Partner und Internationale Organisationen
(Englisch)
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A, B, BG, CY, CZ, DK, EE, FIN, F, D, GR, H, IRL, I, LV, LT, NL, N, PL, RO, SK, SI, E, S, CH, GB
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Abstract
(Englisch)
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Medicinal plants used for the production of evidence based phytotherapy often do not conform to the requirements of the pharmaceutical industry with regard to homogenity and reproducible biological activity. Numerous studies prove that genetic variability also is true for medicinal plant species and greatly affects the quantitative and qualitative composition of active substances; if not addressed properly, this variability may lead to considerable differences in quality between batches of the same extract. However, so far very little efforts have been made to control or to utilize the genetic variability in medicinal plants. Therefore our research is aimed at the development of genetically homogenous varieties with improved biological activity using modern breeding tools. Extracts of the dried rhizome and roots of Valeriana officinalis L. are widely used in Europe as a mild sedative and sleep aid for insomnia, excitability, and exhaustion. So far no homogenous varieties are available for the commercial cultivation. Therefore we aimed at homogenous varieties that can be propagated by seeds. In order to speed up the breeding progress of this outcrossing species we applied gametic embryogenesis. We followed first two different approaches: (i) the in vitro culture of entire anthers and (ii) the culture of isolated microspores. After spontaneous or induced chromosome doubling, these regenerants will be completely homozygous and fertile. Inbred lines produced in this way can also be used as parents for the development of hybrids to avoid inbreeding depression and exploit heterosis. The main advantage of gametic embryogenesis over repeated inbreeding is that homozygous lines can be developed much faster; homozygous plants can be produced in one in vitro steps, while repeated inbreeding would require at least four to five generations (corresponds to 8 to 10 years) until the degree of homozygosity is sufficiently high. We have cultivated the donor plant for the gametic embryogenesis in the greenhouse, nursery and field. The best yield of flowering plants with high level of viable microspores was derived from the field in the season from May till June. After successful establishment of a method for determination of the ploidy level we selected diploid donor plants. We optimized the sterilization method for the anther culture and microspore culture for each experiment. Anther culture resulted in a high rate of calli. However, we were not able to regenerate plantlets from these calli testing 4 different regeneration media. DNA analysis of the calli revealed that they were not derived from microspores but from somatic tissue. Therefore we concentrated on the microspore culture. We tested different pre-treatment and fount best results with a pretreatment at 4°C for 7 days. After optimization of the microspore isolation we tested different basal media, different carbon sources, different osmotic values, different combinations of phytohormons as well as co culture with pistil. Across all experiments conducted from 2003 till 2006 we tested 40 different media for the induction of cell division of the haploid microspores. Best results were received with low microspore density of 10'000 microspores per ml incubated on the Lichter medium supplemented with 130g/l sucrose, 0.8mg/l 2,4-D + 2.7mg/l NAA + 3.9mg/l BA, and antibiotics (Cefotaxin 25mg/l, Vancomycin 25mg/l and Nystatin 25 mg/l). The number of cell colonies per inoculated microspore was 0.007%. This amount of cell colonies is comparable to other microspore cultures of dicotyledonous plant species. The regeneration of these micro calli to fertile plantlet was not successful until now using 12 different regeneration media. In order to gain more experience in the regeneration of calli, we established and optimised the in vitro propagation system for somatic tissue. With this method we could regenerate vigorous plantlets derived from callus cultures. In order to choose the best donor plants for our breeding purpose, we screened 50 accessions for their agronomic performance (root yield, vigour, resistance, root morphology), their secondary metabolites (essential oil content, valerenic acid, valepotriates) and amino acids (glutamine, GABA) and drug extract ratio. The plants with the highest valerenic and essential oil content were tested in in vitro assays for their pharmacological activity. For all the measured traits we found a great genetic variation. We also tested the seasonal effect on root yield and valerenic acid and determined Dezember till March as best harvesting time of valerian roots under our growing conditions. For the production of hybrids we screened several hundred individual plants for male sterility. We found one plant without microspore production but fertile pistil. This plant was cloned and used for crossing experiments.
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