 Sofia, Bulgaria
2Institut fur Entwicklungs und Molecularbiologie der Phlanzen, Heinrich-Heine, Universitat,
Universitatstr.1, Geb.26.13. D-4000 Dusseldorf, Germany

Due to their complex structures alkaloids, saponins, polysaccharides and flavonoids are still most efficiently produced by the plants. However there are several problems connected with this production method. Variable quantities and qualities of the plant material, plants that need to grow several years before they are ready for harvesting and over collecting of endangered species are just a few of the problems connected with the production of these natural products. Therefore, cultured cells rather than plants are as a possible alternative production method.

Genetic manipulation in plant cell cultures by delivering foreign DNA to plant cells allows for stable transgenic integration into recipient genome and permits not only sustained cell proliferation leading to formation of the transgenic event but also to production of plant chemicals of interest. The most commonly used transformation techniques is Agrobacterium-mediated gene delivery. The examples of phytochemical diversity from our investigations on the Astragalus, Althaea, Datura, Hyoscyamus plants will be present.

Four different bacterial strains were used in our experiments - TR 105, R 1601, ATCC 15834, and LBA 9402. The phenotypic response results from the insertion into the plant genome of T-DNA was different. The difference in virulence could be explained by the plasmids harboured by bacterial strains. Our experiments showed that transformation by TR 105 and R 1601 occurs at a low frequency compared to results observed using strains ATCC 15834 and LBA 9402. The last two show good virulence characteristics. The bacterial strains LBA 9402, which contains the plasmid pRi 1855 shows less dependence on the internal auxin levels in the experimental plant, with regard to its ability to produce roots, than several others. Agrobacterium strain had a some effect on growth and content of biologically active compounds. The clones infected by ATCC 15834 and LBA 9402 grew the fastest and the biomass increased 2-3 fold more than the other clones after 28 days of cultures. Moreover, different Agrobacterium strains have an effect on total saponin or alkaloid content in older hairy roots. Maximum saponin and tropan alkaloid content was found in HR, transformed with LBA 9402 bacteria.

A variety of experimental approaches have been used to increase the efficiency of the production of saponins, polysaccharides and alkaloids in different plant hairy roots. A major problem associated with organised cultures remains their growth on a large scale. Growth in liquid medium results in a root "nucleus" with young, rapidly growing roots on the periphery and a core of older tissue inside. Restriction of nutrient and oxygen delivery to the center of the mass gives rise to a growing pocket of senescent tissue. In our experiments, partial success has been achieved, leading to the formation of dense beds of roots with root hairs which provide a large surface for nutrient absorption and release of products into the medium. The excretion of some of this compounds to the liquid medium offers possibilities for continuous cultures. A rapidly growing root lines of Astragalus, Datura, Hyoscyamus and Althaea plants were selected for cultivation in an airlift fermenter .

In general HR are preferable to conventional cultures, because of the higher genetic stability, higher growth rates, and production of pharmacologically active compounds.

[Full paper: P-013]
[P-013]